Surface plasmon resonance measurement of pH-induced responses of immobilized biomolecules: conformational change or electrostatic interaction effects?

Recently, the observation of pH-induced conformational changes of biomolecules supported on carboxymethyldextran (CMD)-coated surfaces measured using surface plasmon resonance (SPR) has been reported. However, it is apparent that the evidence reported in the literature is ambiguous. The research presented in this paper describes investigations to study the changing SPR signal of immobilized biomolecules as a function of varying pH, to provide a detailed understanding of the origin of the pH-induced changes in the SPR profile. SPR measurements were performed with cytochrome c, concanavalin A, and poly-L-lysine, biomolecules that exhibit diverse conformational responses to changing pH, covalently immobilized onto CMD-coated supports. These SPR measurements were supported by circular dichroism (CD) solution studies. The SPR profiles recorded were not consistent with the conformational transitions of the biomolecules as observed using CD. An alternative explanation for the observed shifts in SPR is proposed, which explains the SPR profiles in terms of electrostatic interaction effects between the immobilized biomolecules and the carboxymethyldextran matrix.     

Conducting polyamic acid membranes for sensing and site-directed immobilization of proteins

A biosensor platform based on polyamic acid (PAA) is reported for oriented immobilization of biomolecules. PAA, a functionalized conducting polymer substrate that provides electrochemical detection and control of biospecific binding, was used to covalently attach biomolecules, resulting in a significant improvement in the detection sensitivity. The biosensor sensing elements comprise a layer of PAA antibody (or antigen) composite self-assembled onto gold (Au) electrode via N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) linking. The modified PAA was characterized by Fourier transform infrared (FTIR), (1)H nuclear magnetic resonance (NMR), and electrochemical techniques. Cyclic voltammetry and impedance spectroscopy experiments conducted on electrodeposited PAA on Au electrode using ferricyanide produced a measurable decrease in the diffusion coefficient compared with the bare electrode, indicating some retardation of electron transfer within the bulk material of the PAA. Thereafter, the modified PAA surface was used to immobilize antibodies and then to detect inducible nitric oxide synthase and mouse immunoglobulin G (IgG) using enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), and amperometric techniques. ELISA results indicated a significant amplified signal by the modified PAA, whereas the SPR and amperometric biosensors produced significant responses as the concentration of the antigen was increased. Detection limits of 3.1×10(-3)ng/ml and 2.7×10(-1)ng/ml were obtained for SPR and amperometric biosensors, respectively.     

Label-enhanced surface plasmon resonance applied to label-free interaction analysis of small molecules and fragments

Surface plasmon resonance (SPR) is a well-established method for studying interactions between small molecules and biomolecules. In particular, SPR is being increasingly applied within fragment-based drug discovery; however, within this application area, the limited sensitivity of SPR may constitute a problem. This problem can be circumvented by the use of label-enhanced SPR that shows a 100-fold higher sensitivity as compared with conventional SPR. Truly label-free interaction data for small molecules can be obtained by applying label-enhanced SPR in a surface competition assay format. The enhanced sensitivity is accompanied by an increased specificity and inertness toward disturbances (e.g., bulk refractive index disturbances). Label-enhanced SPR can be used for fragment screening in a competitive assay format; the competitive format has the added advantage of confirming the specificity of the molecular interaction. In addition, label-enhanced SPR extends the accessible kinetic regime of SPR to the analysis of very fast fragment binding kinetics. In this article, we demonstrate the working principles and benchmark the performance of label-enhanced SPR in a model system—the interaction between carbonic anhydrase II and a number of small-molecule sulfonamide-based inhibitors.     

Nanotechnologies in proteomics

Progress in proteomic researches is largely determined by development and implementation of new methods for the revelation and identification of proteins in biological material in a wide concentration range (from 10(-3) M to single molecules). The most perspective approaches to address this problem involve (i) nanotechnological physicochemical procedures for the separation of multicomponent protein mixtures; among these of particular interest are biospecific nanotechnological procedures for selection of proteins from multicomponent protein mixtures with their subsequent concentration on solid support; (ii) identification and counting of single molecules by use of molecular detectors. The prototypes of biospecific nanotechnological procedures, based on the capture of ligand biomolecules by biomolecules of immobilized ligate and the concentration of the captured ligands on appropriate surfaces, are well known; these are affinity chromatography, magnetic biobeads technology, different biosensor methods, etc. Here, we review the most promising nanotechnological approaches for selection of proteins and kinetic characterization of their complexes based on these biospecific methods with subsequent MS/MS identification of proteins and protein complexes. Two major groups of methods for the analysis and identification of individual molecules and their complexes by use of molecular detectors will be reviewed: scanning probe microscopy (SPM) (including atomic-force microscopy) and cryomassdetector technology.     

Surface plasmon resonance biosensors incorporating gold nanoparticles

SPR biosensing is increasingly popular for the detection of a multitude of biomolecules. It offers label-free detection and study of proteins, nucleic acids, and other biomolecules in real time. A recent trend involves incorporation of AuNPs, either within the sensing surface itself or as signal enhancing tagging molecules. The importance of AuNP and detecting agent spacing is described and techniques using macromolecular spacing aids are highlighted. Recent methods to enhance SPR detection capabilities using gold nanoparticles are reviewed, as well as device fabrication and the results of incorporation. SPR detection is a highly versatile method for the detection of biomolecules and, with the incorporation of AuNPs, shows promise in extending it to a number of new applications.     

Sensitivity deviation: instrumental linearity errors that influence concentration analyses and kinetic evaluation of biomolecular interactions.

Many scientific instruments utilise multiple element detectors, e.g. CCD's or photodiode arrays, to monitor the change in a position of an optical pattern. For example. instruments for affinity biosensing based on surface plasmon resonance (SPR) or resonant mirror are equipped with such detectors. An important and desired property of these bioanalytical instruments is that the calculation of the movement or change in shape follows the true change. This is often not the case and it may lead to linearity errors, and to sensitivity errors. The sensitivity is normally defined as the slope of the calibration curve. A new parameter is introduced to account for the linearity errors, the sensitivity deviation, defined as the deviation from the undistorted slope of the calibration curve. The linearity error and the sensitivity deviation are intimately related and the sensitivity deviation may lead to misinterpretation of kinetic data, mass transport limitations and concentration analyses. Because the linearity errors are small (e.g. 10 pg/mm2 of biomolecules on the sensor surface) with regard to the dynamic range (e.g. 30,000 pg/mm2), they can be difficult to discover. However, the linearity errors are often not negligible with regard to a typical response (e.g. 0-100 pg/mm2). and may therefore cause serious problems. A method for detecting linearity errors is outlined. Further on, this paper demonstrates how integral linearity errors of less than 1% can result in a sensitivity deviation of 10%, a value that in our opinion cannot be ignored in biospecific interaction analysis (BIA). It should also be stressed out that this phenomenon also occurs in other instruments using array detectors.     

A novel ultrahigh-resolution surface plasmon resonance biosensor with an Au nanocluster-embedded dielectric film

The detection performance of conventional surface plasmon resonance (SPR) biosensors is limited to a 1 pg/mm(2) surface coverage of biomolecules, and consequently, such sensors struggle to detect the interaction of small molecules in low concentrations. The present study is attempted to propose the use of a novel SPR biosensor with Au nanoclusters embedded in a dielectric film to achieve a 10-fold improvement in the resolution performance. A co-sputtering method utilizing a multi-target sputtering system is used to fabricate the present dielectric films (SiO(2)) with embedded Au nanoclusters. It is shown that the sensitivity of the developed SPR biosensor can be improved by adjusting the size and volume fraction of the embedded Au nanoclusters in order to control the surface plasmon effect. The present gas detection and DNA hybridization experimental results confirm that the proposed Au nanocluster-enhanced SPR biosensor provides the potential to achieve an ultrahigh-resolution detection performance of approximately 0.1 pg/mm(2) surface coverage of biomolecules.     

Analysis of native proteins from biological fluids by biomolecular interaction analysis mass spectrometry (BIA/MS): exploring the limit of detection, identification of non-specific binding and detection of multi-protein complexes

Biomolecular interaction analysis mass spectrometry (BIA/MS) is a two-dimensional analytical technique that quantitatively and qualitatively detects analytes of interests. In the first dimension, surface plasmon resonance (SPR) is utilized for detection of biomolecules in their native environment. Because SPR detection is non-destructive, analyte(s) retained on the SPR-active sensor surface can be analyzed in a second dimension using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The qualitative nature of the MALDI-TOF MS analysis complements the quantitative character of SPR sensing and overcomes the shortcomings of the SPR detection stemming from the inability to differentiate and characterize multi-protein complexes and non-specific binding. In this work, the benefit of performing MS analysis following SPR sensing is established. Retrieval and detection of four markers present in biological fluids (cystatin C, beta-2-microglobulin, urinary protein 1 and retinol binding protein) was explored to demonstrate the effectiveness of BIA/MS in simultaneous detection of clinically related biomarkers and delineation of non-specific binding. Furthermore, the BIA/MS limit of detection at very low SPR responses was investigated. Finally, detection of in-vivo assembled protein complexes was achieved for the first time using BIA/MS.     

Chemoselective surface immobilization of proteins through a cleavable peptide

Surface immobilization of biomolecules is a fundamental step in several experimental techniques such as surface plasmon resonance analysis and microarrays. Oxime ligation allows reaching chemoselective protein immobilization with the retention of native-like conformation by proteins. Beside the need for chemoselective ligation of molecules to surface/particle, equally important is the controlled release of the immobilized molecules, even after a specific binding event. For this purpose, we have designed and assessed in an SPR experiment a peptide linker able to (i) anchor a given protein (enzymes, receptors, or antibodies) to a surface in a precise orientation and (ii) release the immobilized protein after selective enzymatic cleavage. These results open up the possibility to anchor to a surface a protein probe leaving bioactive sites free for interaction with substrates, ligands, antigens, or drugs and successively remove the probe-ligand complex by enzymatic cleavage. This peptide linker can be considered both an improvement of SPR analysis for macromolecular interaction and a novel strategy for drug delivery and biomaterial developments.     

Graphene/Au-Enhanced Plastic Clad Silica Fiber Optic Surface Plasmon Resonance Sensor

Owing to its large surface-to-volume ratio and good biocompatibility, graphene has been identified as a highly promising candidate as the sensing layer for fiber optic sensors. In this paper, a graphene/Au-enhanced plastic clad silica (PCS) fiber optic surface plasmon resonance (SPR) sensor is presented. A sheet of graphene is employed as a sensing layer coated around the Au film on the PCS fiber surface. The PCS fiber is chosen to overcome the shortcomings of the structured microfibers and construct a more stable and reliable device. It is demonstrated that the introduction of graphene can enhance the intensity of the confined electric field surrounding the sensing layer, which results in a stronger light-matter interaction and thereby the improved sensitivity. The sensitivity of graphene-based fiber optic SPR sensor exhibits more than two times larger than that of the conventional gold film SPR fiber optic sensor. Furthermore, the dynamic response analyses reveal that the graphene/Au fiber optic SPR sensor exhibits a fast response (5 s response time) and excellent reusability (3.5% fluctuation) to the protein biomolecules. Such a graphene/Au fiber optic SPR sensor with high sensitivity and fast response shows a great promise for the future biochemical application.     

Surface Plasmon Resonance Imaging Sensors: A Review

Surface plasmon resonance (SPR) imaging sensors realize label-free, real-time, highly sensitive, quantitative, high-throughput biological interaction monitoring and the binding profiles from multi-analytes further provide the binding kinetic parameters between different biomolecules. In the past two decades, SPR imaging sensors found rapid increasing applications in fundamental biological studies, medical diagnostics, drug discovery, food safety, precision measurement, and environmental monitoring. In this paper, we review the recent advances of SPR imaging sensor technology towards high-throughput multi-analyte screening. Finally, we describe our multiplex spectral-phase SPR imaging biosensor for high-throughput biosensing applications.     

Detection of Fusarium culmorum in wheat by a surface plasmon resonance-based DNA sensor

A surface plasmon resonance (SPR) sensor based on DNA hybridization has been developed for the detection of Fusarium culmorum, a fungal pathogen of cereals. A 0.57 kbp DNA fragment of F. culmorum was amplified by specific primers and a 25-mer oligonucleotide probe was selected within the sequence of the PCR amplicon. After biotinilation, the probe was immobilized on a streptavidin sensor chip and tested for biospecific interaction with PCR products of F. culmorum. The effect of denaturating agents (formamide and urea) and ionic strength (NaCl) on hybridization efficiency of double-stranded PCR products with the immobilized probe and the specificity of the probe were investigated. The SPR biosensor was successfully used for the detection of F. culmorum in culture material of different strains and in naturally infected wheat samples. Tested on fungal cultures, it showed a good selectivity for F. culmorum against other species of either Fusarium or other fungal genera. A background signal was observed in wheat samples strictly depending on the DNA amount of the testing matrix. Testing 30 ng of durum wheat DNA the detection limit was 0.06 pg of F. culmorum DNA. The developed PCR-SPR assay allowed to detect F. culmorum with sensitivity and specificity higher than gel-electrophoresis analysis.     

Biotinylated steroid derivatives as ligands for biospecific interaction analysis with monoclonal antibodies using immunosensor devices

Systematic ligand-binding studies of the biospecific interaction between steroids and antisteroid antibodies can be performed in real time using biosensor techniques. In this study, quartz crystal microbalance (QCM) and surface plasmon resonance (SPR) biosensor systems were applied. Different biotinylated testosterone (T) and 17beta-estradiol (E2) derivatives were preincubated with streptavidin and immobilized on the sensor surfaces. We obtained low matrix densities of antigen enabling the investigation of the binding kinetics and position specificities of various anti-E2 and anti-T monoclonal antibodies (mAbs) to these steroidal compounds. The highest immunoreactivity of anti-E2 and anti-T mAbs is not necessarily for the specific modified steroid that was used as a protein-coupled hapten for immunization. The kinetic data confirm that both 3- and 19-specific anti-T mAbs do not discriminate between the 3- and 19-biotinylated T derivatives, whereas the 7alpha-biotinylated T probe showed no affinity to these two anti-T mAbs. In the case of the 3-specific anti-E2 mAb, comparable interaction data were found for 3- and 6alpha-biotinylated E2 compounds. The 6-specific anti-E2 mAb showed comparable ligand binding, but a significant higher dissociation rate to the position-specific antigen. The QCM and SPR results correspond well to the data from cross-reactivity studies in solution as well as to enzyme immunoassay equilibrium measurements.     

Development of the myoglobin sensor based on the surface plasmon resonance

Bioaffinic sensor based on the surface plasmon resonance (SPR) with golden layer as a transducer was developed. This sensor was applied for control of antigen-antibodies (Ag-Ab) interactions. For modification of transducer surface the spontaneously organized molecular assemblies of two types of mercaptan were formed. It was shown that the created bioaffinic sensor allowed to detect minimal concentration of antibodies, about 1 ng/ml, to the human heart myoglobin, which preliminary was immobilized on the sensitive surface. The particularities of the dynamics of protein interaction were observed. Duration of incubation with the biospecific component was no more than 5 minutes. After rupturing of specific binding it was possible to obtain initial signal again during 7-9 cycles.     

Dynamics of complex formation between biological and luminescent conjugated polyelectrolytes--a surface plasmon resonance study

A water-soluble polythiophene, POWT, with zwitterionic peptide like side chains possess good characteristics for biosensor applications. The zwitterionic side chains of the polymer can couple to biomolecules via electrostatic and hydrogen bonding. This creates possibilities to imprint biomolecules to spin-coated polymer films with maintained functionality, and use the resulting matrix as a biosensor. Polymer-biomolecular interaction studies done with surface plasmon resonance (SPR) reveal a well performing sensor matrix with high affinity for DNA hybridizations as well as for protein detection. The responses are distinct and very specific. A directional dependence of antibodies binding to POWT layer has also been observed. The polymer films have also been characterized by optical methods. Emission and absorption measurements in different buffer systems confirm that the polymer matrix can undergo structural and conformational changes on surfaces. The dielectric function in the interval 300-800 nm of POWT is reported, based on variable angle spectroscopic ellipsometry. This modeling reveals that a considerable amount of water is included in the material. The polymer layer possesses the characteristics needed for biochip applications and micro array techniques.     

Modulation of biorecognition of glucoamylases with Concanavalin A by glycosylation via recombinant expression

Various types of glucoamylases were prepared to modulate their biospecific interaction with Concanavalin A. Glucoamylase Glm was isolated from the native yeast strain Saccharomycopsis fibuligera IFO 0111. Two glycosylated recombinant glucoamylases Glu's of S. fibuligera HUT 7212 were expressed and isolated from the strains Saccharomyces cerevisiae and one, nonglycosylated, from Escherichia coli. The biospecific affinity of those preparations to Concanavalin A was investigated and compared with the commercially available fungal glucoamylase GA from Aspergillus niger. All glycosylated enzymes showed affinity to Concanavalin A characterized by their precipitation courses and by the equilibration dissociation constants within the range from 1.43 to 4.17 × 10⁻⁶ M (determined by SPR method). The results suggested some differences in the interaction of Con A with the individual glucoamylases. The highest affinity to Con A showed GA. The recombinant glucoamylase Glu with the higher content of the saccharides was comprised by two binding sites with the different affinity. The glucoamylases with the lowest affinity (Glm and Glu with a lower content of saccharides) also demonstrated a nonspecific interaction with Con A in the precipitation experiments. The minimal differences between the individual glucoamylases were determined by the inhibition experiments with methyl--d-mannopyranoside.     

Photo-immobilization of biological components on gold-coated chips for measurements using surface plasmon resonance (SPR) and a quartz crystal microbalance (QCM)

The photo-immobilization technique is useful for immobilization of various biomolecules on assorted material surfaces, independent of the organic functional groups that may be present. Here, we report a convenient new photo-immobilization technique that was developed by combining a nonbiofouling polymer containing polyethylene glycol and a photoreactive crosslinker for surface plasmon resonance (SPR) and quartz crystal microbalance (QCM) measurements. By this method, nonspecific interactions were reduced and various types of molecules, bovine serum albumin, heparin, dsDNA, phosphatidylserine, Tobacco Mosaic Virus, and norfloxacine, were immobilized on an alkane thiol-modified gold surface by a single method. The interactions of photo-immobilized biomolecules and their corresponding antibodies were investigated by SPR and QCM. In addition, SPR imaging was possible using the present method.     

Study of the interaction between the G-quadruplex-forming thrombin-binding aptamer and the porphyrin 5,10,15,20-tetrakis-(N-methyl-4-pyridyl)-21,23H-porphyrin tetratosylate

The G-quadruplex DNA structure has been suggested to be a potential target for anticancer therapies. Therefore, there is increasing interest in the development of drugs that could modulate the stability of G-quadruplex structures. In the current work, the interaction between the thrombin-binding aptamer (TBA, 5′-GGT TGG TGT GGT TGG-3′), which can form an intramolecular G-quadruplex structure, and the porphyrin 5,10,15,20-tetrakis-(N-methyl-4-pyridyl)-21,23H-porphyrin tetratosylate (TmPyP4) was studied. The application of a high-performance liquid chromatography-photodiode array (HPLC-PDA) detector-based method to study this kind of interaction was tested. Molecular absorption data recorded along the chromatographic runs were analyzed by means of multivariate data analysis methods. Moreover, biospecific interaction analysis (BIA) by surface plasmon resonance (SPR) and melting and mole ratio experiments monitored by UV-visible molecular absorption and circular dichroism spectroscopies, were applied to confirm and expand the chromatographic studies. The results showed the formation of an interaction complex with a stoichiometry 1:1 (TmPyP4/TBA) and logarithm of the equilibrium constant equal to 5.7 ± 0.2. Melting and circular dichroism data reflected that the initial G-quadruplex structure of TBA is stabilized in the interaction complex, being slightly distorted by the presence of the ligand.     

A miniaturized germanium-doped silicon dioxide-based surface plasmon resonance waveguide sensor for immunoassay detection

A surface plasmon resonance (SPR) waveguide immunosensor fabricated by germanium-doped silicon dioxide was investigated in this study. The designed waveguide sensor consisted of a 10 microm SiO(2) substrate layer (n=1.469), a 10 microm Ge-SiO(2) channel guide (n=1.492) and a 50 nm gold film layer for immobilization of biomolecules and SPR signal detection. The resultant spectral signal was measured by a portable spectrophotometer, where the sensor was aligned by a custom-designed micro-positioner. The results of the glycerol calibration standards showed that the resonance wavelength shifted from 628 to 758 nm due to changes of refractive index from 1.36 to 1.418. Flow-through immunoassay on waveguide sensors also showed the interactions of protein A, monoclonal antibody (mAb ALV-J) and avian leucosis virus (ALVs) resulted in wavelength shifting of 4.17, 3.03 and 2.18 nm, respectively. The SPR dynamic interaction could also be demonstrated successfully in 4 min as the sensor was integrated with a lateral flow nitrocellulose strip. These results suggest that SPR detection could be carried out on designed waveguide sensor, and the integration of nitrocellulose strip for sample filtering and fluid carrier would facilitate applications in point-of-care portable system.     

Short order nanohole arrays in metals for highly sensitive probing of local indices of refraction as the basis for a highly multiplexed biosensor technology

A small array of subwavelength apertures patterned in a gold film on glass was characterized for use as a biosensor. It is widely believed that such arrays allow the resonance of photons with surface plasmons in the metallic film. Surface plasmon methods (and other evanescent wave methods) are extremely well suited for the measure of real time biospecific interactions. An extremely high sensitivity of 88,000%/refractive index unit was measured on an array with theoretical active area of .09 microm2. The formation of a biological monolayer was monitored. Both sensitivity and resolution were determined through measurement. The measured resolution, for a sensor with an active area of less than 1.5 microm2, is 9.4 x 10(-8) refractive index units which leads to a calculated sensitivity of 3.45E6%/refractive index unit. These values far exceed theoretical and calculated values of other grating coupled surface plasmon resonance (SPR) detectors and prism based SPR detectors. Because the active sensing area can be quite small (.025 microm2) single molecule studies are possible as well as massive multiplexing on a single chip format.