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.

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.

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.