Nanotechnology in diagnostic techniques for SARS- CoV-2

Summary

The advances of nanotechnology are of significant importance in the diagnosis of COVID-19. Protection and diagnosis are essential for controlling the spread of infection. Nanotechnology offers novel techniques for rapid diagnosis, early-stage infection detection, and identification of COVID-19. Due to their smaller size and larger surface area, nanotechnology products can detect the disease with high precision. As the symptoms of COVID-19 are very comparable to those of other respiratory diseases, it is essential to have precise, sensitive and fast diagnostic tools to detect the infection at an early stage.

Key words/phrases: nanotechnology, COVID-19, diagnosis

1. Introduction

The coronavirus disease 2019 (COVID-19) induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a global health issue that the WHO announced a pandemic. COVID- 19 has resulted in a worldwide lockdown and endangered the global economy. SARS-CoV-2 has spread fast worldwide, generating a global pandemic outnumbering. This virus can be transferred human-to-human via droplets and tight contact, and people of all ages are exposed to this virus. The COVID-19 outbreak put international pressure on modern societies, especially the infrastructure linked to health care. Therefore, diagnostic tests special to this disease are urgently required to ensure positive cases, screen patients and execute viral surveillance. Diagnostics may play an influential role in preventing COVID-19, permitting quick execution of management actions that limit the spread by detecting and isolating cases and via contact tracing. Consequently, the world is encountering a new challenge: to create ultra-rapid, ultra-sensitive devices, and nanoscale analytical tools, or sensing systems (e.g., nanobiosensors) that are highly efficacious at detecting the 2019 novel coronavirus (COVID-19) or severe acute respiratory syndrome (SARS) [3, 10, 17].

With the advancements in nanotechnology, their exceptional properties, including their capability to strengthen signal, can be employed for the development of nanobiosensors and nanoimaging processes that can be utilised for early-stage detection along with other diagnostic tools. nanotechnology is being thoroughly examined for its prospect in the development of , diagnostic techniques, therapeutics, vaccines and strategies to ease the healthcare burden [10].

2. Current laboratory methods for diagnosis of SARS-CoV-2

The diagnosis of COVID-19 relies on the analysis of the patient’s reaction due to the disease or the study of virus contents, e.g., RNA or their protein. The patient’s temperature (boosted temperature), feeling exhausted, and difficulty breathing suggest infection. Nevertheless, these symptoms are lack particularity and may be observed due to the infection with other pathogens. The patient’s pathological modifications in organs such as the chest can be observed via computerised tomography (CT) scan. A CT scan may be a reliable test for screening SARS-COV 2 cases like other pneumonia types. However, the analysis demanded specialised equipment and failed to meet a considerable scale of requirements. COVID-19 can be diagnosed via laboratory measurements. These methods are usually utilised for the study of patients. They cannot be used to analyse contaminated samples such as surface and air [1].

Figure 1. Diagnosis methods for COVID-19.

Several methods have been developed for the diagnosis of COVID-19. The main tests for diagnosis can be classified in three main categories [1]:

1. Genetic tests (viral nucleic acid tests):analysis of viral genome employing methods such as real-time- quantitative reverse transcription-polymerase chain reaction (RT-qPCR), isothermal amplification (e.g., Loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), rolling circle amplification (RCA), Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)), and nanopore targeted sequencing (NTS).
2. Antigen tests:analysis of the viral proteins (mem- brane-bound spike proteins or the nucleocapsid proteins) using techniques such as colorimetric, field-effect transistor (FET), enzyme-linked immuno- sorbent assay (ELISA), and mass spectrometry (MS)
3. Serological tests: analysis of the antibodies (Immu- noglobulin M (IgM) and Immunoglobulin G (IgG)) against the virus [18, 19]. The study of patient’s antibodies can be achieved using methods such as electrical (EC) biosensors, localized surface plasmon resonance (LSPR), surface-enhanced Raman scat- tering (SERS), quartz crystal microbalance (QCM), fluorescence-based biosensor, colorimetric biosensor, gold immunochromatography, ELISA, chemilumi- nescence immunoassay, and piezoelectric microcan- tilever sensors (PEMS).

Unfortunately, many traditional detection methods of respiratory viruses, such as RT-PCN have many disadvantages. These include time-consuming, costly, are not always determinable or reproducible, and demand qualified staff and other technical facilities [3].

Figure 2. Disadvantages of traditional methods.

3. Nanotechnology

Nanotechnology offers new techniques for rapid diagnosis, early-stage infection detection, and identification of virulent pathogens causing the pandemic, particularly in improving the efficiency and quality of the detection process by employing nanobiosensors. Furthermore, new nanostructures and nanosensors display properties and performances unseen at the macroscopic level, significantly for detecting and sensing occasions at a nanoscale level [3].

Nanotechnology can enhance the diagnosis of COVID- 19 and suggest a state-of-the-art diagnostic method based on a Point Of Care (POC) sensing technology. Moreover, it can be interfaced with artificial intelligence (AI) techniques and the internet of medical things (IoMT)-integrated biosensors for studying practical informatics via data storage, sharing, and analytics. Also, they can bypass traditional processes such as low sensitivity, low selectivity, high cost, and extended diagnostic time. New methods can be utilised for no pain sample analysis, such as analysing the patient’s saliva using graphene oxide (GO)/Au/Fiber Bragg grating (FBG) probe. Nanotechnology can advance technologies such as label-free biosensors, paper lateral flow assays, optical technologies, and digital technologies [1].

3.1. Nanobiosensors

The higher preponderance of viral outbreaks can be attributed to the improper detection tools utilised to detect the contagious agents. Consequently, this requires a detection or diagnostic tool that is vigorous, quick, demanding, and precise in its biosensing properties. The biosensors can be characterised as analytical instruments that can assess low concentrations of an analyte in biological samples (like the human serum, blood, tears, saliva, etc.). Compared to traditional qualitative and quantitative test kits, these biosensors are favourably precise and susceptible to the directed target [15].

Figure 3. Classification and applications of various biosensors.

The need for accuracy and rapidity in diagnosing COVID-19 is not fulfilled by the traditional methods of serology-based tests and reverse transcription-polymerase chain reaction (RT-PCR), which are routinely utilised to detect and diagnose COVID-19. That condition can be fulfilled by employing ultrasensitive nanobiosensors that play a significant role in detecting novel coronavirus. Nanobiosensors deliver a quick, cost-effective, precise, and miniaturised platform for the detection of SARS-CoV-2 [10]. Biosensors typically include a biological recognition molecule immobilised onto the surface of a signal transducer and can be utilised for analysis, diagnosis, protection, security, and testing of larger populations [3].

Nanobiosensors provide several advantages that cause detection effective, such as [3]:

• Cost effective;
• Long self-life;
• Easy to use;
• Autonomous;
• Precision;
• Portability;
• Quick response;
• High sensitivity;
• Multiplexing capabilities;
• Viable process.

Nanobiosensors are devices in which the transducer is altered to catch the target component, convert the biological answer into electrical signals, and fast detect it with high precision. The physical responses can be calculated by determining the appropriate bioreceptors, such as nucleic acids, antigens, DNA probe, peptide, whole-cell, micro-organism, and tissue. These receptors are easily recognizable, highly sensitive, and detect specific bioanalyte. Various types of bioreceptors have been investigated to catch the viruses, such as nucleic acids (NA), immunoaffinity and protein in multiple nanobiosensors based on electrochemical, impedance, quartz crystal microbalance, and optical and surface plasmon resonance. The target molecule binds to the bioreceptor to catch a biological molecule by an unusual reaction. Then, the transducer with integrated molecule by a particular response. Then, the transducer with integrated nanostructures transforms the detection into an electrical signal defined by the detector (Fig. 4) [3].

Figure 4. The schematic diagram of different analytes, bioreceptors for biorecognition elements, transducers with integrated nanostructures as parts of a typical nanobiosensor design for respiratory viruses.

Nanobiosensors utilised for the detection of SARS or MERS coronaviruses can be categorised based on the biological molecule of the viral target (nucleic acids, antigens, or antibodies) into nucleic acid-based into nucleic acid- biosensor, antigen-based biosensor, and antibody-based biosensor (Fig. 5) [3].

Figure 5. Schematic representation of different biosensors classifications for the detection of SARS and MERS coronaviruses

3.1.1. Electrochemical Nanobiosensors

Electrochemical biosensors are the most widely used and favourably favoured type of sensing venues. According to the International Union of Pure and Applied Chemistry (IUPAC) definition, electrochemical biosensor is “a self-contained integrated device which is capable of providing specific quantitative or semi-quantitative analytical information using a biological recognition element (biochemical receptor) which is retained in direct spatial contact woth an electrochemical transduction element.” [8, 13].

An electrochemical nanobiosensor is a molecular sensing appliance that pairs a biological recognition event with an electrode transducer to create a usable electrical signal. Because electrochemical nanosensors include electrodes, the semiconductors properties, dielectric properties, and charge distribution are critical elements [3].

Figure 6. Scheme showing biosensor design components employed for sensing of target analyte samples, with special emphasis to utilizing an electrochemical bio- sensing platform that transforms biochemical information into current or voltages signals on an electrochemical transducer surface.

The main benefits of electrochemical biosensors are [5, 11]:

• Easy development;
• Possibility of miniaturization;
• High sensitivity;
• Relatively low cost.

Electrochemical sensors are an appealing choice for detecting a variety of biomolecules because they can be smoothly combined with multiple modules such as a. low-cost microelectronic circuits b. miniaturized lab-on-a-chip, c. interfacing with electronic read-out, and d. a signal processing unit. Electrochemical biosensors being sensitive, easy to miniaturize, require little analyte volumes, the superior limit of analyte detection and display on-site results are most preferred in medical diagnostics and many other research areas, including food safety and environmental monitoring [8].

The electrochemical biosensor can be categorised based on the transducer modes utilised for signal measurements. These include conductometry and surface charge, amperometry and potentiometry transduction platforms. The general principle of electrochemical biosensing (bio-electrochemistry) is established on electrochemical response taking place on or at the proximity of electrode and or between the electrodes that lead to; (i) a measurable current signal (amperometric), (ii) cumulate charge or potential(potentiometric), or (iii) changes in the conductivity of the medium (conductometric). [8].

Figure 7. Classification of the electrochemical biosensors based on type of transducer and signal modes.

Electrochemical nanobiosensors can also be employed to identify viral nucleic acids. An electrochemical genosensor developed for detecting SARS was developed using a monolayer of thiolated oligonucleotides self-assembled on gold nanoparticles-coated carbon electrodes. The oligonucleotide sequences are precise to the nucleocapsid protein of SARS, and the viral infection is detected via enzymatic amplification of viral DNA. The nanobiosensor helps the susceptible detection of SARS. An electrochemical nanobiosensor manufactured utilising gold nanoparticles changed with a carbon electrode and recombinant spike protein S1 as a biomarker was designed to detect MERS-CoVs; nevertheless, this approach also keeps promise for detection coronaviruses. Because of its electrical conductivity, the biosensor was created using fluorine-doped substrate and gold nanoparticles as a signal amplifier. [10].

Modifying electrochemical sensing interfaces with gold nanoparticles (AuNPs) shows improved applications and can be used to detect MERS-CoV. AuNPs act as working interfaces possessing electrocatalytic properties and permit amplification of the electric reaction (Figure 8). An immunosensor was designed to detect the MERS-CoV virus connecting the prospect of electrochemical sensors and gold nanoparticles. The nanobiosensor is developed with a bunch of carbon electrode-coated gold nanoparticles.

Figure 8. Operation steps for the COVID-19 electrochemical sensing platform: (A) sample collection via the nasal swab or saliva, (B) RNA extraction, (C) immobilization of RNA extract on the top of the graphene- ssDNA-AuNP platform, (D) incubation of 5 min, and (E) record the digital electrochemical output.

It has been observed that the recombinant spike (S1) protein gets immobilized to gold nanoparticles and competes with the virus particles for binding to the antibody. When virus infection is absent, it attaches to the immobilized spike protein. Because this nanobiosensor method has a group of electrodes, it can be used to detect various coronaviruses [5, 10].

 Graphene interfaced electrochemical detection of SARS-CoV-2

Electrochemical transducing platforms can detect viruses or any living microbial pathogens using their specific biorecognition elements. There are several alternative ways for electrochemical detection of disease-causing mechanisms. However, detecting genetic markers employing electrochemical sensing platforms is not susceptible for viral detections due to their undetectable viral titers, particularly at the early onset of viral infections [8, 14].

In current years, several attempts have been made toward applying strategies similar to electrochemical glucometers to detect viruses or viral infections. Torrente-Rodríguez et all developed low-cost graphene integrated portable electrochemical biosensor for rapid diagnosis and biochemical monitoring markers in serum and saliva samples for COVID-19 [8, 14].

The electrochemical sensor electrodes were graphene inscribed on a flexible polyimide (PI) polymeric substrate for multiplexed detection of viral infection biomarkers (antigens and antibodies). Torrente-Rodríguez et all demonstrated quantitative detecting specific biomarkers of COVID-19, such as SARS-CoV-2 spike protein (S1), nucleocapsid protein of SARS, CRP, a protein biomarker for inflammation within physiologically relevant ranges in both blood and saliva and specific immunoglobulins (Igs) such as S1-IgM and S1-IgG. This venue uses seized antigens and antibodies on graphene electrodes with increased sensitivity with a multiplexing capacity for sensing multiple SARS-Co- V2 makers, while the resulting response data is transmitted wirelessly to a portable mobile device. This type of miniaturized electrochemical platform shows a grand promise for the future PoC electrochemical and personalized health care devices [8, 14].

3.1.2. Optical Nanobiosensors

Due to the exceptional features of optical biosensors, such as high sensitivity, being label-free, robustness, immunity to electromagnetic interference, having computable optical outputs, being amenable to miniaturisation, integration capabilities, portability, multiplexing capacity and delivering simultaneous detection of various targets, optical biosensors are employed as diagnostic tools for respiratory virus infection. Thus, optical biosensors are eligible for the point-of-care zone [9, 10].

Figure 9. A Wireless Graphene-Based Telemedicine Platform (SARS-CoV-2 RapidPlex) for Rapid and Multiplex Electrochemical Detection of SARS- CoV-2 in Blood and Saliva (A) Schematic illustration of the SARS-CoV-2 RapidPlex multisensor telemedicine platform for detection of SARS-CoV-2 viral proteins, antibodies (IgG and IgM), and inflammatory biomarker C-reactive protein (CRP). Data can be wirelessly transmitted to a mobile user interface. WE, working electrode; CE, counter electrode; RE, reference electrode. (B) Mass-producible laser-engraved graphene sensor arrays. (C) Photograph of a disposable and flexible graphene array. (D) Image of a SARS-CoV-2 RapidPlex system with a graphene sensor array connected to a printed circuit board for signal processing and wireless communication.

Carbon nanotubes, gold nanoislands, and graphene are majorly employed in optical and electrochemical biosensors. Gold nanoislands made of tiny gold nanostructures can be constructed with artificially synthesised DNA receptors and complementary RNA sequences of SARS-CoV-2 on a glass substrate. Because COVID-19 is a single-stranded RNA virus, the receptor of the nanobiosensor acts as a complementary succession to the RNA sequence of the coronavirus and detects the virus. LSPR (localised surface plasmon resonance) was utilised to detect RNA sequence binding to the sensor. After binding the molecules on the surface of the nanobiosensor, the local infrared index changes, and an optical nanobiosensor calculates the modifications and determines the existence of RNA strands [9, 10].

Notably, highly effective optical biosensor-based detection of SARS-CoV-2 has been presented with surface plasmon resonance and fluorescence. When an optical biosensor is conjoined with the surface plasmon resonance method, the resulting method is valuable for rapid diagnosis of SARS infection, more so than enzyme-linked immunosorbent assays (ELISA). A fiber-optic-enabled biosensor based on localized surface plasmon associated fluorescence (LSPCF) can sense the recombinant N protein (SARS-CoV-N) using AuNPs. It was marked that a viral stock as small as 106 particles/mL can be detected by using a fiber-optics-based nano-enabled biosensor within 15 min. These surveys indicate that viral respiratory infections can be diagnosed rapidly and promptly by utilising nanomaterial-enabled [9].

Figure 10. A schematic of an optical biosensor

3.1.3. Graphene-based biosensors

A grapheme-based FET (field effect transistor) device is employed to determine SARS-CoV-2 viral burden in nasopharyngeal swabs of COVID-19 patients. The graphene-based FET nanobiosensor consists of a graphene sheet as the sensing area, moved to a SiO2/Si substrate and SARS-CoV-2 spike antibody immobilized on the graphene sheet. The biosensors help detection of SARS-CoV-2 antigen spike even at the concentration of 1 fg/mL in phosphate buffer [10].

Graphene-based biosensors are valuable for testing and cutting-edge detection of [9]:

• blood glucose;
• respiration rate;
• real-time body temperature;
• blood pressure;
• virus;
• small molecules.

Due to the cost-effectiveness, high association, and ease of fabrication, graphene-based nanomaterials are the most appealing materials for biosensors. For example, a transistor-based biosensor has been successfully developed to detect SARS-CoV-2 (spike protein). The biosensor was manufactured using field-effect transistor (FET) coated graphene sheets with a specific antibody (Figure 9). Graphene and its derivatives show suitable integrity FET-based biosensing devices for capturing viruses as they have benefits over other diagnostic methods which are at the present available [9].

FET-based biosensing devices can make susceptible and instantaneous measurements by using small amounts of analytes. Furthermore, FET-based biosensors have probable and utility in clinical diagnosis, on-sight detection and point-of-care testing. An unamplified and quick nanosensing platform was created to detect SARS-CoV-2 RNA in human throat swab specimens. A graphene field-effect transistor (G-FET) sensor was designed to illustrate gold nanoparticle (AuNP). On the surfaces of AuNPs, complementary phosphorodiamidate morpholino oligo (PMO) probes were immobilized. This sensor directs to a low background signal, as the PMO is highly sensitive to SARS-CoV-2 RdRp. When a graphene field-effect transistor is connected with a CRISPR-Cas9-based biosensor, it will be capable of detecting unamplified target genes, and thus, it could be evaluated for viral targets, such as the nucleic acids of SARS-CoV-2 [9]:

3.1.4. Chiral Nanobiosensors

Chiral biosensors will soon be at the bionanotechnology domain’s vanguard due to their ultra-sensitivity and rapid response time. They will be instrumental in the SARS-CoV-2 pandemic. The popularity of nano-chiroptics has burst because of novel methods to manufacture engineered metallic nanostructures with a tunable surface morphology and complete their nano-assembly. This offers unparalleled power over their electronic and optical properties. The most important benefit of such nanohybrid structures is that they improve the chiroptical reaction, which could be of significant interest in various applications linked to chiral biosensing, opening up new research areas. In comparison to natural chiral molecules, chiral plasmonic nanostructures not only lead to significant chiroptical effects, but also present completely unique ideas of superchiral light in technological applications [2, 5].

Figure 11. Detection of SARS-CoV-2 using FETs: The schematic shows a collection of biological samples from a patient and their application to the graphene-based sensing area of a FET biosensor. Binding events associated with the SAR-CoV2 virus can be captured by the sensor in real time.

Ahmed et al. developed a self-assembled technique for the development of a chiral immunosensor using gold nanoparticles and quantum dots. Zirconium quantum dots and magnetic nanoparticles were conjugated with coronavirus specific antibodies and mixed. In the presence of a viral target, both the quantum dots and nanoparticles will attach to the viral target and develop magneto plasmonic- fluorescent nanohybrids, which an exterior magnet can divide. The analyte concentration was then decided by calculating the fluorescence assertiveness of the diverged nanohybrids. This sensing process has a limit of detection of 79.15 EID/50 μl [2, 5, 10].

3.1.5. Aptamer-Based Biosensor

Due to the robust screening method, aptamers can detect viral genes, proteins, or any other viral infection markers. By adjusting the developed assays, aptamer-based sensors can distinguish between infected and uninfected host cells or active and inactive viral forms. Because of their properties, aptamer-based detection has significant benefits over antibodies, including high resilience at a vast range of temperatures and situations, straightforward synthesis through a systematic evolution of ligands by exponential enrichment (SELEX) method, and easy transformation according to the needs of the assay [4].

Figure 12. Detection of SARS-CoV and SARS-CoV-2 using aptamer-based biosensors.

Biosensors employ antibody- and aptamer-based detection mechanisms. Aptamers are more durable, more affordable and quicker to synthesize than antibodies. Aptamers, also known as ‘chemical antibodies’ or ‘artificial antibodies’, are often analogised to antibodies regarding their critical particularity towards their targets. Some aptamers have been isolated in SARS-CoV-2 and incorporated in aptasensing platforms [7].

Aptamers are oligonucleotide sequences that can be designed to recognize and bind to different biomolecules specifically:

• diminutive molecules such as amino acids, nucleotides, and antibiotics;
• 67,68 macromolecules such as nucleic acids and proteins;
• 69 and even surface-epitope bearing whole bacteria, viruses70 and other cells.

Aptamers form unique three-dimensional (3D) structures while securing specifically to analytes. These can be modelled quickly and can be immobilized stably on the surface of biosensors. Aptamer-based biosensors (aptasensors) can quantitatively detect target analytes by calculating the signal developed from the coupled chemical and/or biochemical surface interactions. Aptamers have been considered as a promising diagnostic tool for detecting viruses [7].

Aptasensors are aptamer-based biosensors designed to explore and quantify target analyte biomolecules via distinct biochemical reactions related to a quantifiable signal generation mechanism. The interchange of specific aptamers with target biomolecules represents the biorecognition and capturing the event, which is additionally transduced into a proportionate signal. Aptasensors recently reported for detecting SARS-CoV-2 can be broadly divided into two categories based on the nature of signal transduction: optical and electrochemical aptasensors [7].

3.2. Point-of-Care Testing

Point-of-Care testing as it is defined by the Centers for Disease Control and Prevention Center are “diagnostic tests performed at or near the place where a specimen is collected, and they provide results within minutes rather than hours. These may be Nucelic Acid Amplification Test (NAAT), antigen, or antibody tests.” [18].

The point-of-care testing (POCT) infectious disease market represents an auspicious and substantial increase in the industry’s global in-vitro diagnostics (IVD). The increasing spread of human immunodeficiency virus (HIV), tuberculosis (TB), and malaria in developing countries, and the danger of emerging and reemerging contagious diseases such as the Middle East respiratory syndrome (MERS), severe acute respiratory syndrome (SARS), ZIKA, a variety of influenza strains, and the West Nile virus are factors that boost the need for POCT [6].

Contagious diseases pose a substantial threat to human health and lead to more than half of deaths worldwide. Further, widespread contagious diseases have continuously increased fatality rates in developing countries. The most efficient way to contain the epidemic is an early diagnosis, which is challenging to utilize common approaches because of expensive and extensive equipment, specialists, and slow data output. Thus, rapid POCT methods are essential for overcoming these burdens by miniaturizing and decreasing the device expense and delivering accessible, quick, easy-to-use diagnostic tests without specialized training [6].

POC testing enables the diagnosis of infected individuals without sending patient specimens to laboratories. This is extremely important for places or residents that lack appropriate laboratory infrastructure for specimen testing. The essential part of PoC testing is the biosensor, which is utilised to achieve a biochemical assay to detect the pathogen.

The advantages of using PoC testing are [4].

1. minimal space condition for testing and storage;
2. wide-scale analysis;

• testing can be achieved in a variety of locations;

1. adaptable in meeting various medical needs.

Figure 13. Schematic illustration of the quantitative evaluation of SARS-CoV-2 using the SERS-based aptasensor. (a) After SARS-CoV-2 lysates release the target spike proteins, they are recognized by the aptamer DNAs on the Au nanopopcorn surfaces. The S protein–bound aptamers move away from the Au nanopopcorn surfaces, leading to a decreased Raman peak intensity of Cy3 reporters. (b) Cy3-tagged aptamer DNAs are hybridized with capture DNAs on the Au nanopopcorn substrate. The internal standard 4-MBAs are immobilized along with aptamer DNAs on the Au nanopopcorn substrate. (c) Recognition of the SARS-CoV-2 S protein induces a conformational change of aptamer DNAs, enabling the aptamer DNAs to bind with the RBD on the spike protein.

One of the most attractive POCTs is the ones that are based on the colometric biosensors as they permit detection of the analyte through easy color changes that are observable to the unassisted eye [5].

Figure 14: Nanoparticle based colorimetric detection of virus. This figure depicts the mechanism by which virus causes aggregation of nanoparticles, leading to color change from red to purple.

Kim et al. created a colorimetric assay employing gold nanoparticles to detect the MERS-CoV virus. They suggested a colorimetric assay based on an extended structure of double-stranded DNA (dsDNA) self-assembly shielded gold. This assay utilises two thiol modified probes and citrate capped gold nanoparticles (AuNPs) nanoparticles (AuNPs) under positive electrolyte (e.g., 0.1 M MgCl2) [5, 6].

The gold nanoparticle-based colorimetric test makes a gold nanoparticle solution that collects the virus and shows an observable colour change in the liquid. This gives a rapid test for COVID-19 by turning the colour of gold nanoparticles. This low-cost test acts much better than the other diagnostic techniques, similar to the standard PCR tests. The main benefit of this test is that gold nanoparticles show specific colours because they absorb particular wavelengths. To the gold nanoparticles, the sample containing SARS-CoV-2 is counted, resulting in the accumulation of the virus, and it provokes a transformation in the absorption height that results in a change of colour of the solution. This shift in the colour will be observable to the naked eye, and the downside is that it is feasible only when a load of the virus is very high [12].

The probes are conjugated to AuNPs via substantial Au-S interchanges. In the absence of a target, the AuNPs total (in a positive electrolyte) leads to colour change, either envisioning with the naked eye or detecting by localised surface plasmon resonance (LSPR) shift. Nonetheless, the existence of viral target causes comprehensive self-assembly of double-stranded DNA, controlling the accumulation of gold nanoparticles in the existence of positive electrolytes, discouraging shift in optical properties of AuNPs [5, 6].

The potential detection limit of this assay is 1 pmol μl−1, permitting the detection of lower amounts of the viral target. Furthermore, using such kind of colorimetric based assay enables low-cost and rapid disease diagnosis without the requirement for sophisticated tools [5].

3.3. Nanopore target sequencing (NTS)

The nanopore metagenome method (NTS) has been demonstrated to detect respiratory bacterial infection and viruses instantly from clinical samples. In addition, pathogens and antibiotic resistance genes can be recognised in several hours, much faster than conventional culture processes as the real-time data generation of nanopore sequencers. Moreover, nanopore sequencing was utilised to direct sequences in the transcriptome of SARS-CoV-2. The NTS method simultaneously detects SARS-CoV-2 and ten other respiratory viruses within only 6–10 hours. Therefore, it is suitable for the current diagnosis of COVID-19; nonetheless, the framework can be extended to diagnose other viruses and pathogens. NTS is based on amplifying 11 SARS-CoV-2 virulence-related and exceptional gene fragments (e.g., orf1ab) utilising an inner primary panel followed by sequencing the boosted fragment on a nanopore platform. This task uses a nanopore platform for sequencing to sequence long nucleic acid fragments and simultaneously analyze the data output in real-time. This allows verifying SARS-CoV-2 infections within minutes of sequencing by mapping the sequence reads to the SARS- CoV-2 genome and analyzing the output sequence’s originality, validity, and read number sequence [16, 17].

4. Challenges and Limitations of Nanotechnology in COVID-19

Nanotechnology-based systems, despite their advantages, encounter multiple barriers before they can be safely presented to the market. The most common issues are:

1. Scalability and production costs;
2. ,Intellectual and regulatory properties;
3. Potential toxicity and environmental effects.

Some problems in nanotechnology applications must be handled before they are widely adopted in the healthcare system. The primary task will be to secure the safety of nanomaterial via in vitro studies of their biocompatibility. The destiny of nanomaterials can be transformed into the body when they travel through blood due to the protein corona formation. Hence, in vivo studies need to be executed carefully to understand better the toxicity of nanoparticles in the body. Because of constraints, generic protocols have been utilised for categorization at an early stage of research and development that miscalculate the chances of failures in clinical translation of nanotechnology-based therapy. Closer cooperation between regulatory agencies, experts in material science, pharmacology, and toxicology are needed to overcome other limitations [10].

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