Smartphone-assisted point-of-care testing of nucleic acids based on hybridization chain reaction, magnetic beads, and gold nanorods etching

Colorimetric biosensors are simple but effective tools that are gaining popularity due to their ability to provide low-cost, rapid, and accurate detection for viruses like the Novel coronavirus, Influenza A, and Dengue virus, especially in point-of-care testing (POCT) and visual detection. In this study, a smartphone-assisted nucleic acid POCT was built using hybridization chain reaction (HCR), magnetic beads (MBs), and oxidized 3,3′,5,5′-tetramethylbenzidine (TMB2+)-mediated etching of gold nanorods (GNRs). The application of HCR without enzyme isothermal characteristics and MBs with easy separation, can quickly amplify nucleic acid signal and remove other reaction components. The blue shift of longitudinal localized surface plasmon resonance (LSPR) based on GNRs showed significant differences in etching color for different concentrations of target nucleic acid, which convert the signal into a visually semi-quantitative colorimetric result, achieving quantitative analysis with the color recognition software built into smartphones. This strategy, which only takes 40 min to detect and is two-thirds less time than the PCR, was successfully applied for the detection of the Dengue target sequence with a detection limit of 1.25 nM and exhibited excellent specificity for distinguishing single-base mutations, indicating broad application prospects in clinical laboratory diagnosis and enriching the research of nucleic acid POCT.

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.

Wide-range direct detection of 25-hydroxyvitamin D3 using polyethylene-glycol-free gold nanorod based on LSPR aptasensor.

Vitamin D is associated with various diseases such as obesity, digestive problems, osteoporosis, depression, and infections, which has emerged as an interest in public healthcare. Recently, vitamin D has received more attention because of the potential implication with coronavirus disease 2019. In this study, we developed a localized surface plasmon resonance (LSPR) aptasensor based on polyethylene-glycol(PEG)-free gold nanorods (AuNRs) for the wide-range and direct detection of 25-hydroxyvitamin D3. The surfactant on AuNRs was removed by exchanging with polystyrene sulfonate (PSS) instead of PEG then the PSS was exchanged with citrate. By exchanging the stabilizer of AuNRs from PEG to PEG-free (i.e., citrate), the sensing efficiency of LSPR aptasensor was significantly improved. Additionally, LSPR aptasensor was functionalized with aptamer and blocking agent to enhance the sensing performance. The LSPR aptasensor achieved the direct, highly sensitive, and selective detection of 25-hydroxyvitamin D3 over a wide concentration range (0.1-105 ng/mL), with a limit of detection of 0.1 ng/mL. This detection range included the concentration of vitamin D from deficiency to excess. The PEG-free AuNR-based LSPR aptasensor affords a new avenue for the development of robust sensing technology for vitamins.

Localized Plasmonic Photothermal Therapy as a Life-saving Treatment Paradigm for Hospitalized COVID-19 Patients.

Lung failure is the main reason for mortality in COVID-19 patients, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To date, no drug has been clinically approved for treatment of COVID-19. Nanotechnology has a great potential in contributing significantly to the fight against COVID-19 by developing effective therapies that can selectively eradicate the respiratory virus load. We propose a novel COVID-19 management approach that is efficient in eliminating the virus load from the airways and protecting the lungs from the fatal effects of the virus. This approach relies on targeting the virus using ACE-2-functionalized gold nanorods (AuNRs) followed by irradiation with near-infrared (NIR) light for the selective eradication of SARS-CoV-2 without off-target effects, i.e., targeted plasmonic photothermal therapy. Using discrete dipole approximation (DDA), we quantitatively determined the efficiency of AuNRs (31 nm × 8 nm) in absorbing NIR when present at different orientations relative to one another on the surface of the virus. The safety and the local administration of AuNRs using a well-tolerated flexible bronchoscopy technique, commonly used for hospitalized COVID-19 patients, ensure feasibility and clinical translation. While requiring further research, we anticipate this approach to result in a first-line treatment for hospitalized COVID-19 patients that are experiencing severe respiratory conditions or belong to a high-risk population, e.g., seniors and diabetic patients.

Gold Nanorod Assisted Enhanced Plasmonic Detection Scheme of COVID-19 SARS-CoV-2 Spike Protein.

The beautiful interplay between light and matter can give rise to many striking physical phenomena, surface plasmon resonance (SPR) being one of them. Plasmonic immunosensors monitor refractive index changes that occur as a result of specific ligand-analyte or antibody-antigen interactions taking place on the sensor surface. The coronavirus disease (COVID-19) pandemic has jeopardized the entire world and has resulted in economic slowdown of most countries. In this work, a model of a sandwich plasmonic biosensor that utilizes gold nanorods (Au NRs) for the detection of COVID-19 SARS-CoV-2 spike protein is presented. Simulation results for different prismatic configurations for the basic Kretschmann layout are presented. It is found that a BK7 glass prism-based SPR sensor has an incremental sensitivity of 111.11 deg RIU-1. Additionally, using Comsol Multiphysics the electric field enhancement observed for various aspect ratios and layouts of Au NRs are discussed in depth.