A Guide to Quantitative Biomarker Assay Development using Whispering Gallery Mode Biosensors

Heather M. Robison1, Ryan C. Bailey2

1 Department of Chemistry, University of Illinois at Urbana‐Champaign, 2 Department of Chemistry, University of Michigan, Ann Arbor
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/cpch.23
Online Posting Date:  September, 2017
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Abstract

Whispering gallery mode (WGM) sensors are a class of powerful analytical techniques defined by the measurement of changes in the local refractive index at or near the sensor surface. When functionalized with target‐specific capture agents, analyte binding can be measured with very low limits of detection. There are many geometric manifestations of WGM sensors, with chip‐integrated silicon photonic devices first commercialized because of the robust, wafer‐scale device fabrication, facile optical interrogation, and amenability to the creation of multiplexed sensor arrays. Using these arrays, a number of biomolecular targets have been detected in both label‐free and label‐enhanced assay formats. For example, sub‐picomolar detection limits for multiple cytokines were achieved using an enzymatically enhanced sandwich immunoassay that showed high analyte specificity suitable for detection in complex, clinical matrices. This protocol describes a generalizable approach for the development of quantitative, multiplexed immunoassays using silicon photonic microrings as an example WGM platform. © 2017 by John Wiley & Sons, Inc.

Keywords: whispering gallery mode sensors; immunoassay; biomarker; protein detection; multiplex; quantitative analysis

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Functionalization of the Sensor Surface with Antibody Capture Probes
  • Basic Protocol 2: Assessing Optimal Conditions, Cross‐Reactivity, and Matrix Effects for Whispering Gallery Mode (WGM) Assay Optimization
  • Basic Protocol 3: Calibrating the Optimized Multiplexed Assay in Complex Matrix for Analyte Quantitation
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Functionalization of the Sensor Surface with Antibody Capture Probes

  Materials
  • Acetone
  • Ιsopropanol
  • 3‐Aminopropyltriethoxysilane (1% APTES solution in acetone)
  • Distilled water or Milli‐Q purified water
  • N 2 source
  • Bissulfosuccinimidyl suberate (5 mM BS3 in 2 mM acetic acid; 2 mg No‐Weigh Format, Thermo Scientific)
  • Acetic acid (2 mM in distilled water)
  • Capture antibody stock solution (at least 0.25 mg/ml, <1% sodium azide)
  • PBS buffer with BSA (10 mM with 0.5% BSA)
  • Glycerol
  • DryCoat assay stabilizer (Virusys, cat. no. AG066‐1)
  • StartingBlock (PBS) buffer (Thermo Scientific)
  • WGM sensor/sensor array (128‐microring sensor arrays, Genalyte Inc.)
  • Tweezers
  • Platform shaker
  • 20‐ml scintillation vials
  • 24‐well plates
  • Stereoscope
  • Clean room cloth
  • Desiccator at 4ºC

Basic Protocol 2: Assessing Optimal Conditions, Cross‐Reactivity, and Matrix Effects for Whispering Gallery Mode (WGM) Assay Optimization

  Materials
  • Standard protein solutions (1 to 100 µg/ml stock)
  • Running buffer: 10 mM PBS, 0.5% BSA, detergents such as Tween20 (optional)
  • Biotinylated tracer antibodies (0.5 to 4 µg/ml in running buffer)
  • Streptavidin‐horseradish peroxidase conjugate (1 to 6 µg/ml in running buffer)
  • 1‐Step chloronapthol solution (4‐CN, Thermo Scientific, containing 4‐chloro‐1‐naphthol and proprietary peroxide‐containing buffer)
  • Capture agent‐functionalized WGM sensor (see protocol 1)
  • Low pH glycine solution (10 mM in Milli‐Q water, pH 2.2)
  • Concentrated matrix (e.g., plasma or serum, aliquotted and stored frozen)
  • Vortex mixer
  • 96‐well plates (400 ml maximum volume)
  • Pre‐cut piercable films for Robotics (X‐Pierce, Excel Scientific)
  • Cartridge assembly (Genalyte Inc.)
  • Teflon sipper tubes (0.01‐in. i.d. Teflon tubing)
  • Precision torque wrench and screwdriver (1/16‐in.)
  • Maverick optical scanning instrumentation (Genalyte Inc.)
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Figures

Videos

Literature Cited

Literature Cited
  Baaske, M. D., Foreman, M. R., & Vollmer, F. (2014). Single‐molecule nucleic acid interactions monitored on a label‐free microcavity biosensor platform. Nature Nanotechnology, 9, 933–939. doi: 10.1038/nnano.2014.180.
  Fan, X. D. & White, I. M. (2011). Optofluidic microsystems for chemical and biological analysis. Nature Photonics, 5, 591–597. doi: 10.1038/nphoton.2011.206.
  Foreman, M. R., Swaim, J. D., & Vollmer, F. (2015). Whispering gallery mode sensors. Advances in Optics and Photonics, 7, 168–240. doi: 10.1364/AOP.7.000632 10.1364/AOP.7.000168.
  Iqbal, M., Gleeson, M. A., Spaugh, B., Tybor, F., Gunn, W. G., Hochberg, M., … Gunn, L. C. (2010). Label‐free biosensor arrays based on silicon ring resonators and high‐speed optical scanning instrumentation. IEEE Journal of Selected Topics in Quantum Electronics, 16, 654–661. doi: 10.1109/JSTQE.2009.2032510.
  Kindt, J. T. & Bailey, R. C. (2012). Chaperone probes and bead‐based enhancement to improve the direct detection of mRNA using silicon photonic sensor arrays. Analytical Chemistry, 84, 8067–8074. doi: 10.1021/ac3019813.
  Kindt, J. T., Luchansky, M. S., Qavi, A. J., Lee, S.‐H., & Bailey, R. C. (2013). Subpicogram per milliliter detection of interleukins using silicon photonic microring resonators and an enzymatic signal enhancement strategy. Analytical Chemistry, 85, 10653–10657. doi: 10.1021/ac402972d.
  Luchansky, M. S. & Bailey, R. C. (2011). Rapid, multiparameter profiling of cellular secretion using silicon photonic microring resonator arrays. Journal of the American Chemical Society, 133, 20500–20506. doi: 10.1021/ja2087618.
  Luchansky, M. S. & Bailey, R. C. (2012). High‐Q optical sensors for chemical and biological analysis. Analytical Chemistry, 84, 793–821. doi: 10.1021/ac2029024.
  McClellan, M. S., Domier, L. L., & Bailey, R. C. (2012). Label‐free virus detection using silicon photonic microring resonators. Biosensors & Bioelectronics, 31, 388–392. doi: 10.1016/j.bios.2011.10.056.
  Qavi, A. J. & Bailey, R. C. (2010). Multiplexed detection and label‐free quantitation of MicroRNAs using arrays of silicon photonic microring resonators. Angewandte Chemie‐International Edition, 49, 4608–4611. doi: 10.1002/anie.201001712.
  Qavi, A. J., Kindt, J. T., Gleeson, M. A., & Bailey, R. C. (2011a). Anti‐DNA:RNA antibodies and silicon photonic microring resonators: Increased sensitivity for multiplexed microRNA detection. Analytical Chemistry, 83, 5949–5956. doi: 10.1021/ac201340s.
  Qavi, A. J., Mysz, T. M., & Bailey, R. C. (2011b). Isothermal discrimination of single‐nucleotide polymorphisms via real‐time kinetic desorption and label‐free detection of DNA using silicon photonic microring resonator arrays. Analytical Chemistry, 83, 6827–6833. doi: 10.1021/ac201659p.
  Scheler, O., Kindt, J. T., Qavi, A. J., Kaplinski, L., Glynn, B., Barry, T., … Bailey, R. C. (2012). Label‐free, multiplexed detection of bacterial tmRNA using silicon photonic microring resonators. Biosensors & Bioelectronics, 36, 56–61. doi: 10.1016/j.bios.2012.03.037.
  Valera, E., McClellan, M. S., & Bailey, R. C. (2015). Magnetically‐actuated, bead‐enhanced silicon photonic immunosensor. Analytical Methods, 7, 8539–8544. doi: 10.1039/C5AY01477H.
  Valera, E., Shia, W. W., & Bailey, R. C. (2016). Development and validation of an immunosensor for monocyte chemotactic protein 1 using a silicon photonic microring resonator biosensing platform. Clinical Biochemistry, 49, 121–126. doi: 10.1016/j.clinbiochem.2015.09.001.
  Wade, J. H., Alsop, A. T., Vertin, N. R., Yang, H. W., Johnson, M. D., & Bailey, R. C. (2015). Rapid, multiplexed phosphoprotein profiling using silicon photonic sensor arrays. ACS Central Science, 1, 374–382. doi: 10.1021/acscentsci.5b00250.
  Wade, J. H., & Bailey, R. C. (2016). Applications of optical microcavity resonators in analytical chemistry. Annual Review of Analytical Chemistry, 9(1)1–25. doi: 10.1146/annurev‐anchem‐071015‐041742.
  Washburn, A. L., Gunn, L. C., & Bailey, R. C. (2009). Label‐free quantitation of a cancer biomarker in complex media using silicon photonic microring resonators. Analytical Chemistry, 81, 9499–9506. doi: 10.1021/ac902006p.
  Washburn, A. L., Luchansky, M. S., Bowman, A. L., & Bailey, R. C. (2010). Quantitative, label‐free detection of five protein biomarkers using multiplexed arrays of silicon photonic microring resonators. Analytical Chemistry, 82, 69–72. doi: 10.1021/ac902451b.
  Washburn, A. L., Shia, W. W., Lenkeit, K. A., Lee, S. H., & Bailey, R. C. (2016). Multiplexed cancer biomarker detection using chip‐integrated silicon photonic sensor arrays. Analyst, 141, 5358–5365. doi: 10.1039/C6AN01076H.
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