Review—Recent Advances in Carbon Nanomaterials as Electrochemical Biosensors

The allotropic modification of Carbon, known as fullerene, was discovered in 1985 by H. W. Kroto, R. F. Curl, and R. F. Smalley.¹⁰ It was the first nanomaterial to be successfully isolated. The characteristic feature of fullerenes is the formation of a number of atomic C_n clusters (n > 20) of carbon atoms on a spherical surface. The carbon atoms form covalent bonds with each other in the sp² hybridization in fullerenes. They are most commonly present on the surface of the sphere at the vertices of pentagons and hexagons. C₆₀ is the fullerene that has been extensively studied and investigated. It has highly symmetric spherical molecules consisting of 60 carbon atoms, present at the vertices of 20 hexagons and 12 pentagons or 60 Carbon atoms comprising of 12-five member rings and 20-six member rings.¹¹ The diameter of fullerene is 0.7 nm.¹² Fullerenes have been used in the medical field such as in cancer therapies, MRI and for gynecological malignancies.^13–17

Synthesis

One of the allotropic modifications of carbon, known as Carbon nanotubes (CNTs) were discovered in 1991 by the Japanese scientist S. Ijima.²⁷ In CNTs, each carbon atom with 3 electrons forms trigonally coordinated $\sigma$bonds to three carbon atoms by using sp² hybridization.^27–29 CNT is basically one layer of Graphene rolled in the form of a hollow tube seamlessly. The rolled Graphene sheets stacked in cylindrical/tubular structures with a diameter of several nanometers is the characteristic feature of carbon nanotubes. The CNTs can have variable length, diameter, the number of layers and chirality vectors (symmetry of the nulled Graphite sheet). Based on their structures, CNTs can be divided into two basic groups: single walled Carbon nanotubes (SWCNTs) and multi-walled Carbon nanotubes (MWCNTs).^30,31 The SWCNTs have a diameter around 1–3 nm and a length of few micrometers whereas MWCNTs have a diameter of 5–25 nm and a length around 10 μm. However, recently the synthesis of CNTs with a length of 550 nm has been investigated and reported.³² The CNTs have excellent physical properties like rigidity, strength and elasticity as compared to other fibrous materials. They do posses high value of aspect ratio (length to diameter ratio) than other materials. The high aspect ratios of CNTs may vary from 10² to 10⁷. The larger aspect ratio comes out for SWCNTs than MWCNTs as a consequence of their smaller diameter. Not only this, they do posses high thermal and electrical conductivities in comparison to other conductive materials. The strength of CNTs is 10–100 times larger than the strong steel at a fraction of steel weight.³³

The one layer of Graphene in CNTs can be rolled in different ways. Based on the rolling of Graphene sheets, the CNTs are classified as zigzag, armchair, chiral, depending on the number of unit vectors in the crystal lattice of Graphene along two directions in honey comb structure. The chirality has a significant effect on the properties of CNTs. The electrical properties of SWCNTs are a function of their chirality or hexagon orientation with respect to the tube axis. The chirality decides whether a particular CNT is metallic or semiconducting in nature.³⁴ The electrochemical properties of SWCNTs depend upon the roll-up vectors (n, m). If the roll-up vectors n-m = 3q where q is any integer/zero, the SWCNTs are metallic. If n-m = 3q, the SWCNTs are semi conductive in nature.^35,36 If n = m, the nanotubes are known as armchair. If m = 0, they are known as zigzag, otherwise they are known as chiral.

Also, the SWCNTs can exhibit electrical conductivity or semi conductive properties that depend upon the diameter of the tubes.^37–41 The armchair SWCNTs have electrical conductivity more than that of copper whereas zigzag and chiral SWCNTs do display semi conductive properties for their use in sensor fabrication.^40,42 The MWCNTs are composed of multiple Carbon layers with inconsistent chirality and can display extraordinary mechanical attributes instead of exceptional electrical characteristics. These nanomaterials do posses such characteristic feature that makes them potential candidates for use in technological fields. The CNTs have been used as an electrode in electrochemical reactions due to their significant electron transfer capabilities.⁴³ They can be used in electrochemical sensors as they do have the ability to make electron transfer possible in chemical reactions at the electrode interface.^44–47 The CNTs find immense applications in the field of nano-electro-mechanical systems.^48–50 Table I shows the values of significant physical, electronic and mechanical characteristic features of CNTs.

Synthesis

It is a 2D allotropic form of Carbon comprising of a single layer of Carbon atoms. The Carbon atoms exhibit a hexagonal crystal lattice joined to each other by $\sigma$and $\pi$ bonds in sp² hybridization with an interatomic distance of 0.142 nm of Carbon hexagons. The Graphene was first explored, searched by a Canadian theoretical physicist, P. R. Wallace in 1947 whereas the samples were later investigated by a Dutch-British physicist A. Geim and a Russian-British physicist K. Novoselov.^82–84 Although, the theoretical investigations on the Graphene have been conducted extensively, the real material has been synthesized only recently. The research on the characteristic features of Graphene is still going on. It do possess extremely high mechanical rigidity and a high thermal stability. The electrical properties of this carbon allotrope basically distinguish from the properties of 3D materials. Graphene is a building block of other allotropes of carbon as it can be wrapped up, rolled up cylindrically or stacked up to get 0D fullerenes, 1D carbon nanotubes and 3D Graphite respectively.⁸⁵ Thus it depicts the structural element of some other Carbon allotropes such as fullerenes, CNTs and Graphite. The Graphene rolled into 0D buckyballs, 1D nanotube and stacked up into 3D graphite is shown in the Fig. 1.⁸⁵

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Graphene is a semiconductor material with zero band gaps, ambipolar electric field with charge carrier mobility more than 15000 to 20000 cm² Vs⁻¹ at room temperature. It possesses excellent mechanical, physical, chemical and thermal properties and is transparent to light up to 97.7%. That's why it is a potential candidate for the application in highly sensitive electrochemical sensors.^86–89 The mobility of electrons in the layers of Graphene is one hundred times more than that in Silicon.⁹⁰ That's why it is predicted that one day it will replace Silicon in the electronic industry. Graphene finds immense application in sensors due to its large specific surface area and high charge carrier mobility.^91,92 Table II shows the values of significant physical, electronic and mechanical characteristic features of Graphene.

Synthesis

Reduced graphene oxide (rGO)

The electrochemical reduction method has also been applied to obtain reduced Graphene oxide (rGO).¹¹⁶ Various reduction methods have been employed to reduce Go partially to form reduced Graphene oxide (rGO) by using laser radiation,¹¹⁷ annealing¹¹⁸ and chemical methods.¹¹⁹ However, the harsh use of chemicals for oxidation degrades the properties of Graphene by damaging the basal plane of the Graphene. That's why peeling off of Graphene from Graphite is done under suitable solvents and surfactants.^94,95 The Graphene has a tendency to aggregate into Graphite in some solvents. Thus it is difficult and challenging to prepare pure and uniformly dispersed single layer Graphene in the solvents. The mechanical peeling of the Graphite is done to obtain pure 2D Graphene by making use of the adhesive tapes⁴ that has lesser density of defects. The structures of Graphene based nanomaterials are shown in the Fig. 2.¹²⁰

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Another allotrope of carbon, Carbon Nanodiamonds (CNDs) is the nanoparticles with the crystal structure of Diamond, and exhibit excellent properties of diamond.^121,122 The CNDs consist of a crystalline Diamond core which is surrounded by a anion like amorphous Graphite shell.¹²³ They do possess very small size, large surface area and large adsorption capacity for the attachment of chemical to biological molecules.^124,125 They exhibit exceptional hardness, thermal conductivity, refractive index, coefficient of friction, insulation characteristics and have very low toxicity.¹²⁶

The CNDs display fluorescence due to the presence of a complex defect N-V, containing nitrogen (N) and a vacancy (V). Since CNDs are chemically stable, their photo luminescent behavior can be used for the several in-vivo and in-vitro applications.¹²⁷ The CNDs are the potential fluorescent probes for use as biomarkers and in bio labeling studies.¹²⁸

Synthesis

Carbon Nanohorns (CNHs) are one of the allotrope of Carbon consisting of closet cages of Carbon atoms with a diameter of 2–5 nm and length 40–50 nm.¹⁵⁹ They are more beneficial to use than CNTs as they can be synthesized at a larger scale at room temperature without any use of metal catalysts. They can be synthesized by using arc discharge of Carbon rods,¹⁶⁰ laser ablation of pure Graphite¹⁶¹ and Joule heating. The CNHs do posses high surface area and good porosity which can be exploited for their potential application in the field of biosensing.^161,162

The Carbon Dots (CDs) are zero-dimensional CNMs consisting of Carbon atoms with a size below 10nm. These materials do possess significant electronic and optical properties as exhibited by Quantum Dots.¹⁶³ They do possess low toxicity, stability and biocompatibility for their application as electrochemical biosensors.^164–166 The CDs have been synthesized by using laser ablation method applied to the Carbon atoms.¹⁶⁷ The various processes like pyrolysis,¹⁶⁸ hydrothermal synthesis,¹⁶⁹ electrochemical methods¹⁷⁰ and microwave synthesis¹⁷¹ have been used to synthesize CDs. They can also be prepared by using the soot of the candle flame.¹⁷²

CDs can be classified into Carbon Quantum Dots (CQDs) and Graphene Quantum Dots (GQDs). The CQDs and GQDs have a diameter range from 1 to 10 nm. The GQDs consist of Graphene layers of size less than 10 nm. They can be synthesized by using thermal plasma jet technique with low fabrication cost. They can be an alternative to the nanodiamonds.^173–176

Carbon Nanofibres (CNFs) are cylindrical wire shaped nanostructures in which graphene sheets are piled in different arrangements such as ribbon-like, platelet or herringbone. The length of CNFs varies in order of micrometers and can be up to 10 μm whereas their diameters vary from 10 to 500 nm. Their mechanical strength and electric properties are just like that of CNTS.¹⁷⁷ As a consequence of stacking of graphene sheets with different shapes in different arrangements, CNFs have more edge sites on their outer walls in comparison to CNTs. The presence of edge sites makes it feasible to transfer electrons with electro active species in solution and the detector substrate.^178,179 The CNFs do possess attributes like good electrical conductivity, large surface area, biocompatibility and easy fabrication process that are vital for electrochemical sensing applications. Moreover, CNFs can be easily functionalized to suit a particular detection mechanism.

Synthesis

The CNFs can be prepared by employing arc discharge¹⁸⁰ and laser ablation¹⁸¹ methods. The thermal chemical vapour deposition (CVD),Plasma enhanced chemical vapour deposition(PECVD) and electro spinning have also been employed for the preparation of CNFs.¹⁸² During thermal CVD method, a compound containing hydrogen and carbon is thermally decomposed by employing a metal catalyst at a constant temperature.¹⁸³ This method is further divided into three types based on the way in which the catalyst is employed i.e. Substrate method, Spray method and Gas phase flow catalytic method.

During PECVD method, the high energy electrons present in the plasma collide with the gas molecules. As a result, they transfer their kinetic energy to them, thereby, causing excitation, ionization and decomposition which results in production of CNFs.^184–186

During electro spinning process, the polymers like silk, DNA, collagen and polyester have been used to obtain CNFs. The polymeric solution is firstly subjected to a potential of very high volts for getting charged, then to a spinning port where it is moved at a very fast rate. As a result, the nanofibres get deposited at the collecting plate in the form of a mat. The fiber mat undergoes oxidation and is carbonized in nitrogen atmosphere to produce CNFs.^187–190 The CVD produces CNFs with impurities which require a further complicated purification process whereas electro spinning produces CNFS through a very easy process with high purity.^191,192 The Fig. 3 shows the SEM images of alignment of CNFs grown on a Silicon substrate in plasma growth process in the presence of electric field (a) & (b) and CNFs exposed density (c).¹⁹³

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The Carbon black (CB) is a nanomaterial prepared from the combustion of petroleum products. They are the nanoparticles spherical in shape and are strongly bonded to each other to form aggregates. The size of Carbon black particles varies from 3.0 to 100nM. The significant physical, electronic and mechanical characteristic features of Carbon black are given in Table III. The conductivity of Carbon black can be enhanced by heating up to 7000 °C because more number of electrons in sp² hybridization state with delocalized pi-bonds is available for the conduction of current.¹⁹⁴ Due to their large surface area; a large number of oxygenated groups are formed at the edges of the Carbon black nanoparticles. It is the presence of sp² hybridized Carbon atom edge planes and oxygenated groups over the Carbon black nanomaterials that make them capable to attach biomolecules on their surface to act as electrochemical biosensors. They can be used for the detection of analytes for the biosensing applications.

The Carbon black nanoparticles can be prepared by employing furnance, channel and acetylene processes.^201,202 The preparation process is very easy and has low cost. The properties of Carbon black can be easily tailored by introducing other materials such as polymers²⁰³ or metallic nanoparticles into them for better electrochemical sensing applications.²⁰⁴

The physical and chemical properties of nanoparticles can be engineered by subjecting them to functionalization, by attaching some molecules on their surface.²²² The CNTs are not soluble in aqueous solutions but when they undergo oxidation in a mixture of acids, the carboxylic groups attach to the surface and side walls of the nanotubes making them soluble in aqueous solutions. Thus, functionalization has proved to be a boon to the CNTs for modifying their physical and chemical properties.^222,223 The functionalization of CNTs with chemicals or suspension in a surfactant containing solution results in decrease in their bundle formation.²²⁴

The most commonly used tailoring technique for CNTs in order to enhance their electrochemical sensing performance (sensitivity and selectivity) are covalent or non-covalent functionalization. The biomolecules are attached to the surface of CNTs by covalent or non-covalent functionalization. In covalent functionalization, the various chemical functional groups like carboxylic and amine groups are attached to the surface and side walls of CNTs by certain chemical processes. These functional groups on the CNTs react with the functional groups present in the bimolecular structure resulting in the formation of a covalent or a non-covalent bond. The CNT functionalized with poly (amidoamine) dendrimer through covalent functionalization has been used for the attachment of glucose oxidase and Horseradish peroxidase.²²⁵ The use of covalent functionalization for the attachment of biomolecules on CNTs for sensing glucose,^225,226 H₂O₂,²²⁷ aflatoxin,²²⁸ carcinoembroyonic antigen detection²²⁹ have been reported. The amine functionalized CNTs interact with the amino groups on biomolecules such as enzymes, proteins and nucleic acids. The glutaraldehyde, active ester or epoxy has been used to attach the amine containing biomolecules to the CNTs.^226,229

An electrochemical immunosensor has been developed for the detection of carcinoembryonic antigen in saliva and serum in which monoclonal anti-carcinoembryonic antigen antibodies are attached on polyethylene amine treated MWCNTs side walls by using covalent functionalization with the help of glutaraldehyde.²²⁹

The vertically aligned SWCNTs are attached on a GCE with a covalent bonding²³⁰ or on a gold surface by diazonium has been studied and investigated. The SWCNTs modified with diazonium have displayed the highest electron transfer in cellobiose hydrogenase from phanerochaete Sordida with small values of lactose oxidation potential.²³¹

However, the covalent functionalization of CNTs has an influence on its intrinsic properties as the change in CNT surface by covalent attachment can cause hybridization to change from sp² to sp³. As a consequence of it, the mechanical strength and electrical properties could be hampered due to the decrease in conjugation abilities of the CNTs.^232,233

The non-covalent functionalization of CNTs has been significant for attaching the biomolecules on CNTs as it does not affect the intrinsic properties of CNTs. As a consequence of that the mechanical and electrical properties are not affected.^232,233 The CNTs are non-covalent functionalized as a result of pi-pi electrostatic interactions between the CNTs and the biomolecules.^233,234 The adsorption of aromatic molecules and benzene derivatives on the surface of SWCNTs has been achieved due to the establishment of pi-pi interactions between the CNTs and the benzene derivatives.²³⁵ The aromatic compounds have been employed for attaching the biomolecules on the surface of CNTs by non-covalent functionalization such as ferrocane,^236–238 anthracene,^239–241 pyrene^242–244 etc for the development of electrochemical biosensors. The non-covalent functionalized CNTs by aromatic compounds have been used for the bioelectrocatalysis of oxygen,^{239,242,245–248} glucose biosensors,^236,237,249 H₂O₂ detection,²⁵⁰ ethanol biosensor,²⁵¹ and trichloroacetic acid biosensor.²⁵² The polymers have been employed for the non-covalent functionalization of CNTs for use in biosensors. The polymers like polypyrroles,^253,254 glycolipids,^255,256 polyethyleneimine^257–260 have been used for the non-covalent functionalization of CNTs. The commonly used tailoring techniques for CNTs have been functionalization with conducting polymers,^261–263 mixing with surfactants or polyelectrolyte,^264–266 using metal oxides or nanoparticles,^267–270 adding enzymes^{56,266,271–275} and doping with heteroatom.^55,276

The first use of CNT electrochemical biosensor was reported in 1996 where it was used for the detection of dopamine.²²⁰ The CNT biosensor exhibited better detection in comparison to other Carbon based sensors due to the fact that there was the presence of pores due to the packaging of CNTs as a result of which its surface area was increased for the attachment of dopamine.²⁷⁷ The formation of oxygen containing groups at the surface of nanotube during oxidation process has also a significant effect on the detection process with CNTs. The multi-walled carbon nanotubes have been mixed with bromoform to obtain a paste which is packed into a glass tube forming a CNT Carbon paste sensor. The CV and DPV characterization techniques have been employed to study the oxidation of dopamine by the traditional Carbon sensor and CNT based carbon paste sensor.²²⁰

The dopamine has also been detected in the presence of ascorbic acid by employing MWCNTs functionalized with Sodium dedecyl sulphate on a Ta substrate. The DPV was employed to detect the dopamine in the presence of ascorbic acid. However, the detection levels were in the micrometer range i.e. limit of detection is 3.75 μM for dopamine in the presence of ascorbic acid with range from 0.02mM to 0.2 mM which restricts the applicability of the sensor for the practical use.²⁶⁴ The modification of a MWCNTs electrode with RuO₂ has an influence on the enhancement of limit of detection using DPV than the MWCNTs. A much higher concentration of dopamine, ascorbic acid and uric acid in the μM to mM range have been tested using this investigation and the electrode is highly selective to the presence of dopamine, ascorbic acid and uric acid.²⁶⁷

The Over Oxidized polypyrrole (OPPY)-MWCNT-modified Glassy Carbon Electrode (GCE) has reported an enhanced sensitivity to the detection of dopamine. The electrochemical deposition has been employed to tailor MWCNTs with cadmium oxide (CdO) nanoparticles of size approx. 50 nm diameters. The resulting CdO/MWCNT sensor does not exhibit any signal response to uric acid, ascorbic acid and dopamine up to 100 μM but it has a better selectivity for H₂O₂.²⁶⁹ The enzyme laccase has also been used to detect the presence of dopamine in the presence of ascorbic acid and 3, 4-dihydroxyphenyl acetic acid (DOPAC). In this technique, the limit of detection of 0.4 μM has been reported with high selectivity.²⁷¹ The Tyrosinase and Nafion has been employed for the detection of dopamine by using MWCNTs but the limit of detection was reported to be very low of 0.5 μM.²⁶⁶ The doping of CNTs with certain elements can customize their electronic properties, conductivity and mechanical strength.²⁷⁶ The doping of CNTs with Boron for the detection of dopamine is significant due to the presence of more functionalized groups at the defect sites of CNTs and presence of extra edge plane sites.⁵⁵ The limit of detection of 1.4 nM has been reported in Boron doped CNTs which has been enhanced by incorporating Boron in CNTs. The SWCNTs functionalized by over-oxidized polypyrrole has been electro-copolymerized on the surface of the electrode.^262,263 The films of over-oxidized polypyrrole allowed the cations of dopamine to pass through them but repelled ascorbic acid and serotonin thus exhibiting better selectivity.²⁶¹ The use of untreated DWCNT has been reported to be the best choice for the development of amperometric sensors. The CV, XPS and BET analysis have shown that the smaller length DWCNTs are more effected by the acid functionalization which decreases the electro active area of the respective modified electrode due to which the amperometric sensitivity in the detection of dopamine and catechol on the functionalized DWCNTs modified electrode is decreased.²⁷⁸

The MWCNTs, SWCNTs, GO, rGO and CQDs have been employed for the electrode modification in the detection of dopamine. However, due to the presence of one type of nanostructures, these materials almost provide same sensitivity and linear range of detection for dopamine. The linear range and lower limit of detection can be improved by employing the hybrid or composite materials of Carbon nanostructures.²⁷⁹ The electrochemical functionalization plays a very important role in increasing limit of detection and sensitivity in dopamine sensor with the SWCNT electrode. The limit of detection of approx. 100 nM and 1 μM has been reported for the functionalized electrode and as-fabricated electrodes respectively. The functionalized SWCNT electrode exhibited better selectivity to dopamine in the presence of ascorbic acid and uric acid than the as-fabricated electrodes.²⁸⁰

The CVD synthesized CNT fibers has been used as a sensing electrode to detect the presence of glucose. The CNT based biosensors have exhibited a very fast amperometric response to the presence of glucose.^281–283 The glucose based CNTs have been extensively investigated and reported.²⁸⁴

A glucose biosensor has been fabricated by using phase separation method by employing MWCNT-grafted chitosan (CS)-nanowire (NW) to which glucose oxidase is attached to obtain the biosensor. The electrochemical detection of glucose was done by employing cyclic voltammetry and amperometry. The fabricated biosensor exhibited a high sensitivity of 5.03 μA/mM in a concentration range of 1–100 mM and a low response time to the detection of glucose. The MWCNTs-CS-NW facilitates the conduction of electrons between glucose oxidase and target molecules.²⁸⁵ In another study, Glucose oxidase was attached on CNT nanoelectrode ensembles by covalent bonding with the formation of amide linkages between their amine residues and the carboxylic acid groups present on the tips of CNTs. The release of H₂O₂ from the enzymatic reaction of glucose oxidase upon the glucose and oxygen on CNT nanoelectrodes causes the detection of glucose.²⁸⁶

In another study, a biosensor employing Ni-nanoparticles dispersed in vertically aligned CNTs grown on Si/SiO2 substrate has been reported in which the Ni-nanoparticles have deposited uniformly inside and on the top of the CNT forest. The fabricated biosensor exhibited a sensitivity of 1433μAmM¹cm² with a limit of detection 2 μM over a linear range between 5 μM to 7 μM.²⁸⁷ In another study, CuO nanoparticles deposited on the side walls and tips of vertically well-aligned MWCNTs array by a two step electro deposition method has been investigated. The CuO-modified MWCNT electrochemical biosensor exhibited a limit of detection of 800nM and a very high sensitivity of 2190 μAmM⁻¹cm⁻² with a linear range response up to 3.0mM glucose concentration with rapid response and good stability.²⁸⁸ The Pt nanoparticles loaded CNTs composites have been prepared under hydrothermal conditions by polymerization reaction of glucose and reduction deposition of a platinum source in the pores of anodic alumina membranes. The fabricated electrode has been used as a amperometric sensor for the low potential detection of H₂O₂. The as-prepared Pt-CNT glucose biosensor displayed a limit of detection of 0.055mM with a linear range of 0.16–11.5 nM glucose concentration with a very high sensitivity and selectivity.²⁸⁹ Another CuO-MWCNTs fabricated biosensor exhibited a high sensitivity of 2596 μAmM⁻¹cm⁻² to the detection of glucose with a limit of detection of 0.22 μM in a linear range over a concentration up to 1.2 mM.²⁹⁰

The CNT based electrochemical biosensors have been fabricated by using dehydrogenase enzymes such as alcohol-dehydrogenase,²⁹¹ phosphatase,²⁹² D-fructose dehydrogenase and glucose dehydrogenase. A biosensor employing alcohol-dehydrogenase enzyme along with MWCNT and poly (vinyl alcohol) has reported a response time of about 8 s for the ethanol detection. The electro oxidation of NADH which is produced during the enzymatic activity produces a current that is taken into account for generating the response by the biosensor.²⁹¹ Other electrochemical biosensors have been fabricated by using acetyl cholinesterase, alkalinephosphatase, organophosphorus hydrolase and urease enzymes. The voltammetric biosensors have been fabricated by attaching urease and acetyl cholinesterase enzymes on the surface of the SWCNT modified electrode by means of sol-gel material for the detection of urea and acetylthiocholine respectively.²⁹¹ In another study, alkaline phosphatase enzyme is attached on the surface of CNTs in the presence of streptavidin by using layer by layer method.²⁹³ The lactate level monitoring is significant for the use in biotechnology, food processing and in sports medicine. The amperometric detection of the level of lactate has been done by using CNT and mineral oil paste having lactate oxidase.²⁹²

The electronic structures of SWCNTs have an influence on the detection in CNTs enzymatic biosensor. The [FeFe]-hydrogenase enzyme from clostridium acetobutylicum when attached to metallic SWCNTs have reported better electro catalytic activity.²⁷³ This is because of the better coordination between clostridium acetobutylicum redox-active sites and the electron surface.²⁷³ The glutamate hydrogenase was attached to the top of CNTs by covalent bonding to fabricate a glutamate biosensor that has been able to display the limit of detection up to 10 nM. The CNT electrodes are better candidates for the fabrication of enzymatic biosensors with a high sensitivity and high selectivity.²⁷⁴

An electrochemical biosensor for the determination of tyrosine has been reported. The sensor was fabricated by dissolving polysulfone (PSF) in dichloromethane and depositing it on a GCE. The nitric acid functionalized MWCNTs are drop coated on the PSF layer. The Tyrosinase enzyme (TyO_x) is deposited on MWCNT/PSF/GCE after cross linking with glutaraldehyde. The characterization of the biosensor has been done by using the CV and electric impedance spectroscopy. The biosensor exhibited a very low limit of detection 0.3 nM and a very high sensitivity 1.988 μAμM⁻¹cm⁻² to detect the tyrosine.²⁹⁴ The doping of CNTs by nitrogen has an influence on the electrochemical performance for the detection of H₂O₂ and has been used to make H₂O₂ based enzymatic biosensor at a lower potential.^295,296

The Hydrogen peroxide biosensors have been fabricated by using enzyme horseradish peroxidase(HRP) in which the enzyme is attached on the surface of MWCNTs with the aid of mediator methylene blue and by crossing linkage between HRP and BSA composite film.^297,298 A hydrogen peroxide detector has been reported by employing CNTs modified with Pt nanoparticles fabricated by means of chemical reduction method on the surface of a waxed graphite electrode.²⁹⁹ The glucose oxidase was attached on the side walls of SWCNTs in order to detect the presence of glucose. The glucose oxidase attached SWCNTs exhibited an enhancement in conductance upon adding glucose and thus acting as a biosensor for the enzymatic activities.⁵⁶

The cellular prion protein has been detected by making use of a fluorescent-label aptamer which involves the quenching of the fluorescence by non-covalent modification of the MWCNTs with the aptamer. But the quenching is restored in the presence of cellular prion protein. The biosensor exhibited a limit of at least 4.1 nM. The sensor demonstrated outstanding selectivity for the cellular prion protein in the presence of amino acid and other proteins.³⁰⁰ The SWCNTs optical biosensor has been reported for the detection of protein-protein interaction with limit of detection 10 pM.³⁰¹ The insulin has been detected by SWCNTs upon functionalization with insulin-binding aptamer.³⁰² The CNT electrochemical biosensors for the detection of nucleic acids have been investigated vastly and have been brought in notice.^303–308

A highly sensitive electrochemical biosensor based on CNT-bioconjugates and SWCNT forest platform has been reported for the detection of prostate specific antigen (PSA) detection by using horseradish peroxidase (HRP)/secondary antibody ratio. The device exhibited a limit of detection of 4 pgmL⁻¹ in 10 μl calf serum without any dilution.³⁰⁹ With the replacement of SWCNTs with gold nanoparticles customized MWCNTs, the limit of detection of PSA changed to 0.40 pgmL⁻¹,thereby, significantly enhancing the sensitivity and selectivity of detection.³¹⁰ The CNT based biosensors have been coated with the antibodies for the detection of the biomolecules/disease causing agents. The CNTS-arrayed electrodes coated with anti-PSA antibodies have been reported to detect PSA.³¹¹ The immunosensors have been reported for the detection of osteopontin that causes prostate cancer. They have been fabricated on the glass substrate which is coated with SWCNTs with attached osteopontin molecules on their surface.³¹² Another biosensor, A CNT (EDC/NHS functionalized) transistor and an antibody which is a hybrid to the genetically engineered antibody have been used for the detection of osteopontin.³¹³ The DNA strands functionalized MWCNTs and SWCNTs have been reported for the detection of PSA in the blood samples with more sensitivity and selectivity.³¹⁴ The electrochemical biosensors used for the detection of prostate cancer biomarker using nanoparticles have been briefly reviewed and reported.³¹⁵

The amount of blood cholesterol level has been detected by the amperometric electrochemical biosensors.^316–318 The amperometric biosensors employing sol gel chitosin/silica and MWCNTs organic-inorganic nanohybrid composite material has been reported for the detection of cholesterol in the blood.³¹⁸ The cholesterol oxidase enzymes are used for the oxidation of cholesterol to 4-cholesten-3-one producing hydrogen peroxide. The electrochemical detection of level of H₂O₂ produced during the enzymatic activity gives the level of cholesterol in the blood. An amperometric cholesterol biosensor has been fabricated through layer by layer deposition of (poly (diallyl dimethyl ammonium) chloride) and cholesterol oxidase enzyme on a MWCNT electrode modified by the gold nanoparticles. A protective coating of non conducting (poly (o-phenylenediamine) film has been produced over it by means of the electrochemical methods. A limit of detection of 0.2 mM to the detection of cholesterol has been reported by the fabricated biosensor.³¹⁶

A poly dopamine coated CNT functionalized by the folic acid has been used for the detection of HeLa and HL60 cancer cells.³¹⁹ The biosensors have been reported to detect O₂ released from HeLa cells which have been fabricated from the hollow Carbon cubic (HCC) and porous Carbon cubic (PCC) nanomaterials. The etching of zeolitic imidazolate framework K-8(ZIF-8) with tannic acid followed by calcinations process produces HCC nanomaterials whereas PCC nanomaterials were obtained by direct pyrolysis of ZIF-8. The HCC and PCC are attached on the surface of Screen printed Carbon electrode (SPCE). The HCC based sensor have exhibited a limit of detection 207 nM whereas the PCC electrode have reported a limit of detection 140 nM to detect the O₂ released from HeLa cells. The selectivity of the biosensors has been examined by studying the amperometric response in the presence of interfering agents like 4-acetaimidophenol, uric acid, ascorbic acid, D-glucose and dopamine. The biosensors displayed very small current response to them, thereby, exhibiting excellent selectivity.³²⁰ The synthesis of Hollow Carbon Cubic (HCC) and Porous Carbon Cubic (PCC) nanomaterial and various steps involved in the detection of superoxide anions in HeLa cell are shown in Fig. 5.³²⁰

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The surface ITO electrode coated with an assembly of CNT multilayer and antibodies to the epithelial cell adhesion molecules have been used to detect the cancer cells of liver.³²¹ The Au-Ag alloy coated MWCNTs have been used for the detection of volatile biomarkers of the gastric cancer cells MGC-803.³²² The femtomolar level gastric cancer biomarker miRNA-106a has also been detected and reported.³²³

The carcinoembryonic antigen has been detected by the CNT based electrochemical immunosensor employing gold nanoparticles- encapsulated dendrimers as a sensor surface with the attachment of two enzymes Glucose oxidase and horseradish peroxidase. The electron transfer is facilitated by the encapsulation of gold nanoparticles in the interior structure of the device. The immunosensor has exhibited a wide concentration range from 10 pgmL⁻¹ to 50 ngmL⁻¹ and the limit of detection has been reported to be lower as compared to the ELISA test.³²⁴

A DNA hybridization biosensor has been reported based on the ssDNA and SWCNT. The ssDNA was wrapped over the SWCNT by the hydrophobic interaction. The DPV response detection technique has been applied during the DNA hybridization process. The detector exhibited a low response between 40–110 nM range and limit of detection 20 nM.³¹⁰ The hybridization of DNA targets have been examined by the CNT modified electrode for the DNA analysis with Ru(bpy) ²⁺ mediated guanine oxidation. By using this method, the hybridization of less than a few attomoles of oligonucleotide targets has been detected. The Fig. 6 shows the fabrication of DNA/Self assembled MWCNTs electrodes for the detection of target DNA sequences.³²⁵ An electrochemical DNA sensor using hollow carbon spheres (HCS) decorated with polyaniline (PANI) has been reported in which thiolated 21-meroligonucleotide is attached by the electrodeposited gold nanoparticles on the HCS-PANI substrate. The biosensor has reported a limit of detection 10fM for the detection of the target sequences of Hepatitis B virus DNA. The selectivity of the biosensor has been studied by using different types of DNA strands which comprises of a non complementary strand and a mutated one. The DNA sensor has exhibited more selectivity to the complementary strand than the other ones.³²⁶

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An electrochemical sensor employing SPCE with 1w% MWCNT and 1w% Graphene has been reported for the detection of H₂O₂ and nicotinamide adenine dinucleotide (NAD⁺/NADH) and has exhibited a limit of detection of 7.1 μM and 3.6 μF for the detection of H₂O₂ and NADH respectively with sensitivity 0.0027 μAμM⁻¹ and 0.0075 μAμM⁻¹ respectively.³²⁷

The DNA aptamer based biosensors has been employed for the detection of metal ion, organic dyes, peptides, proteins, cancer cells and infectious microorganisms.³²⁸ The DNA aptamers are the potential candidates for the biosensors as they do have small size, low cost and large stability.³²⁹ The Au-electrodes coated by MWCNTs have been used as biosensors for detecting the target DNA sequences. The DNA probes are adsorbed on the self assembled MWCNTs and methylene blue has been used as an indicator. The change in the voltammetric peak of the methylene blue has been used to detect the hybridization of DNA on the electrodes with more selectivity.³³⁰ The CV has been used for the detection of DNA with ferrocene labeled complimentary Oligonucleotide which gives reversible electrochemical response.³³¹ The hybridization of the DNA on a DNA- functionalized CNT array has been detected by the DPV with daunomycine as a redox label agent.³³² An electrochemical micro fluidic DNA sensor has been fabricated by employing carboxyl functionalized MWCNTs. The working and counter electrodes have been developed through ITO by using wet chemical etching technique. The working electrode was modified by using electrophoretic deposition of MWCNT and attachment of a DNA probe. The detector exhibited detection at a concentration as low as 1 fM with a response time of 1 min to detect the oligonucleotide. The modification by MWCNTs resulted in enhanced surface area of the electrode for loading DNA which leads to higher sensitivity and higher linear range. The biosensor has exhibited the ability to distinguish mismatch in single nucleotide and non complementary DNA sequence, thereby, exhibiting very good selectivity.³³³ An electrochemical DNA biosensor using ITO modified with curcumin have been reported to treat the Alzheimer's and artery coronary diseases by detecting APOe4 DNA.³³⁴ The various steps involved in the fabrication of electrochemical DNA biosensor employing GQDs as the surface modifier of the ITO electrode to sense Alzheimer's and artery coronary diseases are shown in the Fig. 7.³³⁴

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The CNTs can also be used as an optical biosensor for the detection of damage to the DNA. The fluorescence response of DNA tailored SWCNTs have been researched and reported. The DNA coding at the surface leads to a strong luminescence. However, the luminescence gets decreased upon the damage to the DNA. Thus, the light emitted from the nanotube can be correlated with the concentration of the DNA damaging agent by making use of principal component analytical method.³³⁵

The GCE modified with nafian coated MWCNTs and mesoporous silica MCM41 nanoparticles functionalized with hemoglobin(Hb) hybrid conjugate has been employed as a biosensor for the detection of nitride (NO₂^-) and trichloroacetic acid (TCA) exhibiting higher hypersensitivity and selectively which is attributed to the exceptional biocatlytic activity of Hb and excellent charge transfer capability of the heme group. The biosensor did not exhibit any amperometric response to the addition of cations, anions and biomolecules, thereby, exhibiting very good selectivity.³³⁶ The various steps involved in the preparation of MWCNTs-MCM41-Hb hybrid bioconjugate are shown in the Fig. 8.³³⁶

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An electrochemical phage based biosensor has been reported for the detection of E. coli by employing Polyethyleneimine (PEI)-functionalized CNT. The positively charged PEI-CNT facilitates to place the phage particle in the right orientation on the electrode. The phage detector exhibited a limit of detection of 10³ CFUmL⁻¹ and a range in between 10³ CFUmL⁻¹ and 10⁷ CFUmL⁻¹.³³⁷ The attachment and orientation of bacteriophages onto PEI-functionalized CNT on the surface of electrode and the SEM images of CNT without and with PEI-functionalization is shown in Fig. 9.³³⁷

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The first Graphene based electrochemical biosensor for the detection of glucose was reported in 2009. The device displayed a limit of detection in the range of 2–14 nM and has highly stable output. Another biosensor employing the attachment of Glucose Oxidase in a Graphene-chitosan nanocomposite has been reported which has detected glucose in a range from 0.08 mM to 12 mM with greater sensitivity and selectivity.^356,357

The Horseradish peroxidase (HP) and lactate oxidase have also been attached on the surface of Graphene which have displayed better electrochemical performance.^358,359 A porous Graphene biosensor has been fabricated by the attachment of catalase enzyme.³⁶⁰ The in-situ detection of the glucose and urea within the same sample has been made possible by modifying GCE surface by the chitosan-rGO complex. The pH of the sample gets increased by the oxidation of urea by urease and suppressing the electron transfer of Fe(CN)₆^3- whereas pH is decreased by the oxidation of glucose by Glucose Oxidase, making it feasible to transfer electrons in Fe(CN)₆³⁻.³⁶¹ A glucose oxidase enzyme based biosensor using spatially separated electrochemically reduced Graphene oxide (ERGO) by MWCNTs functionalized with 4-(pyrrole-1-yl) benzoic acid has been reported for the detection of glucose in real food samples. Through CV experiments, it was reported that the presence of ERGO in the conductive material increase the rate of electron transfer between the enzyme redox centre and the electrode surface.³⁶²

The detection of DNA plays a pivotal role in the diagnosis of various genetic or mutated medical ailments in the body. The electrochemical biosensor employed for detection of DNA has attracted the attention of researchers due to its low cost, high sensitivity and high selectivity for the detection of DNA sequences or mutated genes responsible for the various human diseases. The electrochemical DNA sensor working on the principle of direct oxidation of DNA is the simplest one. The Graphene has been used for the direct oxidation of DNA. The Graphene electrode has displayed the well resolved oxidation current signals of the free bases which are guanine, adenine, thymine and cytosine on DPV. Thus the Graphene electrode has displayed a higher electro catalytic activity to the oxidation of DNA. The electrochemical signals for the oxidized single stranded and double stranded DNA at the Graphene electrode have been well resolved.³⁶³ The rGO nanowalls deposited on a Graphite rod by electrophoretic deposition method has also been employed for the detection of four bases of DNA which displayed the highly resolved oxidation signals for the four bases of DNA.³⁶⁴

The anodized epitaxial Graphene with a large number of Oxygen related defects has been reported which has detected all the DNA bases that are well resolved on the DPV. The detection limit for dsDNA has been reported 1 μgmL⁻¹. The anodized epitaxial Graphene can be used to differentiate between the dsDNA and ssDNA and is highly selective.³⁶⁵ The single nucleotide mismatch of DNA by the hybridization can be detected by the anodized epitaxial Graphene on the DPV. The covalent grafting of the DNA sensor on anodized epitaxial Graphene provides higher sensitive and selective response in comparison to pi-stacked DNA sensor.³⁶⁶ The Graphene based sensors have been employed to detect a wide range of DNA molecules.^367,368

The fluorescence resonance energy transfer based process has been used in GO based sensor for the detection of DNA. The detection is highly selective to even a single mismatch in DNA and the limit of detection has been reported to be 40 pM.³⁶⁷ The additional surface area provided by the presence of nanoparticles, enhanced electro affinity for the analyte or improved redox properties has been provided by the Graphene in the presence of substances like gold nanoparticles,³⁶⁹ Fe₃O₄,³⁷⁰ nickel hydroxide,³⁷¹ and cobalt pthalocyanine.³⁷²

The advanced electrochemical detection of dopamine by the Graphene based sensors has been investigated extensively and reported.^373–378 The Fe₃O₄-NH₂ in Graphene has been used to detect ascorbic acid, dopamine, and uric acid which have exhibited enhanced electrochemical oxidation of these acids as compared to the Graphene electrodes.³⁷⁰ The dopamine-graphene-chitosan complex has been synthesized and electrodeposited on a GCE in a molecularly imprinted polymer electrochemical sensor in which the lower limit of detection has been reported to be 10 pM.³⁷⁹

The Graphene has been used to detect the uric acid,^370,371 ascorbic acid,^370,371 L-cysteine,³⁷² glutamate^380,381 and dopamine.^{369–371,382–384} The Graphene nanosheets have been used as immunodetectors for the detection of carcinoembryonic antigen and alpha-fetoprotein in the biological fluids. The simultaneous monitoring of both the antigens has been reported with the wide working ranges from 0.01–200 ngmL⁻¹ and 0.01–80 ngmL⁻¹ for the carcinoembryonic antigen and alpha-fetoprotein respectively with a limit of detection 1 pgmL⁻¹ for both of them.³⁸⁵ A reduced GO based electrochemical immunosensor for the detection of the antigen in Enzyme Linked Immuno Sorbent Assay (ELISA) test has been reported by employing a rGO, poly(benzene-ringpoly(ethyleneglycol)methacrylate-N-acryloxy succinimide)(poly(B.P.N.)layer) and bovine serum albumin (B.S.A.). The electrochemical immunosensor displayed a limit of detection 100 fgmL⁻¹ for the detection of antigen mouse I_gG.³⁸⁶ In another research, the Micro-cystine LR-aptamers are attached onto the Graphene surface to fabricate an aptasensor system. The sensor exhibited a wide linear range of 1–100 pM and a low limit of detection of 0.8 pM to detect the aptamer.³⁸⁷ An electrochemical aptasensor employing rGO has been fabricated for the evaluation of dynamic cell surface of N-glycan in which the concanavalin A (Con A) has been attached on the surface of dendrimer- conjugated rGO modified GCE which causes multivalent bonding between Con A and N-glycan on the surface of cell as shown in the Fig. 10. As a result of which the efficiency of aptasensor for the cell capture has been enhanced.³⁸⁸

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A critical review of CNFs based biosensors has been reported.⁴²¹ The Ni nanoparticles loaded CNFs prepared by electro spinning have been used to detect the presence of glucose. The fabricated biosensor exhibited a limit of detection 1 μM with a linear range of 2–2500 μM.⁴²² In another study, the bimetallic CuCo nanoparticles embedded in CNFs by employing electro spinning has been used to detect the presence of glucose in human urine. The fabricated CuCo-CNFs electrode has exhibited a limit of detection 1 μM with a linear range of 0.02–11 mM.⁴²³ The Pt nanoparticles loaded CNFs have exhibited a linear range of 2–20 mM to the detection of glucose.⁴²⁴ The presence of glucose has also been detected by Copper oxide nanoneedles with reduced graphene oxide with CNFs. The fabricated composite has exhibited a limit of detection 0.1 μMin the linear range of 1–5.3 mM.⁴²⁵ Another research has reported a limit of detection 0.03mM in the linear range of 0.06–6 mM to the detection of glucose for a palladium nanoparticles decorated helical CNF hybrid.⁴²⁶

The palladium nanoparticles loaded CNFs have been used to prepare an electrode which has been used for the detection of dopamine, uric acid and ascorbic acid. The fabricated biosensor has reported a limit of detection of 0.2 μM, 0.7 μM and 15 μM in the linear range of 0.5–160 μM, 2–200 mM and 4 mM for the detection of dopamine, uric acid and ascorbic acid respectively.⁴²⁷ In another study, the GCE modified with CNFs loaded with Ag-Pt alloy nanoparticles have exhibited the detection of dopamine in the presence of uric acid and ascorbic acid with a limit of detection of 0.11 μM with a linear range of 10–500 μM.⁴²⁸ The magnetic composite of polydopamine,laccase and Ni nanoparticles decorated CNFs prepared by electro spinning and high temperature carbonization method have exhibited a limit of detection of 0.69 μM for catechol in the real water samples in a linear range from 1 to 9.1 mM along with good selectivity.⁴²⁹

The vertically aligned CNFs i.e. CNFs grown perpendicular to the substrate is also the potential candidate for use in electrochemical biosensing applications. A nanoelectrode array based on vertically aligned CNFs have been reported for the detection of C-reactive protein with a limit of detection of 90 pM.⁴³⁰ The molybdenum disulphide nanosheet arrays with CNFs have been synthesized by electro spinning and hydrothermal synthesis and are employed as an electrode for the detection of Levodopa and uric acid by using cyclic voltammetry and differential pulse voltammetry.Alimit of detection of 0.91 and 0.73 μAμM⁻¹ to the detection of Levodopa and uric acid respectively in the linear range of 1–60 μM has been reported.⁴³¹

The Vertically aligned CNF arrays grown on silicon wafers has been reported to detect the DNA hybridization by employing AC Voltammetry. The oligonucleotide probe molecules functionalized CNFs hybridize with target DNA strands causing DNA hybrids due to the replacement of guanine bases from the oligonucleotide probes with Inosine groups. The signal of guanine oxidation is amplified by the presence of electrochemical mediator Ru(bpy)₂³⁺ in the solution. The study of scans of sequential AC Voltammetry detects the presence of hybridized DNA with a high sensitivity of approx.1000 PCR amplicon molecules with excellent selectivity.⁴³² Another electrochemical biosensor have been prepared by attaching an anchoring layer of chitosan, carboxyl group functionalized CNFs and glutaraldehyde on a GCE to detect the DNA hybridization. By using cyclic voltammetry, the hybridization is detected by the change in oxidation peak current of ferrocene carboxylic acid at the sensor interface. The sensor exhibited a limit of detection of 88pM in the dynamic range of 0.50 and 40nM with an excellent selectivity by exhibiting insignificant current change for non-complementary DNA targets.⁴³³

The CNFs modified onto carbon paste electrode has been used for the detection of amino acids L-tryptophan-tyrosine and L-Cysteine with a limit of detection 0.1 μM.⁴³⁴ For the early diagnosis of myocardial infection, a CNF based nanoelectrode array for cardiac troponin-I have been reported which exhibited a limit of detection 0.2 ng ml⁻¹ to the detection of cardiac troponin-I within a linear range of 0.25–1.0 and 5.0–100 mg ml⁻¹.⁴³⁵

The wild M13 bacteriophage particles have got the potential to modify the features of electrodes as a consequence of non-specific electrostatic interactions and pi-pi interactions between them and CNFs. This results into uniform distribution of CNFonto electrode surface. The cyclic voltammograms of GCE electrodes with M13 particles along with CNFs have demonstrated an improvement in their electrochemical properties. The addition of bacteriophage particles has a direct influence on the aggregation of CNFs as it prevents their aggregation. Also, with the addition of bacteriophage particles into CNFbased electrodes, the electro active surface is well produced for the electrochemical biosensing applications. The cyclic voltammetry results of electro catalytic response of oxidation of L-Cysteine have exhibited an improvement if M13 bacteriophage particles are incorporated into the CNF based electrodes.⁴³⁶

The application of Carbon black (CB) based nanomaterials for the electrochemical biosensing have been widely reported. The Carbon black has been used for the electrochemical sensing of biomolecules. The Carbon black based sensors has been used for the detection of glucose. A biosensor employing poly (Sodium 4-styrenesulphonate) attached on the CB surface has been reported that can be used for the detection of glucose.⁴³⁷ The glucose oxidase biosensors based on CB nanoparticles in electrode have exhibited a better electron transfer between the glucose oxidase enzyme and the CB electrode in comparison to other electrodes made up of SWCNTs, MWCNTs, C60, GO, rGO and SWCNHs. They have exhibited comparable limit of detection, linear range and better sensitivity.⁴³⁸ A non-enzymatic biosensor has been reported employing carbon nanoparticles functionalized carbon black composite modified GCE fabricated by microwave method to detect the presence of glucose in biological fluids and human serum samples.⁴³⁹

The CB has also been used for the biosensing of DNA molecules. A Tungsten disulphide –acetylene black composite formed by using hydrothermal process has been reported for the DNA biosensing applications. The fabricated nanocomposite with GCE electrode with Au nanoparticles has exhibited a limit of detection of 1.2 × 10⁻¹⁶molL⁻¹ in the range from 1.0 × 10⁻¹⁵molL⁻¹ to 1.0 × 10⁻¹⁰molL⁻¹to the target ssDNA.⁴⁴⁰ Another biosensor has been fabricated by using composite formed by CuS nanosheets and acetylene black(AB) by employing solvothermal process with the help of ethylene glycol. The surface of GCE was modified with CuS-AB film,later gold nanoparticles are deposited on it by the electro deposition method. The detector exhibited a decrease in current for the [Fe (CN)₆]^3-redox couple every time when there is a hybridization between the attached ssDNA probe and target ssDNA.⁴⁴¹ In another significant research, an electrochemical biosensor employing pencil graphite electrodes modified with CB has reported the detection of micro RNA-125a hybridization in samples by using impedance spectroscopy. The biosensor exhibited a limit of detection of 10pM with a linear range of 0.008–15 μgml⁻¹ with good selectivity. The fabricated biosensor has been used for the detection of micro RNA-125a in real serum samples proving its viability for diagnostic purposes.⁴⁴²

The presence of oxygenated functional groups and active sites on the CB particle surface facilitates the biosensing process. A biosensor employing GCE substrate has been modified with a mixture of hemoglobin-Carbon black (Hb-CB) which is anchored by using Nafion film. By using cyclic voltametry, it has been confirmed that there is a direct electron transfer from the Hb protein to the CB modified electrode which is attributed to the presence of oxygenated functional groups and active sites on the CB particle surface.⁴⁴³ The direct electron transfer of different Fe(III) hemeproteins attached on the electrodes modified with CB and didodecycldimethyl ammonium bromide have been thoroughly investigated and reported.⁴⁴⁴ The direct electron transfer of Cytochrome C and Hb biosensor based on CB nanoparticles have also been reported.^445,446

The Carbon black has the potential to substitute graphite powder for the fabrication of enzyme linked biosensors. The enzymatic biosensor constructed by combining carbon black paste with tyrosinase enzyme have demonstrated highest sensitivity of 625nAlμM with a limit of detection of 0.008 μM to the detection of catechol, thereby, showing electrochemical features even better than graphite and carbon nanotube tyrosinase paste electrode.⁴⁴⁷ The Bisphenol A has been used in the coating of metallic cans for storage of tomato juice samples. Its detection is very important as it can cause serious health implications. A biosensor based on laccase-thionine-carbon black –modified Screen printed electrode has been reported to detect the presence of Bisphenol A employing the use of nanostructured carbon black coupled with electrochemical mediator thionine. The fabricated biosensor has demonstrated a sensitivity of approx. 5.0nA μm⁻¹ and limit of detection 0.2 μM.⁴⁴⁸ Another biosensor capable of detecting H₂O₂ in the samples of oxygenated water and milk has been reported in which Zein, a protein in corn seed is combined with carbon black to attach the haemoglobin. The fabricated biosensor has demonstrated a limit of detection 4 × 10⁻⁶molL⁻¹ using differential pulse voltammetry.⁴⁴⁹ The urease enzyme attached to CB paste electrode modified with a flexible membrane based on terylene with polyvinyl alcohol has been used to detect the presence of urea in milk samples. The presence of urea was detected by the amperometric sensing of carbamic acid which is produced during the enzymatic conversion of urea. The biosensor exhibited a linear response in the range 2.0 × 10⁻⁴molL⁻¹ to 4.0 × 10⁻³molL⁻¹.⁴⁵⁰

An enzymatic biosensor to detect the presence of hydroquinone and Catechol has been reported in which quatnernized cellulose nanoparticles with CB and Au nanoparticles composite is used for the attachment of hemocyanin enzyme. The fabricated biosensor has exhibited a limit of detection of 8.0 × 10⁻⁹molL⁻¹ and 8.7 × 10⁻⁷molL⁻¹ in the linear range from 4.5 × 10⁻⁷molL⁻¹to 2.4 × 10⁻⁵molL⁻¹ and 3.7 × 10⁻⁷molL⁻¹ to 5.0 × 10⁻⁵molL⁻¹ for hydroquinone and catechol respectively.⁴⁵¹

Recently, a non-enzymatic biosensor PdCu/Carbon Black based biosensor has been reported that has detected the presence of H₂O₂ released from living cells in real time. The fabricated biosensor exhibited a limit of detection of 54nM with a wide detection range from 0.4 μM to 5.0mM.⁴⁵² Figure 12 shows the TEM image of PdCu particles dispersed on Carbon Black, High Resolution TEM image of PdCu alloy, Histogram graph of PdCu nanoparticles size distribution and TEM image of Carbon Black in the fabricated biosensor.⁴⁵²

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The CB based sensors has also been used for detecting the presence of pesticides in the samples.

The acetyl cholinesterase (AChE) biosensor, using unsubstituted pillar⁵arene as electron mediator, has been fabricated for the detection of organophosphate and carbamate pesticides in the samples of peanut and beetroot. The AChE is attached on the surface of CB placed on GCE by carboimide binding. The fabricated biosensor detected malaoxon,paraoxon,carbofuran and aldicarb with a limit of detection 4 × 10⁻⁶, 5 × 10⁻⁹, 2 × 10⁻¹⁰ and 6 × 10⁻¹⁰ M in the linear range of 1 × 10⁻¹¹- 1 × 10⁻⁶M, 1 × 10⁻⁸- 7 × 10⁻⁶M, 1 × 10⁻¹⁰- 2 × 10⁻⁶M and 7 × 10⁻⁹- 1 × 10⁻⁵M respectively.⁴⁵³

A screen printed electrochemical electrode has been reported for the detection of paraoxon by using enzyme butyrylcholinesterase (BChE). The CB nanoparticles are dispersed on the surface of electrode in a dimethylformamide water mixture. The BChE was attached on the surface of electrode by means of cross-linking. The fabricated biosensor exhibited a limit of detection 5 μgl⁻¹ for a concentration of paraoxon up to 30 μgl⁻¹ and stability up to 78 days of storage at room temperature in waste water samples.⁴⁵⁴