Application of surface plasmon resonance sensing to studying elastohydrodynamic lubricant films

We present a new technique based on the spectral characteristics associated with the surface plasmon resonance (SPR) effect for studying lubricants in elastohydrodynamic (EHD) dimples. The pressure inside the EHD dimple causes a localized change of the refractive index (RI) of the entrapped lubricant. This also results in a shift in the spectral SPR absorption dip. By monitoring the color changes within the SPR image, one can obtain a direct measurement of the RI of the entrapped lubricant, from which the pressure distribution within the elastohydrodynamic lubrication (EHL) dimple can be deduced by means of a predetermined relation of pressure and RI of the tested lubricant. Dimples formed with the lubricants PB 2400 and H 1900 were studied in our experiments. Because SPR is sensitive only to the RI variation within a thin region (approximately one wavelength) close to the sensor's surface, the new technique does not require any measurement of the absolute film thickness of the lubricant. This is much simpler than the existing two-beam interferometric technique for measuring the RI of lubricants in EHD dimples, which requires simultaneous measurements of optical film thickness by use of two beams of different angles of incidence. In light of this advantage we anticipate that the new technique can be applied to pressure field mapping in highly loaded rolling and sliding EHL contacts.     

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.     

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.     

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.     

Fiber-optic surface plasmon resonance sensors in the near-infrared spectral region

The sensitivity of fiber-optic surface plasmon resonance (SPR) sensors was improved by a factor of at least thirteen for aqueous solutions by modifying the tip geometry to allow interrogation of the surface plasmon (SP) band in the near-infrared (NIR) region. This was achieved by tuning the angle at the distal end of the SPR sensor to a dual taper of 71 degrees and 19 degrees . Using a low numerical aperture (NA) fiber-optic sensor, NA = 0.12, is necessary to obtain a functional SPR sensor working in the NIR region. Theoretical simulations using the Maxwell equations demonstrated that even higher enhancement is theoretically possible while maintaining a narrow spectral feature upon the excitation of the SP bands on gold surfaces. The manufacture of the SPR sensors yields good agreement between theoretical simulations and experimental observations. To investigate the properties of these fiber-optic SPR-NIR sensors, sucrose solutions ranging from 0 to 15 x 10(-3) in mole fraction were utilized. The increased sensitivity of the fiber-optic SPR sensors, when used to monitor biomarkers, would yield lower detection limits. The smaller sensing area, compared to planar or other fiber-optic SPR sensors, combined with an improvement of the sensitivity, would yield a dramatic reduction of the absolute amount detected by biosensors.     

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.     

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.     

Systematic characterization of spectral surface plasmon resonance sensors with absorbance measurement

Spectral surface plasmon resonance (SPR) sensors with absorbance measurement were prepared. Resonant wavelengths (lambda) versus effective refractive indexes of the SPR mode were measured with different media in contact with the gold layer. An investigation into the refractive-index sensitivity of the sensor at a fixed angle reveals a linear dependence of lambda(R) on the refractive index of the solution (n(c)), with Dlambda /Dn(c) = 3553.6 nm in a small range of 1.333< or = n(c) < or =1.347. It was observed that the effective refractive index slowly decreases with increasing n(c), attributable to wavelength-induced modulation of optical dielectric constant for the gold layer. Adsorption of bromothymol blue (BTB) on the gold layer leads to a redshift of Dlambda(R) = 3.7 nm, larger than Dlambda(R) = 2.5 nm induced by myoglobin (Mb) adsorption. On the basis of Fresnel equations, calculations with d approximately 1 nm and n=1.69 for BTB and d approximately 3 nm and n = 1.40 for Mb also demonstrate that the SPR band shift induced by full-monolayer adsorption of BTB is larger than that for full-monolayer Mb adsorption. The combination of both measured and calculated results suggests that the contribution of the adlayer index of refraction to the sensitivity of the sensor is greater than that of the adlayer thickness.     

Ultrafast pump-probe surface plasmon resonance spectroscopy of thin gold films

A time-resolved reflection pump-probe method was combined with a surface plasmon resonance technique in Kretschmann geometry for the investigation of ultrafast light-induced processes in thin films. Transient changes in the gold layer's reflectivity were observed when the layer was excited by 3 ps duration pulses with photon energy exceeding the interband transition and by probing with photon energy close to the interband transition. Comparison of the experimental and modeling results has shown that the imaginary part of the dielectric function of gold increases linearly during excitation, whereas the real part remains unchanged. The decay of the light-induced changes has two components. The first component is faster than the pulse duration, and the second is much longer than 1.5 ns; they are related to cooling of the electron plasma and lattice, respectively.     

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.     

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.     

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.     

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.     

Characterization of high refractive index semiconductor films by surface plasmon resonance

Si-based surface plasmon resonance (SPR) in the Kretschmann-Raether geometry is considered as a platform for the optical measurement of high refractive index films. The implementation of the SPR effect becomes possible due to the relatively high index of refraction of Si compared to most materials. As examples we study the SPR responses for some important semiconductor-based films, including laser-ablated porous silicon and thin germanium films. Using SPR data, we determine the refractive indices of these films for different parameters (thickness and porosity) and ambiences. We also discuss novel SPR biosensor architectures with the use of these solid films.     

Quantitative surface plasmon resonance imaging: a simple approach to automated angle scanning

Here we present an automated angle-scanning surface plasmon resonance imaging (SPRi) instrument which provides multiplexed, quantitative reflectance data over a wide angular range. Angle-dependent artifacts, which arise from the simple optical setup, are corrected using software. This enables monitoring of significantly different surface coatings in many solvents, which would be outside the dynamic range of typical fixed-angle instruments. Operation in the visible to near-infrared range without the need for reconfiguration extends the instrument capabilities to increase sensitivity or to investigate the optical properties of surface films. This instrument provides maximum flexibility to study a wide range of systems with full exploitation of the quantitative capabilities of SPRi achieved by fitting data to the Fresnel model.     

Near infrared surface plasmon resonance phase imaging and nanoparticle-enhanced surface plasmon resonance phase imaging for ultrasensitive protein and DNA biosensing with oligonucleotide and aptamer microarrays

The techniques of surface plasmon resonance-phase imaging (SPR-PI) and nanoparticle-enhanced SPR-PI have been implemented for the multiplexed bioaffinity detection of proteins and nucleic acids. The SPR-PI experiments utilized a near-infrared 860 nm light emitting diode (LED) light source and a wedge depolarizer to create a phase grating on a four-element single-stranded DNA (ssDNA) microarray; bioaffinity adsorption onto the various microarray elements was detected via multiplexed real time phase shift measurements. In a first set of demonstration experiments, an ssDNA aptamer microarray was used to directly detect thrombin at concentrations down to 100 pM with SPR-PI. Two different ssDNA aptamers were used in these experiments with two different Langmuir adsorption coefficients, K(A1) = 4.4 × 10(8) M(-1) and K(A2) = 1.2 × 10(8) M(-1). At concentrations below 1 nM, the equilibrium phase shifts observed upon thrombin adsorption vary linearly with concentration with a slope that is proportional to the appropriate Langmuir adsorption coefficient. The observed detection limit of 100 pM is approximately 20 times more sensitive than that observed previously with SPRI. In a second set of experiments, two short ssDNA oligonucleotides (38mers) were simultaneously detected at concentrations down to 25 fM using a three-sequence hybridization format that employed 120 nm DNA-modified silica nanoparticles to enhance the SPR-PI signal. In this first demonstration of nanoparticle-enhanced SPR-PI, the adsorbed silica nanoparticles provided a greatly enhanced phase shift upon bioaffinity adsorption due to a large increase in the real component of the interfacial refractive index from the adsorbed nanoparticle. As in the case of SPR-PI, the detection limit of 25 fM for nanoparticle-enhanced SPR-PI is approximately 20 times more sensitive than that observed previously with nanoparticle-enhanced SPRI.     

Single-nucleotide polymorphism genotyping by nanoparticle-enhanced surface plasmon resonance imaging measurements of surface ligation reactions

A sensitive method for the analysis of single nucleotide polymorphisms (SNPs) in genomic DNA that utilizes nanoparticle-enhanced surface plasmon resonance imaging (SPRI) measurements of surface enzymatic ligation reactions on DNA microarrays is demonstrated. SNP identification was achieved by using sequence-specific surface reactions of the enzyme Taq DNA ligase, and the presence of ligation products on the DNA microarray elements was detected using SPRI through the hybridization adsorption of complementary oligonucleotides attached to gold nanoparticles. The use of gold nanoparticles increases the sensitivity of the SPRI so that single bases in oligonucleotides can be successfully identified at a concentration of 1 pM. This sensitivity is amply sufficient for performing multiplexed SNP genotyping by using multiple PCR amplicons and should also allow for the direct detection and identification of SNP sequences from 1 pM unamplified genomic DNA samples with this array-based and label-free SPRI methodology. As a first example of SNP genotyping, three different human genomic DNA samples were screened for a possible point mutation in the BRCA1 gene that is associated with breast cancer.     

Inverse surface plasmon resonance based effective hydrogen sensing using nanoscale palladium films

We demonstrate a nanoscale palladium (Pd) based inverse surface plasmon resonance (ISPR) setup for sensing highly inflammable hydrogen (H₂) gas. The ISPR setup was employed in Kretschmann configuration to assess the sensitivity of the Pd-films when subjected to H₂ gas exposure. With an adequate broadening of the SPR peak maxima, the SPR angle was found to shift from a value of 46.57° to 50.97°, when the concentration of H₂ was varied between 0% and 0.9%. The shifting can be attributed to the transient development of isolated PdH_x (x < 1) clusters within Pd lattices, resulting in an appreciable change of refractive index locally. The dynamical behaviour of switching on/off states exhibited by a ∼20 nm Pd-film and exposed to 0.1% H₂ gas was monitored over several cycles repetitively. The ISPR based H₂ sensor, as demonstrated in ambient environment, would find scope to detect low level H₂ in industrial and other hazardous areas.     

Evanescent field in surface plasmon resonance and surface plasmon field-enhanced fluorescence spectroscopies

The highly sensitive nature of surface plasmon resonance (SPR) spectroscopy and surface plasmon field-enhanced fluorescence spectroscopy (SPFS) are governed by the strong surface plasmon resonance-generated evanescent field at the metal/dielectric interface. The greatest evanescent field amplitude at the interface and the maximum attenuation of the reflectance are observed when a nonabsorbing dielectric is employed. An absorbing dielectric decreases the evanescent field enhancement at the interface. The SPR curve of an absorbing dielectric is characterized by a greater reflectance minimum and a broader curve, as compared to those of the nonabsorbing dielectric with the same refractive index. For a weakly absorbing dielectric, such as nanometer-thick surface-confined fluorophores, the absorption is too small to induce a significant change in the SPR curve. However, the presence of a minute amount of the fluorophore can be detected by the highly sensitive SPFS. The angle with the maximum fluorescence intensity of an SPFS curve is always smaller than the resonance angle of the corresponding SPR curve. This discrepancy is due to the differences of evanescent field distributions and their decay characteristics within the metal film and the dielectric medium. The fluorescence intensity in an SPFS curve can be expressed in terms of the evanescent field amplitude. Excellent correlations between the experimentally measured fluorescence intensities and the evanescent field amplitudes are observed.