In situ sensing of single binding events by localized surface plasmon resonance

Single binding events of nanoparticle-labeled DNA strands were detected as stepwise peak shifts in localized surface plasmon resonance by single particle measurement. We confirmed the number of binding events by observing label particles by scanning electron microscopy. Our simulation based on a multiple multipole program showed that the peak shift is dependent on interparticle gap size and binding position. The experimental peak shift distribution was also reproduced by simulation.     

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.     

Real-time and full-field detection of near-wall salinity using surface plasmon resonance reflectance

An idea of real-time and full-field detection of near-wall salinity is presented to use the surface plasmon resonance (SPR) reflectance that changes with refractive index variations of the tested saline fluid. The laboratory-designed SPR system, based on the Kretschmann's configuration, uses a 47.5 nm thick gold layer as the SPR resonator, coated on a BK7 prism (n=1.515), and requires a one-time system calibration to establish a correlation of the specified saline mass concentration levels to the corresponding CCD (charge-coupled device) pixel gray levels. As a gravity-falling saline drop in water reaches the bottom and diffuses thereafter, the SPR system quantitatively maps the evolution of the salinity distributions in the near-wall region (less than 1 microm). An elaborate uncertainty analysis shows that the overall measurement uncertainties critically depend on the uniformity of the metal film thickness and the accuracy of its dielectric constant.     

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.     

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.     

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.     

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.     

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.     

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.     

Determination of ribonuclease H surface enzyme kinetics by surface plasmon resonance imaging and surface plasmon fluorescence spectroscopy

The kinetics of the ribonuclease H (RNase H) surface hydrolysis of RNA-DNA heteroduplexes formed on DNA microarrays was studied using a combination of real-time surface plasmon resonance imaging (SPRI) and surface plasmon fluorescence spectroscopy (SPFS). Time-dependent SPRI and SPFS data at various enzyme concentrations were quantitatively analyzed using a simple model that couples diffusion, enzyme adsorption, and surface enzyme kinetics. This model is characterized by a set of three rate constants, enzyme adsorption (k(a)), enzyme desorption (k(d)), enzyme catalysis (k(cat)), and one dimensionless diffusion parameter (beta). Values of k(a) = 3.15 (+/-0.20) x 10(6) M(-1).s(-1), k(d) = 0.10 (+/-0.05) s(-1), and k(cat) = 0.95 (+/-0.10) s(-1) were determined from fitting all of the SPRI and SPFS data sets. One of the most interesting kinetic parameters is the surface RNase H hydrolysis reaction rate constant (k(cat)), which was found to be approximately 10 times slower than that observed in solution, but approximately 100 times faster than that recently observed for the exonuclease III surface hydrolysis of double-stranded DNA microarrays (k(cat) = 0.009 s(-1)). Moreover, the surface coverage of the intermediate enzyme-substrate complex (ES) was found to be extremely small during the course of the reaction because k(cat) is much larger than the product of k(a) and the bulk enzyme concentration.     

Multicolor surface plasmon resonance imaging of ink jet-printed protein microarrays

We report a technique that utilizes surface plasmon resonance dispersion as a mechanism to provide multicolor contrast for imaging thin molecular films. Illumination of gold surfaces with p-polarized white light in the Kretschmann configuration produces distinct reflected colors due to excitation of surface plasmons and the resulting absorption of specific wavelengths from the source light. In addition, these colors transform in response to the formation of thin molecular films. This process represents a simple detection method for distinguishing between films of varying thickness in sensor applications. As an example, we interrogated a protein microarray formed by a commercial drop-on-demand chemical ink jet printer. Submonolayer films of a test protein (bovine serum albumin) were readily detected by this method. Analysis of the dispersion relations and absorbance sensitivities illustrate the performance and characteristics of this system. Higher detection sensitivity was achieved at angles where red wavelengths coupled to surface plasmons. However, improved contrast and spatial resolution occurred when the angle of incidence was such that shorter wavelengths coupled to the surface plasmons. Simplified optics combined with the robust microarray printing platform are used to demonstrate the applicability of this technique as a rapid and versatile, high-throughput tool for label-free detection of adsorbed films and macromolecules.     

Probing molecular junctions using surface plasmon resonance spectroscopy

The optical absorption spectra of nanometer-thick organic films and molecular monolayers sandwiched between two metal contacts have been measured successfully using surface plasmon resonance spectroscopy (SPRS). The electric field within metal-insulator (organic)-metal (MIM) cross-bar junctions created by surface plasmon-polaritons excited on the metal surface allows sensitive measurement of molecular optical properties. Specifically, this spectroscopic technique extracts the real and imaginary indices of the organic layer for each wavelength of interest. The SPRS sensitivity was calculated for several device architectures, metals, and layer thicknesses to optimize the organic film absorptivity measurements. Distinct optical absorption features were clearly observed for R6G layers as thin as a single molecular monolayer between two metal electrodes. This method also enables dynamic measurement of molecular conformation inside metallic junctions, as shown by following the optical switching of a thin spiropyran/polymer film upon exposure to UV light. Finally, optical and electrical measurements can be made simultaneously to study the effect of electrical bias and current on molecular conformation, which may have significant impact in areas such as molecular and organic electronics.     

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.     

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.     

Deep UV nano-microstructuring of substrates for surface plasmon resonance imaging

In this paper, we describe wafer-scale fabrication and characterization of plasmonic chips-containing different sizes and spacings of metallic micro- and nanoline structures-using deep UV lithography. Using a high dose (25 mJ cm( - 2)) and a proper lift-off process, feature sizes as small as 25 nm are obtained. Moreover, we study the dependence of surface plasmon resonance on the angle of incidence and wavelength for different micro- and nanoline size and spacing values, yielding localized to quasi-propagative plasmonic behaviors. Rigorous coupled wave analysis (RCWA) techniques are employed to numerically confirm these experimental observations. Finally, the refractive index of media around the SPRI sensor chips is varied, showing the angulo-spectral regions of higher sensitivity for each type of structure.     

In situ biosensing with a surface plasmon resonance fiber grating aptasensor

Surface plasmon resonance (SPR) biosensors prepared using optical fibers can be used as a cost-effective and relatively simple-to-implement alternative to well established biosensor platforms for monitoring biomolecular interactions in situ or possibly in vivo. The fiber biosensor presented in this study utilizes an in-fiber tilted Bragg grating to excite the SPR on the surface of the sensor over a large range of external medium refractive indices, with minimal cross-sensitivity to temperature and without compromising the structural integrity of the fiber. The label-free biorecognition scheme used demonstrates that the sensor relies on the functionalization of the gold-coated fiber with aptamers, synthetic DNA sequences that bind with high specificity to a given target. In addition to monitoring the functionalization of the fiber by the aptamers in real-time, the results also show how the fiber biosensor can detect the presence of the aptamer's target, in various concentrations of thrombin in buffer and serum solutions. The findings also show how the SPR biosensor can be used to evaluate the dissociation constant (K(d)), as the binding constant agrees with values already reported in the literature.     

Detection of oxidized low-density lipoproteins using surface plasmon resonance

Management of atherosclerosis is a high priority target. If this is to be achieved, the early detection of risk and risk factors are paramount and integrated with this is a need for the detection of the oxidation state of a patient's low density lipoprotein (LDL). Presently no readily usable technique exists for their rapid determination and in order to develop such a technique a monitoring system must be devised which distinguishes a parameter which changes on oxidation and distinguishes critical and noncritical oxidation products. The strategy which is investigated here is based on the use of a heparin-modified Au-surface plasmon resonance (SPR) device as a modulator of LDL binding, according to its oxidation state. Heparin is strongly negatively charged and seven binding sites for heparin have been identified on the LDL apoprotein consisting of arginine and lysine clusters; these are regarded as identical to the LDL receptor binding sites. The heparin-modified surface was calibrated for LDL and a calibration factor of 1.84 × 10(9) particles mm(-)(2) Δ(o)(-)(1) SPR and instrumental resolution of 9 × 10(6) particles mm(-)(2) obtained which gives sufficient scope to distinguish LDL dependent binding. LDL oxidation could involve the protein and/or lipoprotein, the latter being of interest for athersclerosis risk and the LDL binding to heparin was shown to decrease with degree of protein oxidation as determined by the free amino groups (fluorescamine assay), but was not influenced by lipid oxidation (determined by thiobarbituric acid reactive substances assay, TBARS). The SPR based assay was tested for LDL in plasma and the calibration found to follow that obtained in buffer, although the scatter was higher, probably due to interference from other plasma species. Nevertheless, in the context of the normal distribution of LDL in healthy patients, the assay would almost certainly be able to determine Ox-LDL in atherosclerotic patients.     

Detection of PCR products in solution using surface plasmon resonance

Polymerase chain reaction (PCR) products were detected using a flow injection-type sensor based on surface plasmon resonance. Asymmetric PCR was used to amplify the target DNA sequence, and two products with different length were produced. The novelty of our DNA detection system was that our target DNA was double stranded but the probe binding site, located in the 3'-terminus, was single stranded. This avoids the formation of intra- and intermolecular complexes. This novel design permitted us not only to detect PCR product but also to develop a rapid detection system for the detection of the verotoxin 2 gene of Escherichia coli O157:H7.     

Two-dimensional biosensor arrays based on surface plasmon resonance phase imaging

We present a biosensor design based on capturing the two-dimensional (2D) phase image of surface plasmon resonance (SPR). This 2D SPR imaging technique may enable parallel label-free detection of multiple analytes and is compatible with the microarray chip platform. This system uses our previously reported differential phase measurement approach, in which 2D phase maps obtained from the signal (P) and reference (S) polarizations are compared pixel by pixel. This technique greatly improves detection resolution as the subtraction step can eliminate measurement fluctuations caused by external disturbances as they essentially appear in both channels. Unlike conventional angular SPR systems, in which illumination from a range of angles must be used, phase measurement requires illumination from only one angle, thus making it well suited for 2D measurement. Also, phase-stepping introduced from a moving mirror provides the necessary modulation for accurate detection of the phase. In light of the rapidly increasing need for fast real-time detection, quantification, and identification of a range of proteins for various biomedical applications, our 2D SPR phase imaging technique should hold a promising future in the medical device market.     

Detection of protein biomarkers using RNA aptamer microarrays and enzymatically amplified surface plasmon resonance imaging

A methodology for the detection of protein biomarkers at picomolar concentrations that utilizes surface plasmon resonance imaging (SPRI) measurements of RNA aptamer microarrays is developed. The adsorption of proteins onto the RNA microarray is detected by the formation of a surface aptamer-protein-antibody complex. The SPRI response signal is then amplified using a localized precipitation reaction catalyzed by the enzyme horseradish peroxidase that is conjugated to the antibody. This enzymatically amplified SPRI methodology is first characterized by the detection of human thrombin at a concentration of 500 fM; the appropriate thrombin aptamer for the sandwich assay is identified from a microarray of three potential thrombin aptamer candidates. The SPRI method is then used to detect the protein vascular endothelial growth factor (VEGF) at a biologically relevant concentration of 1 pM. VEGF is a signaling protein that has been used as a serum biomarker for rheumatoid arthritis, breast cancer, lung cancer, and colorectal cancer and is also associated with age-related macular degeneration.