IMMUNOASSAY ON COTTON YARN FOR LOW-COST DIAGNOSTICS G.Z. Zhou, R. Safaviah, X. Mao, and D. Juncker

Biomedical Engineering, McGill University, Montreal, CANADA ABSTRACT

We report our findings on the flow properties of cotton yarns for microfluidics, and illustrate the use of yarn to per-form a sandwich immunoassay to detect and quantify C-reactive protein (CRP), an inflammatory and cardiovascular dis-ease biomarker, with a dynamic range of three orders of magnitude, and a sensitivity of 10 ng/ml. KEYWORDS: yarns, microfluidic devices, immunoassay, C-reactive protein, low-cost diagnostics

INTRODUCTION

Paper [1] and most recently yarns [2, 3, 4] have been reported as economic materials for making microfluidic de-vices and performing biomedical assays. Here we examine the fluid flow speed within a cotton yarn, and confirm that the flow within yarns is a capillary flow in a porous material, thereby obeying Washbrun’s equation. We then demon-strate yarn-based immunoassay in two setups: a multistep test, and a single step test with reduced assay time. The multis-tep test is similar to the conventional sandwich assay where the reagents are applied in sequence, with incubation, the as-say time can be long; whereas in the one-step assay, reagents are pre-immobilized on the yarn substrate, with a step of applying the sample, the results can be visualized within minutes. C-reactive protein (CRP), an inflammatory and cardio-vascular disease biomarker, is chosen as the analyte for the immunoassay. CRP has a high concentration in the diseased subject, rising from an average concentration of 0.8 µg/ml in the blood to between 40 µg/ml and 500 µg/ml in response to an infection [5].

THEORY

An ubiquitous diagnostic test is the immunochromatographic test carried out in a lateral flow strip format. For ex-ample, the pregnancy test strip detects hCG (Human Chorionic Gonadotropion) in the urine sample, and provides a vis-ual read-out indicating the presence of the hCG. The strip is composed of a nitrocellulose membrane for immobilizing the capture antibody (cAb), a fiber glass membrane for drying and later releasing of the detection antibody (dAb), and an absorbent membrane for loading the biological sample and absorbing the excessive reagents. This assembly is depicted in the Fig 1.

a) b)

c)

Figure 1: The assembly and working principle of a lateral-flow immunochromatographic test. a) A lateral flow strip is composed of sample, conjugate, membrane, and absorbent pads. b) cAb are pre-immobilized on the membrane, and dAb conjugated with beads and particles are pre-dried on the conjugate pad. c) When sample containing the analyte of inter-est is applied, it releases the dAb from the conjugate pad, flows through the test line, together they bind with the cAb and form a complex that is visible to the eyes.

Developing a similar high-sensitivity yarn-based device faces several challenges: 1) identifying the right yarn mate-

rial to serve different purposes on a flow strip, 2) applying the reagents precisely, 3) controlling the fluid transport within the yarn, 4) choosing a detection technique that allows direct read-out and easy interpretation of the assay results, 5) as-sembling and packaging the yarns for easy handling and manipulation. EXPERIMENTAL

Yarns and Flow Property. After a comparison of multiple yarns, mercerized cotton yarn (Gütermann, Germany) is used for all future devices. Red food dye is used as an indicator to study the capillary flow in the yarns and the effect of O2 plasma on the flow rate.

Multistep Assay Device Assembly. A bundle of short yarns is knotted to the end of a long thread to serve as a res-ervoir that continuously draws the liquid from the well through the long thread, as illustrated in Fig 4a.

Multistep Assay Procedure. A small section of the yarn is coated with 0.6 µl of 1 mg/ml CRP cAb (R&D Systems, Minneapolis, MN, USA). The subsequent samples are flown by capillary effect by sequentially dipping the end of the yarn into Eppendorf tubes containing each of the different solutions (Fig 4a). 1% BSA is flown to block the surface. 10 µl of CRP with desired concentration spiked in PBS (PBS only for the negative control) is flown and incubated at

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room temperature for 30 minutes, followed by 10 µl of dAb conjugated with streptavidin beads. The yarn is rinsed with washing buffer between each step.

dAb-Au NP Conjugate Preparation. The dAb is conjugated to 44 nm Au colloids (Nanopartz Inc, Loveland, CO, USA) according to a published protocol [6] with minor modifications. The functionalized Au NPs are re-suspended in 1 ml of 20 mM of Na3PO4 buffer with 5% BSA, 2.5% Tween, and 10% sucrose.

One-step Assay Device Assembly. This design emulates a lateral flow strip, where two types of yarns are used, a mercerized cotton yarn and a porous fiber. The cotton yarn is coated with 0.6 µl of 1 mg/ml CRP cAb, and the porous fi-ber with 4 µl of Au NPs dAb conjugates, then stored at 4°C in a low humidity condition.

One-step Assay Procedure. The assay test is carried out by first knotting the two yarns to where the Au NPs is dried (Fig 5a), and then applying CRP with a desired concentration at the knot, and flushing with PBS.

Quantitative Analysis. In both cases, the accumulation of beads or particles leads to a color stripe on the yarn visi-ble to the naked eye, indicating successful binding of proteins. A desktop scanner (N6310, Hewlett Packard) was used to record the images, and the binding is quantified as the average color hue change, using ImageJ software. RESULTS AND DISCUSSION

Yarns. The yarns are 250 ± 20 µm in diameter and made up of ~300 fibers, each 12-15 µm thick (Fig 2). a) b) c)

Figure 2: Images of cotton yarn at different scales. a) Photograph of a spool of cotton yarn. b) SEM micrograph of loosely twisted bundles of yarn. c) A close-up view of the fibers and the narrow gaps in which fluid flows.

Flow Property. The red food dye wicks approximately 5 mm after 40 s in a cotton yarn used as received (Fig 3a),

and 25 mm in a yarn made hydrophilic using plasma (Fig 3b). The wetted length L is plotted as a function of wicking time t, and fit to the Washburn’s equation that describes the capillary flow within porous material, Fig 3c [7]. Under the assumption that the plasma treatment only changes the free surface energy of the cotton, but not the geometry, a 14.1-fold increase in the capillary pressure is found.

a) b)

c)

where

Figure 3: Images illustrating the wicking of a red dye in (a) as-received and (b) plasma activated, hydrophilic cotton yarn. (c) The wetted length shown as a function of wicking time, and fitted to Washburn’s equation, shown on the top right.

Multistep Assay. With the knowledge of flow property in the yarn, a “pump” structure is built to enhance the fluid flow in making the multistep yarn-based immunoassay device. Since samples are flown by capillary effect by dipping the yarn into different solutions, the pump not only helps to pull the liquid from the well, but also increases the volume of the sample that could be flushed through the yarn (Fig 4a). A blue color is developed where the target CRP binds. The color intensity is proportional to the analyte concentration, and is visualized and quantified to obtain a calibration curve (Fig 4b and 4c). The color intensity of the yarn with 10 µg/ml is significantly different from the negative control yarn.

There are two limitations of this multistep assay. First, the protocol requires dipping the yarn into each one of the

reagents sequentially, with incubation, the assay can take a long time. To move towards a simple diagnostic device, a lat-eral flow format is more desirable where all reagents are pre-immobilized on the yarn. With a single step of sample flow-ing, results will be available within minutes. The second limitation is that the beads are relatively large (380 nm in di-ameter), so they can be trapped between fibers and result in a high background. Small Au-NPs (44 nm in diameter) are used in the subsequent one-step assay to overcome this problem.

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a) b) c)

Figure 4: Immunoassay on cotton yarns carried out in multiple steps. a) A photograph showing the yarn, with a pump formed by multiple short yarns, dipping into a well to draw the liquid upwards. b) Scanned image of the yarns that de-tects CRP concentration from 1 µg/ml to 320 µg/ml. The color intensity at 10 µg/ml is significantly different from the negative control yarn. c) Binding curve of CRP extracted from b).

One-step Assay. The one-step assay emulates a lateral flow strip, where two types of yarns are used. The sample yarn made of mercerized cotton has high protein binding property, and the conjugate yarn are considerably porous thus can release the Au NPs effectively (Fig 5a). The conjugate yarn is knotted across several sample yarns enabling multi-plexing, only the conjugate zone where the two yarns intersect is coated with dAb-Au NP. The area is defined and sepa-rated by wax. A duplicate of three yarns is tested for each CRP concentration, between 5 ng/ml and 500 ng/ml. A red color is developed on the yarn indicating the protein binding. The color intensity is visible to the eye and increases with the concentration (Fig 5b). The small Au particle size is shown to improve the assay sensitivity down to the ng/ml range.

a) b)

Figure 5: Immunoassay on cotton yarns in a lateral-flow strip format. a) Photo of two yarns and one porous fiber knot-ted, forming a cross. The cAb is pre-immobilized on the yarn, and the porous fiber is patterned with wax to form a well where it intersects the yarn, to contain and to release the dAb-Au NPs. b) Graph showing the color intensity increase for a protein dilution series from 5 ng/ml to 0.5 µg/ml. A red color is distinguishable down to a concentration of 10 ng/ml.

CONCLUSION

This work shows some preliminary results and steps taken towards building a prototype of such sort. First, the flow property in the yarn is studied, and shown to be predictable, obeying Washburn’s equation. Passive microfluidic ele-ments, such as pumps and reservoirs, are constructed to enable yarns to be used as immunoassay substrates. Finally, two assay tests are shown to demonstrate the feasibility of yarn used for high sensitive protein quantification, with a detection limit down to 5 ng/ml. The “microfluidic fibers” introduced here do not require lithographic steps to form fluid channels. Yarns therefore present a new opportunity for developing low-cost diagnostic devices with microfluidic components. REFERENCES [1] Martinez, A.W., Phillips, S.T., Butte, M.J., Whitesides, G.M., Angewandte 2007, 46, 1318. [2] Safavieh, R., Mirzaei, M., Qasaimeh, M.A., Juncker, D., Proceedings of µTAS, Jeju, Korea, 2009; pp685-687. [3] Li, X. , Tian, J. F., and Shen, W. ACS Appl. Mater. Interfaces 2010, 2, 1– 6 [4] Reches, M., Mirica, K.A., Dasgupta, R., Dickey, M.D., Butte, M.J., Whitesides, G.M. ACS Appl Mater. Inter-

faces 2010, 2, 1722-1728. [5] Gervais, L., Delamarche, E., Lab on a Chip 2009, 9, 3330. [6] Lyon, L. A. , Musick, M. D. , and Natan, M. J. Anal. Chem. 1998, 70, 5177– 5183. [7] Washburn E.W., Physical Review 1921, 17, 273. CONTACT - gina.zhou@mcgill.ca and david.juncker@mcgill.ca

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