**3. MXene characterization**

The selective chemical etching of "A" in "MAX" phases have led to successful synthesis of MXenes. MAX phases are found to have elusive properties like stiff elasticity, good thermal and electrical conductivity, as well as relatively low thermal expansion coefficients and resistance towards chemical attack. There is a general formula for MXene synthesis where, "MAX" phases have a formula of M*n+1*AX*n*, with "M" meaning early d-transition metal, "A" representing the main group spelement, and "X" indicates C and/or N [24]. Hence, with this analogy, more than 70 different kinds of MXenes with different M and X are theoretically possible to synthesize. Out of these theoretical MXene types, 20 different combinations of MXenes have been synthesized successfully [25]. MXenes can conduct heat and electricity like metals and are strong and brittle like ceramics with high surface area. Exfoliated MXene exhibits higher pseudocapacitance than most capacitive materials [26]. Additionally, the MXene-have properties like a clay. Furthermore, Ca2+, Mg2+ and Al3+ ions (intercalated polyvalent cations) have all shown a huge storage power capacity [27–29]. It needs to be stressed out that energy storage capacity, high conductivity, photochemical properties, modulated surface chemistry and tunable composition make MXene and their derivatives very perspective to (bio)sensing

2D MXenes are candidates for energy storage [30] (Li-ion batteries, supercapacitors) and electromagnetic interference shielding applications [31–35] and in the form of composites become ever more useful for sensing as *e.g*. gas sensing devices [36, 37], pressure sensor [38, 39] and sensors for various analytes [40–43]. Number of other biomedical applications (such as biosensor, biological imaging, photothermal therapy, drug delivery, theranostic nanoplatforms and antibacterial agents) have become a challenge for MXenes [44]. The antibacterial properties making them potentially appealing for nanomedicine were proved for (Ti3C2Tx) MXene quantum dots [45], MXene-hybridized silane film [46], Cu2O/ MXene [47] and MXene-gold nanoclusters [48] *etc*. The multifunctional MXenes have attracted attention in biosensing [49, 50] with the aim at the ultrasensitive determination of cancer diseases related biomarkers. Examples include biosensors based on Ti3C2 MXenes-Au NPs hybrids, delaminated Ti3C2Tx MXene@AuNPs, nanohybrid of Ti3C2Tx MXene and phosphomolybdic acid (PMo12) embedded with

polypyrrole, MXene-TiO2/BiVO4 hybrid and AuNPs/Ti3C2 MXene three-

dimensional nanocomposite for detection of carcinoembryonic antigen [51], prostate specific antigen [52], osteopontin [53], CD44 [54] and microRNA-155 [55],

Generally, top-down selective etching process is used for the synthesis of MXenes [56]. Strong etching solutions containing a fluoride ion (F) such as hydrofluoric acid (HF), ammonium difluoride (NH4HF2), and a mixture of hydrochloric acid (HCl) and lithium fluoride (LiF) are used for production of MXene in such processes [57]. Since typically, the etching process results in

replacement of the M-A bond by M-O, M-OH, M-H, and M-F bonds on the surface of MXenes, the structure of MXenes can be expressed as M*<sup>n</sup>* + 1X*n*T*<sup>x</sup>* or M*<sup>n</sup>* + 1X*<sup>n</sup>* (M and X are in same form as the MAX phase and T is =O, -OH, -H, or -F) [58, 59]. A single and/or few layers of MXene can be synthetized by exfoliation or delamination of a multilayer structure of a MAX phase. The composition and electrochemical properties of MXene strongly depend on the conditions used during etching procedure [60]. As an example, application of LiF/HCl as etchant led to production of MXene with interlayers intercalated with Li<sup>+</sup> ions. Exfoliation can be

applications.

*Novel Nanomaterials*

respectively.

**220**

**2. MXene synthesis**

Since introduction of nanolayered and machinable MXenes in 2011 by Gogotsi and co-workers through wet-etching process with HF to obtain multilayered flakes of Ti3C2T*<sup>x</sup>* [13], few improvements in MXene synthesis and MXene-nanocomposite preparation resulted in various elemental composition and surface functionality [65]. In last few years the single layers of MXene were isolated adding salts or organic solvents (NH4HF2, tetrabuthylammonium hydroxide, isopropylamine) during synthesis process and resulted in delaminated MXene layers. The significant breakthrough for MXene synthesis named as "clay method" in 2014 was based on *in situ* formation of HF (LiF/HCl). The lattice *c* parameter increased to a value of ≈40 Å by applying LiF-HCl as an etchant to produce Ti3C2T*<sup>x</sup>* instead of HF etchant with a lattice *c* parameter of 20 Å [60]. The battery of techniques were employed to observe variations in the composition of Ti3C2Tx MXene produced either by HF or LiF-HCl method including nuclear magnetic resonance (<sup>1</sup> H, 13C and 19F NMR), scanning electron microscopy (SEM), X-ray diffraction method (XRD), energydispersive X-ray spectroscopy (EDS) techniques [59]. The most suitable combination presented utilization of LiF/HCl as an etchant with minimally intensive layer delamination "MILD" method instead of sonication to produce huge MXene flakes with minimum of defects [66]. Ti3C2Tx MXene has become an attractive subject of interest due its high capacitance (1500 F cm<sup>3</sup> ) in supercapacitors and an excellent high metallic conductivity (15,000 S cm<sup>1</sup> ). On the other hand there is still demand to improve stability of MXene flakes with a poor resistance in aerated aqueous suspensions resulting in oxidized form with loss of its activity for potential applications [67]. The optimization of etching process is cardinal to access single- to few-layer Ti3C2 MXene flakes. SEM technique providing information about flake size and distribution revealed formation of aggregates on the surface varying in size i.e. having few μm in size or with size larger than 10 μm in a lateral dimension. It was found out by atomic force microscopy (AFM), that thickness of single MXene monolayer was (1.1 0.1) nm for Ti3C2Tx [68]. Platinum nanoparticles with average diameter of 3 nm were homogeneously distributed on the MXene sheets surface, that was found out by transmission electron microscopy (TEM) [69]. MXene and oxidized MXene were analyzed and differentiated by applying Raman spectroscopy method providing more detailed information about the characteristic vibrational bands and the dependence thickness of Ti3C2Tx layers on Raman signal enhancement [68–71].

The electrochemical behavior employing methods like cyclic voltammetry (CV), chronoamperometry (CA), differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS) revealed significant findings related to the electrochemical activity of MXene. The electrochemical investigation of Ti3C2Tx MXene to detect significant analytes (O2, H2O2 and NADH) was performed by applying cyclic voltammetry and chronoamperometry techniques, whereas Ti3C2TX demonstrated electrocatalytic activity towards H2O2 reduction with LOD at nanomolar level [68]. Unfortunately, formation of TiO2 layer or domains with

subsequent TiO2 dissolution caused by F ions was observed during oxidation process at anodic potential window in a plain phosphate buffer electrolyte pH 7.0 leading to the decrease in electrochemical activity of Ti3C2Tx MXene.

The improvement of stability and redox behavior was achieved by further modification of MXene with nanoparticles of platinum (Ti3C2Tx/Pt) [69, 72]. The electrocatalytically active sensor based on Ti3C2Tx/Pt nanocomposite successfully determined H2O2 by CA, and moreover small organic molecules (acetaminophen, dopamine, ascorbic acid, uric acid) were selectively determined by DPV [72].

In addition electrochemical study confirmed significant differences in a negative charge density on the MXene surface as well electrocatalytic activity depending on the etchant (HF, LiF/HCl) used during MXene synthesis with preference towards utilization of LiF/HCl [60].

salt modified zwitterions to MXene was feasible, electrochemically triggered grafting of diazonium salts bearing zwitterionic pendants was more effective (**Figure 1**) [73]. Electrochemical characterization tools confirmed a much quicker spontaneous SB grafting compared to spontaneous CB grafting. Zwitterionic modification is considered as a benchmark to design antibiofouling interfaces with such modification offering to reduce dramatically non-specific protein binding compared to an unmodified MXene interface [73]. It is worth mentioning that grafting of a mixed layer composed of CB and SB can be applied to tune density of carboxylic groups and by amine coupling chemistry it is possible to finely tune density of immobilized bioreceptors for effective and efficient recognition of an analyte *via* affinity interactions [79]. Diazonium salts can be utilized in order to achieve stable modification of all surfaces (radical reaction providing most often disordered

*Electrochemically triggered grafting of diazonium salt-containing compounds to conductive surfaces. Electrochemical reduction of diazonium salt-containing compounds is feasible via freely available clouds of*

*electrons (plasmons) present in metallic nanoparticles, but also in MXene.*

*Ti3C2 MXene-Based Nanobiosensors for Detection of Cancer Biomarkers*

*DOI: http://dx.doi.org/10.5772/intechopen.94309*

*Diazonium salts can be easily synthetized from aromatic amines that are commercially available. Modification can be performed by applying different grafting methods like electrochemistry, spontaneous*

Besides application of APTES there are other strategies for modification of MXene such as self-initiated photo-grafting and photopolymerization not requiring an anchor layer, self-assembled monolayer (SAM) and initiator, applying a nature polymer, soy phospholipid (SP) improving permeability, stable cycling, and retention and PEGylation of MXene improving the water dispersibility of MXene

Recently, a novel MXene modification approach was developed by substitution or elimination reactions in molten inorganic salts. Such modification allowed to synthetize MXenes containing = O, -NH, =S, -Cl, -Se, -Br, and -Te surface terminations [82].

The hydrothermal method run in a Teflon-lined stainless steel autoclave (150°C, 5 h; aqueous solution of vitamin C and Fe3+ salt) allowed preparation of composite of MXene

Other promising nanocomposite option is represented by MXene sheets combined with metallic NPs [84–87], which can be further effectively modified by crosslinkers due to their high affinity towards MXene or by other biomolecules for final detection of target molecules/biomarkers. MXene/metallic nanoparticles (NPs) based nanocomposites can be prepared by spontaneous reduction of salts of precious metals or by applying an external reducing agent such as NaBH4. A simple spontaneous reduction of metallic salts to form Ag, Au, and Pd nanoparticles onto

with small magnetic Fe3O4 nanoparticles with an average size of 4.9 nm (TiO2/ Ti3C2Tx/Fe3O4). These hybrid magnetic nanoparticles show a great promise for selective enrichment of various biomolecules/antigens based on affinity interactions [83].

*reduction, by reducing surfaces and reagents, photochemistry etc.*

**4.2 Preparation of hybrid nanoparticles based on MXene**

oligomers ("multilayers")) [80].

**Figure 1.**

by electrostatic adsorption [81].

**223**

Aryldiazonium salts were utilized in modification of Ti3C2Tx MXene either spontaneously by free electrons or electrochemically. Electrochemical modification of Ti3C2Tx MXene by aryldiazonium-based grafting with derivatives bearing a SBor CB- betaine pendant moiety was performed by cyclic voltammetry in a potential window from 0 V to 1 V with a sweep rate of 0.25 V s<sup>1</sup> and 48 cycles. The electrochemical grafting resulted in denser CB or SB layer on MXene interface, lower interfacial resistance and an electrochemically active surface area for SB layer in comparison to CB layer [73].

In the following years the exponential increase in the number of affinity-based MXene biosensors can be expected, though it is necessary to develop advanced strategies for modification of MXene interfaces with an effort to eliminate nonspecific binding of proteins, bring in anti-fouling behavior and immobilize target biomolecules. Electrochemical methods can be employed as a useful tools for interfacial patterning, characterization of MXene-based biosensors and furthermore ultrasensitive detection of cancer related biomarkers [23].

#### **4. MXene functionalization**

#### **4.1 Covalent modification of Ti3C2 MXenes with biomolecules**

Functionalization and various methods for synthesis of MXenes can result in production of the nanomaterial with a diverse range of properties. This is why, it is very important to describe synthesis of MXenes in full details. Another point to focus on is to properly describe delamination conditions since the flake size and density of defects governs MXene's surface properties and stability. It is important to know the molecular structure of MXenes in order to decide the best application of such nanomaterial for catalysis, (bio)sensing or for chemical adsorption of various compounds.

Due to presence of -OH groups on surface, functionalization of MXene employing silylation reagents was developed by a simple reaction with triethoxysilane derivatives [74–76]. Such modification led to production of nanosheets of Ti3C2-MXene uniformly patterned by aminosilane moieties allowing NHS/EDC-based amine coupling for covalent immobilization of bioreceptors such as anti- carcinoembryonic antigen (CEA) antibodies [77].

Another viable surface modification of MXenes can be done by applying zwitterions. It was observed that spontaneous grafting of sulfobetaine (SB) and carboxybetaine (CB) derivatives onto Ti3C2Tx MXene is feasible [73]. The approach is similar to spontaneous grafting of diazonium salt modified zwitterions to gold nanoshell modified particles by consuming surface plasmons (free electron cloud) present within Au nanoshells [78]. Even though spontaneous grafting of diazonium *Ti3C2 MXene-Based Nanobiosensors for Detection of Cancer Biomarkers DOI: http://dx.doi.org/10.5772/intechopen.94309*

**Figure 1.**

subsequent TiO2 dissolution caused by F ions was observed during oxidation process at anodic potential window in a plain phosphate buffer electrolyte pH 7.0

ification of MXene with nanoparticles of platinum (Ti3C2Tx/Pt) [69, 72]. The electrocatalytically active sensor based on Ti3C2Tx/Pt nanocomposite successfully determined H2O2 by CA, and moreover small organic molecules (acetaminophen, dopamine, ascorbic acid, uric acid) were selectively determined by DPV [72].

The improvement of stability and redox behavior was achieved by further mod-

In addition electrochemical study confirmed significant differences in a negative charge density on the MXene surface as well electrocatalytic activity depending on the etchant (HF, LiF/HCl) used during MXene synthesis with preference towards

Aryldiazonium salts were utilized in modification of Ti3C2Tx MXene either spontaneously by free electrons or electrochemically. Electrochemical modification of Ti3C2Tx MXene by aryldiazonium-based grafting with derivatives bearing a SBor CB- betaine pendant moiety was performed by cyclic voltammetry in a potential window from 0 V to 1 V with a sweep rate of 0.25 V s<sup>1</sup> and 48 cycles. The electrochemical grafting resulted in denser CB or SB layer on MXene interface, lower interfacial resistance and an electrochemically active surface area for SB layer

In the following years the exponential increase in the number of affinity-based MXene biosensors can be expected, though it is necessary to develop advanced strategies for modification of MXene interfaces with an effort to eliminate nonspecific binding of proteins, bring in anti-fouling behavior and immobilize target biomolecules. Electrochemical methods can be employed as a useful tools for interfacial patterning, characterization of MXene-based biosensors and furthermore

Functionalization and various methods for synthesis of MXenes can result in production of the nanomaterial with a diverse range of properties. This is why, it is very important to describe synthesis of MXenes in full details. Another point to focus on is to properly describe delamination conditions since the flake size and density of defects governs MXene's surface properties and stability. It is important to know the molecular structure of MXenes in order to decide the best application of such nanomaterial for catalysis, (bio)sensing or for chemical adsorption of vari-

Due to presence of -OH groups on surface, functionalization of MXene employing silylation reagents was developed by a simple reaction with triethoxysilane derivatives [74–76]. Such modification led to production of

terions. It was observed that spontaneous grafting of sulfobetaine (SB) and

nanosheets of Ti3C2-MXene uniformly patterned by aminosilane moieties allowing NHS/EDC-based amine coupling for covalent immobilization of bioreceptors such

Another viable surface modification of MXenes can be done by applying zwit-

carboxybetaine (CB) derivatives onto Ti3C2Tx MXene is feasible [73]. The approach is similar to spontaneous grafting of diazonium salt modified zwitterions to gold nanoshell modified particles by consuming surface plasmons (free electron cloud) present within Au nanoshells [78]. Even though spontaneous grafting of diazonium

ultrasensitive detection of cancer related biomarkers [23].

as anti- carcinoembryonic antigen (CEA) antibodies [77].

**4.1 Covalent modification of Ti3C2 MXenes with biomolecules**

leading to the decrease in electrochemical activity of Ti3C2Tx MXene.

utilization of LiF/HCl [60].

*Novel Nanomaterials*

in comparison to CB layer [73].

**4. MXene functionalization**

ous compounds.

**222**

*Electrochemically triggered grafting of diazonium salt-containing compounds to conductive surfaces. Electrochemical reduction of diazonium salt-containing compounds is feasible via freely available clouds of electrons (plasmons) present in metallic nanoparticles, but also in MXene.*

salt modified zwitterions to MXene was feasible, electrochemically triggered grafting of diazonium salts bearing zwitterionic pendants was more effective (**Figure 1**) [73]. Electrochemical characterization tools confirmed a much quicker spontaneous SB grafting compared to spontaneous CB grafting. Zwitterionic modification is considered as a benchmark to design antibiofouling interfaces with such modification offering to reduce dramatically non-specific protein binding compared to an unmodified MXene interface [73]. It is worth mentioning that grafting of a mixed layer composed of CB and SB can be applied to tune density of carboxylic groups and by amine coupling chemistry it is possible to finely tune density of immobilized bioreceptors for effective and efficient recognition of an analyte *via* affinity interactions [79]. Diazonium salts can be utilized in order to achieve stable modification of all surfaces (radical reaction providing most often disordered oligomers ("multilayers")) [80].

*Diazonium salts can be easily synthetized from aromatic amines that are commercially available. Modification can be performed by applying different grafting methods like electrochemistry, spontaneous reduction, by reducing surfaces and reagents, photochemistry etc.*

Besides application of APTES there are other strategies for modification of MXene such as self-initiated photo-grafting and photopolymerization not requiring an anchor layer, self-assembled monolayer (SAM) and initiator, applying a nature polymer, soy phospholipid (SP) improving permeability, stable cycling, and retention and PEGylation of MXene improving the water dispersibility of MXene by electrostatic adsorption [81].

Recently, a novel MXene modification approach was developed by substitution or elimination reactions in molten inorganic salts. Such modification allowed to synthetize MXenes containing = O, -NH, =S, -Cl, -Se, -Br, and -Te surface terminations [82].

#### **4.2 Preparation of hybrid nanoparticles based on MXene**

The hydrothermal method run in a Teflon-lined stainless steel autoclave (150°C, 5 h; aqueous solution of vitamin C and Fe3+ salt) allowed preparation of composite of MXene with small magnetic Fe3O4 nanoparticles with an average size of 4.9 nm (TiO2/ Ti3C2Tx/Fe3O4). These hybrid magnetic nanoparticles show a great promise for selective enrichment of various biomolecules/antigens based on affinity interactions [83].

Other promising nanocomposite option is represented by MXene sheets combined with metallic NPs [84–87], which can be further effectively modified by crosslinkers due to their high affinity towards MXene or by other biomolecules for final detection of target molecules/biomarkers. MXene/metallic nanoparticles (NPs) based nanocomposites can be prepared by spontaneous reduction of salts of precious metals or by applying an external reducing agent such as NaBH4. A simple spontaneous reduction of metallic salts to form Ag, Au, and Pd nanoparticles onto

the Ti3C2Tx MXene sheets was applied for formation of particles exhibiting surface– enhanced Raman spectroscopy (SERS) phenomenon [85]. Moreover, an AuNP/ MXene composite boosts sensitivity of detection of oncomarker such as microRNA [88]. Similarly, the composite consisting of Ti3C2Tx MXene and PtNPs was prepared by means of *in-situ* reduction of Pt precursor (spontaneously or by external reducing agents) on MXene surface. Composite was used for electrochemical catalysis [69] and sensing of important small bioactive compounds [72]. The negatively charged acetylcholinesterase (AChE) was electrostatically deposited on the hybrid nanocomposite of MXene/AgNPs/chitosan from a mixture of the enzyme and chitosan onto MXene/AuNPs for detection of organophosphate pesticide [86].

Any conductive interface can be patterned by MXene by a simple casting of a MXene dispersion on untreated electrodes with formation of MXene layer after drying [73]. Alternatively, the electrodes can be pretreated in order to make them more adhesive for formation of MXene layer. To make surface of screen-printed electrodes (SPEs) hydrophilic for subsequent deposition of MXene, SPEs were electrochemically activated in 0.1 M NaOH by CV in a potential range from 0.6 V

enhancer were applied for quantifying acetaminophen (ACOP) and isoniazid (INZ) in blood serum samples [99]. The presence of abundant highly active surface sites due to functional groups (=O, -F and -OH) offers additional opportunity for MXene

Besides electrostatic modification of MXene by a modifier applied as glue for subsequent attachment of bioreceptors, electrostatic interactions could be applied also to modify MXene by redox molecules. Methylene blue as a redox probe due to its positive charge can be electrostatically deposited on MXene layer with a final immobilization of the enzyme urease on the surface using glutaraldehyde [50]. Moreover electrostatic interaction was utilized for deposition of positively (CTA<sup>+</sup>

)

to 1.3 V [50]. SPEs patterned with delaminated MXene suspension as signal

*Ti3C2 MXene-Based Nanobiosensors for Detection of Cancer Biomarkers*

*DOI: http://dx.doi.org/10.5772/intechopen.94309*

to interact with various positively charged functional groups of molecules.

charged cetyltrimethylammonium chloride (CTAC) on the negatively (OH) charged Nb2C nanosheets resulting in CTAC-anchored Nb2C nanosheets and subsequently *in situ* formation of mesoporous silica layer (a pore size of 2.9 nm) by codeposition of CTAB and tetraethyl orthosilicate (TEOS) in the next step [100]. Rich surface chemistry of MXenes can be also applied for interaction with a number of molecules. High applicability of exfoliated MXene (e-MXene) has been investigated as a matrix due to its high laser energy absorption, electrical conductivity and photothermal conversion for laser desorption/ionization time-of-flight mass spectrometry (LDI-MS) analysis of various analytes (saccharides - glucose, sorbitol,

sucrose, and mannitol, amino acids - Arg, Phe, His, and Pro, peptide - leu-

combination with other functionalized magnetic nanoparticles [102].

*and in metal ions (Na<sup>+</sup>*

*glycans.*

**225**

*/K+*

*Glycans are biomolecules, both simple and complex carbohydrates playing important roles in molecular recognition, protein conformation, cell proliferation and differentiation. The analysis of glycans and their structure has gained considerable attention because of their close relationship with disease occurrence and progression. Major types of glycans include N-linked glycans attached to the nitrogen atom in the asparagine side chain within a consensus amino acid sequence Asn-X-Ser/Thr (X should not be proline), and O-linked glycans attached to the oxygen atom of several amino acid residues including serine and threonine. Other types of glycans include glycosaminoglycans usually found attached to the proteins (proteoglycans) and also lipid chains as in glycolipids. MXenes can play an important role in the hydrogen-bonding interactions with glycans*

*) enrichment and transfer leading to improvement of the ionization efficiency of*

enkephalin and antibiotics - sulfamerazine and norfloxacin, benzylpyridinium salt (BP), environmental pollutants). Before LDI-MS measurement 1 μL of each small molecule solution was spotted on a target plate, mixed with 1 μL of e-MXene suspension and dried under ambient condition. The e-MXenes exhibiting a high resolution and salt-tolerance demonstrated a strong potential for the development of an efficient analytical platform based on LDI-MS analysis [101]. In addition, Ti3C2 MXene assisted LDI-LIFT-TOF/TOF was utilized for differentiation and relative quantitative analysis of three types of glycan isomers resulting in higher sensitivity, better homogeneity and stable relative peak intensity for glycan analysis. Moreover nine disaccharides, two trisaccharides, three heptasaccharides and ten natural product extractions were resolved by applying MXene with LDI-LIFT-MS/MS. The enhanced sensitivity and background-free nature of the fragment profile obtained by LDI-LIFT-TOF/TOF opens up a new realm for nanomaterial assisted glycan structural analysis and/or enrichment either through MXenes themselves or in

Graphite oxide as another 2D material was used to form composite together with MXene and such a composite led to a stable and efficient electrochemical detection of H2O2 and maintained hemoglobin biological activity even after ink jet printing applied for a sensor-based application [89].

#### **4.3 Electrostatic and other interactions**

MXene surface can be patterned *via* electrostatic interactions between MXene and chitosan making a nanocomposite from a negatively charged Ti3C2Tx MXene and positively charged biopolymer. Chitosan due to beneficial properties i.e. biocompatibility, nontoxicity and film-forming ability was successfully applied in numerous studies for preparation of MXene/chitosan bionanocomposites or hybrid MXene/chitosan-based nanoparticles. Such a bionanocomposite was used for attachment of an enzyme sarcosine oxidase for detection of sarcosine as a potential prostate cancer biomarker. The biosensor could detect the analyte from LOD of 18 nM up to 7.8 μM. The device responded to the analyte in an extremely short time of 2 s and the analyte was detected in a complex sample with recovery index of 102.6% (**Figure 2**) [90]. Glutaraldehyde was not needed for immobilization of the enzyme [90]. Such approach with glutaraldehyde was also used in other studies [91, 92]. In addition Nafion was proved to be an effective "adhesive" to deposit MXene onto the surface, e.g. for a final electrostatic immobilization of glucose oxidase (GOx) [84], to deposit MXene with adsorbed hemoglobin [93, 94], MXene/Mn3(PO4)2 hybrid particles [95] or MXene/TiO2 mixed with hemoglobin on GCE [96].

DNA aptamer activated through EDC/NHS chemistry was covalently immobilized onto MXene electrostatically modified with polyethyleneimine (PEI) [97].

Zheng *et al.* [98] described *in situ* adsorption of DNA on MXene surface through aromatic hydrophobic bases and in further step modified Ti3C2/DNA interface was patterned by PdNPs and PtNPs deposited using NaBH4 as a reducing agent to obtain Ti3C2/DNA/Pd/Pt nanocomposite.

#### **Figure 2.**

*A graphical presentation of a glassy carbon electrode (GCE) modified using a MXene/chitosan nanocomposite as a support for sarcosine oxidase (SOx) immobilization and indirect sarcosine detection in urine, based on hydrogen peroxide electrochemical reduction. SOx structure is adapted from the protein data Bank (code 1EL5). Figure taken from Ref. [90].*

#### *Ti3C2 MXene-Based Nanobiosensors for Detection of Cancer Biomarkers DOI: http://dx.doi.org/10.5772/intechopen.94309*

Any conductive interface can be patterned by MXene by a simple casting of a MXene dispersion on untreated electrodes with formation of MXene layer after drying [73]. Alternatively, the electrodes can be pretreated in order to make them more adhesive for formation of MXene layer. To make surface of screen-printed electrodes (SPEs) hydrophilic for subsequent deposition of MXene, SPEs were electrochemically activated in 0.1 M NaOH by CV in a potential range from 0.6 V to 1.3 V [50]. SPEs patterned with delaminated MXene suspension as signal enhancer were applied for quantifying acetaminophen (ACOP) and isoniazid (INZ) in blood serum samples [99]. The presence of abundant highly active surface sites due to functional groups (=O, -F and -OH) offers additional opportunity for MXene to interact with various positively charged functional groups of molecules.

Besides electrostatic modification of MXene by a modifier applied as glue for subsequent attachment of bioreceptors, electrostatic interactions could be applied also to modify MXene by redox molecules. Methylene blue as a redox probe due to its positive charge can be electrostatically deposited on MXene layer with a final immobilization of the enzyme urease on the surface using glutaraldehyde [50]. Moreover electrostatic interaction was utilized for deposition of positively (CTA<sup>+</sup> ) charged cetyltrimethylammonium chloride (CTAC) on the negatively (OH) charged Nb2C nanosheets resulting in CTAC-anchored Nb2C nanosheets and subsequently *in situ* formation of mesoporous silica layer (a pore size of 2.9 nm) by codeposition of CTAB and tetraethyl orthosilicate (TEOS) in the next step [100].

Rich surface chemistry of MXenes can be also applied for interaction with a number of molecules. High applicability of exfoliated MXene (e-MXene) has been investigated as a matrix due to its high laser energy absorption, electrical conductivity and photothermal conversion for laser desorption/ionization time-of-flight mass spectrometry (LDI-MS) analysis of various analytes (saccharides - glucose, sorbitol, sucrose, and mannitol, amino acids - Arg, Phe, His, and Pro, peptide - leuenkephalin and antibiotics - sulfamerazine and norfloxacin, benzylpyridinium salt (BP), environmental pollutants). Before LDI-MS measurement 1 μL of each small molecule solution was spotted on a target plate, mixed with 1 μL of e-MXene suspension and dried under ambient condition. The e-MXenes exhibiting a high resolution and salt-tolerance demonstrated a strong potential for the development of an efficient analytical platform based on LDI-MS analysis [101]. In addition, Ti3C2 MXene assisted LDI-LIFT-TOF/TOF was utilized for differentiation and relative quantitative analysis of three types of glycan isomers resulting in higher sensitivity, better homogeneity and stable relative peak intensity for glycan analysis. Moreover nine disaccharides, two trisaccharides, three heptasaccharides and ten natural product extractions were resolved by applying MXene with LDI-LIFT-MS/MS. The enhanced sensitivity and background-free nature of the fragment profile obtained by LDI-LIFT-TOF/TOF opens up a new realm for nanomaterial assisted glycan structural analysis and/or enrichment either through MXenes themselves or in combination with other functionalized magnetic nanoparticles [102].

*Glycans are biomolecules, both simple and complex carbohydrates playing important roles in molecular recognition, protein conformation, cell proliferation and differentiation. The analysis of glycans and their structure has gained considerable attention because of their close relationship with disease occurrence and progression. Major types of glycans include N-linked glycans attached to the nitrogen atom in the asparagine side chain within a consensus amino acid sequence Asn-X-Ser/Thr (X should not be proline), and O-linked glycans attached to the oxygen atom of several amino acid residues including serine and threonine. Other types of glycans include glycosaminoglycans usually found attached to the proteins (proteoglycans) and also lipid chains as in glycolipids. MXenes can play an important role in the hydrogen-bonding interactions with glycans and in metal ions (Na<sup>+</sup> /K+ ) enrichment and transfer leading to improvement of the ionization efficiency of glycans.*

the Ti3C2Tx MXene sheets was applied for formation of particles exhibiting surface– enhanced Raman spectroscopy (SERS) phenomenon [85]. Moreover, an AuNP/ MXene composite boosts sensitivity of detection of oncomarker such as microRNA [88]. Similarly, the composite consisting of Ti3C2Tx MXene and PtNPs was prepared by means of *in-situ* reduction of Pt precursor (spontaneously or by external reducing agents) on MXene surface. Composite was used for electrochemical catalysis [69] and sensing of important small bioactive compounds [72]. The negatively charged acetylcholinesterase (AChE) was electrostatically deposited on the hybrid nanocomposite of MXene/AgNPs/chitosan from a mixture of the enzyme and chitosan onto MXene/AuNPs for detection of organophosphate pesticide [86].

Graphite oxide as another 2D material was used to form composite together with MXene and such a composite led to a stable and efficient electrochemical detection of H2O2 and maintained hemoglobin biological activity even after ink jet printing

MXene surface can be patterned *via* electrostatic interactions between MXene and chitosan making a nanocomposite from a negatively charged Ti3C2Tx MXene and positively charged biopolymer. Chitosan due to beneficial properties i.e. biocompatibility, nontoxicity and film-forming ability was successfully applied in numerous

MXene/chitosan-based nanoparticles. Such a bionanocomposite was used for attachment of an enzyme sarcosine oxidase for detection of sarcosine as a potential prostate cancer biomarker. The biosensor could detect the analyte from LOD of 18 nM up to 7.8 μM. The device responded to the analyte in an extremely short time of 2 s and the analyte was detected in a complex sample with recovery index of 102.6% (**Figure 2**) [90]. Glutaraldehyde was not needed for immobilization of the enzyme [90]. Such approach with glutaraldehyde was also used in other studies [91, 92]. In addition Nafion was proved to be an effective "adhesive" to deposit MXene onto the surface, e.g. for a final electrostatic immobilization of glucose oxidase (GOx) [84], to deposit MXene with adsorbed hemoglobin [93, 94], MXene/Mn3(PO4)2 hybrid particles [95]

DNA aptamer activated through EDC/NHS chemistry was covalently immobilized

Zheng *et al.* [98] described *in situ* adsorption of DNA on MXene surface through aromatic hydrophobic bases and in further step modified Ti3C2/DNA interface was patterned by PdNPs and PtNPs deposited using NaBH4 as a reducing agent to obtain

*A graphical presentation of a glassy carbon electrode (GCE) modified using a MXene/chitosan nanocomposite as a support for sarcosine oxidase (SOx) immobilization and indirect sarcosine detection in urine, based on hydrogen peroxide electrochemical reduction. SOx structure is adapted from the protein data Bank (code*

onto MXene electrostatically modified with polyethyleneimine (PEI) [97].

studies for preparation of MXene/chitosan bionanocomposites or hybrid

or MXene/TiO2 mixed with hemoglobin on GCE [96].

Ti3C2/DNA/Pd/Pt nanocomposite.

*1EL5). Figure taken from Ref. [90].*

**Figure 2.**

**224**

applied for a sensor-based application [89].

*Novel Nanomaterials*

**4.3 Electrostatic and other interactions**
