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3D Printing and Assay Development for Point-of-Care Applications
by
SHREESHA JAGADEESH
A thesis submitted in conformity with the requirements for the degree of Masters of Applied Science in
Electrical & Computer Engineering Department University of Toronto
© Copyright by Shreesha Jagadeesh 2016
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3D Printing and Assay Development for Point-of-Care
Applications
Shreesha Jagadeesh
Masters of Applied Science
Department of Electrical & Computer Engineering
University of Toronto
2016
Abstract
Existing centralized labs do not serve patients adequately in remote areas. To enable universal
timely healthcare, there is a need to develop low cost, portable systems that can diagnose
multiple disease (Point-of-Care (POC) devices). Future POC diagnostics can be more multi-
functional if medical device vendors can develop interoperability standards. This thesis
developed the following medical diagnostic modules: Plasma from 25 µl blood was extracted
through a filter membrane to demonstrate a 3D printed sample preparation module. Sepsis
biomarker, C - reactive protein, was quantified through adsorption on nylon beads to demonstrate
bead-based assay suitable for 3D printed disposable cartridge module. Finally, a modular
fluorescent detection kit was built using 3D printed parts to detect CD4 cells in a disposable
cartridge from ChipCare Corp. Due to the modularity enabled by 3D printing technique, the
developed units can be easily adapted to detect other diseases.
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Acknowledgments
My Master’s degree at the University of Toronto has been a great experience to me and has
provided me invaluable insights into academic research at the top Canadian institute of higher
education. The past 2 years have been filled with fond memories of my time at the beautiful St.
George campus in the heart of Toronto, one of North America’s largest metropolises. I would
like to thank the following people without whom this thesis would not be possible.
Professor Stewart Aitchison has been an incredible supervisor not only providing me excellent
guidance for my research work but also giving me valuable career advice on industrial
collaboration. He has provided me with a free and flexible academic environment enabling me
to carry out my thesis research with complete freedom. In addition, he has been available
whenever I have questions about my coursework and research directions. He has provided me
complete freedom in choosing co-curricular activities like training courses, seminars and
conferences. I am grateful to Professor Iain Thayne from the University of Glasgow, my alma
mater, for recommending me to him back in 2013.
I am grateful to my colleagues and other researchers in the Galbraith office and other
researchers including Suthamathy Sathananthan, Matthew Shipton, Pisek Kultavewuti, Arash
Joushagani, Mohammad Alam, Xiao Sun, Kevin Joseph, Kevin De Haan, Yuan Ming Chen and
Zhongfa Liao for providing me tips on good research practices and navigating the University of
Toronto academic system.
I am especially thankful to Dr. Lindsey Fiddes and Dr. Dan Voicu from the MIE’s Center for
Microfluidic Systems for training me on all the cleanroom equipment and supplying me with
consumables. In particular, Lindsey has been an invaluable resource on 3D Printing and Soft
Lithographic techniques on which my thesis is based on. I am also thankful to Dr. Carol
Laschinger and Aric Pahnke from Dr. Milica Radisic’s research group for providing me access to
their 3D printing facilities at short notice. I would like to thank Dr. Douaud Shah from the
Techna Institute for allowing me to use the cleanroom facilities and training me.
My special thanks to ChipCare Corp for providing me space at their lab for conducting
fluorescence assays. I am also indebted to their Dr. Lu Chen, Dr. Susan Bortolin and Rakesh
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Nayyar for guiding me during the assay development process by providing bio-chemical
expertise and troubleshooting my experiments. My special thanks to Jun Yang for helping me to
get started on the experiments by providing technical usage instructions. I am grateful to James
Dou for accepting me as a Mitacs intern and to James Fraser for providing me with a 3D printer
and more than adequate resources to perform whatever experiments I chose to conduct.
On a personal note, I would like to thank my parents and my uncle for not just providing me a
line of credit but also guiding me during the entire degree. To all these people, I am eternally
indebted for giving me a chance to study at the top research institution in Canada.
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Table of Contents
Acknowledgments .................................................................................................................... iii
Table of Contents ....................................................................................................................... v
List of Abbreviations ............................................................................................................. viii
List of Tables ........................................................................................................................... xi
List of Figures ......................................................................................................................... xii
List of Appendices ................................................................................................................... xx
Chapter 1 .................................................................................................................................... 1
Chapter Organization ............................................................................................................ 1
1.1 Aim ................................................................................................................................ 1
1.2 Chapter 2: Introduction .................................................................................................. 3
1.3 Chapter 3: Sample Preparation Module ......................................................................... 4
1.4 Chapter 4: Bead-based Assay ........................................................................................ 4
1.5 Optical Detection System .............................................................................................. 4
1.6 Chapter 6: Future Work and Conclusion ....................................................................... 4
Chapter 2 .................................................................................................................................... 5
Introduction ........................................................................................................................... 5
2.1 Motivation for POC Modular Design ............................................................................ 5
2.2 Advantages of POC ........................................................................................................ 7
2.3 Reason for POC blood tests ......................................................................................... 10
2.4 Advantages of 3D Printers ........................................................................................... 11
2.5 3D Printers in Research ............................................................................................... 13
2.6 Comparison of commercial desktop 3D printers ......................................................... 14
Sample Preparation Module ................................................................................................ 15
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3.1 Background .................................................................................................................. 15
3.2 Methods of Blood Filtration ......................................................................................... 17
3.3 Centrifugal-force based separation .............................................................................. 17
3.4 Filter Membrane-based separation ............................................................................... 28
3.5 Plasma Extraction Experiment ..................................................................................... 29
Chapter 4 .................................................................................................................................. 32
Bead-based Assay Module .................................................................................................. 32
4.1 Sepsis Literature Review ............................................................................................. 32
4.2 Bead-based Assay Review ........................................................................................... 38
4.3 Assay Development ..................................................................................................... 41
4.4 Adsorption on bead surface ......................................................................................... 45
4.5 Streptavidin-Biotin Assay ............................................................................................ 47
4.6 C - reactive Protein assay ............................................................................................. 51
Chapter 5 .................................................................................................................................. 58
Optical Detection System .................................................................................................... 58
5.1 Point-of-Care manufacturer ......................................................................................... 58
5.2 Existing HIV diagnostic methods ................................................................................ 58
5.3 HIV monitoring using CD4 counts .............................................................................. 61
5.4 Fluorescence Microscope Background ........................................................................ 62
5.5 System Description ...................................................................................................... 64
5.6 Issues resolved ............................................................................................................. 68
5.7 Verification by CD4 Cell and fluorescent bead counting ............................................ 69
Chapter 6 .................................................................................................................................. 70
Future work and conclusions .............................................................................................. 70
6.1 Conclusions .................................................................................................................. 70
6.2 Future Work ................................................................................................................. 71
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References or Bibliography ..................................................................................................... 74
Appendix A: Solidworks Drawings .................................................................................... 85
Appendix B: COMSOL Simulation for Chapter 3 ............................................................ 102
Appendix C: Streptavidin and CRP adsorption recipes (Chapter 4) ................................. 120
Appendix D: 3D Printing tips ........................................................................................... 124
viii
List of Abbreviations
ASSURED Affordable Sensitive Specific User-friendly Rapid Equipment-free and Delivered to
those who need it
CDC Centers for Disease Control
CNT Carbon Nano Tubes
CRP- C-Reactive Protein
CSR Corporate Social Responsibility
DA Diagnostic Accuracy
EHR Electronic Health Record
EMR Electronic Medical Record
FDA Federal Drug Administration
FDM- Fused Deposition Modelling
GSID Global Solutions for Infectious Diseases
HRP Horse Radish Peroxidase
ICU- Intensive Care Unit
IEEE Institute of Electrical and Electronics Engineers
IP Intellectual Property
ISO International Organization for Standardization
IVD In vitro Diagnostics
HCV Hepatitis C Virus
HIV Human Immuno Virus
ix
HCV Human Papilloma Virus
LAMP Loop Mediated Isothermal Amplification
LCA Ligase Chain Reaction
LMIC Lower and Middle Income Countries
LOD Limit of Detection
MDA Multiple Displacement Amplification
NASBA Nucleic Acid Sequence-based Amplification
NGO Non-Governmental Organization
NPV Negative Predictive Value
PCR Polymerase Chain Reaction
PDMS Poly-DiMethylSiloxane
POC Point of Care
POCT Point-of-Care Testing
PPV Positive Predictive Value
QA Quality Assurance
R&D Research and Development
RCA Rolling Circle Amplification
SDA Strand Displacement Amplification
SLA- Stereo lithography
TB Tuberculosis
x
TEC – Thermoelectric Controller
TMA Transcription Mediated Amplification
WHO World Health Organization
xi
List of Tables
Table 2-1 Comparison of some popular 3D printers [35,36]. The Form1+ and the Zortrax were
used in this thesis. ......................................................................................................................... 14
Table 4-1 Definitions of Diagnostic Accuracy of Biomarker (Courtesy [59]) ............................. 35
Table 4-2 Diagnostic Accuracy (DA) Values of C-Reactive Protein, Procalcitonin, Serum
Amyloid A, Mannan and Antimannan and IFN-γ -inducible Protein Biomarkers (Courtesy [59])
....................................................................................................................................................... 35
Table 4-3 Commercial CRP detecting systems (Courtesy [59]) All except the last two, use either
plasma or serum instead of whole blood to quantify CRP. ........................................................... 37
Table 7-1 Qualitative Assay for detecting Streptavidin on Nylon beads .................................... 120
Table 7-2 Quantitative assay for fluorescent detection of Streptavidin on Nylon bead ............. 121
Table 7-3 Direct (Non-specific) Adsorption procedure for detecting CRP on Nylon Beads .... 122
Table 7-4 Sandwich assay for CRP detection with coating of antibody C6 on Nylon bead ...... 123
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List of Figures
Figure 1:1 Applications of 3D printing in various stages of Point-of-Care Diagnostics. In this
thesis, a sample preparation module, disposable cartridge module for bead-based assay and an
optical detection system were built using parts created from a 3D printer ..................................... 1
Figure 1:2 The above schematic illustrates the utility of having a diagnostic kit with a constant
detection scheme and a variable bio-marker cartridge. The development of new assays would
require just replacing the cartridge instead of developing an entirely new diagnostic kit .............. 3
Figure 2:1 Typical steps followed in diagnostic instruments. Sampling from the patient (blood,
urine, saliva, etc.) is usually done off-chip. The biological markers present in the sample are
usually detected on a disposable chip after stimulation from an optical source. The signal can be
collected using a camera such as a Charge-Coupled Display (CCD) camera. Finally, the results
are interpreted in the analysis step. ................................................................................................. 5
Figure 2:2 Framework for understanding 3D Printing paths and values ([22]). Various levels of
adoption of 3D printing are seen in businesses. Some businesses tend to replace their entire
supply chain, while others seek out 3D printing capabilities only when rapid prototyping or cost-
effective customization capability is needed. ............................................................................... 11
Figure 3:1 Blood Composition (Reprinted with permission from © M. Kersaudy-Kerhoas and E.
Sollier [39]) Red Blood Cells (RBCs) and Plasma occupy nearly occupy the entire volume of
blood. Plasma contains useful biomarkers for different diseases and needs separation from RBCs
to improve optical detection performance. ................................................................................... 15
Figure 3:2 In helical/spiral channels, centrifugal forces cause recirculation patterns that tend to
concentrate particles within the vortices [47] ............................................................................... 17
Figure 3:3 Geometry of a straight channel developed in COMSOL Multiphysics. The channel
was 20 mm long and had a uniform rectangular cross-sectional area of 2 mm x 2 mm. .............. 19
Figure 3:4 A) (Uniform) Velocity Profile at the inlet of a straight channel (0.05 m/s) and B)
(Parabolic) Fluid velocity profile at the output of the straight channel. The simulation illustrates
the effects of solving a 3-dimensional fluidic equation. ............................................................... 20
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Figure 3:5 Time lapse snapshots of particles at t=0 s and 0.1 s. In A) the particles were uniformly
distributed to the inlet boundary condition and had the same velocity of 0.05 m/s. In B) The
particles had traversed 5 mm along the channel and they had a non-uniform velocity profile as
indicated by the darker red colors in the middle and (slower) blue colored dots in the periphery
Focusing of particles as they traverse the length of the channel was observed. ........................... 21
Figure 3:6 Effects of the particle diameter on the flow trajectories. A) 10 um particle trajectories
and B) 100 µm particle trajectories. Larger particles (100 um) were seen to be significantly
affected by gravity compared to the smaller 10 um particles. ...................................................... 22
Figure 3:7 A 2-loop helix with 2 mm x 2mm channel dimensions, 20 mm helix diameter and 20
mm pitch. (All shown dimensions in the graph are in millimeters). The outlet was geometrically
split into two equal sections to observe the effects of particle separation. The helix was drawn in
Solidworks. ................................................................................................................................... 23
Figure 3:8 Velocity profile at the output of the 2-loop helix. Higher velocity of the fluid was
noticed at the outer edges of the channel compared to the inner side. This is shown by the darker
red regions towards the left side of the output face. ..................................................................... 24
Figure 3:9 Snapshots of particle trajectories at A) t=0 s, and B) t=0.2 s when particles reach the
outlet. As noticed from B), the particles were more concentrated at the (outer) wall of the
channel denoted by Outlet 1. It was also observed that there were particles, which never reached
the output irrespective of how long the simulations were run for. They are seen in B) as blue
particles trailing in the channel. .................................................................................................... 24
Figure 3:10 Excel graph of enrichment factor for different diameters and particle numbers. The
data suggests that the number of particles at the inlet does not have a significant effect on the
enrichment factor. It stays constant at ~1.30. This would be imply that the Outlet 1 has 30%
more particles than Outlet 2. This would be ideal for blood cell separation because the number of
cells would be different for different patients. .............................................................................. 25
Figure 3:11 The particle diameter’s influence on the Enrichment Factor. A rising trend was seen.
Theoretically, infinite Enrichment Factor (100% separation into the Outlet 1) would be seen for
particles diameter larger than 0.140 mm. ...................................................................................... 26
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Figure 3:12 The solution time dependency on the number of (1 µm) particles. Orders of
magnitude increase results in less than a linear increase in the time taken to complete the
simulation. ..................................................................................................................................... 27
Figure 3:13 Design 1 for the filtration module. The capillary channels of 0.5 mm heights and
widths are seen as horizontal stripes in the middle. The hole in the center leads to a collection
well. Note that the filter membrane was transparent and covers an area sli ................................. 29
Figure 3:14 Fluorescent images of A) beads present in the whole blood before filtration and b)
after filtration in the extracted plasma from capillary channels. The presence of beads in B)
indicate the failure of the filtration process through leakage of the whole ................................... 30
Figure 3:15 A) Blood droplets in the six equally spaced inlets on the clamp part for uniform
distribution of the blood and right B) After filtration picture with the clamp removed. The
presence of the clamp secured the membrane filter and reduced the air gap. Thus, plasma was
seen to be more widely dispersed throughout compared to the previous filter design. The
Solidworks drawing files are listed in Appendix A. ..................................................................... 31
Figure 3:16 Fluorescent images of a) remnants of blood on top of the filter and b) extracted
plasma from the capillaries. The second design with the clamp on top of the membrane filter
prevented leakage and hence no fluorescent beads were seen. ..................................................... 31
Figure 4:1 Classification of Inflammatory response ([59]). Sepsis is diagnosed when both
infectious agent and inflammation is present in the patient. The cause of this infection could be
bacteria, fungus, virus, etc and they are denoted by circles indicating the relative frequency at
which they occur. Similarly, inflammation could be because of trauma, burns, pancreatitis or
other causes. .................................................................................................................................. 33
Figure 4:2 Thresholds of different markers for distinguishing infectious pathogens in blood
(Reprinted with permission from © 2011 Taylor & Francis [62]). The bacterial cause of Sepsis is
marked by a high value of CRP beyond 100 mg/l of blood. In contrast, viral causes of Sepsis are
marked by elevated levels of CRP but they tend to stay below 10 mg/l. Both regimes of CRP
levels are quantified using a bead-based assay in section 4.6 ....................................................... 36
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Figure 4:3 BD CBA test with 5 unique (A-E) beads having different fluorescent intensity ([64]).
In commercial bead-based assays, the beads have a unique color as well as a unique capture
antibody on their surface. This allows multiplexed sample diagnostics ....................................... 38
Figure 4:4 Antibody conjugation of the BD’s CBA set using sulfo-SMCC chemistry ([64]). The
conjugation of capture antibodies is performed using chemical bonding techniques. However,
later in this chapter, a technique for attaching antibodies through passive adsorption is
demonstrated. ................................................................................................................................ 39
Figure 4:5 Fluorescence Emission process flow ([73]) Absorption of a suitable photon causes the
electron to jump from the ground state to an excited state. The decay is usually instantaneous and
is accompanied by the emission of longer wavelength light. ....................................................... 42
Figure 4:6 Absorption and Emission spectra of 3 Phycobiliproteins – R-Phycoerythrin (RPE), B-
Phycoerythrin (BPE) and Allophycocyanin (APC) ([72]). R-PE has a broad absorption spectrum
and a narrow emission spectrum centered close to 585 nm .......................................................... 43
Figure 4:7 Antibody structure showing the Heavy chains and the Light chains (Courtesy [74]) 44
Figure 4:8 Large (1.5 mm diameter) Nylon beads can be trapped in 3D printed fluidic channels
within a disposable cartridge. ....................................................................................................... 47
Figure 4:9 Fluorescent intensities of beads with no added Streptavidin and Streptavidin-coated
beads upon the addition of Biotin conjugated with R-PE ............................................................. 48
Figure 4:10 Image of the nylon bead with 8 pico moles of Streptavidin. 3x higher intensity was
observed in the presence of protein. The image was taken with a CCD camera attached to a
fluorescent microscope with each snapshot having a 50 ms exposure and with image ............... 48
Figure 4:11 Image of bead with no protein. Only the circular outline of the bead is visible under
the fluorescent microscope. The non-zero background could possibly be attributed to auto-
fluorescence of the bead itself. ...................................................................................................... 49
Figure 4:12 Variation of Fluorescent Intensity with added Streptavidin (direct adsorption). The
Streptavidin was added in increasing concentrations, incubated and washed. BSA was added to
block unbound sites on the surface of the beads. Finally, Biotin-RPE was added to tubes
xvi
containing the beads to use the fluorescent methods of detection. The images were captured by a
CCD camera and then fluorescent intensity information was extracted using ImageJ by drawing a
rectangular box and using the Analysis-> Measure toolbar. The orange line at approximately
5000 intensity units represents the background fluorescence. ...................................................... 50
Figure 4:13 Flow chart for direct (non-specific) capture of the CRP antigen. Nylon beads were
used as a substrate for capturing antigen through adsorption. They are then bound with primary
antibody C2 followed by secondary antibody (IgG1 conjugated with R-PE). The secondary
antibody has a fluorescent tag that emits strongly at 585 nm. Additional blocking and washing
steps are not shown. ...................................................................................................................... 51
Figure 4:14 Fluorescent intensity emitted on the surface of the bead when coated with CRP. A
linear trend was observed with the emitted fluorescent intensity from the bead increasing with
higher input concentration of CRP on the bead. The orange line at approximately 5000 intensity
units represents the background fluorescence. .............................................................................. 52
Figure 4:15 Flow chart for indirect (specific) capture and fluorescent detection of the CRP
antigen. This is similar to Figure 4:12 except there was an additional step initially to coat the
Capture Antibody. This method is more specific to CRP and has a better Limit of Detection due
to the smaller quantity of CRP required. ...................................................................................... 54
Figure 4:16 Fluorescent intensity vs. concentration of CRP using the indirect capture. The orange
line around 5000 intensity units represents background fluorescent intensity of the bead. A
logarithmic trend in the emitted fluorescent intensity was observed. ........................................... 55
Figure 4:17 Stability of the binding across washing steps. The number of washing steps (to
remove any excess reagents) caused an insignificant change in the emitted intensity. This
suggests that the binding of the protein on the bead surface was relatively stable as was discussed
in section 4.4 ................................................................................................................................. 56
Figure 4:18 Effect of exposure on the emitted fluorescent light intensity. Due to the technical
limitations of the imaging unit, snapshots were taken every 30 seconds until the emitted intensity
faded away to the level of background. ........................................................................................ 57
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Figure 5:1 Epi-fluorescent microscope ([98]). The light source emits all wavelengths which are
filtered by the excitation filter within the filter cube. The sample receives this light and emits a
longer wavelength collected by the objective lens. Then it passes through the emission filter into
either the eyepiece or the camera. ................................................................................................. 63
Figure 5:2 Illustration of the Filter Cube containing the Dichroic mirror and the filters (Courtesy
Nikon [99]) The illumination is from the right side and the light is reflected downwards onto the
specimen using the dichromatic mirror. Then the emitted fluorescence light is allowed to pass
through the same mirror and filtered by the Emission filter before optical detection through a
camera ........................................................................................................................................... 63
Figure 5:3 CD4 cell counting setup prototyped using 3D printed parts. The 3D printed
components are denoted in color. The setup was screwed onto a standard optical base. The other
external components were translation stage for the optical tube, rotary stage for the laser, stepper
motor and the disposable cartridge itself. ..................................................................................... 64
Figure 5:4 Cartridge Module with custom-designed sliding base for easy removal. The parts in
pink were 3D printed while the green layer in the middle was the PCB controlling the Stepper
Motor. The cartridge is shown inserted on the right side with its bellow facing the blue shaft of
the motor. ...................................................................................................................................... 65
Figure 5:5 Resuspension Cartridge Holder. The transparent piece at the top is a representation of
the older Resuspension cartridge. ................................................................................................. 66
Figure 5:6 Laser Module containing custom-designed adapters for the Z-axis stage, Rotary Stage
and the Laser Covers ..................................................................................................................... 66
Figure 5:7 Optical Tube Holder Module with the grey xyz translation stage controlling the
relative position of the tube (not shown) ...................................................................................... 67
Figure 7:1 Drawing of the 3D printed Membrane Filter Holder from Chapter 3 with 0.5 mm
capillaries in the middle ................................................................................................................ 85
Figure 7:2: The cap for filter membrane holder for the sample preparation from chapter 3 ........ 86
Figure 7:3: Design 1 for holding the Nylon beads from Chapter 4 .............................................. 87
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Figure 7:4 Design 1 Bead Well Cover (from Chapter 4) .............................................................. 88
Figure 7:5 Design 2 for holding the Nylon beads from Chapter 4, there is a drainage channel of 1
mm width below the bead well. .................................................................................................... 89
Figure 7:6 Design 2: The cover for the bead well for the bead based assay in Chapter 4. ........... 90
Figure 7:7 3D printed base for the xyz translation stage holding the Optical Tubes in Chapter 5 91
Figure 7:8 Solidworks drawing of the 3D printed Optical Tube Holder ...................................... 92
Figure 7:9 Solidworks drawing of the 3D printed holder for the z translation stage for the Laser
....................................................................................................................................................... 93
Figure 7:10 Solidworks drawing of the 3D printed adapter from the Rotary stage to the
translation stage ............................................................................................................................ 94
Figure 7:11 Solidworks drawing of the 3D printed part for inserting the laser ........................... 95
Figure 7:12 Solidworks drawing of the 3D printed part for enclosing the laser. The screw holes
are M2 and they pass straight through to the Rotart stage ............................................................ 96
Figure 7:13 Solidworks drawing of the 3D printed part that holds both the new cartridge and the
stepper motor. ............................................................................................................................... 97
Figure 7:14 Solidworks drawing of the 3D printed part for holding the black cartridge and
sliding it into the main Cartridge Holder module ......................................................................... 98
Figure 7:15 Solidworks drawing of the 3D printed part for holding the Resuspension cartridge
and sliding it into the base ............................................................................................................ 99
Figure 7:16 Solidworks drawing of the 3D printed part that supports the PCB and has a groove
that slides into the base ............................................................................................................... 100
Figure 7:17 Solidworks drawing of the 3D printed part that connects the Optical bench to the
Cartridge Holder module ............................................................................................................ 101
Figure 10:1 User interface for the Zortrax M200 ....................................................................... 124
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Figure 10:2 The desired model in the middle protected by sacrificial rods of arbitrary dimensions
..................................................................................................................................................... 126
Figure 10:3 Price comparison for top 3D printers [37, 38]. Assuming that printers last just one
year, the above table gives a rough estimate of the costs involved in using these two 3D printers.
..................................................................................................................................................... 127
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List of Appendices
Appendix A: Solidworks drawings
Appendix B: COMSOL parameters
Appendix C: Recipes for Bead-based assay
Appendix D: 3D Printing techniques
1
Chapter 1
Chapter Organization
1.1 Aim
The capability of providing rapid medical test results at the patient’s location is the main
hallmark of Point-of-Care (POC) diagnostic kits. POC kits typically have a series of modules
that work in unison to detect a specific disease.
This thesis proposes to develop device components to be used in the next generation of Point-of-
Care (POC) diagnostics kits. The components that are developed are: a) Sample Preparation
Module b) Bead-based Assay Module and c) Optical Detection Module. A proof-of-concept
protein assay was used to illustrate the general application of this bead-based test. Additionally, a
fluorescent particle detection system was built to count CD4 cells for HIV monitoring.
Figure 1:1 Applications of 3D printing in various stages of Point-of-Care Diagnostics. In
this thesis, a sample preparation module, disposable cartridge module for bead-based assay
and an optical detection system were built using parts created from a 3D printer
The filter membrane separation method for the sample preparation module in Chapter 3 has been
replicated from literature for a smaller quantity of blood. The COMSOL models developed for
the helical geometry are something that is not found in literature. The bead based assay using
2
Nylon beads for fluorescent detection of CRP in Chapter 4 is an advancement over existing
Agarose bead-based adsorption method. The modular fluorescent detection kit designed in
Chapter 5 is an improvement of the bench top unit previously used at ChipCare Corp.
The three modules that were developed and prototyped through 3D printing have the potential to
be adapted into a generalized diagnostic kit. Many Lab-on-a-chip device have the limitation of
only detecting specific diseases. The bead-based assay in a disposable cartridge developed in this
thesis can overcome such limitation by extending the disease detection capability to any blood
protein marker. The advantage of segregating the biomarker identification from the detection
system is that the overall medical kit is now more versatile. The proof-of-concept bead-based
assay module here can be theoretically be used for identifying any blood plasma biomarker
amenable to fluorescent methods of detection. The key idea is that further assays can be
developed relatively easy after the diagnostic platform is validated for the first assay.
The generalized Fluorescent Detection Kit described in Chapter 5 was used to detect
fluorescently tagged cells for HIV monitoring. The utility of this kit can be extended to any
fluorescent marker through the development of a bead-based assay. For example, if future
researchers from ChipCare Corp were to include Sepsis detection, all they would need to do is
swap out the Cell Counting Cartridge and replace it with the newly developed Bead-Based Assay
cartridge as shown in the figure below:
3
Figure 1:2 The above schematic illustrates the utility of having a diagnostic kit with a
constant detection scheme and a variable bio-marker cartridge. The development of new
assays would require just replacing the cartridge instead of developing an entirely new
diagnostic kit
Due to the digital nature of 3D Printing, it has the potential to accelerate the development of new
assays as well as shorten the development cycle for improving existing assays. By having the
designs as a digital copy available for rapid prototyping using 3D Printing, POC device
interoperability will be a step closer.
1.2 Chapter 2: Introduction
Chapter 2 provides an overview to the thesis and the motivation as to why this topic was chosen
for research during this Master’s degree. It provides a general introduction to the Point-of-Care
testing and the challenges to overcome before they are available to a wider market. Statistics on
the impact of Sepsis on the general population is given and current diagnostic techniques are
elaborated. Then, steps required to make this diagnosis on a portable platform are discussed.
Additive manufacturing is explored in greater detail and adopted in this thesis due to the wide
range of benefits and the rapidly advancing technology. Finally, desktop 3D printers are
compared to give researchers a better picture of their options before purchasing one.
4
1.3 Chapter 3: Sample Preparation Module
Chapter 3 introduces sample preparation as an essential step in POC applications. Towards this
goal, separation of blood cells from plasma is explored using a variety of techniques drawn from
literature. Two such techniques, filtration and centrifugation, were found to be compatible for 3D
Printing technology and hence dealt in more detail. The centrifugal approach uses a syringe-
based pumping mechanism for injecting fluid through helical channels exploring size-based
separation of spherical particles. Simulation were performed using COMSOL Multiphysics to
develop a proof-of-concept model for separating particles from liquid. The membrane-filter
based separation technique uses an off-the-shelf membrane filter with pore dimensions smaller
than the blood cells to exclude cells larger than 3 µm. The latter technique was successfully
demonstrated on a 3D printed disposable capillary chip.
1.4 Chapter 4: Bead-based Assay
Chapter 4 deals with the development of a bead-based assay that has the potential to be
integrated into a 3D printed disposable cartridge. A fluorescent test for protein on Nylon
Polyamide beads is elaborated. Through direct adsorption of Streptavidin on the bead, a yes/no
test is initially demonstrated. This is followed by a adapting this test to quantify a Sepsis marker
called C-Reactive Protein (CRP). The specificity of the test is further improved by utilizing
CRP-specific antibodies and the corresponding results are discussed.
1.5 Optical Detection System
Chapter 5 elaborates system-level application of 3D printing through an internship at a POC
medical diagnostics company, ChipCare Corp. In one such application, a compact fluorescence
microscope customized for ChipCare’s bead-based assay is designed and 3D Printed. A
multifunctional lab prototype was developed and used extensively for CD4+ cell detection.
Additionally, the setup is being used to validate the lab results of the commercial CD4+ cell
counting handheld device that the company is planning to commercialize.
1.6 Chapter 6: Future Work and Conclusion
Chapter 6 dwells on ways to improve the modules discussed so far. The findings from the
previous sections are also summarized.
5
Chapter 2
Introduction
2.1 Motivation for POC Modular Design
A Point-of-Care system, which can function as a multipurpose diagnostic platform, is a goal
targeted by the World Health Organization (WHO). Towards this end, a global consultation was
held to discuss interoperability standards between medical devices as well as device connectivity
[1]. The goal of the WHO consultation was to come up with standards so that testing kits from
different vendors would be interoperable. In the diagram below, the general sequence of steps
when performing a medical test is illustrated.
Figure 2:1 Typical steps followed in diagnostic instruments. Sampling from the patient
(blood, urine, saliva, etc.) is usually done off-chip. The biological markers present in the
sample are usually detected on a disposable chip after stimulation from an optical source.
The signal can be collected using a camera such as a Charge-Coupled Display (CCD)
camera. Finally, the results are interpreted in the analysis step.
The key idea is that when scientists develop, say, a new diagnostic test, the bio recognition
module can be replaced keeping the rest of the diagnostic kit intact. Building modules that work
equally well with other modules from different vendors through a plug-and-play approach would
cause an innovation boom that would fuel the rapid adoption of POC diagnostics.
During infectious disease outbreaks, rapid diagnostic tests have to be available so that they are
contained. Tests available only in centralized health facilities require the samples to be
transported to the laboratory and the results sent back to the patient of the particular clinic. In
remote or rural settings, this can take days or even weeks, delaying the identification of the
pathogen and compromising the treatment.
6
Having a different technology for each diagnostic means that the health centers have to train
personnel for each type of device being used. However, if there was a common diagnostic
platform on which a multitude of tests can be performed, local health centers will not have to
struggle to maintain separate equipment for each testing. Chin et al [2] feel that inspite of rapid
advances in individual components of a Lab-on-a-chip components, there is a dearth of system-
level applications relevant for clinical use in a point-of-care setting. In this thesis, work is done
to build both the components as well as a complete system to be used in POC applications.
There are some common characteristics of a POC device, a few of which are listed below [ibid]:
1) Low Power Consumption
2) Low Cost
3) Ability to function at high humidity levels typical of tropical countries.
4) No storage requirements for cartridges/reagents
5) Long Shelf Life for the reagents
6) Portability and ruggedness
7) Minimal training requirements
8) Plug-and-play nature for use with different
The WHO has started encouraging Diagnostics manufacturers to explore modularity. Their intent
is to come up with standards to make cartridges from different vendors interoperable with other
diagnostic platforms. During the recent conference held in Geneva [1], the participants discussed
the plug and play nature of consumer electronics made possible because of standardized
interfaces. Achieving such standards for diagnostics would open up platforms and allow
companies to collaborate seamlessly. Dr. Powers from the International Standards Organization
(ISO) suggested that the interoperability model developed would contain the following three
modules: a) a medical information bus device with a basic instrument supplied by power, b)
unique elements of the assay incorporated in a disposable reagent-filled cartridge and c)
Technology-specific interconnectors that connect the cartridges to the instrument
7
Other benefits include the ability of one reader to be used with multiple cartridges from different
suppliers allowing only relevant tests to be stocked; reduced training for technicians; easy
understanding of the technology and adoption by new diagnostic companies fuelling innovation
and finally the rapid development of unforeseen technologies similar to what occurred after the
adoption of USB standards.
Since the emerging markets are more likely to invest smaller amounts regularly over long
periods instead of large upfront costs, the diagnostic companies with business models relying on
plug and play cartridges will experience higher sales and hence have an incentive to adopt
common standards[ibid]. With a population of 4.5 billion in LMICs, diagnostic firms would find
it increasingly hard to ignore such a large market. In addition, the possibility of increased market
share exists for industrial participants who develop the technology and make it freely available.
For example [3], after Adobe Systems bequeathed their PDF standard to ISO, PDF became a
brand name and standard for document management and counter-intuitively Adobe increased
their market share
The commercial development of POC devices is aimed at not only emerging markets but also
advanced economies. Europe and North America are bound to see a rapid growth in demand for
POC applications in assisted living centers due to ageing populations. The POC market in the US
alone was worth 14 billion dollars in 2014 [4]. All of these suggests that the development of
interoperable POC devices will be the focus of many diagnostics firms.
2.2 Advantages of POC
Current diagnostic tests can take up to 36 hours to recognize bacterial infections due to the time
it takes to culture the strains [5]. This lengthy diagnoses time pressures doctors to prescribe
antibiotics. However if the illness is due to a viral infection, the treatment is ineffective and
counterproductive as the prescribed antibiotics may cause other bacteria in the patient’s body to
develop resistance. This has prompted concern among several developed nations. The British
Government constituted a task force to combat the overuse of antibiotics. This task force
identified several issues including the observation that Drug companies have no incentives to
develop rapid diagnostics, as it would undercut the sale of antibiotics.
8
It is estimated that a 30% reduction in the effectiveness of antibiotics will cause 120,000 new
infections and 6000 deaths in the US every year [6]. To avoid such scenarios, rapid diagnosis is
necessary. There are several markers in the patient’s blood that can be an effective indicator of a
bacterial infection, most notably C - reactive protein. Countries like Netherlands, where
healthcare systems place an emphasis on CRP testing, also have the lowest rates of antibiotic
prescription.
In many countries, due to the nature of the healthcare system, several ailments are categorized as
non-emergencies and the patients are sent home [7]. In the ensuing days or weeks, it is possible
the problems may have worsened and require treatment that is more thorough. The Medical
profession is of the opinion that this a waste of resources and can be rectified by faster diagnosis
[8].
Currently when patients see a doctor or physician, they are prescribed medicines based on what
information they provide and physician’s knowledge and understanding of that information [9].
If a course of treatment does not work in the first instance, then the doctor prescribes more or a
combination of several medicines. This hit-and-miss approach often fails and can be eliminated
if there is a faster way of diagnosing the relevant condition. With the right diagnosis, the doctor
can prescribe treatment to actually attack the relevant medical condition rather than fight the
symptoms and hope it cures the disease. To address diagnostics to assist the doctor in the
prescription of antibiotics, a simple test for sepsis is needed. A proof-of-concept assay for
detecting sepsis using the C - reactive protein is presented in chapter 4.
The POC tests have to be rapid because faced with a choice of administering antibiotics right
away for a patient-suffering from fever and waiting hours to get results from a test, the
healthcare worker from LMICs are more likely to choose the former.
Companies like Stratos are already developing such rapid diagnostics [10]. This company
researched clinics in India, Latin America and Africa and came up with the PanDx concept. This
is a machine with multiple slots that can hold various tests. This allows the user to mix and
match different instruments with multiple cartridges allowing flexibility and modularity. A
breadboard like instrument has been designed so far that can accept cartridges for TB, HIV and
ALT assay from whole blood.
9
Another example of interoperable products being developed is Global Solutions for Infectious
Diseases (GSID) platform [11]. This non-profit organization made use of already existing
products to develop new assays. The kit uses an Android phone operating as a wireless reader
with pre-existing diagnostics.
However, there are several issues to be overcome before plug and play systems are adapted by
consumers in LMICs. For example, a study [ibid] by GSID found out that 50% the people have
never used a touch screen device before. Additionally unlike electronics interoperability
standards that deal with primarily communication standards, POCT interoperability has to be
specific on the hardware technology of both the reader and the cartridge. Since most assays have
common processing steps such as sample preparation, mixing incubation, signal measurement,
etc, the cost of the cartridge will be dependent on how many of these steps are performed in it.
Therefore, an optimum tradeoff exists between cartridge complexity and cost.
Other advantages of modern POC devices include increased efficiency and simpler management
of medical records due to the wireless data transfer, reduced overcrowding in hospital waiting
rooms due to faster tests and allowing paramedics in emergency vehicles to know more about the
patient [12].
Modern POC devices like iStat [13] routinely use electronic data transferring using wireless
technologies increasing efficiency and simplifying the process for creating medical records.
Revue et al [14] analyzed data from POC usage in ambulances in Germany and found that iStat
devices were useful in quick diagnosis and treatment. Studies show that the Abbott i-STAT helps
clinicians to make quick, informed decisions. Dr. Jarvis [15] showed that usage of POC devices
in the Emergency Ward reduced the amount of time the patients spent by an average of 53
minutes which corresponds to more than 40% reduction. For these reasons, developing an
effective POC device and making them available to the wider health community is essential.
10
2.3 Reason for POC blood tests
Samples from the patient could be blood, saliva, urine, sweat, etc. This section elaborates why
assay development aimed at diagnosing conditions through blood samples was chosen. Infectious
and non-communicable diseases result in premature and preventable deaths in the world, reduce
economic growth and limit human development [16]. Factors limiting an effective response
include:
1) Lack of access to diagnostic capacity in remote or rural settings in the Africa and Canada [17]
2) Lack of sufficient number of healthcare providers resulting in overburdened doctors’ offices,
clinics and hospitals [18] and
3) The lack of data available to health systems managers to make informed, evidence based
funding and policy decisions, leading to a disconnected, uncoordinated health system with poor
outcomes.
An easy to use portable diagnostic device that make early detection of a wide range of infectious
or non-communicable diseases will be very beneficial. Microfluidics, which uses smaller sample
volume, less reagents, inexpensive polymer, and low power consumption present the opportunity
to make this a reality [19]. Chapter 4 discusses a bead-based assay that is suitable for
microfluidic test requirements.
Complete Blood Count (CBC) is done in more than 50% of Emergency Room patients and is
done for a range of ailments. These typically give information about the RBC, WBC and platelet
counts [20]. These tests are performed using Flow Cytometers. However, these cumbersome
equipment are not portable or cost-efficient thus ruling them out for POC applications.
Affordable blood tests using small quantities of blood are becoming widespread due to
companies like Theranos [21]. The technology however is only available in specific locations
like pharmacies in the US. A more widely available and portable testing tool for POC
applications is needed. In Chapter 5, a lab prototype is built for counting CD4 cells within a few
minutes and is the first step towards a POC device being built by ChipCare Corp.
11
2.4 Advantages of 3D Printers
The Deloitte white paper on 3D Printing explores the impact of Additive Manufacturing (AM) in
producing prototypes, models, visualization tools, tooling and end-user part production [22].
Popularly known as 3D Printing, this technology enables the production of objects through the
addition of materials instead of conventional manufacturing techniques relying on removal.
Deloittte estimates that this $2 billion industry is growing at an annualized 14.2 %.[23]
Additive Manufacturing helps distributed manufacturers as it reduces the minimum capital
requirements for them to achieve economies of scale in their supply chain [24]. Additionally,
lower capital is required to achieve a wider scope in their capabilities. This can change the
business model pursued by companies as illustrated in the graphic below:
Figure 2:2 Framework for
understanding 3D Printing
paths and values ([22]).
Various levels of adoption of
3D printing are seen in
businesses. Some businesses
tend to replace their entire
supply chain, while others
seek out 3D printing
capabilities only when rapid
prototyping or cost-effective
customization capability is
needed.
3D printing is increasingly
used in tooling for a broad range of applications in aerospace, automotive, defense and
healthcare for producing assembly jigs, fixtures and custom medical guides [25]. The available
materials are plastics, rubber, composites, metal and wax. Though the overall impact of 3D
12
printing for tooling on overall supply chain is small, the advantages include a) lead time & b)
cost reduction, c) improved functionality and c) customizability
a) Lead time reduction: 3D Printing manufacturers advertise that their systems can reduced lead
times from 40 – 90 % [26]. This is due to several reasons: fewer labor inputs and machining
steps, usage of digital design files instead of paper drawings and lastly the ‘in-housing’ of
fabrication that was previously outsourced. The usage of digital design files is especially
important in eliminating the need for a technician to interpret the drawings. In addition, in
housing of the design process eliminates the risk of receiving improperly fabricated tools.
b) Cost Reduction: The costs associated with AM can be lower due to the higher product yield,
reduced labor and minimum scrap material generation. The reduced labor input is due to AM
being an automated process. Additionally, low volume production is especially economical with
AM as the existence of digital design files allows for rapid design changes eliminating expensive
up-front costs [27]. Additive manufacturing can result in substantial reduction in scrap material
due to the very nature of additive manufacturing.
c) Improved functionality: Previously unobtainable designs like complex geometries and free-
form shapes can be created. For example, Citizen (a manufacturer of watches) uses Additive
Manufacturing to create custom jigs for their watch assembly process [28]. They were able to
simplify operations by creating more types of tooling at a lower cost.
d) Customizability: Medical device and health care industries make use of user-specific
customization. Every year, about 50,000 patients are operated on using 3D Printed personalized
instruments and surgical guides [29]. The operating room efficiency and patient outcomes were
improved due to those tools. Dentistry is another medical field where customizability makes
significant difference to the end-user experience. The use of surgical implant guides specific to
the patient’s dental scan reduces surgery time and recovery process [ibid]. 3D printing is better
suited for such low-volume production.
Due to the above reasons, 3D Printing is more suited to keep up with changes in the product
design cycles at a lower cost than other manufacturing techniques. Hence, this technique is
explored in subsequent chapters for a variety of applications.
13
2.5 3D Printers in Research
3D Printers are also increasingly being used in research to produce functional microfluidic
devices. The gradual shift towards 3D printing techniques is because lithography techniques are
expensive for small volume prototyping. 3D printers also allow previously unattainable complex
geometries to be readily created. In this section, a brief review of microfluidic and diagnostic
application developed by other researchers is presented.
Lego-like microfluidic parts were produced by Lee et al and Bhargava et al with the intention of
standardizing fluidic interconnects and components [27, 28]. They designed and characterized
fluidic channels, mixers, reactor chambers, gradient generators and inlet/outlets. Customizable
fluidic interconnects were produced by Paydar et al [29] and characterized for stability under
pressure. Their aim was to develop a reliable packaging technology for microfluidics.
Researchers have developed low-cost lateral flow assays [30] that can be prototyped in under 30
minutes. This has the potential to be adapted for containment of disease outbreaks. Lee et al [31]
created a millifluidic device that performs size-based separation of particular bacteria from other
clusters. They used the device to quantify E.coli from milk. Researchers have also utilized the
transparent nature of resin-based 3D printed parts to create customized 96-well plates [32]. With
the use of simple polishing techniques such as sanding, the surface of the finished material
became clear. This technique has the potential to be adopted in manufacturing disposable
cartridges for fluorescent detection for the assay developed in Chapter 4.
On a system level, researchers have developed a 3D printed fluorescence detection head [33] as
well as chemiluminescence biosensor for smartphone detection [34]. The customizability of the
parts enabled them to produce relatively complex designs for under $50 material cost as opposed
to more than $500 if they were machined. Similar cost advantages led to 3D printing being
adopted as a tool for designing a fluorescent detection kit as discussed in Chapter 5.
With the development of CAD designs suitable for 3D printing, microfluidic researchers will no
longer be restricted to producing their devices in expensive clean rooms. They will be able to
outsource their designs to be created elsewhere through 3D printing service companies [35]
14
2.6 Comparison of commercial desktop 3D printers
This section lists the top 20 printers and their associated specifications [36]
Table 2-1 Comparison of some popular 3D printers (Reprinted with permission from ©
O’Neill et al [36] AIP Publishing). The Form1+ and the Zortrax were used in this thesis.
In this thesis, Form1+ and Zortrax M200 were explored. The latter was extensively used in the
later chapters. The Appendix includes good practices and techniques to achieve failure-free
printing.
15
Chapter 3
Sample Preparation Module
3.1 Background
In this section, information about the composition of the blood is provided and literature on how
researchers have separated plasma from blood.
Whole blood contains nucleated White Blood Cells (WBC), non-nucleated Red Blood Cells
(RBC), Platelets and other molecules suspended in a fluid called Plasma [39]. The composition
of blood is illustrated below:
Figure 3:1 Blood Composition
(Reprinted with permission from
© M. Kersaudy-Kerhoas and E.
Sollier [39]) Red Blood Cells
(RBCs) and Plasma occupy
nearly occupy the entire volume
of blood. Plasma contains useful
biomarkers for different diseases
and needs separation from RBCs
to improve optical detection
performance.
In some flow cytometry applications involving WBC cell counting [40], whole blood is used
with RBCs lysed beforehand. This is to make sure that the data analysis software is not
overburdened with orders of magnitude higher numbers of RBCs. In other applications, where
16
shorter sample preparation times are required (for example, ChipCare’s CD4 cell counter
discussed in more detail in chapter 5), the RBCs are not lysed. To ensure that the optical detector
picks up WBC cell population from among the higher numbers of RBCs, fluorescent particle
counting method is adopted. The subpopulations of the WBCs can be determined by mixing
monoclonal antibody reagents to whole blood. The fluorochrome-labelled antibodies bind only to
the specific antigen sites on the surface of the White Blood Cells. When excited with suitable
wavelengths, these labelled cells fluoresce and are picked up by the tracking software.
There are some applications [41] where fluorescent particle counting is needed for proteins in the
blood (which are much smaller than blood cells). Plasma contains many protein biomarkers as
indicated by Fig 3.1. For example, elevated amounts of Circulating Nucleic Acids (CNAs) can
be linked to cancer; sepsis could be identified by circulating C – reactive proteins, etc. The
advantage of using these kind of biomarkers for diagnosis is that they are cleared soon after the
associated condition is removed. Hence, there is lesser chance of false positives. In such
detection schemes, it becomes necessary to remove the interfering cells during the sample
preparation step. The separation of plasma is useful when the detection scheme relies on specific
proteins/antigens in the plasma to be used in bead-based assays [42]. This is achieved by filtering
blood. Some methods of isolating the plasma from whole blood found in literature are discussed
in the following sections.
17
3.2 Methods of Blood Filtration
One of the common methods in clinical facilities to separate plasma is to use centrifugation [43].
However, commercial devices are expensive, bulky and require electricity, which makes them
unsuitable for many POC applications. Hence, the authors [44] developed a hand-powered
centrifuge based on an eggbeater that could achieve near 100% purity for plasma separation.
Users would then extract the supernatant manually after centrifugation through visual inspection.
However, the system is prone to human errors in handling during the final extraction phase.
For direct integration with a microfluidic channel, a different setup is needed. Researchers [45]
had developed a filtration method consisting of beads packed at the channel inlet. However, this
device is more difficult to manufacture due to vacuum requirements for packing the beads in the
channel. Using Lithographic methods, researchers have created micro posts [46] that can direct
flow causing size-based separation of cells. However, prototyping posts with micron dimensions
are beyond the capabilities of current 3D printers. In this thesis, centrifugal-force based and filter
based separation methods were explored. The former is described in the next section.
3.3 Centrifugal-force based separation
The possibility of separating plasma from blood using 3D printed helixes/spirals is explored in
this section. Particles in a straight channel experience drag forces as well as two types of inertial
lift forces (wall and shear induced) [47]. The two lift forces acting in opposite directions cause
recirculation in a curved channel termed as Dean Forces. The flow patterns are as illustrated
below:
Figure 3:2 In helical/spiral channels, centrifugal forces cause recirculation patterns that
tend to concentrate particles within the vortices [47]
18
The equations for designing a specific helical channel of radius of curvature, R, Length, LD and
Channel Dimensions, Dh is given by the following equations from [48]
Where ρ = fluid density; Uf = fluid velocity; R = radius of curvature; Dh = Hydraulic diameter of
the channel; ap = particle diameter; µ= Dynamic viscosity of the fluid; LM = Migration Length;
LD =Channel Length for Dean Migration;
Solving these equations by fixing the channel dimensions, radius of curvature and fluid velocity
will give the required length of the spiral/helical channels for focusing particles of diameter ap
Researchers have reported the use of centrifugal force for size-based separation of particles [48].
Their microfluidic separation spirals can be used for continuous separation of particles or where
a large volume is used. The inertial forces caused the particles to separate into distinct
streamlines. The above devices were utilized by researchers [49] to separate Circulating Cancer
Cells (CTCs) as well as for chemiluminescent detection of cardiac arrest markers
In the above applications, researchers solved the hydrodynamic equations listed above assuming
that the ratio of the particle diameter to the channel diameter was (ap / Dh ) >0.07. Particles
satisfying this will streamline to the inner side of the channels while smaller particles will be at
the outer streamline within the same channel. However, this condition could not be satisfied for
3D printed channels. Performing the calculation for typical human cells by modelling them as
spheres, it was found that the channel widths required to separate 5 um cells from smaller
particles would be 70 µm. This turns out to be an order of magnitude smaller than what desktop
3D Printers can reliably create. Hence, this suggests that the separation of blood cells from
plasma would not be theoretically possible. Numerical simulations were attempted in COMSOL
Multiphysics (parameters are described in Appendix B) to check if channel dimensions could be
found for plasma separation without satisfying (ap / Dh ) >0.07.
19
In an attempt to understand the flow dynamics associated with particles in a fluid, simulations
were performed. In COMSOL Multiphysics, a model was built with two Physics: Laminar Flow
and Particle Tracing for Fluid Flow. The continuous phase modelled by the Laminar Flow profile
was solved first by using the Stationary Study option. This was followed by the dispersed phase
modelled by the time dependent Particle Tracing for Fluid Flow Module. The two simulations
were decoupled to reduce the computational requirements.
The Particle Tracing model in COMSOL Multiphysics can model particles whose impact on the
fluid flow is negligible. Due to the difference in velocity between the particle and the fluid, the
fluid exerts a drag force.
The Freeze option was used at the Outlet to recover the particles’ velocity profile and positions.
The Bounce parameter for the particles was selected so that everywhere within the channel,
elastic collisions between the wall and the particle would occur.
A flow model for a straight rectangular channel with a single inlet and outlet was initially
developed with the above parameters. The geometry is given below:
Figure 3:3 Geometry of a straight channel developed in COMSOL Multiphysics. The
channel was 20 mm long and had a uniform rectangular cross-sectional area of 2 mm x 2
mm.
20
This rectangular channel was simulated to show the effects of flow. The figure below shows the
velocity profile for a straight channel.
Figure 3:4 A) (Uniform) Velocity Profile at the inlet of a straight channel (0.05 m/s) and B)
(Parabolic) Fluid velocity profile at the output of the straight channel. The simulation
illustrates the effects of solving a 3-dimensional fluidic equation.
The above simulations confirmed that the parameters were correctly set for obtaining the flow
velocity. Subsequently, the particle tracing capability was added to the model and a time
dependent simulation was performed. The results are shown below:
21
Figure 3:5 Time lapse snapshots of particles at t=0 s and 0.1 s. In A) the particles were
uniformly distributed to the inlet boundary condition and had the same velocity of 0.05
m/s. In B) The particles had traversed 5 mm along the channel and they had a non-uniform
velocity profile as indicated by the darker red colors in the middle and (slower) blue
colored dots in the periphery Focusing of particles as they traverse the length of the
channel was observed.
As expected, the parabolic velocity profile observed previously caused the particles to become
focused as they travelled along the channel. The effects of gravity on this flow can be seen in the
next simulation result.
22
Figure 3:6 Effects of the particle diameter on the flow trajectories. A) 10 um particle
trajectories and B) 100 µm particle trajectories. Larger particles (100 um) were seen to be
significantly affected by gravity compared to the smaller 10 um particles.
Though hydrodynamic focusing does take place within straight channels, the manufacturing
requirements for extracting the output limits the utility of such devices. So, helical channels were
next explored.
23
The above models were extended to a helical geometry. A helix with 2 mm x 2 mm channel
dimensions was created in Solidworks and imported to COMSOL using the LiveLink Feature.
The dimensions were chosen so that 3D printing could be potentially be adopted to prototype
these cell separation devices.
Figure 3:7 A 2-loop helix with 2 mm x 2mm channel dimensions, 20 mm helix diameter and
20 mm pitch. (All shown dimensions in the graph are in millimeters). The outlet was
geometrically split into two equal sections to observe the effects of particle separation. The
helix was drawn in Solidworks.
24
Then, simulations were performed as before to characterize the flow velocity first. The results
are shown below:
Figure 3:8 Velocity profile at the output of the 2-loop helix. Higher velocity of the fluid was
noticed at the outer edges of the channel compared to the inner side. This is shown by the
darker red regions towards the left side of the output face.
The curved channels caused a different velocity profile at the output compared to the straight
channels. As noticed from Figure 3:8 B), the velocity was higher at the outer outlets, similar to
predictions illustrated previously in Fig 3:2. This was confirmed by performing particle
trajectory simulations and the results are illustrated below:
Figure 3:9 Snapshots of particle trajectories at A) t=0 s, and B) t=0.2 s when particles reach
the outlet. As noticed from B), the particles were more concentrated at the (outer) wall of
the channel denoted by Outlet 1. It was also observed that there were particles, which
never reached the output irrespective of how long the simulations were run for. They are
seen in B) as blue particles trailing in the channel.
25
From the above simulation graphs, it was observed that there was an increased spatial
distribution at the outer wall of the channel (Outlet 1). To characterize the separation efficiency
of this model, it was necessary to count the number of particles reaching the outlet. However, it
was noticed that a significant number of particles did not reach the outlet. This was possibly due
to meshing inefficiencies or simulation time steps being not precise enough. The transmission
probability was ~40%. To account for just the particles reaching the outlets and splitting into
Outlet 1 and Outlet 2, an Enrichment Factor was defined as
Enrichment Factor = 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑃𝑎𝑟𝑡𝑖𝑐𝑙𝑒𝑠 𝑖𝑛 𝑂𝑢𝑡𝑙𝑒𝑡 1
𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑃𝑎𝑟𝑡𝑖𝑐𝑙𝑒𝑠 𝑖𝑛 𝑂𝑢𝑡𝑙𝑒𝑡 2
Based on this factor, graphs were plotted to see the effect of number of particles injected into the
helix on the separation efficiency. For 100% separation into Outlet 1, the Enrichment Factor
would be infinity. Simulations were performed on 100, 1000 and 10000 particles.
Figure 3:10 Excel graph of enrichment factor for different diameters and particle numbers.
The data suggests that the number of particles at the inlet does not have a significant effect
on the enrichment factor. It stays constant at ~1.30. This would be imply that the Outlet 1
has 30% more particles than Outlet 2. This would be ideal for blood cell separation because
the number of cells would be different for different patients.
0.75
0.9
1.05
1.2
1.35
1.5
0 2000 4000 6000 8000 10000 12000
Enri
chm
ent
Fact
or
Number of 1 um particles
Enrichment Factor vs Number of 1 um particles
26
The influence of the particle diameter on the spatial distribution at the outlet was simulated next.
Figure 3:11 The particle diameter’s influence on the Enrichment Factor. A rising trend was
seen. Theoretically, infinite Enrichment Factor (100% separation into the Outlet 1) would
be seen for particles diameter larger than 0.140 mm.
The enrichment factor increased with increasing diameter, but simultaneously the overall
transmission decreased due to the particles stopping midflow due to reasons possibly associated
with meshing or simulation time steps.
Theoretically, the particles should tend to separate at 𝑝𝑎𝑟𝑡𝑖𝑐𝑙𝑒 𝑑𝑖𝑎𝑚𝑒𝑡𝑒𝑟
𝑐ℎ𝑎𝑛𝑛𝑒𝑙 𝑑𝑖𝑚𝑒𝑛𝑠𝑖𝑜𝑛 > 0.07 but there was
no way to verify this as the particles never reached the outlet.
To make sure all the particles reach the output, a deeper understanding of the underlying
algorithms used by COMSOL will be needed. To guide future research in this area, the
simulation time was plotted against the number of particles to give a better understand how the
algorithm handles complexity,
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
0 10 20 30 40 50 60
Enri
chm
ent
Fact
or
Particle Diameter in um
Enrichment Factor vs Particle Diameter
27
Figure 3:12 The solution time dependency on the number of (1 µm) particles. Orders of
magnitude increase results in less than a linear increase in the time taken to complete the
simulation.
Further optimization for obtaining channel dimensions appropriate for blood plasma separation
was not performed for the following reasons: Particle Tracing in COMSOL would not be a valid
model for blood cells. This is because COMSOL the models assume the dispersed phase (cells)
are less than 1% of the continuous phase. This is not the case with Red Blood Cells because they
occupy nearly as much volume as blood plasma. Moreover, the volumes of fluid needed to
achieve separation at the output would be ~ 1 mL. For the above reasons, the centrifugal force-
based separation of plasma from whole blood through 3D printing was not pursued. Instead a
membrane filter-based approach was undertaken.
y = -2E-06x2 + 0.0449x + 187.13
0
50
100
150
200
250
300
350
400
450
0 2000 4000 6000 8000 10000 12000
Sim
ula
tio
n t
ime
(s)
Number of Particles
Simulation time vs number of particles
28
3.4 Filter Membrane-based separation
An alternative simpler approach would be to use a filter membrane. The authors [51,52] use a
membrane filter to separate plasma from blood cells. They also performed biochemistry tests
where they showed that membrane based filters have low enough adsorption to allow for
downstream disease detection. However, their device was optimized for use with ~1 ml of whole
blood. The fact that a large quantity of blood is required for flow limits the utility of their design
to venous-type blood draws. For many POC devices (discussed in chapter 5), a finger-prick
quantity of blood is sufficient for analysis.
In this section, a proof-of-concept device is presented that is useful for separating plasma from
finger-prick volumes (25 µl) of blood. A similar membrane filter-based approach was adopted in
this thesis but with 3D printed parts as explained in this section.
Filter Selection: Filters can be either Depth or Membrane Filters depending on the underlying
filtration mechanism [53]. Depth filters have a matrix of randomly oriented fibers that can
prevent particles from passing through. They have a higher dirt-holding capacity. However, they
also tend to shed possibly causing contamination in high throughput samples. On the other hand,
membrane filters have a network of pores on their surface, which stop particles bigger than the
pore diameter. They are good for tests requiring higher integrity of the filtrate. For this reason,
membrane filters were chosen over Depth filters.
Extractable are contaminants that may leach from a filtration system to the filtrate. Polyester
Track Etch (PETE) filter have low extractables meaning no extraneous substances will be added
by the filter to the filtrate (eg: manufacturing debris, sterilization residue, adhesives, etc) [54].
This is essential for biological applications as certain contaminants can kill cells by inducing
cytotoxicity. The filter is also thermally stable upto 140o C and has a pH range of 4-8 which is
sufficient for blood filtration done in this thesis.
For the above reasons, the membrane that was used for filtration was PETE polyester membrane
(2.0 µm pore size, 25 mm in diameter) from Sterlitech Corporation [55]. The material has low
non-specific absorption. It has a smooth surface thereby reducing the chance of hemolysis. Other
useful properties include its resistance against a wide range of chemicals, minimum auto
fluorescence and low moisture absorption [56].
29
3.5 Plasma Extraction Experiment
The key idea was to have capillary channels beneath the filter paper, as gravity was observe to
be not sufficient to let the droplets seep through the filter. Various capillary channel dimensions
were tried on a 3D printed platform and it was observed that smaller the capillary channels (the
spacing between the walls as well as the capillary wall thickness), the shorter the filtration time.
However, desktop 3D printers (Form1+ and the Zortrax M200) have a limitation of 0.5 mm wall
thickness and 1 mm wall spacing. Hence, these dimensions were chosen for the capillaries and
manufactured. The Solidworks drawing is attached in Appendix A.
This module initially consisted of a single 3D printed piece with capillary channels with a
collection port in the middle as shown below:
Figure 3:13 Design 1 for the filtration module. The capillary channels of 0.5 mm heights
and widths are seen as horizontal stripes in the middle. The hole in the center leads to a
collection well. Note that the filter membrane was transparent and covers an area sli
Immuno-trol™ cell [57] samples were used instead of real blood to minimize biohazard risk and
comply with lab regulations. They were mixed with fluorescent Spherotech [58] beads of
diameter 3.42 µm to allow the use of fluorescent microscopes. Those beads were diluted 10x and
10 µl of this solution and added to 90 µl of Immunotrol blood to achieve fluorescent bead
concentrations around 1000/µl. These numbers were chosen to be close to the average number of
WBCs in the human blood.
24 µl of this Immunotrol + bead mixture was taken and pipetted into the sample preparation
module. After a few minutes, it was seen that the capillary channels were full and the filter
30
membrane was carefully removed from the filter holder. The plasma was extracted from the
capillary channels using a pipette and analyzed under a fluorescent microscope. However, when
the experiment was performed, it was noted that the filter membrane would sometimes have an
air gap. This would cause the blood drop on the top to roll off to the side and contaminate the
plasma extracted by the capillary channels. The results are shown below
Figure 3:14 Fluorescent images of A) beads present in the whole blood before filtration and
b) after filtration in the extracted plasma from capillary channels. The presence of beads in
B) indicate the failure of the filtration process through leakage of the whole
Design 2: The sample prep module had six equally spaced holes at the top for even distribution
of the pipetted blood as well as a circular rim to clamp the filter membrane to prevent leakage
issues. Similar experiment was performed using the second design. The plasma was extracted
from the capillary channels using a pipette and analyzed under a fluorescent microscope. As
expected, no fluorescent beads were visible indicating that this filter module restricts passage of
particles 3 µm or higher.
31
Figure 3:15 A) Blood droplets in the six equally spaced inlets on the clamp part for uniform
distribution of the blood and right B) After filtration picture with the clamp removed. The
presence of the clamp secured the membrane filter and reduced the air gap. Thus, plasma
was seen to be more widely dispersed throughout compared to the previous filter design.
The Solidworks drawing files are listed in Appendix A.
Figure 3:16 Fluorescent images of a) remnants of blood on top of the filter and b) extracted
plasma from the capillaries. The second design with the clamp on top of the membrane
filter prevented leakage and hence no fluorescent beads were seen.
As shown in the above pictures, the filtration is a 2-step process requiring the use of 3D Printed
Holder containing the capillaries as well as a commercial filter paper with appropriate pore sizes.
Here the application was to filter blood cells and therefore 2 µm would be sufficient to remove
the smallest cells.
This is useful for a significant number of biochemistry applications where ultrapure samples of
plasma are needed for example in quantification of C-Reactive Proteins (Chapter 4).
32
Chapter 4
Bead-based Assay Module
4.1 Sepsis Literature Review
Sepsis is a range of symptoms caused by the host response to infection. A reliable indicator of
Sepsis is the C - reactive protein (CRP) present in the blood plasma. The biochemistry of an
existing bead-based assay from the literature will be adapted to make it suitable for disposable
microfluidics. A brief review of sepsis and its diagnosis in clinical settings is described below:
The Society of Critical Care Medicine Conference proposed the definitions of Sepsis, Systematic
Inflammatory Response Syndrome and Multiple Organ Dysfunction in 1991 [59]. A severity
index was developed during the conference to deal with septic patients to assess their mortality.
The systemic response to an infection was termed as Sepsis and is the most common cause of
ICU deaths. On the other hand, Systemic Inflammatory Response is a generalized inflammatory
response whose cause may not necessarily be an infection. The figure below illustrates the
various definitions:
33
Figure 4:1 Classification of Inflammatory response ([59]). Sepsis is diagnosed when both
infectious agent and inflammation is present in the patient. The cause of this infection
could be bacteria, fungus, virus, etc and they are denoted by circles indicating the relative
frequency at which they occur. Similarly, inflammation could be because of trauma, burns,
pancreatitis or other causes.
If the Systemic Response is characterized by 2 or more of the following symptoms, then it can be
classified as Sepsis: a) Temperature >38o C or < 36o C, b) heart rate > 90 beats/minute c)
Respiratory rates > 20 breaths/ minute d) WBC count > 12000/mm3 or <4000/ mm3.
The presence of viable bacteria in the blood is termed as Bacteremia. Multiple Organ
Dysfunction is the presence of altered organ function in an acutely ill patient whose homeostasis
cannot be maintained without intervention. Any infection can lead to a response from the body
with a continuum of severity possibly leading all the way to mortality.
More than 30,000 patients are hospitalized for sepsis every year in Canada and about a third of
them die [60]. Even the patients who survive end up spending a lot of time in the Intensive Care
Unit (ICU) resulting in high personal and economic costs. The median hospital stay was 12 days,
more than a week longer than for other diseases. Among patients with severe sepsis, organ
dysfunction results with the respiratory system most commonly affected. Patients for whom
34
sepsis was diagnosed had lower odds of dying compared with patients for whom sepsis was not
identified in the Emergency Department.
Due to the above reasons, considering the severity of the infectious process will allow the
healthcare professional to make an informed choice on the allocation of resources and in making
better clinical decisions. Since the symptoms of Sepsis are numerous, several scoring systems
have been developed [61]. A common method will be to evaluate the observed physiological
response of the patient. For example, the Organ-specific scoring system like the Glasgow Coma
Scale (GCS) or general ICU scales (Logistic Organ Failure Score LODS, Multiple Organ Failure
Score MODS, Sequential Organ Failure Assessment SOFA) like the APACHE are highly
dependent on the quality of the input. The Organ Dysfunction scores assess only degree of organ
malfunction. The data collection rules must match exactly those stipulated by the model.
Moreover, the observer reliability must also be taken into account. In addition, many of the
equations underlying the models rely on the limited populations of ICU patients available. And
finally, the use of other measures introduces a bias into the predictive equations.
Given the multitude of non-quantitative options discussed above to diagnose sepsis, there is a
necessity for a more specific test for sepsis. This is achieved through biomarkers [62].
Biomarkers are defined as a ‘characteristic that is objectively measured and evaluated as an
indicator of normal biologic process’. The usefulness of biomarkers are indicated by their
Diagnostic Accuracy (DA). This is characterized by high values of Sensitivity, Specificity,
Positive Predictive Value (PPV), Negative Predictive Value (NPV), Positive Likelihood Ratio
(PLR) and Negative Likelihood Value (NLR). Their definitions are given in the table below:
35
Table 4-1 Definitions of Diagnostic Accuracy of Biomarker (Courtesy [59])
C - reactive protein is one of the most common biomarkers [63] used as the concentration can
increase 1000-fold after infection. As seen from Table below, CRP has favorable detection range
encompassing nearly 2 orders of magnitude allowing the most common bacterial and fungal
infections to be identified. Therefore, CRP was selected as the Sepsis marker in this thesis.
Additionally depending on the commercial test used, the DA values can be close to the ideal
100%. The lower ranges in some of the tests are due to the different population characteristics
like age which influence the cutoff values.
Table 4-2 Diagnostic Accuracy (DA) Values of C-Reactive Protein, Procalcitonin, Serum
Amyloid A, Mannan and Antimannan and IFN-γ -inducible Protein Biomarkers
(Reprinted with permission from © 2011 Taylor & Francis [62])
Researchers have found that levels below 100 mg/L are indicative of fungal infection while
above 100 mg/L occur for bacterial infection as shown in Figure.
36
Figure 4:2 Thresholds of different markers for distinguishing infectious pathogens in blood
(Reprinted with permission from © 2011 Taylor & Francis [62]). The bacterial cause of
Sepsis is marked by a high value of CRP beyond 100 mg/l of blood. In contrast, viral causes
of Sepsis are marked by elevated levels of CRP but they tend to stay below 10 mg/l. Both
regimes of CRP levels are quantified using a bead-based assay in section 4.6
The above table summarizes other sepsis biomarkers as well as thresholds used clinically to
categorize the cause of infection into bacterial, fungal or viral.
The fastest test among the commercial systems is Alere’s Nyocard (3 minutes read time) [ibid].
However, the measuring device is restricted to sequential testing of proteins, multiplexing ability
does not exist. A diagnostic system with minimum sample preparation and parallel testing of
multiple proteins is needed. This is because no single marker has been deemed sufficient to
reliably identify/rule out infection.
37
Table 4-3 Commercial CRP detecting systems (Reprinted with permission from © 2011
Taylor & Francis [62]) All Assays except the last two, use either plasma or serum instead of
whole blood to quantify CRP.
Hence, an attempt will be made in this thesis to develop an assay that is amenable to
multiplexing. In later sections of this chapter, a bead-based assay for detecting CRP is presented
that has the potential to be extended for multiplexed protein assays.
38
4.2 Bead-based Assay Review
In this section, a commercial multiplexed protein test is reviewed to illustrate the general steps
involved. Proteins can be detected using sandwich detection schemes like Enzyme Linked
Immunosorbent Assay (ELISA). Benect Dickson (BD) [64] has an assay kit consisting of a Bead
Array that allows flow cytometric users to quantify several proteins in parallel. This method is
suitable for microfluidic applications due to the significantly reduced sample volumes and time
compared to ELISA and Western Blot tests. This is achieved by the use of beads, which have a
different but unique fluorescent intensity, and on whose surface are coated antibodies capable of
capturing specific analytes.
The BD CBA Flex set is capable of analyzing up to 30 proteins from a sample size as small as 25
µL. ELISA and Western Blot require similar volumes for just a single protein. The pictorial
representation of the assay is given below:
Figure 4:3 BD CBA test with 5 unique (A-E) beads having different fluorescent intensity
([64]). In commercial bead-based assays, the beads have a unique color as well as a unique
capture antibody on their surface. This allows multiplexed sample diagnostics
39
The beads are initially unconjugated allowing antibodies to be attached through sulfo-SMCC
chemistry as shown below:
Figure 4:4 Antibody conjugation of the BD’s CBA set using sulfo-SMCC chemistry ([64]).
The conjugation of capture antibodies is performed using chemical bonding techniques.
However, later in this chapter, a technique for attaching antibodies through passive
adsorption is demonstrated.
Once the conjugation is done, the sample containing the analytes of interest is added. Then
detection antibodies conjugated with a different fluorophore are added to form a sandwich with
the analytical protein in the middle. All beads are then excited with 2 lasers (488 nm or 532 nm
and 633 nm). The beads have different emission intensities allowing a qualitative yes/no test for
the protein. The fluorophore attached to the detection antibody will then give a quantitative
reading for each protein. In the absence of the targeted antigen on the surface of the bead, the
bead will still fluoresce when excited by red laser but there will not be any signal when the
second laser (532 nm) is shone due to the absence of the detection antibody. The detection
algorithm picks up this absence.
Researchers [65,66] have used CRP for detecting upper respiratory tract infections in addition to
sepsis on a bead-based platform. The Christodoulides et al used Agarose beads to capture the
CRP antigen in human saliva in patients with dental inflammation. The beads were porous and
presented a larger surface area for the immobilization of capture antibodies than possible in
ELISA. They coupled the Antibodies onto the bead using reductive amination. Their results
40
indicate that fluorescence detection was more sensitive to the colorimetric detection. The epi-
fluorescent setup allowed the signal coming from the entire Agarose bead to be sensed. Kulla et
al [66] developed a similar assay using the CRP biomarker to detect children who are at a risk of
death from malnutrition. In both the applications. The beads were positioned in microfluidic
channels in the path of the free flowing analytes. Microtainers hold the beads in place while the
fluid flows through them and around them. Agarose beads have diameters under 400 µm, which
makes them less amenable to be used in 3D Printed wells. Therefore, larger beads were
necessary to perform the bead-based assay for which 1/16” Nylon beads were chosen in this
thesis as described in the following section. The beads are well adapted for integration into a
disposable cartridge. Only the biochemistry part of the test will be on the disposable chips. This
allows for potential interoperability with fluorescent optical detection kits from several vendors
in the future as previously discussed in Chapter 2.
41
4.3 Assay Development
To enable the protein assay to be performed on a 3D printed platform, it was necessary to find
suitable beads. Polysciences had large (1/16”) polyamide nylon 6/6 beads [67], which were large
enough to not be washed away during the washing step.
This eliminated the need for external magnetic force generators to immobilize the beads. Before
the 3D Printed well can be used to hold the beads, it was necessary to first develop a reliable
assay protocol using beads incubated in plastic vials. The following sub-topics outline how the
assay was systematically developed to realize an Antigen detection module.
i) Choice of bead material: Nylon beads were chosen because they perform well even under a
chemically aggressive environment [68]. They are insoluble in most organic and diluted
inorganic acids, resistant to alkalis and have a glass transition temperature of 50o C. They are
also readily available in all sizes and forms with worldwide production being 3.4 million tons
[69]. They do not float on water due to their density of 1.19 g/cc making them ideal for fluidic
applications. Beads with lower density would have required external magnetic fields to hold
them in place. Nylon absorbs moisture better than other polymers.
ii) Choice of Blocking Agent: One of the critical steps in most assays is the addition of the
blocking agent after the addition of the antigen [70]. Enzyme Linked Immunosorbent Assay
(ELISA) is a common solid phase immunoassay where biomolecules of interest are selectively
captured on the surface of the bead. Non-Specific Binding (NSB) of other proteins during
subsequent steps can occur on the unoccupied spaces. To prevent this, these unoccupied sites are
saturated with a blocking agent. An ideal blocking agent does not take part in the assay reaction
while simultaneously preventing other NSB [71].
There are two major classes of blocking reagents: proteins and detergents. A good blocking
agent will
1) Prevent Non-specific binding on the unoccupied sites
2) Not exhibit cross-reactivity with biomolecules in subsequent steps
3) Not interfere with the existing binding of the antigen
42
4) Stabilize the existing antigen-bead bonds and prevent denaturation
Bovine Serum Albumin is a commonly used protein blocker. It is a permanent blocker and hence
only needed to be added once. The typical concentrations used is 1 to 3%. BSA of concentration
(50 mg/ml) from Invitrogen was purchases and diluted 2x with Phosphate Buffer Saline (PBS) to
achieve the necessary concentration required for stable blocking.
iii) Choice of Reporter fluorophore: The physical phenomenon where light is absorbed by a
material and subsequently re-emitted is termed as fluorescence [72]. There is a negligible time
delay (microseconds) between the absorption and the emission processes. Fluorescence emission
will always be at a longer wavelength compared to the absorption wavelength. A fluorochrome
is a substance that has a well-defined absorption and emission spectra whose light output is
available at useful intensities. They are highly specific and have a high quantum yield. This
fluorochrome when attached to a binding antibody is termed as fluorophore
Figure 4:5 Fluorescence Emission process flow ([73]) Absorption of a suitable photon
causes the electron to jump from the ground state to an excited state. The decay is usually
instantaneous and is accompanied by the emission of longer wavelength light.
As shown in the figure above, fluorescence is a three-step process. During the excitation phase,
the fluorophore absorbs a photon from the light source and goes from the ground state to the
excited state S2. Then it decays rapidly to state S1. Finally, a photon with a lower energy is
43
emitted as the fluorophore goes back to the ground state. To ensure easier optical filter
requirements, a good spectral separation between the excitation and emission spectra is
necessary. This implies a large Stokes shift. Therefore, dyes with a large Stokes shift are suitable
for easier fluorescent hardware detection requirements.
Phycoerythrin was chosen as the fluorophore in this thesis due to several reasons. The Molecular
Weight of R-Phycoerythrin is 240,000 Daltons [72]. They are classified under Phycobiliproteins,
which have high Quantum Yield. They are derived from cyanobacteria, which have high
absorbance and fluorescence without quenching. Therefore, Phycoerythrin has relatively high
fluorescent yield (comparable to 30 fluorescein or 100 rhodamine molecules). The excitation
spectrum of Phycoerythrin lends itself well to multiplexing with other fluorophore. For example,
a different cell population tagged with Alexa Fluor can be detected by having a filter at 520 nm
while simultaneously having another filter with a >575 nm pass band.
Figure 4:6 Absorption and Emission spectra of 3 Phycobiliproteins – R-Phycoerythrin
(RPE), B-Phycoerythrin (BPE) and Allophycocyanin (APC) ([72]). R-PE has a broad
absorption spectrum and a narrow emission spectrum centered close to 585 nm
Phycobiliproteins, R-PE was chosen in this thesis because of readily available conjugated
proteins (Streptavidin-PE, Biotin-PE and Goat IgG1-PE). The Quantum Yield of R-PE is 0.82.
44
iv) Antibodies:
Antibodies have four polypeptide
chains. These glycoprotein molecules
are also known as Immunoglobulin
(Ig) [15]. The amino acid composition
of the terminal ends are diverse and
hence referred to as the variable (V)
regions. The total molecular weight of
each monomer Immunoglobulin
molecule is approximately 150,000
Daltons (made of two H chains of
50,000 each and two light chains of
25,000 each)
There are two binding sites in each
monomer where the variable regions
occur and can be tailored to be
specific to an antigen. The neck of the V-region is called the hinge region and is held together by
Disulphide bonds. There are five types of Immunoglobulin – IgA, IgD, IgE, IgG and IgM. The
differences arising from the polypeptide regions of the Fc chain.
Immunoglobulin Gamma (IgG) is the most common antibody found in the human serum
constituting nearly 75% of the total Ig. Hence, it is the predominant version used in clinical
diagnostics. Antibodies can be further classified as monoclonal or polyclonal depending on their
specificity to a region on the antigen. Monoclonal antibodies are specific to a single epitope on
the antigen and work well as primary antibody. Polyclonal antibodies on the other hand are better
suited for use as secondary antibodies due to their abilities to target multiple epitope sites on the
antigen. In this assay, monoclonal antibodies were used when specific capture was necessary and
polyclonal antibodies were used for the reporter fluorophore.
Figure 4:7 Antibody structure showing the Heavy
chains and the Light chains (Courtesy [74])
45
4.4 Adsorption on bead surface
Literature [75] exists on protein adsorption on polystyrene beads where the authors noted that the
biological activity is greater for the adsorbed antibodies as opposed to covalently linked
antibodies. This was because the sites on the protein molecule for covalent binding were the
same as those meant for antigen binding. Additionally, those studies also indicated that the
adsorbed protein was stable under dilution and would only be desorbed if a competing protein
with a higher binding energy existed. However, it was found that research on protein adsorption
on polyamide beads used in this thesis was lacking. Therefore, an empirical approach was used
to determine the quantities required for binding.
Guideline amounts of the required protein to form a monolayer was given in the supplier
(Bangslab) literature. Adsorption is through hydrophobic attractions (Van der waals) between the
polybead’s surface and the hydrophobic parts of the protein. The Fc (stem) portion of the
antibody is more hydrophobic and hence attaches more readily than the Fab region. This is ideal
as the biologically active Fab region is exposed and will not be physically hindered. To make
sure that the protein is oriented the right way, an excess amount is recommended to be added
[76]. The base amount needed for a monolayer is given by
S= (C)(6/Dρ)
S = Protein amount for surface saturation; C = Microsphere surface capacity
D = Diameter of beads; ρ = Density of beads
Data already exists [76] on concentration needed for bovine IgG. About 15 mg of Bovine IgG is
needed for 1 g of 1 µm of beads. The density of the polyamide beads is 1.19 g/cc and the
diameter is 1.5875 mm. This corresponds to 8 µg of protein for coating the surface of 1 g of the
bead. Since each bead weighs 2.5 mg, the quantity of protein on the monolayer is 20 ng. Due to
limitations in the amount of reagents available, the amount of protein added in the first
adsorption step was approximately 10x lower.
Researchers [77] have previously investigated the effects of applying an electric field to speed up
the adsorption kinetics. They used a conducting layer on an optical waveguide as the substrate
for protein adsorption. They have found that applying an electric voltage caused a significant
46
increase in adsorption on the conductive layer. Electrostatic attraction between the charged
adsorbent surface and the oppositely charged amino acid favored adsorption.
While this method is suitable for detecting the adsorbed proteins through a direct method such as
through refractive index change or spectroscopic based detection methods, it will not be suitable
for indirect methods such as fluorescence detection.
The functional surfaces of the protein might be de-activated by the change in pH caused by the
application of the electric field. This can lower the antibody binding in subsequent steps making
it more difficult to get a fluorescent signal.
Also improper voltage levels could potentially cause hydrogen bubbles from the electrolysis of
water, that can compete with the protein adsorption on the bead surface. Eliminating the bubbles
would require optimizing the electrical resistance of the buffer solution.
Finally, inclusion of extra electrodes would be disadvantageous to the compactness of the bead-
based assay for POC applications. For these reasons, simple adsorption without any recourse to
electrical voltage has been attempted in this thesis.
47
4.5 Streptavidin-Biotin Assay
A) Qualitative Assay: The aim was to develop a qualitative assay to detect the presence or
absence of an antigen. To characterize the applicability of beads to detect fluorescence, a test
protocol was developed to detect Streptavidin [78] through the use of the fluorescently labelled
Biotin [79].
The primary aim of using large beads for a bead-based assay is that they can be immobilized in a
3D printed channel as shown below
Figure 4:8 Large (1.5 mm diameter) Nylon beads can be trapped in 3D printed fluidic
channels within a disposable cartridge.
The first step was to develop the assay on a general protein. Streptavidin was chosen as the
antigen of choice due to its strong binding kinetics with Biotin. Streptavidin is a large molecule
and has a Molecular Weight of 60,000 g/mole. An assay was developed as follows: A known
amount of Streptavidin was added to the beads. The incubation was overnight followed by the
addition of blocking agent BSA for another 24 hours. After thorough rinsing, the detection
antibody was added (Biotin conjugated with Phycoerythrin). This was flushed before imaging
under a fluorescent microscope having a mercury vapor lamp source fitted with a 530 nm
Excitation filter and the 585 nm Emission wavelength filter. The steps are included in Appendix
C: Table 1. The results are shown below:
48
The vertical axis the made of arbitrary
intensity units when analyzed by ImageJ.
As expected, the bead with Streptavidin
coating fluoresced with higher intensity
than the Control bead, indicating that
successful adsorption of Streptavidin had
taken place on the surface of the bead.
As seen from the bar graphs, the fluorescent intensity of the streptavidin-coated beads was about
3x higher than the control beads. This is sufficient for typical optical detection tools to detect the
presence or absence of the antigen. This would be useful for a simple yes/no tests for diseases
like Ebola.
Figure 4:10 Image of the nylon bead with 8 pico moles of Streptavidin. 3x higher intensity
was observed in the presence of protein. The image was taken with a CCD camera attached
to a fluorescent microscope with each snapshot having a 50 ms exposure and with image
0
500
1000
1500
2000
2500
3000
3500
No Streptavidin Streptavidin-coatedFlu
ore
scen
t In
ten
sity
(A
rbit
rary
U
nit
s)
Fluorescent intensities of beads
Figure 4:8 Fluorescent intensities of beads with no
added Streptavidin and Streptavidin-coated beads
upon the addition of Biotin conjugated with R-PE
Figure 4:9 Fluorescent intensities of beads with no
added Streptavidin and Streptavidin-coated beads
upon the addition of Biotin conjugated with R-PE
49
Figure 4:11 Image of bead with no protein. Only the circular outline of the bead is visible
under the fluorescent microscope. The non-zero background could possibly be attributed to
auto-fluorescence of the bead itself.
50
B) Quantitative Assay:
The next phase was to optimize this assay to enable quantitative enumeration of Streptavidin. A
variable quantity of streptavidin was added onto the beads and incubated. Then the blocking
agent (BSA) was added afterwards to block unbound sites on the bead surface. Finally, Biotin
conjugated with R-PE was added and imaged. The exact recipe is attached at the Appendix. The
results from ImageJ were plotted and shown below:
Figure 4:12 Variation of Fluorescent Intensity with added Streptavidin (direct adsorption).
The Streptavidin was added in increasing concentrations, incubated and washed. BSA was
added to block unbound sites on the surface of the beads. Finally, Biotin-RPE was added to
tubes containing the beads to use the fluorescent methods of detection. The images were
captured by a CCD camera and then fluorescent intensity information was extracted using
ImageJ by drawing a rectangular box and using the Analysis-> Measure toolbar. The
orange line at approximately 5000 intensity units represents the background fluorescence.
The graph indicates that the adsorption of Streptavidin rises monotonically with increasing
concentration. When the amount of Streptavidin is low, there is at most a 2x higher intensity
compared to the control bead intensity of ~5000 units. The brightest beads had 4x more intensity
than the beads without the protein. The lowest fluorescent intensity that could be discerned
corresponded to an initial quantity of 0.66 pico moles of Streptavidin.
y = 952.74x + 7669
0
5000
10000
15000
20000
25000
0 2 4 6 8 10 12 14
Flu
ore
scen
t In
ten
sity
(A
rbit
rary
Un
its)
Streptavidin Quantity (pico moles)
Fluorescent Intensity vs Streptavidin Quantity
Baseline Fluorescent Intensity
51
4.6 C - reactive Protein assay
The subsequent phase was to develop an assay that would detect a specific disease. As discussed
earlier in the literature review section, C - reactive protein (CRP) is an important biomarker for
Sepsis as discussed in earlier sections. Detecting this protein using adsorption test on the beads
would be an important step towards integrating this module on a 3D printed platform.
Researchers [80] have previously found that 75% of the adsorption is completed in the first 5
minutes. Further increase in the time will correspond to more adsorption. In this thesis, the
incubation was done for several hours just to ensure that adsorption would be close to the
theoretical maximum. The assay steps are illustrated in the figure below:
Figure 4:13 Flow chart for direct (non-specific) capture of the CRP antigen. Nylon beads
were used as a substrate for capturing antigen through adsorption. They are then bound
with primary antibody C2 followed by secondary antibody (IgG1 conjugated with R-PE).
The secondary antibody has a fluorescent tag that emits strongly at 585 nm. Additional
blocking and washing steps are not shown.
52
The typical volumes of a pinprick of blood is 25 µl. This corresponds to about 2.5 µg of the CRP
antigen present in that volume when the levels rise beyond 100 mg/L during a bacterial infection.
This corresponds to 0.1 nano moles of CRP.
An experiment was performed with the addition of 8, 16 and 32 µg of CRP (available from
Hytest [81])directly onto the beads (corresponding to 0.32, 0.64 and 1.28 nano moles
respectively) followed by the primary (C2) Antibody and the fluorescently tagged secondary
Antibody (IgG1-PE) from Invitrogen [78]. The complete recipe is attached in the Appendix. The
results are depicted below:
Figure 4:14 Fluorescent intensity emitted on the surface of the bead when coated with
CRP. A linear trend was observed with the emitted fluorescent intensity from the bead
increasing with higher input concentration of CRP on the bead. The orange line at
approximately 5000 intensity units represents the background fluorescence.
Discussion:
From the above graph, it is noted that the complete absence of CRP was marked by fluorescent
intensity level no greater than the background level of approximately 5000 units. However high
CRP concentrations (0.32, 0.64 and 1.28 nano moles) assay were marked by increasing intensity,
which can be approximated by a linear equation. This suggests that the assay developed in this
y = 13748x + 4527.8
0
5000
10000
15000
20000
25000
30000
0 0.2 0.4 0.6 0.8 1 1.2 1.4
Flu
ore
scen
t In
ten
sity
(A
rbit
rary
Un
its)
CRP Quantity (nano moles)
Fluorescent Intensity vs CRP Quantity
53
thesis was capable of detecting CRP concentrations beyond the 100 mg/L seen during bacterial
sepsis. Theoretically [80], the graph would flatten out as the surface adsorbed proteins reach
saturation similar to what was observed for specific capture of CRP
This simple yes/no test could be further improved by lowering the amount of CRP needed to get
a detectable signal. This can be achieved by the use of capture antibodies in the first step
followed by the addition of CRP antigen. This capture antibody coated on the surface of the
beads also ensures that non-specific adsorption does not occur. As noted in the previous sections
on Antibody stability, protein adsorbed on the surface of the beads are stable in solution unless
displaced by higher binding energy protein at a higher concentration. In case this assay was
adapted for commercial applications, large batches of beads would be indicated with an excess of
the capture antibody ready for the assay.
To illustrate this sandwich ELISA-like test, experiments were performed with the following
procedure:
An excess of monoclonal anti-CRP Antibody C6 (from Hytest [81]) was incubated with the
beads to ensure a monolayer was obtained with the above capture antibodies on the bead surface.
After a washing step to remove unbound C6 antibody, CRP was added to the beads and
incubated. After a washing step, a blocking agent (2.5 % BSA) was added to cover any
remaining unbound sites on the bead surface. Subsequently, the monoclonal detection antibody
C2 (from Hytest [81]) was added, incubated and washed. This ensures the CRP is between the
antibodies C6 and C2 as illustrated in the figure below:
54
Figure 4:15 Flow chart for indirect (specific) capture and fluorescent detection of the CRP
antigen. This is similar to Figure 4:12 except there was an additional step initially to coat
the Capture Antibody. This method is more specific to CRP and has a better Limit of
Detection due to the smaller quantity of CRP required.
The last step was to add the fluorescent-tagged antibody that binds with C2. Polyclonal Goat
Anti-mouse IgG1 bound with R-PE was added, incubated and washed. The beads were taken and
imaged. The results are shown below:
55
Figure 4:16 Fluorescent intensity vs. concentration of CRP using the indirect capture. The
orange line around 5000 intensity units represents background fluorescent intensity of the
bead. A logarithmic trend in the emitted fluorescent intensity was observed.
As expected, increasing the concentration of the added antigen caused an increase in the
fluorescent intensity. The data suggests that there is a logarithmic rise in the fluorescent intensity
with CRP quantity. This could be either because the capture antibody sites were nearly saturated
with CRP or the secondary antibody does not have enough space to attach itself due to steric
hindrance.
The advantage of this method over the previous direct adsorption method is that similar output
fluorescent intensities were achieved with nearly 1000x lower CRP concentration. This implies
that using this ELISA scheme to quantify CRP is more sensitive than direct adsorption This
would ease the hardware requirements for lower concentrations typically seen in viral infections.
y = 5603.1ln(x) + 9237.2
0
5000
10000
15000
20000
25000
30000
0 2 4 6 8 10 12 14 16 18
Flu
ore
scen
t In
ten
sity
(A
rbit
rary
Un
its)
CRP Quantity (pico moles)
Fluorescent Intensities vs CRP Quantity
Baseline Fluorescent Intensity
56
Additional tests were perfomed to see if the assay conditions themselves could be optimized. A
bead with the bound fluorophore was taken and imaged. It was imaged again after three more
washing steps. It was noted that the presence of the washing step had negligible effects on the
binding. The fluorescent intensity of a particular bead was compared before and after several
washing steps. There was little change in the bead intensity indicating that the adsorption is
stable.
0
5000
10000
15000
20000
1 Wash 3 Washes
Flu
ore
scen
t In
ten
sity
(A
rbit
rary
Un
its)
Washing Steps
Fluorescent Intensity vs Washing steps
Figure 4:16
Figure 4:17 Stability of the binding across washing steps. The number
of washing steps (to remove any excess reagents) caused an
insignificant change in the emitted intensity. This suggests that the
binding of the protein on the bead surface was relatively stable as was
discussed in section 4.4
57
The effects of the exposure time on the fluorescent intensity were also tested, it was noted that
after 1 minute of constant exposure, the emitted light intensity faded away and was
indistinguishable from a bead that had no antigen. This indicates that care has to be taken to
ensure measurements are taken immediately to avoid photo bleaching.
Figure 4:18 Effect of exposure on the emitted fluorescent light intensity. Due to the
technical limitations of the imaging unit, snapshots were taken every 30 seconds until the
emitted intensity faded away to the level of background.
In this chapter, a bead-based assay was demonstrated for the Sepsis biomarker called CRP. This
assay was performed in tubes instead of fluidic channels. Due to the large diameter of the bead,
no immobilization would be necessary in case this assay were to be performed on a 3D printed
fluidic device. Two designs of proof-of-concept disposable 3D printed modules were developed
for holding the beads and are listed in Appendix A.
y = -4.5329x2 + 41.69x + 19356
0
5000
10000
15000
20000
25000
0 10 20 30 40 50 60 70
Flu
ore
scen
t In
ten
sity
(A
rbit
rary
Un
its)
Expsoure (seconds)
Fluorescent Intensity vs Exposure
Baseline Fluorescent Intensity
58
Chapter 5
Optical Detection System
5.1 Point-of-Care manufacturer
Globally, remote or rural areas lacking access to diagnostic capacity results in preventable
deaths. ChipCare’s device, a lab quality, point-of-care blood testing platform, increases access to
life-saving diagnostics for people without access to primary health care.
ChipCare Corporation is a Toronto-based IVD medical device company [82]. To target POC
applications targeted at linking people with HIV to appropriate treatments, ChipCare Corp will
be rolling out a portable blood testing platform in 2016. The envisioned product entails two
components: handheld reader and disposable test cartridge. Blood sample is drawn on to a
disposable plastic cartridge via a finger prick. Upon the insertion of the cartridge into the
handheld analyzer, blood analysis result is available in 10 – 15 minutes.
Currently, the handheld reader for just the cell count has been designed and assembled.
However, the same reader cannot currently perform protein-based assays for infectious and non-
communicable diseases. The development of the new versatile detection system in this project
was a starting step that will enable both cell and protein based assays to be performed on the
same device. The development of 3D printed bench top prototype developed in this chapter was
undertaken through support from the Mitacs Accelerate internship
5.2 Existing HIV diagnostic methods
In this section, review of HIV diagnostic methods available commercially and under research are
discussed. The WHO review article [83] introduced some common detection methods employed
in diagnostics as well as POC Diagnostics companies adopting these technologies.
Acquired Immunodeficiency syndrome (AIDS) is a disease caused by the Human Immunovirus
(HIV). The CD4+ T lymphocytes cells of the immune system get destroyed and the infected
patient is left vulnerable to other infections [84]. The pandemic nature of AIDS meant that a lot
of resources have been allocated to diagnostics and treatment. The resulting progress has seen a
59
20 % reduction in the number of people newly infected since 1998 [85]. However, the disease
still represents a challenge to economic progress due to the large number of people (34 million
infected as of 2010) infected. Since the disease is highly infectious at an early stage, early and
accurate detection is important. Furthermore, timely access to treatment enables patients to
reduce adverse health outcomes and increases their life expectancy.
The tests for one-off diagnosis are well established (ELISA) [86]. However, ongoing treatment
for HIV requires timely knowledge of the disease progression within a patient. Detecting CD4,
Viral Load and Early Infant diagnosis (EID) present hurdles to be overcome. The present
generation of lab-based platforms are expensive, as they require long-distance specimen
transportation. This makes diagnostics out-of-reach for people in remote areas. Community-level
access to Antiretroviral Treatment (ART) will require simple, affordable POC diagnostics
without the patient having to undergo arduous journey to the clinic. To characterize POC
devices, World Health Organization (WHO) introduced the ASSURED (Affordable, Sensitive,
Specific, User-friendly, Robust, Equipment-free) criteria [87].
The analytical targets for POC devices can be proteins, nucleic acids, human cells, bacteria,
viruses, etc. [86] The Ora Quick Rapid HIV-1/2 Antibody Test is a lateral flow test using oral
fluid specimens. Another POC device is Aware HIV-1/2 U that is an alternative ELISA test and
claims to have 100% specificity and 97% sensitivity. [89] The technologies involving blood
samples are a lot simpler in their implementation compared to the products above.
In general, HIV diagnostics can be divided into three varieties: a) Initial diagnosis, b) tests to
quantify the patient stage c) Tests to monitor the success of ART. HIV testing can be done on
either the anti-HIV anitibody or the p24 antigen [90]. The antibody may not show up in the blood
for several weeks after infection, yet, the person is highly infectious in the first weeks. Tests to
determine both the antibody and the antigen will decrease the probabilities of false negatives and
are required for purposes for accuracy.
P24 antigen can be detected during the early stage HIV infection because it is characterized by
early spike in the antigens. In most patients, this early spike is detectable until the levels decrease
due to the patient’s immune response to the infection. After several years, the antigen levels start
increasing again with the failure of the immune system and the antigens become detectable
again. There are several viral proteins of which p24 is quite prominent as it is the major internal
60
structural protein of HIV-1. Hence, quantifying p24 is useful for a) identifying early HIV
infection b) diagnosing infant infection and c) monitoring the success of ART [91]
HIV viral load tests can quantify the patient’s disease progression stage as well as the ART
treatment success. Reverse Transcriptase (RT) protein serves as a valid indicator of HIV load
and is a cheaper alternative to longer nucleic acid tests. However, RT viral load tests are not as
reliable as results obtained from PCR. They do have the advantage of detecting multiple
subtypes of HIV and also HIV load in pediatric patients [92].
Viral load testing can be further divided into Nucleic Acid-based Tests (NAT) and non-NAT
based technologies. Viral RNAs are used in NAT technologies to quantify and to detect, while
non-NAT technologies use proteins and enzymes from the HIV virus itself. NATs are usually
more reliable as clinicians are familiar with interpreting the results [93]. Some of the RT-PCR
system examples are given below.
Roche Molecular System’s COBAS HIV Monitor, Abbott’s Real-time HIV-1 and Siemen’s
VERSANT HIV-1 RNA assay [94].
RT assays can be correlated to the levels of HIV virus by quantifying the viral enzyme [95].
ExaVir Load from Cavidi AB is an example of RT assay and is generally less expensive.
Moreover, it does not target any specific Nucleic Acid, so the assay can measure any HIV
subtype.
The gold standard for quantitative viral load testing is RT-qPCR. PCR amplifies regions of DNA
in vitro and can create millions of copies by cycling between different temperatures. However,
the test requires trained technicians, expensive reagents and dedicated lab space to run it.
Furthermore, the nature of the PCR reaction means that the simplicity of the PCR chemistry is
overridden by the use of complicated supporting hardware. This makes the hardware unsuitable
for POC settings.
61
5.3 HIV monitoring using CD4 counts
The Human Immuno Virus depletes the patient’s CD4+ T Lymphocyte count, therefore HIV can
also be quantified indirectly by measuring CD4 counts. Antiretroviral Therapy has to be begun to
slow the progression of the disease to AIDS. Additionally, the risk of disease transmission from
the mother to the child can be reduced upon treatment [85]. The WHO recommendation is for the
treatment to begin at counts below 350 cells/µl. With the aim of achieving CD4 cell counts using
easier-to-use POC devices (reduced blood volume, portable instrumentation, rapid test results,
etc), a few diagnostic companies have released their products. There are a few portable devices
that carry this test: PointCare NOW, Partec’s CyFlow, Alere’s Pima and Daktari Diagnostics’
Daktari CD4+. These devices are powered by batteries, require finger pricks of blood (<25 uL)
and provide rapid results and they do not require cold storage for their reagents [84].
The technologies underlying some of the POC devices are discussed in this section. Daktari’s
test [96] uses a chromatography gradient to capture CD4+ cells from whole blood and a non-
optical method of counting. The cartridge is disposable and contains the reagents. The cells are
captured by antibody-coated chamber and then selectively lysed. The cellular ions are imaged by
impedance spectroscopy. This impedance can be correlated to the cell count over the required
range.
The Pima [97] device has two fluorescence channels to detect labelled anti-hCD3 and anti-hCD4.
The cartridge and the detection analyzer are separate. The disposable cartridge has all the
reagents and the specimen. The absolute counts of the CD3+ and CD4+ cells are displayed along
with the Quality Control results.
In addition to the above advantages, 1) Easier technology implementation 2) Wider choice of
reagents 3) Readily available market encourages the development of fluorescent assays, CD4
quantification using fluorescent particle counting was thus chosen by ChipCare Corp as their
choice of POC technology implementation.
62
5.4 Fluorescence Microscope Background
A brief review of the fluorescent microscope is presented in this chapter. Optical detection is the
simplest and therefore the most widely used in immunoassay applications. Optical methods are
again divided into 5 classes: fluorescence, luminescence, absorbance, surface plasmon resonance
(SPR) and surface enhanced Raman Scattering (SERS). Fluorescence has the largest number of
applications due to the sensitivity and range of available colors. They are also suitable for
multiplexing and offer very low Limit of Detection (LOD).
In a fluorescent microscope, the specimen is irradiated with a small band of wavelengths and the
weaker emitted light is collected after separating it from the much stronger excitation light. The
excitation light is 5 -6 orders of magnitude brighter than the emitted light is usually stopped by
the use of optical filters. The limit of detection for the fluorescently detected light is constrained
by how dark the background is. The higher the contrast of the signal, the better.
The epi-fluorescent microscope [98] consists of a high-intensity light source that directs light
onto the specimen through the objective lens (Illuminator). The microscope uses the same
objective lens to capture the emitted light. The advantage of the vertical illumination is that most
of the excitation light is
scattered away and only
a small portion is
collected by the
objective. This improves
the signal-to-noise ratio.
Additionally, the
illumination area is the
same as the collection
area thus utilizing the
full Numerical Aperture
(NA) of the Objective.
63
Figure 5:1 Epi-fluorescent microscope ([98]). The light source emits all wavelengths which
are filtered by the excitation filter within the filter cube. The sample receives this light and
emits a longer wavelength collected by the objective lens. Then it passes through the
emission filter into either the eyepiece or the camera.
Redirection of the illumination into the objective, segregation of the excitation light are both
accomplished by the elements
contained within the filter cube
[99]. This contains the dichromatic
mirror (which has different
reflectance and transmittance for
excitation and emission) and the
emission filter (bandpass filter
with the fluorophore’s emission
spectrum).
The ChipCare CD4 lab prototype uses a variation of the microscope setup with a finite optical
system and the excitation laser source coming at an angle. This simpler setup uses fewer optical
components and is cheaper to build. The description of the system that was built is given in the
next section
Figure 5:2 Illustration of the Filter Cube containing the Dichroic mirror and the filters
(Courtesy Nikon [99]) The illumination is from the right side and the light is reflected
downwards onto the specimen using the dichromatic mirror. Then the emitted fluorescence
light is allowed to pass through the same mirror and filtered by the Emission filter before
optical detection through a camera
64
5.5 System Description
The aim of this part of this project was to come up with a flexible system for the laboratory CD4
cell counting device. The overall schematic for the completed device is shown below
Figure 5:3 CD4 cell counting setup prototyped using 3D printed parts. The 3D printed
components are denoted in color. The setup was screwed onto a standard optical base. The
other external components were translation stage for the optical tube, rotary stage for the
laser, stepper motor and the disposable cartridge itself.
The previous lab setup was heavy and had a large footprint because the xyz adjustment stages for
the Optical Tube was large. Additionally, the laser and the cartridge were held in place by
clamps which made their relative adjustments not user-friendly.
To improve the user-friendliness of the lab setup, a modular approach was adopted for the
individual units as described in this section. The old lab setup was decomposed and each unit
was analyzed for improvements. New external parts were chosen for the external components
chosen for the laser angular stage & z axis translation stage and xyz stage for the optical tube to
be small.
The connecting parts were designed using Solidworks and created using 3D printing on a Zortrax
M200 printer. The turnaround time for each part was a few hours, significantly faster than
65
machining. The other advantage of 3D printing was the lower weight: the use of Plastic (ABS or
PLA) ensured the final device weighed at least 50% less than a similar metal piece. The
difference was due to the lower density of ABS (1.05 g/cc) vs. Aluminum (2.7 g/cc) [100].
The custom-built modules were Stepper Motor, Microfluidic Cartridge Holder, Laser and the
Optical Tube Holder Modules. Their purpose is described below:
Two types of disposable cartridges were being used at ChipCare (a) an old cartridge
(Resuspension) that had just the microfluidic channels and required connection to a black
pumping cartridge and b) A new (blue) cartridge that combined the microfluidic channels and the
bellow). Since experiments were being performed on both types of cartridges, an interchangeable
module was required that made it easy to swap the two types of cartridges.
The other criterion was the Stepper Motor
had to be physically held in position and
aligned with the bellow of the cartridge. The
combined part is connected to the Base Plate
using a custom-designed slider. This slider
design allows the module to be removed to
make space for the Resuspension Cartridge
Holder when necessary
Figure 5:4 Cartridge Module with custom-designed sliding base for easy removal. The parts
in pink were 3D printed while the green layer in the middle was the PCB controlling the
Stepper Motor. The cartridge is shown inserted on the right side with its bellow facing the
blue shaft of the motor.
66
When experiments were required to be
performed on the older Resuspension
cartridge, the Cartridge Module was slid out
and the Resuspension Cartridge holder
inserted. This module was designed so that
the detection spot is at the same location as
the newer cartridge even though the two
cartridges have different sizes and shapes. In
addition, there were two orientations that the
Resuspension Cartridge could be inserted,
vertical and horizontal, having two different
detection spots. Both orientations were
accounted for in this custom-design.
The 530 nm laser required Off-the-Shelf Rotary stage and
a Z-axis translation stage for easy adjustment. The Z-axis
stage was slid into a custom-built holder. The other side
was attached to another custom-built holder for the
Rotary Stage. On top of the Rotary Stage, the machined
Laser cover were attached.
Figure 5:6 Laser Module containing custom-designed
adapters for the Z-axis stage, Rotary Stage and the
Laser Covers
Figure 5:5 Resuspension Cartridge Holder. The transparent piece at the top is a
representation of the older Resuspension cartridge.
67
This module contains a base that holds the xyz stage and two complementary hollow pieces that
enclosed the Optical Tube.
Figure 5:7 Optical Tube Holder Module with the grey xyz translation stage controlling the
relative position of the tube (not shown)
Some optical components were purchased from Thorlabs [101] that were slid into the Optical
Tube Module.
1) SM1A9TS – Thermally-insulated Adapter with external C-Mount threads to internal SM1
threads (from CCD Camera’s CMount to Optical Tube)
2) SM1V10 – Adjustable optical tube with required length 20.1 mm
3) SM1L05 – Fixed Length Optical Tube with length 13.5 mm
4) SM1L40 – Fixed Length Optical Tube of length 102.4 mm
5) SM1A3 – Adapter with external SM1 threads and internal RMS threads (from Optical
Tube to Objective Lens)
These components were determined and the required lengths were calculated from the fact that
the distance between the Objective lens and the active area of the CCD camera was 160 mm.
68
5.6 Issues resolved
Vibrational issues: The hinges of the xyz translation stage that was used for aligning the optical
tube was not rigid enough to withstand the weight of the parts that were being suspended from it.
In previous versions of the setup, it was noticed that the image would drift. This was because the
center of gravity of the module was closer to the camera side causing the optical tube to tilt to
one side. Additional vibrations were noticed when the setup touched.
Changing the following minimized the vibration: the attachment position of the optical tube
holder was moved such that the center of mass was closer to the xyz stage. Additionally, screw
holes were introduced at the corners of the optical tube to seal the two halves together. Finally,
the mass of the plastic module base was increased.
Laser Module: The laser was aimed at an angle to the objective tube. To ensure proper focus, it
was necessary to include an angular adjustment tool and a z axis translation stage. However on
inclusion of these, it was noticed that the laser module spot was too tall. To fix this issue, the
module’s base was hollowed out and the laser holder was made as thin as possible.
Cartridge Holder: The disposable cartridge had to be imaged from one side and pumped from the
opposite side. This meant that the both faces had to be exposed save for a narrow region
surrounding the perimeter that was used for attachment. This was achieved by printing a rim-like
holder for the perimeter of the cartridge.
The robustness of the setup was verified by performing experiments at different orientations
(horizontal, vertical, sideways and at various angles in between) to determine the optimum angle
of the cartridge. The crucial advantage of 3D printed parts was that there was scope for trial and
error. The locking mechanism was through sliding a unit into the appropriate area rather than
using moving parts or clips.
69
5.7 Verification by CD4 Cell and fluorescent bead counting
The disposable cartridge was loaded with blood mixed with fluorescent reagents that bind to
CD4 cells. The cartridge was sealed and inserted into the cartridge module with the transparent
side facing the optics. The system was powered up and the objective focus was adjusted until a
clear view of the detection spot in the cartridge was displayed on the computer screen. To ensure
mixing of the reagents, the blood sample was pumped using a stepper motor. After an
appropriate number of mixing cycles, the sample was allowed to flow through the detection
region within the cartridge. The green laser and the objective were adjusted to point only at the
detection region and the video was recorded.
When the older (Resuspension) version of the cartridge was needed to be tested, the entire
module was taken out from the base through the sliding mechanism. Then the holder for the
other version of the cartridge was inserted and tests would be run as usual. The following
procedure was used to extract relevant data from the captured videos.
The unit was built and tested using various cartridges using blood mixed with Quality Control
Fluorescent Beads. Recorded pictures from the camera were imported to ImageJ. Then a demo
software for particle characterization was used to process the image stack. Important parameters,
such as fluorescent total intensity and particle size, were extracted from the images and analyzed
using Microsoft Excel.
The graphs and data so obtained were consistent with the older (non-3D printed) lab setup.
Hence, the performance of the new modular lab unit was verified.
70
Chapter 6
Future work and conclusions
6.1 Conclusions
This thesis demonstrated the goal of inter-device operability by building modules through 3D
printing suitable for integration with POC medical devices.
An example of a sample preparation module was presented in Chapter 3. Plasma was separated
from whole blood using a PETE filter membrane on a 3D printed capillary channel. The test was
demonstrated by filtering out 3.4 µm fluorescent beads that were mixed with whole blood. This
unit does not require an external power supply to achieve separation in contrast to conventional
centrifuge systems. This module can be the first stage in a multi-step diagnostic device aimed at
Point-of-Care settings. The extracted plasma can be either suctioned off using a pipette from the
capillaries after removing the filter paper or diluted by the addition of a buffer fluid and carried
downstream for the appropriate protein assay when used in an integrated POC device. Its
modular nature offers healthcare providers the flexibility of integrating it as a sample preparation
stage in plug-and-play diagnostic kits.
In Chapter 4, a bead-based assay was demonstrated for the Sepsis biomarker C-Reactive Protein
on the surface of a nylon bead. The quantity of CRP necessary to run the test was found to be
suitable for analysis from finger-prick volumes of blood. The direct adsorption assay gave a
linear trend for high CRP concentrations (>100 mg/l) typically seen in bacterial infections. On
the other hand, the indirect capture of CRP, by coating the bead with specific antibodies, gave a
sufficiently high signal to discern viral infections. A logarithmic trend was observed empirically.
The detection range of the two bead based assays were thus useful for identifying bacterial from
viral causes of Sepsis. By combining both types of beads in a single cartridge, this assay can be a
useful tool for the doctors in prescribing antibiotics in treating infection. This can be adapted for
microfluidic volumes due to the small quantities of sample required to get a relevant signal. The
bead-based assay offers researchers the option of multiplexing several protein detection. This can
71
be achieved by coating the appropriate antibody on the surface of the bead. The large nature of
the bead means that it can be immobilized relatively easily on a 3D printed well-type fluidic
channel.
In Chapter 5, system-level application of 3D printing was demonstrated by prototyping an optical
detection unit for a disposable microfluidic cartridge developed at ChipCare Corp. The
customized setup was modular and weighed at least 50% less than if the parts were machined
because they were plastic and because parts can be hollowed out.
Due to the modularity enabled by 3D printing technique, the designs can be easily adapted to
detect other diseases. For example, to use the fluorescent detection system (demonstrated in
Chapter 5) for diagnosing Sepsis would require swapping out the existing CD4 cartridge and
inserting the Bead-based Assay cartridge. The use of the Blood Plasma filtration module for
sample preparation would be needed only if the fluorescent signal coming the beads are found to
be weak in the presence of interfering blood cells.
To detect diseases other than Sepsis or HIV from blood, the required capture antibody will be
coated on the Nylon bead surface first and then the bead-based assay cartridge can be used with
the existing fluorescent detection system. The applicability of 3D printing in various stages of
assay development and prototyping was demonstrated. The following section describes some
suggestions on further research in this field.
6.2 Future Work
Given the rapidly advancing field of additive manufacturing, the existing limitations on size and
resolution can be easily overcome in the coming years. However to adapt this technology for
POC applications requires further research into the material aspect:
i) Sample contamination: The most popular materials for 3D printing are ABS, PLA
and Methyl Methacrylate. Currently, very little research exists on using these for
handling blood or other biological samples. It remains to be seen if ABS has
particular affinity for specific proteins existing in the blood samples or exhibits
toxicity when exposed to certain reagents. It is hoped that interest in using 3D
72
printing for medical devices will spur advancements in this field and a wider selection
of compatible materials will be available in the coming decade.
ii) Optical Detection: PLA is opaque while ABS and resin-based materials can be up to
90% transparent. However, this transparency is still less than conventional POC
materials such as PMMA thus worsening the Limit of Detection (LOD) for
experiments involving light transmission through the material. Additionally, auto
fluorescence properties of the different materials needs to explored.
iii) Disposal: The plastic materials similar to those used in 3D printing are usually sent to
the landfill as their physical strength degrades. ABS degradation takes hundreds of
years while PLA takes several years. Since, bio-hazardous waste generated by the
disposable medical devices are usually incinerated, research has to be undertaken to
see the effects of burning 3D printed materials.
iv) Material: To take full advantage of the resolution offered by 3D printers, new less
viscous resin have to be developed. Though the highest stated resolution from Table
2-1 above is ~1 µm, in practice this would not be achievable for internal microfluidic
channels. This is because for resin-based laser printers, the limiting factor would be
the viscosity of the resin: when internal channels are being created, how fast the cured
resin can drain out decides the smallest internal cavity feature that can be formed.
For the sample preparation module, the relationship of filtration time to the volume of the input
sample needs to be explored. Due to the small number of capillary channels, a few drops placed
at the right location was found to be sufficient to cover the channels and saturate them with the
collected fluid. With the right microfluidic optimization, the dead volume of the unfiltered
droplets of blood can be reduced. Numerical models could be developed for blood enabling
visualization of the separation process leading to optimization of the capillary designs.
For the bead-based assay module, the passive adsorption process can be replaced by a stronger
(covalent) binding method for antibodies that have poor affinity for the bead surface. The assay
in this thesis was performed using pure CRP samples. Therefore, the assay needs to be validated
with plasma or serum. Multiplexing could be done to detect multiple proteins from the blood
73
plasma to ensure a robust diagnostic test for Sepsis. This could be achieved by coating different
beads with different antibodies.
Agarose beads can be tried instead of nylon beads to get a 3-dimensional adsorption through the
volume of the bead. They can be packed more with the capture antibodies as they are porous than
nylon beads which are non-porous. Flow optimization could also be performed to ensure proper
volumes of reagents are mixed at the right velocity and pressure by performing simulations in a
Multiphysics software package such as COMSOL.
In Chapter 5, the volume of printing area of Zortrax M200 limited the size of the parts created by
the 3D printer. Given a large enough printer, it would be possible to create an enclosure resulting
in further reduction in the mass of the setup. Future work can also be aimed at shrinking the
optical detection unit further by using smaller optical components, shorter focal lengths and laser
diodes instead of a laser. Multiple lenses could be used to achieve the same magnification but
without requiring the 160 mm optical path. One method of achieving this would be to place the
cartridge at the focal point of the first lens. Then the second lens can be placed at the focus of the
camera. The rays will be parallel between the lenses. The use of an integrated laser diode within
the PCB of the Stepper Motor module beneath the cartridge could save space.
In the coming decades, advances in 3D printing will enable the prototyping of microfluidic
cartridges as well as optical components like lenses, etc. This will give researchers the power to
create any component within the confines of their lab if they have access to the design files. The
design of a modular fluorescence detection system allows researchers to focus on developing just
the cartridge side of the assay. With distributed access to manufacturing available through 3D
printing, medical device vendors will start selling their design files instead of their products. It is
hoped that this will give rise to interoperability of devices as envisioned by the WHO.
74
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Appendix A: Solidworks Drawings
Figure 7:1 Drawing of the 3D printed Membrane Filter Holder from Chapter 3 with 0.5
mm capillaries in the middle
86
Figure 7:2: The cap for filter membrane holder for the sample preparation from chapter 3
87
Figure 7:3: Design 1 for holding the Nylon beads from Chapter 4
88
Figure 7:4 Design 1 Bead Well Cover (from Chapter 4)
89
Figure 7:5 Design 2 for holding the Nylon beads from Chapter 4, there is a drainage
channel of 1 mm width below the bead well.
90
Figure 7:6 Design 2: The cover for the bead well for the bead based assay in Chapter 4.
91
The following files are from Chapter 5:
Figure 7:7 3D printed base for the xyz translation stage holding the Optical Tubes in
Chapter 5
92
Figure 7:8 Solidworks drawing of the 3D printed Optical Tube Holder
93
Figure 7:9 Solidworks drawing of the 3D printed holder for the z translation stage for the
Laser
94
Figure 7:10 Solidworks drawing of the 3D printed adapter from the Rotary stage to the
translation stage
95
Figure 7:11 Solidworks drawing of the 3D printed part for inserting the laser
96
Figure 7:12 Solidworks drawing of the 3D printed part for enclosing the laser. The screw
holes are M2 and they pass straight through to the Rotart stage
97
Figure 7:13 Solidworks drawing of the 3D printed part that holds both the new cartridge
and the stepper motor.
98
Figure 7:14 Solidworks drawing of the 3D printed part for holding the black cartridge and
sliding it into the main Cartridge Holder module
99
Figure 7:15 Solidworks drawing of the 3D printed part for holding the Resuspension
cartridge and sliding it into the base
100
Figure 7:16 Solidworks drawing of the 3D printed part that supports the PCB and has a
groove that slides into the base
101
Figure 7:17 Solidworks drawing of the 3D printed part that connects the Optical bench to
the Cartridge Holder module
102
Appendix B: COMSOL Simulation for Chapter 3
An example COMSOL setup is described below for a helical geometry imported from
Solidworks using the LiveLink feature. Since COMSOL, does not have an inbuilt model for
representing blood, simulating particle flowing water was performed. The simulation would
involve using the Laminar Flow and the Particle Tracing capabilities. The Laminar Flow Physics
is solved first by performing a time-dependant study to obtain the flow profile (velocity,
pressure, etc). Then, Particle Tracing Physics will be added and another time-dependant Study
will be performed, but with the results from the previous study as the input conditions. The
parameters corresponding to each study are listed below:
Geometry 1
Geometry 1
Units
Length unit mm
Angular unit deg
Geometry statistics
103
Property Value
Space dimension 3
Number of domains 1
Number of boundaries 16
Number of edges 34
Number of vertices 20
Laminar Flow (spf)
Laminar Flow
Selection
Geometric entity level Domain
Selection Domain 1
104
Equations
Settings
Description Value
Discretization of fluids P1 + P1
Value type when using splitting of complex
variables
{Real, Real, Real, Real, Real, Real, Real,
Real, Real}
Neglect inertial term (Stokes flow) Off
Properties from material
Property Material Property group
Density Water, liquid Basic
Dynamic viscosity Water, liquid Basic
Wall 1
Selection
Geometric entity level Boundary
Selection Boundaries 2–4, 6–15
105
Equations
Settings
Settings
Description Value
Temperature User defined
Temperature 293.15[K]
Electric field User defined
Electric field {0, 0, 0}
Boundary condition No slip
Apply reaction terms on Individual dependent variables
Use weak constraints Off
Inlet 1
106
Inlet 1
Selection
Geometric entity level Boundary
Selection Boundary 16
Equations
Settings
Settings
Description Value
Apply reaction terms on All physics (symmetric)
Use weak constraints Off
Boundary condition Velocity
Velocity field componentwise Normal inflow velocity
107
Description Value
Normal inflow velocity 1
Standard pressure 1[atm]
Standard molar volume 0.0224136[m^3/mol]
Normal mass flow rate 1e-5[kg/s]
Mass flow type Mass flow rate
Standard flow rate defined by Standard density
Outlet 1
Outlet 1
Selection
Geometric entity level Boundary
108
Selection Boundaries 1, 5
Equations
Settings
Settings
Description Value
Boundary condition Pressure
Pressure 0
Normal flow Off
Suppress backflow On
Apply reaction terms on All physics (symmetric)
Use weak constraints Off
Particle Tracing for Fluid Flow (fpt)
109
Particle Tracing for Fluid Flow
Selection
Geometric entity level Domain
Selection Domain 1
Equations
Settings
Description Value
Formulation Newtonian
Relativistic correction Off
Maximum number of secondary particles 10000
110
Description Value
Store particle status data Off
Release type Transient
Compute particle temperature Off
Compute particle mass Off
Wall accuracy order 1
Settings
Description Value
Wall condition Bounce
Primary particle condition None
Include secondary emission Off
Particle Properties
Settings
Description Value
Charge number 0
Particle density 1100[kg/m^3]
Particle diameter 50E-6[m]
111
Description Value
Particle properties 0
Particle property specification Specify particle density and diameter
Inlet 1
Inlet 1
Selection
Geometric entity level Boundary
Selection Boundary 16
Equations
Settings
Settings
112
Description Value
Release times 0
Initial position Density
Number of particles per release 10000
Density proportional to spf.U
Initial velocity Expression
Velocity field Velocity field (spf/fp1)
Outlet 1
Outlet 1
Selection
Geometric entity level Boundary
113
Selection Boundaries 1, 5
Settings
Description Value
Wall condition Freeze
Drag Force 1
Drag Force 1
Selection
Geometric entity level Domain
Selection Domain 1
Equations
114
Settings
Settings
Description Value
Turbulent dispersion Off
Drag law Stokes
Velocity field Velocity field (spf/fp1)
Dynamic viscosity Dynamic viscosity (spf/fp1)
Turbulent kinetic energy User defined
Turbulent kinetic energy 0
Mesh 1
Mesh statistics
Property Value
Minimum element quality 0.05607
Average element quality 0.5872
Tetrahedral elements 42338
Pyramid elements 156
Prism elements 12260
Triangular elements 7142
Quadrilateral elements 276
115
Property Value
Edge elements 990
Vertex elements 20
Mesh 1
Size (size)
Settings
Name Value
Calibrate for Fluid dynamics
Maximum element size 1.47
Minimum element size 0.44
Curvature factor 0.6
116
Name Value
Resolution of narrow regions 0.7
Maximum element growth rate 1.15
Study 1
Stationary Study settings
Property Value
Include geometric nonlinearity Off
Mesh selection
Geometry Mesh
Geometry 1 (geom1) mesh1
Physics selection
Physics Discretization
Laminar Flow (spf) physics
Fully Coupled 1 (fc1)
General
117
Name Value
Linear solver Iterative 1
Iterative 1 (i1)
Error
Name Value
Factor in error estimate 20
Maximum number of iterations 200
Nonlinear based error norm On
Multigrid 1 (mg1)
Coarse Solver (cs)
Direct 1 (d1)
General
Name Value
Solver PARDISO
Study 2
Time Dependent Study settings
118
Property Value
Include geometric nonlinearity Off
Times: range(0,0.01,0.2)
Mesh selection
Geometry Mesh
Geometry 1 (geom1) mesh1
Physics selection
Physics Discretization
Particle Tracing for Fluid Flow (fpt) physics
Time-Dependent Solver 1 (t1)
General
Name Value
Defined by study step Time Dependent
Time {0, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11,
0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2}
Relative tolerance 1.0E-5
119
Absolute tolerance
Name Value
Tolerance 1.0E-6
Time stepping
Name Value
Method Generalized alpha
120
Appendix C: Streptavidin and CRP adsorption recipes (Chapter 4)
Table 9-1 Qualitative Assay for detecting Streptavidin on Nylon beads
Streptavidin coated bead Control bead with no Streptavidin
1) 1 ul of Streptavidin in 14 ul PBS
Buffer to dilute 15x. Stir
2) Wash beads and drain PBS buffer in
20 ul
3) Add the Streptavidin solution onto the
beads and incubate overnight.
4) Dilute 1 % BSA into 0.1% by mixing
5 ul of BSA with 45 ul of buffer.
5) Pour 20 ul of that mixture into the
beads (This blocks the unused STV
binding sites on the bead)
6) Incubate for 1 hour in the fridge
7) 2 ul Biotin (4mg/ml) was taken and
mixed with 8 ul of BSA to bring it up
to volume
8) Then 4 ul of this stock was added to
the Streptavidin-coated beads
9) Incubate for 15 mins with occasional
mixing. Drain the solution
10) Add 0.1% BSA volume up to 15 ul
11) Repeatedly wash the beads to ensure
there isn’t any PE-Biotin left
12) Image it
Add 15 ul of PBS onto the beads and incubate
overnight.
Pour 20 ul of that mixture onto Control beads
Incubate for an hour in the fridge
4 ul of this stock was added to Control
Incubate for 15 mins with occasional mixing
Drain the solution
Repeatedly wash the beads to ensure there
isn’t any PE-Biotin left
Image it
121
Table 9-2 Quantitative assay for fluorescent detection of Streptavidin on Nylon bead
Streptavidin coated beads Control beads
Streptavidin (0.66, 1.33, 2, 2.66, 3.33, 5,
6.67, 8.33, 10, 13 pico moles)
5 ul of 5%BSA
Incubate overnight Incubate Overnight
Pipette out the old sol Pipette out the old sol
5 ul of 2.5%BSA 5 ul of 2.5%BSA
Incubate few hours Incubate few hours
Pipette out the old sol, change vials Pipette out the old sol, change
vials
Biotin-PE (66 pmoles) 4 ul of 4mg/ml Biotin-PE (66
pmoles)
Incubate 2 hours Incubate 2 hours
change vials change vials
Twice add 20 ul and pipette out Twice add 20 ul and pipette out
Image Image
122
Table 9-3 Direct (Non-specific) Adsorption procedure for detecting CRP on Nylon Beads
Beads with CRP Control Beads
CRP (0.32, 0.64, 1.28 nano moles) Just 10 ul of 5% BSA
Incubate @25 C overnight Incubate @25 C overnight
Pipette out the old sol, add 20 ul PBS and
pipette out again
Pipette out the old sol, add 20 ul PBS and
pipette out again
Add 10 ul of 5% BSA Add 10 ul of 5% BSA
Incubate @25 C overnight Incubate @25 C overnight
Pipette out the old sol, change vials, add PBS
and pipette out
Pipette out the old sol, change vials, add PBS
and pipette out
Add 3 ul C2 antibody Add 3 ul C2 antibody
Incubate overnight Incubate overnight
Wash with 10 ul PBS Wash with 10 ul PBS
Add 12.5 pico moles Secondary Ab Goat
Anti-Mouse IgG1
Add 12.5 pico moles Secondary Ab Goat
Anti-Mouse IgG4
Incubate few hours Incubate few hours
Pipette out the old sol, change vials Pipette out the old sol, change vials
add 20 ul PBS and pipette out again add 20 ul PBS and pipette out again
Image Image
123
Table 9-4 Sandwich assay for CRP detection with coating of antibody C6 on Nylon bead
Beads with capture antibody (C6) Control bead with no capture antibody
Monoclonal Antibody C6 (19 pmoles) 5 ul of 5% BSA
Incubate @25 C overnight Incubate @25 C overnight
Pipette out the old sol Pipette out the old sol
Add CRP (0.4, 1, 2, 4, 8, 16 pmoles) Add CRP (8 pmoles)
Incubate overnight Incubate overnight
Pipette out the old sol, Add 5 ul of 2.5% BSA
and incubate for an hour
Pipette out the old sol, Add 5 ul of 2.5% BSA
and incubate for an hour
Pipette out the old sol, add PBS and pipette
out, change vials
Pipette out the old sol, add PBS and pipette
out, change vials
Add monoclonal antibody C2 (0.66, 1.9, 3.8,
6.6, 12.2, 20.6 pmoles)
Add 5 ul of 62 ug/ml C2 antibody (1.9
pmoles)
Incubate 1.5 hours Incubate 1.5 hours
Pipette out old, Wash with 10 ul PBS, change
vials
Pipette out old, Wash with 10 ul PBS, change
vials
Add detection antibody Goat Anti-Mouse
IgG1 (0.625, 2, 3.125, 6.25, 12.5, 25 pmoles)
Add 15 ul of 100 ug/ml Secondary Ab Goat
Anti-Mouse IgG1 (12.5 pmoles)
Incubate 1.5 hours Incubate 1 hours
Pipette out the old sol, change vials Pipette out the old sol, change vials
add 20 ul PBS and pipette out again add 20 ul PBS and pipette out again
Image Image
124
Appendix D: 3D Printing tips
The author is experienced in using the Form1+ Stereolithography-based resin printer and the
Fusion-Deposition-Modelling (FDM) based ABS plastic printer (Zortrax M200). Techniques to
achieve the best results while printing are summarized below:
Zortrax M200 is a Fusion Deposition Modelling (FDM) printer where objects are created out of a
thermoplastic material. This printer uses a proprietary version of the Acrylo Butadiene Styrene
(ABS) called Z-ABS which has a higher melting point (260 C) than other commercially available
ABS. The filament diameter is 1.75 mm. The best vertical z resolution is 90 µm meaning the
thinnest layers are 90 µm. The xy resolution is limited by the size of the nozzle, which is 0.4
mm. The basic procedure to use the printer is as follows:
The user converts their CAD model from either a .stl, .dxf or an .obj format to z-code by using
the proprietary meshing software Z-Suite. The software also allows the user to orient, scale or
split the model into sections. The meshing is done automatically after the user selects the
resolution, material type, infill (density), and support angle needed for the print.
Figure 10:1 User interface for the Zortrax M200
125
During the course of the thesis, the following conditions were optimized and issues resolved. The
printing time is inversely proportional to both the resolution and infill. A poor resolution of 0.29
mm compromised the strength of the material due to adjacent layers being deposited further
apart. Conversely, a high resolution (0.090 mm) gives a relatively smooth exterior indicating the
layers are packed closer at the cost of time. It was found that 0.19 mm resolution and medium
infill gives the optimum tradeoff between speed, strength and aesthetics. For models with small
holes, it is recommended that the “hole offset” option be set as 0.2 mm to account for shrinking
post-printing.
After the printing is complete, the supporting material has to be peeled off from the part. To
enable easier removal, it is recommended to use “Support Lite” with the “Angle” option set at 20
degrees. Since better resolution is available in the z-axis compared to the xy, the part can be
oriented such that the most dimension-sensitive areas are printed vertically.
Warping:
ABS expands upon heating and contracts during the cooling step. This causes the entire part to
shrink slightly. However, the corners of the base tend to cool faster resulting in the corner
sections peeling off from the build platform during the printing causing uneven print jobs. This
uneven shrinkage is termed as warping and is the major cause of failure when printing parts with
a rectangular footprint. To solve this issue, several methods can be adopted:
1) The cooling fan can be turned off
2) The build platform can be coated with a layer of Acetone/office glue prior to starting the print
3) The room can be kept at a warmer temperature/minimizing airflow
4) Enclosing the printer to trap the heat
5) The perforated plate can be taken out and bent inwards before the start of the print
6) The build platform can be sanded to achieve better adhesion and
126
7) Letting the part cool down slowly after printing by allowing it to remain in the printer for 30
more minutes.
Even with all these steps, large parts often fail. To work around this issue, it was discovered that
surrounding the part with sacrificial pieces caused the latter to warp, thereby protecting the
model at the center as illustrated below:
Figure 10:2 The desired model in the middle protected by sacrificial rods of arbitrary
dimensions
Other issues, their potential causes and general techniques are listed below:
Stuck Filaments: If the filament is not extruding from the nozzle, then go to user menu
“filaments” -> “unload filaments” and then allow it to heat. The heating will melt any stuck
pieces. Then the gear bit will rotate anti-clockwise to push the top part of the filament upwards.
Remove the filament. Then press load filament. Then push the filament back in and follow the
usual procedure to load the filament. If these steps fail, the nozzle can be cleaned by detaching it
and dipping it for a few hours in Acetone.
127
Knotty filaments: During the print job, if the filament appears knotted, it is because there is a
slack in the filament spool.
Noise: After several months of usage, it is necessary to grease the rods to keep them moving
smoothly. However if the stop screw has fallen off, extremely loud noise will be heard from the
printer. It is unsafe to use it until the screw is bolted back in.
Smoothening the Surface: To obtain a glossy surface finishing, the printed part can be covered
under a jar with acetone vapors. The acetone dissolves the surface and causes a smooth finish.
The top two [35] 3D printers under $5k are compared below along with their estimated cost of
usage.
Figure 10:3 Price comparison for top 3D printers [37, 38]. Assuming that printers last just
one year, the above table gives a rough estimate of the costs involved in using these two 3D
printers.
Form 1+
Cost Per Unit
Resin [1 year supply]
Resin Tanks [1 year supply]
Build Platform
Isopropyl Alcohol
Shipping and Taxes
Total:
Man-hours (100 hours * $20)
Zortax M200
Cost Per Unit
ABS Filament [1 year supply]
Nozzles [3 * $35]
Hot End [2 * $90]
Build Plate [5 * $45]
Shipping and Taxes
Total:
Man-hours (30 hours * $20)
(UV Laser cures liquid resin)
(High temperature melts plastic, then solidifies)
Average Cost per print: $35
$230.00
$500.00
$3,355.00
$600.00
Build Volume : 200 x 200 x 185 mm
Failure Rate: N/A
Z Resolution: 100 um
XY Resolution: 400 um
Technology: Filament Extrusion
Cost in USD
$1,990.00
$350.00
$105.00
$180.00
$500.00
$6,033.00
$2,000.00
Average Cost per print: $50
Failure Rate: 30%
XY Resolution: 25 um
Build Volume : 125 x 125 x 165 mm
Z Resolution: 25 um
Technology: Stereo Lithography
$250.00
3D Printer price comparison
Cost in USD
$3,300.00
$1,490.00
$295.00
$198.00
128
“It is not the critic who counts; not the man who points out how the strong man
stumbles or where the doer of deeds could have done better. The credit belongs
to the man who is actually in the arena, whose face is marred by dust and sweat and blood,
who strives valiantly, who errs and comes up short again and again, because there is no
effort without error or shortcoming, but who knows the great enthusiasms, the great
devotions, who spends himself for a worthy cause; who, at the best, knows, in the end, the
triumph of high achievement, and who, at the worst, if he fails, at least he fails while
daring greatly, so that his place shall never be with those cold and timid souls who knew
neither victory nor defeat.” – Theodore Roosevelt