key: cord-0820896-ig1npgst
authors: Khaksarinejad, Reza; Arabpour, Zohreh; RezaKhani, Leila; Parvizpour, Farzad; Rasmi, Yousef
title: Biomarker based biosensors: An opportunity for diagnosis of COVID‐19
date: 2022-04-27
journal: Rev Med Virol
DOI: 10.1002/rmv.2356
sha: d8e08d2127bc703e0b05938fb5a7ae5ee10dbbc5
doc_id: 820896
cord_uid: ig1npgst

Early diagnosis and treatment of diseases are crucial research areas of human health. For early diagnosis, one method that has proven efficient is the detection of biomarkers which can provide real‐time and accurate biological information. Most biomarker detection is currently carried out at localised dedicated laboratories using large and automated analysers, increasing waiting time and costs. Smaller, faster, and cheaper devices could potentially replace these time‐consuming laboratory analyses and make analytical results available as point‐of‐care diagnostics. Innovative biosensor‐based strategies could allow biomarkers to be tested reliably in a decentralised setting. Early diagnosis of COVID‐19 patients has a key role in order to use quarantine and treatment strategies in a timely manner. Raised levels of several biomarkers in COVID‐19 patients are associated with respiratory infections or dysfunction of various organs. Through clinical studies of COVID‐19 patient biomarkers such as ferritin, Interleukins, albumin and …are found to reveals significant differences in their excretion ranges from healthy patients and patients with SARS‐CoV‐2, in addition to the development of biomarkers based biosensor such as stated biomarkers can be used and to investigate more specific biomarkers further proteomic analysis can be performed. This review presents several biomarker alterations in COVID‐19 patients such as salivary, circulatory, coagulation, cardiovascular, renal, liver, C‐reactive protein (CRP), immunological and inflammatory biomarkers. Also, biomarker sensors based on electrochemical, optical, and lateral flow characteristics which have potential applications for SARS‐COV‐2 in the recent COVID‐19 pandemic, will be discussed.

A novel human coronavirus, called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing the disease was identified in China in December 2019. 1 Severe acute respiratory syndrome coronavirus 2 has acquired the ability to establish sustained human to-human transmission. Its basic reproductive number, the number of secondary infections generated from one infected individual, is estimated to be between 1.4 and 6.49, with a mean of 3.28. 2 The recent emergence of SARS-CoV-2 in the human population has caused a dramatic and unprecedented impact of the economy and prompted mobilisation of public health authorities around the world to counter the rapid spread of the virus, and a wide variety of methods have been developed for the purpose of the rapid and accurate diagnosis of COVID-19 virus. 3 Several studies have reported haematologic and blood chemistry alterations in patients infected by SARS-CoV-2. 1 Major laboratory findings in COVID-19 patients identified by meta-analysis include leucopenia, leucocytosis, decreased albumin levels, increased levels of C-reactive protein (CRP), lactate dehydrogenase (LDH), creatinine kinase, and bilirubin, and a high erythrocyte sedimentation rate (ESR). 4 A growing body of evidence suggests that SARS-CoV-2 infection can trigger the overproduction of cytokines in some patients, known as a cytokine storm, which is associated with poor outcomes. 5 As for other severe viral infections, the exacerbated production of proinflammatory cytokines may be involved in some of the pathophysiology of COVID-19, including pulmonary oedema, lung failure, and damage to the liver, heart, and kidneys. Compared to healthy adults, COVID-19 patients had higher levels of several biomarkers such as IL-1β, IL-1RA, IL-7, IL-8, IL-9, IL-10, basic fibroblast growth factors, granulocyte colony stimulating factor (G-CSF), granulocytemacrophage colony-stimulating factor, IFN-γ, IP-10, MCP-1, MIP-1A, MIP-1B, platelet-derived growth factor, and VEGF. 5 Serum biomarkers associated with severe disease included IL-2, IL-7, IL-10, G-CSF, IP-10, MCP-1, MIP-1A, and TNF-α. 5 A recent retrospective study of 150 confirmed COVID-19 cases (68 fatal and 82 discharged cases) in Wuhan, China, identified several serological biomarkers that were more elevated in lethal cases than in survivors: elevated ferritin, IL-6, myoglobin, CRP, and cardiac troponin. 6 Together, these findings suggest that COVID-19 makes alterations in biomarkers compared to healthy individuals.

Since stricter requirements regarding human health have led to a rising number of clinical tests, there is an increasing need to develop highly sensitive, fast, and economic methods of analysis. 7 There are several reported biosensors based on biomarkers that accurately detect particular diseases such as non-invasive biosensor developed by Kumar et al., 7 for oral cancer detection which used CYFRA-21 as a specific biomarker for oral cancer, same as these specific biomarkers for COVID-19 can also be used, and biosensor can be made. 8 The development of biosensors is probably one of the most promising ways to solve some of the problems concerning the increasing need to develop highly sensitive, fast, and economic methods of analysis in medical diagnostics. 7 In this review, some consideration will be given to biomarkers levels in COVID-19 patients as well as biomarkers based-biosensors and their application in medical diagnostics, taking into account several crucial features. Researchers can break through bottlenecks of existing biomarker sensors by reviewing previous works and finally meet the various complex detection needs for the early diagnosis of COVID-19. The purpose of this review is to understand the present by reviewing the past.

Early identification and classification of COVID-19 patients is very important in order to use treatment strategies in a timely manner. 9 According to research, several biomarkers have been identified to be increased in COVID-19 patients that are associated with respiratory infections and dysfunction of various organs. Prognostication of intense diseases such as COVID-19 can be possible by Prognostic biomarkers. 10 According to research, markers of the surface and sequence of the genome of the COVID-19 virus have been identified. This data is essential for identifying new biomarkers that can be used to diagnose and predict pandemics. 11

Saliva is a hypotonic fluid secreted by the salivary glands, including the parotid, submandibular, and sublingual glands. Since salivary glands have high permeability and molecular exchange and are also located in an environment rich in capillaries, blood, and acini, they can be an appropriate source for evaluating circulating biomarkers. 12 Human salivary glands secrete 600 ml of serum and mucinous saliva daily containing mucins, minerals, growth factors, cytokines, buffers, electrolytes, enzymes and enzyme inhibitors, immunoglobulins, and glycoproteins. 13 Saliva is currently being considered as a potential diagnostic tool and as an alternative to other biological fluids such as serum or urine for diagnosis. Saliva assessment is a non-invasive, self-collecting method for detecting and monitoring COVID-19. Several salivary biomarkers, including salivary metabolism, have the ability to better detect COVID-19 and possibly identify a disease with the ability to classify the severity of the disease and even identify asymptomatic carriers. 14 It is competitive with nasopharyngeal swabs in terms of sensitivity and properties.

Human saliva is exuded about 600-1000 ml from the salivary glands every day. Saliva, like a serum, contains growth factors, IgA, cytokines, hormones, antibodies, enzymes, and microbes and has the diagnostic ability. Therefore, saliva can be used as a fluid to assess the physiological function of the body. 15 Although it is difficult to evaluate some analytics in saliva due to their low concentration compared to blood, highly sensitive molecular methods and nanotechnology have largely solved this problem. Saliva has been used to diagnose several diseases, including malignancies, autoimmune and hereditary diseases. 16 Saliva could be evaluated in terms of proteomics, transcriptomics, metabolomics, microRNA, microbiome. 17 The viral infections are detectable by evaluating of presence   viral RNA, DNA, microRNA, antigens, or antibodies in saliva, and   some kind of viral infections may be detected up to 29 days after contamination. 18, 19 The potential use of saliva for the diagnosis of SARS-CoV-2 by expression of ACE2 as a SARS-CoV-2 major surface receptor 20 from the salivary gland has been scientifically proven. 21 In addition, recent studies by To et al. Have shown the presence of live SARS-CoV-2 in saliva. 22 The diagnostic use of saliva for several viral infections such as coronavirus has been shown >90% accordance between saliva and pharyngeal swabs. 23 Recently, salivary biomarkers have been considered to use in advanced technologies like electro-mechanical systems, RNAsequencing, fluorescent biosensors, photometric and electrochemical, and lab-on-chips. 24 Reduction or delayed generation of interferon (IFN) after coronavirus infection leads to high inflammatory reactions that lead to severe pulmonary disorders. 25 The inflammatory cytokines production is depends on infection severity. 26 Increased expression of pro-inflammatory chemokines and cytokines, such as chemokine ligand (CC motifs; CCL) -2, CCL-3, Regulated on Activation, Normal T Cell Expressed and Secreted (RANTES), interleukin (IL) -2 and IL-8 have been shown during MERS-CoV infection. 26 According to recent studies in COVID-19 patients with increasing severity of infection, the amount of gamma-induced protein, interferon gamma 10 kDa, IL-2, IL-6, IL-7, IL-10, granulocyte colony-stimulating factor, macrophage chemotactic protein 1, macrophage inflammation of protein-1A, and tumour necrosis factorα (TNFα) in serum were increased to inflammatory response induced of cytokine secretion. 5, 25 Also, inflammatory markers like chemokines and cytokines are present in saliva and this source is available for diagnosis and prognosis of oral and systemic diseases such as COVID-19 infection. 16 Biomarkers such as lactate hydrogenase, CRP, malic acid, platelet degranulation, guanosine monophosphate, and macrophage-related proteins could be detected in saliva as well as in plasma.

Metabolism or the study of small molecules in cells, tissues, or fluids that identify a phenotype. Metabolomics are used to identify biomarkers and describe metabolic pathways in a variety of clinical conditions, including viral pathogens, especially those that affect the respiratory system, such as SARS and influenza. 27, 28 There are studies that suggest the specific regulation of micro-RNA is related to various infections, including respiratory virus infection. 29 The previous study has been demonstrated the effect of upregulation of miR-574-5p and miR214 expression and downregulation of miR-223 and miR-98 expression on pro-inflammatory cytokines generation in coronavirus (SARS-CoV) infection. 30 Recently, the expression potential of microRNAs has been considered as salivary biomarkers because microRNAs in extracellular vesicles are protected from degradation. Therefore, micro-RNAs in the biological fluid can be used to assess the condition of cell infection. 18 SARS-CoV-2 infects the host respiratory tract cells via ACE2 receptors. 31 Also previous studies the expression of ACE2 receptor in epithelial cells of salivary glands, oral and the tongue in humans. 32 So it can be an available source for the detection of SARS-CoV-2 infection. 33 Furthermore, the salivary glands may have hidden COVID-19 infection that may be activated. 34 According to the information obtained, the study of salivary biomarkers is an opportunity to achieve a more complete molecular view of the clinical relationship and risk assessment of COVID-19, as well as the evaluation of new antiviral therapies.

Using of recovered COVID-19 patients serum has been approved as a safe and effective treatment in severe patients or to strengthen instant immunity of high-risk patients. 35, 36 Boostels et al. have shown that a new class of inflammatory dendritic cells (inf-cDC 2) as a virusspecific antibody in serum can increase patient immunity. 37 In clinical trials, collecting enough volunteers for testing is one of the most important limitations.

In an interesting study, patients who recovered from acute respiratory syndrome induced by SARS-CoV were evaluated by metabolic assessment after 12 years. Phospholipids, organic acids, amino acids, carnitine, and inositol in the serum of these patients were different from healthy persons. Therefore, metabolism will be considered to evaluate long-term results. 28 The existence of IgM and IgG blood antibodies in the serum Generally, the presence of IgG or IgM/IgG in the serum shows active immunity. 38 According to research, the diagnosis of IgM was more variable than IgG and the results of both should be analysed to more precisely evaluate. 39 

Evaluation of immune response in 450 COVID-19 patients has been reported that severe cases compared with mild cases had lower lymphocyte counts, higher leucocyte, and lower percentages of eosinophils, basophils, and monocytes. 40 To control viral infection, the role of cytotoxic lymphocytes, like natural killer (NK) cells and cytotoxic T lymphocytes (CTLs), are essential, and decreased cytotoxic lymphocytes potency is associated with exacerbation of the disease. 43 In some studies demonstrated that SARS-CoV-2 patients had a lower amount of T cells, NK and, B cells. 44, 45 According to the research decreased specific subtypes of T lymphocytes such as lymphocyte (<500/µL), B cell (<50/µL), CD3 + T cell (<200/µL), CD4 + T cell (<100/µL) and CD8 + T cell (<100/µL) were observed in patients who died of COVID-19 infection in hospital. 46 Analysis of eosinophil count in COVID-19 patients showed that although in most COVID-19 patients eosinopenia was seen at admission and returned to normal before discharge, in some cases eosinopenia was not reported, so eosinopenia could not be a potential predictor for COVID-19 progression. [47] [48] [49] Neutrophil/ lymphocyte count ratio (NLR) is used as an inflammatory marker to predict mortality in cardiovascular disease 50, 51 Also, NLR is known as a biomarker for severe diseases such as sepsis. 52 In studies in patients with severe COVID-19 infection have been shown that NLR amount was remarkably increased. 53 Platelet Platelet counts are used as available biomarkers to assess disease severity and mortality risk in intensive care units (ICUs). 54 In COVID-19 patients, platelet depletion has been reported to be significantly associated with disease severity and risk of death. 55, 56 Previous studies have shown that COVID-19 patients with higher platelets and platelet/lymphocyte ratio (PLR) stayed longer in the hospital. 57

C-reactive protein is a serum protein generated by the liver through the stimulation of various inflammatory mediators such as IL-6. This biomarker is used to assess various inflammatory conditions and prediction of disease severity. 58 

D-dimer is derived from fibrin lysis and its increase indicates activation of coagulation and fibrinolysis. 63 Because COVID-19 is associated with haemostatic disorders, high levels of D-dimer were observed among patients. 64 D-dimer levels were increased in approximately 90% of patients admitted to pneumonia. It was directly related to the mortality rate. 65 It can be an appropriate marker for predicting severity and mortality in COVID-19 patients. 62 

The most common clinical complications of COVID-19 are acute respiratory distress syndrome and lung disturbance. Also, the cardiovascular disorder is another complication of this viral disease.

Evidence suggests the prediction of COVID-19 severity and mortality by cardiac biomarkers. 10 According to the researches, in COVID-19 patients the number of cardiac markers such as alpha-hydroxybutyrate dehydrogenase (α-HBDH), Lactate dehydrogenase (LDH), creatine kinase (CK), aspartate aminotransferase (AST), N-terminal of the prohormone brain natriuretic peptide (NT-proBNP), creatinine kinase-muscle/brain activity (CK-MB), myoglobin (Mb) and cardiac troponin I (cTnI) were enhanced. 66 Increased in CTnI, NT-proBNP, CK-MB, and Mb biomarker indicate the heart injury ( Table 1 ) but an increase of LDH, CK, α-HBDH, and AST as cardiac enzymes, may not necessarily indicate cardiac damage. 74 The most common clinical complications of COVID-19 are acute respiratory distress syndrome and lung disturbance. Also, the cardiovascular disorder is another complication of this viral disease.

Evidence suggests the prediction of COVID-19 severity and mortality by cardiac biomarkers. 10 According to the researches, in COVID-19 patients the number of cardiac markers such as alpha-hydroxybutyrate dehydrogenase (α-HBDH), Lactate dehydrogenase (LDH), creatine kinase (CK), F I G U R E 1 Schematic of serological antibody testing by a lateral flow assay for COVID-19. Reprinted from 11 aspartate aminotransferase (AST), N-terminal of the prohormone brain natriuretic peptide (NT-proBNP), creatinine kinase-muscle/ brain activity (CK-MB), myoglobin (Mb), and cardiac troponin I (cTnI) were enhanced. 66 Increased in CTnI, CK-MB, NT-proBNP and Mb biomarker indicate heart injury but an increase of LDH, CK, α-HBDH, and AST as cardiac enzymes, may not necessarily indicate cardiac damage. 74 The lungs and heart express the angiotensin-converting enzyme 2 (ACE2). 74 Studies have shown that ACE2 receptor expression is directly associated with SARS virus attack, 77 and SARS-CoV infection can cause ACE2-dependent cardiomyocyte infection. 78 In addition, some studies have confirmed that due to cardiac expression, SARS-CoV-2 viruses easily attack cardiomyocytes and destroy cardiomyocytes, thereby altering cardiac markers. 79 Troponin is a cardiac biomarker that can be used to predict and assess the severity of heart damage. According to the studies, COVID-19 patients who died had a higher amount of troponin than those who survive. 80 Heart injury is a complication of COVID-19 patients, the severity of infection is associated with increase B-type natriuretic peptide (BNP) levels as well as high-sensitivity cardiac troponin I (hs-TnI). So the early detection and severity prediction of COVID-19 can be carried out by measuring Cardiac biomarkers BNP and hs-TnI. 80 CK-MB CK-MB is a biomarker of heart damage and blood flow. CK-MB level is directly related to the severity of the injury. 72, 73 Increased CK-MB is associated with -Acute myocardial damage -ICU admission -In-hospital death 

There is also an indication that kidney injury is related to infection with COVID-19. 41 There are many biomarkers for kidney dysfunction diagnosis which can be divided into urinalysis and blood indicators related to kidney injury. 91 Biomarkers of renal impairment, including an increase in creatinine, blood urine nitrogen (BUN), and the presence of AKI have been reported in most studies. 77 Furthermore, independent of age and sex, a higher baseline creatinine, underlying proteinuria, and haematuria were associated with a higher risk of mortality. 62, 92 In patients with severe disease, creatinine and BUN levels were consistently higher in men compared to women and older males were more likely to have a higher baseline creatinine and develop AKI,17. 92 Although studies have not investigated the effect of sex on renal biomarkers and COVID-19 severity. A woman's hormonal environment, however, is thought to have a protective effect against the development of AKI and females have been previously shown to be at lower risk of AKI compared to males. 93 Similarly, smaller studies of renal transplant patients have suggested that male sex may be a risk factor for AKI in COVID-19. 94 Table 2 lists the types of biomarkers in the diagnosis of Covid-19 related to renal impairment.

Liver dysfunction caused by COVID-19 has also been identified in some cases, which may suggest a risk of liver damage caused by COVID-19. 95 96, 97 In intense COVID-19, hepatic dysfunction is followed by relatively greater activation of coagulative and fibrinolytic pathways, depressed counts of platelets, climbing counts of neutrophils and neutrophils lymphocyte percentages, and elevated amounts of ferritin. 66 Therefore, it is necessary to pay attention to the level of liver tests in Covid-19 patients. Aminotransferase aspartate (AST) and aminotransferase alanine (ALT) are enzymes present in heart cells, muscle tissue, red blood cells, and other tissues, such as the pancreas and kidneys, are found primarily in the liver. AST and ALT were previously referred to as serum glutamic oxaloacetic transaminase (GOT) and serum glutamic pyruvic transaminase (GPT), respectively. 98 In COVID-19 patients, the prevalence of elevated ALT and AST levels ranged from 14% to 53%. 99 However, liver dysfunction tests have concentrated mostly on improvements in the levels of ALT, AST, and total bilirubin (TB). The role of prealbumin has been underestimated as an important biomarker for assessing the liver's protein synthesis function. 100 Nanotechnology-based biosensors are known for their promising results in addition to their advantage of being highly customisable through immobilisation, labelling, and biofunctionalisation. In order to find an efficient biomolecule immobilisation, a surface plasmon resonance (SPR) biosensor was developed for SARS-CoV based on the use of gold binding polypeptide (GBP). 108 GBP was fused to enhanced green fluorescent protein (GBP-E) and to SARS-CoV membrane envelope (SCVme), the latter that can bind to anti-SCVme antibodies.

Blood test Figure 2 ). [112] [113] [114] [115] The development of electrochemical biosensors for COVID-19 detection is now in the early stage. Therefore, thorough review of an electrochemical biosensor for virus detection will help biosensing communities as soon as to develop an effective electrochemical biosensor platform for COVID-19.

The application of immunosensors in clinical diagnosis and monitoring of diseases has been reported for the detection of biomarkers, 114, 116 and viruses. 117 In electrochemical immunosensors, the biological signal is converted into an electrical signal when the antigen-antibody complex is formed. 84 Recently, an electrochemical immunosensor has been developed for the detection of highly pathogenic coronavirus associated with the MERS-CoV. 77 testing on SARS-CoV ( Figure 4 ). 120 However, the DNA-hybridisationbased disease diagnosis requires the extraction of target DNA/RNA from the infected host and the subsequent sample preparation.

In another attempt, label-free electrochemical detection of DNA hybridisation has been presented as a potential approach for COVID-19 diagnosis by using complementary thiolated probes ( Figure 5 ). 121 The methods of electrochemical analysis to be used for data acquisition and subsequent calibration, in relation to target analytic detection. DNA hybridisation can be considered as a portable electrochemical sensor for point mutation detection of COVID-19specific viral RNA/cDNA.

It is predicted that the whole virus SARS-CoV-2 have 28 proteins (Figure 6 ).

The FET system has detected SARS-CoV-2 based on the changes in channel surface potential and its effect on the electrical response. The gate surface of FET is covered with a layer that can be modified with biomolecules for selective detection of targets ( Figure 7) . 127 Graphene FET was decorated with an antibody of SARS-CoV-2 spike S1 subunit protein (CSAb) or angiotensinconverting enzyme 2 (ACE2) to detect SARS-CoV-2 spike protein S1. The binding of the S1 protein that possesses a slightly positive charge with the CSAb/ACE2 receptors on the graphene surface changed the conductance/resistance in graphene-FET which was LFB) and biotin/streptavidin interaction (biotin on the duplex and streptavidin on the polymerase nanoparticle; Figure 9 ). 136 these studies which also shows a potential to detect specifically COVID-19 as there is a difference between secretion range and cut off the range, and also haematological biomarkers which secreted in a more substantial amount in COVID-19 patient as compared to a healthy patient. 5, [139] [140] [141] COVID-19 has become a substantial lethal disease worldwide, and early diagnosis is a significant concern for this virus. Rapid and early diagnosis of any disease is always a major concern for all countries. Currently, the situation related to COVID-19 is enormous, as the globe does not have any rapid system for early and fast detection of this virus. 142 Currently, RT-PCR is being used for testing the virus which is time taking and costly, moreover, some of the research group has recently developed a biosensor for COVID-19

detection through different approach but they all are invasive and lead to virus particles exposers.

There are other techniques that can resolve this problem with a more manageable approach and detect the virus rapidly. One of these techniques is biomarker-based on sensors, terming biosensors. 142 The biomarker-based biosensor can play a pivotal role, as biomarkers are naturally occurring biomolecules specific to particular diseases, such as CYFRA-21 is a protein-based biomarker for oral cancer. Protein-based biomarkers are easy to isolate as compared to a nucleic acid (DNA/RNA) or cell-based biomarker.

Moreover, the biomarkers isolation and sample preparation are much more comfortable in protein-based biomarker as compared to a nucleic acid or other biomarkers. The current detection of COVID-19, which is RT-PCR required RNA isolation, purification, and processing step, which increases the time of detection and cost of testing. It can locate out such biomarkers through proteomics studies from COVID-19 infected patients and find out specific biomarkers for COVID-19.

Furthermore, biosensors based on this approach will be noninvasive that can be user-friendly in use so that the need for highly qualified professional limits can be overcome, apart from these other biomarkers which can also be considered in healthy patients and COVID-19 infected patients. 143 As well, the primary concern related to this virus is early diagnosis, cost-effectiveness and, reducing the chance of spread so that working professionals also do not get affected by human-to-human transmission while testing. Professional working for the diagnosis is in a major threat to get into the contact of this virus and get affected and to subdue this approach this biomarkers based biosensor can be integrated with microfluidics system which will restrict the sample amount as well as the chance of virus transmission, 142 such as Singh et al., has tried to develop a microfluidics-based biosensor for influenza detection. Considering the urgent need for rapid detection of COVID-19 the biomarkers based sensor can play a pivotal role as it will reduce the time to detect, will be cost-effective, and also reduce the chance of virus transmission while diagnosis, we can look forward to the integration of microfluidics system with this biosensor so that a minimal amount of sample is used and the chance of virus transmission remains insignificant. 142 In the other words, in recent years, the development of biosensors for biomarkers of diseases has received a lot of attention.

However, the developments of biomarkers and the innovation of diagnostic tools for early detection of COVOD-19 are still in their early stages. For future works, the development of another problem is that owing to its low accuracy and reliability, few portable electrochemical instruments are in clinical usage. Therefore, robust biosensor-based POCT devices are required of ultrasensitive electrochemical label-free methods will be of great potential. Re- 

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The authors would like to thank all staff who helped us to prepare and publish this article.

All authors declare that there is no conflict of interest.

All authors equally contributed to this work.

Data sharing is not applicable to this article as no new data were created or analyzed in this study.

https://orcid.org/0000-0003-3711-9171