single cell biosensor
DESCRIPTION
Single Cell Biosensor. Allan Fierro David Sehrt Doug Trujillo Evan Vlcek Michael Bretz. Fabrication. Flow Control. Introduction. Optical Detection Circuit. Allan Fierro. Cell Trapping. What is Flow Cytometry?. Technique for counting cells Examining cells Sorting cells. - PowerPoint PPT PresentationTRANSCRIPT
![Page 1: Single Cell Biosensor](https://reader035.vdocuments.mx/reader035/viewer/2022062217/56815003550346895dbdd125/html5/thumbnails/1.jpg)
Single Cell Biosensor
Allan FierroDavid Sehrt
Doug TrujilloEvan Vlcek
Michael Bretz
![Page 2: Single Cell Biosensor](https://reader035.vdocuments.mx/reader035/viewer/2022062217/56815003550346895dbdd125/html5/thumbnails/2.jpg)
Introduction
Allan FierroOptical
Detection Circuit
Flow Control
Cell Trapping
Fabrication
![Page 3: Single Cell Biosensor](https://reader035.vdocuments.mx/reader035/viewer/2022062217/56815003550346895dbdd125/html5/thumbnails/3.jpg)
What is Flow Cytometry?
• Technique for counting cells
• Examining cells• Sorting cells
![Page 4: Single Cell Biosensor](https://reader035.vdocuments.mx/reader035/viewer/2022062217/56815003550346895dbdd125/html5/thumbnails/4.jpg)
Example of Flow Data
Analysis of a marine sample of photosynthetic picoplankton by flow cytometry showing three different populations (Prochlorococcus, Synechococcus and picoeukaryotes)
![Page 5: Single Cell Biosensor](https://reader035.vdocuments.mx/reader035/viewer/2022062217/56815003550346895dbdd125/html5/thumbnails/5.jpg)
What is Optofluidics?• Optofluidics combines microfluidics and optics• Able to see the difference in light refraction
wavelength
wavelength
*diagrams courtesy of L. Shao, Ph.D.from Ph.D. Defense Presentation
![Page 6: Single Cell Biosensor](https://reader035.vdocuments.mx/reader035/viewer/2022062217/56815003550346895dbdd125/html5/thumbnails/6.jpg)
Flow Cytometry vs. Optofluidics
• Flow uses scatter of light
• Flow more expensive to run
• Flow samples take longer to prep
• Can’t use cells after
• Optofluidics uses refraction of light
• No fluorescent dye - cheaper and less prep time
• Optofluidics can keep cells after
Flow Cytometry Optofluidics
![Page 7: Single Cell Biosensor](https://reader035.vdocuments.mx/reader035/viewer/2022062217/56815003550346895dbdd125/html5/thumbnails/7.jpg)
Microscope
Infrared LED
Cell
Light
![Page 8: Single Cell Biosensor](https://reader035.vdocuments.mx/reader035/viewer/2022062217/56815003550346895dbdd125/html5/thumbnails/8.jpg)
Fabrication
David SehrtOptical
Detection Circuit
Flow Control
Cell Trapping
Fabrication
![Page 9: Single Cell Biosensor](https://reader035.vdocuments.mx/reader035/viewer/2022062217/56815003550346895dbdd125/html5/thumbnails/9.jpg)
DEP Chip
Images courtesy of Weina Wang
![Page 10: Single Cell Biosensor](https://reader035.vdocuments.mx/reader035/viewer/2022062217/56815003550346895dbdd125/html5/thumbnails/10.jpg)
DEP Chip Processing
1. 30 nm Chrome Deposition2. 120 nm Gold Deposition3. Spin Photoresist4. Exposure5. Development6. Gold Etch7. Chrome Etch8. Resist Removal
![Page 11: Single Cell Biosensor](https://reader035.vdocuments.mx/reader035/viewer/2022062217/56815003550346895dbdd125/html5/thumbnails/11.jpg)
PDMS Channel
• PDMS- Polydimethylsiloxane• Suitable optical properties• Adhesive to glass• Channel mold made of a
silicon substrate and SU-8 Photoresist
• PDMS poured into mold and baked to form elastic solid
![Page 12: Single Cell Biosensor](https://reader035.vdocuments.mx/reader035/viewer/2022062217/56815003550346895dbdd125/html5/thumbnails/12.jpg)
DEP PDMS Bonding
• Oxygen-plasma treated• Bonding is performed
with a mask aligner• Minute Pressure is
applied for bonding to transpire PDMS Channel
DEP Chip
![Page 13: Single Cell Biosensor](https://reader035.vdocuments.mx/reader035/viewer/2022062217/56815003550346895dbdd125/html5/thumbnails/13.jpg)
Flow Control
Evan VlcekOptical
Detection Circuit
Flow Control
Cell Trapping
Fabrication
![Page 14: Single Cell Biosensor](https://reader035.vdocuments.mx/reader035/viewer/2022062217/56815003550346895dbdd125/html5/thumbnails/14.jpg)
Chip Design for Fluid
• Two drilled holes at each end
• Nanoports for each hole• 200 µm wide, 25 µm
deep, channel• Need flow rate of
approximately 40 µm/s
drilled hole
nanoport
channel
chip
200 µm
flow
*picture courtesy of L. Shao, Ph.D.from Ph.D. Defense Presentation
![Page 15: Single Cell Biosensor](https://reader035.vdocuments.mx/reader035/viewer/2022062217/56815003550346895dbdd125/html5/thumbnails/15.jpg)
Nanoport Assembly
chipdrilled hole
adhesive o ring
nanoport
nanotube
TOP VIEW SIDE VIEWnanotube nanoport
from pump “waste”
adhesive o ring
![Page 16: Single Cell Biosensor](https://reader035.vdocuments.mx/reader035/viewer/2022062217/56815003550346895dbdd125/html5/thumbnails/16.jpg)
Block Diagram
Pump? Off Cycle Stop?On Cycle Stop PumpSet
Parameters
Yes Yes
No
Labviewprogram
OrielController
DB9 RS-232
Actuator Syringe Nanotube
Chip
![Page 17: Single Cell Biosensor](https://reader035.vdocuments.mx/reader035/viewer/2022062217/56815003550346895dbdd125/html5/thumbnails/17.jpg)
Actual Setup• Pump set to move actuator
0.5 µm/s
• On for 0.825 s, off for 7 s (≈10.5% duty cycle)
• π cm2 syringe area * 0.5 * 10-4
cm/s * 0.105 1.66 * 10-5 cm3/s through channel
• This is 1.43 liters being pumped through the channel every 24 hours!
Oriel Controller
nanotube
syringe
actuator
LabviewVI
RS 2320.5 µm/s
![Page 18: Single Cell Biosensor](https://reader035.vdocuments.mx/reader035/viewer/2022062217/56815003550346895dbdd125/html5/thumbnails/18.jpg)
Actual Setupactuator
syringe
Labview program
Orielcontroller
![Page 19: Single Cell Biosensor](https://reader035.vdocuments.mx/reader035/viewer/2022062217/56815003550346895dbdd125/html5/thumbnails/19.jpg)
Optical Detection Circuit
Doug TrujilloOptical
Detection Circuit
Flow Control
Cell Trapping
Fabrication
![Page 20: Single Cell Biosensor](https://reader035.vdocuments.mx/reader035/viewer/2022062217/56815003550346895dbdd125/html5/thumbnails/20.jpg)
Optical Detection of Cells-Purpose-• Detect the presence of a cell in proximity of a trap• Interface with the pump flow controller• Provide triggering for the RF traps
-Implementation-• Photo diode coupled via fiber optic cable from microscope to detect light modulation• Monitor the change of the reverse bias current from the diode through the use of a USB Data Acquisition unit• Digital switch to trigger RF traps
![Page 21: Single Cell Biosensor](https://reader035.vdocuments.mx/reader035/viewer/2022062217/56815003550346895dbdd125/html5/thumbnails/21.jpg)
Optical Detection of Cells-Requirements-• Able to detect light modulation of cells traveling 40 µm/sec• Output a voltage in the range of 2V - 3.8V. Any higher voltage output may damage the
DAQ• Photodiode must be responsive to light source of 860-910 nm
Circuit Design Flow Chart
Microscope output
Photo Diode Low pass filter
Amplifier Buffer
Signal to DAQ
![Page 22: Single Cell Biosensor](https://reader035.vdocuments.mx/reader035/viewer/2022062217/56815003550346895dbdd125/html5/thumbnails/22.jpg)
Optical Detection of Cells
-Light Source-The light source is set up as shown in the figure. The LED is a high-intensity Infrared LED.
Lear, Kevin L., Hua Shao, Weina Wang, and Susan E. Lana. "Optofluidic Intracavity Spectroscopy of Canine Lymphoma and Lymphocytes." IEEE Explore (2007). 4 Dec. 2007.
50/125 multimode fiber
micro-fluidic sample
beam-splitter
infrared source LEDs
D1
PH
OTO
DIO
DE
1 2CircuitCircuit
Labview
DAQ
Traps
DAQ
Labview Traps
![Page 23: Single Cell Biosensor](https://reader035.vdocuments.mx/reader035/viewer/2022062217/56815003550346895dbdd125/html5/thumbnails/23.jpg)
Optical Detection of Cells-Circuit Design-
0
0
0
+5 VDC
-5 VDC
-
+
U1A
LF412
3
21
84
R1250k
R2
3.2k
C19n
-
+
U1B
LF412
5
67
84
A1
C5
100n
0
C6
100n
R4100
0
+5 VDC
0
C7
100n
R5
124k
D1PHOTODIODE
12
C8
1n
0
+5 VDC
C9
100n
-5 VDC
0
C10
100n
~2mV Output from Diode
A1 signal to USB DAQ
![Page 24: Single Cell Biosensor](https://reader035.vdocuments.mx/reader035/viewer/2022062217/56815003550346895dbdd125/html5/thumbnails/24.jpg)
Optical Detection
Figure of Time Response of Optical Detection Circuit
Rise Time: 128 µs
Peak Voltage:2.7 V
![Page 25: Single Cell Biosensor](https://reader035.vdocuments.mx/reader035/viewer/2022062217/56815003550346895dbdd125/html5/thumbnails/25.jpg)
Cell Trapping
Michael BretzOptical
Detection Circuit
Flow Control
Cell Trapping
Fabrication
![Page 26: Single Cell Biosensor](https://reader035.vdocuments.mx/reader035/viewer/2022062217/56815003550346895dbdd125/html5/thumbnails/26.jpg)
Automated Trapping
DetectionCircuit
Data Acquisition
Unit
Triggering Circuit
Analog Output Digital Output
AC SignalThe digital signal from the Data Acquisition Unit is input into an ADG-452 (basically a digital switch) chip. This allows an AC signal to be applied to the DEP traps DEP Traps
![Page 27: Single Cell Biosensor](https://reader035.vdocuments.mx/reader035/viewer/2022062217/56815003550346895dbdd125/html5/thumbnails/27.jpg)
Dielectrophoretic(DEP) Trapping
• Uses theory of electromagnetics to trap cells• A non-uniform electric field causes polarization within individual molecules of spheres/cells.•This polarization along with electric field apply a force that will move spheres to a desired location.
Electric field
![Page 28: Single Cell Biosensor](https://reader035.vdocuments.mx/reader035/viewer/2022062217/56815003550346895dbdd125/html5/thumbnails/28.jpg)
Electromagnetic modeling
Ground
+AC Voltage
fluid flow
GroundAC voltage
The force caused by the electric field will push the spheres into the center of the trap.
Picture Courtesy of Weina Wang et al. powerpoint presentation “Lab-on-a-Chip Single Particle Dielectrophoretic (DEP) Traps” 3-6-2006.
![Page 29: Single Cell Biosensor](https://reader035.vdocuments.mx/reader035/viewer/2022062217/56815003550346895dbdd125/html5/thumbnails/29.jpg)
Requirements1. Sphere must be traveling at 40
micron/second or less2. Signal used to generate the electric
field must be a time varying signal in order to produce a non-uniform electric field.
3. Signal used to generate the electric field must have an amplitude greater than 5 Volts peak to peak
Both of these requirements are necessary in order for the for the force caused by the electric field to overcome the force caused by the flow of water. i.e. an object in motion stays in motion unless acted upon by a net external force.
![Page 30: Single Cell Biosensor](https://reader035.vdocuments.mx/reader035/viewer/2022062217/56815003550346895dbdd125/html5/thumbnails/30.jpg)
Pictures of DEP traps
![Page 31: Single Cell Biosensor](https://reader035.vdocuments.mx/reader035/viewer/2022062217/56815003550346895dbdd125/html5/thumbnails/31.jpg)
Video of DEP Trapping
![Page 32: Single Cell Biosensor](https://reader035.vdocuments.mx/reader035/viewer/2022062217/56815003550346895dbdd125/html5/thumbnails/32.jpg)
Budget
• ADG 452 Digital Switch -- $15
• Various circuit elements -- $10
• Hytek iUSBDAQ U120816 -- $105
• TOTAL EXPENSES = $130
![Page 33: Single Cell Biosensor](https://reader035.vdocuments.mx/reader035/viewer/2022062217/56815003550346895dbdd125/html5/thumbnails/33.jpg)
Future Work
• Improve glass chip fabrication process• Fine-tune optics for better optical detection• Incorporate spectrometer and automate data
acquisition• Research best trap design to allow for faster
flow rate• Explore methods to allow for cells to be
analyzed faster