Two-dimensional transition metal carbides and nitrides (MXenes) based biosensing and molecular imaging

: Asa“starmaterial”,2Dtransitionmetalcarbides and/ornitrides(MXenes)havetremendouspotentialappli-cations in biosensor development and molecular imaging. MXenes have a lot of advantages due to their large specific surface, excellent electrical conductivity, adjustable band gap, and easy modification. MXenes that immobilized with DNA strands, proteins, enzymes, or other bioluminescent materials on the surface, have been used to measure small molecules with extraordinary sensitivity and remarkable limitofdetection.Thisreviewprovidesanoverviewofmost recent development in the synthesis, fundamental properties, biosensing, and molecular imaging applications of MXenes. We focused on molecular detection through MXene-based electrochemical properties their challenges and novel opportunities of MXenes in biological applications. This article will provide a guide for researchers who are interested in the application of MXenes biosensors.


Introduction
Since the two-dimensional (2D) graphene nanosheets were magnificently exfoliated from bulk graphite in 2004 Huiyu Liu and Xiaotong Xing contributed equally. [1,2], 2D nanomaterials have attracted extensive interest and become one of the most dynamic research areas in nanotechnology. Etching the A element from the MAX phase of layered ceramic yielded 2D transition metal carbonitrides, generating a new class of nanomaterials termed MXene materials. The chemical formula of MXene is M n+1 X n T x [3,4], wherein M represents an early transition metal element (Sc, Ti, V, Cr, Y, Zr, Nb, Mo, Hf, Ta, W, etc.), X represents C or N, and n = 1 to 4. T represents a surface end group (-OH, -O, -F, etc.) ( Figure 1A).
MXene materials derived from MAX have become the most prominent family of 2D materials due to the diversity of MAX phase compositions and structures [5]. Gogotsi group successfully prepared Ti 3 C 2 nanosheets by etching Ti 3 AlC 2 with hydrofluoric acid (HF) at room temperature in 2011, which promoted progress in the synthesis and characterization of MXenes [6]. The structure and preparation of M 2 X, M 3 X 2 , and M 4 X 3 are shown in Figure 1B [7].
Considering the research on MXene materials in our group, the goal of this review mainly focused on the summary of the latest progress in the field of biosensing using MXenes. We provide an overview of MXenes and describe the synthesis, properties, and surface modifications. Furthermore, we elucidate the MXenes-based detection of microRNA and other small molecules and biomedical imaging applications. Finally, we illustrate the challenges for MXenes-based analysis. This review will outline the significant developments of MXenes in molecular detection and bioimaging applications. It will also serve as a reference for researchers by highlighting notable instances of their application in microRNA detection (Scheme 1).  MXenes materials considerably improves their chemical and physical reactivity, enabling drug loading and functional modification, and generating unique photonic, catalytic, magnetic, and electronic characteristics not found in bulk materials. MXenes are employed to construct innovative nanoplatforms with intriguing functions for optical imaging, photothermal imaging, biosensing, and targeted drug delivery. Thus, MXenes have great potential in biomedical applications [8,9].

Electronic property
MXenes have either conductor or semiconductor properties due to their ordered arrangement of metal atoms. The various coating groups of MXenes produce distinct electron attraction capabilities, which profoundly influence their electrical properties. A significant influence on the metal layer's electrical properties would occur from the imbalanced distribution of electron density caused by the increase in surface defects created during the synthesis of MXenes, which hinders the free flow of electrons [10]. MXenes have the capacity to produce active electrons and vacancies in response to specific stimuli (such as light excitation), and the interaction between MXene and surrounding environment is able to cause oxidative stress. The electrical conductivity can be accurately determined by measuring the current changing between the electrodes after binding to the testing species. A change in the local charge field initiates the signal accumulation which is reflected by material's conductivity. The main advantage of MXenes as electronic biosensors is that the cross-sectional area is on the same spatial scale as the charge field of the nearby biomolecules, which serve as the smallest spatially limited target to sense electrical changes as a function of the measured conductivity [11]. Moreover, MXenes can produce active electrons under external stimuli such as heat or light.

Optical property
According to "first-principles density functional theory," the calculations show that the optical band gap of MXenes can be changed by modifying the surface group compositions and contents. The linear optical properties (e.g., absorption, photoluminescence) and nonlinear optical properties (e.g., saturable absorption, nonlinear refractive index) highly depended on the structure [12]. Compared to the MXenes with the same thickness, the MAX phase absorbs more light and is transparent under visible light irradiation. Halim group established that MXenes films with larger intercalants were more transparent and less conductive [13]. The optoelectronic properties of the films may be modified by the electrochemical intercalation of the cations, exhibiting reversible transmission in the UV-Vis range, indicating the potential of MXenes to act as transparent conductors [14]. The UV-Vis spectrum shows a clear difference between MAX and MXene that MXene has significant absorption near 300 nm and a broad absorption near 800 nm, while the MAX showed weak absorption of at the range of 200-1000 nm [15,16].
Nonlinear optics relate to the interplay between light and matter interactions in the nonlinear response of materials to electromagnetic fields. This phenomenon is crucial for laser optics, photonic devices, and optical communications applications. The unique photoluminescence lifetime of MXene nanomaterials can be directly used as a valuable probe for sensing and enabling live cell imaging [17]. Additionally, MXenes have potential to generate reactive oxygen species (ROS) under light irradiation, which can be designed as a platform for simultaneous photoacoustic imaging (PAI) and photodynamic therapy (PDT) [18]. However, the photothermal conversion mechanism of MXenes has not yet fully developed. Dong et al. speculated that MXenes like Ti 3 C 2 have a localized surface plasmon resonance (LSPR) effect that similar to the gold nanoparticles [19]. It has the ability of photothermal conversion in the near-infrared (NIR) light region. Similarly, Wang's group demonstrated that MXenes have photothermal conversion ability in NIR because of their excellent electromagnetic interference shielding and LSPR effect [20]. The Mo 2 Ti 2 C 3 T x MXene material reported by Guo et al. exhibited excellent saturable absorption properties, with an enhanced 40% modulation depth observed within the double transition carbide compared to previously reported MXenes, and can be used as a passively Q-switched mid-infrared fiber laser for SAM [21].

Synthesis methods
The efficient synthesis of MXene is the basis for expanding the materials' range of applications. MAX has a crystal structure, and "M" atoms form an octahedral-like structure. "X" atoms are filled in the gaps of the octahedron. "A" intercalated into the lamellae formed with M and X and finally obtained an M n+1 AX n structure. Primarily, the M-X bond belongs to the covalent bond and ionic bond. M-A and A-A belong to metallic bonds, and their bond energy is lower than that of the M-X bond, so the "A" atom is more active and easily stripped. The method for synthesizing MXenes is mainly a chemical etching method. Generally, MXenes materials are fabricated using two different methods: a top-down approach based on multilayer bulk flake exfoliation and a bottom-up approach to growing 2D flakes from their precursor salts. These synthesis methods endow various physical, surface, chemical, and electronic properties that can be used to perceive the connection between structural and functional properties [22]. In a top-down approach, thin layers of MXene films are exfoliated from their MAX phase precursors, primarily by mechanical exfoliation [23,24]. Moreover, this is a liquid strip method with lower production costs and extended production capacity. The bottom-up approach depends on combining appropriate "metal-organic molecules" to make MXene films, mostly by metal-organic decomposition, chemical vapor deposition (CVD), wet chemistry, and other methods. Large areas of two-dimensional defect-free monolayer crystals can also be synthesized [25].

Top-down method
The top-down method for MXenes preparation is selectively etching the A layer in MAX material. It is mainly divided into HF etching, fluoride etching, molten salt, alkali-assisted hydrothermal, and other methods. Because the bond between the M layer and the A layer of the MAX phase is a solid covalent or metallic bond, MXenes was synthesized from the MAX phase by selective etching of the A layer by temperature processing [26]. The smaller the force constant contributed by adjacent atoms to the A atom, the smaller the exfoliation energy and the more manageable the exfoliation. Electrons injected into the MAX phase lead to the elongation of the M-A bond, which further induces the MAX phase's swelling and the layer's exfoliation [27].

HF etching
HF selectively etches metal layers, mainly adopting the following equation of reaction to complete the preparation of MXenes (Figure 2A). The first non-MAX phase precursor synthesized Mo 2 CT x was Mo 2 Ga 2 C by HF etching of Ga [28]. HF etching method has the advantages of simple operation and low reaction temperature and is suitable for etching the MAX phase containing Al and part of non-MAX phases. However, it suffers from the disadvantages of high corrosivity, toxicity, operational risk, and poor energy storage [25].

Acid/fluoride etching
Halim et al. made the first hydrogen fluoride salt etching application using NH 4 HF 2 sputter-deposited epitaxial Ti 3 AlC 2 films [13]. Owing to the change of fluoride salt can adjust the interlayer spacing of MXenes, Ghidiu et al. conducted an etching process with HCl/LiF solution at 40 • C ( Figure 2B) [29]. Additionally, this synthesis strategy can etch Ti 3 AlC 2 without water and apply to water-sensitive MXenes materials. Difluoride salts are solid at room temperature and much safer than HF, and more attention should be paid for their application to exfoliate MAX phases. Multilayer Ti 3 C 2 T x MXenes material reported by Wu et al. was etched by a hydrothermal process using oxalic acid and NH 4 F at different temperatures of 100-180 • C for 24 h ( Figure 2C) [30]. The kinetic of this method depended on the acidity of the dissociated organic anion and its interaction with the dissociated F of the ionic liquid.

Electrochemical etching method
The metal-carbon atoms are selectively extracted from the ternary layered carbides in an electrochemical manner. The carbide was added in a NaCl, HCl, or HF solution, and an anodic potential was applied so that carbide-derived carbon (CDC) is formed ( Figure 2D), and a carbon film with a very narrow pore size distribution was formed [31]. The critical points of this method were the voltage, etching time, and electrolyte concentration. However, this method was unsuitable for large-scale preparation due to its low yield.

Molten salt method and other methods
Carbide MXenes can be easily and successfully prepared by HF or fluoride etching, but nitride MXenes cannot be prepared. The Ti-Al bond in Ti n+1 AlN n is stronger than that in Ti n+1 AlC n , so the preparation of Ti n+1 AlN n requires higher energy. Meanwhile, Ti n+1 AlN n is less stable and easy to degrade in HF.  Figure 2F) [33]. Zada et al. used algae extract to etch bulk V 2 AlC crystals in an aqueous solution ( Figure 2G) [34]. It has also been reported using UV-induced etching [38] and surface acoustic waves for ultrafast one-step synthesis of MXenes [39].

Bottom-up
The bottom-up method for preparing MXenes is a chemical synthesis that includes CVD, atomic layer deposition (ALD), plasma-enhanced pulsed laser deposition (PEPLD), template methods, etc. Xu et al. produced ultrathin α-Mo 2 C crystals of several nanometers by using methane as a carbon source, copper foil on Mo foil as substrate, and a temperature higher than 1085 • C [40]. Geng et al. reported the growth of Mo 2 C crystals with controllable thickness and morphology on a liquid copper surface by ambient pressure CVD ( Figure 2H) [35]. ALD, a variant of CVD, is a gas-phase method based on two successive self-limiting surface reactions. However, the rate of final products is usually low and requires special equipment. Zhang et al. demonstrated a type of Mo 2 C thin films with controlled crystal structure growth on sapphire substrates by PLD at a temperature of 700 • C [41]. Jia et al. used MoO 2 as a template that was also a highly active electrocatalyst to synthesize N-doped Mo 2 C nanosheets ( Figure 2I) [36]. Compared with the top-down method, the bottom-up approach saves raw materials and accurately controls the element composition, size, and surface groups. However, it is a challenge to prepare large-size MXenes. Most of the reported MXenes are prepared by the top-down method, with few reports on the bottom-up approach.

Surface modification
By altering the surface and interlayer spacing of MXenes, the proton transport efficiency will be enhanced, which will be beneficial to the electrochemical properties of MXenes. MXenes can be doped with various elements, nanoparticles, ligands, drugs, and other surface modifications to obtain desired characteristics. Doping with Fe 3+ , Co 2+ , Ni 2+ , Mn 2+ , and other metal ions is helpful to promote intrinsic performance. Zhang et al. replaced a Cu layer electrodeposited thereon by a Pt current using Pt-modified SnO 2 C (Pt/SnO 2 C) nanofibers. Thus, SnO 2 C nanofibers exhibited competitive oxygen reduction reaction catalytic activity, enhanced methanol tolerance, and superior durability [42]. Cao et al. utilized Au nanocrystals to selectively grow on the edges of TiO 2 nanosheets with highly exposed (001) facets to fabricate Au-TiO 2 NSs as an acoustic sensitizer [43]. Polymers such as dextran, cellulose, chitosan, polyethylene glycol (PEG) [44][45][46], polyethyleneimine (PEI) [47], polyvinylpyrrolidone (PVP) [48], polyacrylic acid, and polyvinyl alcohol [49], which are used to improve the stability, hydrophilicity, degradability, and biocompatibility of the MXenes. Pan

MicroRNA detection
As the Mxenes materials process unique metallic conductivity and hydrophilic properties which are fit for microRNA (miRNA) analysis, they are able to adsorb the single-stranded DNA (ssDNA) through weak Van Der Waals force. The biosensor based with fluorescent signals (FL), surface-enhanced Raman spectroscopy (SERS), photoelectrochemical (PEC) and electrochemical (EC) have been exploited [56] (Table 1).
In the presence of miRNA, the duplex DNA will be formed and dissociated from the surface of the Mxene materials. The fluorescence will be recovered that realized rapid, simple, and selective recognition [69]. In Wang's work, a synergistic calibrated SERS strategy based on MXene/MoS 2 @AuNPs with controllable morphology has been presented for detecting miRNA-182. The system has three characteristic Raman peaks (at 382 cm −1 and 402 cm −1 corresponding to MoS 2 and at 611 cm −1 corresponding to MXene) as a benchmark instead of additional beacon molecules. Specifically, the LOD was 6.61 am for miRNA-182 in human serum samples [57]. Various studies focused on signal amplification methods to enlarge the detection sensitivity and accuracy. For example, Wang et al. fabricated novel in situ reductions of gold nanoparticles (AuNPs)-decorated Ti 3 C 2 MXene electrochemical biosensor combined with a cascaded signal amplification strategy for the detection of miRNA-21, MXene served as both the reductant and stabilizer. By cascaded signal amplification, the assessment indicated that this electrochemical biosensor has a detection limit of 50 aM (S/N = 3) ( Figure 3A) [70]. Li's group also constructed a Ti 3 AlC 2 -Au nanocomposite and G-quadruplex nano-amplification-based electrochemical biosensor for miRNA-377 measurement in human serum samples. Specifically, the designed biosensor displayed excellent sensing performance with a limitation of detection (LOD) as low as 1.35 aM ( Figure 3B) [58]. For microRNA-155 detection, a Ti 3 C 2 T x QDs/(001) TiO 2 /FTO platform was constructed. Detailed, the structure was composed of TiO 2 and Ti 3 C 2 T x QDs by a type II heterojunction and the LOD was 25 fM [59]. Xu's group presented a Ti 3 C 2 @ReS 2 via the vertical anchoring flaky ReS 2 on the Ti 3 C 2 backbone for miRNA-141 detection. According to the assessment, the Ti 3 C 2 @ReS 2 sample containing 45 wt% of ReS 2 showed a 2.48-time promotion in the photocurrent compared to ReS 2 owing to the synergistic effects of its photoactive and conductive counterparts. In essence, the estimated LOD was 2.4 aM ( Figure 3C) [60].
There are also numerous examples of the detection of multiple intracellular miRNAs. Liao et al. used folate-adsorbed carbon nitride to create a multipurpose probe for in-situ monitoring of various miRNAs [71]. Lee et al. synthesized AuNP@MXene/Au to modify with vast numbers of DNA probes for miRNA-21 and miRNA-141 detection, and LOD was determined as 204 aM and 138 aM, respectively. Moreover, this device successfully indicated three cancer plasma samples [62]. Tian et al. reported a simple amplification strategy of enzyme-free miRNA target-triggered strand displacement reaction to fabricate a molybdenum carbide (Mo 2 C) biosensor with ferrocene to detect miR-21 [63]. Additionally, Dai et al. also synthesized a class of monolayer Mo 2 C QDs with biocompatibility and water solubility by liquid exfoliation method to deliver the optimized molecular beacons MB into cells for accurate quantitative detection of mature miRNAs [64]. Wang et al. reported a stable luminol-Au NPs-Ti 3 C 2 as an ECL biosensor for miRNA-155 detection. The immobilization of ECL emitters is a versatile strategy   modified Ti 3 C 2 MXenes as an ECL signal emitter to detect miRNA-141 [65]. Jiang et al. reported a hybrid luminescent Co-MOF-ABEI/Ti 3 C 2 T x composite used to estimate miRNA-21 with a detection limit of 3.7 fM [66]. Overall, compared with FL output signals, the biosensor with EC and PEC exhibited much higher sensitive performance.

Detection of other small molecules
The qualitative and quantitative analysis of metabolites is a crucial indicator of physiological and pathological alterations. H 2 O 2 is an essential molecule in many signal transduction processes in vivo and is involved in cellular metabolism. MXene-Ti 3 C 2 nanosheet was designed as a colorimetric strip that reacted with H 2 O 2 and has good performance for free radical scavenging ( Figure 6A) [86]. Ti 3 C 2 T x /PtNP sensor can detect small redox molecules such as ascorbic acid, dopamine, uric acid, and acetaminophen [87]. MoS 2 nanosheets and lactate oxidase biosensors can selectively detect lactate ( Figure 6B) [88]. Moreover, MXenes materials can detect glucose, GSH, proteins, dengue DNA and RNA, circulating tumor DNA, and T4 polynucleotide kinase (T4 PNK) [89][90][91][92][93][94][95]. For example, MoS 2 could specifically detect a minimal amount of target DNA molecules ( Figure 6C) [96]. Very recently, Wang et al. reported Ti 3 C 2 T x as an ultra-efficient hemoperfusion absorber for eliminating the cytokine storm syndrome which induced by COVID-19. The molecular-level investigations demonstrated that Mxenes has strong chemisorption for immobilizing cytokine interleukin-6 and good blood compatibility ( Figure 6D) [97].

Biological imaging and disease diagnosis
MXenes have great potential for the non-invasive imaging, including MRI, positron emission tomography (PET), X-ray computed tomography, FL, PAI, etc. [98][99][100][101][102][103][104][105]. Nanoscale MXenes are also suitable for bioimaging and disease diagnosis [106,107]. First, the nanoscale size enables them to exist in the organism for a longer time, effectively avoiding the self-clearing function of the blood circulation, enabling intermolecular interactions, and inducing luminescence. Secondly, certain hydrophilic functional groups on the surface of MXenes could improve water solubility and have promising biological. Especially, recent studies have shown that MXenes can be degraded and eliminated in organisms. In addition, the near-infrared absorption of MXenes makes them a suitable contrast agent for PAI.

Photoacoustic imaging
As a new diagnostic imaging technique, PAI induces optical imaging by irradiating the tissue with excitation light. Because of its low tissue attenuation coefficient, PAI can achieve the purpose of real-time detection of biological lesions. The spectrum of MXenes extends from ultraviolet-visible to NIR, and MXenes have good photothermal conversion capability, which enables them to be effective PAI contrast agents. The photothermal effect has been demonstrated in various MXene compositions such as Ti 3 C 2 , Nb 2 C, and Ta 4 C 3 [36,108,109]. The strong

SERS and fluorescence imaging
The LSPR effect of MXene nanosheets with semi-metallic properties can enhance the Raman scattering signal and serve as a good building block for SERS. Emitting MXene QDs are reported by fabricating small-size dot phase MXenes. Strong emissions were realized under specific wavelength excitation, which resulted from the quantum confinement caused by size effect and luminescence resulting in induced defects [111,112]. Based on this, many researchers used MXene SERS imaging for sensitive detection applications. For instant, the Yury Gogotsi group reported titanium carbide MXene Ti 3 C 2 T x that can enhance Raman signal from organic dyes on a substrate and in solution ( Figure 7A) [113]. Due to the synergistic effect of the charge transfer resonance and electromagnetic enhancement, Nb 2 C and Ta 2 C MXenes were shown as remarkable SERS enhancement probes for sensitively detecting the SARS-CoV-2 spike protein ( Figure 7B). The results indicated that the detection limit is as low as 5 × 10 −9 M, which is suitable for achieving real-time monitoring and early warning of novel coronavirus [114].
With the recently developed luminescent MXene QDs, MXenes have been applied to luminescent cell imaging. Similar to graphene and carbon dots, MXene QDs exhibited luminescence properties, and their emission is related to excitation. For example, Zhi et al. developed photoluminescent Ti 3 C 2 MXene QDs (MQD) for multicolor cell imaging. Due to the strong quantum confinement, the as-prepared MQD showed excitation-dependent photoluminescence spectra with high quantum yields. The application of MQD as a biocompatible multicolor cell imaging probe and zinc ion sensor was demonstrated in (Figure 7C) [115]. Wang et al. synthesized amphiphilic carbide-derived GQDs combined with layered Ti 3 C 2 X MXene to apply in cellular imaging due to their excellent properties, such as bright and tunable photoluminescence, low cytotoxicity, good photostability, and chemical inertness ( Figure 7D) [116].  [122,123]. Actually, these nitride-based MXenes exhibited better biodegradability in the physiological environment. According to a facile top-down method, Prof. Huang's group synthesized titanium nitride quantum dots (Ti 2 N QDs) in solution. The Ti 2 N QDs exhibited good performance on PAI-guided PTT in both NIR-I/II biowindows for precision cancer treatment [124]. Interestingly, MXenes with enzymatic labels were used to fabricate a versatile multiplexed label-free single-cell detection strategy with high-dimensional imaging. Generally, a set of MXenes is selected to ensure mass detection within the cytometry range while avoiding overlap with more than 70 currently available tags and able to survey multiple biological parameters at the single-cell level or in different organs ( Figure 8B) [125].

Conclusion and outlook
MXenes offer unique properties and enormous potential in biological applications. However, the clinical translation of these compounds still confronts several obstacles. Most of MXenes are prepared by the top-down method, lacking an approach to precisely control the size, layer number distribution, and surface groups of the final products. Large-scale preparation is essential for further commercial applications. However, the current synthesis of MXenes is only in the laboratory stage. There are few bottom-up approaches, developing an effective bottom-up method for MXene synthesis is extremely desirable. In addition to nanosheets, it's also important to construct MXenes with various morphologies such as nanotubes and nanocages. Furthermore, the combination of MXenes with other functional materials to form hybrid materials with attractive properties is also highly needed. Finally, the biosafety remains a key challenging issue. Scientists have conducted short-term toxicity and organ residue distribution studies to confirm that MXenes have excellent ideal short-term biosafety. However, long-term biosafety including genotoxicity, immunotoxicity, and reproductive toxicity are required. We believed that the development of chemical materials science, biology, and medicine and the collaboration among various disciplines would accelerate the bioanalytical and imaging applications of MXenes in the future.
Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.