Real-time protein biosensor arrays based on surface plasmon resonance differential phase imaging

This paper reports the application of differential phase surface plasmon resonance (SPR) imaging in two-dimensional (2D) protein biosensor arrays. Our phase imaging approach offers a distinct advantage over the conventional angular SPR technique in terms of utilization efficiency of optical sensor elements in the imaging device. In the angular approach, each biosensor site in the biosensor array requires a linear array of optical detector elements to locate the SPR angular dip. The maximum biosensor density that a two-dimensional imaging device can offer is a one-dimensional SPR biosensor array. On the other hand, the phase-sensitive SPR approach captures data in the time domain instead of the spatial domain. It is possible that each pixel in the captured interferogram represents one sensor site, thus offering high-density two-dimensional biosensor arrays. In addition, our differential phase approach improves detection resolution through removing common-mode disturbances. Experimental results demonstrate a system resolution of 8.8 x 10(-7)RIU (refractive index unit). Real-time monitoring of bovine serum albumin (BSA)/anti-BSA binding interactions at various concentration levels was achieved using a biosensor array. The detection limit was 0.77 microg/ml. The reported two-dimensional SPR biosensor array offers a real-time and non-labeling detection tool for high-throughput protein array analysis. It may find promising applications in protein therapeutics, drug screening and clinical diagnostics.     

Combination of Digital Image Processing and Statistical Data Segmentation to Enhance SPR and SPRi Sensor Responses

The work reports the combination of basic digital image processing (DIP) techniques and statistical segmentation strategy (SDS) to improve surface plasmon resonance curve (SPRc) and SPR imaging (SPRi) sensors' performance. The SPR image is used for sensing and monitoring biological events in the so-called SPR imaging process. In the traditional SPR process based on the attenuated total reflection (ATR) method, the image is used to create the SPR curve, and the curve features tracking is employed on sensing applications. The SPR curve features are enhanced after the pixels of the SPR image have been processed with low-complexity filters in the spatial domain (brightness, contrast, threshold, and morphological). The bootstrap was used as a statistical processing approach, selecting lines and columns from the image that was less affected by imperfections and noises in the image detector, and consequently reducing the SPR sensor instrumentation disturbances. Experimental tests with reversible binding water-mixture were performed, and both image and statistical processing were reported. The combination of DIP and SDS approaches improves the extraction of the curve features, increasing the performance in terms of resonance position sensitivity to 81%.

     

Surface plasmon resonance sensors in cell biology: basics and application

Optical sensors based on the excitation of surface plasmons, referred to as surface plasmon resonance (SPR) sensors, have become a central analytical tool for characterizing and quantifying a wide variety of macromolecular interactions, like receptor–ligand contacts. Besides this classical field of application, in the last 15 years, the development of SPR sensors aiming for the detection and analysis of ligand/cell or host/pathogen interactions, cell/cell contacts, and cellular reactions gained considerable momentum. The number of publications reporting about applications of SPR sensors implementing vital prokaryotic or eukaryotic cells as biorecognition elements for medical diagnostics, environmental monitoring, or biological safety is steadily growing. This review gives a short introduction to the technique of surface plasmon resonance and the parameters that are important for its application in the field of vital cell sensors. Furthermore, the publications concerning the application of such sensors in the analysis of cellular interactions and cellular reactions to extra- and intracellular stimuli are summarized.     

Angle-scanning SPR imaging for detection of biomolecular interactions on microarrays

In this paper we describe the use of a commercial surface plasmon resonance (SPR) imaging instrument for monitoring the binding of biomolecules on user-defined regions of interest of a microarray. By monitoring the angle shift of the SPR-dip using a continuous angle-scanning mode instead of monitoring the change in reflectivity at a fixed angle, a linear relationship with respect to the mass density change on the surface will remain over a wide dynamic angle range of 8 degrees. Peptides (2.4 kDa) and proteins (150 kDa) were both spotted on the same sensor chip to illustrate that both, low and high molecular weight ligands with initial large differences in off-set SPR angles, can be applied within the same experiment. By using a fluorescently labeled antibody, SPR results can be confirmed by means of fluorescence microscopy after completion of a SPR experiment. SPR imaging in angle-scanning operation provides sensitive, accurate, and label-free detection of analyte binding on microarrays containing different molecular weight ligands.     

Large-area gold nanohole arrays fabricated by one-step method for surface plasmon resonance biochemical sensing

Surface plasmon resonance (SPR) nanosensors based on metallic nanohole arrays have been widely reported to detect binding interactions in biological specimens. A simple and effective method for constructing nanoscale arrays is essential for the development of SPR nanosensors. In this work, we report a one-step method to fabricate nanohole arrays by thermal nanoimprinting in the matrix of IPS (Intermediate Polymer Stamp). No additional etching process or supporting substrate is required. The preparation process is simple, time-saving and compatible for roll-to-roll process, potentially allowing mass production. Moreover, the nanohole arrays were integrated into detection platform as SPR sensors to investigate different types of biological binding interactions. The results demonstrate that our one-step method can be used to efficiently fabricate large-area and uniform nanohole arrays for biochemical sensing.     

Liquid phase SPR imaging experiments for biosensors applications

Surface plasmon resonance (SPR) has recently gained attention as a label-free method for the detection of biological molecules binding onto functionalised surfaces. It is one of the most sensitive detection method for monitor variations in the thickness and refractive index in ultra-thin films. Here, the adsorption processes of oligonucleotides onto gold substrates have been investigated in aqueous buffer solution using SPR imaging measurements. The hybridization of a thiol-modified, single stranded oligonucleotide anchored to a gold surface via thiol group, with its complementary sequence has been observed and characterised monitoring the hybridization process by SPR equipment. In situ investigation of smallest changes in SPR imaging measurements dynamically performed in liquid phase in the presence of DNA complementary probes was performed. Infrared spectroscopy and scanning electron microscopy characterisation of the functionalised gold surfaces of the biosensor were compared with the images obtained by SPR experimental apparatus.     

Analysis of protein interactions on protein arrays by a novel spectral surface plasmon resonance imaging

We presented a novel surface plasmon resonance (SPR) imaging method for analysis of protein arrays based on a wavelength interrogation-based SPR biosensor. The spectral imaging was performed by the combination of position control and resonance wavelengths calculated from SPR reflectivity spectra. The imaging method was evaluated by analyzing interactions of glutathione S-transferase-fusion proteins with their antibodies. Antigen-antibody interactions were successfully analyzed on glutathione S-transferase-fusion protein arrays by using the spectral imaging method, and the results were confirmed by a parallel analysis using a previously used spectral SPR biosensor based on wavelength interrogation. Specific binding of anti-Rac1 and anti-RhoA to Rac1 and RhoA on the protein arrays was qualitatively and quantitatively analyzed by the spectral SPR imaging. Thus, it was suggested that the novel spectral SPR imaging was a useful tool for the high-throughput analysis of protein-protein interactions on protein arrays.     

Comparison of Gold and Silver/Gold Bimetallic Surface for Highly Sensitive Near-infrared SPR Sensor at 1550 nm

A large majority of surface plasmon resonance (SPR) sensors reported in the literature are designed to operate in the visible electromagnetic spectrum. However, the near-infrared, particularly at the telecommunications wavelength of 1550 nm, is also especially attractive for SPR sensing applications. In fact, SPR sensors operating in this region benefit from narrower resonance and deeper field penetration. In this paper, we report a theoretical and experimental study of an SPR sensor operating at a fixed wavelength of 1550 nm. The influence of the choice of metals and the interrogation methods on the sensitivity of the resulting SPR sensor is investigated. Two types of sensor chips (simple gold (Au) and bimetallic silver/Au structure) and three interrogation methods (monitoring of the position of the reflectivity minimum, the position of the centroid, and the intensity evolution of the reflectivity) are examined. We show that a refractive index resolution of 2.7?×?10^?6 refractive index unit can be easily obtained, and with further optimization of the measurement system, the ultimate limit of detection is expected to be even lowered. Therefore, the approach discussed here already shows a promising potential for highly sensitive SPR sensors.     

Sensitivity of ex situ and in situ spectral surface plasmon resonance sensors in the analysis of protein arrays

We have investigated the sensitivity of ex situ (analysis under air condition) and in situ (analysis under liquid condition) spectral SPR sensors, which were self-constructed with fiber optic spectrometers. The sensitivity of SPR sensors was analyzed in the wavelength range of 550-780 nm by the interactions of streptavidin and biotinylated IgG, and the sensitivity was dependent on the wavelength of measurements. The sensitivity of an ex situ SPR sensor operated at the long wavelength range from 712 nm was approximately 2.6 times higher than that at the short wavelength range from 571 nm. In addition, the sensitivity of an ex situ spectral SPR sensor was about twice as high as that of an in situ spectral SPR sensor for the same resonance wavelength range. This was interpreted in that the difference in sensitivity between two SPR sensors was significantly caused by the evanescent field intensity at the metal/dielectric interface. Thus, it was suggested that ex situ spectral SPR sensors operated at the long wavelength range are sensitive biosensors for the high-throughput analysis of protein interactions on protein arrays.     

Surface plasmon resonance for high-throughput ligand screening of membrane-bound proteins

Technologies based on surface plasmon resonance (SPR) have allowed rapid, label-free characterization of protein-protein and protein-small molecule interactions. SPR has become the gold standard in industrial and academic settings, in which the interaction between a pair of soluble binding partners is characterized in detail or a library of molecules is screened for binding against a single soluble protein. In spite of these successes, SPR is only beginning to be adapted to the needs of membrane-bound proteins which are difficult to study in situ but represent promising targets for drug and biomarker development. Existing technologies, such as BIAcoreTM, have been adapted for membrane protein analysis by building supported lipid layers or capturing lipid vesicles on existing chips. Newer technologies, still in development, will allow membrane proteins to be presented in native or near-native formats. These include SPR nanopore arrays, in which lipid bilayers containing membrane proteins stably span small pores that are addressable from both sides of the bilayer. Here, we discuss current SPR instrumentation and the potential for SPR nanopore arrays to enable quantitative, high-throughput screening of G protein coupled receptor ligands and applications in basic cellular biology.     

A new surface plasmon resonance sensor for high-throughput screening applications

We report a new high-throughput surface plasmon resonance (SPR) sensor based on combination of SPR imaging with polarization contrast and a spatially patterned multilayer SPR structure. We demonstrate that this approach offers numerous advantageous features including high-contrast SPR images suitable for automated computer analysis, minimum crosstalk between neighboring sensing channels and inherent compensation for light level fluctuations. Applications of a laboratory prototype of the high-throughput SPR sensor with 108 sensing channels for refractometry and biosensing are described. In refractometric experiments, the noise-limited refractive index resolution of the system has been established to be 3 x 10(-6) refractive index unit (RIU). Experimental data on detection of human choriogonadotropin (hCG) suggest that in conjunction with monoclonal antibodies against hCG, the reported SPR imaging sensor is capable of detecting hCG at concentrations lower than 500 ng/ml.     

Peptide-conjugated gold nanorods for nuclear targeting

Resonant electron oscillations on the surface of noble metal nanoparticles (Au, Ag, Cu) create the surface plasmon resonance (SPR) that greatly enhances the absorption and Rayleigh (Mie) scattering of light by these particles. By adjusting the size and shape of the particles from spheres to rods, the SPR absorption and scattering can be tuned from the visible to the near-infrared region (NIR) where biologic tissues are relatively transparent. Further, gold nanorods greatly enhance surface Raman scattering of adsorbed molecules. These unique properties make gold nanorods especially attractive as optical sensors for biological and medical applications. In the present work, gold nanorods are covalently conjugated with a nuclear localization signal peptide through a thioalkyl-triazole linker and incubated with an immortalized benign epithelial cell line and an oral cancer cell line. Dark field light SPR scattering images demonstrate that nanorods are located in both the cytoplasm and nucleus of both cell lines. Single cell micro-Raman spectra reveal enhanced Raman bands of the peptide as well as molecules in the cytoplasm and the nucleus. Further, the Raman spectra reveal a difference between benign and cancer cell lines. This work represents an important step toward both imaging and Raman-based intracellular biosensing with covalently linked ligand-nanorod probes.     

Chemisorptions of bacterial receptors for hydrophobic amino acids and sugars on gold for biosensor applications: a surface plasmon resonance study of genetically engineered proteins

This paper demonstrates potential applications of two periplasmic receptor proteins from E. coli as sensing elements for biosensors using the surface plasmon resonance (SPR) technique. These molecules, namely the aspartate to cysteine mutant of the leucine-specific receptor (LS-D1C) and the glutamine to cysteine mutant of the D-glucose/D-galactose receptor (GGR-Q26C) proteins, are chemisorbed on a thin (approximately 40 nm) Au film in neutral K2HPO4 buffers. Using angle and time resolved SPR measurements; we show that adsorption behaviors of both proteins are dominated by diffusion-free second order Langmuir kinetics. We also show that the protein-modified Au films exhibit measurable SPR shifts upon binding to their respective target ligands. According to these SPR data, the kinetics of ligand binding for both LS-D1C and GGR-Q26C are governed by irreversible first order diffusion limited Langmuir model. The utility of the SPR technique for studying reactions of biological molecules is further illustrated in this work.     

Sugar Chips immobilized with synthetic sulfated disaccharides of heparin/heparan sulfate partial structure

Carbohydrate chip technology has a great potential for the high-throughput evaluation of carbohydrate-protein interactions. Herein, we report syntheses of novel sulfated oligosaccharides possessing heparin and heparan sulfate partial disaccharide structures, their immobilization on gold-coated chips to prepare array-type Sugar Chips, and evaluation of binding potencies of proteins by surface plasmon resonance (SPR) imaging technology. Sulfated oligosaccharides were efficiently synthesized from glucosamine and uronic acid moieties. Synthesized sulfated oligosaccharides were then easily immobilized on gold-coated chips using previously reported methods. The effectiveness of this analytical method was confirmed in binding experiments between the chips and heparin binding proteins, fibronectin and recombinant human von Willebrand factor A1 domain (rh-vWf-A1), where specific partial structures of heparin or heparan sulfate responsible for binding were identified.     

Comparison of surface plasmon resonance spectroscopy and quartz crystal microbalance techniques for studying DNA assembly and hybridization

In this study we evaluate the strengths and weaknesses of surface plasmon resonance (SPR) spectroscopy and quartz crystal microbalance (QCM) technique for studying DNA assembly and hybridization reactions. Specifically, we apply in parallel an SPR instrument and a 5 MHz QCM device with dissipation monitoring (QCM-D) to monitor the assembly of biotinylated DNA (biotin-DNA) on a streptavidin-modified surface and the subsequent target DNA hybridization. Through the parallel measurements, we demonstrate that SPR is more suitable for quantitative analysis of DNA binding amount, which is essential for interfacial DNA probe density control and for the analysis of its effect on hybridization efficiency and kinetics. Although the QCM is not quantitative to the same extent as SPR (QCM measures the total mass of the bound DNA molecules together with the associated water), the dissipation factor of the QCM provides a qualitative measure of the viscoelastic properties of DNA films and the conformation of the bound DNA molecules. The complexity in mass measurement does not impair QCM's potential for a kinetic evaluation of the hybridization processes. For quantification of target DNA, the biotin-DNA modified SPR and QCM sensors are exposed to target DNA with increasing concentration. The plots of SPR/QCM signals versus target DNA concentration show that water entrapment between DNA strands make the QCM sensitivity for the hybridization assay well comparable with that of the SPR, although the intrinsic mass sensitivity of the 5 MHz QCM is approximately 20 times lower.     

Real-time and Label-free Bio-sensing of Molecular Interactions by Surface Plasmon Resonance: A Laboratory Medicine Perspective

Radioactive, chromogenic, fluorescent and other labels have long provided the basis of detection systems for biomolecular interactions including immunoassays and receptor binding studies. However there has been unprecedented growth in a number of powerful label free biosensor technologies over the last decade. While largely at the proof-of-concept stage in terms of clinical applications, the development of more accessible platforms may see surface plasmon resonance (SPR) emerge as one of the most powerful optical detection platforms for the real-time monitoring of biomolecular interactions in a label-free environment.In this review, we provide an overview of SPR principles and current and future capabilities in a diagnostic context, including its application for monitoring a wide range of molecular markers of disease. The advantages and pitfalls of using SPR to study biomolecular interactions are discussed, with particular emphasis on its potential to differentiate subspecies of analytes and the inherent ability for quantitation through calibration-free concentration analysis (CFCA). In addition, recent advances in multiplex applications, high throughput arrays, miniaturisation, and enhancements using noble metal nanoparticles that promise unprecedented sensitivity to the level of single molecule detection, are discussed.In summary, while SPR is not a new technique, technological advances may see SPR quickly emerge as a highly powerful technology, enabling rapid and routine analysis of molecular interactions for a diverse range of targets, including those with clinical applicability. As the technology produces data quickly, in real-time and in a label-free environment, it may well have a significant presence in future developments in lab-on-a-chip technologies including point-of-care devices and personalised medicine.     

Detection of embryonic stem cell lysate biomarkers by surface plasmon resonance with reduced nonspecific adsorption

Surface plasmon resonance imaging (SPRi) has emerged as a versatile biosensor to detect a wide range of biomolecular interactions with divergent potential applications. However, the use of this advanced-level technology for stem cell lysate study is still not much explored. Cell lysates are significant biological analytes used for disease diagnostics and proteomic studies, but their complex nature limits their use as an analyte for SPRi biosensors. Here, we review the problems associated with the use of SPRi for stem cell lysate study and examine the role of surface chemistry, running buffer, and blocking solution in order to minimize nonspecific adsorption (NSA). We detect the expression of Oct4, Sox2, Nanog, Rex1, and Lin28 biomarkers present in mouse embryonic stem cell (mESC) lysate against their corresponding antibodies immobilized on the sensor surface with reduced NSA. The current study shows that the conjunction of SPRi and microarray can be used as a label-free, high-throughput, and rapid technique for detection of biomarkers and their relative abundance in stem cell lysate study.     

Looking towards label-free biomolecular interaction analysis in a high-throughput format: a review of new surface plasmon resonance technologies

Surface plasmon resonance (SPR) biosensors have enabled a wide range of applications in which researchers can monitor biomolecular interactions in real time. Owing to the fact that SPR can provide affinity and kinetic data, unique features in applications ranging from protein-peptide interaction analysis to cellular ligation experiments have been demonstrated. Although SPR has historically been limited by its throughput, new methods are emerging that allow for the simultaneous analysis of many thousands of interactions. When coupled with new protein array technologies, high-throughput SPR methods give users new and improved methods to analyze pathways, screen drug candidates and monitor protein-protein interactions.     

金纳米棒的表面修饰及其应用于肿瘤诊疗的研究进展

金纳米棒具有独特的光学性质、表面易修饰性、较低的生物毒性和良好的生物相容性,因而在成像、光热治疗和药物载带等方面具有极高的潜在应用价值.本文综述了典型的金纳米棒表面修饰方法及其在生物成像、光热治疗和药物治疗中的应用,重点阐述了通过金纳米棒同时实现肿瘤诊断和治疗相结合的研究进展.