MXene/Carbon Dots Nanozyme Composites for Glutathione Detection and Tumor Therapy

Co-N-CDs-based MXene nanocomposites (MXene@PDA/Co-N-CDs) were constructed by decorating Co-N-CDs on polydopamine-functionalized MXene nanosheets. Both Co-N-CDs and MXene nanosheets have peroxidase-like activity; when the two materials are combined to form MXene@PDA/Co-N-CDs nanocomposites, the peroxide-like activity can be further enhanced. MXene@PDA/Co-N-CDs could oxidize the substrate 3,3′5,5′-tetramethylbenziline (TMB) to form ox-TMB, as confirmed by detecting the absorption of the blue products. A highly selective colorimetric biosensor was developed for the determination of glutathione (GSH) in the concentration range of 0.3 to 20 µM with a lower detection limit (LOD) of 0.12 µM, which realized the accurate detection of GSH in human serum and urine samples. Moreover, in the tumor microenvironment, MXene@PDA/Co-N-CDs could catalyze hydrogen peroxide to produce hydroxyl free radicals and produce a photothermal effect under the exposure of NIR-I irradiation. The catalytic activity of MXene@PDA/Co-N-CD nanocomposites was fully achieved for the death of cancer cells through photothermal/photodynamic synergistic therapy. The MXene@PDA/Co-N-CDs nanozyme offers multiple applications in GSH detection and tumor therapy.


Introduction
The superior biocompatibility, low cytotoxicity, and good photostability of carbon dots (CDs) position them as highly promising candidates for biomedical applications.Their distinctive characteristics and properties offer exciting opportunities for advancements in biosensing, bioimaging, and nanomedicine [1][2][3].Besides that, CDs have also been identified as excellent candidates for nanozymes because of their peroxidase (POD)-like activity [4].Many efforts have been made to improve the catalytic efficiency of carbon dots through surface modification [5,6], heteroatom doping [7], and composite with other nanomaterials [8].In the application of CDs, nitrogen doping significantly increases their peroxidase activity.As an effective material modification method, nitrogen doping provides strong support for peroxidase activity by increasing the quantity of active sites, thus facilitating the transfer of electrons among substrates [9].
MXene is a transition metal carbide containing either nitrides or carbonitrides with a two-dimensional layered structure.Its general formula is M n+1 X n T x , in which M represents the transition metal site [10][11][12][13], X stands for carbon and/or nitrogen, and Tx indicates functional groups on the surface of the outer transition metal layers.It is widely used in catalysis [10,11], energy storage [12], and chemical sensing [13] because it has a high surface area, high catalytic activity, abundant functional groups, and so on [14].MXenebased nanozymes have aroused significant attention because of their distinctive catalytic and physicochemical properties, offering exciting prospects in the domains of biology and nanomedicine, especially pertaining to medical diagnostics [15,16].However, MXene Scheme 1. Illustrative representation of the synthesis process for MXene@PDA/Co-N-CDs nanocomposites and the applications in colorimetric detection of GSH and hyperthermal-enhanced catalytic therapy of tumors.

Synthesis of MXene
During the experiment, 0.8 g LiF was added to a 10 mL HCl (9 M) PTFE reaction kettle, stirring for 5 min to ensure that it was fully dissolved.Then, 0.5 g Ti3AlC2 was slowly added to the previously prepared solution and continuously stirred at 3 °C for 24 h to obtain an adequate reaction.Subsequently, the obtained mixture was rinsed a few Scheme 1. Illustrative representation of the synthesis process for MXene@PDA/Co-N-CDs nanocomposites and the applications in colorimetric detection of GSH and hyperthermal-enhanced catalytic therapy of tumors.

Synthesis of MXene
During the experiment, 0.8 g LiF was added to a 10 mL HCl (9 M) PTFE reaction kettle, stirring for 5 min to ensure that it was fully dissolved.Then, 0.5 g Ti 3 AlC 2 was slowly added to the previously prepared solution and continuously stirred at 3 • C for 24 h to obtain an adequate reaction.Subsequently, the obtained mixture was rinsed a few times with distilled water, centrifuged at 3500 rpm through a centrifuge, and the process was repeated many times until the pH of the supernatant attained a value of 6.Then, water was added to the solid precipitate to 100 mL followed by ultrasonic treatment for 1 h under nitrogen protection.The mixture was centrifuged again at 3000 rpm for 1 h.The supernatant was subjected to freeze-drying within the centrifugal tube to facilitate its subsequent utilization.

Preparation of MXene@PDA
First, 100 mg of MXene was dispersed in 100 mL of Tris-HCl buffer solution, then 100 mg of dopamine hydrochloride was added into the mixture followed by stirring in the dark for 24 h at 45 • C. Finally, in order to obtain dry MXene@PDA, the MXene@PDA precipitate obtained by centrifugation was placed in a freeze dryer, and the moisture in the sample was completely removed by low-temperature freezing and vacuum drying; thus, the dry MXene@PDA powder was obtained.

Synthesis of Co-N-CDs
Here, a hydrothermal technique was employed for the synthesis of Co-N-CDs.First, 2.0 g CA was dissolved in 20 mL distilled water.Then, 0.04 g CoCl 2 •6H 2 O was added, and the mixture was continuously stirred for five minutes until fully dissolved.Then, 85 µL EDA•H 2 O was added to the mixture.After mixing, the resulting mixture was carefully transferred to the PTFE reactor, and the temperature was kept stable at around 160 • C to provide a stable reaction environment.After 8 h of the heating reaction, we obtained the initial product.After the high-temperature reaction, we remove the reactor and allow it to cool naturally to room temperature.Once the product reached room temperature, the mixture was filtered using a 0.22 µm membrane filter (aquo-system) to remove large impurity particles from the mixture and ensure the purity of the product.The filtered solution then underwent dialysis for 48 h to remove unreacted small molecules and further improve the purity of the product.Finally, we freeze-dried the solution after dialysis and successfully obtained a pure and dry product.

Synthetic MXene@PDA/Co-N-CDs
First, 100 mg MXene@PDA and Co-N-CDs were precisely weighed and carefully dispersed in 100 mL of Tris-HCl buffer solution, and then the mixture was agitated at 45 • C for a duration of 24 h to ensure thorough mixing and reaction.After completing the above steps, we centrifuged and washed the mixture.Finally, through freeze-drying technology, we successfully obtained products ready for further use.The schematic fabrication process of MXene@PDA/Co-N-CDs nanocomposites is shown in Scheme 1.

Exploration of the POD-Like Activity of MXene@PDA/Co-N-CDs
Exploiting the catalytic oxidation capacity of TMB in the presence of hydrogen peroxide, we successfully evaluated the peroxide-like activity of MXene@PDA/ Co-N-CDs.MXene@PDA/Co-N-CDs (10 µg•mL −1 ), hydrogen peroxide (50 mM), TMB (5 mM), and NaAC-HAC (pH 3.6) were mixed into a 2 mL centrifuge tube and reacted in a water bath at 40 • C for 12 h.The absorbance at 652 nm was recorded.The impact of pH (from 3.0 to 5.0) and temperature (from 25 • C to 55 • C) were determined to investigate their effects on the POD-like activity of MXene@PDA/Co-N-CDs.

•OH Monitored by Using MB
First, 10 µg•mL −1 of the MXene@PDA/Co-N-CDs nanocomposites was added to a freshly prepared methylene blue solution (MB, H 2 O, 2 µg•mL −1 ) containing 50 mM hydrogen peroxide.After incubation at different time intervals, the absorbance of λ = 664 nm was determined on a UV-visible spectrophotometer.

Steady-State Kinetic Analysis of MXene@PDA/Co-N-CDs
The steady-state kinetics of the MXene@PDA/Co-N-CDs nanocomposites (10 µg•mL −1 ) in the presence of NaAc-HAc (pH 3.6) were investigated.Different concentrations of TMB solution or hydrogen peroxide were added, and the absorbance at 652 nm of the solution was then determined.After further analysis of the experimental data, the Michaelis-Menten saturation curve was obtained.Finally, the Michaelis constant of MXene@PDA/Co-N-CDs when used as peroxidase was obtained through calculation.

H 2 O 2 Detection
To explore the catalytic process of MXene@PDA/Co-N-CDs as a peroxide mimic enzyme and substrate, 200 µL TMB (5 mM), 300 µL NaAC-HAC (pH 3.6), 500 µL of various concentrations of hydrogen peroxide, and 500 µL MXene@PDA/Co-N-CDs (10 µg•mL −1 ) were added to a 2 mL centrifuge tube.After mixing thoroughly and incubating for 12 h at room temperature, a UV-vis spectrophotometer was used to measure the absorbance at λ = 652 nm.

Detection of GSH
First, 200 µL TMB (5 mM), 300 µL NaAC-HAC buffer solution (pH 3.6), MXene@PDA/ Co-N-CDs (10 µg•mL −1 500 µL), and 50 mM H 2 O 2 (500 µL) solution were successively added to 2 mL centrifuge tubes and incubated for 12 h.Then, 500 µL GSH solution in a series of concentration gradients was added to the mixture and incubated at normal temperature for 20 min to ensure that glutathione fully reacted with MXene@PDA/Co-N-CDs.Finally, a UV-vis spectrophotometer was utilized to measure the absorbance at λ = 652 nm accurately.

Detection of Glutathione in Human Urine and Serum Samples
To verify the practicality and applicability of our proposed method for detecting catalytic activity based on MXene@PDA/Co-N-CDs in actual sample detection, we further extended our investigation to the detection of GSH in human urine and serum samples.In this study, we used morning urine provided by 3 volunteers as experimental samples.The urine samples were pretreated, and the solid particles and impurities were removed by centrifugation (1200 rpm, 40 min) to ensure the accuracy and reliability of subsequent analysis.Subsequently, we diluted the urine sample 10-fold with HAC-NaAC solution (pH 3.6) for further use.Serum samples were provided by the Affiliated Hospital of Qingdao University.The experiments on detecting GSH in human serum complied with relevant laws and institutional guidelines and were approved by Qingdao University.The serum samples were centrifuged (12,000 rpm, 5 min), and the supernatant was diluted 50-fold with HAC-NaAC solution (pH 3.6).
A total of 200 µL 5 mM TMB, 300 µL NaAC-HAC buffer solution (pH 3.6), 500 µL 10 µg•mL −1 Co-N-CDs, and 500 µL 50 mM of H 2 O 2 solution were added to 2 mL centrifuge tubes successively.After 12 h of incubation, the diluted real samples and different spiked concentrations of GSH were added into the above tubes for recovery experiments.We then incubated the mixture at normal temperature for 20 min.After incubation, the absorbance at λ = 652 nm was accurately measured.

Photothermic Performance of MXene@PDA/Co-N-CDs In Vitro
The temperature changes and thermal images of Co-N-CDs, MXene, and MXene@PDA/ Co-N-CDs nanocomposites at different times were measured by using an infrared thermal camera.We used an 808 nm laser light (2 W•cm −2 ) to irradiate the samples at normal temperature for 10 min.At the same time, in order to more fully evaluate the impact of laser light irradiation on the properties of the material, we also irradiated purified water under the same conditions.

Cytotoxicity Test by the CCK-8 Assay
The cytotoxicity of MXene@PDA/Co-N-CDs was tested in human breast cancer cells (MCF-7).MCF-7 cells were seeded on a 96-well plate and cultured in the incubator for 24 h.Then, different concentrations of MXene@PDA/Co-N-CDs (0, 100, 250, 500, 750, 1000 µg•mL −1 ) and DMEM high glucose medium were added and incubated for 4 h.For comparison, the light group was illuminated with 808 nm laser light (2 W•cm −2 ) for 10 min, and the medium without MXene@PDA/Co-N-CDs was the blank group.Next, 100 µL 10% CCK-8 was added to all the wells.After incubation in the incubator for 0.5-1 h, the absorbance was measured in a microplate reader, and then the level of cytotoxicity was determined.

Apoptosis Detection
MCF-7 cells were inoculated into Petri dishes and further incubated in an incubator.Once the cells reached the desired state, the original culture medium was removed and replaced with culture medium containing MXene@PDA/Co-N-CDs (500 µg•mL −1 ) or substituted with fresh culture medium, following an additional incubation for 4 h.Subsequently, the cells were digested, centrifuged (1000 rpm, 3 min), washed twice with PBS, redispersed in buffer solution, and stained with Annexin-FITC/PI for 15 min.Finally, the extent of apoptosis was detected using a flow cytometer.

Clonogenic Assay
Initially, MCF-7 cancer cells were seeded at a density of 2000 cells per well for 24 h, followed by the addition of MXene@PDA/Co-N-CDs nanocomposites and incubation for another 4 h.Subsequently, the laser light group was exposed to 808 nm near-infrared laser irradiation.After 8 days of culture, the cells were fixed with paraformaldehyde for 15 min.The plate was then cleaned three times with deionized water and 1 mL crystal violet dye was added for 20 min of dyeing.Then, the plate was washed again, air-dried, and photographed.Finally, the clones from all groups were counted, and the survival rate was plotted.

Characterization
Transmission electron microscopy (TEM) provided a comprehensive visualization of the morphologies of Co-N-CDs, MXene, MXene@PDA, and MXene@PDA/Co-N-CDs separately.By reaction in a mixed fluoride salt/hydrochloric acid system, the Al layer in Ti 3 AlC 2 was successfully etched to obtain an MXene nanosheet with a smooth surface with a single layer (Figure 1a) [29].Figure 1b shows that a light-colored boundary layer was clearly visible along the edges of MXene, unequivocally indicating the successful coating of PDA on the surface of MXene [30].The spherical Co-N-CDs in Figure 1c are uniformly monodisperse, with an average diameter of approximately 3.3 nm.Furthermore, Figure 1d clearly shows that the carbon dots bind tightly on the MXene@PDA surface to form the MXene@PDA/Co-N-CDs.These results demonstrate that MXene@PDA/Co-N-CDs composites were successfully prepared.
Based on the AFM image (Figure S1a) analysis, the thickness of MXene nanosheets obtained was relatively uniform, and the average height of MXene was 1.02 nm, indicating that monolayer MXene nanosheets were successfully obtained.
Using a Zeta analyzer, the potential of MXene was measured to be −27.3mV.Because of the presence of a large number of -OH, -O, and -F groups, MXene exhibits a high electronegativity [10].After coating PDA on MXene, the Zeta potential was obviously decreased to -44.9 mV because PDA contains more electronegativity groups, such as -OH and -NH groups.The Zeta potential of CDs was measured at −0.3 mV.Finally, with the loading of MXene@PDA, the potential of MXene@PDA/Co-N-CDs was -21.7 mV, further indicating the successful preparation of MXene/CD-based nanocomposites (Figure S1c).
XRD was used to confirm the structure of the MXene nanosheet.Compared with Ti3AlC2, the MAX phase of the MXene nanosheet of the (002) peak shifted from 9.5° to 5.91°.The disappearance of the peaks at 39.5° indicated that the Al layer was completely etched away [31,32] and the MXene nanosheet was successfully obtained (Figure 2a).As shown in Figure 2b, compared with pure MXene, the peak value of MXene coated with PDA was reduced to 5.57°, suggesting that the addition of the PDA coating may lead to an increase in interlayer spacing.Figure 3a,b show the full spectrum XPS data for MXene, MXene@PDA, Co-N-CDs, and MXene@PDA/Co-N-CDs.Figure 3a shows the XPS measurement spectra of MXene and MXene@PDA.These spectral data clearly confirm the presence of C, O, F, and Ti elements.Furthermore, the N element was found in the spectrum of MXene@PDA.MXene@PDA/Co-N-CDs revealed the presence of the C, N, Ti, Co, and O elements.The C1s XPS spectrum of MXene@PDA/Co-N-CDs was divided into three peaks (Figure 3c), Figure 1b shows that a light-colored boundary layer was clearly visible along the edges of MXene, unequivocally indicating the successful coating of PDA on the surface of MXene [30].The spherical Co-N-CDs in Figure 1c are uniformly monodisperse, with an average diameter of approximately 3.3 nm.Furthermore, Figure 1d clearly shows that the carbon dots bind tightly on the MXene@PDA surface to form the MXene@PDA/Co-N-CDs.These results demonstrate that MXene@PDA/Co-N-CDs composites were successfully prepared.
Based on the AFM image (Figure S1a) analysis, the thickness of MXene nanosheets obtained was relatively uniform, and the average height of MXene was 1.02 nm, indicating that monolayer MXene nanosheets were successfully obtained.
Using a Zeta analyzer, the potential of MXene was measured to be −27.3mV.Because of the presence of a large number of −OH, −O, and −F groups, MXene exhibits a high electronegativity [10].After coating PDA on MXene, the Zeta potential was obviously decreased to −44.9 mV because PDA contains more electronegativity groups, such as −OH and −NH groups.The Zeta potential of CDs was measured at −0.3 mV.Finally, with the loading of MXene@PDA, the potential of MXene@PDA/Co-N-CDs was −21.7 mV, further indicating the successful preparation of MXene/CD-based nanocomposites (Figure S1c).
XRD was used to confirm the structure of the MXene nanosheet.Compared with Ti 3 AlC 2 , the MAX phase of the MXene nanosheet of the (002) peak shifted from 9.5 • to 5.91 • .The disappearance of the peaks at 39.5 • indicated that the Al layer was completely etched away [31,32] and the MXene nanosheet was successfully obtained (Figure 2a).As shown in Figure 2b, compared with pure MXene, the peak value of MXene coated with PDA was reduced to 5.57 • , suggesting that the addition of the PDA coating may lead to an increase in interlayer spacing.Figure 1b shows that a light-colored boundary layer was clearly visible along the edges of MXene, unequivocally indicating the successful coating of PDA on the surface of MXene [30].The spherical Co-N-CDs in Figure 1c are uniformly monodisperse, with an average diameter of approximately 3.3 nm.Furthermore, Figure 1d clearly shows that the carbon dots bind tightly on the MXene@PDA surface to form the MXene@PDA/Co-N-CDs.These results demonstrate that MXene@PDA/Co-N-CDs composites were successfully prepared.
Based on the AFM image (Figure S1a) analysis, the thickness of MXene nanosheets obtained was relatively uniform, and the average height of MXene was 1.02 nm, indicating that monolayer MXene nanosheets were successfully obtained.
Using a Zeta analyzer, the potential of MXene was measured to be −27.3mV.Because of the presence of a large number of -OH, -O, and -F groups, MXene exhibits a high electronegativity [10].After coating PDA on MXene, the Zeta potential was obviously decreased to -44.9 mV because PDA contains more electronegativity groups, such as -OH and -NH groups.The Zeta potential of CDs was measured at −0.3 mV.Finally, with the loading of MXene@PDA, the potential of MXene@PDA/Co-N-CDs was -21.7 mV, further indicating the successful preparation of MXene/CD-based nanocomposites (Figure S1c).
XRD was used to confirm the structure of the MXene nanosheet.Compared with Ti3AlC2, the MAX phase of the MXene nanosheet of the (002) peak shifted from 9.5° to 5.91°.The disappearance of the peaks at 39.5° indicated that the Al layer was completely etched away [31,32] and the MXene nanosheet was successfully obtained (Figure 2a).As shown in Figure 2b, compared with pure MXene, the peak value of MXene coated with PDA was reduced to 5.57°, suggesting that the addition of the PDA coating may lead to an increase in interlayer spacing.Figure 3a,b show the full spectrum XPS data for MXene, MXene@PDA, Co-N-CDs, and MXene@PDA/Co-N-CDs.Figure 3a shows the XPS measurement spectra of MXene and MXene@PDA.These spectral data clearly confirm the presence of C, O, F, and Ti elements.Furthermore, the N element was found in the spectrum of MXene@PDA.MXene@PDA/Co-N-CDs revealed the presence of the C, N, Ti, Co, and O elements.The C1s XPS spectrum of MXene@PDA/Co-N-CDs was divided into three peaks (Figure 3c), Figure 3a,b show the full spectrum XPS data for MXene, MXene@PDA, Co-N-CDs, and MXene@PDA/Co-N-CDs.Figure 3a shows the XPS measurement spectra of MXene and MXene@PDA.These spectral data clearly confirm the presence of C, O, F, and Ti elements.Furthermore, the N element was found in the spectrum of MXene@PDA.MXene@PDA/Co-N-CDs revealed the presence of the C, N, Ti, Co, and O elements.The C1s XPS spectrum of MXene@PDA/Co-N-CDs was divided into three peaks (Figure 3c), corresponding to C-C (284.28 eV), C-O-Ti (287.4 eV), and C-O/C-N (285.94 eV).The XPS spectra of N 1s (Figure 3d) were consistent with four components, including a primary amine (R=N-R) at 398.7 eV, a tertiary amine (R-NH-R) at 400.45 eV, a primary amine (R-NH 2 ) at 401.05 eV, and Co-N at 399.45 eV [29,33].The O 1s region (Figure 3e) had five characteristic peaks at 530.3, 531.25, 532.15, 532.95, and 533.9 eV, which were ascribed to titanium dioxide, Ti-(O)x, C=O/C-O-Ti, Ti-(OH)x, and C-O/H 2 O, respectively [34].The Ti 2p spectrum (Figure 3f) was divided into three pairs of spin peaks of Ti 2p 3/2 and Ti 2p 1/2 .The four functional components that appeared at 455.75, 460.5, 466.15, and 459.2 eV were correlated with Ti-C 2p 3/2 , Ti-C 2p 1/2 , Ti-X 2p 1/2 , and Ti-X 2p 3/2 , respectively.As evident in Figure 3g, the Co 2p energy spectrum showed significant peaks of Co 2p 3/2 and Co 2p 1/2 at 781.38 and 797.21 eV, respectively.The interval between these two peaks was 15.83 eV, indicating the presence of Co(III).In addition, the presence of Co(II) was confirmed by the XPS spectra of the Co 2p 3/2 peak and the recombinant satellite peak at 786.94 eV, providing the structural foundation for the enzymatic reaction [9]., respectively [34].The Ti 2p spectrum (Figure 3f) was divided into three pairs of spin peaks of Ti 2p 3/2 and Ti 2p 1/2 .The four functional components that appeared at 455.75, 460.5, 466.15, and 459.2 eV were correlated with Ti-C 2p 3/2 , Ti-C 2p 1/2 , Ti-X 2p 1/2 , and Ti-X 2p 3/2 , respectively.As evident in Figure 3g, the Co 2p energy spectrum showed significant peaks of Co 2p 3/2 and Co 2p 1/2 at 781.38 and 797.21 eV, respectively.The interval between these two peaks was 15.83 eV, indicating the presence of Co(III).In addition, the presence of Co(II) was confirmed by the XPS spectra of the Co 2p 3/2 peak and the recombinant satellite peak at 786.94 eV, providing the structural foundation for the enzymatic reaction [9].The FT-IR spectra of MXene, MXene@PDA, Co-N-CDs, and MXene@PDA/Co-N-CDs are shown in Figure 3h.The characteristic absorption bands at 3425, 1779, 1706, 1572, 1173, and 1235 cm −1 belonged to NH/N-H, C=O, C=N, C=C, C-N, and C-O vibrations of Co-N-CDs, respectively, which indicated that the surface contained many hydrophilic groups such as amino and hydroxyl groups.In addition, the peak at 624 cm −1 was attributed to the tensile vibration of Co−N, and this indicated that cobalt doping was successful and provided active sites for nanozymes.[9] The peak at 3418 cm −1 was the tensile vibration of -OH in MXene (curve b), while the tensile vibrations of C=O, C−O, and C-F were located at 1630 cm −1 , 1091 cm −1 , and 1049 cm −1 , respectively.The peak value at 558 cm −1 was the characteristic peak of the Ti-O bond [30].
For MXene@PDA (curve c), two intense characteristic peaks at 3419 and 1618 cm −1 were observed in the FT-IR spectrum.According to previous literature reports [35], these two characteristic peaks correspond to the aromatic ring structure and the catechole-OH group in the PDA molecule, respectively.More abundant characteristic peaks were observed in the FT-IR spectra (curve d) of MXene@PDA/Co-N-CDs composites.These characteristic peaks included both the strong absorption peak of MXene@PDA and the The FT-IR spectra of MXene, MXene@PDA, Co-N-CDs, and MXene@PDA/Co-N-CDs are shown in Figure 3h.The characteristic absorption bands at 3425, 1779, 1706, 1572, 1173, and 1235 cm −1 belonged to NH/N-H, C=O, C=N, C=C, C-N, and C-O vibrations of Co-N-CDs, respectively, which indicated that the surface contained many hydrophilic groups such as amino and hydroxyl groups.In addition, the peak at 624 cm −1 was attributed to the tensile vibration of Co−N, and this indicated that cobalt doping was successful and provided active sites for nanozymes [9].The peak at 3418 cm −1 was the tensile vibration of -OH in MXene (curve b), while the tensile vibrations of C=O, C-O, and C-F were located at 1630 cm −1 , 1091 cm −1 , and 1049 cm −1 , respectively.The peak value at 558 cm −1 was the characteristic peak of the Ti-O bond [30].
For MXene@PDA (curve c), two intense characteristic peaks at 3419 and 1618 cm −1 were observed in the FT-IR spectrum.According to previous literature reports [35], these two characteristic peaks correspond to the aromatic ring structure and the catechole-OH group in the PDA molecule, respectively.More abundant characteristic peaks were observed in the FT-IR spectra (curve d) of MXene@PDA/Co-N-CDs composites.These characteristic peaks included both the strong absorption peak of MXene@PDA and the characteristic absorption peak of Co-N-CDs.The results fully show that Co-N-CDs were successfully introduced into the MXene@PDA system to form MXene@PDA/Co-N-CDs.

Peroxidase-like Activity of MXene@PDA/Co-N-CDs
Co-N-CDs and MXene both showed significant peroxisase-like activity.Specifically, when the two nanomaterials reacted with the TMB substrate in the presence of hydrogen peroxide, they were able to catalyze the oxidation of the TMB substrate.Figure S2 explores in detail how three key factors, i.e., pH, temperature, and TMB concentration, affect the catalytic activity of MXene@PDA/Co-N-CDs composite.The results showed that MXene@PDA/Co-N-CDs had good enzymatic activity at a temperature of 40 • C, the TMB concentration was 5 mM, and the pH value was 3.6.
The UV absorption spectra in the different systems are shown in Figure 4a.After mixed reactions in different systems at the optimal temperature and pH, it was obvious that the UV-visible absorbance of MXene@PDA/Co-N-CDs was higher than the other substances, indicating a higher peroxidase-like activity.
Nanomaterials 2024, 14, 1090 9 of 17 characteristic absorption peak of Co-N-CDs.The results fully show that Co-N-CDs were successfully introduced into the MXene@PDA system to form MXene@PDA/Co-N-CDs.

Peroxidase-like Activity of MXene@PDA/Co-N-CDs
Co-N-CDs and MXene both showed significant peroxisase-like activity.Specifically, when the two nanomaterials reacted with the TMB substrate in the presence of hydrogen peroxide, they were able to catalyze the oxidation of the TMB substrate.Figure S2 explores in detail how three key factors, i.e., pH, temperature, and TMB concentration, affect the catalytic activity of MXene@PDA/Co-N-CDs composite.The results showed that MXene@PDA/Co-N-CDs had good enzymatic activity at a temperature of 40 °C, the TMB concentration was 5 mM, and the pH value was 3.6.
The UV absorption spectra in the different systems are shown in Figure 4a.After mixed reactions in different systems at the optimal temperature and pH, it was obvious that the UV-visible absorbance of MXene@PDA/Co-N-CDs was higher than the other substances, indicating a higher peroxidase-like activity.The catalytic pathways for peroxidase-like activity usually involve two core processes including reactive oxygen species generation and electron transfer processes that occur during this reaction.In the current study system, in order to reveal potentially active intermediates, we used methylene blue (MB) as a probe to monitor the formation of hydroxyl (•OH) radicals [36].The hydroxyl radical, as a highly active oxidant, can react with MB, resulting in a significant decrease in the absorbance intensity of MB.As shown in Figure 4b, we observed the absorbance changes in solutions containing MB, hydrogen peroxide, and MXene@PDA/Co-N-CDs during the reaction.After a reaction period of 6 h, it became evident that the absorbance of the solution underwent a notable decrease.This experimental phenomenon strongly proves the existence of •OH free radicals and further confirms that CD composites can produce •OH free radicals during the catalytic process, thus exhibiting peroxidase activity.CDs activated the decomposition process of hydrogen peroxide, thus promoting the oxidation of TMB.With the addition of GSH, the dark blue hue of oxTMB gradually faded away.The corresponding UV-vis spectra showed that the absorption peak near 652 nm was significantly reduced, indicating that the oxTMB could be reduced by GSH.
To investigate the POD simulation activity of MXene@PDA/Co-N-CDs systematically, we explored the steady-state kinetics at different concentrations of hydrogen peroxide and TMB under the optimal conditions described above.The results showed that both hydrogen peroxide and TMB conform to the standard Michahelis-Menten model (Figure 4c,d).By fitting with the Lineweaver Burk plot as 1/V = (Km/Vmax) (1/[S]) + 1/Vmax, the maximum initial velocity (Vmax) and Miter constant (Km) corresponding to hydrogen peroxide and TMB were obtained, as shown in Table 1.In addition, compared with HRP and the other related nanozymes reported, The catalytic pathways for peroxidase-like activity usually involve two core processes including reactive oxygen species generation and electron transfer processes that occur during this reaction.In the current study system, in order to reveal potentially active intermediates, we used methylene blue (MB) as a probe to monitor the formation of hydroxyl (•OH) radicals [36].The hydroxyl radical, as a highly active oxidant, can react with MB, resulting in a significant decrease in the absorbance intensity of MB.As shown in Figure 4b, we observed the absorbance changes in solutions containing MB, hydrogen peroxide, and MXene@PDA/Co-N-CDs during the reaction.After a reaction period of 6 h, it became evident that the absorbance of the solution underwent a notable decrease.This experimental phenomenon strongly proves the existence of •OH free radicals and further confirms that CD composites can produce •OH free radicals during the catalytic process, thus exhibiting peroxidase activity.CDs activated the decomposition process of hydrogen peroxide, thus promoting the oxidation of TMB.With the addition of GSH, the dark blue hue of oxTMB gradually faded away.The corresponding UV-vis spectra showed that the absorption peak near 652 nm was significantly reduced, indicating that the oxTMB could be reduced by GSH.
To investigate the POD simulation activity of MXene@PDA/Co-N-CDs systematically, we explored the steady-state kinetics at different concentrations of hydrogen peroxide and TMB under the optimal conditions described above.The results showed that both hydrogen peroxide and TMB conform to the standard Michahelis-Menten model (Figure 4c,d).By fitting with the Lineweaver Burk plot as 1/V = (Km/Vmax) (1/[S]) + 1/Vmax, the maximum initial velocity (Vmax) and Miter constant (Km) corresponding to hydrogen peroxide and TMB were obtained, as shown in Table 1.In addition, compared with HRP and the other related nanozymes reported, MXene@PDA/Co-N-CDs had the minimum Km.Km is a key parameter of enzyme kinetics that reflects the ability of an enzyme to bind to a specific substrate.A low Km value signifies that the enzyme exhibits a stronger affinity for the substrate, subsequently indicating a higher catalytic efficiency towards the substrate.
In this study, we observed that the Km value between MXene@PDA/Co-N-CDs and TMB is low.This finding not only confirmed the strong affinity between MXene@PDA/Co-N-CDs and TMB but also revealed its excellent POD-like activity.

Colorimetric Detection of H 2 O 2 and GSH
To improve the catalytic activity of MXene@PDA/Co-N-CDs towards H 2 O 2 and GSH, after optimizing the main parameters, a temperature of 40 • C, pH of 3.6, and TMB concentration of 5 mM were selected as the optimum experimental conditions for detecting H 2 O 2 and GSH (Figure S2).
As the concentration of H 2 O 2 rises, a pronounced shift occurs in the color of the mixture, transitioning smoothly from its initial colorless state to a vibrant blue.Therefore, we performed the colorimetric detection of H 2 O 2 by observing the color change.Furthermore, we further investigated the relationship between the H 2 O 2 concentration and absorbance.The experimental results showed that in the concentration range of 0.4 to 50 mM, the concentration of H 2 O 2 showed a linear correlation with the absorbance at 652 nm (as shown in Figure 5a).The linear regression equation is A = 0.0038 × C + 0.0247, R 2 = 0.998, with the detection limit of 0.23 mM.
MXene@PDA/Co-N-CDs had the minimum Km.Km is a key parameter of enzyme kinetics that reflects the ability of an enzyme to bind to a specific substrate.A low Km value signifies that the enzyme exhibits a stronger affinity for the substrate, subsequently indicating a higher catalytic efficiency towards the substrate.In this study, we observed that the Km value between MXene@PDA/Co-N-CDs and TMB is low.This finding not only confirmed the strong affinity between MXene@PDA/Co-N-CDs and TMB but also revealed its excellent POD-like activity.

Colorimetric Detection of H2O2 and GSH
To improve the catalytic activity of MXene@PDA/Co-N-CDs towards H2O2 and GSH, after optimizing the main parameters, a temperature of 40 °C, pH of 3.6, and TMB concentration of 5 mM were selected as the optimum experimental conditions for detecting H2O2 and GSH (Figure S2).
As the concentration of H2O2 rises, a pronounced shift occurs in the color of the mixture, transitioning smoothly from its initial colorless state to a vibrant blue.Therefore, we performed the colorimetric detection of H2O2 by observing the color change.Furthermore, we further investigated the relationship between the H2O2 concentration and absorbance.The experimental results showed that in the concentration range of 0.4 to 50 mM, the concentration of H2O2 showed a linear correlation with the absorbance at 652 nm (as shown in Figure 5a).The linear regression equation is A = 0.0038 × C + 0.0247, R 2 = 0.998, with the detection limit of 0.23 mM.Upon the addition of GSH, the absorbance of the oxTMB-containing solution system gradually decreased at 652 nm.This occurred because the oxidation process of TMB catalyzed by MXene@PDA/Co-N-CDs was inhibited by the addition of GSH because of the competitive effect.As shown in Figure 5b, the absorbance of the reaction system with TMB was significantly proportional to the concentration of GSH in the concentration range of 0.3-20 µM.In this concentration range, the absorbance of the reaction system also increased with the increase in the GSH concentration.It is worth noting that the detection Upon the addition of GSH, the absorbance of the oxTMB-containing solution system gradually decreased at 652 nm.This occurred because the oxidation process of TMB catalyzed by MXene@PDA/Co-N-CDs was inhibited by the addition of GSH because of the competitive effect.As shown in Figure 5b, the absorbance of the reaction system with TMB was significantly proportional to the concentration of GSH in the concentration range of 0.3-20 µM.In this concentration range, the absorbance of the reaction system also increased with the increase in the GSH concentration.It is worth noting that the detection limit of GSH in this method reached 0.12 µM; this finding was comparable to other previously reported works.(Table 2).The selectivity of the MXene@PDA/Co-N-CDs system for GSH detection was also investigated.Common metal ions, inorganic cations, and other common biological substances were tested including K + , Na + , Zn 2+ , Mg 2+ , Cl − , I − , urea, cysteine, glycine, etc.It can be seen in Figure 5c that the selected interfering substance does not affect the catalytic reaction.Even if the concentration of interfering substances introduced in the experimental system was ten times the concentration of glutathione, it would not affect the catalytic reaction, suggesting that MXene@PDA/Co-N-CDs showed good selectivity and specificity for GSH detection.
To validate the utility of this method, it was used to detect GSH in human urine (Table 3) and serum samples (Table 4).The GSH concentrations of diluted serum samples were measured to be 0.178, 0.179, and 0.25 µM, which were equivalent to 27.3 mg/100 g, 27.5 mg/100 g, and 38.4 mg/100 g in the original serum samples.These results were consistent with the report results.To further confirm the accuracy of the method, spiked recovery experiments were carried out.The spiked recoveries were between 96% and 110%, which demonstrated this proposed assay system could become an effective method for GSH determination in real samples.The photothermal conversion performance of MXene@PDA/Co-N-CDs was evaluated by recording the temperature variations and infrared thermography images while irradiating with various concentrations (250 µg•mL −1 , 400 µg•mL −1 , 500 µg•mL −1 , 750 µg•mL −1 , 1000 µg•mL −1 , and water) under an 808 nm NIR laser irradiation at 2 W•cm −2 .As the concentration increased, the temperature elevated gradually.The temperature could be increased by 67 • C for 10 min with 1000 µg•mL −1 MXene@PDA/Co-N-CDs compared with pure water.So, the optimal concentration of MXene@PDA/Co-N-CDs was chosen at 500 µg•mL −1 .The photothermal conversion performance of MXene@PDA/Co-N-CDs was evaluated by recording the temperature variations and infrared thermography images while irradiating with various concentrations (250 µg•mL −1 , 400 µg•mL −1 , 500 µg•mL −1 , 750 µg•mL −1 , 1000 µg•mL −1 , and water) under an 808 nm NIR laser irradiation at 2 W•cm −2 .As the concentration increased, the temperature elevated gradually.The temperature could be increased by 67 °C for 10 min with 1000 µg•mL −1 MXene@PDA/Co-N-CDs compared with pure water.So, the optimal concentration of MXene@PDA/Co-N-CDs was chosen at 500 µg•mL −1 .As shown in Figure 6c, the photothermal performance at different power densities (1 W•cm −2 , 1.5 W•cm −2 , 2 W•cm −2 , 2.5 W•cm −2 and 3 W•cm −2 ) was also evaluated by keeping the concentration of MXene@PDA/Co-N-CDs at 500.0 µg•mL −1 .With the increase in laser light power density, a more significant temperature elevation was observed, exhibiting a photothermal effect dependent on the power density.
At the same time, as shown in Figure 6f, laser switching experiments were used to study the photothermal stability of MXene@PDA/Co-N-CDs nanocomposites.In four consecutive heating and cooling cycles, the temperature variations remained nearly identical, indicating its ideal photothermal stability.The high stability of MXene @PDA/Co-N-CDs nanocomposites as a durable photothermal agent was highlighted [15].

Anticancer Cell Effects In Vitro
Because of the high POD-like activity of MXene@PDA/Co-N-CDs nanozyme, catalytic therapy in vitro was investigated.ROS production in MCF-7 cells was evaluated by using DCFH-DA as a fluorescent probe.As can be seen in Figure 7a, cells incubated with MXene@PDA/Co-N-CDs exhibited stronger green fluorescence than those in the blank control groups (water and NIR laser light only), indicating that MXene@PDA/Co-N-CDs nanocomposites could effectively catalyze the generation of intracellular •OH.Moreover, the fluorescence signal was stronger in the NIR laser-irradiated group compared with the MXene@PDA/Co-N-CDs group at the concentration of 500 µg•mL −1 , showing that the photothermal effect of MXene@PDA/Co-N-CDs could enhance its POD-like catalytic activity.
At the same time, as shown in Figure 6f, laser switching experiments were u study the photothermal stability of MXene@PDA/Co-N-CDs nanocomposites.I consecutive heating and cooling cycles, the temperature variations remained identical, indicating its ideal photothermal stability.The high stability of M @PDA/Co-N-CDs nanocomposites as a durable photothermal agent was highlighte

Anticancer Cell Effects In Vitro
Because of the high POD-like activity of MXene@PDA/Co-N-CDs nano catalytic therapy in vitro was investigated.ROS production in MCF-7 cells was eva by using DCFH-DA as a fluorescent probe.As can be seen in Figure 7a, cells inc with MXene@PDA/Co-N-CDs exhibited stronger green fluorescence than those blank control groups (water and NIR laser light only), indicating that MXene@PD N-CDs nanocomposites could effectively catalyze the generation of intracellula Moreover, the fluorescence signal was stronger in the NIR laser-irradiated compared with the MXene@PDA/Co-N-CDs group at the concentration of 500 µg showing that the photothermal effect of MXene@PDA/Co-N-CDs could enhance its like catalytic activity.Cytotoxicity evaluation is a key step in the biocompatibility study of MXene@PDA/Co-N-CDs.The cytotoxicity of MXene@PDA/Co-N-CDs to MCF-7 cells was successfully evaluated by the CCK-8 assay.As shown in Figure 7b, the cell viability treated with laser light was like that of the blank control group, and a slight decrease could be observed in these control groups.MXene@PDA/Co-N-CDs showed a concentration-dependent phenomenon on the cell viability of MCF-7.The cell viability was 83% after cultivation with 100 µg•mL −1 MXene@PDA/Co-N-CDs, indicating the biocompatibility of this material.However, it decreased to 72% after cultivation with 500 µg•mL −1 MXene@PDA/Co-N-CDs, indicating that ROS were produced at this concentration.In addition, after incubation with 500 µg•mL −1 MXene@PDA/Co-N-CDs, the MCF-7 cells treated with laser radiation showed a higher killing effect, and the cell viability of MCF-7 further decreased to 37%.Laser light irradiation improved cell death.Figure 7c also shows that under 808 nm laser irradiation, MXene@PDA/Co-N-CDs produced more intracellular ROS and induced cell apoptosis.
At the same time, the photokilling ability of the MXene@PDA/Co-N-CDs complex on cells was evaluated by the clonogenic assay to record both early and late cell death [48].As depicted in Figure 7, the colony formation rates of MCF-7 cells treated with 808 nm laser light irradiation and the MXene@PDA/Co-N-CDs nanocomposite were 89% and 72%, respectively.Further data from the clone survival assay indicated that the combination treatment group using MXene@PDA/Co-N-CDs and laser irradiation (MXene@PDA/Co-N-CDs + Laser light) significantly enhanced the therapeutic effect, resulting in a decrease to 34% in the colony formation rate of MCF-7 cells, which may be due to the fact that MXene@PDA/Co-N-CDs nanocomposites produced a large amount of •OH in cells, which induced the cell death of MCF-7 cells.Additionally, local hyperthermia accelerated the generation of •OH and enhanced the cell death of MCF-7 cells, further confirming that the nanocomposite had a high synergistic therapeutic effect [49], and its photothermal properties promoted the improvement in peroxidase activity under NIR laser irradiation.Meanwhile, autophagy induced more cell death.
Also, the killing capability of MXene@PDA/Co-N-CDs was further evaluated using the Annexin V-FITC/PI double staining method, and the apoptosis status of tumor cells after different treatments was analyzed by flow cytometry (Figure S3).The results indicated that the control group cells exhibited an overall good condition, mostly existing in the Q1-LL region (96.11%,Annexin V-FITC-/PI-).The laser-treated cells displayed a similar overall condition to the control group (95.75%,Annexin V-FITC-/PI-).In contrast, MCF-7 cells treated with MXene@PDA/Co-N-CDs were predominantly located in the Q1-LL region (68.02%,Annexin V-FITC+/PI-), while MCF-7 cells treated with MXene@PDA/Co-N-CDs + laser light were mostly found in the Q1-UR region (41.79%,Annexin V-FITC+/PI+), indicating late apoptosis, which was significantly different from the control group.
Using in vitro experiments, by observing and measuring the response and changes in MCF-7 cells under different treatment conditions, it is possible to evaluate the effect of the synergistic therapeutic effect of PTT and photothermal enhanced POD (peroxidase) catalytic therapy on cells.The results showed that the proliferation of MCF-7 cells was significantly inhibited, and the cell viability was significantly reduced, indicating that the material had a synergistic effect between MXene and Co-N-CDs for enhancing the therapeutic effect.

Conclusions
In summary, we fabricated a novel MXene@PDA/Co-N-CDs nanozyme with high enzyme-mimic activity and excellent photothermal capability by depositing Co-N-CDs onto MXene@PDA nanosheets.The well-encapsulated PDA improved the biocompatibility and hydrophilicity of MXene, while Co-N-CDs endowed the nanozyme with outstanding catalytic properties.By combining MXene with PDA and Co-N-CDs, the POD catalytic activity was remarkably improved.Based on MXene@PDA/Co-N-CDs, a highly sensitive and selective colorimetric detection method for H 2 O 2 and GSH was established, which

Figure 4 .
Figure 4. UV-vis absorption spectra of different catalytic systems (a); UV-vis absorbance spectra of MB (1 mg L −1 ) as substrate catalyzed by MXene@PDA/Co-N-CDs in the presence of H 2 O 2 (50 mM) (b) and steady-state kinetic analysis of MXene@PDA/CDs (c,d).

Figure 5 .
Figure 5.The UV−vis absorption variation photo of MXene@PDA/Co-N-CDs with different concentrations of H2O2 and its calibrations curve toward 0.4−50 mM (a).The correlation between absorbance and GSH concentration with a photo of the corresponding solution (b); and the selectivity of MXene@PDA/Co-N-CDs for GSH detection (c).

Figure 5 .
Figure 5.The UV−vis absorption variation photo of MXene@PDA/Co-N-CDs with different concentrations of H 2 O 2 and its calibrations curve toward 0.4−50 mM (a).The correlation between absorbance and GSH concentration with a photo of the corresponding solution (b); and the selectivity of MXene@PDA/Co-N-CDs for GSH detection (c).

Figure
Figure 6a,e show that the hybrid materials combined with MXene and Co-N-CDs exhibited more excellent photothermal properties than MXene and Co-N-CDs themselves.The photothermal conversion performance of MXene@PDA/Co-N-CDs was evaluated by recording the temperature variations and infrared thermography images while irradiating with various concentrations (250 µg•mL −1 , 400 µg•mL −1 , 500 µg•mL −1 , 750 µg•mL −1 , 1000 µg•mL −1 , and water) under an 808 nm NIR laser irradiation at 2 W•cm −2 .As the concentration increased, the temperature elevated gradually.The temperature could be increased by 67 • C for 10 min with 1000 µg•mL −1 MXene@PDA/Co-N-CDs compared with pure water.So, the optimal concentration of MXene@PDA/Co-N-CDs was chosen at 500 µg•mL −1 .

Figure
Figure 6a,e show that the hybrid materials combined with MXene and Co-N-CDs exhibited more excellent photothermal properties than MXene and Co-N-CDs themselves.The photothermal conversion performance of MXene@PDA/Co-N-CDs was evaluated by recording the temperature variations and infrared thermography images while irradiating with various concentrations (250 µg•mL −1 , 400 µg•mL −1 , 500 µg•mL −1 , 750 µg•mL −1 , 1000 µg•mL −1 , and water) under an 808 nm NIR laser irradiation at 2 W•cm −2 .As the concentration increased, the temperature elevated gradually.The temperature could be increased by 67 °C for 10 min with 1000 µg•mL −1 MXene@PDA/Co-N-CDs compared with pure water.So, the optimal concentration of MXene@PDA/Co-N-CDs was chosen at 500 µg•mL −1 .

Figure 6 .
Figure 6.Temperature change curves of various samples (a).Temperature change curves of water and MXene@PDA/Co-N-CDs with different concentrations as a function of irradiation time (b).NIR irradiation with different power densities (the irradiation wavelength was 808 nm (c).Photostability of MXene@PDA/CDs under 808 nm laser light irradiation for four cycles (2 W•cm −2 ) (d).Infrared thermal images of pure water and MXene@PDA/Co-N-CDs nanocomposites with various concentrations irradiated with 808 nm laser light (2 W•cm −2 ) for 10 min (e).Infrared thermal images of water, MXene, Co-N-CDs, and MXene@PDA/Co-N-CDs nanocomposite aqueous solutions with the same concentration irradiated with 808 nm laser light (2 W•cm −2 ) for 10 min (f).

Figure 6 .
Figure 6.Temperature change curves of various samples (a).Temperature change curves of water and MXene@PDA/Co-N-CDs with different concentrations as a function of irradiation time (b).NIR irradiation with different power densities (the irradiation wavelength was 808 nm (c).Photostability of MXene@PDA/CDs under 808 nm laser light irradiation for four cycles (2 W•cm −2 ) (d).Infrared thermal images of pure water and MXene@PDA/Co-N-CDs nanocomposites with various concentrations irradiated with 808 nm laser light (2 W•cm −2 ) for 10 min (e).Infrared thermal images of water, MXene, Co-N-CDs, and MXene@PDA/Co-N-CDs nanocomposite aqueous solutions with the same concentration irradiated with 808 nm laser light (2 W•cm −2 ) for 10 min (f).

Table 1 .
Comparisons of the kinetic behavior of different nanozymes.

Table 1 .
Comparisons of the kinetic behavior of different nanozymes.

Table 2 .
Comparison of the MXene@PDA/Co-N-CDs colorimetric sensing assay with the reported colorimetric methods for GSH detection.