Patterned resonance plasmonic microarrays for high-performance SPR imaging

We report a novel optical platform based on SPR generation and confinement inside a defined three-dimensional microwell geometry that leads to background resonance-free SPR images. The array shows an exceptionally high signal-to-noise ratio (S/N > 80) for imaging analysis and subnanometric thickness resolution. An angular sensitivity of 1°/0.01 RIU has been achieved and the signal to background ratio (S/B) improves to 20, 1 order of magnitude higher than that of the best literature results. The design proves effective for probing-supported lipid membrane arrays in real time with a thickness resolution of 0.24 nm and allows for imaging analysis of microfluidic circuits where resonant spots are separated by only one pixel (～7 μm). The high image quality and unique chip geometry open up new avenues for array screening and biomicrofluidics using SPRi detection.     

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.     

Development of air-stable, supported membrane arrays with photolithography for study of phosphoinositide-protein interactions using surface plasmon resonance imaging

We report the development of an air-stable, supported membrane array by use of photolithography for label-free detection of lipid-protein interactions. Phosphoinositides and their phosphorylated derivatives (PIPs) were studied for their binding properties to proteins with lipid microarray in combination with surface plasmon resonance imaging (SPRi). We have demonstrated a simple method to fabricate lipid arrays using photoresist and carried out a series of surface characterizations with SPRi, ac impedance, cyclic voltammetry, and fluorescence microscopy to validate the array quality and lipid bilayer formation. A number of lipid compositions have been tested for the robustness of resulting membranes when undergoing dehydration and rehydration procedures, and the 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine/poly(ethylene glycol)-phosphatidylethanolamine (DOPC+/PEG-PE) system stands out as the best performer that yields the recovery to within 2% of the original state according to SPR sensorgrams. Limits of detection on the dehydrated/rehydrated DOPC+/PEG-PE membranes were determined to be 33 nM for avidin binding to biotinylated lipids, 73.5 nM for cholera toxin to GM1, and 25 nM for PtdIns(4,5)P2-binding protein (P(4,5)BP) to PtdIns(4,5)P2 lipid, respectively. These results demonstrate the suitability and sensitivity of this membrane for constructing membrane arrays for SPRi analysis under ambient conditions. With the use of this addressable and functional lipid membrane array, the screening of specific lipid-protein interactions has been conducted. Strong and specific interactions between P(4,5)BP and PtdIns(4,5)P2/DOPC+/PEG-PE membrane were observed as expected, while cross reactions were spotted for P(4,5)BP/PtdIns(4)P and avidin/GM1 at varied degrees. The air-stable membrane array demonstrated here presents a simple, effective approach for constructing functional membrane surfaces for screening applications, which opens a new avenue for the label-free study of membrane proteins and other forms of lipid-membrane interactions.     

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.     

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.     

Integrated label-free protein detection and separation in real time using confined surface plasmon resonance imaging

We demonstrate a label-free protein detection and separation technology for real-time monitoring of proteins in micro/nanofluidic channels, confined surface plasmon resonance imaging (confined-SPRi). This was achieved by fabricating ultrathin fluidic channels (500 nm high, 500 microm wide) directly on top of a specialized SPRi sensor surface. In this way, SPRi is uniquely used to detect proteins deep into the fluidic channel while maintaining high lateral accuracy of separated products. The channel fluid and proteins were driven electrokinetically under an external electric field. For this to occur, the metallic SPR sensor (46 nm of Au on 2 nm of Cr) was segmented into an array of squares (each 200 microm x 200 microm in size and spaced 8 microm apart) and coated with 30 nm of CYTOP polymer. In this work, we track label-free protein separation in real time through a simple cross-junction fluidic device with an 8-mm separation channel length under 30 V/cm electric field strength.     

Investigation on an application of silver substrates for sensitive surface plasmon resonance imaging detection

A surface plasmon resonance (SPR) imaging biosensor based on silver substrates was investigated to demonstrate that silver could be used as a substrate material for sensitive detection of biomolecular interactions, despite its poor chemical stability. The calculation results showed that oxidation of silver film may lead to a decrease in the sensitivity due to a variation in SPR characteristics such as a broader curve width and shallower minimum reflectance at resonance. The effect of a change in the refractive index of target analytes on the sensitivity was also explored. In particular, it is noteworthy that Ag/Au bimetallic substrates with a thin gold protection layer to prevent oxidation of a silver film can provide a significant amplification of SPR imaging signals in comparison with conventional gold substrates.     

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.     

Designed biointerface using near-infrared quantum dots for ultrasensitive surface plasmon resonance imaging biosensors

The surface plasmon resonance imaging chip biointerface is fully designed using near-infrared (NIR) quantum dots (QDs) for the enhancement of surface plasmon resonance imaging (SPRi) signals in order to extend their application for medical diagnostics. The measured SPRi detection signal following the QD binding to the surface was amplified 25-fold for a 1 nM concentration of single-stranded DNA (ssDNA) and 50-fold for a 1 μg/mL concentration of prostate-specific antigen (PSA), a cancer biomarker, thus substantiating their wide potential to study interactions of a diverse set of small biomolecules. This significant enhancement is attributed to the QD's mass-loading effect and spontaneous emission coupling with propagating surface plasmons, which allowed the SPRi limit of detection to be reduced to 100 fM and 100 pg/mL for ssDNA and PSA, respectively. Furthermore, this study illustrates the potential of SPRi to be easily integrated with fluorescent imaging for advanced correlative surface-interaction analysis.     

Hybrid surface platform for the simultaneous detection of proteins and DNAs using a surface plasmon resonance imaging sensor

In this work, we present a novel surface and assay for the simultaneous detection of DNA and protein analytes on a surface plasmon resonance (SPR) imaging sensor. A mixed DNA/oligo (ethylene glycol) (OEG) self-assembled monolayer (SAM) is created using a microarrayer. Thiol-modified single-stranded DNA sequences are spotted onto a gold-coated glass substrate. Backfilling with an OEG-modified alkanethiol creates a protein-resistant surface background. Antibodies conjugated to complementary single-stranded DNA sequences are immobilized on the surface through DNA hybridization. By converting only part of the DNA array into a protein array, simultaneous detections of DNA and protein analytes are possible. A model system of two cDNA sequences and two human pregnancy hormones are used to demonstrate the assay. No cross-reactivity was observed between DNA or protein analytes and nontargeted immobilized cDNA sequence or antibodies. A response from a detection of a single analyte in a mixture of protein and DNA analytes corresponds well with that of a single-analyte solution.     

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.     

Wavelength-scanning surface plasmon resonance imaging

A new surface plasmon resonance (SPR) imaging technique was proposed. After measurements were conducted at varying wavelengths, the wavelength affording the minimum brightness (SPR wavelength) was determined at each pixel of the image. A two-dimensional map of the SPR wavelength could be converted to a thickness profile by use of a nonlinear calibration curve, which was obtained by Fresnel calculation. An array of protein thin layers on a gold film was evaluated in air to present the layers' surface structure in nanometer scale.     

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).     

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.     

Field-matter integral overlap to estimate the sensitivity of surface plasmon resonance biosensors

We have analyzed the effectiveness of field-matter integral overlap between target index distribution and local near-fields to assess detection sensitivity of surface plasmon resonance (SPR) biosensors. The correlation of the overlap with sensitivity was clear. An overlap integral defined with lateral electric field intensity produced the highest correlation due to tangential continuity across a boundary. Among the three detection scenarios considered, the correlation for localized SPR sensing was slightly lower than that of thin film-based detection and improved with an increased fill factor in the structure. The results will be useful to maximize the optical signature created by target interactions and to produce highest sensitivity of SPR detection to variations when target or field distribution is not uniform.     

Tunable surface plasmon resonance and strong SERS performances of Au opening-nanoshell ordered arrays

Au opening-nanoshell ordered arrays with tunable local surface plasmon resonance (SPR) property have been fabricated based on sputtering deposition onto monolayer colloidal crystal. The changes in local SPR peak for the arrays can be well tuned from visible to near-infrared region with decreasing of the spacing between two neighbor opening-nanoshells. It has been revealed that the changes of SPR peak originate from the electromagnetic coupling between two adjacent Au opening-nanoshells. This study is important to design and fabricate surface-enhanced Raman scattering substrates with high activity and practical application.     

Characterization and optimization of peptide arrays for the study of epitope-antibody interactions using surface plasmon resonance imaging

The characterization of peptide arrays on gold surfaces designed for the study of peptide-antibody interactions using surface plasmon resonance (SPR) imaging is described. A two-step process was used to prepare the peptide arrays: (i) a set of parallel microchannels was used to deliver chemical reagents to covalently attach peptide probes to the surface by a thiol-disulfide exchange reaction; (ii) a second microchannel with a wraparound design was used as a small-volume flow cell (5 microL) to introduce antibody solutions to the peptide surface. As a demonstration, the interactions of the FLAG epitope tag and monoclonal anti-FLAG M2 were monitored by SPR imaging using a peptide array. This peptide-antibody pair was studied because of its importance as a means to purify fusion proteins. The surface coverage of the FLAG peptide was precisely controlled by creating the peptide arrays on mixed monolayers of alkanethiols containing an amine-terminated surface and an inert alkanethiol. The mole fraction of peptide epitopes was also controlled by reacting solutions containing FLAG peptide and the non-interacting peptide HA or cysteine. By studying variants based on the FLAG binding motif, it was possible to distinguish peptides differing by a single amino acid substitution using SPR imaging. In addition, quantitative analysis of the signal was accomplished using the peptide array to simultaneously determine the binding constants of the antibody-peptide interactions for four peptides. The binding constant, K(ads), for the FLAG peptide was measured and found to be 1.5 x 10(8) M(-1) while variants made by the substitution of alanine for residues based on the binding motif had binding constants of 2.8 x 10(7), 5.0 x 10(6), and 2.0 x 10(6) M(-1).     

Phase detection sensitivity enhancement of surface plasmon resonance sensor in a heterodyne interferometer system

We propose a method of rotating the analyzer in front of the photodetector in a heterodyne interferometer system to implement optical phase shift and detection sensitivity enhancement of surface plasmon resonance (SPR) sensors. When the analyzer is rotated to shift the phase curve to be near the phase jump point, the phase detection sensitivity of the SPR sensor can be greatly enhanced. Theoretical calculations of a prism-coupled SPR device were performed using this method. Experimental result using an electro-optic heterodyne interferometer is reported.     

Parallel scan spectral surface plasmon resonance imaging

We describe a parallel scan spectral surface plasmon resonance (SPR) imaging technique. We demonstrate experimentally, with a line-shaped light illumination, that an image acquired with an area CCD detector provides both SPR wavelength information and one-dimensional spatial distribution. Thus two-dimensional distribution of the refractive index of the entire sensing plane can be obtained with a one-dimensional optical line parallel scan. The technique offers advantages of both high sensitivity and high throughput, and could have potential applications in biochips analysis.     

Surface plasmon resonance phase imaging measurements of patterned monolayers and DNA adsorption onto microarrays

The optical technique of surface plasmon resonance phase imaging (SPR-PI) is implemented in a linear microarray format for real-time measurements of surface bioaffinity adsorption processes. SPR-PI measures the phase shift of p-polarized light incident at the SPR angle reflected from a gold thin film in an ATR Kretschmann geometry by creating an interference fringe image on the interface with a polarizer-quartz wedge depolarizer combination. The position of the fringe pattern in this image changes upon the adsorption of biomolecules to the gold thin film. By using a linear array of 500 μm biosensor element lines that are perpendicular to the interference fringe image, multiple bioaffinity adsorption measurements can be performed in real time. Two experiments were performed to characterize the sensitivity of the SPR-PI measurement technique: First, a ten line pattern of a self-assembled monolayer of 11-mercaptoundecamine (MUAM) was created via photopatterning to verify that multiple phase shifts could be measured simultaneously. A phase shift difference (Δφ) of Δφ = 182.08 ± 0.03° was observed for the 1.8 nm MUAM monolayer; this value agrees with the phase shift difference calculated from a combination of Fresnel equations and Jones matrices for the depolarizer. In a second demonstration experiment, the feasibility of SPR-PI for in situ bioaffinity adsorption measurements was confirmed by detecting the hybridization and adsorption of single stranded DNA (ssDNA) onto a six-component DNA line microarray patterned monolayer. Adsorption of a full DNA monolayer produced a phase shift difference of Δφ = 28.80 ± 0.03° at the SPR angle of incidence and the adsorption of the ssDNA was monitored in real time with the SPR-PI. These initial results suggest that SPR-PI should have a detection limit roughly 100 times lower than traditional intensity-based SPR imaging measurements.