Ashok Mulchandani

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.

Engineering Plant-Microbe Symbiosis for Rhizoremediation of Heavy Metals

ABSTRACT 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 cell surface display of organophosphorus hydrolase for selective screening of improved hydrolysis of organophosphate nerve agents.

ABSTRACT Organophosphorus hydrolase (OPH) is a bacterial enzyme that has been shown to degrade a wide range of neurotoxic organophosphate nerve agents. However, the effectiveness of degradation varies dramatically, ranging from highly efficient with paraoxon to relatively slow with methyl parathion. Sequential cycles of DNA shuffling and screening were used to fine-tune and enhance the activity of OPH towards poorly degraded substrates. Because of the inaccessibility of these pesticides across the cell membrane, OPH variants were displayed on the surface of Escherichia coli using the truncated ice nucleation protein in order to isolate novel enzymes with truly improved substrate specificities. A solid-phase top agar method based on the detection of the yellow product p-nitrophenol was developed for the rapid prescreening of potential variants with improved hydrolysis of methyl parathion. Two rounds of DNA shuffling and screening were carried out, and several improved variants were isolated. One variant in particular, 22A11, hydrolyzes methyl parathion 25-fold faster than does the wild type. Because of the success that we achieved with directed evolution of OPH for improved hydrolysis of methyl parathion, we believe that we can easily extend this method in creating other OPH variants with improved activity against poorly degraded pesticides such as diazinon and chlorpyrifos and nerve agents such as sarin and soman.

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.

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.

Amperometric Thick-Film Strip Electrodes for Monitoring Organophosphate Nerve Agents Based on Immobilized Organophosphorus Hydrolase

An amperometric biosensor based on the immobilization of organophosphorus hydrolase (OPH) onto screen-printed carbon electrodes is shown useful for the rapid, sensitive, and low-cost detection of organophosphate (OP) nerve agents. The sensor relies upon the sensitive and rapid anodic detection of the enzymatically generated p-nitrophenol product at the OPH/Nafion layer immobilized onto the thick-film electrode in the presence of the OP substrate. The amperometric signals are linearly proportional to the concentration of the hydrolyzed paraoxon and methyl parathion substrates up to 40 and 5 μM, showing detection limits of 9 × 10(-)(8) and 7 × 10(-)(8) M, respectively. Such detection limits are substantially lower compared to the (2-5) × 10(-)(6) M values reported for OPH-based potentiometric and fiber-optic devices. The high sensitivity is coupled to a faster and simplified operation, and the sensor manifests a selective response compared to analogous enzyme inhibition biosensors. The applicability to river water sampling is illustrated. The attractive performance and greatly simplified operation holds great promise for on-site monitoring of OP pesticides.

Enhanced Arsenic Accumulation in Engineered Bacterial Cells Expressing ArsR

ABSTRACT 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 Cd2+ or Zn2+ 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.

Graphene nanomesh as highly sensitive chemiresistor gas sensor

Graphene is a one atom thick carbon allotrope with all surface atoms that has attracted significant attention as a promising material as the conduction channel of a field-effect transistor and chemical field-effect transistor sensors. However, the zero bandgap of semimetal graphene still limits its application for these devices. In this work, ethanol-chemical vapor deposition (CVD) of a grown p-type semiconducting large-area monolayer graphene film was patterned into a nanomesh by the combination of nanosphere lithography and reactive ion etching and evaluated as a field-effect transistor and chemiresistor gas sensors. The resulting neck-width of the synthesized nanomesh was about ∼20 nm and was comprised of the gap between polystyrene (PS) spheres that was formed during the reactive ion etching (RIE) process. The neck-width and the periodicities of the graphene nanomesh (GNM) could be easily controlled depending on the duration/power of the RIE and the size of the PS nanospheres. The fabricated GNM transistor device exhibited promising electronic properties featuring a high drive current and an I(ON)/I(OFF) ratio of about 6, significantly higher than its film counterpart. Similarly, when applied as a chemiresistor gas sensor at room temperature, the graphene nanomesh sensor showed excellent sensitivity toward NO(2) and NH(3), significantly higher than their film counterparts. The ethanol-based graphene nanomesh sensors exhibited sensitivities of about 4.32%/ppm in NO(2) and 0.71%/ppm in NH(3) with limits of detection of 15 and 160 ppb, respectively. Our demonstrated studies on controlling the neck width of the nanomesh would lead to further improvement of graphene-based transistors and sensors.

Single-walled carbon nanotube-based chemiresistive affinity biosensors for small molecules: ultrasensitive glucose detection.

We report for the first time single-walled carbon nanotube (SWNT)-based chemiresistive affinity sensors for highly sensitive and selective detection of small and/or weakly charged or uncharged molecules using a displacement format. The detection of glucose, a small, weakly charged molecule, by displacement of plant lectin (concavalin A) bound to a polysaccharide (dextran) immobilized on SWNTs with picomolar sensitivity and selectivity over other sugars and human serum proteins is demonstrated as a proof of concept.

Sensitive detection of H2S using gold nanoparticle decorated single-walled carbon nanotubes.

Herein, we demonstrate that highly sensitive conductometric gas nanosensors for H(2)S can be synthesized by electrodepositing gold nanoparticles on single-walled carbon nanotube (SWNT) networks. Adjusting the electrodeposition conditions allowed for tuning of the size and number of gold nanoparticles deposited. The best H(2)S sensing performance was obtained with discrete gold nanodeposits rather than continuous nanowires. The gas nanosensors could sense H(2)S in air at room temperature with a 3 ppb limit of detection. The sensors were reversible, and increasing the bias voltage reduced the sensor recovery time, probably by local Joule heating. The sensing mechanism is believed to be based on the modulation of the conduction path across the nanotubes emanating from the modulation of electron exchange between the gold and carbon nanotube defect sites when exposed to H(2)S.