Trends and Innovations in Biosensors for COVID-19 Mass Testing

Abstract

Fast and widespread diagnosis is crucial to fighting against the outbreak of COVID-19. This work surveys the landscape of available and emerging biosensor technologies for COVID-19 testing. Molecular diagnostic assays based on quantitative reverse transcription polymerase chain reaction are used in most clinical laboratories. However, the COVID-19 pandemic has overwhelmed testing capacity and motivated the development of fast point-of-care tests and the adoption of isothermal DNA amplification. Antigenic and serological rapid tests based on lateral-flow immunoassays suffer from low sensitivity. Advanced digital systems enhance performance at the expense of speed and the need for large equipment. Emerging technologies, including CRISPR gene-editing tools, benefit from high sensitivity and specificity of molecular diagnostics and the easy use of lateral-flow assays. DNA sequencing and sample pooling strategies are highlighted to bring out the full capacity of the available biosensor technologies and accelerate mass testing.

Keywords: COVID-19; CRISPR diagnostics; immunoassays; isothermal amplification; molecular diagnostics.

Figure 1

a) Scheme of Covid‐19 diagnostics tools and workflow based on sample type, biomarker (genetic, antigenic or serological), signal amplification and detection method. b) Representation of SARS‐CoV‐2 (adapted from RCSB PDB, credit: Maria Vogt). c) The suitable time window for different test types. The stage of the disease determines the viral load and antibodies present in the patient.10

Figure 2

Steps in the qRT‐PCR test: a) Specimen is taken from the nose or throat of individual, b) RNA is extracted and c) is transcribed into complementary DNA (cDNA). d) Once the primers have bound to the DNA, they provide a starting point for the DNA polymerase to help copy it. DNA polymerase then degrades the bound probe, which results in an increased fluorescence signal. e) The fluorescence signal increases as copies of the virus DNA are made. If the fluorescence level crosses a certain threshold, the test result is positive.

Figure 3

CRISPR‐based diagnosis (DETECTR) by Mammoth Biosciences. a) Schematic of the SARS‐CoV‐2 DETECTR workflow. Conventional RNA extraction is used as an input. It is followed by reverse transcription, loop‐mediated isothermal amplification (LAMP) and Cas12‐based detection of target genes (E, N and RNase P). These are visualised on a lateral‐flow strip. b) Lateral‐flow strip assay read‐out: The line closest to the sample pad is the control line, and the line that appears farthest from the sample pad is the test line. c) Interpretation of results: a positive result requires the detection of at least the two SARS‐CoV‐2 viral gene targets (N gene and E gene). Adapted with permission from ref. [30]'with permission Copyright: 2020, Springer Nature

Figure 4

a) Workflow of whole‐genome sequencing for COVID‐19 diagnostics. The sample is collected, and reverse transcription and PCR amplification of a SARS‐CoV‐2 target region are performed without RNA extraction by direct addition to a one‐step RT‐PCR master mix, together with a synthetic spike‐in strand. After co‐amplification of spike‐in and sample on a thermal cycler, the amplified products are sequenced, and the resulting chromatograms are analysed to determine if the sample contains viral RNA. b) A synthetic spike‐in RNA with a 4 bp deletion is designed with sequence homology to the SARS‐CoV‐2 target so that it co‐amplifies with the SARS‐CoV‐2 target. This enables quantification of relative abundances of spike‐in and SARS‐CoV‐2 DNA from a Sanger sequencing chromatogram. Representative Sanger sequencing traces showing pure genomic sequence (top), pure spike‐in sequence (middle), and sequencing from a mixture of genomic and spike‐in sequences (bottom). When both spike‐in and viral genomic sequence are present, the signals are used to estimate their relative abundances. Adapted with permission from ref. ^[37]. Copyright: 2020, the authors.

Figure 5

Typical lateral‐flow assay for a serological test. a) Inside the cassette is a strip made of filter paper and nitrocellulose. Typically, a drop of blood is added to the cassette through one hole (sample well), and then a number of drops of buffer are added usually through another hole (buffer well). The buffer carries the sample along the length of the cassette to the results window. b) Interpretation of results. c) A schematic of a COVID‐19 lateral‐flow test. The antibody binds to an antigen conjugated to colloidal gold in the conjugation pad, and the resultant complex is captured on the strip by a band of bound antibodies, forming a visible line (T: test line) in the results window for IgM and IgG. A control line (C) gives information on the integrity of the antibody‐gold conjugate. Adapted with permission from ref. [41]. Copyright: 2020, the authors.

Figure 6

a) Scheme of the pooling strategy: individual samples are collected, RNA is extracted, and up to 64 samples are pooled, out of which one individual is infected (red). b) Representative RT‐qPCR fluorescence curves of a positive sample (Pos) diluted in different numbers of negative samples (red: no dilution, blue: dilution in 63 negative samples). Dots represent the cross point of the fluorescence threshold; extracted with permission from ref. [49a]. Copyright: 2020, the authors.