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.

Smartphone-assisted detection of monocrotophos pesticide using a portable nano-enabled chromagrid-lightbox system towards point-of-care application.

With the aim of monocrotophos pesticides detection in environmental and food samples at point-of-care (PoC) application, this research, for the first time, explores silica alcogel as an immobilization matrix to support the development of in-house customized nano-enabled "chromagrid-lighbox" as a sensing system. This system is fabricated using laboratory waste materials and demonstrates the detection of highly hazardous monocrotophos pesticide using a smartphone. Nano-enabled chromagrid is a chip-like assembly filled with silica alcogel -a nanomaterial (hence the name "nano-enabled" chromagrid), and "chromogenic reagents" which is required for the enzymatic detection of monocrotophos. Lightbox is the imaging station fabricated to provide constant lighting conditions to the chromagrid to capture accurate colorimetric data. The silica alcogel used in this system was synthesized from Tetraethyl orthosilicate (TEOS) via a sol-gel method and characterized using advanced analytical techniques. Further, three chromagrid assays were developed for the optical detection of monocrotophos with a low detection limit (LOD) at 0.421 ng ml-1 (by α-NAc chromagrid assay), 0.493 ng ml-1 (by DTNB chromagrid assay) and 0.811 ng ml-1 (by IDA chromagrid assay). The developed novel PoC chromagrid-lightbox system is capable of on-site detection of monocrotophos in environmental as well as food samples. This system is able to be manufacture prudently using recyclable waste plastic. Overall, such developed eco-friendly PoC testing system will surely manage rapid detection of monocrotophos pesticide needed for environmental and sustainable agricultural management.

Emerging 0D, 1D, 2D, and 3D nanostructures for efficient point-of-care biosensing.

The recent COVID-19 infection outbreak has raised the demand for rapid, highly sensitive POC biosensing technology for intelligent health and wellness. In this direction, efforts are being made to explore high-performance nano-systems for developing novel sensing technologies capable of functioning at point-of-care (POC) applications for quick diagnosis, data acquisition, and disease management. A combination of nanostructures [i.e., 0D (nanoparticles & quantum dots), 1D (nanorods, nanofibers, nanopillars, & nanowires), 2D (nanosheets, nanoplates, nanopores) & 3D nanomaterials (nanocomposites and complex hierarchical structures)], biosensing prototype, and micro-electronics makes biosensing suitable for early diagnosis, detection & prevention of life-threatening diseases. However, a knowledge gap associated with the potential of 0D, 1D, 2D, and 3D nanostructures for the design and development of efficient POC sensing is yet to be explored carefully and critically. With this focus, this review highlights the latest engineered 0D, 1D, 2D, and 3D nanomaterials for developing next-generation miniaturized, portable POC biosensors development to achieve high sensitivity with potential integration with the internet of medical things (IoMT, for miniaturization and data collection, security, and sharing), artificial intelligence (AI, for desired analytics), etc. for better diagnosis and disease management at the personalized level.

Ultrasensitive immunosensing of Penicillin G in food samples using reduced graphene oxide (rGO) decorated electrode surface.

The impact of uncontrolled antibiotic use in animals has subsequently led to emergence of antibiotic-resistant bacteria among humans due to consumption of animal by-products. Hence, to investigate antibiotic contamination in animal origin food products, we have developed a reduced graphene oxide (rGO) based immunosensor using fabricated electrode conjugated with anti-Penicillin antibody (rGO/Pen-Ab) for sensitive detection of Penicillin G. To execute this, Penicillin was first conjugated with Bovine Serum Albumin (BSA) which was confirmed via chromatographic, spectroscopic and electrophoretic-based techniques against both the in-house developed Penicillin conjugate (Pen-BSA) as well as the commercial Penicillin conjugate (Com-Pen-BSA). Further, we fabricated electrode based on one step synthesized rGO and immobilized with antibodies generated against Pen-BSA (Pen-Ab), and Com-Pen-BSA (Com-Pen-Ab), separately for detection of Penicillin. Each synthesis and conjugation step was confirmed by different spectroscopic methods. For efficient working of the electrode, various parameters were optimized using Voltammetry. The limit of detection for Penicillin G against Pen-Ab and Com-Pen-Ab was determined as 0.724 pM and 0.668 pM respectively and both displayed negligible cross reactivity against other ß-lactam antibiotics (Cefalexin and Ampicillin). Furthermore, antibiotics were also detected in spiked milk, egg and meat samples and the electrode was evaluated for repeatability and storage stability. In conclusion, in-house developed Pen-Ab showed better sensitivity as compared to Com-Pen-Ab. The fabricated rGO/Pen-Ab biosensor shows future potential for rapid detection of penicillin and other ß-lactam antibiotics for safe consumption of animal by-products in humans.

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.

A Recent Update on Advanced Molecular Diagnostic Techniques for COVID-19 Pandemic: An Overview.

Coronavirus disease 2019 (COVID-19), which started out as an outbreak of pneumonia, has now turned into a pandemic due to its rapid transmission. Besides developing a vaccine, rapid, accurate, and cost-effective diagnosis is essential for monitoring and combating the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its related variants on time with precision and accuracy. Currently, the gold standard for detection of SARS-CoV-2 is Reverse Transcription Polymerase Chain Reaction (RT-PCR), but it lacks accuracy, is time-consuming and cumbersome, and fails to detect multi-variant forms of the virus. Herein, we have summarized conventional diagnostic methods such as Chest-CT (Computed Tomography), RT-PCR, Loop Mediated Isothermal Amplification (LAMP), Reverse Transcription-LAMP (RT-LAMP), as well new modern diagnostics such as CRISPR-Cas-based assays, Surface Enhanced Raman Spectroscopy (SERS), Lateral Flow Assays (LFA), Graphene-Field Effect Transistor (GraFET), electrochemical sensors, immunosensors, antisense oligonucleotides (ASOs)-based assays, and microarrays for SARS-CoV-2 detection. This review will also provide an insight into an ongoing research and the possibility of developing more economical tools to tackle the COVID-19 pandemic.

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.

Microfluidic paper device for rapid detection of aflatoxin B1 using an aptamer based colorimetric assay.

Contamination of milk by mycotoxins is a serious problem worldwide. Herein, we focused on the detection of aflatoxin B1 (AflB1) using a paper microfluidic device fabricated with specific aptamers as biorecognition elements. The fabrication process resulted in the generation of a leak proof microfluidic device where two zones were designed: control and test. Detection is achieved by color change when aflatoxin reacts with an aptamer followed by salt induced aggregation of gold nanoparticles. Specific aptamers for aflatoxin B1 were immobilized successfully onto the surface of gold nanoparticles. Biophysical characterization of the conjugated AuNPs-aptamer was done by UV-vis spectroscopy, DLS (dynamic light scattering), TEM (transmission electron microscopy). Under optimal conditions, the microfluidic device showed an excellent response for aflatoxin B1 detection in the range of 1 pM to 1 µM with a detection limit of up to 10 nM in spiked samples. Disadvantages associated with conventional techniques of ELISA were overcome by this one step detection technique with low operation cost, simple instrumentation, and user-friendly format with no interference due to chromatographic separation. The developed microfluidic paper-based analytical device (µPAD) can provide a tool for on-site detection of food toxins in less than a minute which is the main requirement for both qualitative and quantitative analysis in food safety and environmental monitoring.