Nanomaterials in design and application of SARS-CoV- 2 detection methods

Summary

To battle with the current COVID-19 pandemic, nanomaterials can be deemed excellent candidates against viral infections, particularly CoVs, because of their capability to penetrate cells easily, interact with viruses, and avoid viral genome reproduction. In addition, nanoparticles’ use permits the detection of contagious agents in tiny sample volumes instantly in a susceptible, precise, and quick format at lower costs than current in-use technologies. This advancement in early detection allows accurate and fast treatment.

Key words/phrases: nanomaterials, COVID-19, detection

1. Introduction

The continuing explosion of the novel coronavirus disease COVID-19 attracts worldwide considerations due to its prolonged incubation duration and substantial infectivity. The fast worldwide spread of the pandemic, driven by the harsh acute respiratory SARS-CoV-2, has created a pressing need for its diagnosis and treatment. As a result, many researchers have sought to find the most efficient and suitable methods to detect and treat the SARS-CoV-2. Real-time reverse-transcriptase polymerase chain reaction (RT-PCR) testing is presently used as one of the most reliable approaches to detect the new virus; however, this process is time-consuming, labour-intensive, and demands trained laboratory workers. Moreover, despite its high perceptiveness and specificity, false negatives are documented, particularly in non-nasopharyngeal swab samples that yield lower viral loads. Consequently, developing and utilising faster and more reliable methods seems crucial. In recent years, many attempts have been made to manufacture various nanomaterial-based biosensors to detect viruses and bacteria in clinical samples [27, 46].

A discreet way for diagnosing coronavirus disease COVID-19 is highly demanded to fight the existing and forthcoming global health hazards. Nanoparticles offer favourable implementation and significant prospects to function as a platform for quickly diagnosing viral infection with elevated sensitivity. Nanoparticles such as gold nanoparticles, magnetic nanoparticles, and graphene (G) were applied to detect SARS-CoV 2. They have been employed for molecular-based diagnosis processes and serological approaches. Nanoparticles enhanced explicitness and shortened the time demanded the diagnosis. They may be executed into tiny devices that encourage self-diagnosis at home or in places such as airports and shops. Nanoparticles-based methods can be employed for the analysis of virus-contaminated samples from a patient, surface, and air [1].

2. Current methods of detection of SARS-CoV-2

Conventional methods for the detection of SARS-CoV-2 are the reverse transcription polymerase chain reaction (RT-PCR), computed tomography (CT) scan and next-generation sequencing (NGS) [1, 26, 40] (Fig. 1). RT-PCR and chest CT imaging are the most typical diagnostic techniques in detecting COVID-19. In addition, several diagnostic methods such as clustered regularly interspaced short palindromic repeats (CRISPR)–specific high-sensitivity enzymatic reporter unlocking (SHERLOCK), reverse transcription loop-mediated isothermal amplification (RT-LAMP), enzyme-linked immunosorbent assay (ELISA), and sequencing are under development for enhanced detection of the virus in a minimum amount of time [1, 9]. RT-PCR has been acknowledged as the leader and most effective method for coronavirus detection [1, 26].

Figure 1. Conventional methods currently being used for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection. (A) Reverse transcription polymerase chain reaction (RT-PCR). cDNA, complementary DNA. (B) Computed tomography scan. (C) Enzyme-linked immunosorbent assay.

2.1. Reverse Transcription Polymerase Chain Reaction (RT-PCR)

RT-PCR is vastly used for COVID-19 detection. It is based on cDNA synthesis from genomic RNA and is followed by amplification [26, 38]. Amplification of minimal amounts of viral genetic material in a mixture of other nucleic acid series is effectively done by RT-PCR. It is presently the standard gold technique of SARS-CoV-2 detection in upper respiratory tract samples. Several studies have used serum, ocular, and stool specimens for the RT-PCR-based detection. A contemporary method has used self-collected salivary samples as a non-invasive and secure technique for healthcare providers before doing RT-PCR. In this method, the reverse transcriptase first alters the RNA viral genome into DNA using a small sequence succession primer and the complementary DNA (cDNA) generation. Then, a fluorescent dye or a fluorescent-labelled sequence-specific DNA probe observes the amplification of DNA in real-time. Finally, a fluorescent or electrical signal displays the viral cDNA after successive amplification cycles [1, 9, 22].

Conventional RT-PCR procedures included one-step or two-step approaches. While one-step methods entangle a single primer-contained tube, the two-step procedure utilizes more than one tube to conduct the reactions. Still, it provides a more prudent and flexible track. Also, it can stock cDNA for the quantification of diverse targets with fewer starting materials. However, the standard method in detecting SARS-CoV-2 is the one-step approach since it is swifter, demands less sample handling, reduces bench time, and lowers pipetting errors [9, 43].

RT-PCR-based detection is also linked with false-negative results, which might be due to the low viral load in patients’ throats, improper handling of RNA samples, or lack of sufficient internal controls [8, 9, 16]. The main issue of RT-PCR is its low sensitivity to chest scans due to the inadequate number of viruses in the blood of RT-PCR. In addition, it is low sensitivity to chest scans due to the insufficient number of viruses in the blood or the laboratory kit’s inaccuracy [26].

2.2. Computed tomography scan (CT)

Another method for detecting and managing COVID-19 is the chest CT scan, which applies X-ray imaging of a patient’s chest at different angles. As per radiological reports, any uncommon features on the CT scan print may be due to COVID-19 infestation. Typically observed characteristics on a chest scan of a patient with COVID-19 are ground-glass opacification (GGO), especially on the peripheral and more inferior lobes, consolidations (rise in the opacity of the parenchyma, which results in coverage of the underlying vessels), crazy-paving pattern (GGO with intralobular and interlobular septal thickening), and linear opacities. The high-resolution CT could help detect GGOs in the early stages of infection [9, 26, 40].

CT sensitivity appears to be increased in patients with positive RT-PCR (86–97% in various case studies) and lower in patients with only constitutional and nonrespiratory symptoms (about 50%). Ultrasound has been used as a diagnostic tool in a minimal number of cases. Ultrasound has very low specificity, and, despite being influenced by factors such as disease stringency, patient weight and operator dexterity, sensitivity is estimated to be around 75%. However, ultrasound may play a role in observing the advancement of the disease via detection of interstitial lung disease features [26].

2.3. SHERLOCK

Further than RT-PCR and CT scans, various other detection techniques have also been developed for SARS-CoV- 2 detections. As it is described in Gupta at all SHERLOCK has been developed by Zhang et al. [45] “to detect RNA fragments of SARS-CoV- 2 with 10–100 copies/μl of the input. The basic principle of SHERLOCK-based diagnosis is CRISPR-based detection. This test can be performed in < 60 min, without requiring specific instruments. They chose two targets, the S gene and Orf1ab gene, from the SARS-CoV-2 genome. To minimize cross-reactivity with other respiratory virus genomes, they also selected specific guide sequences.” [9].

2.4. RT-LAMP

An optimized RT-LAMP-based detection method has more sensitivity than traditional PT-PCR methods and needs less time (Fig. 2). As a result, this process can be utilized to rapidly diagnose coronavirus and increase the testing capacity by 2–2.5-fold [9, 13].

Figure 2. Workflow comparison of our RT-LAMP assay relative to qRT-PCR for emergency cases (outpatients) and inpatients. Our RT-LAMP assay is 2–2.5 times faster than the qRT-PCR assays and can be shipped at room temperature.

Gupta et al. summarized the current techniques used to detect SARS-CoV-2 infection in Fig. 3 [9].

Figure 3. Current techniques used for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection.

3. Nanomaterials for SARS-CoV-2 detection

Nucleic acid testing by reverse transcription-polymerase chain reaction (RT-PCR) is the current method for detecting COVID-19 infection. Although RT-PCR is widely used to detect COVID-19, there are several issues [3, 14, 29, 35].

1. False-negative results;
2. Long response times / Time consuming;
3. RT-PCR is unable to detect asymptomatic patients, as it demands the existence of observable SARS-CoV-2 in collected samples;
4. Poor analytical sensitivity;
5. Labor intensive;
6. Healthcare centers in non-urban settings lack adequate PCR infrastructure to accommodate increased sample throughput;
7. Expensive;
8. The availability of RT-PCR kits and reagents cannot meet the augmented demand.

The present situation requires developing detection techniques that are rapid, cost-effective and easy to operate. To overcome the limitations of traditional methods, an improved multidisciplinary approach is needed. Nanomaterial based technological solutions present diverse possible applications to battle against the virus [10, 32].

3.1. Properties of nanomaterials

The unique characteristics of nanoparticles play a critical role in tackling pandemic and mitigating future outbreak. Nanoparticles show distinctive properties such as:

• tiny size;
• solubility;
• multifunctionality;
• target-ability;
• stimulus-responsive features;
• large surface area;
• surface adaptivity.

Therefore, they have been used widely for several applications in a variety of fields such as analytical chemistry, pharmacy, sensing/biosensing, biotechnology, nanomedicine, drug delivery, biological detection, gene transfer, optics, wound healing, energy-based applications, agriculture and environmental applications. Nanoparticles enhanced these applications by delivering increased performance with a significant prospect for enactment into a miniaturized machine, including wearable electronics. Hence, they show the tremendous potential to enhance the quality of life via regulating the viral spread via premature detection. Nanoparticles have at least one dimension in the nanometer range (1 nm =10^-9) (Fig. 4) [1, 15, 30, 36].

Figure 4. Scale of nanoparticles with some examples.

The nanoparticles’ high surface-to-volume ratio, high adsorption, quantum size effects and high reactivity allows for efficient interaction with sample analytes. Furthermore, they have exceptional multiplexing abilities, rendering them appropriate for incorporation into state-of-the-art technologies for virus detection. Moreover, nanoparticles offer ease of surface functionalization, suggesting that multiple ligands can be attached via covalent or noncovalent bonding, which further improves selectivity and particularity and decreases detection time. In addition, nanomaterials can also be used as labels for improving the signals, which helps detect very low-magnitude signals [32].

3.2. Categories of nanomaterials

A variety of nanomaterials for virus detection and tracking have been created, contributing to the illumination of virus infection mechanisms, such as [11]:

• Metallic nanoparticles, e.g., gold nanoparticles (Au NPs), silver nanoparticles (Ag NPs);
• Metal oxide nanoparticles, e.g., iron oxide magnetic nanoparticle (Fe₃O₄NPs);
• Carbon nanomaterials including 0-dimensional (0D, e.g., fullerenes (C60), carbon dots (C-dots)), 1D (carbon nanotunes (CNTs), 2D (e.g., graphene (G), graphene oxide (GO), and 3D (e.g., graphite);
• Quantum dots (QDs): CdS QDs, CdTe QDs, carbon QDs;
• Porous materials: metal–organic frameworks (MOFs), covalent organic frameworks (COFs);
• Polymers: natural polymers (e.g., chitosan, cellulose), and synthetic (e.g., polythiophene, polypyrrole);
• Lipid nanoparticles (LNPs): triglycerides, fatty acids, steroids, and waxes.

Figure 5. Schematic diagrams showing different examples of nanomaterial-based COV detection methods. (a) Fluorescent Zr QDs and magnetic nanoparticles are conjugated with antibodies that specifically bind to COV. In the presence of COV, a magnetic fluorescent complex is formed, which is isolated magnetically and detected by fluorescence measurements. (b) Nanotraps are used to concentrate COV and improve their stability, hence facilitating their detection. (c) Reverse transcription PCR is carried out in the presence nanoparticles, improving the efficacy of the polymerase chain reaction, and resulting in a better detection sensitivity of this method. (d) COV detection method, which is based on the interactions between complementary DNA originating from COV and acpnPNA probe at the surface of Ag NP, which results in a separation between Ag NPs, and a yellow color associated with the luminescence of well dispersed Ag NPs, further revealing COV presence.

Figure 6. Different nanoparticles.

Nanomaterials can be utilized in a variety of roles for COVID-19. Rasmi et all summarize the functions and primary role of nanomaterials in the below table (Fig. 7) [30].

Figure 7. Summary of the role on nanomaterials for COVID-19.

3.2.1. Gold NPs (Au NPs)

Gold nanoparticles (AuNPs) have been increasingly employed in SARS-CoV-2 detection platforms due to their remarkable optical properties such as increased extinction coefficients and tunable localized surface plasmon resonance (LSPR), allowing a separate color readout with a simple equipment or the naked eye. For SARS-CoV-2 antibody detection (IgG, IgM, or IgA), AuNP- and fluorescent nanoparticle-based assays have been suggested [17].

Gold nanoparticle (AuNPs) is one of the most typically utilised nanomaterials for quick diagnostics. The gold nanoparticle was employed to detect target viruses’ double-stranded DNA (dsDNA). Specifically, single-stranded DNA (ssDNA) or ssRNA can interact with citrate ions on the AuNP surface. Adding salt to the solution can stabilise the particles and change colour. Furthermore, a simple colourimetric hybridisation assay was applied to detect dsDNA of SARS-CoV based, developed from ssRNA. This assay can see the target at 4.3 nM in 10 min without needing any cumbersome device [18, 30].

Colour change approach

Another analysis introduced a method to visually detect the COVID-19 virus without sophisticated tools. Colourimetric detection was designed using thiol-modified antisense oligonucleotides (ASOs)-coated AuNPs explicitly intended for the. Thiol-modified ASO-cap AuNPs were selectively aggregated in the existence of the SARS-CoV-2 target RNA sequence and delivered a modification in its surface plasmon resonance. The result can be observed in 10 min with a detection limit of 0.18 ng/μL [23, 30].

Figure 8: Schematic representation for the selective naked-eye detection of SARS-CoV-2 RNA mediated by the suitably designed ASO-Capped AuNPs.

Effective detection of COVID-19 was developed by immobilizing proteins on the surface of Au using the Au-binding polypeptides. Using the improved green fluorescent protein, SARS-CoV-E protein, and core streptavidin of Streptomyces avidinii as examples, the Au-binding polypeptide fusion protein was immobilized explicitly on AuNP, and the protein nanopatterns on the bare Au surface were demonstrated. These complexes interact with the antibody, resulting in absorbance and colour change [25, 30].

 Non-invasive approach

The detection of COVID-19 using non-invasive approaches has been proposed from exhaled breath using an AuNP-based sensor. The sensor consisted of different AuNP attached to organic ligands and inorganic nanomaterial film. The inorganic film is accountable for electrical conductivity. Therefore, when exposed to the volatile organic compounds (VOCs) from exhaled breath, the organic film reacts with the VOCs, resulting in the inorganic film swelling or shrinkage and the changes in electrical conductivity. Therefore, this non-invasive sensor could potentially be used to rapidly screen COVID-19 [30, 32].

Electrochemical hybridization approach

An AuNP-based electrochemical hybridization method was defined using a gene-sensor consisting of a thiolated-DNA probe-immobilized on the AuNPs carbon electrode to hybridize biotinylated-target DNA. An electrochemical chip was presented via a carbon electrode composed of AuNP array. The coronavirus protein was bound on an AuNP-electrode, and both coronavirus protein and free viruses compete for binding sites in the existence of antibodies. There was an excellent linear reaction between the sensor response and the concentrations of coronavirus ranging from 0.001 to 100 ng mL−1. The assay achieved the detection limit of as low as 1.0 pg mL−1. The method was single-step, sensitive and precise (Fig. 9) [17, 30].

Figure 9. COV immunosensor array chip (a), The immunosensor fabrication steps (b), the detection process of the competitive immunosensor for the virus (c).

Immunochromatogrphy approach

A lateral flow assay for the rapid detection of IgM against COVID-19 was designed through the indirect immunochromatography approach. The SARS-CoV-2 nucleoprotein (SARS-CoV-2 NP) was coated on an analytical membrane for target capturing, and anti-human IgM was conjugated to AuNP, operating as a detection reporter. AuNP-LF analysis exhibited remarkable selectivity in the IgM detection without interference from other viruses. Each assay only needs 10–20 μL serum, and the result can be received within 15 min [12, 30].

3.2.2. Quantum Dots (QDs)

Quantum dots (QDs) are multifaceted mechanisms that can battle against COVID-19 virus. Quantum dots (QDs), likewise known as “semiconductor nanomaterials,” play a vital role in COVID-19 detection. QDs have been recognised as a new fluorescent probe for molecular imaging. The size of the QDs varies from 1 to 10 nm. The exceptional characteristics of QDs, including great optical and semiconductor properties, exemplified photo- stability, high quantum yield, and narrow emission spectrum with adjustable size, have made them a significant candidate to operate as a fluorescent label. Because of these outstanding properties, QDs can be considered a great agent to fight against viral infections. Moreover, incorporating possible biocompatible carriers can aid interdisciplinary study and permit clinical approaches to fighting the virus. Owing to their superior properties, QDs are now dominant imaging probes (chemosensors and biosensors) for sensing [21, 30].

Figure 10. Description of Operation Principle of the AuNP=LF Strip.

QDs are employed due to their traceability under a specific wavelength of light. In addition, QDs can be tunable into the desired size (1–10 nm) and shape that efficiently targets/penetrates SARS-CoV-2 with a size span between 60 and 140 nm. Furthermore, the positive surface charge of carbon-based QDs could be utilised to sequester/disable the S protein of SARS-CoV-2. In addition, cationic surface charges exhibited by QDs interact with the negative RNA strand of the virus, directing to the production of reactive oxygen species within SARS-CoV-2 [21].

A QD-conjugated RNA aptamer-based chip was introduced for sensitive and rapid detection of SARS-CoV N protein with a detection limit of 0.1 pg mL−1 on a developed chip. The QD- conjugated RNA aptamer can bind to the SARS-CoV N protein immobilized on the chip, creating an optical signal. The use of fluorescent-based QDs may help researchers in designing an easy, sensitive and rapid diagnostic tool for COVID-19 [30, 31].

Figure 11. Schematic representation of the actions exerted by QDs on SARS-CoV-2. QD, quantum dot; S protein, spike protein; SARS-CoV-2, severe acute respiratory syndrome coronavirus type 2

Carbon quantum dots

Carbon quantum dots (CQDs) can be utilized to sense microbes, biomolecules and infections. In addition, they can be used as biocompatible inactivation systems for pathogenic human coronavirus infections as dominant imaging probes (chemosensors and biosensors) with antiviral activity. The CQDs are about 10 nm with high solubility in water, were fabricated via hydrothermal carbonization of carbon precursors.  Some innovative approaches for detecting coronaviruses have focused on the application of CQDs. In one method, the antiviral activities of seven types of CQDs were used to cure human coronavirus contagions. Different kinds of CQDs by hydrothermal carbonization and conjugation of boronic acid were used. It was disclosed that the virus inhibition is possibly owing to the interchange between CQDs operating groups with entry receptors of the virus [10].

Zirconium quantum dots (Zr QDs)

Zirconium, due to its properties such as mechanical stability, thermal resilience and UV light capture, has been utilised in many biomedical areas as a nontoxic transition. Besides, the nanosize of Zr has unique physical and chemical aspects due to its high surface area and the captivity of electronic states in comparison with its bulk regime [10].

In general, the employment of QDs against coronavirus is one of the most suitable choices due to its outstanding curative efficiency. Moreover, QDs can be employed as a robust imaging probe and sensor in diagnosis and prognostic. In addition, the drugs can be coated on the surface of QDs to target COVID-19. Nevertheless, caution should be exerted to avoid renal filtration and additional side consequences.

3.2.3. Carbon-Based Nanomaterials

Carbon materials are functional in every aspect of our everyday life because they are plentiful and weightless fabrics that can be used for a combination of applications. Carbon-based nanomaterials can be categorised based on their dimensionalities (D) as zero-D (0D) such as carbon dots, one-D (1D) as in CNTs, and two-D (2D) in graphene nanostructures. These nanomaterials maintain more exhaustive operational temperature, perceptiveness and vaster dynamic transducing signal range even in extreme environmental situations [24].

Carbon-based nanomaterials have been extensively employed in developing a platform for COVID-19 detection. Their outstanding physicochemical and antiviral characteristics suggest that nanomaterials play a vital role against COVID-19. These nanomaterials, including graphene and graphene oxide, carbon quantum dot, carbon nanotube, and fullerene with excellent properties mainly sensing, antiviral and antimicrobial properties, are superior options with potential applications against COVID-19 in biosensor for diagnosis, antiviral coating, airborne virus filtration, facemask, and drug delivery [10, 24, 30].

 Graphene and graphene oxide

The antimicrobial and antiviral properties of the nanomaterial graphene and graphene oxide has two dimensions that captured a lot of awareness and examination. First, graphene-based field-effect transistors (FET) as potable sensors have been developed to analyze COVID-19 viral load in clinical nasopharyngeal samples, utilizing unique antibodies against its spike protein. The fabricated FET sensors can catch the SARS- CoV-2 spike protein in phosphate-buffered saline and 100 fg mL− 1 medical transfer system, at the level of 1fgmL− 1 concentration and limit of detection ~1.6 × 101 pfu mL-1 and ~2.42 × 10² pfu mL-1 for the cultured sample and medical test, respectively. This sensor shows is highly sensitive to screening and diagnosis of novel coronavirus disease 2019 without any sample pretreatment. The existence of graphene leads to an improved signal-to-noise ratio [10].

Figure 12. Schematic illustration of certain allotropes of carbon nanomaterials for nanobiosensor development.

Carbon nanotubes

Carbon nanotubes (CNTs) were widely applied for biology and biomedical sciences due to the following properties and open new horizons for scientific development [10]:

• 10− 100 nm dimensions;
• antiviral and antimicrobial activity;
• good light-heat conversion efficiency;
• large surface volume ratio;
• slight density;
• small pore size;
• flexibility;
• resistance to acids and bases;
• great mechanical strength;
• ability to create reactive oxygen species;
• resistance to respiratory droplet;
• biological compatibility with several drugs.

Carbon dots were found in 2004, and they usually have photoluminescence, bio-compatibility, and high resilience, predisposing them to diverse applications, including biosensing and bio-imaging. Carbon nanotubes (CNTs), graphene, and carbon dots (CDs) can be classified as zero-(0D), one-(1D), and two-(2D) dimensional carbon nanomaterials [10, 30].

Figure 13. Future prospects of CNTs in the prevention, diagnosis and treatment of SARS-CoV-2 infection.

High storage space, high surface area, high biocompatibility, excellent permeability of biological barriers, reasonable bio absorption rate, multi-energy surface/tube chemical functional group capability, and targeted biomolecule modification potency are excellent properties of CNTs that provide novel suggestions encountering COVID-19. Similarly, CNTs are used as diagnosis systems, filtering and virus inactivation agent [10].

A CNT size-tunable enrichment microdevice (CNT-STEM) was designed to enrich and concentrate viruses from raw samples. CNTs can be utilised to diagnose respiratory viruses, including SARS-CoV-1 and SARS-CoV-2. The channel sidewall in the microdevice was manufactured by nitrogen-doped multiwalled CNTs, where the intratubular space between CNTs is optimised to correspond to the size of different viruses. By using this device, the avian influenza virus strain was determined. The CNT-STEM significantly improves virus isolation rates and detection perceptiveness. Because of the ease and trustworthiness of this technique, it can be adjusted to detect SARS-CoV-2 RNA or proteins [30, 34].

Figure 14. The working principle of virus enrichment and concertation from field samples. (A) A filed sample containing viruses (purple spheres) is collected by a cotton or as a tissue sample. (B) The supernatant of the field sample flows through the CNT-STEM, and the viruses are enriched within the device. Inset (right): Illustration of size-based virus enrichment by the aligned N-MWCNTs. Inset (bottom right): SEM image (scale bar, 100 nm) of the H5N2 AIV virions trapped inside the aligned N-MWCNTs. Inset (bottom left): Dark-field TEM image (scale bar, 100 nm) of enriched H5N2 AIV after the aligned N-MWCNTs structures were retrieved from the CNT-STEM.

Nanodiamonds

Due to its high stability and low cytotoxicity, nanodiamonds have received significant attention for COVID-19 diagnostics. Therefore, fluorescent nanodiamonds were utilised for COVID-19 lateral flow immunoassay as an ultrasensitive label. These nanodiamonds were immobilised on the test line, and a microwave field was used to selectively split their fluorescence signal from the background signal, which significantly enhanced the detection sensitivity. This assay was 105 more sensitive than the traditional gold-nanoparticle-based lateral flow assay. Carbon-based nanomaterials can be employed as an antiviral therapeutic agent for COVID-19 [30].

3.2.4. Magnetic NPs (MNPs)

Before detection, magnetic NPs (MNPs) are typically used to detect SARS-CoV-2, host antibody response, and nucleic acid separation. It was shown that silica-coated iron oxide NPs have a significant association with SARS-CoV-2 RNA, as the cracked open the virus. The magnet was utilised to isolate the RNA coated NPs from the sample solution. This method is economical and straightforward, enabling to extract RNA from patient samples efficiently [15, 30].

Precise detection demands efficient extraction and separation of nucleic acids from samples, allowing target purification. Superparamagnetic nanoparticles (80 nm) conjugated with a complementary probe to the target sequence SARS-CoVs was employed in one study. Utilizing a magnet, the functionalized superparamagnetic nanoparticles can extract target cDNA from specimens. The amount of extracted DNA was boosted through PCR which was tested employing silica-coated fluorescence nanoparticles conjugated with a complementary sequence. Silica-coated fluorescence NPs produce fluorescence signals directly correlated to the concentration of the target cDNA [30].

The surface functionalized MNP’s adsorbs the nucleic acid from the lysis solution and are fast separated from most of the contaminations with the assistance of an external magnetic field. Following this short procedure, the nucleic acid can be additionally separated from the functionalized surface of MNP’s by the desorption process in the eluent. However, although this process is much easier and shorter than traditional procedures, MNP’s assisted extraction process still consists of several stages, which is inadequate for practical detection. The zinc ferrite nanoparticles were synthesized by discharge, and the nanoparticle surfaces were functionalized with silica and carboxyl-modified polyvinyl alcohol. This platform shows the capability to automatically remove the viral RNA from diverse sample types. It decreases the functional steps, which presents a significant prospect for COVID-19 molecular-level diagnostics [30, 34].

A more straightforward and contemporary MNP’s assisted RNA-extraction protocol is suggested for possible extraction and RT-PCR-based diagnosis of COVID-19. The MNP’s of zinc ferrite (ZNF) were manufactured by the cost-efficient sol-gel auto-combustion route, and after that, its surface was functionalized with carboxyl containing polymers (CPoly). Among the magnetic materials, zinc ferrite was selected due to its high chemical resilience, smooth magnetic behaviour, uncomplicated preparation and biocompatible character. Due to the robust interface among nucleic acids and carboxyl groups, the surface-functionalized MNP’s promote fast and possible viral RNA’s adsorption. This cost-effective and straightforward technique may provide a qualified alternate for conventional methods [34].

Figure 15. Schematic procedure for surface functionalised MNP’s assisted RNA-extraction protocol.

In addition, there is a one-step nucleic acid extraction procedure that particularly ties viral RNA using polycarboxyl-functionalized amino group-modified MNPs (PC-coated NH2-MNP). Nucleic acids were gathered using a magnetic field, and then they were released from the MNPs by adding wash buffer. By catching COVID-19-pseudoviruses, polycarboxyl-functionalized MNPs exhibited perfect absorption and paramagnetic properties via fast capture (30 s magnetic capture) of targets [30, 46].

Figure 16: A schematic representation of the pcMNP-based viral RNA extraction method.

3.2.5. Nanozymes

Nanozymes are unnatural enzymes composed of nanomaterials with similar efficiency as natural enzymes. In addition, nanozymes have superior catalytic activities, quick response and self-assembly capability, extensively employed for disease diagnostics and treatment. A novel nanozyme-based chemiluminescence paper assay for rapid and acute detection of SARS-CoV-2 spike antigen combines nanozyme and enzymatic chemiluminescence immunoassay with the lateral flow strip created.

Figure 17: (A) Schematic illustration of the nano- zyme chemiluminescence paper test for SARS-CoV-2 S-RBD antigen. Recognition, separation and cata- lytic amplification by nanozyme probes.

Conventional chemiluminescence immunodiagnosis utilises natural proteases such as HRP or alkaline phosphatase that reveal constraints such as scarce storage resilience, complicated preparation methods and high cost. The suggested biosensor employed peroxidase-mimic Co-Fe@hemin nanozyme rather than natural horseradish peroxidase (HRP) that could greatly boost the chemiluminescent signal reaching the detection limit of 0.1 ng/mL. The Co-Fe@hemin nanozyme was demonstrated to have better stability for temperature and acerbity or alkalinity as compared to HRP, which can be stably held at room temperature. This testing can be conducted within 16 min, much quicker compared to the usual 1-2 h needed for currently employed nucleic acid tests. Furthermore, signal detection is possible using the camera of a typical smartphone. Components for nanozyme synthesis are easy and readily obtainable, considerably reducing the overall expense [20, 30].

3.2.6. Metal-Organic Framework

Porous nanomaterials can be used for the detection of different pathogens. The analyte, pathogen, does not require to be absorbed by the porous nanomaterials; however, the pathogen needs to interact with the surface of the MOF that different NPs modify. By this interaction, additional Off–On or On–Off optical mechanisms can be optimized to detect the pathogen, and in this case, various optical active components can be employed as quenchers or activators. In the issue of SARS-CoV-2, there is no necessity to detect the same genetic material and genetic sequence on the surface of the mask or even clothes due to the significant discrepancies between the concentrations of the SARS-CoV-2 with others. Instead, using a fingerprint fluorescence pattern, which has been optimized before, the same range of attention of SARS-CoV-2 on the contact surface of the gas and solid phases can be measured by optical changes. Moreover, if the MOF based biosensors successfully work for HIV-1, H1N1, ZIKA, and other pathogens detections with considerable precision and LOD, then the morphology and optical-based biosensor for detection SARS-CoV-2 should function as well. [26].

4. Challenges and Limitations of nanomaterials

Nanomaterials can be significantly valuable for biomedical applications. However, they have some constraints, such as toxicity. One of the significant challenges is to ensure the safe use of nanomaterials. Another challenge is that the behaviour of nanomaterials in the body can change when they reach blood circulation due to protein corona formation. Thus, faithful in vivo models are required to sufficiently comprehend the toxicokinetic behaviour of the nanoparticles in the body, particularly for long-term exposure.

Another problem is the absence of standardized protocols for nanomaterials’ physicochemical and biological definition and the lack of a universally agreed-upon definition of a nanomaterial. Capacity for large-scale manufacturing is another hurdle that needs to be overcome for the broader commercialization of nano-based formulations. Due to the multi-faceted interchanges between nanomaterials and biological systems, it is very demanding to foresee the behaviour of these materials under physiological conditions. Once within the body, the nanoparticles reach the blood circulation, a complex matrix containing ions, small molecules, proteins and cells. [37].

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