Real-time surface plasmon resonance imaging measurements for the multiplexed determination of protein adsorption/desorption kinetics and surface enzymatic reactions on peptide microarrays

The kinetics of protein adsorption/desorption onto peptide microarrays was studied using real-time surface plasmon resonance (SPR) imaging. S protein binding interactions were examined using an array composed of five different peptides: N terminal and C terminal immobilized wild-type S peptide (S1 and S2), an alternate binding sequence derived by phage display (LB2), an NVOC-protected S peptide, and a FLAG peptide control sequence (F). Kinetic measurements of the S protein-S1 peptide interaction were analyzed to determine a desorption rate constant (k(d)) of 1.1 (+/-0.08) x 10(-2) s(-1), an adsorption rate constant (k(a)) of 1.9 (+/-0.05) x 10(5) M(-1) s(-1), and an equilibrium adsorption constant (K(Ads)) of 1.7 (+/-0.08) x 10(7) M(-1). SPR imaging equilibrium measurements of S protein to S1 peptide were performed to independently confirm the kinetically determined value of K(Ads). Rate constants for the S2 and LB2 peptides on the array were measured as follows: 1.6 (+/-0.04) x 10(5) M(-1) s(-1) (k(a)) and 1.1 (+/-0.07) x 10(-2) s(-1) (k(d)) for S2, 1.2 (+/-0.05) x 10(5) M(-1) s(-1) (k(a)) and 1.1 (+/-0.03) x 10(-2) s(-1) (k(d)) for LB2. In addition to S protein adsorption/desorption, real-time SPR imaging of peptide arrays was applied to study the surface enzymatic activities of the protease factor Xa. Enzymatic cleavage of the substrate peptide (P1) was shown to follow first-order kinetics and proceed at a rate 10 times faster than that of the mutant peptide (P2), with cleavage velocities of 5.6 (+/-0.3) x 10(-4) s(-1) for P1 and 5.7 (+/-0.3) x 10(-5) s(-1) for P2.     

Surface plasmon resonance imaging measurements of DNA and RNA hybridization adsorption onto DNA microarrays

Surface plasmon resonance (SPR) imaging is a surface-sensitive spectroscopic technique for measuring interactions between unlabeled biological molecules with arrays of surface-bound species. In this paper, SPR imaging is used to quantitatively detect the hybridization adsorption of short (18-base) unlabeled DNA oligonucleotides at low concentration, as well as, for the first time, the hybridization adsorption of unlabeled RNA oligonucleotides and larger 16S ribosomal RNA (rRNA) isolated from the microbe Escherichia coli onto a DNA array. For the hybridization adsorption of both DNA and RNA oligonucleotides, a detection limit of 10 nM is reported; for large (1,500-base) 16S rRNA molecules, concentrations as low as 2 nM are detected. The covalent attachment of thiol-DNA probes to the gold surface leads to high surface probe density (10(12) molecules/cm2) and excellent probe stability that enables more than 25 cycles of hybridization and denaturing without loss in signal or specificity. Fresnel calculations are used to show that changes in percent reflectivity as measured by SPR imaging are linear with respect to surface coverage of adsorbed DNA oligonucleotides. Data from SPR imaging is used to construct a quantitative adsorption isotherm of the hybridization adsorption on a surface. DNA and RNA 18-mer oligonucleotide hybridization adsorption is found to follow a Langmuir isotherm with an adsorption coefficient of 1.8 x 10(7) M(-1).     

Nonregeneration protocol for surface plasmon resonance: study of high-affinity interaction with high-density biosensors

Surface plasmon resonance (SPR) has been used in determining kinetics and thermodynamics of biological interaction in the past decades. One difficulty encountered in this technology is the need for a proper regeneration, which means the removal of analytes from the bound complexes to regenerate the activity of the ligands. Regeneration is not always practical since the harsh regeneration reagents may destroy the bioactivity of the ligands. It is even more difficult for complexes with high affinity constants. In this paper, we report a nonregeneration protocol for SPR techniques in which subsequent ligand/analyte interactions can be measured without regeneration; thus ligand biological activity could be retained. Kinetics, binding models, and mathematics of this protocol are discussed in detail using rabbit IgG as the analyte and engineered recombinant antibody A10B single-chain fragment variables (scFv) as the ligand. The affinity constant of rabbit IgG binding with A10B scFv measured by using a nonregeneration protocol was (2.5 +/- 0.2) x 10(7) M(-1), which was comparable with the value determined with a conventional regeneration SPR method ((2.2 +/- 1.5) x 10(7) M(-1)) and quartz crystal microbalance (1.9 x 10(7) M(-1)). A paradigm of streptavidin-biotin binding was analyzed to validate this protocol. The affinity constant for each binding subunit of streptavidin to the immobilized biotin was determined to be (7.3 +/- 0.2) x 10(6) M(-1), which was comparable with the solution-based value of 2 x 10(7) M(-1). The nonregeneration protocol requires a relatively high ligand density on the biosensor surface so that more data points can be obtained before surface saturation. The small size of scFv enables them to be constructed in the biosensors for such purpose.     

Development of surface plasmon resonance mass spectrometry array platform

Protein microarrays are the format of choice for high-throughput, high-content protein interaction analysis. In most of the array formats, reporter molecules are used in multistep detection of the protein interactions. Among the few existing label-free detection approaches, surface plasmon resonance (SPR) and mass spectrometry (MS) stand out as most promising for utilization in protein microarrays, albeit both have been used only sporadically for high-content protein arrays. Shown here for the first time is the combination of SPR and MS detection on a single high-content protein microarray. Antibodies to five human plasma proteins were arrayed in a 10 x 10 spot arrangement on a chemically activated gold-coated glass chip. Binding of proteins to their corresponding antibodies was monitored via SPR imaging across the entire surface of the chip. Following protein affinity retrieval, the chip was overlaid with MALDI matrix and MS analyzed, producing protein-specific mass spectra from distinct spots on the array. The SPR-MS dual detection is well suited for high-content protein microarrays and comprehensive protein analysis-from quantitative assessment of the protein concentration to detection of structural protein variants arising from genetic variations and postexpression processing.     

Fabrication of histidine-tagged fusion protein arrays for surface plasmon resonance imaging studies of protein-protein and protein-DNA interactions

The creation and characterization of histidine-tagged fusion protein arrays using nitrilotriacetic acid (NTA) capture probes on gold thin films for the study of protein-protein and protein-DNA interactions is described. Self-assembled monolayers of 11-mercaptoundecylamine were reacted with the heterobifunctional linker N-succinimidyl S-acetylthiopropionate (SATP) to create reactive sulfhydryl-terminated surfaces. NTA capture agents were immobilized by reacting maleimide-NTA molecules with the sulfhydryl surface. The SATP and NTA attachment chemistry was confirmed with Fourier transform infrared reflection absorption spectroscopy. Oriented protein arrays were fabricated using a two-step process: (i) patterned NTA monolayers were first formed through a single serpentine poly(dimethylsiloxane) microchannel; (ii) a second set of parallel microchannels was then used to immobilize multiple His-tagged proteins onto this pattern at discrete locations. SPR imaging measurements were employed to characterize the immobilization and specificity of His-tagged fusion proteins to the NTA surface. SPR imaging measurements were also used with the His-tagged fusion protein arrays to study multiple antibody-antigen binding interactions and to monitor the sequence-specific interaction of double-stranded DNA with TATA box-binding protein. In addition, His-tagged fusion protein arrays created on gold surfaces were also used to monitor antibody binding with fluorescence microscopy in a sandwich assay format.     

Microspotting streptavidin and double-stranded DNA arrays on gold for high-throughput studies of protein-DNA interactions by surface plasmon resonance microscopy

We present two strategies for microspotting 10 x 12 arrays of double-stranded DNAs (dsDNAs) onto a gold-coated glass slide for high-throughput studies of protein-DNA interactions by surface plasmon resonance (SPR) microscopy. Both methods use streptavidin (SA) as a linker layer between a biotin-containing mixed self-assembled monolayer (SAM) and biotinylated dsDNAs to produce arrays with high packing density. The primary mixed SAM is produced from biotin- and oligo(ethylene glycol)-terminated thiols bonded as thiolates onto the gold surface. In the first method, a robotic microspotter is used to deliver nanoliter droplets of dsDNA solution onto a uniform layer of this SA ( approximately 2 x 10(12) SA/cm(2)). SPR microscopy shows a density of (5-6) x 10(11) dsDNA/cm(2) (0.2-0.3 dsDNA/SA) in the array elements. The second method uses instead a microspotted array of this SA linker layer, onto which the microspots of dsDNA are added with spatial registry. SPR microscopy before addition of the dsDNA shows a SA coverage of 2 x 10(12) SA/cm(2) within the spots and a dsDNA density of 8.5 +/- 3.5 x 10(11) dsDNA/cm(2) (0.3-0.7 dsDNA/SA, depending on the length of dsDNA) after dsDNA spotting. We demonstrate the ability to simultaneously monitor protein binding with the SPR microscope in many 200-microm spots with 1-s time resolution and sensitivity to <1 pg of protein.     

Label-free reading of microarray-based immunoassays with surface plasmon resonance imaging

A simple method is presented for patterning of protein antigens at a gold surface for use in surface plasmon resonance (SPR) imaging experiments. Microfluidic devices fabricated from poly(dimethylsiloxane) were used to flow various fluids over a gold substrate in spatially defined channels. This technique was used to pattern the surface chemistry of the gold as well as to adsorb antigens from solution to the modified substrates. The resulting antigen arrays were probed with complementary antibodies in order to demonstrate the effectiveness of the patterning for antibody capture experiments. SPR imaging was used to aid in the optimization of array fabrication and to observe the interactions of unlabeled antibodies with these microarrays. This work presents a means of fabricating microarrays with controlled surface density of antigens. SPR imaging provides both quantitative and qualitative evaluation of antibody binding in a label free format.     

High-throughput immunophenotyping by surface plasmon resonance imaging

Cell binding assays on antibody arrays permit the rapid immunophenotyping of living cells. The throughput of the analysis, however, is still limited due to our inability to perform parallel and quantitative detection of cells captured on the array. To address this limitation, we employed here an imaging technique based on surface plasmon resonance (SPR). SPR has been frequently used to monitor capture of proteins on antibody microarrays, while few cases were reported for capture of cells. Antibody arrays were prepared through the photopatterning of an alkanethiol monolayer on a gold-evaporated glass plate and the subsequent immobilization of various antibodies onto 4-9 separate spots created by photopatterning. A glass slip was mounted onto the array with a thin spacer to construct a parallel-plate chamber. Leukemia cells were injected into the chamber to conduct a binding assay, while refractive index changes at the vicinity of the array surface were monitored by SPR imaging. We observed that SPR signals were intensified on specific antibody spots but not on nonspecific spots. Confocal laser scanning microscopy revealed that the observed SPR signals were attributed to cell deformations caused by multivalent interactions with immobilized antibody, which effectively elevated the refractive index of a medium phase within an evanescent field. This effect could be suitably utilized to monitor quantitatively cell binding to multiple spots from a heterogeneous cell population.     

Carbohydrate-protein interactions by "clicked" carbohydrate self-assembled monolayers

A Huisgen 1,3-dipolar cycloaddition "click chemistry" was employed to immobilize azido sugars (mannose, lactose, alpha-Gal) to fabricate carbohydrate self-assembled monolayers (SAMs) on gold. This fabrication was based on preformed SAM templates incorporated with alkyne terminal groups, which could further anchor the azido sugars to form well-packed, stable, and rigid sugar SAMs. The clicked mannose, lactose, and alpha-Gal trisaccharide SAMs were used in the analysis of specific carbohydrate-protein interactions (i.e., mannose-Con A; ECL-lactose, alpha-Gal-anti-Gal). The apparent affinity constant of Con A binding to mannose was (8.7 +/- 2.8) x 10(5) and (3.9 +/- 0.2) x 10(6) M(-1) measured by QCM and SPR, respectively. The apparent affinity constants of lactose binding with ECL and alpha-Gal binding with polyclonal anti-Gal antibody were determined to be (4.6 +/- 2.4) x 10(6) and (6.7 +/- 3.3) x 10(6) M(-1), respectively by QCM. SPR, QCM, AFM, and electrochemistry studies confirmed that the carbohydrate SAM sensors maintained the specificity to their corresponding lectins and nonspecific adsorption on the clicked carbohydrate surface was negligible. This study showed that the clicked carbohydrate SAMs in concert with nonlabel QCM or SPR offered a potent platform for high-throughput characterization of carbohydrate-protein interactions. Such a combination should complement other methods such as ITC and ELISA in a favorable manner and provide insightful knowledge for the corresponding complex glycobiological processes.     

Long-range surface plasmon resonance imaging for bioaffinity sensors

A novel bioaffinity sensor based on surface plasmon resonance (SPR) imaging measurements of a multiple-layered structure that supports the generation of long-range surface plasmons (LRSPs) at the water-metal interface is reported. LRSPs possess longer surface propagation lengths, higher electric field strengths, and sharper angular resonance curves than conventional surface plasmons. LRSPR imaging is a version of SPR imaging that requires a symmetric dielectric arrangement around the gold thin film. This arrangement is created using an SF10 prism/Cytop/gold/water multilayer film structure where Cytop is an amorphous fluoropolymer with a refractive index very close to that of water. LRSPR imaging experiments are performed at a fixed incident angle and lead to an enhanced response for the detection of surface binding interactions. As an example, the hybridization adsorption of a 16-mer single-stranded DNA (ssDNA) onto a two-component ssDNA array was monitored with LRSPR imaging. The ssDNA array was created using a new fabrication technology appropriate for the LRSPR multilayers.     

Nanohole-based surface plasmon resonance instruments with improved spectral resolution quantify a broad range of antibody-ligand binding kinetics

We demonstrate an affordable low-noise surface plasmon resonance (SPR) instrument based on extraordinary optical transmission (EOT) in metallic nanohole arrays and quantify a broad range of antibody-ligand binding kinetics with equilibrium dissociation constants ranging from 200 pM to 40 nM. This nanohole-based SPR instrument is straightforward to construct, align, and operate, since it is built around a standard microscope and a portable fiber-optic spectrometer. The measured refractive index resolution of this platform is 3.1 × 10(-6) without on-chip cooling, which is among the lowest reported for SPR sensors based on EOT. This is accomplished via rapid full-spectrum acquisition in 10 ms followed by frame averaging of the EOT spectra, which is made possible by the production of template-stripped gold nanohole arrays with homogeneous optical properties over centimeter-sized areas. Sequential SPR measurements are performed using a 12-channel microfluidic flow cell after optimizing surface modification protocols and antibody injection conditions to minimize mass-transport artifacts. The immobilization of a model ligand, the protective antigen of anthrax on the gold surface, is monitored in real-time with a signal-to-noise ratio of ~860. Subsequently, real-time binding kinetic curves were measured quantitatively between the antigen and a panel of small, 25 kDa single-chain antibodies at concentrations down to 1 nM. These results indicate that nanohole-based SPR instruments have potential for quantitative antibody screening and as a general-purpose platform for integrating SPR sensors with other bioanalytical tools.     

Etched glass microarrays with differential resonance for enhanced contrast and sensitivity of surface plasmon resonance imaging analysis

We report the fabrication and characterization of gold-coated etched glass array substrates for surface plasmon resonance imaging (SPRi) analysis with significantly enhanced performance, in particular image contrast and sensitivity. The etching of the glass substrate induces a variation in the resonance condition and thus in the resonance angle between the etched wells and the surrounding area, leading to the isolation of the array spot resonance with a significant reduction of the background signal. FDTD simulations show arrays with large spots and minimal spot-to-spot spacing yield ideal differential resonance conditions, which are verified by experimental results. Simulations also indicate the etched well structure exhibits enhanced SPR electric field intensity by 3-fold as compared to standard planar gold chips. Changes in the bulk sensitivity of the etched arrays have been obtained at the 10(-4) RIU level based on image intensity difference. The strong image contrast allows for improved microarray imaging analysis with easily distinguished signals from background resonance. The etched array chips are demonstrated for SPRi detection of bacterial toxins through the coating of an ultrathin SiO(2) film for direct vesicle fusion that establishes a supported membrane-based biosensing interface. Protein detection with cholera toxin (CT) at 5 nM is obtained, making this chip one of the most sensitive SPR imaging substrates ever reported without a postbinding amplification scheme. Furthermore, the surface can be regenerated by Triton X-100 for repeated cycles of membrane formation, protein binding, and biomolecular removal. The reusability and enhanced performance of the etched glass array chips should find a broad range of applications, opening up new avenues for high-throughput SPR imaging detection with convenience and marked surface sensitivity.     

Detection and quantification of on-chip phosphorylated peptides by surface plasmon resonance imaging techniques using a phosphate capture molecule

We describe herein a detection and quantification system for on-chip phosphorylation of peptides by surface plasmon resonance (SPR) imaging techniques using a newly synthesized phosphate capture molecule (i.e., biotinylated zinc(II) complex). The biotinylated compound is a dinuclear zinc(II) complex that is suitable for accessing phosphate anions as a bridging ligand on the two zinc(II) ions. The compound was exposed on the peptide array and detected with streptavidin (SA) via a biotin-SA interaction by SPR imaging. In the conventional method using antibody, both anti-phosphoserine and anti-phosphotyrosine antibodies were required for phosphoserine and phosphotyrosine detection, respectively. Detection of the phosphate group by the zinc(II) complex, however, was independent of the phosphorylated amino acid residues. The calibration curve for the phosphorylation ratios was established with a calibration chip, on which phosphoserine-containing peptide probes were immobilized. The peptide probes, which were phosphorylated on the surface by protein kinase A, were detected and quantified by SPR imaging using the zinc(II) complex, SA, and anti-SA antibody. The reaction rate and the kinetics of on-chip phosphorylation were also evaluated with the peptide array. The phosphorylation ratio was saturated at approximately 20% in 2 h in this study.     

Differential surface plasmon resonance imaging for high-throughput bioanalyses

A new imaging technique for high-throughput surface plasmon resonance (SPR) measurements is described. It is the application of a CCD camera for simultaneous processing of two images at two different wavelengths provided by two laser diodes. The two lasers are brought to resonance by tuning of the angle of incidence so that the detection power and the dynamic range are optimized for the wavelength pair selected. Applying a special differential processing of the two images, SPR measurements can be performed near the shot noise limit taking into account the number of CCD pixels involved. It is shown that the detection limit of imaging methods can be improved significantly if the working point is set near to the reflection minimum instead of choosing the angle with the steepest slope of the reflection curve. The technique is demonstrated by simultaneous measurement of hybridization reactions of three different types of thiolated oligonucleotides in 30 small areas set by a commercial spotter. A noise level of 1.5 x 10(-6) refractive index units (RIU) was obtained for single, 500 x 500 microm2 reaction areas. The noise level was about 6 x 10(-7) RIU when five areas were taken into account. The present arrangement and the particular spotter applied would allow simultaneous measurements of up to 400 binding reactions with a noise level of about 1.5 x 10(-6) RIU.     

Tuning the autophagy-inducing activity of lanthanide-based nanocrystals through specific surface-coating peptides

The induction of autophagy on exposure of cells to a variety of nanoparticles represents both a safety concern and an application niche for engineered nanomaterials. Here, we show that a short synthetic peptide, RE-1, identified by means of phage display, binds to lanthanide (LN) oxide and upconversion nanocrystals (UCN), forms a stable coating layer on the nanoparticles' surface, and effectively abrogates their autophagy-inducing activity. Furthermore, RE-1 peptide variants exhibit a differentially reduced binding capability, and correspondingly, a varied ability to reduce the autophagic response. We also show that the addition of an arginine-glycine-aspartic acid (RGD) motif to RE-1 enhances autophagy for LN UCN through the interaction with integrins. RE-1 and its variants provide a versatile tool for tuning material-cell interactions to achieve the desired level of autophagy, and may prove useful for the various diagnostic and therapeutic applications of LN-based nanomaterials and nanodevices.     

Analysis of biomolecular interactions using a miniaturized surface plasmon resonance sensor

A commercially available miniaturized surface plasmon resonance sensor has been investigated for its applicability to biological interaction analysis. The sensor was found to exhibit excellent repeatability and linearity for high-refractive index solutions and good reproducibility for the binding of proteins. Its detection limit for the monoclonal antibody M1 was found to be 2.1 fmol, which corresponds to a surface concentration of 21 pg/mm2. Simple surface immobilization procedures relying on biotin/avidin or glycoprotein/lectin chemistry have been explored. Equilibrium dissociation constants for the binding of the FLAG peptide to its monoclonal antibody (M1) and for the binding of concanavalin A to a glycoprotein have been determined. The close agreement of these measurements with values obtained by surface fluorescence microscopy and fluorescence correlation spectroscopy helps to validate the use of this device. Thus, this sensor shows promise as an inexpensive, portable, and accurate tool for bioanalytical applications in laboratory and clinical settings.     

Label-free detection of proteins in crude cell lysate with antibody arrays by a surface plasmon resonance imaging technique

We established a label-free method of measuring proteins in crude cell lysate using antibody arrays and surface plasmon resonance (SPR) imaging. The refractivity of the running buffer was adjusted with that of the lysate to overcome the bulk effect. The chemistries of the fabricated arrays were investigated to reduce nonspecific adsorption on the array surface. We found that the hydrophilicity of the poly(ethylene glycol) moiety and lower electrostatic charge on the surface provided a specific measurement of antigen-antibody interaction. We validated the system by measuring the expression of eight proteins in the mouse brain and comparing the results to those by conventional Western blotting. The detection limit of the antibody array was approximately 30 ng/mL in crude cell lysate, on the same order as that of previous SPR research. The system enabled quick, label-free, and high-throughput analysis of abundant proteins with minimal sample volume ( approximately 200 muL). It is expected that our SPR antibody array will be applicable for direct protein expression profiling of cell lysate, as well as for cell phenotyping, food analysis, discovery of new biomarkers, and immunological disease diagnostics.     

Surface plasmon resonance imaging detection of silver nanoparticle-tagged immunoglobulin

The detection sensitivity of silver nanoparticle (AgNP)-tagged goat immunoglobulin G (gIgG) microarrays was investigated by studying surface plasmon resonance (SPR) images captured in the visible wavelength range with the help of a Kretchmann-configured optical coupling set-up. The functionalization of anti-gIgG molecules on the AgNP surface was studied using transmission electron microscopy, photon correlation measurements and UV–visible absorption spectroscopy. A value of 1.3 × 10⁷ M⁻¹ was obtained for the antibody–antigen binding constant by monitoring the binding events at a particular resonance wavelength. The detection limit of this SPR imaging instrument is 6.66 nM of gIgG achieved through signal enhancement by a factor of larger than 4 owing to nanoparticle tagging with the antibody.     

Biomaterials functionalization using a novel peptide that selectively binds to a conducting polymer

The goal in biomaterial surface modification is to retain a material's bulk properties while modifying only its surface to possess desired recognition and specificity. Here we develop a unique strategy for surface functionalization of an electrically conductive polymer, chlorine-doped polypyrrole (PPyCl), which has been widely researched for various electronic and biomedical applications. An M13 bacteriophage library was used to screen 10(9) different 12-mer peptide inserts against PPyCl. A binding phage (phiT59) was isolated, and its binding stability and specificity to PPyCl was assessed using fluorescence microscopy and titer count analysis. The relative binding strength and mechanism of the corresponding 12-mer peptide and its variants was studied using atomic force microscopy and fluorescamine assays. Further, the T59 peptide was joined to a cell adhesive sequence and used to promote cell attachment on PPyCl. This strategy can be extended to immobilize a variety of molecules to PPyCl for numerous applications. In addition, phage display can be applied to other polymers to develop bioactive materials without altering their bulk properties.