2. Nanobiosensors for SARS-CoV-2 Diagnosis
Countless efforts have been made to develop several nanomaterial-based biosensors to identify various diseases such as the hepatitis C virus, porcine epidemic diarrhea, human immune deficiency virus, and SARS-CoV-2 virus in the last several years
[2][19][2,52]. The nanomaterial-based biosensor has been the focus because of its exclusive detection properties, which allow the sensing of analytes such as aptamers, nucleic acids, and proteins. Nanomaterials, namely carbon-based, polymer-based, quantum-dot, and metal-based nanomaterials, are frequently intended for biosensing applications to diagnose SARS-CoV-2 infection through various modes of detection (
Table 1)—such as viral sequence, antibody, or antigen—as they have already exhibited their potential for detecting other viruses. Thus, nanomaterials and their composites have unique properties such as excellent adsorption, quantum-size effects, and great surface-to-volume ratios, which play a vital role in refining the performance of biosensors
[20][53].
Table 1.
Characteristics of nanobiosensors for detection of SARS-CoV-2 virus.
S. No. |
Type of Sensor |
Core Nanomaterials |
Biomarker |
Limit of Detection |
Detection Time |
Ref. |
1 |
Plasmonic-effect-based colorimetric biosensing |
Gold NPs |
Thiol-modified antisense oligonucleotides (ASOs) specific for nucleocapsid phosphoprotein (N-gene) of SARS-CoV-2 |
0.18 ng/µL |
10 min |
[21][54] |
2 |
Combined effect of plasmonic photothermal (PPT) and localized-surface-plasmon resonance (LSPR) biosensing |
Gold NPs |
SARS-CoV-2 nucleic acid/thiol-cDNA |
0.22 |
15–20 min |
[22][55] |
3 |
LSPR biosensing |
Silver NPs |
SARS-CoV-2 spike RBD protein |
0.83 pM |
20 min |
[23][56] |
4 |
Lateral flow immunoassay based on fluorescence biosensing |
Lanthanide-doped polystyrene NPs |
Protein |
- |
10 min |
[24][57] |
5 |
Electrochemical biosensing |
Cobalt functionalized TiO2 |
SARS-CoV-2 spike RBD protein |
~0.7 nM |
30 s |
[7][8] |
6 |
Electrochemical biosensing |
Graphene oxide |
RNA of SARS-CoV-2 |
200 copies/mL |
- |
[25][58] |
7 |
Nanoresonator biosensing |
Graphene |
SARS-CoV-2 spike S1 antigen |
10 copies per test |
- |
[26][59] |
8 |
Field-effect transistor-based amperometric biosensing |
Graphene |
SARS-CoV-2 spike protein S1 |
1.6 × 10 1 pfu/mL in spike culture 2.42 × 10 2 copies/mL in clinical samples |
5–8 min |
[1] |
9 |
Electrochemical biosensing |
Carbon nanotube |
SARS-CoV-2 spike S1 antigen |
4.12 fg/mL |
2–3 min |
[27][60] |
10 |
Electrochemical biosensing |
Quantum dots |
RNA of SARS-CoV-2 |
- |
- |
[28][61] |
11 |
Electrochemical biosensing |
Polymer nanomaterials |
SARS-CoV-2 nucleoprotein |
15 fM |
- |
[29][62] |
12 |
Electrochemical biosensing |
Polymer nanomaterials |
SARS-CoV-2 spike protein subunit S1 |
15 fM in buffered saline and 64 fM in nasopharyngeal |
- |
[30][63] |
13 |
Circle-to-circle-amplification-based optomagnetic biosensing |
Iron oxide NPs |
DNA of SARS-CoV-2 |
0.4 fM |
~100 min |
[31][64] |
14 |
LSPR biosensing |
Gold NPs |
SARS-CoV-2 nucleocapsid (N) protein |
150 ng/mL |
5 min |
[32][65] |
2.1. Metal and Metal Oxides-Based Nanobiosensors
Metal nanoparticles have created enormous potential for treating and diagnosing pathogenic viruses, as nanoparticles, namely copper (Cu), Gold (Au), iron (Fe), and silver (Ag), display excellent antibacterial and antiviral activity, contrary to other nanoparticle types
[6][33][7,66]. NP biosensors comprise a biological substrate or immobilized ligand, which undergoes conversion with the occurrence of an analyte. The analyte induces modification in the substrate composition, thus imparting a change in the surroundings of the NPs, resulting in changes in the aggregation status of the NPs and leading to detectable color changes
[34][67]. Gold nanoparticles (AuNPs) have recently garnered consideration by virtue of their unique optical, physical, catalytical, biocompatible, and electrical properties; furthermore, the biosensing of virus infection is mostly performed by gold nanoparticles
[35][36][68,69]. NPs could conjugate with receptors to form aggregates. AuNPs couple with antibodies specific to the SARS-CoV-2 antigens, and in the presence of antigens, AuNPs aggregate. The aggregation of AuNPs leads to the optical absorption spectrum, which undergoes a red shift (larger wavelength) and the solution changes to a blue spectrum
[37][70]. Therefore, various gold-nanoparticle-based biosensors can determine the SARS-CoV-2 infection via the colorimetric method. For instance, Moitra, et al. conducted a colorimetric bioassay with gold-nanoparticle-capped thiol-modified antisense oligonucleotides (ASOs) to determine SARS-CoV-2 by considering nucleocapsid phosphoprotein (N-Gene) as an analyte of interest. The methodology uses a comprehensive targeting method facilitated by four of the ASO sequences (which are selected based on their relative binding energies with the target sequence and binding disruption energies) covering two regions of the target gene sequence (N-gene) simultaneously. Additionally, with the occurrence of the target RNA sequence of SARS-CoV-2, the thiolated ASO-capped AuNPs agglomerate and a modification in its SPR is demonstrated; this is further augmented with the addition of RNaseH (which cleaves the RNA strand from the RNA–DNA hybrid), leading to the visual naked-eye detection of AuNPs. The identification is made through surface plasmon resonance with a red shift of approx. 40 nm in the spectra of absorbance. Further, this same method was carried out for MERS-CoV viral RNA determination, but no alteration in absorbance was observed
[21][54].
Additionally, an unusual effort was made with gold nanoparticles to identify the disease and bacteria in laboratory and medical samples. Gasbarri, et al. illustrated the antiviral activity of the sulfonated compound against SARS-CoV-2 by using gold nanoparticles coated with decanesulfonic acid in the micromolar and nanomolar range. Previously, sulfonated compounds were shown to be active against the heparan sulfates (HS) virus
[38][71]. Further, Li, et al. fabricated a gold-nanoparticle-based LFIA strip that can concurrently diagnose IgM and IgG antibodies for SARS-CoV-2, which might be employed to quickly screen asymptomatic and symptomatic patients of SARS-CoV-2 in laboratories and hospitals in real-time, POC diagnostics. Additionally, a combined IgG-IgM antibody test kit for SARS-CoV-2 has numerous advantages; for example, it saves time, is easy to carry out, additional equipment is not required, and it requires little training
[39][72]. Further, a single line at the control (C) validates the authenticity of the test; thus, this line is a must during analysis, and G and M, respectively, represent IgG and IgM. Further, the single line at G and M represents previous and recent infection with SARS-CoV-2, respectively. In addition, lines at both G and M represent SARS-CoV-2 infection, and the absence of lines in both places represents no infection or early infection due to antibodies not being available in traceable amounts
[39][72].
Qui, et al. reported a two-dimensional gold-nanoisland (AuNIs)-based nucleic acid sensor for detecting SARS-CoV-2. A dual-functional plasmonic biosensor was successfully fabricated and incorporated with localized surface plasmon resonance (LSPR) and a plasmonic photothermal (PPT) effect as transduction principles for sensing. This biosensor was fabricated using a 2D AuNIs chip following the self-assembly of thermally de-wetted Au nanofilm on the BK7 glass surface. The Au-film was firstly prepared via magnetron-sputtering. The LSPR and PPT can be excited at two different wavelengths through two different rent angles of incidence, which considerably amplifies the sensitivity, reliability, and sensing stability. Therefore, the suggested biosensor can provide an easy and reliable diagnosis platform; moreover, it alleviates the pressure of PCR-based tests. The proposed LSPR biosensor has superb sensitivity for detecting SARS-CoV-2 nucleic acid, with a detection limit of 0.22 pM
[22][55]. Similarly, silver nanoparticles (AgNPs) are taken into consideration as one of the potential antiviral agents that can act against the lethal virus. AgNPs can be incorporated to the viral genome, obstructing the interaction and activity of numerous virus and cellular factors responsible for repetition and resulting in the prohibition of viral replication. By evaluating various properties such as shape, size, and surface charge, the interaction of AgNPs with viruses is enhanced
[40][73]. AgNP-based biosensors could be employed in diagnostics and in the sensing of viral infection. Due to their catalytic, photonic, electronic, optical, and clinical applications, AgNPs are crucial in combating the SARS-CoV-2 virus
[22][41][55,74]. Yang, et al. developed an ACE2-functionalized silver-nanotriangle (AgNT)-based LSPR sensor for detecting SARS-CoV-2 spike RBD with a detection limit of 0.83 pM. The fabricated biosensor consists of four steps: the fabrication of AgNT, the immobilization of ACE2 on AgNT, BSA blocking, and then, the detection of the virus or RBD protein. The developed sensor shows high specificity and sensitivity to CoV NL63 and SARS-CoV-2
[23][56]. Ghisetti, et al. reported on the POCT STANDARD Q COVID-19 Ag for rapid SARS-CoV-2 nucleoprotein detection in NP swabs, with the result appearing in less than 30 min; this was applied to 330 patients of two different populations from phase 1 and 3 of the pandemic. The main advantage of POCT for antigen testing is that it requires limited technical skill and infrastructure; furthermore, it is certainly preferable in point-of-care centers for mass screening of the SARS-CoV-2 virus. The study reported that 70.6% of the positive RT-PCR samples were not false-positive results, which were identified via STANDARD Q COVID-19 Ag
[42][75]. Teengam, et al. reported AgNP paper-based colorimetric DNA sensors for detecting Middle East respiratory syndrome (MERS). MERS-CoV DNA was effortlessly determined using a cationic pyrrolidinyl peptide nucleic acid (acpcPNA) probe. The probe was positively charged with a lysine moiety present at the carboxyl terminus so it could interact with the negatively charged targeted DNA or AgNPs. In the case of DNA binding, it prevents aggregation without any color change, while in the case of AgNP binding, it leads to NP aggregation with a significant color change. This fabricated paper-based colorimetric DNA sensor could be another approach for easy, quick, sensitive, and selective DNA detection, and therefore, the fabricated sensor can serve as a POC diagnostic tool for viral DNA detection. In the same way, SARS-CoV-2 DNA may be detected by AgNPs
[43][44][76,77]. One more work by Chen, et al. developed a simple and rapid lateral flow immunoassay based on lanthanide-doped nanoparticles (LNPs) to diagnose anti-SARS-CoV-2 IgG in serum samples within 10 min. This biosensing platform was fabricated through lanthanide-doped polystyrene NPs, developed using the mini-emulsion polymerization method. By using rabbit IgG (R-IgG) following a EDC/NHS chemical reaction and mouse anti-human IgG antibody (MH-IgG), the surface modification of LNPs was performed. LNPs have demonstrated several optical properties, as they possess remarkable electronic configurations along with high and sharp emission bands, which are extensively used for sensitive biosensing applications
[24][57]. Likewise, various metal oxides were recently turned into significant materials for identifying several viral diseases, including influenza, ebola, and current COVID-19
[45][46][78,79]. Ishikawa, et al. reported that In
2O
3 nanowires were grown on Si/SiO
2 substrate via the laser ablation CVD method to detect the SARS-CoV-2 N protein. The fabricated FET biosensor could detect N protein at subnanomolar concentrations, with a response time of ~10 min
[47][80]. Similarly, transition metal oxide is extensively reviewed for its antiviral nature due to its stability, physiochemical properties, cost-effectiveness, and reasonable stability rate. Vadlamani, et al. developed a cobalt-functionalized TiO
2-nanotube-(Co-TNTs)-based electrochemical sensor for detecting the SARS-CoV-2 spike-receptor-binding domain protein with a detection limit of 0.7 nM. The TNTs were fabricated via one-step electrochemical anodization of the Ti sheet and Co functionalization was performed using the wet ion-exchange method
[7][8]. Neal, et al. synthesized two sliver-modified cerium oxide nanoparticles by using the base-mediated forced hydrolysis method for SARS-CoV-2, as the cerium oxide nanomaterial has demonstrated considerable biomedical efficiency
[48][81]. The SARS-CoV-2 virus spreads through surfaces (such as handles, gas pumps, doors, and elevator buttons), and remains on the surface for up to 1 week; thus, surface transmission can be inhibited by using a coating. Coating the surface with cuprous oxide (Cu
2O) inactivates 99.9% of SARS-CoV-2
[49][82]. For example, Hosseini, et al. fabricated cupric oxide (CuO), which diminishes infection from SARS-CoV-2. The porous film creates a good connection between the microbe and the CuO solid because of the highly sintered area. A thin coating of CuO, developed via the thermal oxidation of Cu
2O, succeeded via sintering. The inactivity of the virus due to the coating of CuO was examined using Vero E6 cells
[50][83]. It has become known that the SARS-CoV-2 virus is mainly transmitted from an infected person’s nose or mouth in small liquid particles when they cough, speak, sneeze, and breathe; these pass-through air and are small enough to remain airborne for hours. Therefore, there have been several reviews reported on breath analysis due to its potential for the screening, treatment, and diagnosis of SARS-CoV-2. Moreover, it could be used at home, in central facilities, and in POC, which lowers the burden on hospitals
[51][52][84,85]. Thus, Haick, et al., in 2022, reported a AuNP-based sensor for screening and detecting the SARS-CoV-2 virus directly from exhaled breath. Recent studies have suggested that the microenvironments emitting volatile organic compounds were created by the viral agents of SARS-CoV-2. The various sensing layers developed by the link of AuNPs to organic ligands shrink and bulge upon exposure to VOCs; this causes changes in the electric resistance, which can be measured digitally. The proposed device needs frequent washing with alcohol and the sensor needs to be flushed with air after every use. The VOCs can target breath diagnostics without being invasive to patients. The study cohort included 14 symptomatic negative cases, 47 asymptomatic controls, and 41 confirmed cases of the SARS-CoV-2 virus
[53][86].
2.2. Carbon-Based Nanobiosensor
Carbon-based nanomaterials, namely carbon nanotubes, graphene, and fullerene, are fascinating for antimicrobial application. They are favorable materials for combating SARS-CoV-2 viruses via various mechanisms because of their exceptional electrical conductivity, optical and biocompatible performance, etc.
[54][55][87,88].
2.2.1. Graphene
Graphene is an intriguing two-dimensional (2D) carbon material in which each carbon in graphene is sp2 hybridized, and the carbon atoms are covalently linked in a honeycomb lattice. Furthermore, it has attracted much consideration owing to its promising antiviral and antimicrobial applications. However, the interactions among graphene derivatives and viruses are still disputable. Graphene materials are highly suitable for biosensing to detect viruses due to their unique characteristics and properties such as electrical and thermal conductivity, light weight, chemical stability, high optical transparency, their function as a zero-band-gap semiconductor, non-toxicity, and mechanical stability. Additionally, the large surface-area-to-volume ratio of graphene is advantageous for drug carrier applications, but it can also play a vital role in biosensing applications
[56][57][58][89,90,91]. In biosensors, graphene-based nanomaterials are employed as transducers that convert the interactions among the target molecule and the receptor molecules into measurable signals
[59][92]. For this to occur, molecules such as ssDNA, enzymes, and antibodies must be attached to the transducer surface. The most common approach for enzyme immobilization onto graphene, graphene oxide, and reduced graphene is physisorption, while ssDNA and antibodies are most commonly immobilized using EDC/NHS chemistry
[60][93]. In several studies, graphene-based biosensing devices have already been reported to screen various infectious diseases, including SARS-CoV-2. Two-dimensional-material-based biosensors can be classified as optical-based devices, mainly relying on sensing via SPR, surface-enhanced Raman scattering (SERS), and fluorescence and electrode-based devices, including field-effect transistors (FET) and electrochemical biosensors
[61][94]. Graphene-based FETs have mainly been significant due to their low noise detection, rapid estimation, even in the presence of small amounts of the analyte, and ultra-sensitivity
[62][95]. For instance, by utilizing the properties of graphene, Seo, et al. fabricated a FET-based biosensor with a LOD of 1.6 × 10
1 pfu/mL, and 2.24 × 10
2 copies/mL in the case of SARS-CoV-2 spiked culture and clinical samples. The FET-based biosensing device was developed by coating graphene surface with a specific antibody of the SARS-CoV-2 spike protein. The fabricated graphene-based biosensor could detect the SARS-CoV-2 virus because the functionalized graphene-based biosensor provides rapid, highly responsive, and simple detection
[1]. Huang, et al.
[63][96] developed ultrasensitive (low limit of detection of 1.6 pg/mL) and selective graphene@chitosan@Au nanoparticles (G@CS@Au NPs) and G@Ag NPs@CS-based sandwich-type biosensors (immunosensor) for diagnosing avian influenza virus H7. In this sandwich-type immunosensor, H7-monoclonal antibodies (MAbs) are attached with G@CS@Au NPs, and H7-polyclonal antibodies (PAbs) are attached with G@Ag NPs@CS; this fabrication of a diagnostic approach for the detection of the virus led to the development of biosensors for virus determination. Further, Palmieri, et al. demonstrated that graphene could be one of the prime materials for combating the SARS-CoV-2 virus by interacting with the surface of the virus, its genetic material, and its proteins via diffusion of these materials to inhibit functions such as genome replication and protein synthesis, which damages the integrity of the virus
[64][97]. Layqah, et al. developed an electrochemical immunosensor using a carbon electrode with gold nanoparticles to detect the MERS-CoV virus
[65][98]. Yusof, et al. showed that graphene is an appropriate material to fight the spread of the SARS-CoV-2 virus, as biomass-derived graphene has enormous potential to be implemented as a sterilizer and can be used in face masks to inhibit any droplets from entering our body
[66][99].
2.3. Quantum-Dot-Based Nanobiosensors
Quantum dots (QDs) were first ascertained around the beginning of the 1980s. Quantum dots are inorganic semiconductor materials conjugated with high fluorescent probes, usually in the range of 1 to 10 nm with a tunable optical wavelength. Furthermore, quantum dots are often cited as artificial atoms
[67][124]. Quantum dots have captured enormous attention in nanotechnology, biotechnology, and bioimaging applications, with high sensitivity, selectivity, and rapidity because of their unique optical and electronic properties
[68][69][125,126]. Quantum dots have been considered promising for sensing and treating SARS-CoV-2 and other viral infections, as their low toxicity and high surface performance make them supreme amongst other nanomaterials
[70][127]. Quantum-dot-based nanomaterials, for example, fluorescent, graphene, and carbon quantum dots, have also attracted considerable attention for developing an efficacious biosensor for detecting various viral infections due to their unique properties
[71][128].
Boroushaki, et al. fabricated a carbon electrochemical sensor to fabricate a screen-printed electrode reinforced by boron nitride quantum dots/a flower-like gold nanostructure. The developed electrochemical biosensor has the potential for the sensitive and accurate diagnosis of SARS-CoV-2 in laboratory samples
[28][61]. Further, Zheng, et al. developed a fluorescent quantum dot coupled with the B-cell epitopes of SARS-CoV-2 to determine SARS-CoV-2 with a detection limit of 100 pM. B-cell-epitope-based quantum dot biosensors can vigorously separate coronavirus diseases compared to the conventional ELISA. Fluorescent quantum dots have the potential to detect antigens and antibodies quickly
[72][129]. Graphene quantum dots (GQDs) are nanoscale structures of graphene with a size of less than 100 nm, which show outstanding properties such as exceptional biocompatibility; electronic, optical, and quantum confinement; low toxicity; high water solubility; and multicolored emission
[73][74][130,131]. Owing to the aforementioned properties, graphene quantum dots have potential in biomolecule detection, drug delivery, and the biological imaging of numerous viral infections, and are promising for nanotechnological development
[75][132]. Graphene quantum dots can be synthesized either via bottom-up or top-down methods
[76][133]. Song, et al. synthesized N-doped graphene quantum dots (NGQDs) via a microwave-assisted hydrothermal reaction for the transfection of several genes, as the graphene quantum dots provide a promising platform for drug delivery
[77][134]. Ray, et al. synthesized human cathelicidin LL-37 and neutrophil α-defensin HNP1 (a human host defense peptide) attached to GQDs. GQDs inhibit the delta variant’s virus by accessing host cells and prohibiting the spike protein RBD from binding with ACE2 due to the GQDs’ unique properties
[78][135]. Li., et al. fabricated a magnetic relaxation switch (MRSw) to detect and eradicate the antigen S protein of SARS-CoV-2 based on GQDs. This approach gives quick, safe, and extremely sensitive identification of the SARS-CoV-2 virus
[79][136]. Carbon quantum dots (CQDs), also called carbon dots, are carbon-based nanomaterials that are small nanoparticles with an average size below 10 nm. CQDs have shown tremendous potential in various applications such as photothermal therapy, drug delivery, bioimaging, and biosensing, due to their state-of-the-art properties for example, conductivity, optical detection, high chemical stability, outstanding biocompatibility, and low cost. Furthermore, with brilliant accuracy, they have demonstrated the incredible capability to detect and exterminate several pathogenic viruses, including the SARS-CoV-2
[80][137]. Loczechin, et al. developed carbon-based quantum dots for the potential diagnosis of human coronavirus (HCoV-229E), and boronic acid improved CQDs
[81][138]. Thus, QDs can play a significant role in determining SARS-CoV-2, but are not explored fully in terms of SARS-CoV-2 diagnosis
[82][83][139,140]. Hence, researchers focusing on the applications of various QDs, such as CQDs and GQDs, can explore their utility for determining contagious viral infections such as SARS-CoV-2 and MERS-CoV.