Elsevier

Biosensors and Bioelectronics

Volume 25, Issue 2, 15 October 2009, Pages 275-281
Biosensors and Bioelectronics

Spectral-domain optical coherence phase microscopy for label-free multiplexed protein microarray assay

https://doi.org/10.1016/j.bios.2009.06.028Get rights and content

Abstract

Quantitative measurement of affinities and kinetics of various biomolecular interactions such as protein–protein, protein–DNA and receptor–ligand is central to our understanding of basic molecular and cellular functions and is useful for therapeutic evaluation. Here, we describe a laser-scanning quantitative imaging method, referred to as spectral-domain optical coherence phase microscopy, as an optical platform for label-free detection of biomolecular interactions. The instrument is based on a confocal interferometric microscope that enables depth-resolved quantitative phase measurements on sensor surface with high spatial resolution and phase stability. We demonstrate picogram per square millimeter surface mass sensitivity, and show its sensing capability by presenting static and dynamic detection of multiplexed protein microarray as immobilized antigens capture their corresponding antibodies.

Introduction

Sensitive and selective methods to detect molecular affinity and kinetics are important tools in molecular and cell biology, medicine, and environmental monitoring. For instance, knowledge of multitudes of biomolecular interactions is essential to understanding signal transduction pathways of cells (Cooper and Hausman, 2006). Medicine has an obvious need for highly sensitive detection methods for various molecular disease signatures and viruses, and early detection of chemicals and pathogens (e.g., anthrax) that could trigger corrective action is also of significance in environmental applications.

The vast majority of sensing schemes typically employ secondary agents conjugated to captured molecules, which are subsequently detected with either fluorescent or enzyme-linked reagents. These methods have been widely utilized and successful for the detection of nucleic acid interactions in microarrays (Heller, 2002), since the structure and reactivity of nucleic acids are relatively uniform and minimally influenced by the secondary agents. However, the structure and reactivity of proteins are much more complex and diverse than those of DNA, and the attachment of the secondary agents to proteins is likely to alter kinetic characteristics of the proteins to other molecules (MacBeath, 2002). The efficiency of labeling may also vary from protein to protein, making it difficult to realize high-throughput protein assay (Mitchell, 2002). Noting these issues, there is a rising need for label-free techniques that enable direct monitoring of biomolecular interactions without secondary agents.

Various types of label-free optical biosensors have been investigated, which include sensors based on surface plasmon resonance (SPR) (Homola, 2006), reflectometric interference spectroscopy (Birkert et al., 2002), ring micro-resonators (Armani et al., 2007, Vollmer and Arnold, 2008), and interferometry (Bornhop et al., 2007, Yalcin et al., 2006). Among these detection methods, interferometric measurements can potentially provide high sensitivity to phase change, achieving better than 10−10 rad resolution (Ando et al., 2001). Mach–Zehnder interferometers have been integrated with SPR for differential phase measurements (Wu et al., 2004), and Young interferometers were implemented with optical waveguides of which surfaces were activated with probe molecules (Ymeti et al., 2007). Common-path in-line shearing interferometer demonstrated ∼pg/mm2 level surface mass sensitivity (Zhao et al., 2008). Recently, spectral reflectance imaging biosensor (SRIB) has demonstrated high-throughput label-free protein assay based on the change of interference signature from reflected light upon the protein absorption (Özkumur et al., 2008).

Spectral-domain optical coherence phase microscopy (SD-OCPM) (Joo et al., 2005, Joo et al., 2007) is a quantitative phase imaging modality capable of generating depth-resolved amplitude and quantitative phase images of transparent specimens. Based on a common-path low-coherence interferometer, it has a picometer-level optical path-length sensitivity, and has been employed for quantitative visualization of structures and dynamics of cellular specimens without any exogenous contrast agents. Unlike other quantitative phase imaging techniques based on the transmitted light through a specimen (Choi et al., 2007, Popescu et al., 2004), SD-OCPM operates in reflection and allows simultaneous phase measurements on multiple interference signals of interest along the optical axis.

Here, we describe the first, to our knowledge, demonstration of the utility of a quantitative phase microscope, or SD-OCPM as a label-free screening tool for multiplexed protein microarray. Interference spectrum produced by the superposition of the reflected light from the sensor chips is measured and converted into complex-valued depth profile. We measure the shift of the interference signal of interest to quantify the molecular absorption and desorption on the sensor surface. We demonstrate picogram per square millimeter protein mass sensitivity, and present its detection capability through dynamic binding measurements of multiple analytes on immobilized probes. The advantages and the aspects for the improvement will also be discussed.

Section snippets

SD-OCPM principle of operation

The basic principle of SD-OCPM is identical to that of spectral-domain optical coherence tomography (SD-OCT), as detailed elsewhere (Fercher et al., 1995, Nassif et al., 2003). Briefly, SD-OCT is based on a low-coherence spectral interferometer in which interference of reference and measurement light is spectrally dispersed, detected, and converted into path-length resolved amplitude and phase distribution of a specimen.

For optical path-length differences between reference and measurement

SD-OCPM performance

We first quantified performance of SD-OCPM by imaging a SiO2 etch pattern array designed and fabricated to model a protein array chip. In a typical biochip assay, the analytes bind to the ligands on sensor surface, and the average signal change over the multiple activated sites is measured to detect binding events.

To mimic this scenario, we fabricated a ∼10 μm thick SiO2 substrate into which a 5 × 5 square pattern was etched using standard photolithography and wet etching techniques. In practice,

Discussion

Based on a short coherence length and high phase stability provided by a common-path low-coherence interferometer, SD-OCPM measures the phase delay of the surface of interest without the effect of other surfaces and solution temperature/concentration fluctuations. This feature is highly attractive in sensor applications, since refractive index change in bulk solutions due to a temperature drift is a significant noise factor in other surface-sensitive sensor methods based on SPR and evanescent

Conclusion

We presented SD-OCPM as an optical method for label-free, multiplexed assay of protein microarray. We demonstrated picometer-level thickness sensitivity of SD-OCPM, and employed the method to measure specific binding of streptavidin to bBSA spots. SD-OCPM was further utilized to detect dynamic molecular interactions of multi-analytes in a fluidic format with better than ∼7 pg/mm2 mass sensitivity per spot, and ∼5 pg/mm2 by averaging over 5 spots.

As a functional derivative of SD-OCT, SD-OCPM has

Acknowledgements

This work was supported by grants from National Institute of Health (R01 RR19768, EY14975 to J.F.dB), the U.S. Department of Defense (F4 9620-01-1-0014 to M.S.U.), Army Research Laboratory (W911NF-06-2-0040 to M.S.U.), and the Center for Integration of Medicine and Innovative Technology. The authors are grateful to Drs. Ki Hean Kim and Conor Evans for their contributions to the system setup. C.J. would like to thank the support through Wellman Graduate Fellowship and Hatsopoulous Innovation

References (30)

  • A.F. Fercher et al.

    Optics Communications

    (1995)
  • M. Ando et al.

    Physics Review Letters

    (2001)
  • A.M. Armani et al.

    Science

    (2007)
  • O. Birkert et al.

    Analytical Chemistry

    (2002)
  • D.J. Bornhop et al.

    Science

    (2007)
  • W. Choi et al.

    Nature Methods

    (2007)
  • G.M. Cooper et al.

    The Cell: A Molecular Approach

    (2006)
  • A.F. Fercher et al.

    Reports on Progress in Physics

    (2003)
  • T. Gao et al.

    Analytical Chemistry

    (2006)
  • G. Gauglitz

    Review of Scientific Instruments

    (2005)
  • M.J. Heller

    Annual Review of Biomedical Engineering

    (2002)
  • J. Homola

    Surface Plasmon Resonance Based Sensors

    (2006)
  • C. Joo et al.

    Optics Letters

    (2005)
  • C. Joo et al.

    Optics Letters

    (2007)
  • G. MacBeath

    Nature Genetics

    (2002)
  • View full text