Antibacterial Activity of Ti3C2Tx MXene

MXenes are a family of atomically thin, two-dimensional (2D) transition metal carbides and carbonitrides with many attractive properties. Two-dimensional Ti3C2Tx (MXene) has been recently explored for applications in water desalination/purification membranes. A major success indicator for any water treatment membrane is the resistance to biofouling. To validate this and to understand better the health and environmental impacts of the new 2D carbides, we investigated the antibacterial properties of single- and few-layer Ti3C2Tx MXene flakes in colloidal solution. The antibacterial properties of Ti3C2Tx were tested against Escherichia coli (E. coli) and Bacillus subtilis (B. subtilis) by using bacterial growth curves based on optical densities (OD) and colonies growth on agar nutritive plates. Ti3C2Tx shows a higher antibacterial efficiency toward both Gram-negative E. coli and Gram-positive B. subtilis compared with graphene oxide (GO), which has been widely reported as an antibacterial agent. Concentration dependent antibacterial activity was observed and more than 98% bacterial cell viability loss was found at 200 μg/mL Ti3C2Tx for both bacterial cells within 4 h of exposure, as confirmed by colony forming unit (CFU) and regrowth curve. Antibacterial mechanism investigation by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) coupled with lactate dehydrogenase (LDH) release assay indicated the damage to the cell membrane, which resulted in release of cytoplasmic materials from the bacterial cells. Reactive oxygen species (ROS) dependent and independent stress induction by Ti3C2Tx was investigated in two separate abiotic assays. MXenes are expected to be resistant to biofouling and offer bactericidal properties.


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Recently, the family of 2D materials has been augmented by a large group of early transition metal carbides. [1][2][3][4][5][6] This new family of 2D materials has been labeled "MXenes", where M is an early transition metal and X is carbon and/or nitrogen. Ti 3 C 2 T x (T is standing for the surface termination, such as -O, -OH or -F) is the most studied MXene and recently we reported the selective ion sieving of micrometer-thick Ti 3 C 2 T x membranes. 7 The hydrophilic nature of Ti 3 C 2 T x , together with the hydrated interlayer spacing, promotes ultrafast water flux and differential sieving towards single-, double-and triple-charged metal cations of different sizes. Several studies have compared the antibacterial activity of 2D graphene-based materials (graphite oxide, GO and reduced GO (rGO)) against Gram-negative (Gram (-)) and Grampositive (Gram (+)) bacteria through direct contact [8][9][10][11][12][13][14] . The antibacterial activity of metal and metal oxide nanoparticles (e.g., Ag, ZnO and TiO 2 ) have also been well documented by a sizable number of studies. 15,16 Antibacterial activity of these nanoparticles has been associated with production of reactive oxygen species (ROS) and direct contact with bacteria membrane, penetrating into the bacteria and interacting with sulfur-containing proteins as well as phosphorus-containing DNA, leading to bacterial cell death. [17][18][19][20][21] Similarly, the antimicrobial activities of graphene have been found to be the synergy of both "chemical" and "physical" effects. 14,19 Most of the studies have attributed the antibacterial activity of GO and rGO to oxidative and physical stress induced by sharp edges of graphene nanosheets, which may result in mechanical damage of cell membranes, leading to a loss of their integrity. 14,[22][23][24] Moreover, 4 several mechanisms have been proposed to explain the antimicrobial properties of carbon nanotubes (CNT) based composite films including inhibition of electron transports, leakage and penetration of cell membrane and generation of ROS. [25][26][27][28][29] Despite antimicrobial properties of MXenes have never been examined before, it is reasonable to assume that at least some of those mechanisms may work in MXenes, which were shown to destroy dye molecules in solution. 9 Therefore, investigations of the mechanism of MXene's interaction with bacterial cell membranes and its bactericidal activity are needed to determine the range of potential applications of these new materials. 30,31 Here we present for the first time a report on the antibacterial behavior of Ti 3 C 2 T x MXene in the colloidal suspension. To better understand the health and environmental impacts of the new 2D carbides, the antibacterial activity of Ti 3 C 2 T x MXene toward two bacterial models -Escherichia coli (E. coli) and Bacillus subtilis (B. subtilis), was studied and compared with GO.
The concentration dependent antibacterial activities were evaluated by cell viability assays together with scanning electron microscopy (SEM), transmission electron microscopy (TEM) and lactate dehydrogenase (LDH) release assay. On the basis of these results, we introduce MXenes as a new family of 2D antimicrobial nanomaterials. This will open a door for MXenes in the antibacterial applications and water purification industry.

Synthesis and Characterization of Ti 3 C 2 T x
Ti 3 C 2 T x suspension was prepared from multilayer (ML) Ti 3 C 2 T x "clay" by ultrasonication under flow of Argon (Ar) gas as described in the experimental section. Ti 3 C 2 T x synthesis was described in details elsewhere. 32 GO was also synthesized by oxidizing natural graphite powders 5 using H 2 SO 4 and KMnO 4 according to the modified Hummers method and was used as a reference in this study. 33 Figure 1 shows SEM images of Ti 3 AlC 2 ( Figure 1A), ML-Ti 3 C 2 T x ( Figure 1B), and Ti 3 C 2 T x ( Figure 1C) nanosheets dried on alumina wafer and photographs of their corresponding colloidal suspensions in water. The images clearly show the different appearance of the three materials after 10 minutes sonication. Both Ti 3 AlC 2 and as-produced ML-Ti 3 C 2 T x show opaque gray color and their particles precipitated after 1 h and the SEM revealed well stacked nanosheets. On the other hand, delaminated Ti 3 C 2 T x formed dark green colloidal solution and the stacked layers were delaminated as observed from SEM ( Figure 1C).
The TEM micrograph in ( Figure 1D) revealed thin, transparent flakes of delaminated Ti 3 C 2 T x nanosheets. Fluorine and oxygen were confirmed by energy-dispersive spectroscopy (EDS), suggesting O-and F-containing surface terminations. Delaminated Ti 3 C 2 T x has highly exfoliated and smaller sheets, which are expected to provide a significantly higher surface area than Ti 3 AlC 2 and ML-Ti 3 C 2 T x and an improved antimicrobial performance. 4,24 A typical XRD pattern of air-dried Ti 3 C 2 T x powder is shown in Figure 1E. The presence of peaks corresponding to basal-plane reflections (00l) with c lattice parameter of 27-28 Å suggests the presence of water, and possibly Li ions, between the hydrophilic and negatively charged Ti 3 C 2 T x MXene nanosheets. 32 The sharp and intense peak (002) at 6.17 o is at a much lower angle that typical of Ti 3 C 2 T x produced by etching in HF. Peaks around 40 o are still observed, which suggests a good periodicity between the stacked MXene layers.
6 Figure 1: SEM images of Ti 3 AlC 2 (A) and ML-Ti 3 C 2 T x (B) and Ti 3 C 2 T x nanosheets on an alumina filter (C), and their corresponding photographs showing Ti 3 AlC 2 , ,ML-Ti 3 C 2 T x and Ti 3 C 2 T x solution, respectively; D, TEM image of the pristine Ti 3 C 2 T x flake; E, typical XRD pattern of ML-Ti 3 C 2 T x .

Antibacterial Activity
In order to investigate the effect of delamination on the antibacterial efficiency of MXene, the inhibition effect of three materials (Ti 3 AlC 2 (MAX), as-produced ML-MXene, and 7 delaminated Ti 3 C 2 T x nanosheets) were examined against both E. coli and B. subtilis. The bacterial growth inhibition was determined by the colony counting method. Figure S1A (Supporting Information) shows the photographs of agar plates onto which control and bacterial cells were re-cultivated after treatment for 4 h with the same concentration of 100 µg/mL of nanomaterial. Figure S1B (Supporting Information), depicts the percentage growth inhibition of both bacterial strains exposed to the materials under study. MAX dispersion showed growth inhibition of only 14.39±1.43% and 18.34±1.59% for E. coli and B. subtilis, respectively. The ML-Ti 3 C 2 T x dispersion showed a little higher antibacterial activity compared with MAX with E.coli and B. subtilis growth inhibition of 30.55±2.56% and 33.60±2.89%, respectively. Whereas for the cells exposed to the colloidal solution of delaminated Ti 3 C 2 T x MXene, the loss of E. coli and B. subtilis cells viability increases to 97.70±2.87% and 97.04±2.91%, respectively, exhibiting much stronger inhibition. The three materials showed significant differences in their antibacterial activities against both bacterial strains. In particular, delaminated Ti 3 C 2 T x MXene has a much more pronounced antibacterial activity compared with those of MAX and ML-Ti 3 C 2 T x MXene and was used for further studies.

Concentration Dependent Antibacterial Activity of Ti 3 C 2 T x
The antibacterial activity of Ti 3 C 2 T x against Gram (+) B. subtilis and Gram (-) E. coli was evaluated by measuring the growth curve and the cell viability after exposure of the bacteria to increasing concentrations of Ti 3 C 2 T x colloidal solutions. The optical density (OD) was monitored spectrophotometrically at 600 nm for pristine bacteria and bacteria treated with Ti 3 C 2 T x by over different time intervals from lag phase (when individual bacteria are adjusting to the environment) to stationary phase (when their growth and death rates are equivalent). Bacteria (at h, re-cultivated on agar plates, and evaluated by using the bacteria counting method. Figure 2 shows the typical photographs of E. coli or B. subtilis bacteria colonies after treatment with various concentrations of bacteria. As can be seen from both panels, the number of colonies significantly decreases with increasing concentration of Ti 3 C 2 T x . The obtained results indicate the dose-dependent antimicrobial activity of Ti 3 C 2 T x .  9 50 µg/mL (D), 100 µg/mL (E), and 200 µg/mL (D) of Ti 3 C 2 T x , respectively. Bacterial suspensions in deionized water without Ti 3 C 2 T x MXene material was used as control. Figure 3 shows the bacterial cells viability exposed to Ti 3 C 2 T x and GO concentrations in the range of 2-200 µg/mL for 4 h. Ti 3 C 2 T x showed excellent antimicrobial activity for both Gram (+) and Gram (-) bacteria. The bacterial cell loss gradually ascended with the increasing concentration of Ti 3 C 2 T x . E. coli and B. subtilis showed 92.53% and 93.96% survival rate, respectively, at the lowest Ti 3 C 2 T x concentration of 2 µg/mL. By increasing the Ti 3 C 2 T x MXene concentration from 2 µg/mL to 20 µg/mL, the survival rate of E. coli and B. subtilis was decreased to 35.31% and 28.21%, respectively. More than 96% bacterial viability loss for both bacterial strains was observed at 100 µg/mL of Ti 3 C 2 T x and bacterial inhibition was increased to more than 99% at 200 µg/mL of Ti 3 C 2 T x ( Figure 3). Additionally, the Ti 3 C 2 T x dispersions revealed a stronger influence on B. subtilis than E. coli at lower concentrations.
The obtained results are in agreement with previously reported data, where several nanomaterials showed a higher antibacterial activity against Gram (+) bacterial strains than Gram (-) bacteria and differences of the cell wall structure of two bacterial strains were reported as a possible reason for different sensitivities. 9, 34 Gram (-) E. coli cells have negatively charged cellular membranes, as function of the isoelectric point (pI) = 4-5. For the G-positive B. subtilis cells, the pI value of the membranes can reach 7, which produces a more negatively charged surface in culturing medium. 35,36 Therefore, the higher negative charges of E. coli cells at pH 7 could explain their higher resistance against the direct exposure to Ti 3 C 2 T x substrate than B.  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60 10 one of the most popular research subjects. 10,37,38 Moreover, E. coli, as Gram (-) bacteria, are covered by a much thinner layer of peptidoglycan (thickness of 7-8 nm), but have an external protective lipid membrane. 39 Whereas, Gram (+) B. subtilis lacks the external lipid membrane, but its thicker peptidoglycan cell walls are in the range of 20-80 nm. It was reported that the cell membrane of Gram (+) bacteria lacking the outer membrane were more easily damaged by direct contact with graphene nanowalls, as compared to the Gram (-) E. coli with the outer membrane. 10,39 The hydrophilic Ti 3 C 2 T x could effectively attach to bacteria, facilitating their inactivation by direct contact interaction.
In order to compare antibacterial activity of Ti 3 C 2 T x with GO, both bacterial strains were treated with different concentrations of GO under the same experimental conditions. Figure 3 shows the viability of both E. coli and B. subtilis bacteria in control, which was taken as 100%, and exposed to 0-200 µg/mL of GO. For both bacterial strains, there were substantial differences in bacteria colonies on agar plates, indicating that the Ti 3 C 2 T x MXene has a higher antibacterial activity as compared to GO in our experimental setup. Ti 3 C 2 T x showed more than 98% cell inactivation to both bacterial strains at 200 µg/mL of Ti 3 C 2 T x , whereas, GO induces about 90 % inactivation at the same concentration ( Figure 3).
To further evaluate the bactericidal properties of Ti 3 C 2 T x MXene, the antibacterial activity is  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60   11 To evaluate the antibacterial activity of Ti 3 C 2 T x MXene in growth media, both bacterial strains were exposed to 200 µg/mL of Ti 3 C 2 T x in LB media for 4 h. Figure S3 (Supporting Information) depicts the growth of bacterial cells in LB media in presence of Ti 3 C 2 T x . Figure   S3A shows a significant decrease in log of bacterial growth when exposed to Ti 3 C 2 T x . The viable cells count in growth media were 33.32% and 27.34% for E. coli and B. subtilis, respectively, in presence of Ti 3 C 2 T x as compared to that of control (see Figure S3B).
The effect of contact time on bactericidal activity of Ti 3 C 2 T x (200 µg/mL) was further examined during the 4 h incubation period. Figure S4 (Supporting Information) shows the kinetics of antibacterial activity in terms of cell viability and log reduction. The antibacterial activity increased with increasing contact time and cell viability decreased to 50% within 2 h of contact time and more than 98% cells viability loss was observed after 4 h. This relatively short contact time might also be advantageous for the application of Ti 3 C 2 T x as antibacterial agent.  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60 Gentamicin at concentration of 50 µg/mL was used as positive control. Error bars represent the standard deviation.
The antimicrobial activity of Ti 3 C 2 T x nanosheets was further confirmed by bacterial regrowth curves using a second assay. Figure 4 shows the OD growth curves of E. coli and B. subtilis cells incubated with different concentrations of Ti 3 C 2 T x . It was found that inhibition of both bacterial strains growth was dose dependent and the bactericidal activity increased with increasing Ti 3 C 2 T x concentration, which was in line with the number of colonies grown on the LB plates.

Bacterial Membrane Morphology Changes
To understand the antibacterial effect of Ti 3 C 2 T x MXene, changes of morphology and membrane integrity of E. coli and B. subtilis cells, due to the interaction with Ti 3 C 2 T x , were further evaluated by SEM and TEM. As depicted by SEM images in Figure 5a, bacterial cells for both E. coli and B. subtilis cultured in the absence of Ti 3 C 2 T x were viable with no observed membrane damage or cell death. The higher magnification in lower panels shows that the bacterium is protected by intact cytoplasmic membrane. 40 On the other hand, most bacterial cell suffered from a prevalent membrane damage and cytoplasm leakage in the presence of 50 µg/mL of Ti 3 C 2 T x , which is clearly observed at high magnifications ( Figure 5B). Some bacterial cells still maintained the membrane integrity, but they were deformed. At 100 µg/mL of Ti 3 C 2 T x , both bacteria suffered from prevalent cell lysis indicated by a severe membrane disruption and cytoplasm leakage (see the red circles at high magnification in Figure 5C).  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58 Figure 5C).
Significant morphological changes in the cell structure could be attributed to detachment of the cytoplasmic membrane from the cell wall as confirmed by LDH release assay. The SEM observations were consistent with the bacteria colonies numbers in Figure 3. It is suggested that  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60 16 with increasing Ti 3 C 2 T x concentration both E. coli and B. subtilis were trapped or wrapped by the thin sheets of Ti 3 C 2 T x and subsequently formed agglomerates. This has been confirmed by spot EDS analysis on the surface of the bacterium (see Figure S5, (Supporting Information)).   LDH release assay was used to quantitatively determine the extent of cell damage. Figure   7 shows the LDH activity in the supernatants after 4 h of incubation. Concentration dependent LDH release was observed as bacterial cells were exposed to Ti 3 C 2 T x nanosheets dispersions ( Figure 7). The bacterial cells exposed to 2 and 10 µg/L of 18 shows that both the walls and the inner contents of the cell were damaged, suggesting that membrane disruption might be a major cell inhibitory mechanism. Figure 7: Ti 3 C 2 T x cytotoxicity measured by LDH release from the bacterial cells exposed to different concentrations of Ti 3 C 2 T x for 4 h.

Oxidative-Stress and Antimicrobial Activity of Ti 3 C 2 T x MXene
Some earlier studies have proposed oxidative stress as a common mechanism of antibacterial activity of several metal, metal oxide and carbon based nanomaterials. 8 1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60   19 nanoparticles suspensions under dark conditions, 43,44 whereas other studies did not. 45 A similar mechanism has been thought for antibacterial activity of iron oxide nanoparticles in which reduced iron species (Fe 3+ /Fe 2+ ) reacted with oxygen to create ROS. 46 To identify if cellular oxidative stress may be induced by Ti 3 C 2 T x , ROS dependent and independent oxidative stress was investigated in two separate abiotic assays. First, the production of superoxide anion (O 2 •-) at different Ti 3 C 2 T x concentrations was monitored using XTT assay. As shown in the Figure S8 (Supporting Information), no noticeable absorption was detected at different Ti 3 C 2 T x concentrations revealing that MXene mediated no or negligible superoxide anion production and their role in Ti 3 C 2 T x antibacterial activity could be minimal.
However, the production and impact of ROS other than superoxide anion needs to be discreetly examined in future studies.
Second, oxidative stress mediated by Ti 3 C 2 T x was examined using glutathione oxidation assay. Glutathione is a tripeptide with a thiol group, which serves as one of the major cellular antioxidant enzymes in bacteria. It is involved in the intracellular oxidative balance and protects the cells against external electrophilic compounds. The oxidation of glutathione has been widely used as an indicator of the oxidative stress induced by different nanomaterials. Thiol groups (-SH) in glutathione can be oxidized to disulfide converting glutathione to glutathione disulfide.
Moreover, direct contact of glutathione with nanoparticle surface also logically could lead to loss of glutathione by adsorption, or binding. 47 In this study glutathione was exposed to increasing concentrations of Ti 3 C 2 T x in a bicarbonate buffer and incubated for 4 h, after which the concentration of thiol groups was quantified by Ellman's assay.  As shown in Figure 8, glutathione depletion was dependent on both Ti 3 C 2 T x concentration and incubation time. While negligible glutathione loss was observed for the control samples in the absence of Ti 3 C 2 T x , glutathione concentration was reduced to 97.5 to 61.7 % when Ti 3 C 2 T x concentration was increased from 2 to 200 µg/mL, respectively ( Figure 8B). It is unlikely that Ti 3 C 2 T x MXene itself can work as an oxidant for glutathione, but Ti 3 C 2 T x has reactive Ti-F groups on its surface, which are not stable at high pH, and also Ti 3 C 2 T x possesses a high negative surface charge, as shown by its -30 to -40 mV zeta-potential in aqueous solutions. 48 Thus, both chemical reactions and physisorption are potentially possible. However, at the moment, there is no published data on interaction of Ti 3 C 2 T x with thiols.

Proposed Inhibition Mechanism of Ti 3 C 2 T x MXene
Strong antibacterial property of Ti 3 C 2 T x may be partially attributed to the anionic nature of its surface. Ti 3 C 2 T x nanosheets have negatively charged surfaces. In addition, its high hydrophilicity may enhance bacterial contact to membrane surface resulting in inactivation of adhered microorganisms according to direct contact-killing mechanism. Morover, hydrogen bonding between oxygenate groups of Ti 3 C 2 T x MXene and the lipopolysaccharide strings of the cell membrane could result in bacterial inhibition by preventing nutrient intake as recently proposed for GO nanosheets 10,50 . It is important to understand the interaction of MXene with cell membranes for the evaluation of MXene's health and environmental impacts and to utilize it as biocide in disinfection industry. We have found the interesting antibacterial activity of Ti 3 C 2 T x ; however, still the interaction between MXene and bacterial cell membrane has to be investigated and fully understood. From the above LDH release assay, SEM and TEM images, as well as glutathione oxidation assays, the antimicrobial mechanism of Ti 3 C 2 T x MXene nanosheets can be explained as follows: First of all, delaminated Ti 3 C 2 T x nanosheets with sharp edges have the capacity of adsorbing on the surface of microorganisms. It is also suggested that with increasing Ti 3 C 2 T x concentration, both E. coli and B. subtilis were trapped or wrapped by the nanometer-thin sheets of Ti 3 C 2 T x and subsequently formed agglomerates. Moreover, exposure of bacterial cells to sharp edges of Ti 3 C 2 T x , as shown by the TEM image in Figure 1A, may induce membrane damage. The water contact angle on Ti 3 C 2 T x films was found to be 37°  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60   22 and its hydrophilicity may result in effective attachment of bacteria to Ti 3 C 2 T x . 51 The antibacterial effects may also be attributed to strong reducing activity of MXene and its reactive surfaces. 52 The smallest Ti 3 C 2 T x nanosheets could permeate into the microorganism cell through direct physical penetration or via endocytosis. Finally, Ti 3 C 2 T x may also react with some  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59

Cell preparation
The antibacterial properties of Ti 3 C 2 T x and GO colloids were evaluated using E. coli and B. subtilis as the model gram negative and gram-positive bacteria, respectively. Glycerol stocks were used to inoculate defined overnight cultures in LB medium at 35°C. Following that, 1 mL volumes of cell suspensions were sub-cultured and harvested at the exponential growth phase.
Cultures were centrifuged at 5000 rpm for 5 min and pellets obtained were washed three times

Antibacterial activity of Ti 3 C 2 T x (MXene) nanosheets dispersions
Antibacterial activity against each strain was determined by the colony count method and the measurement of OD. Batch assays were performed to compare the antibacterial activity of delaminated Ti 3 C 2 T x in colloidal solution with that of dispersions of ML-Ti 3 C 2 T x and Ti 3 AlC 2 . The log reduction was calculated using the following equation: where A is the number of viable microorganisms before treatment and B is the number of viable microorganisms after treatment.
Additionally, batch assays were performed with different Ti 3 C 2 T x concentrations. To examine the effect of MXene on bacterial growth, the batch assays were subjected to 2, 10, 20,

Abiotic thiol oxidation and quantification
The Ti 3 C 2 T x (MXene)-mediated abiotic oxidation of glutathione was studied by quantifying thiol concentration following Ellman's assay as described earlier 49