Determination of glycated hemoglobin with special emphasis on biosensing methods.

The glycated hemoglobin (HbA1c) level in blood is a measure of long-term glycemic status in patients with diabetes mellitus. Current clinical methods for determination of the HbA1c level include electrophoresis/electroendosmosis, ion exchange chromatography, high-performance liquid chromatography, boronate affinity chromatography, immunoassay, and liquid chromatography-tandem mass spectroscopy in addition to fluorometry and colorimetry. These methods have certain drawbacks such as being complex, time-consuming, and requiring expensive apparatus and trained persons to operate. These drawbacks were overcome by biosensing methods. We review these biosensors, which are based on (i) measurement of electrons, that is, current generated from splitting of hydrogen peroxide released during oxidation of fructosyl valine by immobilized fructosyl amino acid oxidase, which is directly proportional to HbA1c concentration, and (ii) direct measurement of HbA1c by some specific reaction. HbA1c biosensors work optimally within 4 to 1800 s, between pH 7.0 and 9.0 and between 25 and 45 °C, and in the range of 1 to 10,000 µM, with a detection limit between 20 and 500 µM and sensitivity between 4.6 nA and 21.5 µA mM⁻¹ cm⁻² and stable over a period of 5 to 90 days. We suggest the ways to modify existing HbA1c biosensors, leading to simple, reliable, and economical sensors ideally suited for point-of-care treatment.

Covalent immobilization of lipase, glycerol kinase, glycerol-3-phosphate oxidase & horseradish peroxidase onto plasticized polyvinyl chloride (PVC) strip & its application in serum triglyceride determination.

BACKGROUND & OBJECTIVES: Reusable biostrip consisting enzymes immobilized onto alkylamine glass beads affixed on plasticized PVC strip for determination of triglyceride (TG) suffers from high cost of beads and their detachments during washings for reuse, leading to loss of activity. The purpose of this study was to develop a cheaper and stable biostrip for investigation of TG levels in serum. METHODS: A reusable enzyme-strip was prepared for TG determination by co-immobilizing lipase, glycerol kinase (GK), glycerol-3-phosphate oxidase (GPO) and peroxidase (HRP) directly onto plasticized polyvinyl chloride (PVC) strip through glutaraldehyde coupling. The method was evaluated by studying its recovery, precision and reusability. RESULTS: The enzyme-strip showed optimum activity at pH 7.0, 35 o C and a linear relationship between its activity and triolein concentration in the range 0.1 to 15 mM. The strip was used for determination of serum TG. The detection limit of the method was 0.1 mM. Analytical recovery of added triolein was 96 per cent. Within and between batch coefficients of variation (CV) were 2.2 and 3.7 per cent, respectively. A good correlation (r=0.99) was found between TG values by standard enzymic colrimetric method employing free enzymes and the present method. The strip lost 50 per cent of its initial activity after its 200 uses during the span of 100 days, when stored at 4 o C. INTERPRETATION & CONCLUSIONS: The nitrating acidic treatment of plasticized PVC strip led to glutaraldehyde coupling of four enzymes used for enzymic colourimetric determination of serum TG. The strip provided 200 reuses of enzymes with only 50 per cent loss of its initial activity. The method could be used for preparation of other enzyme strips also.

Determination of sulfite with emphasis on biosensing methods: a review.

Sulfite is used as a preservative in a variety of food and pharmaceutical industries to inhibit enzymatic and nonenzymatic browning and in brewing industries as an antibacterial and antioxidizing agent. Convenient and reproducible analytical methods employing sulfite oxidase are an attractive alternative to conventional detection methods. Sulfite biosensors are based on measurement of either O2 or electrons generated from splitting of H2O2 or heat released during oxidation of sulfite by immobilized sulfite oxidase. Sulfite biosensors can be grouped into 12 classes. They work optimally within 2 to 900 s, between pH 6.5 and 9.0, 25 and 40 °C, and in the range from 0 to 50,000 µM, with detection limit between 0.2 and 200 µM. Sulfite biosensors measure sulfite in food, beverages, and water and can be reused 100-300 times over a period of 1-240 days. The review presents the principles, merits, and demerits of various analytical methods for determination of sulfite, with special emphasis on sulfite biosensors.

An Amperometric Acetylcholine Biosensor Based on Co-Immobilization of Enzyme Nanoparticles onto Nanocomposite.

An electrochemical biosensor was fabricated using nanoparticles of acetylcholinesterase (AChE) and choline oxidase (ChO)/Pt nanoparticles (PtNPs)/porous graphene oxide nanosheet (GONS) composite. A pencil graphite electrode (PGE) was used for the electrodeposition of nanocomposite and the determination of acetylcholine (ACh), a neurotransmitter. Various techniques such as scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier-transform infrared (FTIR) spectra and cyclic voltammetry (CV) were used for characterization. This biosensor (AChENPs-ChONPs/GONS/PtNPs/PGE) indicated a very short response time (3 s), a lower limit of detection (0.001 µM), good linearity (0.001-200 µM), longer storage stability (6 months) and better reproducibility. The percent analytical recoveries of added acetylcholine in serum (5.0 and 10 µM) were found to be 97.6 ± 0.7 and 96.5 ± 0.3 for the present biosensor. The coefficients of variation were obtained to be 8% and 3.25%, correspondingly. The biosensor was applied to measure the ACh amount in the serum of healthy individuals and patients with Alzheimer's disease. The number of interferents had no effect on the biosensor at their physiological concentrations.

Acetylcholinesterase inhibition-based biosensors for pesticide determination: a review.

Pesticides released intentionally into the environment and through various processes contaminate the environment. Although pesticides are associated with many health hazards, there is a lack of monitoring of these contaminants. Traditional chromatographic methods-high-performance liquid chromatography, capillary electrophoresis, and mass spectrometry-are effective for the analysis of pesticides in the environment but have certain limitations such as complexity, time-consuming sample preparation, and the requirement of expensive apparatus and trained persons to operate. Over the past decades, acetylcholinesterase (AChE) inhibition-based biosensors have emerged as simple, rapid, and ultra-sensitive tools for pesticide analysis in environmental monitoring, food safety, and quality control. These biosensors have the potential to complement or replace the classical analytical methods by simplifying or eliminating sample preparation and making field-testing easier and faster with significant decrease in cost per analysis. This article reviews the recent developments in AChE inhibition-based biosensors, which include various immobilization methods, different strategies for biosensor construction, the advantages and roles of various matrices used, analytical performance, and application methods for constructing AChE biosensors. These AChE biosensors exhibited detection limits and linearity in the ranges of 1.0×10(-11) to 42.19 µM (detection limits) and 1.0×10(-11)-1.0×10(-2) to 74.5-9.9×10(3)µM (linearity). These biosensors were stable for a period of 2 to 120days. The future prospects for the development of better AChE biosensing systems are also discussed.

An amperometric hemoglobin A1c biosensor based on immobilization of fructosyl amino acid oxidase onto zinc oxide nanoparticles-polypyrrole film.

Measurement of hemoglobin A1c (HbA1c, glycated hemoglobin) level in blood provides the long-term glucose level in diabetic patients without the influence of short-term fluctuations. The existing methods for HbA1c determination, including biosensors, suffer from insufficient sensitivity, detection limit, response time, and storage stability. These problems were overcome in the current biosensor. A method is described for construction of an amperometric HbA1c biosensor by immobilizing a fructosyl amino acid oxidase (FAO) onto zinc oxide nanoparticles/polypyrrole (ZnONPs/PPy) hybrid film deposited onto gold (Au) electrode and using it as working electrode, Ag/AgCl as reference electrode, and platinum (Pt) as auxiliary electrode. The whole blood samples were hemolyzed and digested by protease before measuring their HbA1c level by the biosensor. The enzyme electrode detected fructosyl valine (FV) as low as 50µM at a signal-to-noise ratio of 3 within 2s at +0.27V versus Ag/AgCl, pH7.0, and 35°C with a linear working range of 0.1 to 3.0mM for FV and sensitivity of 38.42µAmM(-1). The electrode showed only a 30% loss of its initial response over a period of 160days when stored at 4°C. The biosensor measured HbA1c in whole blood of apparently healthy individuals and diabetic patients and found it to be in the ranges of 4.0% to 5.6% and 5.7% to 12.0%, respectively.

Amperometric determination of total phenolic content in wine by laccase immobilized onto silver nanoparticles/zinc oxide nanoparticles modified gold electrode.

A method is described for construction of a highly sensitive amperometric biosensor for measurement of total phenolic compounds in wine by immobilizing laccase covalently onto nanocomposite of silver nanoparticles (AgNPs)/zinc oxide nanoparticles (ZnONPs) electrochemically deposited onto gold (Au) electrode. Scanning electron microscopy, X-ray diffraction, and electrochemical impedance spectroscopy were applied for characterization of the surface morphology of the modified electrode, and cyclic voltammetry was used to investigate the electrochemical properties of the proposed electrode toward the oxidation of guaiacol. The linearity between the oxidation current and the guaiacol concentration was obtained in a range of 0.1 to 500µM with a detection limit of 0.05µM (signal-to-noise ratio (S/N)=3) and sensitivity of 0.71µAµM(-1)cm(-2). The electrode showed increased oxidation and reduced reduction current with the deposition of AgNPs/ZnONPs on it. R(CT) values of ZnONPs/Au, AgNPs/ZnONPs/Au, and laccase/AgNPs/ZnONPs/Au electrode were 220, 175, and 380Ω, respectively. The biosensor showed an optimal response within 8s at pH 6.0 (0.1M acetate buffer) and 35°C when operated at 0.22V against Ag/AgCl. Analytical recovery of added guaiacol was 98%. The method showed a good correlation (r=0.99) with the standard spectrophotometric method, with the regression equation being y=1.0053x-3.5541. The biosensor lost 25% of its initial activity after 200 uses over 5months.

Development of an amperometric polyphenol biosensor based on fungal laccase immobilized on nitrocellulose membrane.

A method is described for construction of an amperometric polyphenol biosensor employing nitrocellulose membrane-bound laccase purified from cell-free extract of Ganoderma lucidum onto a Pt electrode. The biosensor showed optimum response within 10s, at 0.4 V in 0.1M acetate buffer, pH 6.0, and 35°C. Detection limit of the biosensor was 3.0 × 10(-8)M. Analytical recovery of added guaiacol was 97.00%. Within batch and between batch coefficients of variation were <0.97% and <1.26%, respectively. The sensor measured total phenolic content in fruit juices and alcoholic beverages. The enzyme electrode was used 100 times over 4 months, when stored at 4°C.

Electrochemical Sensor for Bilirubin Detection Using Paper-Based Screen-Printed Electrodes Functionalized with Silver Nanoparticles.

A notable diagnostic for the detection of hemolytic diseases is bilirubin, a by-product of haemoglobin breakdown. The concentration of bilirubin ranges from 0.3 to 1.9 mg in 100 mL of blood. Low blood bilirubin levels are associated with a greater risk of coronary heart disease and anaemia. Hyperbilirubinemia results from a serum bilirubin level of more than 2.5 mg/100 mL. Therefore, it is very crucial to check the serum bilirubin level. Analytical equipment for point-of-care testing must be portable, small, and affordable. A unique method is used to detect bilirubin selectively using paper-based screen-printed carbon electrodes that were covalently linked with nanoparticles, that serves as a key biomarker for jaundice. In order to create an electrochemical biosensor, bilirubin oxidase was immobilised on electrodes modified with AgNPs. The morphology of Ag nanoparticles in terms of size and shape was determined using both UV- Vis Spectroscopy and transmission electron microscopy (TEM). The biosensor's analytical response was assessed using potentiostat (Cyclic voltammetry (CV) and linear sweep voltammetry (LSV)). The developed paper-based sensor provided optimum feedback and a broad linear range of 1 to 9 µg/mL for bilirubin, with a lower LOD of 1 µg/mL. Through tests of bilirubin in artificial blood serum, the viability is confirmed. The method that is being used makes it possible to create and use an inexpensive, miniature electrochemical sensor.

An amperometric uric acid biosensor based on multiwalled carbon nanotube-gold nanoparticle composite.

An amperometric uric acid biosensor was fabricated by immobilizing uricase (EC 1.7.3.3) onto gold nanoparticle (AuNP)/multiwalled carbon nanotube (MWCNT) layer deposited on Au electrode via carbodiimide linkage. Determination of uric acid was performed by oxidation of enzymically generated H(2)O(2) at 0.4V. The sensor showed optimal response within 7s at 40°C in 50mM Tris-HCl buffer (pH 7.5). The linear working range of the biosensor was 0.01-0.8mM. The limit of detection (LOD) was 0.01mM. The sensor measured uric acid levels in serum of healthy individuals and persons suffering from gout. The analytical recoveries of the added uric acid, 10 and 20mgL(-1), were 98.0% and 96.5%, respectively. Within- and between-batch coefficients of variation were less than 5.6% and less than 4.7%, respectively. A good correlation (r=0.998) was obtained between serum uric acid values by the standard enzymic colorimetric method and the current method. A number of serum substances had practically no interference. The sensor was used in more than 200 assays and had a storage life of 120 days at 4°C.

Development of an amperometric sulfite biosensor based on a gold nanoparticles/chitosan/multiwalled carbon nanotubes/polyaniline-modified gold electrode.

A sulfite oxidase (SOx) purified from leaves of Syzygium cumini (Jamun) was immobilized covalently onto a gold nanoparticles (AuNPs)/chitosan (CHIT)/carboxylated multiwalled carbon nanotubes (cMWCNTs)/polyaniline (PANI) composite that was electrodeposited onto the surface of a gold (Au) electrode. A novel and highly sensitive sulfite biosensor was developed that used this enzyme electrode (SOx/AuNPs/CHIT/cMWCNT/PANI/Au) as the working electrode, Ag/AgCl as the standard electrode, and Pt wire as the auxiliary electrode. The modified electrode was characterized by Fourier transform infrared (FTIR) spectroscopy, cyclic voltammetry (CV), scanning electron microscopy (SEM), and electrochemical impedance spectroscopy (EIS) before and after the immobilization of the SOx. The sensor produced its optimum response within 3 s when operated at 50 mVs(-1) in 0.1 M phosphate buffer, pH 7.0, and at 35 °C. The linear range and detection limit of the sensor were 0.75-400 µM and 0.5 µM (S/N = 3), respectively. The biosensor was employed to determine sulfite levels in fruit juices and alcoholic beverages. The enzyme electrode was used 300 times over a period of three months when stored at 4 °C.

A changing trend in diagnostic methods of Influenza A (H3N2) virus in human: a review.

The influenza virus is classified into four types A, B, C, and D, but type A and B are responsible for major illnesses in people with influenza A being the only virus responsible for flu pandemics due to the presence of two surface proteins called hemagglutinin (H) and neuraminidase (N) on the virus. The two subtypes of influenza A virus, H1N1 and H3N2, have been known to cause many flu pandemics. Both subtypes change genetically and antigenically to produce variants (clades and subclades, also know as groups and subgroups). H3N2 tends to change rapidly, both genetically and antigenically whereas that of H1N1 generally tends to have smaller changes. Influenza A (H3N2) viruses have evolved to form many separate, genetically different clades that continue to co-circulate. Influenza A(H3N2) viruses have caused significant deaths as per WHO report. The review describes methods for detection of influenza A(H3N2) viruses by conventional serological methods as well as the advanced methods of molecular biology and biosensors. All these methods are based on different parameters and have different targets but the goal is to improve specificity and increase sensitivity. Amongst the molecular methods, real-time polymerase chain reaction (RT-PCR) is considered a gold standard test due to its many advantages whereas a number of other molecular methods are time-consuming, complex to perform or lack specificity. The review also considers bio-sensing methods for simple, rapid, highly sensitive, and specific detection of H3N2. The classification and principle of various H3N2 biosensors are also discussed.

An improved enzyme nanoparticles based amperometric pyruvate biosensor for detection of pyruvate in serum.

The nanoparticles of commercially available pyruvate oxidase (POx) from Aerococcus species were prepared by desolvation method, which were then characterized and covalently immobilized onto gold electrode (AuE) to construct an improved model of amperometric pyruvate biosensor. The POxNPs/Au electrode was analyzed morphologically by scanning electron microscopy (SEM). On the other hand, cyclic voltammetry studies (CV) and electrochemical impedance spectroscopy (EIS) helped in deciphering the electrochemical properties of the electrode at different stages of construction. The biosensor showed optimum response within 7.5 s, at a potential of 0.28 V, pH 5.5 and 35 °C. A linear relationship was observed between biosensor response i.e. current (µA) and pyruvate concentration in the range, 0.01 µM - 5000 µM, with a lower detection limit of 0.67 µM. The analytical recovery of added pyruvate in sera was 99.0% and 99.5% within and between batch coefficient of variation (CV) were 0.045% and 0.040% respectively. The working electrode displayed an excellent correlation coefficient (R2 = 0.99%) between levels of pyruvate in sera, as detected by the standard spectrophotometric method and the present biosensor. The biosensor was utilized for detection of total pyruvate level in sera of apparently healthy individuals and patients suffering from cardiogenic stress, more specifically cardiac failure. The activity of the biosensor deteriorated by 25%, after its regular use over a period of 240 days, while being stored dry at 4°C.

Activation of polyvinyl chloride sheet surface for covalent immobilization of oxalate oxidase and its evaluation as inert support in urinary oxalate determination.

Polyvinyl chloride (PVC) sheets are a promising material for enzyme immobilization owing to the PVC's properties such as being chemically inert, corrosion free, weather resistant, tough, lightweight, and maintenance free and having a high strength-to-weight ratio. In this study, this attractive material surface was chemically modified and exploited for covalent immobilization of oxalate oxidase using glutaraldehyde as a coupling agent. The enzyme was immobilized on activated PVC surface with a conjugation yield of 360 microg/cm(2). The scanning electron micrographs showed the microstructures on the PVC sheet surface revealing the successful immobilization of oxalate oxidase. A colorimetric method was adopted in evaluating enzymatic activity of immobilized and native oxalate oxidase. The immobilized enzyme retained 65% of specific activity of free enzyme. Slight changes were observed in the optimal pH, incubation temperature, and time for maximum activity of immobilized oxalate oxidase. PVC support showed no interference when immobilized oxalate oxidase was used for estimation of oxalic acid concentration in urine samples and showed a correlation of 0.998 with the values estimated with a commercially available Sigma kit. The overall results strengthen our view that PVC sheet can be used as a solid support for immobilization of enzymes and in the field of clinical diagnostics, environmental monitoring and remediation.

Prussian blue nanocubes/carbon nanospheres heterostructure composite for biosensing of metformin.

This paper reports the fabrication of highly sensitive metformin sensor based on Prussian blue (PB) nanocubes/carbon nanosphere (CNS) heterostructures composed of a perfect cube and spherical composite on a fluorine-doped tin oxide surface. Due to the excellent biocompatibility of PB nanocubes the PB/CNS based Mf sensor exhibited a wide linear range of 0.001-10 mM with a response duration of less than 5 s and a detection limit (based on signal to noise ratio) of 0.1 µM.

Morphology-Preferable MoSe2 Nanobrooms as a Sensing Platform for Highly Selective Apta-Capturing of Salmonella Bacteria.

The present report employed nanobroom (NB)-shaped two-dimensional molybdenum diselenide (MoSe2) for the preparation of a sensing matrix for the detection of Salmonella paratyphi. An aptamer specific to salmonella was immobilized onto MoSe2NB-modified fluorine-doped tin oxide via glutaraldehyde cross-linking. Structural and morphological characterizations were performed using UV-vis spectroscopy, scanning electron microscopy, Fourier transform infrared spectroscopy, and X-ray diffraction techniques. Characterizations confirmed the nanobroom morphology and nanosize of the MoSe2 material. Electrochemical studies revealed a good linear detection range of 10-2-10-10 CFU/mL with low detection limit of 1 × 10-10 CFU/mL and with R 2 = 0.98. The developed preferable nanobroom-shaped sensing matrix can provide a promising platform for rapid and accurate detection of Salmonella in real samples due to its tremendous stability and sensitivity.

Development of MoSe2 Nano-Urchins as a Sensing Platform for a Selective Bio-Capturing of Escherichia. coli Shiga Toxin DNA.

The present study was aimed to develop "fluorine doped" tin oxide glass electrode with a MoSe2 nano-urchin based electrochemical biosensor for detection of Escherichia. coli Shiga toxin DNA. The study comprises two conductive electrodes, and the working electrodes were drop deposited using MoSe2 nano-urchin, and DNA sequences specific to Shiga toxin Escherichia. coli. Morphological characterizations were performed using Fourier transforms infrared spectrophotometer; X-ray diffraction technique and scanning electron microscopy. All measurements were done using methylene blue as an electrochemical indicator. The proposed electrochemical geno-sensor showed good linear detection range of 1 fM⁻100 µM with a low detection limit of 1 fM where the current response increased linearly with Escherichia. coli Shiga toxin dsDNA concentration with R2 = 0.99. Additionally, the real sample was spiked with the dsDNA that shows insignificant interference. The results revealed that the developed sensing platform significantly improved the sensitivity and can provide a promising platform for effective detection of biomolecules using minute samples due to its stability and sensitivity.

Immobilization of grain-sorghum (Sorghum bicolor, var. CSH-14) leaf oxalate oxidase on to a modified mica chip and its application in the determination of urinary oxalate.

A new method is described for immobilization of oxalate oxidase purified from grain sorghum (Sorghum bicolor, var. CSH-14) leaves on to mica chip through MnCl(2) x 4H(2)O coupling with a conjugation yield of 0.36 mg of protein/cm(2) and 65.3% retention of the initial activity of the free enzyme. The immobilized enzyme showed maximum activity at pH 6.5 when incubated at 40 degrees C for 15 min. K(m) for oxalate was decreased from 0.7 to 0.53 mM after immobilization. The immobilized enzyme lost 12% of its initial activity after storage at 4 degrees C in reaction buffer. The oxalate in urine was analysed using this immobilized enzyme. The minimum detection limit was 2.5 mg/l of urine. Analytical recovery of added oxalate (10, 20 and 30 mg/l) was 89.2, 91 and 92.1% respectively. Within- and between-batch coefficients of variation were <1.3% and <4.1% respectively. A good correlation (r=0.92) was found between oxalate values obtained using a commercial enzymic colorimetric kit (Oxalate Urinalysis Diagnostic kit; procedure no. 591-C; Sigma) based on free barley oxalate oxidase and the present method.

Biosensing methods for xanthine determination: a review.

Xanthine (3,7-dihydro-purine-2,6-dione) is generated from guanine by guanine deaminase and hypoxanthine by xanthine oxidase (XOD). The determination of xanthine in meat indicates its freshness, while its level in serum/urine provides valuable information about diagnosis and medical management of certain metabolic disorders such as xanthinuria, hyperurecemia, gout and renal failure. Although chromatographic methods such a HPLC, capillary electrophoresis and mass spectrometry are available for quantification of xanthine in biological materials, these suffer from certain limitations such as complexity, time consuming sample preparation and requirement of expensive apparatus and trained persons to operate. Immobilized XOD based biosensors have emerged as simple, rapid, sensitive and economic tools for determination of xanthine in food industries and clinical diagnosis. This review article describes the various immobilization methods of XOD and different matrices used for construction of xanthine biosensors, their classification, analytical performance and applications along with their merits and demerits. The future perspectives for improvement of present xanthine biosensors are also 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).