The Zanthoxylum armatum fruit’s oil exterminates Candida cells by inhibiting ergosterol biosynthesis without generating reactive oxygen species

Candida spp. is a significant cause of topical and fungal infections in humans. In addition to Candida albicans, many non-albicans species such as C. krusei, C. glabrata, C. parapsilosis, C. tropicalis, C. guilliermondii cause severe infections. The main antifungal agents belong to three different classes, including azoles, polyenes, and echinocandins. However, resistance to all three categories of drugs has been reported. Therefore, there is an urgent need to search for other alternatives with antifungal activity. Many herbal extracts and compounds from natural sources show excellent antifungal activity. In this study, we used an oil extract from the fruits of Zanthoxylum armatum, which showed significant antifungal activity against various Candida spp. by two different methods—minimum inhibitory concentration (MIC) and agar diffusion. In addition, we attempted to explore the possible mechanism of action in C. albicans. It was found that the antifungal activity of Z. armatum oil is fungicidal and involves a decrease in the level of ergosterol in the cell membrane. The decrease in ergosterol level resulted in increased passive diffusion of a fluorescent molecule, rhodamine6G, across the plasma membrane, indicating increased membrane fluidity. The oil-treated cells showed decreased germ tube formation, an important indicator of C. albicans’ virulence. The fungal cells also exhibited decreased attachment to the buccal epithelium, the first step toward invasion, biofilm formation, and damage to oral epithelial cells. Interestingly, unlike most antifungal agents, in which the generation of reactive oxygen species is responsible for killing, no significant effect was observed in the present study. (AU)

The Zanthoxylum armatum fruit's oil exterminates Candida cells by inhibiting ergosterol biosynthesis without generating reactive oxygen species.

Candida spp. is a significant cause of topical and fungal infections in humans. In addition to Candida albicans, many non-albicans species such as C. krusei, C. glabrata, C. parapsilosis, C. tropicalis, C. guilliermondii cause severe infections. The main antifungal agents belong to three different classes, including azoles, polyenes, and echinocandins. However, resistance to all three categories of drugs has been reported. Therefore, there is an urgent need to search for other alternatives with antifungal activity. Many herbal extracts and compounds from natural sources show excellent antifungal activity. In this study, we used an oil extract from the fruits of Zanthoxylum armatum, which showed significant antifungal activity against various Candida spp. by two different methods-minimum inhibitory concentration (MIC) and agar diffusion. In addition, we attempted to explore the possible mechanism of action in C. albicans. It was found that the antifungal activity of Z. armatum oil is fungicidal and involves a decrease in the level of ergosterol in the cell membrane. The decrease in ergosterol level resulted in increased passive diffusion of a fluorescent molecule, rhodamine6G, across the plasma membrane, indicating increased membrane fluidity. The oil-treated cells showed decreased germ tube formation, an important indicator of C. albicans' virulence. The fungal cells also exhibited decreased attachment to the buccal epithelium, the first step toward invasion, biofilm formation, and damage to oral epithelial cells. Interestingly, unlike most antifungal agents, in which the generation of reactive oxygen species is responsible for killing, no significant effect was observed in the present study.

Exploring structural antigens of yellow fever virus to design multi-epitope subunit vaccine candidate by utilizing an immuno-informatics approach.

BACKGROUND: Yellow fever is a mosquito-borne viral hemorrhagic disease transmitted by several species of virus-infected mosquitoes endemic to tropical regions of Central and South America and Africa. Earlier in the twentieth century, mass vaccination integrated with mosquito control was implemented to eradicate the yellow fever virus. However, regular outbreaks occur in these regions which pose a threat to travelers and residents of Africa and South America. There is no specific antiviral therapy, but there can be an effective peptide-based vaccine candidate to combat infection caused by the virus. Therefore, the study aims to design a multi-epitope-based subunit vaccine (MESV) construct against the yellow fever virus to reduce the time and cost using reverse vaccinology (RV) approach. METHODS: Yellow fever virus contains 10,233 nucleotides that encode for 10 proteins (C, prM, E, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) including 3 structural and 7 non-structural proteins. Structural proteins-precursor membrane protein (prM) and envelope protein (E)-were taken as a target for B cell and T cell epitope screening. Further, various immunoinformatics approaches were employed to FASTA sequences of structural proteins to retrieve B cell and T cell epitopes. MESV was constructed from these epitopes based on allergenicity, antigenicity and immunogenicity, toxicity, conservancy, and population coverage followed by structure prediction. The efficacy of the MESV construct to bind with human TLR-3, TLR-4, and TLR-8 were evaluated using molecular docking and simulation studies. Finally, in-silico cloning of vaccine construct was performed withpBR322 Escherichia coli expression system using codon optimization. RESULTS: Predicted epitopes evaluated and selected for MESV construction were found stable, non-allergenic, highly antigenic, and global population coverage of 68.03% according to in-silico analysis. However, this can be further tested in in-vitro and in-vivo investigations. Epitopes were sequentially merged to construct a MESV consisting of 393 amino acids using adjuvant and linkers. Molecular docking and simulation studies revealed stable and high-affinity interactions. Furthermore, in-silico immune response graphs showed effective immune response generation. Finally, higher CAI value ensured high gene expression of vaccine in the host cell. CONCLUSION: The designed MESV construct in the present in-silico study can be effective in generating an immune response against the yellow fever virus. Therefore, to prevent yellow fever, it can be an effective vaccine candidate. However, further downstream, in-vitro study is required.

In silico and in vitro evaluation of designed fluconazole analogues as lanosterol 14α-demethylase inhibitors.

The drugs fighting against aggressive fungal infections are in limited number, therefore, extensive research is obligatory to develop new therapeutic strategies. Fluconazole (FLZ) is a clinically approved drug, but resistant drug against most fungal pathogens, thus it is vital to identify more compounds that can better check the fungal growth. Analogue-based drug designing is a quick and economical way since it has inherent drug-like properties of marketed drugs. This study aims to generate and evaluate analogues of FLZ with better potency against fungal-borne infections. A total of 3307 analogues of FLZ were developed from six scaffold structures. Only 390 compounds passed Lipinski's rule, of which 247 analogues exhibited lower docking scores than FLZ with 5FSA. These inhibitors were further subjected to pharmacokinetics property evaluation and cytotoxicity test and it was found that only 46 analogues were suitable for further evaluation. Based on the molecular docking score of the best two analogues, 6f (-12.7 kcal/mol) and 8f (-12.8 kcal/mol) were selected for molecular dynamics and in-vitro studies. Antifungal activities of both compounds against 4 strains of Candida albicans were evaluated by disc diffusion assay and micro broth dilution assay and Minimum inhibitory concentrations (MICs) for 6f and 8f were observed as 256 µg/ml against 4719, 4918 and 5480 strains but the MIC was extended to 512 µg/ml for strain 3719. Both analogues exhibited low antifungal activities as compared to FLZ (8-16 µg/ml). The interaction of 6f with Mycostatin was also performed using a chequerboard assay that was found additive.Communicated by Ramaswamy H. Sarma.

Comparative genome analysis of Streptococcus strains to identify virulent genes causing neonatal meningitis.

AIM: To determine Streptococcus agalactiae genes responsible for causing neonatal meningitis. BACKGROUND: Streptococcus agalactiae strain 2603 V/R is causative agent of neonatal meningitis, maternal infection and sepsis in young children. World health organisation reported high burden of new born death caused by this bacterium. Streptococcus agalactiae colonizing epithelial cells of vagina and endothelial cells have high resistance to available antibiotic drugs which makes it essential to determine new drug targets. OBJECTIVES: To compare the genome of selected strain with the non-pathogenic strains of streptococcus and identify the virulent and antibiotic resistant genes for adaptation in host environment. METHOD: The whole genome of human pathogen Streptococcus agalactiae strain 2603 V/R was analysed and compared with Streptococcus dysgalactiae strains using visualization and annotation tools. Genomic islands, mobile genetic elements, virulent and resistant genes were studied. RESULTS: Genetically pathogenic strain is most similar to Streptococcus dysgalactiae subsp. equisimilis strain NCTC 7136. Comparative analysis revealed the importance of capsular polysaccharides and surface proteins responsible for avoiding immune system attachment to host epithelial cells and virulent behaviour. High number of genes coding for antibiotics resistance may provide a competitive advantage for survival of pathogenic Streptococcus agalactiae strain 2603 V/R in its niche. CONCLUSIONS: The comparative analysis of pathogenic strain Streptococcus agalactiae with non-pathogenic strains of Streptococcus dysgalactiae provided new insights in pathogenicity that could aid in recognization for new regions and genes for development of new drug development strategies considering presence of high number of resistance genes.

A computational approach to discover antioxidant and anti-inflammatory attributes of silymarin derived from Silybum marianum by comparison with hydroxytyrosol.

Medicinal plants possess therapeutic potential for reducing reactive oxygen species (ROS)-mediated cellular damage. Hydroxytyrosol is one of the most potent antioxidants that served as control in the current study, including other synthetic antioxidants to computationally identify the antioxidant properties of Silymarin. The sequences of the receptors IκB kinase (IKK), Kelch-like ECH-associated protein 1 (Keap-1) and mitochondrial transcription factor A (Tfam) were retrieved from UniProtKB and homology modeling was performed using Swiss-Model server. Thereof the molecular docking and dynamic simulation studies were performed using Schrödinger's software version 11.5. From the current study, it was reported that on comparison of the binding energy of silymarin, hydroxytyrosol, α-tocopherol, ascorbic acid, butylated hydroxy anisole (BHA) and butylated hydroxytoluene (BHT), Silymarin exhibited better affinities with IKK receptor followed by Hydroxytyrosol suggesting it as the best or comparable of all other known antioxidants that could potentially suppress inflammation and other diseases. Also, Silymarin exhibited poorest binding affinity with Tfam promoting mitochondrial biogenesis, thereby scavenging ROS. However, with Keap-1, Silymarin is ranked 4th in the list, whereas hydroxytyrosol exhibited highest binding affinity to release oxidative stress. The stability of docked complexes made us conclude that Silymarin has comparable antioxidant properties to hydroxytyrosol, better anti-inflammatory potential and mitochondrial biogenesis enhancing properties to ultimately reduce oxidative stress. Now it can be tested further for in vitro or in vivo studies as potential drug against oxidative insult.Communicated by Ramaswamy H. Sarma.

Development of pharmacophore model to identify potential DNA gyrase inhibitors.

There is great concern in the medical community due to rapid increase in antibiotic resistance, causing 700,000 deaths annually worldwide. Therefore, there is paramount need to develop novel and innovative antibacterial agents active against resistant bacterial strains. DNA gyrase is a crucial enzyme in bacterial replication that is absent in eukaryotes, making it effective curative target for antibacterials. To identify potential DNA gyrase inhibitors by virtual screening of NCI database using a 3-step approach. A total of 271 compounds with known IC50 values against Escherichia coli DNA GyrA were selected to develop a pharmacophore model for dual screening approach to identify new potential hits from the NCI database. In the second step, the NCI database was also screened using in-house built NN-QSAR model. Molecular docking of common 5298 compounds screened from both methods were performed against E. coli DNA GyrA (PDB id- 6RKU), and 3004 compounds are reported to exhibit lower binding energies than ciprofloxacin (-6.77 Kcal/mol). The top three compounds (NCI371878, NCI371876 and NCI142159) reported with binding energy of -13.5, -13.19 and -13.03 Kcal/mol were further subjected to MD simulation studies for 100 ns supporting the stability of the docked complexes.

Computational Design for Identification of Human Anti-MUC1 Heteroclitic Peptides in the Treatment of HER2-Positive Breast Cancer through Neural Network Training and Monomeric based Design.

AIMS: Generation of the human anti-MUC1 peptide through neural network training and monomeric design method. Analyzing 9-mer peptide potential computationally for treatment of HER2-positive breast cancer. BACKGROUND: With the advancements of cancer genome atlas project (TCGA), cancer dependancy project (DepMap) and human protein atlas (HPA), large-scale datasets are generated for oncology studies. However, after development of redefined breast cancer drug targets, there are key issues in successful breast cancer treatments that needed to be pursued which paved the pathway for new approaches or strategies. In that respect, our research data aimed to represent a new aspect of breast cancer drug development studies. OBJECTIVE: Extract human MUC1 sequences from various databases. Perform neural networking method for novel peptides sequences. Analyze the potentiality of generated heteroclitic peptide sequences for suitable vaccine candidate for breast cancer treatment. METHODS: Input scaffolds of protein database (PDB) files for human MUC1 were retrieved and loaded into Evo design server with monomeric based design option. Further, neural network training approaches were followed and other computational tools were used for alignment-independent prediction of protective antigens and subunit vaccines potency of designed heteroclitic peptides. RESULTS: Study findings revealed two human anti-MUC1 heteroclitic peptides of 9mers (WAVWTYVSV, FMSFYIMNL), which showed the lowest energy cluster and sequence identity, normalized relative error rate of secondary structure, solvent accessibility, backbone torsion angles for neural networking and RMSD values in evolutionary profiling, and online MHCPred IC50 interaction values. VaxiGen v2.0 server revealed subunit vaccine potency values of in-silico designed two heteroclitic peptides were 0.1551 (WAVWTYVSV) and 0.3508 (FMSFYIMNL) with a threshold value of 0.5 followed by AllerTOP v2.0 for their allergenicity nature in immunogenic reactions. CONCLUSION: Computationally designed heteroclitic peptide WAVWTYVSV indicated promising values which can be utilised as drug delivery or tumour marker candidate in the treatment of human breast cancer by eliciting lyse of tumor cells.

Discovery of Novel Inhibitors of Bacterial DNA Gyrase Using a QSAR-Based Approach.

Type II topoisomerases like DNA gyrase initiate ATP-dependent negative supercoils in bacterial DNA. It is critical in all of the bacteria but is missing from eukaryotes, making it a striking target for antibacterials. Ciprofloxacin is a clinically approved drug, but its clinical effectiveness is affected by the emergence of resistance in both Gram-positive and Gram-negative bacteria. Thus, it is vital to identify novel compounds that can efficiently inhibit DNA gyrase, and quantitative structure-activity relationship (QSAR) modeling is a quick and economical means to do so. A QSAR-based virtual screening approach was applied to identify new gyrase inhibitors using an in-house-generated combinatorial library of 29828 compounds from seven ciprofloxacin scaffold structures. QSAR was built using a data set of 271 compounds, which were identified as positive and negative inhibitors from existing data reported in in vitro studies. The best QSAR model was developed using the 5-fold cross-validation Neural Network in Orange, and it was based on five PaDEL descriptors with an accuracy and sensitivity of 83%. As a result of screening of an in-house-built combinatorial library with the best-developed QSAR model, 675 compounds were identified as potential inhibitors of DNA gyrase. These inhibitors were further docked with DNA gyrase using AutoDock to compare the binding mode and score of the selected/screened compounds, and 615 compounds exhibited a docking score comparable to or lower than that of ciprofloxacin. Out of these, the top five analogues 902b, 9699f, 4419f, 5538f, and 898b reported in our study have binding scores of -13.81, -12.95, -12.52, -12.43, and -12.41 kcal/mol, respectively. The MD simulations of these five analogues for 100 ns supported the interaction stability of analogues with Escherichia coli DNA gyrase. Ninety-one per cent of the analogues screened by the QSAR model displayed better binding energy than ciprofloxacin, demonstrating the efficacy of the generated model. The NN-QSAR model proposed in this manuscript can be downloaded from https://github.com/ritu225/NN-QSAR_model.git.

Therapeutic approaches for combating Pseudomonas aeruginosa infections.

Pseudomonas aeruginosa is an opportunistic pathogen responsible for acute hospital-acquired infections. This review described various therapeutic approaches to treat infections caused by P. aeruginosa, including conventional therapy, novel antibiotic treatments and treatments other than antibiotics. Most of the developments are still in research that will provide novel treatment options in future.

Chemotherapeutic Strategies for Combating Staphylococcus aureus Infections.

Staphylococcus aureus is a prominent human pathogen that causes nosocomial and community acquired infections. The accelerating emergence and prevalence of staphylococcal infections have grotesque health consequences which are mostly due to its anomalous capability to acquire drug resistance and scarcity of novel classes of antibacterials. Many combating therapies are centered on primary targets of S. aureus which are cell envelope, ribosomes and nucleic acids. This review describes various chemotherapeutic strategies for combating S. aureus infections including monotherapy, combination drug therapy, phage endolysin therapy, lysostaphins and antibacterial drones. Monotherapy has dwindled in due course of time, but combination therapy, endolysin therapy, lysostaphin and antibacterial drones are emerging alternatives which efficiently conquer the shortcomings of monotherapy. Combinations of more than one antibiotic agents or combination of adjuvant with antibiotics provide a synergistic approach to combat infections causing pathogenic strains. Phage endolysin therapy and lysostaphin are also presented as possible alternatives to conventional antibiotic therapies. Antibacterial Drones go a step further by specifically targeting the virulence genes in bacteria, giving them a certain advantage over existing antibacterial strategies. But the challenge remains on the better understanding of these strategies for executing and implementing them in the health sector. In this day and age, most of the S. aureus strains are resistant to an ample number of antibiotics, so there is an urgent need to overcome such multidrug-resistant strains for the welfare of our community.

Nanotechnology in Cancer Diagnostics and Therapeutics: A Review.

Cancer is the uncontrolled proliferation of cells that involves accumulation of genetic mutations by different types of mutagens including physical, chemical, and biological. Consequently, normal cell cycles get interrupted. Immunological assays, histopathological tests, polymerase chain reaction, computed tomography, magnetic resonance, and radiation therapy are some conventional techniques for cancer diagnostics. However, these techniques are not only expensive, time-consuming, tedious but also toxic to healthy cells. Therefore, these limitations are overcome by nanodevices that show high sensitivity, selectivity, rapidity, and cost-effectiveness in the detection of cancer biomarkers. Electrochemical biosensors are more efficient in the early diagnosis of cancers that help in patients' effective and timely treatment. Distinct types of nanotools viz. inorganic, organic, and polymeric nanomaterials are used in cancer therapeutics. Nano approaches have shown many advantages: they are site-specific, require meager amounts of drugs, limited toxicity, avoid drug resistance, and are more efficient, sensitive, and reliable. Therefore, future research should focus on developing highly inventive nanotools for the diagnosis and therapeutics of cancers.

Detection and remediation of pollutants to maintain ecosustainability employing nanotechnology: A review.

Environmental deterioration due to anthropogenic activities is a threat to sustainable, clean and green environment. Accumulation of hazardous chemicals pollutes soil, water and air and thus significantly affects all the ecosystems. This article highlight the challenges associated with various conventional techniques such as filtration, absorption, flocculation, coagulation, chromatographic and mass spectroscopic techniques. Environmental nanotechnology has provided an innovative frontier to combat the aforesaid issues of sustainable environment by reducing the non-requisite use of raw materials, electricity, excessive use of agrochemicals and release of industrial effluents into water bodies. Various nanotechnology based approaches including surface enhance scattering, surface plasmon resonance; and distinct types of nanoparticles like silver, silicon oxide and zinc oxide have contributed significantly in detection of environmental pollutants. Biosensing technology has also gained significant attention for detection and remediation of pollutants. Furthermore, nanoparticles of gold, ferric oxide and manganese oxide have been used for the on-site remediation of antibiotics, organic dyes, pesticides, and heavy metals. Recently, green nanomaterials have been given more attention to address toxicity issues of chemically synthesized nanomaterials. Hence, nanotechnology has provided a platform with tremendous applications to have sustainable environment for present as well as future generations. This review article will help to understand the fundamentals for achieving the goals of sustainable development, and healthy environment.

An overview of nanomaterial based biosensors for detection of Aflatoxin B1 toxicity in foods.

Aflatoxin B1 (AFB1) is one of the most potent mycotoxin contaminating several foods and feeds. It suppresses immunity and consequently increases mutagenicity, carcinogenicity, teratogenicity, hepatotoxicity, embryonic toxicity and increasing morbidity and mortality. Continuous exposure of AFB1 causes liver damage and thus increases the prevalence of cirrhosis and hepatic cancer. This article was planned to provide understanding of AFB1 toxicity and provides future directions for fabrication of cost effective and user-friendly nanomaterials based analytical devices. In the present article various conventional (chromatographic & spectroscopic), modern (PCR & immunoassays) and nanomaterials based biosensing techniques (electrochemical, optical, piezoelectrical and microfluidic) are discussed alongwith their merits and demerits. Nanomaterials based amperometric biosensors are found to be more stable, selective and cost-effective analytical devices in comparison to other biosensors. But many unresolved issues about their stability, toxicity and metabolic fate needs further studies. In-depth studies are needed for development of advanced nanomaterials integrated biosensors for specific, sensitive and fast monitoring of AFB1 toxicity in foods. Integration of biosensing system with micro array technology for simultaneous and automated detection of multiple AFs in real samples is also needed. Concerted efforts are also required to reduce their possible hazardous consequences of nanomaterials based biosensors.

CRISPR: A new paradigm of theranostics.

Infectious and hereditary diseases are the primary cause of human mortality globally. Applications of conventional techniques require significant improvement in sensitivity and specificity in therapeutics. However, clustered regularly interspaced short palindromic repeats (CRISPRs) is an innovative genome editing technology which has provided a significant therapeutic tool exhibiting high sensitivity, fast and precise investigation of distinct pathogens in an epidemic. CRISPR technology has also facilitated the understanding of the biology and therapeutic mechanism of cancer and several other hereditary diseases. Researchers have used the CRISPR technology as a theranostic approach for a wide range of diseases causing pathogens including distinct bacteria, viruses, fungi and parasites and genetic mutations as well. In this review article, besides various therapeutic applications of infectious and hereditary diseases we have also explained the structure and mechanism of CRISPR tools and role of CRISPR integrated biosensing technology in provoking diagnostic applications.

Advances in tuberculous meningitis diagnosis.

Introduction: Tuberculous meningitis (TBM) is the most devastating form of central nervous system tuberculosis (TB) and causes high mortality worldwide. Nonspecific clinical manifestations and limited sensitivity of existing laboratory methods make the diagnosis elusive due to the paucibacillary nature of the infection. Areas Covered: We reviewed current literature on the adequacy and limitations of globally existing laboratory methods for diagnosing TBM. Expert opinion: TBM is deadliest among all TB forms, as the outcome may lead to death in 50% of cases, and survivors undergo irreversible neurological disorders. Therefore, early diagnosis and prompt treatment are cornerstones of effective disease management. Conventional microscopy and culture are widely used modalities but remain inadequate in most TBM cases. Although expanded use of rapid molecular tests such as real-time PCR and Xpert Ultra, even in resource-limited settings, hold promising results for TB diagnosis but need optimization for early detection of TBM. Moreover, CSF IGRA is also used but unable to differentiate between active and latent TB. Overall no single test for diagnosing TBM has adequate accuracy so, there is an urgent need to devise a point-of-care test.

Bioactive constituents of Emblica officinalis overcome oxidative stress in mammalian cells by inhibiting hyperoxidation of peroxiredoxins.

Emblica officinalis (Amla) is a renowned fruit having nutritional and medicinal traits mostly linked to its antioxidants content. In the current study, the methanolic crude extract of amla fruit is subjected to sequential fractionation to get its partially purified fractions. The ethyl acetate (EA) and butanol (BUT) fractions of amla showed maximum antioxidant potential. The ferric reducing capability and nitric oxide scavenging activity were highest in EA fraction. One of the highlights of the study is the cellular antioxidant assay conducted in HeLa cells. Additionally, HeLa cells pre-treated with EA and BUT fractions were able to combat oxidative stress via total reduction in hyperoxidation of intracellular peroxiredoxin enzyme. Gallic acid, ascorbic acid, ellagic acid, rutin, quercetin, and catechol are the major compounds present in these fractions as identified by LC-ESI-MS followed by their quantification by HPLC. These findings indicate that components of E. officinalis can protect intracellular oxidative stress-mediated degeneration. PRACTICAL APPLICATIONS: The study highlighted that E. officinalis is a promising source of phenolics and flavonoids acting as natural antioxidants, which showed varied potential to scavenge ROS. Also, the plant fractions were able to fight intracellular oxidative stress via total reduction in hyperoxidation of the human peroxiredoxin. In conclusion, we can say that the regular intake of such food supplements that affect important antioxidant enzymes can be of special interest in the management of oxidative stress-mediated human ailments.

Enzyme nanoparticles and their biosensing applications: A review.

Enzyme nanoparticles (ENPs) are the aggregates of enzymes in the nano scale (10-100 nm). These ENPs have been characterized by transmission electron microscopy (TEM), fourier transformation infrared spectroscopy (FTIR), UV-Visible spectroscopy, X-ray diffraction (XRD), atomic force microscopy (AFM), scanning electron microscopy (SEM) and electrochemical impedance spectroscopy (EIS). Enzymes are the biocatalysts exhibiting substrate specificity, catalytic activity, regulator of various metabolic reactions under mild conditions and hence, used as industrial catalysts efficiently. Due to these extraordinary properties of enzymes, they have been used for the diagnosis and treatment of various diseases. However, direct application to enzymes has increased the chances of degradation and thus lowers the performance of analytical devices. Therefore these limitations have been overcome by synthesizing the nanoparticles (NPs) of enzyme. As ENPs have exhibited extraordinary properties such as large surface to volume ratio, unique optical, thermal and chemical than native enzymes. ENPs based biosensor work optimally within 2-300s, pH range 5.0-6.0 and temperature 3.5-45°C. The linear range of ENPs based biosensor varies between 0.0001 and 100000 µM and detection limit 0.0002-550 µM. These biosensors have been used for the diagnosis of various diseases such as cardiovascular diseases, renal disorders, diabetes, environment monitoring, and biochemical engineering and reused up to 8-200 times over a period of 60-240 days while stored dry at 4°C. Therefore, the future research should be focused to understand the interaction of ENPs with their analyte and further improvement and commercialization of ENPs based biosensor are discussed. Present review article deciphers the various methods of ENPs preparation, their characterization and ENPs based biosensors.

Preparation, Characterization, and Application of Enzyme Nanoparticles.

Enzymes are fundamental biocatalysts, which regulate various metabolic reactions. They exhibit high substrate specificity, sensitivity, and exceptional catalytic activity under ideal conditions and, hence, have been used as industrial catalysts. Enzyme nanoparticles have attracted the scientific community as they can be used for environment protection, biochemical engineering, and biomedicine. It is necessary to understand the nature of enzyme nanoparticles interactions with their analyte. Various types of enzyme/protein nanoparticles have been immobilized onto a matrix (electrode or membrane) for the fabrication of biosensors. Among the various nanoparticles and nanomaterials, organic nanoparticles have received more attention due to their fascinating properties. Therefore, the future research should be focused to develop advanced biosensors based on enzyme nanoparticles that could be used for early diagnosis and management of chronic diseases. This chapter explains various enzyme nanoparticles-based biosensors, such as GOX, HRP, uricase, cholesterol oxidase, hemoglobin, and their biomedical applications.

Preparation, characterization and application of haemoglobin nanoparticles for detection of acrylamide in processed foods.

The nanoparticles of haemoglobin (HbNPs) were prepared by desolvation method and characterized by transmission electron microscopy (TEM),UV-vis spectroscopy, Fourier transformation infra red (FTIR) spectroscopy and X-ray diffraction (XRD) and atomic force microscopy (AFM). Protein profile of HbNPs was also studied by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). An amperometric acrylamide biosensor was constructed by immobilizing covalently HbNPs onto polycrystalline Au electrode. The Au electrode was characterized by scanning electron microscopy (SEM) and electrochemical impedance spectra (EIS) before and after immobilization of HbNPs. The biosensor showed optimum current response within 2s at 0.26V, pH 5.0 at room temperature (20°C). The biosensor measured the acrylamide concentration in processed foods. The working range of biosensor was 0.1nm-100mM with a limit of detection (LOD) as low as 0.1nM. The biosensor measured acrylamide concentration in various processed foods such as biscuits, bread, potato crisps, "kurkure", nuts and fried cereals. The analytical recovery of added acrylamide in aqueous extract of food at 5 and 10mM was 99% and 98% respectively. Within-and between-batch, co-efficient of variations were 3.85% and 4.67% respectively. The structural analogs of acrylamide such as acrylic acid and propionic acid had practically no interference on the biosensor.