key: cord-0993696-1nogvgdt
authors: Wang, Shiwen; Liu, Ying; Qiu, Yang; Dou, Qian; Han, Yang; Huang, Muhan; Hong, Ke; Yang, Bei; Zhou, Xi; Dai, Qing
title: Saliva-based point-of-care testing techniques for COVID-19 detection
date: 2022-05-01
journal: Virol Sin
DOI: 10.1016/j.virs.2022.04.004
sha: 018fa0981faebcc1b21411477642624b9abf22ee
doc_id: 993696
cord_uid: 1nogvgdt

nan

the shortcomings of nucleic-acid-based COVID-19 detection. As the unique evidence of a 61 COVID-19 infection, the detection of related antibodies or antigens in saliva can help to 62 confirm the infection status. Isho et al. observed a significant positive correlation of various 63 SARS-CoV-2-related antibodies in saliva and serum (Isho et al., 2020) . In our recent study, 64 the correlation between IgG antibody levels in both saliva and serum was tested using an 65 enzyme-linked immunosorbent assay (ELISA) kit. One hundred patients infected with 66 COVID-19 were enrolled from Wuhan Jinyintan hospital. Among them, 94% of the serum 67 samples tested positive for IgG antibodies, while 83% of the saliva samples were positive. 68 The sensitivity and specificity of IgG antibody detection in saliva were 87. 23% and 83.33%, 69 respectively. Notably, the IgG antibody titers in saliva were positively correlated with those in 70 serum (r = 0.579, P < 0.001) (Fig. 2) . These results indicated that a comparable accuracy of 71 antibody detection for COVID-19 can be achieved in saliva and serum. However, a time delay 72 exists in antibody detection compared to nucleic-acid-based methods since it takes 3-7 days 73 for a virus to activate sufficient immune responses, thus limiting the feasibility to use 74 antibody detection for early diagnosis. Hence, the combined detection of both antibody and 75 nucleic acid is more desirable to effectively improve the test precision for viral diseases. 76

As already mentioned, saliva specimens can be effectively used for the mass screening of 77 COVID-19. However, the detection of targeted biomarkers largely depends upon traditional 78 techniques, e.g., real-time polymerase chain reaction (RT-PCR) and ELISA, which have 79 stringent requirements for processing procedures, duration, and equipment. In contrast, 80 saliva-based POCT devices have emerged as promising alternatives. Various technical routes 81 for saliva-based POCT techniques have been developed for COVID-19 detection, including 82 colorimetric, optical, electrochemical, and piezoelectric biosensors (Table 1) . Each technique 83 has its own merits and limitations. Colorimetric biosensors are portable, easy to use and rapid 84 in response, with potential for large-scale POCT in remote locations (Li et al., 2020; Alafeef 85 et al., 2021; dos Santos et al., 2021; Ferreira et al., 2021) . As a typical example, the lateral 86 flow assay (LFA) is a type of these biosensors that uses conjugated gold nanoparticles, 87 fluorescent molecules, or quantum dots for visual sensing of color changes. LFA has been 88 demonstrated to detect IgM and IgG antibodies of COVID-19 within 15 minutes (Li et al., 89 so it is almost impossible to use LFA to detect the low concentrations of viral nucleic acid or 91 antibodies in saliva (Ning et al., 2021) . Very recently, an innovative method called Low-cost Optodiagnostic for Rapid testing (COLOR) has been developed by Ferreira et al., 93 which could detect SARS-CoV-2 within five minutes using a smartphone (Ferreira et al., 94 2021) . The COLOR test was found to be highly sensitive, with a detection limit of 0.154 95 pg/mL for spike protein (SP) and an accuracy of 90% in 100 clinical samples. Alafeef et al. 96 also designed an RNA-extraction-free nano-amplified colorimetric test for rapid and 97 naked-eye molecular diagnosis of COVID-19, with an accuracy, sensitivity, and specificity of > 98 98.4%, > 96.6% and 100%, respectively, and a detection limit of 10 copies/μL (Alafeef et al., 99 2021) . Another important type of biosensor commonly used in COVID-19 detection is based 100 on electrochemical principles, with distinct advantages of high sensitivity, user-friendliness, 101 and robustness, thus providing a reliable method for clinical diagnosis. Electrochemical 102 principles including current, impedimetric, potentiometric, or field-effect transduction (FET) 103 have been applied to develop biosensors to monitor the variations of viral nucleic acids or 104 antigens/antibodies. The FET biosensor could detect SARS-CoV-2 antigen in saliva or a 105 nasopharyngeal swab within one minute, with a detection limit of 0.2 pmol/L (Seo et al., 106 2020). Torres et al. developed a handheld electrochemical impedance spectroscopy (EIS, 107 RAPID) biosensor for rapid detection of SARS-CoV-2, exhibiting a sensitivity and specificity 108 of 85.3% and 100%, 100% and 86.5%, respectively, for nasopharyngeal/oropharyngeal swab 109 and saliva samples (Torres et al., 2021) . This type of electrochemical biosensors have rapidly 110

Wang developed a nanowell-based QCM aptasensor to detect H5N1 and avian influenza virus 121 (AIV), which significantly reduced the test time to a few minutes (Wang et al., 2017). 122 However, QCM sensors suitable for COVID-19 detection have not been reported, which 123 requires an elaborately designed COVID-19-specific probe to be developed at first. 124

Though these saliva-based POCT techniques are promising, it is difficult to use them for 125 clinical diagnoses due to challenges in both sample treatment and POCT device development. 126 On the one hand, there is an urgent need to standardize the whole procedure of saliva sample 127 treatment including collection, storage, transportation, and preparation. First, saliva is a 128 relatively heterogenic biofluid compared to blood since it is an exocrine secretion. This 129 renders it more sensitive to external stimuli such as smoking, eating and drinking, which may 130 induce distinct changes in its composition that potentially interfere with the subsequent 131 detection and analysis (Heikenfeld et al., 2019) . Thus, some standardized procedures should 132 be established before sample collection to maintain oral hygiene and eliminate possible 133 interferences, such as fasting for 12 h, gargling 3-4 times, avoiding gum bleeding, etc. 134 Second, appropriate sample collection method, including coughing out, saliva swabs, and 135 direct collection from the salivary gland duct, ought to be pre-determined depending on the 136 targeted biomarkers, since it may also affect the accuracy of final detection results. For 137 respiratory virus detection, the patients should be instructed to expectorate saliva from the 138 lower respiratory tract since the viral loads in saliva cough production were higher than that in 139 saliva swabs (Xu et al., 2020) . Third, to protect the salivary components from degradation, the 140 collected samples should be stored in a sterile container between −20 ℃ and −80 ℃ during 141 both transportation and storage processes. On the other hand, the concentration of targeted 142 biomarkers in saliva is often much lower than that in blood, which poses additional challenges 143 for the testing and analytical techniques. For POCT devices, ultra-sensitive biological probes 144 are indispensable for accurate saliva detection, where some strategies including signal 145 amplification and antifouling are required to achieve high specificity and sensitivity. 146

Elaborately engineered nanomaterials or biomaterials have often been adopted to design 147 sensors in POCT devices to achieve or improve specific biorecognition. Besides, anti-fouling 148 materials are necessary to prevent nonspecific adsorption. For example, a key challenge in 149 saliva glucose detection is to develop biosensors that could simultaneously enable both specific recognition of small glucose molecules at low concentrations and avoid nonspecific 151 adsorption of larger proteins. This is usually achieved by designing selective and antifouling 152 films immobilized on biosensors, containing chemical groups that can reversibly bind to 153 glucose along with some coatings resistant to proteins. For instance, Dai's group has been 154 devoted to designing QCM-based biosensors to detect saliva glucose, in which antifouling 155 hydrogel films with boric acid groups were fabricated and tested, enabling high accurate 156 glucose detection in 50% human saliva within five minutes (Dou et al., 2020) . Moreover, 157 stable conditions for the transportation and storage of these ultra-sensitive probes in POCT 158 are required to maintain their biological activity. 159

Saliva-based POCT techniques have emerged as a burgeoning field due to the perfect 160 match between noninvasive saliva collection and convenient POCT testing, both of which 161 permit self-operation by patients and eliminate the temporal and spatial limitations of 162 traditional diagnostics. This feature renders saliva-based POCT techniques particularly 163 suitable for large population-level screening of highly contagious pandemics as and also daily health monitoring of chronic diseases such as diabetes. Although challenges 165 remain at every step from sample collection, storage, transportation, preparation, detection to 166 final diagnosis, the unlimited potential and urgency will definitely drive their rapid progress 167 towards the development of commercial saliva-based POCT devices. We can expect these 168 devices to further develop into personalized and intelligent devices combined with electronics 169 or new apps that promote POCT platform as a smart detector and health-keeper. Moreover, 170 multiplexed detection can be achieved to synchronously monitor various diseases using a 171 single sample, and highly accurate identification of a specific disease can be conducted by 172 detecting pre-defined biomarker collections. The sensor detects target SARS-CoV-2 antigen protein with a limit of detection (LOD) of 1 fg/mL, which is able to detect SARS-CoV-2 virus in clinical samples.

Colorimetric sensor (Moitra et al., 2020) Nasopharyngeal swab N gene The sensor exhibits a linear range of 0. 

RNA-extraction-free nano

colorimetric test for point-of-care clinical diagnosis of COVID-19

Saliva sampling: Methods and devices. An overview

CRISPR-Cas12-based detection of 192 SARS-CoV-2

Minute-scale detection of SARS-CoV-2 using a low-cost biosensor composed of pencil 195 graphite electrodes

Detection of SARS-CoV-2 in Saliva by RT-LAMP 198

During a Screening of Workers in Brazil, Including Pre-Symptomatic Carriers

A highly 201 sensitive quartz crystal microbalance sensor modified with antifouling microgels for saliva 202 glucose monitoring

Accessing analytes in biofluids for peripheral biochemical monitoring

Persistence of serum and saliva antibody responses to SARS-CoV-2 spike antigens in 220 COVID-19 patients

Development and clinical application of a rapid IgM-IgG combined antibody test 224 for SARS-CoV-2 infection diagnosis

Nanozyme 226 chemiluminescence paper test for rapid and sensitive detection of SARS-CoV-2 antigen

Selective Naked-Eye Detection of 229

SARS-CoV-2 Mediated by N Gene Targeted Antisense Oligonucleotide Capped Plasmonic 230