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 antigen selectivity, and rapid testing capability, this work offers a promising alternative to current SARS-CoV-2 antigen tests.
more »
« less
The rapid diagnosis and effective inhibition of coronavirus using spike antibody attached gold nanoparticles
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 and 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.
more »
« less
- NSF-PAR ID:
- 10216692
- Date Published:
- Journal Name:
- Nanoscale Advances
- ISSN:
- 2516-0230
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
more » « less
In the context of continued spread of coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 and the emergence of new variants, the demand for rapid, accurate, and frequent detection is increasing. Moreover, the new predominant strain, Omicron variant, manifests more similar clinical features to those of other common respiratory infections. The concurrent detection of multiple potential pathogens helps distinguish SARS-CoV-2 infection from other diseases with overlapping symptoms, which is significant for providing tailored treatment to patients and containing the outbreak. Here, we report a lab-on-a-chip biosensing platform for SARS-CoV-2 detection based on the subwavelength grating micro-ring resonator. The sensing surface is functionalized by specific antibody against SARS-CoV-2 spike protein, which could produce redshifts of resonant peaks by antigen–antibody combination, thus achieving quantitative detection. Additionally, the sensor chip is integrated with a microfluidic chip featuring an anti-backflow Y-shaped structure that enables the concurrent detection of two analytes. In this study, we realized the detection and differentiation of COVID-19 and influenza A H1N1. Experimental results indicate that the limit of detection of our device reaches 100 fg/ml (1.31 fM) within 15 min detecting time, and cross-reactivity tests manifest the specificity of the optical diagnostic assay. Furthermore, the integrated packaging and streamlined workflow facilitate its use for clinical applications. Thus, the biosensing platform presents a promising approach for attaining highly sensitive, selective, multiplexed, and quantitative point-of-care diagnosis and distinction between COVID-19 and influenza.
-
Development of effective vaccines against coronavirus disease 2019 (COVID-19) is a global imperative. Rapid immunization of the entire human population against a widespread, continually evolving, and highly pathogenic virus is an unprecedented challenge, and different vaccine approaches are being pursued. Engineered filamentous bacteriophage (phage) particles have unique potential in vaccine development due to their inherent immunogenicity, genetic plasticity, stability, cost-effectiveness for large-scale production, and proven safety profile in humans. Herein we report the development and initial evaluation of two targeted phage-based vaccination approaches against SARS-CoV-2: dual ligand peptide-targeted phage and adeno-associated virus/phage (AAVP) particles. For peptide-targeted phage, we performed structure-guided antigen design to select six solvent-exposed epitopes of the SARS-CoV-2 spike (S) protein. One of these epitopes displayed on the major capsid protein pVIII of phage induced a specific and sustained humoral response when injected in mice. These phage were further engineered to simultaneously display the peptide CAKSMGDIVC on the minor capsid protein pIII to enable their transport from the lung epithelium into the systemic circulation. Aerosolization of these “dual-display” phage into the lungs of mice generated a systemic and specific antibody response. In the second approach, targeted AAVP particles were engineered to deliver the entire S protein gene under the control of a constitutive CMV promoter. This induced tissue-specific transgene expression, stimulating a systemic S protein-specific antibody response in mice. With these proof-of-concept preclinical experiments, we show that both targeted phage- and AAVP-based particles serve as robust yet versatile platforms that can promptly yield COVID-19 vaccine prototypes for translational development.more » « less
-
null (Ed.)The COVID-19 pandemic demonstrated the critical need for accurate and rapid testing for virus detection. This need has generated a high number of new testing methods aimed at replacing RT-PCR, which is the golden standard for testing. Most of the testing techniques are based on biochemistry methods and require chemicals that are often expensive and the supply might become scarce in a large crisis. In the present paper we suggest the use of methods based on physics that leverage novel nanomaterials. We demonstrate that using Surface Enhanced Raman Spectroscopy (SERS) of virion particles a very distinct spectroscopic signature of the SARS-CoV-2 virus can be obtained. We demonstrate that the spectra are mainly composed by signals from the spike (S) and nucleocapsid (N) proteins. It is believed that a clinical test using SERS can be developed. The test will be fast, inexpensive, and reliable. It is also clear that SERS can be used for analysis of structural changes on the S and N proteins. This will be an example of application of nanotechnology and properties of nanoparticles for health and social related matters.more » « less
-
The COVID-19 pandemic caused by SARS-CoV-2 sparked intensive research into the development of effective vaccines, 50 of which have been approved thus far, including the novel mRNA-based vaccines developed by Pfizer and Moderna. Although limiting the severity of the disease, the mRNA-based vaccines presented drawbacks, such as the cold chain requirement. Moreover, antibody levels generated by these vaccines decline significantly after 6 months. These vaccines deliver mRNA encoding the full-length spike (S) glycoprotein of SARS-CoV-2, but must be updated as new strains and variants of concern emerge, creating a demand for adjusted formulations and booster campaigns. To overcome these challenges, we have developed COVID-19 vaccine candidates based on the highly conserved SARS CoV-2, 809-826 B-cell peptide epitope (denoted 826) conjugated to cowpea mosaic virus (CPMV) nanoparticles and bacteriophage Qβ virus-like particles, both platforms have exceptional thermal stability and facilitate epitope delivery with inbuilt adjuvant activity. We evaluated two administration methods: subcutaneous injection and an implantable polymeric scaffold. Mice received a prime–boost regimen of 100 μg per dose (2 weeks apart) or a single dose of 200 μg administered as a liquid formulation, or a polymer implant. Antibody titers were evaluated longitudinally over 50 weeks. The vaccine candidates generally elicited an early Th2-biased immune response, which stimulates the production of SARS-CoV-2 neutralizing antibodies, followed by a switch to a Th1-biased response for most formulations. Exceptionally, vaccine candidate 826-CPMV (administered as prime-boost, soluble injection) elicited a balanced Th1/Th2 immune response, which is necessary to prevent pulmonary immunopathology associated with Th2 bias extremes. While the Qβ-based vaccine elicited overall higher antibody titers, the CPMV-induced antibodies had higher avidity. Regardless of the administration route and formulation, our vaccine candidates maintained high antibody titers for more than 50 weeks, confirming a potent and durable immune response against SARS-CoV-2 even after a single dose.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1039/D0NA01007C
