Impedimetric cell-based biosensor for real-time monitoring of cytopathic effects induced by dengue viruses.

We describe an impedimetric cell-based biosensor constructed from poly-l-lysine (PLL)-modified screen-printed carbon electrode for real-time monitoring of dengue virus (DENV) infection of surface-immobilized baby hamster kidney (BHK-21) fibroblast cells. Cytopathic effects (CPE) induced by DENV-2 New Guinea C strain (including degenerative morphological changes, detachment, membrane degradation and death of host cells), were reflected by drastic decrease in impedance signal response detected as early as ~30 hours post-infection (hpi). In contrast, distinct CPE by conventional microscopy was evident only at ~72 hpi at the corresponding multiplicity of infection (MOI) of 10. A parameter that describes the kinetics of cytopathogenesis, CIT50, which refers to the time taken for 50% reduction in impedance signal response, revealed an inverse linear relationship with virus titer and MOI. CIT50 values were also delayed by 31.5h for each order of magnitude decrease in MOI. Therefore, based on the analysis of CIT50, the virus titer of a given sample can be determined from the measured impedance signal response. Furthermore, consistent impedance results were also obtained with clinical isolates of the four DENV serotypes verified by RT-PCR and cycle sequencing. This impedimetric cell-based biosensor represents a label-free and continuous approach for the dynamic measurement of cellular responses toward DENV infection, and for detecting the presence of infectious viral particles.

Novel biosensing methodologies for ultrasensitive detection of viruses.

Various infectious diseases caused by the spread of viruses create adverse implications on global biosecurity. Increasing demands for virus surveillance and effective control of the spread of diseases reveal the need for rapid and sensitive virus diagnostic devices. Due to the remarkable sensitivity and specificity of biosensors, they appear as a potential and promising tool for accurate and quantitative detection of viruses. Furthermore, recent advancements in transduction systems, nanotechnology and genetic engineering offer various strategies to improve the detection performance of biosensors. This review presents an overview of the current states of novel biosensing methodologies for the ultrasensitive detection of viruses with highly promising applications for future disease diagnosis. Additionally, a brief summary of the recent state-of-the-art virus diagnostic molecular technologies is included.

Impedimetric DNA biosensor based on a nanoporous alumina membrane for the detection of the specific oligonucleotide sequence of dengue virus.

A novel and integrated membrane sensing platform for DNA detection is developed based on an anodic aluminum oxide (AAO) membrane. Platinum electrodes (~50-100 nm thick) are coated directly on both sides of the alumina membrane to eliminate the solution resistance outside the nanopores. The electrochemical impedance technique is employed to monitor the impedance changes within the nanopores upon DNA binding. Pore resistance (Rp) linearly increases in response towards the increasing concentration of the target DNA in the range of 1 × 10⁻¹² to 1 × 10⁻6 M. Moreover, the biosensor selectively differentiates the complementary sequence from single base mismatched (MM-1) strands and non-complementary strands. This study reveals a simple, selective and sensitive method to fabricate a label-free DNA biosensor.

Impedimetric microbial sensor for real-time monitoring of phage infection of Escherichia coli.

We describe an impedimetric microbial sensor for real-time monitoring of the non-lytic M13 bacteriophage infection of Escherichia coli cells using a gold electrode covalently grafted with a monolayer of lipopolysaccharide specific antibody. After infection, damage to the lipopolysaccharide layer on the outer membrane of E. coli causes changes to its surface charge and morphology, resulting in the aggregation of redox probe, Fe(CN)6(3-/4-) at the electrode surface and thereby increases its electron-transfer rate. This consequent decrease of electron-transfer resistance in the presence of bacteriophage can be easily monitored using Faradaic impedance spectroscopy. Non-lytic bacterium-phage interaction which is hardly observable using conventional microscopic methods is detected within 3h using this impedimetric microbial sensor which demonstrates its excellent performance in terms of analysis time, ease and reduced reliance on labeling steps during in-situ monitoring of the phage infection process.

Fuel cell virus sensor using virus capture within antibody-coated nanochannels.

We report a unique fuel cell sensor system for the first time direct detection of unlabeled virus particles based on the formation of antibody-virus complexes within the sensor's membrane nanochannels. This strategy exploits the change in the membrane resistance of the powered system, comprising a Prussian blue nanotubes (PB-nt) membrane cathode and a platinum mesh anode. The method reports an impressive shortest response time of ∼5 min toward the specific virus target, at low concentration values of 3-45 plaque-forming units per milliliter (pfu mL(-1)) with detection limit of 0.04 pfu mL(-1), comparable to state-of-the-art polymerase chain reaction (PCR)-based methods. The sensor can clearly differentiate dengue virus serotype 2 from serotype 3. When filled with Nafion perfluorinated resin, the PB-nt membrane demonstrates powerful utilization as a stand-alone fuel cell based virus sensor, and thus offers the outstanding promise of a sustainable, low-cost, and rapid low-power virus detection tool.

Electrochemical nanoporous alumina membrane-based label-free DNA biosensor for the detection of Legionella sp.

An electrochemical nanoporous alumina membrane-based label free DNA biosensor is developed using 5'-aminated DNA probes immobilized into the nanochannels of alumina. Alumina nanoporous membrane-like structure is carved over platinum wire electrode of 76 µm diameter dimension by electrochemical anodization. The hybridization of complementary target DNA with probe DNA molecules attached inside the nanochannels influences the pore size and ionic conductivity. Electrochemical biosensing signal is derived from only redox species Fe(CN)(6)(4-) across single wire Pt electrode. The biosensors sensing mechanism relies on the monitoring of electrode's Faradaic current response toward redox species, Fe(CN)(6)(4-), which is sensitive toward the hybridization of complementary target with probe DNA immobilized into the alumina nanochannels. The biosensor demonstrates wide linear range over 7 orders of magnitude with ultrasensitive detection limit 3.1×10(-13) M for the quantification of ss 21 mer DNA sequence and selectively differentiates the complementary sequence from target sequences with single base mismatch (MM1) and triple bases mismatch (MM3) of different strain of Legionella sp. Its applicability is also challenged against real time Legionella pneumophila genomic DNA sample derived from the asymmetric PCR method.

Ultrasensitive cDNA detection of dengue virus RNA using electrochemical nanoporous membrane-based biosensor.

A nanoporous alumina membrane-based ultrasensitive DNA biosensor is constructed using 5'-aminated DNA probes immobilized onto the alumina channel walls. Alumina nanoporous membrane-like structure is carved over platinum wire electrode of 76 µm diameter dimension by electrochemical anodization. The hybridization of complementary target DNA with probe DNA molecules attached inside the pores influences the pore size and ionic conductivity. The biosensor demonstrates linear range over 6 order of magnitude with ultrasensitive detection limit of 9.55×10(-12) M for the quantification of ss-31 mer DNA sequence. Its applicability is challenged against real time cDNA PCR sample of dengue virus serotype1 derived from asymmetric PCR. Excellent specificity down to one nucleotide mismatch in target DNA sample of DENV3 is also demonstrated.

Electrochemical impedance spectroscopy characterization of nanoporous alumina dengue virus biosensor.

The Faradaic electrochemical impedance technique is employed to characterize the impedance change of a nanoporous alumina biosensor in response towards the specific binding of dengue serotype 2 (Denv2) viral particles to its serotype 2-specific immunoglobulin G antibody within the thin alumina layer. The optimal equivalent circuit model that matches the impedimetric responses of the sensor describes three distinct regions: the electrolyte solution (R(s)), the porous alumina channels (including biomaterials) (Q(1), R(1)) and the conductive electrode substrate layer (Q(2), R(2)). Both channel resistance R(1) and capacitance Q(1) change in response to the increase of the Denv2 virus concentration. A linear relationship between R(1) and Denv2 concentration from 1 to 900 plaque forming unit per mL (pfu mL(-1)) can be derived using Langmuir-Freundlich isotherm model. At 1pfu mL(-1) Denv2 concentration, R(1) can be distinguished from that of the cell culture control sample. Moreover, Q(1) doubles when Denv2 is added but remains unchanged in the presence of two other non-specific viruses - West Nile virus and Chikungunya virus indicates biosensor specificity can be quantitatively measured using channel capacitance.

Development of an electrochemical membrane-based nanobiosensor for ultrasensitive detection of dengue virus.

A sensitive membrane-based electrochemical nanobiosensor is developed for the detection of dengue type 2 virus (DENV-2) using nanoporous alumina-modified platinum electrode. Its sensing mechanism relies on the monitoring of electrode's Faradaic current response toward redox probe, ferrocenemethanol, which is sensitive toward the formation of immune complexes within the alumina nanochannels. Anti-DENV-2 monoclonal antibody (clone 3H5, isotype IgG) is used as the biorecognition element in this work. The stepwise additions of antibody, bovine serum albumin (BSA) and DENV-2 are characterized by differential pulse voltammetry (DPV). A low detection limit of 1 pfu mL(-1) with linear range from 1 to 10(3) pfu mL(-1) (R(2)=0.98) can be achieved by the nanobiosensor. The nanobiosensor is selective toward DENV-2 with insignificant cross reaction with non-specific viruses, Chikungunya virus, West Nile virus and dengue type 3 virus (DENV-3). Relative standard deviation (RSD) for triplicate analysis of 5.9% indicates an acceptable level of reproducibility. The first direct quantitation of DENV-2 concentration in whole mosquito vector is demonstrated using this electrochemical nanobiosensor.

Electrochemically amplified molecular beacon biosensor for ultrasensitive DNA sequence-specific detection of Legionella sp.

An electrochemically amplified molecular beacon (EAMB) biosensor is constructed using thiolated hairpin DNA-ferrocene probes on gold electrode. The switching from "on" to "off" states of individual probes in the presence of complementary DNA target influences the electrode potential, besides the current, owing to changes in surface density of the electroactive hairpin DNA-ferrocene probes. The EAMB biosensor demonstrates linear range over 8 orders of magnitude with ultrasensitive detection limit of 2.3 × 10(-14)M for the quantification of a 21-mer DNA sequence. Its applicability is tested against PCR amplicons derived from genomic DNA of live Legionella pneumophila. Excellent specificity down to one and three nucleotides mismatches in another strain of L. pneumophila and a different bacterium species, respectively, is demonstrated.

Membrane-based electrochemical nanobiosensor for Escherichia coli detection and analysis of cells viability.

A sensitive and selective membrane-based electrochemical nanobiosensor is developed for specific quantitative label-free detection of Escherichia coli (E. coli) cells and analysis of viable but nonculturable (VBNC) E. coli cells which remain mostly undetected using current methods. The sensing mechanism relies on the blocking of nanochannels of a nanoporous alumina-membrane modified electrode, upon the formation of immune complexes at the nanoporous membrane. The resulting obstacle to diffusive mass transfer of a redox probe in the analysis solution to the underlying platinum electrode reduces the Faradaic signal response of the biosensor, measured using cyclic voltammetry. Antibody loading under conditions of varying antibody concentrations and pHs are optimized. The biosensor gives a low detection limit of 22 cfu mL(-1) (R(2) = 0.999) over a wide linear working range of 10 to 10(6) cfu mL(-1). It is specific toward E. coli with minimal cross-reactivity to two other pathogenic bacteria (commonly found in waters). Relative standard deviation (RSD) for triplicate measurements of 2.5% indicates reasonably useful level of reproducibility. Differentiation of live, VBNC, and dead cells are carried out after the cell capture and quantitation step, by simple monitoring of the cells' enzyme activity using the same redox probe in the analysis solution, in the presence of glucose.

Current and nano-diagnostic tools for dengue infection.

Dengue is one of the infectious diseases that is widespread over global regions with yearly occurrence of epidemics and could be deadly in some cases. Thus the developments of rapid and specific diagnostic tools which can achieve early detection of dengue infection for disease control during epidemic situations and before complications occur are deemed highly desirable. This paper describes the current and advanced methods for diagnosis of dengue infection and discusses the advantages and disadvantages of these methods in terms of their analytical performances and clinical applicabilities. The current methods discussed herein include enzyme-linked immunosorbent assay and reverse transcriptase polymerase chain reaction. In addition, recent instrumental methods such as quartz crystal microbalance, surface plasmon resonance, photonic crystal and electrochemical impedance spectroscopy have shown promising results. Interesting developments in detection of dengue infection using nanosized materials including liposomes, nanowires and nanopores, coupled to conventional fluorescence, potentiometry and voltammetry methods are also described and could possibly point the way forward for the development of inexpensive diagnostic tools for use at point-of-care and in events of epidemic scale.

Nanoarray membrane sensor based on a multilayer design for sensing of water pollutants.

A ubiquitous electrochemical sensor which can detect pollutants in nonconducting aqueous solutions is prepared using a triple layer design, comprising a polyelectrolyte entrapped within micrometer-length nanochannels and sandwiched between two nanometer-thick electrode layers. Replacement of the polyelectrolyte with an enzyme-polyelectrolyte mixture within the nanochannels confers excellent biosensing characteristics. Its superior analytical performance of quantitating copper ions and formaldehyde at trace levels without additional sample treatment steps is demonstrated in freshwater samples derived from a local reservoir.

Membrane-based electrochemical nanobiosensor for the detection of virus.

A membrane-based electrochemical nanobiosensor sensitive toward whole viral particles is fabricated by forming a submicrometer thick nanoporous alumina membrane over a platinum disk electrode. Antibody probe molecules are physically adsorbed onto the walls of the membrane nanochannels. The sensing signal is based on the monitoring of the electrode's Faradaic current response toward ferrocenemethanol, which is extremely sensitive to the formation of immunocomplex within the nanoporous membrane. This nanobiosensor is demonstrated for the sensing of West Nile virus protein domain III (WNV-DIII) and the inactivated West Nile viral particle, using anti-WNV-DIII immunoglobulin M (IgM) as the biorecognition probe. The detection of the viral protein and the particle are logarithmically linear up to 53 pg mL(-1) (R(2) = 0.99) and 50 viral particles per 100 mL (R(2) = 0.93) in pH 7, with extremely low detection limits of 4 pg mL(-1) and ca. 2 viral particles per 100 mL, comparable to sensitivities of polymerase chain reaction techniques. The relative standard deviation (RSD) of whole viral particle detection in whole blood serum is 6.9%. In addition, the simple nanobiosensor construction procedure, minimal sample preparation, and short detection time of 30 min are highly attractive properties and demonstrate that the detection of a wide range of proteins and viruses can be achieved.

Development of a biomimetic nanoporous membrane for the selective transport of charged proteins.

We report the use of a micrometer-thick platinum-coated nanoporous membrane for the separation of differently charged proteins. A high field strength of about 25 kV m(-1) was applied, using very low transmembrane potentials of +/-1.5 V between the platinum-coated membranes. The system mimics the cell membrane function of facilitated transport for specific solutes. The selectivity for Lys:BSA:Mb in a mixed protein solution could be tuned readily between the flux ratios of 2:2:1 and 96:1:12 respectively, by simple variation of the transmembrane potentials from +1.5 V to -1.5 V. The experimental fluxes agreed closely with calculated fluxes derived from a simple electrophoresis-potential shielding model at favourable transmembrane potentials.

Recent advances in analytical chemistry--a material approach.

Advancements of materials research have profound direct impacts on developments in analytical chemistry and may hold the key to improvement of existing or new techniques at present times and near future. Applications of materials in analytical chemistry are reviewed, with focus on sensors, separations and extraction techniques. This review aims to survey examples of interesting works carried out in the last five years over a broad spectrum of materials classified as hybrids, nanomaterials and biomolecular materials.

Electrochemical polymerization of aniline monomers infiltrated into well-ordered truncated eggshell structures of polyelectrolyte multilayers.

The use of nanosphere lithography to construct two-dimensional arrays of polystyrene (PS) particles coated with multilayered polyelectrolyte (PE) shells and truncated eggshell structures composed of PE thin layers is reported. The truncated eggshell PE structures were produced by extraction of the PS particle cores with toluene. The core-extraction process ruptures the apex of the PE coating and causes a slight expansion of the PE thin layers. Aniline hydrochloride was infiltrated into the PE shells and subsequently electropolymerized to yield an array of a composite containing polyaniline (PAni) and PE thin shells. Voltammetric, quartz crystal microbalance, and reflectance Fourier transform infrared spectroscopic measurements indicate that aniline monomers were confined within the thin PE shells and the electropolymerization occurred in the interior of the PE shell. The PE thickness governs the amount of infiltrated monomer and the ultimate loading of the PAni in the truncated eggshell structure. Surface-structure imaging by atomic force microscopy and scanning electron microscopy, carried out after each step of the fabrication process, shows the influence of the PE thickness on the organization and dimensions of the arrays. Thus, the PE thin shells composed of different layers can function as nanometer-sized vessels for the entrapment of charged species for further construction of composite materials and surface modifications. This approach affords a new avenue for the synthesis of new materials that combine the unique properties of conductive polymers and the controllability of template-directed surface reactions.

A method for the determination of enzyme mass loading on an electrode surface through radioisotope labelling.

A direct method has been developed for the quantitation of the amount of immobilised enzymes on biosensor surfaces. This quantity is of key importance in establishing the activity, kinetics and optimal immobilisation conditions in the construction of both amperometric and optical biosensors. Recombinant L-lactate dehydrogenase incorporating both a biosynthetically introduced radiolabel, 3H-leucine, and a hexahistidine peptide tag was immobilised on a poly(aniline) composite film and then quantitated by liquid scintillation counting. It was found that enzyme mass loading was proportional to the concentration of LDH in solution, and also depended on the morphology of the composite film. The LDH mass loading on the composite film doubled when a surface cysteine containing variant was used, possibly due to the covalent attachment of the cysteine to the diiminoquinoid rings of the poly(aniline).

The design of dehydrogenase enzymes for use in a biofuel cell: the role of genetically introduced peptide tags in enzyme immobilization on electrodes.

The immobilization of the mutants of L-lactate dehydrogenase (LDH) on poly(aniline) (PANi) composite films has been investigated. Mutants possessing peptide tags of varying charge and nucleophilicity were created to probe the nature of the interaction between the protein and PANi. These results are significant for the development of a 'generic' approach to the immobilization of enzymes and other proteins.

Immobilisation of enzymes on poly(aniline)-poly(anion) composite films. Preparation of bioanodes for biofuel cell applications.

Immobilisation of enzymes is important for applications such as biosensors or biofuel cells. A poly(histidine) tag had been introduced on the C terminus of a lactate dehydrogenase enzyme. This mutant enzyme was then immobilised onto poly(aniline) (PANi)-poly(anion) composite films, PANi-poly(vinylsulfonate) (PVS) or PANi-poly(acrylate) (PAA). The NADH produced by the immobilised enzyme in the presence of beta-nicotinamide adenine dinucleotide (NAD(+)) and lactate is oxidised at the poly(aniline)-coated electrode at 0.05 to 0.1 V vs. saturated calomel electrode (SCE) at 35 degrees C.