• Hyperspectral Mapping for the Detection of SARS-CoV-2 Using Nanomolecular Probes with Yoctomole Sensitivity

      Alafeef, Maha; Moitra, Parikshit; Dighe, Ketan; Pan, Dipanjan (American Chemical Society, 2021-07-19)
      Efficient monitoring of SARS-CoV-2 outbreak requires the use of a sensitive and rapid diagnostic test. Although SARS-CoV-2 RNA can be detected by RT-qPCR, the molecular-level quantification of the viral load is still challenging, time-consuming, and labor-intensive. Here, we report an ultrasensitive hyperspectral sensor (HyperSENSE) based on hafnium nanoparticles (HfNPs) for specific detection of COVID-19 causative virus, SARS-CoV-2. Density functional theoretical calculations reveal that HfNPs exhibit higher changes in their absorption wavelength and light scattering when bound to their target SARS-CoV-2 RNA sequence relative to the gold nanoparticles. The assay has a turnaround time of a few seconds and has a limit of detection in the yoctomolar range, which is 1 000 000-fold times higher than the currently available COVID-19 tests. We demonstrated in ∼100 COVID-19 clinical samples that the assay is highly sensitive and has a specificity of 100%. We also show that HyperSENSE can rapidly detect other viruses such as influenza A H1N1. The outstanding sensitivity indicates the potential of the current biosensor in detecting the prevailing presymptomatic and asymptomatic COVID-19 cases. Thus, integrating hyperspectral imaging with nanomaterials establishes a diagnostic platform for ultrasensitive detection of COVID-19 that can potentially be applied to any emerging infectious pathogen.
    • Monitoring the Viral Transmission of SARS-CoV-2 in Still Waterbodies Using a Lanthanide-Doped Carbon Nanoparticle-Based Sensor Array.

      Alafeef, Maha; Dighe, Ketan; Moitra, Parikshit; Pan, Dipanjan (American Chemical Society, 2021-12-29)
      The latest epidemic of extremely infectious coronavirus disease 2019 (COVID-19) has created a significant public health concern. Despite substantial efforts to contain severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within a specific location, shortcomings in the surveillance of predominantly asymptomatic infections constrain attempts to identify the epidemiological spread of the virus. Continuous surveillance of wastewater streams, including sewage, offers opportunities to track the spread of SARS-CoV-2, which is believed to be found in fecal waste. To demonstrate the feasibility of SARS-CoV-2 detection in wastewater systems, we herein present a novel facilely constructed fluorescence sensing array based on a panel of three different lanthanide-doped carbon nanoparticles (LnCNPs). The differential fluorescence response pattern due to the counterion-ligand interactions allowed us to employ powerful pattern recognition to effectively detect SARS-CoV-2 and differentiate it from other viruses or bacteria. The sensor results were benchmarked to the gold standard RT-qPCR, and the sensor showed excellent sensitivity (1.5 copies/μL) and a short sample-to-results time of 15 min. This differential response of the sensor array was also explained from the differential mode of binding of the LnCNPs with the surface proteins of the studied bacteria and viruses. Therefore, the developed sensor array provides a cost-effective, community diagnostic tool that could be potentially used as a novel epidemiologic surveillance approach to mitigate the spread of COVID-19.
    • A rapid RNA extraction-free lateral flow assay for molecular point-of-care detection of SARS-CoV-2 augmented by chemical probes.

      Dighe, Ketan; Moitra, Parikshit; Alafeef, Maha; Gunaseelan, Nivetha; Pan, Dipanjan (Elsevier Ltd., 2021-12-18)
      The coronavirus disease 2019 (COVID-19) pandemic has highlighted the major shortcoming of healthcare systems globally in their inability to diagnose the disease rapidly and accurately. At present, the molecular approaches for diagnosing COVID-19 primarily use reverse transcriptase polymerase chain reaction (RT-PCR) to create and amplify cDNA from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. Although molecular tests are reported to be specific, false negatives are quite common. Furthermore, literally all these tests require a step involving RNA isolation which does not make them point-of-care (POC) in the true sense. Here, we report a lateral flow strip-based RNA extraction and amplification-free nucleic acid test (NAT) for rapid diagnosis of positive COVID-19 cases at POC. The assay uses highly specific 6-carboxyfluorescein (6-FAM) and biotin labeled antisense oligonucleotides (ASOs) as probes those are designed to target N-gene sequence of SARS-CoV-2. Additionally, we utilized cysteamine capped gold-nanoparticles (Cyst-AuNPs) to augment the signal further for enhanced sensitivity. Without any large-stationary equipment and highly trained staffers, the entire sample-to-answer approach in our case would take less than 30 min from a patient swab sample collection to final diagnostic result. Moreover, when evaluated with 60 clinical samples and verified with an FDA-approved TaqPath RT-PCR kit for COVID-19 diagnosis, the assay obtained almost 99.99% accuracy and specificity. We anticipate that the newly established low-cost amplification-free detection of SARS-CoV-2 RNA will aid in the development of a platform technology for rapid and POC diagnosis of COVID-19 and other pathogens.
    • Rapid, Ultrasensitive, and Quantitative Detection of SARS-CoV-2 Using Antisense Oligonucleotides Directed Electrochemical Biosensor Chip.

      Alafeef, Maha; Dighe, Ketan; Moitra, Parikshit; Pan, Dipanjan (American Chemical Society, 2020-10-20)
      A large-scale diagnosis of the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) is essential to downregulate its spread within as well as across communities and mitigate the current outbreak of the pandemic novel coronavirus disease 2019 (COVID-19). Herein, we report the development of a rapid (less than 5 min), low-cost, easy-to-implement, and quantitative paper-based electrochemical sensor chip to enable the digital detection of SARS-CoV-2 genetic material. The biosensor uses gold nanoparticles (AuNPs), capped with highly specific antisense oligonucleotides (ssDNA) targeting viral nucleocapsid phosphoprotein (N-gene). The sensing probes are immobilized on a paper-based electrochemical platform to yield a nucleic-acid-testing device with a readout that can be recorded with a simple hand-held reader. The biosensor chip has been tested using samples collected from Vero cells infected with SARS-CoV-2 virus and clinical samples. The sensor provides a significant improvement in output signal only in the presence of its target-SARS-CoV-2 RNA-within less than 5 min of incubation time, with a sensitivity of 231 (copies μL-1)-1 and limit of detection of 6.9 copies/μL without the need for any further amplification. The sensor chip performance has been tested using clinical samples from 22 COVID-19 positive patients and 26 healthy asymptomatic subjects confirmed using the FDA-approved RT-PCR COVID-19 diagnostic kit. The sensor successfully distinguishes the positive COVID-19 samples from the negative ones with almost 100% accuracy, sensitivity, and specificity and exhibits an insignificant change in output signal for the samples lacking a SARS-CoV-2 viral target segment (e.g., SARS-CoV, MERS-CoV, or negative COVID-19 samples collected from healthy subjects). The feasibility of the sensor even during the genomic mutation of the virus is also ensured from the design of the ssDNA-conjugated AuNPs that simultaneously target two separate regions of the same SARS-CoV-2 N-gene.
    • RNA-extraction-free nano-amplified colorimetric test for point-of-care clinical diagnosis of COVID-19

      Alafeef, Maha; Moitra, Parikshit; Dighe, Ketan; Pan, Dipanjan (Springer Nature, 2021-04-30)
      The global pandemic of coronavirus disease 2019 (COVID-19) highlights the shortcomings of the current testing paradigm for viral disease diagnostics. Here, we report a stepwise protocol for an RNA-extraction-free nano-amplified colorimetric test for rapid and naked-eye molecular diagnosis of COVID-19. The test employs a unique dual-prong approach that integrates nucleic acid (NA) amplification and plasmonic sensing for point-of-care detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with a sample-to-assay response time of <1 h. The RNA-extraction-free nano-amplified colorimetric test utilizes plasmonic gold nanoparticles capped with antisense oligonucleotides (ASOs) as a colorimetric reporter to detect the amplified nucleic acid from the COVID-19 causative virus, SARS-CoV-2. The ASOs are specific for the SARS-CoV-2 N-gene, and binding of the ASOs to their target sequence results in the aggregation of the plasmonic gold nanoparticles. This highly specific agglomeration step leads to a change in the plasmonic response of the nanoparticles. Furthermore, when tested using clinical samples, the accuracy, sensitivity and specificity of the test were found to be >98.4%, >96.6% and 100%, respectively, with a detection limit of 10 copies/μL. The test can easily be adapted to diagnose other viral infections with a simple modification of the ASOs and primer sequences. It also provides a low-cost, rapid approach requiring minimal instrumentation that can be used as a screening tool for the diagnosis of COVID-19 at point-of-care settings in resource-poor situations. The colorimetric readout of the test can even be monitored using a handheld optical reader to obtain a quantitative response. Therefore, we anticipate that this protocol will be widely useful for the development of biosensors for the molecular diagnostics of COVID-19 and other infectious diseases. © 2021, The Author(s)