skip to main content

This content will become publicly available on December 1, 2023

Title: Quantum optical immunoassay: upconversion nanoparticle-based neutralizing assay for COVID-19
Abstract In a viral pandemic, a few important tests are required for successful containment of the virus and reduction in severity of the infection. Among those tests, a test for the neutralizing ability of an antibody is crucial for assessment of population immunity gained through vaccination, and to test therapeutic value of antibodies made to counter the infections. Here, we report a sensitive technique to detect the relative neutralizing strength of various antibodies against the SARS-CoV-2 virus. We used bright, photostable, background-free, fluorescent upconversion nanoparticles conjugated with SARS-CoV-2 receptor binding domain as a phantom virion. A glass bottom plate coated with angiotensin-converting enzyme 2 (ACE-2) protein imitates the target cells. When no neutralizing IgG antibody was present in the sample, the particles would bind to the ACE-2 with high affinity. In contrast, a neutralizing antibody can prevent particle attachment to the ACE-2-coated substrate. A prototype system consisting of a custom-made confocal microscope was used to quantify particle attachment to the substrate. The sensitivity of this assay can reach 4.0 ng/ml and the dynamic range is from 1.0 ng/ml to 3.2  $$\upmu$$ μ g/ml. This is to be compared to 19 ng/ml sensitivity of commercially available kits.
Authors:
; ; ; ; ; ; ; ; ;
Award ID(s):
2013771
Publication Date:
NSF-PAR ID:
10348557
Journal Name:
Scientific Reports
Volume:
12
Issue:
1
ISSN:
2045-2322
Sponsoring Org:
National Science Foundation
More Like this
  1. The COVID-19 pandemic demonstrated the public health benefits of reliable and accessible point-of-care (POC) diagnostic tests for viral infections. Despite the rapid development of gold-standard reverse transcription polymerase chain reaction (RT-PCR) assays for SARS-CoV-2 only weeks into the pandemic, global demand created logistical challenges that delayed access to testing for months and helped fuel the spread of COVID-19. Additionally, the extreme sensitivity of RT-PCR had a costly downside as the tests could not differentiate between patients with active infection and those who were no longer infectious but still shedding viral genomes. To address these issues for the future, we propose a novel membrane-based sensor that only detects intact virions. The sensor combines affinity and size based detection on a membrane-based sensor and does not require external power to operate or read. Specifically, the presence of intact virions, but not viral debris, fouls the membrane and triggers a macroscopically visible hydraulic switch after injection of a 40 μL sample with a pipette. The device, which we call the μSiM-DX (microfluidic device featuring a silicon membrane for diagnostics), features a biotin-coated microslit membrane with pores ∼2–3× larger than the intact virus. Streptavidin-conjugated antibody recognizing viral surface proteins are incubated with the samplemore »for ∼1 hour prior to injection into the device, and positive/negative results are obtained within ten seconds of sample injection. Proof-of-principle tests have been performed using preparations of vaccinia virus. After optimizing slit pore sizes and porous membrane area, the fouling-based sensor exhibits 100% specificity and 97% sensitivity for vaccinia virus ( n = 62). Moreover, the dynamic range of the sensor extends at least from 10 5.9 virions per mL to 10 10.4 virions per mL covering the range of mean viral loads in symptomatic COVID-19 patients (10 5.6 –10 7 RNA copies per mL). Forthcoming work will test the ability of our sensor to perform similarly in biological fluids and with SARS-CoV-2, to fully test the potential of a membrane fouling-based sensor to serve as a PCR-free alternative for POC containment efforts in the spread of infectious disease.« less
  2. Abstract Rapid, inexpensive, and easy-to-use coronavirus disease 2019 (COVID-19) home tests are key tools in addition to vaccines in the world wide fight to eliminate national and local shutdowns. However, currently available tests for SARS-CoV-2, the virus that causes COVID-19, are too expensive, painful, and irritating, or not sufficiently sensitive for routine, accurate home testing. Herein, we employ custom-formulated graphene inks and aerosol jet printing to create a rapid electrochemical immunosensor for direct detection of SARS-CoV-2 spike receptor-binding domain (RBD) in saliva samples acquired noninvasively. This sensor demonstrated limits of detection that are considerably lower than most commercial SARS-CoV-2 antigen tests (22.91 ± 4.72 pg ml −1 for spike RBD and 110.38 ± 9.00 pg ml −1 for spike S1) as well as fast response time (∼30 min), which was facilitated by the functionalization of printed graphene electrodes in a single-step with SARS-CoV-2 polyclonal antibody through the carbodiimide reaction without the need for nanoparticle functionalization or secondary antibody or metallic nanoparticle labels. This immunosensor presents a wide linear sensing range from 1 to 1000 ng ml −1 and does not react with other coexisting influenza viruses such as H1N1 hemagglutinin. By combining high-yield graphene ink synthesis, automated printing, high antigenmore »selectivity, and rapid testing capability, this work offers a promising alternative to current SARS-CoV-2 antigen tests.« less
  3. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of the coronavirus disease that began in 2019 (COVID-19), has been responsible for 1.4 million deaths worldwide as of 13 November 2020. Because at the time of writing no vaccine is yet available, a rapid diagnostic assay is very urgently needed. Herein, we present the development of anti-spike antibody attached gold nanoparticles for the rapid diagnosis of specific COVID-19 viral antigen or virus via a simple colorimetric change observation within a 5 minute time period. For rapid and highly sensitive identification, surface enhanced Raman spectroscopy (SERS) was employed using 4-aminothiophenol as a reporter molecule, which is attached to the gold nanoparticle via an Au–S bond. In the presence of COVID-19 antigen or virus particles, owing to the antigen–antibody interaction, the gold nanoparticles undergo aggregation, changing color from pink to blue, which allows for the determination of the presence of antigen or virus very rapidly by the naked eye, even at concentrations of 1 nanogram (ng) per mL for COVID-19 antigen and 1000 virus particles per mL for SARS-CoV-2 spike protein pseudotyped baculovirus. Importantly, the aggregated gold nanoparticles form “hot spots” to provide very strong SERS signal enhancement from anti-spike antibody andmore »4-aminothiophenol attached gold nanoparticles via light–matter interactions. Finite-difference time-domain (FDTD) simulation data indicate a 4-orders-of-magnitude Raman enhancement in “hot spot” positions when gold nanoparticles form aggregates. Using a portable Raman analyzer, our reported data demonstrate that our antibody and 4-aminothiophenol attached gold nanoparticle-based SERS probe has the capability to detect COVID-19 antigen even at a concentration of 4 picograms (pg) per mL and virus at a concentration of 18 virus particles per mL within a 5 minute time period. Using HEK293T cells, which express angiotensin-converting enzyme 2 (ACE2), by which SARS-CoV-2 enters human cells, we show that anti-spike antibody attached gold nanoparticles have the capability to inhibit infection by the virus. Our reported data show that antibody attached gold nanoparticles bind to SARS-CoV-2 spike protein, thereby inhibiting the virus from binding to cell receptors, which stops virus infection and spread. It also has the capability to destroy the lipid membrane of the virus.« less
  4. Highly sensitive, specific, and point-of-care (POC) serological assays are an essential tool to manage coronavirus disease 2019 (COVID-19). Here, we report on a microfluidic POC test that can profile the antibody response against multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigens—spike S1 (S1), nucleocapsid (N), and the receptor binding domain (RBD)—simultaneously from 60 μl of blood, plasma, or serum. We assessed the levels of antibodies in plasma samples from 31 individuals (with longitudinal sampling) with severe COVID-19, 41 healthy individuals, and 18 individuals with seasonal coronavirus infections. This POC assay achieved high sensitivity and specificity, tracked seroconversion, and showed good concordance with a live virus microneutralization assay. We can also detect a prognostic biomarker of severity, IP-10 (interferon-γ–induced protein 10), on the same chip. Because our test requires minimal user intervention and is read by a handheld detector, it can be globally deployed to combat COVID-19.
  5. Abstract

    Monoclonal antibodies that target SARS-CoV-2 with high affinity are valuable for a wide range of biomedical applications involving novel coronavirus disease (COVID-19) diagnosis, treatment, and prophylactic intervention. Strategies for the rapid and reliable isolation of these antibodies, especially potent neutralizing antibodies, are critical toward improved COVID-19 response and informed future response to emergent infectious diseases. In this study, single B cell screening was used to interrogate antibody repertoires of immunized mice and isolate antigen-specific IgG1+memory B cells. Using these methods, high-affinity, potent neutralizing antibodies were identified that target the receptor-binding domain of SARS-CoV-2. Further engineering of the identified molecules to increase valency resulted in enhanced neutralizing activity. Mechanistic investigation revealed that these antibodies compete with ACE2 for binding to the receptor-binding domain of SARS-CoV-2. These antibodies may warrant further development for urgent COVID-19 applications. Overall, these results highlight the potential of single B cell screening for the rapid and reliable identification of high-affinity, potent neutralizing antibodies for infectious disease applications.