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).     

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.     

Quantitative comparison of background rejection, signal-to-noise ratio, and resolution in confocal and full-field laser scanning microscopes

Development of a laser scanning microscope for simultaneous three-dimensional imaging in both a full-field laser scanning mode (FLSM) and a confocal laser scanning mode (CLSM) permits the direct comparison of axial resolution and out-of-focus background rejection as a function of sample thickness for both FLSM and CLSM with varying detector aperture (pinhole) radii. The sample-dependent detector aperture radii that optimize the signal-to-noise ratio (S/N) in the CLSM are experimentally determined. The results verify earlier calculations [Appl. Opt. 33, 603 (1994)]. Using these results, we discuss the practical and theoretical limits on the S/N in the CLSM and compare them with those of a full-field epifluorescence microscope (FEM) that is enhanced by image deconvolution. The specimen volume over which the FLSM exhibits imaging properties that are equivalent to a FEM is calculated in the appendices.     

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.     

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.     

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.     

Phase-conjugate holographic system for high-resolution particle-image velocimetry

A novel holographic particle-image velocimeter system has been developed for the study of threedimensional (3-D) fluid velocity fields. The recording system produces 3-D particle images with a resolution, a signal-to-noise ratio, an accuracy, and derived velocity fields that are comparable to high-quality two-dimensional photographic particle-image velocimetry (PIV). The high image resolution is accomplished through the use of low f-number optics, a fringe-stabilized processing chemistry, and a phase conjugate play-back geometry that compensates for aberrations in the imaging system. In addition, the system employs a reference multiplexed, off-axis geometry for the determination of velocity directions with the cross-correlation technique, and a stereo camera geometry for the determination of the three velocity components. The combination of the imaging and reconstruction subsystems makes the analysis of volumetric PIV domains feasible.     

薄壁钢板自冲铆接受剪性能及承载力计算方法研究

为将机械工程领域中效率与自冲化程度高的自冲铆接应用于冷弯薄壁型钢结构,对51组薄壁钢板自冲铆接进行了受剪性能试验。研究了钢板厚度、厚度比及铆钉长度对其受剪性能和破坏机理的影响规律,拟合出了钢板组合厚度与铆钉长度间的经验公式;分析了现有自冲铆接受剪强度计算方法和各国规范中自攻螺钉连接受剪承载力计算方法的适用性;基于试验和分析结果,提出了薄壁钢板自冲铆接受剪承载力计算方法。结果表明:钢板厚度和板厚比分别是影响受剪性能与破坏机理的关键因素,铆钉长度对受剪性能影响较大且存在较优长度,其可通过拟合出的经验公式快速确定;针对不同破坏模式提出的自冲铆接受剪承载力计算方法,与现有方法相比更适用于设计,且其精度更高、稳定性更好。     

Large improvement of the electrical impedance of imaging and high-intensity focused ultrasound (HIFU) phased arrays using multilayer piezoelectric ceramics coupled in lateral mode

With a change in phased-array configuration from one dimension to two, the electrical impedance of the array elements is substantially increased because of their decreased width (w)-to-thickness (t) ratio. The most common way to compensate for this impedance increase is to employ electrical matching circuits at a high cost of fabrication complexity and effort. In this paper, we introduce a multilayer lateral-mode coupling method for phased-array construction. The direct comparison showed that the electrical impedance of a single-layer transducer driven in thickness mode is 1/(n2(1/(w/t))2) times that of an n-layer lateral mode transducer. A large reduction of the electrical impedance showed the impact and benefit of the lateral-mode coupling method. A one-dimensional linear 32-element 770-kHz imaging array and a 42-element 1.45-MHz high-intensity focused ultrasound (HIFU) phased array were fabricated. The averaged electrical impedances of each element were measured to be 58 Ω at the maximum phase angle of -1.2° for the imaging array and 105 Ω at 0° for the HIFU array. The imaging array had a center frequency of 770 kHz with an averaged -6-dB bandwidth of approximately 52%. For the HIFU array, the averaged maximum surface acoustic intensity was measured to be 32.8 W/cm2 before failure.     

Optical properties and instrumental performance of thin gold films near the surface plasmon resonance

The optical properties of very thin gold films have been evaluated by Fresnel analysis, with optical boundary conditions pertaining to the surface plasmon resonance (SPR) at the gold-water interface. The experimental SPR characteristic was evaluated in the angular interrogation mode. Film morphology was characterized by high resolution transmission electron microscopy. The magnitude of the resonance, i.e., the SPR signal, sensitively depends on, and is affected by film thickness and morphology. A sharply defined thickness of 55 ± 5 nm is required, to achieve optimum SPR excitation conditions, and instrumental sensitivity. With decreasing film thickness, below 40 nm, the resonance angle starts to shift to larger values. A substantial increase of the intrinsic resonance broadening parameter is observed below 70 nm, associated with an increasingly asymmetric SPR line shape. A similar effect occurs in the presence of a very thin chromium adhesion layer. Surface roughness and film thickness modulations determine the experimentally observed line broadening parameter. Instrumental noise levels largely depend on accuracy and quality at which the resonance angle can be determined. Substantial improvement and instrumental sub-pixel resolution is achievable by optimum fitting routines, accounting for drastic noise reduction and improved instrumental sensitivity, up to two orders of magnitude over the inherent geometric sensor pixel resolution.     

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.     

Surface plasmon resonance imaging using a high numerical aperture microscope objective

We designed, constructed, and tested a surface plasmon resonance (SPR) microscope using a high numerical aperture objective from a commercially available inverted optical microscope. Such a configuration, combined with various methods to shorten the surface plasmon propagation length, achieves diffraction-limited spatial resolution in the transverse direction and near-diffraction-limited resolution in the longitudinal direction. A virtue of the objective-type SPR imaging is that we achieve distortion-free angle-resolved SPR imaging, allowing the angle-dependent reflectivity of the sample to be examined on a pixel-by-pixel basis, thus offering high-resolution information about surface properties.     

Computational ghost imaging for remote sensing

Computational ghost imaging is a structured-illumination active imager coupled with a single-pixel detector that has potential applications in remote sensing. Here we report on an architecture that acquires the two-dimensional spatial Fourier transform of the target object (which can be inverted to obtain a conventional image). We determine its image signature, resolution, and signal-to-noise ratio in the presence of practical constraints such as atmospheric turbulence, background radiation, and photodetector noise. We consider a bistatic imaging geometry and quantify the resolution impact of nonuniform Kolmogorov-spectrum turbulence along the propagation paths. We show that, in some cases, short-exposure intensity averaging can mitigate atmospheric-turbulence-induced resolution loss. Our analysis reveals some key performance differences between computational ghost imaging and conventional active imaging, and identifies scenarios in which theory predicts that the former will perform better than the latter.     

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.     

Volumetric real-time imaging using a CMUT ring array

A ring array provides a very suitable geometry for forward-looking volumetric intracardiac and intravascular ultrasound imaging. We fabricated an annular 64-element capacitive micromachined ultrasonic transducer (CMUT) array featuring a 10-MHz operating frequency and a 1.27-mm outer radius. A custom software suite was developed to run on a PC-based imaging system for real-time imaging using this device. This paper presents simulated and experimental imaging results for the described CMUT ring array. Three different imaging methods--flash, classic phased array (CPA), and synthetic phased array (SPA)--were used in the study. For SPA imaging, two techniques to improve the image quality--Hadamard coding and aperture weighting--were also applied. The results show that SPA with Hadamard coding and aperture weighting is a good option for ring-array imaging. Compared with CPA, it achieves better image resolution and comparable signal-to-noise ratio at a much faster image acquisition rate. Using this method, a fast frame rate of up to 463 volumes per second is achievable if limited only by the ultrasound time of flight; with the described system we reconstructed three cross-sectional images in real-time at 10 frames per second, which was limited by the computation time in synthetic beamforming.     

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.     

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.     

Characterization of the spatial resolution of different high-frequency imaging systems using a novel anechoic-sphere phantom

The spatial resolution of high-frequency ultrasound (HFU, >20 MHz) imaging systems is usually determined using wires perpendicular to the beam. Recently, two tissue-mimicking phantoms (TMPs) were developed to estimate three-dimensional (3-D) resolution. Each TMP consists of nine 1-cm-wide slabs of tissue-mimicking material containing randomly distributed anechoic spheres. All anechoic spheres in one slab have the same dimensions, and their diameter is increased from 0.1 mm in the first slab to 1.09 mm in the last. The scattering background for one set of slabs was fabricated using 3.5-μm glass beads; the second set used 6.4-μm glass beads. The ability of a HFU system to detect these spheres against a speckle background provides a realistic estimation of its 3-D spatial resolution. In the present study, these TMPs were used with HFU systems using single-element transducers, linear arrays, and annular arrays. The TMPs were immersed in water and each slab was scanned using two commercial imaging systems and a custom HFU system based on a 5-element annular array. The annular array had a nominal center frequency of 40 MHz, a focal length of 12 mm, and a total aperture of 6 mm. A synthetic-focusing algorithm was used to form images with an increased depth-of-field. The penetration depth was increased by using a linear-chirp signal spanning 15 to 65 MHz over 4 μs. Results obtained with the custom system were compared with those of the commercial systems (40-MHz probes) in terms of sphere detection, i.e., 3-D spatial resolution, and contrast-to-noise ratio (CNR). Resulting B-mode images indicated that only the linear-array transducer failed to clearly resolve the 0.2-mm spheres, which showed that the 3-D spatial resolution of the single-element and annular-array transducers was superior to that of the linear array. The single-element transducer could only detect these spheres over a narrow 1.5 mm depth-of-field, whereas the annular array was able to detect them to depths of at least 7 mm. For any size of the anechoic spheres, the annular array excited by a chirp-coded signal provided images of the highest contrast, with a maximum CNR of 1.8 at the focus, compared with 1.3 when using impulse excitation and 1.6 with the single-element transducer and linear array. This imaging configuration also provided CNRs above 1.2 over a wide depth range of 8 mm, whereas CNRs would quickly drop below 1 outside the focal zone of the other configurations.     

Multi-wavelength raman imaging using a small-diameter image guide with a dimension-reduction imaging array

A commercially available fiber-optic Raman probe was modified for high-resolution spectral Raman imaging using a 350 microm diameter optical fiber image guide coupled to a dimension-reduction imaging array (DRIA). The DRIA comprised 672 optical fibers, arranged as a square array (21 x 32 fibers) on one end and a linear array (672 x 1 fibers) on the other. An imaging spectrograph was used with the DRIA to acquire multi-wavelength Raman images from -250 to 1800 cm(-1) at a spectral resolution of approximately 5 cm(-1). The utility of this technique for in situ and remote Raman imaging is demonstrated by monitoring the polymerization of a model polymer, dibromostyrene (DBS), while simultaneously measuring the Raman Stokes/ anti-Stokes ratio as a function of sample heating time, over a sample area of approximately 4 x 1.6 mm.     

System-on-Chip Circuit Architecture for Eliminating Interferents in Surface Plasmon Resonance Sensing Systems

This paper presents a system-on-chip circuit architecture that enables the extraction of concentration information directly from a surface plasmon resonance (SPR) probe, independent of ambient fluctuations in the reference medium, temperature, and background light. Compensation for these baseline (bulk) interferences is embedded into the baseline integration state of the photodetectors in the optical path, creating a ldquoflat linerdquo for the baseline [no analyte present/bulk refractive index (RI)] condition and the characteristic SPR dips for the measurement (analyte present) condition. A resolution of 2 times 10^-4 RI units is possible with this system, comparable to the 5 times 10^-4 RI unit resolution of conventional signal processing (software-based) approaches to processing the same data using a similar framework. This approach demonstrates experimentally the capability of the dip-based SPR probe in a portable footprint for detecting RI at resolution levels suitable for practical applications of these probes to field environments.