COLLECTIVE TRANSFER OF BIOMOLECULES FROM GEL DROPLET MICROARRAY-TO-GEL DROPLET MICROARRAY: APPLICATION TO HIGH SENSITIVITY MULTIPLEXED BEADS-IN-GEL IMMUNOASSAYS

Huiyan Li1,2*, David Juncker1,2,3

1 Biomedical Engineering Department, McGill University, Montreal, Quebec, Canada 2 McGill University and Genome Quebec Innovation Centre, Montreal, Quebec, Canada

3 Department of Neurology and Neurosurgery, McGill University, Montreal, Quebec, Canada ABSTRACT

We present a novel and simple approach for transferring a microarray of detection antibodies (dAbs) in agarose gel spots to a microarray of alginate droplets containing beads coated with the matched capture antibody (cAb). Cross-reactivity among antibody pairs was thus eliminated. We developed an alignment and transfer apparatus and process to achieve precise mirror symmetric alignment. We illustrate the potential of this platform by profiling 9 cancer-related proteins with limits of detections as low as 3 pg/ml. KEYWORDS: transfer, gel droplet microarray-to-gel droplet microarray, immunoassays INTRODUCTION

The performance of multiplexed assays is severely limited owing to cross-reactivity between antibodies and antigens which occurs because detection antibodies are applied as a mixture. We have developed antibody colocalization microarrays to eliminate cross reactivity by spotting each dAb on the spot of the corresponding cAb on a nitrocellulose slide [1]. Recently we also introduced beads-in-gel droplet microarrays that allowed for more sensitive multiplexed protein assays in serum [2]. Here, we introduce microarray-to-microarray transfer of antibodies and combine the advantages of antibody colocalization microarray and of beads-in-gel droplet microarrays while producing a handheld, highly sensitive and scalable multiplex immunoassay chip. EXPERIMENTAL The process is schematized in Fig. 1. A commercial inkjet spotter was used to spot 0.65% alginate solutions mixed with cAb-coated polystyrene microbeads onto aminosilane slides at precise coordinates, and fluorescently labeled dAbs in an agarose solution on another slide in a mirrored pattern. The alginate cAb droplets were gelated immediately by adding a calcium solution and the agarose dAb droplets by cooling the slide to 4°C. Next, the cAb slide was blocked with bovine serum albumin for 1 h, and incubated with a sample for 1 h, and briefly dried. The two slides were then clamped together in a custom designed alignment and transfer apparatus, brought into contact, and incubated for 1 h. Finally, the assay was read out using a microarray scanner. RESULTS AND DISCUSSION The procedure to fabricate gel droplet transfer microarrays is shown in Figure 1, step a to b.

Figure 1: Beads-in-gel droplet immunoassays using microarray-to-microarray transfer. Hydrogel solutions of cAbs and dAbs

were spotted at precise coordinates in a mirror pattern while using back-side alignment for the dAbs. The assay slide was

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October 2-6, 2011, Seattle, Washington, USA

incubated with sample and briefly dried. The two slides were aligned, and brought into contact for the collective transfer of dAbs using the transfer apparatus, followed by incubation with streptavidin-Cy5 and fluorescent imaging.

An important challenge was to achieve homogeneous transfer of antibodies and precise mirror alignment, Figure 2.

Alignment between both microarrays was achieved using mirror symmetric patterns, and a custom built alignment and transfer apparatus with a vacuum chuck, Figure 2 (a). The alignment accuracy was less than 150 µm.

Figure 2: Alignment and transfer apparatus. (a) Photograph of the two vacuum chucks comprising a recess for mechanical

slide alignment and a precision machined support plate for the transfer. (b) Schematic of the apparatus and contact between gel droplets during droplets during transfer. A rubber backing and spacers ensure a homogeneous transfer across the slide.

Another challenge is the reagent confinement during the transfer process. Confinement of transferred antibodies was

achieved using microscale spacers to ensure chip-scale droplet-to-droplet transfer and was confirmed using a one-step assay with two fluorescently labeled antibodies, Figure. 3.

Figure 3: Confinement of transferred antibodies onto their corresponding spots using a one-step assay. (a) Assay schematic. Alexa 532 labeled goat IgG (Ab 1) were coated on beads, and Alexa 633 labeled anti-goat IgG (Ab 2) were dissolved in an agarose solution and spotted on every second spot. (b) Scan of the beads-in-gel slide after transfer using 532 nm laser and

633 nm laser.

We then used the beads-in-gels microarray-to-microarray transfer chips for a multiplexed immunoassay of 9 proteins, Tumor necrosis factor receptor-II (TNF RII), granulocyte macrophage colony-stimulating factor (GM-CSF), chemokine (C-

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C motif) ligand 2 (CCL2), interleukin-1 beta (IL-1β), chemokine (C-C motif) ligand 4 (CCL4), interleukin-5 (IL 5), tumor necrosis factor receptor-I (TNF RI), interleukin-18 (IL 18), and tumor necrosis factor alpha (TNF α), Figure 4. Upon optimization, the binding curves for these assays reach a low pg/mL range, which is below the physiological levels in the blood of healthy people, and indicates that our platform would be suitable for profiling these proteins in clinical studies.

Figure 4: Binding curves of 9 proteins measured simultaneously shown in the inset. The LOD for TNF RII is 3 pg/mL. The

error bars are standard deviations between triplicate experiments. CONCLUSION

We introduced a novel microarray-to-microarray antibody transfer and illustrated its potential with high sensitivity, multiplex protein assays using beads-in-gel droplet microarrays. This system may readily be scaled up, slides prepared ahead of time, stored, and then unpacked and used when needed. We believe that this method will be particularly useful for the discovery of protein biomarkers for disease and for their subsequent clinical validation. ACKNOWLEDGEMENTS We thank the Canadian Institutes for Health Research (CIHR), Genome Canada, Genome Quebec, and the Canada Foundation for Innovation (CFI) for financial support. D.J. acknowledges support from a Canada Research Chair. REFERENCES [1] Pla-Roca et al. Antibody Colocalization Microarray: A Scalable Technology for Multiplex Protein Analysis in Complex Samples, submitted for publication. [2] H. Li et al. Hydrogel droplet microarrays with trapped antibody-functionalized beads for multiplexed protein analysis. Lab on a Chip, 11, 528-534, (2011). CONTACT *Huiyan Li, tel: +1-514-398-4400; huiyan.li@mail.mcgill.ca

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