A Method Based on a Modified Fluorescence In Situ Hybridization (FISH) Approach for the Sensing of Staphylococcus aureus from Nasal Samples.

Staphylococcus aureus is a major source of bacteremia and develops several complications, causing high morbidity and mortality. Rapid identification and detection of these bacteria have become an important issue for biomedical applications. Herein, an optical method based on a modified fluorescence in situ hybridization (FISH) approach has been established using DNA hybridization technology for the swift detection of pathogenic S. aureus from clinical samples. The platform was constructed with single-stranded genomic DNA and microbial colony by directly immobilizing in agarose-polyvinyl alcohol (AG-PVA) hydrogel on the surface of a glass slide. The probe was based on an elongation factor encoding the tuf gene, which binds with equal affinity to single-stranded DNA targets as well as surface proteins on microbial cells. The probe was labeled with MFP488 fluorophore having excitation wavelength 501 nm. The hybridization of the labeled probe with the target DNA and surface proteins was carried out under optimal FISH conditions, and the detection of bacteria was based on temporary field excitation of the labeled probe under a fluorescence microscope. Positive hybridization signals were detected by high fluorescence intensity. In comparison to genomic DNA, robust signals were observed with microbial cells, perhaps due to the moonlighting effect of the elongation factor Tu (Ef-Tu) expressed on the surface of bacterial cells. The applicability of the developed platform was tested on pediatric nasal samples, and results were verified with real-time qPCR. The designed platform is stable and sensitive, and after detailed optimization, a portable structure for on-site detection of pathogenic bacteria from clinical samples can be produced.

Host-Guest Mechanism via Induced Fit Fullerene Complexation in Porphin Receptor to Probe Salivary Alpha-Amylase in Dental Caries for Clinical Applications.

Salivary α-amylase is the most abundant protein of human saliva that potentially binds to streptococcus and other bacteria via specific surface-exposed α-amylase-binding proteins and plays a significant role in caries development. The detection of α-amylase in saliva can be used as a bioindicator of caries development. Herein, a facile strategy has been applied, tailoring the photochemical properties of 5,10,15,20-tetrakis(4-hydroxyphenyl)-21H,23H-porphine (TPPOH) and the fullerene C60 complex. The fluorescence emission of TPPOH is quenched by starch-coated fullerene C60 via charge-transfer effects, as determined by UV absorption and fluorescence spectroscopic studies. The starch-coated C60 has been thoroughly characterized via Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), field emission scanning electron microscopy (FE-SEM), optical microscopy, thermal gravimetric analysis (TGA), static water contact angle measurements, and zeta potential measurements. The analytical response of the assay showed a linear fluorescent response in α-amylase concentrations ranging from 0.001-0.1 Units/mL, with an LOD of 0.001 Units/mL. The applicability of the method was tested using artificial saliva with quantitative recoveries in the range 95-100%. The practicability of the procedure was verified by inspecting saliva samples of real clinical samples covering all age groups. We believe that the proposed method can serve as an alternative analytical method for caries detection and risk assessment that would also minimize the cost of professional preventive measures and treatments.

Rhodamine B functionalized silver nanoparticles paper discs as turn-on fluorescence sensor, coupled with a smartphone for the detection of microbial contamination in drinking water.

The precise detection of pathogenic microorganisms is crucial for the reduction of water-borne diseases. Herein, a filter-paper-based florescent chemosensor was fabricated for the detection of Escherichia coli and Staphylococcus aureus contamination exploiting protein-DNA interaction between the target and a specific probe. The sensing mechanism involved the self-assembly of Rhodamine B (RhB) on silver nanoparticles (AgNPs) surface that was labeled with a single-stranded DNA probe. This causes the fluorescence quenching of RhB by a distant-dependant process. The hybridization between pathogen-specific probe and bacterial surface protein causes the release of fluorescence of RhB, which was observed under UV light. For paper-based bio-surface preparation, the mixture comprising RhB-AgNP-ssDNA was drop-casted on filter paper discs. The conditions were optimized using isolated genomic DNA of the microbes. The method was applied for E.coli detection using an eae gene-based probe targeting intimin protein and S. aureus detection using tuf gene-based probe targeting EF-tuf protein on the microbe's surface. The chemosensor had a notable specificity and selectivity for E.coli, and S. aureus, with detection limits of 0.6 × 108 and 0.37 × 103 CFU/mL respectively. Moreover, the sensor was tested on real water samples, which presented excellent reproducibility of results (RSD ≤ 0.24%). Furthermore, the gradient change of fluorescence was captured by a smartphone, which allows direct detection of pathogens in a sensitive semi-quantitative way without the need for expensive instruments. The designed chemosensor can serve as a simple, inexpensive, and rapid method for the on-site detection of microbial contamination in drinking water.

A novel construct of an electrochemical acetylcholinesterase biosensor for the investigation of malathion sensitivity to three different insect species using a NiCr2O4/g-C3N4 composite integrated pencil graphite electrode.

Herein, an electrochemical biosensor has been prepared to assess the sensitivity of an organophosphate insecticide, malathion, to acetylcholinesterase (AChE) enzyme of three insects including Apis mellifera (honeybee), Tribolium castaneum (red flour beetle), and Zootermopsis nevadensis (dampwood termite). A composite of nickel chromite (NiCr2O4) and graphitic carbon nitride (g-C3N4) was prepared and characterized for its morphological, chemical and electrical properties. The NiCr2O4/g-C3N4 composite integrated pencil graphite electrodes were used to covalently immobilize insect AChE enzymes and amperometric response of bioelectrodes was determined through cyclic voltammetry. The prepared bioelectrodes exhibited high enzyme immobilization efficiency and electro-catalytic performance. The integrated bioelectrodes could efficiently detect malathion induced inhibition of insects' AChEs. The linear ranges for malathion were found to be 0.1-1.6 µM, 1-40 nM and 2-100 nM, and LODs were 2 nM, 0.86 nM and 2.3 nM for A. mellifera, T. castaneum, and Z. nevadensis, respectively. Additionally, the biosensing platform developed using A. mellifera AChE was found highly sensitive and effective for malathion recoveries from spiked wheat flour samples with high recovery rates. Moreover, the proposed method was adequately reproducible and selective. The results revealed that A. mellifera AChE is less sensitive to inhibition by malathion as compared to T. castaneum, and Z. nevadensis AChE. The experimental results were validated through computational docking of malathion with insect AChEs and the results were in correspondence to experimental outcomes. The proposed method can be a plausible alternate to conventional analytical methods to assess the pesticide sensitivity and toxicity of various compounds against insect enzymes.

Efficient Preparation of a Nonenzymatic Nanoassembly Based on Cobalt-Substituted Polyoxometalate and Polyethylene Imine-Capped Silver Nanoparticles for the Electrochemical Sensing of Carbofuran.

The ever-growing exploitation of pesticides and their lethal effects on living beings have made it a dire need of the day to develop an accurate and reliable approach for their monitoring at trace levels. The designing of an enzyme-free electrocatalyst to electrochemically detect the pesticide residues is currently gaining much importance. In this study, a novel redox-sensing film was constructed successfully based on cobalt-substituted Dawson-type polyoxometalate [P2W17O61 (Co2+·OH2)]7- (Co-POM) and polyethylene imine (PEI)-capped silver nanoparticles (AgNPs). A nanohybrid assembly was fabricated on a glassy carbon electrode's surface by alternately depositing Co-POM and PEI-AgNPs using the layer-by-layer self-assembly method. The surface morphology of the immobilized CoPOM/AgNP multilayer nanoassembly was analyzed through scanning electron microscopy along with energy-dispersive spectroscopy for elemental analysis. The redox properties and surface morphologies of fabricated assemblies were evaluated by cyclic voltammetry and electrochemical impedance spectroscopy. The practicability and feasibility of the proposed sensing layer was tested for the detection of a highly toxic insecticide, that is, carbofuran. The fabricated sensor exhibited a limit of detection of 0.1 mM with a sensitivity of 13.11 µA mM-1 for carbofuran. The results depicted that the fabricated nonenzymatic hybrid film showed excellent electrocatalytic efficiency for the carbofuran oxidation. Furthermore, the obtained value of "apparent Km", that is, 0.4 mM, illustrates a good electro-oxidation activity of the sensor for the detection of carbofuran. The exceptionally stable redox activity of Co-POM, high surface area and greater conductivity of AgNPs, and the synergistic effect of all components of the film resulted in an excellent analytical performance of the proposed sensing assembly. This work provides a new direction to the progress and designing of nonenzymatic electrochemical sensors for pesticide determination in real samples.

Assessment of pesticide induced inhibition of Apis mellifera (honeybee) acetylcholinesterase by means of N-doped carbon dots/BSA nanocomposite modified electrochemical biosensor.

This work describes the development and optimization of an electrochemical method to evaluate pesticide induced inhibition of honey bee (Apis mellifera) acetylcholinesterase (AChE) by means of acetylcholinesterase biosensor. The inhibition assay was based on the detection of changes in electrochemical activity of the enzyme caused by pesticide. As transducer, nitrogen doped carbon dots BSA (N-CD/BSA) nanocomposite electrodeposited on pencil graphite electrode was used to covalently immobilize AChE. The as-synthesized nanocomposite and fabricated electrodes were characterized for the structural, functional and electrochemical properties. Nanocomposite promoted the electron transfer reaction to catalyze the electro-oxidation of thiocholine and a large current response was obtained by cyclic voltammetry at 0.77 V, indicating successful immobilization of AChE. The sensitivity of Diazinon, an OP insecticide, for honeybee AChE was tested under optimal conditions and a linear response ranging 10-250 nM was obtained with a detection limit of 8.9 nM, and sensitivity 9 uA/nM/cm2. The method showed a good operational reproducibility and selectivity of biosensor. Further, the molecular docking provided additional support to the experimental data suggesting irreversible nature and contact toxicity of the pesticide for honey bee AChE. The developed biosensor has proved useful for the diazinon detection in wheat samples with 99% recovery rate.

An insect acetylcholinesterase biosensor utilizing WO3/g-C3N4 nanocomposite modified pencil graphite electrode for phosmet detection in stored grains.

This study was undertaken to assess the potential of Tribolium castaneum (Red flour beetle) acetylcholinesterase (Tc-AChE) based electrochemical biosensor integrating WO3/g-C3N4 nanocomposite modified Pencil graphite electrode to detect an organophosphate insecticide, Phosmet. The WO3/g-C3N4 nanocomposite provides a non-toxic, biocompatible surface for binding the enzyme on the electrode surface, attributed to its large surface area, high conductivity, and low ohmic resistance. The proposed biosensor shows a very good analytical performance with LOD 3.6 nM for Phosmet and effectively determined Phosmet in wheat with a 99% recovery rate. Furthermore, molecular docking deciphers the binding interactions of Phosmet with Tc-AChE using a modified AutoDock LGA algorithm and an AMBER03 force field in YASARA. The kinetic parameters strongly suggest the high potency of inhibitor with the enzyme. This study presents an adaptable, rapid, and straightforward approach that opens ways towards real progress in developing commercial biosensors for pesticide detection.