Salivary Detection of Dengue Virus NS1 Protein with a Label-Free Immunosensor for Early Dengue Diagnosis.

Dengue virus (DENV) is a highly pathogenic, arthropod-borne virus transmitted between people by Aedes mosquitoes. Despite efforts to prevent global spread, the potential for DENV epidemics is increasing world-wide. Annually, 3.6 billion people are at risk of infection. With no licensed vaccine, early diagnosis of dengue infection is critical for clinical management and patient survival. Detection of DENV non-structural protein 1 (NS1) is a clinically accepted biomarker for the early detection of DENV infection. Unfortunately, virtually all of the laboratory and commercial DENV NS1 diagnostic methods require a blood draw for sample analysis, limiting point-of-care diagnostics and decreases patient willingness. Alternatively, NS1 in human saliva has been identified for the potential early diagnosis of DENV infection. The collection of saliva is simple, non-invasive, painless, and inexpensive, even by minimally trained personnel. In this study, we present a label-free chemiresistive immunosensor for the detection of the DENV NS1 protein utilizing a network of single-walled carbon nanotubes functionalized with anti-dengue NS1 monoclonal antibodies. NS1 was successfully detected in adulterated artificial human saliva over the range of clinically relevant concentrations with high sensitivity and selectivity. It has potential application in clinical diagnosis and the ease of collection allows for self-testing, even within the home.

An oligonucleotide-functionalized carbon nanotube chemiresistor for sensitive detection of mercury in saliva.

Divalent mercuric (Hg(2+)) ion and monomethyl mercury (CH3Hg(+)) are two forms of mercury that are known to be highly toxic to humans. In this work, we present a highly selective, sensitive and label-free chemiresistive biosensor for the detection of both, Hg(2+) and CH3Hg(+) ions using DNA-functionalized single-walled carbon nanotubes (SWNTs). The SWNTs were functionalized with the capture oligonucleotide, polyT, using a linker molecule. The polyT was hybridized with polyA to form a polyT:polyA duplex. Upon exposure to mercury ions, the polyT:polyA duplex dehybridizes and a T-Hg(2+)-T duplex is formed. This structure switch leads to the release of polyA from the SWNT surface and correspondingly a change in the resistance of the chemiresistive biosensor is observed, which is used to quantify the mercury ion concentration. The biosensor showed a wide dynamic range of 0.5 to 100 nM for the detection of CH3Hg(+) ions in buffer solution with a sensitivity of 28.34% per log (nM) of CH3Hg(+). Finally, real world application of the biosensor was demonstrated by the detection of Hg(2+) and CH3Hg(+) ions in simulated saliva samples spiked with a known concentration of mercury ions.

Poly(3-aminophenylboronic acid)-functionalized carbon nanotubes-based chemiresistive sensors for detection of sugars.

The poly(aniline boronic acid) (PABA)-functionalized single-walled carbon nanotube (SWNT) non-enzymatic sensor was developed for the detection of saccharides. The work involved the electrochemical polymerization of 3-aminophenylboronic acid (3-APBA) in the presence of fluoride on the surface of SWNTs and their subsequent evaluation as chemiresistive sensors towards the detection of d-fructose and d-glucose. By varying the sensor's synthesis conditions by charge-controlled electropolymerization, the sensing performance was systematically optimized. Through electrical characterization in terms of change in resistance, cyclic voltammetry confirmed the electrochemical deposition of the PABA coating on the SWNTs. The optimized sensors showed sensing response over a wide dynamic range of concentrations and a limit of detection of 2.92 mM for D-fructose and 3.46 mM for D-glucose. The hybrid sensors could be regenerated on the basis of the reversible nature of the binding between PABA and 1,2- or 1,3-diols at lower values of pH.

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 chemoresistive label-free immunosensor for salivary stress biomarkers.

The present work is focused on the development, analytical characterization and evaluation of selective and sensitive SWNT-chemiresistor immunosensor for the label-free detection of salivary α-amylase (SAA). SWNTs were aligned to bridge lithographically patterned gold microelectrodes using AC dielectrophoresis followed by functionalization with anti-SAA antibodies. The nano-immunosensors exhibited excellent sensitivity over the clinically relevant range (19 to 308 U ml(-1)) with a limit of detection (LOD) of 6 µg ml(-1) (0.6 U ml(-1)) and 7.8 µg ml(-1) (0.78 U ml(-1)) in phosphate buffer and artificial saliva, respectively, and no interference from other components of saliva. This label-free nano-immunosensor technology has potential application in clinical diagnosis for stress biomarkers expressed in human saliva at the point-of-care.

Label-free chemiresistive immunosensors for viruses.

We report development, characterization, and testing of chemiresistive immunosensors based on single polypyrrole (Ppy) nanowire for highly sensitive, specific, label free, and direct detection of viruses. Bacteriophages T7 and MS2 were used as safe models for viruses for demonstration. Ppy nanowires were electrochemically polymerized into alumina template, and single nanowire based devices were assembled on a pair of gold electrodes by ac dielectrophoretic alignment and anchored using maskless electrodeposition. Anti-T7 or anti-MS2 antibodies were immobilized on single Ppy nanowire using EDC-NHS chemistry to fabricate nanobiosensor for the detection of corresponding bacteriophage. The biosensors showed excellent sensitivity with a lower detection limit of 10(-3) plaque forming unit (PFU) in 10 mM phosphate buffer, wide dynamic range and excellent selectivity. The immunosensors were successfully applied for the detection of phages in spiked untreated urban runoff water samples. The results show the potential of these sensors in health care, environmental monitoring, food safety and homeland security for sensitive, specific, rapid, and affordable detection of bioagents/pathogens.

Single conducting polymer nanowire chemiresistive label-free immunosensor for cancer biomarker.

A simple and cost-effective, all-electrochemical method to fabricate and assemble single conducting polymer nanowire based biosensors was developed. Polypyrrole (Ppy) nanowires were synthesized by electrochemical polymerization using an alumina template. The single-nanowire chemoresistive sensor device was assembled using ac dielectrophoretic alignment followed by maskless anchoring on a pair of gold electrodes separated by 3 microm. To establish an efficient covalent surface biofunctionalization route, glutaraldehyde (GA) and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) chemistries were compared. EDC was established to be the most effective chemistry and was used to surface-functionalize a single Ppy nanowire with cancer antigen (CA 125) antibody to fabricate a nanoimmunosensor for CA 125 biomarker detection and quantification. The immunosensor had excellent sensitivity with a lower detection limit of 1 U/mL CA 125 and dynamic range up to 1000 U/mL in 10 mM phosphate buffer. Furthermore, there was no loss of performance upon exposure to CA 125 in spiked human blood plasma. This demonstrates the clinical importance of these sensors for cancer marker detection with cost benefits and great portability for diagnosis of patients at the point of care.

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.

Development of an alginate hydrogel to deliver aqueous bait for pest ant management.

BACKGROUND: Insecticide sprays used for ant control cause environmental contamination. Liquid bait is a safe and effective alternative, but it requires bait stations to dispense the toxicant. We developed a biodegradable hydrogel to deliver liquid bait obviating the need for bait stations. RESULTS: Alginate hydrogel beads with preferred rigidity and maximum hydration in 25% sucrose solution were engineered by optimizing a crosslinking process. The moisture content of the substrate on which the beads were placed and the relative atmospheric humidity significantly influenced water loss dynamics of the hydrated hydrogel beads. Laboratory choice studies indicated that hydrated hydrogel beads had reduced palatability to foraging ants when they lost ≥50% water. An enzyme-linked immunosorbent assay (ELISA) indicated that the insecticide thiamethoxam added to sucrose solution was absorbed into the hydrogel beads. Hydrogel beads conditioned in sucrose solution with 1 mg L-1 thiamethoxam provided complete control of all castes of Argentine ant Linepithema humile (Mayr) colony by 14 days post treatment in the laboratory trial and provided a 79% reduction in ant activity after 8 weeks in the field trial. CONCLUSION: Alginate hydrogel beads provided an effective delivery system for liquid baits laced with low concentrations of insecticide to control Argentine ants. © 2017 Society of Chemical Industry.

A miniature chemiresistor sensor for carbon dioxide.

A carpet-like nanostructure of polyaniline (PANI) nanothin film functionalized with poly(ethyleneimine), PEI, was used as a miniature chemiresistor sensor for detection of CO2 at room temperature. Good sensing performance was observed upon exposing the PEI-PANI device to 50-5000 ppm CO2 in presence of humidity with negligible interference from ammonia, carbon monoxide, methane and nitrogen dioxide. The sensing mechanism relied on acid-base reaction, CO2 dissolution and amine-catalyzed hydration that yielded carbamates and carbonic acid for a subsequent pH detection. The sensing device showed reliable results in detecting an unknown concentration of CO2 in air.

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.

Hexavalent chromium removal mechanism using conducting polymers.

We report detoxification of Cr(VI) into Cr(III) using electrochemically synthesized polyaniline (PANI), polypyrrole (PPY), PANI nanowires (PANI-NW) and palladium-decorated PANI (PANI-Pd) thin films. Percent Cr(VI) reduction was found to be decreased with an increase in pH from 1.8 to 6.8 and with initial Cr(VI) concentration ranging from 2.5 to 10mg/L. Efficacy of PANI increased at higher temp of 37 °C as compared to 30 °C. PANI-Pd was found to be most effective for all three initial Cr(VI) concentrations at pH 1.8. However, efficacy of PANI-Pd was significantly reduced at higher pHs of 5 and 6.8. Efficacy of PANI and PANI-NW was found to nearly the same. However, there was a significant reduction in effectiveness of PANI-NW at 10mg/L of Cr(VI) at all the three pHs studied, which could be attributed to degradation of PANI-NW by higher initial Cr(VI) concentration. PPY and PANI-NW were found to be highly sensitive with respect to pH and Cr(VI) initial concentration. Chromium speciation on PANI film was carried out by total chromium analysis and XPS, which revealed Cr(III) formation and its subsequent adsorption on the polymer. PANI-Pd and PANI are recommended for future sensor applications for chromium detection at low pH.

Conducting polymer nanowires-based label-free biosensors.

Label-free sensing technologies have recently attracted a great deal of interest for sensitive, rapid and facile analysis for applications in health care, environmental monitoring, food safety and homeland security. One-dimensional (1-D) nanostructures such as nanowires, configured as field-effect transistors (FETs)/chemiresistors that change conductance upon binding of charged macromolecules to receptors linked to the device surfaces are extremely attractive for label-free biosensors. Herein, we review recent advances in label-free biosensors based on conducting polymer nanowires based FET/chemiresistor. Specifically, we address the fabrication, functionalization, assembly/alignment and sensing applications of FET/chemiresistor based on these nanomaterials. The advantages and disadvantages of various fabrication, functionalization, and assembling procedures of these nanosensors are reviewed and discussed.

Label-free, chemiresistor immunosensor for stress biomarker cortisol in saliva.

Salivary cortisol is commonly used as a bioindicator of the psychobiologic response to environmental and psychological stressors. Current analytical approaches rely on immunoassays performed at distant, centralized laboratories and involve an elaborate specimen collection-processing-transportation-storage-analysis-reporting cycle. To facilitate point-of-use measurement of salivary cortisol levels, we describe the development and proof-of-concept testing of an ultrasensitive, label-free immunosensor based on a single-walled, carbon nanotube-based chemiresistive transducer. Carbon nanotubes were functionalized with a cortisol analog [cortisol-3-CMO-NHS ester] and a monoclonal anti-cortisol antibody was ligated to this receptor. Addition of phosphate buffer as well as artificial saliva spiked with varying cortisol concentrations displaced the anti-cortisol antibody producing corresponding decreases in the resistance/conductance of the nanotube-biomolecule hybrid. The immunosensor demonstrated an ultralow detection limit of 1 pg/ml and excellent binding selectivity for cortisol even in the presence of structurally similar steroids such as 21-hydroprogesterone. The nanotube immunosensor offers attractive prospects for the development of highly sensitive biosensor for rapid, label-free measurement of salivary cortisol in a variety of clinical and research settings.

Conducting polymer nanowire-based chemiresistive biosensor for the detection of bacterial spores.

Polypyrrole nanowires (Ppy) were assembled onto microfabricated gold interdigitated microelectrodes, to construct a chemiresistive biosensor for the detection of Bacillus globigii, used as simulant of the threatening bioterrorism agent B. anthracis. The fabricated biosensor showed good linear correlation (r(2)=0.992) for low spore concentrations ranging from 1 to 100 CFU (colony forming units)/mL, a concentration that could be used in a bioterrorism attack, with a response time of 30 min, after which the sensor was saturated. The performance of the biosensor was also assessed in the absence of anti-B. globigii antibodies and in the presence of non-target bacterial cells (Escherichia coli) showing no significant non-specific interactions. We believe that Ppy nanowires are a good platform for the detection and also quantification of large molecules and biocomponents even at low concentrations.

Label-free detection of cupric ions and histidine-tagged proteins using single poly(pyrrole)-NTA chelator conducting polymer nanotube chemiresistive sensor.

Novel chemical and biological sensors based on a single poly(pyrrole)-NTA chelator nanotube for sensitive, selective, rapid and real-time detection of histidine-tagged protein and cupric ions are reported. NTA groups on the nanotube surface provided a simple mechanism for metal ion sensing via the high-affinity interaction between NTA and the subsequent detection of histidine-tagged protein through the coordination with metal chelated nanotube. Poly(pyrrole)-NTA chelator nanotubes of 190 nm outside diameter, 35 nm wall thickness and 30 microm long were synthesized by electrochemical polymerization of pyrrole-NTA inside a 200 nm diameter alumina template and assembled as a chemoresistive device by bottom-up contact geometry on a pair of parallel gold electrodes with a gap distance of 3 microm. The chemoresistive sensors based on single poly(pyrrole)-NTA chelator nanotube exhibited detection as low as one-hundredth attomolar (0.6 ppt) cupric ions and 1 ng/ml of penta-histidine tagged syntaxin protein.

Biomolecules-carbon nanotubes doped conducting polymer nanocomposites and their sensor application.

A simple method for preparing bio-functionalized soluble single-walled carbon nanotubes (SWNTs) is described. Different proteins such as bovine serum albumin (BSA), cytochrome c and horseradish peroxidase (HRP) were used to solubilize low functionality SWNTs in water aided by sonication. The unbound proteins were removed by column chromatography and the SWNT-protein conjugate was used as the sole anionic dopant in electropolymerization of polypyrrole from polymerization solution at pH above the isoelectric point of the protein to provide a negative charge. The morphology of the polypyrrole with SWNT-protein dopant was found to be three-dimensional and fibrous with wide open interlocking pores in contrast to smooth and cauliflower-like for chloride doped polypyrrole. Enhanced sensor performance was demonstrated for hydrogen peroxide detection on polypyrrole/SWCNT-HRP nanocomposites modified electrode. Such nanocomposites can be potentially applied for other biosensor and bio-fuel cell applications.

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.

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.