Surface Plasmon Resonance Sensor Chip Research Articles

SPR Signals of Three-dimensional Antibody-immobilized Gel Layers Formed on Sensor Chips by Atom Transfer Radical Polymerization

Abstract We prepared antibody-immobilized gel layers on surface plasmon resonance (SPR) sensor chips by atom transfer radical polymerization (ATRP). In a buffer solution with a target antigen, the SPR signal of the antibody-immobilized gel layer chip with an optimal thickness was much larger than that of a directly antibody-immobilized chip prepared by the standard amine coupling method. The antibody-immobilized gel layer chips are candidates as highly sensitive bioconjugated devices for SPR sensing systems. This paper focuses on the relationship between the thickness of the gel layer and the SPR signal in the detection of a target antigen.

ANT-VDAC1 interaction is direct and depends on ANT isoform conformation in vitro

The voltage-dependent anion channel (VDAC) and the adenine nucleotide translocase (ANT) have central roles in mitochondrial functions such as nucleotides transport and cell death. The interaction between VDAC, an outer mitochondrial membrane protein and ANT, an inner membrane protein, was studied in isolated mitochondria and in vitro. Both proteins were isolated from various mitochondrial sources and reconstituted in vitro using a biomimetic system composed of recombinant human VDAC isoform 1 (rhVDAC1) immobilized on a surface plasmon resonance (SPR) sensor chip surface. Two enriched-preparations of (H)ANT (ANT from heart, mainly ANT1) and (L)ANT (ANT from liver, mainly ANT2) isoforms interacted differently with rhVDAC1. Moreover, the pharmacological ANT inhibitors atractyloside and bongkrekic acid modulated this interaction. Thus, ANT-VDAC interaction depends both on ANT isoform identity and on the conformation of ANT.

Titanium nitride thin film as an adhesion layer for surface plasmon resonance sensor chips

Optical properties, surface plasmon resonance (SPR) response characteristics, and the adhesion properties of the plasmonic stacks with titanium nitride (TiNx) adhesion layers were analyzed and compared with those of the Au single stack and the plasmonic stacks with conventional titanium adhesion layer. All the films were deposited by radio frequency magnetron sputtering. TiNx single layer exhibited higher electrical conductivity and reflectance in long wavelength range than Ti single layer of similar thickness. When compared with the plasmonic stacks with Ti adhesion layer, the plasmonic stacks with TiNx adhesion layer showed higher peak transmittance and less absorption loss in wavelength range longer than 500nm. Examination of the SPR response curves revealed that much improved SPR characteristics could be attained if conventional Ti adhesion layer were replaced by TiNx layer, and it was attributed to the lower damping of TiNx film than Ti film at corresponding thickness. Also, it was proved that TiNx layer could provide sufficient adhesion strength at the glass/TiNx and TiNx/Au interfaces, which is comparable with that of Ti layer.

Solution-based fabrication of gold grating film for use as a surface plasmon resonance sensor chip

Abstract Imprinted solution-processible gold nanoparticle (AuNP) grating films were fabricated for use as a grating-coupled surface plasmon resonance (SPR) excitation substrate. The imprinted AuNP grating pattern, which consists of a 1.72 μm grating pitch, was fabricated on flat glass substrates by imprinting AuNP on silicon grating templates. In this study, the SPR measurements were carried out on an SPR device in the grating-coupling configuration with a He–Ne laser ( λ = 632.8 nm) as the excitation source, and multimode surface plasmon excitations were observed upon irradiation with white light. SPR excitation of our substrate was observed at the incident angle of 47.1°. For SPR measurements using white light irradiation, multimode surface plasmon excitation resulting from several diffraction orders was observed in the wavelength region of 500–850 nm. The surface plasmon dispersion branches were calculated to obtain the diffraction order in this region. SPR excitation of the imprinted AuNP grating substrate was further studied by fabrication of a layer-by-layer ultrathin film of iron containing bis(terpyridine) polymer (Fe(II)-BTP) and poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS). A shift of the SPR excitation spectrum was observed when the ultrathin film was deposited on the imprinted AuNP grating surface. Thus, this substrate should be a useful SPR substrate in a variety of applications such as photoelectric conversions and sensors.

Cancer Biomarker Detection in Serum Samples Using Surface Plasmon Resonance and Quartz Crystal Microbalance Sensors with Nanoparticle Signal Amplification

Early detection of cancer is vital for the successful treatment of the disease. Hence, a rapid and sensitive diagnosis is essential before the cancer is spread out to the other body organs. Here we describe the development of a point-of-care immunosensor for the detection of the cancer biomarker (total prostate-specific antigen, tPSA) using surface plasmon resonance (SPR) and quartz crystal microbalance (QCM) sensor platforms in human serum samples. K(D) of the antibody used toward PSA was calculated as 9.46 × 10(-10) M, indicating high affinity of the antibody used in developing the assay. By performing a sandwich assay using antibody-modified nanoparticles concentrations of 2.3 ng mL(-1) (Au, 20 nm) and 0.29 ng mL(-1) (8.5 pM) (Au, 40 nm) tPSA in 75% human serum were detected using the developed assay on an SPR sensor chip. The SPR sensor results were found to be comparable to that achieved using a QCM sensor platform, indicating that both systems can be applied for disease biomarkers screening. The clinical applicability of the developed immunoassay can therefore be successfully applied to patient's serum samples. This demonstrates the high potential of the developed sensor devices as platforms for clinical prostate cancer diagnosis and prognosis.

Detection of TNT by a molecularly imprinted polymer film-based surface plasmon resonance sensor

A surface plasmon resonance (SPR) sensor using a molecularly imprinted polymer (MIP) film as a recognition element for detection of 2,4,6-trinitrotoluene (TNT) was developed. The TNT-imprinted polymer film was prepared by thermo initiated polymerization on the bare Au surface of an SPR sensor chip. Template TNT molecules were quickly removed with an organic solution of acetonitrile/acetic acid (9:1, v/v), causing a shift of 0.7° in SPR angle. The limit of detection for TNT was demonstrated to be as low as 1×10−8 mol/L. In the concentration range of 1×10−8−1×10−5 mol/L, the change of SPR angle was linear with the negative logarithm of TNT concentration. Selectivity studies showed that the SPR sensor had no response to TNT analogues 2,4,5-trinitrotoluene and 1,3,5-trinitrohexahydro-1,3,5-triazine at a concentration of 1×10−4 mol/L. The results suggest that the SPR sensor combined with a MIP film has the advantages of high sensitivity and selectivity, and long-term stability toward the analyte TNT.

Size-exclusion SPR sensor chip: application to detection of aggregation and disaggregation of biological particles

We propose a novel surface plasmon resonance (SPR) sensor chip with a microfabricated slit array. The microslit excludes micrometre-size objects larger than its slit size from the SPR sensing area, so that it functions as an in situ filter. We demonstrated the sensing of microparticles of different diameters using the chip, and the results show a successful size-exclusion effect. As a demonstration of the biological application, we performed the detection of aggregation and disaggregation of biological particles using sugar-chain-immobilized gold nanoparticles as a test sample.

Development of a label-free competitive ligand binding assay with human serum albumin on a molecularly engineered surface plasmon resonance sensor chip

The binding interactions between ligands and receptors play a vital role in their biological functions. In this work, a label-free indirect competitive binding assay based on surface plasmon resonance (SPR) detection was developed, using fatty acid–human serum albumin (HSA) as a model system. In the assay, a fatty acid analog was immobilized on the surface of an SPR gold chip by self-assembly, and was allowed to bind with HSA in solution. Addition of a ligand under investigation into the solution set up a competition with the analog for the protein, causing a reduction in the SPR signal. Due to the asymmetric structure of the ligand binding channel, HSA exhibited remarkable selectivity for the orientation of the fatty acid analog on a gold surface. When the carboxylic acid group of the analog was exposed on the sensor surface, the amount of bound HSA was very low. However, it increased by more than 7 fold when the alkyl chain was exposed. Dilution of the surface coverage of the fatty acid analog also enhanced HSA binding by reducing steric hindrance on the surface. Using a 1 : 4 mercaptoundecanoic acid–mercaptohexanol modified surface derivatized with octylamine, competitive binding assays for octanoic acid and perfluorooctane sulfonate with HSA were conducted. Using a derived kinetic equilibrium model, the equilibrium association constant was determined to be 4.57 × 105 M−1 and 2.11 × 105 M−1, respectively, which are consistent with the values measured by other established methods. The engineered sensor chip surface can be used to study the binding interactions of other chemicals with this important serum transport protein.

Signal enhancement of protein binding by electrodeposited gold nanostructures for applications in Kretschmann-type SPR sensors

We propose a novel Kretschmann-type surface plasmon resonance (SPR) sensor chip having a surface covered with electrodeposited gold nanostructures to enhance the sensitivity of SPR biosensing. The nanostructure is three-dimensional and has a larger surface area than a conventional flat surface chip, which increases the amount of protein binding and also induces a large change in the effective dielectric constant of the sensing area. The gold nanostructures were formed by electrodeposition under galvanostatic conditions, so their size could be controlled by manipulating the deposition time and current. The sensing characteristics, including the concentration dependence and noise level, indicated that the performance of the resulting chip (called a Au-black chip) was equivalent to that of a conventional sensor chip. We also determined the optimal electrodeposition conditions to obtain a sharp SPR curve for protein detection assay; the optimal thickness of the gold layer was 50-60 nm. Enhanced protein sensing was demonstrated by using a binding assay of anti-BSA antibody and BSA molecules. The protein binding signal was several times higher than that of the conventional assay. The insights into electrodeposition for SPR sensing presented here will enable improved sensitivity for detecting low-concentration and small proteins.

Dielectric coated plasmonic interfaces: their interest for sensitive sensing of analyte-ligand interactions

Surface plasmon resonance (SPR) sensors have matured over the last 2 decades into very powerful tools for the study of biomolecular interactions, chemical detection and immunoassays. The performance of the sensor depends on several parameters, such as the choice of the metal thin fi lm where the plasmonic wave propagates, the excitation wavelength and the refractive index (RI) of the glass prism. Next to these physical parameters, the strategy selected to bind the desired receptors to the SPR chip, has a strong infl uence on the overall sensitivity and selectivity of the device. This review focuses on the advancement made using lamellar SPR structures, where a thin dielectric layer is deposited onto the surface plasmon active metal thin fi lm. Silver-based SPR interfaces can be developed using this approach, as these overlayers allow an effi cient protection of the underlying silver fi lm. At the same time, these interfaces open the scope for new surface functionalization schemes, which can be employed for anchoring ligands to the SPR sensor chip. While self-assembled monolayers (SAMs) are widely used, due to the possibility of easily incorporating carboxylate, amine or hydroxyl groups, the drawbacks of such fi lms include limited chemical and electrochemical stability. Moreover, a poor orientation and potential problems of protein adsorption and fouling, is often encountered if no synthetic effort in the synthesis of more sophisticated thiols is made. In addition, while the surface chemistry developed on gold has been of great value, the limitations of working on gold are becoming more noticeable, with increasingly complex fabrication requirements for biometric systems and arrays. Lamellar SPR interfaces represent an alternative route. Finally, the contribution of the thin dielectric top layer to the sensitivity of SPR sensors will be discussed.

On-Line Monitoring 1H-Indole-3-Acetic Acid in Plant Tissues Using Molecular Imprinting Monolayer Techniques on a Surface Plasmon Resonance Sensor

A surface plasmon resonance (SPR)-based detection methodology, which allows the identification and quantification of the plant hormone, 1H-indole-3-acetic acid (IAA) in Peach, Rosa flower, and Crape myrtle bud is described. To achieve high selectivity, a molecularly imprinted monolayer (MIM) was prepared from alkanethiols self-assembled on an SPR sensor chip with preadsorbed template (IAA). The association constant of the MIM to IAA was calculated to be 2.8 × 1011 M−1 by analysis of the SPR wavenumber changes induced by different concentrations of IAA. A good calibration curve was obtained in a low concentration range of IAA. Moreover, the MIM revealed impressive selectivity with the selectivity factors of 1.0, 0.12, and 0.20 for IAA and other related analogues. Compared to the MIM sensor chip, the affinity of the self-assembled monolayer (SAM) control sensor chip for IAA was significantly low. The single SPR sensor decorated with the MIM was used to determine IAA concentrations in peach, Rosa flower, and Crape myrtle bud, and satisfactory results were obtained. Based on a signal to noise ratio of 3, the detection limits were 0.23 pM for Crape myrtle, 0.20 pM for Peach, and 0.32 pM for Rosa. The method showed good recoveries and precision, 95.7% (RSD 0.54%) for Peach, 98.1% (RSD 3.4%) for Rosa, and 98.1% (RSD 2.5%) for Crape myrtle, spiked with 0.4171 µg, respectively. These results indicate that a combination of SPR sensing and the MIM provides a simple, promising alternative method for determining endogenous IAA in plant tissue in a real-time manner.

Novel Polyphosphate-Binding Proteins in Human Plasma and Platelet Releasates

Abstract 2270 Introduction:Inorganic polyphosphates (polyP) are negatively charged, linear phosphate polymers found throughout biology. PolyP is secreted in substantial amounts by activated human platelets and it also accumulates in many infectious microorganisms. We have shown that polyP is a potent hemostatic regulator, acting at multiple points in the clotting cascade; in particular, polyP triggers the contact pathway, enhances FXI activation by thrombin, accelerates factor V activation, and modulates fibrin clot structure. Since there may be other, as-yet undiscovered (patho)physiologic roles for polyP, we undertook a study to identify the major polyP-binding proteins in human plasma and in releasates from activated human platelets. Methods:PolyP was end-labeled by biotinylation, then bound to streptavidin coated onto magnetic beads (Dynabeads), polystyrene microtiter plates, or surface plasmon resonance sensorchips. PolyP-Dynabeads were exposed to plasma or to releasates from platelets activated by TRAP; the beads were then washed repeatedly, and polyP-binding proteins were eluted with 1 M LiCl. Eluted proteins were separated by 2-D gel electrophoresis and were identified by mass spectrometry. Some additional proteins known or suspected to bind to polyP were also analyzed in parallel. Binding affinities for selected proteins were quantified by microtiter plate assays or by surface plasmon resonance approaches. Results:Candidate poly-P binding proteins identified in plasma pull-downs included parvulin, factor H, complement C1q, complement C1q tumor necrosis factor-related protein, C4 binding protein, and apolipoprotein E. Candidate poly-P binding proteins identified in pull-downs of platelet-releasate included platelet factor 4, multimerin, thrombospondin, and cyclophilin. High affinity binding to polyP was also quantified for thrombin, FXI, FXIa, kallikrien, histones and the isolated polyphosphate-binding domain of E. coli exopolyphosphatase. Conclusions:PolyP binds with high affinity to certain proteins that participate in the coagulation cascade as well as several additional proteins. Some of these are involved in reactions known to be modulated by polyP, but several have yet to be evaluated. Further investigation is indicated into potential roles for polyP in modulation of the complement cascade, prolyl isomerase activity, angiogenesis, and platelet responses. Disclosures:Smith:University of Illinois: Patents & Royalties. Choi:University of Illinois: Patents & Royalties. Morrissey:University of Illinois: Patents & Royalties.

Kinetic Analyses of Keap1–Nrf2 Interaction and Determination of the Minimal Nrf2 Peptide Sequence Required for Keap1 Binding Using Surface Plasmon Resonance

The Keap1-Nrf2 interaction plays important roles in regulation of Nrf2 activity and induction of chemopreventive enzymes. To better understand the interaction and to determine the minimal Nrf2 sequence required for Keap1 binding, we synthesized a series of Nrf2 peptides containing ETGE motif and determined their binding affinities to the Kelch domain of Keap1 in solution using a surface plasmon resonance-based competition assay. The equilibrium dissociation constant for the interaction between 16mer Nrf2 peptide and Keap1 Kelch domain in solution (K(solution)(D)) was found to be 23.9 nM, which is 10× lower than the surface binding constant (K(surface)(D)) of 252 nM obtained for the direct binding of Keap1 Kelch domain to the immobilized 16mer Nrf2 peptide on a surface plasmon resonance sensor chip surface. The binding affinity of Nrf2 peptides to Keap1 Kelch domain was not lost until after deletion of eight residues from the N-terminus of the 16mer Nrf2 peptide. The 9mer Nrf2 peptide has a moderate binding affinity with a (K(solution)(D)) of 352 nM and the affinity was increased 15× upon removal of the positive charge at the peptide N-terminus by acetylation. These results suggest that the minimal Nrf2 peptide sequence required for Keap1 binding is the 9mer sequence of LDEETGEFL.

Surface characterization and efficiency of a matrix-free and flat carboxylated gold sensor chip for surface plasmon resonance (SPR)

We report the preparation and characterization of a matrix-free carboxylated surface plasmon resonance (SPR) sensor chip with high sensing efficiency by functionalizing a bare gold thin film with a self-assembled monolayer of 16-mercaptohexadecanoic acid (SAM-MHDA chip). The self assembled monolayer surface coverage of the gold layer was carefully evaluated and the SAM was characterized by infrared reflection absorption spectroscopy, X-ray photoemission spectroscopy, atomic force microscopy, X-ray reflectivity-diffraction, and SPR experiments with bovine serum albumin. We compared the SPR signal obtained on this chip made of a dense monolayer of carboxylic acid groups with commercially available carboxylated sensor chips built on the same gold substrate, a matrix-free C1 chip, and a CM5 chip with a ~100 nm dextran hydrogel matrix (GE Healthcare). Two well-studied interaction types were tested, the binding of a biotinylated antibody (immunoglobulin G) to streptavidin and an antigen-antibody interaction. For both interactions, the well characterized densely functionalized SAM-MHDA chip gave a high signal-to-noise ratio and showed a gain in the availability of immobilized ligands for their partners injected in buffer flow. It thus compared favourably with commercially available sensor chips.

Orientation and capturing of antibody affinity ligands: Applications to surface plasmon resonance biochips

A surface plasmon resonance (SPR) sensor chip with immobilized protein G was used for simultaneously capturing, purifying and orienting antibody ligands. The ligands were further stabilized by chemical cross-linking. This procedure of designing the sensor chip improved efficient use of the ligands and could prolong the analytical use. The procedure was evaluated on standard dextran-coated sensor chips onto which commercial semi-purified antibodies towards human serum albumin and human troponin where captured and used for analysing their antigens. The procedure demonstrates a general design approach for presenting the biorecognition element on a biosensor surface which enhances sensitivity, stability and selectivity at the same time as an impure ligand is purified.

A novel method to prepare SPR sensor chips based on photografting molecularly imprinted polymer

Abstract A novel method to prepare surface plasmon resonance (SPR) sensor chips based on grafted imprinted polymer is explored. Benzophenone photografting system is used to grow molecularly imprinted polymer (MIP) films from the modified surface of gold substrate. The surface morphology and thickness of MIP films were investigated by scanning electronic microscope (SEM). The adsorption properties of sensor chip were studied by SPR spectroscopy. The results demonstrate that nano-MIP films can be constructed on the surface of gold substrate with the good adsorption of template molecules.

Comparative Assessment of Different Histidine-Tags for Immobilization of Protein onto Surface Plasmon Resonance Sensorchips

Surface plasmon resonance (SPR) is widely used to assess the kinetics and thermodynamics of binding of two molecules. The major challenge is immobilization of one molecule onto the sensorchip for robust detection of binding of the other molecule. We have compared a number of immobilization strategies for noncovalent attachment of an example protein (the substrate binding protein SiaP) by hexa-histidine (His), deca-His, and double-His tags to a nickel-nitrilotriacetic acid (NTA) surface. The stability of immobilization was assessed, and the binding of two low molecular weight ligands, Neu5Ac and 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN), at different temperatures studied. The hexa-His tagged SiaP washed off from the surface too rapidly for ligand binding to be measured reliably. Systematic variation of chip loading identified conditions under which the deca-His tagged SiaP could generate reliable results. The double-His tagged protein performed as well as covalently attached deca-His tagged protein at 15, 25, and 35 °C. The observed ligand binding kinetics were comparable for all immobilization strategies, and thermodynamic values calculated from SPR are in agreement with solution-based isothermal titration calorimetry measurements. Extended trials suggest that covalent attachment is preferable for screening campaigns, whereas the double-His-tag strategy allows rapid regeneration of the chip, for example, when tight binding compounds are assessed.

Spin-coated methacrylic acid copolymer thin films for covalent immobilization of small molecules on surface plasmon resonance substrates

Hydrophilic copolymers of methacrylic acid and ethylene glycol dimethacrylate are simply and rapidly prepared as thin films by spin-coating on gold-coated glass slides with a concurrent photo-crosslinking step. Coating techniques were optimized for use on gold surfaces both separately and as part of surface plasmon resonance (SPR) sensor chips. The population of carboxylic acid functional groups as binding sites in the polymer matrix, as reflected in the corresponding hydrophilicity, could be easily adjusted through changes to the stoichiometric ratio of the monomers, allowing for good control of immobilization capacity. The polymers used adhered to the gold surfaces both with and without use of thiol moieties. Coating thickness was measured by ellipsometry and coatings of 30–40 nm thickness were routinely achieved on gold-coated slides. This dimension is dependent on the spin speed and the viscosity of the polymerization mixture applied. The polymers were further characterized by contact angle measurements and infrared spectroscopy before being applied to immobilization of the steroid cortisol in a BIAcore SPR instrument, where binding to a monoclonal antibody was studied and the surface coatings optimized for maximum specific binding capacity. Optimized surfaces could be regenerated and re-used, and have potential applications as immobilization matrices in plasmonic biosensors with a very rapid coating technique.

Comparative surface plasmon resonance and enzyme-linked immunosorbent assay characterisation of a monoclonal antibody with N-acyl homoserine lactones

This study shows the detection of ( N-acyl) homoserine lactones (AHLs or HSL) with monoclonal antibodies via a surface plasmon resonance (SPR)-based immunosensor in comparison to conventional microtiter plate-based enzyme-linked immunosorbent assay (ELISA). An HSL derivative, named HSL2 ( Table 1), was attached to bovine serum albumin (BSA) and the conjugate (HSL2-BSA-r2) was either covalently immobilised on the SPR sensor chip surface via free amino groups or via adsorption on the ELISA polystyrene plate surface. With a newly developed rat monoclonal antibody (mAb HSL1/2 2C10), AHLs were detected sensitively in a competitive format with SPR and ELISA. Well comparable experiments between SPR and ELISA could be obtained in buffers. Moreover, the SPR sensor surface with the immobilised conjugate HSL2-BSA-r2 could be regenerated at least 340 times (regeneration cycles) without loss of activity. The measurement time per cycle was approximately 15 min. The competitive detection format for SPR and ELISA allowed the detection in the μg L −1 range.

Double recognition of oligonucleotide and protein in the detection of DNA methylation with surface plasmon resonance biosensors

DNA methylation plays an essential role in maintenance of cellular function. A growing number of human diseases have been found to be associated with aberrant DNA methylation, especially cancer. However, current technologies used in DNA methylation detection are complicated and time consuming. A promotor of the Adenomatous polyposis coli (APC) gene, a well-studied tumor suppressor gene, was used as the detection target DNA sequence. The double recognition mechanism was realized with oligonucleotide probe hybridization and specific protein binding. First, complementary target DNA was captured by the probe immobilized onto a surface plasmon resonance (SPR) sensor chip. Then, the recombinant methyl-CpG binding domain (MBD) protein was passed over the surface to recognize and bind to methylated CpG sites. Binding resulted in an increase in the refractive index, and a detectable optical signal was generated. Five picomoles of methylated APC promotor DNA could be easily detected with this method. The entire detection could be completed within 1 h. This work represents the first SPR based biosensor technology, which achieves simple and specific DNA methylation detection and avoids complicated bisulfite treatment and methylation-sensitive restriction digestion. It will improve our ability to detect DNA methylation specifically and rapidly, and promote our understanding of the role of DNA methylation in gene regulation and diseases.