**5.2 MXene-based electrochemical nanobiosensors**

Electrochemical biosensors are prospective tool of choice for an early-stage diagnostics of cancer diseases [114]. Electrochemical methods such as CV, CA, DPV, EIS, square wave voltammetry (SWV) provide a number of advantages. They are reliable, easy-to-use, affordable and highly sensitive and reliable [107, 115, 116]. Lab-on-chip biosensors are compact and portable miniaturized devices that can be employed in cancer biomarkers research leading to potential clinical applications. Biosensors employing surface nanoarchitectures with this type of detection offer attractive features including straightforward miniaturization, excellent LODs, robustness, small analyte volumes and the ability to be applied in turbid biofluids with optically absorbing and fluorescing compounds.

Single/few-layered MXene (Ti3C2) nanosheets were functionalized with (3 aminopropyl)triethoxysilane (APTES) to enable covalent attachment of bioreceptor onto *f*-Ti3C2-MXene for electrochemical detection of carcinoembryonic antigen (CEA) as a widely used tumor marker [77]. The ultrathin 2D nanosheets of single/multilayer MXene Ti3C2 with high density of functional groups brought in improved antibodies anchoring and faster access to analyte. The label-free aminosilane and bio-functionalized *f*-Ti3C2-MXene-based biosensor (BSA/anti-CEA/f-Ti3C2-MXene/GC) demonstrated LOD of 0.000018 ng mL<sup>1</sup> with sensitivity of 37.9 μA ng<sup>1</sup> mL cm<sup>2</sup> *per* decade (a linear detection range of 0.0001– 2000 ng mL<sup>1</sup> ) for CEA determination using hexaammineruthenium ([Ru(NH3)6] 3+) as a preferable redox probe and CV as a detection technique [77].

*Carcinoembryonic antigen (CEA, molecular mass of 180–200 kDa) is a highly glycosylated cell surface protein consisting of approx. 60% carbohydrates, which attains elevated levels in a number malignancies, such as colorectal, breast and ovarian, gastric, liver and pancreatic cancer. Serum CEA is used in clinical research to identify early stages of disease, monitor tumor recurrence and metastatic disease. The normal range of serum CEA in healthy adults of non-smokers is below 2.5 ng mL<sup>1</sup> and in the serum of smokers below 5.0 ng mL<sup>1</sup> , but increases rapidly when normal cells become cancerous.*

Due to their excellent electrical conductivity and large specific surface area with a large number of potential attachment binding sites, 2D MXenes are also applied as

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

a conductive support for immobilization of aptamer probes. Wang *et al*. modified electrode surface with MXene for development of a MUC1 biosensor [117]. The ferrocene-labeled complementary DNA was bound onto MXene nanosheets to design a detection probe for electrochemical signal amplification. GCE was modified by electrodeposited AuNPs with MUC1 aptamer attached to the modified electrode *via* Au-S bonds. The modified electrode was blocked using bovine serum albumin (BSA) in order to resist non-specific interactions. Next, a detection probe was attached to the modified electrode *via* hybridization between complementary DNA and a MUC1 aptamer. Upon interaction of MUC1 with such an electrode, the detection probe was detached from the working electrode resulting in a decrease of an electrochemical signal (a signal-off response). This competitive aptasensor detected MUC1 with LOD of 0.33 pM with a linear range up to 10 mM. The relative standard deviation (RSD) of the peak current difference response was 1.43%, indicating that the aptasensor had good reproducibility. The peak current difference response of the aptasensor did not change much in ten days, indicating its acceptable stability [117].

*Currently, there are more than 20 known types of mucins. They are encoded by MUC genes and represent high molecular weight glycoproteins expressed on epithelial cells. Aberrantly glycosylated mucins are expressed in cancer cells and serve as oncogenic molecules.*

*MicroRNAs (miRNAs) overexpression is a biomarker for a number of diseases including cardiovascular disorders, cancer, rheumatic diseases, diabetes, neurological disorders, liver diseases, kidney diseases, and immune dysfunction. The microRNAs (miRNAs) are biomolecules composed of 18–24 nucleotides and they play a key role in biological processes such as cell proliferation, apoptosis and tumorigenesis. Abnormal expression has been monitored in breast cancer as well as in other cancer types with observed blood stability. The miRNA-182 demonstrates tissue specificity and sequential expression in the different stages during lung cancer development or evolution. The miRNA-155 is overexpressed in human breast cancers.*

The label-free strategy for the ultrasensitive detection of miRNA-182 was based on glassy carbon electrode (GCE) modified step-by-step by van der Waals forces and electrostatic interactions with MoS2/Ti3C2, AuNPs, ssRNA [118]. BSA was used to block unbound gold particles surface and avoid nonspecific adsorption. The biosensor was able to determine miRNA-182 with LOD of 0.43 fM (a linear range of 1 fM - 0.1 nM) by DPV method [118]. The recovery was 105%, 95.3% and 93.0% for the concentration of 10<sup>10</sup> M, 10<sup>12</sup> M and 10<sup>14</sup> M of the analyte respectively, manifesting its effective detection of miRNA-182 in real sample [118].

Duan with co-workers [119] developed an impedimetric aptasensing strategy based on a novel zero dimensional (0D)/2D nanohybrid of Ti3C2Tx nanosheets decorated with FePc QDs (denoted as Ti3C2Tx@using iron phthalocyanine quantum dots (FePcQDs)) for miRNA-155 detection. The miRNA-155 was established by applying impedimetric aptasensor with LOD of 4.3 aM (S/N = 3, a linear concentration range from 0.01 fM to 10 pM). The observed relative standard deviation (RSD) of the five aptasensors for detection of miRNA-155 was as low as 2.98**%**, demonstrating good reproducibility of the proposed aptasensor. Moreover, the signal remained 104**%** of the original signal after 15 days of storage, revealing a satisfactory stability of the present aptasensor [119].

Multiple (miRNA-21 and miRNA-141) and rapid (80 min) analysis of onco microRNAs in total plasma was carried out with combination of AuNPs (5 nm) decorated MXene as an electrode interface and a duplex-specific nuclease (DSN) as an amplification system applied onto home-made screen-printed gold electrode

low level or even absent, while increased values can reveal development and/or progression of a disease [112]. Serum biomarkers providing key information about the disease are important for management of cancer patients since blood aspiration is only a moderately invasive procedure. There is clear need for early-stage cancer diagnostics, efficient treatment and posttreatment monitoring to avoid progress of the disease into advanced stages. Therefore there is an enormous demand for efficient less-invasive investigation *i.e.* analysis of cancer biomarkers in plasma/

*Limit of Detection (LOD): the level of analyte that leads to a sensor signal which is statistically significantly different from the background signal obtained in the absence of the analyte. A frequently used definition of LOD is a concentration that gives a signal greater than three times the standard deviation of a blank sample*

The unique physico-chemical properties of MXenes make them a significant tool that can be employed in the cancer therapy (photothermal therapy, photodynamic therapy, radiation therapy, chemotherapy), cancer imaging (CT/MRI/PA imaging)

Electrochemical biosensors are prospective tool of choice for an early-stage diagnostics of cancer diseases [114]. Electrochemical methods such as CV, CA, DPV, EIS, square wave voltammetry (SWV) provide a number of advantages. They are reliable, easy-to-use, affordable and highly sensitive and reliable [107, 115, 116]. Lab-on-chip biosensors are compact and portable miniaturized devices that can be employed in cancer biomarkers research leading to potential clinical applications. Biosensors employing surface nanoarchitectures with this type of detection offer attractive features including straightforward miniaturization, excellent LODs, robustness, small analyte volumes and the ability to be applied in turbid biofluids

Single/few-layered MXene (Ti3C2) nanosheets were functionalized with (3 aminopropyl)triethoxysilane (APTES) to enable covalent attachment of bioreceptor onto *f*-Ti3C2-MXene for electrochemical detection of carcinoembryonic antigen (CEA) as a widely used tumor marker [77]. The ultrathin 2D nanosheets of single/multilayer MXene Ti3C2 with high density of functional groups brought in improved antibodies anchoring and faster access to analyte. The label-free aminosilane and bio-functionalized *f*-Ti3C2-MXene-based biosensor (BSA/anti-CEA/f-Ti3C2-MXene/GC) demonstrated LOD of 0.000018 ng mL<sup>1</sup> with sensitivity

*Carcinoembryonic antigen (CEA, molecular mass of 180–200 kDa) is a highly glycosylated cell surface protein consisting of approx. 60% carbohydrates, which attains elevated levels in a number malignancies, such as colorectal, breast and ovarian, gastric, liver and pancreatic cancer. Serum CEA is used in clinical research to identify early stages of disease, monitor tumor recurrence and metastatic disease. The normal range of serum CEA in healthy adults of non-smokers is below 2.5 ng mL<sup>1</sup> and in the serum of smokers below 5.0 ng mL<sup>1</sup>*

Due to their excellent electrical conductivity and large specific surface area with a large number of potential attachment binding sites, 2D MXenes are also applied as

) for CEA determination using hexaammineruthenium ([Ru(NH3)6]

3+)

*,*

of 37.9 μA ng<sup>1</sup> mL cm<sup>2</sup> *per* decade (a linear detection range of 0.0001–

as a preferable redox probe and CV as a detection technique [77].

serum samples at low limit of detection [113].

as well as cancer theranostic applications [21].

**5.2 MXene-based electrochemical nanobiosensors**

with optically absorbing and fluorescing compounds.

*but increases rapidly when normal cells become cancerous.*

2000 ng mL<sup>1</sup>

**228**

*consisting entirely of a matrix (S/N) = 3*.

*Novel Nanomaterials*

(SPGE) [88]. As the initial step functionalization of two magnetic particles (MPs) with two different single-stranded DNAs (ssDNAs) was performed through labeling with methylene blue (MB) and ferrocene (Fc) that were partially complementary to the target miRNA. After the invasion of targets and amplification cycle, the released uncleaved DNA sequences harboring redox labels were hybridized with the electrochemical sensor platforms for subsequent measurements. To enhance the electrochemical signal, the SPGE was modified with the synthesized MXene-Ti3C2Tx and patterned with AuNPs and further loaded with abundant ssDNAs (base) to provide a significantly higher electrochemical signal compared to the AuNP/Au electrodes (almost 4 orders of magnitude increase). The LODs of the biosensor exhibiting multiplex ability, antifouling activity and single mutation recognition for microRNA-21 and microRNA-141 detection reaching low LOD levels down to 204 aM and 138 aM (a wide linear range up to 50 nM), respectively. The synergic effect of combining MXene based electrochemical amplification and DSN target recycling, resulted in a short assay time of 80 min, a good assay reproducibility (RSD ≈ 4.7%) and stability of 95.2% and 97.1% of its initial signal values assigned to MB and Fc, respectively, after 4 weeks of storage [88].

Ti3C2/BiVO4 nanocomposite covered the surface of the electrode to produce an initial photocurrent signal. The T7 Exonuclease (T7 Exo)-assisted dual signal amplification strategy was applied to achieve improved sensitivity of the PEC sensor. With the target VEGF165, the hairpin DNA (HP2), containing the aptamer of VEGF165 can be specifically identified and opened to specifically recognize the exposed toehold of S1 on the magnetic bead, releasing the output DNA S2, S3, and S4. Further, T7 Exo was used to digest the recessed 5<sup>0</sup> termini of double-stranded DNA (dsDNA). VEGF165-HP2 complex was released for the next cycle, which can be converted to multiple output DNAs. Next, the output DNA hybridized with hairpin DNA (HP1) on the electrode to form a double-stranded structure, which provided a wonderful platform for the intercalation of methylene blue. Methylene blue effectively increased light absorption and promoted the electron transfer along the dsDNA, resulting in an enhanced PEC signal. The inter-assay and intra-assay RSD values were calculated to be 2.42% and 2.26%, respectively, illustrating the

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

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

*The vascular endothelial growth factor (VEGF) is a biomarker with a molecular mass of 18–27 kDa and can be related to various cancer types for example brain, lung, gastrointestinal, hepatobiliary, renal, breast,*

An impedimetric aptasensor based on the nanostructured multicomponent hybrid of Ti3C2Tx nanosheets and phosphomolybdic acid (PMo12) nanoparticles integrated by embedding within the polypyrrole (PPy) matrix (PPy@Ti3C2Tx/ PMo12) was utilized for detection of osteopontin (OPN) [53]. The PPy@Ti3C2Tx/ PMo12-based aptasensor estimated OPN with LOD of 0.98 fg mL�<sup>1</sup> in a linear range

*Osteopontin (OPN, 41–75 kDa) known as a phosphoprotein regulates tumor metastasis and leads to cancer progression (breast, colon, liver, lung, ovarian, prostate). OPN plays an important role in tumor invasion, growth, angiogenesis, and metastasis by upregulating several signaling pathways. Normal level in serum is*

Surface plasmon resonance (SPR) is a principal technique for *in situ* bioaffinity assays of various target (bio)molecules without a need for fluorescent or enzymatic labeling. SPR (bio)sensors could be developed with improved operational parameters by applying nanomaterials [123]. SPR detection platform offers beneficial advantages for the biosensing including label-free and real-time detection, high sensitivity and selectivity, ease of miniaturization and rapid detection making the

*Surface plasmon resonance (SPR) is optical sensing technology which can be used for health monitoring, early disease diagnosis, and environment safety. It has become a valuable tool for biological, chemical, and biomedical applications. It has been widely used in various biochemical and biosensing applications, particularly for enzyme detection, drug diagnostic, dsDNA hybridization, and applied as an immune sensor. SPR sensors are refractive index based sensors that can be experimentally implemented for real-time*

1.7% and during the biosensor offered also good operational stability [53].

*.*

. The biosensor exhibited low RSD of the assays of around

outstanding reproducibility of the biosensor [122].

*ovarian. Normal level of VEGF in serum is* � *220 pg mL*�*<sup>1</sup>*

**5.3 MXene-based optical nanobiosensors**

technique well suited for bioassays.

*biosensing without the labeling of the analytes or bioreceptors.*

of 0.05–10,000 pg. mL�<sup>1</sup>

*16 ng mL*�*<sup>1</sup> .*

**231**

Xu *et al*. [120] treated Ti3C2 MXene with NaOH and hydrogen peroxide in a Teflon lined stainless-steel autoclave by a simultaneous oxidation and alkalization resulting in the synthesized 3D sodium titanate nanoribbons (M-NTO) in order to overcome restacking of MXenes flakes. Such a composite offered fast electron transfer ability, high specific surface area and excellent biocompatibility by connection of 3D M-NTO with conductive poly(3,4-ethylenedioxythiophene) (PEDOT). AuNPs were electrodeposited in the next step on the surface of M-NTO-PEDOT for immobilization of antibodies against prostate specific antigen (PSA) for PSA detection. Assay reproducibility was high with RSD of 1.89% with satisfactory biosensor stability (84.2% of its original response after 2 weeks storage at 4°C). The label-free immunosensor could detect PSA with LOD of 0.03 pg. L<sup>1</sup> (S/N = 3) by DPV [120].

*The prostate-specific antigen (PSA, 28.4 kDa) belongs to the tissue kallikrein-related family of peptidases and is also known as g-seminoprotein, kallikrein-3 or KLK3. PSA presenting a single-chain glycoprotein containing approximately 8% (by mass) of N-glycan with a single glycosylation site is produced by vesicles in prostate epithelial cells. Prostate cancer (PCa, adenocarcinoma or glandular cancer of the prostate gland) is the 2nd most abundant cancer type in men worldwide, with an estimated 1.1 million cases diagnosed in 2012 alone. The PSA level in health body is lower than 4 ng mL<sup>1</sup> .*

In addition, PSA was sensitively detected with capacitance-based enzyme immunosensor [121] based on enzymatic biocatalytic precipitation of precipitate on interdigitated micro-comb electrode (IDE). AuNPs heavily functionalized with HRP and detection antibodies (HRP-Au-Ab2) were utilized as the signal generating probe. Firstly, MXene dispersion in 1.0 wt % Nafion ethanol solution was dropped onto IDE to modify it. Next anti-PSA capture antibodies (Ab1) were physically adsorbed onto the nanosheets. Subsequently PSA, HRP-Au-Ab2 conjugates, H2O2 and HRP-tyramine conjugates were incubated step-by-step with the immunosensor at room temperature. The target PSA was determined with LOD of 0.031 ng mL<sup>1</sup> in a linear range up to 50 ng mL<sup>1</sup> with RSD of 10.7%, indicating good reproducibility [121].

Liu *et al*. [122] designed a "signal-on" photoelectrochemical (PEC) biosensor employing a Ti3C2/BiVO4 Schottky junction for a signal generation for ultrasensitive detection of vascular endothelial growth factor165 (VEGF165) with LOD of 3.3 fM (a linear range of 10 fM - 100 nM). First, *in situ* synthesized

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

Ti3C2/BiVO4 nanocomposite covered the surface of the electrode to produce an initial photocurrent signal. The T7 Exonuclease (T7 Exo)-assisted dual signal amplification strategy was applied to achieve improved sensitivity of the PEC sensor. With the target VEGF165, the hairpin DNA (HP2), containing the aptamer of VEGF165 can be specifically identified and opened to specifically recognize the exposed toehold of S1 on the magnetic bead, releasing the output DNA S2, S3, and S4. Further, T7 Exo was used to digest the recessed 5<sup>0</sup> termini of double-stranded DNA (dsDNA). VEGF165-HP2 complex was released for the next cycle, which can be converted to multiple output DNAs. Next, the output DNA hybridized with hairpin DNA (HP1) on the electrode to form a double-stranded structure, which provided a wonderful platform for the intercalation of methylene blue. Methylene blue effectively increased light absorption and promoted the electron transfer along the dsDNA, resulting in an enhanced PEC signal. The inter-assay and intra-assay RSD values were calculated to be 2.42% and 2.26%, respectively, illustrating the outstanding reproducibility of the biosensor [122].

*The vascular endothelial growth factor (VEGF) is a biomarker with a molecular mass of 18–27 kDa and can be related to various cancer types for example brain, lung, gastrointestinal, hepatobiliary, renal, breast, ovarian. Normal level of VEGF in serum is* � *220 pg mL*�*<sup>1</sup> .*

An impedimetric aptasensor based on the nanostructured multicomponent hybrid of Ti3C2Tx nanosheets and phosphomolybdic acid (PMo12) nanoparticles integrated by embedding within the polypyrrole (PPy) matrix (PPy@Ti3C2Tx/ PMo12) was utilized for detection of osteopontin (OPN) [53]. The PPy@Ti3C2Tx/ PMo12-based aptasensor estimated OPN with LOD of 0.98 fg mL�<sup>1</sup> in a linear range of 0.05–10,000 pg. mL�<sup>1</sup> . The biosensor exhibited low RSD of the assays of around 1.7% and during the biosensor offered also good operational stability [53].

*Osteopontin (OPN, 41–75 kDa) known as a phosphoprotein regulates tumor metastasis and leads to cancer progression (breast, colon, liver, lung, ovarian, prostate). OPN plays an important role in tumor invasion, growth, angiogenesis, and metastasis by upregulating several signaling pathways. Normal level in serum is 16 ng mL*�*<sup>1</sup> .*
