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Biomedical subjects

Ashok Mulchandani

Publications and source records attributed to Ashok Mulchandani.

At least 37 records · Page 2Linked to original sources
PubMed2004

Amperometric microbial biosensor for p-nitrophenol using Moraxella sp.-modified carbon paste electrode.

An amperometric microbial biosensor for highly specific, sensitive and rapid quantitative determination of p-nitrophenol was developed. The biosensor takes advantage of the ability of Moraxella sp. to specifically degrade p-nitrophenol to hydroquinone, a more electroactive compound than p-nitrophenol. The electrochemical oxidation current of hydroquinone formed in biodegradation of p-nitrophenol was measured at Moraxella sp.-modified carbon paste electrode and correlated to p-phenol concentrations. The optimum response was realized by electrode constructed using 15 mg of dry cell weight per 1 g of carbon paste and operating at 0.3 V (versus Ag/AgCl reference) in pH 7.5, 20 mM sodium phosphate buffer. Operating at these optimum conditions the biosensor had excellent selectivity against phenol derivatives and was able to measure as low as 20 nM (2.78 ppb) p-nitrophenol with very good accuracy and reproducibility. The biosensor was stable for approximately 3 weeks when stored at 4 degrees C. The applicability of the biosensor to measure p-nitrophenol in lake water was demonstrated.

Biological Assay↗
PubMed2004

Microchip capillary electrophoresis with electrochemical detection of thiol-containing degradation products of V-type nerve agents.

A microchip protocol for the capillary electrophoresis separation and electrochemical detection of thiol-containing degradation products of V-type nerve agents is described. The microchip assay relies on the derivatization reaction of 2-(dimethylamino)ethanethiol (DMAET), 2-(diethylamino)ethanethiol (DEAET), and 2-mercaptoethanol (ME) with o-phthaldialdehyde in the presence of the amino acid valine along with amperometric monitoring of the isoindole derivatives. Both off-chip and on-chip derivatization reactions have led to highly sensitive and rapid detection of the thiol degradation products. Various parameters influencing the derivatization, separation, and detection processes were examined and optimized. These include the amino acid co-reagent, reagent-mixing ratio, reaction time, injection time, separation voltage, and detection potential. The chip microsystem offers a rapid (<4 min) simultaneous detection of micromolar concentrations of DMAET, DEAET, and ME. Linear calibration plots were observed for the V-type nerve agent thiol degradation products, along with good stability and reproducibility (RSD < 8.0%). Detection limits of 5 and 8 microM were obtained for the off-chip reaction of DMAET and DEAET, respectively, following a 2-s injection. The suitability for assays of environmental matrixes was demonstrated for the determination of DMAET and DEAET in untreated tap and river water samples. The favorable analytical performance makes the new microfluidic device attractive for addressing the needs of various security scenarios.

Biodegradation, Environmental↗
PubMed2004

Cadmium removal from contaminated soil by tunable biopolymers.

An elastin-like polypeptide (ELP) composed of a polyhistidine tail (ELPH12) was exploited as a tunable, metal-binding biopolymer with high affinity toward cadmium. By taking advantage of the property of ELPH12 to undergo a reversible thermal precipitation, easy recovery of the sequestered cadmium from contaminated water was demonstrated as the result of a simple temperature change. In this study, batch soil washing experiments were performed to evaluate the feasibility of using ELPH12 as an environmentally benign strategy for removing cadmium from contaminated soil. The stability constant (log KL) for the cadmium-ELPH12 complex was determined to be 6.8, a value similar to that reported for the biosurfactant rhamnolipid. Two washings with 1.25 mg/mL of ELPH12 were able to remove more than 55% of the bound cadmium as compared to only 8% removal with ELP containing no histidine tail or 21% removal using the same concentration of EDTA. Unlike rhamnolipid from Pseudomonas aeruginosa ATCC 9027, which adsorbs extensively to soil, less than 10% of ELPH12 was adsorbed under all soil washing conditions. As a result, a significantly lower concentration of ELPH12 (0.036 mM as compared to 5-10 mM of biosurfactants) was required to achieve similar extraction efficiencies. However, cadmium recovery by simple precipitation was incomplete due to the displacement of bound cadmium by zinc ions present in soil. Owing to its benign nature, ease of production, and selective tailoring of the metal binding domain toward any target metals of interest, ELP biopolymers may find utility as an effective extractant for heavy metal removal from contaminated soil or ore processing.

Adsorption↗
PubMed2004

Whole cell-enzyme hybrid amperometric biosensor for direct determination of organophosphorous nerve agents with p-nitrophenyl substituent.

In this paper, we reported the construction of a hybrid biosensor for direct, highly selective, sensitive, and rapid quantitative determination of organophosphate pesticides with p-nitrophenyl substituent using purified organophosphorus hydrolase (OPH) for the initial hydrolysis and Arthrobacter sp. JS443 for subsequent p-nitrophenol oxidation. The biocatalytic layer was prepared by co-immobilizing Arthrobacter sp. JS443 and OPH on a carbon paste electrode. OPH catalyzed the hydrolysis of organophosphorus pesticides with p-nitrophenyl substituent such as paraoxon and methyl parathion to release p-nitrophenol that was oxidized by the enzymatic machinery of Arthrobacter sp. JS443 to carbon dioxide through electroactive intermediates 4-nitrocatechol and 1,2,4-benzenetriol. The oxidization current of the intermediates was measured and correlated to the concentration of organophosphates. The best sensitivity and response time were obtained using a sensor constructed with 0.06 mg dry weight of cell and 965 IU of OPH operating at 400 mV applied potential (vs. Ag/AgCl reference) in 50 mM citrate-phosphate pH 7.5 buffer at room temperature. Using these conditions, the biosensor measured as low as 2.8 ppb (10 nM) of paraoxon and 5.3 ppb (20 nM) of methyl parathion without interference from phenolic compounds, carbamate pesticides, triazine herbicides, and organophosphate pesticides that do not have the p-nitrophenyl substituent. The biosensor had excellent operational life-time stability with no decrease in response for more than 40 repeated uses over a 12-h period when stored at room temperature, while its storage life was approximately 2 days when stored in the operating buffer at 4 degrees C.

Actinobacteria↗
PubMed2004

Affinity purification of plasmid DNA by temperature-triggered precipitation.

This report describes a new plasmid DNA purification method, which takes advantage of the DNA-binding affinity and specificity of the bacterial metalloregulatory protein MerR, and of the temperature responsiveness of elastin-like proteins (ELPs). Upon increasing the temperature, ELP undergoes a reversible phase transition from water-soluble forms into aggregates, and this property was exploited for the precipitation of plasmid DNA containing the MerR recognition sequence by a simple temperature trigger. In one purification step, plasmid DNA was purified from E. coli cell lysates to a better purity than that prepared by a standard alkaline purification method, with no contaminating chromosomal DNA and cellular proteins. This protein-based approach, in combination with the reversible phase transition feature of ELP, makes the outlined method a promising candidate for large-scale purification of plasmid DNA for sensitive applications such as nonviral gene therapy or DNA vaccines.

Chemical Precipitation↗
PubMed2004

Vapor and liquid phase detection of cyanide on a microchip.

A capillary electrophoresis microchip is used to selectively and sensitively monitor cyanide levels in both vapor (HCN((g))) and aqueous (NaCN in drinking water) phases. Laser-induced fluorescence detection is applied using a violet diode laser to monitor the fluorescent isoindole derivative formed by the reaction of cyanide with 2,3-naphthalenedicarboxaldehyde (NDA) and taurine. Air sampling of hydrogen cyanide is achieved using a miniature impinger (2 mL), giving collection efficiencies as high as 79% for a sampling rate of 1.0 L/min and a 10 s sampling time (relative standard deviation RSD: 2.7% for n = 5). Following the addition of NDA and taurine to either the vapor phase impinger sample or an aqueous drinking water sample, the NDA/cyanide derivative can be detected in just over 40 s on the microchip, giving a detection limit of 0.56 microg/L and a linear dynamic range from 0.56 microg/L-2.4 mg/L. The detection limit for hydrogen cyanide in air was determined to be 2.3 ppb (mole%). On-chip derivatization of cyanide by NDA was successful, although a 50% decrease in signal intensity was observed due to insufficient time for completion of the reaction on the microchip. A number of different interferents were examined, and only iron(II) and chlorine showed any interference due to their capability for masking the presence of cyanide by reacting with free cyanide in solution.

Air Pollution↗
PubMed2004

Combined immunomagnetic separation-molecular beacon-reverse transcription-PCR assay for detection of hepatitis A virus from environmental samples.

In this study, a molecular-beacon-based real-time reverse transcription (RT)-PCR assay was developed to detect the presence of hepatitis A virus (HAV) in environmental samples. A 125-bp, highly conserved 5' noncoding region of HAV was targeted. The sensitivity of the real-time RT-PCR assay was tested with 10-fold dilutions of viral RNA, and a detection limit of 1 PFU was obtained. The specificity of the assay was demonstrated by testing with other environmental pathogens and indicator microorganisms, and only HAV was positively identified. When combined with immunomagnetic separation, the real-time RT-PCR assay successfully detected as few as 20 PFU in seeded groundwater samples. Because of its simplicity and specificity, this assay has broad applications for the rapid detection of HAV in contaminated foods or water.

Fresh Water↗
PubMed2004

Enhanced arsenic accumulation in engineered bacterial cells expressing ArsR.

The metalloregulatory protein ArsR, which offers high affinity and selectivity toward arsenite, was overexpressed in Escherichia coli in an attempt to increase the bioaccumulation of arsenic. Overproduction of ArsR resulted in elevated levels of arsenite bioaccumulation but also a severe reduction in cell growth. Incorporation of an elastin-like polypeptide as the fusion partner to ArsR (ELP153AR) improved cell growth by twofold without compromising the ability to accumulate arsenite. Resting cells overexpressing ELP153AR accumulated 5- and 60-fold-higher levels of arsenate and arsenite than control cells without ArsR overexpression. Conversely, no significant improvement in Cd(2+) or Zn(2+) accumulation was observed, validating the specificity of ArsR. The high affinity of ArsR allowed 100% removal of 50 ppb of arsenite from contaminated water with these engineered cells, providing a technology useful to comply with the newly approved U.S. Environmental Protection Agency limit of 10 ppb. These results open up the possibility of using cells overexpressing ArsR as an inexpensive, high-affinity ligand for arsenic removal from contaminated drinking and ground water.

Arsenic↗
PubMed2004

Altering the substrate specificity of organophosphorus hydrolase for enhanced hydrolysis of chlorpyrifos.

Chlorpyrifos is one of the most popular pesticides used for agriculture crop protection, and widespread contamination is a potential concern. However, chlorpyrifos is hydrolyzed almost 1,000-fold slower than the preferred substrate, paraoxon, by organophosphorus hydrolase (OPH), an enzyme that can degrade a broad range of organophosphate pesticides. We have recently demonstrated that directed evolution can be used to generate OPH variants with up to 25-fold improvement in hydrolysis of methyl parathion. The obvious question and challenge are whether similar success could be achieved with this poorly hydrolyzed substrate, chlorpyrifos. For this study, five improved variants were selected from two rounds of directed evolution based on the formation of clear haloes on Luria-Bertani plates overlaid with chlorpyrifos. One variant, B3561, exhibited a 725-fold increase in the k(cat)/K(m) value for chlorpyrifos hydrolysis as well as enhanced hydrolysis rates for several other OP compounds tested. Considering that wild-type OPH hydrolyzes paraoxon at a rate close to the diffusion control limit, the 39-fold improvement in hydrolysis of paraoxon by B3561 suggests that this variant is one of the most efficient enzymes available to attack a wide spectrum of organophosphate nerve agents.

Amino Acid Substitution↗
PubMed2003

An immunoassay for atrazine using tunable immunosorbent.

A novel, simple, economical, and environmentally friendly tunable immunosorbent-based immunoassay for sensitive and selective determination of atrazine is reported. Tunable immunosorbents consisting of a fusion between an elastin-like polypeptide made up of 77 repeating units of the pentapeptide VPGVG and a single-chain Fv of an anti-atrazine antibody were synthesized biologically and purified by temperature-triggered phase transition. A competitive immunoassay based on the competition of atrazine-horseradish peroxidase and atrazine was established with IC(50) and lower detection limit of 0.16 and 0.01 ppb, respectively. Excellent recoveries (mean values ranging between 92 and 104%) were demonstrated in simulated atrazine-contaminated water samples.

Antibodies↗
PubMed2003

A tubulin-based fluorescent polarization assay for paclitaxel.

This paper reports the development, characterization, and application of a separation-free, homogeneous, and simple to perform fluorescence polarization bioassay for paclitaxel. The bioassay is based on the binding interaction of paclitaxel to tubulin, the receptor protein on which this drug acts. The bioassay was carried out in a competitive format where the paclitaxel and a synthetic fluorescent-labeled paclitaxel (Rh-Tx) competed for tubulin binding, causing a change in fluorescence polarization, which was an inverse function of the paclitaxel concentration. The bioassay had a dynamic range from 0.03 to 0.35 microM, which falls in the therapeutic range (0.01-10 microM), and a limit of detection of 23 nM. The bioassay was selective against other naturally occurring taxanes such as baccatin III and 10-deacetylbaccatin III. However, there was interference from cephalomannine. The excellent sensitivity, accuracy, reproducibility, and simplicity make this analytical technique suitable for routine and high-throughput analyses and might be helpful in providing better care for patients. The bioassay was successfully applied to the determination of paclitaxel in human plasma.

Animals↗
PubMed2003

A temperature responsive biopolymer for mercury remediation.

Tunable biopolymers based on elastin-like polypeptides (ELP) were engineered for the selective removal of mercury. ELP undergoes a reversible thermal precipitation within a wide range of temperatures and was exploited to enable easy recovery of the sequestered mercury. A bacterial metalloregulatory protein, MerR, which binds mercury with an unusually high affinity and selectivity, was fused to the ELP to provide the highly selective nature of the biopolymers. Selective binding of mercury was demonstrated at an expected ratio of 0.5 mercury/biopolymer, and minimal binding of competing heavy metals (cadmium, nickel, and zinc), even at 100-fold excess, was observed. The sequestered mercury was extracted easily, enabling continuous reuse of the biopolymers. In repeating cycles, mercury concentration was reduced to ppb levels, satisfying even drinking water limits. Utility of the biopolymers with mercury-contaminated Lake Elsinore water was demonstrated with no decrease in efficiency. The nanoscale biopolymers reported here using metalloregulatory proteins represent a "green" technology for environmentally benign mercury removal. As nature offers a wide selection of specific metalloregulatory proteins, this technology offers promising solutions to remediation of other important pollutants such as arsenic or chromium.

Adsorption↗
PubMed2003

One-step metal-affinity purification of histidine-tagged proteins by temperature-triggered precipitation.

The feature of elastin-like proteins (ELPs) to reversibly precipitate above their transition temperature was exploited as a general method for the purification of histidine (His)-tagged proteins. The principle of the single-step metal-affinity method is based on coordinated ligand-bridging between the modified ELPs and the target proteins. ELPs with repeating sequences of [(VPGVG)(2)(VPGKG)(VPGVG)(2)](21) were synthesized and the free amino groups on the lysine residues were modified by reacting with imidazole-2-carboxyaldehyde to incorporate the metal-binding ligands into the ELP bio- polymers. Biopolymers charged with Ni(2+) were able to interact with a His tag on the target proteins based on metal coordination chemistry. Purifications of two His-tagged enzymes, beta-D-galactosidase and chloramphenicol acetyltransferase, were used to demonstrate the utility of this general method and over 85% recovery was observed in both cases. The bound enzymes were easily released by addition of either EDTA or imidazole. The recovered ELPs were reused four times with no observable decrease in the purification performance.

Affinity Labels↗
PubMed2003

A novel bioassay for screening and quantification of taxanes.

We report a novel method for screening and quantifying tubulin-binding antimitotic agents that promote microtubule formation. The method is based on the shift in the peak of the fluorescence emission difference spectrum of tubulin complexed to metal free tetrakis(4-carboxyphenyl)porphyrin (TCPP) in the presence of antimitotic agents. Taxol (paclitaxel), an anti-tumor drug approved for the treatment of a variety of cancers, caused the appearance of a new fluorescence peak at 645 nm at concentrations as low as 125 nM, the intensity of which was a function of the paclitaxel concentration. Cephalomannine caused the induction of a new fluorescence peak at 651 nm only above 1 muM. Baccatin did not induce the appearance of any new peak within detectable operating measurement conditions. These observations are in accordance with the biological activities/cytotoxicities of these compounds. Accordingly, it is proposed that the new method can be used for high throughput screening of antimitotic compounds.

Antineoplastic Agents, Phytogenic↗
PubMed2003

Thermally triggered purification and immobilization of elastin-OPH fusions.

A bifunctional fusion protein consisting of organophosphorus hydrolase (OPH) and elastin-like polypeptide (ELP) was synthesized for the detoxification of organophosphorus compounds. ELPs undergo a reversible phase transition upon an increase in temperature, forming hydrophobic aggregates. This thermally triggered property of phase transition allows for a simple and rapid means of purifying the fusion protein. Over 1,300-fold purification was achieved after only 2 cycles of inverse phase transition. The purified fusion protein showed identical kinetic properties as the native OPH with only a modest 10% increase in K(m) and a 5% decrease of K(cat). The ability of the ELP domain to form collapsed aggregates also improved long-term stability of the fusion enzyme. Aggregated ELP-OPH retained nearly 100% activity over a span of three weeks. In addition to facilitating purification and stability, the ELP moiety served as a hydrophobic tag for one-step immobilization of the fusion protein onto hydrophobic surfaces. The ELP-OPH was capable of rapidly degrading paraoxon while immobilized. The protein also retained ELP functionality of reversible phase transition thereby allowing for the regeneration of the treated surface. This technology offers a swift and convenient means for purification, immobilization, and regeneration of OPH onto a variety of hydrophobic surfaces by simple environmental triggers.

Aryldialkylphosphatase↗
PubMed2003

Flow injection amperometric detection of OP nerve agents based on an organophosphorus-hydrolase biosensor detector.

A flow-injection system with an organophosphorus-hydrolase (OPH)-biosensor detector has been developed and characterized for the rapid detection of organophosphorus (OP) nerve agents. The enzyme was immobilized onto a thin-film gold detector through a cystamine-glutaraldehyde coupling. Factors influencing the performance were optimized. The resulting flow system offered a fast, sensitive, selective, and stable response. The peak current increased linearly with the concentration of paraoxon and methyl parathion over the 1-10 microM range (sensitivity, 2.29 and 1.04 nA/microM, respectively). The OPH-biosensor flow injection systems offered low detection limits (e.g. 0.1 microM paraoxon), along with a good precision (R.S.D. of 3.6% for 20 successive injections of a 1.0 microM paraoxon solution). The OPH-biosensor flow detector offers great promise for rapid field screening of OP pesticides and nerve agents.

Aryldialkylphosphatase↗
PubMed2003

Novel synthetic phytochelatin-based capacitive biosensor for heavy metal ion detection.

A novel capacitance biosensor based on synthetic phytochelatins for sensitive detection of heavy metals is described. Synthetic phytochelatin (Glu-Cys)(20)Gly (EC20) fused to the maltose binding domain protein was expressed in Escherichia coli and purified for construction of the biosensor. The new biosensor was able to detect Hg(2+), Cd(2+), Pb(2+), Cu(2+) and Zn(2+) ions in concentration range of 100 fM-10 mM, and the order of sensitivity was S(Zn)>S(Cu)>S(Hg)>>S(Cd) congruent with S(Pb). The biological sensing element of the sensor could be regenerated using EDTA and the storage stability of the biosensor was 15 days.

Biosensing Techniques↗