Development of evanescent wave all-fiber immunosensor for environmental water analysis

A compact and portable evanescent wave all-fiber immunosensor is developed, which employs a novel single-multi-mode fiber optic coupler for exciting and collecting fluorescence emission from the fiber optic probe. Combination tapered fiber probes are produced by tube-etching method and the best tapered ratio of the probe determined is approximately 0.37. Calibration curves obtained for 2,4-dichlorophenoxyacetic acid (2,4-D) and Microcystin-LR (MC-LR) have detection limits of 0.09 microgL(-1)and 0.03 microgL(-1), respectively. The 50% inhibition concentration (IC(50)) for MC-LR and 2,4-D were 1.12+/-0.01 microgL(-1)and 3.81+/-0.03 microgL(-1), respectively. A reusable immunosurface is provided via the covalent attachment of the analyte derivative to a self-assembled monolayer formed onto the fiber optic probe. The regeneration of the sensor surface allows the performance of more than 100 assay cycles within an analysis time of about 20 min for each assay cycle.     

Novel miniature SPR immunosensor equipped with all-in-one multi-microchannel sensor chip for detecting low-molecular-weight analytes

A simple and versatile miniaturized surface plasmon resonance (SPR) immunosensor enabling parallel analysis of multiple analytes or multiple samples of an analyte has been investigated for detection of a low-molecular-weight (lmw) toxin, 2,4-dichlorophenoxyacetic acid (2,4-D). A specially designed multi-microchannel SPR sensor module, integrating an optical-prism coated with an array of thin Au-films, a multi-microchannel plate (eight channels) and a flow-cell together, has been fabricated. The sensing surface was fabricated simply by physical adsorption of a protein conjugate of 2,4-D, and an indirect competitive immunoassay principle has been applied for the quantification of 2,4-D. Multiple 2,4-D samples were analyzed in a single step and a low-detection-limit (LDL) of 0.1 ppb (ng ml(-1)) 2,4-D was established. Competence of the portable SPR immunosensor for selective detection of 2,4-D despite the presence of various structurally resemblant interferents and from river-water samples has been demonstrated. The independent all-in-one sensor module highly favors shelf-storage between multiple determinations, and reusability of a same multi-microchannel flow-module for more than 35 days with intermittent storage (4-8 degrees C) has been confirmed. The LDL of 2,4-D could be enhanced further by introducing a simple avidin-biotin interaction-based sandwich immunoassay, with which the sensor signal multiplied enormously by a factor of ca. 10 and the LDL enhanced to 0.008 ppb. The miniature SPR sensor demonstrated here for simultaneous analysis of multiple samples with reusability and good storage ability is an important consideration for the advancement of biosensor technology.     

An impedimetric immunosensor based on interdigitated microelectrodes (IDmicroE) for the determination of atrazine residues in food samples

A novel impedimetric immunosensor for atrazine detection has been developed. The immunosensor is based on an array of interdigitated micro-electrodes (IDmicroE) and immunoreagents specifically developed to detect this pesticide. Immunochemical determination of atrazine is possible without the use of any label. An atrazine-haptenized protein was covalently immobilized on the surface of the interdigitated mu-electrodes area (interdigits space) previously activated with (3-glycidoxypropyl)trimethoxysilane. Before, the gold electrodes were blocked using N-acetylcysteamine to prevent non-specific adsorptions. All biofunctionalization steps were characterized by chemical affinity methods and impedance spectroscopy. Immunosensors measures are made by exposing the sensor to solutions containing a mixture of the analyte and the specific antibody. With this configuration, the immunosensor detects atrazine with a limit of detection of 0.04 microg L(-1) without the use of any label. The potential of the immunosensor to analyze pesticide residues in complex sample matrices, such as red wine, has been evaluated. The results shown that after solid-phase extraction atrazine can be determined in this type of sample with a limit of detection of 0.19 microg L(-1), far below the Maximum Residue Level (MRL) established by EC for residues of this herbicide in wine.     

Fiber-optic immunosensor for mycotoxins

Evanescent wave-based fiber-optic immunosensors were studied for the detection of fumonisins and aflatoxins in maize. Two formats, competitive and non-competitive, were used. A competitive format was used to measure fumonisin B1 (FB1) in both spiked and naturally contaminated maize samples. Fumonisin monoclonal antibodies were covalently coupled to an optical fiber and the competition between FB1 and FB1 labeled with fluorescein (FB1-FITC) for the limited number of binding sites on the fiber was assessed. The signal generated in the assay was inversely proportional to the FB1 concentration. For samples, the concentration causing an inhibition of binding by 50% (IC50) was dependent upon the clean-up procedure used. Simple dilution of methanolic maize extracts yielded an assay with an IC50 equivalent to 25 microg FB1 g(-1) maize with a limit of detection of 3.2 microg g(-1) maize. Affinity column clean-up yielded an assay with an IC50 equivalent to 5 microg FB1 g(-1) maize (limit of detection 0.4 microg FB1 g(-1)). An HPLC method and the immunosensor method agreed well for naturally contaminated maize samples except when large amounts of other fumonisins that cross-react with the immunosensor were present. The second sensor format, for the mycotoxin aflatoxin B1 (AFB1), was a non-competitive assay using the native fluorescence of this mycotoxin. Because the fluorescence of AFB1 itself was detected, the response of the sensor was directly proportional to the toxin concentration. The sensor, while capable of detecting as little as 2 ng ml(-1) of AFB1 in solution was technically not an immunosensor, since the attachment of aflatoxin specific antibodies was not required. Sensors of the formats described have the potential to rapidly screen individual maize samples but require coupling with a clean-up technique to be truly effective.     

A SPR-based immunosensor for the detection of isoproturon

The proof of principle of a reusable surface plasmon resonance (SPR)-based immunosensor for the monitoring of isoproturon (IPU), a selective and systemic herbicide, is presented. The detecting rat monoclonal anti-isoproturon antibody (mAb IOC 7E1) was reversibly immobilized through the use of a capture mouse anti-rat (kappa-chain) monoclonal antibody (mAb TIB 172), which was covalently immobilized on the sensor chip surface. Such strategy features a controlled binding of the captured detecting antibody as well as facilitates the surface regeneration. The capture of the anti-IPU mAb by the antibody (TIB 172) coated sensor surface could be carried out up to 120 times (immobilization/regeneration cycles) without any evidence of activity loss. With a high test midpoint and a low associated SPR signal, the direct detection format was shown to be unsuitable for the routine analysis of isoproturon. However, the limit of detection (LOD) could be easily enhanced by using a strategy based on a surface competition assay, which improved all immunosensor parameters. Moreover, the sensitivity and working range of the indirect format were found to be dependent on the surface density of the anti-IPU mAb IOC 7E1. As expected for competitive formats, the lowest surface coverage (0.5 ng/mm(2)) allowed a lower detection of the herbicide isoproturon with a calculated LOD of 0.1 microg/l, an IC(50) (50% inhibition) of 5.3+/-0.6 microg/l, and a working range (20-80% inhibition) of 1.3-16.3 microg/l.     

Determination of carbaryl in natural water samples by a surface plasmon resonance flow-through immunosensor

The analysis of carbaryl in natural water samples was accomplished using a portable immunosensor based on surface plasmon resonance (SPR) technology. The assay was based on a binding inhibition immunoassay format with the analyte derivative covalently immobilized on the sensor surface. An alkanethiol self-assembled monolayer (SAM) was formed onto the gold-coated sensor surface to allow the reusability of the same sensing surface during 220 regeneration cycles. Reproducibility was evaluated by performing three independent assays in triplicate on 3 different days. The batch-assay variability was also calculated using three different gold-coated sensor surfaces. The intra- and inter-day relative standard deviation were 8.6 and 15.3%, respectively, whilst a variation of 7.4% in assay sensitivity was obtained by employing different sensor chips. The lowest detection limit, calculated as the concentration providing a 10% decrease of the blank signal, was of 1.38 microg L(-1). Matrix effects were also evaluated in different water types, showing I50 values (carbaryl concentrations that produced a 50% decrease of the blank signal) within the range of carbaryl standard curves in distilled water (2.78-3.55 microg L(-1)). The carbaryl immunoassay performance was validated with respect to conventional high-performance liquid chromatography-mass spectrometry (HPLC-MS). The correlation between methods was in good agreement (r2 = 0.998, 0.999 and 0.999) for the three types of natural water samples tested. A complete assay cycle, including regeneration, is accomplished in 20 min. All measurements were carried out with the SPR sensor system (beta-SPR) commercialised by the company SENSIA, SL (Spain). The small size and low-time of response of the beta-SPR platform would allow its utilization in real contaminated locations.     

Investigation of highly sensitive piezoelectric immunosensors for 2,4-dichlorophenoxyacetic acid

The improved highly sensitive piezoelectric immunosensor has been developed and evaluated using a model interaction of antibody with the model hapten-herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). For immobilization of 2,4-D, the self-assembled layers of cystamine, 4-aminothiophenol or 3,3'-dithio-bis(propionic acid N-hydroxysuccinimide ester) were formed on smooth and rough crystals coated with gold or silver electrodes. The immunochemical interactions performed well in all cases, the aminothiophenol on gold was chosen as the optimum with regard to regeneration of immunosensing surfaces. The kinetics of interaction of surface-bound 2,4-D with free antibody provided significantly higher kinetic parameters (kinetic association rate constant) when using optically smooth crystals compared to common rough crystal. Therefore, the smooth crystal should be preferred for future kinetic studies. The competitive assay of the herbicide 2,4-D achieved a limit of detection of 10 ng/l using the monoclonal anti-2,4-D antibody F6C10. Finally, a direct assay format has been evaluated using a thicker layer of glutaraldehyde-crosslinked antibody on the sensing surface. The direct binding of a small herbicide molecule was followed in real time. The detected concentration of 2,4-D (5 microg/l) was low enough for future direct monitoring of this herbicide in water.     

Surface plasmon resonance immunosensor for highly sensitive detection of 2,4,6-trinitrotoluene

We have examined the sensing characteristics of a surface plasmon resonance (SPR) immunoassay for the detection of 2,4,6-trinitrotoluene (TNT) using an immunoreaction between 2,4,6-trinitrophenol-ovalbumin (TNP-OVA) conjugate and anti-2,4,6-trinitrophenol antibody (anti-TNP antibody). TNP-OVA conjugate was attached to a SPR-gold sensing surface by means of physical immobilization, which undergoes binding interaction with anti-TNP antibody. Both the immobilization and binding processes were studied from a change in the SPR-resonance angle. The quantification of TNT is based on the principle of indirect competitive immunoassay, in which the immunoreaction between the TNP-OVA conjugate and anti-TNP antibody was inhibited in the presence of free TNT in solution. The decrease in the resonance angle shift is proportional to an increase in concentration of TNT used for incubation. The immunoassay exhibited excellent sensitivity for the detection of TNT in the concentration range from 0.09 to 1000 ng/ml with good stability and reproducibility. The immunosensor developed could detect TNT as low as 0.09 ng/ml, within a response time of approximately 22 min. The sensor surface was regenerated by a brief flow of pepsin solution, which disrupts the antigen-antibody complex without destroying the conjugate biofilm. Cross-reactivity of the SPR sensor to some structurally related nitroaromatic derivative and the detection of TNT in the presence of these nitroaromatic compounds were investigated. The cross-reactivity of the SPR sensor to 2,4-dinitrotoluene (2,4-DNT), 1,3-dinitrobenzene (1,3-DNB), 2-amino-4,6-dinitrotoluene (2A-4,6-DNT) and 4-amino-2,6-dinitrotoluene (4A-2,6-DNT) were very low (< or =1.1%). The analytical characteristics of the proposed immunosensor are highly promising for the development of new field-portable sensors for on-site detection of landmines.     

A novel piezoelectric quartz micro-array immunosensor based on self-assembled monolayer for determination of human chorionic gonadotropin

A novel multi-channel 2 x 5 model of piezoelectric quartz micro-array immunosensor has been developed for quantitative detection of human chorionic gonadotropin (hCG) in serum or urine samples. Every crystal unit of the fabricated piezoelectric hCG micro-array immunosensor can oscillate independently without interfering each other. A 2 x 5 model of micro-array immunosensor as compared with a one-channel immunosensor can provide eight times higher detection speeds for hCG assay. The anti-hCG antibody is deposited on the gold electrode's surface of 10 MHz quartz AT-cut crystal by self-assembled technique using sulfosuccinimidyl 6-[3'-(2-pyridyldithio) propionamido] hexanoate (Sulfo-LC-SPDP), and serves as an antibody recognizing layer. The highly ordered self-assembled monolayers (SAM) ensure well-controlled surface structure and offer many advantages to the performance of the sensor. Compared with conventional antibody immobilization methods, the amount and the reaction activity of antibody monolayer coated by the SAM binding are bigger than those by the SPA method, and less non-specific binding caused by other analytes in sample is found. Under the optimized experimental conditions, the results showed that micro-array immunosensor quantitatively detected serum or urine hCG in the range of 2.5-500 mIU/ml with high precision (CV<5%); other hormones in human serum and urine did not interfere with the determination markedly. Serum and urine samples of 60 patients were detected by the micro-array immunosensor, and the results agreed well with those given by the commercial radioimmunoassay test kit, with correlation coefficient of 0.92. After regeneration with urea solution the coated immunosensor can be reused five times without appreciable loss of activity.     

A novel sandwich immunosensing method for measuring cardiac troponin I in sera

Common methods for monitoring human cardiac troponin I (cTn I) are based on using antibodies against cTn I labeled with horseradish peroxidase, radioactive isotopes, or other labels. In this study, a novel label-free sandwich immunosensing method for measuring cTn I was developed. Three monoclonal antibodies (mAbs 9F5, 2F11, and 8C12) against human cTn I were generated by the commonly used hybridoma technique and characterized by a surface plasmon resonance (SPR) biosensor. An optimal pair of mAbs for measuring human cTn I was selected, as both mAbs have high affinities for cTn I and do not compete against each other for cTn I binding. An optical immunosensor for measuring cTn I in sera based on SPR was developed by using avidin as an intermediate layer and biotinylated-2F11 as the capturing antibody. Two detection methods for cTn I with the immunosensor were performed: (1) the direct detection of cTn I with a detection range of 2.5 to 40 microg/L and (2) the sandwich immunosensing method. In the sandwich assay mode, the second antibody 9F5 biologically amplified the sensor response. As a result, the sandwich assay showed a sensitivity of 0.25 microg/L and a detection range of 0.5 to 20 microg/L with within-run variation of 4.9 to 6.7% and between-run variation of 5.2 to 8.4%. This method has greatly enhanced the sensitivity for detection compared to that previously reported in the literatures.     

Development of an immunosensor for the detection of vitellogenin using impedance spectroscopy

A simple and direct immunosensor for the determination of carp (Carassius auratus) vitellogenin (Vtg), a female-specific protein, has been proposed based on an antibody-captured conducting polymer-coated electrode. The monoclonal antibody specific to carp (C. auratus) Vtg was immobilized by covalent coupling to the carboxylic acid group on the polymer. The antibody immobilization and antibody-antigen interaction have been demonstrated by means of quartz crystal microbalance and impedance spectroscopic techniques. The impedance change occurred at the sensor surface due to the specific immuno-interaction was utilized to determine Vtg. The sensor showed high selectivity and sensitive response to Vtg in a buffered medium without redox probe. Vtg was determined in the linear range from 1.0 to 8.0 microg/l with the standard deviation of +/-0.13 (n =3) and the detection limit was determined to be 0.42 microg/l. This method was applied to the determination of Vtg in real male and female carp (C. auratus) serum samples.     

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.     

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.     

Self-assembled PEG monolayer based SPR immunosensor for label-free detection of insulin

A simple and rapid continuous-flow immunosensor based on surface plasmon resonance (SPR) has been developed for detection of insulin as low as 1 ng ml-1 (ppb) with a response time of less than 5 min. At first, a heterobifunctional oligo(ethyleneglycol)-dithiocarboxylic acid derivative (OEG-DCA) containing dithiol and carboxyl end groups was used to functionalize the thin Au-film of SPR chip. Insulin was covalently bound to the Au-thiolate monolayer of OEG-DCA for activating the sensor surface to immunoaffinity interactions. An on-line competitive immunosensing principle is examined for detection of insulin, in which the direct affinity binding of anti-insulin antibody to the insulin on sensor surface is examined in the presence and absence of various concentrations of insulin. Immunoreaction of anti-insulin antibody with the sensor surface was optimized with reference to antibody concentration, sample analysis time and flow-rate to provide the desired detection limit and determination range. With the immunosensor developed, the lowest detectable concentration of insulin is 1 ng ml-1 and the determination range covers a wide concentration of 1-300 ng ml-1. The developed OEG-monolayer based sensor chip exhibited high resistance to non-specific adsorption of proteins, and an uninterrupted highly sensitive detection of insulin from insulin-impregnated serum samples has been demonstrated. After an immunoreaction cycle, active sensor surface was regenerated simply by a brief flow of an acidic buffer (glycine.HCl; pH 2.0) for less than 1 min. A same sensor chip was found reusable for more than 25 cycles without an appreciable change in the original sensor activity.     

A chemiluminescence flow immunosensor based on a porous monolithic metacrylate and polyethylene composite disc modified with protein G

A generic, fast, sensitive and new type of flow immunosensor has been developed. The basis is a monolithic porous poly(glycidyl methacrylate-co-trimethylolpropane trimethacrylate) polymer disc modified with protein G, placed in a fountain type flow cell compartment, in close proximity to a photomultiplier tube (PMT). Analyte and HRP labelled analyte derivative (tracer) compete for anti-analyte antibody binding sites. The mixture is then injected into the flow immunosensor system where the formed analyte- and tracer-antibody complexes are trapped by the monolithic protein G disc. The amount of bound tracer, inversely related to the concentration of analyte in the sample, is determined in a second step by injection of luminol, p-iodophenol and H2O2, generating enhanced chemiluminescence (CL) with horseradish peroxidase (HRP). A third and final step is need for regeneration of the protein G disc so that a new analysis cycle can take place. The performance of the disc immunosensor system was compared with a one step continuous flow injection immunoassay (FIIA) system, using the same reagents and a protein G column, in terms of assay sensitivity and influence of matrix effects from various water samples (millipore-, tap- and surface water). The detection limit for the analyte atrazine in PBS and surface water (SW) was 0.208 +/- 0.004 microg l(-1) (PBS) and 0.59 +/- 0.120 microg l(-1) (SW) for the FIIA and 0.033 +/- 0.003 microg l(-1) (PBS) and 0.038+/-0.003 microg l(-1) (SW) for the disc immunosensor. Statistical comparison of the two systems shows that the disc immunosensor results were significantly less influenced by the sample matrix, which is explained by the fact that the sample in the FIIA arrives simultaneously with the matrix to the detector, whereas these are separated in time in the disc immunosensor system.     

The development of a real-time biosensor for the detection of trace levels of trinitrotoluene (TNT) in aquatic environments

This paper describes the development of a highly sensitive TNT immunosensor consisting of a highly specific monoclonal antibody coupled with a prototype fluorescence-based detector system (KinExA Inline Biosensor, Sapidyne Instrument Inc). The antibody developed possesses a high affinity for TNT (association constant, aK 8.2) with minimal cross reactivity with other compounds such as tetryl, 2,4-dinitrotoluene and 2-amino-4,6-dinitrotoluene. This system provides sample assessment within 160 s from source acquisition and possesses sensitivity for TNT of 0.05 microg/L in ground water. The sensor can be regenerated in 8 min, allows a minimum of 40 repeated readings, and has a standard error of 0.1-0.4% between repeat readings. The fluidics and software allow samples to be obtained from up to eight different sources allowing the user to examine the stratification of the pollutant in the water column. We believe that this immunosensor can be used to rapidly assess trace levels of TNT in environmental water samples.     

A self-assembled monolayer-based piezoelectric immunosensor for rapid detection of Escherichia coli O157:H7

A piezoelectric immunosensor was developed for rapid detection of Escherichia coli O157:H7. It was based on the immobilization of affinity-purified antibodies onto a monolayer of 16-mercaptohexadecanoic acid (MHDA), a long-chain carboxylic acid-terminating alkanethiol, self-assembled on an AT-cut quartz crystal's Au electrode surface with N-hydroxysuccinimide (NHS) ester as a reactive intermediate. The binding of target bacteria onto the immobilized antibodies decreased the sensor's resonant frequency, and the frequency shift was correlated to the bacterial concentration. The stepwise assembly of the immunosensor was characterized by means of both quartz crystal microbalance (QCM) and cyclic voltammetry techniques. Three analytical procedures, namely immersion, dip-and-dry and flow-through methods, were investigated. The immunosensor could detect the target bacteria in a range of 10(3)-10(8)CFU/ml within 30-50 min, and the sensor-to-sensor reproducibility obtained at 10(3) and 10(5) colony-forming units (CFU)/ml was 18 and 11% R.S.D., respectively. The proposed sensor was comparable to Protein A-based piezoelectric immunosensor in terms of the amount of immobilized antibodies and detection sensitivity.     

Herbicide 2,4-dichlorophenoxyacetic acid (2,4-D)-induced cytogenetic damage in human lymphocytes in vitro in presence of erythrocytes

The genotoxic effects of 2,4-D and its commercial derivative 2,4-D DMA were studied by measuring sister chromatid exchange (SCE), cell-cycle progression and mitotic index in human whole blood (WBC) and plasma leukocyte cultures (PLC). Concentrations of 10, 25, 50 and 100 microg herbicide/ml were used during 72 h. In WBC, a significant increase in SCE frequency was observed within the 10-50 microg 2,4-D/ml and 25-100 microg 2,4-D DMA/ml dose range. Contrarily, in PLC, none of the concentrations employed affected the SCEs frequency. A significant delay in cell proliferation was observed in WBC after treatments with 25 and 50 microg 2,4-D/ml and 50 and 100 microg 2,4-D DMA/ml. In PLC, only 100.0 microg 2,4-D/ml altered cell-cycle progression. For both chemicals, a progressive dose-related inhibition of mitotic activity was observed. The results demonstrated that the presence of erythrocytes in the culture system modulated the DNA and cellular damage inflicted by 2,4-D and 2,4-D DMA into human lymphocytes in vitro as well as both 2,4-D and 2,4-D DMA were more potent genotoxic agents in the presence of human red cells.     

Development of an electrochemical immunosensor for alanine aminotransferase

Alanine aminotransferase (ALT) has been regarded as one of the most sensitive indicators of hepatocellular damage. While ALT is widely used in the practice of medicine, few attempts have been made to develop biosensors applicable to the on-site diagnosis of liver diseases. In the hope of developing an immunosensor for measurement of ALT activity, we have generated monoclonal antibodies to human recombinant ALT and fabricated them for use in a sensor. The ALT immunosensor was composed of the followings: (1) anti-ALT antibody-immobilized outer membrane; (2) pyruvate oxidase-absorbed inner membrane; (3) a self assembled monolayer mediator-coated gold working electrode and an Ag/AgCl reference electrode. The chronoamperometric measurement of the immunosensor was performed with 40 microl of PBS containing substrates and ALT without a washing step in less than 5 min. The dynamic range of ALT immunosensor was presented as five orders of magnitude, ranging between 10 pg/ml and 1 microg/ml. The detection limit and the sensitivity were 10 pg/ml and 26.3 nA/(ng/ml), respectively. In the meantime, the enzyme sensor fabricated without anti-ALT antibody showed much poorer analytical values. The dynamic range, the detection limit, and the sensitivity were 10 ng/ml-100 microg/ml, 10 ng/ml and 11.4 nA/(ng/ml), respectively. The presented results indicated that the immunosensor system provided much better technical performance in all of the aspects evaluated than did the enzyme sensor without the immobilized-antibody.     

A magnetic molecular imprinting-based fluorescence probe for sensitive and selective detection of 2,4-D herbicide

A new magnetic molecular imprinting-based turn-on fluorescence probe (Fe₃O₄NPs@SiO₂@NBD@MIPs) has been synthesized via a facile sol–gel polymerization for the detection of 2,4-dichlorophenoxyacetic acid (2,4-D). Based on the photoinduced electron transfer (PET) of nitrobenzoxadiazole (NBD), 2,4-D can be recognized by enhancement of NBD fluorescence. With the presence of Fe₃O₄ in the core of the probe, this sensor can also be reused many times using magnetic aggregation methods. After the addition of various concentrations of 2,4-D, the fluorescence peak at 530 nm (excitation of 468 nm) increased linearly ranging from 0.1 to 3 μM with a detection limit of 0.023 μM. This sensing system is believed to be available for detecting 2,4-D in real samples, with high recovery rates ranging from 94% to 108% for three spike levels of 2,4-D with precisions below 5%.