Laminated Paper-Based Analytical Devices (LPAD) with Origami

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Laminated Paper-Based Analytical Devices (LPAD) with OrigamiEnabled Chemiluminescence Immunoassay for Cotinine Detection in Mouse Serum Wei Liu,*,†,‡ Christopher L. Cassano,† Xin Xu,†,⊥ and Z. Hugh Fan*,†,§,∥ †

Interdisciplinary Microsystems Group, Department of Mechanical and Aerospace Engineering, University of Florida, P.O. Box 116250, Gainesville, Florida 32611, United States ‡ Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an, Shaanxi 710062, P. R. of China § J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, P.O. Box 116131, Gainesville, Florida 32611, United States ∥ Department of Chemistry, University of Florida, P.O. Box 117200, Gainesville, Florida 32611, United States S Supporting Information *

ABSTRACT: Laminated paper-based analytical devices (LPAD) with origami-enabled chemiluminescence immunoassay have been developed for the detection of cotinine, a secondhand smoke (SHS) biomarker. The devices were fabricated by a craft-cutter to define flow channels, followed by lamination. This approach of cutting/lamination to fabricate LPAD is very similar to making an identification card, offering advantages in simplicity and rugged backing when compared to the common method of patterning paper using SU-8 or wax. We also developed a protocol of localized incision and paperfolding to isolate the detection zone from flow channels; the simple origami step eliminated possible reagent diffusion and flow during antibody immobilization steps and numerous washings. By incorporating luminol-based chemiluminescence for detecting horseradish peroxidase-conjugated cotinine, we employed origami-enabled LPAD to detect cotinine in mouse serum using competitive immunoassay. The detection limit was determined to be 5 ng/mL, a clinically relevant concentration. We believe that LPAD with chemiluminescence detection provides a new platform of low cost and sensitive assays for cotinine detection.

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operation and sufficient detection sensitivity for cotinine is desirable and would be very beneficial at the point of care. Paper-based microfluidic devices have recently gained significant interest because of their small size, easy operation, low-cost, and other advantages.11−15 The paper devices have been fabricated primarily by two methods.14,15 One is to define hydrophobic zones by impregnating SU-8 into paper and then photopatterning, or printing wax and then melting it into paper.11−18 The other method is to cut paper to form physical boundaries using a laser, cutter, or knife plotter.19−23 Paper devices fabricated using these methods (particularly the second method) tend to have low mechanical strength, especially when wet. To address this common problem, we developed a simple fabrication method by craft-cutting and lamination, producing laminated paper-based analytical devices (LPADs) in a way similar to making an identification (ID) card.24 LPADs have increased mechanical strength and overall durability while avoiding the shortcomings of other backing methods such as clamping down a paper device to a glass substrate or taping the paper device to plastic surfaces.25,26

econdhand smoke (SHS), a mixture of side stream smoke from cigarettes and the smoke exhaled by smokers, has been associated with a variety of adverse health outcomes in nonsmokers, including lung cancer, respiratory illness, and cardiovascular diseases.1,2 SHS is designated as a known human carcinogen by the U.S. Environmental Protection Agency as well as the World Health Organization.2 To detect exposure to SHS, biomarkers are often employed by measuring their levels in the bodily fluids of test subjects. Among those biomarkers associated with SHS, cotinine (a metabolite of nicotine) is most commonly used because of its relatively long half-life in human bodies (16−19 h) and high specificity.3 Since the concentration of cotinine in SHS samples is typically on the order of 10 ng/ mL (e.g., 2−7 ng/mL in one study), sensitive detection methods are needed for the verification of SHS exposure.4,5 High-performance liquid chromatography or gas chromatography coupled with mass spectrometry has been developed for the detection of cotinine as well as other SHS biomarkers.6−9 Online electrokinetic concentration with capillary electrophoresis has also been employed for cotinine detection in SHS samples.10 However, all of these methods require bulky and sophisticated instruments. For some situations, for example, a pediatrician wants to determine whether a child’s asthma results from SHS, a portable device with simple © 2013 American Chemical Society

Received: July 5, 2013 Accepted: September 26, 2013 Published: October 11, 2013 10270

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Figure 1. Schematics of the LPAD fabrication process. Drawings are not to scale. (a) A paper strip, a cover film with an opening similar to the pattern of the paper strip, and a bottom film were aligned and passed through a laminator. (b) Layout of a device, showing the reagent pad, flow channel, and detection zone. (c) Picture of laminated devices. (d) Device with an incision along the black line indicated, near the detection zone. (e) Picture of a device with an incision. (f) Device with the detection zone folded back to the bottom side of the device, allowing numerous applications of reagents and washings on the detection zone without worrying about diffusion and flow into the channel. The device is shown upside down. (g) Picture of a device with folded detection zone.

produce redox molecules. Other methods such as chemiluminescence and fluorescence have been developed for paperbased analytical devices.28−30 We chose chemiluminescence due primarily to the high sensitivity requirement imposed by the low abundance of cotinine in clinical samples.3,4 In this work, we employed a commercial instrument to measure chemiluminescence to demonstrate the concept, though the potential integration of a photodiode detector with LPAD should be feasible. Here, we report the development of laminated paper-based analytical devices (LPAD) for the detection of SHS exposure by measuring cotinine with immunoassay. The format of competitive immunoassay was employed because cotinine is a small molecule and it does not have multiple epitopes that can be used for binding with both capture antibodies and detection antibodies. A similar format has been reported for cotinine detection, but it was in a 96-well plate.33 Immobilization of the capture antibody was achieved by modifying paper surfaces with chitosan and a cross-linking reagent. A simple origami step of local incision and paper-folding was exploited to isolate the detection zone from flow channels. Detection was achieved by using luminol-based chemiluminescence generated from horseradish peroxidase (HRP) conjugated with cotinine. After the optimization of various assay conditions, LPADs were used to determine cotinine in mouse serum.

Immunoassay has been implemented in paper-based microfluidic devices by several research groups.27−29 A typical immunoassay format such as ELISA (enzyme-linked immunosorbent assay) requires numerous steps, including the immobilization of capture antibodies, dispensing samples, application of detection antibodies, as well as several washings after each step. One challenge resulting from these numerous steps is the necessary separation of the detection zones from the sample flow channels. During each reagent application or washing step, solutions dispensed to the detection zone have a tendency to diffuse and spread into the flow channels, especially when the reagents from the previous step have not completely dried. To address the issue, we developed a protocol of localized incision and paper-folding to isolate the detection zone from flow channels. This simple origami step eliminated possible reagent diffusion and flow during the immobilization and numerous washing steps. Note that origami has been used for fabricating three-dimensional paper-based microfluidic devices by other research groups.29−31 Colorimetric detection is the most used method in paperbased analytical devices because of its simplicity.11−19 In general, however, only qualitative or semiquantitative measurement can be obtained. Electrochemical detection is another common method for paper microfluidic devices as is commonly found in commercial sensors.31,32 However, it is only useful for electrochemically active species or those compounds that can 10271

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Immobilization and Immunoassay. The immobilization steps for the competitive immunoassay are illustrated in Figure 2a. First, the detection zone of a paper device was coated with

EXPERIMENTAL SECTION Reagents and Materials. Luminol (3-aminophthalhydrazide), H2O2, chitosan, acetic acid, glutaraldehyde, and phosphate buffered saline (PBS, 1X) were purchased from Fisher Scientific (Pittsburgh, PA, U.S.A.). Cotinine was obtained from Alfa Aesar (Ward Hill, MA), while HRP conjugated cotinine (cotinine-HRP) was from CalBioreagents (San Mateo, CA). A luminol stock solution of 25 mM was prepared by dissolving 4.43 g of luminol in 20 mL of 0.10 M NaOH, followed by adding water to 1.0 L. The luminol solution was stored in the dark for 1 week to ensure that the reagent was stabilized. Working solutions of luminol were obtained by diluting the stock solution with a buffer of 25 mM NaHCO3/NaOH at pH 11.0 (the optimal pH value for luminol chemiluminescence). Cotinine is not soluble in water, thus a stock solution of cotinine (1.0 mg/mL) was prepared in methanol and refrigerated at 4 °C while a solution of cotinineHRP (50 μg/mL) was in PBS and refrigerated at −20 °C. Mouse monoclonal cotinine antibody (anticotinine) was bought from Abcam (Cambridge, MA) and bovine serum albumin (BSA) was from Sigma-Aldrich (St. Louis, MO). Mouse serum samples were purchased from Equitech Bio (Kerrville, TX). Except where specified otherwise, solutions were prepared using deionized water from a Millipore Nanopure water system (Barnstead, Dubuque, IA). Whatman chromatography paper (CHR1, 2-cm-wide roll) was obtained from Fisher Scientific (Pittsburgh, PA) while a roll of 75-μm-thick polyester thermal bonding lamination films was from Lamination Plus (Kaysville, UT). Device Fabrication and Origami Procedures. The device fabrication process has been discussed previously.24 In brief, the pattern of a device was designed using AutoCAD, and the design was then exported into a craft cutting-plotter (Craft Robo Pro-S, Graphtec Corporation). Chromatography paper was affixed to a carrier sheet before cutting. After removing the unwanted parts, paper strips were obtained. The paper strips were then laminated with a cover film and a bottom film as shown in Figure 1a. The cover film has a cutout that is slightly smaller than the paper strip so that the paper strip can be accessed for reagent dispensing and sample applications. Both cover and bottom films were made from polyester, providing mechanical backing to the paper device. The lamination process is similar to making an identification card. The device layout for this work is shown in Figure 1b, consisting of a reagent pad, flow channel, and detection zone. Except where specified otherwise, the flow channel is 1.6 mm wide and 10 mm long. The sample pad is in diamond shape with a side length of 2.5 mm. The detection zone is a circle with a diameter of 3.6 mm. A picture of several laminated devices is shown in Figure 1c. After lamination, an incision was created at 1 mm away from the detection zone of the device as indicated by the black line in Figure 1d. The incision was only made in the middle, leaving ∼3 mm of the plastic films uncut on both sides to facilitate subsequent operations. The incision would separate the detection zone from the flow channel after it was folded (Figure 1f), so that the solution dispensed to the detection zone would not spread into the channel during immobilization steps and washings as discussed in the next section. At the completion of the reagent loading, the detection area was folded back, and the flow channel sections were aligned with each other.

Figure 2. Device preparation and chemiluminescence-based immunoassay protocol. (a) The folded detection zone of a laminated device was modified by chitosan, then cross-linked with glutaraldehyde, followed by conjugation with anticotinine. Nonspecific binding sites were then blocked by BSA. (b) A mixture containing a cotinine sample and a fixed amount of cotinine-HRP was applied to the device before the detection zone was folded back to the normal position. The device was then placed into a microplate reader, and chemiluminescence was measured after adding luminol-based assay reagents.

chitosan, followed by cross-linking using an amine-reactive bifunctional molecule (glutaraldehyde) in a way similar to those reported in the literature.28 A chitosan solution of 3.0 μL at 0.25 mg/mL was dispensed onto the detection zone, followed by drying at ambient temperature. Chitosan adhered to paper due to electrostatic interactions between positively charged chitosan and negatively charged cellulose under wet conditions.34 The second step was to apply a solution of 2.5% glutaraldehyde in 10 mM PBS to the detection zone, modifying the surface through amino groups of chitosan. After 2 h of reactions, the detection zone was washed twice using 10 μL of water. The washing solution was removed by laboratory Kimwipe, followed by drying at ambient temperature. The third step was to dispense 3 μL of 5 μg/mL anticotinine in PBS to the detection zone, followed by 30 min of incubation. At the end of covalent binding of the antibodies to glutaraldehyde on paper surfaces, excess antibodies were washed away using 5 μL of PBS (three times). Step four was to apply 3 μL of 1% BSA in PBS to block possible nonspecific binding sites, followed by 15 min of incubation. The detection zone was then ready for chemiluminescence assay after two washings using 5 μL of PBS. The protocol to implement competitive chemiluminescence immunoassay on LPAD is illustrated in Figure 2b. Step one was to add 3 μL of a sample solution containing cotinine and cotinine-HRP to the detection zone. After 4 min of incubation, the detection zone was washed twice. Step two was to fold back the detection zone and align it to the flow channel. The paper device was then fixed onto a dummy 96-well plate with the detection zone aligned with a well (say E7). After dispensing 10 μL of 2.0 mM luminol and 20 mM hydrogen peroxide to the reagent pad, the plate with the device was placed in a microplate reader (Mithras LB 940, Berthold Technologies, Germany). The luminol-based chemiluminescent assay reagents 10272

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could be folded back and readily aligned along the flow channel to ensure the fluidic connection between the detection zone and the reagent pad. Our experiments confirmed that this simple origami step was easy to carry out, reagent diffusion and flow during the immobilization and numerous washing steps were eliminated, and immobilization/assay can be successfully realized on every device. Assay Condition Optimization. Device design parameters affecting chemiluminescence-based immunoassay were studied first. All optimization results discussed below were based on three repeat experiments except where specified otherwise. Since the analysis time is very important in point-of-care testing, we investigated the effects of the channel length on the time needed by the assay reagents to flow to the detection zone. With the channel width fixed at 1.8 mm, chemiluminescence signals were detected at 50, 74, and 120 s after reagent dispensing for channel lengths of 10, 15, and 20 mm, respectively. We chose 10 mm as the channel length because it is very close to the pitch of a 96-well plate, has a short flow time, and has a good relative standard deviation of the flow rate (4.3%, n = 5). We also studied the effects of the channel width by varying it from 1.0, 1.3, 1.6, 1.9, to 2.2 mm with the channel length fixed at 10 mm. We then chose 1.6 mm as the optimal width. Too narrow a channel allowed only a limited amount of assay reagents to pass through, whereas too wide a channel consumed excessive assay reagents in the channel itself with an inadequate amount of reagents reaching the detection zone. The effects of the assay reagent concentration on chemiluminescence signals were investigated. Figure 3a shows the luminescence intensity as a function of the luminol

flowed through the channel and then reacted with cotinineHRP in the detection zone while the microplate reader was programed to measure the signal at the predetermined well location. A waiting period of 30 s was used to allow the assay reagents to flow and for reactions, followed by luminescence measurement over 90 s. The arrangement of placing chemiluminescent assay reagents in a separate well is to simulate the real world applications in resource-poor regions. Surface modification and antibody immobilization should be completed during device manufacture. The chemiluminescence assay reagents should also be deposited in the reagent pads during the manufacturing process. The only step needed in the field is to apply a sample to the detection zone and add pure water to the reagent pad to reconstitute the assay reagents.35 Relatively clean water such as bottled drinking water is likely accessible in the field, and it is easier than carrying the assay reagents. An acceptable shelf life of antibodies and reagents on a paper device should be possible as proved in commercially available pregnancy test strips.

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RESULTS AND DISCUSSION Device Fabrication and Origami Folding. As mentioned above, paper-based microfluidic devices have been fabricated primarily by two methods: creating either hydrophobic boundaries or physical boundaries. They generally suffer from the common problem that paper has relatively low mechanical strength, especially when wet. To address this concern, lamination has been proposed as one of the methods to increase mechanical strength and durability of the devices.24 In a way similar to making an identification (ID) card as shown in Figure 1a, a digital craft cutter was used to create paper strips based on the design, followed by a roll laminator to produce laminated paper-based analytical devices (LPADs). By encapsulating the paper strip between layers of thermally bonded polymer films, low-cost and rugged devices can be produced. To implement immunoassays on paper-based microfluidic devices, one common requirement is an effective washing to remove extra or nonreacted reagents as demonstrated by several research groups.27−29 Since the amount of the washing solution is often in excess, the solution tends to spread from the desired location into other areas. For example, a washing solution dispensed onto the detection zone in Figure 1b would flow into the channel. One way to address the challenge was to encircle the immunoassay area using SU-8 or wax.27,28 However, this approach is only applicable to discrete spot assays without channels or other fluidic connections. Another way was to create a three-dimensional device that contained waste pads to suck away the washing solutions.29 A switch may also be created by inserting a paper flap through a channel that essentially functions as a valve.36 However, these methods make device fabrication and assay operations much more complicated. To address this challenge, we developed a protocol of localized incision and paper-folding to isolate the detection zone from flow channels. As shown in Figure 1d, an incision line was created through the flow channel. Note that the lamination layers were not cut all the way to the edge of the device so that two sections of the channel remained linked, allowing for easy alignment later on. The flexible plastic films can then be folded to physically separate the detection zone from the channel, as shown in Figure 1f. After antibody immobilization and numerous washings, the detection zone

Figure 3. Parameter optimization for chemiluminescence-based immunoassay. Each datum represents the average of three repeat experiments, while the error bars indicate one standard deviation. (a) The ratio of the chemiluminescence signal to the background (signal/ noise ratio) is plotted as a function of the luminol concentration. The dashed line represents the mathematically best-fit regression. (b) Effects of the hydrogen peroxide (H2O2) concentration on the signal/ noise ratio of chemiluminescence detection. (c) Effects of the incubation time on the chemiluminescence signal. 10273

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concentration when the concentration of H2O2 was fixed at 50 mM (pH 11.0). A solution of 10 μg/mL cotinine-HRP was used for these experiments. Because the background signal changed for different luminol concentrations, we used the signal-to-noise ratio to search for the optimum concentration. The results suggest that we should choose the luminol concentration at 2.0 mM for immunoassay in LPAD. Similarly, the effects of H2O2 concentration on chemiluminescence signals were investigated with the luminol concentration fixed at 2.0 mM, and the result is shown in Figure 3b. The data indicate that the optimum concentration of H2O2 is 20 mM. We also studied two parameters related to immunoassay. With the optimum conditions mentioned above and 10 μg/mL cotinine-HRP, we evaluated the concentration of anticotinine at 1.0, 2.0, 5.0, and 10 μg/mL (Figure S1 in Supporting Information). On the basis of the results, we chose 5.0 μg/ mL as the optimal anticotinine concentration. Another parameter we examined is the incubation time, which is often from half to a few hours for traditional immunoassays. The incubation time for a cotinine assay was 24 h in one report.33 The assay time is expected to be much shorter due to microscale diffusion distances and favorable reaction kinetics than in a macroscale container.37 With the concentration of anticotinine at 5.0 μg/mL, the effects of incubation time on immunoassay were examined, and the results are shown in Figure 3c. The luminescence signal initially increased with increasing incubation time, and then reached a plateau at 240 s (4 min). We chose 4 min of incubation time for the experiments discussed below. Calibration Curve. Under the optimum conditions, we obtained the calibration curve of cotinine detection using chemiluminescence-based competitive immunoassays in LPAD as shown in Figure 4. Since cotinine and cotinine-HRP

relevant since the concentration of cotinine in SHS samples is typically on the order of 10 ng/mL.4,5 We also found that the relative standard deviation of seven repeated measurements of 1 μg/mL cotinine was 5.0%. Mouse Serum Analysis. The performance of LPAD for the determination of cotinine was evaluated in mouse serum samples. We purchased mouse serum from a commercial source, and it was used after 50 times dilution with PBS. Dilution was to reduce the interference of some serum components with the chemiluminescence system we used. With cotinine-HRP fixed at 5.0 μg/mL, we studied the amount of cotinine detected when different amounts of cotinine were spiked into the diluted serum sample. The recovery rate in percentage was obtained by dividing the amount of cotinine calculated from the measured luminescence signal and the calibration curve by the amount of cotinine spiked into the sample. We obtained a recovery rate from 97.1% to 103.5% as shown in Table 1. Each value represents an average of the results from three repeat experiments. We compared our chemiluminescence immunoassay in LPAD with two other methods in the literature for cotinine recovery studies. One was high-performance liquid chromatography (HPLC)8 for measuring cotinine in either plasma or urine, while the other was gas chromatography coupled with mass spectrometry (GC-MS) for quantification of urinary cotinine.9 The recovery rate as well as the precision (relative standard deviation) of our method is comparable to, if not better than, two other methods that require bulky benchtop instruments. We have focused on lower concentrations of cotinine since it is more relevant to SHS exposure. The results suggest that LPAD with chemiluminescence immunoassay has the potential to become an alternative method for SHS detection.

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CONCLUSION We have developed laminated paper-based analytical devices (LPAD) with origami-enabled chemiluminescence immunoassay for the detection of cotinine, a secondhand smoke (SHS) biomarker. To our knowledge, this is the first report on applying paper-based microfluidic devices for the detection of cotinine. Integration of LPAD with chemiluminescence immunoassay brought about advantages in simplicity and low cost of the devices and in sensitivity and selectivity of the detection method. Using a craft-cutter to define flow channels and using lamination films to seal/support paper devices are very similar to making a photo identification card. As a result, the device fabrication process is simple, and the cost of manufacturing a large quantity of devices is low. To enable chemiluminescence immunoassay in LPAD, we developed a protocol of localized incision and paper-folding to isolate the detection zone, enabling antibody immobilization and numerous washings without worrying about reagent diffusion and spreading. The origami-enabled, chemiluminescence-based competitive immunoassay in LPAD was demonstrated for the detection of cotinine in mouse serum with the limit of detection at 5.0 ng/ mL, a clinically relevant concentration for SHS exposure. SHS has been associated with a variety of adverse health outcomes in nonsmokers, including lung cancer, respiratory illness, and cardiovascular diseases, and it is designated as a known human carcinogen.2 Therefore methods for detecting the exposure to SHS are urgently needed. However, the instruments used to determine cotinine or other SHS

Figure 4. Calibration curve for cotinine analysis using chemiluminescence-based competitive immunoassays in LPAD. Each data point represents an average from five repeat experiments, and the error bars indicate one standard deviation.

compete with each other for the fixed amount of anticotinine immobilized on the surfaces of the detection zone and the chemiluminescence intensity represents the amount of cotinine-HRP, the decrease in signal from the blank sample indicates the presence of cotinine in a sample, and the degree of the decrease is proportional to the concentration of cotinine. The results show that a linear detection range of 0.01−1 μg/ mL was obtained. The limit of detection was determined to be 5.0 ng/mL using the definition of 3 times the standard deviation of a blank solution. These results are clinically 10274

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Table 1. Cotinine Recovery Rate and Assay Precision detection methods

samples

chemiluminescence immunoassay in LPAD (this work)

mouse serum

HPLC8

human plasma

HPLC8

human urine

GC-MS9

human urine

added cotinine concentration (ng/mL)

measured concentration (ng/mL)

recovery (%)

precision (%)

70.0 80.0 90.0 500 2000 4000 500 2000 4000 0.5 2500 5000

72.5 ± 2.5 77.7 ± 2.3 88.4 ± 5.0

103.5 97.1 98.2 93.6 98.0 95.8 85.5 85.8 101.5 104.4 101.8 95.1

3.5 3.0 5.6 6.0 1.0 1.7 4.8 0.1 1.6 8.5 4.0 5.9

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biomarkers are generally bulky and require skilled personnel. LPAD in this work could provide an alternative that is portable, simple to operate, and with sufficient detection sensitivity. Moreover, the design of LPAD and origami-enabled chemiluminescence immunoassay can be readily adapted for other biomarkers and molecules of interest for a variety of applications. The LPAD platform is especially useful for point of care, environmental testing, and medical diagnostics in remote regions and resource-poor countries.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: hfan@ufl.edu. Present Address

⊥ Columbus Police Department Crime Laboratory, 1501 Main St., Columbus, Mississippi 39701, United States.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported in part by the Flight Attendant Medical Research Institute (FAMRI-082502) and the National Science Foundation (OISE-0968313). W.L. acknowledges the support of the National Natural Science Foundation of China (No. 21005048). We thank Weian Sheng of the University of Florida for helpful discussion. W.L. is grateful to the China Scholarship Council for funding her stay in the U. S. A.

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0.52 ± 0.04 2777.4 ± 111.1 4906.7 ± 288.1

REFERENCES

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Analytical Chemistry

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dx.doi.org/10.1021/ac402055n | Anal. Chem. 2013, 85, 10270−10276