TWO-DIMENSIONAL METAL CARBIDE ANTIMICROBIAL MEMBRANE AND ANTIMICROBIAL AGENT

Abstract
The antimicrobial agent includes at least one two-dimensional metal carbide layer. The two-dimensional metal carbide has the formula Tin+1CnTx, where T represents a terminal functional group and x represents the number of terminal functional groups. The two-dimensional metal carbide is preferably Ti3C2Tx. The terminating group may be oxygen, hydroxide (OH), fluorine or combinations thereof. The antimicrobial agent may be used as a two-dimensional metal carbide antimicrobial membrane (10) or filter for removal of harmful bacteria, such as E. coli and B. subtilis. A stack of two-dimensional metal carbide layers (14) may be supported on a polymer filter substrate (12), such as a polyvinylidene fluoride (PVDF) supporting substrate.
Description
TECHNICAL FIELD

The present invention relates to the antimicrobial properties of two-dimensional metal carbides, and particularly to a two-dimensional metal carbide antimicrobial membrane and an antimicrobial agent that include a membrane or agent formed from one or more layers of a two-dimensional metal carbide, particularly the MXene Ti3C2Tx, where T represents a terminating functional group (O, OH and/or F) and x represents the number of terminating groups.


BACKGROUND ART

Biofouling is a major challenge in the membrane separation industry. Bacteria and other microorganisms adhere to the membrane surface and form a viscous gel-like biofilm, causing a severe decline in the flux. Biofouling of membranes in wastewater treatment and water purification facilities decreases operational efficiency and may result in down time for cleaning or replacing the membranes. Thus, there is great interest in membranes and coatings for membranes that exhibit antimicrobial activity, as well as antibacterial agents that can be used to treat water to reduce populations of bacteria that may cause biofouling.


Although there are some materials known to exhibit some antibacterial properties, there is a need for improved antimicrobial filtering and coating materials that are less subject to biofouling. Thus, a two-dimensional metal carbide antimicrobial membrane and antimicrobial agent solving the aforementioned problems is desired.


DISCLOSURE OF INVENTION

The two-dimensional metal carbide antimicrobial agent includes at least one layer of MXenes. MXenes include atomically thin, two-dimensional (2D) transition metal carbides and carbonitrides. Preferably, the two-dimensional metal carbide has the formula Ti3C2Tx, where T represents a terminating or terminal functional group, and x represents a number of the terminating functional groups. The terminating group may be oxygen, hydroxide (OH), fluorine or combinations thereof.


At least one of the two-dimensional metal carbide layers may be supported on a polymer substrate, including, but not limited to, polyethersulfone (PES), polypropylene (PP), cellulose acetate (CA) or polyvinylidene fluoride (PVDF), to form an antimicrobial membrane filter for removal of harmful gram negative (G−) and gram positive (G−) bacteria, such as E. coli and B. subtilis, from water. Further, by contacting or coating a substrate with an effective amount of the two-dimensional metal carbide, the MXene metal carbide may be used as an agent for controlling biofilm formation on the substrate.


These and other features of the present invention will become readily apparent upon further review of the following specification and drawings.





BRIEF DESCRIPTION OF THE DRAWINGS


FIG. 1A is a graph showing cell viability measurements of E. coli and B. subtilis bacteria treated with Ti3C2Tx, in water for 4 hours as a function of concentration.



FIG. 1B is a graph comparing cell viability measurements of the E. coli and B. subtilis bacteria after incubation with 100 μg/mL of Ti3C2Tx, and 100 μg/mL of graphene oxide, respectively.



FIG. 2A is an optical density growth curve of E. coli bacteria in Luria-Bertani broth (LB) medium at 35° C. following treatment of the bacterial cells with different concentrations of Ti3C2Tx in water for four hours.



FIG. 2B is an optical density growth curve of B. subtilis bacteria in Luria-Bertani broth (LB) medium at 35° C. following treatment of the bacterial cells with different concentrations of Ti3C2Tx in water for four hours.



FIG. 3 is a graph showing growth behavior of E. coli and B. subtilis bacteria incubated on uncoated polyvinylidene fluoride (PVDF) control membranes, PVDF membranes modified with a coating of graphene oxide (GO), and PVDF membranes coated with Ti3C2Tx.



FIG. 4 is a partial side view in section of a two-dimensional metal carbide antimicrobial membrane according to the present invention.



FIG. 5 is a photograph showing a comparison of both E. coli and B. subtilis grown on uncoated PVDF (control) membrane filters, PVDF membrane filters coated with graphene oxide (GO), and Ti3C2Tx (MXene)-coated membranes incubated on agar plates.



FIG. 6A shows a scanning electron microscope (SEM) photomicrograph of E. coli bacterial colonies grown on the uncoated PVDF (control) membrane filter.



FIG. 6B shows a scanning electron microscope (SEM) photomicrograph of E. coli bacterial colonies grown on the Ti3C2Tx (MXene)-coated membrane.



FIG. 7A shows a scanning electron microscope (SEM) photomicrograph of B. subtilis bacterial colonies grown on the uncoated PVDF (control) membrane filter.



FIG. 7B shows a scanning electron microscope (SEM) photomicrograph of B. subtilis bacterial colonies grown on the Ti3C2Tx, (MXene)-coated membranes.



FIG. 8 is a graph comparing growth inhibition of E. coli and B. subtilis bacterial colonies treated with 100 μg/mL of delaminated Ti3C2Tx (MXene) nanosheets, multilayered Ti3C2Tx MXene ((ML)-MXene), and Ti3AlC2 (MAX), respectively.



FIG. 9A is a scanning electron microscope (SEM) photomicrograph of E. coli bacterial cells cultured in the absence of Ti3C2Tx, at a scale of 5 μm.



FIG. 9B is a scanning electron microscope (SEM) photomicrograph of E. coli bacterial cells cultured in the absence of Ti3C2Tx, at a scale of 500 nm.



FIG. 9C is a scanning electron microscope (SEM) photomicrograph of E. coli bacterial cells cultured in the presence of 50 μg/mL of Ti3C2Tx, at a scale of 5 μm.



FIG. 9D is a scanning electron microscope (SEM) photomicrograph of E. coli bacterial cells cultured in the presence of 50 μg/mL of Ti3C2Tx, at a scale of 500 nm.



FIG. 9E is a scanning electron microscope (SEM) photomicrograph of E. coli bacterial cells cultured in the presence of 100 μg/mL of Ti3C2Tx, at a scale of 5 μm.



FIG. 9F is a scanning electron microscope (SEM) photomicrograph of E. coli bacterial cells cultured in the presence of 100 μg/mL of Ti3C2Tx, at a scale of 500 nm.



FIG. 10A is a scanning electron microscope (SEM) photomicrograph of B. subtilis bacterial cells cultured in the absence of Ti3C2Tx, at a scale of 5 μm.



FIG. 10B is a scanning electron microscope (SEM) photomicrograph of B. subtilis bacterial cells cultured in the absence of Ti3C2Tx, at a scale of 500 nm.



FIG. 10C is a scanning electron microscope (SEM) photomicrograph of B. subtilis bacterial cells cultured in the presence of 50 μg/mL of Ti3C2Tx, at a scale of 5 μm.



FIG. 10D is a scanning electron microscope (SEM) photomicrograph of B. subtilis bacterial cells cultured in the presence of 50 μg/mL of Ti3C2Tx, at a scale of 500 nm.



FIG. 10E is a scanning electron microscope (SEM) photomicrograph of B. subtilis bacterial cells cultured in the presence of 100 μg/mL of Ti3C2Tx, at a scale of 5 μm.



FIG. 10F is a scanning electron microscope (SEM) photomicrograph of B. subtilis bacterial cells cultured in the presence of 100 μg/mL of Ti3C2Tx, at a scale of 500 nm.



FIG. 11A is an energy dispersive spectroscopy (EDS) spectrum for treated E. coli bacteria.



FIG. 11B is an energy dispersive spectroscopy (EDS) spectrum for treated B. subtilis bacteria.



FIG. 12A is a transmission electron microscope (TEM) photomicrograph of E. coli bacterial cells treated with 200 μg/mL of Ti3C2Tx, for 4 hours at low magnification.



FIG. 12B is a transmission electron microscope (TEM) photomicrograph of E. coli bacterial cells treated with 200 μg/mL of Ti3C2Tx, for 4 hours at high magnification.



FIG. 13A is a transmission electron microscope (TEM) photomicrograph of B. subtilis bacterial cells treated with 200 μg/mL of Ti3C2Tx, for 4 hours at low magnification.



FIG. 13B is a transmission electron microscope (TEM) photomicrograph of B. subtilis bacterial cells treated with 200 μg/mL of Ti3C2Tx, for 4 hours at high magnification.



FIG. 14A is a transmission electron microscope (TEM) photomicrograph of B. subtilis bacterial cells exposed to 200 μg/mL of Ti3C2Tx at 37° C. for four hours at a direct magnification of 10000×.



FIG. 14B is a transmission electron microscope (TEM) photomicrograph of E. coli bacterial cells exposed to 200 μg/mL of Ti3C2Tx at 37° C. for four hours at a direct magnification of 8000×.



FIG. 15 is a graph showing cytotoxicity in both E. coli and B. subtilis as a function of concentration of Ti3C2Tx MXene as measured by LDH assay of the supernatants after 4 hours of incubation.



FIG. 16 is a graph showing the change in absorbance at 470 nm as a function of time during an XTT assay of bacterial dispersions treated with Ti3C2Tx MXene colloidal solutions at different concentrations for 5 hours at a pH of 7.5 in the dark, with TiO2 (80 μg/mL) used as a control.



FIG. 17A is a graph showing the results of a glutathione oxidation assay showing oxidation of glutathione in a colloidal suspension of Ti3C2Tx MXene at a concentration of 200 μg/ml as a function of incubation time.



FIG. 17B is a graph showing the results of a glutathione oxidation assay showing oxidation of glutathione in a colloidal suspension of Ti3C2Tx MXene as a function of Ti3C2Tx MXene concentration.





Similar reference characters denote corresponding features consistently throughout the attached drawings.


BEST MODES FOR CARRYING OUT THE INVENTION

The two-dimensional metal carbide antimicrobial membrane and antimicrobial agent includes an antimicrobial agent that has at least one two-dimensional metal carbide layer. The two-dimensional metal carbide layer has a two-dimensional transition metal carbide, such as MXene. Preferably, the MXene has the formula Ti3C2Tx, where T represents a terminating or terminal functional group (O, OH and/or F) and x represents the number of terminating groups. Both Ti3C2Tx in aqueous suspension and a Ti3C2Tx coated membrane have exhibited biocidal effects against both gram-negative E. coli and gram-positive B. Subtilis bacteria in water. The antimicrobial agent may be coated on or incorporated into membranes used in water treatment, coatings for surgical materials, and/or paint for biomedical devices.


Transition metal carbides called MXenes are a new family of 2D materials. “MXenes” denote the loss of the A element from the MAX phases and have 2D structure. A MAX-phase composition typically has the empirical formula Mn+1AXn, where M is an early transition metal; A is an element selected from Al, Si, P, S, Ga, Ge, As, Cd, In, Sn, Tl, and Pb; X is either C or N; and n is 1, 2, or 3. An MXene may be formed by removing the A element from the MAX-phase composition, leaving a two-dimensional crystal array wherein each X is positioned within an octahedral array of M. Thus, the MXene has the formula Mn+1Xn, where M is an early transition metal; X is either C or N; and n is 1, 2, or 3. In contrast to other layered materials, such as graphene, where weak Van der Waals interactions hold the structure together, the bonds between the layers in the MAX phases are too strong to be broken by shear or other mechanical means. Chemical etching with subsequent intercalation and sonication is required to produce a colloidal solution of MXene flakes. The edges of the two-dimensional MXene layer usually do not terminate in an M, but with an oxide or fluoride, giving the formula Mn+1XnTx, where x is the number of terminal groups.


In order to prepare Ti3C2Tx in suspension form, the present inventors obtained flakes of Ti3C2Tx (with a few layers or in single layers) by delaminating a multilayer (ML)-Ti3C2Tx powder by ultrasonication after etching Ti3AlC2 (MAX) with NaF/HCl solution. The resultant (ML)-Ti3C2Tx powder was dispersed in de-aerated water with a weight ratio of (ML)-Ti3C2Tx powder:water of 250:1. The suspension was sonicated under flowing argon, and then centrifuged for one hour at 3,000 rpm to obtain a supernatant containing Ti3C2Tx flakes. Transmission electron microscopy (TEM), scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), and X-ray diffraction (XRD) were each used to confirm the morphology of the flakes. The structural morphology of the delaminated MXene sheets included a thin, transparent and smooth film with small wrinkles. Fluorine and oxygen were observed in energy-dispersive spectroscopy.


A two-dimensional metal carbide antimicrobial membrane, i.e., a Ti3C2Tx, membrane, was fabricated by a vacuum-assisted filtration method, where colloidal solutions of delaminated Ti3C2Tx, were filtered to obtain membranes with controllable mass-loadings and thicknesses. The delaminated Ti3C2Tx, solution was diluted to 0.01 mg/ml. The dilute colloidal solution contained single-layer Ti3C2Tx, sheets with thicknesses on the order of 1 nm and lateral sizes on the order of hundreds of nanometers to several microns. The high aspect ratio of the nanosheets ensures uniform and narrow two-dimensional (2D) nanochannels and mitigates the presence of meso- and macro-pores across the membrane.


Vacuum-assisted filtration (VAF) was used to filter 500 mL of the diluted solutions through a commercial polyvinylidene fluoride (PVDF) membrane (hydrophilic, 0.45 μm pore size, EMD Millipore Durapore, US) with a diameter of 47 mm. As shown in FIG. 4, the two-dimensional metal carbide antimicrobial membrane 10 can be formed as a thin film layer 14 of Ti3C2Tx, formed on a PVDF filter substrate/membrane 12.


A glass microfiltration apparatus, with a fritted alumina supported base, was used for the vacuum filtration. The filtered membrane was air dried and retained on the PVDF support. The hydrophilicity of the membrane was evaluated by measuring the water contact angle of pristine PVDF membranes and the water contact angle after coating with Ti3C2Tx, film. The PVDF was relatively hydrophobic with a contact angle of 81°. On the other hand, the Ti3C2Tx, coated membrane functionalization significantly increased the hydrophilicity of the membrane, decreasing the water contact angle to 38°.


The delaminated Ti3C2Tx, sheets formed thin, transparent and smooth films with small wrinkles. Fluorine and oxygen were observed using energy-dispersive spectroscopy (EDS), suggesting O- and F-containing surface terminations. XRD was performed on both air-dried Ti3AlC2 and Ti3C2Tx powders, and typical (000ι) peaks from Ti3AlC2 were observed. Specifically, only peaks corresponding to basal-plane oriented Ti3AlC2 with a c lattice parameter of 18.6 Å were found. However, Ti3C2Tx showed a shift to lower angles of the (000ι) peaks and a large increase of c lattice value to 27-28 Å. These large shifts are suggestive of the presence of water, and possibly cations, between the hydrophilic and negatively charged Ti3C2Tx sheets.


As will be described in greater detail below, the antibacterial properties of Ti3C2Tx and Ti3C2Tx membranes 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 a Luria-Bertani broth (LB) medium at 35° C. Following this, 1 mL aliquots of cell suspensions were subcultured and harvested at the exponential growth phase. Cultures were centrifuged at 5,000 rpm for five minutes, and the resultant pellets were washed three times with saline buffer (pH 7.2) to remove residual macromolecules and other growth medium constituents. The cell pellets collected by centrifugation were re-suspended in sterilized deionized water (DW) and diluted to a cell concentration of approximately 108 colony forming units (CFU)/ml.


The antibacterial activity of each strain was determined by the measurement of optical density (OD) and the colony count method. Batch assays were performed with different Ti3C2Tx concentrations in 1.5 mL tubes containing 500 μL of reaction mixture. In order to examine the effect of Ti3C2Tx concentration on bacterial growth, the batch assays were subjected to 2, 10, 20, 50, 100 and 200 μg of Ti3C2Tx/mL, respectively. The reactors were exposed to continuous shaking at 150 rpm and a constant mesophilic temperature of 35° C. for four hours. For controls, DW was added instead of Ti3C2Tx. The reaction mixture was then transferred to 15 mL tubes, each containing 10 mL of LB medium, and the tubes were inoculated on a shaking incubator at 150 rpm at 35° C. Aliquots of the samples were withdrawn at specific time intervals and the value of optical density (OD) at a wavelength of 600 nm was measured on an ultraviolet-visible light (UV-VIS) spectrometer. Bacterial growth curves were created by plotting OD values versus time, and bacterial growth kinetics were studied. All experiments were performed as duplicates and average values were reported.


A second set of experiments was performed for antibacterial activity. The bacteria (about 108 CFU/mL, where CFU=colony forming units) were incubated with different concentrations of Ti3C2Tx for four hours. Aliquots of the samples were withdrawn, and the CFU were counted by plating 40 μL of 106 dilutions of the bacterial suspensions onto LB agar plates. Colonies were counted after incubation at 35° C. and the cell survival rate was expressed as the percentage of the control. The following equation was used to represent cells' relative viability:








Relative





cells





vaibility

=


(


N
c


N
m


)

×
100


,




where Nc is the number of bacterial colonies of the control sample and Nm is the number of colonies of cells treated with Ti3C2Tx. The antibacterial effect of Ti3C2Tx was compared with that of graphene oxide (GO), which was supplied as stock suspensions of 2 mg/ml. Ti3C2Tx concentrations of 100 μg/mL were applied to both E. coli and B. subtilis and cell survival rates were counted by CFU/mL, as described above. The GO used for comparison in the experiments was synthesized by oxidizing natural graphite powders using H2SO4 and KMnO4 according to the modified Hummers method.


For testing the antibacterial activity of Ti3C2Tx and GO membranes, bacterial cells were diluted to 105 CFU/mL in sterilized DW, and 40 μL of suspensions were pipetted and spread onto a nutritive agar plate, followed by air drying for five minutes. The Ti3C2Tx and GO membranes were placed on the surfaces of agar plates and incubated at 35° C. for 12 hours. Finally, the growths of the colonies on the membrane surface were observed. The membrane samples were sterilized with ultraviolet (UV) radiation for 30 minutes before the experiments.


The antibacterial activity of Ti3C2Tx toward selected bacteria strains was investigated by measuring the growth and the cell viability after the contact of the bacteria with increasing concentrations of Ti3C2Tx suspensions. The pristine bacteria and bacteria treated with Ti3C2Tx were cultivated in LB medium, and the value of optical density (OD) at 600 nm (OD600) was monitored by UV-Vis spectrometer over different time intervals from lag to stationary phases. FIG. 1A is a graph showing cell viability measurements of E. coli and B. subtilis bacteria treated with Ti3C2Tx for four hours in water as a function of Ti3C2Tx concentration, with Ti3C2Tx concentrations ranging between 0 and 200 μg/ml. Here, the survival rates were obtained by the colony forming count method. FIG. 1B is a graph showing cell viability measurements of the E. coli and B. subtilis bacteria after incubation with 100 μg/mL of Ti3C2Tx, as compared to incubation with 100 μg/mL of a graphene oxide (GO). Control for both E. coli and B. subtilis bacteria was taken to be 100%. It can be seen that Ti3C2Tx shows excellent antimicrobial activity for both gram (+) and gram (−) bacteria. The agar plate growth indicated that the bacterial cell loss gradually ascended with increasing concentrations of Ti3C2Tx. The E. coli and B. subtilis showed 92.53% and 93.96% survival rates, respectively, at the lowest Ti3C2Tx concentration of 2 μg/ml. By increasing the Ti3C2Tx concentration from 2 μg/mL to 20 μg/mL, the survival rates of E. coli and B. subtilis were decreased to 35.31% and 15.21%, respectively, as shown by the consecutively decreasing number of colonies grown on the LB plates. More than 99% bacterial viability loss was observed at the highest Ti3C2Tx concentration of 200 μg/ml.


Two-dimensional transition metal carbides have graphene-like morphology. In order to compare antibacterial activity of Ti3C2Tx with GO, both bacterial strains were treated with 100 μg/ml of GO under similar experimental conditions as that of the Ti3C2Tx. FIG. 1B shows the comparison between E. coli and B. subtilis bacterial colonies exposed to 100 μg/mL of Ti3C2Tx or GO, respectively, against a control take to be 100% for both E. coli and B. subtilis bacteria. For both bacterial strains, there was a substantial difference in bacteria colonies on the agar, significantly indicating that the Ti3C2Tx, has higher antibacterial activity when compared against GO. Ti3C2Tx, showed more than 98% cell death to both bacterial strains at 100 μg/mL concentrations of Ti3C2Tx, whereas GO induced about 90% at the same concentration. In FIGS. 1A and 1B, the error bars represent the standard deviation.


SEM was performed to observe the effect of Ti3C2Tx, on morphology and surface structure of the bacterial cells. SEM imaging of samples was accomplished as follows. After the antibacterial studies, cells from the treated samples and on the membrane surfaces were fixed with 2.5% glutaraldehyde overnight at 4° C., followed by washing with 0.1 M phosphate buffer (pH 7.4) and dehydration with a graded ethanol series (25, 50, 80, 100%). Samples were allowed to dry completely at room temperature and were then coated with gold by sputtering. Using SEM, the bacterial cells in the absence of Ti3C2Tx presented as viable and intact rod-shape bacteria and showed relatively smooth cellular membranes. Following cell exposure to 10 μg/mL of Ti3C2Tx, the morphology of a significant fraction of E. coli cells exhibited cell surface damage. As the Ti3C2Tx concentration increased to 50 μg/mL, the morphology of a large fraction of both strains was significantly damaged, and these cells were characterized by noticeable holes or wrinkles on their surfaces. This suggests that with an increase in Ti3C2Tx concentration, both E. coli and B. subtilis are trapped or wrapped by the thin sheets of Ti3C2Tx and subsequently form agglomerates. After exposure to 200 mg/mL of Ti3C2Tx, the bacteria cells became highly deformed and collapsed, with some of the bacteria presenting as being burst, as indicated by scattered cellular debris.


To further evaluate the antibacterial effects of Ti3C2Tx, OD growth curves (after treatment of the cells with the Ti3C2Tx) were studied. FIG. 2A is an optical density growth curve of E. coli bacteria in Luria-Bertani broth (LB) medium at 35° C., following treatment of the bacterial cells with differing concentrations of Ti3C2Tx in water for four hours. Similarly, FIG. 2B is an optical density growth curve of B. subtilis bacteria in Luria-Bertani broth (LB) medium at 35° C., following treatment of the bacterial cells with differing concentrations of Ti3C2Tx in water for four hours. Here, control samples were the respective bacteria untreated with Ti3C2TX.


It can be seen that for both bacterial strains, growth inhibition is dose dependent and bactericidal activity increases with increasing Ti3C2Tx concentration, as is expected from the number of colonies grown on the LB plates. Growth kinetics constants and doubling time for both bacterial strains were evaluated and are shown below in Table 1. The specific growth constant for E. coli e) decreased from 0.277 h−1 to 0.068 h−1 with increasing Ti3C2Tx concentration from 0 to 200 μg/ml. For B. subtilis, a decrease in the growth rate constants (μb) from 0.347 h−1 to 0.134 h−1 was observed with increasing Ti3C2Tx, concentration. Additionally, by increasing the Ti3C2Tx, concentration from 0 to 200 μg/mL, bacterial doubling time (Td) was increased from 2.496 h to 10.114 h and 1.996 h to 5.158 h for E. coli and B. subtilis, respectively, showing a strong bactericidal effect.









TABLE 1







Growth constants and doubling times of bacteria treated at


different Ti3C2Tx concentrations









Sub-
Con-
Ti3C2Tx (μg/mL)















strate
stant
0
2
10
20
50
100
200



















E. coli

μe
0.277
0.271
0.261
0.239
0.168
0.087
0.068



(h−1)



Td
2.496
2.550
2.654
2.897
4.124
7.917
10.114



(h)



B.

μb
0.347
0.319
0.306
0.264
0.240
0.190
0.134



subtilis

(h−1)



Td
1.996
2.251
2.259
2.617
2.878
3.629
5.158



(h)









In order to investigate the successful fabrication of antimicrobial Ti3C2Tx, membrane filters and to understand the interaction of the membrane surface with bacteria, the surface morphologies of uncoated and coated PVDF membrane filters, before and after exposure to bacteria, were examined using SEM. The viability and potential regrowth of bacteria retained on the membrane surface was further evaluated by the agar assay to assess the inactivation of E. coli and B. subtilis.



FIG. 5 shows a comparison of both E. coli and B. subtilis grown on uncoated PVDF (control) membrane filters, PVDF membrane filters coated with graphene oxide (GO), and two-dimensional metal carbide antimicrobial membranes coated with MXene (specifically, Ti3C2Tx), incubated on agar plates. After 24 hours of incubation at 35° C., the control membranes were covered by dense E. coli and B. subtilis bacterial colonies. By contrast, as can be seen in FIG. 5, only a limited number of bacterial colonies were found on the Ti3C2Tx MXene and GO membranes. However, fewer colonies were found on the Ti3C2Tx membranes compared to the GO membranes, thus showing the superior antibacterial effect of Ti3C2Tx.


Additionally, FIG. 6A shows a scanning electron microscope (SEM) image of E. coli bacterial colonies grown on the uncoated PVDF (control) membrane filter, which is compared against FIG. 6B, which shows a scanning electron microscope (SEM) image of E. coli bacterial colonies grown on the MXene (Ti3C2Tx) coated membrane. Similarly, FIG. 7A shows a scanning electron microscope (SEM) image of B. subtilis bacterial colonies grown on the uncoated PVDF (control) membrane filter, which is compared against FIG. 7B, which shows a scanning electron microscope (SEM) image of B. subtilis bacterial colonies grown on the MXene (Ti3C2Tx) membrane. It can be clearly seen that bacterial cell density growth on the Ti3C2Tx, modified membrane is far less when compared to that of the control PVDF membrane. Moreover, the cell colonies consist of intact cell surfaces, whereas the cells grown on the Ti3C2Tx, MXene membrane were flattened and lost their cellular integrity after exposure to the MXene. Thus, not only do fewer bacterial colonies grow on the MXene membrane surface, but damage is also induced to the bacterial cells following direct contact with the MXene.



FIG. 3 is a graph showing growth behavior of E. coli and B. subtilis bacteria incubated on untreated polyvinylidene fluoride (PVDF) membranes, graphene oxide (GO)-modified membranes, and the present Ti3C2Tx-modified membrane. The results showed that membrane filters modified with Ti3C2Tx coatings presented a reduced bacterial growth when compared to GO-modified and unmodified PVDF (control) membrane filters. After 24 hours of incubation at 35° C., control membranes were completely covered by dense E. coli and B. subtilis bacterial colonies. In contrast, only a limited number of bacterial cell colonies were found on the Ti3C2Tx and GO membranes. However, fewer colonies were found on Ti3C2Tx membranes compared to the GO membranes, thus showing the superior antibacterial effect of Ti3C2Tx. In FIG. 3, the data was normalized based on controls. The error bars in FIG. 3 indicate the standard deviation of duplicate measurements.


Further, FIG. 8 compares growth inhibition of E. coli and B. subtilis bacterial colonies, respectively, treated with 100 μg/mL of delaminated Ti3C2Tx (MXene) nanosheets, multilayered Ti3C2Tx MXene ((ML)-MXene), and Ti3AlC2 (MAX). MAX dispersion showed growth inhibition of 14.39±1.43% and 18.34±1.59% for E. coli and B. subtilis, respectively. The (ML)-MXene dispersion showed slightly 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. For the cells exposed to delaminated MXene, the loss of E. coli and B. subtilis cell viability increased to 97.70±2.87% and 97.04±2.91, respectively, exhibiting much stronger activity. The three forms of material showed significant differences in their antibacterial activities against both bacterial strains. In particular, delaminated MXene has a much more pronounced antibacterial activity compared with those of MAX and (ML)-MXene.


In order to understand the antibacterial effect of MXene, changes of morphology and membrane integrity of E. coli and B. subtilis cells due to the interaction with MXene were further evaluated by SEM and TEM. FIGS. 9A and 9B are scanning electron microscope (SEM) images of E. coli bacterial cells cultured in the absence of Ti3C2Tx, at low magnification and high magnification, respectively. Similarly, FIGS. 10A and 10B are scanning electron microscope (SEM) images of B. subtilis bacterial cells cultured in the absence of Ti3C2Tx, at low magnification and high magnification, respectively. The bacterial cells for both E. coli and B. subtilis cultured in the absence of Ti3C2Tx, were viable with no observed membrane damage or cell death. The higher magnification of FIGS. 9B and 10B shows that the bacteria are protected by intact cytoplasmic membranes.



FIGS. 9C and 9D are SEM images of E. coli bacterial cells cultured in the presence of 50 μg/mL of Ti3C2Tx, at low magnification and high magnification, respectively. Similarly, FIGS. 10C and 10D are SEM images of B. subtilis bacterial cells cultured in the presence of 50 μg/mL of Ti3C2Tx, at low magnification and high magnification, respectively. Most bacterial cells in the visible field at low magnification suffered from prevalent membrane damage and cytoplasm leakage in the presence of 50 μg/ml of Ti3C2Tx, which can be clearly observed at the high magnifications. Some bacterial cells still maintained the membrane integrity, but they were deformed. FIGS. 9E and 9F are SEM images of E. coli bacterial cells cultured in the presence of 100 μg/mL of Ti3C2Tx, at low magnification and high magnification, respectively. Similarly, FIGS. 10E and 10F are SEM images of B. subtilis bacterial cells cultured in the presence of 100 μg/mL of Ti3C2Tx, at low magnification and high magnification, respectively. At 100 μg/ml of Ti3C2Tx, both bacteria suffered from prevalent cell lysis, indicated by a severe membrane disruption and cytoplasm leakage. 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.


As confirmed by the spot energy dispersive spectroscopy (EDS) results for the surface of E. coli bacteria and B. subtilis bacteria of FIGS. 11A and 11B, respectively, increasing Ti3C2Tx, concentration causes both E. coli and B. subtilis to be trapped or wrapped by the thin sheets of Ti3C2Tx, and subsequently form agglomerates.



FIG. 12A is a transmission electron microscope (TEM) image of E. coli bacterial cells cultured with 200 μg/ml of Ti3C2Tx, for 4 hours at low magnification. FIG. 12B is a TEM image of E. coli bacterial cells cultured with 200 μg/mL of Ti3C2Tx, at high magnification. Similarly, FIG. 13A is a TEM image of B. subtilis bacterial cells cultured with 200 μg/ml of Ti3C2Tx, for 4 hours at low magnification, and FIG. 13B is a TEM image of B. subtilis bacterial cells cultured with 200 μg/mL of Ti3C2Tx, at high magnification. One can clearly see the cell wall and membrane damage, as well as the change of the inner structures of the cells. TEM analysis of E. coli and B. subtilis before and after being exposed to 200 μg/ml of MXene nanosheets shows a decrease in the number of bacterial cells in the MXene treated groups compared to the control. As FIGS. 12B and 13B show, the MXene nanosheets were tightly adsorbed around the cells and even entered into the cells (arrows “a” and “c”). At the same time, the intracellular densities of both E. coli and B. subtilis decreased, revealing that they lost some intracellular substance. In both E. coli and B. subtilis, the cell wall was stripped down after exposure to MXene nanosheets (arrows “b” and “d”). Significant inner cell structure leakage can also be observed due to cell wall and membrane damage.



FIGS. 14A and 14B are TEM images of B. subtilis and E. coli bacterial cells, respectively, exposed to 200 μg/mL of Ti3C2Tx at 37° C. for four hours. In agreement with the cells viability assay, more B. subtilis cells with altered cell morphology were found than with E. coli, where cell damage intensity was lower. Ti3C2Tx MXene exhibited a higher antibacterial activity towards gram-positive B. subtilis as compared to gram-negative E. coli. The E. coli bacteria, as gram-negative bacteria, are covered by a much thinner layer of peptidoglycan (thickness of 7-8 nm), but have an external protective lipid membrane. Gram-positive B. subtilis lacks the external lipid membrane, but its thicker peptidoglycan cell walls are in the range of 20-80 nm. The hydrophilic Ti3C2Tx MXene could effectively attach to bacteria, facilitating their inactivation by direct contact interaction.



FIG. 15 is a graph showing cytotoxicity in both E. coli and B. subtilis as a function of the concentration of Ti3C2Tx MXene. Lactate dehydrogenase (LDH) release assays were used to quantitatively determine the extent of cell damage. FIG. 15 shows the LDH activity in the supernatants after four hours of incubation. Concentration dependent LDH release was observed as bacterial cells were exposed to MXene nanosheet dispersions. The bacterial cells exposed to 2 and 10 μg/L of MXene nanosheets exhibited minimal LDH release for both E. coli and B. subtilis. However, LDH release increased significantly when bacterial cells were exposed to a 200 μg/L solution of MXene, which showed cytotoxicity of 38.41% and 55.24% for E. coli and B. subtilis, respectively. This dose dependent cytotoxicity 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.


Further, the possibility of MXene-induced reactive oxygen species (ROS) dependent and independent oxidative stress was studied in two separate abiotic assays. First, superoxide anion (O2.) production at different MXene concentrations was monitored using an XTT assay. As shown in FIG. 16, no noticeable absorption was detected at different MXene concentrations, thus revealing that MXene mediated no or negligible superoxide anion production and their role in MXene antibacterial activity could be minimal. The likelihood of ROS-independent oxidative stress mediated by MXene dispersions was examined using a 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 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. Glutathione was exposed to MXene nanosheets at different concentrations in a bicarbonate buffer and incubated for four hours, after which the unoxidized fraction of glutathione was measured spectrophotometrically using Ellman's reaction. Glutathione oxidation was found to be dependent on the MXene concentration.



FIGS. 17A and 17B show that as the MXene concentration in colloidal solution was increased from 2 μg/mL to 200 μg/mL, glutathione oxidation increased from 2.5 to 38.3%. Two possible paths of glutathione oxidation by MXene could be possible: First, oxidation can occur either via direct oxidation of a specific microbial process, or a vital component, as previously shown for fullerenes and CNT. The second mechanism could involve the reduction of surface oxides formed on MXene surface in the presence of oxygen by electron transfer from the glutathione enzymes. Furthermore, the reactive oxygen species (H2O2 or O2) may be released by the O2 molecules adsorbed on the MXene surface, which could later be reduced by the action of glutathione.


The surfaces of MXene sheets suggest O- and F-containing surface terminations, thus giving MXenes negatively charged surfaces. In the case of MXene, its strong antibacterial property can be partially attributed to the anionic nature of the membrane surface. In addition, the antimicrobial activity of MXene can also be due to its high hydrophilicity, which can enhance bacterial contact to the membrane surface, thus resulting in inactivation of adhered microorganisms according to a direct contact-killing mechanism. Moreover, hydrogen bonding between oxygenate groups of MXene and the lipopolysaccharide strings of the cell membrane could result in bactrial inhibition by preventing nutrient intake.


It is to be understood that the present invention is not limited to the embodiments described above, but encompasses any and all embodiments within the scope of the following claims.

Claims
  • 1. An antimicrobial agent, comprising at least one two-dimensional metal carbide layer, the two-dimensional metal carbide having the formula Mn+1XnTx, where M is an early transition metal, X is either C or N, n is 1, 2, or 3, T represents a terminal functional group, and x represents the number of terminal functional groups.
  • 2. A method of protecting a substrate from bacterial contamination, comprising the step of coating the substrate with at least one layer of the antimicrobial agent according to claim 1.
  • 3. The antimicrobial agent according to claim 1, wherein the two-dimensional metal carbide has the formula Ti3C2Tx.
  • 4. The antimicrobial agent as recited in claim 3, wherein the terminal functional group comprises at least one functional group selected from the group consisting of O, OH, and F.
  • 5. A method of protecting a substrate from bacterial contamination, comprising the step of coating the substrate with at least one layer of the antimicrobial agent according to claim 3.
  • 6. The method of protecting a substrate according to claim 5, wherein said at least one layer comprises stacked layers of the antimicrobial agent according to claim 1.
  • 7. The method of protecting a substrate according to claim 5, wherein said substrate comprises a polymer.
  • 8. The method of protecting a substrate according to claim 5, wherein said substrate comprises a polyvinylidene fluoride (PVDF) membrane support.
  • 9. A method of treating water for reduction of bacterial contamination, comprising the step comprising the steps of: adding nanosheets of the antimicrobial agent according to claim 3 to the contaminated water to form a colloidal suspension; andallowing the colloidal suspension to incubate for a period of time sufficient to reduce levels of bacterial contamination in the water.
  • 10. The method of treating water according to claim 9, wherein said step of adding nanosheets of the antimicrobial agent according to claim 3 further comprises adding the nanosheets to obtain a concentration of at least 100 μg/ml.
  • 11. The method of treating water according to claim 9, wherein said step of adding nanosheets of the antimicrobial agent according to claim 3 further comprises adding the nanosheets to obtain a concentration of about 200 μg/ml.
  • 12. The method of treating water according to claim 9, wherein said step of allowing the colloidal suspension to incubate for a period of time further comprises allowing the colloidal suspension to incubate for about 4 hours.
  • 13. An antimicrobial membrane, comprising: a polyvinylidene fluoride (PVDF) membrane support; andat least one two-dimensional metal carbide layer coated on the PVDF membrane support, the two-dimensional metal carbide having the formula Ti3C2Tx, wherein T represents a terminal functional group, and x represents the number of the terminal functional groups.
  • 14. The antimicrobial membrane as recited in claim 13, wherein the terminal functional comprises at least one functional group selected from the group consisting of O, OH, and F.
  • 15. The antimicrobial membrane as recited in claim 13, wherein said at least one two-dimensional metal carbide layer comprises a stack of layers of the two-dimensional metal carbide.
  • 16. A method of making the antimicrobial membrane of claim 13, comprising the step of filtering a dilute colloidal solution of nanosheets of the two-dimensional metal carbide on the polyvinylidene fluoride (PVDF) membrane supports by vacuum filtration.
  • 17. The method of making the antimicrobial membrane according to claim 16, wherein the nanosheets of the two-dimensional metal carbide have a thickness of about 1 nm and a lateral size greater than 450 nanometers.
  • 18. The method of making the antimicrobial membrane according to claim 16, wherein said polyvinylidene fluoride (PVDF) membrane supports have a pore size of 450 nanometers.
  • 19. A method of controlling biofilm formation on a substrate, comprising the step of coating the substrate with an antimicrobial agent in an amount effective to inhibit biofilm formation, the antimicrobial agent including at least one two-dimensional metal carbide layer, the two-dimensional metal carbide layer having the formula Ti3C2Tx, wherein T represents a terminal functional group and x represents the number of terminal functional groups.
  • 20. The method of controlling biofilm formation on a substrate as recited in claim 19, wherein the terminal functional group comprises at least one functional group selected from the group consisting of O, OH, and F.
PCT Information
Filing Document Filing Date Country Kind
PCT/US16/57029 10/14/2016 WO 00
Provisional Applications (1)
Number Date Country
62244432 Oct 2015 US
Continuation in Parts (1)
Number Date Country
Parent 62080967 Nov 2014 US
Child 15769632 US