Biomedical subjects

A Mulchandani

Publications and source records attributed to A Mulchandani.

At least 19 recordsLinked to original sources

PubMed2001

Flow injection amperometric enzyme biosensor for direct determination of organophosphate nerve agents.

A flow injection amperometric biosensor for the determination of organophosphate nerve agents was developed. The biosensor incorporated an immobilized enzyme reactor that contains the enzyme organophosphorus hydrolase covalently immobilized on activated aminopropyl controlled pore glass beads and an electrochemical flow-through detector containing carbon paste working electrode, a silver/silver chloride reference electrode, and stainless steel counter electrode. The organophosphorus hydrolase catalyzed the hydrolysis of organophosphate with nitrophenyl substituent to generate p-nitrophenol which is then detected downstream electrochemically at the carbon paste electrode poised at 0.9 V vs the reference electrode. The amperometric response of the biosensor was linear up to 120 microM and 140 microM, with lower detection limits of 20 nM and 20 nM, for paraoxon and methyl parathion, respectively. The response was very reproducible (RSD 2%, n = 35) and stable for over 1 month when the immobilized enzyme column was stored at 4 degrees C. Each assay took ca. 2 min giving a sample throughput of 30 h(-1). The applicability of the biosensor to monitor paraoxon and methyl parathion in distilled water and simulated well water was demonstrated.

Aryldialkylphosphatase↗

PubMed2001

Capillary electrophoresis microchips for separation and detection of organophosphate nerve agents.

A miniaturized analytical system for separating and detecting toxic organophosphate nerve agent compounds, based on the coupling of a micromachined capillary electrophoresis chip with a thick-film amperometric detector, is described. Factors influencing the on-chip separation and detection processes have been optimized. Using a MES buffer (20 mM, pH 5.0) running buffer, a 72-mm-long separation channel, and a separation voltage of 2000 V, baseline resolution is observed for paraoxon, methyl parathion, fenitrothion, and ethyl parathion in 140 s. Such miniaturization and speed advantages are coupled to submicromolar detection limits and good precision. Applicability to spiked river water samples is demonstrated, and the implications for on-site environmental monitoring and rapid security screening/warning are discussed.

Chemical Warfare Agents↗

PubMed2001

Use of real-time polymerase chain reaction and molecular beacons for the detection of Escherichia coli O157:H7.

Molecular beacons (MBs) are oligonucleotide probes that fluoresce upon hybridization. In this paper, we described the development of a real-time PCR assay to detect the presence of Escherichia coli O157:H7 using these fluorogenic reporter molecules. MBs were designed to recognize a 26-bp region of the rfbE gene, coding for an enzyme necessary for O-antigen biosynthesis. The specificity of the MB-based PCR assay was evaluated using various enterohemorrhagic (EHEC) and Shiga-like toxin-producing (STEC) E. coli strains as well as bacteria species that cross-react with the O157 antisera. All E. coli serotype O157 tested was positively identified while all other species, including the closely related O55 were not detected by the assay. Positive detection of E. coli O157:H7 was demonstrated when >10(2) CFU/ml was present in the samples. The capability of the assay to detect E. coli O157:H7 in raw milk and apple juice was demonstrated. As few as 1 CFU/ml was detected after 6 h of enrichment. These assays could be carried out entirely in sealed PCR tubes, enabling rapid and semiautomated detection of E. coli O157:H7 in food and environmental samples.

Animals↗

PubMed2001

Simultaneous degradation of organophosphorus pesticides and p-nitrophenol by a genetically engineered Moraxella sp. with surface-expressed organophosphorus hydrolase.

Moraxella sp., a native soil organism that grows on p-nitrophenol (PNP), was genetically engineered for the simultaneous degradation of organophosphorus (OP) pesticides and p-nitrophenol (PNP). The truncated ice nucleation protein (INPNC) anchor was used to target the pesticide-hydrolyzing enzyme, organophosphorus hydrolase (OPH), onto the surface of Moraxella sp., alleviating the potential substrate uptake limitation. A shuttle vector, pPNCO33, coding for INPNC-OPH was constructed and the translocation, surface display, and functionality of OPH were demonstrated in both E. coli and Moraxella sp. However, whole cell activity was 70-fold higher in Moraxella sp. than E. coli. The resulting Moraxella sp. degraded organophosphates as well as PNP rapidly, all within 10 h. The initial hydrolysis rate was 0.6 micromol/h/mg dry weight, 1.5 micromol/h/mg dry weight, and 9.0 micromol/h/mg dry weight for methyl parathion, parathion, and paraoxon, respectively. The possibility of rapidly degrading OP pesticides and their byproducts should open up new opportunities for improved remediation of OP nerve agents in the future.

Aryldialkylphosphatase↗

PubMed2001

Biosensors for direct determination of organophosphate pesticides.

Direct, selective, rapid and simple determination of organophosphate pesticides has been achieved by integrating organophosphorus hydrolase with electrochemical and opitical transducers. Organophosphorus hydrolase catalyzes the hydrolysis of a wide range of organophosphate compounds, releasing an acid and an alcohol that can be detected directly. This article reviews development, characterization and applications of organophosphorus hydrolase-based potentiometric, amperometric and optical biosensors.

Biosensing Techniques↗

PubMed2001

Amperometric microbial biosensor for direct determination of organophosphate pesticides using recombinant microorganism with surface expressed organophosphorus hydrolase.

An amperometric microbial biosensor for the direct measurement of organophosphate nerve agents is described. The sensor is based on a carbon paste electrode containing genetically engineered cells expressing organophosphorus hydrolase (OPH) on the cell surface. OPH catalyzes the hydrolysis of organophosphorus pesticides with p-nitrophenyl substituent such as paraoxon, parathion and methyl parathion to p-nitrophenol. The later is detected anodically at the carbon transducer with the oxidation current being proportional to the nerve-agent concentration. The sensor sensitivity was optimized with respect to the buffer pH and loading of cells immobilized using paraoxon as substrate. The best sensitivity was obtained using a sensor constructed with 10 mg of wet cell weight per 100 mg of carbon paste and operating in pH 8.5 buffer. Using these conditions, the biosensor was used to measure as low as 0.2 microM paraoxon and 1 microM methyl parathion with very good sensitivity, excellent selectivity and reproducibility. The microbial biosensor had excellent storage stability, retaining 100% of its original activity when stored at 4 degrees C for up to 45 days.

Aryldialkylphosphatase↗

PubMed2001

Continuous-flow fluoro-immunosensor for paclitaxel measurement.

A fluorescence-based continuous-flow immunosensor for sensitive, precise, accurate and fast determination of paclitaxel was developed. The sensor utilizes anti-paclitaxel antibody immobilized through its Fc region and crosslinked by dimethylpimelimidate to protein A attached covalently onto the silanized inner walls of a glass capillary column followed by saturation of the paclitaxel-binding sites with rhodamine-labeled paclitaxel. The assay is based on the displacement and detection downstream of the rhodamine-labeled paclitaxel, by a flow-through spectrofluorometer, as a result of the competition with paclitaxel introduced as a pulse into the stream of carrier buffer flowing through the system. The peak height of the fluorescence intensity profile of the displaced rhodamine-labeled paclitaxel was directly proportional to the concentration of paclitaxel applied and was a function of the carrier buffer flow rate. The sensitivity of the immunosensor response ranged from 0.31 relative fluorescence units (RFU)/ng/ml at a flow rate 0.1 ml/min to 0.52 RFU/ng/ml at 1 ml/min, while the lower detection limit ranged from 1 ng/ml at 0.1 ml/min to 4 ng/ml at 1 ml/min. The immunosensor response was very reproducible (RSD=4.8%; n=10) and linear up to 100 ng/ml. The assay time ranged from 2 min at 1 ml/min to 8 min at 0.1 ml/min. A technique developed to resaturate the antigen binding sites of the immobilized antibody with rhodamine-labeled paclitaxel was successful in regenerating the capillary column without affecting its performance, thus enhancing the economic viability of the immunosensor. The immunosensor was successfully applied for the determination of paclitaxel in human plasma.

Antineoplastic Agents, Phytogenic↗

PubMed2001

Genetic engineering of Escherichia coli for enhanced uptake and bioaccumulation of mercury.

Synthetic phytochelatins (ECs) are a new class of metal-binding peptides with a repetitive metal-binding motif, (Glu-Cys)(n)Gly, which were shown to bind heavy metals more effectively than metallothioneins. However, the limited uptake across the cell membrane is often the rate-limiting factor for the intracellular bioaccumulation of heavy metals by genetically engineered organisms expressing these metal-binding peptides. In this paper, two potential solutions were investigated to overcome this uptake limitation either by coexpressing an Hg(2+) transport system with (Glu-Cys)(20)Gly (EC20) or by directly expressing EC20 on the cell surface. Both approaches were equally effective in increasing the bioaccumulation of Hg(2+). Since the available transport systems are presently limited to only a few heavy metals, our results suggest that bioaccumulation by bacterial sorbents with surface-expressed metal-binding peptides may be useful as a universal strategy for the cleanup of heavy metal contamination.

Chelating Agents↗

PubMed2000

Enhanced bioaccumulation of heavy metals by bacterial cells displaying synthetic phytochelatins.

A novel strategy using synthetic phytochelatins is described for the purpose of developing microbial agents for enhanced bioaccumulation of toxic metals. Synthetic genes encoding for several metal-chelating phytochelatin analogs (Glu-Cys)(n)Gly (EC8 (n = 8), EC11 (n = 11), and EC20 (n = 20)) were synthesized, linked to a lpp-ompA fusion gene, and displayed on the surface of E. coli. For comparison, EC20 was also expressed periplasmically as a fusion with the maltose-binding protein (MBP-EC20). Purified MBP-EC20 was shown to accumulate more Cd(2+) per peptide than typical mammalian metallothioneins with a stoichiometry of 10 Cd(2+)/peptide. Cells displaying synthetic phytochelatins exhibited chain-length dependent increase in metal accumulation. For example, 18 nmoles of Cd(2+)/mg dry cells were accumulated by cells displaying EC8, whereas cells exhibiting EC20 accumulated a maximum of 60 nmoles of Cd(2+)/mg dry cells. Moreover, cells with surface-expressed EC20 accumulated twice the amount of Cd(2+) as cells expressing EC20 periplasmically. The ability to genetically engineer ECs with precisely defined chain length could provide an attractive strategy for developing high-affinity bioadsorbents suitable for heavy metal removal.

Bacterial Outer Membrane Proteins↗

PubMed2000

Expression, immobilization, and enzymatic characterization of cellulose-binding domain-organophosphorus hydrolase fusion enzymes.

Bifunctional fusion proteins consisting of organophosphate hydrolase (OPH) moieties linked to a Clostridium-derived cellulose-binding domain (CBD) were shown to be highly effective in degrading organophosphate nerve agents, enabling purification and immobilization onto different cellulose materials in essentially a single step. Enzyme kinetics studies were performed for the CBD-OPH fusions using paraoxon as the substrate. The kinetics values of the unbound fusion enzymes were similar to OPH with a modest increase in K(m). Immobilization of the enzymes onto microcrystalline cellulose resulted in a further increase in the K(m) values of approximately twofold. The pH profile of the cellulose-immobilized enzymes was also only minimally affected. The CBD-OPH fusion proteins could be immobilized onto a variety of cellulose matrixes, and retained up to 85% of their original activity for 30 days. The durability of the bound fusions increased with the amount of Avicel used, suggesting that protein/cellulose interactions may have a dramatic stabilizing effect. Repeated hydrolysis of paraoxon was achieved in an immobilized enzyme reactor with 100% degradation efficiency over 45 days. These fusion proteins should prove to be invaluable tools for the development of low cost, OPH-based cellulose materials for the simultaneous adsorption and degradation of stored or spilled organophosphate wastes.

Aryldialkylphosphatase↗

PubMed2000

Development of a fluorescence immunoassay for measurement of paclitaxel in human plasma.

A new fluorescence immunoassay for the quantitative determination of paclitaxel (Pac) under equilibrium conditions was developed. Anti-Pac IgG2a antibody was immobilized through its Fc region to protein A covalently bound to the inside surface of a silanized glass capillary column and the antigen-binding sites of anti-Pac saturated with rhodamine-labeled Pac (Rh-Pac). Analyte Pac was circulated through the column in a closed loop and the steady-state fluorescence of the Rh-Pac displaced from the immobilized antibody was recorded after 6 min. The Rh-Pac fluorescence emission intensity was directly related to the concentration of the Pac analyte over a broad dynamic range of up to 400 ng/ml with a linear range up to 200 ng/ml and lower detection limit of 5.85 ng/ml. While there was no interference from the baccatin III and 10-deacetylbaccatin III, cephalomannine was found to interfere in Pac determination. When applied for measurement of Pac in human plasma, the concentration of Pac determined by the fluorescence assay was found to be in excellent agreement with the Pac added, confirming the potential of the fluorescence immunoassay for clinical application.

Alkaloids↗

PubMed2000

Molecular beacons: a real-time polymerase chain reaction assay for detecting Salmonella.

Molecular beacons are oligonucleotide probes that become fluorescent upon hybridization. We developed a real-time PCR assay to detect the presence of Salmonella species using these fluorogenic reporter molecules. A 122-base-pair section of the himA was used as the amplification target. Molecular beacons were designed to recognize a 16-base-pair region on the amplicon. As few as 2 colony-forming unit (CFU) per PCR reaction could be detected. We also demonstrated the ability of the molecular beacons to discriminate between amplicons obtained from similar species such as Escherichia coli and Citrobacter freundii in real-time PCR assays. These assays could be carried out entirely in sealed PCR tubes, enabling fast and direct detection of Salmonella in a semiautomated format.

Bacterial Proteins↗

PubMed1999

Detoxification of organophosphate nerve agents by immobilized Escherichia coli with surface-expressed organophosphorus hydrolase.

An improved whole-cell technology for detoxifying organophosphate nerve agents was recently developed based on genetically engineered Escherichia coli with organophosphorus hydrolase anchored on the surface. This article reports the immobilization of these novel biocatalysts on nonwoven polypropylene fabric and their applications in detoxifying contaminated wastewaters. The best cell loading (256 mg cell dry weight/g of support or 50 mg cell dry weight/cm2 of support) and subsequent hydrolysis of organophosphate nerve agents were achieved by immobilizing nongrowing cells in a pH 8, 150 mM citrate-phosphate buffer supplemented with 1 mM Co2+ for 48 h via simple adsorption, followed by organophosphate hydrolysis in a pH 8, 50 mM citrate-phosphate buffer supplemented with 0.05 mM Co2+ and 20% methanol at 37 degrees C. In batch operations, the immobilized cells degraded 100% of 0.8 mM paraoxon, a model organophosphate nerve agent, in approximately 100 min, at a specific rate of 0.160 mM min-1 (g cell dry wt)-1. The immobilized cells retained almost 100% activity during the initial six repeated cycles and close to 90% activity even after 12 repeated cycles, extending over a period of 19 days without any nutrient supplementation. In addition to paraoxon, other commonly used organophosphates, such as diazinon, coumaphos, and methylparathion were hydrolyzed efficiently. The cell immobilization technology developed here paves the way for an efficient, simple, and cost-effective method for detoxification of organophosphate nerve agents.

Aryldialkylphosphatase↗

PubMed1999

Ferrocene-conjugated m-phenylenediamine conducting polymer-incorporated peroxidase biosensors.

The development and characteristics of a reagentless amperometric biosensor employing horseradish peroxidase incorporated in an electrochemically deposited ferrocene-modified phenylenediamine film on a glassy carbon electrode is reported. The horseradish peroxidase/poly(m-aminoanilinomethylferrocene)- modified glassy carbon electrode reagentless biosensor measured hydrogen peroxide and other organic peroxides in both aqueous and organic medium by reduction at a low applied potential of -0.05 V (vs Ag/AgCl) without interference from molecular oxygen. When modified with glucose oxidase, the new bienzyme electrode measured glucose sensitively and selectively, demonstrating the suitability of the above peroxide biosensor for other oxidoreductase enzyme-based biosensors.

Biosensing Techniques↗

PubMed1999

Biosensor for direct determination of organophosphate nerve agents. 1. Potentiometric enzyme electrode.

A potentiometric enzyme electrode for the direct measurement of organophosphate (OP) nerve agents was developed. The basic element of this enzyme electrode was a pH electrode modified with an immobilized organophosphorus hydrolase (OPH) layer formed by cross-linking OPH with bovine serum albumin (BSA) and glutaradehyde. OPH catalyses the hydrolysis of organophosphorus pesticides to release protons, the concentration of which is proportional to the amount of hydrolysed substrate. The sensor signal and response time was optimized with respect to the buffer pH, ionic concentration of buffer, temperature, and units of OPH immobilized using paraoxon as substrate. The best sensitivity and response time were obtained using a sensor constructed with 500 IU of OPH and operating in pH 8.5, 1 mM HEPES buffer. Using these conditions, the biosensor was used to measure as low as 2 microM of paraoxon, ethyl parathion, methyl parathion and diazinon. The biosensor was completely stable for at least one month when stored in pH 8.5, 1 mM HEPES + 100 mM NaCl buffer at 4 degrees C.

Animals↗

PubMed1999

Engineering of improved microbes and enzymes for bioremediation.

Bioremediation with microorganisms is an attractive alternative to conventional techniques, such as incineration and chemical treatment, for disposing of pollutants. Recent progress in molecular biology, microbiology, and genetics is providing the driving force towards engineering improved microbes and enzymes for bioremediation. A number of genetic engineering approaches have been developed in the past several years that have proven useful in introducing/evolving the desired properties for different biodegradative pathways or enzymes. The initial excitement generated in this area should continue to pave the way for rational or irrational design of microbes or enzymes with novel remedial properties.

Amino Acid Sequence↗

PubMed1999

Organophosphorus hydrolase-based assay for organophosphate pesticides

We report a rapid and versatile organophosphorus hydrolase (OPH)-based method for measurement of organophosphates. This assay is based on a substrate-dependent change in pH at the local vicinity of the enzyme. The pH change is monitored using fluorescein isothiocyanate (FITC), which is covalently immobilized to the enzyme. This method employs the use of poly(methyl methacrylate) beads to which the FITC-labeled enzyme is adsorbed. Analytes were then measured using a microbead fluorescence analyzer. The dynamic concentration range for the assay extends from 25 to 400 &mgr;M for paraoxon with a detection limit of 8 &mgr;M. Organophosphorus insecticides measured using this technique included ethylparathion, methylparathion, dursban, fensulfothion, crotoxyphos, diazinon, mevinphos, dichlorvos, and coumaphos. This technique was used to measure coumaphos in biodegradation samples of cattle dip wastes and showed a high correlation (r2 = 0.998) to an HPLC method.

Journal Article↗

PubMed1998

Biosensor for direct determination of organophosphate nerve agents using recombinant Escherichia coli with surface-expressed organophosphorus hydrolase. 2. Fiber-optic microbial biosensor.

A fiber-optic microbial biosensor suitable for direct measurement of organophosphate nerve agents was developed. The unique features of this novel microbial biosensor were the recombinant Escherichia coli cells expressing the enzyme organophosphorus hydrolase on the cell surface and the optical detection of the products of enzyme-catalyzed organophosphate hydrolysis. The use of cells with the metabolic enzyme expressed on the cell surface as a biological sensing element provides advantages of no resistance to mass transport of the analyte and product across the cell membrane and low cost due to elimination of enzyme purification, over the conventional microbial biosensors based on cells expressing enzyme intracellularly and enzyme-based sensors, respectively. The use of an optical transducer allows the detection of different organophosphates in a mixture, presently not feasible with acetylcholinesterase-based biosensors. E. coli cells expressing organophosphorus hydrolase (OPH) on the cell surface were immobilized in low melting temperature agarose on a nylon membrane and attached to the common end of a bifurcated fiber-optic bundle. OPH-expressing E. coli cells catalyzed the hydrolysis of organophosphorus pesticides to form stoichiometric amounts of chromophoric products that absorb light at specific wavelengths. The backscattered radiation of the specific wavelength incident light was measured using a photomultiplier detector and correlated to the organophosphate concentration. The best sensitivity and response time were obtained using a sensor constructed with 1.5 mg of cells operating in pH 9, 50 mM HEPES buffer with 100 mM NaCl and 0.05 mM CoCl2 at 30 degrees C. At optimized conditions, the biosensor measured paraoxon, parathion, and coumaphos pesticides with high selectivity against triazine and carbamate pesticides in approximately 10 min. The lower detection limits were 3 microM for paraoxon and parathion and 5 microM for coumaphos. When stored in the buffer at 22 degrees C, the biosensor was stable for over a 1-month period and showed no decline in the response for over 75 repeated usages. The new fiber-optic microbial biosensor is an ideal tool for on-line monitoring of the detoxification process for organophosphate pesticides-contaminated wastewaters but may not be suitable for environmental monitoring.

Aryldialkylphosphatase↗