fabrication of a textile-based platform for rapid analyte detection
TRANSCRIPT
Fabrication of a Textile-Based Platform for Rapid Analyte Detection
Sakip Önder 1, a, Fatma Neşe Kök 2,b, Levent Trabzon 3,c, Hüseyin Kızıl 4,d,
Burçak Karagüzel Kayaoğlu 1,e and İkilem Göcek 1,f 1Istanbul Technical University, Department of Molecular Biology and Genetics, 34469 Maslak
Istanbul, Turkey 2Istanbul Technical University, Department of Mechanical Engineering, MEMS Lab, 34437
Gumussuyu-Beyoglu Istanbul, Turkey 3Istanbul Technical University, Department of Materials and Metallurgical Engineering, MEMS Lab,
34469 Maslak Istanbul, Turkey 4Istanbul Technical University, Department of Textile Engineering, 34437 Gumussuyu-Beyoglu
Istanbul, Turkey a [email protected], [email protected], [email protected], [email protected],
[email protected], [email protected]
Keywords: Analyte detection, fabrication, nonwoven, photolithography, spunbond.
Abstract. A novel textile-based analytical device with a simultaneous, rapid, sensitive and qualitative
response for analyte detection that may have a potential use in different body fluids such as sweat,
blood, saliva and urine is proposed in this study as an alternative to its paper-based counterparts. A
porous polypropylene spunbond nonwoven was used as base fabric which is superior to paper with
higher tear and crinkle resistance, flexibility and wearability.
Introduction
By developing effective technologies in health-related diagnostic devices for developing countries,
healthcare may be possible in areas without access to trained medical personnel [1]. Practical
methods for detecting and quantifying analytes in the developing world are different from those in the
developed world: suitable detection technologies must be robust, lightweight, easy to use, and above
all, low-cost [2].
Analytical devices have been successfully applied in the detection of many small molecules such
as glucose, lactose, uric acid, cholesterol, alcohol, antigen and antibody. Different fabrication
methods for analytical devices have been used such as screen (wax) printing, ink-jet printing,
plotting, plasma treatment and photolithography. It is difficult to form hydrophobic barriers through
the entire thickness of substrate with uniform width, using simple printing technologies [3, 4].
Colorimetric assays have been most commonly used for detecting urinary protein and glucose on
the paper based microfluidic devices which have been fabricated via photolithography and ink-jet
printing technologies. Inkjet printing device was used to print chemical sensing inks, comprising the
necessary reagents for colorimetric analytical assays. Analyte solutions have been spotted on the
substrates and allowed to wick down into the detection zone where it comes into contact with the
detection reagent. The color changes in the chemical sensing areas have been monitored by the naked
eye and measured digitally with specific software after recording a color scan. The patterned paper
fabricated by photolithography has been reproduced for biological assays by adding appropriate
reagents to the testing areas [5, 6].
In addition to glucose assays, colorimetric assays were conducted for ketones and nitrite analyses.
Paper based microfluidic chip was designed for the determination of acetoacetate and nitrite which
were detected in artifical urine and saliva, respectively. Chip was fabricated using photolithography
technique. Reagents were spotted on the detection zones of the paper device and were dried prior to
spotting of analytes. Following the spotting of the reagents, sample solution was added onto the paper
device. After drying, a flatbed scanner was used to record color images on the testing zones [7].
Applied Mechanics and Materials Vols. 490-491 (2014) pp 1611-1616© (2014) Trans Tech Publications, Switzerlanddoi:10.4028/www.scientific.net/AMM.490-491.1611
All rights reserved. No part of contents of this paper may be reproduced or transmitted in any form or by any means without the written permission of TTP,www.ttp.net. (ID: 160.75.58.45-13/01/14,09:42:10)
In the current study, photolithography was used to pattern a simple reservoir design on nonwoven
in order to obtain a device that is capable of detecting various kinds of analytes such as glucose,
lactose, uric acid, cholesterol, alcohol in different body fluids. For demonstrating the developed
device’s capability of analyte detection, simulated sweat was used as an example. SU-8 was selected
as an epoxy-based negative photoresist due to its high optical transparency, chemical resistance and
biocompatibility. Hydrophobic photoresist polymer provided a good physical barrier defining the
reservoir areas (detection zones) where the simulated sweat was deposited for analyte detection.
Experimental
Materials. A 45 g/m2 polypropylene spunbond nonwoven fabric coated with a 35 g/m
2 polyethylene
film was used as a platform in the fabrication of textile-based analytical device. The uncoated side of
the spunbond fabric was used for enzyme immobilization after plasma treatment. However, the
hydrophobic film coated surface acted as a barrier for leak-proofing of the simulated sweat solution.
This was critical to immobilize the enzymes on the surface of the substrate and to clearly observe the
color change due to the interaction between the simulated sweat and the enzymes.
Nonwoven was spin coated using SU-8 3050, an epoxy based negative photoresist, which was
purchased from MicroChem Corporation (Newton, MA). SU-8 was selected due to its high optical
transparency, chemical resistance and biocompatibility. Unpolymerized photoresist which was not
exposed to UV light was removed from the fabric by submerging in a developer
(1-methoxy-2-propanol acetate solution) which was purchased from MicroChem Corperation
(Newton, MA). Iso-Propyl alcohol 2-Propanol was supplied from Zag Chemical Industry Research,
Development and Calibration Laboratory.
Lactate detection reservoirs were functionalized with the co-immobilization of Lactate Oxidase
(L0638 Sigma) (LOX), and peroxidase (P8375 Sigma) (POX). Simulated sweat, L-(+)-Lactic acid
(L1750 Sigma), was used as substrate for LOX, and
2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (A1888 Sigma) (ABTS )
was used as a substrate for POX.
Method. Textile-based analytical device was fabricated using photolithography technique. Patterns
of a simple reservoir design with different diameters of 4 mm, 6 mm and 8 mm were produced on the
nonwoven substrate. Different reservoir diameters were utilized in order to achieve a proper
immobilization on the surface and to deposit sufficient amount of solution yielding a clear, visual
color change.
In the fabrication process, due to the fabric’s hydrophobic nature, firstly fabric was exposed to the
oxygen plasma treatment for 15 minutes to ensure the adhesion of SU-8 on the fabric surface. Fabric,
attached on the silisium wafer, was placed on the spincoater and approximately 50µm SU-8 was
coated at once. Spunbond fabric coated with photoresist was baked at 95 °C for 15 min to complete
soft bake process. In order to create desired pattern, transparent film photomask was placed on the
surface of the textile and irradiated with a UV lamp for 8 seconds (Figure 1(a)). The pattern of mask
was designed with Tanner Tools L-Edit 13.0 drawing programme. After photolithography, textile
surface with the silisium base was kept for 1-2 minutes at 65°C on the hot plate which was followed
by 5 minutes of post exposure bake at 95°C. Then wet etching process was applied. Unpolymerized
SU-8 was removed by submerging the textile into a developer (1-methoxy-2-propanol acetate)
solution about 5 minutes. Nonwoven surface coated with SU-8 was hard baked at 45°C for 2 hours in
a convection oven to further cross link the resist. Schematic cross sectional view of reservoirs after
etching process of SU-8 obtained on the spunbond nonwoven fabric are shown in Fig. 1(b).
1612 Mechanical Design and Power Engineering
(a)
(b)
Fig. 1. (a) An image of SU-8 coated fabric under UV exposure and (b) Spunbond non-woven
fabric coated with a single layer SU-8 and cross-sectional view of the sample
Two layered and three layered SU-8 photoresist coatings were applied on the spunbond surface to
obtain reservoirs with desired depths applying the same process for each layer. The images of
spunbond non-woven fabrics which were coated with double and three layers of SU-8 are shown in
Fig. 2(a) and 2(b), respectively. Photolithography steps and production parameters for spunbond
fabric are shown in Fig. 3(a) and 3(b).
(a) (b)
Fig. 2. Spunbond non-woven fabric coated with double (a) and three layers (b) of SU-8 and
cross-sectional views of the samples with reservoirs.
Fig. 3. (a) Photolithography steps and (b) production parameters of spunbond fabric.
Since polypropylene fibers in the reservoir areas were highly hydrophobic, an oxygen plasma
treatment was applied to render the surfaces hydrophilic. Through this process, the surfaces of the
reservoir areas were prepared for the enzyme immobilization.
Enzymes were added one by one into the reservoir, meaning that first enzyme solution was
completely absorbed by the surface before the addition of the next one. 4 µL of ABTS (2.67 µg), 2 µL
of LOX (0.004 units) and 2 µL of POX (3 units) solutions were added into reservoirs respectively.
LOX activity led to the production of hydrogen peroxide by the oxidation of lactic acid. POX, then,
oxidized 2,2’-Azino-bis(3-Ethylbenzthiazoline-6-Sulfonic Acid) (ABTS) in the presence of
hydrogen peroxide and color formation (green-brown) was observed. Different substrate
concentrations (lactate, 1-100 µM) were added into enzyme immobilized reservoir areas. However,
ABTS and substrate optimization studies were done in tubes before starting the studies on the textiles.
Test and Characterization. An oxygen plasma treatment was applied for 15 minutes to render the
surfaces hydrophilic before leak-proofing control on the nonwoven substrates. A red colored solution
was prepared by dissolving a red ink in deionized water to observe fluid absorption on the reservoirs.
After plasma treatment, in order to control the hydrophilicity of the zones and test leak-proofing, 2 µL
of the colored solution was dispended using micropipette on each reservoir. Pattern obtained by
photolithography was characterized utilising a Zeiss EVO MA10 SEM (Scanning Electron
Applied Mechanics and Materials Vols. 490-491 1613
Microscope). SEM analysis was used for analyzing the formation of reservoirs and the penetration of
photoresist material into the surface of textile material.
In order to determine the color change based on the lactate level, ABTS and analyte optimization
studies were conducted with free enzymes in tubes and spectrophotometric measurements were done
at 404 nm with Biorad Model 3559 UV Microplate. An enzyme solution was prepared by using 20 µL
LOX (0.04 unit), 30 µL POX (5 unit) and 400 µL ABTS (0.27 mg) and 50 µL of this solution was put
into different measurement tubes. Then 10 µL of the analyte solutions with different concentrations
(lactic acid, 1-100 mM) were added into these tubes for analyte optimization studies and color
densities were measured spectrophotometrically. Enzyme solutions were kept at 37 °C and
measurements were taken after 10 min. Different ABTS concentrations (0.013-6.67 mg/ ml) were
also studied and color densities in tubes were measured similarly. After optimization studies made in
tubes, studies on enzyme immobilized reservoirs were done to obtain a color-scale for different
lactate levels on textiles. Different analyte solutions, 2 µL, were added into each reservoir and color
changes depending on the lactate levels were observed.
Results and Discussions
As can be seen from the figures below, the reservoir patterns were successfully fabricated on the
uncoated side of the spunbonded nonwoven fabric. It was also shown that by the application of
multiple layers of SU-8, reservoirs with different depths could be fabricated. This may allow the
deposition of different volumes of analyte solution onto the reservoirs. Reservoir patterns on the
fabric surface which was coated with a single layer, double layer and three layers of SU-8 are shown
in Fig. 4 and Fig. 5, respectively.
(a) (b)
Fig. 4. Reservoirs with (a) 8 mm, (b) 4 and 6 mm diameters patterned on the fabric coated with a
single layer SU-8.
(a) (b)
Fig. 5. Reservoirs with 4 and 6 mm diameters patterned on the fabric coated with double (a), three
(b) layers of SU-8.
In order to test the hydrophilicity of the reservoir areas which is required for the enzyme
immobilization and to verify the leak-proofing of the hydrophobic barriers, a red dye solution (red ink
in deionized water) was dispensed and the in-plane penetration of the solution was observed (Fig. 6).
No leakage of dye solution was observed. This may be attributed to the fact that good penetration of
SU-8 into the nonwoven was achieved, and the hydrophobic barriers of the SU-8 polymer were well
defined around the hydrophilic reservoirs. After the creation of reservoirs on the surface of the fabric
to the desired height, material thickness and penetration of photoresist material into the textile
substrate were characterized using SEM (Scanning Electron Microscopy) (Fig. 7).
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Fig. 6. Top view of reservoirs with 6mm and 4mm diameters and liquid absorption to monitor
reservoirs hydrophilicity.
Fig. 7. SEM image of PP spunbond nonwoven after double layer SU-8 coating (Measurement
results from two different points Pa1: 83.11 µ and Pa2: 78.74 µ).
In the enzymatic reactions to determine lactate in sweat, color change corresponding to different
substrate concentrations was measured spectrophotometrically at 404 nm for the solutions (Fig. 8 (a))
and was observed visually on the textile samples (Fig. 8 (b)). It was observed that color change
became more evident with the increasing substrate concentration until 20 mM, remained almost
constant between 20-50 mM and started to decrease with further increase are. Visual observation was
in accordance with the spectrophotometric measurements as different color densities on reservoirs
were obtained depending on the lactate level compared with the control group (K,
LOX+POX+ABTS) (Fig. 8 (b)).
(a) (b)
Fig. 8. (a) Spectrophotometric measurements of solutions, (b) color optimization studies on
textiles.
Conclusions
Preliminary results showed that the developed analytical device having lightweight, portable,
disposable, easy to use, and store characteristics may be potentially used as point-of-care and
inexpensive platform for disease diagnostics and for environment supervision as well as for the
detection of various kinds of analytes such as glucose, lactose, uric acid, cholesterol, alcohol in
different body fluids.
Acknowledgements
The authors acknowledge The Scientific and Technological Research Council of Turkey (TUBITAK)
for the financial support (No. 111M483).
Applied Mechanics and Materials Vols. 490-491 1615
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