Sequential detection of Salmonella typhimurium and Bacillus anthracis spores using magnetoelastic biosensors

Multiple phage-based magnetoelastic (ME) biosensors were simultaneously monitored for the detection of different biological pathogens that were sequentially introduced to the measurement system. The biosensors were formed by immobilizing phage and 1mg/ml BSA (blocking agent) onto the magnetoelastic resonator's surface. The detection system included a reference sensor as a control, an E2 phage-coated sensor specific to S. typhimurium, and a JRB7 phage-coated sensor specific to B. anthracis spores. The sensors were free standing during the test, being held in place by a magnetic field. Upon sequential exposure to single pathogenic solutions, only the biosensor coated with the corresponding specific phage responded. As the cells/spores were captured by the specific phage-coated sensor, the mass of the sensor increased, resulting in a decrease in the sensor's resonance frequency. Additionally, non-specific binding was effectively eliminated by BSA blocking and was verified by the reference sensor, which showed no frequency shift. Scanning electron microscopy was used to visually verify the interaction of each biosensor with its target analyte. The results demonstrate that multiple magnetoelastic sensors may be simultaneously monitored to detect specifically targeted pathogenic species with good selectivity. This research is the first stage of an ongoing effort to simultaneously detect the presence of multiple pathogens in a complex analyte.     

Variable wavelength surface plasmon resonance (SPR) in biosensing

In this study, we fabricated a novel variable wavelength surface plasmon resonance (SPR) sensor, which detects resonance conditions such as a maximum attenuation wavelength, measuring change of microscopic refractive index. Such a change was measured to detect a salmonella antigen–antibody reaction and a penicillinase–penicillin reaction. Our experiments were performed after immobilizing a salmonella antibody on the sensor chip. We measured the shift in resonant wavelength during the antigen–antibody reaction for 30 min by injecting 5 × 10⁷ cells/ml concentration of salmonella antigen solution into the sample chamber. Also, after immobilizing penicillinase on the sensor chip, we measured the shift in resonant wavelength during the reaction. Penicillin solution at 10 mM was injected into the sample chamber. The shift of resonant wavelength for each experiment was measured using a white light source, multimode optical fiber, a part of sensor chip and an optical spectrum analyzer.As a result, the resonant wavelength shifted about 0.26 nm/min owing to the salmonella antibody–antigen reaction. Thus, we could detect the change in wavelength (0.8 nm/min) through the interaction of penicillin and penicillinase for 15 min using variable wavelength SPR sensor.     

Continuous flow immunosensor for highly selective and real-time detection of sub-ppb levels of 2-hydroxybiphenyl by using surface plasmon resonance imaging

A biosensor based on surface plasmon resonance (SPR) is developed for the detection of 2-hydroxybiphenyl (HBP). A monoclonal antibody against HBP (abbreviated hereafter as HBP-mAb) is developed and used for the detection of HBP by competitive SPR-based immunoassay and enzyme linked immunosorbent assay (ELISA) methods. A novel HBP-hapten compound, HBP-bovine serum albumin conjugate (HBP-BSA), derived by binding several HBP units with BSA by an aliphatic chain spacer is used in the development of antibody and for the functionalization of immunoprobes. HBP-BSA linked to the Au surface of the SPR sensor chip undergoes inhibitive immunoreaction with HBP-mAb in the presence of free HBP. The SPR-based immunoassay provides a rapid determination (response time: approximately 20 min) of the concentration of HBP in the range of 0.1-1000 ppb (ng/ml). Regeneration of the sensor chip is gained by treating the antibody-anchored SPR sensor chip with a pepsin solution (100 ppm (microg/ml); pH 2.0) for few minutes. The SPR sensor chip is reusable for the detection of HBP for more than 20 cycles with average loss of 0.35% reactivity per regeneration step. HBP concentration is determined as low as 0.1 and 3 ppb using the SPR sensor and ELISA measurements, respectively. The developed SPR sensor for HBP is free from interference by coexisting benzo[a]pyrene (BaP), 2,4-dichlorophenoxyacetic acid (2,4-D) and benz[a]anthracene; SPR angle shift obtained to the flow of HBP is almost same irrespective to the presence or absence of a same concentration of these carcinogenic polycyclic aromatic hydrocarbons together. The SPR sensor for HBP is proved to be applicable in simultaneous detection of HBP and BaP in parallel with another SPR sensor for BaP.     

Highly sensitive and selective surface plasmon resonance sensor for detection of sub-ppb levels of benzo[a]pyrene by indirect competitive immunoreaction method

A surface plasmon resonance (SPR)-immunosensor for detection of benzo[a]pyrene (BaP) is developed by using a model BaP-hapten compound, BaP-bovine serum albumin conjugate (BaP-BSA), and an anti-BaP-BSA monoclonal antibody. BaP-BSA conjugate is immobilized on a gold thin-film sensor chip by means of simple physical adsorption. The number of BaP-hapten units in BaP-BSA conjugate is estimated to be 28 from the difference in molecular weight (MW) between BaP-BSA conjugate and BSA based on the results of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) measurement. Anti-BaP-BSA antibody on contact with the BaP-BSA conjugate immobilized sensor chip causes an increase in the incident angle of the sensor chip. Binding of anti-BaP-BSA antibody with surface-immobilized BaP-BSA conjugate is inhibited by the presence of BaP in analyte solution, because of the inhibition effect of BaP. The SPR immunosensor for BaP functioning with the indirect competitive immunoreaction of anti-BaP-BSA antibody between the analyte (BaP) in testing solution and the BaP-BSA conjugate immobilized on the sensor chip provides a rapid determination (response time: ca. 15 min) of BaP in the concentration range of 0.01-1000 ppb. The antibody anchored to the sensor chip by antigen-antibody binding is removed on treatment with a pepsin solution (pH 2.0) for few minutes. The SPR sensor chip is found to be reusable for more than 20 times with a little decrease (<7%) in the sensor response. Detection of BaP by direct competitive immunoreactions is also carried out by enzyme-linked immunosorbent assay (ELISA). The concentration of BaP could be determined as low as 0.01 ppb and 2 ppb using the SPR sensor and the ELISA method, respectively. The SPR sensor is found to detect BaP selectively in the presence of 2-hydroxybiphenyl (HBP); the incident angle shift of the SPR sensor for BaP is found to be same irrespective to the presence or the absence of a same concentration (as much as 30 ppb) of HBP together.     

BSA–boronic acid conjugate as lectin mimetics

We report bovine serum albumin (BSA)–boronic acid (BA) conjugates as lectin mimetics and their glyco-capturing capacity. The BSA–BA conjugates were synthesized by amidation of carboxylic acid groups in BSA with aminophenyl boronic acid in the presence of EDC, and were characterized by Alizarin Red S (ARS) assay and SDS–PAGE gel. The BSA–BA conjugates were immobilized onto maleimide-functionalized silica beads and their sugar capturing capacity and specificity were confirmed by ARS displacement assay. Further, surface plasmon resonance (SPR) analysis of the glyco-capturing activity of the BSA–BA conjugates was conducted by immobilizing BSA–BA onto SPR gold chip. Overall, we demonstrated a BSA–BA-based lectin mimetics for glyco-capturing applications. These lectin mimetics are expected to provide an important tool for glycomics and biosensor research and applications.     

界面上配基种类对BSA吸附行为的影响

利用双偏振极化干涉测量仪(DPI)研究了界面上配基种类对BSA吸附行为的影响。采用3-氨基丙基三乙氧基硅烷(APTES)、3-(甲氨基)丙基三甲氧基硅烷(MAPTMS)和N,N-二乙基-3-氨基丙基三甲氧基硅烷(DAPTMS)对DPI芯片进行了修饰,利用X射线光电子能谱比较了芯片上不同配基的密度,采用原子力显微镜(AFM)和DPI对界面上BSA吸附行为进行了研究。结果表明APTES修饰界面上BSA呈饼状,高配基密度易导致BSA多位点吸附。相同偶联密度条件下BSA在DAPTMS修饰芯片的吸附量高于MAPTMS修饰芯片,但吸附层厚度一致,表明DAPTMS表面上BSA存在聚集现象;AFM扫描结果与DPI分析结果一致,证明了配基密度和种类不仅影响界面上蛋白质吸附量,而且影响蛋白质吸附密度和表面聚集行为。     

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.     

Characterization of the surfaces generated by liposome binding to the modified dextran matrix of a surface plasmon resonance sensor chip

The dextran matrix of a surface plasmon resonance (SPR) sensor chip modified with hydrophobic residues (BIAcore sensor chip L1) provides an ideal substrate for liposome adsorption. Liposomes of different lipid compositions are captured on the sensor chips by inserting these residues into the liposome membrane, thereby generating stable lipid surfaces. To gain a more detailed understanding of these surfaces, and to prove whether the liposomes stay on the matrix as single particles or form a continuous lipid layer by liposome fusion, we have investigated these materials, using atomic force microscopy (AFM) and fluorescence microscopy. Force measurements with AFM probes functionalized with bovine serum albumin (BSA) were employed to recognize liposome adsorption. Analysis of the maximal adhesive force and adhesion energy reveals a stronger interaction between BSA and the dextran matrix compared to the lipid-covered surfaces. Images generated using BSA-coated AFM tips indicated a complete and homogeneous coverage of the surface by phospholipid. Single liposomes could not be detected even at lower lipid concentrations, indicating that the liposomes fuse and form a lipid bilayer on the dextran matrix. Experiments with fluorescently labeled liposomes concurred with the AFM studies. Surfaces incubated with liposomes loaded with TRITC-labeled dextran showed no fluorescence, indicating a complete release of the encapsulated dye. In contrast, surfaces incubated with liposomes containing a fluorescently labeled lipid showed fluorescence.     

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.     

Surface plasmon resonance imaging biosensor for cathepsin G based on a potent inhibitor: development and applications

A specific surface plasmon resonance imaging (SPRI) array biosensor for the determination of the enzymatically active cathepsin G (CatG) has been developed. For this purpose, a specific interaction between an inhibitor immobilized onto a chip surface and CatG in an analyzed solution was used. The MARS-115 CatG peptidyl inhibitor containing the 1-aminoalkylphosphonate diaryl ester moiety at the C terminus and N-succinamide with a free carboxylic function was synthesized and covalently immobilized onto the gold chip surface via the thiol group (cysteamine). Atomic force microscopy was used for the observation of surface changes during the subsequent steps of chip manufacture. Optimal detection conditions were chosen. High specificity of synthesized inhibitor to CatG was proved. The precision, as well as the accuracy, was found to be well suited to enzyme determination. The sensor application for the determination of CatG in white blood cells and saliva was shown for potential diagnosis of leukemia and oral cavity diseases during the early stages of those pathological states.     

The Surface Plasmon Resonance Imaging sensor for papain based on immobilized cystatin

The Surface Plasmon Resonance Imaging (SPRI) sensor for papain determination based on the interaction between the immobilized cystatin and aqueous papain solution has been developed. The development of the sensor is a stage in the development of the sensor for the determination of cystein proteinases of the papain group. Cystatin was immobilized onto a gold chip by means of a thiol underlayer (cysteamine) and EDS/NHS reaction. Conditions of cystatin- papain interaction were optimized (pH, time of interaction and cystatin concentration). The developed sensor works within the range of 1 - 10 ng ml(-1). However, the precision of measurements (RSD equal to 45%) should be improved. The response of the sensor to papain is specific. This was checked using BSA as a reference.     

Novel approach to analyzing RNA aptamer-protein interactions: toward further applications of aptamers

Surface plasmon-resonance analysis using a Biacore biosensor is a powerful tool for the detailed study of biomolecular interactions. The authors examined the methods of immobilizing proteins on the surface of NTA, SA, and CM5 sensor chips to study RNA aptamer-protein interactions. RNA aptamers and their deletion variants were loaded onto a protein-immobilized sensor chip, and their binding affinities were analyzed. Immobilizing the protein on a CM5 sensor chip via an anti-His-tag antibody was the only strategy that clearly detected the kinetic parameters of the interactions. DeltaNEO-III-14U, one of the deletion variants of the NS3 aptamer, had the highest binding affinity for the deltaNS3 protein in this study (KD = 4 x 10(-8)). Moreover, the 29-amino-acid spacer fragment was essential for protein immobilization using this strategy. This novel method will be useful in comparing the affinity of various RNA aptamers and selecting the most suitable candidates for a given target, as well as facilitating the in vitro selection procedure itself.     

Surface plasmon resonance-based detection of ladder-shaped polyethers by inhibition detection method

Ladder-shaped polyether (LSP) compounds represented by brevetoxins and ciguatoxins were largely discovered in association with seafood poisoning. Thus, a quick quantification method for LSPs is potentially important. We examined a surface plasmon resonance method using desulfated-yessotoxin (dsYTX) immobilized on a sensor chip and phosphodiesterase PDEII in a inhibition detection mode. Yessotoxin, brevetoxin B and synthetic LSP derivatives showed clear inhibition against PDEII binding to the immobilized dsYTX, by which their half inhibitory concentrations were successfully estimated. This inhibition method appeared to be superior in specificity to direct binding assays where binding proteins to LSP was immobilized on a sensor chip.     

Binding properties of antimicrobial agents to dipeptide terminal of lipid II using surface plasmon resonance

We developed a surface plasmon resonance (SPR) assay to estimate the interactions of antimicrobial agents with the dipeptide terminal of lipid II (d-alanyl-d-alanine) and its analogous dipeptides (l-alanyl-l-alanine and d-alanyl-d-lactate) as ligands. The established SPR method showed the reproducible immobilization of ligands on sensor chip and analysis of binding kinetics of antimicrobial agents to ligands. The ligand-immobilized chip could be used repeatedly for at least 200 times for the binding assay of antimicrobial agents, indicating that the ligand-immobilized chip is sufficiently robust for the analysis of binding kinetics. In this SPR system, the selective and specific binding characteristics of vancomycin and its analogs to the ligands were estimated and the kinetic parameters were calculated. The kinetic parameters revealed that one of the remarkable binding characteristics was the specific interaction of vancomycin to only the d-alanyl-d-alanine ligand. In addition, the kinetic binding data of SPR showed close correlation with the antimicrobial activity. The SPR data of other antimicrobial agents (e.g., teicoplanin) to the ligands showed correlation with the antimicrobial activity on the basis of the therapeutic mechanism. Our SPR method could be a valuable tool for predicting the binding characteristics of antimicrobial agents to the dipeptide terminal of lipid II.     

Rapid analysis of protein interactions: On-chip micropurification of recombinant protein expressed in Esherichia coli

We describe a rapid analysis of interactions between antibodies and a recombinant protein present in total cell lysates. Using a surface plasmon resonance biosensor, a low concentration of glutathione-S-transferase (GST) fused protein expressed in small scale Esherichia coli culture was purified on an anti-GST antibody immobilized sensor chip. The 'on-chip purification' was verified using matrix-assisted laser desorption/ionization-time of flight mass spectrometry by measuring the molecular masses of recombinant proteins purified on the sensor chip. The specific binding of monoclonal antibodies for the on-chip micropurified recombinant proteins can then be monitored, thus enabling kinetic analysis and epitope mapping of the bound antibodies. This approach reduced time, resources and sample consumption by avoiding conventional steps related to concentration and purification.     

Immobilization of bovine serum albumin as a sensitive biosensor for the detection of trace lead ion in solution by piezoelectric quartz crystal impedance

A biosensor based on bovine serum albumin (BSA) for the detection of lead (Pb(2+)) ion was developed and characterized. BSA was immobilized onto a colloidal Au-modified piezoelectric quartz crystal (PQC) as a biosensor for the detection of Pb(2+) ion by piezoelectric quartz crystal impedance (PQCI). Calibration curves for the quantification of Pb(2+) ion showed excellent linearity throughout the concentration range from 1.0 x 10(-7) to 3.0 x 10(-9)mol/L. The interaction between the Pb(2+) ions and the sensor chip is influenced significantly by the pH of the reaction buffer, and the optimal pH for the experiment was 5.4. Under the optimal conditions, the detection limit of 1.0 x 10(-9)mol/L for Pb(2+) was obtained. Kinetic parameters of the Pb(2+)-BSA interactions were also determined by using this chip. The sensor chip could be regenerated for use by dipping in the ethylenediaminetetraacetic acid (EDTA) solution for approximately 2h, and the chip was used to detect Pb(2+) ion for eight times without obvious signal attenuation.     

A vesicle capture sensor chip for kinetic analysis of interactions with membrane-bound receptors

A novel sensor chip for use in surface plasmon resonance (SPR) biosensors has been developed to capture vesicles which may contain membrane-bound receptors. Sulforhodamine-containing vesicles were shown by fluorescence microscopy to be immobilized intact on the sensor chip. Binding of cholera toxin to captured vesicles containing ganglioside GM(1) was demonstrated using SPR, and the derived kinetic and affinity constants were similar to literature values. Biotinylated vesicles captured on the sensor chip were used to bind streptavidin and then biotinylated ss-DNA. The hybridization of complementary ss-DNA to the immobilized ss-DNA was then analyzed using SPR. The values obtained were similar to those obtained for an identical interaction analyzed using a commercially available streptavidin-containing sensor chip. Binding of vancomycin-group antibiotics to captured vesicles containing a bacterial cell wall mucopeptide analogue was demonstrated. No binding of the bacterial endotoxin Cry1A(c) to captured vesicles containing its cell surface receptor could be demonstrated.     

Rapid and specific detection of herbicides using a self-assembled photosynthetic reaction center from purple bacterium on an SPR chip

In this study, a direct detection system for herbicides inhibiting photosynthetic electron transfer was developed using the photosynthetic reaction center (RC) from the purple bacterium, Rhodobacter sphaeroides, and surface plasmon resonance (SPR) apparatus. The heavy-subunit-histidine-tagged RCs (HHisRCs) were immobilized on an SPR sensor chip via nickel chelation chemistry as a binder for one of the triazine herbicides, atrazine. Immediately after injection of atrazine solution on the HHisRCs-immobilized chip, the SPR responses increased and reached plateaus within 1 min. The SPR signals were proportional to the sample concentrations of atrazine in the range 1-100 microg/ml. To evaluate the binding specificity to atrazine, chlorinated aromatic herbicides, DCMU and MCPP, were investigated using the HHisRCs-immobilized chip. An RC inhibitor, DCMU, could also be detected with a higher detection limit of 20 microg/ml than atrazine (1 microg/ml). MCPP showed no signals because its inhibition mechanism against plants is different from that of atrazine and DCMU. These results indicated that the sensor chip immobilized RCs could be used for the specific detection of photosynthetic inhibitors.     

A surface plasmon resonance immunosensor based on the streptavidin-biotin complex.

It is shown that a streptavidin monolayer immobilized onto an evaporated gold film with biotin forms the basis of a highly specific sensing element. As an example, we show that by immobilizing the biotinylated antibody sex hormone binding globulin (alpha-SHBG) to the bound streptavidin monolayer a specific sensor for the antigen SHBG is readily fabricated. The interaction between immobilized antibody and corresponding antigen is monitored by surface plasmon resonance spectroscopy and is shown to follow a classic Langmuir isotherm. Detection of SHBG at nanomolar concentrations is demonstrated.     

Intact-cell-based surface plasmon resonance measurements for ligand affinity evaluation of a membrane receptor

Toward future applications to the discovery of drugs against membrane receptors on pathological cells, an intact-cell-based surface plasmon resonance (SPR) methodology has been developed. The injection of a suspension of epidermal carcinoma A431 cells (5×10(7)cells/ml), as an analyte, generated clear SPR responses to epidermal growth factor (EGF) immobilized on the sensor chip. Because the responses were competitively reduced by the free ligand EGF, added to the analyte cell suspension, they certainly reflect the specific interaction of the immobilized EGF with the extracellular region of its receptor, which is highly expressed on the surface of the A431 cells.