Nidhi Chauhan,Kirti Saxena,Mayukh Tikadar,and Utkarsh Jain

ABSTRACT Biosensors have acquired much importance in drug discovery,medical diagnostics,food safety,defense,security,and monitoring of environmental conditions.Furthermore,there has been great progress in the potential applications of advanced nanomaterials in biosensors.Every year there are several advances in sensing techniques that can be attributed to nanomaterials,biorecognition elements,or their related fabrication techniques.The further development of nanotechnology-based sensors provides a wide variety of opportunities to modern research.Advanced nanomaterials can provide remarkable optical,electrical,mechanical,and catalytic properties.For example,transition metals and organic polymers have been used in the fabrication of powerful,sensitive,and precise biosensors.The distinctive properties of advanced nanomaterials have been widely incorporated into biosensors.However,fabrication techniques also play important roles in the development of these devices.Therefore,we present a review of some of the advanced nanomaterials that have been widely used over the last few years and discuss their fabrication techniques.The focus of this review is to provide a directional perspective of recently fabricated advanced nanomaterial-based biosensors in the diagnosis of various diseases.

KEYWORDS Biosensors,Advanced nanomaterials,Transition metals,Fabrication of biosensors,Fabrication techniques

I.INTRODUCTION

Nanomaterial-based biosensors have gradually emerged as a highly advanced and well-developed class of biosensors.A biosensor consists of three major components(i)a biorecognition element for sensing the presence of an analyte and its concentration (ii) a transducer surface that functions according to an optical,electrochemical,piezoelectric,or thermal principle (iii) and a detector to measure the generated signals.1The components of a nanomaterial based biosensor are summarized in Fig.1.

The incorporation of nanomaterials into a biosensor can amplify the signals and achieve more accurate and specifi recognition of an analyte.2The biorecognition element can be immobilized on the surface of the transducer using various methods.These immobilized biorecognition elements are specifi to their analyte,and this results in highly selective binding occurring on the sensing platform.The analyte could be an ion,molecule,cell,nucleic acid,or a protein,while the biorecognition elements can involve enzymes,nucleic acids,whole cells,aptamers,or polymers.3In a biosensor,the transducer surface contains an electrode as a sensing platform,and this is where the interaction of an analyte with a biorecognition element takes place.Interaction between a bio-receptor and an analyte generates an optical,electrochemical,thermal,or piezoelectric signal that can be quantified4In recent years,several new strategies have been adopted by modifying the structure,combinations,and other parameters of nanomaterials to improve the quality of these signals.5,6Depending on their distinct properties,advanced nanomaterials may be categorized as being biomimetic optical nanomaterials,polymer electrolyte emulsions,nanostabilizers,biomedical materials,or bio-inspired mechanically adaptive nanomaterials.

The development of nanomaterial-based biosensors opens a new path for the detection—as well as real-time monitoring—of biomarkers associated with various diseases and the treatment thereof.7In last few years,biosensors have become highly attractive to researchers on account of their ease of fabrication,sensitivity,very small size,and reproducibility.Presently,surface-modifie working electrodes are widely used for developing biosensors with high selectivity.Biosensors show high sensitivity and specificit towards biomolecules;therefore,they are projected to have applications in healthcare devices for the detection and treatment of various diseases.8,9There is also a wide range of biosensing devices that have been developed for agriculture and environmental monitoring.With the help of such biosensors,the recognition—as well as quantification—o particular biomolecules can be easily achieved.10

FIG.1.Schematic of the main components of a nanomaterial-based biosensor,showing its biorecognition elements,nanomaterials,transducer,and signal processor.

Biosensors have a broad range of applications from the health industry to environmental safety,food-quality monitoring,defense,security,and agriculture.Biosensors are widely used in diagnostics and monitoring11as they are cost-effective,miniaturized,and easy to use.Thus,in recent years,the focus has shifted toward the development of point-of-care devices for healthcare management.Various new fabrication techniques have also come to light in the past few years,and this gives a new direction for the advancement of biosensors.The focus of this review is on recent advances in the fabrication of biosensing devices.New fabrication techniques are discussed,along with the recent progress in the modificatio and synthesis of new forms of nanomaterials.

II.EMERGING NANOMATERIALS USED IN FABRICATION OF BIOSENSORS

Nanomaterials are usually synthesized through specifi selfassembly processes at the nanoscale level.They have several unique attributes,including distinct electronic,magnetic,optical,and mechanical properties.Recently,advanced nanomaterials have started to play an essential role in drug delivery,tissue engineering,battery materials,multifunctional materials,and the fabrication of biosensors.Nanomaterial-based biosensors have altered the outlook for conventional biosensors by improving their performance in terms of sensitivity,stability,and selectivity for the detection of various biomolecules.Nanomaterials include single or combinatorial(hybrid)nanostructures of varied forms that can be synthesized with distinctive features,including nanotubes,nanorods,nanospheres,nanoreefs,nanoclusters,tetrapods,12nanopores,and nanochannels.Nanochannels are protein-based synthetic ion channels that provide an interesting platform for biosensing applications by mimicking natural systems.Nanochannels are a very promising research area,and they exhibit extensive potential applications in diagnostics,security,and nanoimprinting.Advanced nanomaterials can bring novel opportunities for the mass production of advanced and robust biosensors.13–17

Various types of advanced nanomaterials and their properties are listed in Table I.These advanced nanomaterials can be categorized as being either 2D transition metals or 2D organic polymers.Materials in both categories have been used extensively in the last few years due to their distinct properties (Fig.2).The 2D transition metal materials are light and stable,and they exhibit high catalytic and optical activity as well as good electrical conductivity.Transition metal dichalcogenides (TMDCs) and metal oxides show unique polymorphism,and they are able to mimic biocatalytic activity;thus,these nanomaterials are widely used by researchers for diagnosis as well as treatment of several diseases.In comparison,2D organic polymers are widely used for their remarkable properties,which include high photothermal activity,abundant electroactive sites,and an excellent ability to capture electrolyte ions.These are properties that help with the development of the most advanced biosensing platforms for disease detection.Table I describes the uses of advanced nanomaterials in biosensor development and gives a comparison of their operational characteristics.As shown,the available data regarding sensitivity and detection limits show that 2D transition metals have better sensitivity and lower detection limits(∼3.4 fM).In particular,biosensors fabricated using black phosphorous have exhibited very low detection limits,specificall 8 pM or 7.8 pg/mL.

A.2D transition metals

The elements of groups 4 to 11 in the periodic table are considered as transition metals.Transition metal nanomaterials are useful in the fabrication of biosensors due to their interesting properties such as a large surface area and an ultrathin planar structure.Both TMDCs and transition metal oxides(TMOs)exhibit remarkable nanomaterial properties;they both have important nanomaterial-based qualities that can be used for the fabrication of biosensors.They also possess the ability for fluorescen quenching and have significan biocompatibility.

TABLE I.Uses of advanced nanomaterials in biosensor development and a comparison of their operational characteristics.

1.Transition metal dichalcogenides

The TMDCs are known as an advanced family of 2D graphene cognate/nanosheets,and they are generally represented by the formula MX2,indicating a hexagonal layer of a transition metal(M) and two layers of chalcogen (X).They can be synthesized by using several methods,including mechanical cleavage,epitaxial growth,and chemical vapor deposition (CVD).A TMDCbased enzyme biosensor using titanium disulfid (TiS2) has been fabricated for detection of glucose.18TiS2possesses high conductivity and has unique polymorphism;it was doped with niobium to form TiS3nanobelts to fabricate a selective and sensitive second-generation electrochemical biosensor.In the same study,the researchers also used Ti0.95Nb0.05S2/GOx/GTA nanomaterial,and they claimed that this is a superior material for glucose detection.

A surface plasmon resonance (SPR)-based biosensor employing a hybrid structure of blue phosphorene (BlueP)/TMDCs and graphene with Ag–Au bimetallic film was recently fabricated.The results of this study indicated that the monolayer BlueP/MoS2and graphene structure enhances the sensitivity of the biosensor by 19.73% in comparison to conventional biosensors.19It is easy to synthesize MoS2,and it exhibits remarkable optical and electrical properties.Recently,an acetylcholine (Ach)-based biosensor was fabricated by using MoS2and palladium nanoparticles.This biosensor showed good reliability,accuracy,and the potential for clinical diagnosis of Ach in real samples.The biosensor was found to have an excellent linear range of 1.0 nM to 10 mM with a detection limit of 1.0 nM.Figure 3 shows plots of the current–voltage(C–V)responses of AChe-Cho/Pdnano/MoS2-modifie gold electrodes with different substrate concentrations.20

In another study,tungsten disulfid (WS2) nanosheets were used to fabricate a photoelectrochemical immunosensor for the detection of carcinoembryonic antigen.21A gold nanoparticle(GNP)/WS2nanocomposite was developed.The conducted study described that WS2integrated with GNPs showed enhanced photoelectrochemical responses.The nanocomposite was coated onto a glass surface followed by immobilization of antibodies specifi to carcinoembryonic antigens to obtain photoelectrical immunosensing of the antigen in clinical samples.The detection limit of the biosensor was calculated as 0.5 pg/mL[at S/N(signal to noise ratio)=3].Glycated hemoglobin has also been recognized by a WS2nanosheet-based biosensor.22The work showed that a B-PVA-WS2[Boronic acid modifie poly(vinyl alcohol)] nanohybrid is fluores cently quenched in the presence of HbA1c.Recently,another interesting work reported the use of diblock molecular beacons with polycytosine(polyC)tails,which were attached with MoS2nanosheets as probes for microRNA detection.23It was found that the combination of polyC-mediated molecular beacons and MoS2resulted in very low background-noise signals.High sensitivity was shown,with a detection limit ∼3.4 fM.According to the findings this will be helpful in the quantitative analysis of microRNA in clinical research.

FIG.3. (a) Biosensing response (C–V) plots of AChe-Cho/Pdnano/MoS2@Au-ET with different acetylthiocholine chloride (ATCl) concentrations.(b) 3D representation of the cyclic voltammogram data of AChe-Cho/Pdnano/MoS2@Au-ET obtained with different ATCl concentrations.(c)Standard curve of AChe-Cho/Pdnano/MoS2@Au-ET at 50 mV/s between −0.4 V and 0.6 V.Reproduced with permission from Ref.20.

Two-dimensional TMDCs are also capable of antibody-free recognition of pathogens.This was studied recently,and three types of 2D TMDCs namely WS2,WS3,and MoSe2with dextran were used.24The dextran/TMDC hybrid expressed a higher affinit forE.colibacteria thanE.coli-specifi antibodies and aptamers.A biosensor coated with vanadium disulfid (VS2)nanosheets has been designed to detect the glucose concentration in fruit juices colorimetrically.25The researchers found that 2D nanosheets of VS2mimic stable peroxidase activity;therefore,they proposed that these nanosheets could be used to replace horseradish peroxidase because of their robustness,availability,and reusability.A combination of two different TMDCs can enhance the performance of a biosensor.This can be seen in a study in which WS2and MoS2were used to fabricate a biosensor with a non-ssDNA probe for utilization in biomedical applications.26The WS2and MoS2quench the fluores cence of a constructed Arg-rich probe peptide,and the hybridization between this probe peptide and analyte collagen sequences causes fluorescenc recovery.Quantum dots made from 2D TMDCs have also shown their significanc in the optical sensing of neurotransmitters without any antibodies.27

2.Advanced transition metal oxides

Similar to TMDCs,TMOs exhibit various unique properties,including both electrocatalytic and magnetic properties,which make them a great option for use in the fabrication of biosensors.In recent years,several studies have demonstrated their exploitation as an nanomaterials.Among the various TMOs,manganese oxides are the most-used advanced materials in sensing applications due to their unique electrical,optical,and chemical properties when compared with other TMOs.A recently conducted study used a combination of TMOs and 2D transition metal carbides to fabricate a biosensor.28The fabricated biosensor was based on metal–oxide framework-derived 3D MnO2/Mn3O4and 2D Ti3C2MXene/Au nanoparticle nanocomposites for the determination of organophosphorus pesticides.These nanocomposites showed high environmental biocompatibility and remarkable electrochemical performance.A low detection limit of 1.34×10−13M was found.

A molecular-imprinted polymer-based biosensor has been designed for protein separation and purification29In this study,MnO2nanosheets were grown on the surface of carboxyl-modifie tubular carbon fibers The results showed that the formation of MnO2shells enhances the number of protein-imprinting sites.For the detection of hydrogen peroxide,an electrochemical biosensor was fabricated with a MnO2–graphene nanosheet nanocomposite on a glassy electrode.30The results of this study demonstrated that the electrocatalytic activity of TMOs is helpful in the oxidization of H2O2,resulting in the catalytic activity of the biosensor,which can be used to detect H2O2even at very low concentrations.

Recently TMOs have been used by several researchers for detection and therapy of tumors,and they are known to have excellent photocatalytic activity.In one study,a new photosensitizer was developed for photodynamic therapy of cancer.31MnO2nanosheets,along with selenophenol—a substitute for BODIPY—were used for the fabrication of a biosensor to provide selective activation in the tumor environment.Similarly,in another study,MnO2on polydopamine nanospheres was used as a nanotheranostic agent for cancer.32The researchers designed a nanoflower of Polydopamine@ultrathin manganesedioxide/methylene blue(PDA@ut-MnO2/MB) and these are capable of modulating the tumor microenvironment through oxygen generation and glutathione depletion.In this study,the intramural blood flo and glucose distribution were boosted by the photothermal effect,which was responsible for increasing efficac of photodynamic therapy.Nanoflowe composite-based magnetic resonance imaging was also suggested in this study.In a similar study,a nanoflowe composite was synthesized by coating polyphosphazene onto Fe3O4nanoclusters,followed by fabrication of MnO2nanosheets on the outer shell.33Interestingly,the researchers named this an“intelligent nanoflower because of its great performance in bothin vivoandin vitrostudies.

A nanohybrid has been constructed using a TMDC and a TMO for cellular imaging and fluorometri detection of glutathione.34The study suggests that this nanohybrid will be effective in bioanalysis and intracellular imaging.In another study,MnO2nanosheets were used in combination with polyethylene glycol-cyclic arginineglycine aspartic acid tripeptide as photodynamic and photothermal agents for the treatment of cancer.35These biodegradable MnO2nanosheets exhibited high photothermal conversion performance and remarkable colloidal stability for use in synergetic photothermal/photodynamic cancer therapy.A newly developed point-ofcare device for detection of urolithiasis has been synthesized by fabrication of 3,3′,5,5′-tetramethylbenzidine-manganese dioxide nanosheets,and this can measure oxalate in urine samples within 10 s.36The results showed a detection limit of 0.91μM.It has also been found that TMOs can exhibit biomimetic properties,and MnO2was used in a recent study to fabricate a sensitive biosensor for the detection of acetylcholine,a neurotransmitter.37This was an enzyme-based biosensor that showed a linear range of 0.1 to 500μM.

Nanoparticles of another TMO,copper oxide,were synthesized by direct pyrolysis of metal–organic gels,and these were found to be capable of mimicking peroxidase activity and can thus be used for glucose and cholesterol detection.38The fabricated nanoparticles showed detection limits of 0.59μM and 0.43μM for glucose and cholesterol,respectively.

B.2D organic polymers

Organic polymers have been used very frequently in recent years,and they are continuously in demand due to their remarkable properties such as high flexibilit and tunability,ultrathin structure,and adaptivity.Among this group,the most widely used organic polymers are modifie metal–organic frameworks and polypeptides;they have emerged as the materials of choice in the fabrication of biosensors.

1.Metal–organic frameworks

Metal–organic frameworks (MOFs) have a cage-like structure that consists of crystalline porous hybrid materials made up of inorganic and organic materials.Generally,organic“linker”molecules are found all around positively charged metal ions.The hollow structure of an MOF provides an extremely huge internal surface area,and this makes them extraordinarily useful.

To use the properties of MOFs,advanced polymer nanosheets have been designed using [Ba(DPA)2(H2O)2]nnanostructures (in which DPA refers to dipicolinic acid) doped with Eu3+and Tb3+ions.39The results showed a high relative sensitivity of 2.42% K−1,and they can be reused.These nanostructures are useful in the detection of anthrax biomarkers with a detection limit of 0.03 nM.Organic polymer-based MOFs can also be used in photodynamic therapy for tumors.40A MOF-based biosensing device has also been developed for the detection of neurotransmitters by incorporating platinum nanoparticles to amplify the signals.41The fabricated biosensor exhibited a good linear range(0.01–500μM)with a very low detection limit(0.01μM),and the biosensor showed half-life of four months when stored at 4○C.

A theranostic nanoplatform was fabricated using an MOF encapsulating 2D palladium nanosheets and doxorubicin with polydopamine to increase biocompatibility.42This combination exhibited remarkable photothermal conversion and optoacoustic effects and was used as a drug-delivery component.In combination with other materials,these nanomaterials can therefore effectively be used in sensing,imaging,and drug-delivery applications.They can also be used in bacterial eradication and therapy.MOF-derived 2D carbon nanosheets have been exploited to fabricate a biosensor for augmented anti-infective therapy.43In this work,a ZnOdoped carbon-on-graphene sheet was synthesized and anchored with phase-transformable thermally responsive brushes byin situpolymerization.The fabricated device could easily be switched on and off,and it possessed high photothermal activity,showing the remarkable properties of the nanostructure for broad-spectrum eradication of bacteria at very low concentrations.

2D organic polymers can also mimic many biomolecules,and they are therefore used in the synthesis of various nanostructures for drug delivery,therapy,and the monitoring of various diseases.A 2D MOF was used as a nanozyme sensor for detection of phosphatase and its enzymatic hydrolysis.44Phosphatase is an important enzyme that is actively involved in several biological activities,and it can be harnessed for the identificatio of diseases.

Ni-MOF nanosheets have been used for fabrication of a colorimetric H2O2sensor for human serum,showing a wide linear range of 0.04–160μM and a low detection limit of 8 nM.45These sensors have promising roles in food and environmental analysis and clinical medicine.Thus,2D MOFs have been used as advanced nanomaterials in distinct biosensor applications.For example,MOF-based aptasensors and DNA sensors have been found to be useful for the detection of heparin and cocaine.46–48

2.Black phosphorous

Black phosphorous (BP) is the most thermodynamically stable allotrope of phosphorous.A monolayer of BP—also known as phosphorene—has significan properties as a nanomaterial exhibiting good electroconductivity,an ambipolar fiel effect,high carrier mobility,and an adjustable band gap.They are frequently used in the fabrication of nanobiosensor devices.In recent years,many researchers have used BP to fabricate biosensors for detection of and therapy for various diseases.In 2020,a novel work was presented in which a 3D BP nanoscaffold was applied for restoring neurogenesis,angiogenesis,immune homeostasis,and post-trauma tissue regeneration.49Numerous studies have applied BP as a photothermal agent.For example,2D BP nanosheets(BPNSs)have been designed with cellulose hydrogels for photothermal therapy against cancer.50These 3D networks of hydrogels and BPNSs exhibit good flexibility good photothermal response,and enhanced stability.Moreover,this nanoplatform is completely harmless and biocompatible,showing its distinctive applications in this clinical study.In another interesting work,a 3D-printed scaffold was designed with BP for the treatment of osteosarcoma by photothermal ablation,and this takes part in bone regeneration.51Thus,the photothermal properties of BP make it very popular among researchers for the development of nanodevices for disease management.

Near-infrared-II-responsive carbon dots coated onto BP nanosheets have been used for cancer therapy.52There are several studies indicating modificatio in BP for fabrication of sensing devices;for instance,BP–gold nanocomposites have been used in the fabrication of microfluidi aptasensors for detection of okadaic acid,53as well as for detection of bisphenol A and 17β-estradiol.54In the firs study,the detection limit was found to be 8 pM with a linear range of 10–250 nM,and a point-of-care device to perform on-farm assays in fishin units was proposed.In the second study,cDNAs were self-assembled on a 2D nanohybrid consisting of BP and gold to give BP-Au@T-cDNA nanostructures,which showed detection limits of 7.8 pg/mL and 92 pg/mL for bisphenol A and 17β-estradiol,respectively.

An advanced fabrication technique for biological Surface-Enhanced Raman Scattering (SERS) analysis using a 2D finger print nanoprobe based on BP has been demonstrated.55In this study,flake-shape BP–gold nanoparticle nanohybrid theranostic nanoplatforms for drug delivery,bioimaging,and phototherapy were developed.Biosensors incorporating BP have also been used in genome editing,56antibacterial performance,57photodynamic antitumor therapy,58imaging,and cancer theranostics.59

III.DISTINCT PLATFORMS IN FABRICATION OF ADVANCED BIOSENSORS

In the past few years,more advanced biosensors have been developed using several techniques.They can be categorized into four types based on their operating principle:electrochemical,optical,thermal,or piezoelectric.These can be further divided into immunosensors,aptasensors,DNA sensors,cell-based sensors,and enzyme-based sensors by the biorecognition element they use.Techniques including focused ion beams,microfluidics near-fiel electrospinning,lab on a chip,and chip calorimetry are widely used for the development of biosensors based on different principles.The most advanced techniques and their principles,advantages,and disadvantages are summarized in Table II.

The fabrication of integrated electrochemical sensors is an important step towards the development of fully integrated and truly wireless platforms for many local,real-time sensing applications.Micro-and nanoscale patterning of small surface areas of electrochemical sensors enhances their performance by overcoming surface-based limitations.Thus,this is the key to the successful miniaturization of integrated platforms.Several types of biosensors based on these techniques have recently been described,and they exhibit remarkable sensitivity with very low detection limits.These are reviewed and summarized in Table III.They have been fabricated for the detection of various biomarkers/diseases including cancer antigens,cholesterol,cortisol,viruses/bacteria,microRNA,and exosomes.

A.Focused ion beam

The focused ion beam (FIB) technique is particularly used in site-specifi analysis,the deposition of nanomaterials,and the cutting of materials.Recently,the use of the FIB technique in the development of nanosensors has been growing continuously.In this technique,an ion beam attached to a probe is used for the manipulation and development of various nanotechnological sensing devices.It is a highly reliable technique that shows several advantages in the fabrication process.60There are distinct modification that have been performed to develop more reliable nanosensors using the FIB technique.One study described the fabrication mechanism and resolution limit of nanogaps that were designed using FIB CVD.61A 5-nm-long and 42-nm-high nanogap was synthesized for use in fabrication.In another study,FIB milling was used for writing surfacerelief gratings onto the surface of a multimode optical-fibe core.62The fabricated device was compact,of low cost,and could be used as a refractive-index sensor in various biological and chemical sensing applications.The refractive index unit(RIU)sensitivity of the device was calculated to be 70 nm/RIU for a refractive index range of 1.333 to 1.345.In recent years,helium FIB milling has also been used in the fabrication of sensors,achieving a high milling resolution.63,64

B.Near-field electrospinning

Although the near-fiel electrospinning technique has been used for more than ten years,researchers have made several modifi cations to develop biosensors according to their requirements.With this technique,one can shorten the electrode gap distance and successfully deposit a nanomaterial on a sensing platform.This technique has evolved from electrospray technology,in which a polymer melt is drawn by an electrical force.65,66M13 bacteriophage nano-and microfiber have been designed using this technique.67The phage fibe pattern of choice was fabricated with tunable direction and spacing with the help of a field-electrospinnin technique,with the aim of developing a future phage sensor with uses in various applications such as tissue regenerative medicine and water purification

In 2017,a biosensor was fabricated for tissue engineering.68The study described the use of 3D near-fiel electrospinning to construct a 3D polymeric fiber—poly(methy methacrylate)—pattern on nonconductive materials including 3D collagen gels loaded with cells in an automated manner.Various fibe patterns for translation of unique cellular patterns were also discussed.

The near-fiel electrospinning technique has also been used for the alignment of dipoles in poly(γ-methyl L-glutamate)(PMLG),a biocompatible material.69The piezoelectric properties of the resulting fibrou materials were enhanced.In this study,a biosensor was fabricated on a glass substrate to observe the effect of fibrou PMLG with poly(ethylene oxide) on the proliferation of mouse fibroblas cells,resulting in a decrease in cell proliferation.An electrospinning technique was also used in the fabrication of low-cost and highly sensitive biosensors for the detection of cancer-related antigens.70

TABLE II.Summary of advanced biosensor techniques and their principles,advantages and disadvantages.

TABLE II.(Continued.)

C.Laser-based nanofabrication of photonic structures

A laser can be employed in the fabrication of highly sensitive multidimensional photonic nanostructures,providing higher resolution power.Dual-wavelength femtosecond laser irradiation has been used in scalable manufacturing of patterned functional plasmonic nanostructures.71This is an advanced technique for the formation of plasmonic nanostructures involving abnormal doublepeak energy deposition using dual-wavelength synthesis of the firs fundamental frequency (ω) and the second-harmonic frequency(2ω)of a femtosecond laser.Deposition of nanoparticles and sensor fabrication were performed in a controlled manner using this dual laser peak.

Laser irradiation of 1064 nm at a power density of 1 W/cm2has been used to increase the temperature at a tumor site to almost 70○C.72A soluble polyvinylpyrrolidone microneedle was created from 2D Nb2C nanosheets,and this may function in medical implantation and photothermal removal of superficia tumors.The experiments were carried outin vivoin a mouse model,effectively indicating suppression of tumors with the survival of mice in comparison to a control group.Thus,the technique could be used for the management of various diseases.

D.Paper-based microfluidics

Paper-based microfluidi devices are based on the principle of fluidi actuation.These devices are easy to use and very low in cost.Cellulose paper is used to fabricate integrated printed microfluidi paper devices,and this involves designing them using a computer followed by printing.The printing can be achieved through several methods,including wax printing,laser cutting,and using polydimethylsiloxane(Fig.4).The wax printing method is of comparatively lower cost than other methods,and it is therefore widely applied.Paper-based microfluidi devices can perform all the sensing steps on paper,acting as a“lab-on-paper”technique.73This technique has been used for the fabrication of paper-based CMOS photodiode sensing devices for the diagnosis of sepsis.74In this work,laser cutting was used for the fabrication of microfluidi channels on paper strips,and the device was designed in such a manner that multiple metabolites could be detected simultaneously.With the help of this device,glucose and lactate biomarkers have been studied in human serum and quantifie for diseases associated with sepsis.

A 3D paper-based device having a glucose meter has also been developed for biosensing of silver ions.75This glucose meter was used for sensitive and quantitative monitoring of the output signals generated by the biocatalytic reaction between glucose and glucose oxidase enzymes.The detection limit was calculated as ∼58.1 pM (3σ) for the specifi detection of an analyte.Another work described the fabrication of a microfluidi device for simultaneous detection of multiple cancer-related biomarkers on a single device.76This study was dependent on aptasensor-based electrochemical detection of both carcinoembryonic antigen and neuronspecifi enolase on a single device for a clinical sample of lung cancer.The device was fabricated by two printing methods—wax printing and screen printing—and it was coated with aminofunctional graphene,i.e.,Nitrogen doped graphene-thionine-gold nanoparticle (NG)/(THI)/(AuNPs) and Prussian blue/Poly(3,4-ethylenedioxythiophene)/gold nanoparticles (PB/PEDOT/AuNPs)nanocomposites,respectively.The nanocomposite coating was followed by immobilization of the concerned aptamers of carcinoembryonic antigen (CEA) and neuron-specifi enolase (NSE).The results showed that the detection limit of the device was 2 pg/mL for CEA and 10 pg/mL for NSE.

FIG.4.Steps involved in the fabrication of a paper-based microfluidic device.

Recently,the focus has been shifted towards paper-based pointof-care biosensing devices due to their cost effectiveness,reusability,and high selectivity for detection and quantificatio of glucose in oral fluids77,78A paper-based microfluidi aptasensor device was fabricated for the colorimetric detection of cocaine,and the study indicated the significanc of this device in the screening of forensic samples.79Additionally,a pen-on-paper strategy for writing a paperbased microfluidi biosensor was demonstrated for point-of-care testing.80The proposed strategy involved two custom-made pens,which were respectively used for the fabrication of wax channels and electrode writing.The device showed a colorimetric detection limit of 1 nM forSalmonella typhimuriumDNA and 1 mM for electrochemical detection of glucose.

E.MEMS

Microelectromechanical systems (MEMS),nanoelectromechanical systems (NEMS),and bionanoelectromechanical systems(BioNEMS)are fourth-generation biosensor fabrication techniques working at micro-nanoscale levels.These are emerging techniques that are widely used in nanotechnology,providing important features in the fabrication of biosensors for the determination of chemical and biological components.81,82BioNEMS are believed to represent an important approach to the fabrication of biosensors for the sensing of highly complex biological components.In contrast,MEMS are applied more broadly in silicon-based microelectronics and micromachining technology.

MEMS technology has been used in the development of sensing platforms for diagnosis of diseases,therapeutics,and monitoring of health and the environment.A miniaturized silicon chip has been fabricated for the detection of the genomes of bacteria and viruses using real-time polymerase chain reactions(RT-PCR).83This device exhibited accurate thermal control and an enhanced optical signal when compared to conventional RT-PCR.A MEMS-based biosensor for cholesterol detection has been reported showing an immobilized selective enzyme,cholesterol oxidase,on carbon interdigitated electrodes having gold nanoparticles on their surfaces.84In this study,interdigitated 3D carbon electrodes were fabricated and further coated with gold nanoparticles through electrodeposition.The detection limit and sensitivity of the device were reported as∼1.28μM and ∼993.91μA mM−1cm−2,respectively.To detect antibiotic sulfapyridine,which is used in prophylactic and therapeutics,a biosensor was developed utilizing a fully integrated electrochemical Bio-MEMS.This biosensor included gold microelectrodes modifie with magnetic nanoparticles and further coated with poly(pyrrole-co-pyrrole-2-carboxylic acid).The device was reported as a useful tool in industrial food control.

BioNEMS techniques can also be used for the fabrication of various useful devices to detect pathogens,biomolecules,environmental pollutants,and cancer.Approaches based on MEMS can be used to perform chemical analysis on a single chip by combining laboratory processes,and this is known as“lab-on-a-chip”technology.Their fabrication involves the scaling of single or multiple laboratory processes into a chip format,and MEMS is the basis for most lab-on-a-chip fabrication processes.

1.Lab on a chip

Lab-on-a-chip technology is a subset of MEMS devices,and devices using this technology are sometimes known as micro total analysis systems (μTAS).A lab on a chip is a device with a size of the order of a few centimeters that integrates laboratory techniques for one or more analyses.Multiple applications,including clinical diagnostics,DNA analysis(sequencing),electrophoretic separations,and biochemical detection,can be performed using labon-a-chip techniques.85When focused on biochemical operations,research into these miniaturized devices can minimize their cost and increase their sensitivity and diagnostic speed.This can also potentially minimize exposure to dangerous chemicals.The development of lab-on-a-chip techniques has mainly relied on core techniques such as microfluidic and molecular biology.The developed devices are composed of a network of electrodes,millions of microchannels,and electrical circuits(Fig.5).86The microchannels provide simple handling of fluid in very low quantities(of the order of picolitres)and they can be used to achieve biochemical reactions at these very limited volumes.Previously,microfluidi techniques have also been used to fabricate MEMS.

In a recent study,the quantity of unbound cortisol—a hormone that is linked to mental health—present in saliva was determined by a polymer-based lab-on-a-chip system based on a microfluidi capillary flo assay.87A fluorescenc analyzer was used for quantitative detection.The results showed a linear range of 7.0 pg/mL–16.0 ng/mL.In another study,a device mimicking the movement mechanism,microenvironment,and multicellular construction of a human lung was developed.88The device combines several techniques like microbiology,polymer science,bioengineering and microfluidic to mimic microenvironment,physiochemical features,cell-cell interfaces,multicellular construction of a human lung.The study aims to evaluate tissue and organ functionality since it shall be creating great potential for advancing the studies about drug discovery,disease etiology and organ physiology compared with two dimensional(2D)and three dimensional(3D)cell culture models and current organoids.In this study,history of research on lung anatomy and physiology,techniques of recreating lung functionality such as cell cultures in 2D and 3D models.Similar to this“lung on a chip”,“heart on a chip”devices have also been developed on sensor-free or sensor-based chip platforms.89

F.Chip calorimetry

Calorimetry is the science of the measurement of the heat produced or absorbed by a system.The use of calorimeters is continuously increasing due to their increasingly diverse range of applications.Through advances in fabrication techniques,calorimeters have been miniaturized to the form of a chip.Chip calorimeters have been used as label-free biosensors,and they exhibit high-throughput capabilities,functioning as an insulator and thermometer.90Chip calorimetry was used for the determination of anaerobe involvement in the energy metabolism ofTrypanosoma cruzi.91Enzymatic activity has also been accurately measured using a chip-based microflu idic calorimeter.92Such miniaturization of devices may cause problems such as a decrease in volumetric heat generation and incomplete mixing of samples.Therefore,the researchers introduced a new calibration method that makes it simple to use the device for enzyme engineering and drug-discovery applications.

G.SPR-based biosensor

Surface plasmon resonance is a technique based on the stimulation of electrons at the interfaces of materials by incident light.The phenomenon is based on the resonance and oscillation of these electrons in response to a particular wavelength which further enhance the light intensity (Fig.6).93An SPR biosensor was recently fabricated for application in the real-time monitoring of a gluten-free diet using the urine of people with Celiac disease(an autoimmune disease).94Monoclonal antibodies specifi to the 33-merα2-gliadin antigen (the main immunogenic peptide of wheat gluten) were immobilized on the surface of the device for the detection of gluten immunogenic peptides.The detection limit of this device was calculated as 1.6–4.0 ng/mL.Furthermore,a biosensor using the SPR technique for detection of the Ebola virus has also been fabricated.95To develop this sensor,a gold SPR chip was coated with 4-mercaptobenzoic acid,followed by immobilization of the recombinant nucleoprotein of Ebola.This was then used for the detection of the interaction of the monoclonal antibodies mAb1,mAb2,and mAb3 with the immobilized nucleoproteins.The researchers found that,in comparison to other techniques,mAb3 has a higher affinit when using SPR.This study revealed that SPR can also be used for screening other monoclonal antibodies.

FIG.6. Diagrammatic representation of the basic principle involved in surface plasmon resonance based transducer.

The fabrication of a highly sensitive biosensor for detection of a biomarker microRNA was achieved in another study.96In this process,an enzyme-free SPR-imaging technique was exploited for microRNA detection based on a catalytic hairpin assembly.The resulting detection limit was 53.7 fM.Other SPR techniques have been used for fabrication of DNA sensors,97biomimetic sensors for detection of urinary tract infections,98and sensors for the detection of pathogens99and the sickle hemoglobin allele.100A pointof-care device was developed for the detection of stroke biomarkers through modificatio of SPR by a coating of functionalized SiO2.101The detection limit of this device was less than 1 ng/mL.An SPR technique was also used to fabricate a biosensor coated with Au nanofilm-graphene/D-typ fibe for detection of nucleotide bonding between dsDNA structures and distinguishing mismatched DNA sequences.102

H.Whispering-gallery mode biosensors

Whispering-gallery mode(WGM)biosensors are a highly sensitive technology for the detection of various biomolecules and chemical ions.They can be used for the analysis of surface geometries and for examining any modificatio occurring on the surfaces materials.They are based upon the principle of waves traveling on a concave surface.103The WGM technique can be used for the ultrasensitive detection of biomolecules.

An optical immunosensor device using a WGM resonator technique was fabricated for the detection of grapevine virus A-type(GVA)proteins.104In this device,ZnO nanorods(NRs)were coated onto a silicon surface using atmospheric-pressure metal organic CVD.The silicon/ZnO-NRs structure can act as a WGM resonator by generating quasi WGMs which were observed experimentally in infrared and visible ranges of photoluminescence spectra.In this study,the surface of silicon/ZnO-NRs was modifie with anti GVA antibodies to develop an immunosensor.the interaction of analyte with this developed platform resulted into an opposite shifts of the WGMs peaks obtained in the visible range.The device exhibited high sensitivity in an analyte concentration range of 1–200 ng/mL.A WGM device using an optofluidi microbubble resonator was developed for the detection of lead ions in drinking water.105GR-5 DNAzyme was coated onto the microbubble with the help of auxiliary piranha and ploy-l-lysine solution.The detection limit was calculated as approximately 15 fM,and the refractive-index sensitivity was 265.2 nm/RIU.In another study,urea was detected by a liquid-crystal 4-cyano-4′-pentylbiphenyl microdroplet-based biosensor based on a WGM lasing technique.106Detection of heavy-metal ions has also been achieved using the same strategy.107

With the help of WGM,protein conformation and monitoring has also been achieved.108In this study,WGM was combined with microfluidic for the real-time detection of protein secretion.Digital microfluidic has also been combined with WGM for labelfree detection of biomolecules.109This approach could help in the detection of cancer biomarkers,potentially playing a key role in determining different stages of cancer.

I.SERS-based biosensors

Biosensors based on SERS show remarkable sensitivity and produce high analytical signals.The SERS technique is based on the principle of surface plasmons.110Many modification have been made to this technique to make it more sensitive for detection of exosomes.

A SERS technique was used in an immunoassay for detection of prostate cancer antigen.111In this study,an Fe3O4@TiO2@Au nanocomposite was used for the fabrication of an immunosensor.The detection limit of this device was calculated to be as low as 0.014 mM.The study concluded that this recyclable SERS technique exhibited an ultralow detection limit of 1.871 pg/mL.Thus,the device could potentially be used in clinical cancer diagnosis.

An application of SERS to the visualization of antidepressant analogues in the brain has been described,and this technique is potentially beneficia in imaging.112In brief,the SERS technique combined with fluorescenc microscopy allows visualization of alkynylated S-citalopram,an inhibitor in the neuronal membrane,brain vessels,and parenchyma.A label-free SERS platform with a high-dimensionality artificia nose-based biosensor has been used for detection and measurement of complex biological matrices.113Another modifie technique is tip-enhanced Raman spectroscopy for fabrication of a biocompatible mica/gold surface,and this was used for DNA and protein detection.114SERS can also be used inin situbioanalysis of the safety of certain foods in fiel applications.115

The applications of aptamer-based SERS sensors in quantitative analysis and biotherapy have been demonstrated.116The fabricated biosensor exhibited various applications for the analysis of biomolecules,pathogens,biomarkers,and fungal toxins.As noted earlier,SERS technology is commonly used in the detection of exosomes.Recently,a modifie structure of gold as“nanostars”(NSs)was used to form a nanocomposite AuNS@4-mercaptobenzoic acid @Au with a B-Chol-labelled DNA anchored to form a SERS nanoshell.117The interactions between exosomes and the nanocomposite occurred by forming an immunocomplex that ultimately produced enhanced SERS signals.The detection limit was calculated to be as low as 27 particles perμL.

IV.CONCLUSION

Over the past few years,there have been significan achievements in the fabrication of biosensors to provide better health management.However,there are still opportunities for the further development,implementation,and acquisition of unique properties in nanomaterials in various disciplines.The exploitation of advanced nanomaterials in the fabrication of biosensors has presented new opportunities for diagnosis.Advanced 2D transition metals and metal oxides could be used to improve the stability and sensitivity of biosensors.Owing to their fluorescen quenching ability,modifie TMDCs and TiS2have shown significan biocompatibility and are perfect for the fabrication of biosensors for the detection of various disease biomarkers.Nanomaterials including vanadium disulfid and copper oxide,which can mimic peroxidase activity,can also improve the performance of a biosensor.The biocompatible nature of these nanomaterials results in better conductivity and sensitivity,making it easier to develop point-of-care devices.

Advanced nanomaterials such as MoS2,WS2,and MOF have high conductivity and,in novel combinations with other nanomaterials,they may lead to biosensors with very low detection limits.Moreover,various forms of nanomaterial have been integrated into controlled drug-delivery systems and used as biocompatible materials in nerve and tissue repair and the development of better and more miniaturized sensors.Several new techniques such as nearfiel electrospinning present as potentially useful tools for the future fabrication of phage-based chemical sensors and tissue regenerative materials.The advantages of lab-on-a-chip techniques include automation,high-throughput portability,and reduced sample size,along with much shorter reaction and analysis times.Similarly,SERS and WGM techniques have remarkable technical advantages in newly developed biosensors.These newly developed fabrication techniques can be used for point-of-care devices,and their miniaturized structure and easy handling make them convenient for health-monitoring applications.

Various developments can be anticipated to result from the use of these new fabrication techniques using advanced nanomaterials.In the future,several modification of nanomaterials and biosensors are likely to accelerate healthcare diagnosis,treatment,and monitoring.Diseases such as cancer are a major challenge because they can lack early detection methods and reliable therapies.Research has been carried out into the development of nano-biosensors for early detection of cancer and other diseases,and with continuous modification in nanomaterials and fabrication techniques,it is hoped that advanced therapy for successful eradication of diseases can be achieved.

The future focus should be on developing point-of-care devices for diagnosing multiple diseases on a single platform.Point-of-care diagnosis,along with desirable measurement of the clinical characteristics of dangerous diseases such as malaria,HIV,cancer,and diabetes,has now been made possible with the development of nanobiosensors.At present,biosensors that have been developed for various diseases to facilitate proper diagnosis of medical conditions have been successfully miniaturized for diagnosis of disease biomarkers in real samples.However,there is still much research required into the point-of-care diagnosis of cancers,and no commercial biosensors have yet been reported for this purpose.In this review,we have reported the use of various advanced nanomaterialbased biosensors for disease diagnosis.Yet,there is still a lack of availability of commercialized biosensors.Therefore,we suggest that future research should be focused on developing and commercializing more advanced and easier-to-handle devices.

ACKNOWLEDGMENTS

This work was supported by the Department of Science&Technology (DST) (Grant No.TDP/BDTD/33/2019),the Science and Engineering Research Board (SERB) (Grant Nos.EMR/2016/007564 and YSS/2015/000023),and the Biotechnology Industry Research Assistance Council (BIRAC) (Grant No.BT/IIPME0211/02/16).

AUTHOR DECLARATIONS

Conflict of Interest

The authors have no conflict to disclose.