Silver-doped graphite oxide composites used as antimicrobial agents against Staphylococcus aureus, Escherichia coli and Tatumella terrea evaluated by direct TLC bioautography

Silver-doped graphite oxide composites presented mixed Ag and Ag2O crystalline phases accompanied by C/O and ID/IG average ratios of 2.13 and 1.16, associated with a good oxidation degree and high structural disorder or defects in the carbon material, respectively. The low-silver-doped GrO composite showed homogenous silver particle dispersion and a low particle size distribution (96 nm). However, high-silver-doped GrO composites generated materials with high relative crystallinity, silver particle agglomeration, and Ag metal phase promotion. At 3 and 5 mg ml−1 per chromatographic plate, the silver-doped graphite oxide composites were tested by direct TLC bioautography against Staphylococcus aureus, Escherichia coli, and Tatumella terrea microorganisms, where the minimum inhibitory concentration was 3 mg ml−1 per chromatographic plate. At 3.0 mg ml−1 per chromatographic plate, high-silver-doped GrO composites exhibited a 39%, 3.2-fold, and 83% higher retention factor (R f ) compared with the composites with low-silver-doped GrO composites against S. aureus, E. coli, and T. terra microorganisms, respectively. However, both composites showed similar inhibition capacities at 5.0 mg ml−1 per chromatographic plate against the three microorganisms. This behavior may be associated with both composites reaching the threshold limit. In general, the silver acetate amount used in the silver-doped GrO composites influenced the dispersion, crystalline phase promotion, particle size distribution, and the silver particle release capacity, which modified the electrostatic adsorption type between the composites and the bacterial cell walls.


Introduction
The development of new antimicrobial materials is a continuous process due to microorganism resistance, excessive use of antibiotics, and the misuse of antibiotics.Since ancient times, silver-based materials have been used mainly to maintain the quality of water in silver containers (Ag°) and for treating infections, e.g., Hippocrates (5th-4th century BC) prescribed remedies made from silver preparations.In this sense, silver nitrate (AgNO 3 ) was administered as medicine by the Romans, which was still used to treat injuries and skin ulcers during the Middle Ages [1].Later, silver sulfadiazine (C 10 H 9 AgN 4 O 2 S) emerged, which is currently used to treat infections caused by skin burns [2].Silver (I) salts give rise to high concentrations of silver ions with astringent effects, which are used in caustic AgNO 3 pens used for cauterization of small injuries and removal of granulation tissue.Also, silver acetate (CH 3 COOAg) has shown antimicrobial activity attributed to silver ions in solution.Silver ions are capable of binding to the bases of DNA and RNA, generating mutations and replication problems [3].Silver ions also cause alterations of proteins within the cell, where silver ions bind to sulfhydryl groups in amino acids, altering the Fe-S groups and thiol groups and metal sulfhydryl ligands [4,5].However, these properties are undesirable for an antibacterial material and have been replaced by other antibiotics [6].Currently, silver nanoparticles (SNP) have been shown to be potent antimicrobial and antifungal materials because SNP frequently release silver ions.The ability of Ag + ions to bind and complex with nucleic acids has been compared to that of phosphate groups.In this sense, silver-based materials are sources of Ag + ions, where the main interaction between negatively charged microbial cells and positively charged particles is an electrostatic attraction.This promotes affinity towards sulfur proteins, where metal ions adhere to the cytoplasm and cell wall and significantly improve permeability, disrupting the bacterial wall.Finally, cells absorb free Ag + ions, causing respiratory enzymes to deactivate, generating reactive oxygen species (ROS) and a disruption in the release of adenosine triphosphate (ATP) [7,8].However, one of the limitations of SNP is the matrix in which they are deposited in such a way that the modulation between the matrix and the metallic particle facilitates the interaction with the bacteria.In this sense, carbon-based materials have demonstrated antibacterial activity and have support or matrix properties that facilitate the incorporation or arrangement of metals to increase the inhibition of the growth of bacteria, fungi, and viruses.Therefore, carbon-based materials (some allotropes) turn out to be a stable matrix that allows silver compounds located at the boundaries, peripheries, or internally to favour the synergy between functional groups of carbon-based material and release active silver ions.Some examples are silver-modified graphene oxide nanosheets [9], silver/reduced graphene oxide [10], C70 fullerene and SNP [11], SNP grafted onto multiwalled carbon nanotubes [12], carbon nanoscrolls filled with SNP [13], carbon dots doped with AgNO 3 [14], and carbon/silver thin films [15].
The antibacterial activity of all these materials is attributed to the direct physicochemical interaction between transgenics and bacteria, which causes deadly deterioration of cellular components, mainly proteins, lipids, and nucleic acids.In fact, graphene-derived materials have a high affinity for membrane proteoglycans, where they accumulate and cause membrane damage; in the same way, after internalization, these materials can interact with the hydrogen groups of the RNA/DNA of the bacteria, which disrupts the replicative stage [16].In this way, graphite oxide (GrO), also known as graphitic oxide, is an oxidized bulk product of graphite with a variable composition.However, it did not receive much attention until it was identified as an important and readily available precursor for the preparation of graphene.Recent developments in this type of material suggest that its intercalation implies applications in multifaceted areas such as catalysis, sensors, supercapacitors, water purification, hydrogen storage, and magnetic shielding, among others [17].The properties of GrO vary according to its composition, since it can act as a 'p' type semiconductor due to the high electronegativity of oxygen with respect to carbon, or it can act as an 'n' type semiconductor when doped with nitrogenous compounds [18,19].Another application of GrO is to act as an antimicrobial agent, where the carboxyl, epoxy, carbonyl, and hydroxyl functional groups cause cellular oxidative stress by interacting with microorganisms such as S. aureus, E. coli, and P. aeruginosa.Graphite exhibits slightly weaker cytotoxicity than GrO and graphene oxide has much stronger bacterial activity compared to GrO (4-fold) tested for loss of viability against E. coli [20].In this sense, GrO doped with AgNO 3 showed antimicrobial activity against the microorganisms B. subtilis, C. albicans, E. coli, and S. aureus.In the adsorption mechanism between the materials and bacteria, the functional groups of GrO and free silver particles caused the permeability of the cell wall, increasing oxidative stress and stopping bacteria growth [21].Also, partially reduced GrO material with infrared-irradiated bis (lysinate)zirconium(IV)phthalocyanine complex nanoparticles and silver was tested against S. aureus, P. aeruginosa, and E. coli microorganisms, which are responsible for many infections and are one of the most difficult pathogens to treat.The antibacterial photoactivity obtained showed high inhibition values of the P. aeruginosa bacterium, associated with the cooperative action of GrO, SNP, phthalocyanine molecules, and irradiation in the near infrared range [22].GrO with phthalocyanine (Pc) derivatives of zirconium (IV) employed as a bacteriostatic photosensitive additive and as an antibacterial agent was tested.These materials were tested against the E. coli 6.2E strain isolated from the root canal of a previously endodontically treated tooth without showing activity.Additionally, reinfection of the root canal system and periapical formation consequently produced lesions, and E. coli J53 (resistant to antibiotics and silver ions) was also tested without success under 10 min of irradiation.Therefore, for this type of material, the minimum inhibitory concentration must be above 8 mg ml −1 since the patient cannot be exposed to long exposure times [23].
In general, GrO is a material that has been shown to have antimicrobial properties and that, when doped or irradiated, considerably increases its activity.This antimicrobial behavior is due to its high adsorption capacity, which allows it to bind to microorganisms and disrupt their cell membrane.However, the antimicrobial mechanism of interaction between doped or irradiated GrO and the cell wall of microorganisms is not clear to the scientific community.In this sense, the main interaction mechanisms between the bacteria and the medicament or material are the limitation in absorption, the modification of the target, the inactivation, and the antibacterial active release [24].To solve this and other problems, the type of evaluation of an antimicrobial material or agent helps us to understand the interaction mechanism between the bacteria and the agent and to be able to predict the behavior of the proposed material in the field.Paper chromatography (PC)-based bioautography and thin-layer chromatography (TLC)-based bioautography techniques have become tools for the detection of antimicrobial agents.Three bioautographic methods are mainly used: (I) agar diffusion or contact bioautography; (II) direct TLC bioautographic detection; and (III) agar immersion or overlay bioautography to detect antimicrobial agents in a mixture of compounds [25,26].Direct bioautography technique turns out to be a fundamental evaluation technique that accurately determines the substance or substances responsible for the antimicrobial activity in a complex mixture while optimizing its chromatographic profile.Antimicrobial compounds from plant extracts have been successfully detected by thin layer chromatography [27][28][29].The main bioautographic techniques are (1) agar diffusion with carrier culture medium, where the culture condition is diffusion-dependent, and incubation for several hours at 0 to 4 °C, followed by culture time at 37 °C.(2) On a thin-layer plate with direct bioautography culture conditions, the incubation temperature is generally slightly higher than room temperature, and the incubation time is 2-3 days.It can also be grown overnight at around 30 °C, sometimes with light.Finally, the (3) supported agar overlay bioautography was grown overnight at approximately 30 °C on a thin-layer plate with the culture conditions after solidification of the agar.The direct TLC bioautography technique exhibits the best sensitivity and strongest specificity among the bioautographic techniques [30].
Therefore, the objective of this work was to calculate the inhibition percentage of silver-doped graphite oxide composites against S. aureus, E. coli, and T. terrea microorganisms by direct TLC bioautography.Additionally, the silver-doped graphite oxide composites were characterized by x-ray diffraction, FTIR, Raman, and x-ray photoelectron spectroscopies, and scanning electron microscopy (SEM) with energy dispersive x-ray spectroscopy (EDS).

Experimental section
2.1.Silver-doped graphite oxide composites synthesis Graphite powder was chemical oxidized by the modified Hummers method [31,32].The GrO impregnation with 0.5 wt% (GrO-0.5Ag)and 1.5 wt% CH 3 COOAg (GrO-1.5Ag)was carried out by the incipient impregnation method, considering the experimental GrO pore volume of ca.2.0 cm 3 g −1 .A similar pore volume value was obtained by the He buoyancy gravimetric method for GrO synthesized with tetraethylammonium tetrafluoroborate intercalated by the Brodie method [33].Subsequently, the GrO-0.5Agand GrO-1.5Agcomposites were dried at 150 °C for 2 h, where the theoretical metal loading was 0.32 wt% and 0.97 wt% silver, respectively.

Microorganisms and growth medium
The species used for testing antagonism are presented in table 1.The multidrug-resistant strains were grown routinely in Luria Bertani (LB) medium at 37 °C and T. terrea at 30 °C.The strains were stored in 35% glycerol at −70 °C, using the previously described methodology [34].

Direct TLC bioautography detection 2.3.1. Impregnation of petri dishes
All composites were placed on 20 × 20 mm chromatographic plates at the bottom of the Petri dish, where the diffusion halos can be observed at concentrations of 3.0 and 5.0 mg ml −1 silver-doped graphite oxide composite per chromatographic plate.The Petri dish was covered with a layer of agar inoculated with S. aureus, E. coli, and T. terrea microorganisms, respectively.Each bacterium was allowed to grow in each Petri dish at 37 °C ± 0.5 or 30 °C ± 0.5 for 72 h for subsequent TLC bioautography [35].Afterward, MTT compound Sigma ® (3-[4,5dimethyl-thiazol-2-yl]−2,5-diphenyltetrazolium bromide) was added at 0.8 g L −1 in newt X-100 (Sigma ® ) at 0.1% in water, inoculated into agar, and then layered on chromatographic plates at 37 °C ± 0.5 until complete bacterial growth and assimilation of the enzyme substrate by each microorganism.The bleached areas (marked with a blue circle) of the Petri dishes 72 h after starting the antimicrobial test revealed the inhibition zone or halo of each microorganism measured in mm.

Measurement of inhibition halos
TLC bioautography is a method that combines chromatographic separation with in situ localization of compounds with biological activity.The chromatographic plate is exposed to the bioassay and bioactive compounds, which results in a visible effect including inhibition of microbial growth or enzyme activity that is displayed as zones or halos [36].The retention factor (R f ) value is a relationship that represents the inhibition halo formed by the silver-doped graphite oxide composites with respect to the maximum diameter of the halo formed when the bacteria proliferate freely (physical barrier), as described in equation (1).
= R halo diameter formed by the composites halo diameter generated by cell proliferation The R f value in each test was calculated from the diameter (mm) obtained after the reduction of MTT with the composites.The measurement range of the inhibition zones was from 0 to 50 mm (0 to 1.97'), where the R f values are between 0 and 1.The R f values obtained have no units and should always be considered approximate (each experiment was performed in triplicate).However, this value can indicate the antibacterial efficacy of the silver-doped graphite oxide composites, as inferred by the radial diffusion zone in the Petri dish.

Composites characterization
The characterization equipment and operating conditions of each technique used for the characterization of silver-doped graphite oxide composites have been described in previous works [19,21,32].

Thin layer chromatography (TLC) bioautography tests
The antimicrobial activity of the silver-doped graphite oxide composites was first tested by the agar well diffusion method shown in the support information.However, agar-well diffusion tests present limitations of effective diffusion, precipitation, and composite incompatibility with the medium, affecting the release and diffusion of silver particles in the medium.In contrast, the TLC bioautography test, allows target-directed isolation and diffusion of the active components of the composites on the surface of the chromatographic plate.Furthermore, Direct bioautography is very sensitive and provides accurate localization of active components inside the composites.However, is limited to microorganisms able to grow directly on the chromatographic layer.In contact bioautography, the transfer process directs to larger inhibition zones which decrease the sensitivity and the ability to discriminate between active compounds with similar R f values [37].
The inhibition zones produced by the contact of GrO, GrO-0.5Ag, and GrO-1.5Agcomposites with the S. aureus (figure 1), E. coli (figure 2), and T. terrea (figure 3) microorganisms at 3 (left) and 5 (right) mg ml −1 per chromatographic plate, respectively.The bacteria grow directly on the surface of the plate, excluding the places where silver-doped graphite oxide composites are found.The revelation zone after the reduction of MTT is white, associated with inhibition of bacterial growth (indicated with a blue circle).
At 3 and 5 mg ml −1 concentrations, silver acetate showed similar inhibition zones against E. coli and for S. aureus and T. terrea microorganisms.According to the literature, only CH 3 COOAg has demonstrated antimicrobial activity against five strains of Acinetobacter baumannii [38].
At 3 and 5 mg ml −1 , GrO showed no significant growth inhibition against S. aureus and T. terrea microorganisms.However, for the E. coli microorganism, the inhibition zone for GrO increased by 50% at 5 mg ml −1 compared to 3 mg ml −1 .Regarding S. aureus, silver acetate has basically shown higher inhibition zones than GrO and similar behavior for the GrO-0.5Agcomposite.However, at 3 mg ml −1 and 5 mg ml −1 per chromatographic plate, the net charge was 0.015 mg and 0.025 mg silver acetate for the GrO-0.5Agcomposite, so the precursor salt has an antagonistic effect on the evaluation of the growth inhibition of E. coli and T. terrea bacteria.Therefore, a synergistic relationship between the silver ions release and GrO functional groups can be responsible for the antimicrobial characteristics of the silver-doped graphite oxide composites.
For GrO-0.5Ag and GrO-1.5Agcomposites, the minimum inhibitory concentration was 3 mg ml −1 per chromatographic plate, where the inhibition zone will extend radially outward from the thin layer towards the Petri dish in circular motion and/or where the antimicrobial agent is suspended, inferring the antibacterial efficacy [39].It is known that high areas infer a high level of antimicrobial activity, and the behavior observed in a circle and/or square area per Petri dish (off-white color) represents the composite inhibition zone.In particular, the mechanism of similar carbon-based material toxicity in various E. coli strains is not associated with oxidative stress or significant mechanical disruption of the treated bacteria.Antimicrobial activity is related to neutralization dose, surface charge transport, direct membrane interaction, internalization, and membrane stress [40].Figure 4 shows the R f values as a function of silver-doped graphite oxide composite concentration graphs with linear regression.At 3 mg ml −1 per chromatographic plate, the R f values for the GrO-1.5Agcomposite were 39%, 3.2-fold, and 83% higher than those for the GrO-0.5Agcomposite for the S. aureus, E. coli, and T. terrea  microorganisms, respectively.Also, at this concentration, the GrO-1.5Agcomposite showed 2.7-fold, 6.2-fold, and 6.0-fold higher growth inhibition than GrO against S. aureus and T. terrea microorganisms, respectively.However, at 5 mg ml −1 per chromatographic plate, GrO-0.5 and GrO-1.5 composites had similar antimicrobial activity against S. aureus, E. coli, and T. terrea microorganisms because both composites saturate the bacteria binding sites, affecting their growth.This would mean that once composites reach a minimum effective concentration, neither GrO-0.5 nor GrO-1.5 can inhibit bacterial growth any more effectively.However, it is important that the silver available on the composite surface counteract the electrostatic forces between the composite and the cell wall so that silver ions can be deposited in the cell wall and cause poisoning by interfering with metabolic processes.It is known that the high silver ion dispersion increases the bactericidal efficacy of carbon-silver-based composites.Similar carbon-based materials act as a general enhancer of cellular growth by increasing cell attachment and proliferation due to the content of functional groups [41].
On the one hand, the slopes calculated indirectly provide the growth inhibition capacity of the silver-doped graphite oxide composites against S. aureus, E. coli, and T. terrea microorganisms.GrO-1.5Agagainst S. aureus, E. coli, and T. terrea microorganisms showed 22%, 20%, and 12% higher inhibition capacities than GrO-0.5Ag,respectively.The high antimicrobial activity of the GrO-1.5Agcomposite is a function of the silver acetate addition, which allowed more free metal ions that did not chemically interact with the GrO functional groups.The effectiveness of free silver particles is not only due to their size but also to their large surface-to-volume ratio.These can produce ROS and disrupt deoxyribonucleic acid replication by releasing silver ions [42].In particular, the inhibition percentage of T. terrea microorganism growth was a function of the silver acetate concentration in the GrO doping, presenting a higher amount of silver available to diffuse or deposit on the cell membrane.Also, excess silver on the composites can inhibit the proton motive force and electron transport in the respiratory chain, which can affect membrane permeability and cause the bacterial cell death [43].Free metal ions that involve the interaction of silver with biological macromolecules such as enzymes and DNA through an electronrelease mechanism [44].The free silver ions are not chemically bound with GrO, resulting in silver conglomerates with high cohesion forces to one another, where metallic saturation on the surface improves the synergy between the GrO functional groups and the silver ions release with the culture medium [21].
The XRD spectra of the GrO-0.5Agand GrO-1.5Agcomposites are shown in figures 5(b) and (c), respectively.Also, GrO-0.5Ag and GrO-1.5Agcomposites showed elemental sulfur with two peaks at 2θ = 18.0°a nd 27.8°, associated with low sulfur elimination during synthesis by the Hummers and incipient impregnation methods, respectively.GrO-0.5Ag and GrO-1.5Agcomposites showed three diffraction peaks with the '´' tag observed at 2θ = 31.0°,54.6°, and 67.3°related to the (111), (220), and (013) planes assigned to the cubic Ag 2 O phase, verified with ICDD-76-1393 [47].Also, both composites showed three diffraction peaks with the '*' tag observed at 2θ = 32.2°,37.0°, and 46.3°related to the (122), (111), and (231) planes, assigned to the elemental Ag with system face-centered cubic and verified with the cards ICDD-04-0783 and ICDD-65-2871.In this sense, the silver acetate comprises the often-observed Ag 2 (carboxylate) 2 dimer unit connected by Ag-O interdimer bonds to form infinite chains that align in parallel, forming stacks.During the thermally induced reduction of silver carboxylates, the Ag(I)-Ag(I) bond can promote the SNP formation [48].During the composite impregnation, the silver acetate dissociates into the silver ions and the acetate counterions (carboxylates), which are dispersed into the GrO.After this process, the composites were dried at 150 °C for 3 h, where the silver oxide was formed.It is known that AgO is less stable than Ag 2 O, as described in the following reaction: ( ) from 83 to 134 °C, with 30 kcal mol −1 activation energy [49].Also, the Ag 2 O phase presented an increment in the toxicity of bacteria observed when the particle size decreased, which can be explained by an increase in the release of Ag + and Ag°species.The nanoparticles from the Ag 2 O phase can surround the cell due to an increase in the area/volume ratio [50].Furthermore, the Ag 2 O phase on antimicrobial activity indicates at least 53 microbial species, including 21 Gram-negative bacterial species, 15 Gram-positive bacterial species, and 17 fungal species.Among the most frequent organisms are the bacteria E. coli, P. aeruginosa, K. pneumoniae, S. aureus, and B. subtilis, as well as the Aspergillus and C. albicans fungi.The Ag and Ag 2 O coexistence on the composite surface can increase or limit the permeability, modify the structure of the membranes, and cause cell wall damage at different exposure times [51].In our case, GrO-0.5Ag and GrO-1.5Agcomposites presented the mixed Ag 2 O and Ag crystalline phases, and the percentage of relative crystallinity was 16% higher for GrO-1.5Agthan for GrO-0.5Ag.Also, the crystallite size was calculated with the Debye-Scherrer equation for the Ag 2 O phase; the values were c.a. 40 and 29 nm for GrO-0.5Ag and GrO-1.5Agcomposites, respectively [52].On the other hand, for the Ag phase, the crystal size was 21 and 29 nm for GrO-0.5Ag and GrO-1.5Agcomposites, respectively.It is known that materials with small crystallite sizes have higher antimicrobial activity than those with large crystallite sizes [53,54].

FTIR spectroscopy
The FTIR spectrum of GrO is shown in figure 6(a), where the broad band at 3375 cm −1 corresponds to the O-H stretching vibration associated with the −OH and/or −COOH functional groups from GrO material [55].The other absorption bands present at 2860, 2925, 1700, 1580, 1405, 1220, and 1025 cm −1 wavelengths were associated with weak symmetric and asymmetric stretching -CH 2 vibration, −C=O stretching vibration, C=C stretching, O-H bending, C-O stretching of epoxy groups, and C-O stretching of an alkoxy group, respectively.
The FTIR spectra of the GrO-0.5Agand GrO-1.5Agcomposites are shown in figures 6(b) and (c), respectively.Both FTIR spectra show a strong absorption band at 3200-2750 cm −1 associated with O-H stretching vibration.This absorption band is associated with the free water in the composites.During the impregnation of the silver-doped graphite oxide composites, the CH 3 COOAg salt was dissolved in water, generating solvation spheres of the Ag + ion and the CH 3 COO -counterion.For monovalent silver carboxylates, it is observed that the asymmetric COO− stretching frequency decreases while the symmetric -COO stretching frequency increases with respect to that observed for the free carboxylate ion [56].Additionally, the GrO-0.5Agand GrO-1.5Agcomposites show two bands at 1560 cm −1 and 1415 cm −1 associated to C-OH bending vibrations [57].For both composites, FTIR spectra presented one band at 550 cm −1 related to the characteristic lattice vibration of the Ag 2 O phase [58].

Raman spectroscopy
The Raman spectra of the silver-doped graphite oxide composites showed two representative bands, the D-and G-bands.The D-band is attributed to defects such as dislocations, cracks, or vacancies created during the graphite oxidation process, and the intensity of this band is proportional to the number of defects in the A 1g vibrational zone-edge mode.Meanwhile, the G-band is related to E 2g in the plane vibrational mode, commonly used for similar carbon-based materials with sp 2 hybridization [59].On the one hand, the G-band for GrO (figure 7(a)) was found at 1578 cm −1 , while for the GrO-0.5Ag(figure 7(b)) and GrO-1.0Ag(figure 7(c)) composites, it appeared at 1584 and 1582 cm −1 , respectively.On the other hand, the I D /I G relationship can be associated with the degree of disorder caused by the oxidation process occasioned by oxygen-carbon functional group incorporation [60].The I D /I G ratios founded were 1.10, 1.15, and 1.17 for GrO, GrO-0.5Ag, and GrO-1.5Agcomposites, respectively.Additionally, the full width at half maximum (FWHM) of the D-band for the GrO-0.5Agand GrO-1.5Agcomposites was narrower than GrO.This region can be fixed by sub-bands in each D band associated with external and internal processes in the 'phonon first' or 'defect first' process caused by edge defect creation, vacancies, and distortions in the carbon material [61].

X-ray Photoelectron spectroscopy (XPS)
Figure 8(a) presents the survey XPS spectra of the GrO, GrO-0.5Ag, and GrO-1.5Agcomposites with associated peaks from the C 1 s, Ag 3d, and O 1 s regions, respectively.The 3d Ag peaks intensity in the survey spectra appear as a function of the salt precursor concentration, for GrO-0.5Ag, and GrO-1.5Agpresented 0.29 and 0.90 atomic % silver, respectively.The high-resolution C 1 s spectra for GrO (figure 8(b)), GrO-0.5Ag(figure 8(d)), and GrO-1.5Ag(figure 8(f)) composites show four peaks centred at 284.5 eV (C=C), 286.5 eV (C-OH), 288 eV (C=O), and 288.7 eV (O=C-OH) that indicate the presence of different functionalities (hydroxyl group, epoxide group, and carbonyl group) located in the edges and faces of the GrO [62].The GrO-0.5Ag intensity of the C-OH peak is significantly reduced, accompanied by an increase in the sp 2 carbon peak, revealing that the epoxy group helps to anchor silver particles in the GrO surface and restore sp 2 carbon networks [63].In the GrO composite, the sp 2 carbon was 62.9% and the sum of C=O and O=C-OH functional groups was 12.1%; after doping with silver acetate, the sp 2 carbon percentage increased by 16.8% and 11.3% for GrO-0.5Ag and GrO-1.5Agcomposites, respectively.The presence or abundance of the carbonyl group of GrO was reduced by 17% and 51% for the GrO-0.5Agand GrO-1.5Agcomposites, respectively, associated with chemical interaction between carbonyl groups and silver precursor.It is known that in similar carbon-based materials, these functional groups are mostly present at the edges of the carbon material lamellae.In GrO doping, two types of deposition or anchoring of the silver particles can occur due to the carboxylic acid, epoxy, and hydroxyl groups and may be involved in the nucleation process, offering sites for the anchoring and growth of the metallic particles in the GrO.(1) Silver particles can be on the GrO surface modifying the distance between the GrO sheets.(2) Ag + ions interact with the carboxylic groups at the edges of GrO, and when this deposition occurs, it makes bacterial cells more susceptible to interacting with silver particles [64].Table 2 summarizes the XPS binding energies, FWHM, and the chemical percent of each high-resolution regions present in the GrO, GrO-0.5Ag, and GrO-1.5Agcomposites.
The O 1 s spectrum of GrO (figure 8(c)) shows one broad band associated with the carbon-oxygen functional groups C-OH, C=O and O=C-OH, respectively [65].Figures 8(e) and (g) show the O 1 s highresolution spectra for the GrO-0.5Agand GrO-1.5Agcomposites, respectively.These spectra show two peaks at 530 eV and 531 eV, which can be attributed to surface lattice oxygen (O latt ) and surface adsorbed oxygen (O ads ), respectively [66,67].The maximum O ads /O latt ratio indicates the number of surface oxygen vacancies on the composite surface, which were 0.60 and 0.45 for the GrO-0.5Agand GrO-1.5Agcomposites, respectively.The decrease in the O ads /O latt ratio is attributed to the different in Ag°and/or Ag + and silver incorporated into the GrO matrix.Also, the increase in O latt species is related to oxygen vacancy sites that are generated by the release of lattice oxygen during the oxidation reaction and are filled by molecular oxygen, improving surface charge transport [68].
Finally, the high-resolution Ag 3d spectra for the GrO-0.5Agand GrO-1.5Agcomposites are shown in figure 9.In general, silver-doped graphite oxide composites showed Ag 3d spectra with two zones attributed to Ag (3d 5/2 ) and Ag (3d 3/2 ) and can be fitted by Ag°and Ag + curves [69].However, both Ag 3d 3/2 and Ag 3d 5/2 shifted slightly towards a higher energy level, suggesting that the interaction between the GrO and the silver particles led to a decrease in the electron density, probably due to the conjugation between the d-orbit of the silver atom and the π-bond of GrO [70].Also, the low intensity in the Ag 3d region is caused by the morphological change of the GrO-0.5Agand GrO-1.5Agcomposites that have undergone agglomeration as a function of silver acetate concentration.
GrO-0.5Ag and GrO-1.5Agcomposites showed 53.4% and 51.1% Ag + species associated with the Ag 2 O phase, confirming the XRD results.Ag and Ag 2 O phase coexistence is due to the absence of a reducing agent during silver-doped graphite oxide composites synthesis and the abundance of hydroxyl groups CO OH -(-) that have a binding affinity for Ag + ions.On the other hand, the CO OH -(-)groups facilitate the complexity of the silver ions in the molecular matrix [71].According to the literature, the high-resolution Ag 3d spectra in similar carbon-based materials show bonds of molecule-bridged graphene/Ag that are shifted at higher binding energies, implying Ag-S bond formation due to efficient interfacial interaction between graphene and cysteamine [72].

Scanning electron microscopy with energy dispersive x-ray spectroscopy (SEM-EDS)
Figures 10(a) and (b) show SEM micrographs of GrO at 10,000 and 5,000x magnification with a lower electron detector (LED), respectively.GrO morphology presented randomly aggregated nanosheets closely related to each other obtained by the Hummer method; this composite usually showed flake and stacked cloud-like structures [73].SEM micrographs of similar carbon-based materials show a reconstruction of the sp 2 network through the reduction of sp 3 oxygenated sites [74].oxidation process with aggregates generated by a chemical intercalation between each carbon stack caused by a strong π-π interaction.In our case, the doping process contributes to the oxidation degree of the silver-doped graphite oxide composites.Also, from EDS analysis GrO presented 19.7 wt% sulfur due to the partial remotion of the precursors used in the chemical oxidation of the material.The sulfur persisted after oxidation, doping, and heat treatments in all composites (also observed in XRD).
To improve the antimicrobial activity of carbon-based materials usually are doped with different metals, and the explanation of the mechanism is not entirely clear to the scientific community.In this way, various studies show that the synergy between carbon-based materials and metal doping considerably improves the final activity.However, the use of high composite concentrations does not always favour the inhibition of bacterial growth and is a function of other variables such as active metal dispersion, particle size, precursors used, and the release active metal ions [75].For example, SNP can interact with the bacterial cell membrane, simultaneously releasing biochemically active silver ions.These ions induce the formation of holes, which damage the cell membrane and cause leakage of cellular contents, obstructing vital transport processes, denaturing proteins, blocking DNA functions, and altering enzymatic processes [76].In this sense, the inhibitory mechanism in spherical metal nanoparticles is size-and concentration-dependent.The mechanism is related to the metal ions released and the ROS production that interacts with the cell membrane and produces severe cell damage.[77].Therefore, the silver acetate concentration in GrO modulated the GrO-Ag chemical interaction, silver dispersion, and number of active silver ions in the final composite.This conformation can influence the antimicrobial activity, and backscattered electron micrographs can aid in the visualization and discrimination of the disposition of silver ions in the GrO matrix.Hence, SEM micrographs with COMPO configuration for GrO-0.5Ag and GrO-1.5Agcomposites are shown in figures 10(c)-(d) and (e)-(f), respectively.
On the one hand, the SEM micrograph of the GrO-0.5Agcomposite with a 5 μm scale mark (figure 11(c)) showed the EDS analysis (figure 11(d)).
On the other hand, the SEM micrograph of the GrO-1.5Agcomposite with a 5 μm scale mark (figure 11(e)) showed the EDS analysis (figure 11(f)).The EDS analysis of GrO-0.5Ag and GrO-1.5Agcomposites showed 7.0 and 7.86 wt% S, respectively.The EDS analysis of GrO-0.5Ag and GrO-1.5Agcomposites showed 0.95 and 2.22 wt% silver, respectively.However, not all silver interacts with the GrO functional groups; some silver ions were dispersed in the GrO matrix.All the silver that does not interact with the functional groups of GrO is available in Ag°and Ag + form for its interaction with the cell walls of bacteria.It is known that high silver concentrations can change the intermolecular interactions between the GrO functional groups, and the interlayer adhesion forces [78].The silver that did not chemically interact with the GrO functional groups was oxidized, and this could limit the antimicrobial activity.Monovalent cations in microorganisms cause toxicity, which eventually affects the cellular metabolism of the cell.Several biological processes, including cell adhesion, intracellular and intercellular communication, protein folding, maturation, apoptosis, ionic transport, enzyme regulation, and lead release, are significantly altered by the ionic mechanism [79].During the oxidation and doping processes, the precursor salt directly increases particle agglomeration and chemical binding capacity.For example, in the synthesis of SNP using chitosan as a stabilizer, ascorbic acid as a reducing agent and AgNO 3 as a precursor.The SNP sizes obtained ranged between 400 and 420 nm depending on the concentration of the precursor salt [80].Furthermore, spherical SNP anchored on reduced graphene oxide sheets presented size distributions ranging from 108 to 126 nm, synthesized by a chemical reduction method [81].In our case, SEM micrographs show silver spherical particles with smooth surfaces, which can cause damage to the external cell wall of each microorganism [82].The particle size distribution of the GrO-0.5Agand GrO-1.5Agcomposites (figure 12) During the of ca.96 and 293 nm, respectively.The size of the metal particles plays an important role in their antibacterial activity.For example, small particles are more easily absorbed by bacterial cell membranes because low-particle sizes give microorganisms more space to interact.This accelerates the ROS generation that is responsible for the cytotoxic effect, which kills injured or microbial cells [83].However, spherical SNP with 16 to 23 nm particle diameters presented similar antibacterial activities, where the factor responsible for their activity was the high dispersion and uniformity of SNP on the surface of the aminopolyacrylonitrile nanofibrous [84].The antimicrobial activity of the silver-doped graphite oxide composites against S. aureus, E. coli, and T. terrea microorganisms can be described according to the type of adsorption between the bacteria and the composite.The adsorption types that can occur between antimicrobial materials and bacteria are electrostatic adsorption, chemical adsorption (covalent chemical bonds between the material and the cell wall), and physical adsorption (van der Waals forces or hydrophobic interaction) [85].In this sense, the S. aureus bacterium has a thick and dense cell wall composed mainly of peptidoglycan (net positive charge), in which silver-doped graphite oxide composites can interact with the cell wall through physical adsorption.GrO has a negative charge due to the carbon-oxygen functional groups present on the surface; this modifies the van der Waals forces between the composite and bacterial cell wall [86].Additionally, free silver particles can be attached to the cell wall surface through electrostatic adsorption due to the presence of phosphate groups in peptidoglycan, allowing electrostatic interaction with the positively charged silver particles.In this way, silver particles in the composites can deposit on the periphery of bacteria, affecting the integrity of the cell membrane [87].
Moreover, E. coli and T. terrea bacteria cell walls are made up of a peptidoglycan layer surrounded by an outer membrane of lipopolysaccharides (LPS) and proteins.This outer membrane is negatively charged due to the presence of LPS.Therefore, the silver particles can be adsorbed in the outer membrane of bacteria and create pores in the cell membrane that alter its integrity and eventually trigger cell death [88].In addition, free silver

Conclusions
Graphite oxide (GrO) and GrO doped with 0.5 wt% (GrO-0.5Ag)and 1.5 wt% silver acetate (GrO-1.5Ag)composites presented between 7 and 19 wt% S, associated with a partial sulfur remotion during the chemical oxidation.This impurity presented diffraction peaks in all composites associated with elemental sulfur segregated in the GrO matrix.GrO-0.5Ag and GrO-1.5Agcomposites presented a mixture of Ag and Ag 2 O crystalline phases.For both composites, silver impregnation reduced the -OH (B) domains in the FTIR bands with respect to the GrO bands.In this sense, the average I D /I G ratio was 5.5% higher for the GrO-0.5Agand GrO-1.5Agcomposites than for GrO, which is related to defects in the carbon material.After doping, C-OH and O-C=O functional groups were more susceptible to interacting with silver particles, while the GrO-1.5Agcomposite presented high O lattice species associated with silver incorporation into the GrO matrix and oxygen vacancy sites.This silver conformation generated a 293 nm particle size distribution and high-release silver particles reaching the minimum inhibitory concentration at 3 mg ml −1 per chromatographic plate.At similar concentrations, the GrO-1.5Agcomposite showed inhibition halos of 2.7-fold, 6.2-fold, and 6-fold higher than the values found for GrO against S. aureus, E. coli, and T. terrea, respectively.However, at 5.0 mg ml −1 per chromatographic plate, the GrO-0.5 and GrO-1.5 composites had similar antimicrobial activity against the three bacteria due to the threshold effect.Both composites achieved this effect by saturating the bacterial binding sites, affecting their growth.In general, silver acetate doping in the GrO matrix can modulate the silver particle dispersion, crystalline phase promotion, particle size distribution, release capacity of the silver particles, and antimicrobial activity.Therefore, high-silver-doped GrO composites improve the electrostatic interaction between the composite and the cell surface.On the interphase, the composites can interfere with the internal functions of the cell, break the cell membrane, and cause a loss of cellular integrity.Therefore, the GrO-1.5Agcomposite can be considered a viable alternative to inhibit the growth of Gram-positive and Gram-negative bacteria.

Figure 4 .
Figure 4. Retention factor (R f ) values as a function of the silver-doped graphite oxide composites concentration for the (a) Staphylococcus aureus, (b) Escherichia coli, and (c) Tatumella terrea microorganisms with the parameters obtained by linear regression.
Figure 11(a)  shows a SEM micrograph with a 5 μm scale mark, and figure 11(b) shows EDS analysis.The elemental analysis showed C/O ratios of 2.2, 2.0, and 2.3 for GrO, GrO-0.5Ag, and GrO-1.5Agcomposites, respectively.Values of C/O  2.0 are associated with a good

Table 1 .
Concentration and control used in the culture media per microorganism.

Table 2 .
XPS binding energies, full width at half maximum (FWHM), and chemical percent for high-resolution C 1 s, O 1 s, and Ag 3d regions for the graphite oxide (GrO), GrO-0.5Ag, and GrO-1.5Agcomposites.