Wandoo Walk 1

Cervical cancer is a life-threatening complication, appearing as the uncontrolled growth of abnormal cells in the lining of the cervix. Every year, increasing numbers of cervical cancer cases are reported worldwide. Different identification strategies were proposed to detect cervical cancer at the earlier stages using various biomarkers. Squamous cell carcinoma antigen (SCC-Ag) is one of the potential biomarkers for this diagnosis. Nanomaterial-based detection systems were shown to be efficient with different clinical biomarkers. In this study, we have demonstrated strontium oxide-modified interdigitated electrode (IDE) fabrication by the sol-gel method and characterized by scanning electron microscopy and high-power microscopy. Analysis of the bare devices indicated the reproducibility with the fabrication, and further pH scouting on the device revealed that the reliability of the working pH ranges from 3 to 9. The sensing surface was tested to detect SCC-Ag against its specific antibody; the detection limit was found to be 10 pM, and the sensitivity was in the range between 1 and 10 pM as calculated by 3σ. The specificity experiment was carried out using major proteins from human serum, such as albumin and globulin. SCC-Ag was shown to be selectively detected on the strontium oxide-modified IDE surface

Encapsulation of fungal extracellular enzyme cocktail in cellulose nanoparticles: enhancement in enzyme stability

We demonstrated the nano-immobilization of fungal enzymes through their encapsulation in cellulose nanoparticles (CNPs). An extracellular enzyme cocktail (a mixture of amylase, protease, lipase, and cellulose) was produced from Aspergillus niger and Phanerochaete chrysosporium through submerged fermentation. The process of encapsulation was carried out through a microemulsion nanoprecipitation method in the presence of a lipid, a surfactant, and a co-surfactant. The morphology of CNPs was determined by field-emission scanning electron microscopy and transmission electron microscopy; CNPs were less than 100 nm in diameter. Fourier transform infrared spectroscopy (FTIR) and energy dispersive spectroscopy demonstrated the successful encapsulation of the fungal enzyme cocktail and revealed C and O as its major components. FTIR peaks of CNPs with encapsulated enzymes occurred at 3421.80, 2828.91, 1649.29, 1450.24, and 1061.61 cm−1 as well as in the range of 1050–1150 cm−1. Encapsulated enzymes showed excellent stability with a peak at −70.91 mV in zeta potential studies. Thermogravimetric analysis proved that the CNP-encapsulated enzymes had an initial weight loss at 250C. The encapsulated fungal enzyme cocktail exhibited higher catalytic performance and stability than the free enzymes. The encapsulated fungal enzyme cocktail derived from A. niger at the concentration of 100 µg/mL, showed the highest amylase activity with a clear zone of 2.5 cm. Overall, the results of this research reveal the enhancement in the activity of fungal extracellular enzyme cocktail through nanoencapsulation

Amino acid analysis of lipases from oil pollutant isolates: Cunninghamella verticillata and Geotrichum candidum

Lipase is an enzyme commonly used in food, dairy, and other industries. Fungal lipases are more commonly used due to their secretion and easier production. Analyses of the amino acid composition of microbial lipases will hasten their potential usage in industrial applications. In this study, the major amino acid compositions of lipases secreted by oil pollutant isolates (Cunninghamella verticillata and Geotrichum candidum) enriched with fatty substances were analyzed by high performance liquid chromatography. Among eight major amino acids found in these lipases, histidine and ornithine were predominant. Lysine was absent from lipase generated by C. verticillata, while glutamine was absent from that produced by G. candidum. Conversely, glutamic acid, asparagine, histidine and arginine were present in slightly higher amounts in G. candidum. However, a slight decrease in aspartic acid and ornithine was observed in G. candidum. Analyses of the amino acids composition of lipase can potentially facilitate to predict the nature of this enzyme

Amine-Aldehyde Chemical Conjugation on a Potassium Hydroxide-Treated Polystyrene ELISA Surface for Nanosensing an HIV-p24 Antigen

Abstract The enzyme-linked immunosorbent assay (ELISA) has been widely used for disease surveillance and drug screening due to its relatively higher accuracy and sensitivity. Fine-tuning the ELISA is mandatory to elevate the specific detection of biomolecules at a lower abundance. Towards this end, higher molecular capture on the polystyrene (PS) ELISA surface is crucial for efficient detection, and it could be attained by immobilizing the molecules in the correct orientation. It is highly challenging to immobilize protein molecules in a well-aligned manner on an ELISA surface due to charge variations. We employed a 3-(aminopropyl) triethoxysilane (APTES)- and glutaraldehyde (GLU)-coupled PS surface chemical strategy to demonstrate the high performance with ELISA. A potassium hydroxide treatment followed by an equal ratio of 1% APTES and GLU attachment was found to be optimal, and a longer incubation with GLU favored maximum sensitivity. p24 is a vital early secreting antigen for diagnosing human immunodeficiency virus (HIV), and it has been used for efficient detection with the above chemistry. Three different procedures were followed, and they led to the improved detection of the HIV-p24 antigen at 1 nM, which is a 30-fold higher level compared to a conventional ELISA surface. The surface chemical functionalization shown here also displays a higher specificity with human serum and HIV-TAT. The above approach with the designed surface chemistry could also be recommended for disease diagnosis on other sensing surfaces involving the interaction of the probe and the analyte in heterogeneous test samples

Strategies to Characterize Fungal Lipases for Applications in Medicine and Dairy Industry

Lipases are water-soluble enzymes that act on insoluble substrates and catalyze the hydrolysis of long-chain triglycerides. Lipases play a vital role in the food, detergent, chemical, and pharmaceutical industries. In the past, fungal lipases gained significant attention in the industries due to their substrate specificity and stability under varied chemical and physical conditions. Fungal enzymes are extracellular in nature, and they can be extracted easily, which significantly reduces the cost and makes this source preferable over bacteria. Soil contaminated with spillage from the products of oil and dairy harbors fungal species, which have the potential to secrete lipases to degrade fats and oils. Herein, the strategies involved in the characterization of fungal lipases, capable of degrading fatty substances, are narrated with a focus on further applications

Red Spectral Shift in Sensitive Colorimetric Detection of Tuberculosis by ESAT-6 Antigen-Antibody Complex: a New Strategy with Gold Nanoparticle

Abstract Tuberculosis (TB) is a highly contagious life-threatening disease caused by the bacterial pathogen Mycobacterium tuberculosis. ESAT-6, an abundant early secretory antigenic target protein by M. tuberculosis, found to play a vital role in virulence. Developing a friendly method for the detection of ESAT-6 at the lower concentration facilitates to treat TB at an earlier stage and helps to control the spreading of disease. Herein, a new single-step approach was designed and was done by pre-mixing ESAT-6 and antibody before being added to the gold nanoparticle (GNP) followed by the salt-induced aggregation. We could attain the detection limit of 1.25 pM, showing the aggregation of GNP and the red spectral shift. Further, a higher specificity was demonstrated with the lack of electrostatic biofouling by ESAT-6 on GNP and retained the dispersed GNP in the presence of 10-kDa culture filtrate protein from M. tuberculosis. The required precise antibody concentration for this assay was found to be 60 nM. The increment in the antibody concentration from 75 nM drastically diminishes the sensitivity to ~ 680-fold, due to the crowding effect. With this assay, we attested the suitability of colorimetric assay for efficiently detecting the smaller-sized protein

Biotin-Streptavidin Competition Mediates Sensitive Detection of Biomolecules in Enzyme Linked Immunosorbent Assay.

Enzyme Linked Immunosorbent Assay (ELISA) is the gold standard assay for detecting and identifying biomolecules using antibodies as the probe. Improving ELISA is crucial for detecting disease-causing agents and facilitating diagnosis at the early stages of disease. Biotinylated antibody and streptavidin-conjugated horse radish peroxide (streptavidin-HRP) often are used with ELISA to enhance the detection of various kinds of targets. In the present study, we used a competition-based strategy in which we pre-mixed free biotin with streptavidin-HRP to generate high-performance system, as free biotin occupies some of the biotin binding sites on streptavidin, thereby providing more chances for streptavidin-HRP to bind with biotinylated antibody. ESAT-6, which is a protein secreted early during tuberculosis infection, was used as the model target. We found that 8 fM of free biotin mixed with streptavidin-HRP anchored the higher detection level of ESAT-6 by four-fold compared with detection without free biotin (only streptavidin-HRP), and the limit of detection of the new method was 250 pM. These results suggest that biotin-streptavidin competition can be used to improve the diagnosis of analytes in other types of sensors