Micropreparative ligand fishing with a cuvette-based optical mirror resonance biosensor

We have previously demonstrated the role of an optical biosensor (BIAcore 2000) as a specific detector to monitor chromatographic fractions during the purification and characterisation of ligands for orphan biomolecules. We have now extended this application to perform micropreparative ligand fishing directly on the sensor surface using an automated cuvette-based optical biosensor (Iasys Auto+) equipped with a high-capacity carboxymethyldextran surface (surface area 16 mm2). Using a F(ab)2' fragment of the A33 monoclonal antibody as bait, we have recovered microgram quantities of essentially homogeneous A33 ligand from the sensor surface in a form suitable for subsequent sensitive and specific down stream analysis (micropreparative HPLC, sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting). The design of the cuvette-based system facilitates recovery of desorbed material from the constrained workspace in small volumes at high concentration. The use of on-surface detection allows the surface viability to be continuously monitored and permits direct quantitation of both bound and recovered material.     

Chromatographic separation simulation of metal‐chelating peptides from surface plasmon resonance binding parameters

Some metal‐chelating peptides have antioxidant properties, with potential nutrition, health, and cosmetics applications. This study aimed to simulate their separation on immobilized metal ion affinity chromatography from their affinity constant for immobilized metal ion determined in surface plasmon resonance, both technics are based on peptide‐metal ion interactions. In our approach, first, the affinity constant of synthetic peptides was determined by surface plasmon resonance and used as input data to numerically simulate the chromatographic separation with a transport‐dispersive model based on Langmuir adsorption isotherm. Then, chromatographic separation was applied on the same peptides to determine their retention time and compare this experimental t_R with the simulated t_R obtained from simulation from surface plasmon resonance data. For the investigated peptides, the relative values of t_R were comparable. Hence, our study demonstrated the pertinence of such numerical simulation correlating immobilized metal ion affinity chromatography and surface plasmon resonance.     

Detection of microcystins in environmental samples using surface plasmon resonance biosensor

An indirect inhibitive surface plasmon resonance (SPR) immunoassay was developed for the microcystins (MCs) detection. The bioconjugate of MC-LR and bovine serum albumin (BSA) was immobilized on a CM5 sensor chip. A serial premixture of MC-LR standards (or samples) and monoclonal antibody (mAb) were injected over the functional sensor surface, and the subsequent specific immunoreaction was monitored on the BIAcore 3000 biosensor and generated a signal with an increasing intensity in response to the decreasing MCs concentration. The developed SPR immunoassay has a wide quantitative range in 1-100 μg L⁻¹. Although not as sensitive as conventional enzyme-linked immunosorbent assay (ELISA), the SPR biosensor offered unique advantages: (1) the sensor chip could be reusable without any significant loss in its binding activity after 50 assay-regeneration cycles, (2) one single assay could be accomplished in 50 min (including 30-min preincubation and 20-min BIAcore analysis), and (3) this method did not require multiple steps. The SPR biosensor was also used to detect MCs in environmental samples, and the results compared well with those obtained by ELISA. We conclude that the SPR biosensor offers outstanding advantages for the MCs detection and may be further developed as a field-portable sensor for real-time monitoring of MCs on site in the near future.     

Surface plasmon resonance spectroscopy-based high-throughput screening of ligands for use in affinity and displacement chromatography

We describe an approach that uses surface plasmon resonance (SPR) spectroscopy and self-assembled monolayers (SAMs) for the high-throughput screening of ligands for use in displacement and affinity chromatographic processes. We identified a set of commercially available organic amines and allowed them to react with SAMs presenting interchain carboxylic anhydride groups; the resulting surfaces presented ligands of interest in a background of carboxylic acid groups. We used SPR spectroscopy to determine the extent of adsorption of two model proteinslysozyme and cytochrome conto these "multimodal" surfaces and to select promising "affinity" ligands for further characterization. The attachment of selected ligands to UltraLink Biosupport resulted in beads with a significantly greater affinity for lysozyme than for cytochrome c that would be suitable for use in affinity chromatographic processes. Furthermore, we also used the screens to design "affinity displacers"small molecules that selectively retain lysozyme on chromatographic resins, while displacing cytochrome c. The combination of SPR spectroscopy and SAMs represents a powerful technique for identifying novel ligands that enable the purification of complex protein mixtures.     

Real-time immunoassay of antibody activity in serum by surface plasmon resonance biosensor

A surface plasmon resonance biosensor has been used to determine antibody activity in serum. As a model system, the interaction of mouse IgG and sheep anti-mouse IgG polyclonal antibody was investigated in real time. The factors, including pH value, ionic strength, protein concentration, influencing electrostatic adsorption of mouse IgG protein onto carboxylated dextran-coated sensor chip surface, were studied. The procedures of mouse IgG protein immobilization and immune reaction were monitored in real time. The regeneration effect using the different elution reagents was also investigated. The same mouse IgG immobilized surface can be used for 100 cycles of binding and elution with only 0.38% loss per regeneration in reactivity. The results show that the surface plasmon resonance biosensor is a rapid, simple, sensitive, accurate and reliable detection technique for real-time immunoassay of antibody activity. The assay allows antibodies to be detected and studied in their native form without any purification.     

Purification and characterization of a high specificity polyclonal antibody to the adenomatous polyposis coli tumour suppressor protein

Recombinant proteins, commonly expressed in fusion with an affinity tag to facilitate purification, are often used as immunogens for polyclonal antibody production. Careful immunopurification of the antibody product is often the key to obtaining a high-specificity polyclonal antibody against the protein domain of interest. This study describes the purification and characterization of such an antibody directed against the adenomatous polyposis coli (APC) tumour suppressor. We used a combination of affinity chromatography and biosensor analysis to optimize and monitor antibody purification. This antibody was then characterized by immunoprecipitation, proteomic analyses and immunofluorescence staining and shown to be a valuable reagent for the study of APC biology. Using this antibody we successfully isolated and identified APC, using MS/MS, from transfected cell lines. A novel phosphorylation site on APC was identified at ser 1436. Similar strategies involving multiple immuno-affinity steps coupled with surface plasmon resonance (SPR), immunoprecipitation proteomic and immunofluorescence analyses should be generally applicable for the purification and characterization of other polyclonal antibodies.     

Present and future of surface plasmon resonance biosensors

Surface plasmon resonance (SPR) biosensors are optical sensors exploiting special electromagnetic waves—surface plasmon-polaritons—to probe interactions between an analyte in solution and a biomolecular recognition element immobilized on the SPR sensor surface. Major application areas include detection of biological analytes and analysis of biomolecular interactions where SPR biosensors provide benefits of label-free real-time analytical technology. This paper reviews fundamentals of SPR affinity biosensors and discusses recent advances in development and applications of SPR biosensors.     

Kinetic Analysis of the Interaction between Nonsteroidal Anti-inflammatory Drugs and Cyclooxygenase-2Using Wavelength Modulation Surface Plasmon Resonance

A surface plasmon resonance (SPR) biosensor based on wavelength modulation technology was developed and validated for the kinetic analysis of the interactions between two nonsteroidal anti‐inflammatory drugs (NSAIDs) and cyclooxygenase‐2 (COX‐2). After the effect of different concentration COX‐2 on the binding capacity of the SPR biosensor surface was studied, the COX‐2 was immobilized covalently onto the biosensor surface using a standard amine coupling method. The affinity constants for indomethacin, ketoprofen binding to COX‐2 are 7.5×10³ L/mol and 9.25×10³ L/mol, respectively. The biosensor surface can be regenerated after being rinsed with 0.01 mol/L NaOH, and the biosensor can be used repeatedly. These indicated that the wavelength modulation SPR biosensor has the potential application in the fields of pharmacokinetics, pharmacodynamics and drug discovery.     

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.     

Detection of bisphenol A using a novel surface plasmon resonance biosensor

We present a compact surface plasmon resonance (SPR) biosensor for the detection of bisphenol A (BpA), an endocrine-disrupting chemical. The biosensor is based on an SPR sensor platform (SPRCD) and the binding inhibition detection format. The detection of BpA in PBS and wastewater was performed at concentrations ranging from 0.05 to 1,000 ng/ml. The limit of detection for BpA in PBS and wastewater was estimated to be 0.08 and 0.14 ng/ml, respectively. It was also demonstrated that the biosensor can be regenerated for repeated use. Results achieved with the SPR biosensor are compared with those obtained using ELISA and HPLC methods.     

Comparison of reflectometric interference spectroscopy with other instruments for label-free optical detection

On the basis of kinetic measurements of biomolecular interactions, a reflectometric interference spectroscopy (RIfS) setup is compared with two commercial instruments. These instruments are based on evanescent wave techniques, surface plasmon resonance (SPR) (represented by BIAcore 2000) and resonant mirror (RM) technique (using IAsys plus). All methods allow a label-free and time-resolved optical detection of biomolecular interaction. These methods are mainly used in biomolecular interaction analysis (BIA). They provide practical techniques for quantifying equilibrium constants and rate constants over several orders of magnitude. The general parameters of the three detectors, namely baseline noise and drift as well as overall sensitivity and limits of detection were compared. The fluid handling and the related implications on the measurements have also been considered. The interaction between thrombine and thrombine inhibitor (TI) was investigated as a test system with the three different methods and the kinetic rate constants were determined and compared. For this TI was immobilized on the surface and binding of thrombine was monitored time-resolved. Determination of the kinetic rate constants could prove that the RIfS set-up is comparable with SPR using BIAcore 2000 and RM technique represented by IAsys plus.     

Recognition of peptides by antibodies and investigations of affinity using biosensor technology

The study of peptide-antibody interactions has many applications in biology and medicine. Synthetic peptides corresponding to single protein epitopes are used instead of intact proteins as reagents for the diagnosis of viral and autoimmune diseases. Furthermore, antibodies raised against peptides are useful reagents for isolating and characterizing gene products. In this review, methods for analysing the molecular basis of peptide-antibody interactions are described, such as amino acid replacement studies, X-ray crystallography of peptide-antibody complexes and biosensor technology based on surface plasmon resonance. The importance of peptide conformation in antibody recognition is discussed, and the antigenic reactivity of epitopes in synthetic peptides and in cognate, intact proteins is compared.     

Applications of silica supports in affinity chromatography

The combined use of silica-based chromatographic supports with immobilized affinity ligands can be used in many preparative and analytical applications. One example is the use of silica-based affinity columns in HPLC, giving rise to a method known as high-performance affinity chromatography (HPAC). This review discusses the role that silica has played in the development of affinity chromatography and HPAC and the applications of silica in these methods. This includes a discussion of the types of ligands that have been employed with silica and the methods by which these ligands have been immobilized. Various formats have also been presented for the use of silica in affinity chromatographic methods, including assays involving direct or indirect analyte detection, on-line or off-line affinity extraction, and chiral separations. The use of silica-based affinity columns in studies of biological systems based on zonal elution and frontal analysis methods will also be considered.     

Discovery of G-quadruplex stabilizing ligands through direct ELISA of a one-bead-one-compound library

We describe the identification of small-molecule G-quadruplex ligands using a direct ELISA screen of a one-bead-one-compound library of unnatural polyamides displayed on a branched linker with a biotin tag. This general purpose parallel screen for small molecule-oligonucleotide interactions was validated by surface plasmon resonance and ELISA of resynthesized compounds. Linear polyamides displayed similar rankings in their affinity for quadruplex as their branched counterparts. Quadruplex affinity as judged by these surface based techniques was a useful predictor of the ability of the ligands to stabilize the quadruplex to thermal unfolding in solution.     

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.     

表面等离子体共振技术在电化学反应过程研究中的应用

简述了表面等离子体共振(SPR)的基本原理,并综述了表面等离子体共振技术在电化学反应过程中的应用。SPR技术可以无需任何标记原位实时地检测分子间的相互作用,也可用于连续监测吸附/脱附和缔合/解离过程。表面等离子共振光谱(SPRS)与电化学技术结合可用来同时表征和处理电极/溶液的界面,在电化学掺杂/去掺杂过程、吸附/脱附反应的研究、痕量物质的检测、薄膜厚度、介电常数的测定等方面的应用已取得了很大的进展。     

Biospecific interaction analysis using surface plasmon resonance detection applied to kinetic, binding site and concentration analysis.

A system for real-time biospecific interaction analysis using biosensor technology based on the optical phenomenon surface plasmon resonance is described. The biospecific interface is a sensor chip covered with a hydrogel matrix. One component of the interaction to be studied is immobilized covalently to the hydrogel and other interactants are passed over the chip in solution. The mass change at the sensor surface, reflecting the progress of the interaction studied, is monitored in real time. The technique, which does not require molecular labels for detection, can measure mass changes down to 10 pg/mm2. Repeated analyses can be performed on the same sensor chip. Applications shown include kinetic measurements, binding site analysis and concentration determination.     

High-sensitivity biosensors fabricated by tailoring the localized surface plasmon resonance property of core-shell gold nanorods

An enhanced sensitive biosensor has been developed to detect biological targets by tailoring the localized surface plasmon resonance property of core–shell gold nanorods. In this new concept, a shell layer is produced on gold nanorods by generating a layer of chalcogenide on the gold nanorod surface after attachment of the recognition reagent, namely, goat IgG and antigen of schistosomiasis japonica. The bioactivity of these attached biomolecules is retained and the sensitivity of this biosensor is thus enhanced significantly. The plasmonic properties of the gold nanorods attached with the biomolecules can be adjusted and the plasmon resonance wavelength can be red-shifted up to several hundred nanometers in the visible or near infrared (NIR) region, which is extremely important to biosensing applications. This leads to a lager red-shift in the localized surface plasmon resonance absorption compared to the original gold nanorod-based sensor and hence offers greatly enhanced sensitivity in the detection of schistosomiasis japonica. The human serum infected with schistosomiasis japonica diluted to 1:50,000 (volume ratio, serum/buffer solution) can be detected readily. The technique offers enhanced sensitivity and can be easily extended to other sensing applications based on not only immuno-recognition but also other types of specific reactions.     

用多克隆抗体构建的夹心免疫传感器检测心肌肌钙蛋白I

用表面等离子体子共振生物传感器构建对心肌肌钙蛋白I特异性的免疫传感器检测心肌肌钙蛋白I,并建立两种检测方法:直接法的最低检测限为2.5μg/L,基于传感膜上的夹心免疫法的灵敏度为0.5μg/L,检测范围为0.5～20μg/L,批内及批间精密度分别为3.5%～4.9%,6.1%～7.4%;用夹心法及国外试剂盒对40名健康献血者和20例急性心肌梗死患者血清心肌肌钙蛋白I水平进行检测,两者符合率为95%.     

Multimodal electrochemical and nanoplasmonic biosensors using ferrocene-crowned nanoparticles for kinase drug discovery applications

The use of on-chip multimodal sensing approaches is very promising towards integrated biosensing systems, which measure different parameters involved in biomolecular interactions and provide automated validation of true positives. In this report, we investigate a proof of concept that enables multiple detection technologies for screening inhibitors of kinase activity, which is a crucial process in drug discovery applications. We demonstrate the integration of electrochemical techniques on the same chip, namely, differential pulse voltammetry, impedance spectroscopy, and direct open circuit potential measurements. Gold nanoparticles that attach to the thio-phosphorylated proteins facilitate localised surface plasmon resonance detection. The addition of thiolated ferrocene, which attaches to the nanoparticles like a crown, enables sensitive electrochemical amperometric detection of kinase activity. This novel multimodal biosensor provides a more rigorous measurement of biomolecules, with wide significance in biomedical, environmental, and pharmaceutical applications.