Review—Role of Nanomaterials in Screenprinted Electrochemical Biosensors for Detection of Covid-19 and for Post-Covid Syndromes

Covid-19 still remains as global pandemic surging with different variants like alpha, beta, gamma, delta and most omicronin various countries resulted into SARS-CoV-2. An escalation in random testing for Covid-19 is considered as golden standard by World Health Organization (WHO) to control the spread of Corona virus. RT-PCR and nucleic acid hybridization strategies are the two highly sensitive outstanding tests used for the detection of SARS-CoV-2 in the DNA by the former and RNA/DNA by the later. Apart from the test for virus, antigen and antibodies other alternative hematological tests like CRP, Ferritine, IL-6, Blood Platelets and WBC and plasma became essential for the detection and management of Covid-19. Overcoming the limitations a rapid, efficient, low-cost and easy-to-implement test kits are in imperative need to meet the requirement. Electrochemical sensors are considered as potential, ultra-fast and point-of-care sensors used as self-testing kits. In this work, a meta review of various modified electrodes with different nanomaterials such as graphene, CNTs, and nano-dopants used as electrochemical sensors in association with testing various different parameters for detection of Covid-19. A detailed discussion on the merits and demerits of present testing patterns and scope of screen-printed electrochemical sensors modified with different nanomaterials and their advantages are presented.

The novel coronavirus disease 2019 was caused by SAR-CoV-2 (Severe Acute Respiratory Syndrome)reported for the first in December 2019 by China in Wuhan and followed with several viral pneumonia cases. 1 The covid−19 created several unprecedented disruptions over the world both economically and societally. 2 Every year the virus is challenging the scientific society with new mutations from the deadliest delta variant in the second wave to the highly contagious omicron variant in the third wave. SARS-CoV-2 hit India in early March 2020 and reached a maximum spike of ninety-seven thousand infections in September 2020 regarded as the first wave. 3 The second wave hit the country with an average spike of three lakhs in the first week of May 2021. 4 The gimmer Covid-19 strain of the second wave in India is classified as "Variant of Global Concern" by World Health Organisation (WHO) and declared to be the first found in India. The said mutation was initially termed as "Variant of Interest (VoI)" and was elevated to "Variant of Concern (VoC)" as it fulfilled one of the criterias which include (a) easy transmission, (b) severe illness, and (c) reduced neutralization by antibodies or reduced effectiveness of treatment and vaccines. 5,6 The world has witnessed 219,000,000 corona cases and 4,550,000 death cases. In India, it is reported about 37,500,000 effected by a coronavirus while 447,000 lost their lives batting with covid-19. 6 Numerous literature testified that asymptomatic carriers play a crucial role in transmitting the disease as the virus incubates and becomes highly infectious over a wide range in 0-24 days. 7 From retrospective studies, the principal source of coronavirus SARS-CoV-2 is through aerosolized droplets and direct contact, oral-fecal route,mother-to-child transmission,and some other possible transmission routes. 8 Asymptomatic or mild disease symptoms constitute majority of the cases of Covid-19 where patients with good immunity were affected with mild disease symptoms. The disease progressed to pneumonia, Acute Respiratory Syndrome (ARDS) with multi-organ dysfunction in elderly patients and patients with other comorbidities affected with coronavirus SARS-CoV-2. 9 Patients affected with coronavirus SARS-CoV-2 reports initial symptoms such as fever, cold, cough, sore throat, breathlessness, fatigue, and malaise. Gastrointestinal symptoms, anosmia and dysgeusia, dermatological symptoms, olfactory and gustatory disorder, anxiety, and depression are reported with the severity of the virus replications. 10 Hypoglycemic respiratory failure requiring prolonged mechanical ventilatory support with critical illness is found the only insubstantial percentage of patients who reported respiratory illness. The autopsy studies of severe Covid-19 subjugated with acute pneumonia demonstrated alveolar damage, inflammatory infiltrates, and microvascular thrombosis. 11 The world is now witnessing a new massive surge of coronavirus (omicron variant). Testing and isolating are the two golden rules devised by various health agencies to break the chain of spreading the coronavirus. 12 Various testing patterns like the most effective RTPCR, rapid antigen test, and serological tests approved by the WHO are in wide practice. In spite of these robust testing practices, every test draws few limitations in practical use. 13 RT-PCR requires a specialized technician and a minimum of 5 h to get the results. 13 Rapid antigen test suffers from false negative reports and can serve a purpose only when the patient is on maximum viral replication load. Antibody/serological tests can only provide the information of the past infection as it provides backdated data of about 4 to 14 days, but cannot give the imprint of the active virus at the earliest stage. 14 In the present review, we summarise the importance of testing which helps in limiting the surge of coronavirus. The present testing patterns like RT-PCR, Antigen test, and Antibody tests are explained along with their demerits.
The role of disposable screen-printed electrochemical sensors was highlighted as an alternative rapid testing sensor for Covid- 19. The role of nanomaterials in enhancing the electrochemical sensing properties is disclosed.
In the management of Covid-19 patients, the biomarkers like CRP, IL-6, Ferritin, Blood platelets, are to be evaluated on daily basis in order to assess the prognosis of the corona disease. 15 There is a need for an array of sensors to evaluate all the important biomarkers with a single blood sample on daily basis. 16 In this review, we have given a cognitive explanation of the role of different z E-mail: sundeepdola@gmail.com * Electrochemical Society Student Member. biomarkers associated with covid-19 management, like the development of mass production of Point of Care (PoC) glucose sensor, an array of sensors for biomarkers with a PoC covid-19 sensor has to fabricate on mass-production. 17 In the management of a covid-19 patient, around 30% of the hospitals bill is charged on the diagnosis of the patients for different hematological tests some daily and some twice to thrice in a day. 18 Getting infected with Covid-19 is itself a great economic burden on a patient for which the diagnostic should not be an additional burden. To summarize, robust testing patterns need to be developed to eradicate the spread of the virus. The aim of this review is to create a thrust on the research community to develop the ultra-fast, highly sensitive and selective, and highperformance disposable Screen Printed Electrochemical Sensors that can be deployed for detection of Covid-19. [19][20][21] A variety of nanomaterials are always available which can be used to enhance the efficacy of the SPEs. Very low research has been reported on the disposable SPEs for SARS-CoV-2 and no mass production of SPEs for Covid-19 is available to date. The research should not to be restricted to the laboratory but rather transformed to the industry for the fabrication of economical, easy, and PoC sensors.

Severe Acute Respiratory Syndrome SARS-CoV-2
It is believed that the SARS-CoV-2 has emerged from a zoonotic source having a genome size near to 30 kb. 22 Figure 1 shows the structure of coronavirus with Spike (S), Membrane (E), Membrane protine (M), and Neculeocapsid (N). Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS) are other two human viruses responsible for severe pandemics. 24,25 Like other corona viruses the new SARS-CoV-2 virus causes respiratory diseases amongst other symptoms. 25 This single stranded RNA virus is identical to SARS-CoV having 75 to 90 nm virion size. 26 There are four structural proteins in SARS-CoV-2 genome virus namely Spike (S), Envelope (E), Membrane (M) and Nucleocapsid (N) proteins. 24 From Fig. 1 it is clearly seen that at the core of the corona virus, it contains RNA which is the blueprint similar to the DNA. The single stranded RNA acts as the molecular messenger, enabling the production of proteins needed for virus and other elements bound to the string of RNA on nuclear proteins. 27 The ability to replicate and structure of the virus are obtained with the help of proteins. When the virus is out of the host cell, the envelope encapsulating the RNA genome protected the virus. 28 The genome is comprised of a minimum 6 to 11 open reading frames (ORFs) and the flanking untranslated regions (UTR)s are about 50 and 30. 29 This outer envelope is made up of a layer of lipids and a wax barrier containing fat molecules. It also protects the precious genetic cargo and helps in anchoring the different structural proteins needed by the virus to infect the cells. [7][8][9][10][11][12][13][14][15]18,22,[24][25][26][27][28][29] The envelope proteins embedded in this layer aid the assembly of new virus particles once it infected a cell. The bulbous projection seen on the out side of the corona virus are the spike proteins. 8,30 The crown or halo like structure which is the corona moniker is offered by these spike proteins. They act as grappling hooks allowing the virus to latch on the host cells and crack them open for infection. Like all viruses the corona viruses are unable to thrive and reproduce outside of a living host. 29 The SARS-CoV-2 covid-19 have a less similarity in comparing genetically with SARS-CoV about 79% and MERS about 50%. 30 All the three virues are structurally differ in the arrangment of the nucleocapsid protine (N), envelope protine (E) and membrane protine (M). Using the spike glycoprotine the SARS-CoV-2 bind to the Angiotensin-Converting-Enzyme-2 (ACE-2) which helps to affect the cells. Protease an enzyme which is coupled with the spike protein paves the virus to enter the cell completely and finalised with the help of TMPRSS2 protease. This protease activates the attachment order of spike protein to the cellular ligand (ACE2). 31

Need for Testing
The mitigation and suppression of community transmission of covid-19 is only possible with extensive testing. 32 The role of testing comes into a sustainable obligation when a surge of new variant of corona virus is seen. Even after the wide availability of the vaccine for SARS-CoV-2 is available and nearly 1.5 billion population of India is administered with a double jab of vaccine, still the vaccine resistant variants of virus like Mu variants arecontinue to emerge. 33 Eventually testing for detection of virus comes into significant role in management of the disease which is the omicron variant. Testing for Covid-19 virus is not only essential for the infected patient but testing the immediate and asymptomatic contacts of the patient plays a crucial role in ceasing the spread of virus. 34 Immediate testing, treatment, disease surveillance and reopening the economy is highly depended on rapid and ultra-fast testing patterns. Identification of the virus infected patient and restraining such patient from entering into workplace is considered as the effective strategy to reopening the educational institutions and other organizations. The governments should focus on the rapid acceleration of the mass production of the ultra-fast, point-of-care and highly sensitive diagnosing kits for the detection of the virus. To achieve such massive testing patterns, a minimum of 5-10 million tests needto be performed on daily basis in a dense country like India with 138 crores.
Management of public health in a hugely populated country is always a challenge to the doctors. Testing for corona virus is a major task, due to ignorance, low severity and asymptomatic condition of the virus affected host. One key issue pertaining to rapid spread of the virus is the severity and infectivity of the disease in the asymptomatic patients which is comparatively lower than in the symptomatic patients. 35 However, the virus effected asymptomatic patient may not be an asymptomatic or have mere severity and infectivity of the disease. Not less than five incubation days required by the grievous virus to exhibit its symptoms, during which a considerable replication of virus is noted to be the highest in the lungs. 36 The rapid, ultra-fast and highly sensitive tests are indeed, to identify such patients with decent immunity and asymptomatic patients to cease the spread of virus to immunocompromised patients. In several instances, the World Health Organization (WHO) insisted that testing is the only key to arrest the spread of virus and to end the pandemic. 37 To achieve such higher testing for virus, a broad class of testing devices must come into accessibility for instantaneous tracing for virus.

WHO Accepted Testing Patterns
A comprehensive approach to strike and sojourn the spread of SARS-CoV-2 is screening for symptoms, testing for symptoms and tracing the symptom contact. The testing for the Covid-19 is classified as diagnostic tests and antibodies test. 38 Nucleic Acid Amplification Tests (NAATs) like Reverse Transcription Polymerase Chain Reaction (RT-PCR) is considered as a golden test strategy for detection of SARS-CoV-2 by WHO. 13 Apart from RT-PCR test, antigen test is also introduced for rapid detection of virus classified under immediate and PoC testing pattern. Both the RT-PCR and Antigen test falls under the category of diagnostic tests while some clinicians also recognized the serology test for detection of Antibodies of past infection mostly inclined to asymptomatic patients, as the antibody test. 8,39 The diagnostic tests are used to detect the active virus infection used to identify the infected patient to isolate and quarantine them. A brief technical explanation of the present testing patterns is presented.
Diagnostic tests.-Reverse transcription polymerase chain reaction (RT-PCR) 40 .-The standard test to detect Covid-19 are as shown in Figure 2. Direct detection of the viral Ribonucleic acid (RNA), Deoxyribonucleic acid (DNA) and proteins is a molecular diagnostic technique involved in detecting a specific nuclear sequence of the genetic material of the virus for identifying the covid-19. 40 The diagnostic test samples are collected either by a nasopharyngeal swab or by an oropharyngeal swab. In the NAAT, the initial procedure is to amplify the genetic material into numerous copies. This amplification of nucleic acid paves the way to detect even a minute RNA of SARS-CoV-2 in the specimen. 41 Hence, the NAAT procedure is a highly sensitive and reliable test for Covid-19 as it can detect the minute presence of virus in the sample, which restrains the false result. Apart from RT-PCR different nucleic acid different amplification methods like Transcription Mediated Amplification (TMA), Nicking Endonuclease Amplification Reaction (NEAR), Loop-mediated isothermal Amplification (LAMP), Helicase-Dependent Amplification (HDA), Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and Strand Displacement Amplification (SDA) can also be employed to amplify the nucleic acids for detecting the virus. 37,42 Although the RT-PCR is considered as the golden standard detection technique for SARS-CoV-2, false case reports were testified rarely. These false reports landed the clinicians into dilemma and misperception and showed a major impact on the health statistics. However, these false reports were considered as two sides of a coin in which one side to appreciate that very rare false results were reported in view that no test gives a 100 percent accuracy and another side is to consider the anxiety and agony of the patient with a false result. Other limitations include the long turnouts, the test has to be performed by a designated skilled technician in a dedicated laboratory and the difficulties to give the swabs by the elderly and children. 37,41 Rapid antigen test (RAT).-The rapid antigen test is a diagnostic test that emerged as an alternative to the RTPCR test for the detection of SARS-CoV-2. RATs gained potential as they as economic, easy to use, point-of-care tests, and are rapid tests where the results are demonstrated within 15 min compared to RT-PCR which takes at least 4-5 h. 43 Unlike the chemiluminescence-based RT-PCR, the RAT is a lateral flow test with a rapid result. Most RATs are based on immunochromatography which goals the nucleocapsid (N) protein using the antibodies. Differing from the detection of antibodies like in serological tests and nucleic acid in RT-PCR, the RAT is used to detect the availability of antigen. 44 Even though the sensitivity and false results with RAT are much compromised with RT-PCR, the majority reported that the positive for virus by RAT was always in conformity with RT-PCR. RAT is only disregarded for its false-negative reports as it can only detect the virus infection at peak of the viral load. Out of all the dissociations on the utilization of RAT as a standard testing pattern for detecting covid-19, the WHO upholds the employment of RAT as significant in disease surveillance and patient management with a sensitivity and specificity ⩾80% for the former and ⩾97% for the later. 45 The actual performance value in correlation with the manufacturer's analytical results always contrasts and need not be resembled necessarily.
Antibody test 46 .-While RT-PCR and RAT are used to detect the present virus, the Antibody or immunoglobin test is used to trace the past infection associated with SARS-CoV-19. It detects the antibodies that were produced by the body in providing the defense against the disease. Antibodies are Y-shaped proteins produced by the immune system in response to an infection and are specified to a particular infection. If an individual is exposed to the virus, one shows positive to the antibody test. 46 There are two types of antibodies namely immunoglobin M (IgM) and immunoglobin G (IgG) tested for Covid-19. The presence of IgM antibodies has resulted in the presence of active viruses and the presence of IgG antibodies results in the presence of past infection. The IgM are short-lived antibodies detectable after 4 to 7 days later of an infection flinch and IgG are long-lived and are produced after 7 to 14 days after the presence of infection. 47,48 They show the body's effort (past infection) or preparedness (past infection or vaccination) to fight a specific disease. According to FDA (U.S. Food and Drug Administration), the antibodies are part of the body's immune response to exposure and not from the virus itself and devaluated the antibody test for diagnosis of infection. 49 The merits and demerits of antibody test are summarized in Table I.

Role of Haematological Tests in Covid-19 Detection
The role of biomarkers from a peripheral blood sample in the diagnosis of Coronavirus disease is inevitable. Biomarkers like CRP: C-Reactive Protein, IL-6: Interleukin-6, Blood Platelets, WBC: White Blood Cells, Plasma levels, and Blood Platelets provide quantitative and qualitative information regarding the disease intensity. 59 Figure 3 shows the biomarkers used for Covid-19 management.
C-reactive protein (CRP).-The C-Reactive Protein (CRP) belongs to the family of pentraxin protein found in blood plasma which is synthesized in the liver along with a few minor extrahepatic productions. 60 This hepatic synthesis site bestowed the CRP into a new superfamily of proteins. The role of CRP biomarkers is inevitable in monitoring and diagnosing the Covid -19 virus. The annular hemopentameric structured protein comprises five subunits attached noncovalently. 61 This extremely preserved major acutephase protein was primarily isolated in 1941 by Macleod and Avery. They discovered the utilization of calcium ions by CRP to react with C-polysaccharide (CPS). CRP has a significant biological role as it provides a constructive resistance to pathogens. 60 In response to the inflammation, the CRP concentration in blood plasma increases to 3000 folds. In the incidence of innate immune system response, the cytokine Interleukin-1 and Interleukin-6 control the CRP. CRP corelates with immunoglobin molecule especially with Immunoglobin G (IgG) in numerous ways like promoting quellung reaction in bacteria, phagocytosis, and agglutination but differ largely in structural shape. 62 With an exceptional binding specificity, the CRP is used as a sensitive biomarker for inflammation and has a wide clinical use. 63 CRP is used as a prognostic biomarker for Covid-19 affected patients. It is an important indicator of inpatient management for triage, diagnostics, and prognostication. 60 Within 4 to 10 h of inflammation attack, the secretion of CRP potentiates and reaches the threshold at 48 h. The half-life of CRP is 18 to 19 h and the healing of a patient is associated with a decrease in the CRP concentration levels. CRP is found on the surface of the damaged cells, bind to the phosphocholine. 61,63 The normal CRP levels of a healthy person are less than 10 mg l −1 and exceptionally elevated levels range to 170+10 mg l −1 . Since the advent of the covid 19 numerous reports were available on the use of CRP as an important biomarker in analyzing the severity of the disease and management. All the studies reported an upsurge in the levels of CRP in blood serum concentration on an average of 20-50 mg l −1 . Elevated levels of CRP ranged between 18.8 to 25 mg l −1 for mild and 39.4 to 46 mg l −1 for severe symptomatic cases. It also found that the patients with low oxygen saturation (SpO 2 < 90) patients have a median increase in CRP levels to 12 to 14 mg l −1 , 64 Interleukin -6 (IL-6).-Interleukin -6 (IL-6) is secreted by T cells and helps the B-cells to mature the antibodies. T-Cells, B-Cells, mesangial cells, dendritic cells, fibroblasts, mast cells, keratinocytes, and vascular endothelial cells share in the production of this pleiotropic cytokine. 65 It is rapidly produced in response to pathogen-associated molecular patterns like infections and tissue injuries and offers acute phase reactions. It plays a major biological activity in immune regulation, oncogenesis, inflammation, and haematopoiesis. 66 Leukaemia Inhibitory Factor (LIF), Ciliary Neurotrophic Factor (CNTF), and Oncostatin (M) belong to the IL-6 family and share their work. The interleukin IL-6 plays a dual role as a pro-inflammatory cytokine and an anti-inflammatory myokine. It is a cytokine that features pleiotropic activity and involves the production of acute-phase proteins like CRP, serum amyloid, fibrinogen and also restricts albumin production. Hence, IL-6 is considered a unique receptor system for its pleiotropic and redundancy functions. 67 In Covid-19 affected patients, IL-6 acts as a mediator of inflammation and viral cytokine. Out of several algorithms to assess the severity of the patients, IL-6 plays an immensely appreciable role. The orderly upsurge of IL-6 in a covid-19 patient relates to the correlation of exponential increase of the viral load in the patient. 68 Ferritin.-Ferritin is an iron-binding protein that stores (Fe 3+ ) and releases (Fe 2+ ), thus, helping in regulating Iron Homeostasis. It is a spherical multi-metric protein composed of 24 H and Lsubunits which form an apoferritin shell. 69 Ferroxidase enzyme present in H -subunit catalyzes the conversion of Fe 2+ to Fe 3+ and L -subunit promotes the incorporation of Fe 3+ into the apoferritin shell. 70 Depending on the tissue type, expression of H and L subunits varies and in serum, it is predominantly of L type. Ferritin expression is regulated by Iron response element/Iron regulatory protein (IRE/IRP) dependent mechanisms and Ferritin synthesis takes place at level. A cytoplasmic 5 l -untranslated mRNA. Cytokines like IL-6, TNF -alpha, IFN -gamma stimulate the production of Ferritin, the reason behind increased ferritin levels during inflammatory conditions. 71 Studies reported that Ferritin mediates the inflammatory process through its binding with T -Cell immunoglobulin by advancing the expression of pro-inflammatory mediators. The normal level of Ferritin depends on age and gender, males-30-300 ug l −1 , females-15-200 ug l −1 . Ferritin levels of 500 ug l −1 indicate up to 58% of morality. Even though increased levels of Ferritin are biomarkers of inflammation, they are important host defense mechanism as high levels of iron is toxic and inhibits the growth of pathogens. 72 Blood platelets.-Blood Platelets count is a reliable marker in COVID-19 patients as a strong association is found between PLT  -Antibody test is used for detection of antibodies produced in resistance to virus, so the antibody test shows the result only after a week to detect the antibodies. 58 and mortality. Also, mean platelet volume (MPV) is studied which is a contemplation of the average size of platelets. Mean platelet volume/platelet count ratio is an independent risk determining factor. 73 Studies showed that COVID-19 patients had high levels of IPF (immature platelet function) of young platelets with the exaggerated ability to aggregate. Increased IPF was thought of as a compensatory response to excessive PLT consumption. Also, evidence shows elevated levels of P-selectin (CD62P) not only on the surface of circulating PLTs but also in the serum of the patients in a soluble form (sP-selectin) indicating disease mortality. 74 Pathophysiology:.-SARS-CoV-2 activates B cells to produce pro-inflammatory cytokines and autoantibodies that interact with the PLTs' glycoproteins, The pro-inflammatory cytokines can stimulate the coagulation system also attracts neutrophils, macrophages, and expose the tissue factor on their surface which can subsequently activate thrombin. Thereby, coagulation cascade is initiated, leading to thrombocytopenia. SARS-CoV-2 could directly affect hematopoietic cells and bone marrow stromal cells inducing thrombocytopenia. 75 Bacterial sepsis in hospitalized patients may hamper the expression of plasminogen activator inhibitor-1 (PAI-1), therefore, fibrinolysis is interrupted, and more vigorously the number of PLTs will drop in Covid-19 patients. Persistent hypertension, low oxygen levels, and diffused alveolar damage alters the integrity of pulmonary capillary beds and suppresses PLT release. Thrombotic thrombocytopenic purpura, atypical hemolytic uremia syndrome, drug-induced Heparin, antibiotics, and antiviral agents such as azithromycin and hydroxychloroquine thrombocytopenia might also contribute to this drop. Apart from the drugs, hemodialysis and extracorporeal membrane oxygenation (ECMO) could also induce thrombocytopenia in Covid-19 patients. FDA-approved approaches such as tocilizumab, eculizumab, and rovelizumab were proven to improve the PLT count; but no drug or treatment has yet been discovered to substantially increase their numbers. 76,77 White blood cells (WBC).-An elevated neutrophil to lymphocyte ratio was identified as a biomarker for Covid-19 disease. Few cases reported a significant reduction in granulocytes, eosinophils in severe cases of Covid-19. The reference levels for neutrophilia (absolute neutrophil count > 3-7.5 × 10 9 /L) and for lymphocytopenia is (lymphocyte count < 1.5 × 10 9 /L). 78 The possible mechanism for lymphopenia is invasion of lymphocytes by the virus, by binding to ACE2 receptors expressed on lymphocytes, bone marrow, hematopoietic stem cells etc. causing apoptosis, invasion and their destruction. Direct effects on the hematopoietic stem cells affects haematopoiesis. 79 ACE2 receptors on CD68 and CD169 macrophages in the spleen and lymph nodes were targeted resulting in cell degeneration/necrosis in the spleen which impacts immune cells, particularly a decrease in CD4 + and CD8 + T lymphocytes. Abnormalities of hematopoiesis could be the result of activation of nucleotide-binding domain (NOD)-like receptor protein 3 (Nlrp3) inflammasome expressed on hematopoietic stem cells. Increased lactic acid levels in Covid-19 may lead to reduced lymphocyte proliferation. Neutrophilia may be due to viral-induced inflammation or due to secondary bacterial infections. 80,81 Blood plasma.-Blood plasma factors are a valuable diagnostic tool in the management of Covid-19. Cytokine storm-induced in Covid -19 disease led to derangement in vascular function and blood composition. 82 Patients present elevated circulating D-dimer, a biomarker of fibrin formation and degradation, disharmony of the balance in fibrin-forming [TG]and fibrin-dissolving [PG] pathways are a major area of Covid-19 pathogeneses is a plasma protein secreted by the liver and induced by pro-inflammatory mediators. 83 progressing to severe disease is noted in patients with CRP levels >41.8 mg l −1 . Some cases of infection showed changes in serum amyloid A (SAA]. Covid-19 patients have elevated levels of PAI-1, t-PA, and fibrinogen, as well as vWF and the IL-33 receptor, ST2, responsible for fibrin-mediated hypercoagulability and thrombosis. homeostatic equilibrium between coagulation and fibrinolysis is present generally. Increased production of fibrinogen, but not plasminogen, indicates the increase of extra-hepatically synthesis of fibrinogen in response to inflammation. PAI-1 is the most important inhibitor of this fibrinolytic system, and elevated levels of PAI-1 are reported in severely ill patients. Also, an increase in t-PA levels, the enzyme responsible for the conversion of plasminogen into plasmin. PAI-1, t-PA, is produced and released from the vascular endothelium, but PAI-1 can also be produced by extravascular tissues, where its expression can be induced via activation of Nuclear Factor κB (NF-κB) by pro-inflammatory mediators in a cytokine storm. 84 The IL-33 acts on its cognate receptor ST2 and the IL-33/ST2 axis was proposed to mediate cytokine storm. Mast cells induce the recruitment of inflammatory cells during vascular injury and the production of PAI-1. Tryptase is generally measured as a proxy indicator of mast cell degranulation. It has been proposed that the correlation between inflammation and thrombosis in Covid-19 is induced by endothelium injury and platelet aggregation. 85 A high level of vWF may lead to platelet aggregation and the formation of microthrombi. vWF is part of an endothelium-mediated inflammatory response. Determining levels of t-PA and PAI-1 and calculating the ratio between t-PA and PAI-1 may help to some degree in identifying patients at risk for bleeding. Elevated PAI-1 results in an imbalance between coagulation and fibrinolysis, contributing to persistent coagulopathy. elevated levels of biomarkers like t-PA, ST2, and vWF, as well as abnormal coagulation, may be related to the incidence of thrombosis, renal failure, etc. 84

Sensors
Biosensors.-The invention of the world's first biosensor remarks to L.L Clark an American biochemist in late 1950. The principle behind the biosensor lies in biochemical analysis, where the receptors, enzymes or antibodies like biological components interactions. 85 The concept of biosensors was introduced since 1962 to the conventional, standard and established enzymatic or immunoassay based photometric detection for soluble enzymes for the former and fluorescence or radioactivity detection for the later. Biosensors are based on immobilizing the biological component and combining with suitable sensor, either physical or chemical. Biosensors are based on two types namely biological element and transducing method. The biosensors based on antibody, DNA, Enzyme, Biomimetic and Phage falls under biological element biosensors. Biosensors based on transducing method are classified into three types as (i) Optical biosensors, (ii) Electrochemical biosensors, and (iii) Mass-based biosensors. 86,87 The sensors based on fiberoptics, surface plasmon resonance (SPR), FTIR and Raman falls under optical sensors. Sensors based on potentiometric, amperometic, conductometric and impedimetric are classified under electrochemical sensors. The mass-based sensors are in turn classified into magnetoelectric and piezoelectric sensors like quartz crystal microbalance and surface acoustic wave. This biosensor revolution genesis began for their usage as point of care glucose sensors. 88 It offered a great relief to the diabetic patients for instance simple test for glucose levels in the blood sample. Apart from the glucose sensors, the disposable screen-printed electrodes use for various applications like environment pollutants (water monitoring, soil quality monitoring and environment monitoring), medical diagnosing, food analysis, drug discovery, prosthic devices and defense interested toxins. 89 Electrochemical (EC) sensors.-A device responds to a physical stimulus in material and if it transmits an electric impulse as the result of a change of the intrinsic property of that material, then such device is called a sensor. In the class of chemical sensors, electrochemical sensors derived a special stimulus for their extensive use as portable sensors for sensing various day-to-day constraints. 90 Out of several applications of EC sensors in health, defense, instrumentation, environment, etc., these sensors play a crucial role in various health monitoring applications for their miniature size and are economically offered with ultra sensitivity and selectivity. 91 Analyte, Sensory layer, Transducer, and Detector are the four prime components of an EC sensor. The response between the analyte and the sensory layer is measured and this chemical response is converted into an electrical signal by a transducer which, in turn, the detector amplifies the detected signal and displays. 92 In an EC sensor the Potentiometry, Conductometry, Amperometric, voltammetric, and Coulometry are the important EC methods used as detection techniques. 93 Not only viruses but the EC sensors are also used to detect bacteria, fungi, protozoa, and influenza, etc., at the earliest stage. Including SAR-CoV-2, various EC sensors are also developed in other viruses' classes such as HIV and influenza. 94 A wide range of EC biosensors was reported to detect glucose, cholesterol, urea, uric acid, DNA, hemoglobin, lactate, antibiotics, ketones, pathogens, amino acids, etc. 95 Screen-printed electrochemical sensors (SPECs).- Figure 4A shows the schematic diagram of the Screen printed Electrode and Fig. 4B represents the modified screen printed electrode. In the development of electrochemical sensor devices, disposable screenprinted technology has gained potential for their economic, ultrafast, and simple production. Different printing technologies were experimented with since the 1990s for the fabrication of electrodes via screen-printing, pad-printing, and roll-to-roll. 95,96 Only the screenprinted electrodes have overcome all the limitations by contributing the ability for mass-production. The SPEs are known for their sensitivity and selectivity, low signal-to-noise ratio, very small sample consumption, and reusability of the electrodes. 97 The introduction of screen-printed electrodes transfigured the EC research society by supplementing the gap between the laboratory experiments to mass-production. Commercially the SPEs are used widely for glucose sensing and also for detection of uric acid in human urine, cholesterol, lactate, diazepam, and codeine in soft drinks and DNA sensing. 98,99 Every year the role of SPEs intensifies massively for a variety of applications in the biomedical arena. During the past decade, the SPEs took over the biosensors for determining the damaged DNAs, cancer cells, and biomarkers as shown in Fig. 5. The SPEs also have an embracing application in photocatalytic experiments, fuel cells, and solar cells. 100 The present review confines the role of Screen-Printed Electrochemical Sensors (SPES) and nanomaterials modified SPESs for ultra-fast, highly sensitive determination of Covid-19.

Nanomaterials to Modify Screen Printed Electrodes
The unique properties of nanomaterials paved way for their effective usage in diagnosis, drug delivery, and antiviral therapy of novel corona virus. Recent years have seen a significant increase in the use of electrochemical sensor and biosensor platforms that incorporate nanostructured materials as powerful analytical methods due to their ease of handling, affordability, high sensitivity and selectivity, quick response, ease of fabrication, and portability. 101 Nanomaterials for used to screen printed electrochemical sensors can be classified as noble metal nanomaterials, metal oxide nanomaterials, carbon nanomaterials and polymer and bio-nanomaterials.
Nobel metal nanomaterials.-The Nobel metal nanoparticles such as gold, silver, platinum, and palladium are widely used nanomaterials to modify the screen printed electrode. 102,111 Along with the superior electrochemical properties noble metals are also used for their unique size, and shape dependent physical and chemical properties. These noble nanomaterials plays a crucial role in enhancement of electrochemical reactions which throughout the sensitivity, selectivity, and signal amplification.
Gold nanoparticles.-Gold nanoparticles are broadly used to modify the screen printed working electrode, as the gold nanoparticles offer distinctive high surface area, tunable optical properties and electro-catalytic activity. 101 Gold nanoparticles modified screen printed electrodes are used in various detection applications. Many researchers applied gold nanoparticles to modify the working electrode for detection of CRP, IL-6, WBC, D-Dimer, proteins, DNA etc. Modifying the electrode with gold nanopartices to detect corona virus is a new research. Alafeef et al., developed graphenebased biosensor in which the electrochemical response is recorder form the graphene-ss DNA-AuNP surface. 103 Silver nanoparticles.-An extensive research is Silver nanoparticles were used in biosensors for their exceptional biocompatibility and offers high conductivity and capability of amplifying the electrochemical signal. Numerous efforts have been made over the past two decades to develop novel analytical techniques based on Ag nanoparticles and their nanocomposites for biosensor Platinum nanoparticles.-Because of their unique electrical and electrocatalytic capabilities, Pt nanoparticles have gained a lot of attention in the last ten years in the field of electrochemical sensors for various environmental applications. 107,108 Chemical composition, surface quality, crystal structure, crystallographic axis orientation, and other factors can have a big impact on how the Platinum nanoparticles transmit electrons. Jinji Tian et al., reported the electrochemical dual-aptamer biosensor based on metal-organic frameworks MIL-53 decorated with Au@Pt nanoparticles and enzymes for detection of COVID-19 nucleocapsid protein. 109,110 Palladium nanoparticles.-Due to its numerous catalytic and sensor uses for gases, biomolecules, and dangerous poisonous compounds, palladium nanoparticles have garnered significantly more interest. The electrode materials made of Pd nanoparticles show strong electrocatalytic activity toward a variety of analytes.
The mass diffusion of the analytes can be enhanced by palladiumbased nanocomposites, which also provide electron tunnelling to allow electron transfer between the active site and the electrode, resulting in good electrochemical sensing performance. Chirlene N. Botelho and co-workers reported the use of palladium nanoparticles for detection of SARS-CoV-2 spike glycoprotein S1. 111 Metal oxide nanomaterials.-For the development of electrochemical sensor systems, metal oxide nanoparticles with large surface areas, strong adsorptive capacities, distinctive electrochemical activity, and stability are crucial. Uniformed nanostructures like nanorods, nanofibers, nanobelt, nanocomb and nanotubes are constructive metal oxide nanomaterials for modifying the electrode. Cu 2 O, TiO 2 , Fe 3 O 4 , metal oxide nanostructures are new perspectives in modifying the electrode for sensing covid-19. Copper is less expensive and has been used for a very long time compared to noble metals. Copper and copper oxide nanoparticles are employed in many applications and have good antibacterial and antiviral capabilities. The coronavirus 229E has also been demonstrated to be extremely susceptible to copper. Zeinab et al., reported the Electrochemical immunosensor with Cu 2 O nanocube coating for detection of SARS-CoV-2 spike protein. 112 Magnetic Fe 3 O 4 nanoparticles are excellent carriers with respectable separation ability due to their various benefits, including high surface area, exceptional magnetism, facile modification, etc. In this regard, a novel approach for the detection of COVID-19-specific IgG and IgM with good sensitivity, precision, and stability was devised. 113 In a different investigation, a test strip that can identify SARS-CoV-2 in 15 min was created with the aid of Fe3O4. Specific antibodies against coronavirus were combined with magnetic Fe3O4 nano-beads, and the spike protein of SARS-CoV-2 served as a coating antigen. 114 Another excellent metal oxide is titanium dioxide, which exhibits excellent antibacterial, antiviral, and photocatalytic efficacy and can be used to combat COVID-19. By sensing the spike protein (RBD) on the virus surface, a sensitive and affordable electrochemical bio-sensor based on cobalt-functionalized nano-TiO 2 was created for the quick diagnosis of SARS-CoV-2 infection in nasal/saliva secretions. 115 This prepared bio-sensor successfully detected SARS-CoV-2 protein in low amounts and showed a linear response for a short time of approximately 30 s.
Carbon based nanomaterials.-Excellent characteristics of carbon based materials which include great stability, low cost, vast potential windows, and ease of surface functionalization made them to use as sensing materials by electrochemical analytical methods. Carbon nanotubes (CNTs), graphene and nano/mesoporous carbon are few carbon based materials makes the surface groups to be exposed in a way that is favourable for the binding of analyte molecules to transduction material for sensing applications. 116 Based on their dimensionalities (D), these carbon-based nanomaterials can be categorised as zero-D (0D) materials like carbon dots, one-D (1D) materials like CNTs, and two-D (2D) materials like graphene nanostructures. Even in difficult environmental circumstances, this carbon based nanomaterials have a larger operating temperature, sensitivity, and dynamic transducing signal range. Both graphene and CNTs are significant polymorphic forms of carbon that have high mechanical, electrical (electron transport), and biocompatibility features. Graphene and CNTs are suitable carbon-based materials for the production of sensor electrodes due to their advantageous properties. 117 Because of its exceptional physico-chemical and biological capabilities, graphene has been used as an electrochemical nanomaterial for the creation of sensors including gas, chemical, and biosensors. Because of its versatility, compactness, and cost-effectiveness, graphene can be used to develop electrical sensors that are analogues to silicon. The lack of an energy band gap in graphene's electronic energy structure and its low chemical activity, however, are the biggest obstacles to manufacturing sensor devices employing this material. Carbon nano tubes are one-dimensional structures and due to its high length-to-diameter ratio and structural division into single-walled CNTs (SWCNTs) and multi-walled (MWCNTs) CNTs based on the number of graphene sheet planes.
Integrating carbon nanostructure, such as graphene and carbon nanotubes, can increase the sensitivity and performance of these electrochemical platforms (CNTs). These nanostructures provide the superior electrical properties, biocompatibility, chemical stability, mechanical strength, and wide surface area that are most sought after in building PoC diagnostic tools for quickly, accurately, and affordably detecting viral infections. Torrente-Rodrguez et al. describe a portable electrochemical biosensor with low cost graphene integration for quick diagnosis and tracking biochemical indicators in serum and saliva samples for COVID-19. 118 The screen-printed electrodes can be modified using a variety of nanomaterials like nanoparticles, nanotubes, nanospheres, nano polymers, nanocrystals, and quantum dots as shown in Fig. 6. 119 The nanomaterials modification of electrodes is done for enhancing the electroanalytical efficiency and to improve the immobilization of the biological element. In particular the doping of nanomaterial into the electrode enhances the detection application area and improves the mass transfer rate. Different modification techniques like ink mixing, drop-casting (Fig. 4B), and electrochemical deposition were employed to couple the nanomaterials on the electrode. 120 The present review focus on the various nanomaterials used to detect Covid-19 through screen-printed electrochemical sensors. The virus partticles and the nanomaterials are closely related to their size with different morphologiocal structures ranging from 20 to 800 nm. Different nanomaterials were applied to screen printed electrodes to enhace the electrode properties. 121,122 Very few research is contributed till todate on improving the SP electrodes which are as shown in Table II(B). nanomaterials like graphene oxide, 127 gold and graphene, 129 cobalt and titanium nanorods 130 were used to improve the immobilisation of antibody, enhance the surface are of the electrode and to obtain an over all eficacy. Table II is divided into two parts were part A is tabulated with the nanomaterials modifies screen printed electrode for biomarkers detection. Biomarkers like CRP, IL-6, White Blood Cells and D-dimer are detect on SPEs using different electrochemical sensing techniques. Part B is tabulated with nanomaterials modified SPEs for detection of Covid-19. Bhaskar S. Vadlamani et al. synthesised functionalised titanium nanorods coupled with cobalt used to modifie the SPE. 130 They reported a linear response of detecting S-RDB viral protein in 30 s at a very low concentration over 14 to 1400 nano molar. Others used cotton tipped SPE, reduced Graphene Oxide (rGO) 3D printed electrode, MIP/Au-TFE, Graphene-Gold Nanoparticles etc., 104,[127][128][129][130][131][132][133] and every nanomaterial treatment offered an excellent enhancemnt of sensing properties. A thrust for utilisation of nanomaterials should be focused in development of PoC Covid-19 sensors for a radip sensing is needed accross the world.

Conclusion Remarks
In the view of further waves of SARS-CoV-2 -Omicron variant prevalence across the world, rapid and random screening for the virus is the only means forward to break the chain of spreading the virus. Identifying the virus at an early stage and abstaining the asymptomatic patients can only reopen the economy and public places. Even though the RTPCR test for detecting the virus remains as the standard test, ultra-rapid, highly sensitive, and point-of-care (PoC) tests are required to identify the virus-affected person on an immediate basis to restrain the asymptomatic patients entering into  public places. Disposable Screen-Printed Electrochemical Sensors (SPES's) plays a vital role in the diagnosis of different diseases and are known for offering the PoC tests for Covid-19. The main drawback of SPES is the quality which varies from the screen-printed electrode to electrode and batch to batch. SPES is often convinced for single-use measurements, the required stability of the reference electrode is limited to one unique measurement and not a large number as in the case of conventional electrodes. Nanomaterials modified electrodes overcome a majority of the demerits pertaining to conventional electrodes. The coupling of nanomaterials with SPES improves the sensitivity and stability of the bio element in biosensors and detection possibilities. Yet significant research has to be done to elevate the Screen-Printed Electrochemical Biosensors status to one of the standard testing approaches for Covid-19which needs to be ratified by the World Health Organization (WHO). To summarize, we presented a detailed discussion on the merits and demerits of the present testing patterns and presented the scope of SPES modified with different nanomaterials and their advantages in using them as a commercial disposable sensor to detect the Covid-19 virus.