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Biomedical subjects

Ashok Mulchandani

Publications and source records attributed to Ashok Mulchandani.

At least 19 recordsLinked to original sources
PubMed2006

Organophosphorus hydrolase multilayer modified microcantilevers for organophosphorus detection.

We report a biosensor based on organophosphorus hydrolase (OPH) multilayer modified microcantilever (MCL) for detection of organophosphorus compounds (OPs). The assay is based on substrate-dependent bending of the OPH functionalized MCLs. The cantilever bending amplitude at equilibrium was a function of the concentration of paraoxon with the dynamic range extending from 10(-7) to 10(-3)M. The lower detection limit of approximately 10(-7)M for paraoxon was an order of magnitude better than the OPH-based potentiometric and optical biosensors based on pH modulation. There was a good intra-sensor and an acceptable inter-sensor reproducibility as evidenced by the standard errors of 5% and 15%, respectively. OPs measured using this technique included parathion and diisopropyl fluorophosphate (DFP) in the order of paraoxon>DFP>parathion. The conformational change of the OPH was most likely the main origin of MCL bending.

Aryldialkylphosphatase↗
PubMed2006

Simple conjugation and purification of quantum dot-antibody complexes using a thermally responsive elastin-protein L scaffold as immunofluoresecent agents.

Fluorescent quantum dots (QDs), because of their tunable spectral properties, are ideal for simultaneous multiplexed detection in an antibody array format. Despite these advantages, their widespread usage is limited by the costly and tedious conjugation and separation protocol. Herein, we report a simple platform for the direct conjugation and separation of highly luminescent CdSe-ZnS QD-antibody complexes using a genetically engineered polyhistidine tagged elastin-protein L fusion (His-ELP-PL). The principle of immunoassay-ready conjugates was to take advantage of the direct conjugation of QDs via metal coordination with the His tag, the unique temperature-responsive property of ELP, and the high affinity of the antibody-binding protein L domain toward IgGs. Simple separation of the QD- His-ELP-PL-IgG complex was achieved by thermally triggered precipitation without any interference on the QD functionality. The utility of the biofunctionalized OD probes was demonstranted in an antibody array for the detection of carcinoembryonic antigen.

Antibodies↗
PubMed2006

Fabrication of antibody arrays using thermally responsive elastin fusion proteins.

A general method has been developed to immobilize antibodies onto an array surface by employing fusion proteins consisting of an elastin domain with tunable hydrophobic properties and an antibody-binding domain with high binding affinity and specificity for antibodies. Antibodies conjugated with the elastin fusion proteins can be directly printed on a self-assembled monolayer-modified glass slide in a functionally active orientation with a spatially defined pattern. An antibody array sensor for detection of tumor markers was fabricated to demonstrate the utility of the method. We expect that the method presented here could be a simple and universal platform to immobilize antibodies for the fabrication of a variety of antibody array sensors.

Animals↗
PubMed2006

Functional analysis of organophosphorus hydrolase variants with high degradation activity towards organophosphate pesticides.

Organophosphorus hydrolase (OPH, also known as phosphotriesterase) is a bacterial enzyme that is capable of degrading a wide range of neurotoxic organophosphate nerve agents. Directed evolution has been used to generate one variant (22A11) with up to 25-fold improved hydrolysis of methyl parathion. Surprisingly, this variant also degraded all other substrates (paraoxon, parathion and coumaphos) tested 2- to 10-fold faster. Since only one mutation (H257Y) is directly located in the active site, site-directed mutagenesis and saturation mutagenesis were used to identify the role of the other distal substitutions (A14T, A80V, K185R, H257Y, I274N) on substrate specificity and activity. Sequential site-directed mutagenesis indicated that K185R and I274N are the most important substitutions, leading to an improvement not only in the hydrolysis of methyl parathion but also the overall hydrolysis rate of all other substrates tested. Using structural modeling, these two mutations were shown to favor the formation of hydrogen bonds with nearby residues, resulting in structural changes that could alter the overall substrate hydrolysis.

Amino Acid Substitution↗
PubMed2006

V-type nerve agent detection using a carbon nanotube-based amperometric enzyme electrode.

An enzyme electrode for the detection of V-type nerve agents, VX (O-ethyl-S-2-diisopropylaminoethyl methylphosphonothioate) and R-VX (O-isobutyl-S-2-diethylaminoethyl methylphosphonothioate), is proposed. The principle of the new biosensor is based on the enzyme-catalyzed hydrolysis of the nerve agents and amperometric detection of the thiol-containing hydrolysis products at carbon nanotube-modified screen-printed electrodes. Demeton-S was used as a nerve agent mimic. 2-(Diethylamino)ethanethiol (DEAET) and 2-(dimethylamino)ethanethiol (DMAET), the thiol-containing hydrolysis product and hydrolysis product mimic of R-VX and VX, respectively, were monitored by exploiting the electrocatalytic activity of carbon nanotubes (CNT). As low as 2 microM DMAET and 0.8 microM DEAET were detected selectively at a low applied potential of 0.5 V vs Ag/AgCl at a CNT-modified mediator-free amperometric electrode. Further, the large surface area and the hydrophobicity of CNT was used to immobilize organophosphorus hydrolase mutant with improved catalytic activity for the hydrolysis of the P-S bond of phosphothiolester neurotoxins including VX and R-VX nerve gases to develop a novel, mediator-free, membrane-free biosensor for V-type nerve agents. The applicability of the biosensor was demonstrated for direct, rapid, and selective detection of V-type nerve agents' mimic demeton-S. The selectivity of the sensor against interferences and application to spiked lake water samples was demonstrated.

Biosensing Techniques↗
PubMed2006

Engineering plant-microbe symbiosis for rhizoremediation of heavy metals.

The use of plants for rehabilitation of heavy-metal-contaminated environments is an emerging area of interest because it provides an ecologically sound and safe method for restoration and remediation. Although a number of plant species are capable of hyperaccumulation of heavy metals, the technology is not applicable for remediating sites with multiple contaminants. A clever solution is to combine the advantages of microbe-plant symbiosis within the plant rhizosphere into an effective cleanup technology. We demonstrated that expression of a metal-binding peptide (EC20) in a rhizobacterium, Pseudomonas putida 06909, not only improved cadmium binding but also alleviated the cellular toxicity of cadmium. More importantly, inoculation of sunflower roots with the engineered rhizobacterium resulted in a marked decrease in cadmium phytotoxicity and a 40% increase in cadmium accumulation in the plant root. Owing to the significantly improved growth characteristics of both the rhizobacterium and plant, the use of EC20-expressing P. putida endowed with organic-degrading capabilities may be a promising strategy to remediate mixed organic-metal-contaminated sites.

Bacterial Proteins↗
PubMed2005

Detoxification of the organophosphate nerve agent coumaphos using organophosphorus hydrolase immobilized on cellulose materials.

Neurotoxic organophosphates (OPs) are widely used as pesticides and for public health purposes, as well as being nerve gases. As a result of the widespread use of these compounds for agriculture, large volumes of wastewater are generated. Additionally, there are large stockpiles of the nerve gases soman, sarin and VX in the United States and elsewhere around the world. Organophosphorus hydrolase (OPH) is an enzyme that catalyzes the hydrolysis of OP nerve agents. To date, however, the use of this enzyme in detoxification processes has been rather limited due to the high cost of its purification and short catalytic half-life. This paper reports the development of a cost-effective method for the production and immobilization of OPH in a pilot application in an enzyme bioreactor column for detoxification of paraoxon and coumaphos in contaminated wastewaters. A fusion between OPH and a cellulose binding domain that binds selectively to cellulose was generated to allow one-step purification and immobilization of OPH on cheap and abundantly available cellulose immobilization matrices. When packed in a column bioreactor, the immobilized fusion enzyme was able to completely degrade coumaphos up to a concentration of 0.2 mM. However, stirring of OPH immobilized on cellulose materials resulted in complete OP degradation of 1.5 mM coumaphos. The bioreactor column degraded the compounds tested at high concentration, rapidly, and without loss of process productivity for about 2 months.

Aryldialkylphosphatase↗
PubMed2005

Removal of estrogenic pollutants from contaminated water using molecularly imprinted polymers.

A synthetic molecularly imprinted polymer (MIP) sorbent for estrogenic compounds was prepared using a noncovalent imprinting technique. MIP microspheres sized from 1 to 2 microm were synthesized in acetonitrile by using alpha-estradiol as the template, acrylamide as the functional monomer, and trimethylpropanol trimethacrylate as the cross-linker. When compared with the nonimprinted polymer (NIP), the MIP showed outstanding affinity toward alpha-estradiol in aqueous solution with a binding site capacity (B(max)) of 380 nmol mg(-1) MIP, imprinting effect of 35, and a dissociation constant (Kd) of 38 microM. The MIP exhibited significant binding affinity toward other related estrogenic compounds such as beta-estradiol, diethylstilbestrol, estriol, and estrone, suggesting that this material may be appropriate for treating a complex mixture of estrogenic pollutants. The feasibility of removing estrogenic compounds from environmental water by the MIP was demonstrated using lake water spiked with alpha-estradiol. In addition, the MIP reusability without any deterioration in performance was demonstrated for at least five repeated cycles.

Environmental Monitoring↗
PubMed2005

Highly sensitive and selective amperometric microbial biosensor for direct determination of p-nitrophenyl-substituted organophosphate nerve agents.

We report herein a whole cell-based amperometric biosensor for highly selective, highly sensitive, direct, single-step, rapid, and cost-effective determination of organophosphate pesticides with a p-nitrophenyl substituent. The biosensor was comprised of a p-nitrophenol degrader, Pseudomonas putida JS444, genetically engineered to express organophosphorus hydrolase (OPH) on the cell surface immobilized on the carbon paste electrode. Surface-expressed OPH catalyzed hydrolysis of the p-nitrophenyl substituent organophosphorus pesticides such as paraoxon, parathion, and methyl parathion to release p-nitrophenol, which was subsequently degraded by the enzymatic machinery of P. putida JS444. The electrooxidization 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 0.086 mg dry weight of cells operating at 600 mV applied potential (vs Ag/AgCl reference) in 50 mM citrate--phosphate pH 7.5 buffer with 50 microM CoCl2 at room temperature. Under optimum operating conditions the biosensor measured as low as 0.28 ppb of paraoxon, 0.26 ppb of methyl parathion, and 0.29 ppb parathion. These detection limits are comparable to cholinesterase inhibition-based biosensors. Unlike the inhibition-based format, this biosensor manifests a selective response to organophosphate pesticides with a p-nitrophenyl substituent only, has a simplified single-step protocol with short response time, and can be used for repetitive/multiple and on-line analysis. The service life of the microbial amperometric biosensor was 5 days when stored in the operating buffer at 4 degrees C. The new biosensor offers great promise for rapid environmental monitoring of OP pesticides with nitrophenyl substituent.

Aryldialkylphosphatase↗
PubMed2005

Detoxification of organophosphate nerve agents by immobilized dual functional biocatalysts in a cellulose hollow fiber bioreactor.

A whole-cell technology for detoxification of organophosphates based on genetically engineered Escherichia coli cell expressing both cellulose-binding domain (CBD) and organophosphorus hydrolase (OPH) onto cell surface was reported recently (Wang et al., 2002). This study reports the application of these biocatalysts when immobilized in a cellulose hollow fiber bioreactor (HFB) for the biodetoxification of a model organophosphate, paraoxon, in a continuous flow mode. In 24 h, 0.79 mg wet cell/cm2 fiber surface were immobilized onto cellulose fibers specifically and strongly through the cellulose binding domain, forming a monolayer demonstrated by Scanning Electronic Micrograph, and essentially no cell was washed away by washing buffer. The immobilized biocatalyst had a high performance of detoxifying paraoxon solution of 5,220 mumol/h x L reactor or 990 mumol/h x m2 reactor. The immobilized biocatalysts maintained a stable degradation capacity for 15 uses over a period of 48 days with only 10% decline in degradation efficiency under operating and storage conditions. In addition, the bioreactor was easily regenerated by washing with 1% sodium dodecyl sulfate (SDS), with 86.7% immobilization capacity and 93.9% degradation efficiency recovery. This is the first report using the HFB in a non-traditional way, immobilizing whole-cell biocatalysts by specific adhesion thus rendering the catalysis operation the advantages of low pressure drop, low shear force, and low energy requirement. The successful application of this genetically engineered dual functional E. coli strain in a model bioreactor shows its promise in large-scale detoxification of organophosphate nerve agents in bulk liquid phase.

Aryldialkylphosphatase↗
PubMed2005

Temperature-triggered purification of antibodies.

In this article the unique capability of elastin-like protein (ELP) to reversibly precipitate was combined with the high affinity and specificity of antibody-binding domains such as Protein G, Protein L, or Protein LG as a general method for antibody purification that combines in a unique manner the simplicity and robustness of temperature-triggered precipitation with the selectivity of affinity interactions. In a single precipitation step, antibodies derived from different sources (animal sera or hybridoma cell cultures) were selectively recovered by a simple temperature trigger. Due to the versatility of the binding ligands toward different classes of antibodies, we believe that this technology will be useful as an economical, highly efficient, and universal platform for the purification of antibodies.

Antibodies↗
PubMed2005

Genetically engineered elastin-protein A fusion as a universal platform for homogeneous, phase-separation immunoassay.

A simple and universal platform for competitive phase-separation immunoassay is reported based on a fusion protein composed of a temperature-responsive elastin-like polypeptide (ELP) and the antibody-binding staphylococcal protein A (SpA). The basic principle is to take advantage of the ability of SpA to bind a variety of antibodies with high affinity, allowing simple separation of antigen-antibody complex by thermal precipitation. The resulting ELP-SpA fusion was shown to preserve the ability to reversibly precipitate as well as its high affinity toward different IgGs and IgMs. As a model system, a competitive phase-separation immunoassay based on the ELP-SpA format was established for paclitaxel (taxol) with IC(50) (20.18 nM) and the lower detection limit (2.94 nM) very similar to those reported for the ELISA format. Unlike the heterogeneous interaction in ELISA, which decreases the antibody-binding activity, the reported homogeneous immunoassay not only alleviates this problem but also enables the potential for high-throughput automation. We believe that the reported ELP-SpA fusion will find applications not only as a powerful diagnostic tool for diverse analytes but also a potential useful tool for purification and immobilization of antibody.

Elastin↗
PubMed2005

Direct determination of p-nitrophenyl substituent organophosphorus nerve agents using a recombinant Pseudomonas putida JS444-modified Clark oxygen electrode.

A microbial biosensor for rapid, sensitive, selective, and cost-effective determination of the total content of organophosphorus nerve agents with p-nitrophenyl substituent is reported. The biosensor consisted of genetically engineered PNP-degrader Pseudomonas putida JS444 expressing organophosphorus hydrolase (OPH) on its cell surface immobilized on a dissolved oxygen electrode. Surface-expressed OPH catalyzed the hydrolysis of organophosphorus pesticides with p-nitrophenyl substituent such as paraoxon, methyl parathion, and parathion to release p-nitrophenol that was oxidized by the enzymatic machinery of Pseudomonas putida JS444 to carbon dioxide while consuming oxygen. The oxygen consumption was measured and correlated to the concentration of organophosphates. The sensor signal and response time were optimized with 0.086 mg dry weight of cell and operating in 50 mM pH 7.5 citrate-phosphate buffer with 50 microM CoCl(2) at room temperature. When operated at optimized conditions, the biosensor measured as low as 55 ppb of paraoxon, 53 ppb of methyl parathion, and 58 ppb of parathion without interference from most phenolic compounds and other commonly used pesticides, such as atrazine, coumaphos, sutan, sevin, and diazinon. The operational life of the microbial biosensor was approximately 5 days when stored in the operating buffer at 4 degrees C.

Aryldialkylphosphatase↗
PubMed2005

Bioaffinity sensing using biologically functionalized conducting-polymer nanowire.

A simple, one-step method for fabricating single biologically functionalized conducting-polymer (polypyrrole) nanowire on prepatterned electrodes and its application to biosensing was demonstrated. The biologically functionalized polypyrrole was formed by the electropolymerization of an aqueous solution of pyrrole monomer and the model biomolecule, avidin- or streptavidin-conjugated ZnSe/CdSe quantum dots, within 100 or 200 nm wide by 3 mum long channels between gold electrodes on prefabricated silicon substrate. When challenged with biotin-DNA, the avidin- and streptavidin-polypyrrole nanowires generated a rapid change in resistance to as low as 1 nM, demonstrating the utility of the biomolecule-functionalized nanowire as biosensor. The method offers advantages of direct incorporation of functional biological molecules into the conducting-polymer nanowire during its synthesis, site-specific positioning, built-in electrical contacts, and scalability to high-density nanoarrays over the reported silicon nanowire and carbon nanotube biosensors.

Biosensing Techniques↗
PubMed2005

Aqueous sol-gel encapsulation of genetically engineered Moraxella spp. cells for the detection of organophosphates.

An all-aqueous sol-gel method for encapsulation of bacterial cells in porous silicate matrices towards the development of a biosensor is described. The sol-gel encapsulation of cells is achieved at room temperature and neutral pH. Furthermore, use of sodium silicate as precursor avoids generation of alcohol that can be detrimental to cells in contrast to the traditional alkoxide sol-gel encapsulation process. Moraxella spp. cells engineered to express recombinant organophosphorus hydrolase (OPH) on the cell surface were encapsulated and OPH enzymatic activity was measured for paraoxon hydrolysis. Kinetic parameters (Km and Vmax) as well as pH behavior of surface-expressed OPH were determined to evaluate the effect of encapsulation. Cells encapsulated by the sodium silicate method displayed higher activity retention compared to those by the traditional alkoxide process. Time-course studies over a 2-month period indicate that immobilization through the sodium silicate process led to a reduction in activity of approximately 5% as compared to approximately 30% activity reduction in case of free cells in buffer indicating that immobilization leads to stabilization, a key parameter in biosensor development.

Biosensing Techniques↗
PubMed2005

Real-time nucleic acid sequence-based amplification assay for detection of hepatitis A virus.

A nucleic acid sequence-based amplification (NASBA) assay in combination with a molecular beacon was developed for the real-time detection and quantification of hepatitis A virus (HAV). A 202-bp, highly conserved 5' noncoding region of HAV was targeted. The sensitivity of the real-time NASBA 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, with only HAV positively identified. When combined with immunomagnetic separation, the NASBA assay successfully detected as few as 10 PFU from seeded lake water samples. Due to its isothermal nature, its speed, and its similar sensitivity compared to the real-time RT-PCR assay, this newly reported real-time NASBA method will have broad applications for the rapid detection of HAV in contaminated food or water.

Animals↗
PubMed2005

Visualization and detection of infectious coxsackievirus replication using a combined cell culture-molecular beacon assay.

Rapid detection of infectious viruses is of central importance for public health risk assessment. By directly visualizing newly synthesized viral RNA with molecular beacons (MBs), we have developed a generalized method for the rapid and sensitive detection of infectious viruses from cell culture. An MB, CVB1, specifically targeting the 5' noncoding region of the enterovirus genome was designed and synthesized. Introduction of MB CVB1 into permeabilized cells highly infected with coxsackievirus B6 resulted in brightly fluorescent cells that can be easily visualized with a fluorescence microscope. In contrast, no detectable signal was observed with noninfected cells or with nonspecific MBs. The number of fluorescent cells also increased in a dose-responsive manner, enabling the direct quantification of infectious viral dosages by direct counting of fluorescent foci. As little as 1 PFU of infectious coxsackievirus B6 was detected within 6 h postinfection. When combined with nuclease-resistant MBs, this method could be useful not only for the real-time detection of infectious viruses but is also useful to study the life cycle of viral processing in vivo.

Animals↗