**5.3 MXene-based optical nanobiosensors**

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 technique well suited for bioassays.

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

(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].

DPV [120].

*Novel Nanomaterials*

ibility [121].

**230**

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

*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*

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 reproduc-

Liu *et al*. [122] designed a "signal-on" photoelectrochemical (PEC) biosensor

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

employing a Ti3C2/BiVO4 Schottky junction for a signal generation for

*.*

*alone. The PSA level in health body is lower than 4 ng mL<sup>1</sup>*

*It is an established high sensitivity platform for measuring minute concentrations of analyte and kinetics of biomolecular interactions. SPR generates an evanescent wave at the interface of two materials, when properly polarized incident light excites charge density oscillation (also called surface plasmons, SPs) supported by thin metal film deposited on the prism. However, SPR condition is established only after proper coupling of ppolarized incident wave with surface plasmon wave (SPW), when the frequency of evanescent wave matches the natural frequency of the SPW. SPR in reflection mode measures the resonance angle at a dip in reflectivity and a complete energy transfer from evanescent wave to SPW is achieved. Resonance angle is very sensitive to alteration of sensing medium refractive index (RI), i.e. on adsorption of analytes, which changes SPR condition. The sensitivity of the sensor is directly related to the resonance angle shift, which is sensitive to the modification of the RI of the sensing medium. In conventional SPR, a thin film of noble metal is used for SPs generation as well as adsorption of biomolecules or other analytes. Gold is a preferred interface, as it is nonoxidizing, corrosion-free, with substantial chemical stability, and shows stable adsorption of analytes with high sensitivity. However it shows a broad resonance curve causing reduction in detection accuracy. Silver (Ag) on the other hand shows higher accuracy through sharper reflectance curve but a poor chemical stability, as it is assumed to oxidize quickly on direct exposure to the atmosphere. Ag can be utilized efficiently in the SPR sensor if its oxidation can be avoided by using some protective layer over it. Conventional SPR sensor utilizing prism, metal layer and sensing medium offers smaller sensitivity.*

after storage for 28 days), reproducible (average assay RSD less than 5%) and offering operational stability (84% of its initial activity after five reuse

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

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

*Schematic of detection procedure of the prepared SPR biosensor. Reprinted with permission from ref. [127].*

acid to achieve high stabilization and dispersion of MXene with delivering functional groups were employed for covalent linkage of the bioreceptor (a dual

sis of alkaline phosphatase (ALP) activity with LOD of 0.02 U L<sup>1</sup>

Guo *et al*. [129] fabricated Ti3C2 QDs (4.2 nm in diameter) by a hydrothermal treatment with beneficial and excellent salt tolerance, anti-photobleaching and dispersion stability in aqueous solution. Ti3C2 QDs were applied as the fluorescent markers for fluorescent signal readout without and for sensitive fluorimetric analy-

accurate analysis of ALP by applying Ti3C2 QDs-based strategy for assays of AFP in the lysates of embryonic stem cells was also achieved by such a biosensor

*addition a high ALP activity is the traditional biomarker of pluripotent embryonic stem cells.*

*Alkaline phosphatase (ALP), as an essential enzyme in phosphate metabolism, responsible for catalysis of the dephosphorylation of a variety of substrates. The abnormal level of serum ALP, as a crucial biomarker for clinical diagnostics, is closely related to various diseases, such as diabetes, hepatitis and prostatic cancer. In*

A strand displacement dual amplification (SDDA) strategy was developed by Chen *et al*. [130] for simultaneous detection of multiple miRNAs analytes in a cell lysate using a unique single strand-double strand-single strand DNA (sdsDNA) probe, which was generated by the target recognition probe hybridizing with the site region probe. The fluorescence resonance energy transfer (FRET) assay was used for highly photostable, specific and sensitive detection of miRNAs with LOD

. Moreover, an

signal-tagged chimeric DNA probe (dcDNA)) [128].

Among various investigation methods for detection of cancer biomarkers, fluorescence analysis methods, especially fluorescent nanoprobes based on "turn on" mechanism, are regarded as sensitive and reliable analytical tools for cancer diagnosis. The nanoprobes can be ideally stabilized in both extracellular and intracellular microenvironment and respond to multi-biomarkers with different spatial distributions to achieve multilayer information of diverse biomarkers range from cell membrane to the cytoplasm at a cellular level [128]. Wang with colleagues [128] investigated fluorescence quenching capacity of Ti3C2 MXenes for biosensing of dual biomarkers in single (MCF-7) living cells. A chimeric DNA-functionalized Ti3C2 probe was employed for real-time and multilayer simultaneous fluorescent imaging of plasma membrane glycoprotein MUC1 and cytoplasmic microRNA-21 at nM concentration *in vitro (***Figure 4***)*. Ti3C2 MXene was decorated with polyacrylic

cycles) [127].

*Copyright ACS, 2020.*

**Figure 3.**

device [129].

**233**

Due to its absorption, a few-layer Ti3C2Tx MXene can contribute to the improved sensitivity of SPR biosensors. Enhanced sensitivity by 16.8%, 28.4%, 46.3% and 33.6% was achieved for the proposed SPR biosensors based on Au with 4 layers, Ag with 7 layers, Al with 12 layers and Cu with 9 layers of Ti3C2Tx, respectively [124].

The platform based on prism/gold layer/MXene/WS2/black phosphorus using monolayer of each nanomaterial was proved as a novel SPR sensing material with enhanced sensitivity of 15.6% compared to a bare metal film [125]. MXene-based composite, g-C3N4/MXene-AgNPs, including g-C3N4 as a photocatalyst, MXene as a co-catalyst and AgNPs as an electron mediator offered enhanced photocatalytic activity. The increased optical absorption and reduced band-gap energy due to the SPR effect of AgNPs deposited on such nanocomposite modified interface was observed [126].

Wu with co-workers [49] took advantage of hydrophilic and biocompatible Ti3C2 surface as a platform for making a nanohybrid consisting of multi-walled carbon nanotubes (MWCNTs)-polydopamine (PDA)-Ag nanoparticles (AgNPs) as a signal probe to develop SPR biosensor, that is easy to prepare, convenient to operate, and provides high sensitivity and selectivity. In order to obtain good orientation and immobilization of monoclonal anti-CEA antibody (Ab1), synthesized Ti3C2/AuNPs composite was firstly decorated with staphylococcal protein A (SPA) to which Ab1 was captured by affinity interaction through its Fc region. Polyclonal anti-CEA antibodies (Ab2) were conjugated with a nanohybrid through Schiff-base reaction between amino residues and quinone groups of PDA. By introducing a MWPAg-Ab2 conjugate to form a sandwich format, LOD of 0.07 fM was achieved for CEA detection (a dynamic range of 2 <sup>10</sup><sup>16</sup> - 2 <sup>10</sup><sup>8</sup> M). However, there are some limitations of such biosensing platform including time-consuming fabrication of the interfacial layer *prior* to analysis, but the biosensor exhibited good assay reproducibility with RSD below 5%. The stability of the fabricated sensing platform was also investigated by measuring the SPR responses to 10<sup>12</sup> M CEA concentration over the period of 7 days, during which the sensing platform was stored at 4°C. The developed biosensor lost 13% of its initial activity after storing for 7 days [49].

Wu *et al*. [127] utilized amino-functionalized N-Ti3C2-MXene-hollow gold nanoparticles (HGNPs)-staphylococcal protein A (SPA) complexes as a signal enhancer for CEA detection with LOD of 0.15 fM (a linear range of 0.001–1000 pM) at SPR (**Figure 3**). The SPR biosensor was stable (80% of the initial response *Ti3C2 MXene-Based Nanobiosensors for Detection of Cancer Biomarkers DOI: http://dx.doi.org/10.5772/intechopen.94309*

**Figure 3.**

*It is an established high sensitivity platform for measuring minute concentrations of analyte and kinetics of biomolecular interactions. SPR generates an evanescent wave at the interface of two materials, when properly polarized incident light excites charge density oscillation (also called surface plasmons, SPs) supported by thin metal film deposited on the prism. However, SPR condition is established only after proper coupling of ppolarized incident wave with surface plasmon wave (SPW), when the frequency of evanescent wave matches the natural frequency of the SPW. SPR in reflection mode measures the resonance angle at a dip in reflectivity and a complete energy transfer from evanescent wave to SPW is achieved. Resonance angle is very sensitive to alteration of sensing medium refractive index (RI), i.e. on adsorption of analytes, which changes SPR condition. The sensitivity of the sensor is directly related to the resonance angle shift, which is sensitive to the modification of the RI of the sensing medium. In conventional SPR, a thin film of noble metal is used for SPs generation as well as adsorption of biomolecules or other analytes. Gold is a preferred interface, as it is nonoxidizing, corrosion-free, with substantial chemical stability, and shows stable adsorption of analytes with high sensitivity. However it shows a broad resonance curve causing reduction in detection accuracy. Silver (Ag) on the other hand shows higher accuracy through sharper reflectance curve but a poor chemical stability, as it is assumed to oxidize quickly on direct exposure to the atmosphere. Ag can be utilized efficiently in the SPR sensor if its oxidation can be avoided by using some protective layer over it. Conventional SPR sensor utilizing*

Due to its absorption, a few-layer Ti3C2Tx MXene can contribute to the improved sensitivity of SPR biosensors. Enhanced sensitivity by 16.8%, 28.4%, 46.3% and 33.6% was achieved for the proposed SPR biosensors based on Au with 4 layers, Ag with 7 layers, Al with 12 layers and Cu with 9 layers of Ti3C2Tx, respec-

The platform based on prism/gold layer/MXene/WS2/black phosphorus using monolayer of each nanomaterial was proved as a novel SPR sensing material with enhanced sensitivity of 15.6% compared to a bare metal film [125]. MXene-based composite, g-C3N4/MXene-AgNPs, including g-C3N4 as a photocatalyst, MXene as a co-catalyst and AgNPs as an electron mediator offered enhanced photocatalytic activity. The increased optical absorption and reduced band-gap energy due to the SPR effect of AgNPs deposited on such nanocomposite modified interface was

Wu with co-workers [49] took advantage of hydrophilic and biocompatible Ti3C2 surface as a platform for making a nanohybrid consisting of multi-walled carbon nanotubes (MWCNTs)-polydopamine (PDA)-Ag nanoparticles (AgNPs) as a signal probe to develop SPR biosensor, that is easy to prepare, convenient to operate, and provides high sensitivity and selectivity. In order to obtain good orientation and immobilization of monoclonal anti-CEA antibody (Ab1), synthesized Ti3C2/AuNPs composite was firstly decorated with staphylococcal protein A (SPA) to which Ab1 was captured by affinity interaction through its Fc region. Polyclonal anti-CEA antibodies (Ab2) were conjugated with a nanohybrid through Schiff-base reaction between amino residues and quinone groups of PDA. By introducing a MWPAg-Ab2 conjugate to form a sandwich format, LOD of 0.07 fM was achieved for CEA detection (a dynamic range of 2 <sup>10</sup><sup>16</sup> - 2 <sup>10</sup><sup>8</sup> M). However, there are some limitations of such biosensing platform including time-consuming fabrication of the interfacial layer *prior* to analysis, but the biosensor exhibited good assay reproducibility with RSD below 5%. The stability of the fabricated sensing platform was also investigated by measuring the SPR responses to 10<sup>12</sup> M CEA concentration over the period of 7 days, during which the sensing platform was stored at 4°C. The developed biosensor lost 13% of its initial activity after storing for

Wu *et al*. [127] utilized amino-functionalized N-Ti3C2-MXene-hollow gold nanoparticles (HGNPs)-staphylococcal protein A (SPA) complexes as a signal enhancer for CEA detection with LOD of 0.15 fM (a linear range of 0.001–1000 pM) at SPR (**Figure 3**). The SPR biosensor was stable (80% of the initial response

*prism, metal layer and sensing medium offers smaller sensitivity.*

tively [124].

*Novel Nanomaterials*

observed [126].

7 days [49].

**232**

*Schematic of detection procedure of the prepared SPR biosensor. Reprinted with permission from ref. [127]. Copyright ACS, 2020.*

after storage for 28 days), reproducible (average assay RSD less than 5%) and offering operational stability (84% of its initial activity after five reuse cycles) [127].

Among various investigation methods for detection of cancer biomarkers, fluorescence analysis methods, especially fluorescent nanoprobes based on "turn on" mechanism, are regarded as sensitive and reliable analytical tools for cancer diagnosis. The nanoprobes can be ideally stabilized in both extracellular and intracellular microenvironment and respond to multi-biomarkers with different spatial distributions to achieve multilayer information of diverse biomarkers range from cell membrane to the cytoplasm at a cellular level [128]. Wang with colleagues [128] investigated fluorescence quenching capacity of Ti3C2 MXenes for biosensing of dual biomarkers in single (MCF-7) living cells. A chimeric DNA-functionalized Ti3C2 probe was employed for real-time and multilayer simultaneous fluorescent imaging of plasma membrane glycoprotein MUC1 and cytoplasmic microRNA-21 at nM concentration *in vitro (***Figure 4***)*. Ti3C2 MXene was decorated with polyacrylic acid to achieve high stabilization and dispersion of MXene with delivering functional groups were employed for covalent linkage of the bioreceptor (a dual signal-tagged chimeric DNA probe (dcDNA)) [128].

Guo *et al*. [129] fabricated Ti3C2 QDs (4.2 nm in diameter) by a hydrothermal treatment with beneficial and excellent salt tolerance, anti-photobleaching and dispersion stability in aqueous solution. Ti3C2 QDs were applied as the fluorescent markers for fluorescent signal readout without and for sensitive fluorimetric analysis of alkaline phosphatase (ALP) activity with LOD of 0.02 U L<sup>1</sup> . Moreover, an accurate analysis of ALP by applying Ti3C2 QDs-based strategy for assays of AFP in the lysates of embryonic stem cells was also achieved by such a biosensor device [129].

*Alkaline phosphatase (ALP), as an essential enzyme in phosphate metabolism, responsible for catalysis of the dephosphorylation of a variety of substrates. The abnormal level of serum ALP, as a crucial biomarker for clinical diagnostics, is closely related to various diseases, such as diabetes, hepatitis and prostatic cancer. In addition a high ALP activity is the traditional biomarker of pluripotent embryonic stem cells.*

A strand displacement dual amplification (SDDA) strategy was developed by Chen *et al*. [130] for simultaneous detection of multiple miRNAs analytes in a cell lysate using a unique single strand-double strand-single strand DNA (sdsDNA) probe, which was generated by the target recognition probe hybridizing with the site region probe. The fluorescence resonance energy transfer (FRET) assay was used for highly photostable, specific and sensitive detection of miRNAs with LOD

collect the visual thermal data for semi-quantitative analysis of target PSA

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

*Liposome, a target-responsive nanomaterial containing a bilayer of phospholipids with the spherical structure, is promising due to its superior biocompatibility, versatility of surface modification, operability of dimensional control and large-volume internal loading. The functional liposome acts as the biological signal amplifier by encapsulating numerous signal molecules and binding with biological recognition molecules like DNA,*

**5.4 Detection of exosomes as a source of cancer biomarkers by applying 2D**

Exosomes as type of endosome-derived cell-secreted vesicles with the structure of a lipid bilayer membrane are responsible for signal transduction in intercellular communication and extracellular matrix remodeling. In addition exosomes can also carry cargo affecting neighboring cells and they can form pre-metastatic niches [115]. Thus, exosomes are behind localized tumor development, progression and induction of distant tumors forming metastasis. The fact, that a substantially higher cellular activity of tumor cells results in the production of a greater number of exosomes than in normal/healthy cells, makes them hot candidates for cancer diag-

*Exosomes are naturally produced biological nanoparticles, with their size usually defined in the range from 30 nm up to 100 nm or sometimes up to 200 nm. Other types of extracellular vesicles (EVs) include microvesicles (50–1000 nm, which bud directly off the plasma membrane), ectosomes (vesicles assembled at and released from a plasma membrane), shedding vesicles, microparticles and apoptotic vesicles (500–*

Electrochemiluminescence (ECL) as an upcoming technique joining the benefits

, which was more than 100 times lower than the conventional ELISA

of both electrochemistry and chemiluminescence, has been widely applied for biomarker analysis thanks to its high sensitivity, short response time and low background signal [132]. A biosensor based on the application of MXene and ECL

(*ζ* 50 mV) was modified by polyethyleneimine (PEI) (*ζ* +55 mV) through electrostatic interactions to prepare an MXene/PEI nanocomposite (*ζ* 80 mV). This positively charged nanocomposite was subsequently used in covalent immobilization of an aptamer against CD63 protein, which is present on the surface of the exosomes using an amine-coupling chemistry. In an effort to detect exosomes, the GCE was modified by AuNPs, which were next modified by ethylenediamine. In addition, free -NH2 groups of ethylenediamine were activated by EDC/NHS to deposit a polymer, which was finally used for covalent immobilization of an aptamer against the EpCAM protein present on the surface of the exosomes. The signal was generated upon completion of the sandwich configuration as shown in **Figure 5**. The biosensor was most sensitive towards exosomes produced by a breast cancer cell line MCF-7, followed by a human liver cancer cell line HepG2 and a melanoma cell line B16. Exosomes released from the MCF-7 cell line were detected in the concentration range from 500 to 5 106 particles <sup>μ</sup><sup>L</sup><sup>1</sup> with LOD of 125

method. The biosensor exhibited an excellent performance by analysis of spiked serum samples with recovery indices of 95–104% [133]. In the later research of the same group, it was shown that, besides CD63 and EpCAM, other proteins

was developed for sensitive detection of exosomes [133]. First, MXene

*2000 nm, which bud off the membrane of cells undergoing apoptosis).*

within 3 min [131].

**MXenes**

nostics in itself [115].

particles μL<sup>1</sup>

**235**

*enzyme, protein and nanomaterial.*

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

**Figure 4.**

*(a) Illustration of the fabrication of the Ti3C2 MXenes and PAA-Ti3C2. (b) the construction of the dcDNA-Ti3C2 composite nanoprobe. (c) Multilayer imaging of plasma membrane glycoproteins MUC1 and cytoplasmic miR-21 using the dcDNA-Ti3C2 composite nanoprobe. Reprinted with permission from ref. [128]. Copyright ACS, 2019.*

of 0.5 fM and 0.85 fM for miRNA-21 and miRNA-10b, respectively (a linear range from 5 fM to 100 pM) [130].

PSA was the both qualitatively and quantitatively examined through a sandwich-type immunoreaction and a photothermal measurement by applying Ti3C2 MXene quantum dots (QDs)-encapsulated liposome with a high photothermal efficiency [131]. Ti3C2 MXene QDs as the innovative photothermal signal beacons were entrapped in the liposome for the labeling of the secondary antibody on the surface. The sandwich-type assay was carried out by coupling a low-cost microplate with a homemade 3D printed device. Under NIR-laser irradiation of 808 nm, Ti3C2 MXene QDs converted the light energy into heat, and the shift in the temperature correlating with the analyte concentration. LOD of 0.4 ng mL<sup>1</sup> for PSA was obtained by a near-infrared (NIR) photothermal immunoassay (a linear range of 1.0 ng mL<sup>1</sup> - 50 ng mL<sup>1</sup> ). The portable equipment employing a portable NIR imaging camera was able to

collect the visual thermal data for semi-quantitative analysis of target PSA within 3 min [131].

*Liposome, a target-responsive nanomaterial containing a bilayer of phospholipids with the spherical structure, is promising due to its superior biocompatibility, versatility of surface modification, operability of dimensional control and large-volume internal loading. The functional liposome acts as the biological signal amplifier by encapsulating numerous signal molecules and binding with biological recognition molecules like DNA, enzyme, protein and nanomaterial.*
