Flow immunoassay of trinitrophenol based on a surface plasmon resonance sensor using a one-pot immunoreaction with a high molecular weight conjugate

A surface plasmon resonance (SPR) immunosensor based on a competitive immunoreaction for the determination of trinitrophenol (TNP) is described. A goat anti-mouse IgG (1st antibody), which recognizes an Fc moiety of an antibody, was immobilized on a gold film of an SPR sensor chip by physical adsorption. A TNP solution containing a fixed concentration of a mouse anti-TNP monoclonal antibody (2nd antibody) and a TNP-keyhole limpet hemocyanin (KLH) conjugate was incubated in one-pot and introduced into the sensor chip. The TNP-KLH conjugate competes with TNP for binding with the 2nd antibody. The resulting complex of the 2nd antibody with the TNP-KLH conjugate was bound to the 1st antibody, which is immobilized on the sensor chip. The SPR sensor signal based on resonance angle shift is dependent on the concentration of TNP in the incubation solution in the range from 25 ppt to 25 ppb, and the coefficient of variation of the SPR signals for the 25 ppb TNP solution was determined to be 13% (n = 4). The experimental results for the adsorption constant of the 1st antibody on the sensor chip and the binding constant of the 1st antibody complex with the 2nd antibody are discussed, together with theoretical considerations.     

An optical immunosensor based on surface plasmon resonance for determination of bFGF

An optical immunosensor based on surface plasmon resonance (SPR) has been developed for immunosensing. The sensor is designed on the basis of fixing incident angle of light and measuring the reflected intensities in the wavelength range of 400-800 nm simultaneously. The SPR spectrum was shown in terms of reflected light intensities versus wavelengths of incident light. The intensity of the reflected light reaches the minimum at the resonant wavelength. Molecular self-assembling in solution is used to form the sensing membrane on gold substrate. The kinetic processes of sensing monolayer formation were studied. The basic fibroblast growth factor, a kind of basic polypeptide, was determined in the concentration range of 0.24-9.6 μg/ml. Under optimum experimental conditions, the sensor has a good repeatability, reversibility and selectivity.     

Immobilization of antibody fragment for immunosensor application based on surface plasmon resonance

Biosurface fabrication using the Fab′ fragment of immunoglobulin (IgG) was carried out by self-assembly (SA) technique. The pepsin-digested monoclonal antibody (Mab) against bovine insulin containing the F(ab′)₂ fragment and residual proteins was separated using affinity chromatography and dialysis. To prevent the nonspecific binding of F(ab′)₂ onto gold (Au) substrate, the native disulfide bridge was reduced using dithiothreitol (DTT) to convert F(ab′)₂ into Fab′, which made the immobilization to be carried out via the native thiol (–SH) group. The fabricated biosurface using SA technique showed the formation of stable thin film through AFM topography. Through the concentration change of DTT and Fab′, the absorption characteristics against the Au surface were investigated using surface plasmon resonance (SPR) with the flow cell. The amount of immobilized antibody fragment and the antigen binding capacity were regulated with respect to the reduction state and concentration of F(ab′)₂. Based on the biosurface of the fabricated Fab′, the insulin-detection was carried out by the measurement of SPR. The proposed antibody surface could successfully detect the bovine insulin at the concentration from 100 ng/mL to 10 μg/mL.     

Recent advances in surface plasmon resonance based techniques for bioanalysis

Surface plasmon resonance (SPR) is a powerful and versatile spectroscopic method for biomolecular interaction analysis (BIA) and has been well reviewed in previous years. This updated 2006 review of SPR, SPR spectroscopy, and SPR imaging explores cutting-edge technology with a focus on material, method, and instrument development. A number of recent SPR developments and interesting applications for bioanalysis are provided. Three focus topics are discussed in more detail to exemplify recent progress. They include surface plasmon fluorescence spectroscopy, nanoscale glassification of SPR substrates, and enzymatic amplification in SPR imaging. Through these examples it is clear to us that the development of SPR-based methods continues to grow, while the applications continue to diversify. Major trends appear to be present in the development of combined techniques, use of new materials, and development of new methodologies. Together, these works constitute a major thrust that could eventually make SPR a common tool for surface interaction analysis and biosensing. The future outlook for SPR and SPR-associated BIA studies, in our opinion, is very bright. Surface plasmon resonance (SPR) is a powerful and versatile spectroscopic method for biomolecular interaction analysis (BIA) and has been well reviewed in previous years. This updated 2006 review of SPR, SPR spectroscopy, and SPR imaging explores cutting-edge technology with a focus on material, method, and instrument development. A number of recent SPR developments and interesting applications for bioanalysis are provided. Three focus topics are discussed in more detail to exemplify recent progress. They include surface plasmon fluorescence spectroscopy, nanoscale glassification of SPR substrates, and enzymatic amplification in SPR imaging. Through these examples it is clear to us that the development of SPR-based methods continues to grow, while the applications continue to diversify. Major trends appear to be present in the development of combined techniques, use of new materials, and development of new methodologies. Together, these works constitute a major thrust that could eventually make SPR a common tool for surface interaction analysis and biosensing. The future outlook for SPR and SPR-associated BIA studies, in our opinion, is very bright.      

Surface plasmon resonance biosensor based on water-soluble ZnO-Au nanocomposites

A wavelength modulation surface plasmon resonance biosensor based on ZnO-Au nanocomposites for the detection of human IgM was developed. Self-assembly technique has the advantages of flexibility, simplicity and the precise control of film component and was applied to the building of the sensor. The ZnO-Au nanocomposites are in a dumbbell-like shape and can be immobilized on the Au film through 1,6-hexanedithiol by covalent attachment. Meanwhile the activated ZnO nanocrystals can be used to connect protein. The biosensor based on ZnO-Au nanocomposites was used to detect human IgM. Some experimental conditions were examined and optimized. In the selected conditions, the modified biosensor exhibits a satisfactory response for human IgM in the concentration range of 0.30-20.00 μg mL⁻¹. However, the biosensor without ZnO-Au nanocomposites shows a response for human IgM in the concentration range of 1.25-20.00 μg mL⁻¹. Compared with the biosensor based on Au film, when the biosensor based on the ZnO-Au nanocomposites was applied, the sensitivity for determination of human IgM is significantly enhanced.     

Surface plasmon resonance biosensor for enrofloxacin based on deoxyribonucleic acid

A DNA-based surface plasmon resonance biosensor for enrofloxacin was developed. Heating denatured DNA immobilized on the gold-coated glass surface was exploited. The immobilization was performed by a layer-by-layer co-deposition with a cationic polymer. The sensor performance was tested with real biological probes. Direct and simple determination of enrofloxacin in milk samples was demonstrated. The sensor response obeys Langmuir binding isotherm being almost linear until about 20 μg mL⁻¹. The detection limit in milk samples was estimated to be 3 μg mL⁻¹.     

Enhanced surface plasmon resonance immunoassay for human complement factor 4

Using an enhanced surface plasmon resonance (SPR) immunosensor, we have determined the concentration of human complement factor 4 (C4). Antibody protein was concentrated into a carboxymethyldextran-modified gold surface by electrostatic attraction force and a simultaneous covalent immobilization of antibody based on amine coupling reaction took place. The sandwich method was applied to enhance the response signal and the specificity of antigen binding assay. The antibody immobilized surface had good response to C4 in the range of 0.02-20 μg/ml by this enhanced immunoassay. The regeneration effect by pH 2 glycine-HCl buffer was also investigated. The same antibody immobilized surface could be used more than 80 cycles of C4 binding and regeneration. In addition, the ability to determinate C4 directly from serum sample without any purification was investigated. The sensitivity, specificity and reproducibility of the enhanced immunoassay are satisfactory. The results clearly demonstrate the advantages of the enhanced SPR technique for C4 immunoassay.     

Surface plasmon resonance biosensor for genetically modified organisms detection

The development of a surface plasmon resonance (SPR) affinity biosensor based on DNA hybridisation is described. This biosensor has been applied to genetically modified organisms (GMOs) detection. Single stranded DNA (ssDNA) probes were immobilised on the sensor chip of an SPR device and the hybridisation between the immobilised probe and the complementary sequence (target) was monitored. The probe sequences were internal to the sequence of 35S promoter and NOS terminator which are inserted sequences in the genome of GMO regulating the transgene expression. The system has been optimised using synthetic oligonucleotides, then applied to real samples analysis. Samples, containing the transgenic target sequences, were amplified by polymerase chain reaction (PCR) and then detected with the SPR biosensor.     

Surface plasmon resonance

During last decade there has been significant progress in the development of analytical techniques for evaluation of receptor-ligand iteraction. Surface plasmon resonance (SPR)-based optical biosensors are now being used extensively to defined the kinetics of wide variety of macromolecular interactions and high- and low-affinity small molecule interactions. The experimental design data analysis methods are evolving along with widespread applications in ligand fishing, microbiology, virology, host-pathogen interaction, epitope mapping and protein-, cell-, membrane-, nucleic acid-protein interactions. SPR based biosensors have strong impact on basic and applied research significantly. This brief review describes the SPR technology and few of its applications in relation to receptor-ligand interaction that has brought significant change in the methodology, analysis, interpretation, and application of the SPR technology.     

Surface plasmon resonance-based trace detection of small molecules by competitive and signal enhancement immunoreaction

A surface plasmon resonance (SPR)-immunosensor for detection of the low molecular weight compound 2,4-dinitorophenol (DNP) at ultra-low concentration has been developed. The sensor strategy is based on a competitive immunoreaction between DNP and a DNP-protein conjugate, namely DNP-bovine serum albumin conjugate (DNP-BSA). Anti-DNP monoclonal antibody was immobilized on a gold thin-film coated SPR-sensor chip by means of a chemical coupling process. DNP-BSA, on contact with the anti-DNP antibody immobilized SPR-immunosensor chip causes an increase in the resonance angle of the sensor chip. The optimum concentration of immobilized antibody on the SPR-sensor chip is 100 μg mL⁻¹. The SPR-immunosensor response for free DNP determination using the competitive immunoreaction had a response time of ca. 15 min. Using this method, DNP could be determined in the concentration range 1 ppt to 1 ppb. The SPR signal for ppt levels of DNP was enhanced by a factor of three by subsequently treating immuno-bound DNP-BSA with a secondary anti-DNP antibody.     

Single-crystal sapphire-fiber optic sensors based on surface plasmon resonance spectroscopy for in situ monitoring

Single-crystal sapphire-fiber optic sensors based on surface plasmon resonance (SPR) for refractive index (RI) measurements of aqueous and hydrothermal water solutions are described. Accurate measurement of RIs is essential to efficient operation and control of broad range of engineering processes. Some of these processes are carried out with harsh environments, such as high-temperature, high pressure, and chemical corrosion. These extreme physical conditions are proving a limiting factor in application of the conventional silica-based optical sensors. Single-crystal sapphire is an ideal material for sensor applications, where reliable performance is required in the extreme environment conditions. With regard to the liquid species detection, most applications of SPR sensors are designed to function near the refractive index of water (1.3330 RI). The RI changes of aqueous solution can be easily monitored by silica-fiber (RI, 1.4601 at 550 nm) based SPR sensor. However, the sapphire waveguide has a prohibitively high RI (1.7708 at 546 nm) for unmodified monitoring of the RI changes of aqueous solutions. For that purpose, a practical SPR probe geometry has been applied to the ability to tune the SPR coupling wavelength/angle pair with sapphire-fiber based SPR probe.     

The effect of low pH on the glycitein-BSA conjugate interaction with specific antiserum: competitive inhibition study using surface plasmon resonance technique

Competitive inhibition serological assay for detection of the phytoestrogen glycitein (Glyc) was developed using surface plasmon resonance (SPR) technique with protein conjugates and polyclonal antibodies initially designed for the enzyme-linked immunosorbent assays (ELISA). The efficiency of the approach to the quantification of the soy isoflavone glycitein in water was investigated using the competitive reaction of analyte (free Glyc)and immobilized Glyc-BSA-conjugate with polyclonal antibodies. It was shown that the efficiency to detect Glyc drastically depends on the pH level of the probe solution. With the decrease in pH from 7.4 to 4.0, (i) the affinity of the specific reaction increases and (ii) the level of unspecific sorption becomes saturated. Non-specific adsorption to a SPR sensor surface obscures the specific component and shaded specific response at higher pH (6.0-7.4) when used serum for the quantification of specific analytes. The standard curves obtained in acidic solutions (pH 4-5) indicate that the linear part of the dependence completely covers the range between detection limit (0.1 μg/ml) and Glyc solubility in water (0.9 μg/ml). The difference in SPR- and ELISA-based analytical protocols as well as the requirements for increasing the efficiency in quantitative SPR analysis using purified antibodies is discussed.     

Surface plasmon resonance sensing of nucleic acids: A review

Biosensors based on surface plasmon resonance (SPR) have become a central tool for the investigation and quantification of biomolecules and their interactions. Nucleic acids (NAs) play a vital role in numerous biological processes and therefore have been one of the major groups of biomolecules targeted by the SPR biosensors. This paper discusses the advances of NA SPR biosensor technology and reviews its applications both in the research of molecular interactions involving NAs (NA–NA, NA–protein, NA–small molecule), as well as for the field of bioanalytics in the areas of food safety, medical diagnosis and environmental monitoring.     

Organic vapour sensing using localized surface plasmon resonance spectrum of metallic nanoparticles self assemble monolayer

The response of localized surface plasmon resonance (LSPR) spectra of gold and silver nanoparticles, and gold nanoshells to organic vapors was investigated. The surface area of nanomaterials was sufficiently high for quantitative adsorption of volatile organic compounds (VOCs). Surface adsorption and condensation of VOCs caused the environmental refractive index to increase from n = 1.00 in pure air to as high as n = 1.29 in near saturated toluene vapor. The extinction and wavelength shift of the LSPR spectra were very sensitive to changes in the surface refractive index of the nanoparticles. Responses of the LSPR band were measured with a real-time UV-vis spectrometer equipped with a CCD array detector. The response of silver nanoparticles to organic vapors was most sensitive in changes in extinction, while gold nanoshells exhibited red-shifts in wavelength (∼250 nm/RIU) when exposed to organic vapors. The LSPR spectral shifts primarily were determined by the volatility and refractive indices of the organic species. The T₉₀ response time of the VOC-LSPR spectrum was less than 3 s and the response was completely reversible and reproducible.     

Surface plasmon resonance study on the interaction between the fructose and phenylboronic acid monolayer

Phenylboronic acid derivatives were synthesized and their self-assembled monolayers (SAMs) were formed on a gold surface. The interaction between fructose and phenylboronic acid monolayers was evaluated using surface plasmon resonance (SPR). These phenylboronic acid monolayers showed good sensitivity to fructose at a low concentration range and the resonance angle shifts increased in accordance with the alkyl chain length.     

Surface plasmon resonance immunosensor for ErbB2 breast cancer biomarker determination in human serum and raw cancer cell lysates

A highly sensitive surface plasmon resonance (SPR) immunosensor for the important ErbB2 breast cancer biomarker has been developed. Optimization of the assay has been carried out, including signal enhancement employing gold nanoparticles (GNPs). The effect of the signal amplification of the GNPs has been also studied. The assay has been tested with clinically relevant matrices. Results in 50% human serum yielded a LOD of 180 pg mL⁻¹ which is a concentration 83 times lower than the clinical cut-off. Raw lysates from model breast cancer cell lines (SK-BR-3, MCF-7 and MDA-MB-436) have been also assayed and higher quantities of the ErbB2 protein were clearly observed in the ErbB2 over-expressing case (SK-BR-3). The results confirmed that the simple and highly sensitive SPR immunosensor represents a feasible tool for ErbB2 detection.     

Surface plasmon resonance sensor for lysozyme based on molecularly imprinted thin films

Molecularly imprinted polymers (MIPs) selective for lysozyme were prepared on SPR sensor chips by radical co-polymerization with acrylic acid and N,N′-methylenebisacrylamide. Gold-coated SPR sensor chips were modified with N,N′-bis(acryloyl)cystamine, on which MIP thin films were covalently conjugated. The presence of NaCl during the polymerization and the re-binding tests affected the selectivity and the optimization of NaCl concentration in the pre-polymerization mixture and the re-binding buffer could enhance the selectivity in the target protein sensing. When the lysozyme-imprinted polymer thin films were prepared in the presence of 40 mM NaCl, the selectivity factor (target protein bound/reference protein bound) of MIP in the re-binding buffer containing 20 mM NaCl was 9.8, meanwhile, that of MIP in the re-binding buffer without NaCl was 1.2. A combination of SPR sensing technology with protein-imprinted thin films is a promising tool for the construction of selective protein sensors.     

Surface plasmon resonance sensors for real-time detection of cyclic citrullinated peptide antibodies

Surface plasmon resonance (SPR) sensors have been used for detection of various biomolecules because of their simplicity, high specificity and sensitivity, real-time detection, low cost, and no requirement of labeling. Recently, molecularly imprinted polymers that are easy to prepare, less expensive, stable, have talent for molecular recognition and also are used for creation selective binding sites for target molecule on the SPR sensors. Here, we show that preparation of cyclic citrullinated peptide antibody (anti-CCP) imprinted SPR sensor to detect CCP antibodies. For this purpose, anti-CCP/AAm pre-complex was synthesized by interacting acrylamide (AAm) monomer with anti-CCP. Then, anti-CCP imprinted (anti-CCP/PAAm) SPR sensor was obtained by reacting with anti-CCP/AAm pre-complex in the presence of the crosslinker, and initiator/activator pair. Besides this, non-imprinted (PAAm) SPR sensor was also prepared without using anti-CCP template. The SPR sensors were characterized and then adsorption-desorption studies were performed with pH 7.0 phosphate buffer (10 mM) and acetic acid (10%) with Tween 20 (1%) in pH 7.0 phosphate buffer. Selectivitiy of sensors was investigated by using immunoglobulin M (IgM) and bovine serum albumin (BSA). To determine the adsorption model of interactions between anti-CCP solutions and anti-CCP/PAAm SPR sensor, different adsorption models were performed. The calculated maximum reflection, detection limit, association and dissociation constants were 1.079 RU/mL, 0.177 RU/mL, 0.589 RU/mL and 1.697 mL/RU, respectively. Repeatability experiments of anti-CCP/PAAm SPR sensor was performed four times with adsorption-desorption-regeneration cycles without any performance losing. Results showed that anti-CCP/PAAm SPR sensor had high selectivity and sensitivity for detection of CCP antibodies.     

Ultrasensitively sensing acephate using molecular imprinting techniques on a surface plasmon resonance sensor

An ultrathin molecularly imprinted polymer film was anchored on an Au surface for fabricating a surface plasmon resonance sensor sensitive to acephate by a surface-bound photo-radical initiator. The polymerization in the presence of acephate resulted in a molecular-imprinted matrix for the enhanced binding of acephate. Analysis of the SPR wavenumber changes in the presence of different concentrations of acephate gave a calibration curve that included the ultrasensitive detection of acephate by the imprinted sites in the composite, K_ass for the association of acephate to the imprinted sites, 7.7 × 10¹² M⁻¹. The imprinted ultrathin film revealed impressive selectivity. The selectivity efficiencies for acephate and other structurally related analogues were 1.0 and 0.11-0.37, respectively. Based on a signal to noise ratio of 3, the detection limits were 1.14 × 10⁻¹³ M for apple sample and 4.29 × 10⁻¹⁴ M for cole sample. The method showed good recoveries and precision for the apple and cole samples spiked with acephate solution. This suggests that a combination of SPR sensing with MIP film is a promising alternative method for the detection of organophosphate compounds.     

Surface plasmon resonance study on HIV-1 integrase strand transfer activity

Understanding the molecular mechanism of HIV-1 integrase (IN) activity is critical to find functional inhibitors for an effective AIDS therapy. A robust, fast, and sensitive method for studying IN activity is required. In this work, an assay for real-time label-free monitoring of the IN activity based on surface plasmon resonance was developed. This assay enabled direct monitoring of the integration of a viral doubled-stranded (ds) DNA into the host genome. The strand transfer reaction was detected by using two different DNA targets: supercoiled plasmid (pUC 19) and short palindrome oligonucleotide. The effect of the length of the DNA target on the possibility to monitor the actual process of the strand transfer reaction is discussed. The surface density of integrated ds-DNA was determined. IN binding to the oligonucleotide complexes and model DNA triplexes in the presence of various divalent ions as metal cofactors was investigated as well. The assay developed can serve as an important analytical tool to search for potential strand transfer reaction inhibitors as well as for the study of compounds interfering with the binding of ds long terminal repeats–IN complexes with the host DNA. HIV-1 integrase strand transfer activity was monitored in real time using a multichannel surface plasmon resonance biosensor. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.