Comparison of different carbon ink based screen-printed electrodes towards amperometric immunosensing

The aim of the present work was to make amperometric immunosensors based on the principle of enzyme-linked immunosorbent assay (ELISA). For this purpose, screen-printed electrodes (SPEs) were fabricated using various carbon inks (commercially available inks Gwent, Acheson, Eltecks and two homemade inks PSG & PVCG) to determine the best ink in realizing immunosensors. Amperometric immunosensors made by different carbon inks were compared with standard ELISA in terms of total assay time, amount of biological materials used and sensitivity of detection. A model system containing rabbit anti-mouse immunoglobulin G (RαMIgG) as the capturing antibody, mouse IgG (MIgG) as antigen and alkaline phosphatase conjugated RαMIgG as revealing antibody was used. In these studies, 1-naphthyl phosphate was used as substrate. The experiments done include electrochemical characterization of electrodes, optimization of dilutions of antibodies, immobilization of antibody on the electrode were carried out. The minimum detection limit for the best results of MIgG determination were obtained on screen-printed electrode made by Gwent carbon ink and PSG carbon ink, with a detection limit of 1.0 and 2.0 ng/ml respectively. The time required for detection of mouse IgG was 40 min for SPEs. By using the conventional spectrophotometric method (ELISA method), the minimum detection limit for the MIgG (antigen) detection was 50 ng/ml and the time required for analysis was found to be 140 min.     

Functional properties of proteins immobilized on albumin microspheres

Bovine serum albumin (BSA) microspheres with an average diameter of 12.5 micron were prepared by crosslinking of BSA molecules with glutaraldehyde in the presence of polymethylmethacrylate dissolved in chloroform-toluene. Trypsin and anti-human IgG antibody were immobilized onto their surfaces by the glutaraldehyde-activation method. The catalytic activity and storage stability of the immobilized trypsin were satisfactorily high. The enzyme immunoassay (EIA) method using BSA-microspheres as a solid phase has a high sensitivity (the minimum concentration of detectable antigen in the sample: 0.2 ng/ml) and a wide concentration range (final concentration 0.027-3000 ng/ml) for the detection of human IgG.     

A label‐free immunoassay using eggshell membrane as matrix and poly(diallyldimethylammonium chloride) as light‐scattering enhancer

A label‐free immunoassay system using eggshell membrane as a matrix was developed. A common spectrofluorometer was used to collect light‐scattering signals. The rabbit anti‐human IgG (Ab) was first immobilized on the eggshell membrane with glutaraldehyde. Then, based on the immunoreactions and electrostatic interaction, the target human IgG antigen (Ag) and poly(diallyldimethylammonium chloride) (PDDA) were captured on the eggshell membrane. It was found that the light‐scattering signal resulting from the PDDA immunotargeted on modified eggshell membrane was related to the concentration of target antigen. Under the optimal conditions, the light scattering intensity is directly proportional to the concentration of Ag in the range of 5.00–500 ng/mL (r = 0.995) with the limit of detection of 2.31 ng/mL [signal:noise ratio (S:N) = 3]. The proposed method was successfully applied to the determination of IgG in human serum, and the results were in agreement with those obtained by a general immunonephelometric method. Copyright © 2011 John Wiley & Sons, Ltd.     

A novel reagentless amperometric immunosensor based on gold nanoparticles/TMB/Nafion-modified electrode

A novel reagentless immunosensor was fabricated by immobilization of redox mediator 3,3',5,5'-tetramethylbenzidine (TMB) on the Nafion (Nf) film modified glassy carbon electrode. Gold nanoparticles were assembled onto the TMB/Nafion film modified electrode to provide active sites for the immobilization of antibody molecules. The antibody (anti-MIgG), in the present study, was fixed on the electrode for the rapid detection of antigen molecules (MIgG as a model analyte). The results showed that the immunosensor based on the immobilized TMB redox mediator exhibited good electrochemical response. A good linear relationship between peak current and the concentration of the MIgG was obtained in the concentration range from 4 to 120ng/mL. The detection limit was estimated to be 1ng/ml. Under the optimized conditions, the immunosensor exhibits good sensitivity, reproducibility and stability.     

New antibodies immobilization system into a graphite-polysulfone membrane for amperometric immunosensors

Polysulfone membrane is used for the first time for the preparation of electrochemical immunosensors. A disposable immunosensor based on a porous conductor polymer graphite-polysulfone-electrode has been developed using a phase inversion technique for the determination of anti-rabbit IgG (anti-RIgG) as a model analyte. To construct the sensor, a conductor membrane was deposited on the surface of working graphite-epoxy composite (GEC) electrode. The membrane was characterized by SEM. This sensor was based on the competitive assay between free and labeled anti-RIgG for the available binding sites of immobilized rabbit IgG (RIgG). Incubation parameters were optimized in this work. The immunological reaction was detected using an enzymatic-labeling procedure (HRP enzyme) combined with the amperometric detection using H(2)O(2) as substrate and hydroquinone as mediator. This sensor shows stability during a week and a good reproducibility. The current was monitored amperometrically at -0.1 V versus SCE and this method showed a linear range of the anti-RIgG from 1 to 6 microg/ml. The detection limit was determined to be 0.77 microg/ml.     

Urease-gelatin interdigitated microelectrodes for the conductometric determination of protease activity

Conductometric microbiosensors for the determination of trypsin were elaborated via the modification of microfabricated interdigitated gold electrodes by a cross-linked urease/BSA coating covered by a gelatin film. The resulting microelectrodes were exposed to different trypsin concentrations ranging from 100pg/mL to 1mg/mL (1mU/mL to 10,000U/mL) for selective proteolytic degradation of the gelatin film. Then, the conductometric response of the microbiosensors to urea (33muM) was recorded as a function of the trypsin concentration, the gelatin amount (8-80ng) and the incubation time (40s, 100min). The optimum incubation time for each trypsin concentration was determined leading to a detection limit of 100pg/mL (1mU/mL). In these optimized conditions, the proof of concept of this sensitive, disposable, low-cost and label-free trypsin biosensors based on a conductometric transducer was demonstrated for the first time.     

抗吡虫啉单克隆抗体的制备及鉴定

为制备灵敏度高,特异性强的抗吡虫啉单克隆抗体,建立经济、快速、准确的吡虫啉残留免疫学分析方法,采用分子模拟技术分析吡虫啉及其类似农药的电荷分布后,选择1-[6-(2-羧乙硫基-3-吡啶)甲基]-N-硝基-2-咪唑啉亚胺 (H1) 作为免疫半抗原,1-(6-氯-3-吡啶甲基)-3-羧丙基-N-硝基-2-咪唑啉亚胺 (H2) 作为包被半抗原,利用NHS酯法将H1和H2分别与牛血清蛋白 (BSA) 和卵清蛋白 (OVA) 偶联合成免疫原与包被原。免疫BALB/c小鼠后,采用常规杂交瘤技术共获得2株稳定分泌抗吡虫     

Flow-injection resonance light scattering detection of proteins at the nanogram level.

A resonance light scattering (RLS) detection method for protein was developed, using a flow-injection system based on the enhancement of RLS signals from Biebrich scarlet (BS) by protein. The enhanced RLS intensities at 286.0 nm, in acidic aqueous medium, were proportional to the protein concentration over the range 0.005-18 microg/mL and 0.008-16 microg/mL for human serum albumin (HSA) and bovine serum albumin (BSA), respectively, with corresponding limits of detection (3sigma) of 5.00 ng/mL for HSA, and 7.80 ng/mL for BSA. The method was successfully applied to the quantification of total proteins in human serum samples.     

A biosensor based on co-immobilized L-glutamate oxidase and L-glutamate dehydrogenase for analysis of monosodium glutamate in food

A monosodium glutamate (MSG) biosensor made by co-immobilized L-glutamate oxidase (L-GLOD) and L-glutamate dehydrogenase (L-GLDH) as the bio-component based on substrate recycling for highly sensitive MSG or L-glutamate determination, has been developed. Regeneration of MSG by substrate recycling provided an amplification of the sensor response. Higher signal amplification was found in the presence of ammonium ion. The sensor was standardized to determine MSG in the range of 0.02-3.0 mg/L. Linearity was obtained from 0.02 to 1.2 mg/L in presence of ammonium ion (10 mM) and NADPH (reduced nicotinamide adenine dinucleotide phosphate) (2 mM), but in absence of L-GLDH, the detection limit of MSG is confined to 0.1 mg/L. The apparent Km for MSG with L-GLOD-L-GLDH coupled reaction was 0.4451 mM but 1.9222 mM when only L-GLOD was immobilized. Cross linking with glutaraldehyde in the presence of bovine serum albumin (BSA) as a spacer molecule has been used for the method of immobilization. The response time of the sensor was 2 min. The optimum pH and temperature of the biosensor has been determined as 7+/-2 and 25+/-2 degrees C, respectively. The enzyme immobilized on the membrane was used for over 50 measurements. The standard error of the sample measurement was 4-5%. The activity of the enzyme-immobilized membrane was tested over a period of 60 days.     

Determination of echinacoside in rat serum by reversed-phase high-performance liquid chromatography with ultraviolet detection and its application to pharmacokinetics and bioavailability

A rapid and simple high-performance liquid chromatographic (HPLC) method has been developed and validated for the determination of echinacoside (ECH) in rat serum. After protein precipitation of serum sample with trichloroacetic acid, the supernatant was directly injected and analyzed on a C(18) CapcellACR analytical column (150 mm x 4.6mm I.D. 5 microm) with a mobile phase consisting of acetonitrile-0.5% acetic acid (15.5:84.5, v/v). The UV detector was set at 330 nm. The lower limit of detection and quantification were 9 and 29.2 ng/mL, respectively, and the calibration curves were linear over the concentration range of 29.2-18250 ng/mL. The assay method was successfully applied to the study of the pharmacokinetics and bioavailability of ECH in rat.     

Simultaneous determination of doxifluridine and 5-fluorouracil in monkey serum by high performance liquid chromatography with tandem mass spectrometry

A reverse-phase high performance liquid chromatography method with electrospray ionization and detection by mass spectrometry is described for the simultaneous determination of doxifluridine and its active metabolite 5-fluorouracil in monkey serum. A liquid/liquid extraction with ethyl acetate (90%) and isopropyl alcohol (10%) was used to extract simultaneously doxifluridine and 5-FU which have considerable difference in the polarity. Optimum chromatographic separation was achieved on a Agilent Zorbax C(18) (100 mm x 2.1mm, 3.5 microm) column with a mobile phase of methanol-water (20:80, v/v). The flow rate was 0.2 mL/min with total cycle time of 5 min. The lower limit of quantification (LLOQ) was validated at 10.0 ng/mL of serum for both doxifluridine and 5-FU. Accuracy and precision of quality control (QC) samples for both compounds met FDA Guidance criteria of +/-15% with average QC accuracy of 95.5-105.0% and coefficients of variation of 1.1-9.5% in the 10-2000 ng/mL concentration range. This method demonstrated adequate sensitivity, specificity, accuracy, precision, stability to support the analysis of monkey serum samples.     

Cross-talk-free multiplexed immunoassay using a disposable electrochemiluminescent immunosensor array coupled with a non-array detector

A disposable electrochemiluminescent (ECL) immunosensor array was fabricated on a screen-printed carbon electrode (SPCE) substrate to perform multiplexed immunoassay (MIA) for the first time. The SPCE substrate was composed of an array of four carbon working electrodes, one common Ag/AgCl reference electrode, and one common carbon counter electrode. The immunosensor array was constructed by site-selectively immobilizing multiple antigens on different working electrodes of the SPCE substrate. With a competitive immunoassay format, the immobilized antigens competed with antigens in the sample to capture their corresponding tri(2,2'-bipyridyl)ruthenium(II)-labeled antibodies. The ECL signals from the immunosensors in this array were sequentially detected by a photomultiplier with the aid of a homemade single-pore-four-throw switch. Due to the ECL readout mechanism and the sequential detection mode, it could avoid the cross-talk between the adjacent immunosensors, which was common in other reported immunosensor array. Human, rabbit and mouse immunoglobulin Gs were near-simultaneously assayed as the model analytes. The linear ranges for them were 10-400, 20-400, and 20-400 ng/mL, with detection limits of 2.9, 6.1 and 6.5 ng/mL (S/N=3), respectively. This novel ECL strategy based on immunosensor array coupled with non-array detector provided a simple, sensitive, low-cost and time-saving approach for MIA. It showed great application potential in point-of-care test and field analysis of bio-agents, with mass production potential and high throughput.     

A disposable amperometric immunosensor for alpha-1-fetoprotein based on enzyme-labeled antibody/chitosan-membrane-modified screen-printed carbon electrode

A screen-printed three-electrode system is fabricated to prepare a novel disposable screen-printed immunosensor for rapid determination of alpha-1-fetoprotein (AFP) in human serum. The immunosensor is prepared by entrapping horseradish peroxidase (HRP)-labeled AFP antibody in chitosan membrane to modify the screen-printed carbon electrode. The membrane is characterized with scanning electron microscope and electrochemical methods. After the immunosensor is incubated with AFP at 30 degrees C for 35 min, the access of the active center of HRP catalyzing the oxidation reaction of thionine by H(2)O(2) is partly inhibited. In presence of 1.2 mM thionine and 6 mM H(2)O(2), the electrocatalytic current decreases linearly in two concentration ranges of AFP from 0 to 20 and from 20 to 150 ng/mL with a detection limit of 0.74 ng/mL. The immunosensor shows an acceptable accuracy compared with those obtained from immunoradiometric assays. The interassay coefficients of variation are 6.6 and 4.2% at 10 and 100 ng/mL, respectively. The storage stability is acceptable in pH 7.0 phosphate buffer solution at 4 degrees C for more than 10 days. The proposed method can detect the AFP through one-step immunoassay and would be valuable for clinical immunoassay.     

Label-free immunosensor for prostate-specific antigen based on single-walled carbon nanotube array-modified microelectrodes

We have fabricated a label-free electrochemical immunosensor using microelectrode arrays modified with single-walled carbon nanotubes (SWNTs). Label-free detection of a cancer marker, total prostate-specific antigen (T-PSA), was carried out using differential pulse voltammetry (DPV). The current signals, derived from the oxidation of tyrosine (Tyr), and tryptophan (Trp) residues, increased with the interaction between T-PSA on T-PSA-mAb covalently immobilized on SWNTs. The selectivity of our biosensor was challenged using bovine serum albumin (BSA) as the target protein. The detection limit for T-PSA was determined as 0.25 ng/mL. Since the cut-off limit of T-PSA between prostate hyperplasia and cancer is 4 ng/mL, the performance of our label-free electrochemical immunosensor seems promising for further clinical applications.     

Method for printing functional protein microarrays

Piezoelectric dispensing of proteins from borosilicate glass capillaries is a popular method of protein biochip fabrication that offers the advantages of sample recovery and noncontact with the printing substrate. However, little regard has been given to the quantitative aspects of dispensing minute volumes (1 nL or less) at the low protein concentrations (20 micrograms/mL or less) typically used in microprinting. Specifically, loss of protein sample due to nonspecific adsorption to the glass surface of the dispensing capillaries can limit the amount of protein delivered to the substrate. We demonstrate the benefits of a low ionic strength buffer containing the carrier protein BSA that effectively minimizes the ionic strength-dependent phenomenon of nonspecific protein adsorption to borosilicate glass. Over the concentration range of 20-2.5 micrograms/mL, the dispensing of a reference IgG in 10 mM PBS including 0.1% BSA resulted in the deposition of 3.6- to 44-fold more IgG compared to the deposition of IgG in standard 150 mM PBS in the absence of BSA. Furthermore, when the IgG was dispensed with carrier protein, the resulting spots exhibited a more uniform morphology. In a direct immunoassay for cholera toxin, capture antibody spots dispensed in 10 mM PBS containing 0.1% BSA produced fluorescent signals that were 2.8- to 4.3-fold more intense than antibody spots that were dispensed in 150 mM PBS without BSA. Interestingly, no differences were observed in the specific activities of the capture antibodies as a result of printing in the different buffers. The implications of these results on the future development of protein biochips are discussed.     

Monitoring glutamine in mammalian cell cultures using an amperometric biosensor.

An amperometric biosensor has been developed for monitoring glutamine in the pulsed-batch cultivation of murine hybridoma cells. Glutamine oxidase was cross-linked with bovine serum albumin (BSA) via glutaraldehyde activation and deposited on a preactivated nylon membrane. Glutaminase was then immobilized on the protein layer and the resulting membrane was attached to the sensing area of a hydrogen peroxide probe (platinum vs silver/silver chloride polarized at +0.7 V). An orthogonal test was performed to optimize the activity of the membrane for glutamine with respect to the concentrations of glutamate oxidase, BSA, glutaminase and glutaraldehyde. There was an excellent linear relationship between the biosensor's response and glutamine in the range 0.1-3 mM. The determination of glutamine could be performed in 2 min and each membrane was reused for at least 300 consecutive analyses. The data obtained also agreed well with those high-performance liquid chromatography, thus validating the applicability of the biosensor.     

Determination of acyclovir in human serum by high-performance liquid chromatography using liquid-liquid extraction and its application in pharmacokinetic studies

A fast, simple and sensitive high performance liquid chromatographic (HPLC) method has been described for determination of acyclovir in human serum. Since acyclovir is a polar compound and soluble in aqueous medium and practically insoluble in most of organic solvents, its analysis in biological fluids in currently published HPLC methods, involve pre-treatment of acyclovir plasma sample including deproteinization or solid phase extraction. In present method liquid-liquid extraction of acyclovir and internal standard (vanillin) is achieved using dichloromethane-isopropyl alcohol (1:1, v/v) as an extracting solvent. Analysis was carried out on ODS column using methanol-phosphate buffer (0.05 M) containing sodium dodecyl sulfate (200 mg/L) and triethylamine (2 mL/L, v/v) as mobile phase (pH=2.3; 5:95, v/v) at flow rate of 2 ml/min. The method was shown to be selective and linear into the concentration range of 10-2560 ng/mL. Accuracy and precision of the method were also studied. The limit of quantitation was evaluated to be 10 ng/mL. This method was applied in bioequivalence study of two different acyclovir preparations after administration of 400mg in 12 healthy volunteers.     

Detection of Bacillus anthracis spores and a model protein using PEMC sensors in a flow cell at 1 mL/min

Piezoelectric-excited millimeter-sized cantilever (PEMC) sensors of 4mm(2) sensing area were immobilized with antibody specific to Bacillus anthracis (anti-BA) spores or bovine serum albumin (anti-BSA). Detection of pathogen (Bacillus anthracis (BA) at 300 spores/mL) and BSA (1 mg/mL) were investigated under both stagnant and flow conditions. Two flow cell designs were evaluated by characterizing flow-induced resonant frequency shifts. One of the flow cells labeled SFC-2 (hold-up volume of 0.3 mL), showed small fluctuations (+/-20 Hz) around a common resonant frequency response of 217 Hz in the flow rate range of 1-17 mL/min. The total resonant frequency change obtained for the binding of 300 spores/mL in 1h was 90+/-5 Hz (n=2), and 162+/-10 Hz (n=2) under stagnant and flow conditions, respectively. Binding of antibodies, anti-BA and anti-BSA, were more rapid under flow than under stagnant conditions. The sensor was repeatedly exposed to BSA with an intermediate release step. The first and second responses to BSA were nearly identical. The total resonant frequency response to BSA was 388+/-10 (n=2) Hz under flow conditions. Kinetic analysis is carried out to quantify the effect of flow rate on antibody immobilization and the two types of detection experiments.     

Dye-binding protein assay using a long-wave-absorbing cyanine probe

A simple and fast protein assay that involves the binding of water-soluble sulfonate heptamethylene cyanine to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 778 to 904 nm, and the increase in absorption at 904 nm is monitored. This assay is very reproducible, of good color stability for at least 80 min, and sensitive at the 100 ng/mL level of human serum albumin (HSA) when a spectrophotometer with near-infrared wavelength is used to measure absorbance. Few chemicals except ionic surfactants such as cetyltrimethylammonium bromide and sodium dodecyl sulfonate interfere with the assay. Purified proteins have different capacities to interact with the dye; under the experimental conditions, the linear ranges of bovine serum albumin (BSA), HSA and gamma-IgG were 200-2000, 100-2400, and 200-3000 ng/mL, respectively. The relative standard deviation for the five replicate determinations of 1200 ng/mL BSA is 2.1%.     

Carbon nanospheres-promoted electrochemical immunoassay coupled with hollow platinum nanolabels for sensitivity enhancement

Two nanostructures including carbon nanospheres-graphene hybrid nanosheets (CNS-GNS) and hollow platinum nanospheres (HPtNS) were first synthesized by using direct electrolytic reduction and wet chemistry methods, respectively. Thereafter, a specific sandwich-type electrochemical immunoassay was designed for determination of carcinoembryonic antigen (CEA) by using HPtNS-labeled horseradish peroxidase-anti-CEA conjugates (HRP-anti-CEA) as molecular tags and anti-CEA-assembled CNS-GPS as sensing probes. Compared with pure graphene nanosheets, the presence of carbon nanospheres on the graphene increased the surface coverage of the substrate, and enhanced the immobilized amount of primary antibodies. Several labeling protocols, such as HRP-anti-CEA, solid platinum nanoparticle-labeled HRP-anti-CEA, and hollow platinum nanospheres-labeled HRP-anti-CEA, were investigated for determination of CEA and improved analytical features were obtained with hollow platinum nanosphere labeling. With the HPtNS labeling method, the effects of incubation time and pH on the current responses of the immunosensors were also studied. The strong attachment of biomolecules to the CNS-GPS and HPtNS resulted in a good repeatability and intermediate precision down to 10.2%. The dynamic concentration range spanned from 0.001 ng mL(-1) to 100 ng mL(-1) CEA with a detection limit of 1.0 pg mL(-1) at the 3S(blank) level. No significant differences at the 0.05 significance level were encountered in the analysis of 10 clinical serum samples between the developed immunoassay and the commercially available electrochemiluminescent method for determination of CEA.