Sensing of trans-cleavage activity of CRISPR/Cas12a for detection of Salmonella.

Salmonella typhimurium (S. typhi) a predominant foodborne pathogen, significantly impacting global public health. Therefore, timely diagnosis is imperative to safeguard overall human health. To address this, we developed a novel CRISPR/Cas12a-mediated electrochemical detection system (biosensor) for targeting the SifA gene of S. typhi. To construct the biosensor, we utilized a screen-printed gold electrode (SPGE) as an electrochemical transducer and CRISPR/Cas12a for detection of SifA gene of S. typhi. The developed electrochemical biosensor exhibited an exceptional detection limit of 0.634 ± 0.029 pM, which was determined through differential pulse voltammetry (DPV) by utilizing a potentiostat. We compared the fabricated biosensor with gold standard RT-PCR and the visual detection limit of SifA was found to be 10 µM (in spiked buffer samples). The lower detection limit of fabricated biosensor provides an upper edge over the RT-PCR. Further, the fabricated biosensor also has the potential to serve as a rapid, stable, efficient, and early detection tool for S. typhi, offering promising advancements in diagnostic realms.

Immunochromatographic assay for the point-of-care diagnosis of food borne Salmonella strains using smartphone application.

Salmonella strain is a prevalent pathogen, affecting poultry industry and hence human population around the world. Host-specific pathogen infections including fowl typhoid, pullorum disease and typhoid fever affects poultry birds, causing huge economic loss worldwide. This study explored the fabrication of immunochromatographic (ICG) strip by colorimetric method integrated with smartphone ColorGrab application for the detection of Salmonella using in-house generated antibodies (Abs) conjugated with gold nanoparticles. The developed point-of-care diagnostic platform was fabricated in-house and tested to detect the presence of Salmonella in a linear range of 107-100 CFU/mL with the limit of detection (LOD) of 103, 102 and 104 CFU/mL respectively, for Salmonella gallinarum (S.gal), Salmonella pullorum (S.pul) and Salmonella enteritidis (S.ent), which was further confirmed by smartphone-based ColorGrab application. The fabricated ICG strips were further validated using spiked fecal, meat, and milk samples which provided results in 10 mins with stability at 4 °C and 37 °C up to 28 days. Hence, the fabricated in-house ICG strip can be used as a portable, cost-effective diagnostic device for rapid detection of Salmonella strains in food samples.

Recent Advances in Electrochemical Biosensors for the Detection of Salmonellosis: Current Prospective and Challenges.

Salmonellosis is a major cause of foodborne infections, caused by Salmonella, posing a major health risk. It possesses the ability to infiltrate the food supply chain at any point throughout the manufacturing, distribution, processing or quality control process. Salmonella infection has increased severely and requires effective and efficient methods for early monitoring and detection. Traditional methods, such as real-time polymerase chain reaction and culture plate, consume a lot of time and are labor-intensive. Therefore, new quick detection methods for on-field applications are urgently needed. Biosensors provide consumer-friendly approaches for quick on-field diagnoses. In the last few years, there has been a surge in research into the creation of reliable and advanced electrochemical sensors for the detection of Salmonella strains in food samples. Electrochemical sensors provide extensive accuracy and reproducible results. Herein, we present a comprehensive overview of electrochemical sensors for the detection of Salmonella by focusing on various mechanisms of electrochemical transducer. Further, we explain new-generation biosensors (microfluidics, CRISPR- and IOT-based) for point-of care applications. This review also highlights the limitations of developing biosensors in Salmonella detection and future possibilities.

Label free detection of SARS CoV-2 Receptor Binding Domain (RBD) protein by fabrication of gold nanorods deposited on electrochemical immunosensor (GDEI).

Coronavirus Disease 2019 (COVID-19) pandemic has shown the need for early diagnosis to manage infectious disease outbreaks. Here, we report a label free electrochemical Fluorine-Doped Tin Oxide (FTO) Immunosensor coupled with gold nanorods (GNRs) as an electron carrier for ultrasensitive detection of the Receptor Binding Domain (RBD) of SARS CoV-2 Spike protein. The RBD gene was cloned, and expressed in-house with confirmed molecular weight of ∼31 kDa via Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Matrix-Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF). RBD antibodies (Ab) were generated to be used as a bioreceptor for sensor fabrication, and characterized using SDS-PAGE, Western Blot, and Enzyme-Linked Immunosorbent Assay (ELISA). GNRs were fabricated on the electrode surface, followed by immobilization of RBD Ab. The conjugation steps were confirmed by UV-Vis Spectroscopy, Dynamic Light Scattering (DLS), Atomic Force Microscopy (AFM), Transmission Electron Microscopy (TEM), Cyclic Voltammetry (CV), and Differential Pulse Voltammetry (DPV). The fabricated electrode was further optimized for maximum efficiency and output. The detection limit of the developed electrode was determined as 0.73 fM for RBD antigen (Ag). Furthermore, the patient nasopharyngeal samples were collected in Viral Transport Media (VTM), and tested on the sensor surface that resulted in detection of SARS CoV-2 within 30 s, which was further validated via Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Moreover, the immunosensor showed good repeatability, storage stability, and minimal cross reactivity against Middle East Respiratory Syndrome (MERS) spike protein. Along with ease of fabrication, the electrodes show future miniaturization potential for extensive and rapid screening of populations for COVID-19.

Probe-free nanosensor for the detection of Salmonella using gold nanorods as an electroactive modulator.

Salmonellosis is a symptomatic infection, a foodborne disease, caused by Salmonella that enters the body through the ingestion of contaminated food. In this study, a novel electrochemical biosensor integrated with gold nanorods (GNRs) was used to explore the interaction between in-house generated antibodies with Salmonella serovars. Under optimal conditions, the proposed immunosensor depicted a linear range of detection (1-1 × 105) CFU/mL witha detection limit of 105 and 23 colony forming units (CFU) ofS. entandS. typhirespectively. The designed GNR/S. ent/S. typhi/Ab immunosensor was able to successfully detectS. ent/S. typhiin spiked meat and milk samples respectively, with a long shelf life, good repeatability, as well as reproducibility under optimised conditions. Along with the ease of fabrication, the developed electrode produced a highly specific response, and displayed negligible cross reactivity with other Salmonella species. Moreover, the established detection technique may be used as an alternative to conventional analytical approaches for rapid and sensitivediagnosis of Salmonellosis.

Electrochemical Detection of Alzheimer's Disease Biomarker, ß-Secretase Enzyme (BACE1), With One-Step Synthesized Reduced Graphene Oxide.

ß-Secretase1 (BACE1) catalyzes the rate-limiting step in the generation of amyloid-ß peptides, that is, the principal component involved in the pathology of Alzheimer's disease (AD). Recent research studies show correlation between blood and cerebrospinal fluid (CSF) levels of BACE1 with the pathophysiology of AD. In this study, we report one-step synthesized reduced graphene oxide (rGO), activated via carbodiimide chemistry, conjugated with BACE1 antibody (Ab), and immobilized on fluorine-doped tin oxide (FTO) electrodes for rapid detection of BACE1 antigen (Ag) for AD diagnosis. The synthesis and fabrication steps were characterized using different types of spectroscopic, X-ray analytic, microscopic, and voltametric techniques. Various parameters including nanomaterial/Ab concentration, response time, pH, temperature, and rate of scan were standardized for maximum current output using the modified electrode. Final validation was performed via detection of BACE1 Ag ranging from 1 fM to 1 µM, with a detection limit of 0.64 fM in buffer samples and 1 fM in spiked serum samples, as well as negligible cross-reactivity with neurofilament Ag in buffer, spiked serum, and spiked artificial CSF. The proposed immunosensor gave a quick result in 30 s, and good repeatability and storage stability for a month, making it a promising candidate for sensitive, specific, and early diagnosis of AD. Thus, the fabricated electrochemical biosensor for BACE-1 detection improves detection performance compared to existing sensors as well as reduces detection time and cost, signifying its potential in early diagnosis of AD in clinical samples.

Electrochemical immunosensor for detection of avian Salmonellosis based on electroactive reduced graphene oxide (rGO) modified electrode.

A novel reduced graphene oxide based (rGO) fluorine doped tin oxide (FTO) electrode was fabricated to explore the interaction of Salmonella serovars (Salmonella gallinarum, and Salmonella pullorum) with specific antibodies. Reduced graphene oxide (rGO) was labelled with S. gal and S. pul-Ab via carbodiimide activation. The biophysical characterization of fabricated electrode was done by Fourier-transform infrared spectroscopy (FT-IR), Raman Spectroscopy, X-ray Diffraction (XRD), Scanning Electron Microscopy (SEM), Energy Dispersive X-Ray Analysis (EDX), cyclic voltammetry (CV), and differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS). The optimization of fabricated electrode was done for various physico-chemical parameters. Under optimum conditions, the immunosensor exhibited a linear detection range (1- 1 × 105 cells) with 37 and 25 viable cells of S. gal and S. pul, respectively. The developed FTO/rGO/S.gal or S.pul-Ab/Ag immunosensor successfully detected S. gal or S. pul up to 51 and 37 cells, respectively in faecal samples and 218 and 173 cells, respectively in meat samples. FTO/rGO/S.gal or S.pul-Ab/Ag immunosensor revealed satisfactory response, and exhibited relatively low detection limit along with reproducibility. The proposed sensing model can be used as an alternative quantitative tool for the rapid and sensitive detection of Salmonellosis in meat and faecal samples.

Label-free detection of SARS-CoV-2 Spike S1 antigen triggered by electroactive gold nanoparticles on antibody coated fluorine-doped tin oxide (FTO) electrode.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, also known as 2019-nCov or COVID-19) outbreak has become a huge public health issue due to its rapid transmission making it a global pandemic. Here, we report fabricated fluorine doped tin oxide (FTO) electrodes/gold nanoparticles (AuNPs) complex coupled with in-house developed SARS-CoV-2 spike S1 antibody (SARS-CoV-2 Ab) to measure the response with Cyclic Voltammetry (CV) and Differential Pulse Voltammetry (DPV). The biophysical characterisation of FTO/AuNPs/SARS-CoV-2Ab was done via UV-Visible spectroscopy, Dynamic Light Scattering (DLS), and Fourier Transform Infrared Spectroscopy (FT-IR). The fabricated FTO/AuNPs/SARS-CoV-2Ab immunosensor was optimised for response time, antibody concentration, temperature, and pH. Under optimum conditions, the FTO/AuNPs/Ab based immunosensor displayed high sensitivity with limit of detection (LOD) up to 0.63 fM in standard buffer and 120 fM in spiked saliva samples for detection of SARS-CoV-2 spike S1 antigen (Ag) with negligible cross reactivity Middle East Respiratory Syndrome (MERS) spike protein. The proposed FTO/AuNPs/SARS-CoV-2Ab based biosensor proved to be stable for up to 4 weeks and can be used as an alternative non-invasive diagnostic tool for the rapid, specific and sensitive detection of SARS-CoV-2 Spike Ag traces in clinical samples.

Molecular prospect of type-2 diabetes: Nanotechnology based diagnostics and therapeutic intervention.

About ninety percent of all diabetic conditions account for T2D caused due to abnormal insulin secretion/ action or increased hepatic glucose production. Factors that contribute towards the aetiology of T2D could be well explained through biochemical, molecular, and cellular aspects. In this review, we attempt to explain the recent evolving molecular and cellular advancement associated with T2D pathophysiology. Current progress fabricated in T2D research concerning intracellular signaling cascade, inflammasome, autophagy, genetic and epigenetics changes is discretely explained in simple terms. Present available anti-diabetic therapeutic strategies commercialized and their limitations which are needed to be acknowledged are addressed in the current review. In particular, the pre-eminence of nanotechnology-based approaches to nullify the inadequacy of conventional anti-diabetic therapeutics and heterogeneous nanoparticulated systems exploited in diabetic researches are also discretely mentioned and are also listed in a tabular format in the review. Additionally, as a future prospect of nanotechnology, the review presents several strategic hypotheses to ameliorate the austerity of T2D by an engineered smart targeted nano-delivery system. In detail, an effort has been made to hypothesize novel nanotechnological based therapeutic strategies, which exploits previously described inflammasome, autophagic target points. Utilizing graphical description it is explained how a smart targeted nano-delivery system could promote ß-cell growth and development by inducing the Wnt signaling pathway (inhibiting Gsk3ß), inhibiting inflammasome (inhibiting NLRP3), and activating autophagic target points (protecting Atg3/Atg7 complex from oxidative stress) thereby might ameliorate the severity of T2D. Additionally, several targeting molecules associated with autophagic and epigenetic factors are also highlighted, which can be exploited in future diabetic research.

Graphene functionalized field-effect transistors for ultrasensitive detection of Japanese encephalitis and Avian influenza virus.

Graphene, a two-dimensional nanomaterial, has gained immense interest in biosensing applications due to its large surface-to-volume ratio, and excellent electrical properties. Herein, a compact and user-friendly graphene field effect transistor (GraFET) based ultrasensitive biosensor has been developed for detecting Japanese Encephalitis Virus (JEV) and Avian Influenza Virus (AIV). The novel sensing platform comprised of carboxy functionalized graphene on Si/SiO2 substrate for covalent immobilization of monoclonal antibodies of JEV and AIV. The bioconjugation and fabrication process of GraFET was characterized by various biophysical techniques such as Ultraviolet-Visible (UV-Vis), Raman, Fourier-Transform Infrared (FT-IR) spectroscopy, optical microscopy, Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM). The change in the resistance due to antigen-antibody interaction was monitored in real time to evaluate the electrical response of the sensors. The sensors were tested in the range of 1 fM to 1 µM for both JEV and AIV antigens, and showed a limit of detection (LOD) upto 1 fM and 10 fM for JEV and AIV respectively under optimised conditions. Along with ease of fabrication, the GraFET devices were highly sensitive, specific, reproducible, and capable of detecting ultralow levels of JEV and AIV antigen. Moreover, these devices can be easily integrated into miniaturized FET-based real-time sensors for the rapid, cost-effective, and early Point of Care (PoC) diagnosis of JEV and AIV.

Author Correction: Fabrication of microfluidic device for Aflatoxin M1 detection in milk samples with specific aptamers.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Fabrication of microfluidic device for Aflatoxin M1 detection in milk samples with specific aptamers.

This study describes the colorimetric detection of aflatoxin M1 (Afl M1) in milk samples using a microfluidic paper-based analytical device (µPAD). Fabrication of µPADs was done using a simple and quick approach. Each µPAD contained a detection zone and a sample zone interconnected by microchannels. The colorimetric assay was developed using unmodified AuNPs as a probe and 21-mer aptamer as a recognition molecule. The free aptamers were adsorbed onto the surface of AuNPs in absence of Afl M1, even at high salt concentrations. The salt induced aggregation of specific aptamers occurred in presence of Afl M1. Under optimum conditions, the analytical linear range was found to be 1 µM to 1 pM with limit of detection 3 pM and 10 nM in standard buffer and spiked milk samples respectively. The proposed aptamer based colorimetric assay was repeatable, quick, selective, and can be used for on-site detection of other toxins in milk and meat samples.