Graphene oxide reinforced Ni–P coatings for bacterial adhesion inhibition

Abstract

Bacterial adhesion on the surfaces of medical devices, food processing equipment, heat exchangers and ship hulls has been recognized as a widespread problem. Bacterial adhesion on a surface is influenced by surface physical and chemical properties of the surface. In this paper, polyethyleneimine (PEI) grafted graphene oxide (GO) nanosheets were incorporated into Ni–P coatings by an electroless plating technique to modify the surface energy, and therefore to influence the interactions between the prepared surfaces and the tested bacteria. Ni–P–GO coatings were investigated by SEM, AFM, Raman spectrum and XPS. Contact angles of Ni–P–GO coatings with different GO loadings were tested and the surface free energies and their dispersive and polar components were calculated using the van Oss acid–base approach. The results showed that the surface free energies of the coatings had a significant influence on S. aureus adhesion. The Ni–P–GO coatings showed excellent antibacterial adhesion activity. Extended DLVO theory is used to explain the antibacterial adhesion behavior. The novel coatings reported here can be used in controlling bacterial adhesion and biofilm formation for various applications.

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Introduction

Bacteria and other microorganisms have a natural tendency to adhere to the surfaces of medical devices, food-processing equipment, heat exchangers and ship hulls as a survival mechanism.¹ Bacterial adhesion to the surfaces can cause various problems including infections on biomaterials and implanted medical devices, contamination of water resources and biofouling in food-processing equipment, disturbances of heat transfer processes and corrosion in metallic surfaces in many engineered and marine systems.² When bacteria attach to a surface, a multistep process starts leading to the formation of a complex, adhering microbial community that is termed a biofilm. Once a biofilm has formed, it is very difficult to treat clinically because the bacteria on the interior of the biofilm are protected from phagocytosis and antibiotics.³ Thereby, an alternative strategy is required to control biofilm formation. Since bacterial adhesion to surfaces is the essential step, modifications to surfaces are considered to inhibit initial bacterial adhesion. Bacterial adhesion to a material surface is influenced by many physicochemical factors of the surface including chemical composition, roughness, surface energy, water interfacial energy, surface charge and mechanical strength.^4,5 Surface free energy is regarded to be one of the most important parameters for predicting initial bacterial adhesion that lower surface energy materials have been shown to inhibit bacterial adhesion, and it is easier to detach bacteria from these surfaces because of weaker binding at the interface.^4,6 However, there are also a number of contrary findings. It also has been reported that higher surface energy materials have a smaller biofouling tendency.^4,7,8 Contrary to these results, Baier et al. (1980) reported that there is an optimum range of surface free energy (about 25 mN m⁻¹) at which bacterial adhesion is minimal, which can partially explain the above inconsistent conclusion on the effects of surface free energy on bacterial adhesion.^9,10

Electroless Ni–carbon coatings have been extensively applied in numerous fields because of their uniform deposition, excellent wear resistance, good corrosion resistance and electrical conductivity.^11–13 In order to extend the application of Ni–carbon coatings, novel coatings with highly antibacterial adhesion and antifouling performances have become the main requirement. Graphene, a well-known monolayer of carbon atoms that form dense honeycomb structures with higher specific surface area than other carbon materials, which is suitable for the formation a hydrophobic surface, has attracted great attention recently. Water contact angle of epitaxial graphene was reported to be 92° and CA of reduced graphene could reach 127°.¹⁴ Due to its unique properties including electronic, mechanical, and thermal properties, graphene has been extensively applied in many fields, such as transistors, transparent conductors, polymer reinforcement, bioengineering and biomaterials areas.^15–17 The excellent properties of graphene nanosheets are considered to enhance bacterial adhesion inhibition property of Ni–P coatings significantly. However, graphene nanosheets with large surface areas tend to agglomerates through strong π–π stacking and van der Waals interaction.¹⁸ Therefore, it is crucial to prevent the aggregation for graphene nanosheets. Graphene oxide (GO) nanosheet is a graphene derivative with abundant functional groups containing epoxy, hydroxyl and carboxyl groups which provide hydrophilic and reactive sites properties.¹⁹ But the stability and aggregation behaviors of GO nanosheets in aqueous solutions are pH dependent and GO nanosheets aggregate in acidic conditions.²⁰ Functionalization of GO is the ideal solution to disperse GO in acidic solutions. Polyethyleneimine (PEI) is a water-soluble polymer with amine groups in the molecular backbone, providing a positive charged structure in the acid solution. Due to its active amine groups, PEI can react with carboxyl or epoxy groups of GO.²¹ In general, GO compatibility is hypothesized to be governed by the type of functionalization and its oxidation state.²² Many previous reports have indicated the promise of PEI conjugated GO which can be applied for drug carriers, phototherapy, and orthopedic devices with significantly low cytotoxicity.^23–25

The objectives of the present research are to fabricate Ni–P–GO coatings and to gain insight on the influence of surface-energy components on bacterial adhesion. Surface free energy and the total interaction energies of S. aureus ATCC 6538 on Ni–P–GO coatings with different GO contents are measured and calculated. The result of bacterial adhesion was explained by the extended DLVO theory. This study may provide a novel approach to design and synthesize Ni based coatings to eliminate bacteria adhesion.

Experimental

Materials

N-Ethyl-N′-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC·HCl) and poly(ethylene imine) (PEI, MW = 70 000 g mol⁻¹) were purchased from Aladdin Chemical Co., China. A common Hummers method was used to synthesize GO nanosheets using graphite powder provided by XFNANO Materials Tech Co., Ltd. The other chemicals used in the tests were purchased from Sinopharm Chemical Reagent Co., Ltd. All reagents were of analytical grade and used as received without further purifying.

Preparation of PEI grafted GO nanosheets

In this study, 0.08 g GO nanosheets were added into 40 mL de-ionized water and treated by ultrasonication at supersonic power of 500 W for 5 min under ice-water bath. 0.8 g PEI was added into GO suspension and heated to 60 °C. Then EDC·HCl was added to reach a concentration of 20 mM and kept for 24 h with continuous stirring. The obtained PEI grafted GO nanosheets was centrifugated at 10 000 rpm for 10 min and repeated washing for three times using de-ionized water. The resulting suspension was further dialyzed in a dialysis bag (MWCO: 2000 Da) for 3 days to remove free PEI.

Preparation of Ni–P–GO coatings

Ni–P–GO coatings were prepared on 20 mm × 20 mm × 0.5 mm copper plates by electroless plating method. The copper plates were firstly cleaned in an alkaline solution at 90 °C for 2 min. The compositions of the alkaline solution include NaOH 40 g L⁻¹; Na₂CO₃ 30 g L⁻¹; Na₃PO₄ 15 g L⁻¹ and Na₂SiO₃ 5 g L⁻¹. Then they were dipped into 10% HCl solution for 10 s. The plates were rinsed with de-ionized water after each step. The compositions of electroless Ni–P–GO solution used in the present study are shown in ESI Table S1.† The prepared PEI grafted GO was added into the plating solution and the pH of the electroless plating solution was adjusted to 4.8 with 2% H₂SO₄. The final concentrations of PEI grafted GO in the plating solution were 0.1 g L⁻¹, 0.2 g L⁻¹, 0.3 g L⁻¹ and 0.4 g L⁻¹ (marked as Ni–P–GO_0.1, Ni–P–GO_0.2, Ni–P–GO_0.3 and Ni–P–GO_0.4, respectively). Then the plating solution was stirred for 1 h before use. As GO nanosheets were dispersed uniformly in the plating bath, no mechanical agitation was used during the plating process. The plating was performed at 88 °C for 1 h.

Characterization

Fourier-transform infrared (FTIR) spectra were recorded on a Spectrum Two Spectrometer (Perkin Elmer, USA) with the wavenumber range of 4000–400 cm⁻¹ at a resolution of 4 cm⁻¹ in order to characterize PEI grafted GO. A JSM-7600F Scanning Electron Microscope (SEM) equipped with energy dispersive X-ray spectrometer (EDS) operating at an accelerating voltage of 10–15 kV was used to investigate the surface morphologies and their distribution within the coatings. Meanwhile, Ni–P–GO_0.4 coating was immersed into 10% HNO₃ for 10 s to corrupt the surface in order to prove GO was successfully incorporated into Ni–P coatings. Atomic force microscope (AFM) (Bruker Dimension Icon, German) was used to investigate the surface topography of neat Ni–P and Ni–P–GO_0.4 coating. Raman spectra were recorded on a DXR Smart Raman spectrometer (Thermo Fisher, USA) with 532 nm laser excitation. X-ray photoelectron spectroscopy (XPS) measurements were carried out with Thermo Escalab 250Xi instrument (Thermo Fisher, USA) using Al Ka radiation (1486.6 eV, 150 W). The base pressure was less than 10⁻⁸ Torr. The background in XPS data was subtracted using Shirley method²⁶ and the XPS curve-fitting was performed using a Gaussian function at high resolution. Zeta (ζ) potentials of Ni–P–GO coatings were determined by SurPASS Instrument (Anton Paar). Specimens were studied inside the adjustable gap cell in contact with the electrolyte (1 mM KCl, pH = 7).

Antibacterial adhesion

The antibacterial adhesion activities of the coatings were investigated against S. aureus ATCC 6538. The strain was cultured in tryptone soya agar plates in an incubator overnight at 37 °C, respectively. A single colony was inoculated in 20 mL tryptone soya broth and grown statically overnight at 37 °C. Then, 100 μL of this bacterial suspension was transferred into 100 mL TSB in a conical flask and grown in a shaker incubator at 150 rpm at 37 °C until the bacteria grew to mid-exponential phase. The required volume of bacterial suspension was centrifuged at 5000 rpm for 5 min at −4 °C, washed twice with sterile de-ionized water, and then re-suspended in the 60 mL 100 mM PBS (pre-warmed to 37 °C) into a glass tank to make a bacterial solution with a concentration of 5 × 10⁷ CFU mL⁻¹.

In this investigation, Ni–P–GO coatings and Ni–P coating (the control) were sterilized by ultraviolet lamp for 30 min. Then the sterilized samples were put into the tank and then the tank was put in a shaker incubator at 37 °C under a gentle stirring at 20 rpm. After 1 h, the samples were taken out and dipped into a de-ionized water to remove any non-adsorbed bacteria. Then, the bacteria adhered to each sample was removed to sterile glass beakers containing 10 mL sterile de-ionized water by ultrasonication (KH3200B with 40 kHz, Hechuang Ultrasonic, China) for 5 min. 100 μL of the sonication suspension and 10⁻¹, 10⁻² and 10⁻³ dilutions were plated out on TSA plates and incubated overnight at 37 °C. The colonies were counted on the following day. The total number of bacteria in 100 μL of bacterial suspension was obtained for each concentration and hence the total number of bacteria as colony-forming units (CFU) cm⁻² attached to the tested sample as CFU was obtained. The experiments were carried out in triplicate to confirm reproducibility.

Contact angle and surface energy

Contact angles were measured using the sessile drop method with a JC2000D contact angle analyzer (Powereach, China). Eight to ten contact angle measurements were made on each sample for all probe liquids including distilled water and ethylene glycol (Aladdin), and diiodomethane (Aladdin). The surface tension components of the test liquids are shown in ESI Table S2.†³

All measurements were made at 25 °C. The contact angles of bacterial cells were measured by producing the bacteria lawns deposited on membrane filters with a pore diameter of 0.45 μm and applying negative pressure.³ Prior to contact angle measurement, the bacterial lawns were allowed to air-dry until a certain state, indicated by stable water contact angles.²⁷ The surface energies of the prepared samples were calculated using van Oss acid–base approach. The surface energy is seen as the sum of a Lifshitz–van der Waals apolar component γ^LW_i and a Lewis acid–base polar component γ^AB_i:

γ^TOT_i = γ^LW_i + γ^AB_i (1)

The acid–base polar component γ^AB_i can be further subdivided by using specific terms for an electron donor (γ⁻_i) and an electron acceptor (γ⁺_i) subcomponent:

The relation between the measured contact angle and the solid and liquid surface energy terms are described as:

In order to determine the surface energy components (γ^LW_S) and parameters γ⁺_S and γ⁻_S of a solid, the contact angles of at least three liquids with known surface tension components (γ^LW_L, γ⁺_L, γ⁻_L), two of which must be polar, have to be determined.²⁸

Results

FTIR characterization of PEI grafted GO nanosheets

FTIR measurements were carried out to investigate the interactions between PEI and GO in the PEI grafted GO (Fig. 1). In the case of GO, the broad and intense peak centered at 3416 cm⁻¹, which is related to the OH groups, and the strong peak at 1728 cm⁻¹ corresponds to the stretching vibrations of C=O carboxylic moieties.²⁹ The peak at 1621 cm⁻¹ is associated with the skeletal vibrations of aromatic C=C bond or intramolecular hydrogen bonds.³⁰ Other bands at 1365, 1215 and 1054 cm⁻¹ correspond to C–O–H deformation, C–H stretching (epoxy groups) and C–O stretching vibrations (alkoxy groups), respectively.³¹ Therefore, it confirms the existences of the abundance of hydroxyl groups and oxygenous groups on the surface of GO, which makes GO to be convenient for further modification with PEI.³² For PEI grafted GO, almost all the peak positions of the functional groups on GO still remained. In particular, the intensity of C=O carbonyl stretching (1736 cm⁻¹) decreased and hardly can be observed. Compared with the original GO, additional peaks at 2944 cm⁻¹, 2892 cm⁻¹ and 1633 cm⁻¹ emerged in the spectrum. The new peaks 2944 cm⁻¹ and 2892 cm⁻¹ corresponded to the symmetric and asymmetric stretching bands of CH₂ vibrations.³³ While the new peak at 1633 cm⁻¹ corresponded to the C=O stretching vibration of –NHCO– (amide I), which proves that –NH₂ groups on the PEI have already reacted with the –COOH groups on the surfaces of GO by forming –NHCO– groups.³⁴ The results prove the amidation reaction that PEI was successfully grafted onto the surface of GO nanosheets via amido bonds.

Fig. 1 FTIR analysis of GO and PEI grafted GO nanosheets.

Morphology

Fig. 2a–d shows the SEM morphologies of Ni–P coating and Ni–P–GO_0.4 coating. The microstructures between pure Ni–P coating and Ni–P–GO_0.4 coating are different significantly. The Ni–P coating has a rather regular structure with large grain size in the range of 2–5 μm (Fig. 2a). The Ni–P–GO_0.4 coating shows much smaller gain sizes (Fig. 2b and c), which is due to the addition of GO nanosheets disorders the regular crystal structure. It shows that Ni–P–GO_0.4 coating has a fairly uniform, continuous and compact morphology. In order to further prove the existence of GO nanosheets in the Ni–P–GO_0.4 coating, 10% HNO₃ was applied to corrupt the surface of Ni–P–GO_0.4 coating for 10 s and followed by rinsing with de-ionized water for 3 times. Fig. 2d displayed the SEM image of the corrupted surface and an almost-transparent single layer GO nanosheet can be observed. The EDS element maps of Ni–P–GO_0.4 coating were displayed in Fig. 2e and f, which showed that the C and N elements from the PEI grafted GO were distributed uniformly. Herein, the PEI grafted GO nanosheets were proven to distribute uniformly in the Ni–P–GO_0.4 coating. Surface topographies of pure Ni–P coating and Ni–P–GO_0.4 coating were also studied by AFM (Fig. S1†). The surface roughness of pure Ni–P coating and Ni–P–GO_0.4 coating was 41.8 nm and 29.5 nm, respectively. The incorporation of GO nanosheets clearly reduced the surface roughness of Ni–P coating, which is consistent with SEM result.

Fig. 2 SEM image of Ni–P coating (a), Ni–P–GO_0.4 coating (b) magnification of ×2000 and (c) magnification of ×10 000, Ni–P–GO_0.4 coating after being corrupted by 10% HNO₃ (d), C element map (e) and N element map (f) of Ni–P–GO_0.4 coating.

Raman spectroscopy

Raman spectroscopy is one of the most powerful and informative techniques to investigate disorder sp² carbon material. Raman spectra of GO nanosheets and Ni–P–GO_0.4 coating were shown in Fig. 3. Both GO (Fig. 3a) and GO–PEI in the Ni–P–GO_0.4 coating (Fig. 3b) exhibited two peaks. The prominent D peak at 1320 cm⁻¹ assigns to the breathing mode of κ-point phonons with A_1g symmetry.³⁵ And the G band at 1570 cm⁻¹ attributes to the tangential stretching mode of the E_2g phonon of the carbon sp² atoms.³⁶ Both D and G bands belong to the GO nanosheets and proves the existence of PEI grafted GO nanosheets in the Ni–P–GO_0.4 coating. The intensity ratio of D to G band (I_D/I_G) increases from 0.86 to 1.19 for GO after grafted with PEI. The increase may be related to an increase in the degree of disorder of GO matrix, in part due to amidation reaction between amine (PEI) and epoxy groups in GO.

Fig. 3 Raman spectra of GO nanosheets (a) and PEI grafted GO in the Ni–P–GO_0.4 coating (b).

XPS spectroscopy

XPS analysis was performed to investigate the chemical state of Ni–P–GO_0.4 coating. Fig. 4a shows the full survey of XPS spectra for Ni–P–GO_0.4 coating. It is clear that there is a peak for N confirming the presence of N element in the coating. The N 1s XPS spectrum was shown in Fig. 4b. The N 1s peak appearing near 400.3 eV, which indicates that a relative high N composition is realized by a PEI-grafting process.³⁷ The characteristic Ni peaks are observed at 855.9 and 873.5 eV, corresponding to Ni 2p_3/2 (main peak) and Ni 2p_1/2 (main peak) photoelectron line of nickel (Fig. 4c). The other two peaks that appeared at 861.2 and 881.0 eV which are attributed to the shake-up satellite peaks of Ni 2p_3/2 and Ni 2p_1/2 spin–orbit doublet.³⁸ There is a controversy on the peak positions of oxide/hydroxide state. Early study suggested the presence of Ni₂O₃, while, more recent work proposed that it may be assigned to Ni(OH)₂.³⁹

Fig. 4 XPS survey scans of Ni–P–GO_0.4 coating (a), high-resolution XPS spectra of N 1s (b) Ni 2p (c), P 2p (d), O 1s (e) and C 1s (f).

The high-resolution of P 2p spectrum was displayed in Fig. 4d. The coating revealed two chemical states of the phosphorus species, which are elemental phosphorus (P₀) and phosphite/phosphate (P⁺). The elemental phosphorus 2p_3/2 and 2p_1/2 core levels are observed at 129.1 eV and 129.9 eV, indicating that P is in the form of elemental state in the Ni–P–GO_0.4 coating. The phosphite/phosphate state of the phosphorus 2p core level present as a single broad peak which can be deconvoluted into two components with binding energy at 132.4 eV (2p_3/2) and 133.4 eV (2p_1/2), which are assigned to the presence of NaH₂PO₂.⁴⁰ The characteristic O 1s signature with a binding energy of 531.7 eV (Fig. 4e) can be attributed to the oxygen from Ni₂O₃ or Ni(OH)₂.⁴¹ The C 1s XPS spectra of Ni–P–GO_0.4 coating is shown in Fig. 4f. The C 1s core level XPS spectrum can be deconvoluted into four components with binding energies at 284.6 eV, 285.2 eV, 286.4 eV and 288.1 eV. The peaks at binding energies of 284.6 eV, 285.2 eV and 286.4 eV correspond to the graphitic sp²-hybridized carbon atoms (C=C/C–C in aromatic ring), the epoxy, amino groups (C–O–C/C–NH₂) and the hydroxyl (C–OH) group, respectively.^32,37 The peak emerged at a binding energy of 287.7 eV attributed to the peak of amide (O–C–NH) as a result of amidation reaction between amine (PEI) and carboxyl groups in GO.⁴² This demonstrates that PEI grafted GO has been successfully incorporated into Ni–P coating.

Contact angle and surface free energy

The contact angles and surface energy components of Ni–P–GO coating and S. aureus ATCC 6538 are given in Table 1. Obviously the addition of GO had an influence on surface energies, including Lifshitz–van der Waals component γ^LW, electron donor component γ⁻ and total surface energy γ^TOT. The water contact angle increases with the GO loading increasing, which means Ni–P–GO coating is becoming more hydrophobic. The surface free energy of Ni–P coating is quite high (35.06 mN m⁻¹), and with GO loadings increasing, the surface free energy of Ni–P composite coating decreased. For Ni–P–GO_0.4 coating, the surface free energy decreased to 26.11 mN m⁻¹.

Table 1 Contact angles and surface free energy components of Ni–P–GO composites and S. aureus ATCC 6538

Materials	Contact angle, θ (°)		Surface free energy (mN m^−1)
	H2O	CH2I2	C2H6O2	γ^LW	γ^+	γ^−	γ^TOT
Ni–P	81.6 ± 0.5	51.1 ± 0.4	58 ± 0.6	33.66	0.07	6.92	35.06
Ni–P–GO0.1	84.7 ± 0.5	56.9 ± 0.3	65.3 ± 0.7	30.36	0.01	7.22	30.86
Ni–P–GO0.2	88.1 ± 0.6	62.6 ± 0.6	73.4 ± 0.8	27.08	0.02	7.76	27.91
Ni–P–GO0.3	92.2 ± 0.7	67.9 ± 0.7	82.2 ± 0.8	24.05	0.20	8.12	26.63
Ni–P–GO0.4	98.5 ± 0.8	74.7 ± 0.7	95.7 ± 0.9	20.29	0.96	8.77	26.11
S. aureus ATCC 6538	40.6 ± 1.4	51.4 ± 1.2	65.3 ± 1.6	33.49	1.78	75.06	56.59

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Effect of surface free energy on bacterial adhesion

Antibacterial adhesion property of Ni–P–GO coatings was investigated by counting the numbers of bacteria colonies adhered to the Ni–P–GO coatings with different GO loadings at 37 °C after a contact time of 1 h, as shown in Fig. 5. The Ni–P–GO coatings performed much better than Ni–P coating in reducing bacterial attachment, and it was found that bacterial adhesion decreased with increasing GO content in the coatings. The Ni–P–GO_0.4 coatings reduced S. aureus attachment by 96.45% at 1 h compared with Ni–P coating, respectively. Table 1 indicated that there was a significant difference in the electron donor components γ⁻ between GO loaded Ni–P coatings. The γ⁻ values of Ni–P–GO coatings were much higher than that of Ni–P coating. Fig. 5a shows the effect of the electron donor component γ⁻ values on bacterial adhesion. The equation applied to for the curve fit was y = ax² + bx + c since it fit well to both the Baier curve and the experimental data in this investigation.⁴³ The correlation coefficient R² = 0.992 for S. aureus adhesion, which was displayed in Fig. 5a. The number of adhered bacteria declined with γ⁻ values increasing. The number of adhered bacteria to Ni–P coating with the lowest γ⁻ value was the highest and the number of bacteria attached to Ni–P–GO coating with higher γ⁻ value was the lower. S. aureus ATCC 6538 has a very high value of γ⁻ component (75.06 mN m⁻¹) and a low value of γ⁺ component (1.78 mN m⁻¹) (Table 1), and was negatively charged with the zeta potential of −6.28 mV (Table S3†). If a material is negatively charged, the material will be repellent to the bacteria. Chibowski et al.⁴⁴ investigated the changes in zeta potential and surface energy components of calcium carbonate due to exposure to a radiofrequency electric field. They observed that the zeta potential decreased with an increase in the electron donor component γ⁻ of the surface energy. The observed changes in zeta potential and surface free energy components were believed to result from changes in the surface charge of calcium carbonate.³ The larger the electron donor component γ⁻ of a surface, the more negatively charged the surface and the more repellent to bacteria. This may explain why bacterial adhesion decreased with increasing electron donor γ⁻ values of the coatings.

Fig. 5 Effects of electron donor components γ⁻ (a) and surface free energies γ^TOT (b) on S. aureus ATCC 6538 adhesion.

Fig. 5b shows that there was a strong correlation between total surface free energy γ^TOT and bacterial adhesion (R² = 0.999 for S. aureus adhesion). With the surface free energies γ^TOT decreasing, the number of adhered bacteria to Ni–P based coatings decreased. Baier gave a relationship between surface free energy and relative bacterial adhesion^9,43 that there exists an optimum value of the surface free energy (about 25 mN m⁻¹) for which bacterial adhesion is minimal. It can well explain the above consistent conclusion on the effects of surface free energy on bacterial adhesion clearly in this study. These results indicate that Ni–P–GO coatings have excellent antibacterial adhesion activities against S. aureus. Combining all beneficial qualities, make the prepared Ni–P–GO coatings excellent antibacterial materials that can be applied in many applications requiring antibacterial adhesion.

Discussion

The theory used to explain bacterial adhesion was the extended DLVO theory,⁵ which states that the total interaction energy ΔE^TOT between a bacterium and a solid surface immersed in an aqueous medium is the sum of the Lifshitz–van der Waals interactions ΔE^LW, the Lewis acid–base interactions ΔE^AB, electrical double layer ΔE^EL interactions and Brownian motion ΔE^BR.

The Lifshitz–van der Waals interaction energy is defined as:

where R is radius of bacterium, A is Hamaker constant and H is the separation distance between the bacterium and the surface. van Oss⁷ presented a very simple method for the calculation of Hamaker constant based on the surface energy of the interaction materials:

A_ii = 24πH₀²γ^LW_i (5)

where γ^LW_i is the Lifshitz–van der Waals apolar component of the surface energy and H₀ is the minimum equilibrium distance between the two interacting bodies. The Hamaker constant for interaction between bacterium 1 and a solid surface 2 in water 3 is given by:

Combining the above equations, the LW interaction energy can be expressed as:

In the equation, H₀ is 0.157 nm.⁴⁴ γ^LW₁ used in this study was for S. aureus ATCC 6538 and γ^LW₂ is for Ni–P based coating, which are given in Table 1, and γ^LW₃ is for water, which is 21.8 mJ m⁻² and given in ESI Table S2.† The radius of S. aureus can be calculated based on the diameter data in ESI Table S3.†

The electrostatic double-layer interaction ΔE^EL between bacterium 1 and a flat solid surface 2 can be described as:⁴⁵

In this equation, ζ₁ is zeta potential of S. aureus ATCC 6538, which can be found in ESI Table S3.† ζ₂ is zeta potential of solid materials, which is measured and listed in ESI Table S4.† ε and ε₀ are relative dielectric permittivity of water (78.55 for water at 25 °C) and the permittivity under vacuum (8.854 × 10⁻¹² C V⁻¹ m⁻¹) respectively, κ is Debye–Hǔckel length and also an estimation of the effective thickness of the electrical double layer (1/κ = 1.1 nm).

The Lewis acid–base interaction ΔE^AB between bacteria 1 and flat surface 2 in water 3 is given by:⁴⁵

where λ is the characteristic wavelength of the interaction between 0.2 and 1.0 nm. For pure water, λ = 0.2 nm.¹⁴ λ value was taken as 0.6 nm in this study.⁴³

ΔE^BR is given by:

ΔE^BR = 0.414 × 10⁻²⁰ J (11)

The total interaction energy ΔE^TOT between S. aureus ATCC 6538 and the different solid surfaces in water was calculated using the following equation:

ΔE^TOT = ΔE^LW + ΔE^AB + ΔE^EL + ΔE^BR (12)

Fig. 6 shows the effect of the separation distance H on the total interaction energies ΔE^TOT between S. aureus ATCC 6538 and investigated surfaces in 100 mM PBS. The bottom line is Ni–P coating that has the lowest interaction energy profiles, then followed by Ni–P–GO coatings with increasing GO loadings. The top line is Ni–P–GO_0.4 coating which has the highest interaction energy profile. Clearly when H is equal to around 8 nm, the total interaction energies ΔE^TOT between bacteria and the substrates are the minimum. In order to investigate the effect of total interaction energies ΔE^TOT on bacterial adhesion, the minimal total interaction energies ΔE^TOT when H that equals to 8 nm was adopted.

Fig. 6 Effects of the separation distance H on the total interaction energies ΔE^TOT between S. aureus ATCC 6538 and investigated surfaces.

Fig. 7 shows that there is very strong correlation between the total interaction energies ΔE^TOT and S. aureus ATCC 6538 adhesion (R² = 0.963). The number of bacteria attached to the surfaces decreases with total interaction energy ΔE^TOT increasing. According to the extended DLVO theory, if the total interaction energy ΔE^TOT is negative, adhesion is favorable.^29,45 If ΔE^TOT is positive, adhesion is unfavorable. Bacterial adhesion should decrease with ΔE^TOT increasing. The experimental results are in accordance with the extended DLVO theory.

Fig. 7 Effects of total interaction energies ΔE^TOT on S. aureus ATCC 6538 adhesion.

Conclusions

In summary, Ni–P–GO coatings are prepared by electroless plating method. PEI was successfully grafted onto GO and the grafted GO nanosheets can be dispersed in the acidic electroless plating solution uniformly. The coatings showed excellent bacterial adhesion inhibition rates as high as 96.45% against S. aureus, indicating effective bacterial adhesion inhibition ability. The extended DLVO theory is used to well explain the antibacterial adhesion behavior. This novel coatings reported here can be used in controlling bacterial adhesion and biofilm formation for different applications. Therefore, the calculation of the total interaction energies could be applied for quantitatively predicting bacterial adhesion.

Acknowledgements

The work was financially supported by the National Natural Science Foundation of China (51401109) and the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). The authors would like to acknowledge the Advanced Analysis & Testing Center of Nanjing Forestry University.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra04408e

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