Characterization and synergy studies of Caudoviricete Escherichia phage FS2B infecting multi-drug resistant uropathogenic Escherichia coli isolates.

Escherichia coli is one of the most common causes of urinary tract infections. However, a recent upsurge in antibiotic resistance among uropathogenic E. coli (UPEC) strains has provided an impetus to explore alternative antibacterial compounds to encounter this major issue. In this study, a lytic phage against multi-drug-resistant (MDR) UPEC strains was isolated and characterized. The isolated Escherichia phage FS2B of class Caudoviricetes exhibited high lytic activity, high burst size, and a small adsorption and latent time. The phage also exhibited a broad host range and inactivated 69.8% of the collected clinical, and 64.8% of the identified MDR UPEC strains. Further, whole genome sequencing revealed that the phage was 77,407 bp long, having a dsDNA with 124 coding regions. Annotation studies confirmed that the phage carried all the genes associated with lytic life cycle and all lysogeny related genes were absent in the genome. Further, synergism studies of the phage FS2B with antibiotics demonstrated a positive synergistic association among them. The present study therefore concluded that the phage FS2B possesses an immense potential to serve as a novel candidate for treatment of MDR UPEC strains.

An impedimetric immunosensor based on chitosan-Au nanoparticles-reduced graphene oxide nanosheet composite modified PG electrode for detection of brain natriuretic peptide.

An ultrasensitive impedimetric immunosensor was developed to detect brain natriuretic peptide (BNP) for early diagnosis of heart failure. To construct this immunosensor, anti-BNP antibodies were immobilized covalently onto nanocomposite of chitosan-Au nanoparticles and reduced graphene oxide nanosheets (CHIT-Au@rGONs) electrodeposited onto pencil graphite electrode. This approach impedes charge transfer resistance (Rct) value proportionally to the BNP captured by antigen-antibody interactions. The observed Rct values by this immunosensor, were correlated with linear concentrations of BNP in the range, 1 × 10-2 to 1 × 103 pg/mL, with a limit of detection of 12 pg/mL and limit of quantification of 36.3 pg/mL. The immunosensor detected BNP in spiked human sera. The analytic recovery of added BNP in human sera was 97.04%. The present method was fairly consistent with commercial approach. The working electrode was stored for 2 months in cold. BSA-IgG had no interference in the electrode activity showing its high specificity for BNP. This novel approach provided a new POC-diagnostics, as direct sample measurements are easier and more efficient by this immunosensor compared to existing immunosensors. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03704-x.

Complete genome annotation data of Mycobacteriophages Prann and LeoAvram: New members of the family Siphoviridae.

Two novel mycobacteriophages (Prann and LeoAvram) belonging to the family Siphoviridae were isolated from soil samples of Northern India. Genomic DNA of both the phages was extracted, and further sequenced using Illumina technology. Complete genome annotation of both the isolates was performed using DNA Master. Prann and LeoAvram had linear genomes of 68398bp and 47079bp, respectively, with G+C contents of 60-70%. A total of 99 and 75 ORFs were predicted in Prann and LeoAvram, respectively. Based on sequence similarity to known phage proteins, functions were assigned to 44 and 53 genes, respectively. These proteins could be classified into five major groups, viz., phage structural proteins, proteins for recombination, lytic enzymes, proteins involved in DNA / RNA metabolism, and in regulation. Mycobacterium smegmatis was used in this work as a safe surrogate for Mycobacterium tuberculosis, the causative agent for tuberculosis, a major infectious disease worldwide with developing antibiotic resistance. This is the first report of M. smegmatis phages from Northern India.

Immobilization of malate dehydrogenase on carbon nanotubes for development of malate biosensor.

An amperometric malic acid biosensor was developed by immobilizing malate dehydrogenase on multi-walled carbon nanotubes (MWCNT) coated on screen printed carbon electrode. The screen printed carbon electrode is made up of three electrodes viz., carbon as working, platinum as counter and silver as reference electrode. Detection of L-malic acid concentration provides important information about the ripening and shelf life of the fruits. The NADP specific malate dehydrogenase was immobilized on carboxylated multiwalled carbon nanotubes using cross linker EDC [1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide] on screen printed carbon electrode. An amperometric current was measured by differential pulse voltammetry (DPV) which increases with increasing concentrations of malic acid at fixed concentration of NADP. Enzyme electrode was characterized by scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectroscopy. The detection limit of malic acid by the sensor was 60 - 120 µM and sensitivity of the sensor was 60 µM with a response time of 60s. The usual detection methods of malic acid are nonspecific, time consuming and less sensitive. However, an amperometric malic acid nanosensor is quick, specific and more sensitive for detection of malic acid in test samples.

Bacteriophage cocktail and phage antibiotic synergism as promising alternatives to conventional antibiotics for the control of multi-drug-resistant uropathogenic Escherichia coli.

Infections related to antibiotic resistant bacteria are accelerating on a global scale, and hence to encounter this problem in case of urinary tract infections; bacteriophages were isolated for biocontrol of multi-drug resistant (MDR) uropathogenic Escherichia coli (UPECs) isolates. Four lytic phages were purified, characterized, and evaluated for their effectiveness in the form of cocktail and in synergy with antibiotics. Morphological features and other life cycle specifications of phages revealed that two phages Escherichia phage FS11 and Escherichia phage FS17 belonged to Myoviridae and the other two phages Escherichia phage PS8 and Escherichia phage PS6 belonged to Siphoviridae family of order Caudovirales. One step growth curve analysis demonstrated that phage FS11 and phage FS17 had latent time of 24 min and 26 min, and a burst size of ~121 and 98 phage particles/ cell respectively; while for phage PS8 and phage PS6, the latent time was 42 min and 35 min, and the burst size was 87 and 78 particles/ cell, respectively; depicting the lytic nature of phages. The use of all four phages together in the form of a cocktail resulted into a considerable enhancement in the lytic ability; the phage cocktail lysed 86.7% of the clinical isolates, compared to lysis in the range of 50%-66% by individual phages. Studies on in vitro evaluation of phage-antibiotic combinations revealed synergism between antibiotics and the phage cocktail (phage PS6 and phage FS17), wherein the phage cocktail was observed to efficiently inhibit the strains in the presence of sub-lethal doses of antibiotics. The study thus concludes that the use of multiple phages and phage-antibiotic combinations could prove beneficial in the era of rapidly increasing drug-resistant strains.

Antibiotic resistance in agriculture: Perspectives on upcoming strategies to overcome upsurge in resistance.

Antibiotic resistance is a massive problem rising constantly and spreading rapidly since the past decade. The major underlying mechanism responsible for this problem is an overuse or severe misuse of antibiotics. Regardless of this emerging global threat, antibiotics are still being widely used, not only for treatment of human infections, but also to a great extent in agriculture, livestock and animal husbandry. If the current scenario persists, we might enter into a post-antibiotic era where drugs might not be able to treat even the simplest of infections. This review discusses the current status of antibiotic utilization and molecular basis of antibiotic resistance mechanisms acquired by bacteria, along with the modes of transmittance of the resultant resistant genes into human pathogens through their cycling among different ecosystems. The main focus of the article is to provide an insight into the different molecular and other strategies currently being studied worldwide for their use as an alternate to antibiotics with an overall aim to overcome or minimize the global problem of antibiotic resistance.

Whole genome sequencing data of Klebsiella aerogenes isolated from agricultural soil of Haryana, India.

Klebsiella aerogenes, is a Gram-negative bacterium, which was previously known as Enterobacter aerogenes. It is present in all environments such as water, soil, air and hospitals; and is an opportunistic pathogen that causes several types of infections. As compared to other clinically important pathogens included in the ESKAPE category (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species), the pangenome and population structure of Klebsiella aerogenes is still poorly understood. For the present study, the bacterial sample was isolated from agricultural soils of Haryana, India. With an aim to identify the occurrence of multi-drug resistance genes in the agricultural field soil bacterial isolate, whole genome sequencing (WGS) of the bacteria was performed; and the antibiotic resistance causing genes, along with the genes responsible for other major functions of the cell; and the different Single Nuceotide Polymorphisms (SNPs) and Insertions and deletions (InDels) were identified. The data presented in this manuscript can be reused by researchers as a reference for determining the antibiotic resistance genes that could be present in different bacterial isolates, and it would also help in determination of functions of various other genes present in other genomes of Klebsiella species.

Physical inactivity, abdominal obesity and risk of coronary heart disease in apparently healthy men and women.

OBJECTIVE: To test the hypothesis that for any given body mass index (BMI) category, active individuals would have a smaller waist circumference than inactive individuals. Our second objective was to examine the respective contribution of waist circumference and physical inactivity on coronary heart disease (CHD) risk. DESIGN: Prospective, population-based study with an 11.4-year follow-up. SUBJECTS: A total of 21 729 men and women aged 45-79 years, residing in Norfolk, UK. METHODS: During follow-up, 2191 CHD events were recorded. Physical activity was evaluated using a validated lifestyle questionnaire that takes into account both leisure-time and work-related physical activity. Waist circumference was measured and BMI was calculated for each participant. RESULTS: For both men and women, we observed that within each BMI category (<25.0, 25-30 and >or=30.0 kg m(-2)), active participants had a lower waist circumference than inactive participants (P<0.001). In contrast, within each waist circumference tertile, BMI did not change across physical activity categories (except for women with an elevated waist circumference). Compared with active men with a low waist circumference, inactive men with an elevated waist circumference had a hazard ratio (HR) for future CHD of 1.74 (95% confidence interval (CI), 1.34-2.27) after adjusting for age, smoking, alcohol intake and parental history of CHD. In the same model and after further adjusting for hormone replacement therapy use, compared with active women with a low waist circumference, inactive women with an elevated waist circumference had an HR for future CHD of 4.00 (95% CI, 2.04-7.86). CONCLUSION: In any BMI category, inactive participants were characterized by an increased waist circumference, a marker of abdominal adiposity, compared with active individuals. Physical inactivity and abdominal obesity were both independently associated with an increased risk of future CHD.

Efficiency of SCNT buffalo (Bubalus bubalis) embryos in different culture medium and analysis of mRNA expression of insulin-like growth factors during embryogenesis.

Growth factors in culture media are known to affect the embryo production rates in in vitro production cultures. To improve the efficiency of somatic cell nuclear transfer (SCNT) derived embryos in Indian buffaloes (Bubalus bubalis), embryos were cultured in three different culture mediums viz. Group-A; TCM-199 + FBS, Group-B; TCM-199 + Poly vinyl alcohol (PVA) and Group-C; CR1aa + BSA. Embryo production rate and expression level of insulin-like growth factor genes (IGF-1, IGF-1R, IGF-2 and IGF-2R) were analysed in embryo culture. Cleavage and blastocyst production rates were 62.5% and 22.3% in Group-A, 53.8% and 13.0% in Group-B and 62.0% and 19.2% in Group-C respectively, whereas in in vitro fertilization (IVF) control cultured in TCM-199 plus 10% FBS, rates were 79.1% and 29.4%. Relative gene expression of SCNT embryos was compared with that in IVF control. IGF-1 and IGF-2 mRNA expression at blastocyst stage was up-regulated (p ≤ 0.05) in all culture groups, while IGF-1R and IGF-2R expression was down regulated (p ≤ 0.05) in Group-B and Group-C. In conclusion, the higher mRNA levels at certain stages in different culture conditions affected in vitro development of SCNT embryos. These results show that the transcript level of the insulin-like growth factor genes was significantly altered by in vitro culture condition. Culture medium TCM-199 with 10% FBS produced higher number of embryos and was able to co-op with gene expression of IVF control. Differences continue to be observed between SCNT cultured and IVF embryos, and until these differences are minimized, aberrations in SCNT embryonic development will continue to arise.

Managing urinary tract infections through phage therapy: a novel approach.

Upsurge in the instances of antibiotic-resistant uropathogenic Escherichia .coli (UPECs) strains has repositioned the attention of researchers towards a century old antimicrobial approach popularly known as phage therapy. Rise of extended spectrum beta lactamase (ESBL) and biofilm producing strains has added another step of hurdle in treatment of uropathogens with conventional antibiotics, thus providing a further impetus for search for exploring new therapeutic measures. In this direction, bacteriophages, commonly called phages, are recently being considered as potential alternatives for treatment of UPECs. Phages are the tiniest form of viruses which are ubiquitous in nature and highly specific for their host. This review discusses the possible ways of using natural phages, genetically engineered phages, and phage lytic enzymes (PLEs) as an alternative antimicrobial treatment for urinary tract infections. The review also sheds light on the synergistic use of conventional antibiotics with phages or PLEs for treatment of uropathogens. These methods of using phages and their derivatives, alone or in combination with antibiotics, have proved fruitful so far in in vitro studies. However, in vivo studies are required to make them accessible for human use. The present review is a concerted effort towards putting together all the information available on the subject.

Relative expression of cell growth regulatory genes insulin-like growth factors (IGF-1 and IGF-2) and their receptors (IGF-1R and IGF-2R) in somatic cell nuclear transferred (SCNT) and in vitro fertilized (IVF) pre-implantation buffalo embryos.

Relative mRNA transcript expression of insulin-like growth factors, IGF-1, IGF-2 and their receptors, IGF-1R and IGF-2R, was studied in SCNT and IVF buffalo embryos at different developmental stages using SYBR green with real-time PCR. SCNT embryos were produced by enucleating IVM oocytes and transferring granulosa cells (passage 5) followed by the electrofusion and chemical activation method. IVF embryos were produced by culturing 15-20 COCs in BO capacitated sperms from frozen and thawed buffalo semen. SCNT embryo production rate was lower than IVF. IGF-1 mRNA expression was significantly upregulated at 2-cell, 16-cell, morula and blastocyst stages of SCNT embryos than in IVF embryos. IGF-1R expression declined from the 2-cell to the 16-cell stage, which increased in later stages and was highest in IVF blastocysts. Similar regulation was observed at different stages in SCNT embryos, except at the 4-cell stage where expression was higher. IGF-2 expression decreased up to the 8-cell stage and increased until the blastocyst stage, being higher in SCNT than IVF embryos. IGF-2R mRNA transcript expression was consistently lower in SCNT than in IVF embryos. Reprogramming of IGF-1, IGF-1R, IGF-2 and IGF-2R expression may have a significant role in cell proliferation in cloned embryos and is developmentally regulated.

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.

Comparative study of antifungal effect of green and chemically synthesised silver nanoparticles in combination with carbendazim, mancozeb, and thiram.

This study reports synthesis and characterisation of silver nanoparticles and their effect on antifungal efficacy of common agricultural fungicides. Silver nanoparticles were synthesised using biological and chemical reduction methods employing Elettaria cardamomum leaf extract and sodium citrate, respectively. Nanoparticles were then characterised using UV-Visible spectroscopy, X-ray diffraction (XRD), transmission electron microscopy, and dynamic light scattering (DLS). While XRD assigned particles size of 31.86 nm for green and 41.91 nm for chemical silver nanoparticles with the help of the Debye-Scherrer formula, DLS specified monodisperse nature of both suspensions. Nanoparticles were tested individually and in combination with fungicides (carbendazim, mancozeb, and thiram) against fungal phytopathogens. Silver nanoparticles exhibited good antifungal activity and minimum inhibitory concentration (MIC) was observed in the range of 8-64 µg/ml. Also, they positively influenced the efficacy of fungicides. The mean MIC value (mean ± SD) for combination of all three fungicides with green AgNPs was 1.37 ± 0.6 µg/ml and for chemical AgNPs was 1.73 ± 1.0 µg/ml. Hence, it could be concluded that green AgNPs performed better than chemical AgNPs. Synergy was observed between green AgNPs and fungicides against Fusarium oxysporum. In conclusion, this study reports synthesis of monodisperse silver nanoparticles which serve as efficient antifungal agents and also enhance the fungicidal action of reported agricultural fungicides in combination studies.

Differential leucocyte count and the risk of future coronary artery disease in healthy men and women: the EPIC-Norfolk Prospective Population Study.

BACKGROUND: We examined the relationship between granulocyte, lymphocyte and monocyte counts and risk of coronary heart disease (CHD) and cardiovascular disease (CVD) in men and women. There is paucity of data on the differential leucocyte count and its relationship with the risk of CHD and CVD. METHODS: This prospective study comprised 7073 men and 9035 women who were 45-79 years of age and were residents of Norfolk. United Kingdom. RESULTS: During an average of 8 years of follow-up we identified 857 incident CHD events and 2581 CVD incident events. Increased total leucocyte count was associated with increased risk for both CHD and CVD. The highest quartile of granulocyte count was associated with increased risk when compared to lowest quartile for CHD (men HR 1.70 95% CI: 1.30-2.21; women HR 1.24 95% CI: 0.91-1.69) and for CVD (men HR 1.46 95% CI: 1.24-1.71; women HR 1.20 95% CI: 1.02-1.42). The association remained unchanged when the analyses were restricted to nonsmokers and when risk was assessed for every 1000 cells L(-1) increase in cell count. In multivariable models, despite adjusting for C-reactive protein (CRP), the granulocyte count remained an independent predictor of CHD and CVD risk, especially amongst men. Lymphocyte or monocyte counts were not significantly associated with increased risk. In all analyses, additionally adjusting for CRP did not affect the results materially. CONCLUSIONS: In conclusion, we found that the higher risk for CHD and CVD associated with increased total leucocyte count seems to be accounted for by the increased granulocyte count.

Association of white blood cell count with systolic blood pressure within the normotensive range.

Hypertension and inflammation promote cardiovascular disease (CVD). Even high normal systolic blood pressure (SBP) is associated with increased CVD risk. We assessed the relationship of elevated SBP within the normotensive range and white blood cell (WBC) count. This is a cross-sectional study of 3484 white asymptomatic individuals (mean age: 43+/-8 years, 79% males) without hypertension with SBP<140 mm Hg. White blood cell count >or=75th percentile (8.35 x 10(9) cells/l) was considered cutoff for elevated WBC. Subjects were classified into three levels of SBP (first: <120 mm Hg, n=1,176, 34%; second: 120-129 mm Hg, n=1,654, 47%; third: 130-139 mm Hg, n=654, 19%). Mean WBC count increased linearly across SBP categories (first: 6.14+/-1.54, second: 6.20+/-1.52, third: 6.41+/-1.62, P=0.02 for trend). There was a linear increase in prevalence of elevated WBC across higher SBP categories (22, 24 and 28%, P=0.02). As compared to those with SBP<120 mm Hg, in multivariate linear regression analyses (adjusting for age, gender, smoking status, diabetes, body mass index, physical activity, cholesterol/high-density lipoprotein cholesterol ratio) WBC count was significantly higher among participants with SBP 130-139 mm Hg (regression coefficient: 2.64, 95% confidence interval: 1.04-4.24, P=0.001). Odds ratio for prevalence of elevated WBC with SBP<120 mm Hg as reference group was 1.14 (0.92-1.41) for SBP 120-129 mm Hg and 1.50 (1.15-1.92) for SBP 130-139 mm Hg. In conclusion, Higher SBP within the normotensive range is also associated with elevated WBC count. Further studies are needed to clarify the role of inflammation in high normal SBP and associated CVD risk.

Clinico-pathological studies on the effect of different anti-neoplastic chemotherapy regimens on transmissible venereal tumours in dogs.

Thirty-two dogs affected with transmissible venereal tumour (TVT) were divided into three treatment groups. In group I vincristine sulphate at 0.025 mg/kg body weight, in group II vinblastine sulphate at 0.150 mg/kg body weight, and in group III vinblastine sulphate at 0.100 mg/kg body weight plus methotrexate at 0.35 mg/kg body weight were given intravenously at weekly intervals. Biopsies were performed on days 0, 3, 7 and 14. The tissues were preserved in 10% neutral buffered formalin and processed routinely for haematoxylin and eosin staining. Histopathologically, the untreated TVT was characterized by sheets or bundles of mostly rounded cells having a large, highly basophilic nucleus with a prominent, highly basophilic necleolus. Both vincristine and vinblastine primarily affected the nuclei of neoplastic cells, causing condensation, karyorrhexis and karyolysis within 3 days of chemotherapy. The regressing tumour mass showed marked infiltration by lymphocytes, lymphoblasts and macrophages by day 7. There was nearly complete regression of the tumour by day 14, as shown by the almost complete loss of neoplastic cells, with fibrous tissue substitution. However, in group III, the changes occurred more slowly and more injections were needed for complete regression. In both groups I and II, 11/12 of the animals responded completely to the chemotherapy within 3 weeks, while in group III, 6/8 of the dogs responded to the treatment by 21-28 days.

Biochemical changes of the milk in experimental caprine mastitis induced by Mycoplasma serogroup 11 (2-D).

Biochemical changes of the milk were studied in 7 lactating goats. The right udder halves of the animals were inoculated through the teat canal with 1 ml of Mycoplasma ovine/caprine serogroup 11 (2-D) culture containing 6x10(6) colony forming units ml-1. The left udder halves of these goats were kept as control. The experiment was continued for 35 days during which period one goat was killed every 5th day. All the goats developed clinical mastitis within 24 h and the infected udder halves remained enlarged for 4-5 days. The milk from these halves was yellow-tinged and showed slight separation of proteins. Subsequently, there was marked reduction in the size and secretion of the infected udder halves, followed by agalactia from post-inoculation (PI) day 22. The mycoplasmas were reisolated from milk samples of the infected halves up to PI day 20. Histopathological examination of these halves revealed changes indicative of acute and chronic mastitis. Biochemical analysis of mastitic milk/mammary secretions showed a marked increase in the concentration of total proteins, immunoglobulins, free fatty acids and phospholipids, while the concentration of total lipids and glycerides initially underwent a considerable decrease as compared to the basal and control values. However, the level of total cholesterol decreased sharply during the initial days while later, from PI day 5, it markedly increased. This study shows that Mycoplasma ovine/caprine serogroup 11 is pathogenic to the lactating udder of goats and produces marked biochemical alterations in the milk.

Intrauterine growth restriction: a perspective for Pakistan.

BACKGROUND: Intrauterine growth restriction (IUGR) is the second leading contributor to the prevailing perinatal mortality and morbidity. It affects 23.8% of newborns around the world and 75% of these affected infants are born in Asia. In Pakistan the incidence of IUGR is around 25%, more than the WHO criteria for triggering a public health action. INTRODUCTION: IUGR is implicated with profound long-term impacts in adult life; like coronary heart disease, NIDDM and abnormal cortisol levels. The effects of the short and long term sequelae are reviewed. ETIOLOGY: IUGR is associated with a wide variety of etiological factors. But the factor unique in its importance to Pakistan is maternal malnutrition. The fetal gene expression is under the influence of nutrition. Growth projection curves showing the interaction between the genetic and environmental factors are discussed. SURVEILLANCE: Identification of IUGR baby in a primary care setting and the options in diagnosis in secondary and tertiary care settings are overviewed. CONCLUSION: The roots of this problem, with multi factorial causes are in the socioeconomic infrastructure. This calls for a synergistic approach of reducing this public health issue. Women empowerment can help us to get out of this intergenerational cycle of growth failure. Availability of resources aside, it is also a matter of political will to change things for the better.

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).