Immunosensor for detection of inhibitory neurotransmitter gamma-aminobutyric acid using quartz crystal microbalance

A piezoelectric immunosensor for sensing the low molecular weight neurotransmitter gamma-aminobutyric acid (GABA), one of two major inhibitory neurotransmitters in the central nervous system, is described. The sensing interface consists of a dextran layer covalently attached to a self-assembled monolayer of thiolamine compound on the surface of gold electrodes of the crystals. The dextran layer is further modified with GABA molecules to act as the biosensing layer. The affinity binding of monoclonal anti-GABA antibody on the modified piezoelectric crystals is studied in real time without any additional labels. The equilibrium association constant, K(eq) for binding between anti-GABA antibody and GABA molecules is 14.5 microg x mL (-1). The detection limit for anti-GABA is approximately 10 nM. The sensitivity of the sensor at a concentration corresponding to half-maximal response is 13.6 ng/mL x Hz. The functionalized sensor substrate is subsequently used for competitive determination of different concentrations of free GABA (range of 5 microM-50 mM) in PBS-BSA buffer. The detection limit of the immunosensor for sensing GABA with maximum sensitivity is approximately 42 microM.     

Prion protein detection using nanomechanical resonator arrays and secondary mass labeling

Nanomechanical resonators have shown potential application for mass sensing and have been used to detect a variety of biomolecules. In this study, a dynamic resonance-based technique was used to detect prion proteins (PrP), which in conformationally altered forms are known to cause neurodegenerative diseases in animals as well as humans. Antibodies and nanoparticles were used as mass labels to increase the mass shift and thus amplify the frequency shift signal used in PrP detection. A sandwich assay was used to immobilize PrP between two monoclonal antibodies, one of which was conjugated to the resonator's surface while the other was either used alone or linked to the nanoparticles as a mass label. Without additional mass labeling, PrP was not detected at concentrations below 20 microg/mL. In the presence of secondary antibodies the analytical sensitivity was improved to 2 microg/mL. With the use of functionalized nanoparticles, the sensitivity improved an additional 3 orders of magnitude to 2 ng/mL.     

Development of a flow injection capillary chemiluminescent ELISA using an imprinted polymer instead of the antibody

A flow injection competitive assay analogous to enzyme immunoassays has been developed using a molecularly imprinted polymer instead of the antibody. A glass capillary was modified by covalently attaching an imprinted polymer to the inner capillary wall. The herbicide 2,4-dichlorophenoxyacetic acid was used as a model analyte. The analyte was labeled with tobacco peroxidase, and chemiluminescence was used for detection in combination with a photomultiplier tube or a CCD camera. In a competitive mode, the analyte-peroxidase conjugate was passed together with the free analyte through the polymer-coated capillary mounted in a flow system. After a washing step, the chemiluminescent substrate was injected and the bound fraction of the conjugate was quantified by measuring the intensity of the emitted light. Calibration curves corresponding to analyte concentrations ranging from 0.5 ng mL(-1) to 50 microg mL(-1) (2.25 nM-225 microM) were obtained. A lowered detection limit by 2 orders of magnitude was obtained when detection was done in discontinuous mode and the chemiluminescence light was conducted inside the photomultiplier tube by an optical fiber bundle, thus yielding a dynamic range of 5 pg mL(-1)-100 ng mL(-1) (22.5 pM-450 nM).     

Plasmonic detection of a model analyte in serum by a gold nanorod sensor

We describe the fabrication of a label-free, chip-based biosensor based on the localized surface plasmon resonance (LSPR) of gold nanorods. Gold nanorods were chemisorbed onto a mercaptosilane-modified glass substrate, followed by conjugation of biotin to the nanorods. Streptavidin binding to biotin was monitored by the wavelength shift of the LSPR peak in the UV-vis extinction spectrum of the immobilized gold nanorods due to the change in local refractive index at the gold nanorod surface induced by streptavidin binding. The limit of detection of the sensor is 0.005 microg/mL (94 pM) in PBS and 1 microg/mL (19 nM) in serum, and the dynamic range spans 94 pM to 0.19 microM. The advantages of the nanorod-based sensor over an LSPR sensor that we had previously fabricated from gold nanospheres (Nath, N.; Chilkoti, A. Anal. Chem. 2002, 74, 504-509; J. Fluoresc. 2004, 14, 377-389; Anal. Chem. 2004, 76, 5370-5378) are the significantly lower detection limit and the internal self-reference that the signal of the nanorod sensor provides based on the measurement of peak wavelength shift.     

Determination of boron by using a quartz crystal resonator coated with N-methyl-D-glucamine-modified poly(epichlorohydrin)

A nongravimetric quartz crystal resonator for determination of boron was proposed. The key step is the preparation of a polymer that forms a complex with boron (from borate ion). The polymeric film is deposited on one face of an electrode-separated quartz crystal. The backbone of the polymer is poly(epichlorohydrin), which is modified to anchor N-methyl-D-glucamine. After reticulation and reduction, the film presents high stability and sensitivity to boron at pH 8.5. A carrier solution containing 50 mM EDTA ensures high conductivity and the elimination of several interfering metal ions. Boron is strongly retained by the film, and a positive shift of the oscillating frequency is proportional to its concentration. Boron is eluted with 1 mL of a 1 M mannitol solution. For a 0.160-mL sample loop and concentration up to 600 microM, the calibration sensitivity was 1.67 Hz/microM and the LOD was 2 microM. This limit could be lowered to 0.3 microM by using a 1.00-mL sample loop. In both cases, it was possible to detect 3 ng of boron. It was estimated that the nongravimetric sensor is at least 10 times more sensitive that a hypothetical gravimetric sensor.     

Amperometric responses of CYP2D6 drug metabolism nanobiosensor for sertraline: a selective serotonin reuptake inhibitor

Sertraline is selective serotonin reuptake inhibitor drug marketed as Zoloft by Pfizer and used mainly for the treatment of depression, anxiety and obsession. A number of side effects are associated with the use of the drug including gastrointestinal complaints, nervousness and sexual dysfunction. This means that a reliable fast method (such as biosensing) for determining sertraline metabolic profile of patients is essential for adequate dosing. Nanobiosensor for the determination of sertraline biotransformation was prepared with cytochrome P450-2D6 (CYP2D6) and poly(8-anilino-1-napthalene sulphonic acid) nanotubes (90 nm in diameter and 600-800 nm in length) potentiodynamically deposited on gold. The biosensor gave a linear response over the concentration range of 0.2 and 1.4 microM with a sensitivity value of 0.301 microA/microM and a detection limit of 0.13 microM. The nanobiosensor exhibited substrate inhibition response profile for sertraline biotransformation at high concentrations. Analysis of the Michaelis-Menten region of the nanosensor response curve for sertraline gave an apparent Michaelis-Menten constant (K(m)) value of 0.75 microM, which is higher than the peak plasma concentration (C(max)) value of 0.55 microM, thereby making the sensor suitable for sertraline determination in serum.     

A planar amperometric creatinine biosensor employing an insoluble oxidizing agent for removing redox-active interferences.

A planar microchip-based creatinine biosensor employing an oxidizing layer (e.g., a PbO2 film), where interfering redox-active substances are broken (i.e., oxidized) to redox-inactive products, was developed to facilitate the microfabrication of the sensor and to provide improved, reliable determination of creatinine in physiological samples. The feasibility of using hydrophilic polyurethanes in permselective barrier membranes for creatinine biosensors and the effect of adding a silanizing agent (adhesion promoter) on the sensor performance (e.g., sensitivity, stability, and lifetime) are described. The proposed creatinine microsensor with a three-layer configuration, i.e., enzyme, protecting, and oxidizing layers, exhibits good electrochemical performance in terms of response time (t95% = 98 s at 100-->200 microM creatinine change), linearity (1-1000 microM, r = 0.9997), detection limit (0.8 microM), and lifetime (approximately 35 days). The creatinine biosensor devised in a differential sensing arrangement that compensates the erroneous results from creatine is considered to be suitable for assay of serum specimens.     

Comparison of glycosphingolipids and antibodies as receptor molecules for ricin detection

Glycosphingolipids (GSLs) have been shown to undergo strong interactions with a number of protein toxins, including potential bioterrorism agents such as ricin and botulinum neurotoxin. Characterization of this interaction in recent years has led to a number of studies where GSLs were used as the recognition molecules for biosensing applications. Here, we offer a comparison of quartz crystal microbalance (QCM) sensors for the detection of ricin using antibodies and the GSLs GM1 and asialoGM1, which have been shown to undergo strong interactions with ricin. The presence, orientation, and activity of the GSL and antibody films were confirmed using ellipsometry, Fourier transform infrared spectroscopy (FT-IR), and QCM. It was found that the GSLs offered more sensitive detection limits when directly compared with antibodies. Both GSLs had lower detection limits at 5 microg/mL, approximately 5 times lower than were found for antibodies (25 microg/mL), and their linear detection range extended to the highest concentrations tested (100 microg/mL), almost an order of magnitude beyond the saturation point for the antibody sensors. Potential sites for nonspecific adsorption were blocked using serum albumin without sacrificing toxin specificity.     

A simple and selective spectrophotometric flow injection determination of trace amounts of ruthenium by catalytic oxidation of safranin-O

In this work, a simple, selective and rapid flow injection method has been developed for determination of ruthenium. The method is based on its catalytic effect on the oxidation of safranin-O by metaperiodate. The reaction was monitored spectrophotometrically by measuring safranin-O absorbance at lambdamax=521. The reagents and manifold variables, which have influences on the sensitivity, were investigated and the optimum conditions were established. The optimized conditions made it possible to determine ruthenium in the ranges of 0.4-20.0 ng/mL (DeltaA=0.2819CRu+1.1840) and 20.0-100.0 ng/mL (DeltaA=0.0984CRu+7.9391) with a detection limit of 0.095 ng/mL and a sample rate of 30+/-5 samples/h. Relative standard deviation for the five replicate measurements was less than 1.84%. The proposed method has been successfully applied for analysis of ultra trace amounts of ruthenium in real samples.     

Aptamer-based detection and quantitative analysis of ricin using affinity probe capillary electrophoresis

The ability to detect sub-nanomolar concentrations of ricin using fluorescently tagged RNA aptamers is demonstrated. Aptamers rival the specificity of antibodies and have the power to simplify immunoassays using capillary electrophoresis. Under nonequilibrium conditions, a dissociation constant, Kd, of 134 nM has been monitored between the RNA aptamer and ricin A-chain. With use of this free-solution assay, the detection of 500 pM (approximately 14 ng/mL) or 7.1 amol of ricin is demonstrated. The presence of interfering proteins such as bovine serum albumin and casein do not inhibit this interaction at sub-nanomolar concentrations. When spiked with RNAse A, ricin can still be detected down to 1 nM concentrations despite severe aptamer degradation. This approach offers a promising method for the rapid, selective, and sensitive detection of biowarfare agents.     

Design and fabrication of a microimpedance biosensor for bacterial detection

A biosensor for bacterial detection was developed based on microelectromechanical systems, heterobifunctional crosslinkers and immobilized antibodies. The sensor detected the change in impedance caused by the presence of bacteria immobilized on interdigitated gold electrodes and was fabricated from (100) silicon with a 2-/spl mu/m layer of thermal oxide as an insulating layer. The sensor active area is 9.6 mm/sup 2/ and consists of two interdigital gold electrode arrays measuring 0.8 /spl times/ 6 mm. Escherichia coli specific antibodies were immobilized to the oxide between the electrodes to create a biological sensing surface. The impedance across the interdigital electrodes was measured after immersing the biosensor in solution. Bacteria cells present in the sample solution attached to the antibodies and became tethered to the electrode array, thereby causing a change in measured impedance. The biosensor was able to discriminate between different cellular concentrations from 10/sup 5/ to 10/sup 7/ CFU/mL in pure culture. The sample testing process, including data acquisition, required 5 min. The design, fabrication, and testing of the biosensor is discussed along with the implications of these findings toward further biosensor development.     

Spectroelectrochemical sensing based on multimode selectivity simultaneously achievable in a single device. 17. Improvement in detection limits using ultrathin perfluorosulfonated ionomer films in conjunction with continuous sample flow

We report herein an attenuated total reflectance (ATR) absorbance-based spectroelectrochemical sensor for tris(2,2'-bipyridyl)ruthenium(II) ion [Ru(bpy)(3)(2+)] that employs ultrathin (24-50 nm) Nafion films as the charge-selective layer. This film serves to sequester and preconcentrate the analyte at the optically transparent electrode surface such that it can be efficiently detected optically via electrochemical modulation. Our studies indicate that use of ultrathin films in tandem with continuous flow of sample solution through the cell compartment leads to a 100-500-fold enhancement in detection limit (10 nM) compared to earlier absorbance-based spectroelectrochemical sensors ( approximately 1-5 microM); markedly shorter analysis times also result. We report the dependence of the measured absorbance on sample flow rate and Nafion film thickness, and also provide calibration curves that illustrate the linear range and detection limits of the sensor using a 24 nm film at a constant sample flow rate of 0.07 mL/min.     

Homogeneous immunoassay for detection of TNT and its analogues on a microfabricated capillary electrophoresis chip

A homogeneous immunoassay for TNT and its analogues is developed using a microfabricated capillary electrophoresis chip. The assay is based on the rapid electrophoretic separation of an equilibrated mixture of an anti-TNT antibody, fluorescein-labeled TNT, and unlabeled TNT or its analogue. The band intensities of the free fluorescein-labeled TNT and of the antibody-antigen complex reveal the relative equilibrated concentrations. Titration of the anti-TNT antibody with a fluorescein-labeled TNT derivative yields a binding constant of (3.9 +/- 1.3) x 10(9) M(-1). The dissociation rate constant of the complex is determined by kinetic capillary electrophoresis using a folded channel and a rotary scanner to interrogate the separation at multiple time points. The dissociation rate constant is found to be 0.035 +/- 0.005 s(-1), and the resulting binding rate constant is (1.4 +/- 0.7) x 10(7) M(-1) s(-1). Binding constants of TNT and five of its analogues are determined by competitive assays: TNT (4.3 +/- 2.6) x 10(8) M(-1); 1,3,5-trinitrobenzene (5.1 +/- 3.3) x 10(7) M(-1); picric acid (7.5 +/- 4.4) x 10(6) M(-1); 2,4-dinitrotoluene (7.9 +/- 4.0) x 10(6) M(-1); 1,3-dinitrobenzene (1.0 +/- 0.7) x 10(6) M(-1); and 2,4-dinitrophenol (5.1 +/- 3.0) x 10(4) M(-1). TNT and its analogues can be assayed with high sensitivity (LOD 1 ng/mL) and with a wide dynamic range (1-300 ng/mL) using this chip-based method.     

Oxidation of ethyl ether on borate glass: chemiluminescence, mechanism, and development of a sensitive gas sensor

A gas sensor was developed by using the chemiluminescence (CL) emission from the oxidation of ethyl ether by oxygen in the air on the surface of borate glass. Theoretical calculation, together with experimental investigation, revealed the main CL reactions: ethyl ether is first oxidized to acetaldehyde and then to acetic acid, during which main luminous intermediates such as CH 3CO (*) are generated and emit light with a peak at 493 nm. At a reaction temperature of 245 degrees C, the overall maximal emission was found at around 460 nm, and the linear range of the CL intensity versus the concentration of ethyl ether was 0.12-51.7 microg mL (-1) ( R = 0.999, n = 7) with a limit of detection (3sigma) of 0.04 microg mL (-1). Interference from foreign substances including alcohol (methanol, ethanol and isopropanol), acetone, ethyl acetate, n-hexane, cyclohexane, dichloromethane, or ether ( n-butyl ether, tetrahydrofuran, propylene oxide, isopropyl ether and methyl tert-butyl ether) was not significant except a minimal signal from n-butyl ether (<2%). It is a simple, sensitive and selective gas sensor for the determination of trace ethyl ether.     

Graphene-based biosensors

Results of developing and testing graphene-based sensors capable of detecting protein molecules are presented. The biosensor operation was checked using an immunochemical system comprising fluorescein dye and monoclonal antifluorescein antibodies. The sensor detects fluorescein concentration on a level of 1–10 ng/mL and bovine serum albumin–fluorescein conjugate on a level of 1–5 ng/mL. The proposed device has good prospects for use for early diagnostics of various diseases.     

An on-line pre-concentration system for determination of cadmium in drinking water using FAAS

In the present paper, a minicolumn of polyurethane foam loaded with 4-(2-pyridylazo)-resorcinol (PAR) is proposed as pre-concentration system for cadmium determination in drinking water samples by flame atomic absorption spectrometry. The optimization step was performed using two-level full factorial design and Doehlert matrix, involving the variables: sampling flow rate, elution concentration, buffer concentration and pH. Using the established experimental conditions in the optimization step of: pH 8.2, sampling flow rate 8.5 mL min(-1), buffer concentration 0.05 mol L(-1) and elution concentration of 1.0 mol L(-1), this system allows the determination of cadmium with detection limit (LD) (3sigma/S) of 20.0 ng L(-1) and quantification limit (LQ) (10sigma/S) of 64 ng L(-1), precision expressed as relative standard deviation (R.S.D.) of 5.0 and 4.7% for cadmium concentration of 5.0 and 40.0 microg L(-1), respectively, and a pre-concentration factor of 158 for a sample volume of 20.0 mL. The accuracy was confirmed by cadmium determination in the standard reference material, NIST SRM 1643d trace elements in natural water. This procedure was applied for cadmium determination in drinking water samples collected from Salvador City, Bahia, Brazil. For five samples analyzed, the achieved concentrations varied from 0.31 to 0.86 microg L(-1).     

Spectrofluorometric determination of vanadium based on the formation of a ternary complex between vanadium, peroxides, and 2-alpha-pyridylthioquinaldinamide. Application to the determination of hydrogen peroxide and peroxy acids.

A selective and sensitive method for the determination of the total amount of vanadium in nutritional and biological substrates is proposed. The method is based on the reaction of vanadium with 2-alpha-pyridylthioquinaldinamide (PTQA) in the presence of H2O2. The product of this reaction emits constant fluorescence, in a sulfuric acid environment, at 490 nm, with the exciting radiation set at 340 nm. Various parameters such as acidity, flow rate, solvents, and temperature were studied. The presence of a surface-active agent was also considered in order to increase sensitivity. At the optimal conditions, a calibration curve was constructed, revealing a linear range of 2-100 microg L(-1) and a detection limit as low as 0.5 microg L(-1) while the RSD ranged in the area of 0.1-1.8%, depending on vanadium concentration. The method was successfully applied to the analysis of a wide variety of food samples, which are known to contribute to the dietary required amount of vanadium and to relevant biological matrixes. Reversing the conditions of the above reaction, the effect of the peroxy group on the vanadium-PTQA system was examined. The formation of a vanadyl complex was revealed which was suitable for the determination of hydrogen peroxide and peroxy acids. Linear calibration curves in the range of 0.2-50 microM for H2O2 and 0.1-2 microM for a respective peroxy acid were obtained, yielding detection limits of 0.05 and 0.03 microM, respectively.     

Array biosensor for simultaneous identification of bacterial, viral, and protein analytes.

The array biosensor was fabricated to analyze multiple samples simultaneously for multiple analytes. The sensor utilized a standard sandwich immunoassay format: Antigen-specific "capture" antibodies were immobilized in a patterned array on the surface of a planar waveguide and bound analyte was subsequently detected using fluorescent tracer antibodies. This study describes the analysis of 126 blind samples for the presence of three distinct classes of analytes. To address potential complications arising from using a mixture of tracer antibodies in the multianalyte assay, three single-analyte assays were run in parallel with a multianalyte assay. Mixtures of analytes were also assayed to demonstrate the sensor's ability to detect more than a single species at a time. The array sensor was capable of detecting viral, bacterial, and protein analytes using a facile 14-min assay with sensitivity levels approaching those of standard ELISA methods. Limits of detection for Bacillus globigii, MS2 bacteriophage, and staphylococcal enterotoxin B (SEB) were 10(5) cfu/mL, 10(7) pfu/mL, and 10 ng/mL, respectively. The array biosensor also analyzed multiple samples simultaneously and detected mixtures of the different types of analytes in the multianalyte format.     

Preparation of a single-chain variable fragment and a recombinant antigen-binding fragment against the anti-malarial drugs, artemisinin and artesunate, and their application in an ELISA

Two different recombinant antibodies, a single-chain variable fragment (scFv) and an antigen-binding fragment (Fab), were prepared against artemisinin (AM) and artesunate (AS) and were developed for use in an enzyme-linked immunosorbent assay (ELISA). The recombinant antibodies, which were derived from a single monoclonal antibody against AM and AS (mAb 1C1) prepared by us, were expressed by Escherichia coli cells and their reactivity and specificity were characterized. As a result, to obtain sufficient signal in indirect ELISA, a much greater amount of a first antibody was needed in the use of scFv due to the differences of the secondary antibody and conformational stability. Therefore, we focused on the development of the recombinant Fab antibodies and applied it to indirect competitive ELISA. The specificity of the Fab was similar to that of mAb 1C1 in that it showed specific reactivity toward AM and AS only. The sensitivity of the icELISA (0.16 μg/mL to 40 μg/mL for AM and 8.0 ng/mL to 60 ng/mL for AS) was sufficient for analysis of antimalarial drugs, and its utility for quality control of analysis of Artemisia spp. was validated. The Fab expression and refolding systems provided a good yield of high-quality antibodies. The recombinant antibody against AM and AS provides an essential component of an economically attractive immunoassay and will be useful in other immunochemical applications for the analysis and purification of antimalarial drugs.     

Immobilization of antibodies on polyaniline films and its application in a piezoelectric immunosensor

Conducting polymers, especially polyaniline (PAni), have been extensively used in biosensor applications. A protocol for covalent immobilization of human IgG on polyaniline using glutaraldehyde as the cross-linker is described in this report and utilized in development of a piezoelectric immunosensor. Here, PAni was used as the substrate for immobilization. The electropolymerization parameters were optimized to get suitable thickness and surface morphology of the PAni for obtaining high density and uniformity of immobilized antibodies on the surface of our films. Possible reaction between PAni thin films and glutaraldehyde was explored using FT-IR characterization in grazing angle mode and XPS. The protocol has been characterized with the help of quartz crystal microbalance analysis. An antibody surface density of 4.86 ng/mm2 was obtained. A piezoelectric biosensor developed for detection of IgG with the proposed protocol was capable of differentiating the target analyte concentrations between 500 ng/mL and 25 microg/mL with nonspecific binding of approximately 10%.