A portable surface plasmon resonance sensor system for real-time monitoring of small to large analytes

Many environmental applications exist for biosensors capable of providing real-time analyses. One pressing current need is monitoring for agents of chemical- and bio-terrorism. These applications require systems that can rapidly detect small organics including nerve agents, toxic proteins, viruses, spores and whole microbes. A second area of application is monitoring for environmental pollutants. Processing of grab samples through chemical laboratories requires significant time delays in the analyses, preventing the rapid mapping and cleanup of chemical spills. The current state of development of miniaturized, integrated surface plasmon resonance (SPR) sensor elements has allowed for the development of inexpensive, portable biosensor systems capable of the simultaneous analysis of multiple analytes. Most of the detection protocols make use of antibodies immobilized on the sensor surface. The Spreeta 2000 SPR biosensor elements manufactured by Texas Instruments provide three channels for each sensor element in the system. A temperature-controlled two-element system that monitors for six analytes is currently in use, and development of an eight element sensor system capable of monitoring up to 24 different analytes will be completed in the near future. Protein toxins can be directly detected and quantified in the low picomolar range. Elimination of false positives and increased sensitivity is provided by secondary antibodies with specificity for different target epitopes, and by sensor element redundancy. Inclusion of more than a single amplification step can push the sensitivity of toxic protein detection to femtomolar levels. The same types of direct detection and amplification protocols are used to monitor for viruses and whole bacteria or spores. Special protocols are required for the detection of small molecules. Either a competition type assay where the presence of analyte inhibits the binding of antibodies to surface-immobilized analyte, or a displacement assay, where antibodies bound to analyte on the sensor surface are displaced by free analyte, can be used. The small molecule detection assays vary in sensitivity from the low micromolar range to the high picomolar.     

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.     

A DNA sensor based on surface plasmon resonance for apoptosis-associated genes detection

A practical and simple DNA sensor based on surface plasmon resonance (SPR) had been developed to determine apoptosis-associated genes, bcl-2 and bax. This SPR sensor was designed on the basis of simultaneous multi-wavelength detection. The complementary sequences of bcl-2 or bax oligonucleotide labeled with biotin were used as the probes. Biotin-avidin system was used to immobilize the bio-DNA on the sensor surface. The assembling processes and conditions for the DNA sensor were examined. The SPR sensor could be used to monitor the process of the immobility of the bio-DNA and DNA hybridization in real-time. The determination range of bcl-2 and bax oligonucleotide (20 bases) were 50-400 ng/mL. The determination range of polymerase chain reaction (PCR) product of bcl-2 (405 bases) was 5-60 ng/mL and PCR product of bax (538 bases) was 5-40 ng/mL. The stability, reversibility and specificity of the DNA sensor were also investigated. It was found from the experiment that the sensor could be applied for a quite long time (about 90 times). The relative standard deviation (R.S.D.) for determination oligonucleotides and PCR products of bcl-2 were 1.2 and 1.3%, respectively. The interference of noncomplementary DNA sequence with the determination of DNA was examined and it was found that noncomplementary 20-mer and 21-mer DNA (p53 and p21) do not affect the determination of bcl-2 or bax. This device could be used to study apoptosis and signal transduction routine genes. The sensor was shown to be of simplicity, sensitivity, selectivity, rapid response and cost effectiveness.     

A surface plasmon resonance probe with a novel integrated reference sensor surface

A surface plasmon resonance (SPR) sensor probe with integrated reference surface is described. In order to fabricate the integrated reference surface, two dielectric layers with different thickness were deposited on the single gold SPR sensor surface via plasma polymerization of hexamethyldisiloxane. The working sensor surface was a 34 nm dielectric layer with immobilized bovine serum albumin (BSA) antigen and an adjacent thin 1 nm dielectric layer without BSA provided reference surface. A specific immunoreaction of anti-BSA antibody was detected after immersion of the SPR probe into sample solution. Simultaneous observation of reference and working surface response enabled determination of the immunoreaction without the need for the baseline measurement. Moreover, compensation of nonspecific adsorption could be confirmed using anti-human serum albumin antibody.     

Preliminary approach of real-time monitoring in vitro matrix mineralization based on surface plasmon resonance detection

Matrix mineralization is a terminal process in osteoblast differentiation, and several approaches have been introduced to characterize the process in tissues or cultured cells. However, an analytical technique that quantitates in vitro matrix mineralization of live cells without any labeling or complex treatments is still lacking. In this study, we investigate a simple and enhanced optical method based on surface plasmon resonance (SPR) detection that can monitor the surface-limited refractive index change in real-time. During monitoring MC3T3-E1 cells in vitro culture every 2 days for over 4 weeks, the SPR angle is shifted with a greater resonance change in cells cultured with osteogenic reagents than those without the reagents. In addition, the SPR results obtained have a close relevance with the tendency of conventional mineralization staining and an inductively coupled plasma-based calcium content measure. These results suggest a new approach of a real-time SPR monitoring in vitro matrix mineralization of cultured cells.     

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.     

Surface plasmon resonance assay for real-time monitoring of somatic coliphages in wastewaters

The surface plasmon resonance (SPR) technique is a well-established method for the measurement of molecules binding to surfaces and the quantification of binding constants between surface-immobilized proteins and proteins in solution. In this paper we describe an extension of the methodology to study bacteriophage-bacterium interactions. A two-channel microfluidic SPR sensor device was used to detect the presence of somatic coliphages, a group of bacteriophages that have been proposed as fecal pollution indicators in water, using their host, Escherichia coli WG5, as a target for their selective detection. The bacterium, E. coli WG5, was immobilized on gold sensor chips using avidin-biotin and bacteriophages extracted from wastewater added. The initial binding of the bacteriophage was observed at high concentrations, and a separate, time-delayed cell lysis event also was observed, which was sensitive to bacteriophage at low concentrations. As few as 1 PFU/ml of bacteriophage injected into the chamber could be detected after a phage incubation period of 120 min, which equates to an approximate limit of detection of around 10(2) PFU/ml. The bacteriophage-bacterium interaction appeared to cause a structural change in the surface-bound bacteria, possibly due to collapse of the cell, which was observed as an increase in mass density on the sensor chip. These results suggest that this methodology could be employed for future biosensor technologies and for quantification of the bacteriophage concentration.     

Imaging surface plasmon resonance system for screening affinity ligands

A surface plasmon resonance (SPR) system for screening ligands for application in affinity chromatography is described. A combinatorial library of 13 ligands was synthesised, characterised and immobilised to agarose beads and gold SPR devices. Binding and elution behaviour and a range of K(AX) values (10(3) to 10(5) M(-1)) were measured against two target proteins, an insulin analogue (MI3) and a recombinant clotting factor (rFVIIa), in order to create a relational database between the traditional chromatographic format and the new SPR screening system. The SPR transducer surface was fabricated with affinity ligands in a two-dimensional, spatially addressable format, which was durable (>100 cycles) and stable over 6 months. The imaging SPR system comprised a direct optical, CCD-based, instrument capable of imaging the change in refractive index created by biochemical interactions and allowed affinity ligands to be evaluated 15-fold faster with 130-fold less target protein than conventional chromatographic methods. The binding and elution data from both the SPR and chromatographic systems for both target proteins were comparable, with the K(AX) value generating a nearly linear correlation (R(2)=0.875) and a slope bias of approximately 2.5+/-0.25-fold higher for the SPR system. The imaging SPR system has proven capable of screening and evaluating affinity ligands for potential use in the recovery of biopharmaceutical proteins.     

A miniature fiber optic surface plasmon resonance sensor for fast detection of Staphylococcal enterotoxin B

A fiber optic surface plasmon resonance (SPR) biosensor for detection of Staphylococcal enterotoxin B (SEB) is reported. The sensor is based on spectral interrogation of surface plasmons in a miniature sensing element based on a side-polished single-mode optical fiber with a thin metal overlayer. For specific detection of SEB, the SPR sensor is functionalized with a covalently crosslinked double-layer of antibodies against SEB. The SPR biosensor is demonstrated to be able to detect ng/ml concentrations of SEB in less than 10 min.     

Numerical simulations of surface plasmon resonance system for monitoring DNA hybridization and detecting protein-lipid film interactions

This paper presents a simple method to extract information about thin organic films from surface plasmon resonance (SPR) spectra. From numerical simulations it was found that a shift (Δθ _SPR) of an absorption peak in the SPR spectrum was directly proportional to the product of the thin organic film thickness and the refractive index difference between the thin organic film and a buffer soaking the sample. It was also found that Δθ _SPR was not sensitive to the thin organic film support of a gold film and a glass cover slip. Relationships between Δθ _SPR and distributions of macromolecule structures, in the thin organic films were theoretically established. Formulae were derived for a homemade SPR system to calculate length, transverse area, density and surface concentration of macromolecules in the thin organic film. The validity of these treatments was checked by precisely measuring the size of a single distearoylphosphatidylcholine molecule on a gold-supported phospholipid film; by quantitatively monitoring hybridization of synthesized oligonucleotides strands based on a biotin/avidin system; and by quantitatively detecting the steric hindrance of rabbit C-reactive protein specifically bound to phospholipid monolayers composed of synthesized lipids. Received: 4 May 1998 / Revised version: 27 July 1998 / Accepted: 27 August 1998     

Evaluation of surface hydrophobicity of immobilized protein with a surface plasmon resonance sensor

Immobilization is widely used to isolate agglutinative and associative proteins with large hydrophobic surfaces. Surface hydrophobicities of immobilized proteins were quantified by measuring the adsorption amounts of Triton X-100 as a hydrophobic probe with a biosensor that utilizes the phenomena of surface plasmon resonance (SPR). We measured SPR signal changes derived from adsorption of Triton X-100 to five kinds proteins and calculated the monolayer adsorption capacity using the Brunauer-Emmett-Teller equation, partly modified with a term for correcting an influence of the net charge of immobilized protein. SPR signal changes obtained by this method correlated with the values of surface hydrophobicities obtained by conventional assay using a hydrophobic probe. Thus this measuring method using an SPR sensor and Triton X-100 is expected to be a tool for quantifying surface hydrophobicities of immobilized proteins.     

Colloidal Au replacement assay for highly sensitive quantification of low molecular weight analytes by surface plasmon resonance

A novel sensing method based on surface plasmon resonance (SPR) was developed for the highly sensitive quantification of low molecular weight (LMW) analytes (colloidal Au replacement assay). Gold nanoparticles (diameter = 20 nm) functionalized with lactosyl-poly(ethylene glycol) (PEG) were prepared and were specifically adsorbed onto a Ricinus communis agglutinin (RCA120)-immobilized SPR sensor chip surface. Subsequent injection of free d-galactose elicited the elution of the preadsorbed lactosyl-PEGylated gold nanoparticles in a manner proportional to the galactose concentration, achieving a substantial and quantitative analysis over a wide range of galactose concentrations (0.1-50 ppm). This method of d-galactose sensing through the substituted elution of preadsorbed nanoparticles from the sensor chip surface would be applicable for the highly sensitive SPR quantification of various LMW analytes, which are known to be difficult to detect by the conventional SPR sensing regime.     

Real-time monitoring of odorant-induced cellular reactions using surface plasmon resonance

Surface plasmon resonance (SPR) is a powerful technique for measuring molecular interaction in real-time. SPR can be used to detect molecule to cell interactions as well as molecule to molecule interactions. In this study, the SPR-based biosensing technique was applied to real-time monitoring of odorant-induced cellular reactions. An olfactory receptor, OR I7, was fused with a rho-tag import sequence at the N-terminus of OR I7, and expressed on the surface of human embryonic kidney (HEK)-293 cells. These cells were then immobilized on a SPR sensor chip. The intensity of the SPR response was linearly dependent on the amount of injected odorant. Among all the aldehyde containing odorants tested, the SPR response was specifically high for octanal, which is the known cognate odorant for the OR I7. This SPR response is believed to have resulted from intracellular signaling triggered by the binding of odorant molecules to the olfactory receptors expressed on the cell surface. This SPR system combined with olfactory receptor-expressed cells provides a new olfactory biosensor system for selective and quantitative detection of volatile compounds.     

Nanodiscs allow the use of integral membrane proteins as analytes in surface plasmon resonance studies

Nanodiscs are small-sized and flat model membranes that provide a close to native environment for reconstitution of integral membrane proteins. Incorporation of membrane proteins into nanodiscs results in water-soluble proteolipid particles making the membrane proteins amenable to a multitude of bioanalytical techniques originally developed for soluble proteins. The transmembrane domain of the human CD4 receptor was fused to ubiquitin with a preceding N-terminal decahistidine tag. The resulting integral membrane protein was incorporated into nanodiscs. Binding of the nanodisc-inserted histidine-tagged protein to a monoclonal anti-pentahistidine antibody was quantified using surface plasmon resonance (SPR) experiments. For the first time, a membrane-inserted transmembrane protein was employed as analyte while the antibody served as ligand immobilized on the sensor chip surface. SPR experiments were conducted in single-cycle mode. We demonstrate that the nanodisc-incorporated membrane protein showed nearly identical affinity toward the antibody as did the soluble decahistidine-tagged ubiquitin studied in a comparative experiment. Advantages of the new experimental setup and potential applications are discussed.     

Sensitivity enhancement of surface plasmon resonance biosensing of small molecules

Surface plasmon resonance (SPR) biosensor formats using gold nanoparticle or protein signal amplification for the sensitive assay of small molecules were developed using progesterone as a model compound. Progesterone was immobilized to a dextran surface in the Biacore biosensor through in situ covalent immobilization using an oligoethylene glycol linker attached to the 4 position of the steroid. This surface produced stable antibody binding for in excess of 1100 assay cycles. Using this surface, assays were developed for progesterone using 10- and 20-nm gold-streptavidin labels attached to biotinylated monoclonal antibody in both label prebinding and sequential binding formats. Prelabeling formats gave no signal enhancement but produced assays with limits of detection of 143 pg/ml, compared with approximately 1 ng/ml in previous studies. Sequential binding formats gave signal enhancements of 2.2-fold over the monoclonal antibody and a limit of detection of 23.1 pg/ml. It was found that secondary antibody labeling gave 8.1-fold signal enhancements and a limit of detection of 20.1 pg/ml, whereas use of secondary antibody-25 nm gold complexes provided more signal enhancement (13-fold) and a further improvement in limit of detection of 8.6 pg/ml.     

Detection of TP53 mutation using a portable surface plasmon resonance DNA-based biosensor

A DNA-based surface plasmon resonance (SPR) biosensor has been developed for the detection of TP53 mutation using the inexpensive and commercially available instrument, SPREETA SPR-EVM-BT, from Texas Instruments. A direct immobilisation procedure, based on the coupling of thiol-derivatised oligonucleotide probes (Probe-C6-SH) to bare gold sensor surfaces, was optimized using synthetic oligonucleotides. Hybridisation reactions between the immobilised probe and a short sequence (26 mer) complementary, non-complementary and one-point mutation DNA were then investigated. The main analytical parameters of the sensor system were studied in detail including selectivity, sensitivity, reproducibility and analysis time. Finally, the sensor system was successfully applied to polymerase chain reaction (PCR)-amplified real samples, DNA extracted from both normal, wild-type, (Jurkat) and mutated (Molt 4), carrying the mutation at codon 248 of the TP53 cell lines. The results obtained demonstrate that the DNA-based SPR biosensor was able to distinguish sequences present in the various samples that differ only by one base; and hence, it appears to be a strong candidate technique for the detection of gene mutation.     

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.     

Imaging of electrochemical enzyme sensor on gold electrode using surface plasmon resonance

Three types of imaging, namely layer structure, electrochemical reaction, and enzyme sensor response, were achieved by applying surface plasmon resonance (SPR) measurement to an electrochemical biosensor. We constructed glucose oxidase based mediator type sensors on a gold electrode by spotting the mediator that contained horseradish peroxidase and spin coating the glucose oxidase film. The layer structure of the sensor was imaged by means of angle scanning SPR measurement. The single sensor spot (about 1 mm in diameter) consisted of about 100 x 100 pixels and its spatial structure was imaged. The multilayer structure of the enzyme sensor had a complex reflectance-incident angle curve and this required us to choose a suitable incident angle for mapping the redox state. We chose an incident angle that provided the most significant reflection intensity difference by using data obtained from two angle scanning SPR measurements at different electrode potentials. At this incident angle, we controlled the electrochemical states of the spotted mediator in cyclic voltammetry and imaged the degree to which the charged site density changed. Finally, we mapped the enzymatic activity around the mediator spot by the enzymatic reoxidation of pre-reduced mediator in the presence of glucose.     

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.     

Compact, high performance surface plasmon resonance imaging system

We report the construction and characterization of a new compact surface plasmon resonance imaging instrument. Surface plasmon resonance imaging is a versatile technique for detection, quantification and visualization of biomolecular binding events which have spatial structure. The imager uses a folded light path, wide-field optics and a tilted detector to implement a high performance optical system in a volume 7 in. x 4 in. x 2 in. A bright diode light source and an image detector with fast frame rate and integrated digital signal processor enable real-time averaging of multiple images for improved signal-to-noise ratio. Operating angle of the imager is adjusted by linear translation of the light source. Imager performance is illustrated using resolution test targets, refractive index test solutions, and competition assays for the antiepileptic drug phenytoin. Microfluidic flowcells are used to enable simultaneous assay of three sample streams. Noise level of refractive index measurements was found to decrease proportional to the square root of the number of pixels averaged, reaching approximately 5 x 10(-7) refractive index units root-mean-square for 160 x 120 pixels image regions imaged for 1s. The simple, compact construction and high performance of the imager will allow the device to be readily applied to a wide range of applications.