key: cord-0838238-1pt6tf2r
authors: Amara, Umay; Rashid, Sidra; Mahmood, Khalid; Nawaz, Mian Hasnain; Hayat, Akhtar; Hassan, Maria
title: Insight into prognostics, diagnostics, and management strategies for SARS CoV-2
date: 2022-03-11
journal: RSC advances
DOI: 10.1039/d1ra07988c
sha: 47d5fa1846ca67f5740feb6acf829a1db6590596
doc_id: 838238
cord_uid: 1pt6tf2r

The foremost challenge in countering infectious diseases is the shortage of effective therapeutics. The emergence of coronavirus disease (COVID-19) outbreak has posed a great menace to the public health system globally, prompting unprecedented endeavors to contain the virus. Many countries have organized research programs for therapeutics and management development. However, the longstanding process has forced authorities to implement widespread infrastructures for detailed prognostic and diagnostics study of severe acute respiratory syndrome (SARS CoV-2). This review discussed nearly all the globally developed diagnostic methodologies reported for SARS CoV-2 detection. We have highlighted in detail the approaches for evaluating COVID-19 biomarkers along with the most employed nucleic acid- and protein-based detection methodologies and the causes of their severe downfall and rejection. As the variable variants of SARS CoV-2 came into the picture, we captured the breadth of newly integrated digital sensing prototypes comprised of plasmonic and field-effect transistor-based sensors along with commercially available food and drug administration (FDA) approved detection kits. However, more efforts are required to exploit the available resources to manufacture cheap and robust diagnostic methodologies. Likewise, the visualization and characterization tools along with the current challenges associated with waste-water surveillance, food security, contact tracing, and their role during this intense period of the pandemic have also been discussed. We expect that the integrated data will be supportive and aid in the evaluation of sensing technologies not only in current but also future pandemics.

An initial insight into the current novel infectious ailment, namely, COVID-19, appeared on 31 st shortness of breath, and fever were hospitalized at the start. A primary evaluation by compound tomography indicated divers as compared to healthy lungs. 3 This nding directed to an early diagnosis of pneumonia. Further, nucleic acid investigations via multiplex real-time polymerase chain reaction (PCR) of already known pathogens led to negative outcomes, indicating that the actual cause of pneumonia was unfamiliar (1) . A genetic sequence parallel to that of the beta coronavirus B was discovered from a sample of patients suffering from Bronchoalveolar Lavage (BAL) by 10 th January 2020. This study also revealed a genomic similarity of 50%, 80%, and 96% to the Middle East Respiratory Syndrome virus (MERS-CoV), Severe Acute Respiratory Syndrome virus (SARS-CoV), and bat coronavirus (RaTG13), respectively. 4, 5 Therefore, this unknown pathogen was named as SARS-CoV-2, which is responsible for COVID-19. By April 2, 2020, this infection had blown out to almost 202 countries, infecting approximately 1 million people with 45 526 deaths worldwide. Consequently, on March 11, 2020 , this novel COVID-19 outbreak was declared as a pandemic by the World Health Organization (WHO). 6, 7 However, the total number of coronavirus cases till December 29, 2021 were 283 374 722 with 5 434 143 deaths by the Worldometer website (https:// www.worldometers.info) (Scheme 1).

The percentage of individuals affected by SARS CoV-2 infection that remained asymptomatic has not yet been fully assessed. On the other hand, in symptomatic individuals, clinical manifestations of this infection start usually within a week including cough, fatigue, fever, and upper respiratory tract infection. 8 It can also progress to several other diseases such as dyspnoea and other chest infections in almost 75% of the affected persons. It was also reported by one of the studies that the interaction with the laryngopharynx, nasal mucosa, and trachea of patients can develop innate-immune response for instance tissue damage in different parts of the body. It was also found that this infection may affect the brain via different passages also including peripheral nerves. 9 More recently, the investigation of 425 conrmed infected cases demonstrate that the current pandemic may double the number of affected individuals every seven days and that each patient spreads the infection to 2.2 to 3.58 other individuals on an average. 10 Standing against these current circumstances, scientists are actively probing for the important attributes accountable for this pandemic. However, examining the target sequences carried by exogenous nucleic acids along with CT scans 11 suffers from serious limitations produced by nonspecic hybridization or amplication, as well as overpriced infrastructure and Dr Khalid got his PhD degree from College of chemistry Beijing Normal University, Beijing China (2014) in the eld of organic solar cells synthesis. Aer that he got 2-years post-doctoral fellowship from South China University of Technology, Guangzhou China. Currently, he is working as an assistant professor at the Institute of Chemical Sciences, Bahauddin Zakariya University, Multan. The main area of his research is in the synthesis and characterization of novel organic materials, nanocomposites and pharmaceutical compounds for their applications in energy conversion, sensors and drug delivery.

Dr Hasnain earned his PhD in Materials Science and Engineering from East China University of Science and Technology, Shanghai (2013). During his doctoral dissertation, he mainly focused on polymer chemistry and porphyrinfullerene nanocomposites. Later, he joined IRCBM as Assistant Professor to focus on the sensing and biosensing aspects of different carbon-based nanocomposites. He earned CAS Presidential Postdoctoral Fellowship to work on the synthesis and characterization of metal nanoparticle-decorated carbon interfaces and their biofunctionalization towards sensing and energy applications. He has published several articles in internationally recognized journals. Currently, he has multidisciplinary research interests in supramolecular chemistry, advance materials, metal nanoparticles, and biomaterials including their potential applications as sensors and biosensors, bioresorbable medical devices, and bio-fuel cells.

Dr Akhtar Hayat is currently working as Assistant Professor at IRCBM, COMSATS Lahore, Pakistan. He received MS/Ph.D. Degree in biosensors from Universite de Perpignan, France and post-doc from Clarkson University, USA in nanobiosensors. He is the founder of the sensors/ biosensors research group at IRCBM, COMSATS, Lahore, and is acting as the head of this group. Dr Akhtar Hayat is playing a key role towards the development of this group based on his international collaborations and national research funding. difficult work. 12, 13 To mitigate such challenges, protein-based sensing assays that principally depend on identifying antibodies released by individuals on contact with SARS CoV-2 have been introduced. 2,14-16 Certain groups have employed plasmonic sensors as robust and economically viable substitute with much lower detection limits compared to the abovementioned techniques. 17, 18 In parallel, nanoscale visualization procedures such as electron microscopy (EM), atomic force microscopy (AFM), and X-ray diffraction (XRD) have also been exploited for timely analysis and enlightening prognostic topographies of a specic virion. 19, 20 Despite huge successes in vaccine development, the COVID-19 variants, especially the most recent one called Omicron, have raised severe concerns as it is signicantly limiting antibodybased neutralization and elevated the chances of re-infection. 21 Also, an increased number of cases reported have put the efficacy of functional vaccinations in question for emerging alternates. 13 , 22 To solve such challenges, there is a need to improve the management strategies by moving toward tele-health care and tele-medicine 23 to provide care from a distance. 24 It would help to decrease the burden on the healthcare system and preventing exposure to highly vulnerable patients. Numerous clinics have implemented this practice globally. The current review aims to report the importance of the state-of-art instruments and technology employed so far to lessen the current and additional waves of COVID-19. The study also underlines the positive impact of contact tracing and quarantine in this critical pandemic situation. [25] [26] [27] [28] 2 Sequential and molecular data analysis patients by Han et al. 31 Almost >99.9% of the sequential homology was found for CoV-2 from various other corners of the world. Further, a similar kind of resemblance was also observed by Sah et al. in a 32 years old Nepalese patients 32 that is similar to the previously reported viral genome obtained from Wuhan. However, seven new sequences were observed, which vary from MERS and SARS-CoV-1 on the basis of structural evaluation based on biochemical and structural experiments. Meanwhile, a comparative study between a and b coronaviruses revealed that SARS-CoV-2 may have the ability to bind effectively with the human-receptor gene called Angiotensin-Converting-Enzyme-2 (ACE-2). 33 However, computational studies did not prove any linkage between the spike protein and ACE-2. 34 It led the scientists to believe that a greater attraction between ACE-2 and the CoV-2 gene may be the result of mutation to ACE-2. 35 It was also found that this mutation can easily alter the phenotype of SARS-CoV-2, which in turn can alter the diagnosis of the virus. 36 In addition, the mutated portion of the respective genome can forecast the linkage between probe binding and the primer. 37 3 Prognostic strategies using biomarkers for COVID-19

The exploration of various risk factors caused by COVID-19 at different intervals is essential on an urgent basis due to its severity. It would be of great help to take timely actions and reasonable intervention to enhance the cure rate and the prognosis quality. 38 Various biomolecules have been utilized for the effective prognosis evaluation of COVID-19 in laboratory tests. In the following section, we carefully evaluated the utilization of different prognostic strategies including D-dimer, IL-6, and some other biomarkers particularly during the early stages of infection. 39 3.1 C-Reactive protein (CRP) for COVID -19 This protein is developed by liver and various inammatory mediators such as IL-6. Its higher level is parallel to the disease intensity. 40,41 CRP, as a biomarker in disease diagnostics, has been highlighted by a retrospective center in Wuhan. An increased level of CRP (57.9 mg L À1 ) was reported in the patients in Wuhan. 42 Another study reported CRP levels >41.8 mg L À1 and found the likelihood of COVID-19 progression. 43, 44 Pathologically, CT scans have been employed to identify lung lesions during COVID-19 progression but are unable to differentiate between mild and severe cases, as we reported previously. 45 The outstanding activity of CRP as a biomarker was revealed in the 'area under the curve' in the receiver operating examination of 0.87 (95% CI, 0.10-1.00), where 83% sensitivity and 91% specicity were reported. Henceforth, CT scans alone along with CRP values have been proved to be authentic for the earlier detection of cases. Many other studies also corroborated the abovementioned ndings. 46, 47 3.2 Interleukin-6 (IL-6) for COVID-19 diagnostics IL-6 is a primary trigger for cytokine storms. Wan et al. 48 found that cytokine storm is crucial to the progression of COVID-19 and can lead to severe complications even death. The 5 th edition of "Diagnosis and Treatment of COVID-19" 49 suggests that observation of the cytokine level helps to improvise the treatment efficacy with reduced mortality. According to Yang et al., this inammatory factor plays a crucial role in the progression of the disease from a mild to a severe level. 50, 51 Coxproportional-hazard-model-analysis (CPHMA) showed that IL-6 could be utilized as an independent factor to predict the severity of SARS-COVID-19 hematological diseases. 38 Thus, we can conclude that an increased level IL-6 can be a sign of a higher inammatory cytokine storm. Thus, the role of IL-6 in this disease deserves special attention. 52, 53 One meta-analysis recounted that the mean IL-6 concentrations were 2.9 times greater in complicated COVID-19 patients in comparison to non-complicated disease, in which (n ¼ 1302; 95% CI 1. 17-7.19) . 54 The ndings of the study include ICU admittance, onset of acute respiratory distress syndrome (ARDS), and deaths. Groundbreaking conclusions suggested that the proportional upsurge of IL-6 is directly allied with disease severity. Another group proposed that SARS CoV-2 affects both the upper and lower respiratory tract and causes a high or mild acute respiratory syndrome with the consequent release of IL-6. 55 This enhanced appearance of IL-6 in the serum possibly indicates the severity and prognosis of the disease in SARS COVID-19 patients. 56 In line with the ndings of many researchers, the dynamic changes in the level of IL-6 have proven to be an important biomarker for disease diagnostics in severe COVID-19 patients. 57,58

Leucocytes, also known as white blood cells (WBCs), are constituents of blood generated from lymphoid tissue and bone marrow. They are divided into two main categories, i.e., granulocytes and agranulocytes. Eosinophils, neutrophils, and (NC) basophils come under the granulocytes group, while monocytes and lymphocytes (LC) are agranulocytes. Their inconsistent number may cause infection and can be analyzed by blood tests, producing a white blood cell count. A survey was made and numerous differences in the WBC of severe and non-severe COVID-19 patients were noted. 42 Patients of both cases have been reported to have increased leucocytes numbers, while the severe group experienced a comparatively large rise (5.6 vs. 4.9 Â 10 9 L À1 ; P < 0.001). NCs were found to be primarily driving this increase. More interestingly, the level of monocytes, lymphocytes, eosinophils, and basophils were less, causing a larger neutrophil-to-lymphocyte ratio (NLR; 5.5 vs. 3.2; P < 0.001). Also, this NLR has been reported as a biomarker that reects the disease severity.

Another study conducted in China also corroborated the ndings that higher NC and low LC count is associated with the severity of affected patients, signifying WBCs as a potent biomarker for the timely recognition of severe COVID-19. 59 Nevertheless, glucocorticoid and other viral and bacterial infections disturb the accuracy of WBCs ndings. 60 A specic study made in China reported the depleted LC level in most of the COVID-19 affected patients. 53 Another research group reported low blood lymphocyte percentage (LBL%) in critical cases, proposing that a lower LC count is indicative of a poor prognosis. Many other groups justied the same ndings. 61,62 However, the virus can target the mechanisms of IL-6 and lymphoid tissue. Meanwhile, additional reasons for low LC count need to be examined to determine the effect of WBC on COVID-19 severity. 18 

In the glucose metabolic pathway, the enzyme LDH catalyzes the conversion of pyruvate to lactate.

LDH is produced by cell membrane necrosis owing to lung damage or viral infection such as pneumonia caused by SARS CoV-2. Many evidences suggest the strong relationship between the LDH levels and the growth of COVID-19 disease. 63 One study proposed enhanced serum LDH values as one of the unusual diagnostic parameters in SARS CoV-2 patients with severe or fatal development of the disease. Pulmonary injury along with pervasive organ damage are of potential biological and clinical signicance at elevated LDH levels. 64 One meta-study reported signicantly greater levels of LDH in ICU patients, i.e., 248 U L À1 than in non-ICU patients 151 U L À1 (p ¼ 0.002). Higher LDH level is sustained in ICU patients even aer the several postadmission days (160 U L À1 vs. 218 U L À1 , p ¼ 0.002). Therefore, LDH may be a prognostic biomarker of severe disease. Multi-center research involving 1099 COVID-19 patients supported the evidence associating inammation and tissue damage with enhanced LDH levels. 6 Moreover, when LDH was at higher levels, as seen in the associated CT scans, the severity of pneumonia was revealed. 65 Another study was carried out with 87 conrmed COVID-19 patients, assessed by the LDH level at diagnosis and followups. 66 The enhancement or reduction in the LDH level was noted to be representative of radiographic progress. A positive correlation was observed between the time to LDH normalization (5.67 AE 0.55, days) and the time to radiographic absorption (5.57 AE 0.65 days). The study validates the potential utilization of serum LDH as a biomarker for assessing the severity and observing treatment feedback in COVID-19 pneumonia. 67 Many other studies by different research groups also supported the above ndings. 68, 69 3.5 D-Dimer D-Dimer released from the degradation of the cross-linked brin signies the activation of brinolysis and coagulation. 70 Early research has linked elevated levels of D-dimer and hemostatic abnormalities in non-survivors of COVID-19 compared to survivors. 71 A survey performed by a group of researchers on 191 cases reported that a D-dimer level greater than 1.0 mg mL À1 (p ¼ 0.0033) resulted in greater mortality among COVID-19 cases. Moreover, they optimized 2.0 mg mL À1 or more as a cut-off to predict the mortality in SARS CoV-2 patients in hospital. 72 Another group reported a marked rise in the D-dimer level and enhanced coagulation in almost 90% of patients with pneumonia. 73 The researchers also reported the elevated D-dimer level of 2.4 mg L À1 in ICU patients in contrast to non-ICU specimens 0.5 mg L À1 (p ¼ 0.0042). Another study by Sakkat et al. with 1355 patients also justies the previous ndings. 74 Yumeng et al.

reported a D-dimer elevation of 74.6% (185/248) in patients. 75 Furthermore, another research group suggested that the enhanced level of the D-dimer on admittance can be used as a marker to refer patients to critical care. This, along with the many reported studies, proposed that D-dimer levels should be employed as a prognostic marker and aid doctors to monitor and risk-stratify those who are probable to expire earlier. [76] [77] [78] 3.6 Platelet count

As we have discussed in the previous section, the COVID-19 virus leads to severe hematological variation, resulting in thrombocytopenia (Fig. 2) . A survey of 1799 CoV-2 victims has revealed a fewer platelet count (WMD À31 Â 10 9 L À1 ; 95% CI, À35 to À29 Â 10 9 L À1 ) in patients with severe infection. 79 Likewise, the mortality rate is higher in those with fewer platelets count (WMD À31 Â 10 9 L À1 ; 95% CI, À35 to À29 Â 10 9 L À1 ). Considering thrombocytopenia as an endpoint, a vefold higher possibility of SARS CoV-2 (OR, 5.13; 95% CI, 1.81-14.58) has also been observed. Reconsidering research that castoff Cox proportional hazard regression analysis revealed the platelet count as an independent risk factor for deaths amongst CoV-2 patients, where a 50 Â 10 9 L À1 rise is related to 40% mortality. 80 Another study reports the 5.45% death rate (29 subjects died) and decreased platelet count among nonsurvivors compared to survivors aer one week of admission. The difference was enhanced by 50 Â 10 9 L À1 , as reported by previous research. 81 Another group endorsed the previously discussed work by suggesting that the lowest platelet count was directly related to death and lower the count, more the association. 82 Hence, the literature and many ndings by researchers suggested that the platelet count could be exploited feasibly to clinically analyze the severity of infection. [83] [84] [85] 

There are increasing shreds of evidence of higher mortality rates in COVID-19 infected patients with underlying cardiovascular disease. 87, 88 Few research groups have examined the utilization of high-sensitivity cardiac troponin I (hs-TnI) as a biomarker of infection development and transience. [89] [90] [91] [92] [93] A study conducted on COVID-19 patients exploiting SARS CoV-2 RNA detection depicted an invariable odd ratio for death at 80.1 (95% CI 10.3-620.4, p < 0.0001) for hs-TnI. 94 Another study on 416 hospitalized patients with SARS CoV-2 conveyed that hs-TnI was higher in 1 among 5 patients. 95 These patients were more likely to develop infection (59% vs. 15%, p < 0.001) along with acute kidney injury (9% vs. 0%, p < 0.001) and needed noninvasive (46% vs. 4%, p < 0.001) or invasive (22% vs. 4%, p < 0.001) ventilation. Prompt analysis of myocardial damage identied by raised hs-TnI helps in suitable triage to enlighten the use of vasopressors and inotropes. In addition, the enhanced procoagulant, prothrombotic and inammatory responses following COVID-19 infection enhanced the likelihood of acute myocardial infection and acute ischemic myocardial injury owing to respiratory failure with hemodynamic instability and hypoxia in severely sick patients. The use of hs-Tn, as discussed above, has the ability to aid in risk stratication and disease phenotyping in hospitalized SARS CoV-2 patients. 96 The probable pathophysiology and anticipated guideline for identication and management has been reported by Bhurint et al., as shown in (Fig. 3 ).

Several pieces of evidence have also been reported on the association between the severity of COVID-19 infection and chronic kidney diseases. 98 A study on 28 patients has conrmed suggestively that greater levels of renal biomarkers, for example, creatinine, serum urea, and markers of glomerular ltration have been associated with severe cases. 99 A retrospective study on 701 patients exposed raised serum creatinine levels on admittance, associated with severity, owing to substantial anomalies in the coagulation pathway. 100 The group reported that these cases require intensive care and mechanical ventilation. Univariate Cox regression investigation observing higher creatinine was also related to the in-hospital death rate (HR 2.99, 95% CI: 2.00, 4.47). Likewise, haematuria, proteinuria, and raised urea levels had parallel, if not superior, threat effects. Remarkably, another group of researchers reported a superior role for urinalysis over serum markers for kidney function and suggested the disease severity in case of any abnormalities in the urine test at the time of admission. 101 Another meta-analysis by Michael et al. on 3615 patients reported that acute kidney injure is related to poor outcomes in patients with SARS CoV-2. 102 Hence, renal abnormalities in COVID-19 patients at the time of admission might specify greater risks of deterioration, conrming suitable triaging. 103 

The control of viral infection necessarily depends on natural killer cells and cytotoxic T lymphocytes. A functional collapse of antiviral lymphocytes has been noted in patients with COVID-19. 104 It has been observed that the degree of pro-inammatory cytokine storm lymphopenia is greater in severe SARS COV-2 patients than in mild cases, and is related to disease severity. 105, 106 The N8R, NLR, and lymphocyte percentage are considered to be useful reliable prognostic factors for the timely diagnosis and progress of severe COVID-19 cases. Literature reports the survey of 12 patients (death case) with an average age of 76 years, in which lymphocyte percentage decreased up to 5% in 2 weeks aer disease inception. However, when a survey was performed for 7 patients with severe symptoms within a therapeutic window of 35 days and an average age of 35 years, the lymphocyte percentage was reduced at the start but raised to greater than 10% at the time of discharge. Conversely, another survey with 11 patients having a therapeutic time of 26 days and an average age of 49 years was performed in patients with moderate indications. The lymphocyte percentage was found to be persistent and greater than 20% at the time of discharge. The whole study elucidates the practical applicability of lymphopenia-based prognosis for early SARS COV-19 detection. 94 

Chest CT scan or computer tomography is an important technique employed for the detection of COVID-19. It is being utilized either alone or in combination with RT-PCR. 32, 107, 108 The methodology involves a large number of chest X-rays from different dimensions and angles to obtain 3D images, which are later examined by some radiologists to validate the presence of the SARS-CoV2 virus. Consolidation of uid in lungs along with ground glass opacication are observed in patients of COVID-19 associated with pneumonia. Other abnormalities observed in the CT-scan of COVID-19 patients are bilateral lung, diffused dissimilar airspace, or opaque cavity-like patch, involving lower lobes along with peripheral distribution. For instance, at the initial stages (0 to 2 days) of the infection, the CT scan bears a resemblance to a typical chest condition. However, as the infection develops more, the impermeability of the scan rises, and aer 4 days, ground-glass opacity has been observed, which seems to show irregular patterns in the scan images. Fig. 4 illustrates the CT scan image of altered sufferers on different days. Likewise, oddity can be noticeably witnessed. 109, 110 Lately, a 3D framework built on chest CT scan images was constructed for CoV-2 analysis. 112 The described model accomplished a high sensitivity and specicity of 90% and 96%, respectively. Therefore, CT scan images have been employed as a prognostic tool for SARS CoV-2 detection. However, high prices, the requirement of large medical instrumentation, and presence in very few hospitals have limited the use of CT scan technology. Meanwhile, this technology is also unable to distinguish different viruses and unable to distinguish between pre-symptomatic, asymptomatic, and some milder symptomatic patients without pneumonia. 

Aer the recognition of SARS CoV-2 as the main agent for the pandemic, the genome of the virus was sequenced rapidly and distinctive sequences have been recognized for analysis, and different strategies have been proposed for CoVID-19 detection based on the nucleic acid.

4.2.1 Reverse transcription-polymerase chain reaction (RT-PCR)-based SARS COVID-2 detection. Quantitative RT-qPCR has been extensively exploited by Disease Control and Prevention Centre along with the other divisions universally to analyze viruses. This detection strategy has been designed for COVID-19 detection based on the close genetic proximity of 2019-nCoV with other SARS coronavirus, thus exploiting the nucleic acid technology. For detection, rst, biological uids with virus strains are collected from oropharyngeal and nasopharyngeal swabs. 58 Then, viral RNA are isolated through ltration and separation steps. Reverse transcriptase enzyme has been utilized to generate complementary viral DNA (cDNA) from the RNA. It amplies the specied DNA section through a polymerase chain reaction. The conventional method involves radioactive isotopes as markers to analyze particular nucleic acids (certain noncoding RNAs, mRNA, and pre-mRNAs); however, currently, the real-time detection of DNA probes involves uorophores and a quencher.

For the SARS-CoV-2 detection process, initially, DNA polymerase enzyme screened envelope gene (E gene) nucleotides, followed by the verication of the nucleocapsid gene (N gene) to the particular fragment of the virus cDNA. Polymerase enzyme encounters the double-stranded DNA and through exonuclease activity, it parts the quencher and uorophore molecules for the real-time recognition of the viral cDNA using the RT-PCR approach. If the system is well-calibrated, a large number of cDNA copies along with uorescence signals are formed aer a few series of polymerase reactions. The uorescence intensity and virus concentration are directly proportional. If negative results are obtained, further analysis will be performed by sequencing the RNA polymerase (RdRp) gene. Aer that analysis, if a positive outcome is achieved, it conrms the existence of SARS-CoV-2 in the suspected patients, and is labeled as COVID-19 positive clinically (Fig. 5) . Currently, three regions of the cDNA, i.e., RdRP, E, and N genes have been recognized for the SARS-CoV2 virus detection. The high specicity and sensitivity of (500 or 1000 copies mL À1 of viral RNA) are the main causes of the commercialization of PCR-based diagnostic SARS CoV-2 kits. 113 However, this technique has inherent challenges such as extended turnaround time and a sampling procedure that is affected by the viral load (VL). Meanwhile, SARS-CoV2 destroys the targeted RNA while opening a viral capsid that results in the discharge of small RNA fragments into the bloodstream that challenges detection by RT-PCR. Numerous testing kits have been established worldwide on the principle of RT-PCR.

4.2.2 Digital PCR (dPCR)-based SARS COVID-2 detection. To circumvent the difficult and time-consuming operation of RNA extraction, a new strategy has been developed for viral RNA extraction. 115 This technique employs carboxyl group (PC)coated magnetic nanoparticles. The conventional columnbased extraction of nucleic acid consumes a larger time of 2 h. Meanwhile, pcMNPs-based methodology combines RNA binding and virus lysis into a single step, and these developed pcMNPs-RNA complexes were introduced directly into consequent RT-PCR reactions. This technique requires only 30 min compared to the conventional time consuming RT-PCR. It gives the high sensitivity of 10-copies and wide linear bounds over 5 logs gradient for SARS COV-19 RNA detection. The detection limit of the dPCR technique is approximately 10 folds lower than conventional RT-qPCR. 116 The obtained sensitivity, accuracy, and specicity for RNA extraction utilizing RT-dPCR protocol were 90%, 93%, and 100% respectively.

The obstacles of great cost and time consumption associated with the previously discussed techniques have been countered by the development of LAMP. 117 The technique can detect nucleic acids. The designed methodology utilizes the chain displacement reaction by DNA polymerase as an alternative to heat to produce the singlestranded template. 118, 119 The specically designed 4 to 6 primers along with DNA polymerase bind to the target genome at different regions, thus amplifying the DNA signal in a short interval of time (Fig. 6 ). The amplied DNA signal is perceived by variation in turbidity that can be observed by the change in the color owing to pH or uorescent molecule change, added at the double-stranded DNA. This reaction takes approximately 1 h to complete at 60-65 C with a sensitivity of $75 copies along with easy detection. 120 It was potentially implemented for the point-of-care test (POCT). 121 In addition, impure specimens could also be directly sensed by LAMP. 122 Combining impure specimens with non-instrumental calorimetric analysis highthroughput tests is probable. 123 For the robust calorimetric detection of COVID-19, an isothermal LAMP-based method has been developed. [124] [125] [126] The reported read-out time was approximately 30 min along with a sensitivity of 97.6% (42/43). The LAMP-based technique is very simple, specic, and easy to visualize owing to the use of a specic number of primers. 127 The technique has the advantage of multiplexing at the ampli-cation or reading step by functionalizing the beads with genes or optical signals to circumvent the likelihood of mutation that leads to false-negative tests. However, the optimization of the primer and other conditions are the challenges associated with LAMP. 118, 128 Besides LAMP, many other isothermal amplication approaches for POCT-based nucleic acid detection have been explored at a lower scale, including rolling circle amplication (RCA), recombinase polymerase amplication (RPA), multiple displacement amplication (MDA), and nucleic acid sequence-based amplication (NASBA).

The turnaround statistics and capacity have limited RT-PCR and many other similar techniques for application in clinical laboratories in this ongoing pandemic, thus forcing scientists to approach new and quick etection strategies. Clustered-Regularly Interspaced Short Palindromic Repeats (CRISPR), along with CRISPR-associated (Cas) protein (CRISPR/Cas) methodologies, were rstly reported in 1987 to protect prokaryotic cells from foreign plasmids and harmful pathogens by identifying and cutting nucleic acid sequences of foreign species that hold short palindromic repeating spacer sequences. 130 To date, numerous genome-editing methodologies have been introduced but the latest one is recognized as CRISPR/Cas. Therefore, CRISPR/Cas has enticed much importance in the scientic community for disease diagnosis and management owing to its robust, less expensive, and precise genome editing approaches. 131 CRISP works similar to genomic programming by manipulating isothermal reactions to synthesize and amplify cDNA that again reconvert to RNA. The CRISP editor is programmed to bind to amplied RNA with specied genetic coding to edit at a precise location, thereby cutting off the quencher from uorescence for signal detection for genotyping, POC virus analysis, and disease monitoring. A number of laboratories have developed CRISPbased SARS COV-2 detection tests with promising LOD, higher sensitivity, and lesser assay time. It includes a lateral ow assay that extracts RNA from respiratory swabs and is labelled as DNA Endonuclease-Targeted CRISPR Trans Reporter (DETECTR) for SARS-CoV-2 analysis. 132 This protocol works by synchronizing reverse transcription and RT-LAMP for NP and OP swabextracted RNA that was further followed by the detection of Cas12-veried coronavirus sequences. Aerward, corresponding molecule cleavage conrms the occurrence of the virus in the patient (Fig. 7) . Furthermore, the reported LOD was 10 copies per mL input with an assay time of 30 to 40 min. Few other diagnostics studies have also been conducted exploiting similar methodology along with relatable ndings. 133 Few researchers have freshly reported a FnCas9 Editor Linked Uniform Detection Assay (FELUDA) that utilizes a highly sensitive FnCas9 protein to identify the mismatched region and specic position in the reverse transcribed DNA. 134 Another group reported nucleic acid SARS-CoV-2 detection methodology, involving the PCR amplication of target sequences, employing biotinylated primers that were formerly attached to streptavidin-coated beads. Later on, single guide RNAcontaining uorescently-labeled Cas9 complexes interacted with immobilized target sequences, thereby producing analytical signals. This approach has also been improvised to produce streptavidin-containing lateral ow strips that binds to the biotinylated targets, giving a low detection limit of 110 femtomolar for COVID-19 identication.

The antibodies and antigens that are released in reaction to SARS CoV-2 have been exploited as a source to assess COVID-19. However, viral load variations during disease progression have limited the long scale application of methodology.

4.3.1 Serological-based tests (SB-T) for SARS COVID-2 detection. SB-T is another currently studied and explored option for COVID-19 detection. It is a blood-based method, which works by measuring protein and antibodies in blood when the body is countering infection (immune response), caused by COVID-19 rather than the virus itself. 136 Presently, serological tests have been employed on asymptomatic patients. [137] [138] [139] Nucleocapsid protein is highly abundant in viruses and this immunogenic phosphoprotein is used as a biomarker because of its rare mutation property. A group of scientists has exploited Rp3 nucleocapsid protein to detect IgM and IgG antibodies from SARS CoV-2-suspected patients owing to the 90% similarity of the mentioned protein with SARS viruses. 80 The SB tests involve the adsorption of recombinant protein to a multi-well plate. Later on, the dilute serum of the suspected patient is mixed to conduct ELISA. The detection signals are obtained by adding horseradish peroxidasefunctionalized anti-human IgG antibody. If anti-COVID 19 IgG exists, it will be sandwiched between the adsorbed protein and the antihuman IgG probe. Correspondingly, IgM has also been exploited for sandwiched assay. As the antibody level increases with each passing day in COVID-19 patients, so the reported hike in IgM and IgG was found to be 81% and 100% in COVID-19 positive patients, respectively, aer 5 days, compared to 51% and 81% on day 0 of SARS COV-2 contagion. The sensitivity of COVID-19 serological assays is in the linear range of 0-100% for both IgG 140-142 and IgM. 143, 144 The determinants for the sensitivity of the SB test include the test and patient factors. Meanwhile, the reported specicity of the COVID-19 serological assays are in the range of 6.9-100% (ref. [145] [146] [147] and 0-100% (ref. 140, 143) for IgM and IgG, respectively. However, an excellent specicity has been exhibited by several commercial assay kits. Similar to sensitivity, various determinants affect the specicity for serology tests such as test factors and patient factors. This methodology is promising to obtain the prevalence of COVID-19 in the population. Currently, FDA has approved a number of serological tests for early detection and emergency analysis. However, low sensitivity have been reported for the SBbased assay compared to LAMP and rRT-PCR. 128 4.3.2 Enzyme-linked immunosorbent assay (ELISA)-based SARS COVID-2 detection. Enzyme immunoassays (EIA) along with enzyme-linked immunosorbent assays (ELISA) are widely employed techniques for the quantication and detection of viral antibodies or proteins produced by the body in response to COVID-19 infection for the detection of COVID-19 disease. Both techniques work on the same basic principle and are derived from radioimmunoassay (RIA), which have been employed to measure small quantities of biological molecules such as peptides, protein, and hormones. RIA was modied to current ELISA and EIA owing to safety concerns by replacing the radioisotope with friendly enzymes. Both assays utilize the basic idea of an antigen-binding to its specic targeted antibody for the detection of antibodies and antigens in the uid samples (Fig. 8 ). These assays also use enzyme-labeled antibodies and antigens to detect biological molecules. 148 A chromogenic substrate for the enzyme produces a uorescent, colorimetric, or luminescence detection aided by enzymes used in the reaction. Both qualitative and quantitative measures can be measured based on such detection. 149 However, sometimes the enzyme-mediated color change will keep on reacting indenitely, thus leading to false-positive results. Likewise, the amount of viral load changes during infection progression, also producing difficulty, thereby leading to the detection of low concentrations of the viral protein. 58 A study was made on COVID-19 diagnosed cases, in which a group collected ELISA IgM and IgG antibodies from 63 samples. Meanwhile, certain researchers collected 91 plasma specimens for the colloidal gold-immuno-chromatographic assay (GICA). Meanwhile, the sensitivity of ELISA (IgM and IgG) was found to be 55/63 (87.3%). However, the sensitivities of the GICA IgM and IgG were found to be 75/91 (82.4%). Both methodologies showed negative results for healthy controls with 100% specicity. 150 Another study was made for antibody screening to identify SARS-CoV-2 in which IgG and IgA ELISAs were evaluated in unication with the EURO-Lab workstation. The premediated specicities were 91.9% for IgG and 73.0% for IgA. 151 4.3.3 Lateral ow immune-chromato-graphic strip (LFICS)based SARS COVID-2 detection. FICS works on the principle of pregnancy tests and is built up of conjugate pad (CP), sample pad (SP), nitrocellulose membrane (NC), and absorbent pad. An immunouorescent assay-based methodology was also proposed for the rapid diagnosis and sensitive sensing of IgM and IgG of SARS COVID-2 in human serum specimens within the duration of 10 min. The COVID-19 recombinant nucleocapsid protein was utilized to detect the antigen. Meanwhile, lanthanide-uorescent microspheres were utilized to evaluate solid-phase immuno-chromatographic assay qualitatively or semi-quantitatively. The specicity and sensitivity of immunochromatographic assays were found to be 98.68% and 93.10% for IgM and 98.72% and 100% for IgG in 28 positive and 77 negative clinical serum samples, respectively. 152 Another (LFICS) has been sanctioned in China for point-ofcare disease COVID-19 diagnosis. 153 The colloidal gold immunochromatographic assay (CGICA) based on gold nanoparticles (Au NPs) can identify IgM and IgG antibodies concurrently within 15 min, against the SARS COVID-2 virus in the blood. The gross sensitivity of the LFICS assay was found to be 88.66% with 90.63% specicity. 99 The results demonstrate that immunoassay-based COVID-19 detection can also be used as a tool owing to its portable and economically viable infrastructure, along with good sensitivity and specicity. The inclusive process and data analysis of the abovementioned immunodiagnostic assay are revealed in Fig. 9 . The immunoassay involves a few drops of blood to detect a virus qualitatively in a miniaturized set-up. Nonetheless, the analysis of the exact titer is not probable using this methodology. 

The ongoing SARS-COV-2 pandemic situation highlights the need for reliable and fast sensing strategies. Deep down, this pandemic is improving virus sensing to some extent, particularly plasmonic techniques, as portable, cheap, reliable, and fast sensors are in demand now. PCR is considered as the most effective and real time detection methodology for SARS-CoV-2 with almost 100% selectivity. 155 However, it still has several drawbacks including cost and long response time along with a complex system. 156 Another major drawback of RT-PCR-based detection has been associated with a large amount of falsepositive and negative cases, thus limiting its practical applicability. In this regard, plasmonic detection techniques integrated with lateral ow processes have tremendously increased the detection of viruses in a limited amount of time. 157 Apart from the fastest way with higher sensitivity and selectivity for different applications, 158 plasmonic sensors in point-of-care (POC) devices for the early detection of viral diseases remain nascent. 159 Efforts are being made for the improved functionality of such sensors by implementing various techniques, namely, interference lithography, nanoimprinting microsphere lithography, roll-to-roll pattering, and oblique angle deposition. These methods not only advance the accuracy of the sensors but also bringing the cost down. 157, 160, 161 Besides, such modications also simplify the detection of coronavirus and thus may help in disease control. 162 Recently, a plasmonic sensor with dual functionality combining the plasmonic photothermal (PPT) as well as surfaced plasmonic resonance effects has been employed as a robust and cost-effective substitute to RT-PCR. A selective sequence of CoV-2 has been analyzed via nucleic acid hybridization by exploiting complementary DNA-functionalized 2-D gold nanoislands (AuNIs). The two dissimilar incident angles were employed for the excitation of LSPR and plasmonic resonance of PPT, which desirably improved the sensing stability, reliability, and sensitivity, as shown in Fig. 10 . A label-free and real-time recognition of viral sequences comprising ORF1ab, RdRp, and the E genes from the COVID-19 has been made owing to the attained ideal conguration. In addition, the enhanced specicity along with the hybridization kinetics about nucleic acid-based recognition have also been achieved by the utilization of PPT enhancement on AuNPs-chip. Such a biosensor exhibited a low detection limit of 0.22 pM along with multigene combination. 163 Another group developed a selective methodology for the Ngene detection of SARS CoV-2 by the naked eye. They reported a colorimetric assay using AuNPs integrated with thiolated antisense oligonucleotides (ASOs). This protocol was employed for conrming positive COVID-19 cases in a short period of 10 min. The capped AuNPs showed aggregation, while its targeted RNA sequence came into the picture. Furthermore, the existence of RNaseH (enzyme) enhances the splits of the RNA, particularly in RNA-DNA fusion, directing the additional accumulation of AuNPs, as shown in (Fig. 11) . The assay worked selectively for MERS-CoV viral RNA having an LOD of 0.18 ng mL À1 . Consequently, the current work reported an effective reproducible and reliable methodology for the naked eye detection of CoV-2 without the need for any advanced instrumental techniques. 164 

For the evaluation of organic and inorganic compounds, Raman spectroscopy (RS) is a well-known technique. 165 It has been employed for the recognition of bacteria in food 22 as well as for HIV-1 detection. [166] [167] [168] Recently, RS was applied for SARS-COVID-19 detection using Au nanoparticles. 169 However, signal ampli-cation was required due to the very low signal intensity, for which the plasmonic effects of different metallic nanoparticles can be applied. This plasmonic eld is able to enhance the signal by $10 8 to 10 9 times, based on the chemical composition or the aspect ratio (shape). 170 Further, various 2-D materials can easily provide an additional signal amplication of approximately 10 2 because of the effective transfer of electrons. 171 This signal enhancement is known as Surface-Enhanced Raman Spectroscopy (SERS) for the detection of viruses as well as other pathogens 172 with improved specicity and sensitivity. 173 According to Sanchez et al., 175 Raman-SERS spectral analysis of any viral particle is complex and can give broad peaks due to multiple chemical species (Fig. 12) . Lee et al. 176 reported a similar trend in the spectra of HIV-1 viral infection. An interesting fact regarding virion and S-protein spectra is that they lack the peaks characteristic (1640-1678 cm À1 ) of amide-I bonds. Kuroski et al. explained this with reference to the SERS studies of various homopeptides composed of Tyr-, Ala-, Trp-, and Gly-chains with a variety of investigational circumstances. 177 In this study, it was found that the lack of some bands is due to amino side chains. This side chain increased the distance among the particles and the peptide bond. However, another possibility might be the deactivation that can disrupt amino acids. In another study, Zhang et al. employed SERS based on silver nanorods, which were functionalized by binding protein and cellular-receptor angiotensin converting enzyme 2 (ACE2) (Fig. 13 ). 174 A very weak peak for amide I was found in this case, which was closer to the noise. They used the 1189 cm À1 peak in ACE-2 with a peak shi of 1182 cm À1 as the spike protein was attached. Then, they used a ratio of these two peaks in order to predict viral presence. Their SARS-CoV-2 spectra showed very few weak peaks.

Besides, SERS amplication requires a molecule, which should be in contact with any suitable metallic nanoparticle. This method was used by Jhon et al. by combining SERS with a particular substrate, which is able to combine plasmonic amplication with excitation amplication and provides us with a greater number of peaks. A consistent spectrum with almost 5 peaks was obtained by this methodology. These peaks were connected to S and N proteins. However, fewer peaks were assigned to the particles deactivated due to the destruction of some viral parts. The range of 744-7 nM was obtained in the SERS-spectra of InBios-Spike-Protein. Aerward, for the calibration curve, PLSR (Partial Least Squares Regression) was employed with the variance of 99.9%. Finally, a limit of detection (LOD) of 8.89 Â 10 À9 M with a Root Mean Square Error (RMSE) of z2.27 Â 10 À9 was obtained, which is a good approximation for concentrations in the nM range. Meanwhile, we have summarized the pros and cons of all the discussed techniques as shown in (Table 1) .

The FETs and amperometric systems are important electronic and digital devices that are broadly employed for the sensing of biomolecules and pathogens. The remunerations of low cost, mass manufacturing, and miniaturization make these systems most desirable and applicable. FET biosensors have also been applied for the clinical diagnosis of COVD-19. The sensor was established by integrating the COVD-19 spike antibody with the graphene sheets of FET. The developed transducing interface detected the COVID-19 protein in phosphate buffer saline (PBS) in the concentration range of 1-100 fg mL À1 . Meanwhile, the OFET was also apt to analyze COVID-19 in clinical trials with an LOD of 2.42 Â 10 2 copies mL À1 and a detection bound (DB) of 1.6 Â 10 1 pfu mL À1 . 178 Thus, the developed FET-based interfaces can be employed for the production of compact POC devices and provide the advantages of robust, real-time, selective, and label-free probing of SARS Cov-2 at an ultralow concentration. The working methodology of FET-based COVD-19 detection has been shown in Fig. 14. 

These basic techniques are usually employed separately or with the combination of other detection techniques for the viral structure. The outstanding prociencies of these techniques provide a deep examination of the viral structure and function along with a detailed examination of viral inuence on the extracellular environment and host cells, which are benecial in drug discovery applications. [180] [181] [182] [183] [184] [185] [186] [187] [188] [189] [190] 4.7.1 Electron microscopy (EM). An EM utilizes an electronic beam as an illumination source. The resolving power of the EM is much greater compared to optical microscopes owing to a small wavelength of electrons than visible light photons. Therefore, EM has the ability to visualize the particle structure at the nanoscale, thus making it a potent apparatus for viral diagnosis. The most employed molecular and serological approaches need a specied probe to identify the virus; the EM approach does not require organism-specic reagents to detect pathogenic agents. As for anonymous disease analysis, molecular tests require details about potential agents, whereas EM gives an open outlook of the unknown given sample under analysis. 183 Besides the direct analysis of the virus by visualization, it also gives directions regarding the ultrastructure and structural dynamics of the virus associated to attachment and replication. These traits make EM a worthwhile technique for the detection and design of antiviral agents and vaccines. 186, 188, 191, 192 To examine the viral structure, three main techniques are mostly employed. In the negative staining method, the sample remains untouched with a stained background, making the sample visible. Cryo-EM is an image-based technique that is utilized to obtain a high resolution three dimensional image of cells and other biological constituents. 193, 194 SEM has been extensively employed for virus quan-tication. SEM, when combined with TEM, is also be able to provide a high resolution image of the viral structure. 183 Recently, an image of SARS CoV-2 was taken via TEM, 183, 195 as shown in Fig. 15 .

Recently, cryo-EM was utilized for the identication of multiple mono-clonal antibodies, aiming at the coronavirus, particularly using S-protein from memory B-cells of the infected person. It was found that an antibody called S309 has the potential to neutralize CoV-2 and SARS-CoV. 196 The methodology has been utilized for the investigation of RNA-dependent RNA polymerase nsp12 of the COVID-19 structure, which catalyzes the synthesis of viral RNA in complexation with 2 cofactors, namely, nsp7 and nsp8. 197 In ref. 198 , detailed information about the 3D structure and the morphological surface of the causative agent in SARS-CoV was achieved. These results suggest that such techniques can easily be exploited for the detailed analysis of SARS COVID-19. 4.7.2 X-ray crystallography. To achieve highest the resolution image for the viral structure along with macro-molecular associations at the atomic level, single crystal XRD is the most promising methodology. Five steps are involved in identifying the viral structure via XRD, including virus particles' preparation and purication, crystallization, diffracted data analysis, phase calculation through isomorphous replacement or molecular replacement (MR), 198, 200 and, lastly, building the model. [200] [201] [202] Kits are freely available commercially for the preparation and purication of viruses from the extracellular matrix. 203 Handling should be performed as gently as possible to uphold the icosahedral symmetry of the virus. Consequently, crystallization is mandatory to achieve the supersaturation of the aqueous protein solution. Crystallization involves nucleation by developing a crystal nucleus, followed by a growth process. Batch crystallization, dialysis, liquid-liquid diffusion, and vapor diffusion are the major four steps involved in growing the crystals of virus samples. 203 Furthermore, the crystallographic data is collected at 100 K, i.e., cryogenic temperature, to prevent secondary radiation damage to the virus specimen and to improve the resolution. However, growing high-quality virus crystal via X-ray crystallography is quite challenging and the currently used methods are slow and centered on trial and errors.

Nonetheless, this cryo technique could be an analogous process for viral protein crystallography. 204 The details provided by 3D cryo-EM could be assimilated with the obtained X-ray data to ameliorate the constructed model of the virus particles. 205 Serial femtosecond X-ray crystallography (SFX) also uses X-ray free-electron lasers (XFELs). It has been exploited to scrutinize the viruses as single particles and crystals owing to its unmatched brilliance of XFEL beams and the pulse duration in a femtosecond. The variations that occurs in the life cycles of viruses, such as response to the changes in pH and interaction of the viral protein with the receptors, could be sensed using time-resolved SFX. [206] [207] [208] Recently, a 3D crystal structure of unliganded CoV-2 was determined by a resolution power of 1.75 A, as shown in Fig. 16 (ref. 209) and was utilized to determine the optimization of a-ketoamide inhibitor series.

However, EM and XRD methodologies are established on the average of countless particles that exist in the crystal or electron micrographs. Consequently, it generates inadequacy in the data acquired from the structural differences between the discrete particles present in a large population. Moreover, the abovementioned methodologies require an atmosphere that is far from the physiological conditions of viral functioning and prevent the characterization of vibrant properties in real-time. Thus, it obscures the study of the viruses that lack distinct structural symmetry, such as SARS CoV-2, which is an enveloped virus. Lastly, AFM is also a direct imaging technology that offers noteworthy impression of the virus study. It offers prospects for studying the COV-2 virus particles. 4.7.3 Atomic force microscopy. AFM has developed into a signicant instrument for the characterization and visualization of nanoscale images of specimens in air as well as liquid. 210 Normally, the deection of the microcantilever is utilized in AFM measurement to examine the contact between the nanometric tip located at the end of the microcantilever and the surface of the sample. A broad information pool including mechanical, thermal, chemical, physical, atomistic details of the surface, and viscoelastic characteristics of nanobiomaterials entities could be achieved based on time scales, range, and types of interactions. [211] [212] [213] [214] [215] [216] [217] [218] [219] [220] [221] [222] [223] The very rst invented mode of AFM is "contact mode", in which the tip is raster scanned on the specimen interface by sustaining a constant force generated on the sample through deection recognition by regulating the height of the tip. However, the foremost challenge associated with contact mode utilization while imaging so entities such as biological specimens and viruses is the force (applied) to circumvent undesirable friction, impairment to the sample, and reversible or irreversible deformation. 224 Dynamic AFM was invented to minimize the applied force and friction and to enhance image resolution. 225 In dynamic SFM, while scanning the sample surface, the microcantilever oscillates near its resonance frequency. The two main dynamic AFM modes are tapping mode, also known as amplitude modulation atomic force microscopy (AM-AFM), and the second one is frequency modulation atomic force microscopy (FM-AFM). 225 The friction is reduced and the risk of the damage to the specimen is signicantly lessened in the dynamic mode owing to the reduction of the contact time amid the tip and the specimen to the small fraction of the oscillation time. Recently, some radical techniques have been established to enhance the resolution and nd more detailed information on these materials. These dynamic advanced methodologies can be categorized as multifrequency methods 226 as the cantilever is excited at many different frequencies and altered signals are utilized as feedback parameters. AFM can also quantify and measure the structural and mechanical properties of the viruses, in addition to visualizing and imaging the virus particles. 227 In addition, it can also be exploited to operate and dissect biological entities along with viruses. [228] [229] [230] [231] Furthermore, to examine the chemical, physical, and viscoelastic traits of the viruses, the force-distance curve-based AFM (FD-based AFM) and currently, multifrequency methods, are the frequently employed approaches. In the latest FD curve-based AFM, while imaging the biological sample, several FD curves are recorded. 232 Meanwhile, chemical and biological properties mapping methodology has been developed on the idea of FDbased imaging. 233, 234 In the above methodology, the tips are modied by particular ligands. Furthermore, based on the mechanical strength and adhesion of bonds developed among the modied tips and the receptors of the specimen, the biological traits can be investigated during imaging. 235 The time of data acquisition and high volume are the major challenges associated with FD-based AFM. Numerous multifrequency AFM approaches have been projected to decrease the amount of data and increase the imaging speed. 236, 237 However, their complicated physical principle, mechanism of data interpretation, and theoretical development require more research.

The former research on coronaviruses exploiting AFM techniques [238] [239] [240] [241] indicates the substantial potential of AFM to visualize, image, and investigate the morphological topographies of the SARS-CoV virus, as shown in Fig. 17 . Generally, characterization/visualization tools are valuable as they deliver surface and subsurface information of COVID-19 affected cells, perceive multiple versatile interactions amid host cell and SARS CoV-2 virus, explore the mechanism of the COVID-19 virus to pass the cellular membrane and transport its genome into the host cell, study its replication into the cell along with SARS-CoV2 virus nucleic acids characterization, and recognize the way it is packaged and condensed inside capsids. Recently, anticipated techniques at the nanoscale map that the directional ow patterns can also be castoff to examine the effects of ion-specic sieving traits of the host cell and different pH on the binding of the SARS CoV-2 S protein with the receptor at the interface of the cell. 242 Currently, Yang et al. characterized molecular binding and studied the inhibition relation of SARS CoV-2 with ACE2 receptor by exploiting AFM. 243 4.8 Detection of COVID-19 from common sources 4.8.1 Saliva-based detection. Salivary glands release an exocrine secretion named saliva that has multiple functions such as protection and cleansing of oral cavity, aid in the digestion, and most importantly, the antimicrobial activity. Speedy progress in the eld of saliva omics resulted in the acceptance of saliva as a biological biomarker that uctuates from changes in the nucleic acid, proteins, and microora. Saliva has an edge over other diagnostic uids owing to its economic perspective, reliable collection methodology, and non-invasive procedure for monitoring systematic health. Formerly, it has been previously manifested that saliva has a higher consistency rate (90%) with nasopharyngeal specimens for the sensing of coronaviruses. 244 The systematic study publicized 96 records aer the removal of duplicates and among those, 5 records were included for quantitative and 26 records for qualitative synthesis. The ndings proposed 91% (CI 80-99%) sensitivity of saliva-based diagnostics and 98% (CI 89-100%) sensitivity for nasopharyngeal-based diagnostics for CoV-2 patients with moderate heterogeneity in the studies. The same group also recognized 18 registered patients with ongoing clinical trials of saliva-centered prognosis for COVID-19 diagnosis. 245 In Hong Kong, a study was made, in which patients were considered to be diseased if SARS CoV-2 was identied in their sputum or nasopharyngeal specimen. Saliva of a total of 12 patients was obtained and subjected to nucleic acid extraction and RT-PCR for COVID-19 prognosis. Saliva samples were collected at an average of almost 2 days aer hospitalization (range, 0-7 days) and initially, SARS CoV-2 was found to be present in saliva samples of 110 patients (91.7%). Meanwhile, there were 33 cases whose nasopharyngeal samples were found to be negative for COVID-19. Their saliva specimen also showed the same negative response. Recently, a group of scientists has effectually endorsed saliva as a viable source compared to oropharyngeal swabs for COVID-19 detection. They reported the utilization of saliva as a robust source to extract viral RNA for COVID-19 detection. Saliva-based tests will help with the global scarcity of swab-based sampling for COVID-19 analysis. 246 Likewise, many other groups reported the early detection of COVID-19 based on saliva specimen ndings. 247, 248 However, the low concentration of the analyte in the saliva compared to blood limits its practical application, which is circumvented by the development of precise molecular methodologies and nanotechnology.

4.8.2 Wastewater-based detection. Sewage or waste waterbased epidemiology (WBE) has been employed to track and give timely forewarnings of outbreaks of pathogenic diseases such as poliovirus, norvirus, hepatitis, salivirus, enterovirus, rotavirus, and astravirus, and for the regular monitoring of the diversity and concentration of viruses in wastewater. 249 Commonly, WBE evaluates the spatial and temporal trend of virus in wastewater inside the catchment of sewage treatment plants. As we all know, the outburst of COVID-19 has brought a substantial threat to human health. Recently, SARS CoV-2 has been identied in fecal samples of COVID-19 patients from various regions such as China, Korea, 250 United States, 251 Singapore, 252 and Germany. 253 Another research including 10 pediatric COVID-19 patients provided proof for the occurrence of SARS CoV-2 in feces. 254 Even in most negatively reported nasopharyngeally tested cases, rectal testing was found to give positive results for COVID-19, demonstrating the shedding of the gastrointestinal and fecal-oral pathway. Current evidence suggest that analysis of SARS CoV-2 in wastewater is useful to scrutinize the viral transfer in humans as SARS CoV-2 can be released via urine or feces. 255 Medema et al. revealed the detection of SARS CoV-2 from sewage in the Netherlands. 256 Likewise, many other attempts have been conducted to analyze SARS CoV-2 through wastewater in countries such as the United States, 257 Australia, 258 France, 259 and Sweden.

RT-qPCR has been utilized to test N(N1-N3) and gene E, and they all were identied at different sites and validated the presence of SARS CoV-2. In the United States, the SARS-CoV2 at high titers was also detected in wastewater specimens by RT-PCR. 260 Their work further revealed that the longitudinal examination of wastewater can give an approximation of the population level without onsite analysis. The existence of SARS CoV-2 RNA was also conrmed in 6 WWTP wastewater specimens from a lower frequency area in Spain. 261 Environmental surveillance outcomes elucidated that SARS CoV-2 had been transmitted among the population before initial reporting by the municipality. Therefore, WBE can be employed as a surveillance tool for the detection and pervasiveness of COVID-19 and gives a detailed insight into the development magnitude of COVID-19 than clinical analysis. RT-PCR assays have been executed for SARS CoV-2 assessment in many disease control and research centers, as we have discussed previously. 58, 262 However, the requirement of skilled technicians, longstanding data processing, and examination limits its practical application globally. Digital PCR has also been utilized and its sustainability for certain samples has been evaluated. 263 Therefore, it is of paramount importance to manufacture efficient robust and transportable analytical tools to analyze low-level SARS CoV-2 specimens through WBE to validate doubtful cases and monitor asymptomatic patients without the need for centralized laboratories. Thus, exploiting the WBE approach for timely warning and intervention needs a prompt biosensing approach for the on-site recognition of all viruses. The latest progress in sensing devices makes eld analysis possible so that the system can deliver real-time monitoring and public health information, as shown in Fig. 18 . Recently, it has been proposed by a few scientists that paper-based biosensors can be employed for WBE. 262 Owing to the merits such as budget-friendliness, robustness, accuracy, specicity, and sensitivity, these biosensors have been employed previously in the clinical analysis of different analytes, 264 food protection, and environmental monitoring systems. Recently, biosensors have been exploited for electrochemical, acoustic, thermal, electrical, and piezoelectric analysis of contagious diseases. The indicators of these contagious diseases are typically recognized in the nasal mucosa, plasma, sputum, serum, blood, urine, feces, and saliva. Exploiting high-affinity probes for sensing wastewater specimens can also decrease the matrix effects. 265 In addition, biosensors can also be employed for the multiplex analysis of contagious ailments. Moreover, biosensors as medical tools can be established for the POC recognition of contagious ailments in resource-limited areas. For instance, Yang et al. introduced a fast "sample-toanswer" detection process, which can offer quantitative analysis of nucleic acids along with the genetic data by the exploration of sewage waste, 267 and these ndings were further validated by agarose gel image assay and robust electrophoresis, displaying good consistency for wastewater examination. The anticipated biosensors will display advantages such as excellent sensitivity, affordability, superior specicity, rapid sensing time, and low sample consumption for the user-friendly recognition of SARS CoV-2 in sewage water despite conventional detection methods. Under this standpoint, the advanced study on the environment for the examination of disease biomarkers is needed to efficiently exploit WBE sensors for the updated status of COVID-19 in a community (Fig. 19) .

4.8.3 Food and other surface-based detection. As the human health system is globally affected by the current pandemic, adverse effects on the food system and its associated population are also evident. Therefore, to guarantee food safety and to avert the interruption of food supply chains, the development of SARS CoV-2 recognition tools for food analysis is a mandatory need. However, a reliable detection strategy in food is challenging owing to the heterogeneous distribution of virus particles, non-optimal tedious isolation, and low viral load. 269 Previously RT-qPCR, ELISA, 270 and nanoELISA 271 methodologies have been employed for this purpose, but currently, molecular and serological tests are in practice for SARS CoV-2 identication. Most studies recommend that the immune response to the virus commences aer 7 days of the beginning of symptoms. 272, 273 As we discussed, considering the recognition of CoV-2 in food, on surfaces and adjacent mediums, or surrounding is a major concern as currently not much research has been performed and no evidence exists that coronavirus spreads through surfaces, surroundings, and food. Nevertheless, there are maximum chances of the virus spreading from diseased workers via surfaces and adjacent milieu of the food industries and the food supply chain. Cai et al. reported that the transmission of the virus by faucet taps and elevator buttons has also contributed to a number of cases in China. 274 RT-qPCR have been employed to detect COVID-19 from all fomite samples. [275] [276] [277] Though these are preliminarily ndings, some companies have designed commercially available kits for environmental swabs. [278] [279] [280] Meanwhile, sampling kits for surfaces are also offered by few companies. 281 However, their high prices limit their broad application, especially in the food sector.

As mentioned previously, the rst technique employed for the detection of SARS-CoV-2 and to gain information regarding primers, genetic biomarkers, molecular probes, and different concentration of antibodies in samples was rRT-PCR. But the complex infrastructure, longstanding time requirements, 282, 283 and excessive use of reagents for the diagnostic test are the major drawbacks of the technique. Therefore, simplistic diagnostic tools with robust response are the immediate requirement. In this regard, nanotechnology has played an important role to counter the aforementioned challenges. For instance, nanodiagnostics works on the principle of the binding capacity of the nanomaterials and the biomolecules under consideration to generate a quantiable signal for pathogen detection. 284 Meanwhile, nanotechnology has also aided in providing effectual results with prompt and timely diagnosis of the disease. 285 In addition, the role of nanomaterials in bioengineering can also be of signicant importance for SARS-CoV-2 detection as this virus itself has a core shell nanostructure with the size ranging from 60 nm to 140 nm. 286, 287 Thus, it allows the bioengineered nanomaterials to specically bind with the virus, 288 permitting the evaluation, engineering, and development of procedures for diagnosis, treatment, as well as the preventive measures of SARS-COV-2. 289, 290 These traits of nanotechnology also lead to the development of handheld devices/tools that can be commercialized with additional benets of stability, sensitivity, and high accuracy (Tables 2-8 ).

The below table contains the data regarding the conrmed in vitro detection tests, which can be exploited for COVID-19 patients' management.

Still various rational and distinguishing management strategies are critical even aer the development of intensive detection methodologies to overcome the aer effects from this pandemic situation. 291 For intense cases, survivors of SARS COV-19 could 293 Recently, the application of contact tracing has been developed and employed as shown in Fig. 20 .

The main idea of this application is to substitute manual contact tracing with an immediate broadcast of signal toward central/main server. Principally, the information of virus patients has been transferred to the server that recommends risk-stratied quarantine along with robust social distancing to 294 Nevertheless, to achieve the complete exploitation of this technology, the tests number also needs to be increased. Meanwhile, privacy consideration also limits the application of this soware-based technology. 26 However, as the virus responsible for COVID-19 is continuously emerging, thus, such an improved developed system of contact tracing could play an important role in lowering the intensive impact generated by COVID-19. 295 

Intense shallow breathing has been commonly observed aer severe SARS-COVID-19 infection. The Pulse Oximeter (PO) can play an important role in monitoring different respiratory symptoms aer SARS-COVID-19 without any extreme anxiety or stress. 297 Infected persons should be given an observation diary with a pulse oximeter and proper guideline to self-monitor. Normally, in this practice, a daily reading would be taken by a neat and warm nger aer an interval of 20 min. However, the important thing to keep in mind while taking the reading is that the applied device should be allowed to stabilize rst and then the highest reading should be considered. 298 Another major 

Most of the COVID-19 patients suffered from prolonged recovery without any special input through a holistic approach. Much of it could be achieved via community rehabilitation strategies and inter-professional services, which improve a person's self-management and harness the possible video and related technologies. Also, an informative as well as rehabilitation platforms should be established at the community level. 300 In this time of uncertainty, the major role of these platforms should be the witness of "honoring the story" of a patient whose recovery was found to be unexpected. 301 6 Recent trends and future perspective

To date, no accurate treatment/therapy has been proved to circumvent the mortality from this pandemic completely.

Researchers are continuously struggling to invent a universal treatment to reduce the severity of disease. Various COVID-19 vaccines across the world have been developed, as listed in Table 9 . Experts consider Remdesivir as a signicant and clearcut vaccine for reducing the recovery time by blocking this virus. It could be considered as a magic bullet. Further, Favilavir has been considered as an antiviral drug by China. However, its sample size is still very small. Experts also consider that any new compound could take a decade from its initial discovery to reach the nal marketplace; many compounds would never be able to make it that far. This pandemic situation needs intensive actions in various areas from prevention to detection and then to nal treatment. A cheap, simple, and fast testing methodology is required to detect the antibodies able to neutralize COVID-19. In this regard, some of the opportunities have to intervene in long as well as short terms to save lives. But before that, we need a guideline from science. Besides all these scientic efforts, everyone has to contribute to control this uncertain situation to lessen the risk assessment. At this stage, food, shelter, and healthcare for the needy sections of the community are massive challenges. Everyone including migrants and refugees need special attention.

Shaping the future as a global nation is anticipated. The education sector is one of the highly affected areas by SARS-COV-2. All the nations must have strategies to protect children's education. Teenagers are the major group affected by this pandemic-isolation. As the situation prolongs, the need for healthcare is increasing, which also includes the counseling of young generation missing out on school meals amid school closures. 

The SARS-CoV-2 pandemic offers an incomparable global humanitarian and medical trial. Although this has impelled unprecedented growth in the development of therapeutics and vaccines, it has also highlighted the vulnerability of limited resources. The limited testing capacity along with the infrastructure to produce therapeutic drugs was largely absent to combat COVID-19 in the beginning. Efforts were made to exploit available resources to manufacture cheap and robust diagnostic methodologies. The review discusses in detail the standard diagnostic methods along with their pros and cons for the detection of COVID-19 as well as to avoid consequent secondary blow-out. Nucleic acid plus protein-based diagnosis along with biomarkers to evaluate the disease severity have been employed at the start to sense SARS CoV-2. Furthermore, as research proceeded, non-invasive sensing protocols were explored for wastewater surveillance for early detection. Similarly, nanoscale tools have also been discussed to examine viral morphology. Although various nanobiosystems have clearly revealed the viral inhibitory properties of COVID-19, still, most studies have shown the signicance of initial ndings of the virus. The perseverance of nanosystems has not fully been evaluated, regardless of their endpoint applications including therapeutics, surface protection, and water disinfection. Further, the administration of investigative nanobiosystems to animal models should be validated along with its development. This validation would lead to subsequent antiviral coatings for medical devices. The review was an effort to provides insights into the diagnostics and prognostic approaches for current and future pandemics.

Winners and Losers of EU Integration, Policy Issues for Central and Eastern Europe

Advances in Biomarker Sciences and Technology

Medecine et maladies infectieuses

Medecine et maladies infectieuses

Methods in cell biology

Structural biology of viruses

Molecular Biomethods Handbook

Proc. Natl. Acad. Sci

Proc. Natl. Acad. Sci

Nanoscience and Technology: A Collection of Reviews from Nature Journals

Proc. Natl. Acad. Sci

Women in Water Quality: Investigations by Prominent Female Engineers

Community sewage sensors for monitoring public health

The authors declare that they have no known competing nancial interests or personal relationships that could have appeared to inuence the work reported in this paper.