Ti3C2 MXene-Based Nanobiosensors for Detection of Cancer Biomarkers

This chapter provides information about basic properties of MXenes (2D nanomaterials) that are attractive for a design of various types of nanobiosensors. The second part of the chapter discusses MXene synthesis and various protocols for modification of MXene making it a suitable matrix for immobilization of bioreceptors such as antibodies, DNA aptamers or DNA molecules. The final part of the chapter summarizes examples of MXene-based nanobiosensors developed using optical, electrochemical and nanomechanical transducing schemes. Operational characteristics of such devices such as sensitivity, limit of detection, assay time, assay reproducibility and potential for multiplexing are provided. In particular MXene-based nanobiosensors for detection of a number of cancer biomarkers are shown here.


MXenes: their precursors, characterization, unique properties and applications
Nanomaterials of the 2D kind are in the research spotlight due to their superior properties like ultrathin structure and intriguing physico-chemical properties [1-3]. Graphene has made researchers believing in extracting single layer transition metal dichalcogenides, which in turn has led to extensive research dedicated towards 2D nanomaterials [4,5]. Since their inception, 2D nanomaterials have been characterized to have exceptional electronic, mechanical, and optical properties. These outstanding characteristics have driven research to use them in almost all fields of materials science and nanotechnology [6][7][8]. Rather recently in 2011 and 2012, Gogotsi, Barsoum, and colleagues have successfully prepared a new kind of 2D nanomaterial -MXenes, composed of a large group of transition metal carbides and carbonitrides [9][10][11][12][13]. These 2D nanomaterials are found to possess many striking properties and boost attraction in applications such as energy storage [14][15][16], electromagnetic shielding [17,18], water treatment [19,20], disease treatment [21] and (bio)sensing [22,23], MXenes are made up of atomic layers of different materials like transition metal carbides, nitrides, or carbonitrides. All MAX phases consist of twodimensional slabs of close-packed alternating layers of M and A, where M is a transition metal, A is an A-group element and X is C and/or N [23].
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  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 + ions. Exfoliation can be done by a simple shaking or by sonication and prolonged sonication time results in production of MXene with small size of nanosheets and high density of defects [61]. An alternative to use of highly corrosive and harmful HF is to employ small organic molecules or ions such as urea [62], dimethyl sulphoxide (DMSO) [12] (only for Ti 3 C 2 T x MXene) or isopropylamine as etchants [63]. MAX phase containing Si can be also exfoliated using tetrabutylammonium hydroxide (TBAOH) and tetramethylammonium hydroxide (TMAOH) [64].

MXene characterization
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 Ti 3 C 2 T x [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 (NH 4 HF 2 , 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 Ti 3 C 2 T x 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 Ti 3 C 2 T x MXene produced either by HF or LiF-HCl method including nuclear magnetic resonance ( 1 H, 13 C and 19 F 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]. Ti 3 C 2 T x MXene has become an attractive subject of interest due its high capacitance ($1500 F cm À3 ) in supercapacitors and an excellent high metallic conductivity ($15,000 S cm À1 ). 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 Ti 3 C 2 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 AE 0.1) nm for Ti 3 C 2 T x [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 Ti 3 C 2 Tx 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 Ti 3 C 2 T x MXene to detect significant analytes (O 2 ,H 2 O 2 and NADH) was performed by applying cyclic voltammetry and chronoamperometry techniques, whereas Ti 3 C 2 T X demonstrated electrocatalytic activity towards H 2 O 2 reduction with LOD at nanomolar level [68]. Unfortunately, formation of TiO 2 layer or domains with subsequent TiO 2 dissolution caused by F À 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 Ti 3 C 2 T x MXene.
The improvement of stability and redox behavior was achieved by further modification of MXene with nanoparticles of platinum (Ti 3 C 2 T x /Pt) [69,72]. The electrocatalytically active sensor based on Ti 3 C 2 T x /Pt nanocomposite successfully determined H 2 O 2 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].
Aryldiazonium salts were utilized in modification of Ti 3 C 2 T x MXene either spontaneously by free electrons or electrochemically. Electrochemical modification of Ti 3 C 2 T x 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 À1 V with a sweep rate of 0.25 V s À1 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].

Covalent modification of Ti 3 C 2 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][75][76]. Such modification led to production of nanosheets of Ti 3 C 2 -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 Ti 3 C 2 T x 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 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].

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 Fe 3+ salt) allowed preparation of composite of MXene with small magnetic Fe 3 O 4 nanoparticles with an average size of $4.9 nm (TiO 2 / Ti 3 C 2 T x /Fe 3 O 4 ). 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 NaBH 4 . A simple spontaneous reduction of metallic salts to form Ag, Au, and Pd nanoparticles onto 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.

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].