Prospects of Biosensors Based on Functionalized and Nanostructured Solitary Materials: Detection of Viral Infections and Other Risks.

Advances in nanotechnology over the past decade have emerged as a substitute for conventional therapies and have facilitated the development of economically viable biosensors. Next-generation biosensors can play a significant role in curbing the spread of various viruses, including HCoV-2, and controlling morbidity and mortality. Pertaining to the impact of the current pandemic, there is a need for point-of-care biosensor-based testing as a detection method to accelerate the detection process. Integrating biosensors with nanostructures could be a substitute for ultrasensitive label-free biosensors to amplify sensing and miniaturization. Notably, next-generation biosensors could expedite the detection process. An elaborate description of various types of functionalized nanomaterials and their synthetic aspects is presented. The utility of the functionalized nanostructured materials for fabricating nanobiosensors to detect several types of viral infections is described in this review. This review also discusses the choice of appropriate nanomaterials, as well as challenges and opportunities in the field of nanobiosensors.

Introduction

Viruses have been posing a constant threat to humanity over the last several decades. Thousands of people have died as a result of various types of microbial infections over the years. Methodologies for early detection and diagnosis were critical in halting the spread of these rapidly transmitted diseases and controlling morbidity and mortality in these conditions. Conventional methods to detect the virus are real-time polymerase chain reaction (qPCR) tests and serological assays. The conventional methodologies for viral infection diagnosis are colossal and, thus, need adroit staff. The conventional methods reported so far are time-consuming, expensive, and labor-intensive.[1] These limitations can be overcome by using nanotechnology in the diagnosis of viral infections. Recently, nanotechnological advances have emerged as a substitute for conventional therapies.

Previous decades have noticed a high surge in biosensor development for detecting several kinds of disorders,[2] fatal diseases,[3] medical conditions,[4] and infectious diseases[5] because they are found to be rapid, simple, sensitive, and accurate. They established their importance by detecting cancer in early stages, checking diabetes, confirming pregnancy, detecting highly transmissible virulent viruses, and many more.[6,7] Biosensor-based devices are composed of bioentities, and the detection mechanism for the bioentities has been represented schematically (Figure ).[8] The desired features of biosensors are stability, affordability, sensitivity, and accuracy.[9,10] Biosensors detect analytes in the form of electrical, thermal, or optical signals mediated by enzymes or antibodies (Abs).[11,12] These devices have a plethora of applications ranging from clinical to environmental.

Figure 1

Overview of biosensors representing their ideal characteristics and applications.

Reducing the dimensions to the nano range, i.e., 1–100 nm, could extend the utility of nanomaterials (NMs) because they offer miniaturization and better surface area, which has grabbed immense attention. Due to their nanosize, the ratio of surface to volume increases, and it offers efficient interactions between sensor and analyte. By designing effective interactions between analyte and biosensor, NM-based biosensors have been widely used for the detection of biomolecules and disease diagnostics. A brief discussion of different applications is given in the Supporting Information (SI.1).

The present review summarizes different NM-based biosensors such as metal oxide, single-walled carbon nanotubes (SWCNTs), multi-walled carbon nanotubes (MWCNTs), Au nanoparticles (NPs), and SiO2-based biosensors and their probable utilization in biomedical applications such as drug delivery, RNA detection, DNA detection, nCoV-2 detection, etc. In addition, the challenges and opportunities available with the nanosensors are also discussed to give insight into the study.

Different Types of Biosensors for Detection

Biosensors have multiple advantages when used with nanotechnology, such as real-time analysis, detection using a smaller amount of sample, high-throughput screening, label-free detection, and low limit of detection (LOD).[13−15] Different types of biosensors are known, and a few of them are mentioned in Table .

Table 1

Comparative Analysis of Various Types of Biosensors

biosensors	optical	electrical	mechanical	electrochemical	photoelectrochemical
principle	optical property changes on sensor’s surface after analyte binding	current or voltage changes on electrode after analyte binding	oscillation at specific resonant frequency that changes by increasing mass on surface	transduce target molecule into digital electrochemical signals	charge-transfer process between photoactive material, an electrode, and an analyte under light illumination
analytes	enzymes, antibodies, nucleic acids, aptamers	antibody, affirmer, or aptamer	microbial cell, antibody, nucleic acids	enzymes, antibodies, DNA, aptamers	enzymes, antibodies, DNA, aptamers
change detected	optical properties, fluorescence, and absorbance	current or voltage changes on the electrode	resonant frequency of crystal due to change in mass	redox reaction/electrical conductivity as a result of change in ion concentration	charge transfer under light illumination
technique used	SPR or LSPR	potentiometric	QCM	amperometric
	colorimetric	conductometric	cantilever technology	impedimetric
		FET
		voltammetric
		impedance
advantages	simple	multiplex label-free detection	high sensitivity	high selectivity and sensitivity over optical biosensors	high sensitivity and lower background signals to analyze the target
	cost-effective	high specificity and sensitivity	instant response time	lower purchase cost
	rapid detection method without analytical equipment		simple miniaturization process	rapid results
			high throughput	better signal-to-noise ratio
limitations	low sensitivity	translation to clinical samples	requires temperature control	low LOD over other biosensors
	limited capability of a multiplex	buffered solution may interfere	sensitive to sample matrix effect	inconsistent reproductivity
				no specific binding
sensitivity and precision	depends on NMs binding with biosensor	depends on NMs binding with biosensor	depends on NMs binding with biosensor	depends on NMs binding with biosensor	depends on NMs binding with biosensor

Optical Biosensors

Optical biosensors provide the opportunity to change the optical properties of the outer surface of the sensor after binding with the analyte, which is then transduced to the detector for sensing (Figure A). In the early 1990s, the first surface plasmon resonance (SPR)-based commercial optical biosensor was used. Further, SPR was integrated with optical waveguide grating and biolayer interferometry, making it more effective. They all followed the same principle as affixing targets on the biosensor’s surface.[16] The modified biosensor surface was challenged with solutions containing molecules to observe the consequences of binding. The types of optical biosensors, such as colorimetric detection and plasmonic biosensors, are described in the Supporting Information (SI.2). A few examples are label-free miRNA detection,[17] detection of foodborne bacteria,[18] etc.

Figure 2

Schematic of (A) optical biosensor and (B) FET biosensor for detection.

Electrical Biosensor

The working of an electrical biosensor is based on the generation of current or voltage on the electrode due to the interaction of the biological sample with an Ab, affirmer, or aptamer. The current and potential values of semiconductors, which were generated by the interaction process at the gate electrode or already have a current-carrying element, were measured with the help of an field-effect transistor (FET) biosensor (Figure B). Eveness et al. developed an electric biosensor based on a polyethylene terephthalate-ZnO co-planar electrode to quantify variable amounts of C-reactive protein (CRP). The biosensor included a PET insulating layer between the electrodes and the active ZnO sensor surface.[19] In phosphate-buffered saline (PBS) solution, an In2O3 electrically modulated FET biosensing device with great durability was developed and can be employed as a biosensor to respond to miR-21.[20]

Mechanical Biosensor

Mechanical biosensors have various advantages in detection, such as having a high sensitivity value and being able to instantly process samples without the use of any reagents.[21] A quartz crystal microbalance (QCM) biosensor is a piezoelectric-type sensor that detects the whole bacterial cell by altering resonance frequency. When modified with NPs, it generates an amplified signal to be used in detection (Figure A). Zhang et al. fabricated an integrated optomechanical cantilever sensor that contained a rib waveguide. The rib waveguide cantilever was connected with hidden waveguides on silicon.[22] For human serum albumin detection, a surface stress mechanical biosensor based on a double-sided Au NP-coated grid-type polydimethylsiloxane membrane and 3D printing has been developed.[23]

Figure 3

Schematic of (A) QCM and (B) electrochemical biosensor.

Electrochemical Biosensor

An electrochemical biosensor is used to transduce the sensing target molecule into digital electrochemical signals (Figure B).[24,25] When combined with NMs, the electrochemically active surface area of the biosensor increases, and also, the electron-transfer efficiency was accelerated.[25,26] The key advantages were high sensitivity, simplicity, low cost, label-free sensors, and ease of miniaturization, and none of these were affected by the analyte present in the matrix. However, certain drawbacks remain, such as high LOD, inconsistent reproductivity, and no specific binding.[27,28] Recently, prussian blue/reduced graphene oxide (RGO) films co-assembled through electrostatic interaction have been successfully synthesized for wearable biosensors and were reported to be used for the detection of glucose.[29] Moreover, a multi-channel sweat biosensor technology for quantitative analysis of sweat that was based on a fully integrated patch-type array was reported.[30]

Photoelectrochemical Biosensor

A photoelectrochemical (PEC) biosensor is a new analytical approach that is associated with the charge-transfer process between a photoactive material, an electrode, and an analyte under light illumination. The completely separated and different energy forms of light and detection signals result in potentially high sensitivity and lower background signals to analyze the target.[31] It consists of three essential components: (a) a photocatalyst semiconductor immobilized on a transparent substrate having high conductivity as the working electrode, (b) an electrocatalyst counter electrode, and (c) an appropriate electrolyte. The photocatalyst semiconductor absorbs light radiation and generates charge carriers at the working electrode (photoanode). The charge collector receives electrons injected from the photocatalyst’s conduction band (CB), while biological organisms present in the electrolyte scavenge holes in the valence band (VB) of the photocatalyst.[32] Zhao et al. constructed a PEC biosensor based on a Co3O4–Au polyhedron to detect miR-141. Through the particular interaction of streptavidin–biotin, biotin-modified DNA was deposited on ITO/TiO2/Bi2S3 to link streptomycin-modified Co3O4–Au.[33] Moreover, it was constructed based on a TiO2/2D coordination polymer CuCl(4-mercaptobenzoic acid) photoelectrode by Yang et al. for the detection of miR-21.[34]

Functionalized Nanomaterial-Based Biosensors

Nanotechnology and nanoscience deal with the properties of materials utilized for various applications via miniaturization. Integrating nanotechnology with medical science opens a new dimension for new therapeutic approaches at the nanoscale. NPs can be used as a vehicle to carry the drug-like molecule to the target site. These possess a variety of compositions and structures that allow them to possess unique physicochemical properties and open new horizons for various biomedical applications.[35] The properties of functionalized NMs as biosensors can be enhanced by surface modification.[36] They are next-generation smart materials with myriad applications. The following section explores several functionalized NM-based biosensors for biosensing capabilities.

Metal Oxide-Based Biosensors

Metal oxides are versatile materials possessing features like high surface-to-volume ratio, light excitation, and wide bandgap semiconductors[37,38] and can be easily integrated into the development of biosensors. In the past few years, biosensors based on metal oxides were developed for various purposes such as detection of H2O2, urea, and glucose[39] and even for cancer cell and virus detection.[40,41]

In the case of enzyme-immobilized biosensors, an enzyme was immobilized on the surface of nanostructured metal oxide (NMO)-based electrodes without affecting the configuration of the enzyme–matrix binding conformation. This leads to increased signal transduction and stability of biosensors.[42,43] In the case of glucose-based biosensors, glucose oxidase (GO) was immobilized on NMOs.[44,45] In another case, GO was immobilized by the chemical etching of ZnO nanorods through the cross-linking process and electrochemically impregnated with Au NPs.[46] GO and horseradish peroxidase (HRP) immobilized on a carbon-based ZnO (C-ZnO) nanowire array have been reported to perform at low LOD with high sensitivity.[47] Glucose biosensors based on CeO2 films on platinum-coated glass plates using pulsed laser deposition were also used.[48] Immobilized GO on Fe3O4 by a physical adsorption process was developed to detect mediator-free glucose, and it showed high sensitivity.[49] Another glucose biosensor was based on the electrochemical deposition of GO and NiO NPs on a glassy carbon electrode (GCE), providing real-time glucose monitoring.[50] The nanocomposite of ZnO NPs on a GCE was developed with high sensitivity.[51]

In the case of an optical immunosensor, the efficiency is dependent on the silanized TiO2. Fabrication of an anti-α-fetoprotein (AFP) sensor was developed using immobilized AFP on a nanocomposite of TiO2 and CH, providing a low detection limit of 0.1 ng mL–1. On the other hand, an AFP sensor based on immobilizing anti-AFP onto a Au NP surface electrodeposited on a CH-MnO2/MWCNT-Ag nanocomposite on GCE exhibited a 0.08 ng mL–1 LOD.[52,53] CeO2 film and ZnO NP on indium tin oxide (ITO) glass were used to co-immobilize rabbit IgG and bovine serum albumin (BSA) for ochratoxin A (OTA) detection using electrochemical impedance spectroscopy (EIS). This provides improved sensitivity and rapid response time.[43] Currently, NMO-based biosensors have attracted much attention as immobilizing matrixes to develop various biosensors. NMO such as Zn, Fe, Mg, etc. possess various properties such as non-toxic, biocompatibility, etc. A recent study showed that biosensors’ improved characteristics could be achieved by incorporating NPs with NMOs.[54] The exceptional properties of NMOs could offer many prospects for the fabrication of biosensing devices to address future needs for diagnostic tools. The unique properties are due to the high surface-to-volume ratio, the ability of adsorption, etc. It is important to select a suitable NMO for fabrication of an efficient biosensor because the binding between NMO and the biomolecule determines the performance of the biosensor. This binding is crucial for establishing a biocompatible environment as the biomolecule activity is highly stable. This provides opportunities for biosensor development with low cost, high sensitivity, long shelf life, and low LOD.[55]

SWCNT-Based Biosensors

Zhang et al. demonstrated functionalization with polyethylene glycol (PEG). First, SWCNTs were coated with a −COOH functional group followed by SOCl2 that generated a −COCl group on the surface of the SWCNTs. Further, it was mixed with PEG to form PEG-SWCNTs and thereby reduced cytotoxicity, caused less membrane damage, and increased biocompatibility. The SWCNTs had a unique surface chemistry and fibrous structure that helped in the growth of SWCNTs in the cell membrane and then transferred them to the cytoplasm. The nanotubes interacted with the mitochondria in the PC12 neuronal cell and controlled the gene present in the metabolic process, and they showed good drug delivery results.[56] Bhirde et al. also reported modification with PEG used to deliver cisplatin anti-cancer drugs in the infected mice. For the modification, carboxylated SWCNTs were reacted with amine-terminated PEG with the help of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS) as an amidization promoter, as shown in Figure SI.3.[57] Yan et al. modified it with cetyltrimethylammonium bromide (CTAB) via a sonication process (Figure SI.4) that caused binding with small interfering RNA (siRNA).[58] Krajcik et al. proposed functionalization with poly(diallyldimethylammonium chloride) (PDDA) and hexamethylenediamine (HMDA) (Figure SI.5). The desired nanotube was oxidized via a reflux process followed by the treatment of carboxylic acid-activated SWCNTs with HMDA, SOCl2, and NEt3 in a dimethylformamide (DMF) solution. HMDA has protonated amines (−NH4+Cl–) that generates a positive charge on the nanotube, while PDDA generates a more positive charge on SWCNTs than HMDA. siRNA was bound with PDDA-HMDA-SWCNTs (1), which helped as a carrier to enter siRNA in the cell, and siRNA was liberated into the cytosol, enhancing gene silencing. Also, 1 decreased the cytotoxic side effect as compared to other liposomal transfection solutions.[59] See Table for details. More SWCNT-based biosensors are provided in the Supporting Information (SI.3).

Table 2

Functionalized SWCNT Biosensor for the Detection and/or Conjugation with Biomolecules

s. no.	materials used for surface modification	synthesis method	application	ref
1	PEG	RF-CCVD method	drug delivery	(56)
2	PEG		cancer drug delivery and imaging tool	(57)
3	human serum protein		biological application and binds with siRNA	(60)
4	CTAB		binding with siRNA oligonucleotides	(58)
5	HMDA and PDDA		delivery of siRNA	(59)
6	PL–PEG		delivery of siRNA and gene silencing	(61)
7	–NH3+		gene delivery and delivery of plasmid DNA	(62)
8	–Lys-NH3+		gene delivery and delivery of plasmid DNA	(62)
9	biotin		tumor-targeted drug delivery	(63)
10	DNA	reflux process followed by addition of DNA	determination of daunorubicin anti-cancer drug	(64)
11	mesoporous silicon	facile stain-etching and sonothermal process	detection of non-enzymatic glucose	(65)

MWCNT-Based Biosensors

Singh et al. reported ammonium functionalized MWCNTs (f-MWCNTs) (Figure SI.8) for effective performance in gene delivery. f-MWCNTs similarly condensed DNA as f-SWCNTs did. Among these three f-CNTs, MWCNT-NH3+ condensed a larger amount of DNA than the other two functionalized CNTs because MWCNTs provide a larger surface area to interact with DNA.[62] Tao et al. reported the metal templating using poly(amidoamine) dendrimer (PAMAM) f-MWCNTs. For synthesis, MWCNTs were reacted with nitric acid to give a carboxyl group at the surface. Further, it reacted with thionyl chloride to form their acid chloride derivative followed by conjugation via ester linkage with hydroxyl-functionalized dendrons to give PAMAM-MWCNTs (Figure ).[66] Yu et al. published the condensation of DNA and increased gene-transfer process using a polyethyleneimine (PEI)-activated MWCNT heterostructure. The heterostructure was formed when MWCNTs reacted with acid to coordinate the carboxylic group, followed by PEI with the help of EDC/NHC coupling that covalently bound MWCNTs.[67]

Figure 4

Schematic of the synthesis of PAMAM-MWCNTs.

Au NP Based Biosensors

Yu et al. reported Env plasmid DNA vaccine delivery for HIV treatment in infected mice using poly(diallydimethylammonium chloride) (PDDA) and PEI f-Au nanorings (NRs). To synthesize f-Au NRs, Au NRs were mixed with a precursor to form the seed solution of CTAB-Au NRs. After that, CTAB-Au was coated with poly(styrene sulfonate) (PSS), which led to forming negatively charged Au NRs followed by a reaction with PDDA or PEI to get f-Au NRs. For Au NRs as vaccine adjuvants, NRs were coated with contrasting surfaces, and Env led to the formation of a Au-Env complex (Figure ).

Figure 5

Schematic of Au NRs to form a Au-Env complex.

Here, transmission electron microscopy (TEM) images revealed that the sizes of the Au NRs were similar in all three cases; thus, the physicochemical properties of CTAB, PEI, and PDDA affected the overall biosensor performance (Figure SI.9). The author examined the improvement of Env DNA’s immunogenicity with these two f-NRs and found that f-NRs with Env gave less immune response, even lower than that of naked Env DNA.[68] Lee et al. proposed mono f-Au NPs with packaging RNA (pRNA) to sense miRNA using the electrochemical surface-enhanced Raman spectroscopy (SERS) technique. In this, one copy of a pRNA three-way junction (RNA 3WJ) with Sephadex G100 aptamer and biotin on each arm was immobilized to Sephadex G100 resin, resulting in conjugation with one Au NP. The formed complex was bound to streptavidin-coated Au NP via streptavidin–biotin interaction followed by purification and reassembly with one more 3WJ, leading to the formation of 3WJ/Au NP.[69] Hwu et al. demonstrated activation of Au NPs with DNA on a GCE for optimized biosensor-based miRNA detection (Table ). The conjugated DNA-Au NPs were prepared by mixing reduced DNA and sodium dodecyl sulfate (SDS) with Au NPs and retaining it for incubation. After that, NaCl and PBS were also mixed with the sonicating method. The principle of the assay was to remove DNA attached to the surface of Au NPs by dissolving it in potassium cyanide. Then, these strands were hybridized with their complementary DNA (cDNA), and a fluorescence readout was done by staining double-stranded DNA (dsDNA) with dye (Figure ).[70] More Au NP-based biosensors are provided in the Supporting Information (SI.4).

Table 3

Activation of a Au Molecule for Sensing and/or Conjugation with Biomolecules

s. no.	materials used	synthesis method	application	ref
1	PDDA	electrostatic layered assembly	DNA vaccine delivery for HIV treatment	(68)
2	PEI	electrostatic layer assembly	DNA vaccine delivery for HIV treatment	(68)
3	pRNA		biosensor for sensing of miRNA	(69)
4	aptamer		biosensor for sensing of adenosine	(71)
5	DNA		optimized biosensor for detection of miRNA	(70)
6	silsesquioxane	layer-by-layer deposition method	label-free DNA biosensor for Zika virus detection	(72)
7	DNA		amplified electrogenerated chemiluminescence, biosensing for the detection of thymine DNA glycosylase	(73)
8	PEG	chemical reduction method	biosensor for the detection of ssDNA	(74)
9	citrate	citrate reduction method	sensing of hepatitis C RNA virus by colorimetric gene sensor	(75)
10	core–shell Fe3O4–Au NPs/PNA		biosensor for detection of miRNA	(76)

Figure 6

Principle of assay for the staining of dsDNA dye for quantification.

SiO2 NP-Based Biosensors

The SiO2 NPs tend to form aggregates due to their high surface energy. This results in improper distribution into the polyurethane (PU) matrix. To overcome this, Gao et al. modified the SiO2 NPs surface with poly(propylene glycol) phosphate ester (PPG-P) (Figure A); PPG-P was prepared via the esterification process of polyphosphoric acid and PPG. The f-SiO2 NPs showed a decrease in surface energy, increase in stability, no aggregation, and uniformly distributed SiO2 NPs into the PU matrix.[77]

Figure 7

Schematics of (A) dispersion of modified SiO2 into the PU matrix and (B) synthesis of ssDNA f-SiO2 NPs.

Naka et al. published the organo f-SiO2 NPs as 3-aminopropyltriethoxysilane (APTES), 3-mercaptopropyltrimethoxysilane (MPTMS), phenyltrimethoxysilane (PTMS), and vinyltriethoxysilane (VTES), which led to enhanced mechanical strength, thermal stability, and gas permeability of the polymer material. The f-SiO2 NPs are denoted as SNP, AP-SNP, MP-SNP, P-SNP, and V-SNP, respectively. TEM images of organo f-SiO2 NPs are shown in Figure SI.12. The Dmax values measured for the SNP, AP-SNP, MP-SNP, P-SNP, and V-SNP were 33, 63, 63, 50, and 36 nm, respectively. The functionalization of SiO2 led to enhanced NP dispersion uniformly in the polymer matrix, a strong force of interaction to solid materials, and a selective bond formed with certain functional groups of the polymer chain to increase the polymer’s mechanical property and thermal stability.[78] Zhou et al. proposed 5′-phosphorylated single-stranded DNA (ssDNA)-modified SiO2 NPs with T4 RNA ligase to seize and detect 20–24 base subpicomolar concentrations of ssRNA from target solution and remain stable in the presence of dithiothreitol (DTT). The modified ssDNA-SiO2 NPs can be denatured and hybridized again and again. ssDNA-SiO2 NPs were synthesized by a reaction of aminopropyl silane with a silanol group in which attachment of the amino group to the surface of the Si NPs occurred. After that, amino-modified SiO2 NPs adsorbed the monolayer of poly-l-glutamic acid (pGlu) via electrostatic interaction on its surface. In the next step, the carboxylic functional group of pGlu formed a covalent bond with amino groups on the surface of SiO2 NPs and 5′-phosphorylated, 3′-amino-activated ssDNA to form an amide with the help of EDC/NHS in a single step (Figure B).[79] See Table for details.

Table 4

Various Applications of Engineered SiO2 Particles

s. no.	materials used for surface modification	particle size	synthesis method	application	ref
1	PPG-P	30 nm	in situ polymerization	uniformly distributed the NPs in polyurethane matrix	(77)
2	APTES	68 nm	water-in-oil microemulsion method	mechanical strength, thermal stability, and gas permeability	(78)
3	MPTMS	32 nm	water-in-oil microemulsion method	enhancement in mechanical strength, thermal stability, and gas permeability	(78)
4	PTMS	34 nm	water-in-oil microemulsion method	enhancement in mechanical strength, thermal stability, and gas permeability	(78)
5	VTES	38 nm	water-in-oil microemulsion method	enhancement in mechanical strength, thermal stability, and gas permeability	(78)
6	5′-phosphorylated ssDNA	120 ± 5 nm	layer-by-layer electrostatic adsorption	capture and detection of single-stranded RNA	(79)

BNNT-Based Biosensors

Lahiri et al. functionalized boron nitride nanotubes (f-BNNTs) with polylactide–polycaprolactone copolymer (PLC), enhancing the biocompatibility and mechanical strength and providing applications in orthopedic scaffolds. The mechanical properties of f-BNNTs were enhanced due to increased tensile strength up to 109% and elastic modulus up to 1370%. Also, functionalization enhanced the uniform distribution along with excellent interaction with the polymer matrix. The pure BNNT cytotoxicity investigated with macrophage and osteoblast cells resulted in no increase in the number of cell deaths.[80] Ciofani et al. demonstrated the cytocompatibility of human neuroblastoma cells by BNNTs. The authors showed no inauspicious effect of BNNTs on cell replication, viability, and metabolism. The functionalization of BNNTs with PEI enhanced the interaction of the nanotube with the living cells. The material was conjugated with fluorescent probes for incubation with the cells. The internalization of the f-BNNTs by the cells was confirmed by an uptake assay, which illustrated that the mechanism of uptake of the material was dependent on the energy and chemical reactivity of PEI. The enhancement in the chemical reactivity of the nanotube by coating with PEI is what makes the interaction of the material with biological complexes possible.[81] Emanet et al. proposed the modification of BNNTs with glucose, starch, and lactose to study the increment of their diffusibility in an aqueous solution for biocompatibility and cellular uptake. In the synthesis of modified BNNTs, the nanotube that was reacted with hydrogen peroxide resulted in the attachment of −OH groups on the outer part of the nanotubes. Further, carbohydrates and −OH groups were attached to hydroxylated BNNTs by using glutaraldehyde as a cross-linker (Figure ). Among all of the carbohydrates, glucose was very small and bound strongly with the −OH group of hydroxylated BNNTs. Unmodified BNNTs had a hydrophobic nature, which caused toxicity at high doses on adenocarcinoma human alveolar basal epithelial cells (A549) and human dermal fibroblasts cells. This toxicity of unmodified BNNTs was reduced to a specific value by modifying it with carbohydrates even at a high number of doses.[82] See Table for details.

Figure 8

Schematic of a modification of BNNTs with carbohydrate.

Table 5

Modified BNNTs Used in Various Applications

s. no.	materials used	application	ref
1	PLC	biocompatibility, mechanical properties	(80)
2	PEI	biomedical applications	(81)
3	glucose	biomedical applications and interaction with protein	(82)
4	lactose	biomedical applications and interaction with protein	(82)
5	starch	biomedical applications and interaction with protein	(82)
6	glycol–chitosan	biomedical applications	(83)

Upconverting Nanoparticle-Based Biosensors

Jiang et al. demonstrated the modification of silica-coated upconverting nanoparticles (UCNPs) with the amino group and their application in siRNA delivery to the target cell and fluorescence imaging. Modified UCNPs were conjugated with the Her2 Ab with covalent bond formation between carboxylic acid moieties and amino moieties of Ab. They modified NPs to fluorescence labeling of Her2 receptors on SK-BR-3 cells and siRNA delivered to SK-BR-3 cells. Also, the modified NPs conjugated with folic acid on their surface were used to label the folate receptors fluorescently on HT-29 cells.[84] Mi et al. demonstrated the formation of a nanocomposite via conjugation of carboxylic group-functionalized Fe3O4 and amine-functionalized UCNPs coated with silica, with applications in biomedicine. For the synthesis of the nanocomposites, Fe3O4 was functionalized with the carboxylic group on its surface via the coprecipitation method, and silica-coated UCNPs were functionalized with an amine group on their surface via the Stober process. Further, these two NPs conjugated with each other with the help of EDC and NHS by the formation of a covalent bond and formed an Fe3O4/UCNP nanocomposite (Figure ). The nanocomposites had a chemically active amine group on UCNPs and carboxylic groups on Fe3O4, which conjugated with a protein such as transferrin. It was used to acknowledge that transferrin receptors were overexpressed on HeLa cells and for biolabeling and fluorescent imaging of HeLa cells (Figure SI.13).[85]

Figure 9

Schematic of the synthesis of Fe3O4/UCNP nanocomposite.

Yuan et al. synthesized the UCNP@SiO2@Ag nanocomposite and modified it with DNA to enhance its biocompatibility and reduce the cytotoxicity; it was used in cell imaging applications. First, the UCNPs were coated with the silica shell on their surface via a reverse microemulsion process. Further, Ag NPs were grown on Si-coated UCNPs. For Ag NPs, Si-coated UCNPs were reacted with (3-mercaptopropyl)triethoxysilane (MPS) to form a thiol bond on the surface, followed by reduction of AgNO3 to introduce the Ag NPs (Figure ). The silica prevented contact of the Ag NPs to the UCNPs as a spacer molecule in the nanocomposites. DNA was used for the modification of the nanocomposite to enhance the biocompatibility. To reduce the cytotoxicity of Ag NPs, DNA was used as a capping agent that covered the positively charged Ag NP surface with its negatively charged surface.[86] See Table for more details. More UCNP-based biosensors are provided in the Supporting Information (SI.5).

Figure 10

Schematic of the synthesis of UCNP@SiO2@Ag nanocomposite.

Table 6

Activated UCNPs with Application in Sensing and/or Conjugation with Biomolecules

s. no.	surface modification	materials used	synthesis method	particle size	application	ref
1	NaYF4:Yb,Er	streptavidin			DNA sensor	(87)
2	NaYF4:Yb,Er@SiO2	amino group		30 nm	delivery of siRNA	(84)
3	NaYF4:Yb,Tm@SiO2	PEG spacer carrying NHS groups	reverse microemulsion method	38 nm	protein conjugation	(88)
4	NaYF4:Yb,Er@SiO2	amino-ethoxy silane		50 nm	bioimaging and conjugate with biomolecules	(89)
5	NaYF4:Yb,Er@SiO2	antibody	glutaraldehyde spacer method		bioimaging and conjugate with biomolecules	(89)
6	NaYF4:Yb,Er@SiO2	anti-Cx43	glutaraldehyde spacer method		bioimaging and conjugate with biomolecules	(89)
7	NaYF4:Yb,Er	Fe3O4		100–150 nm	biolabeling, imaging of cancer cells, and conjugation with biomolecules	(85)
8	NaYF4:Yb,Er@SiO2@Ag	DNA	reverse microemulsion process		conjugate with DNA and cell imaging	(86)

Other Materials-Based Biosensors

Yang et al. synthesized phytic acid (PA)-functionalized polyaniline (PANI/PA) polymer hydrogel via an electrochemical copolymerization process for the electrochemical detection of miRNA (Figure ).

Figure 11

Schematic of the synthesis of biosensors based on PANI/PA polymer hydrogel.

In the synthesis of a functionalized polymer, 3-aminophenylboronic acid (ABA) and aniline were reacted and a boronic acid species was inserted into PANI chains. Boronic acid acted as a cross-linker between PANI and PA in the preparation of PANI/PA. The polymer was deposited on the GCE surface. For electrochemical biosensors, DNA was covalently attached through a −NH2 group on the surface of the polymer. PA had high biocompatibility and hydrophilicity, while PANI had higher electrochemical properties; these properties of both PA and PANI make a copolymer that is a good electrochemical biosensor.[90] Fan et al. proposed the functionalization of CdS NRs with beta-cyclodextrin (β-CD) (f-CdS NRs) for electrochemical HIV DNA detection by connecting with a catalytic hairpin assembly mediated with DNAzyme catalyst, forming an insoluble precipitate. CdS NRs were synthesized via the hydrothermal method by adding Cd(NO3)2·4H2O and SC(NH2)2 in ethylenediamine in the Teflon autoclave reactor, leading to the formation of a yellow suspension. For f-CdS NRs, synthesized CdS NRs with carboxymethyl-β-cyclodextrin were uniformly dispersed in PBS (5.3 pH) with EDC/NHS (Figure ).[91]

Figure 12

Schematic of the synthesis of β-CD-functionalized CdS NRs.

Wang and Hui synthesized PEG-activated polypyrrole nanowires (Ppy NWs) via electrochemical oxidation of NH2 present on PEG, which covalently attached with Ppy NWs. PEG-Ppy NWs were immobilized on GCE followed by binding with a DNA probe. Methylene blue was used as a redox indicator to detect signals through the differential pulse voltammetry method. PEG-Ppy NWs were used for a biosensor to detect miRNA (Figure ).[92]

Figure 13

Schematic of the detection of microRNA using PEG-activated Ppy NWs.

Ppy NWs were immobilized on GCE via electrochemical polymerization containing PBS, Py, and p-toluene sulfonate acid at a 0.75 V constant potential value. Further, PEG-NH2 was mixed with LiClO4 to attach through covalent bonding on the Ppy NWs surface. Lastly, the product was mixed with 1.0 μM carboxyl-functionalized ssDNA and EDC/NHS solution to covalently immobilize ssDNA on the PEG-NH2.[92] Scanning electron microscopy (SEM) images of f-Ppy revealed that the Ppy NWs were arranged in a vertical position on the surface of the electrode to form one-dimensional NWs (Figure SI.17).[92] See Table for more details. Other materials-based biosensors are provided in the Supporting Information (SI.6).

Table 7

Modification of Various Particles Used for Sensing and/or Conjugation with Biomolecules

s. no	surface modification	materials used	particle size	synthesis method	application	ref
1	PANI	PA		electrochemical copolymerization	electrochemical detection of miRNA	(90)
2	CuO NPs	dopamine	30 nm	microwave-assisted methodology	colorimetric biosensor for detection of cysteine	(93)
3	Gd2O3 NRs	aspartic acid	14.26 ± 0.13 nm		electrochemical biosensor for detection of vitamin D3	(94)
4	CdS NRs	β-CD	25–40 nm	solvothermal method	PEC biosensor for detection of HIV DNA	(91)
5	PtCo mesoporous nanosphere	DNA	43.82 nm		electrochemical immunodetection of N6-methyladenosine RNA	(95)
6	Ppy NW	PEG	300 nm	electrochemical oxidation	biosensor for detection of miRNA	(92)
7	CuO NPs	streptavidin			lateral flow strip biosensor for the sensing of human papillomavirus (HPV) type 16 DNA	(96)
8	dopamine	aggregate induced emission dye (TPE-BTD)			for detecting H2O2, G-quadruplex DNA, glucose, and Dam MTase solid	(97)
9	CRISPR/Cas 13a system	catalytic hairpin DNA circuit			detection of multiple RNA	(98)
10	CRISPR/Cas 13a	Ti3C2Tx/PEDOT:PSS			detection of nucleic acid using wireless sensor	(99)

Utilization of Biosensors in Detection of COVID-19

Conventional methods for the detection of COVID-19 were based on reverse transcriptase quantitative polymerase chain reaction test (RT-qPCR), chest X-ray, computed tomography (CT) scan, and serological assessment of elevated levels of CRP, interleukin 6 and 10, etc. (Figure ).[100,101] However, in some cases, these methods do not give accurate results, which is a serious limitation in exploring these methods or protocols. To overcome this issue, there was a dire need for point-of-care biosensors as a detection strategy to control morbidity and mortality when no drug or vaccine specific for the virus was available.[102,103] Other methods to detect COVID-19 are described in the Supporting Information (SI.7). Biomarkers in blood can be used as a prognostic for disease detection. Recently, smell dysfunction as a biomarker was proposed for COVID-19; therefore, biosensors for mucus protein detection can be helpful for early diagnosis.[104] It should be noted that other diseases also had reported nasal dysfunction, and therefore, it may not be specific for COVID-19.[105] In previous years, some biosensors had been developed for ROS detection, leading to another route for COVID-19 detection.[106]

Figure 14

Schematic of currently used diagnostic techniques and possible biosensing platforms for COVID-19. Reprinted with permission from ref (107). Copyright 2020 Elsevier B.V.

Development of Biosensors

An important plan of action could be the invention of NM-based biosensors to recognize and neutralize an epidemic caused by microbes. It would open new prospects to stop an outbreak of various viruses by identifying molecular species in real time. Patra and co-workers are working on sensor development based on semiconductors or hybrid NMs that show rapid viral identification of ultralow concentrations.[108−110] Hwang et al. fabricated FETs with a graphene channel (gFETs) to recognize DNA/RNA with accuracy and high sensitivity.[111] The NMs of quantum dot (QD)-RNA oligonucleotide can diagnose hepatitis C virus NS3 protein selectively and can act as inhibitors to the virus precisely.[112] Ping et al. explored gFETs functionalized with probe DNA for the fabrication of DNA biosensors effectively and scalably.[113] Chen et al. investigated an AlGaN/GaN high electron mobility transistor for miRNA identification that acted as an indicator and was highly selective for cardiovascular diseases.[114] Gao et al. fabricated nanoelectronic transistor sensors by 1D and 2D NMs.[115] A broad range of biological species was identified with high sensitivity. The researcher developed a gFET biosensor that was used for sensitive and selective recognition of E. coli with pyrene-DNA aptamer and exosome sensing.[116] Recently, a gFET-based biosensor was fabricated for a specific Ab against CoV-2 spike protein,[117] whereas Chen et al. found human mAb that blocked binding to the ACE2 receptor.[118]

Currently, 2D graphene has been used significantly in biosensing.[119−122] A magnetically controlled 2D nano-DNA fluorescence sensor was developed for detecting alkaline phosphatase, having a detection limit of 0.02 mU/mL. They utilized λ-exonuclease and a δ-FeOOH nanosheet, which possessed a fluorescence-quenching ability that differed with different DNA structures (ssDNA or dsDNA). Therefore, this sensor was comparatively better than the other 2D nano-DNA fluorescent sensors.[123,124] Further, the 3D graphene nanocomposite possessed a large surface area, biocompatibility, and electrical conductivity. These advantages attracted extensive interest in developing incensing and energy devices.[125,126] A one-step laser induction method was developed to prepare Au NP-3D graphene nanocomposites. They fabricated a flexible impedimetric immunosensor for E. coli detection, enhancing performance such as flexibility, sensitivity, and low detection limit.[125,127]

With the ongoing COVID-19 pandemic and the contagious nature of the HCoV-2, extreme protective measures are crucial to take while testing for COVID-19. The COVID-19 pandemic could be contained with the development of point-of-care diagnostic biosensors. However, due to restricted research practices, only certain laboratories could minimize COVID-19 spread, although development of point-of-care biosensors for accurate and sensitive diagnosis of COVID-19 could escalate specific testing that would limit the spread of infection.

Researchers also used an optomechanical device attached to a smartphone’s camera to detect the viruses (Figure SI.18). The fluorescent imager on the smartphone had a laser diode with 450 nm excitation, a thin-film interference filter, a coarse numerical aperture lens, and a focusing and depth-adjustment coarse mechanical translation stage. A low-pass (LP) filter blocked the scattered excitation beam and formed a proficient background rejection mechanism to separate the very weak fluorescent signal that occurred from the viruses.[128]

Challenges

A major challenge was concerned with the translation of research into commercial prototypes. Reliability of sensors, manufacturing protocols, and practical hands-on skills are fundamentally distant from each other and are limitations that need to be addressed. Automation might come up with solutions whereby rapid troubleshooting calibration is provided to users easily. Innovative biosensing technologies have future prospects in revolutionizing the healthcare system. There were ethical arguments like confidentiality, ownership, and privacy that were challenging to resolve in a short time frame, resulting in decreased technology adoption. The risks associated with self-use biosensors were unclear, and users were less inclined to read terms and conditions while using the applications. Still, laboratory-based testing eliminates ethical issues, and thus, exhaustive testing can be accomplished on sensing platforms. Therefore, biosensors and nanoscale visualization techniques can be encouraging tools, leading to a better understanding of pandemic strains. It can also provide on-site identification of viral strains for point-of-care prognosis and prevention of future pandemics at an early stage.[103] Despite several advantages of next-generation biosensors, only a limited number of NM-based biosensors are currently available. Therefore, further effort is critical for the widespread application of biosensors in the real world.

Future Perspectives

Next-generation biosensors have gained significance in a wide range of applications across many interdisciplinary research areas. Pathogen detection is a very crucial aspect in healthcare management. However, available conventional approaches are bulky, expensive, and time-consuming, and only trained or skilled people can operate them. Hence, they are available at centralized laboratories.[129,130] Compared with the available diagnostic approaches, much attention has been given to electrochemical and FET biosensors because of miniaturization, point-of-care detection, on-site diagnosis, sensitivity, and low cost. Furthermore, multiplex biosensors could be developed for accurate detection of multiple biomarkers. Biosensors can ultimately lead to early diagnosis of COVID-19. Similarly, NP-based, smartphone-based, and wearable biosensors should be developed to control disease outbreak to contain human loss as early as possible.

Currently, a new horizon of NMO-based biosensors is established for clinical and non-clinical diagnosis. The exceptional properties of NMOs provide a huge scope for interdisciplinary research and have led researchers to establish interactions among several biomolecules–transducers using NMs. The possibility of developing biosensors based on NMOs for biomarkers, early detection, etc. is expected to be useful. To achieve enhanced charge transfer, fabrication of NMOs with desired groups may lead to the invention of a new approach for biosensor development. Also, the interface of NMO-based sensors could be utilized for real-time analyte monitoring.[131,132] The system can be extended to monitor public health, including constant recording and feedback approach, and can trace infections to ensure public health protection (Figure SI.19).[133] Functionalized NM-based biosensors may be used to detect bacteria in environmental and water resources. Moreover, functionalized biosensors can detect bacteria and heavy metals present in water resources.[134]

Conclusion

In this review, reports of various types of functionalized NMs used in the fabrication of different types of biosensors and prevalence in various biological and environmental monitoring in real time are discussed. Further, there is an emphasis on different types of biosensors and their unique properties, merits, and demerits. As illustrated, the integration of NMs has led to new perspectives in innovation of next-generation biosensors. Next-generation biosensors are prevalent in several areas of environmental monitoring such as detection of bacteria and pathogens present in environmental surroundings. Furthermore, an emphasis was given to biosensors’ invention to curb the recent pandemic outbreak of nCoV and their utilization to control future pandemics similar to COVID-19.