Electrochemical sensing of cytochrome c using Graphene Oxide nanoparticles as platform.

An improved cytochrome c (Cyt c) biosensor based on immobilization of cytochrome c oxidase (COx) on the surface of graphene oxide nanoparticles (GONPs) electrodeposited onto pencil graphite (PG) electrode. Characterization of graphene oxide nanoparticle was done by Transmission electron microscopy (TEM), Fourier transform infra-red spectroscopy (FTIR) and X-ray diffraction study (XRD). The working electrode (COx/GONPs/PG) was characterized at its different stages of fabrication by scanning electron microscopy (SEM) and FTIR. Fabrication of Cyt c biosensor was done by connecting COx/GONPs/PG as working electrode, Ag/AgCl as reference electrode and Pt as auxiliary electrode to potentiostat. The mechanism of detection of present biosensor was based on oxidation of Cyt c (reduced) to Cyt c (oxidized) by COx resulting in flow of electrons through GONPs to the PG electrode, hence current generated is proportional to the concentration of Cyt c. Present biosensor exhibited optimum potential at 0.49 V with optimum pH 7.5 and optimum temperature 35°C. Biosensor showed linearity within 40-180 ng/ml having 40 ng/ml limit of detection. The precision i.e. within and between-batch coefficients of variation (CVs) were found <0.04% and <0.21% respectively. The enzyme electrode lost 50% of its initial activity when operated for more than 6 months on weekly basis. It was applied for detection of Cyt c level in in apparently healthy and diseased human sera. The present biosensing method was co-related with standard colorimetric method and co-relation coefficient was found 0.99.

Cholesterol biosensors: A review.

Cholesterol is the most important sterol synthesized by most of the human cells majorly in the liver. It is a necessary constituent of cell membranes, it acts as a precursor for the synthesis of steroid hormones, vitamin D, and bile acids. Cholesterol is transported in plasma primarily in the form of low-density lipoproteins (LDL), the principal route for its removal from tissues to the liver is in high-density lipoproteins (HDL), followed by excretion in the bile. Cholesterol level is less than 200 mg/dL in healthy persons. 200 and 239 mg/dL is considered borderline high and 240 mg/dL and above is considered a biomarker for cardiovascular diseases, heart attack, strokes, peripheral arterial disease, type 2 diabetes and high blood pressure. Several methods are available for detection of cholesterol, among them, most are burdensome, time-consuming, require sample pre-treatment, high-cost instrumental set-up, and experienced personnel to operate. Biosensing approach overcomes these disadvantages, as these are highly specific, fast, easy, cost-effective, and highly sensitive. The review describes the various cholesterol biosensors. Cholesterol biosensors work ideally within 1 to 300 s, in pH range, 7.0-8.6, temperature 25-37 °C and cholesterol concentration range, 0.000025-700 mM, the detection limits being in the range, 0.000002-4 mM, with working potential -0.05 to 0.65 V. These biosensors measured cholesterol level in fruit juices, beverages, sera and urine samples and reused up to 200 times over a period of 15 to 50 days, while stored dry at 4 °C (Table 1). Future perspective for further improvement and commercialization of cholesterol biosensors are discussed.

Determination of lactic acid with special emphasis on biosensing methods: A review.

Lactic acid (2-Hydroxypropanoic acid) is generated from pyruvic acid under anaerobic condition in skeletal muscles, brain, red blood cells, and kidney. Lactate in normal human subjects get cleared very quickly at a rate of 320mmol/L/hr, mostly by liver metabolism and re-conversion of lactate back to pyruvate. Measurement of lactate level in serum is required for the differential diagnosis and medical management of hyperlactatemia, cardiac arrest and resuscitation, sepsis, reduced renal excretion, hypoxia induced cancer, decreased extra hepatic metabolism, intestinal infarction and lactic acidosis. Determination of lactate is also important in dairy products and beverages to access their quality. Among the various methods available for detection of lactate, most are complicated, nonspecific, less sensitive and require time-consuming sample pretreatment, expensive instrumental set-up and trained persons to operate, specifically for chromatographic methods. Biosensing methods overcome these drawbacks, as these are simple, fast, specific and highly sensitive. Lactate biosensors reported so far, work optimally within 3-180s, between pH, 5.5-8.5 and temperature 22°C to 37°C and lactate concentration ranging from 10 to 2000µM. These biosensors have been employed to measure lactate level in embryonic cell culture, beverages, urine, and serum samples and reused upto 200-times within a period of 7-216 days. This review presents the principles, merits and demerits of various analytical methods for lactate determination with special emphasis on lactate biosensors. The future perspective for improvement of analytic performance of lactate biosensors are discussed.

Construction of glutamate biosensor based on covalent immobilization of glutamate oxidase on polypyrrole nanoparticles/polyaniline modified gold electrode.

A method is described for construction of a highly sensitive electrochemical biosensor for detection of glutamate. The biosensor is based on covalent immobilization of glutamate oxidase (GluOx) onto polypyrrole nanoparticles and polyaniline composite film (PPyNPs/PANI) electrodeposited onto Au electrode. The enzyme electrode was characterized by cyclic voltammetry (CV), scanning electron microscopy (SEM), X-ray diffraction (XRD), transmission electron microscopy (TEM), Fourier transform infra-red spectroscopy (FTIR) and electrochemical impedance spectroscopy (EIS). The biosensor showed optimum response within 3s at pH 7.5 (0.1 M sodium phosphate) and 35 °C, when operated at 50 mV s⁻¹. It exhibited excellent sensitivity (detection limit as 0.1 nM), fast response time and wider linear range (from 0.02 to 400 µM). Analytical recovery of added glutamate (5 mM and 10 mM) was 95.56 and 97%, while within batch and between batch coefficients of variation were 3.2% and 3.35% respectively. The enzyme electrode was used 100 times over a period of 60 days, when stored at 4 °C. The biosensor measured glutamate level in food stuff, which correlated well with a standard colorimetric method (r=0.99).

An amperometric glutamate biosensor based on immobilization of glutamate oxidase onto carboxylated multiwalled carbon nanotubes/gold nanoparticles/chitosan composite film modified Au electrode.

A method is described for the construction of a novel amperometric glutamate biosensor based on covalent immobilization of glutamate oxidase (GluOx) onto, carboxylated multi walled carbon nanotubes (cMWCNT), gold nanoparticles (AuNPs) and chitosan (CHIT) composite film electrodeposited on the surface of a Au electrode. The GluOx/cMWCNT/AuNP/CHIT modified Au electrode was characterized by scanning electron microscopy (SEM), fourier transform infra-red (FTIR) spectroscopy, electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The biosensor measured current due to electrons generated at 0.135V against Ag/AgCl from H2O2, which is produced from glutamate by immobilized GluOx. The biosensor showed optimum response within 2s at pH 7.5 and 35°C. A linear relationship was obtained between a wide glutamate concentration range (5-500µM) and current (µA) under optimum conditions. The biosensor showed high sensitivity (155nA/µM/cm(2)), low detection limit (1.6µM) and good storage stability. The biosensor was unaffected by a number of serum substances at their physiological concentrations. The biosensor was evaluated and employed for determination of glutamate in sera from apparently healthy subjects and persons suffering from epilepsy.

Fabrication of a cytochrome c biosensor based on cytochrome oxidase/NiO-NPs/cMWCNT/PANI modified Au electrode.

An amperometric biosensor for determination of Cytochrome c (Cyt c) was fabricated by immobilizing Cytochrome c oxidase (COx) onto nickel oxide nanoparticles (NiO-NPs) decorated carboxylated multiwalled carbon nanotubes/polyaniline (NiO-NPs/cMWCNT/PANI) film electrodeposited on the surface of a gold (Au) electrode. The electrochemical characteristics of immobilized COx were investigated by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), Scanning Electron Microscopy (SEM) and Fourier transform Infra-red spectroscopy (FTIR). Cyclic voltammetric (CV) studies of the electrode at different stages of construction of enzyme electrode demonstrated that the modified Au electrode had enhanced electrochemical oxidation of H2O2, which offers a number of attractive features to develop amperometric biosensors based on split of H2O2. There was a good linear relationship between the current (mA) and Cyt c concentration in the range 5 x 10(-12) M to 5 x 10(-7) M. The sensor had a detection limit of 5 x 10(-12) M (S/N = 3) with a high sensitivity of 3.7 mA cm(-2) nM(-1). The sensor gave accurate and satisfactory results, when employed for determination of Cyt c in different serum samples.

Construction of an improved amperometric acrylamide biosensor based on hemoglobin immobilized onto carboxylated multi-walled carbon nanotubes/iron oxide nanoparticles/chitosan composite film.

A method is described for construction of an improved amperometric acrylamide biosensor based on covalent immobilization of hemoglobin (Hb) onto nanocomposite of carboxylated multi-walled carbon nanotubes (cMWCNT) and iron oxide nanoparticles (Fe3O4NPs) electrodeposited onto Au electrode through chitosan (CHIT) film. The Hb/cMWCNT-Fe3O4NP/CHIT/Au electrode was characterized by scanning electron microscopy, Fourier transform infra-red spectroscopy, electrochemical impedance spectroscopy, and differential pulse voltammetry at different stages of its construction. The biosensor was based on interaction between acrylamide and Hb, which led to decrease in the electroactivity of Hb, i.e., current generated during its reversible conversion [Fe(II)/Fe(III)]. The biosensor showed optimum response within 8 s at pH 5.0 and 30 °C. The linear working range for acrylamide was 3-90 nM, with a detection limit of 0.02 nM and sensitivity of 36.9 µA/nM/cm(2). The biosensor was evaluated and employed for determination of acrylamide in potato crisps.

Construction of an amperometric bilirubin biosensor based on covalent immobilization of bilirubin oxidase onto zirconia coated silica nanoparticles/chitosan hybrid film.

A method is described for the construction of a highly sensitive electrochemical biosensor for the detection of bilirubin. The sensor is based on covalent immobilization of bilirubin oxidase (BOx) onto zirconia coated silica nanoparticles (SiO2@ZrONPs)/chitosan (CHIT) composite electrodeposited onto Au electrode. The enzyme electrode was characterized by scanning electron microscopy (SEM), Fourier transform infra-red spectroscopy (FTIR), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The biosensor showed optimum response within 2s at pH 8.5 (0.1M Tris-HCl) and 35°C, when operated at 20 mV s(-1). The biosensor exhibited excellent sensitivity (detection limit as 0.1 nM), fast response time and wider linear range (from 0.02 to 250 µM). Analytical recovery of added bilirubin was 95.56-97.0%. Within batch and between batch coefficients of variation were 3.2% and 3.35% respectively. The enzyme electrode was used 150 times over a period of 120 days, when stored at 4°C. The biosensor measured bilirubin levels in sera of apparently healthy and persons suffering from jaundice, which correlated well with a standard colorimetric method (r=0.99).

Construction of an amperometric D-amino acid biosensor based on D-amino acid oxidase/carboxylated mutliwalled carbon nanotube/copper nanoparticles/polyalinine modified gold electrode.

An improved D-amino acid biosensor was constructed based on covalent immobilization of D-amino acid oxidase onto carboxylated mutliwalled carbon nanotube/copper nanoparticles/polyalinine hybrid film electrodeposited on gold electrode. The biosensor exhibited an optimal response within 2s at pH 8.0 and 30°C when polarized at 0.09 V. There was a linear relationship between biosensor response (µA) and D-alanine concentration ranging from 0.001 to 0.7 mM. The sensitivity of the biosensor was 54.85 µA cm(-2) mM(-1) with a lower limit of detection of 0.2 µM (signal/noise=3). The enzyme electrode was used 150 times over a period of 4 months. The biosensor measured the d-amino acid level in fruit juices.

An acrylamide biosensor based on immobilization of hemoglobin onto multiwalled carbon nanotube/copper nanoparticles/polyaniline hybrid film.

A method is described for the construction of a highly sensitive electrochemical biosensor for the detection of acrylamide, based on covalent immobilization of hemoglobin (Hb) onto carboxylated multiwalled carbon nanotube/copper nanoparticle/polyaniline (c-MWCNT/CuNP/PANI) composite electrodeposited onto pencil graphite (PG) electrode. The enzyme electrode was characterized by cyclic voltammetry (CV), scanning electron microscopy (SEM), X-ray diffraction (XRD), transmission electron microscopy (TEM), Fourier transform infrared (FTIR) spectroscopy, and electrochemical impedance spectroscopy (EIS). The biosensor showed an optimal response at pH 5.5 (0.1 M sodium acetate buffer) and 35 °C when operated at 20 mV s(-1). The biosensor exhibited low detection limit (0.2 nM) with high sensitivity (72.5 µA/nM/cm(2)), fast response time (<2 s), and wide linear range (5 nM to 75 mM). Analytical recovery of added acrylamide was 95.40 to 97.56%. Within- and between-batch coefficients of variation were 2.35 and 4.50%, respectively. The enzyme electrode was used 120 times over a period of 100 days, when stored at 4 °C.