Simultaneous determination of renal clinical analytes in serum using hydrolase- and oxidase-encapsulated optical array biosensors

An optical array biosensor encapsulated with hydrolase and oxidoreductase using sol-gel immobilization technique has been fabricated for simultaneous analysis and screening of multiple samples to determine the presence of multianalytes which are clinically important in relation to renal failure. Urease and creatinine deiminase were used to detect urea and creatinine, while glucose oxidase and uricase were coimmobilized with horseradish peroxidase to quantify glucose and uric acid. Moreover, the concentrations of analytes in fetal calf serum were measured and quantified using the developed sensing system. The array biosensor showed good specificity for the simultaneous analysis of multiple samples for multianalytes without obvious cross-interference. The analytical ranges of the four analytes were between 0.01 and 10mM with detection limits of 2.5-80 microM. High precision with relative standard deviations of 3.8-9.2% (n=45) was also demonstrated. The reproducibility of array-to-array in 3 consecutive months was 5.4% (n=3). Moreover, the concentrations of analytes in fetal calf serum were 5.9 mM for urea, 0.13 mM for creatinine, 3.3mM for glucose, and 0.15 mM for uric acid, which were in good agreement with results obtained using the traditional spectroscopic methods. These results demonstrate the first use of a sol-gel-derived optical array biosensor for simultaneous analysis of multiple samples for the presence of multiple clinically important renal analytes.     

Verification of performance with the automated direct optical TIRF immunosensor (River Analyser) in single and multi-analyte assays with real water samples

In order to verify the reproducibility, precision, and robustness of the optical immunosensor River Analyser (RIANA), we investigated two common statistical methods to evaluate the limit of detection (LOD) and the limit of quantification (LOQ). Therefore, we performed a simultaneous multi-analyte calibration with atrazine, bisphenol A, and estrone in Milli-Q water. Using an automated biosensor, it was possible for the first time to achieve a LOD below 0.020 microg L(-1) using a common statistically based method without sample pre-treatment and pre-concentration for each of the analytes in a simultaneous multi-analyte calibration. This biosensor setup shows values comparable to those obtained by more classical analytical methods. Based on this calibration, we measured spiked and un-spiked real water samples with complex matrices (samples from different water bodies, from ground water sources, and tap water samples). The comparison between our River Analyser and common analytical methods (like GC-MS and HPLC-DAD) shows overall comparable values for all three analytes. Furthermore, a calibration of isoproturon (IPU) (in single analyte mode) resulted in a LOD of 0.016 microg L(-1), and a LOQ of 0.091 microg L(-1). In compliance with guidelines of the Association of Analytical Communities International (AOAC), six out of nine recovery rates (recovery rate: measured concentration divided by real concentration in percent) for three surface water samples with different matrices (spiked and un-spiked) could be obtained between 70 and 120% (recovery rates between 70 and 120%, as demanded by the guidelines of the AOAC International). The reproducibility was checked by measuring replica of each sample within independent repetitions. Robustness could be demonstrated by long-term stability tests of the biosensor surface. These studies show that the biosensor used offers the necessary reproducibility, precision, and robustness required for an analytical method.     

Simultaneous detection of six biohazardous agents using a planar waveguide array biosensor

Recently, we demonstrated that an array biosensor could be used with cocktails of fluorescent antibodies to perform three assays simultaneously on a single substrate, and that multiple samples could be analyzed in parallel. We extend this technology to demonstrate the simultaneous analysis of six samples for six different hazardous analytes, including both bacteria and protein toxins. The level of antibody cross-reactivity is explored, revealing a possible common epitope in two of the toxins. A panel of environmental interferents was added to the samples; these interferents neither prevented the detection of the analytes nor caused false-positive responses.     

Detection of Salmonella enterica serovar typhimurium by using a rapid, array-based immunosensor

The multianalyte array biosensor (MAAB) is a rapid analysis instrument capable of detecting multiple analytes simultaneously. Rapid (15-min), single-analyte sandwich immunoassays were developed for the detection of Salmonella enterica serovar Typhimurium, with a detection limit of 8 x 10(4) CFU/ml; the limit of detection was improved 10-fold by lengthening the assay protocol to 1 h. S. enterica serovar Typhimurium was also detected in the following spiked foodstuffs, with minimal sample preparation: sausage, cantaloupe, whole liquid egg, alfalfa sprouts, and chicken carcass rinse. Cross-reactivity tests were performed with Escherichia coli and Campylobacter jejuni. To determine whether the MAAB has potential as a screening tool for the diagnosis of asymptomatic Salmonella infection of poultry, chicken excretal samples from a private, noncommercial farm and from university poultry facilities were tested. While the private farm excreta gave rise to signals significantly above the buffer blanks, none of the university samples tested positive for S. enterica serovar Typhimurium without spiking; dose-response curves of spiked excretal samples from university-raised poultry gave limits of detection of 8 x 10(3) CFU/g.     

Exploring "one-shot" kinetics and small molecule analysis using the ProteOn XPR36 array biosensor

A ProteOn XPR36 parallel array biosensor was used to characterize the binding kinetics of a set of small molecule/enzyme interactions. Using one injection with the ProteOn's crisscrossing flow path system, we collected response data for six different concentrations of each analyte over six different target protein surfaces. This "one-shot" approach to kinetic analysis significantly improves throughput while generating high-quality data even for low-molecular-mass analytes. We found that the affinities determined for nine sulfonamide-based inhibitors of the enzyme carbonic anhydrase II were highly correlated with the values determined using isothermal titration calorimetry. We also measured the temperature dependence (from 15 to 35 degrees C) of the kinetics for four of the inhibitor/enzyme interactions. Our results illustrate the potential of this new parallel-processing biosensor to increase the speed of kinetic analysis in drug discovery and expand the applications of real-time protein interaction arrays.     

Array biosensor for detection of biohazards

A fluorescence-based biosensor has been developed for simultaneous analysis of multiple samples for multiple biohazardous agents. A patterned array of antibodies immobilized on the surface of a planar waveguide is used to capture antigen present in samples; bound analyte is then quantified by means of fluorescent tracer antibodies. Upon excitation of the fluorophore by a small diode laser, a CCD camera detects the pattern of fluorescent antibody:antigen complexes on the waveguide surface. Image analysis software correlates the position of fluorescent signals with the identity of the analyte. This array biosensor has been used to detect toxins, toxoids, and killed or non-pathogenic (vaccine) strains of pathogenic bacteria. Limits of detection in the mid-ng/ml range (toxins and toxoids) and in the 10(3)-10(6) cfu/ml range (bacterial analytes) were achieved with a facile 14-min off-line assay. In addition, a fluidics and imaging system has been developed which allows automated detection of staphylococcal enterotoxin B (SEB) in the low ng/ml range.     

A "do-it-yourself" array biosensor

We have developed an array biosensor for the simultaneous detection of multiple targets in multiple samples within 15-30 min. The biosensor is based on a planar waveguide, a modified microscope slide, with a pattern of small (mm2) sensing regions. The waveguide is illuminated by launching the emission of a 635 nm diode laser into the proximal end of the slide via a line generator. The evanescent field excites fluorophores bound in the sensing region and the emitted fluorescence is measured using a Peltier-cooled CCD camera. Assays can be performed on the waveguide in multichannel flow chambers and then interrogated using the detection system described here. This biosensor can detect many different targets, including proteins, toxins, cells, virus, and explosives with detection limits rivaling those of the ELISA detection system.     

Pseudomonas aeruginosa immobilized multiwalled carbon nanotubes as biosorbent for heavy metal ions

Pseudomonas aeruginosa immobilized multiwalled carbon nanotubes has been used as biosorbent for the solid phase extraction of some heavy metal ions in environmental samples. Cobalt(II), cadmium(II), lead(II), manganese(II), chromium(III) and nickel(II) ions have been selected as analytes for the presented study, due to their important negative and positive roles in human life. In order to investigate quantitative biosorption conditions of the analytes, the influences of pH of the aqueous solution, eluent type, eluent volume, samples volume, etc. were examined. The effects of alkaline, earth alkaline and some transitions metals on the biosorption of analyte ions on P. aeruginosa immobilized multiwalled carbon nanotubes were also investigated. The presented biosorption procedure was applied to the determination of analytes in tomato leaves, bovine liver, boiled wheat, canned fish, black tea, lichen and natural water samples.     

An ultrasensitive and stable potentiometric immunosensor

We describe a novel quantitative polypyrrole based potentiometric biosensor that provides broad-spectrum assay capability. The biosensor allows for capture of analytes of interest from complex real samples such as serum and whole blood, and subsequent measurement in a controlled matrix environment. The technology is rapid (<15 min), ultrasensitive (<50 fM) and reproducible (CV<5% at 0.1 ng/ml). In addition the system has shown a wide dynamic range (four to five orders of magnitude), and good stability, 37 degrees C for at least 4 months. This potentiometric biosensor detects enzyme labelled immuno-complexes formed at the surface of a polypyrrole coated, screenprinted gold electrode. Detection is mediated by a secondary reaction that produces charged products (a 'charge-step' procedure). A shift in potential is measured at the sensor surface, caused by local changes in redox state, pH and/or ionic strength. The magnitude of the difference in potential is related to the concentration of the formed receptor-target complex. The potentiometric sensing technology has been demonstrated in assays for hepatitis B surface antigen (HBsAg) (Mw>300 kDa), Troponin I (Mw approximately 23 kDa), Digoxin (Mw 780 Da) and tumour necrosis factor (hTNF-alpha) (Mw approximately 23 kDa). These model targets were chosen to represent analytes of a range of molecular weights, and because of their requirement for assays of high analytical sensitivity and precision. All these assays were performed using complex fluid samples and the presence of any non-specific binding has no significant effect on the final measurement. New assays can be transferred and optimised readily.     

Biosensor arrays for simultaneous measurement of glucose, lactate, glutamate, and glutamine

For simultaneous measurement of glucose, lactate, glutamine, and glutamate a biosensor array is implemented in a micro flow-system thus giving a microsystem. The microsystem consists of a glass chip with the integrated biosensor array and a bottom part, which comprises a gold counter electrode, a 300 microm thick seal, and electrical interconnection lines. The flow device has a total internal volume of 2.1 or 6 microl when integrated with a mixer on chip. The biosensors with no crosstalking and high long term stability were produced by modifying the electrochemical transducers and utilizing photopatternable enzyme membranes. The use of appropriate miniaturization technology leads to mass producable devices for in vivo and ex vivo applications in whole blood and fermentation broth. Due to a novel glutaminase with an activity optimum in the neutral pH range direct and simultaneous monitoring of glutamine together with glucose, lactate, and glutamate could be performed.     

Improved multianalyte detection of organophosphates and carbamates with disposable multielectrode biosensors using recombinant mutants of Drosophila acetylcholinesterase and artificial neural networks

Engineered variants of Drosophila melanogaster acetylcholinesterase (AChE) were used as biological receptors of AChE-multisensors for the simultaneous detection and discrimination of binary mixtures of cholinesterase-inhibiting insecticides. The system was based on a combination of amperometric multielectrode biosensors with chemometric data analysis of sensor outputs using artificial neural networks (ANN). The multisensors were fully manufactured by screen-printing, including enzyme immobilisation. Two types of multisensors were produced that consisted of four AChE variants each. The AChE mutants were selected in order to obtain high resolution, enhanced sensitivity and minimal assay time. This task was successfully achieved using multisensor I equipped with wild-type Drosophila AChE and mutants Y408F, F368L, and F368H. Each of the AChE variants was selected on the basis of displaying an individual sensitivity pattern towards the target analytes. For multisensor II, the inclusion of F368W, which had an extremely diminished paraoxon sensitivity, increased the sensor's capacity even further. Multisensors I and II were both used for inhibition analysis of binary paraoxon and carbofuran mixtures in a concentration range 0-5 microg/l, followed by data analysis using feed-forward ANN. The two analytes were determined with prediction errors of 0.4 microg/l for paraoxon and 0.5 microg/l for carbofuran. A complete biosensor assay and subsequent ANN evaluation was completed within 40 min. In addition, multisensor II was also investigated for analyte discrimination in real water samples. Finally, the properties of the multisensors were confirmed by simultaneous detection of binary organophosphate mixtures. Malaoxon and paraoxon in composite solutions of 0-5 microg/l were discriminated with predication errors of 0.9 and 1.6 microg/l, respectively.     

Co-activation of antibody-responsive, enzymatic sensors by a recombinant scFv antibody fragment produced in E. coli

The biophysical nature of the signal transduction in enzymatic biosensors through which the paratope-epitope interaction enhances enzyme activity, is essentially unknown. Fab fragments of efficiently activating antibodies are, in general, poor sensor activators suggesting that the bivalent antibody binding could contribute to the sensing process. We have cloned and produced in E. coli a recombinant SD6 scFv fragment directed against a sensing peptide, displayed on a model -galactosidase-based biosensor. While the enzymatic response to scFv-binding is not detectable, the simultaneous presence of scFv and the poorly-activator SD6 Fab fragment results in a non-additive, efficient sensor response, enhancing the enzyme activity up to about 200%. This co-operative effect, which is also observed by combining SD6 scFv and the non-homologous anti-peptide 4C4 Fab, confirms that the enzyme up-regulation requires multiple and probably heterogeneous contacts between the sensor molecule and the analytes, but not necessarily done by bivalent molecular ligands.     

Multi-analyte surface plasmon resonance biosensing

Surface plasmon resonance (SPR) biosensors are affinity sensing devices exploiting a special mode of electromagnetic field-surface plasmon-polariton-to detect the binding of analyte molecules from a liquid sample to biomolecular recognition elements immobilized on the surface of the sensor. In this paper, we review advances of SPR biosensor technology towards detection systems for the simultaneous detection of multiple analytes (multi-analyte detection). In addition, we report application of a recently developed multichannel SPR sensor based on spectroscopy of surface plasmons and wavelength division multiplexing of sensing channels to multi-analyte detection.     

Development of a biosensor for urea assay based on amidase inhibition,using an ion-selective electrode

Abstract

A biosensor for urea has been developed based on the observation that urea is a powerful active-site inhibitor of amidase, which catalyzes the hydrolysis of amides such as acetamide to produce ammonia and the corresponding organic acid. Cell-free extract from Pseudomonas aeruginosa was the source of amidase (acylamide hydrolase, EC 3.5.1.4) which was immobilized on a polyethersulfone membrane in the presence of glutaraldehyde; an ion-selective electrode for ammonium ions was used for biosensor development. Analysis of variance was used for optimization of the biosensor response and showed that 30 μL of cell-free extract containing 7.47 mg protein mL^?1, 2 μL of glutaraldehyde (5%, v/v) and 10 μL of gelatin (15%, w/v) exhibited the highest response. Optimization of other parameters showed that pH 7.2 and 30 min incubation time were optimum for incubation of membranes in urea. The biosensor exhibited a linear response in the range of 4.0–10.0 μM urea, a detection limit of 2.0 μM for urea, a response time of 20 s, a sensitivity of 58.245 % per μM urea and a storage stability of over 4 months. It was successfully used for quantification of urea in samples such as wine and milk; recovery experiments were carried out which revealed an average substrate recovery of 94.9%. The urea analogs hydroxyurea, methylurea and thiourea inhibited amidase activity by about 90%, 10% and 0%, respectively, compared with urea inhibition.     

Detection of Salmonella enterica Serovar Typhimurium by Using a Rapid, Array-Based Immunosensor

The multianalyte array biosensor (MAAB) is a rapid analysis instrument capable of detecting multiple analytes simultaneously. Rapid (15-min), single-analyte sandwich immunoassays were developed for the detection of Salmonella enterica serovar Typhimurium, with a detection limit of 8 × 10⁴ CFU/ml; the limit of detection was improved 10-fold by lengthening the assay protocol to 1 h. S. enterica serovar Typhimurium was also detected in the following spiked foodstuffs, with minimal sample preparation: sausage, cantaloupe, whole liquid egg, alfalfa sprouts, and chicken carcass rinse. Cross-reactivity tests were performed with Escherichia coli and Campylobacter jejuni. To determine whether the MAAB has potential as a screening tool for the diagnosis of asymptomatic Salmonella infection of poultry, chicken excretal samples from a private, noncommercial farm and from university poultry facilities were tested. While the private farm excreta gave rise to signals significantly above the buffer blanks, none of the university samples tested positive for S. enterica serovar Typhimurium without spiking; dose-response curves of spiked excretal samples from university-raised poultry gave limits of detection of 8 × 10³ CFU/g.     

Urea cycle of Fasciola gigantica: purification and characterization of arginase

The ornithine-urea cycle has been investigated in Fasciola gigantica. Agrinase had very high activity compared to the other enzymes. Carbamoyl phosphate synthetase and ornithine carbamoyltransferase had very low activity. A moderate enzymatic activity was recorded for argininosuccinate synthetase and argininosuccinate lyase. The low levels of F. gigantica urea cycle enzymes except to the arginase suggest the urea cycle is operative but its role is of a minor important. The high level of arginase activity may benefit for the hydrolysis of the exogenous arginine to ornithine and urea. Two arginases Arg I and Arg II were separated by DEAE-Sepharose column. Further purification was restricted to Arg II with highest activity. The molecular weight of Arg II, as determined by gel filtration and SDS-PAGE, was 92,000. The enzyme was capable to hydrolyze l-arginine and to less extent l-canavanine at arginase:canavanase ratio (>10). The enzyme exhibited a maximal activity at pH 9.5 and Km of 6 mM. The optimum temperature of F. gigantica Arg II was 40 degrees C and the enzyme was stable up to 30 degrees C and retained 80% of its activity after incubation at 40 degrees C for 15 min and lost all of its activity at 50 degrees C. The order of effectiveness of amino acids as inhibitors of enzyme was found to be lysine>isoleucine>ornithine>valine>leucine>proline with 67%, 43%, 31%, 25%, 23% and 15% inhibition, respectively. The enzyme was activated with Mn2+, where the other metals Fe2+, Ca2+, Hg2+, Ni2+, Co2+ and Mg2+ had inhibitory effects.     

Separation and simultaneous detection of anticancer drugs in a microfluidic device with an amperometric biosensor

A simple and highly sensitive method for simultaneous detection of anticancer drugs is developed by integrating the preconcentration and separation steps in a microfluidic device with an amperometric biosensor. An amperometric detection with dsDNA and cardiolipin modified screen printed electrodes are used for the detection of anticancer drugs at the end of separation channel. The preconcentration capacity is enhanced thoroughly using field amplified sample stacking and field amplified sample injection techniques. The experimental parameters affecting the analytical performances, such as pH, temperature, buffer concentration, water plug length, and detection potential are optimized. A reproducible response is observed during multiple injections of samples with a RSD <5%. The calibration plots are linear with the correlation coefficient between 0.9913 and 0.9982 over the range of 2-60 pM. The detection limits of four drugs are determined to be between 1.2 (± 0.05) and 5.5 (± 0.3) fM. The applicability of the device to the direct analysis of anticancer drugs is successfully demonstrated in a real spiked urine sample. Device was also examined for interference effect of common chemicals present in real samples.     

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.     

A fluorescent biosensor based on acetylcholinesterase and 5-oxazolone derivative immobilized in polyvinylchloride (PVC) matrix

A new 2-phenyl-4-[4-(1,4,7,10-tetraoxa-13-azacyclopentadecyl)benzylidene]-5-oxazolone (CPO) derivative was utilized to develop an optical acetylcholinesterase (AChE) biosensor in which the azlactone derivative was embedded in plasticized polyvinylchloride (PVC) matrix. The sensor system was calibrated for the detection of acetylcholine (ACh) and donepezil which is a competitive cholinesterase (ChE) inhibitor. Two different biosensing systems were developed by using AChE enzyme in solution and immobilized together with the fluorescent derivative (CPO) doped in transparent PVC membrane. The enzymatic hydrolysis of ACh was monitored by following changes in the pH induced fluorescence intensity. When AChE enzyme was immobilized in PVC matrix together with CPO, the sensitivity of the measuring system has increased approximately three times for ACh, in comparison to the sensing system where AChE enzyme was in solution phase.

The photophysical parameters of CPO were also examined in solvents of tetrahydrofuran (THF), acetonitrile (ACN) and dichloromethane (DCM) and in solid matrix of PVC. The azlactone derivative exhibits excellent photostability in PVC matrix.     

Optical biosensor for urea with improved response time

An optical biosensor for urea measurements was developed. The operation of the sensor is based on the well-known urease enzyme-catalyzed hydrolysis of urea. The ammonium ions liberated in the reaction are detected with an ion selective optode membrane containing nonactin as ion selective ionophore and ETH 5294 chromoionophore in a thin (1 microm) plasticized poly(vinylchloride) film. The basic sensing element was home made of a microscope glass slide, a HeNe laser light source, photodiode light detector and light in coupling, de-coupling elements. The transducer membrane and the enzyme containing reaction layer were sandwich-cast with spin coating onto the surface of the sensing slide. The attenuation of the laser light propagating inside the glass wave-guide was used as signal for urea measurements. With this arrangement membranes provided good sensitivity (0.05 absorption unit when going from 0.1 to 1 mM urea) and short (16-20 s) response time. Taking advantage on the improved response time, flow injection urea measurements were made in the 0.01-2 mM concentration range. Thirty sample/hour analysis-rate, good peak-to-peak reproducibility (RSD=0.02) and recovery (95-104%) was achieved with buffer diluted urea solutions. Applications for the analysis of real samples are planned to do in the future.