Biomedical subjects

Wilfred Chen

Publications and source records attributed to Wilfred Chen.

At least 37 records · Page 2Linked to original sources

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

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↗

PubMed2005

Protein engineering of epoxide hydrolase from Agrobacterium radiobacter AD1 for enhanced activity and enantioselective production of (R)-1-phenylethane-1,2-diol.

DNA shuffling and saturation mutagenesis of positions F108, L190, I219, D235, and C248 were used to generate variants of the epoxide hydrolase of Agrobacterium radiobacter AD1 (EchA) with enhanced enantioselectivity and activity for styrene oxide and enhanced activity for 1,2-epoxyhexane and epoxypropane. EchA variant I219F has more than fivefold-enhanced enantioselectivity toward racemic styrene oxide, with the enantiomeric ratio value (E value) for the production of (R)-1-phenylethane-1,2-diol increased from 17 for the wild-type enzyme to 91, as well as twofold-improved activity for the production of (R)-1-phenylethane-1,2-diol (1.96 +/- 0.09 versus 1.04 +/- 0.07 micromol/min/mg for wild-type EchA). Computer modeling indicated that this mutation significantly alters (R)-styrene oxide binding in the active site. Another three variants from EchA active-site engineering, F108L/C248I, I219L/C248I, and F108L/I219L/C248I, also exhibited improved enantioselectivity toward racemic styrene oxide in favor of production of the corresponding diol in the (R) configuration (twofold enhancement in their E values). Variant F108L/I219L/C248I also demonstrated 10-fold- and 2-fold-increased activity on 5 mM epoxypropane (24 +/- 2 versus 2.4 +/- 0.3 micromol/min/mg for the wild-type enzyme) and 5 mM 1,2-epoxyhexane (5.2 +/- 0.5 versus 2.6 +/- 0.0 micromol/min/mg for the wild-type enzyme). Both variants L190F (isolated from a DNA shuffling library) and L190Y (created from subsequent saturation mutagenesis) showed significantly enhanced activity for racemic styrene oxide hydrolysis, with 4.8-fold (8.6 +/- 0.3 versus 1.8 +/- 0.2 micromol/min/mg for the wild-type enzyme) and 2.7-fold (4.8 +/- 0.8 versus 1.8 +/- 0.2 micromol/min/mg for the wild-type enzyme) improvements, respectively. L190Y also hydrolyzed 1,2-epoxyhexane 2.5 times faster than the wild-type enzyme.

DNA Shuffling↗

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

Active site engineering of the epoxide hydrolase from Agrobacterium radiobacter AD1 to enhance aerobic mineralization of cis-1,2-dichloroethylene in cells expressing an evolved toluene ortho-monooxygenase.

Chlorinated ethenes are the most prevalent ground-water pollutants, and the toxic epoxides generated during their aerobic biodegradation limit the extent of transformation. Hydrolysis of the toxic epoxide by epoxide hydrolases represents the major biological detoxification strategy; however, chlorinated epoxyethanes are not accepted by known bacterial epoxide hydrolases. Here, the epoxide hydrolase from Agrobacterium radiobacter AD1 (EchA), which enables growth on epichlorohydrin, was tuned to accept cis-1,2-dichloroepoxyethane as a substrate by accumulating beneficial mutations from three rounds of saturation mutagenesis at three selected active site residues, Phe-108, Ile-219, and Cys-248 (no beneficial mutations were found at position Ile-111). The EchA F108L/I219L/C248I variant coexpressed with a DNA-shuffled toluene ortho-monooxygenase, which initiates attack on the chlorinated ethene, enhanced the degradation of cis-dichloroethylene (cis-DCE) an infinite extent compared with wild-type EchA at low concentrations (6.8 microm) and up to 10-fold at high concentrations (540 microm). EchA variants with single mutations (F108L, I219F, or C248I) enhanced cis-DCE mineralization 2.5-fold (540 microm), and EchA variants with double mutations, I219L/C248I and F108L/C248I, increased cis-DCE mineralization 4- and 7-fold, respectively (540 microm). For complete degradation of cis-DCE to chloride ions, the apparent Vmax/Km for the Escherichia coli strain expressing recombinant the EchA F108L/I219L/C248I variant was increased over 5-fold as a result of the evolution of EchA. The EchA F108L/I219L/C248I variant also had enhanced activity for 1,2-epoxyhexane (2-fold) and the natural substrate epichlorohydrin (6-fold).

Agrobacterium tumefaciens↗

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

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↗