key: cord-0788349-81f9esin
authors: Yuan, Xi; Yang, Chengming; He, Qian; Chen, Junhu; Yu, Dongmei; Li, Jie; Zhai, Shiyao; Qin, Zhifeng; Du, Ke; Chu, Zhenhai; Qin, Peiwu
title: Current and Perspective Diagnostic Techniques for COVID-19
date: 2020-07-17
journal: ACS Infect Dis
DOI: 10.1021/acsinfecdis.0c00365
sha: 84cac7e07587ad42a520bc6cf687015f42285dc3
doc_id: 788349
cord_uid: 81f9esin

[Image: see text] Since late December 2019, the coronavirus pandemic (COVID-19; previously known as 2019-nCoV) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been surging rapidly around the world. With more than 1,700,000 confirmed cases, the world faces an unprecedented economic, social, and health impact. The early, rapid, sensitive, and accurate diagnosis of viral infection provides rapid responses for public health surveillance, prevention, and control of contagious diffusion. More than 30% of the confirmed cases are asymptomatic, and the high false-negative rate (FNR) of a single assay requires the development of novel diagnostic techniques, combinative approaches, sampling from different locations, and consecutive detection. The recurrence of discharged patients indicates the need for long-term monitoring and tracking. Diagnostic and therapeutic methods are evolving with a deeper understanding of virus pathology and the potential for relapse. In this Review, a comprehensive summary and comparison of different SARS-CoV-2 diagnostic methods are provided for researchers and clinicians to develop appropriate strategies for the timely and effective detection of SARS-CoV-2. The survey of current biosensors and diagnostic devices for viral nucleic acids, proteins, and particles and chest tomography will provide insight into the development of novel perspective techniques for the diagnosis of COVID-19.

T he rapid increase in confirmed cases of COVID-19 remains uncontrollable, with an increase of approximately 200,000 diagnosed patients globally per day. So far, according to the WHO official counts, this widespread outbreak of coronavirus has over 1,700,000 infected cases with more than 670,000 deaths. 1 SARS-CoV-2 is the pathogen that causes COVID-19. SARS-CoV-2 is named because of its genetic similarity to the severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) discovered in 2003. Belonging to the coronavirus (CoVs) clade, SARS-CoV-2 has a single-stranded positive-sense RNA genome with 30 kilobases in length and is about 80−120 nm in diameter. 2 SARS-CoV-2 is the seventh CoVs known to cause infections in humans. Of the six previously discovered coronaviruses, four of them (HCoV-OC43, -229E, -NL63, and -HKU1) caused common cold symptoms in immunocompetent individuals, 3 while the remaining two (SARS-CoV-1 and MERS-CoV) had high mortality rates of zoonotic origin. 4 Early symptoms in COVID-19 patients include fever, dry cough, shortness of breath, headache, muscle soreness, and fatigue. 5, 6 However, the symptoms are not deterministic due to the identification of asymptomatic SARS-CoV-2 carriers and the overlapping features with other acute respiratory viral infections such as influenza. 5−7 Therefore, highly sensitive and specific diagnostic methods that can distinguish COVID-19 cases from healthy or other virus-infected individuals are essential for disease management and therapeutics. Currently, various organizations have reported a variety of methods for the clinical diagnosis of COVID-19, which have different principles, operations, costs, and sensitivities. Here, we thoroughly review current diagnostic techniques for researchers and clinicians to develop appropriate methods for the timely and effective diagnosis of COVID-19 or the detection of SARS-CoV-2. The principles learned from other viral diagnostics will guide the SARS-CoV-2 diagnostics development. The review of other viral particle, nucleic acid, and protein detection methods provides insight into the development of novel SARS-CoV-2 diagnostic techniques.

In the early stage of the COVID-19 outbreak in Wuhan, researchers isolated the virus from infected pneumonia patients and characterized the pathogen using metagenomic nextgeneration sequencing (mNGS) and electron microscopy. 8, 9 SARS-CoV-2 is a pathological nanoparticle composed essentially of protein and RNA. The first draft of the SARS-CoV-2 genome was released on January 10, 2020 (GenBank: MN908947). The SARS-CoV-2 genome is 29,891 nucleotides in length, encoding 9,860 amino acids that are homologous to lineage B β-CoVs. 8, 10, 11 SARS-CoV-2 is 96.3% homologous to BatCoV RaTG13 but discordant with SARS-CoV-1 and MERS-CoV. 12 The SARS-CoV-2 genome contains five major open reading frames (ORFs), arranged in the order of the 5′ untranslated region (UTR)-replicase complex (ORF1ab)-Spike (S)-Envelope (E)-Membrane (M)-Nucleocapsid (N)-3′ UTR and accessory genes such as 3a, 6, 7a, 7b, and 8 ( Figure 1 ). 8, 10 The ORF1ab gene encodes the nonstructural proteins that aid viral genome replication and transcription, which has about 90% nucleotide sequence (nts) identity to SARS-CoV-1. 11 The E gene, which encodes the membrane protein involving virus assembly, budding, envelop formation, and pathogenesis, 13 has the highest (93%) nts identity with SARS-CoV-1. The S gene, responsible for virus binding and cell entry, 14 shares less than 75% nts identity with other SARS-CoVs, except for RaTG13 (93%). 11 Compared to S and E proteins, M and N proteins are more abundant, which bind to the RNA genome and participate in virus assembly and budding, respectively, where the M and N genes share approximately 90% nts identity with SARS-CoV-1. 11 SARS-CoV-2 infection is initiated with viral entry, in which the S protein first recognizes and binds to angiotensinconverting enzyme 2 (ACE2), a host membrane receptor, and then induces fusion of the virus membrane with the host cell membrane. 8 The next step is the entry of the virion or its RNA genome. The viral antigen is then presented by the antigenpresenting cells, which subsequently stimulates humoral and cellular immunity. The primary humoral immune response has a typical pattern of IgA, IgM, and IgG production. IgG antibodies are primarily S-and N-specific antibodies, 15 which can be utilized as antigens for SARS-CoV-2 antibody development.

Diagnosis of COVID-19 by Viral Cytopathic Effects. According to the Koch's postulates, virus isolation from clinical samples remains the "gold standard" for diagnosing viral infections. The enriched virus minimizes the perturbation from the patients' samples and increases the signal-to-noise ratio for virus detection. Nasopharyngeal swab (NPS), oropharyngeal swab (OPS), bronchoalveolar lavage fluid (BALF), sputum, and stool samples collected from suspicious cases are used for virus isolation. 16 Sampling operations, specimen source, and sampling time all affect diagnosis to a certain extent. 17 The extracted virus shows obvious cytopathogenic effects in both Vero and Huh7 cells, resulting in observable morphological changes visualized by electron microscopy after nucleocapsid staining. 8 A TMPRSS2-expressing VeroE6 cell line is highly susceptible to SARS-CoV-2 infection, making it ideal for virus isolation and cytopathic testing. 18 Photoclickable fluorogenic probes enable the visualization of specific DNA or RNA with precise spatiotemporal control in their native cellular environment, 19 facilitating the diagnosis of the viral cytopathic effect. However, virus isolation and visualization are impractical for large-scale diagnosis of COVID-19, since SARS-CoV-2 requires at least 3 days to inflict obvious cytopathic effects on selected cell lines. 8 In addition, virus isolation and culture require well-trained personnel and a Biosafety Level 3 environment, which are not available in most healthcare clinics. 20 Detection of SARS-CoV-2 Intact Particles. The assimilated principles from other virus diagnostic techniques lay a solid foundation for the development of novel diagnostic techniques for SARS-CoV-2. A portable platform captures viruses by their sizes, coupled with Raman spectroscopy for identification. 21 The SARS-CoV-2 particles in aerosol can be characterized with on-chip spectroscopy using a photonic cavity enhanced silicon nitride (Si 3 N 4 ) racetrack resonatorbased sensor. 22 The electrochemical biosensor based on surface imprinted polymers and graphene oxide composites can identify isolated virus, which has a limit of detection (LoD) similar to RT-PCR for Zika virus detection. 23 Solidstate micro-and nanopores monitor viral particles in the air by cross-pore ionic current. 24 Capillary biosensor, which is employed for the detection of bacterial and viral pathogens, uses a multicolumn capillary for easy separation, Fe nanoclusters for signal amplification, and a smartphone for image analysis. 25 After capturing the virus, microbeads generate a unique diffraction pattern of "blurry beads" on a smartphone. 26 The smartphone-based colorimetric assay utilizes 3D nanostructures as a scaffold for conjugating antibodies to capture virus particles, where the 3D structure provides more virus binding sites. 27 DNA origami-based aptasensors for virus, viral RNA, and protein detection need further attention due to their 3D structure, simple fabrication, and rapid response. 28 Interdigitated organic electrochemical transistors function as transducers to enhance the sensitivity and dynamic detection range of aptasensors. 29 Moreover, a viral imprinted polymerbased biosensor detects the virus at ng/mL. 30 An ultrasensitive electrolyte-gated field-effect transistor (FET) transduces biorecognition events into virus concentrations. 31 Lab-on-achip having a specific biosensor and sensitive detector is desirable for SARS-CoV-2 diagnostics without nucleic acid isolation and amplification. 32 Direct Detection of SARS-CoV-2 RNA. Detection techniques for mRNA or miRNA can be adapted for the detection of SARS-CoV-2 RNA. The miRNA logic sensing techniques amplify signal gain with a programmed nanoparticle network, which is composed of DNA-bridged gold nanoparticles and quantum dots (QDs). 33 Chemiluminescence with ACS Infectious Diseases pubs.acs.org/journal/aidcbc Review a smartphone is capable of detecting miRNA through RNAtriggered in situ growth of nanoprobes in spherical nucleic acid enzymes, such as nanolabels and amplifiers. 34 Another miRNA biosensor uses generic neutravidin combined with electrochemically encoded responsive nanolabels to measure the opening of molecular beacons by means of a square-wave stripping voltammetry. 35 The biosensor that combines cyclic amplification with hairpin DNA detects attomolar miRNA via surface plasmon-enhanced electrochemiluminescence. 36 The gold nanoparticle with an RNA three-way junction motif can sense miRNA using electrochemical surface-enhanced Raman spectroscopy. 37 The photoelectrochemical biosensor, based on a MoS 2 /g-C 3 N 4 /black TiO 2 heterojunction as the photoactive material and gold nanoparticles carrying Histostar antibodies for signal amplification, captures miRNA that leads to horseradish peroxidase (HRP) immobilization, whose insoluble products in the electrode reduce photocurrent. 38 The dual signal amplification system with the hybridization chain reaction and enzyme-induced metallization with anodic stripping voltammetry measures miRNA in the femtomole range. 39 The polydiacetylene microtube waveguide with catalytic hairpin assembly as a signal amplification strategy shows femtomolar sensitivity for RNA detection. 40 The amperometric genosensor based on peptide nucleic acid (PNA) probes immobilized on carbon nanotube-screen printed electrodes determines trace levels of viral RNA. 41 The plasmonic nanoparticle-based optical biosensor array functionalized with capture DNA binds to complementary viral RNA, which can be reversed by chemical treatment (10 mM HCl). 35 FET with a two-dimensional channel made of a single graphene layer achieves label-free detection of RNA down to attomolar concentration. 42 Single-molecule fluorescence resonance energy transfer remains a promising tool for multiplex viral RNA detection (∼200 pM) with interconvertible hairpin-based sensors. 43 Nanoporous anodic alumina supports oligonucleotides as gated ensembles to detect the presence of viral RNA. 44 The 3D nanostructure has a high surface-to-volume ratio and well-controlled shape factors for virus and nucleic acid capture. 45 Previously, a bead-stacked nanodevice has been developed for pathogen trapping 46 and micropillar polydimethylsiloxane, for rapid viral DNA sensing. 47 Protein nanopore detects viral RNA at the singlemolecule level using specific DNA probes. 48 Digital-resolution detection of viral RNA by photonic resonator absorption microscopy relies on RNA-activated nanoparticle−photonic crystal hybrid coupling. 49 Polyadenine-mediated spherical nucleic acid probes on gold nanoparticles facilitate the programmable detection of RNA in saliva or serum samples. 50 Carbon and graphene quantum dots have unique electronic and fluorescent properties, which can be conjugated to RNA or DNA substrate for viral RNA detection. 51 Upconversion nanoprobes with external luminescence quenchers are expected to be used for viral RNA detection due to their high energy transfer efficiency. 52 Carbon nanotube-based sensors have demonstrated the potential of viral optical detection without purification, amplification, and labeling. 53 Nanoparticles fabricated or printed on a 3D structure and functionalized with PNA or DNA efficiently capture SARS-CoV-2 RNA, where single molecule or signal amplification cascade enables the ultrasensitive label-free detection.

SARS-CoV-2 Detection with Genomic Sequencing. For an unknown virus, genomic sequencing is important for its rapid and complete identification. Genomic sequencing has many advantages in virus detection. First, it enables investigation of the SARS-CoV-2 evolution during transmission and tracking of coronavirus movements. 54 Second, sequencing can effectively exclude other pathogens. Third, genomic sequencing can identify multiple pathogens existing in a single patient, which helps implement reasonable treatments. However, operation and data analysis require expensive equipment, long turnaround time, and professional bioinformatics personnel. Droplet-based single-cell sequencing technology, such as inDrop, Drop-seq, and 10× Genomics with barcoded beads, can deliver primers to the beads and achieve high detection efficiency. 55 Currently, viral genome sequencing has top priority at the current stage of the outbreak, but patients' genome sequencing will be informative in understanding human susceptibility to SARS-CoV-2.

SARS-CoV-2 Detection with Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR). Currently, qRT-PCR is commonly used for the clinical diagnosis of SARS-CoV-2 infection. 56 The steps of qRT-PCR are as follows: (1) extract RNA from patient samples; (2) reversely transcribe viral RNA into cDNA and then amplify the specific cDNA fragment by PCR; (3) read the fluorescent signal. Smaller Cycle threshold (C t ) values indicate positive diagnostic results. The C t value of qPCR is the cycle number at which the fluorescent signal of a qPCR product rises above the background threshold.

Some qRT-PCR methods achieve single molecular sensitivity (within 10 molecules) in the laboratory (Table 1) . Regarding clinical diagnosis, 11 fluorescent qRT-PCR kits have received emergency approval from China's National Medical Products Administration (NMPA), 57 and 53 qRT-PCR kits have received an Emergency Use Authorization (EUA) by the Food and Drug Administration (FDA) ( Table 2 ). 58 These SARS-CoV-2 qRT-PCR kits targeting the S, E, N, or ORF1ab genes are a single-or multiple-target system. The PerkinElmer New Coronavirus Nucleic test kit has the lowest LoD (N gene: 24.884 copies/mL; ORF1ab gene: 9.307 copies/mL).

qRT-PCR requires a 2−4 h turnaround time and a laboratory certified under the Clinical Laboratory Improvement Amendments (CLIA) or a clinical laboratory with trained personnel, whose efficiency is low to satisfy the screening requirements of the COVID-19 pandemic. The automated qRT-PCR instrument, which integrates RNA extraction, concentration measurement, reagent preparation, and target sequence amplification/detection steps, has achieved significant improvements in detection efficiency and assay throughput ( Table 2 ). The US FDA has approved five automated PCR-based systems that can return diagnostic results in less than an hour: the Accula SARS-CoV-2 test on Accula dock or Silaris dock (30 min), the BioFire COVID-19 test and BioFire Respiratory Panel 2.1 on the FilmArray system (50 and 45 min), the QIAstat-Dx Respiratory SARS-CoV-2 Panel on the QIAstat-Dx Analyzer System (1 h), and the NeuMoDx SARS-CoV-2 Assay on the NeuMoDx 288 Molecular System (1 h). The Accula integrates PCR with lateral flow technologies, while the ePlex combines DNA hybridization with electrochemical detection. QIAstat-Dx, and BioFire Respiratory Panel 2.1 (RP2.1) are multiplexed and can distinguish SARS-CoV-2 from other bacteria and viruses (more than 21 types). Fully automated diagnostic devices have a LoD of 125 to 1 × 10 5 copies/mL, which seems less sensitive than nonfully automated diagnostic devices with a LoD of 10 to 5.5 × 10 4 copies/mL. qRT-PCR-based diagnostic techniques have several advantages. First, it is more suitable for early stage diagnosis than the antigen−antibody reaction, since it takes time to trigger the immune response. Second, qRT-PCR can dynamically monitor the degree of viral infection and assess the prognosis. However, the COVID-19 diagnostic FNR of qRT-PCR exceeded 30%. 17,59 Some asymptomatic COVID-19 patients show abnormal manifestations on their CT images before positive qRT-PCR. 60 Here are the possible reasons that can explain the FNR. First, the mutation leads to the off-target mismatch between the primer and the target sequence. The ORF1a, ORF 8, and N gene contain hot-spot loci such as 28 144 in ORF 8 (30.53%), 8782 in ORF1a (29.47%), and 29 095 in N (11.58%). 61 Second, for asymptomatic, mildly symptomatic, or discharged patients, low viral loads may be insufficient for qRT-PCR detection. Third, the presence of interfering substances may generate "false-positive" results. Furthermore, the conditions of specimen transportation and preservation as well as the quality of the test kits may significantly affect the diagnostic accuracy. The integration of the qRT-PCR with clinical manifestations and imaging examinations is more efficient and accurate in diagnosing suspected COVID-19 patients. Furthermore, microfluidic digital array PCR that provides gene quantification at the single-molecule level may reduce the false-positive rate. 62 SARS-CoV-2 RNA Detection with Isothermal Amplification. Loop-mediated isothermal amplification (LAMP) for SARS-CoV-2 detection avoids the high-temperature melting step in PCR by using strand displacement DNA polymerase in conjunction with 4 to 6 specially designed primers to achieve highly specific DNA amplification, which can realize 10 9 −10 10fold amplification in 15−60 min at 65°C. The one-step singletube reverse transcription LAMP (RT-LAMP) can detect as low as 10 copies of RNA fragments within 15−40 min ( Figure  2a) . 63 The final amplification products can be visualized by agarose gel electrophoresis or real-time monitoring by fluorescence, turbidity, or colorimetry. Silicon bead-based microfluidics can detect the amplification product in the femtomolar range. 64 Nanopores fabricated in the silicon nitride membrane can quantify DNA concentration in attomoles. 65 A label-free impedimetric electrochemical biosensor identifies Zika DNA in a nanomolar range. 66 The DNA sensor based on the synergistic enhancement strategy of Zn-doped MoS 2 quantum dots and reductive Cu(I) particles determines the DNA in a nanomolar concentration. 67 Universal DNA biosensing based on electrostatic attraction between hexaammineruthenium(III) and DNA molecules detects DNA in solution without the immobilization of capture probes on the electrode. Stepping gating of ion channels on the nanoelectrode via DNA hybridization, carbon dot stabilized silver−lipid nanohybrids, and carbon nanotube-based FET shows label-free DNA detection. 68−70 Digital LAMP on a track-etched polycarbonate membrane can quantify DNA fragments at 10 copies/μL. 71 qLAMP with bisintercalating DNA redox reporters amplifies DNA in 10 min. 72 Fractal LAMP automatically detects label-free DNA in subnanoliter droplets. 73 "LAMP-seq" combines RT-LAMP with next-generation sequencing for large-scale COVID-19 diagnostics. 74 LAMP and a graphene-based screen-printed electrochemical sensor can lead to on-site detection of the pathogen's nucleic acid. 75 The LAMP-based ID NOW system automatically returns the result in 13 min, which has a LoD of 125 copies/mL for SARS-CoV-2 detection, and has been approved by the US FDA for emergency use. The droplet microfluidic LAMP is high throughput for viral RNA detection. 76 The use of eosin photopolymerization for the detection of colorimetric LAMP products reduces falsepositive risk. 77 The recombinase polymerase amplification (RPA) uses recombinases, single-stranded DNA-binding proteins (SSB), and displacement enzymes to replace the high-temperature step in PCR. RPA achieves 10 4 -fold amplification in 10 min at a temperature between 25 and 42°C. 78 Two-stage Penn-RAMP combines LAMP with RPA for the diagnosis of COVID-19: RPA at 38°C for 15−20 min and LAMP at 63°C for 40 min (Figure 2b ). 79 The signal readout is either fluorescent or colorimetric, which is 10 times more sensitive than LAMP and qRT-PCR for detecting SARS-CoV-2 mimics. Rolling circle amplification (RCA)-actuated LAMP measures miRNA in the attomolar range. 80 RCA-based nicking-assisted enzymatic cascade amplification captures amplicons with magnetic nanoparticles through real-time optomagnetic measurement, which can detect dengue virus at the femtomolar (fM) level. 81 Microfluidic-circular fluorescent probe-mediated isothermal amplification can detect viruses up to 10 copies/μL. 82 With the naked eye or microphone readout, LAMP, RPA, and RCA permit the diagnosis of SARS-CoV-2 in the clinic or at home by minimally trained personnel with a simple temperature control device. However, commonly used LAMP requires a complex design of six primers with significant restrictions on their respective position and orientation. Besides, LAMP is not appropriate for multiplex detection due to primer−primer interactions. RPA is inhibited by genomic DNA (20−100 ng/μL); thus, RNA purification is required. The combination of digital, automatic, quantitative, and droplet microfluidic isothermal amplification and other techniques such CRISPR and nanotechnology represents the vigorous future research direction.

SARS-CoV-2 RNA Detection with CRISPR/Cas System. The CRISPR/Cas system, as a potent technology for genome editing, 86, 87 has recently been transformed into virus detection. 88,89 Elements of this system include Cas proteins, guide RNAs (gRNAs), and target RNA/DNA fragments. Both Cas13a and Cas12a have collateral cleavage activity after target binding, where nonspecific single-stranded RNA or DNA labeled with a fluorescent reporter and quencher are digested and released, respectively (10 4 -fold). 89, 90 CRISPR/Cas13a combined with RPA can achieve a LoD of 10−100 copies/ μL. 91 Previously, we integrated CRISPR/Cas13a with microfluidics and custom-designed a mini-fluorometer to detect Ebola virus at 20 plaque forming units (pfu)/mL. 90 The assay combining CRISPR/Cas13a with isothermal amplification for the detection of SARS-CoV-2 has been released, 83 demonstrated in clinical scenarios, 92 and approved by the US FDA for emergency use. Cas12a combined with RT-LAMP exhibits a LoD of 10 copies/μL with 90% sensitivity and 100% specificity in clinical samples. 84 We have applied the Cas12a assay to detect Africa Swine Fever Virus DNA up to 100 fM. 89 Viral RNA or transcribed cDNA passing through nanopores will generate double or triple peaks for viral nucleic acid characterization if the Cas13a or Cas12a complex is associated with the target sequence. 93 CRISPR is the RNA or DNA detection platform that can be integrated with any nucleic acid amplification systems. 83, 84, 91 SARS-CoV-2 RNA Detection by Microarray. Microarray is a powerful analytical tool, which can simultaneously detect multiple targets in a single sample. A DNA microarray consists of a DNA probe array on a supporting material, where the DNA sequence and location are known to characterize unknown RNA/DNA samples. Today, microarray-based platforms can be classified into different families on the basis of the type of matrix (solid or liquid), the size and density of the probe, the method used for visualizing the hybridization results, and the relative costs. Microarray has the advantages of requiring a small sample volume, a rapid reaction in 15 min, and multiple probes per target, which are widely used in the diagnosis of respiratory tract viral infections. 94 High-performance nanoplasmonics and bioprinted microarrays can quantify label-free biomass, with a sample-to-data turnaround time of 40 min. 95 The optical path difference based on the transmission characteristics of the plasmonic gold nanohole determines the pathogen concentration. However, the paperbased microarray integrated with carbon nanodots, which is used for analyte recognition and the smartphone reader, exhibits great potential for viral RNA detection. 96 The coronavirus protein microarray harboring the SARS-CoV-2 and other coronavirus proteins can classify multiple types of infections. 97 Oligonucleotide microchips are hybridized with fluorescently labeled viral RNA, immobilized on a gel, fixed on a glass plate, and imaged by fluorescence microscopy. 98 SARS-CoV-2 Detection by Immunochemistry. SARS-CoV-2 carries a variety of structural proteins on the surface, containing multiple epitopes for antibody or nanobody recognization. 99 There are various techniques for the detection of antigens and antibodies, where a single-shot mass spectrometry can distinguish viral subtypes. 100 A sandwichtype electrochemical immunosensor, which is used for the detection of virus-specific antigen based on a Pd nanoparticle (NP) functionalized electroactive Co-containing metal-organic framework (Co-MOF) for signal amplification, detects viral antigen up to 100 fg/mL. 101 An impedance-based biosensor identifies SARS-CoV-2 pathogens with an ACE2-decorated electrode via a self-assembled monolayer, while the performance usually improves after the insertion of redox-active alkanethiol. 102 A sensor array based on carbon or carbon polymer dots with antibody functionality can detect aqueous proteins at 100 nM. 103, 104 Furthermore, the highly active Fe 3 O 4 nanozyme amplifies the signals of the photoelectrochemical immunoassay. 105 A set of surface-enhanced Raman spectroscopy nanotags can detect multiple viral antigens through a sandwich immunoassay. 106 Moreover, 3D total internal reflection scattering defocus microscopy with wavelength-dependent transmission grating increases the spectral resolution and detects viral capsids at 820 μM. 107 In addition, the digital microfluidics platform based on electrowetting-ondielectric separates antibody-bound microbeads for immunoassay. 108 The microfluidic method, using a monolithic disposable cartridge, facilitates hands-free saliva analysis by integrating sample pretreatment with electrophoretic immunoassays to measure viral antigens. 109 These available methods are important learning examples for developing the SARS-CoV-2 immunosensor.

Human SARS-CoV-2-specific antibody measurement is an alternative diagnostic method. IgM antibodies are detected after 5−7 days of infection, followed by IgG. 110 A colloidal gold immunochromatographic assay (GICA) strip and a peptide-based magnetic chemiluminescence enzyme immunoassay (MCLIA) are used to detect SARS-CoV-2 IgM and IgG antibodies (Figure 2c) . 85, 111 Compared to GICA and MCLIA, the SARS-CoV-2 IgG/IgM ELISA kits display a sensitivity of 87.3% with a longer time and more sophisticated operations. 112 Immunoassays can be digital and multiplexing for the detection of multiple SARS-CoV-2 subtypes by using a smartphone fluorescent reader. The reader utilizes an optoelectromechanical hardware that snaps at the back of a smartphone for data collection, interpretation, display, and communication. 113 The sensitivity of the immunosensor can be increased by means of a triple signal amplification strategy based on (i) carboxylic ester hydrolysis, (ii) electrochemical− chemical redox cycling, and (iii) electrochemical−enzymatic redox cycling. 114 NMPA (8) and US FDA (12) have approved a variety of antibody-based kits, which can be used for the emergency detection of SARS-CoV-2 antibodies in human serum or plasma samples (Table 2) . Besides, a test kit for detecting SARS-CoV-2 antigen in OPS and NPS samples has been approved by US FDA for emergency use. The immunological method circumvents the extraction of virus nucleic acid and shortens the detection time, which is safer. However, the immunological method is not applicable for the early diagnosis of COVID-19 due to the low antibody concentration at the beginning. Besides, antibody detection is susceptible to the presence of interfering substances (such as rheumatoid factor and nonspecific IgM) in blood samples 85, 115 and antibody responses to SAR-CoV-2 in COVID-19 recovered patients. The SARS-CoV-2 antibody test kits are allowed to be used as a supplementary diagnostic method to nucleic acid test kits, but they cannot be used to confirm SARS-CoV-2 infection.

COVID-19 Diagnosis by Chest Computed Tomography (CT). COVID-19 is a fetal viral pneumonia, where breath chemical and respiratory behavior sensors may be helpful to diagnose and evaluate prognosis through a time-series readout.

The chemoresistive orthogonal sensor array enables a simple and portable breath analyzer in daily settings. 116 A wireless wearable sensor on a paper substrate can continuously monitor respiratory behavior and transmit ultrasound data to a smartphone. 117 Chest CT demonstrates typical radiographic features in most patients with COVID-19, including groundglass opacity, multifocal patchy consolidation, and/or interstitial changes with a peripheral distribution. 118 Typical CT features are visible in some patients with negative qRT-PCR results but with clinical symptoms. 17 C-reactive protein, erythrocyte sedimentation rate, and lactate dehydrogenase were positively correlated with the severity of pneumonia assessed on CT. 119 However, the limited number of radiologists is insufficient to accommodate a large number of patients in some regions. Deep learning (DL) typically extracts abstract features in CT images to segment the lesion region and classifies COVID-19 into 6 categories: healthy, ultraearly, early, rapid progression, consolidation, and dissipation stages. 120 Different CT learning models have been developed to aid the diagnosis of COVID-19. The UNet++ takes the raw CT scan images as input and the segmented map as output, which is trained in Keras in an image-to-image manner. The sensitivity (true positive/positive), accuracy (true predictions/total number of cases), specificity (true negative/ negative), positive prediction value (PPV), and negative prediction value (NPV) of the network were 100%, 92.59%, 81.82%, 88.89% and 100%, respectively. The average prediction time per patient is 41.34 s, and the reading time of the radiologist is reduced by 65%. 121 The 3D deep learning model with the classic ResNet-18 network can achieve CT image classification and segmentation with an accuracy rate of 86.7%. 122 Deep pneumonia can assist the COVID-19 diagnosis in three steps. The first step is to extract the main regions of the lungs, and the second step is to design a Details Relation Extraction neural network (DRE-Net) to obtain image-level predictions. Finally, DRE-Net, which starts with a pretrained ResNet50 and an extra Feature Pyramid Network (FPN), is used to extract top-K details from each image and understand the importance of each detail through the attention module. The image level predictions can achieve 93% diagnostic sensitivity and can distinguish SARS-CoV-2 from bacterialrelated pneumonia with 96% sensitivity. Moreover, their models were able to locate the main lesion features in 30 s, especially the ground-glass opacity. 123 The transfer learning based on the modified Inception (M-Inception) consists of two parts: the first part uses a pretrained inception network to convert image data into one-dimensional feature vectors, and the second part uses a fully connected network for classification and prediction. The M-Inception reduces the dimension of the features before the classification layer and combines the Decision tree and AdaBoost as classifiers to improve the classification accuracy. 124 The Human-in-the-loop (HITL) strategy assists radiologists in performing automatic annotation and segmentation. The Dice similarity coefficient generated by the system between automatic and manual segmentation is 91.6% ± 10.0%. The Dice similarity coefficient is an index to measure the spatial overlap between the segmentation result and the ground truth. Fully manual delineation takes 1 to 5 h, but HITL reduces delineation time to 4 min after 3 iterative model updates. 125 DeepLabv3 segmented CT images have a higher accuracy compared to U-net, SegNet, FCN, and DRUNET. 126 However, the CT features of COVID-19 can easily be confused with other infections such as SARS-CoV-1 and seasonal flu. Integrating hierarchical CT features with other factors such as genetic, epidemiological, and clinical information for multimodal analysis will definitely further improve the efficiency and accuracy of COVID-19 diagnosis. 127 

Lessons learned from the past play a vital role to combat against the COVID-19 pandemic. In comparison with the SARS-CoV-1 outbreak in 2003, artificial intelligence (AI) has played a more important role, including contact tracing, evidence-based policymaking, and detection of COVID-19 infection in medical images (chest X-rays and CT scans). AIenabled technologies, such as the AI robot, smart toilet, and smartphone, could provide valuable support for tracing, diagnostics, and therapeutics ( Figure 3) . The smart toilet, as a noninvasive device that can collect and analyze urine and stool, may become an important strategy for the diagnosis of COVID-19. Systems with cameras, pressure and motion sensors, and urinalysis strips have been used for health monitoring. 128 The robot can work in risky environments, thereby relieving the pressure on the frontline medical staff. The AI robot is capable of collecting throat swabs. The diagnostic and service robots will be deployed in crowded public places, such as customs, airports, ports, railway stations, etc. The mobile robot increases the efficiency and coverage of COVID-19 screening. Hopefully, with the consistent development of AI technologies, smarter and more powerful robotic systems under remote control with the ability to disinfect, deliver medications and food, measure temperature and vital signs, diagnose, and assist in transmission control will revolutionize weapons to fight against future viral epidemic outbreaks.

Asymptomatic and discharged COVID-19 patients are likely to transmit the virus to persons who are in close contact, making it challenging for the prevention and control of epidemics and requiring long-term monitoring and tracking. SARS-CoV-2 RNA has been detected in urine and stool samples of certain confirmed patients, even in asymptomatic cases, 129 indicating the high demand for ultrasensitive diagnostic techniques. A highly sensitive diagnostic platform can be developed by combining fluorescence resonance energy transfer, aggregation-induced emission molecules, and luminescent upconverting nanoparticles with unique optical properties ( Figure 3 ). The magnetic properties of some nanoparticles, which enable the enrichment of fluorophores, offer the convenience of developing a smartphone-based readout system. The smartphone equipped with different optomechanical accessories, which enable the performance of brightfield, fluorescence, spectroscopy, or phase imaging, is a promising read-out platform. These devices make precise diagnostics possible in remote areas. The miniaturized sequencing devices make it available outside the laboratory for on-site applications. Synthetic biology has created novel molecular functions that can bring new capabilities to molecular diagnostics. 130 The development of DNA aptamers targeting viral proteins and conjugation with magnetic particles enable the enrichment of viruses during sampling and diagnostics. 131 The QDs with small volume and strong fluorescence are promising reporters for various fluorescencebased detection systems. The fluorescence polarization assay (FPA) takes advantage of molecular rotational properties to measure antigen−antibody or probe-target binding 132 with a small volume (2 μL) and a short diagnosis time (<20 min). 133 With a deeper understanding of viral−host interaction, novel dual biosensors, and the combination with isothermal amplification or immunoassay technology, the next-generation diagnostic techniques will be more robust, rapid, accurate, and easy to operate.

The SARS-CoV-2 genome undergoes rapid evolution during its transmission. 134−136 According to the available SARS-CoV-2 genome sequenced up to June 1, 2020, globally, 8309 single mutations have been observed in 15 140 SARS-CoV-2 isolates. 135 These mutations might affect the efficiency of oligonucleotide annealing and compromise the sensitivity and specificity of RT-PCR assays. 137, 138 The target genes selected for molecular diagnosis should be relatively conserved. Meanwhile, multiple-target detection can minimize the risk of sensitivity loss due to unknown mutations. The kits of the early versions should be used with caution for the detection of prevalent SARS-CoV-2 subtypes. Genomic sequencing guides the design of nucleic acid-based diagnosis. The combination of multiple assays including viral RNA detection, lateral flow immunoassay, NGS, clinical symptoms, and CT imaging will provide a more accurate diagnosis. However, under real clinical conditions, there should be a balance between cost, time, accuracy, and specificity.

Vaccines and drugs for SARS-CoV-2 are the goals of global scientific competition. DNA vaccines are leading ahead of protein vaccines. The nanoparticle vaccine composed of a stabilized form of the respiratory syncytial virus (RSV) fusion (F) protein is fused to ferritin nanoparticle (Pre-F-NP) and modified with glycans to mask poorly neutralizing epitopes, thereby eliciting a potent immune response in mice and primates. 139 Sinovac Biotech has announced a vaccine that can protect monkeys from new coronavirus infections. Moreover, Moderna and CanSino have launched small vaccine clinical trials against COVID-19. Broad-spectrum antivirals inhibit SARS-CoV-2 in human airway epithelial cells and in mice. 140 Furthermore, a systematic study of viral−host interactions identifies 332 high-confidence SARS-CoV-2 human protein− protein interactions (PPI) and 66 druggable PPIs targeted by 69 compounds. 141 The receptor-binding domain is implicated in a viral attachment inhibitor and vaccine. 142 Besides, the structure of the main protease of SARS-CoV-2 has led to the development of two lead compounds with good pharmacokinetic properties in cell assays. 143 A new α-ketoamide inhibitor reveals a pronounced lung tropism and suitability for administration by inhalation. 144 CIRSPR is an alternative strategy for viral RNA inhibition and degradation. 145 The race to find COVID-19 treatments is accelerating and may take a few months or a few years or never succeed. The development of AI-assisted plug-and-read diagnostic robots and the enforcement of social contact patterns will be critical in preventing COVID-19 epidemics before the appearance of vaccines and drugs. 146 Author Contributions † X.Y. and C.Y. contributed equally to this work. All authors were involved in the discussion and writing of the manuscript. All authors have read and agreed to the published version of the manuscript.

The authors declare no competing financial interest.

This research is funded by Zhejiang University special scientific research fund for COVID-19 prevention and control and SUSTech Intelligent Health Engineering Platform (G02326403). The authors thank Wenrong He for the schematic design.

■ ABBREVIATIONS COVID-19, coronavirus disease 2019; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; FNR, false-negative rate; SARS-CoV-1, severe acute respiratory syndrome coronavirus 1; CoVs, coronavirus; mNGS, metagenomic next-generation sequencing; ORFs, open reading frames; UTR, untranslated region; nts, nucleotide sequence; ACE2, angiotensin-converting enzyme 2; NPS, nasopharyngeal swab; OPS, oropharyngeal swab; BALF, bronchoalveolar lavage fluid; LoD, limit of detection; FET, field-effect transistor; QDs, quantum dots; HRP, horseradish peroxidase; PNA, peptide nucleic acid; qRT-PCR, quantitative reverse transcriptionpolymerase chain reaction; C t , cycle threshold; NMPA, National Medical Products Administration; EUA, Emergency Use Authorization; FDA, Food and Drug Administration; CLIA, clinical laboratory improvement amendments; LAMP, loop-mediated isothermal amplification; RT-LAMP, reverse transcription LAMP; RPA, recombinase polymerase amplification; SSB, single-stranded DNA-binding protein; RCA, rolling circle amplification; fM, femtomolar; gRNAs, guide RNAs; pfu, plaque forming units; NPs, nanoparticles; GICA, gold immunochromatographic assay; MCLIA, magnetic chemiluminescence enzyme immunoassay; CT, computed tomography; DL, deep learning; PPV, positive prediction value; NPV, negative prediction value; HITL, Human-in-the-loop; AI, artificial intelligence; PPI, protein−protein interactions; Hel, helicase; ABI 7500, Applied Biosystems Real time PCR system 7500; GEC, genomic equivalents copies; TCID50, median tissue culture infectious dose per milliliter

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