Point of Care, rapid testing for SARS CoV2 - Single molecule plasmonic sensor for amplification-free detection

Aim of the project: The recent outbreak caused by a novel coronavirus (SARS CoV-2) poses a great threat to global public health, particularly in going forward, from community transmission. It becoming increasingly important to establish a rapid diagnostic test for the detection of SARS CoV2 to prevent community spread from a case, given the R0 value is 2.2 to 2.6 on current estimates. Current methods for detecting COVID-19 is to use laboratory base, gold standard, qPCR which detects viral RNA or lateral flow devices that detect a viral infection indirectly by quantifying the presence of antibodies as a result of the immune system's response to an infection. The drawback of the qPCR is the logistics of transporting samples to the laboratory. The drawbacks of the lateral flow devices are firstly they are insensitive such that high viral loads are needed before they will respond, typically 7 days after infection for COVID-19, and a positive would still typically require qPCR to be performed as it is an indirect method of detecting an infection. Thus the unmet need is a rapid test that detects viral RNA that ideally does not require amplification. The primary challenge is the low concentration of the virus genomic material especially during early stages of infection. We have developed a new approach to detecting viral RNA directly from nasopharyngeal swabs using plasmonic nanoparticles that allow single molecule detection and is compatible with a handheld portable device. The optical properties of the plasmonic nanoparticles are dependent on the size, shape and the refractive index surrounding the nanoparticle. Using dark-field optical microscopy, we examine scattering arising from individual nanoparticles. The wide field nature of this measurement allows simultaneous characterization of 1,000 nanoparticles. With the help of computer algorithms, we track the colour of each nanoparticle and their optical signatures are represented in a digital array format. The proof-of concept of the proposed assay platform has been established by the existing collaborative group for detection of Influenza virus (H1N1 subtype) to allow subtype differentiation and quantification by plasmonic sensor using dark field microscopy (Technology patent under progress through UNSW knowledge exchange). The robust design of the sensor allowed direct detection of various clinical samples including nasopharyngeal aspirates and nasopharyngeal swabs with sensitivity comparable to reference techniques. This unique potential of the sensor is now being applied towards development of a rapid diagnostic platform for SARS CoV-2 virus. Methods: The assay consists of a hairpin probe sequence targeting the unique 20-24 bp genome sequence of the virus. The designed target sequence will form the loop region of the probe and the stem region of 7 bases will be added followed by polyA sequence. The designed polyA hairpin sequence will be conjugated to the satellite gold nanoparticles (40 nm). The core gold nanoparticles (80 nm) were conjugated to polyT sequence and anchored on to the substrate via aminosilane chemistry. The poly A hairpin probe conjugated satellite particles were hybridized to the polyT modified core particles to form the hairpin linked dimers. The target is detected by the viral RNA hybridizing to the loop region of the hairpin, leading to the opening of hairpin which increases the particles distance leading to a change in colour towards the blue part of the spectrum (Figure 1). The spectral shift of individual nanoparticles are captured by a digital camera and processed to obtain the pseudospectra of individual particles. The power of this construct over other plasmonic systems is the blue shift makes it far less prone to nonspecific protein adsorption from biological samples and hence makes it compatible for use in biological samples without any sample preparation steps. Any positive response corresponds to single molecule measurement allowing amplification free and calibration free sensor. Expected outcomes of the project: A portable analytical device for detecting viral RNA from SARS CoV-2. The proof of concept system was tested on several strains of influenza virus. The proposed research is expected to show similar sensitivity in detecting SARS CoV-2. The plasmonic nanoparticles exhibit strong optical properties that can be detected by a digital camera or smartphone camera. The sensor surface could be used on direct lysate of clinical samples in 30 min. The above benefits reduce turnaround assay time with ultrasensitive detection limit of 10-100 copies /mL of virus genome in the test sample. The integration of the portable device and digital quantification of the dark field image obtained from the sensor allows automated analysis of the presence of virus minimizing the risk for healthcare workers. Through point-of-care, self sampling applications, this will provide safer, reduced turnaround for SARS CoV-2 diagnosis, applicable to developing country applications through our clinical partners.