Main

In the span of a few months, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was identified as the aetiological agent of coronavirus disease 2019 (COVID-19). Weeks later, viral diagnostic measures were deployed1. This served to supplement the common disease signs and symptoms of COVID-19 of cough, fever and dyspnoea. As all are seen during seasonal upper respiratory tract infections2, precise diagnostic tests detect viral nucleic acids, viral antigens or serological tests are required to affirm SARS-CoV-2 infection3. Chest computed tomography (CT) or magnetic resonance imaging (MRI) confirm disease manifestations2,3. The signature of COVID-19 is the life-threatening acute respiratory distress syndrome (ARDS)4. While the lung is the primary viral target, the cardiovascular, brain, kidney, liver and immune systems are commonly compromised by infection5. Thus, due to significant COVID-19 morbidity and mortality, containment of viral transmission through contact tracing, clinical assessment and virus detection was implemented through social distancing, face masks, contact isolation and hand hygiene to limit SARS-CoV-2 transmission6.

Overview of SARS-CoV-2 detection

The first step in managing COVID-19 is the rapid and accurate detection of SARS-CoV-2 enabled by real-time reverse transcription–polymerase chain reaction (RT–PCR)7. RT–PCR detects SARS-CoV-2 nucleic acids present in nasopharyngeal fluids8. Testing is used to prevent infectious spread between persons and communities that include asymptomatic infected persons, whose viral shedding can inadvertently spread the infection to the elderly and those with disease comorbidities9. Accurate viral detection is a starting point to contain the COVID-19 pandemic10. Lapses affect public safety, enabling infection spread aided by false-negative test results11. Improving test sensitivity and specificity remains an urgent need7. Serological testing complements virus detection, indicating past infection, which could be harnessed for therapeutic gain. Antibodies are detected by enzyme-linked immunosorbent assay using a qualitative detection of IgG or IgM antibodies12. Such tests determine an immune response against the viral spike (S) protein and may be helpful to assess protection against subsequent viral exposure and/or for contact tracing purposes13. Thus, the importance of such tests cannot be overstated. This is also true for epidemiological evaluations and broad global therapeutic needs14. Future work includes the development of diagnostic tests to improve immunoassay sensitivity and specificity13. Indeed, such testing will ultimately reveal viral protection as reinfections emerge15. Inducing immunity against SARS-CoV-2 is the next frontier for COVID-19 control15,16. To this end, our intent in this Review is to summarize the clinical disease presentation with a focus on how to best deploy nanomaterial-based and other diagnostic tests at individual, community and societal levels. The Review outlines current and future nanomaterial diagnostics for COVID-19. The intent is to facilitate the containment of the virus’s global spread12,15.

SARS-CoV-2 body fluid and tissue distribution

SARS-CoV-2 viral load and respiratory tract viral particles parallel virus dynamics in body fluids and tissue (Box 1). All affect concomitant host immune responses5,38. Quantum dots (QDs) could serve as ideal detection tools to study S protein–ACE2 binding dynamics and internalization due to their relatively small size, photostability and the ease of surface functionalization with biological molecules for Förster resonance energy transfer biosensing systems with various energy transfer partners39, such as AuNPs that are characterized by absorption of electromagnetic radiation in the visible region of the spectrum40. A colorimetric assay was developed based on thiol-modified antisense oligonucleotides conjugated with AuNPs for detection of SARS-CoV-2 N-gene RNA. This is used for rapid diagnosis and can be performed within 10 min. The lower limit of detection is 0.18 ng μl−1 RNA particles41. A recombinant S receptor binding domain conjugated to fluorescent QDs was created as an imaging probe for energy transfer quenching with ACE2-conjugated AuNPs. Upon binding of the S to the ACE2 receptor, fluorescence is quenched by the nearby AuNPs to enable monitoring of the binding events in the solution. QD probes can also facilitate cell-based assay identification and validation of inhibitors of the SARS-CoV-2 S protein and ACE2 receptor binding42. The QDs are used as probes to investigate other viral receptors43. This system can identify neutralizing antibodies and recombinant proteins for SARS-CoV-2 and other viruses with S-mediated cell recognition and entry.

Biosensors have been developed for detecting influenza, the human immunodeficiency virus and other viral diseases44. Initially marred by low sensitivity and specificity, limitations were overcome by plasmonic (gold and silver), metal oxide nanoparticle and field effect transistor (FET) bio- and graphene sensors44,45. Graphene has wide application; it consists of hexagonal carbon structures arranged in a two-dimensional sheet. This gives it a large surface area, high electronic conductivity and high carrier mobility, and graphene biosensors are highly sensitive. When develo** a graphene-based biosensor to detect SARS-CoV-2, coronavirus S antibody was immobilized on a graphene surface using 1-pyrenebutyric acid N-hydroxysuccinimide ester linkers. This graphene was used as a sensing material in a FET device to detect the S up to 1 fg ml−1 concentration46 (Fig. 3, top). The optical property of AuNPs and silver nanoparticles conjugated to antibodies, when they are bound to the viral antigens or RNA, causes a detectable signal, which can be used to detect SARS-CoV-2 (ref. 47). Toroidal plasmonic metasensors were developed that detect a femtomolar concentration of the viral S protein. They showed that monoclonal antibody conjugation on functionalized AuNPs could be detected up to 4.2 fM concentration (lower limit of detection). Transmission spectra of metasensors can shift the excitation with a polarized beam of light at terahertz frequency. Metasensors can be very useful in point of care (PoC) testing, where a rapid and sensitive assay is required48. Recently, researchers have devised a single-step, optical S-protein-specific nanoplasmonic resonance sensor that requires minimal sample preparation and provides fast and direct virus detection. In such a system, highly specific antibodies to SARS-CoV-2 were immobilized on nanosensor chip surfaces to which intact coronavirus particles bind through S protein, leading to plasmon resonance or intensity changes that can be optically measured through a sensing system49,50. For this assay, the lower limit of detection is 30 virus particles. The assay can be completed in 15 min. The assay can quantify virus below standard nasopharyngeal swab and saliva viral concentrations51. On analysing the specificity of the sensor for binding SARS-CoV-2 in comparison to SARS-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV) and vesicular stomatitis pseudoviruses, nanoplasmonic sensor chips demonstrated very high specificity (>1,000:1) in detecting the SARS-CoV-2 (ref. 49). The nanoplasmonic sensor chips have the advantage of being low cost and scalable while maintaining uniformity and repeatability. The design of periodic nanostructures, without any external coupling optics52, allows sensor chips to be integrated with a standard 96-microwell plate or microfluidic cuvettes. This allows standard microplate reader measurements53. A low-cost, portable device controlled using a smartphone application can analyse SARS-CoV-2 in one step within 15 min with sensitive viral detection. Although the detection limit is 370, the virus can be quantified linearly from 0 to 107 virus particles per millilitre and it may find application in clinics, roadside screening sites and homes49. AuNP-based sensors coupled with artificial intelligence can detect volatile organic compounds associated with SARS-CoV-2 in exhaled breaths. The assay is able to detect virus on the basis of the change in resistance of the nanomaterial biosensor layer. The methods can be optimized in future months by using other nanomaterials and larger cohort testing54.

Fig. 3: Nanomaterial-based SARS-CoV-2 detections.
figure 3

Representative illustration of nanomaterial-based biosensors for SARS-CoV-2 detection. Top: a FET-based biosensing device for detecting SARS-CoV-2. The sensor was produced using graphene sheets with specific antibodies against the SARS-CoV-2 S protein. Bottom: the dual-functional plasmonic photothermal biosensor and localized surface plasmon resonance biosensor on two-dimensional gold nanoislands functionalized with either cDNA receptors for detection of the selected SARS-CoV-2 sequences by fluorescence and FRET-based nucleic acid hybridization or with nucleic acid or antibody-functionalized nanomaterials for SARS-CoV-2 detection by colorimetric and antigen-binding assays. Ab, antibody; Ag, antigen; FRET, Förster resonance energy transfer; NPs, nanoparticles. Schematic ideas and technical methodological details were followed as represented in previously published reports36,37,45.

Full size image

A clinical diagnostic sensor was developed that combines a dual-functional plasmonic photothermal effect with localized surface plasmon resonance sensing transduction. Tests are done on two-dimensional gold nanoislands (Fig. 3, bottom). The gold nanoislands contain complementary DNA receptors, which hybridize to SARS-CoV-2 nucleic acids. This system can be excited at two different wavelengths as it uses two different angles of incidence, one from a plasmonic photothermal biosensor and the other from localized surface plasmon resonance. It can detect RdRp-COVID, F1ab-COVID and envelope (E) genes from SARS-CoV-2. The dual-functional localized surface plasmon resonance biosensor has a lower detection limit of 0.22 pM and allows precise detection of selected SARS-CoV-2 sequences in a multigene mixture. The plasmonic sensing system can significantly reduce the rate of false-positive results55. Similarly, others developed a plasmonic nanohole array used to transmit light for the label-free detection of the pathogen in biological media without sample preparation. It can quantitate intact virions by capturing them on group-specific antiviral immunoglobulins immobilized at the surface of the sensor. The intact virus binds to a suspended nanohole array grating that couples incident light to surface plasmons, causing a redshift in surface plasmonic resonance frequency. The assay could detect small (vesicular stomatitis virus and pseudotyped Ebola) and large (vaccinia virus) enveloped viruses. The non-destructive nature of the assay allows for further analysis of progeny virions50. Overall, the biosensors and other material-science-based detection techniques can enable rapid and portable diagnostic SARS-CoV-2 testing.

Detection of SARS-CoV-2 antibodies

The synthesis of antibodies against SARS-CoV-2 is a primary immune response to infection. Neutralizing antibodies are found in up to 50% of infected individuals by day 7 and in all infected individuals by day 14. Serological studies are an alternative to RT–PCR for SARS-CoV-2 diagnostics. Combining real-time PCR and serological testing significantly increases positive viral detection rates. IgM levels increase during the first week after SARS‐CoV‐2 infection, peak after 2 weeks and then fall back to near-background levels in most individuals. IgG is detectable after 1 week and is maintained at a high level for a long period56. In contrast, IgG becomes detectable after 1 week, remains elevated for an extended period, sometimes even more than 48 d, and may serve to protect against reinfection. IgA responses appear between 4 and 10 d after infection. Notably, a diagnostic predictor is the presence of serum IgA57 as well as IgG and IgM58. The spectrum of SARS-CoV-2 antibodies is explained, in part, by divergent target antigens. Antibody titres can decrease 7 d after infection56. Recent studies have identified SARS-CoV-2-specific antibodies in the saliva59,60. Multiplex SARS-CoV-2 antibody immunoassays were investigated to determine differences between antibody levels in saliva and sera. Antibodies in saliva consistent with those in sera suggest parallel compartmental humoral immune responses60. A parallel study developed rapid immunoassay using the BreviTest platform technology for measuring salivary IgA, which correlates with COVID-19 disease severity.

Interestingly, low levels of IgA were seen in individuals with IgG without known exposure to the virus, and suggest that it may represent an indicator of herd immunity59. SARS-CoV-2-specific antibody detection, especially that in saliva, may be useful for surveillance. Questions remain as to which antigens are the best candidates for serological testing. While the viral S is perhaps the strongest candidate, what remains unresolved is what part of the S should be developed. Alternatively, multiple isoforms of the S protein, such as those found in variant strains, may be used to ensure assay reproducibility61. Time to results can vary from 13 min (Abbott ID NOW) to 45 min (Cepheid Xpert Xpress)62. Of the five antibody-based tests available, two are lateral-flow immunoassays (BioMedomics rapid test and SureScreen rapid test cassette), one is a time-resolved fluorescence immunoassay (Goldsite diagnostics kit) and two are colloidal gold immunoassays (Assay Genie rapid PoC kit and VivaDiag COVID-19 IgG–IgM based).

Clinical studies will be needed to determine their clinical relevance63. For N-based immunoassays, SARS-CoV-2 IgG (Abbott) shows a sensitivity of up to 100% (ref. 64). For S-based immunoassays, Liaison SARS-CoV-2 S1/S2 IgG and the combination S- and N-based platform COVID-19 VIRCLIA IgG MONOTEST demonstrated equivalent sensitivities. The plaque reduction neutralization test showed a sensitivity of 93.3%. To evaluate specificity, all of the tests except one, the enzyme-linked immunosorbent assay (IgG) (EUROIMMUN), produced at least one positive result for the negative SARS-CoV-2 antigen control. This probably represents large discrepancies between the testing platforms and the assay sensitivity relative to time. Although the plaque reduction neutralization test is the gold standard for immunoglobin-based detection, the test has constraints, including a limited number of sample analyses, and requires a biosafety level 3 laboratory. The titres obtained from the assays correlate well with the plaque reduction neutralization test. Currently, antibody assays are applied principally for epidemiological testing65.

SARS-CoV-2 antigens

A rapid diagnostic assay was also developed to detect the presence of viral antigens expressed by SARS-CoV-2 in samples from the respiratory tract of infected individuals66. For this assay, antigen present in the sample binds to antibodies affixed to a paper strip enclosed in a plastic casing. This reaction generates a visually detectable signal within half an hour. The detected antigen(s) are expressed only if the virus is actively replicating; therefore, the tests can be used to identify acute or early infection66. Also, a more common type of rapid diagnostic assay, which detects the presence of antibodies in the blood of infected individuals, has been marketed for COVID-19 by Abbott. Abbott’s test can detect the SARS-CoV-2 antibody on ARCHITECT i1000SR and i2000SR laboratory instruments, which can run ~100–200 tests per hour67. Antibodies against SARS-CoV-2 are produced after one week of infection68. The strength of any antibody response depends on age, nutritional status, disease severity, comorbid conditions and medications.

Saliva testing

The presence of SARS-CoV-2 RNA in saliva samples is not always associated with disease severity, compared to nasopharyngeal swabs. Nevertheless, human saliva has gained attention as an alternative diagnostic medium for detecting infections69. Naso- or oropharyngeal swabs show limitations in sample collection and present a risk to healthcare workers through sneeze or cough and transmission of virus particles by aerosols10. In addition, in cases of thrombocytopenia or any other coagulation disorders the collection procedure can precipitate bleeding. These complications have led to testing sputum collection for diagnostic purposes. Sputum is an easy directed and non-invasive method of sampling. However, one limitation is that 72% of individuals with COVID-19 are unable to produce sufficient sample volume18. As a multiconstituent oral fluid, saliva has demonstrated high potential for the surveillance of general health and disease70. The ease of collection for diagnostics and monitoring without the need for medical staff can lead to ease of sample collection (Fig. 2c). Saliva is a useful biological medium, as it comprises proteins, nucleic acids, electrolytes and hormones originating from multiple local and systemic sources. Saliva is known to contain approximately 30% of biomolecules found in blood and harbours viral microorganisms71. Moreover, saliva samples can be stored in stabilizing solutions and posted several days later in the testing centre. Saliva collection is less invasive to the donor than blood collection and can permit home sampling69,70. Analysis of saliva samples in individuals with COVID-19 may facilitate the detection of both the virus itself and the antibodies, and as such shows potential as a diagnostic medium. Human saliva sampling may have a major potential for COVID-19 screening51,72. There is a concordance between detecting respiratory pathogens, including two seasonal human coronaviruses, in saliva using RT–PCR22. Indeed, mean SARS-CoV-2 titres (virus copies ml−1) were five times higher (P < 0.05) in saliva (n = 37) compared with nasopharyngeal swabs (n = 46). Furthermore, none of the negative saliva samples became positive. In contrast, in five instances, nasopharyngeal swabs first tested negative for SARS-CoV-2, followed by a positive test result when repeated51,72. However, ever more reliable sample collection that can be self-administered is still needed, with a significant directive of current research activities.

Faecal tests

Knowledge regarding virus incubation, transmission and shedding is crucial for protecting healthcare professionals and stop** the spread of SARS-CoV-2. High incidence and viral persistence in faeces have been observed when nasopharyngeal swab samples were virus negative103. BioFire Diagnostics is develo** FilmArray respiratory panels (RP and RP2), also referred to as BioFire respiratory panels, which will help clinicians rapidly diagnose SARS-CoV-2 and other respiratory infections104. Meridian Biosciences has created a Master Mix containing the building blocks for rapid testing by eliminating the RNA extraction, which facilitates conventional molecular procedural steps. This can significantly reduce the assay cost and time105. Similarly, Cepheid has also announced its SARS-CoV-2 test kit, which can be run on any of its 23,000 GeneXpert systems placed worldwide to deliver PoC results in 30 min (ref. 106). Recently, Abbott received US FDA EUA for its BinaxNOW COVID-19 Ag card, which depends on flow technology to detect SARS-CoV-2 antigen in a nasal swab from individuals with suspected COVID-19 with a sensitivity of 97.1% and specificity of 98.5%. The test can provide results in just 15 min at a cost of US$5 (ref. 107). Abbott also launched the NAVICA app, which allows people to display negative test results obtained from the healthcare provider in the form of a QR code to enter the organization that requires proof of testing. People with positive test results receive a message to quarantine and contact a healthcare provider for treatment107.