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The aggregation of gold nanoparticles (AuNPs) is known to induce an enhancement of localized surface plasmon resonance due to the coupling of plasmonic fields of adjacent nanoparticles. Here we show that AuNPs aggregation also causes a significant enhancement of chemiluminescence in the presence of luminophores. The phenomenon is used to introduce a rapid and sensitive DNA detection method that does not require amplification. DNA probes conjugated to AuNPs were used to detect a DNA target sequence specific to the fungus Ceratocystis fagacearum, causal agent of oak wilt. The hybridization of the DNA target with the DNA probes results in instantaneous aggregation of AuNPs into nanoballs, leading to a significant enhancement of luminol chemiluminescence. The enhancement reveals a linear correlation (R 2  = 0.98) to the target DNA concentration, with a limit of detection down to 260 fM (260 × 10 -15  M), two orders of magnitude higher than the performance obtained with plasmonic colorimetry and absorption spectrometry of single gold nanoparticles. Furthermore, the detection can be performed within 22 min using only a portable luminometer.

Reduced graphene oxide (RGO) has recently gained considerable attention for use in electrochemical biosensing applications due to its outstanding conducting properties and large surface area. This report presents a novel microfluidic chip integrated with an RGO-based electrochemical immunosensor for label-free detection of an influenza virus, H1N1. Three microelectrodes were fabricated on a glass substrate using the photolithographic technique, and the working electrode was functionalized using RGO and monoclonal antibodies specific to the virus. These chips were integrated with polydimethylsiloxane microchannels. Structural and morphological characterizations were performed using X-ray photoelectron spectroscopy and scanning electron microscopy. Electrochemical studies revealed good selectivity and an enhanced detection limit of 0.5 PFU mL -1 , where the chronoamperometric current increased linearly with H1N1 virus concentration within the range of 1 to 10 4  PFU mL -1 (R 2  = 0.99). This microfluidic immunosensor can provide a promising platform for effective detection of biomolecules using minute samples.

Osteopontin (OPN) is involved in almost all steps of cancer development, and it is being investigated as a potential biomarker for a diagnosis and prognosis of prostate cancer. Here, we report a label-free, highly sensitive and transparent immunosensor based on single-walled carbon nanotubes (SWCNTs) for detection of OPN. A high density of COOH functionalized SWCNTs was deposited between two gold/indium tin oxide electrodes on a glass substrate by dielectrophoresis. Monoclonal antibodies specific to OPN were covalently immobilized on the SWCNTs. Relative resistance change of the immunosensors was measured as the concentration of OPN in phosphate buffer saline (PBS) and human serum was varied from 1 pg mL(-1) to 1 μg mL(-1) for different channel lengths of 2, 5, and 10 μm, showing a highly linear and reproducible behavior (R(2)>97%). These immunosensors were also specific to OPN against another test protein, bovine serum albumin, PBS and human serum, showing that a limit of detection for OPN was 0.3 pg mL(-1). This highly sensitive and transparent immunosensor has a great potential as a simple point-of-care test kit for various protein biomarkers., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Reusability of a biosensor has recently received considerable attention, and it is closely related with the effective desorption of probe molecules. We present a novel mechanical desorption technique to reuse biosensors by using periodic jets of carbon dioxide (CO2) aerosols (a mixture of solid and gaseous CO2), and demonstrate its feasibility by removing physically adsorbed and covalently bonded fluorescent proteins i.e., Escherichia coli fluorescein isothiocyanate antibody and bovine serum albumin (E. coli FITC-Ab and FITC-BSA) from silicon chips. The proteins on the chip surfaces were measured by fluorescent images before and after applying the aerosols. The removal efficiency of the aerosol treatment was measured for various concentrations (1-20 μg mL(-1)) of E. coli FITC-Ab and FITC-BSA with two different removal cycles (5 and 11 cycles; each cycle: 8s). We observed high removal efficiencies (>93.5% for physically adsorbed Ab and >84.6% for covalently bonded Ab) at 11 cycle aerosol treatment. This CO2 aerosol treatment did not undermine re-functionalization, which was confirmed by the fluorescent images of FITC-Abs for fresh and reused chips. Desorption of the immobilized layers was validated by Fourier transform infrared and X-ray photoelectron spectroscopic analyses. We also conducted an experiment on the regeneration of E. coli sensing chips using this aerosol treatment, and the chips were re-used 5 times successfully. This mechanical desorption technique is a highly effective and novel strategy for reusable biosensors., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The influenza virus has received extensive attention due to the recent H1N1 pandemics originating from swine. This study reports a label-free, highly sensitive, and selective electrical immunosensor for the detection of influenza virus H1N1 based on dielectrophoretically deposited single-walled carbon nanotubes (SWCNTs). COOH-functionalized SWCNTs were deposited on a self-assembled monolayer of polyelectrolyte polydiallyldimethyl-ammonium chloride (PDDA) between two gold electrodes by dielectrophoretic and electrostatic forces, which resulted in reproducible, uniform, aligned, and aggregation-free SWCNT channels (2-10 μm in length). Avidin was immobilized onto the PDDA-SWCNT channels, and viral antibodies were immobilized using biotin-avidin coupling. The resistance of the channels increased with the binding of the influenza viruses to the antibodies. These immunosensors showed linear behavior as the virus concentration was varied from 1 to 10(4) PFU ml(-1) along with a detection time of 30 min. The immunosensors with a 2 μm channel length detected 1 PFU ml(-1) of the influenza virus accurately (R(2) = 0.99) and selectively from MS2 bacteriophages. These immunosensors have the potential to become an important component of a point-of-care test kit that will enable a rapid clinical diagnosis.

Early diagnosis of gonococcal infections is important with regard to a patient's health and stage of infection. In this context, we report the development of an opa-gene-based electrochemical DNA biosensor for detection of Neisseria gonorrhoeae by monitoring redox peak of methylene blue indicator. The fabricated biosensor has been shown to be highly sensitive and specific when evaluated with complementary, non-complementary, and 1-base mismatch DNA sequences and polymerase chain reaction (PCR) amplified products (amplicons) of standard strain of N. gonorrhoeae (ATCC49226). The biosensor has been further evaluated using amplicons of known positive and negative clinical samples, and cut-off for positives has been determined using receiver operating characteristic curve. The sensitivity (SN), specificity (SP), positive predictive value, and negative predictive value of the biosensor have been found to be 96.2%, 88.2%, 92.6%, and 93.8%, respectively. We conclude that the combination of PCR amplification with electrochemical detection shows distinct advantage of high SN and increased SP for gonococcal detection., (© 2013.)

The nanocomposite based on polyaniline (PANI)-iron oxide nanoparticles (nFe(3)O(4)) and multi walled carbon-nanotubes (CNT) has been fabricated onto indium tin oxide (ITO) coated glass plate via facile electrochemical synthesis of polyaniline in presence of nFe(3)O(4) (~20 nm) and CNT (20-80 nm in diameter). The results of transmission electron microscopic studies show evidence of coating of PANI and nFe(3)O(4) onto the CNT. The PANI-nFe(3)O(4)-CNT/ITO nanoelectrode has been characterized by Fourier transform infrared spectroscopy, X-ray diffraction and scanning electron microscopy studies. The biotinylated nucleic acid probe sequence consisting of 20 bases has been immobilized onto PANI-nFe(3)O(4)-CNT/ITO nanoelectrode using biotin-avidin coupling. It is shown that the PANI-nFe(3)O(4)-CNT platform based biosensor can be used to specifically detect bacteria (N. gonorrhoeae) at minute concentration as low as (1×10(-19) M) indicating high sensitivity within 45 s of hybridization time at 298 K by differential pulse voltammetry using methylene blue as electroactive indicator. This bacterial sensor has also been tested with 4 positive and 4 negative PCR amplicons of gonorrhoea affected patient samples. The results of these studies have implications towards the fabrication of a handheld device for Neisseria gonorrhoeae detection that may perhaps result in a decrease in the human immunodeficiency virus infections., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Electrochemically fabricated nano-composite film of chitosan (CH)-iron oxide (Fe(3)O(4)) has been used to detect gonorrhoea, a sexually transmitted disease (STD) via immobilization of biotinylated probe DNA (BDNA) using avidin-biotin coupling for rapid and specific (mismatch-discriminating) DNA hybridization. The presence of Fe(3)O(4) nanoparticles (∼18nm) increases the electro-active surface area of the nano-biocomposite that provides desirable environment for loading of DNA with better conformation leading to increased electron transfer kinetics between the medium and electrode. The differential pulse voltammetric (DPV) studies have been conducted using BDNA/avidin/CH-Fe(3)O(4)/ITO electrode owing to the reduction of the methylene blue (MB) indicator and investigate electron transfer between MB moieties and electrode for one and two-bases mismatch. This STD biosensor is found to have a detection limit (1 × 10(-15)M) and a wide dynamic range (from 1 × 10(-16)M to 1 × 10(-6)M) using the complementary target DNA. In addition, the sensing system can be utilized to accurately discriminate complementary sequence from mismatch sequences., (Copyright © 2010 Elsevier B.V. All rights reserved.)

A sequence-specific electrochemical sexually transmitted disease (STD) sensor based on self-assembled monolayer of thiolated DNA probe specific to target opa gene for detection of Gonorrhoea, a sexually transmitted disease has been fabricated. 6-Mercapto-1-hexanol (MCH) has been used as a blocking agent to facilitate oligos "stand" up at the surface, a configuration favoring subsequent DNA hybridization and to repel non-specific adsorption of undesired DNA. The results of differential pulse voltammetric studies of this STD sensor reveal low detection limit (1.0 × 10(-18)M) and a wide dynamic range (from 1.0 × 10(-6)M to 0.5 × 10(-18)M) arising due to the stable hybridization using methylene blue as an electro-active DNA hybridization indicator. The experimental results with genomic DNA, clinical patient sample of Neisseria gonorrhoeae, culture of non-N. gonorrhoeae Neisseria species (NgNS) and gram negative bacteria indicate that the fabricated sensor is specific to this STD., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Polyaniline/carbon nanotubes composite (PANI-CNT) electrochemically deposited onto indium-tin-oxide (ITO) coated glass plate has been utilized for Neisseria gonorrhoeae detection by immobilizing 5'-amino-labeled Neisseria gonorrhoeae probe (aDNA) using glutaraldehyde as a cross-linker. PANI-CNT/ITO and aDNA-Glu-PANI-CNT/ITO electrodes have been characterized using scanning electron microscopy (SEM), Fourier Transform Infrared (FT-IR) spectroscopy, cyclic voltammetry (CV), and differential pulse voltammetry (DPV). This bioelectrode can be used to detect N. gonorrhoeae using methylene blue (MB) as redox indicator with response time of 60 s and stability of about 75 days when stored under refrigerated conditions. DPV studies reveal that this bioelectrode can detect complementary DNA concentration from 1 x 10(-6) M to 1 x 10(-17) M with detection limit of 1.2 x 10(-17) M. Further, this bioelectrode (aDNA-Glu-PANI-CNT/ITO) exhibits specificity toward N. gonorrhoeae species and shows negative response with non-Neisseria gonorrhoeae Neisseria species (NgNS) and other gram negative bacteria (GNB).

STD (sexually transmitted disease, Gonorrhoea) sensor based on nucleic acid probe (from Opa, a multi-copy gene of Neisseria gonorrhoeae) functionalized nanostructured-polyaniline coated onto indium-tin-oxide-coated glass plate has been fabricated using avidin-biotin as cross-linking agent. This DNA functionalized electrode can specifically detect upto 0.5 x 10(-15)M of complementary target within 60s of hybridization time at 25 degrees C by differential pulse voltammetry (DPV) using methylene blue as electro-active DNA hybridization indicator. This highly sensitive and specific nucleic acid functionalized nanostructured-polyaniline electrode can distinguish presence of N. gonorrhoeae from Neisseria meningitidis and Escherichia coli culture and spiked samples from the urethral swabs of the patients.