Dual-modality microfluidic biosensor based on nanoengineered mesoporous graphene hydrogels.

A dual-modality microfluidic biosensor is fabricated using a mesoporous nanostructured cysteine-graphene hydrogel for the quantification of human cardiac myoglobin (cMb). In this device, the nanoengineered mesoporous l-cysteine-graphene (Cys-RGO) hydrogel performs the role of a dual-modality sensing electrode for the measurements conducted using differential pulse voltammetry and surface plasmon resonance (SPR) techniques. High surface reactivity, mesoporous structure and fast electron transfer combined with good reaction kinetics of the graphene hydrogel in this device indicate excellent performance for the detection of human cardiac myoglobin in serum samples. In electrochemical modality, this microfluidic chip exhibits a high sensitivity of 196.66 µA ng-1 mL cm-2 for a linear range of concentrations (0.004-1000 ng mL-1) with a low limit of detection (LOD) of 4 pg mL-1 while the SPR technique shows a LOD of 10 pg mL-1 for cMb monitoring in the range 0.01-1000 ng mL-1. The intra-assay coefficient of variation was less than 8% for standard samples and 9% for real serum samples, respectively. This Cys-RGO hydrogel-based microfluidic SPR chip allows real-time dynamic tracking of cMb molecules with a high association constant of 4.93 ± 0.2 × 105 M-1 s-1 and a dissociation constant of 1.37 ± 0.08 × 10-4 s-1, self-verification, reduced false readout, and improved detection reliability.

Fabrication of sensitive bioelectrode based on atomically thin CVD grown graphene for cancer biomarker detection.

Motivation behind the present work is to fabricate a cost effective and scalable biosensing platform for an easy and reliable detection of cancer biomarker Carcinoembryonic antigen (CEA). Here, we report the sensitive and selective detection of CEA using graphene based bio-sensing platform. Large sized (~ 2.5 × 1.0cm2), uniform, continuous, single and few layers graphene films have been grown on copper (Cu) substrate employing chemical vapor deposition (CVD) technique using hexane as a liquid precursor. Functional group has been created over Graphene/Cu substrate through π-π stacking of 1- pyrenebutanoic acid succinimidyl ester (PBSE). Further, to make the sensor specific to CEA, antibody of CEA (anti-CEA) has been covalently immobilized onto PBSE/Graphene/Cu electrode. Selective and sensitive detection of CEA is achieved by anti-CEA/PBSE/Graphene/Cu electrode through electrochemical impedance spectroscopy (EIS) measurements. Under optimal condition, the fabricated sensor shows linear response in the physiological range 1.0-25.0ngmL-1 (normal value ~ 5.0ngmL-1), revealing sensitivity 563.4Ωng-1mLcm-2 with a correlation coefficient of 0.996 and limit of detection (LOD) 0.23ngmL-1. In this way, one step electrode fabrication with high specific surface area provides a light weight, low cost, reliable and scalable novel biosensing platform for sensitive and selective detection of CEA. We believe that this bioelectrode equipped with specific recognition elements could be utilized for detection of other biomolecules too.

Microporous Nanocomposite Enabled Microfluidic Biochip for Cardiac Biomarker Detection.

This paper demonstrates an ultrasensitive microfluidic biochip nanoengineered with microporous manganese-reduced graphene oxide nanocomposite for detection of cardiac biomarker, namely human cardiac troponin I. In this device, the troponin sensitive microfluidic electrode consisted of a thin layer of manganese-reduced graphene oxide (Mn3O4-RGO) nanocomposite material. This nanocomposite thin layer was formed on surface of a patterned indium tin oxide substrate after modification with 3-aminopropyletriethoxysilane and was assembled with a polydimethylsiloxane-based microfluidic system. The nanoengineered microelectrode was functionalized with antibodies specific to cardiac troponin I. The uniformly distributed flower-shaped nanostructured manganese oxide (nMn3O4) onto RGO nanosheets offered large surface area for enhanced loading of antibody molecules and improved electrochemical reaction at the sensor surface. This microfluidic device showed an excellent sensitivity of log [87.58] kΩ/(ng mL-1)/cm2 for quantification of human cardiac troponin I (cTnI) molecules in a wide detection range of 0.008-20 ng/mL. This device was found to have high stability, high reproducibility, and minimal interference with other biomarkers cardiac troponin C and T, myoglobin, and B-type natriuretic peptide. These advantageous features of the Mn3O4-RGO nanocomposite, in conjunction with microfluidic integration, enabled a promising microfluidic biochip platform for point-of-care detection of cardiac troponin.

Recent advances in mycotoxins detection.

Mycotoxins contamination in both food and feed is inevitable. Mycotoxin toxicity in foodstuff can occur at very low concentrations necessitating early availability of sensitive and reliable methods for their detection. The present research thrust is towards the development of a user friendly biosensor for mycotoxin detection at both academic and industrial levels to replace conventional expensive chromatographic and ELISA techniques. This review critically analyzes the recent research trend towards the construction of immunosensor, aptasensor, enzymatic sensors and others for mycotoxin detection with a reference to label and label free methods, synthesis of new materials including nano dimension, and transuding techniques. Technological aspects in the development of biosensors for mycotoxin detection, current challenges and future prospects are also included to provide a overview and suggestions for future research directions.

A chitosan modified nickel oxide platform for biosensing applications.

We present a highly sensitive and selective electrochemical sandwich immunosensor (the analyte is "sandwiched" between two antibodies) based on chitosan (CH) modified nickel oxide (NiO) nanoparticles for the detection of Vibrio cholerae (Vc). The primary monoclonal antibodies specific to Vibrio cholerae (Ab-Vc) and bovine serum albumin (BSA) were co-immobilized on a CH-NiO surface deposited onto an indium tin oxide (ITO) coated glass electrode. The specific binding of Ab-Vc towards Vc was confirmed by interaction of secondary antibodies conjugated with protein [horse radish peroxidase (HRP)], with varying concentrations of hydrogen peroxide (H2O2), via electrochemical as well as optical techniques. The CH-NiO/ITO and Ab-Vc/CH-NiO/ITO electrodes have been characterized using X-ray diffraction, Fourier transform infrared spectroscopy, scanning electron microscopy, high-resolution transmission electron microscopy and electrochemical techniques. This immunoelectrode (BSA/Ab-Vc/CH-NiO/ITO) exhibits a detection range of 20-700 ng mL-1 with a sensitivity of 0.644 µA ng mL-1 cm-2 and a low detection range of 0.108 ng mL-1 to Vc concentration. Besides this, the electrochemical response of the sandwich immunosensor (HRP-Ab-Vc/Vc/BSA/Ab-Vc/CH-NiO/ITO) towards H2O2 concentration is found to be linear in the range of 10-50 mM with excellent sensitivity (2.95 mA mM-1 cm-2).

Graphene oxide-based biosensor for food toxin detection.

We report results of the studies relating to the fabrication of a highly sensitive label free biosensor based on graphene oxide (GO) platform for the detection of aflatoxin B1 (AFB1) which is most toxic and predominant food toxin, using electrochemical impedance spectroscopy. The structural and optical characterization of GO/Au and anti-AFB1/GO/Au has been done by electron microscopy, Raman, X-ray diffraction (XRD), UV-vis and electrochemical impedance spectroscopy (EIS). The impedimetric sensing response of immunoelectrode as a function of AFB1 concentration reveals wider linear detection range (0.5-5 ng/ml), high sensitivity (639 Ω ng(-1) ml), improved detection limit (0.23 ng ml(-1)) and good stability (5 weeks) for the label-free detection. Association constant (k a) for antigen-antibody interaction obtained as 0.46 ng ml(-1) indicates high affinity.

Protein-conjugated quantum dots interface: binding kinetics and label-free lipid detection.

We propose a label-free biosensor platform to investigate the binding kinetics using antigen-antibody interaction via electrochemical and surface plasmon resonance (SPR) techniques. The L-cysteine in situ capped cadmium sulfide (CdS; size < 7 nm) quantum dots (QDs) self-assembled on gold (Au) coated glass electrode have been covalently functionalized with apolipoprotein B-100 antibodies (AAB). This protein conjugated QDs-based electrode (AAB/CysCdS/Au) has been used to detect lipid (low density lipoprotein, LDL) biomolecules. The electrochemical impedimetric response of the AAB/CysCdS/Au biosensor shows higher sensitivity (32.8 kΩ µM(-1)/cm(2)) in the detection range, 5-120 mg/dL. Besides this, efforts have been made to investigate the kinetics of antigen-antibody interactions at the CysCdS surface. The label-free SPR response of AAB/CysCdS/Au biosensor exhibits highly specific interaction to protein (LDL) with association constant of 33.4 kM(-1) s(-1) indicating higher affinity toward LDL biomolecules and a dissociation constant of 0.896 ms(-1). The results of these studies prove the efficacy of the CysCdS-Au platform as a high throughput compact biosensing device for investigating biomolecular interactions.

A dual enzyme functionalized nanostructured thulium oxide based interface for biomedical application.

In this paper, we present results of the studies related to fabrication of a rare earth metal oxide based efficient biosensor using an interface based on hydrothermally prepared nanostructured thulium oxide (n-Tm2O3). A colloidal solution of prepared nanorods has been electrophoretically deposited (EPD) onto an indium-tin-oxide (ITO) glass substrate. The n-Tm2O3 nanorods are found to provide improved sensing characteristics to the electrode interface in terms of electroactive surface area, diffusion coefficient, charge transfer rate constant and electron transfer kinetics. The structural and morphological studies of n-Tm2O3 nanorods have been carried out by X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared (FTIR) spectroscopic techniques. This interfacial platform has been used for fabrication of a total cholesterol biosensor by immobilizing cholesterol esterase (ChEt) and cholesterol oxidase (ChOx) onto a Tm2O3 nanostructured surface. The results of response studies of the fabricated ChEt-ChOx/n-Tm2O3/ITO bioelectrode show a broad linear range of 8-400 mg dL(-1), detection limit of 19.78 mg (dL cm(-2))(-1), and high sensitivity of 0.9245 µA (mg per dL cm(-2))(-1) with a response time of 40 s. Further, this bioelectrode has been utilized for estimation of total cholesterol with negligible interference (3%) from analytes present in human serum samples. The utilization of this n-Tm2O3 modified electrode for enzyme-based biosensor analysis offers an efficient strategy and a novel interface for application of the rare earth metal oxide materials in the field of electrochemical sensors and bioelectronic devices.

Tin oxide quantum dot based DNA sensor for pathogen detection.

We report the application of nano crystalline tin oxide quantum dots (SnO2-QDs) for electrochemical detection of Vibrio cholerae based on DNA hybridization technique. SnO2-QDs (- 1-5 nm) have been synthesized by laser ablation technique in liquid (LAL) and electrophoretically deposited onto hydrolyzed surface of indium tin oxide (ITO) coated glass electrode. A single stranded oligonucleotide probe (23 bases) have been designed form the virulent gene sequence of V. cholerae and has been immobilized onto SnO2-QDs/ITO surface for the fabrication of ssDNA/SnO2-QDs/ITO bioelectrode and these bioelectrode have been further used for DNA hybridization (dsDNA/SnO2-QDs/ITO). The electrochemical response studies have been carried out with different concentration genomic DNA (100-500 ng/microL), which indicated that SnO2 provides an effective surface to bind with the phosphate group of DNA, thus resulting in an enhanced electron transport. The hybridized electrode exhibits linear response with regression coefficient (R) 0.974, high sensitivity 35.20 nA/ng/cm2, low detection limit (31.5 ng/microL), faster response time (3 s) and high stability of 0-120 days when stored under refrigerated conditions.

Molecularly imprinted polyaniline-polyvinyl sulphonic acid composite based sensor for para-nitrophenol detection.

We report results of the studies relating to the fabrication and characterization of a conducting polymer based molecularly imprinted para-nitrophenol (PNP) sensor. A water pollutant, para-nitrophenol is electrochemically imprinted with polyvinyl sulphonic acid (PVSA) doped polyaniline onto indium tin oxide (ITO) glass substrate. This PNP imprinted electrode (PNPI-PANI-PVSA/ITO) prepared via chronopotentiometric polymerization and over-oxidation is characterized by Fourier transform infra-red spectroscopy (FT-IR), UV-visible (UV-vis) spectroscopy, contact angle (CA), scanning electron microscopy (SEM), cyclic voltammetry (CV) and differential pulse voltammetry (DPV) studies. The response studies of PNPI-PANI-PVSA/ITO electrode carried out using DPV reveal a lower detection limit of 1×10(-3) mM, improved sensitivity as 1.5×10(-3) A mM(-1) and stability of 45 days. The PNPI-PANI-PVSA/ITO electrode shows good precision with relative standard deviation of 2.1% and good reproducibility with standard deviation of 3.78%.

Electrophoretically deposited reduced graphene oxide platform for food toxin detection.

Reduced graphene oxide (RGO) due to its excellent electrochemical properties and large surface area, has recently aroused much interest for electrochemical biosensing application. Here, the chemically active RGO has been synthesized and deposited onto an indium tin oxide (ITO) coated glass substrate by the electrophoretic deposition technique. This novel platform has been utilized for covalent attachment of the monoclonal antibodies of aflatoxin B1 (anti-AFB1) for food toxin (AFB1) detection. The electron microscopy, X-ray diffraction, and UV-visible studies reveal successful synthesis of reduced graphene oxide while the XPS and FTIR studies suggest its carboxylic functionalized nature. The electrochemical sensing results of the anti-AFB1/RGO/ITO based immunoelectrode obtained as a function of aflatoxin concentration show high sensitivity (68 µA ng(-1) mL cm(-2)) and improved detection limit (0.12 ng mL(-1)). The association constant (ka) for antigen-antibody interaction obtained as 5 × 10(-4) ng mL(-1) indicates high affinity of antibodies toward the antigen (AFB1).

A highly efficient rare earth metal oxide nanorods based platform for aflatoxin detection.

The nanostructured rare earth metal oxide (samarium oxide, n-Sm2O3) nanorods, prepared using a forced hydrolysis technique, have been electrophoretically deposited (EPD) onto an indium-tin-oxide (ITO) glass substrate. This novel platform has been utilized for co-immobilization of monoclonal antibodies of aflatoxin B1 (Ab-AFB1) and bovine serum albumin (BSA) via electrostatic interactions for food toxin (AFB1) detection. Thus prepared n-Sm2O3 nanorods have been characterized by X-ray diffraction (XRD), atomic force microscopy (AFM), transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared (FTIR) spectroscopic techniques. The results of electrochemical response studies of the BSA/Ab-AFB1/n-Sm2O3/ITO immunoelectrode obtained as a function of aflatoxin concentration reveal a linearity of 10-700 pg mL-1, a detection limit of 57.82 pg mL-1 cm-2, a response time of 5 s and a sensitivity of 48.39 µA pg-1 mL-1 cm-2 with a regression coefficient of 0.961. The association constant (Ka) for antigen-antibody interactions obtained is 47.9 pg mL-1, which indicates high affinity of antibodies towards the antigen (AFB1). The application of n-Sm2O3 modified electrode for immunosensor analysis offers a novel platform and efficient strategy for the application of rare earth metal oxide materials in bioelectronics.

Bienzyme-functionalized monodispersed biocompatible cuprous oxide/chitosan nanocomposite platform for biomedical application.

The ultrafine monodispersed cuprous oxide (Ufm-Cu(2)O) nanoparticles have been successfully synthesized by a facile wet chemical method using poly-N-vinylpyrrolidone (PVP) as a capping agent. This colloidal solution of Ufm-Cu(2)O and chitosan (CS) is electrophoretically deposited (EPD) onto the indium tin-oxide (ITO) glass substrate. Thus synthesized nanocomposite has been characterized by X-ray powder diffraction (XRD, ∼6 nm), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and Fourier transform infrared (FTIR) spectroscopic techniques. This novel biomedical nanocomposite platform has been explored to fabricate a cholesterol biosensor by immobilizing cholesterol esterase (ChEt) and cholesterol oxidase (ChOx) onto Ufm-Cu(2)O-CS/ITO electrode surface. The seed germination tests of these biomaterials (Ufm-Cu(2)O-CS nanocomposite and ChOx-ChEtUfm-CuO(2)-CS nanobiocomposite), conducted using the disc diffusion method, reveal strong activity against the common pathogens and crops, indicating biocompatibility of the nanocomposite. Under optimized conditions, the linearity between the current response and the cholesterol concentration has been obtained in the range of 10-450 mg/dL, with detection limit of 15.9 mg/dL cm(-2) and a high sensitivity of 0.895 µA/(mg/dL cm(-2)). The proposed biocompatible ChEt-ChOx/Ufm-Cu(2)O-CS/ITO bioelectrode shows fast response time (<5 s), good reproducibility, and long-term stability. This biocompatible biosensor has been used to determine the total cholesterol levels in human serum samples. Investigated antimicrobial activities of bienzyme-functionalized Ufm-Cu(2)O-CS nanocomposite are the potential platform for biomedical applications.

Nanobiocomposite platform based on polyaniline-iron oxide-carbon nanotubes for bacterial detection.

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.

Mediator free cholesterol biosensor based on self-assembled monolayer platform.

Self-assembled monolayer (SAM) of 4-aminothiophenol (4-ATP) has been investigated for immobilization of bi-enzymes (ChOx and ChEt) towards development of enzyme biosensors for detection of free and total cholesterol. This enzyme immobilized SAM surface has been characterized by scanning electron microscopy and electrochemical measurements. The results of electrochemical response studies reveal fast enzymatic reaction in phosphate buffer saline solution without using any artificial mediator. This may be attributed to the molecular wire type behavior of short 4-ATP molecule that promotes electron transfer between enzyme and the electrode surface due to its conjugated structure. Interference free estimation of free and total cholesterol has been realized at low operating potential of 0.33 V with range of detection as 25 to 400 mg dl(-1), sensitivity of 542.3 nA mM(-1) (for ChOx/4-ATP/Au) and 886.6 nA mM(-1) (for ChEt-ChOx/4-ATP/Au) with a response time of 20 s at pH 7.4.

Synthesis of optically active silica-coated NdF3 core-shell nanoparticles.

Silica surface-modified NdF(3) core-shell nanoparticles were prepared by sol-gel route. The prepared core-shell nanoparticles were characterized using X-ray diffraction (XRD), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), UV-vis absorption and photoluminescence (PL) spectroscopy studies. Phase identification of the NdF(3) and silica-coated NdF(3) core-shell nanoparticles which was carried-out by XRD, confirms the formation of a well-crystallized hexagonal phase structure. Due to the silica-surface modification, the nanoparticles were not found to be well-separated (agglomerated) in ethanol solvent as scanned by TEM. The results of the FTIR studies conducted on these core-shell reveal the binding of silica with the NdF(3) nanoparticles. The largest intensity and shape variation were observed in all transitions as compared to non-silica modified NdF(3) nanoparticle spectra, and were attributed to the environment around the Nd(III) ion due to coordination of silica molecule(s). A significant enhancement in the emission intensity was measured in silica surface modified NdF(3) core-shell nanoparticles due to the successful silica coating on the surface of nanoparticles. The results of these studies suggest that these nanoparticles may find potential applications in the areas of bioimaging, protein-labeling, optical biosensors and drug delivery, etc.

A self assembled monolayer based microfluidic sensor for urea detection.

Urease (Urs) and glutamate dehydrogenase (GLDH) have been covalently co-immobilized onto a self-assembled monolayer (SAM) comprising of 10-carboxy-1-decanthiol (CDT) via EDC-NHS chemistry deposited onto one of the two patterned gold (Au) electrodes for estimation of urea using poly(dimethylsiloxane) based microfluidic channels (2 cm × 200 µm × 200 µm). The CDT/Au and Urs-GLDH/CDT/Au electrodes have been characterized using Fourier transform infrared (FTIR) spectroscopy, contact angle (CA), atomic force microscopy (AFM) and electrochemical cyclic voltammetry (CV) techniques. The electrochemical response measurement of a Urs-GLDH/CDT/Au bioelectrode obtained as a function of urea concentration using CV yield linearity as 10 to 100 mg dl(-1), detection limit as 9 mg dl(-1) and high sensitivity as 7.5 µA mM(-1) cm(-2).

Polyaniline Langmuir-Blodgett film based aptasensor for ochratoxin A detection.

Ochratoxin A (OTA) produced by Aspergillus Ochraceus and Penicillium verrucosum is a very dangerous toxin due to its toxic effects in human beings and its presence in a wide range of food products and cereals. A Langmuir-Blodgett (polyaniline (PANI)-stearic acid (SA)) film based highly sensitive and robust impedimetric aptasensor has been developed for ochratoxin A (OTA) detection. DNA Aptamer (Apt-DNA) specific to OTA has been covalently immobilized onto mixed Langmuir-Blodgett (LB) monolayer comprising of PANI-SA deposited onto indium tin-oxide (ITO) coated glass plates. This Apt-DNA/PANI-SA/ITO aptaelectrode has been characterized using scanning electron microscopy, Fourier transform-infrared spectroscopy, contact angle measurements, cyclic voltammetry and electrochemical impedance spectroscopy, respectively. The Apt-DNA/PANI-SA/ITO aptasensor shows detection of OTA by electrochemical impedance spectroscopy in the linear range of 0.0001 µg/ml (0.1 ng/ml) to 0.01 µg/ml (10 ng/ml) and 1 µg/ml-25 µg/ml with detection limit of 0.1 ng/ml in 15 min. The Apt-DNA/PANI-SA/ITO aptasensor can be reused ∼13 times. The binding or affinity constant (K(a)) of aptamer with OTA, calculated using Langmuir adsorption isotherm, is found be 1.21×10(7) M(-1).