Elsevier

Biomaterials

Volume 28, Issue 15, May 2007, Pages 2380-2392
Biomaterials

Review
SPR microscopy and its applications to high-throughput analyses of biomolecular binding events and their kinetics

https://doi.org/10.1016/j.biomaterials.2007.01.047Get rights and content

Abstract

Surface plasmon resonance (SPR) sensing has long been used to study biomolecular binding events and their kinetics in a label-free way. This approach has recently been extended to SPR microscopy, which is an ideal tool for probing large microarrays of biomolecules for their binding interactions with various partners and the kinetics of such binding. Commercial SPR microscopes now make it possible to simultaneously monitor binding kinetics on >1300 spots within a protein microarray with a detection limit of ∼0.3 ng/cm2, or <50 fg per spot (<1 million protein molecules) with a time resolution of 1 s, and spot-to-spot reproducibility within a few percent. Such instruments should be capable of high-throughput kinetic studies of the binding of small (∼200 Da) ligands onto large protein microarrays. The method is label free and uses orders of magnitude less of the precious biomolecules than standard SPR sensing. It also gives the absolute bound amount and binding stoichiometry.

Introduction

Surface plasmon resonance (SPR) spectroscopy is a popular surface analysis method based on changes in the optical reflectivity of a thin metal film (typically gold) when species adsorb or bind to its surface or to any material coated onto its surface. Specifically, it detects with high sensitivity (<10−6) and fast time resolution (∼1 s) changes in refractive index of any surface coating or solution near the SPR-active metal surface [1], [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13]. By “SPR spectroscopy”, we refer here to measurements of reflectivity both versus wavelength at fixed angle and versus angle at fixed wavelength (also called SPR reflectometry). Functionalization of the metal surface with specific binding sites with bioaffinity creates a biosensor that can detect biomolecular interactions in real time with no labeling requirements. Therefore, SPR spectroscopy has become a common tool in biochemistry and bioanalytical chemistry, especially for determining the on- and off-rates and equilibrium binding constants which describe the interactions between proteins, DNAs or RNAs and a wide variety of other biomolecules or ligands, or for investigating the effects of various cofactors or inhibitors on these binding constants. Many commercial instruments are available for these applications, the most common of which is the Biacore system [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30]. Our group has studied the kinetics of protein–ligand, protein–dsDNA and protein–vesicle interactions using a home-built SPR spectrometer [8], [9], [10], [11].

More recently, SPR microscopy (SPRM), also referred to as “SPR imaging” (SPRI), has started to be used for the same types of measurements, but with high spatial resolution. Since the measurements are done simultaneously over the entire area radiated by the light or imaged onto the detector array (typically a charge-coupled device (CCD) array), combining SPRM with patterned microarrays of biomolecules allows for very high throughput analyses of biomolecular binding. Thus, SPRM has been used to measure the binding of DNAs and RNAs to DNA arrays, of DNA-binding proteins to dsDNA arrays, of proteins to protein and peptide arrays, and even of small ligands to protein arrays. Just like SPR spectroscopy, SPRM can be used for determining the on- and off-rates and equilibrium binding constants of all these types of interactions, only now in high throughput (>1000 interactions simultaneously). Since the amount of biomaterial needed to make one 100 μm spot on an SPR active surface is tiny, and since the same solution can be used to monitor >1000 spots simultaneously, this provides not only a huge savings in time, but also a tremendous cost savings for the precious biomolecules used for these assays. More importantly, since the buffer and temperature and many other variables are exactly the same for each spot in such an analysis, this approach offers an improvement in measurement reliability relative to 1000 independent measurements by the simpler SPR spectroscopy. Table 1 summarizes some of the advantages of SPRM and SPRI for such analyses.

There are several disadvantages shared by both SPR and SPRM. Perhaps the most important is that, without signal amplification in any way, one requires a minimum of ∼0.1% of the surface receptors to be occupied to detect their presence. Thus, to achieve the advantages in Table 1, one requires a concentration of the biomolecular binding partner in solution that is at least Kd/1000, where Kd is the equilibrium dissociation constant for the interaction of interest. However, a number of methods have been developed for amplification that dramatically relax this limitation, as outlined below. Another disadvantage is that the metal surface must be functionalized with a bioreceptor in a way that avoids non-specific adsorption. Non-specific adsorption can lead to false signals and biosensor fouling. Finally, biomolecules immobilized onto SPR sensor surfaces do not always retain their native bioactivity and, with some immobilization schemes, only a small fraction of the immobilized biomolecules are active. This can effect the determination of binding stoichiometry (biomolecules bound per receptor). The stoichiometry can also be influenced by the proximity of other receptors in the adlayer, especially when the analyte is a large biopolymer [31].

Table 2 lists just a few of the potential applications of SPRM.

Here we will present first a brief review of SPR spectroscopy, and then present a review of SPRM and its applications in microarray-based biaffinity analyses. Because the basis for absolute quantitative analysis in SPRM is the same as that is SPR spectroscopy, we first will present a detailed description of the methods for absolute quantitative analysis in SPR spectroscopy, and explain how these are easily extended to SPRM.

Section snippets

Surface plasmon resonance (SPR) spectroscopy

Typically in SPR spectroscopy, a polarized monochromatic light beam is passed through a prism and its attached, gold-coated glass slide, and reflected off the thin gold coating, which is in contact with the liquid solution of interest (Fig. 1). Excitation of surface plasmons at the gold/solution interface results in nearly complete attenuation of the specularly reflected light intensity for incident angles very near the SPR angle, which depends on wavelength. It can be monitored by following

Quantitative SPR spectroscopy: absolute surface concentrations

We have proven that SPR spectroscopy measures an “effective index of refraction”, ηeff, which is a weighted average of the liquid solution in contact with the gold surface plus any coatings or adsorbed films on the gold surface [8]. We have shown that an SPR system can be calibrated easily with different solutions of known index of refraction (under conditions where the contribution to changes in ηeff are dominated by changes in the bulk liquid's refractive index and not by changes in any

SPR microscopy (SPRM) or “SPR imaging” (SPRI)

SPRM, also referred to as SPRI, provides the same type of quantitative data as obtained in biosensing with SPR spectroscopy (i.e., amount adsorbed versus time), but it has the very important added feature of monitoring adsorption with a spatial resolution down to ∼4 μm over a large area of a sensing surface [36], [41], [42], [43], [44], [45], [46], [47], [48].

In most SPR microscopes, an expanded and collimated, polarized and monochromatic light beam (often a He–Ne laser, but sometimes a

Quantitative SPR microscopy: absolute surface concentrations with spatial resolution

We now review the method we recently developed for the conversion of light intensities in SPRM (or SPRI) to absolute adsorbate coverages (mass or number of molecules per unit area) [36]. For this purpose, it is best first to set up the angle of light incidence (and detection) of the microscope so that it is operating in the “linear” response region, as defined in Fig. 4. In this range of angles, the change of light intensity is proportional to the change in ηeff. The SPR microscope is

Example applications

SPRM has been investigated as a promising tool for simultaneously monitoring binding events across functionalized surface microarrays. Early work in this arena was pioneered by the research groups of Wolfgang Knoll [41], [42], [45], [68], [71], [72], [73] and Robert Corn [46], [53], [74], [75]. By creating 1-D or 2-D arrays of binding sites on an SPR-active surfaces, DNA–DNA binding, RNA–DNA binding, protein–DNA binding and other bioploymer interactions have been studied in a parallel fashion

Acknowledgements

This work was made possible by funding from the Institute for Systems Biology, the University of Washington Center for Nanotechnology, the National Science Foundation, the Department of Energy Office of Basic Energy Sciences, and Lumera Corp. CTC thanks Dr. Ruedi Aebersold, Prof. Jennifer Shumaker-Parry, Dr. Hann Wen Guan, Dr. Pradip Rathod, Dr. Michael Gelb, Dr. Leroy Hood, Prof. Rob Corn and Prof. Wolfgang Knoll for helpful and enjoyable discussions over the years.

References (87)

  • P. Mistrik et al.

    BiaCore analysis of leptin–leptin receptor interaction: evidence for 1:1 stoichiometry

    Anal Biochem

    (2004)
  • D.G. Myszka

    Analysis of small-molecule interactions using Biacore S51 technology

    Anal Biochem

    (2004)
  • P.S. Katsamba et al.

    Kinetic analysis of a high-affinity antibody/antigen interaction performed by multiple Biacore users

    Anal Biochem

    (2006)
  • P. Safsten et al.

    Screening antibody–antigen interactions in parallel using Biacore A100

    Anal Biochem

    (2006)
  • P.G. Wu et al.

    Effect of ethidium on the torsion constant of linear and super-coiled DNA

    Biophys Chem

    (1991)
  • D. Piscevic et al.

    Oligonucleotide hybridization observed by surface-plasmon optical techniques

    Appl Surf Sci

    (1995)
  • M.J. O’Brien et al.

    A surface plasmon resonance array biosensor based on spectroscopic imaging

    Biosens Bioelectron

    (2001)
  • U. Fernandez et al.

    Surface-plasmon microscopy with grating couplers

    Opt Commun

    (1993)
  • D.K. Kambhampati et al.

    Surface-plasmon optical techniques

    Curr Opin Colloid Interface Sci

    (1999)
  • B. Liedberg et al.

    Biosensing with surface plasmon resonance—how it all started

    Biosens Bioelectron

    (1995)
  • A.M. Hutchinson

    Evanescent wave biosensors: real-time analysis of biomolecular interactions

    Mol Biotechnol

    (1995)
  • P.B. Garland

    Optical evanescent wave methods for the study of biomolecular interactions

    Quart Rev Biophys

    (1996)
  • A. Huber et al.

    The use of biosensor technology for the engineering of antibodies and enzymes

    J Mol Recog

    (1999)
  • S.Y. Rabbany et al.

    Optical immunosensors

    Crit Rev Biomed Eng

    (1994)
  • U. Jonnson et al.

    Real time biospecific interaction analysis: the integration of surface plasmon resonance detection, general biospecific interface, and microfluidics into one analytical system

  • L.S. Jung et al.

    Quantitative interpretation of the response of surface plasmon resonance sensors to adsorbed films

    Langmuir

    (1998)
  • L.S. Jung et al.

    Binding and dissociation kinetics of wild-type and mutant streptavidins on mixed biotin-containing alkylthiolate monolayers

    Langmuir

    (2000)
  • L.S. Jung et al.

    Quantification of tight binding to surface-immobilized phospholipid vesicles using surface plasmon resonance: binding constant of phospholipase A(2)

    J Am Chem Soc

    (2000)
  • L.S. Jung et al.

    Sticking probabilities in adsorption of alkanethiols from liquid ethanol solution onto gold

    J Phys Chem B

    (2000)
  • L.S. Jung et al.

    Sticking probabilities in adsorption from liquid solutions: alkylthiols on gold

    Phys Rev Lett

    (2000)
  • K.K. Jensen et al.

    Kinetics for hybridization of peptide nucleic acids (PNA) with DNA and RNA studied with the BIAcore technique

    Biochemistry

    (1997)
  • M.B. Medina et al.

    Real-time analysis of antibody binding interactions with immobilized E. coli O157:H7 cells using the BIAcore

    Biotechnol Tech

    (1997)
  • M. Malmqvist

    BIACORE: an affinity biosensor system for characterization of biomolecular interactions

    Biochem Soc Trans

    (1999)
  • R.L. Rich et al.

    BIACORE J: a new platform for routine biomolecular interaction analysis

    J Mol Recogn

    (2001)
  • A. Naslund et al.

    Rapid, reproducible screening of drug compound–target protein interactions using Biacore A100

    Nat Methods

    (2006)
  • Shumaker-Parry JS, Campbell CT, Stormo GD, Silbaq FS, Aebersold RH. Probing protein: DNA interactions using a uniform...
  • R.A. Innes et al.

    J Phys F: Met Phys

    (1987)
  • E.D. Palick

    Handbook of Optical Constants of Solids

    (1985)
  • S.H. Armstrong et al.

    J Am Chem Soc

    (1947)
  • J.S. Shumaker-Parry et al.

    Quantitative methods for spatially resolved adsorption/desorption measurements in real time by surface plasmon resonance microscopy

    Anal Chem

    (2004)
  • J.E. Darnell et al.

    Molecular cell biology

    (1990)
  • T.E. Leslie et al.

    Aqueous-solutions containing amino-acids and peptides. 20. Volumetric behaviour of some terminally substituted amino-acids and peptides at 298.15 K

    Biopolymers

    (1985)
  • Cited by (379)

    • Sensor principles and basic designs

      2023, Fundamentals of Sensor Technology: Principles and Novel Designs
    View all citing articles on Scopus
    View full text