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A highly sensitive time-resolved fluorescence lateral flow immunoassay (TRF-LFIA) was developed to quantify glial fibrillary acidic protein (GFAP), a trauma brain injury (TBI) biomarker in blood, for the purpose of providing a diagnosis of mild brain injury.

© 2022, The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.This study aimed to establish a Europium label time-resolved fluorescence immunoassay (TRFIA) to detect the chronic kidney disease (CKD) biomarker Cystatin-C. An Europium based Time resolved fluorescence immunoassay was developed to detect the concentration of Cystatin-C in a urine sample to increase the sensitivity with captured anti-Cystatin-C antibodies immobilized on nitrocellulose membrane and then bonded with detection anti-Cystatin-C labelled with CM-EU, followed by fluorescence measurement using time-resolved fluorometry in 15 min. The performance of this TRFIA was evaluated using the clinical urine serum and compared with the ELISA assays. The linear calibration range was 0.015–32 µg/ml, and the limit of detection (LOD) quantified was 0.0001 µg/ml. This current work has improved the LOD of our previous work from 0.013 µg/ml to 0.001 µg/ml. These results indicated that the CM-EU nanoparticle-based LFIA is rapid, more sensitive, reliable, and reproducible for point-of-care testing of Cys-C concentrations in urine.

Tea (Camellia sinensis, Family: Theaceae) is one of the extremely consumed beverages around the world, behind to water. The brew tea is the merely food product contains abundant quantity of the catechins. Green tea is the least processed and thus contains rich antioxidant, polyphenols, especially catechin called epigallocatechin-3-gallate (EGCG), which is whispered to be responsible for a wide range of the health benefits. The key to the amazing health benefits that are derived from green tea is that the leaves are steamed which preserves the EGCG compound from being oxidized. However, the other varieties of teas are under go fermentation process, which breaks down the potential EGCG and destroy from its healing properties. In reality, green tea has very extensive history dating back thousands and thousands of years ago. However, the pharmacological efficacy and stability of green tea catchiness are primarily depended on the formulation and way to drink to alleviate the deadly diseases with scientific evidence. Nanotechnology is a vibrantly emerging field especially in the pharmaceutical industry to explore a lot of application. The promising nano-delivery system used to enhance the therapeutic efficacy with a minimal dose, minimize the dose-related toxicity, target delivery, site-specific delivery, and controlled/sustain the delivery application. In recent decades, the application of nanotechnology has been utilized for phytopharmaceutical industry including green tea catechins to maximize the health benefits. In this review, we tried our level best retrieve the value of information on nanodelivery application of green tea catchiness for various devastating diseases.

Background: Tea (Camellia sinensis, Theaceae) is the second most consumed beverage in the world. Green tea is the least processed and thus contain rich antioxidant level, and believed to have most of the health benefits. Methods: We commenced to search bibliographic collection of peer reviewed research articles and review articles to meet the objective of this study. Results: From this study, we found that the tea beverage contains catechins are believed to have a wide range of health benefits which includes neuroprotective, anti-inflammatory, antiulcer, antiviral, antibacterial, and anti-parasitic effects. The four major catechin compounds of green tea are epigallocatechin (EGC), epicatechin (EC), epigallocatechin gallate (EGCG), and epicatechin gallate (ECG), of which EGCG is the major constituent and representing 50-80% of the total catechin content. And also contain xanthine derivatives such as caffeine, theophylline, and theobromine, and the glutamide derivative theanine. It also contains many nutritional components, such as vitamin E, vitamin C, fluoride, and potassium. We sum up the various green tea phytoconstituents, extraction methods, and its medicinal applications. Conclusion: In this review article, we have summarized the pharmacological importance of green tea catechin which includes antioxidant potential, anti-inflammatory, antimicrobial, anticancer, antidiabetic and cosmetic application.

Obesity is a pandemic disease with immense magnitude and it is due to escalating death with co-morbidity in this century. Obesity is caused by combinatorial factors including genetic, metabolic, behavioral, environmental, cultural, and socioeconomic factors that contribute to a person’s body weight. This condition parse is alarmingly big magnitude problem and it is conformingly with the co-morbidities. An elevated body mass index (BMI) increases the prevalence, morbidity, and mortality of type 2 diabetes mellitus, hypertension, heart disease, stroke, osteoarthritis, respiratory tract disorders, gallstones, certain types of cancer, and psychological disorders. It is striking that only a few therapeutic agents are available to treat obesity including orlistat, sibutramine, lorcaserin, phentermine-topiramate available to treat obesity. These agents can reduce body weight by decreasing the consumption or absorption of food or by augmenting energy disbursement. In recent decades, the limelight of nanotechnology has opened numerous new vistas in medical sciences, especially in the area of drug delivery. The nanoparticulate drug delivery system can transport specific anti-obesity drugs to the white adipose tissue in the body, aiding to evade potential side effects that can occur if the drugs find their way to other parts of the body. In this chapter, we highlighted the various nano-drug delivery systems including polymeric nanoparticles, chitosan nanoparticles, Polyethylene glycol-based nanoparticles, gold nanoparticles, liposomes, transfersomes, and microparticles to enhance the therapeutic efficacy against obesity treatment.

Time resolved acquisition of fluorescence provides advantages of reduced background reflection and reduced analyte autofluorescence, considerably improving the signal to background ratio. Europium chelate with its relatively longer fluorescence decay in milliseconds can be analyzed with inexpensive detectors with simpler timing considerations. In this work we present a Time Resolved Fluorescence (TRF) reader using a monochrome camera and its associated exposure trigger circuit to capture TRF from Europium chelate striped on nitrocellulose membrane. The reader is imaging based and does not require complex mechanical moving parts to read the test strip under investigation. Ten different concentrations of Europium between 100 ng/ml and 1000 ng/ml were imaged and the output of the developed TRF reader was found to be linear (R2=0.9873) over the range of concentrations. An improvement of 70% was observed in the signal to background ratio using TRF technique.

Diabetic wounds (DW) are a chronic, non-healing wound on the feet of diabetic patients that pose a serious challenge to world health. Around 84% of diabetic patients undergo lower leg amputations. Though numerous topical and systemic drugs have been used to heal the DW, these drugs have led to the emergence and subsequent rapid overgrowth of resistant bacterial strains, side effects and toxicity. Many herbal plants have very important role in wound healing because they promote the natural repair mechanisms. Moringaoleifera (MO) is an important medicinal plant which has an impressive range of medicinal uses including antimicrobial, anti-inflammatory, antidiabetic, antioxidant and anticancer activities. Recently few researchers reported that MO extracts have effective wound healing property due to the presence of rich flavonoids and vicenin-2. The objective of the present study was to develop hydrogel formulations loaded with Moringaoleifera leaves extract. The prepared hydrogels were evaluated for physical appearance, rheological behavior, skin irritation and wound-healing power in streptozotocin-induced diabetic male wistar albino rats. Results showed that all hydrogel formulations exhibited good and acceptable physical properties. All the animals tolerated the applied gels and no signs of irritations were noticed during the skin irritation study. The in-vivo wound healing studies showed a time dependent increase in percentage of wound, a contraction which is higher than that produced by the control groups. These contractions were statistically significant (P

In March 2020, SARS-CoV-2 based infections were declared ‘COVID-19 pandemic’ by the World Health Organization (WHO). Pandemic raised the necessity to design and develop genuine and sensitive tests for precise specific SARS-CoV-2 infections detection. Nanotechnological methods offer new ways to fight COVID-19. Nanomaterials are ideal for unique sensor platforms due to their easy manufacturing, chemically versatile properties. In this context, selected examples for integrating nanomaterials and distinct biosensor platforms are given to detect SARS-CoV-2 biological materials and COVID-19 biomarkers, giving researchers and scientists more goals and a better forecast to design more relevant and novel sensor arrays for COVID-19 diagnosis., Graphical abstract Image 1

A wide range of microorganisms can infect the central nervous system (CNS). The immune response of the CNS provides limited protection against microbes penetrating the blood-brain barrier. This results in a neurological deficit and sometimes leads to high morbidity and mortality rates despite advanced therapies. For the last two decades, different studies have expanded our understanding of the molecular basis of human neuroinfectious diseases, especially concerning the contributions of mast cell interactions with other central nervous system compartments. Brain mast cells are multifunctional cells derived from the bone marrow and reside in the brain. Their proximity to blood vessels, their role as "first responders" their unique receptors systems and their ability to rapidly release pathogen responsive mediators enable them to exert a crucial defensive role in the host-defense system. This review describes key biological and physiological functions of mast cells, concerning their ability to recognize pathogens via various receptor systems, followed by a coordinated and selective mediator release upon specific interactions with pathogenic stimulating factors. The goal of this review is to direct attention to the possibilities for therapeutic applications of mast cells against bacterial and viral related infections. We also focus on opportunities for future research activating mast cells via adjuvants.

In the emergency diagnosis of patients, acute myocardial infarction (AMI) is always time-consuming to diagnose, and the process requires multiple laboratory procedures, expensive equipment and skilled workers. Herein, we developed an easy-to-use, low-cost and portable fluorescent lateral flow immunoassay based on paper microfluidics for the point-of-care diagnostics of non-communicable diseases. The fluorescent lateral flow immunoassay can produce results in less than 10 minutes, and the limit of detection (LOD) is 0.019 ng ml−1. The slope was linear from 0 to 100 ng ml−1; the equation is y = 0.0342e2.1181x and R2 = 0.9618, which are distinctive features that ensure maximum amplification of the signal and recording of quantitative values by an analyser. The detection sensitivity showed an exceptional increase to 0.01 ng ml−1. Compared with conventional bioassay readers, our analyser shows some advantages to easily, clearly and effectively read data. The present point-of-care test for cardiac troponin I decreases the turnaround time and has a high coefficient of variation even at lower concentrations of troponin. So, the development of lateral flow assay-based point-of-care assays with higher analytical performance for real world samples can decrease the rule-out time for AMI in emergency departments and other fields.

An amino acid Schiff base (R) capable of recognizing Zn 2+ ions selectively and sensitively in an aqueous medium was prepared and characterized. Upon addition of Zn 2+ ions, the receptor exhibits fluorescence intensity enhancements (~ 40 fold) at 460 nm (quantum yield, Φ = 0.05 for R and Φ = 0.18 for R–Zn 2+ ) and can be detected by naked eye under UV light. The receptor can recognize the Zn 2+ (1.04 × 10 − 8 M) selectively for other metal ions in the pH range of 7.5–11. The Zn 2+ chelation with R decreases the loss of energy through non-radiative transition and leads to fluorescence enhancement. The binding mode of the receptor with Zn 2+ was investigated by 1 H NMR titration and further validated by ESI-MS. The elemental color mapping and SEM/EDS analysis were also used to study the binding of R with Zn 2+ . Density functional theory calculations were carried out to understand the binding mechanism. The receptor was applied as a microbial sensor for Escherichia coli and Staphylococcus aureus .

Several mononuclear complexes [Cu(L1), Cu(L2), Ni(L1) and Ni(L2)] containing N,O-donor Schiff base [L1 = 2,2′-{cyclohexane-1,2-diylbis[nitrilo(E)methylylidene]}bis[5-(prop-2-yn-1-yloxy)phenol] and L2 = 2,2′-{1,2-phenylenebis[nitrilo(E)methyllidene]}bis[5-(prop-2-yn-1-yloxy)phenol]] have been synthesized and characterized by elemental analyses and spectral techniques. The solid-state structures of Cu(L1) and Ni(L1) were determined using single-crystal X-ray crystallography reveal distorted square planar geometry around the metal ions. The binding ability of the complexes with DNA and BSA were investigated. DNA cleavage activity reveals that the complexes cleaved the plasmid DNA via hydrolytic path way. In vitro cytotoxicity assays indicate that these complexes exhibit anticancer activity against human cervical carcinoma cell line (HeLa). Flow cytometric analysis and Annexin V/PI double staining assays suggest that these complexes induce cell apoptosis. The comet assay has been employed to find out the extent of DNA fragmentation in cancer cells. The catecholase activity of the complexes has been investigated by UV-vis, cyclic voltammetry and EPR using 3,5-DTBC as model substrate reflect that the complexes are effective in mimicking catecholase like activity. DFT calculation on the mechanistic insights of catalytic activity suggests a ligand-centred radical mechanism.

A thiazolidine carboxylic acid derivative (L) was conveniently synthesized and characterized by spectral techniques and single crystal X-ray crystallography. The complexation of L with zinc (L–Zn2+) was studied by scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) and further substantiated by 1H NMR and ESI-MS analysis. The L showed excellent specificity and sensitivity towards a zinc-induced fluorescence response by forming a 1 : 1 complex (Job's plot) in a 95% (v/v) water–methanol mixture. The presence of zinc ions causes ∼40 fold fluorescence enhancement at 481 nm (quantum yield, Φ = 0.19) and can be detected with the naked eye under a UV-lamp. The L can detect Zn2+ in nanomolar levels (13.90 nM) with good tolerance in the presence of other interfering metal ions. The reversibility of L–Zn2+ complexation was checked by EDTA titration. The maximum fluorescence enhancement by Zn2+ binding of L was observed in the pH range of 7.0–9.0. The dissociation constants of L and the stability constant of the L–Zn2+complex in 0.15 M NaClO4 were determined by pH metrically. Theoretical calculations were done using density functional theory (DFT) to support the above findings. L was successfully applied for the determination of Zn2+ in water samples. The compound (L) detects the presence of, Klebsiella pneumoniae and E. coli in water samples.

Four mononuclear metal complexes (Cu(II) (1), Ni(II) (2), Zn(II) (3) and V(IV) (4)) were synthesized using the Schiff base ligand 2,2′-{cyclohexane-1,2-diylbis[nitrilo(1E)eth-1-yl-1-ylidine]}bis[5-(prop-2-yn-1-yloxy)phenol] and structurally characterized by various spectral techniques. The catecholase-mimicking activities of 1–4 were investigated and the results reveal that all the complexes have ability to oxidize 3,5-di-tert-butylcatechol (3,5-DTBC) to 3,5-di-tert-butylquinone in aerobic conditions. Electrospray ionization mass spectrometry studies were performed for 1–4 in the presence of 3,5-DTBC to determine the possible complex–substrate intermediates. X-band electron paramagnetic resonance spectroscopy results indicate that the metal centres are involved in the catecholase activity. Ligand-centred radical generation was further confirmed by density functional theory calculation. The phosphatase-like activity of 1–4 was investigated using 4-nitrophenylphosphate as a model substrate. All the complexes exhibit excellent phosphatase activity in acetonitrile medium. The interactions of 1–4 with calf thymus DNA (CT-DNA) and bovine serum albumin (BSA) protein were investigated using absorption and fluorescence titration methods. All the complexes strongly interact with CT-DNA and BSA protein. The complexes exhibit significant hydrolytic cleavage of supercoiled pUC19 DNA. Complexes 1–4 exhibit significant in vitro cytotoxicity against MCF7 (human breast cancer) and MIA-PA-CA-2 (human pancreatic cancer) cell lines. Moreover, the molecular docking technique was employed to determine the binding affinity with DNA and protein molecules.

Colorectal cancer (CRC) is the leading cause of cancer-related mortality, annually responsible for around 655,000 deaths worldwide. CRC usually develops very slowly, over a period of 10 to 20 years and typically begin as a non-cancerous polyp that develops on the inner layer of the colon or rectum. However, CRC is curable if detected at an early stage. The current treatment of colorectal cancer primarily depends on surgery, chemotherapy, radiotherapy and targeted therapy. A combination of two or more treatments is often recommended to achieve the best outcome, depending on the stage of cancer development. In this review, we summarized the challenges of using 5-Fluorouracil as chemotherapy to treat colorectal cancer, and discussed the implications of nano drug delivery system for improved therapeutic outcome of 5-Flurouracil.

Lung development in the mammal carries series of steps starting from simple lung buds to the elaborately branched organ that fulfil the vital function of gaseous exchange. Strict transcriptional control is essential for lung development. To screen the genes responsible for the regional growth and patterning we used RNA Insitu hybridization to identify genes that show restricted expression patterns in the embryonic lung. During organogenesis, the expression of Foxf1 becomes restricted to the splanchnic mesoderm, which provides the mesenchymal cells of the intestinal tract and gut derivates such as lungs and liver. Surfactant is a lipoprotein substance that is synthesized and secreted by alveolar type II epithelial cells and acts to reduce surface tension at the air-alveolar interface. SP-C a 5 kDa, hydro-phobic, surfactant-associated protein. We used the technique of in situ hybridization to localize SP-C mRNA in mice lung tissue. SP-C mRNA was present in all of the epithelial cells of the prealveolar region of day 13 gestational age mice lung tissue. Our data are suggestive that SP-C is present in fetal lung prealveolar epithelial cells much earlier in gestation than are differentiated, surfactant-producing alveolar type II cells.

Human immunodeficiency virus (HIV) infections in sub-Saharan Africa represent about 56% of global infections. Many studies have targeted HIV-1 protease for the development of drugs against AIDS. Recombinant HIV-1 protease is used to screen new drugs from synthetic compounds or natural substances. Along with the wild type (C-SA) we also over-expressed and characterized two mutant forms from patients that had shown resistance to protease inhibitors. Using recombinant DNA technology, we constructed three recombinant plasmids in pGEX-6P-1 and expressed them containing a sequence encoding wild type HIV protease and two mutants (I36T↑T contains 100 amino acids and L38L↑N↑L contains 101 amino acids). These recombinant proteins were isolated from inclusion bodies by using QFF anion exchange and GST trap columns. In SDS-PAGE, we obtained these HIV proteases as single bands of approximately 11.5, 11.6 and 11.7 kDa for the wild type, I36T↑Tand L38L↑N↑L mutants, respectively. The enzyme was recovered efficiently (0.25 mg protein/L of Escherichia coli culture) and had high specific activity of 2.02, 2.20 and 1.33 μmol min(-1) mg(-1) at an optimal pH of 5 and temperature of 37 °C for the wild type, I36T↑T and L38L↑N↑L, respectively. The method employed here provides an easy and rapid purification of the HIV-1(C-SA) protease from the inclusion bodies, with high yield and high specific activities.

The enzyme CP4 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS; EC 2.5.1.19) from Agrobacterium tumefaciens CP4, encoded by the aroA gene, has been used for the construction of genetically modified crops resistant to total herbicide glyphosate. During the study of possible horizontal gene transfer of aroA CP4 gene from genetically modified food in gastrointestinal tract to bacterial community living in the animal gut, we have discovered and characterized truncated form of aroA CP4 within the cloning experiments in Escherichia coli. We have compared properties of the recombinant E. coli strains with both CP4 EPSPS enzyme forms.

The main task in the development of transgenic plants is the capability to distinguish between plant cells with an integrated transgene and the bulk of non-transformed cells. Selectable marker genes are required to achieve this goal within the transgene, and to select for their expression. These selectable markers are mostly based on genes conferring antibiotic or herbicide resistance. The presence of the marker gene will lead to unpredictable environmental hazards, so on the basis of economic incentives and safety concerns, several methods, such as site-specific recombination, homologous recombination and co-transformation, have been developed to eliminate these genes from the genome after successful transformation has been achieved. Gene transfer without the incorporation of an antibiotic-resistance marker or herbicide-resistance marker in the host genome should convince the public with regard to the field release of transgenic organisms. Moreover, it would obviate the need for different selectable markers in subsequent rounds of gene transfer into the same host. Copyright © 2007 Society of Chemical Industry.

An amino acid Schiff base (R) capable of recognizing Zn(2+) ions selectively and sensitively in an aqueous medium was prepared and characterized. Upon addition of Zn(2+) ions, the receptor exhibits fluorescence intensity enhancements (~40 fold) at 460 nm (quantum yield, Φ=0.05 for R and Φ=0.18 for R-Zn(2+)) and can be detected by naked eye under UV light. The receptor can recognize the Zn(2+) (1.04×10(-8) M) selectively for other metal ions in the pH range of 7.5-11. The Zn(2+) chelation with R decreases the loss of energy through non-radiative transition and leads to fluorescence enhancement. The binding mode of the receptor with Zn(2+) was investigated by (1)H NMR titration and further validated by ESI-MS. The elemental color mapping and SEM/EDS analysis were also used to study the binding of R with Zn(2+). Density functional theory calculations were carried out to understand the binding mechanism. The receptor was applied as a microbial sensor for Escherichia coli and Staphylococcus aureus.

Genetically modified (GM) foods are the product of one of the most progressive fields of science-biotechnology. There are major concerns about GM foods in the public; some of them are reasonable, some of them are not. Biomedical risks of GM foods include problems regarding the potential allergenicity, horizontal gene transfer, but environmental side effects on biodiversity must also be recognized. Numerous methods have been developed to assess the potential risk of every GM food type. Benefits of the first generation of GM foods were oriented towards the production process and companies, the second generation of GM foods offers, on contrary, various advantages and added value for the consumer. This includes improved nutritional composition or even therapeutic effects. Recombinant probiotics and the principle of alternative gene therapy represent the latest approach of using GM organisms for biomedical applications. This article tries to summarize and to explain the problematic topic of GM food.

Several protein expression systems for construction of protein arrays have been established in recent years. However, current protocols for protein synthesis are still time consuming, laborious and expensive. This study has established an alternative workflow that covers rapid construction of expression cassettes, in-tube and on-membrane synthesis of recombinant proteins, and straightforward screening of synthesized proteins. Eighteen membrane associated eukaryotic proteins and two secretory complement regulators (C1 inhibitor and vitronectin) were included in the study. To generate hybrid genes, double-overlap extension PCR was employed to fuse the 5' fragment (consisting of a T7 promoter and a species independent translation sequence), ORFs of the target proteins, and the 3' fragment (encompassing GFP fusion, Myc-tag and stop codon). OE-PCR generated fragments were directly mixed with the Leishmania torentolae lysate (translation mix) for protein synthesis. In order to establish a cheap and user-friendly alternative to existing cell-free protein array techniques, PCR products were spotted on the hydrophobic substrate (PVDF membrane), air-dried and covered with only 2 μL of translation mix. All synthesized proteins were spontaneously immobilized on the membrane due to the hydrophobic interaction between C-terminally fused GFP and PVDF. Synthesis and immobilization of proteins were confirmed simply by assessing the GFP chromophore under a laser scanner or a fluorescent microscope.

ACP4 gene encoding CP4 EPSPS from genetically modifiedfeed through gastrointestinal tract to bacteria living in ani-mal gut we have observed beside full length gene alsofunctional truncated one present in bacteria [1]. Thecodon usage of both forms was originally optimized forplant [2]. Therefore we have used two different