Graphene-Based Field-Effect Transistor for Ultrasensitive Immunosensing of SARS-CoV-2 Spike S1 Antigen.

Coronavirus disease (COVID-19) is an infectious disease that has posed a global health challenge caused by the SARS-CoV-2 virus. Early management and diagnosis of SARS-CoV-2 are crucial for the timely treatment, traceability, and reduction of viral spread. We have developed a rapid method using a Graphene-based Field-Effect Transistor (Gr-FET) for the ultrasensitive detection of SARS-CoV-2 Spike S1 antigen (S1-Ag). The in-house developed antispike S1 antibody (S1-Ab) was covalently immobilized on the surface of a carboxy functionalized graphene channel using carbodiimide chemistry. Ultraviolet-visible spectroscopy, Fourier-Transform Infrared Spectroscopy, X-ray Photoelectron Spectroscopy (XPS), Atomic Force Microscopy (AFM), Optical Microscopy, Raman Spectroscopy, Scanning Electron Microscopy (SEM), Enzyme-Linked Immunosorbent Assays (ELISA), and device stability studies were conducted to characterize the bioconjugation and fabrication process of Gr-FET. In addition, the electrical response of the device was evaluated by monitoring the change in resistance caused by Ag-Ab interaction in real time. For S1-Ag, our Gr-FET devices were tested in the range of 1 fM to 1 µM with a limit of detection of 10 fM in the standard buffer. The fabricated devices are highly sensitive, specific, and capable of detecting low levels of S1-Ag.

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.

Molecular Diagnostic of Ochratoxin A With Specific Aptamers in Corn and Groundnut via Fabrication of a Microfluidic Device.

Ochratoxin A (OTA) is one of the predominant mycotoxins that contaminate a wide range of food commodities. In the present study, a 36-mer aptamer was used as a molecular recognition element coupled with gold nanoparticles (AuNPs) for colorimetric detection of OTA in a microfluidic paper-based analytical device (µPADs). The µPADs consisted of three zones: control, detection, and sample, interconnected by channels. UV-vis spectroscopy (UV-vis), Dynamic Light Scattering (DLS), and Transmission Electron Microscopy (TEM) were used for characterization of AuNPs and AuNPs/Aptamer. According to the colorimetric assay, limit of detection (LOD) was found to be 242, 545.45, and 95.69 ng/mL in water, corn, and groundnut, respectively. The HPLC detection method achieved acceptable coefficient in standard curves (r 2 = 0.9995), improved detection range, and recovery rates in spiked corn and groundnut samples as 43.61 ± 2.18% to 87.10 ± 1.82% and 42.01 ± 1.31% to 86.03 ± 2.64% after multiple sample extractions and cleanup steps. However, the developed µPADs analytical device had the potent ability to rapidly detect OTA without any extraction pre-requirement, derivatization, and cleanup steps, thus illustrating its feasibility in the animal health sector, agricultural, and food industries.

Polymeric biocompatible iron oxide nanoparticles labeled with peptides for imaging in ovarian cancer.

Compared with other nanomaterials, surface-modified iron oxide nanoparticles (IONPs) have gained attraction for cancer therapy applications due to its low toxicity, and long retention time. An innocuous targeting strategy was developed by generation of fluorescein isothiocyanate (FITC)-labeled peptide (growth factor domain (GFD) and somatomedin B domain (SMB)) functionalized, chitosan-coated IONPs (IONPs/C). It can be used to target urokinase plasminogen activator receptor (uPAR), which is a surface biomarker, in ovarian cancer. Binding affinity between uPAR and peptides (GFD and SMB) were revealed by in-silico docking studies. The biophysical characterizations of IONPs, IONPs/C, and IONPs/C/GFD-FITC or SMB-FITC nanoprobes were assessed via Vibrating Sample Magnetometer (VSM), Transmission Electron Microscopy (TEM), Dynamic Light Scattering (DLS), and Fourier Transform Infrared Spectroscopy (FT-IR). Prussian Blue staining, fluorescence spectroscopy, and fluorescence imaging were performed to confirm the targeting of nanoprobes with the surface receptor uPAR. The combination of IONPs/C/GFD+SMB showed efficient targeting of uPAR in the tumor microenvironment, and thus can be implemented as a molecular magnetic nanoprobe for cancer cell imaging and targeting.

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.

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.

Urokinase Plasminogen Activator Receptor-Mediated Targeting of a Stable Nanocomplex Coupled with Specific Peptides for Imaging of Cancer.

Targeting peptides are a promising tool for early diagnosis and therapy of cancer. Overexpression of urokinase plasminogen activator receptor (uPAR) leads to the progression of tumors including prostate, colorectal, ovarian, and breast cancers. To improve the diagnosis and imaging efficiency, herein we report a stable nanocomplex comprising methoxy-PEG-hydrazide (mPEG-H-M)-modified gold nanoparticles (AuNPs) conjugated to uPAR (urokinase plasminogen activator receptor)-targeting peptides GFD (growth factor domain-G) and SMB (somatomedian B-S) for efficient imaging of uPAR-overexpressing cancer cells. Fluorescently labeled targeting peptides were covalently linked to mPEG-H coated AuNPs, characterized, and analyzed by UV-vis spectroscopy, diffraction light scattering (DLS), transmission electron microscopy (TEM), energy-dispersive X-ray spectroscopy (EDX), and fluorescence spectroscopy. In vitro evaluation was assessed with a fluorescence-activated cell sorter (FACS), cell adhesion, and fluorescence microscopy. The peptide-functionalized nanocomplex showed a higher uptake of AuNPs@MGS in comparison with AuNPs@G or AuNPs@S alone in uPAR-overexpressing cells and exhibits no toxicity when analyzed with MTT assay. Our results demonstrated that the developed nanocomplex can be used as a platform for imaging and diagnosis of metastatic tumors.

Self-assembled chitosan polymer intercalating peptide functionalized gold nanoparticles as nanoprobe for efficient imaging of urokinase plasminogen activator receptor in cancer diagnostics.

Targeting cell surface receptors for specific drug delivery in cancer has garnered lot of attention. Urokinase plasminogen activator receptor (uPAR), a surface biomarker, is overexpressed on many tumours including breast, colorectal, prostate, and ovarian cancers. Binding of growth factor domain (GFD) of urokinase plasminogen activator (uPA) with uPAR lead to its close conformation, and allow somatomedin B domain (SMB) of vitronectin binding by allosteric modulation. In-silico docking of uPAR with GFD and SMB peptides was performed to identify potential binding affinity. Herein, we report fluorescently labeled peptide functionalized AuNPs with a mixed self-assembled monolayer of intercalating chitosan polymer for efficient targeting and imaging of uPAR-positive cells. The biophysical characterization of nanoconjugates and uPAR-specific targeting was assessed by FACS, cell adhesion, and fluorescence imaging. AuNPs/chitosan/GFD+SMB peptides showed higher uptake as compared to AuNPs/chitosan/GFD, and AuNPs/chitosan/SMB that can be utilized as a tool for molecular targeting and imaging in metastasis.

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.