key: cord-0060526-pi4ghmqb
authors: Yadav, Shalu; Sadique, Mohd. Abubakar; Ranjan, Pushpesh; Kumar, Neeraj; Singhal, Ayushi; Srivastava, Avanish K.; Khan, Raju
title: SERS Based Lateral Flow Immunoassay for Point-of-Care Detection of SARS-CoV-2 in Clinical Samples
date: 2021-03-17
journal: ACS Appl Bio Mater
DOI: 10.1021/acsabm.1c00102
sha: 61582547190943057a69d8d74cd01bc0acd1c5c5
doc_id: 60526
cord_uid: pi4ghmqb

[Image: see text] The current scenario, an ongoing pandemic of COVID-19, places a dreadful burden on the healthcare system worldwide. Subsequently, there is a need for a rapid, user-friendly, and inexpensive on-site monitoring system for diagnosis. The early and rapid diagnosis of SARS-CoV-2 plays an important role in combating the outbreak. Although conventional methods such as PCR, RT-PCR, and ELISA, etc., offer a gold-standard solution to manage the pandemic, they cannot be implemented as a point-of-care (POC) testing arrangement. Moreover, surface-enhanced Raman spectroscopy (SERS) having a high enhancement factor provides quantitative results with high specificity, sensitivity, and multiplex detection ability but lacks in POC setup. In contrast, POC devices such as lateral flow immunoassay (LFIA) offer rapid, simple-to-use, cost-effective, reliable platform. However, LFIA has limitations in quantitative and sensitive analyses of SARS-CoV-2 detection. To resolve these concerns, herein we discuss a unique modality that is an integration of SERS with LFIA for quantitative analyses of SARS-CoV-2. The miniaturization ability of SERS-based devices makes them promising in biosensor application and has the potential to make a better alternative of conventional diagnostic methods. This review also demonstrates the commercially available and FDA/ICMR approved LFIA kits for on-site diagnosis of SARS-CoV-2.

In past decades, three highly pathogenic human coronaviruses including severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2 have emerged and triggered global pandemics that resulted in high morbidity and mortality globally. 1,2 SARS-associated coronavirus, SARS-CoV, was the first outbreak in China, 2002 and spread swiftly to over 30 countries, resulting in more than 8000 cases of SARS-CoV. 3 The outbreak of zoonotic coronavirus in Saudi Arabia in 2012 known as MERS-CoV transferred to humans from dromedary camels. Over 27 countries have reported cases of MERS-CoV, leading to 858 total demises worldwide. 4 In December 2019, the first case of severe acute respiratory syndrome coronavirus (SARS-CoV-2) having pneumonia-like symptoms was identified in China. Since then, the COVID-19 epidemic has speedily spread throughout the world and has undergone serious health concerns and socio-economic burdens. 5 The WHO announced COVID-19 as a pandemic on Mar. 11, 2020. 6 Globally, as of Jan. 16, 2021, there have been over 92 million confirmed cases of COVID-19 out of which approximately 1.9 million demises have been reported and the numbers keep growing. 7 The vital cause of the rapid spread of SARS-CoV-2 is the highly infectious nature of SARS-CoV-2 and transmission of the virus through physical contact. The most common symptoms reported in COVID-19 patients are fever, cough, running nose, difficulty in breathing, muscular pain, and diarrhea. 8 The incubation period of SARS-CoV-2 before the onset of symptoms is 2−7 days. This stage is mainly asymptomatic and infectious because the infected person shows non-specific symptoms. It can easily be transmitted from an infected person to a non-infected person. 9 Approximately 45% of the reported cases are asymptomatic. The main challenge in preventing and suppressing the viral spread is to track and monitor the COVID-19 patients (symptomatic and asymptomatic) who are staying in close contact with healthy individuals. 10 The infected person with particularly SARS-CoV-2 virus and other upper tract respiratory infections suffer a high number of complexities that lead to morbidities mainly in people who have prior health problems. 11 The increasing numbers of cases are the major challenge for the healthcare sector, and they are also imposing a socio-economic burden globally.

The massive growing threat of SARS-CoV-2 could be related to the scarcity of effective point-of-care testing (POCT) assessments for speedy and accurate diagnosis of SARS-CoV-2 infection. There is an urgent need for a fast and accurate laboratory method for the diagnosis of SARS-CoV-2 in an early stage. Since the outbreak of COVID-19, there have been endless efforts to develop effective diagnostic techniques for sensitive and specific identification of SARS-CoV-2. 12 The major challenges in diagnostics are sample collections, false positive and negative results, required sophisticated instruments, well-settled laboratories, skilled personnel, and expensive and time-consuming procedures.

The outbreak of SARS-CoV-2 came as an opportunity for research and development in various healthcare, science, and technology sectors all around the globe. The aim is to come up with accurate, rapid, and reliable detection techniques, which would be distributed on a global scale to control the pandemic. Because one sector of research is dedicated toward the curing and vaccination part, the other research community is coming up with better and better ways to help detect the virus since it has symptomatic as well as asymptomatic transmission which makes it hard to detect. PCR and RT-PCR tests are commonly used as a diagnostic technique that involves the detection of viral genetic material. They are gold-standard tests, since the demand is high, the processing time would delay the tests in a larger population of the developing nations. Another diagnostic technique that shows better results is ELISA test kits, but the time consumption and high cost of the test is a major drawback for the majority of the economically disadvantaged population.

Some advances have been made for detection through stripbased tests which will be cheap and effective to a large number of people. One of the most popularly used techniques is lateral flow immunoassay (LFIA), which can be used either alone or in a combination with other techniques to get better and more reliable results. One of advanced characterization techniques is surface-enhanced Raman spectroscopy (SERS), which is an advanced form of Raman spectroscopy. SERS makes use of unique properties of nanostructures to give enhanced and optimized results for extremely low levels of detection.

The use of SERS in combination with LFIA could become the right combination for reliable and accurate testing, with the merits of a quick and large number of tests. The representation of the fabrication and working principle of conventional LFIA is given in Figure 1A , and SERS-based LFIA is shown in Figure  1B , along with the SERS technique integrated with LFIA, which results in the enhancement of sensitivity and low detection limit of biomolecules. For instance, Lim et al. reported the label-free SERS-based biosensor of gold nanoparticles for the detection of the influenza virus. 13, 14 Similarly, Kukushkin SERS-based aptasensor. SERS sensor reaches a 10 6 to 10 9 times higher signal intensity, which results in the enhancement of the sensitivity of the sensor. This biosensor has an LOD of 10 4 virus particles per clinical sample. 15 In another work, Xiao et al. reported the SERS integrated LFIA strip using gold and silver nanomaterials for the detection of avian influenza virus (H7N9) within 20 min. The LFIA strips provide the qualitative information, but after its integration with the SERS techniques, it delivers the quantitative information of the analyte in clinical samples which can be portable as well. 16 This review will be focusing on the SERS-based lateral flow immunoassay for the detection of antigen and/or antibodies biomarkers against SARS-CoV-2 in clinical specimens such as whole blood, plasma, serum, and saliva. Along with this, the overview on coronavirus and its associated biomarkers will also be discussed in brief. Further, the advantages, challenges, and future opportunities for applications of SERS-based lateral flow immunoassay in viral pathogen detection will be described.

Coronaviruses belong to the large family of viruses; generally, four subdivisions are included in this coronavirinae subclass, that is α-coronavirus, β-coronavirus, γ-coronavirus, and δcoronavirus, out of which SARS-CoV-2 comes under the genus β-coronavirus of subclass coronavirinae in the family Coronaviridae of the Nidovirales order. 17 Until now, seven coronaviruses species have been reported which can be transferable through human contact, and out of these, HCoV-229E, HCoV-NL63, HCoV-HKU1, and HCoV-OC43 cause less harm. However, the other three β-coronaviruses, SARS-CoV, MERS-CoV, and SARS-CoV-2, cause serious harm and show deadly pathogenicity. 18, 19 Most researchers found that the genome sequence of the SARS-CoV-2 virus is approximately alike that of the previously emerged SARS-CoV-like genome and looks diverse from SARS-CoV and MERS-CoV viruses. 19 Usually, the rate of transmission of the virus is evaluated on the basis of the reproduction number (R0) variable. R0 is considered a crucial threshold parameter for measuring viral transferability. 20 The range of basic reproductive number (R0) between 2.2 and 5.7 is proposed for SARS-CoV-2 virus transmission. This parameter is a bit higher than the estimated value for the SARS-CoV virus; i.e., R0 ranges between 2.0 and 5.0, indicating that the transmissibility of SARS-CoV-2 is faster than the SARS-CoV virus. 20 SARS-CoV-2 virus has a single-stranded positive sense RNA genome having the length of approximately 30,000 nucleotides and is a relatively large virus of virion diameter 70−140 nm with 9−12 nm identifiable spikes. 19 ,21 SARS-CoV-2 genome has four structural proteins, i.e., spike (S), envelope (E), membrane (M), and nucleocapsid (N). 22 In addition, the SARS-CoV-2 genome has five main open reading frames (ORFs), organized in the way of the 5′ untranslated (UTR)replicase complex. 23 Tang et al. reported that 149 mutation sites are present in SARS-CoV-2. These are further evolved in two types such as L-and S-types which are responsible for 70% and 30% of the viral population, respectively, and make them highly contagious. 24 25, 26 Currently, in the U.K., over 60% of COVID-19 cases are reported due to infection of a new variant. 27 Commercial molecular assays for SARS-CoV-2 target the ORF region, as well as ES and/or N protein. Therefore, the accuracy of such assays would be theoretically affected by the new mutations in the SARS-CoV-2. 28 There is a dropout in Sgene-based assay that laboratories need to monitor and implement other genomic targets (e.g., E or RdRP genes), based on assays not previously incorporated in current diagnosis methods. 29 The SARS-CoV-2 binds to receptors on the cell surface via a receptor-binding domain (RBD) present in their S1 subunit and enters cells by binding spike proteins to ACE2 receptor. 30 The M-protein has been found in two adaptations that are compact and elongated and hence support in assembling the virus; the E-protein interacts with the protein of host cells and performs the channelization of viroporin. 31 The different proteins of SARS-CoV-2 and their binding mechanism are shown in Figure 2 .

Biomarkers are defined as the measurable biological indicators of the presence, severity, or type of disease. Biomarkers play a vital role in predicting the present, historical, and future status of infections and help in early diagnosis. 32 The prognosis of SARS-CoV-2 can be made by detection of certain biomarkers related to viral infection of SARS-CoV-2. The direct sensing of associated markers can help in the early detection of SARS-CoV-2.

The various inflammatory biomarkers associated with the SARS-CoV-2 infection are C-reactive proteins (CRP), interleukin-6, procalcitonin, and ferritin. CRP is produced in the liver in response to inflammation. A higher level of CRP indicates the viral infection. In a study, the CRP level in COVID-19 patients was found 100-fold higher than normal and it can increase about 1000 times in several other cases. In another study, the IL-6 values were also found 3-fold higher than normal. Ferritin is a widely researched protein; its value was found as high as 1000 ng/mL which was many folds higher than the normal individuals. The detection of these inflammatory biomarkers can help in the diagnosis of the virus in those patients who have been diagnosed with false negative results by traditional diagnostic approaches. 33, 34 On the other hand, antibodies are produced in the body to safeguard against harmful foreign substances. The various serological antibodies are widely used as an efficient biomarker. Upon exposure to SARS-CoV-2 viral infection, first the immunoglobulin (IgM) is produced to defend against the viral infection and later on the immunoglobulin IgG is generated which acts as humoral immunity and keeps the memory of this encounter for the long term. The immunoglobulin IgM and IgG can be detected for SARS-CoV-2 within a week before the onset of symptoms. The presence of a number of immunoglobulins predicts the future complexity and seriousness of SARS-CoV-2 infection. 35

Several diagnostics methods have been developed for the diagnosis of viral infections depending upon the category of virus and their characteristics and also on the collected sample from infected patients. Currently, two diagnostic methods are available for the diagnosis of SARS-CoV-2 based upon the detection of the viral RNA and antibodies produced upon the disclosure of the viral infection. 36 At present, the polymerase chain reaction (PCR) is used for the detection and amplification of deoxyribonucleic acid (DNA), having high sensitivity and specificity. 37 The conceptual simplicity of this method has made a widely used detection technique in molecular biology, and it can detect as little as a single fragment of DNA. The coronavirus genome contains RNA instead of DNA. 38 RT-PCR technique is most commonly used for the detection of viral RNA genome due to its high sensitivity and specificity. In RT-PCR, it isolates the RNA transcripts from biological samples and polymerases into complementary DNA (cDNA). Although, this method directly detects the nucleic acid, i.e., viral RNA, as well it accelerates and multiplies the genomic material. It can even utilize a small amount of viral RNA present in collected biological specimens. 39 Nonetheless, in many cases, the sensitivity of RT-PCR has been poor because of some concerns in performing the assay, such as changeable and low viral loads depending upon the duration of infection, sample classification (nasal swab vs oral swab, upper respiratory tract vs lower respiratory tract), collection of the sample, conservation, and transportation of specimens, etc. 40 In addition, they require a highly pure sample; otherwise, it produces a false negative result. In the case of antigen testing, the majority of patients who tested positive for COVID-19 by RT-PCR later tested negative using the rapid serological assay alone; this emphasized the potential for misdiagnosis of COVID-19 using the assay in isolation. 41 Moreover, the greater time consumption, low availability of trained personnel, and high ratio of false negative and false positive results make them a limitation in diagnostic applications.

On the other hand, a technique similar to RT-PCR known as reverse transcription loop-mediated isothermal amplification (RT-LAMP) has also been utilized for the detection of SARS-CoV-2. 42 RT-LAMP assay is used for detection of specifically the viral RNA of SARS-CoV-2 instead of DNA. 43 It amplifies RNA rather than DNA. This assay is similar to the traditional PCR, but, exceptionally, amplification of nucleic acids is done at a particular temperature rather than the thermal cycler required in PCR as compulsory equipment. 39 For instance, Lamb et al. reported development of a quick screening LAMP test for detection of SARS-CoV-2 infection within 30 min with visualization of the color. 43 RT-LAMP has no cross-reactivity with similar infectious viruses such as SARS-CoV, MERS-CoV, HCoV, and influenza viruses (type B, H1N1pdm, H3N2, H5N1, H5N6, H5N8, and H7N9) as well. 44 In addition, this assay is easy to perform, is cost-effective, gives quick results, and can be performed in laboratories for initial detection of SARS-CoV-2. Over the advantages, they have some drawbacks such as false positive results, high risk of contamination during handling of assay, and requirement of maintaining constant temperature (60−65°C), which may limit their application. 45 Colorimetry is another popular diagnostic platform for the detection of the SARS-CoV-2 virus. Since it is easy to interpret due to the development of color on the strip which is observed through naked eye. In addition, it is easy-to-use, less-expensive, requires minimum technical expertise, and less instrumentation facilities. 46 Over the advantages of a colorimetric assay, the sample contamination, false positive, false negative results, and the lower intensity of color on a strip of the test zone limits the applicability in biosensor applications.

Other complementary or alternative techniques for screening of SARS-CoV-2 are the serological-based techniques including enzyme-linked immunosorbent assay (ELISA), immunofluorescent assay (IFA), and neutralization assays. 47 The serological tests are effectively used for the diagnosis of asymptomatic patients as well as for patients with negative RT-PCR results. 48 ELISA is a sensitive diagnostic method for detecting the presence of antibodies produced against the SARS-CoV-2 antigen. It is specifically designed for the identification of IgM and IgG antibodies. The principle of ELISA is based on the interaction of antibody−antigen binding mechanism followed by enzyme reaction which produces the color. On the basis of these binding interactions, there are several formats such as direct, indirect, and sandwich, etc., which are performed by this assay. The advantageous role of this assay is that there is no requirement for sophisticated facilities and results are seen with naked eyes. Moreover, the major drawback of ELISA includes the lengthy processes, expensive kits, and complicated washing procedures. 49−51 The clustered regularly-interspaced short palindromic repeats (CRISPR) is a powerful technology that has been used to engineer (cut, delete, and repeat) the genome. It has been modified from the prokaryotic adaptive immune system and is known to be a CRISPR (-Cas) system. Currently, it has provided a faster and cheaper diagnostic platform for virus detection in an ongoing pandemic situation. The CRISPR-Cas system focuses upon degrading the viral genome into the cell and its resulting viral mRNAs. Hence, CRISPR is used to identify the SARS-CoV-2 molecular properties and its therapeutic choice. 52 These systems have Cas proteins such as Cas12, Cas13, and so on, known for their collateral, nonspecific activities in succeeding recognition and cleavage of a specific target. In addition to this, collateral activities are responsible for nuclear detection methods with high specificity and sensitivity. 53 To provide the rapid and accurate result for COVID-19, different laboratories explored the capability of CRISPR-associated techniques for the detection of SARS-CoV-2. 54 Some examples of CRISPR-associated approaches such as SHERLOCK (specific, high-sensitivity enzymatic reporter unlocking) and DETECTR (DNA endonucleasetargeted CRISPR trans reporter) methods have gained much popularity for detection of SARS-CoV-2 viral genome in the recent scenario. 53 Despite many advantages such as specificity and flexibility, these existing CRISPR-based diagnostic platforms have some limitations such as the prerequisite of upfront extraction of nucleic acid, a voluminous amount of sample, and several physical steps involved in performing the test. 55 One of the rarely commercialized techniques, mass spectroscopy (MS) is one of the promising techniques for the detection of viral infections. Recently, Nikolaev et al. reported the detection of the N-protein of SARS-CoV-2 in nasopharynx epithelial swabs through the mass spectroscopy technique. They deactivated the viral protein through heating and further added isopropanol for tryptic digestion. They have done the mass analysis which results in the fragmentation peak of viral protein, which confirms the presence of N-protein. 56 Similarly, Ihling et al. and Cardozo et al. reported the mass spectroscopybased analysis of SARS-CoV-2 proteins in clinical samples. 57, 58 MS analysis allows multiplex detection and ease of processing. But the complexity of data interpretation, a requirement of an expert technician, a well-equipped laboratory, costly setup, and the time consumption of about 3−4 h limit their diagnostics application. To eradicate the technological drawbacks such as labor-intensiveness, time-consumption, and complexity in sample preparation, etc., of conventional methods such as RT-PCR, RT-LAMP, and ELISA, other substitute modalities can be developed. 59 After the emerging of a new variant of SARS-CoV-2, several challenges are faced as they affected the accuracy of diagnostic tests and significantly reduced the sensitivity, which leads to the false negative result in several molecular tests. 60 In this scenario, affordable, sensitive, specific, user-friendly, rapid, and robust, equipment-free, and deliverable platforms are required. Hence, POCT is relatively easy to perform and is a small, self-sufficient test for the detection of SARS-CoV-2 viral infection. Apart from these, they do not require trained personnel and other healthcare facilities and can be installed for on-site triage. Moreover, they do not require tedious sample preparation and the clock workload for performing the test. 61 Mujawar et al. in 2020 discussed COVID-19 management with miniaturized nanoenabled sensors embedded with artificial intelligence (AI) and Internet of Things (IoT) for POC diagnostics. They reported the potential advantages of AI that helps in fighting against pandemics at the global level. 62 Therefore, POCT is the need of the hour platform to curb the swiftly growing pandemic across the globe. Consequently, the POC diagnostics device can fulfill a requirement for the detection of SARS-CoV-2 as well as in the surveillance of asymptomatic individuals. Since there is an urgent need for mass testing at a high ratio which could efficiently help to prevent the virus from spreading in a large chunk of the population from infected people. Recent POCT platforms such as LFIA and paper-based devices have the advantages to provide simpler, rapid, and economical platforms for the diagnosis of SARS-CoV-2. But they struggle with their challenges such as insufficient sensitivity, overall reliability, and selectivity, etc. 63, 64 Besides, biosensing technique is the emerging advanced sensing technique to propose the development of an effective diagnostic tool in a miniaturized shape, which is economical and easy to operate without high-level skills. The working of the biosensor propagates in such a manner that target analytes bind to biorecognition elements; a transducer amplifies the signal and generates the quantitative or semiquantitative information which records on a device. The classification of biosensors, based upon the class of transducer used, are named as optical, electrochemical, and mass-based biosensors. 65 All perspectives focus on the affinity of three main targets, i.e., nucleic acids, antibodies, and proteins.

Electrochemical biosensors gained much attention among analytical techniques due to their several advantages. They are superior in sensing biomolecules, because they can detect biomarkers with high specificity, sensitivity, and reliable accuracy. In addition, the ease-of-fabrication, miniaturization ability, budget-friendliness, simplicity, and user-friendly nature are attracting research toward them. They also have been used as POC testing at home and clinics. 66 A concise report on AIbased electrochemical sensing of SARS-CoV-2 at POC by Kaushik et al. describes a nanoenabled biosensor that aids in managing the pandemic. Their work shows the use of bioinformatics, machine learning, data exchange, and use of information with AI at the site of the epidemic to diagnose the disease. 67 Moreover, the short shelf life, lower stability, and lower conductivity of nanomaterials fabricated on sensor surfaces make them a limitation in biosensor application. Over the past few years, plasmonic-based techniques have come out as an important contender in the growth of next level disease diagnosis platforms. It would significantly reduce the burden of contagious diseases in the developing sphere. They can overlap the gap between analytical chemistry and optics and provide inexpensive, real-time, sensible, and label-free detection of biomarkers of infectious diseases. 68 Recent work of Ahmadivand et al. in 2021 has developed a highly sensitive miniaturized THz toroidal plasmonic metasensor to detect SARS-CoV-2 spike proteins at femtomolar concentrations. Their proposed non-invasive, non-contact, and rapid modality research permits the analysis of SARS-CoV-2 spike protein at early stages with high accuracy. 69 Several biosensing modalities such as surface-enhanced raman spectroscopy (SERS), surface plasmon resonance (SPR), localized surface plasmon resonance (LSPR), and colorimetric plasmonic assays have come out for early detection of biomarkers of infectious diseases. These depend on the principle of plasmonic-based optical detection platforms, i.e., absorption of light. 70 Their main advantages are high specificity, sensitivity, and ease of handling. At present, the working principle, advantages, and limitations of several diagnostics' platforms for the detection of SARS-CoV-2 are shown in Figure 3 .

Earlier use of technology was done individually for only one application. Later advancements led to multi-application-based techniques for the same detection setup. Even that had some limitations, which were later overcome by the integration of two or more techniques. With the recent discoveries and developments, some detection tools have optimized working. There is a huge scope of introduction of new methods with improved testing and enhanced results.

One such example is Raman spectroscopy. It enables the identification of molecular vibrations giving characteristic specific signals of each molecule. However, it has some limitations of its own such as low-density scattering signals are very weak and they get capped with other noise signals, which Fleischmann et al. discovered in 1974 and was later individually confirmed by Van Duyne and Creighton in 1977. Pyridine Raman spectra on a small roughened silver electrode had an enhanced spectrum which was later termed as surface-enhanced Raman scattering. 71−73 Nanoparticle size and surface make SERS is a distance dependent phenomenon. In 2015, Kumari et al. investigated this distance dependence of SERS as a function of nanoparticle size. Detection of biomolecules in an aqueous medium requires careful control over nanoparticle size. This directly affects the enhancement of Raman signal and filtering useful information. 74 SERS is a powerful technique which gains much attention in the research community due to its several advantages. Among emerging novel detection techniques, SERS has come out as a robust analytical tool for analysis of single-molecule level sensitivity and capacity of quantitative detection. They can be used for diagnosis purposes, when Raman spectroscopy is incorporated with intrinsic optical and chemical characteristics of plasmonic nanoparticles. 75, 76 Classical plasmonic nanomaterials for SERS applications encompass colloidal nanoparticles, two-dimensional (2D) materials and arranged in a three-dimensional (3D) hierarchy. 77 The historical overview and wide applications of SERS in numerous fields are shown in Figure 4 .

In the SERS sensing technique, an amplified inelastic light scatter from the molecules after being absorbed on the surface of metals. 78 The frequency modulation of excitation light and localized surface plasmonic resonance (LSPR) of the metallic nanomaterial could enhance the scattering signal by a thousand times. In addition to Raman spectroscopy, SERS has a highly sensitive and more specific detection and quantification platform. 79 The potential of SERS significantly increased after the discovery of its detection of microorganisms. The future clinical applications paved the way in healthcare for the rapid detection of pathogens, especially viruses. 80−82 SERS require a specific substrate but the sample preparation is simple, this helps in the safe detection of fragile and sensitive specimen. This was evident from the trace amount detection of viruses in very low concentration which is usually found underwater or in air. 81 SERS plays an important role in basic sciences research. It contributes toward clinical practices for disease prognosis, designing theragnostic agents, drug delivery monitoring, even diagnosis of diseases with real-time therapeutic response, which is possible due to the high enhancement factor of SERS. 79 Metallic nanoparticles show enhanced scattering and can be used as SERS nanotags. It makes them a good candidate as labels, in which the signal of Raman dyes (RDs) with large Raman scattering cross-sections are enhanced by plasmonic nanoparticles of gold or silver. 82, 83 "Hot spots" are the active sites on the substrate, where incident light gets greatly enhanced when exposed to a laser light due to the LSPR effect of SERS nanotags. The amplification of incident light intensity is observed when the target molecules are placed near tiny nanogaps due to electromagnetic effects. 84, 85 Even after considering several advantages of SERS, commercialization of SERS nanotags and surface is challenging due to the practicality of cost-effective fabrication, being highly sensitive, thermally and chemically stable, reusable, and reproducible SERS substrates. 86 Currently, SERS-based assay platforms have gained much awareness among medical sections because of their capacity of multiplex detection and comparative lower limit of detection than other techniques. 87 Since 2007, making use of SERS for LFIA has emerged as highly specific and sensitive for viral pathogen detection methods. Precisely SERS tags can encapsulate with distinct Raman molecules that are used for conjugated detection antibodies for multiple target analytes and lead to multiplex detection on LFIA. 88 The current scenario of healthcare technology significantly influenced the diagnostic methods such as LFIA to get integrated with SERS to compensate for the drawbacks of conventional colloidal gold based LFIA. 89, 90 The diverse applications of SERS-based LFIA include the detection of various target analytes, including proteins, nucleic acids, and infectious pathogens. Different elements, such as antibodies, aptamers, and DNA oligonucleotides, are also used as recognition receptors. The measurement principle in a SERS-based LFIA strip is the same as that in a conventional colorimetric LFIA strip, except for the use of Raman reporterlabeled SERS nanotags 91−104

The LFIA is an attractive detection tool that qualifies all required merits for colorimetric assays. A commonly used lateral flow device consists of four constituents that are sample pad, conjugate pad, detection membrane, and absorbent pad. It is fabricated in such a way that the continuous flow of samples through channels is done, where the flow is based on the mechanism of the capillary action. 105 The sample pad is made up of glass and/or cellulose, and the detection sample is applied to it. It is responsible for the transportation of samples to other components of the assay a smoothly and continuously way. A conjugate pad is placed where labeled biorecognition molecules are distributed. When the running sample is in contact with the material of the conjugate pad, it releases conjugate labels which are generally colloidal nanoparticles commonly AuNPs and AgNPs in the case of a traditional LFIA setup. These labeled conjugates affect the sensitivity of assays. The detection membrane is highly important in deciding the sensitivity of LFIA assay. Test lines and control lines are drawn on this membrane, and it supplies the support to better binding to capture probes such as antibodies, antigens, and so on. The adsorbent pad is connected to the detection membrane at the end of the assay, which works like the sink, also maintains the flow rate of the sample, and prevents the backward flow of sample. Moreover, the adsorption capacity of the holding sample plays a crucial role in the results of assays. 106 Traditional LFIA provides qualitative or semiquantitative results on the basis of color visualization that can be observed by the naked eye. When color is visualized on both test line and control line, they indicate a positive result. However, if color is seen only on the control line, this indicates the negative result for the target analyte present in the sample. 107 In addition, LFIA does not require well-equipped laboratories and trained personnel for performing the test and provides a faster and reliable result.

Furthermore, LFIA tests are capable of denoting the existence of pathogens in various clinical samples, such as whole blood, plasma, serum, and saliva, etc., and are used as an ideal diagnostic assay for POC tests in limited resource surroundings and emergency divisions. In the need of antibody testing of SARS-CoV-2, LFIA raised as a rapid, accurate POCT method that is capable of detecting specific antibodies such as IgM, IgG, and IgA in whole blood, serum, and plasma and also provide distinguished results with specific antibodies detection. 108 For instance, Li et al. developed a LFIA prepared by using gold nanoparticles which are used for simultaneous detection of both antibodies IgM and IgG in the clinical sample within a few minutes. 109 Undoubtedly, LFIA has a broader set of applications in both clinical and non-clinical setups. But, the limited sensitivity upon the higher concentration of analyte and poor reproducibility are the major drawbacks. Additionally, LFIA gives a rapid result, i.e., 10−30 min, and is simpler to use but it provides only qualitative facts and not so always with sufficient accuracy. Therefore, fast and delicate diagnostic methods are required to enlarge the capability of SARS-CoV-2 virus testing which are capable of high-throughput diagnosis and do not need high technical expertise or advanced tools. 110 To overcome the aforementioned issues, LFIA has been integrated with other techniques such as SERS, CRISPR, RT-PCR, RT-LAMP, and ELISA, etc., which are capable of resolve these problems. The potential of integration of LFIA with the other techniques is shown in Figure 5 .

Since the first reported case of COVID-19 last year, many publications and patents have been made in healthcare to develop the diagnostic kits for the detection of SARS-CoV-2. Some of the LFIA having significant advancement as biosensors for SARS-CoV-2 diagnosis is discussed in this section, in which some are either under R&D or commercialized. 111 For instance, Cavalera et al. used a multitarget LFIA and gold nanoparticles conjugate which gave better response and detection. The overall sensitivity for all antibodies combined was 94.6%. The response time after the sample was dropped on the LFIA strip was about 20 min. They used two test lines for the "total antibodies" detection of the SARS-CoV-2 antibodies. 112 In the publication of Journal of Clinical Virology, Nicol et al. did the comparative assessment of three commercially available immunoassays. They reported that the assays give better results for samples 14 days after symptoms onset. They did not observe any significant difference between the sensitivity of IgA ELISA and IgM LFIA which target two different proteins. Their study showed equivalent clinical performance for IgG of three immunoassays, and NG-Test is reliable and accurate for routine use in clinical laboratories. 113 In their research, Flower et al. did the evaluation of LFIA for seroprevalence survey which is widespread at the global scale. They took RT-PCR tested samples and did LFIA in the clinic and laboratory. Further, the results were compared with a sensitivity of 84.4% and specificity of 98.6% and moderate concordance. However, the sensitivity of all of the LFIA kits was comparatively less than either PCR or ELISA, but they all had sufficient specificity to be considered for survey studies. They summarized the results with emphasis on the need to evaluate and develop new more optimized tests for the population. 114 The specificity of LFIA kits was more after considering weak bands as negative, but it decreased the detection of antibodies (sensitivity) in RT-PCR positive cases. 117 Grant et al. developed a half-strip LFIA for the nucleocapsid antigen detection of SARS-CoV-2 and measured the response by two commercially available optical LFIA readers. The limits of detection of N-protein were 0.65 and 3.03 ng/mL for Genemedi and Genscript assays, respectively. The antibodies used for testing in this half-strip assay were originally developed for SARS-CoV. Moreover, the lack of specificity of assay due to cross-reactivity with SARS-CoV is not considered. The clinical sensitivity for SARS-CoV-2 in real samples has not been studied. It has been made for antigen detection as a possible diagnostic tool. Moreover, it opens new ways for further development on a clinical scale. 118 Lyu et al. used commercially available POC test kits for SARS-CoV-2 in pre-COVID-19 serums in Japan (400 healthy subjects in 2012−2015). LFIA and ELISA showed 1.5% and 1.75% for IgG positives, respectively. COVI040 LFIA kit was used for IgM or IgG detection, where weak signals were considered positive. RAI010R ELISA kit used the N-protein of SARS-CoV-2 as an antigen. The test was conducted for 40 pooled samples from 400 clinical samples. The sensitivity of the assay was 99.75%. The possibility of LFIA integration with other diagnostic methods could give better results. There is a need for careful interpretation of results and to adopt far better techniques. 119 Hoste et al. used two serological methods, NovaLisa ELISA kit, namely, "DR-ELISA", and 2019-nCoV LFA, namely, "DR-LFA", for detection of antibodies of SARS-CoV-2. A total of 1056 human serum samples were tested for this study. The potential cross-reactivity of antibodies induced by infection 123 Another development of LFIA with nanomaterial was reported by Chen et al., where they used lanthanide-doped polystyrene nanoparticles (LNPs) to detect SARS-CoV-2 IgG in human serum. The LNPs were functionalized with protein molecules for specific target binding. The use of nanoparticles influenced significantly the response of fluorescence results. The clinical testing was done on 51 normal serum samples and 7 post RT-PCR samples. The assay was reproducible and gave the results in 10 min. Thus, this rapid and sensitive test is essential to control the outbreak and diagnose the bulk population for early detection and prevention. 124 One of the very first research studies was done for the detection of IgM antibodies against the SARS-CoV-2 virus by Huang et al.; they developed a lateral flow assay using colloidal gold nanoparticles (AuNP-LF) to enhance the binding and detection of the virus from human serum. They used COVID-19 patient samples and normal humans then cross-checked their results with RT-PCR. The response time for the test was 15 min, and a sensitivity of 100% was achieved. Even the cross-reactivity of the test was validated. Hence, the feasibility of the test is recommended for use in a large number of clinical testings. 125 Very recently, Wang et al. reported lateral flow immunosensor for the diagnosis of IgG and IgM against SARS-CoV-2 in a microliter of serum specimen within 15 min. Herein, they utilized the SiO 2 @AuNPs@QD nanobeads functionalized with S-protein to target IgG and IgM. The proposed biosensor displays dual-mode signals as colorimetric and fluorescence. Moreover, this biosensor has 100 times higher sensitivity than the gold nanoparticles-based immunoassay strip. They utilized the quantum dots in biosensor fabrication since they possess excellent optical properties due to their quantum confinement effect. In addition, it has long-term light stability and a wide excitation range. Further, they validate the biosensor through the examination of 57 patient samples (16 positive and 41 negative) and found satisfactory performance with almost 100% specificity. 126 Recently, Baker et al. developed a paper-based POC LFIA for the detection of the spike protein of SARS-CoV-2 within 30 min. Herein, they utilized the glycopolymer functionalized gold nanoparticles-based biosensor platform, where they studied that the functional chain of N-acetylneuraminic acid efficiently targets capture of the spike protein of SARS-CoV-2. They also tested that there is no such type of cross-reactivity over spike protein of SARS-CoV in a clinical sample. The LOD of this LFIA was 5.0 μg/mL. 127 Black et al. utilizing a commercial LFIA named "Biolidics 2019-nCoV IgG/IgM detection kit" for the diagnosis of IgG/IgM antibodies against SARS-CoV-2 in serum/plasma/whole blood sample within 10 min. They evaluate that the sensitivity of the test strip is 92.0% while specificity is 92.0% and 100% for IgM and IgG, respectively. They validate the test through the examination of 62 patients (both positive and negative) which were earlier confirmed by RT-PCR and also compared to gold-standard ELISA test. There was no significant alteration of the result of the Biolidics kit as compared to ELISA at day-0 and day-7. But the sensitivity of Biolidics at initial diagnosis of antibodies at day-0 was 29.0% and increases to 97.0% at day-7. So, the large variation of sensitivity at day-0 to day-7 may limit their application. 128 Similarly, McAulay et al. validated the four commercial LFIA test kits (BTNX kit 1, BTNX kit 2, ACON, and SD) from BTNX Inc., ACON Laboratories, and SD Biosensor Companies. They tested 352 patients of both positive and negative samples to clinically validate detection of the IgG antibodies against SARS-CoV-2 (anti-SARS-CoV-2 IgG) in plasma, serum, whole blood samples. Moreover, some of these test kits are also offered to detect IgM in the same strip. They dropped the 10.0−15.0 μL of the clinical sample followed by the 1−2 drops of buffer solution to dilute the sample which helps to flow the sample on the strip. After 10− 15 min the result is shown for IgG/IgM detection. They evaluated that the sensitivity of BTNX 1 and BTNX 2 was 16.0% and 13.0% in the first week and increases to 95.0% and 91.0% after the second week, respectively. Moreover, the sensitivity for ACON and SD was found to be 5.0% and 92.0%, respectively. In addition, they evaluated that the specificities of biosensors are 98.0% for BTNX 1, 98.0% for ACON, and 100% for BTNX 2 and SD test kits. Moreover, few falsepositive reports were also examined through the whole determination of IgG. 129 96 .1%, respectively. The company applied for the emergency approval for detection of antibodies (IgG/IgM) against SARS-CoV-2. But they failed to get approval due to poor diagnosis performance of the kit for IgM detection. 130 Liu antibodies to detect SARS-CoV-2 after three week onset of symptoms. 134 Wang et al. assembled a LFIA for the detection of SARS-CoV-2 IgM and IgG in serum and blood samples within 10 min. Herein, they prepared the selenium nanoparticles and then conjugated by nucleoprotein. They fabricated the conjugate pad of assay by N-protein functionalized selenium nanoparticles while the two test lines (M and G), and control line by antihuman IgM antibodies, antihuman IgG antibodies, and antihis antibodies, respectively. When a drop of clinical sample is dropped on the conjugate pad, it flows on the strip and color develops on M, G, and C lines, which indicate the presence of IgM and IgG in the sample. With simplicity, the result is easy to observe through the naked eye. The sensitivity of LFIA is 93.33% and specificity of 97.34%. The LODs of the test kit are 20.0 ng/mL and 5.0 ng/mL for IgM and IgG, respectively. In addition, the shelf life of the kit is 30 days when it is stored at 37°C temperature. Further, they validate the kit by examination of 90 positive and 263 negative samples and result in the satisfactory performance. They also tested against rheumatoid factor, antinuclear antibodies, influenza A, and influenza B and found no cross-reactivity against such diseases (Figure 6 A,B) . 135 Zeng et al. reported an immunosensor chip for simultaneous detection of IgM and IgG in a single test kit in blood specimen and could detect antibodies within 15 min. Herein, they fabricated the LFIA kit by the separate immobilization of gold nanoparticles labeled S-protein and rabbit IgG antibodies on sample pad. However, the two-test line (M and G) and control lines were fabricated by their respective anti-antibodies. When the red line appears on M line or G line with C line, they indicate the presence of IgM or IgG in a clinical sample. Moreover, the red color on both M and G line with C line indicates both antibodies in the sample. They further tested the 80 (positive and negative) patients samples and found that the sensitivity of the kit is 85.29%. However, specificity is 100%. They also revealed that the sensitivity of immunoassay was low when it detected either IgM or IgG, but it dramatically increases when both antibodies are present. 136 Recently, Guedez-Lopez et al. validated the three lateral flow immunoassays (Sienna, Wondfo, and Prometheus) for the detection of IgM and IgG against SARS-CoV-2 in serum specimens. The overall sensitivities of the three LFIAs are 64.4%, 45.2%, and 75.5%, and specificities are 75.0%, 81.8%, and 12.5% for Sienna, Wondfo, and Prometheus, respectively. Moreover, the sensitivities of these immunoassays are enhanced to 100%, 83.3%, and 100%, respectively, after the third week of onset on disease. 137 Similarly, Gutierrez et al. validated the ten immunoassays (three LFIA rapid tests, four ELISA, and three chemiluminescence assays) for the detection of antibodies (IgM, IgG, and IgA) specific to SARS-CoV-2. The three LFIA kits are Wondfo, SGTi-Flex and Innovita; the four ELISA kits are COVID-19 ELISA IgG, COVID-19 ELISA IgM+IgA (Vircell, Spain, S.L.), EUROIMMUN ELISA Anti SARS-CoV-2 IgG, and Anti SARS-CoV-2 IgA (Medizinische Labordiagnostika AG). The first two ELISA kits utilized the S-protein and N-protein while the last two only the S-protein to specifically detect antibodies. Moreover, three chemiluminescence kits are VIRCLIA IgG Monotest, VIRCLIA IgM+A Monotest (Vircell, Spain, S.L.), and SARS-CoV-2 IgG Architect (Abbott). The first two chemiluminescence kits utilized the S-protein and N-protein while the third only the N-protein to specifically detect antibodies. 138 Fabian et al. validated two commercial test kits, anti-SARS-CoV-2 IgA EUROIMMUN and ENZY-WELL SARS-CoV-2 IgA, for the detection of IgA specific to SARS-CoV-2, instead of IgG/IgM in 65 clinical samples (39 positive and 26 negative) which are tested to PCR. 139 Very recently, Lee et al. reported the LFIA for the detection of S1 protein of SARS-CoV-2 in clinical sample within 20 min. ACE2 receptor potentially binds to the S1 protein of SARS-CoV-2 on a LFIA strip. They have high sensitivity up to 1.0 ng/reaction to the RBD and below 5.0 ng/reaction S1 protein of SARS-CoV-2. The LOD of the assay was 1.86 × 10 5 copies/ mL in sample. They further tested the cross-reactivity of LFIA and found that there are no such properties for S1 protein of SARS-CoV and MERS-CoV. Cellular receptor (ACE2)-based LFIA is shown in Figure 7A ,B. 140 Recently reported SERS and LFIA-based biosensor for detection of COVID-19 in clinical specimens are listed in Table 1 .

The LFIA in itself is a rapid and POC diagnostic tool that has been used for quick diagnosis. It has its own merits and demerits. To enhance the performance and overcome the demerits, several nanotechnology-based enhancements have been made for internal optimization. There are few other methods like the integration of LFIA kit with other diagnostic tools to improve performance; still, there are a few drawbacks for them. One of the recent advancements is the development and utilization of the SERS technique. The applications of SERS are numerous and very promising to be coupled with another basic technique to achieve qualitative as well as quantitative results. SERS-based LFIA makes use of properties of a nanomaterial as a SERS tag and advanced characterization technique being highly specific. Along with rapid detection assay which collectively gives an enhanced signal response, accuracy and high sensitivity in results. Further, SERS dramatically enhanced the intensity up to 10 6 times and even it could detect the single molecule. 141 One novel research is from the group of Liu et al. where they developed a SERSbased LFIA biosensor. They used dual-layer Raman moleculeloaded Ag-coated SiO 2 NPs as advanced SERS tags. The SERS tag was then conjugated with the spike protein of the SARS-CoV-2 virus for the simultaneous detection of IgG/IgM antibodies. SERS measurements were performed on 19 COVID-19 positive serum and 49 COVID-19 negative serum samples by using 785 nm excitation with a power of 10.0 mW for 1.0 s. The results were out in 25 min (optimized time), and LOD was found to be 1.0 ng/mL of S-protein antibody in the case of 240 nm SiO 2 @Ag tag that gave the best results. Further for the clinical samples, the LOD was 1.0 pg/ mL from the SERS results, which is 1000 times more than the visualization results. Considering such enhanced results, the SERS-LFIA technique is proposed for rapid screening and bulk diagnosis at ultralow detection levels when other commonly used methods are not able to detect the virus. The schematic of the preparation of the dual-layers DTNB-modified SiO 2 @ Ag NPs based LIFA is shown in Figure 8A −C. 131 

Since the outbreak of the COVID-19 pandemic, there has been a tremendous amount of research and development for the testing of the clinical samples either in laboratory or POC test kits. Initial testing had several issues such as false negative or false positive results due to inaccurate detection techniques and incomplete knowledge about the virus. This leads to the spread of the virus on a global scale that leads to the high demand for the testing required in developing as well as developed nations. In the foresight of the above, many of the commercial kits were given emergency approval. However, the licenses of few distributors have been canceled. The approving agencies are responsible for validating the test kits which have clinically passed the sample testing and the chances of false results are null. Some of the commercially available approved kits are listed in Table 2 . 142−146

The diagnostic techniques generally implied in the laboratory go toward the commercial market after considering their advantages with respect to other similar techniques available. About the SERS technique, it has overcome many drawbacks of previous methods and presented an advancement in technology, and as an output, a significantly optimized and improved result is achieved. First, the applicability of such integration of techniques which can make the current diagnosis error free and more sensitive and quicker is worth mentioning among the advantages. In this review, we have discussed the integration of SERS with LFIA, which makes the POC diagnosis and at the same time gives accurate results even at multiplexed detection. To mention some of the main advantages of SERS, it has increased sensitivity which means However, at the same time, there are a few challenges when making use of the same technique in various applications. One such example is when the analysis of a protein is to be made; the surface treatment of the substrate to make it SERS active can cause conformation changes in the protein. This can be overcome by another merit of SERS being highly selective. It can still observe the reaction change with respect to the environment change, giving unique spectra for each of them. 148 With regard to the mentioned SERS-based LFIA technique, especially in the case of SARS-CoV-2, the specificity of the analyte after careful sample collection is of utmost importance since the technique is being used in healthcare. This highlights the need for the instrumental part to have the biocompatible components optimized for clinical usage. The use of rough surfaces at the nanoscale or metallic surfaces as SERS active surfaces is an advantage for the detection of other molecules. This gives rise to one of the inherent drawbacks of this technique; that is, it cannot be used for metals or alloys, since the sensitivity is very low and fluorescence of some materials can dim and cover the response of others. Moreover, nonspecific binding is one of the limitations which gives false results. This could be easily extended in terms of clinical samples and diagnosis of analytes at extremely lower concentrations. It makes it harder for each analyte to be detected specifically. 149 Significantly, this can be extended to the healthcare applications of SERS in the disease's regime for the pathogen, bacterial, and viruses' diagnostics. Moreover, this can be termed as a significant disadvantage of SERS in terms of the recent use of this technique in the medical field. Since the equipment needs to be modified with the compatibility of biological samples, currently physicists are more inclined toward use and operation.

Clinical communities need to adopt this technology for use in wide-ranging studies. The principle is the same independently and can govern the testing when coupled with other standard techniques like PCR instruments in clinical areas. 150 One of the main areas which need the attention of researchers and is responsible for signal response and enhancement is the development of substrate properties. There is a need to develop proper fabrication methods for the special purpose of SERS tag synthesis. Properties of well-dispersed nanoparticles show a weak but homogeneous enhancement which needs to experimentally be controlled. Optimized conditions would give spectral reproducibility and even enhance the response effects of nanoparticle aggregates which are already highly ordered with surface uniformity. 151 Few other technical limitations of the SERS technique include the contact between the enhancing surface and the analyte. This causes the substrates to degrade with time, resulting in a decrease in signal and hence making the substrate poorly reusable, and one main issue is having a reproducible SERS signal within a substrate. 152 Concerning the extremely small cross-sectional area of the samples, this leads to a longer acquisition period of analysis and a high concentration of the sample may lead to noise or irregular results. 153,154

After analyzing the advantages and disadvantages of the SERS technique, one must have some future direction for the required modification and scope of the method to be made useful when integrated with other techniques. One of the highly focused clinical applications of LFIA with regard to its usefulness as a detection kit for SARS-CoV-2 and ease the diagnosis efficiently. The prospects of SERS-based LFIA or individually any technique lies in the possible use of the method. Furthermore, it strongly needs improvements in its integral setup or methodology for broader applications. One of the most required developments is making the laboratory scale SERS technique into portable SERS. This would be an opportunity to use it as POC device and as a rapid diagnostic tool for clinical application. Normally the SERS signals for most metals are found near the visible range or near-infrared wavelength. It gives a nudge to explore the ultraviolet frequency range and can be desirable for biological molecules and lead in applications such as disease diagnosis, food quality control, forensics, and others. Another area where potential improvement can be made is the testing time a SERS-based test would take, since currently the scan is time-consuming and requires molecule-by-molecule scanning for higher sensitivity and specificity. The efficient fast spectroscopies are the need of the hour for integrated techniques. One such example is the femtosecond stimulated Raman spectroscopy. The least explored area in SERS-based AFM extended development is TERS, which will make use of sub-diffraction-limited imaging for a wide variety of numerous applications. 

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Complete contact information is available at: https://pubs.acs.org/10.1021/acsabm.1c00102

The authors declare no competing financial interest.

We thank the Director of CSIR-AMPRI Bhopal, India for his interest in this work. S.Y. thanks CSIR, India for the award of a JRF. M.A.S. thanks DST-SERB for a JRF. P.R. thanks CSIR, India for an SRF. N.K. thanks UGC, India for an SRF. R.K. acknowledges SERB for providing funds in the form of the IPA/2020/000130 project.