[System]
You are a **scientific‐rigor auditor**. You will receive the parsed contents of a research paper. Your job is to identify **only** those errors or flaws that directly undermine the **scientific validity** of the paper’s methods, analyses, or conclusions.

Your sole focus is to identify flaws—such as errors in experimental design, data integrity, calculations, statistical inference, or reproducibility—that directly call into question the validity of a specific claim, paragraph, or the paper as a whole.

**Do not** report issues that are purely presentational, rhetorical, stylistic, or related to citation practices.

---

After you’ve done a **detailed walkthrough** of the paper, output exactly in this format—no extra keys or commentary:

```
<analysis>
{detailed walk‑through of how you checked each section/figure and why you flagged (or did not flag) any flaw}
</analysis>

<response>
{
  "has_error": <true|false>,
  "errors": [
    {
      "location": "Section 2.1",
      "description": "Claim that ‘all X are Y’ is contradicted by citation [5], which shows examples of non‑Y X."
    },
    {
      "location": "Figure 3",
      "description": "XAxis labeled ‘Time (s)’ but units appear to be milliseconds; scale bar mismatches caption."
    }
    // …more entries…
  ]
}
</response>
```

- **Do not** include any other keys or prose outside these two tagged blocks.
- **Do not** report stylistic or citation issues.
- Be as precise as possible about **where** (section ID or figure/table) and **what** the scientific flaw is.
- Each description for the errors must be rooted in a scientific rationale explaining why it is 'wrong' (not how it could be improved).

Begin your analysis now.

[User]
View the article online for updates and enhancements.

To cite this article: M. M. Abdelhamied et al 2024 ECS J. Solid State Sci. Technol. 13 063003

Usable inside a glove box. Full multi-user, multi-device control via LAN.

Plan, conduct and analyze experiments with EL-Software.

Suited for all 3-electrode, optical, dilatometry or force test cells from EL-CELL.

+49 40 79012-734

Sudip Bhowmick, Joy Mukherjee, Manorama Ghosal et al.

Reem Altuijri, M. M. Abdelhamied, A. Atta et al.

Each with a fully equipped Potentiostat, Galvanostat and EIS!

Sudip Bhowmick, Joy Mukherjee, Manorama Ghosal et al.

1. The last several decades have seen the emergence of polymer nanocomposites as a promising field for study and development because of their special qualities and extensive range of applications.¹ ² These materials provide a new class of materials with improved physical, thermal, electronic, and optical properties by combining nanoscale fillers, with long chains of repeating subunits, known as polymers.³ ⁴ Because of the addition of nanoscale fillers to polymers results in materials with enhanced stiffness, strength, and toughness.⁵ ⁶ This produces materials that are lighter and stronger at the same time, which makes them very desirable for a variety of applications.⁷ Polymer nanocomposites are perfect for usage in the electronics industry since they have improved thermal stabilities in addition to their improved mechanical qualities.⁸

2. Because of its special qualities and range of uses, PVA is widely utilized, and has become extremely popular across a number of sectors.⁹ PVA is easily manipulated to create films, fibers, and coatings, which makes it a great option for a variety of uses.¹⁰ PVA is a great material for textiles, concrete reinforcement, and packaging because it has good dimensional stability and can be used with other polymers.¹¹ PVA is also ideal for use in agriculture because of its outstanding transparency, strong mechanical strength and gas barrier qualities.¹² CuO nanofiller has been extensively studied for its potential as a catalyst in a variety of chemical processes, including reduction and oxidation.¹³ CuO nanofiller's distinctive structure and large surface area make it useful for uses in chemical synthesis and energy conversion.¹⁴ CuO is a superior option for optical, electrical, and sensing devices due to their low production cost and ideal electrochemical properties.¹⁵ ¹⁶

3. One of the main advantages of an ion beam irradiation is increase the interfacial contact of nanofillers and the polymer matrix.¹⁷ ¹⁸ Additionally, ion irradiation has been shown to be more successful than electrons in the field of particles beam approaches at altering a polymer surface. This is a result of the increased ionization cross-section of the energetic ions as compared to electron. The dispersion and interlocking of the nanofillers inside the polymer matrix can be efficiently promoted by the energetic ions.¹⁹ ²⁰ Ion beams irradiation can affect the nanocomposite’s electrical and optical characteristics in addition to its mechanical and thermal properties.²¹ Electrical conductivity qualities can be altered by changing the structure of the irradiated samples, which opens up new possibilities in industries like electronics, photonics, and sensing. The objective in this study was to present the PVA/CuO composite’s synthesis, characterization, and application procedures. The present investigation involved subjecting the composite PVA/CuO films to a range of argon ion beam. The resulting alterations in the composite’s structural characteristics were made evident using the XRD and FTIR methods. The dielectric properties of these exposed films were computed. The utilization of these bombarded PVA/CuO films can be applied in dielectric applications as indicated by these outcomes.

4. In this research, the casting solution manufacturing approach was used to mix polyvinyl alcohol (PVA) and copper oxide (CuO) to create the composite (PVA/CuO). X-ray diffraction analysis and Fourier transform infrared spectroscopy were applied to record the successful fabrications of the composites. Next, argon ion beams at fluencies of 2.5 × 10¹⁷, 5 × 10¹⁷, and 7.5 × 10¹⁷ ions·cm⁻² were used to irradiate the composites. In frequencies of 50 Hz to 6 MHz, the dielectric characteristics of PVA/CuO were modified by the ion irradiation. The dielectric constant was enhanced from 39 for unmodified PVA/CuO to 356 for the irradiated composite by 7.5 × 10¹⁷ ions·cm⁻², and the conductivity changed from 0.05 × 10⁻⁶ S cm⁻¹ to 2.9 × 10⁻⁶ S cm⁻¹. However, the potential barrier decreased from 0.24 eV for PVA/CuO to 0.21, 0.16, and 0.15 eV, respectively, for 2.5 × 10¹⁷, 5 × 10¹⁷, and 7.5 × 10¹⁷ ions·cm⁻², and the relaxation time decreased from 9.36 × 10⁻⁸ sec for PVA/CuO, to 6.58 × 10⁻⁸ sec for 7.5 × 10¹⁷ ions·cm⁻². The results indicate that the irradiated PVA/CuO nanocomposite can be used in a number of devices such as capacitors and batteries.

5. Manuscript submitted March 22, 2024; revised manuscript received May 10, 2024. Published June 4, 2024.

6. The used substances; CuO (99.5%) and PVA (MW 79000 g mol⁻¹), were given by Sigma-Aldrich company. The PVA fabricated by dissolving 0.8 gm of PVA for 80 ml in distilled water, with constant stirring to produce a homogenous solution.²² In order to create PVA/CuO films, a solution containing the PVA was mixed with 2.0 weight percent CuO powder utilizing the solution casting process previously discussed.²³ After that, the mixture was constantly swirled for 120 min. To create the films, the PVA/CuO combination was cast into a Petri dish. To facilitate analysis, the films were removed from the Petri plates and cutted into 1.8 cm by 1.8 cm pieces. A thickness gauge (7301 dial thickness gage, Mitutoyo, Japan) was used to measure the produced films average thickness of 50 μm.

7. After removal from the Petri dishes, argon beams with fluencies of 2.5 × 10¹⁷, 5.0 × 10¹⁷, and 7.5 × 10¹⁷ ions cm⁻² were used to irradiate the produced PVA/CuO films using the ion source, as illustrated in Fig. 1. As we previously described.²⁴ The ionization region, where a plasma is created, and the extraction region, where ions are extracted and accelerated to produce a beam, are the two components of the source. The chamber is surrounded by permanent magnets, and a tiny aperture allows gas to enter. The energy of the extracted ions was 3 keV, the pressure was about 1.3 × 10⁻⁴ mbar, and the beam intensity was 145 μA cm⁻². The XRD (XRD-6000, Shimadzu, Japan) of Cu kα and λ = 1.54056 Å, was used to examine the structure of the PVA/CuO samples. The produced films’ chemical compositions were ascertained by The FTIR (ATI Mattson, England) was used to study the bonding interactions. Using the LCR meter with a frequency in range of 20 Hz to 5.5 MHz, the dielectric characteristics were determined.

8. ¹Radiation Physics Department, National Center for Radiation Research and Technology (NCRRT), Egyptian Atomic Energy Authority (EAEA), Cairo, Egypt

9. ²Department of Physics, College of Science, Princess Nourah bint Abdulrahman University, P.O. Box 84428, Riyadh 11671, Saudi Arabia

10. ³Physics Department, College of Science, Jouf University, Sakaka, Saudi Arabia

11. M. M. Abdelhamied,¹ Reem Altuijri,² A. Atta,³,z and Mohammed Ezzeldien³

12. © 2024 The Electrochemical Society (“ECS”). Published on behalf of ECS by IOP Publishing Limited. [DOI: 10.1149/2162-8777/ad4f71]

The SRIM simulating method²⁵ was utilized to record the ion beam interaction with the composite, as shown in Fig. 2. The energy used is of 3 keV argon ions interacted with the PVA/CuO at depths ranging from 0 to 1000 Å. As shown in Fig. 2a, the distribution of the argon ion beam forms a path for the bombarded ions in the composite PVA/CuO. Following the irradiation of the ion beam with the target, the penetrating argon ions distributed within a mean range of 5.6 nm. The atoms of carbon and hydrogen recoil when they come into contact with argon ions. Figure 2b illustrates the energy absorbed by the target atoms; this energy allowed the recoiled atoms to escape their locations within the structure and interact with other atoms. Figure 2c depicts the target damage caused by the collisions events of the argon ions colliding with the target.

The XRD of the pristine and bombarded PVA/CuO is displayed in Fig. 3 in the 2θ range of 4°–88°. The PVA/CuO formation is shown by peak of 2θ at 19.6° for PVA (001) diffraction and an additional peak at 39° for CuO. Moreover, it is noted that scission and radical production resulted in a drop in the intensity of the PVA with irradiation. Additionally, after exposed to the ion beam, a slight shifting of the principal peaks occurred, which was brought by the formation of crystal structure defects. According to,^26 the crystallite size (D) is given by:

D = 0.94λ / (β cosθ)   [1]

vacancies. The target material was expected to develop interface or near-surface defects when exposed to ion beams. Ion bombardment can alter targets due to certain defects, such as vacancies and interstitial atoms.

Further, the diameter (R) was computed by Eq. 2.^27

R = λ / (sinβ cos2θ)   [2]

After irradiating PVA/CuO by 7.5 × 10^17 ions cm⁻², the D and R dropped from 17.4 nm and 230 μm for the PVA/CuO film to 13.1 nm and 174 μm, correspondingly. The dislocation density (δ) is calculated by:^27

δ = 1 / D²   [3]

The dislocation density improved from 3.11 × 10⁻³ nm⁻² for PVA/CuO to 5.82 × 10⁻³ nm⁻² for 7.5 × 10^17 ions cm⁻², as listed in Table I. By using the Stokes-Wilson equalization, the lattice strain (ε) was calculated by Eq. 4.^28

ε = β / (4 tanθ)   [4]

The strain ε grew from 0.075 × 10⁻³ for the PVA/CuO film to 0.10 × 10⁻³ for the 7.5 × 10^17 ions cm⁻² bombarded composites. Lastly, the distortion coefficients (g) of the pristine and bombarded sheets were calculated by Eq. 5.^28

g = β / tan(θ)   [5]

Following radiation exposure by 7.5 × 10^17 ions cm⁻², the g of the PVA/CuO film rose from 11.1% to 16.3%, confirming the disordered network in the irradiated composites.

The FT-IR spectrum of PVA/CuO is shown in Fig. 4. The stretching band of CH₂ is for the peaks at 2920 cm⁻¹. The peaks for C=O appear at 1715 cm⁻¹, and the peak at 1255 cm⁻¹ is for C–H vibration. The asymmetric vibrations of C–O and C–C are associated with the peaks at 1092 and 827 cm⁻¹, correspondingly. The peak at nearly 3200 cm⁻¹ is for hydrogen bonds of the OH groups for the composite PVA/CuO. Due to the chemical linkages in the polymer nanocomposite, the intensity of this spectrum was reduced for the bombarded samples relative to that of the PVA/CuO. Another probable reason for the drop in peak intensity could be the production of free radicals, which induce electrons to hopping of valence to conduction states.^29

The irradiated composite showed changes in peak intensity, showing that ion irradiation affected the composite PVA/CuO bonding interaction. The figure shows that the broad peak (3100–3400 cm⁻¹) was moved by irradiation, indicating the bonding of the oxygen CuO and the OH group of PVA. The PVA/CuO sample showed just a minor shift in peak intensity and location when subjected to ion radiation. In addition, there was no noticeable change in the carbonyl group (C=O) at 1715 cm⁻¹, suggesting that the composite PVA/CuO films is stable under irradiation.

Figure 5a investigates the SEM images of the untreated composite PVA/CuO, and Figs. 5b–5d display the images of the irradiated samples. Figure 5a shows the microstructures of the PVA/CuO film as a smooth and regular surface. As shown in Figs. 5b–5d for irradiated composites with fluences of 2.5 × 10^17, 5 × 10^17, and

7.5 × 10¹⁷ ions.cm⁻², correspondingly, were found in close contact to each other. This is because shorter inter-particles distances were induced by irradiation, with more rough fracture surfaces. The irradiated films also showed no signs of surface cracking in the images, which is consistent with the results of the FTIR analyses, suggesting that the films were more stable after ion irradiation.

Analyzing the dielectric is a very sensitive way of finding relevant details concerning a composite's structure. The dielectric permittivity (ε*) is given by Eq. 6.³⁰

ε* = ε' − iε''       [6]

The real constant ε' can be estimated by Eq. 7.³⁰

ε' = (c·t)/(ε₀·A)     [7]

where A is the surface area, t is the thickness and c is the capacitance of the film. Figure 6 shows the ε' for both bombarded and virgin PVA/CuO, with respect to the frequency. At low frequencies, the ε' for all samples were decreasing rapidly with increasing frequency, and at high frequency (above 3 MHz), it approached to a constant. This could be due to the fact that the dipoles were not given a enough amount of time to orient themselves.³¹,³² According to Table II the ε' at frequency 50 Hz rose from 39 for pure PVA/CuO to 71 after exposed to 2.5 × 10¹⁷ ions.cm⁻² to 356 for 7.5 × 10¹⁷ ions.cm⁻². Due to the polarization and imperfections, the irradiated films’ dielectric constant were improved.³³,³⁴

The dielectric loss, ε'', is given by Eq. 8.³⁴

ε'' = ε' tanδ    [8]

Figure 7 shows the plots of ε'' against frequency of the pure and treated PVA/CuO composites. In particular, as the frequency rose, the ε'' declined, which is due to a decrease in the space charge polarization contributions.³⁵ In addition, faults resulting from chain scissioning caused a very substantial increase in ε'' by ion beam at various dosages of irradiation. As indicated in Table II, the ε'' rose from 19 for unirradiated PVA/CuO to 72 for 2.5 × 10¹⁷ ions.cm⁻² and 1042 for 7.5 × 10¹⁷ ions.cm⁻². The cause of this rise was due to the interfacial polarization with the ion irradiation at the nonconductor/conductor contacts.³⁶

The modulus (M*) is determined by:³⁷

\[ M^* = \frac{1}{\varepsilon^*} = M' + iM'' \] [9]

where M′ and M″ are the real and imaginary complex moduli, respectively, and ε* is the complex permittivity. The M′ and M″ were given by Eqs. 10 and 11.³⁸

\[ M' = \frac{\varepsilon'}{\varepsilon'^2 + \varepsilon''^2} \] [10]

\[ M'' = \frac{\varepsilon''}{\varepsilon'^2 + \varepsilon''^2} \] [11]

Figure 8 displays M′ with the frequency of the pristine and the bombarded PVA/CuO sheets by 2.5 × 10¹⁷, 5 × 10¹⁷, and 7.5 × 10¹⁷ ions.cm⁻² of argon beam. The M′ was very small in the low frequency zone, as shown in Fig. 8. The M′ rose with the frequency, reaching a maximum value and then decreased at greater frequencies.³⁹ This phenomenon occurs because electrical dipoles in polymers are more likely to align at low frequencies than at high frequencies.⁴⁰ According to Table II, for un-irradiated PVA/CuO, the M′ value at 50 Hz decreased from 20.1 × 10⁻³ to 7.2 × 10⁻³ for 2.5 × 10¹⁷ ions.cm⁻² and to 0.32 × 10⁻³ for 7.5 × 10¹⁷ ions.cm⁻². The modulus drops after treatment because the charge localization efficiency was enhanced by the dipolar contribution of charge carriers. Furthermore, this is because ion irradiation changes the composite structure and increases the defect density in the films that are exposed to irradiation.

Figure 9 illustrates how the M″ varies with frequency in both the irradiated and pure PVA/CuO films. The figure displays 2 peaks of M″ that indicates the occurrence of a relaxation behavior. The M″ is near to zero at the lower frequencies and by increasing the frequency, it enhances to reach a maximum value. The M″ decreased from 10.4 × 10⁻³ for un-irradiated PVA/CuO to 7.5 × 10⁻³ for 2.5 × 10¹⁷ ions.cm⁻² and to 0.88 × 10⁻³ for 7.5 × 10¹⁷ ions.cm⁻² at 50 Hz. The relaxation time (τₛ) is given by Eq. 12.⁴⁰

\[ \tau_s = \frac{1}{2\pi f_p} \] [12]

The chain relaxation period corresponding to the peak of frequency fₚ is denoted by τₛ. When PVA/CuO is exposed to radiation, its intensity peak shifts to a greater frequency, indicating a reduction in the relaxation time (τₛ). The relaxation time was 9.36 × 10⁻⁸ sec for PVA/CuO, decreased to 7.96 × 10⁻⁸ sec, 7.23 × 10⁻⁸ sec, and to 6.58 × 10⁻⁸ sec for 2.5 × 10¹⁷, 5 × 10¹⁷, and 7.5 × 10¹⁷ ions.cm⁻². The exposure to radiation reduced the relaxation time, which accounts for the drop in τₛ of modified PVA/CuO. Additionally, exposure lowers τₛ, due to the conductivity rising with greater mobility.⁴¹

given by Z". As shown in Fig. 10, the magnitudes of Z' for the films fell as the frequency increased and became nearly zero value at higher frequencies. As previously mentioned, as ion beam irradiation rises, the Z' continuously drop.

Figure 11 displays the variation in Z" of both the bombarded and unirradiated PVA/CuO nanocomposite sheets with respect to frequency. It is evident that the Z" exhibit the same decreasing tendency with frequency. Moreover, because the free carriers were increased, the Z' decrease by irradiation.⁴³ The CuO is responsible for the occurrence of dielectric relaxation peaks in the Z' and Z", of the pristine PVA/CuO and the irradiated 2.5 × 10¹⁷ ions·cm⁻², confirming that the composites were suitable for energy storage devices.⁴⁴ As previously mentioned, with increasing irradiation magnitude, the values of Z' and Z" fall because there are more free charge carriers. This is due to increased free charge availability, which improves conduction and maintains impedance.

The energy density (U) was estimated for practical applications by Eq. 14.⁴⁵

\[ U = \frac{1}{2} \varepsilon' \varepsilon_0 E^2 \] [14]

The U is changes with frequency, as given in Fig. 12. PVA/CuO exhibited an energy density of 1.77 × 10⁻⁴ J/m³ for the untreated sample as shown in Table II, which was modified to 3.16 × 10⁻⁴ J·m⁻³ at 2.5 × 10¹⁷ ions·cm⁻² and to 15.7 × 10⁻⁴ J·m⁻³ at 7.5 × 10¹⁷ ions·cm⁻². This is due to, when the composites were exposed to ion beam irradiation, the charges were transferred more quickly. The findings validate that ion treatment of PVA/CuO enhanced the dielectric characteristics of the energy storage.

The conductivity, σ_ac, is given by Eq. 15.⁴⁶

\[ \sigma_{ac} = 2\pi f \varepsilon_0 \varepsilon'' \] [15]

Figure 13 illustrates the change in σ_ac of both bombarded and pure PVA/CuO composite films with frequency. Interestingly, for every sample, the σ_ac rises with frequency; this is because the variations of the applied electric fields changes the charge carriers.⁴⁷,⁴⁸ To be more specific, the σ_ac at frequency 50 Hz increased from 0.05 × 10⁻⁶ S/cm for the PVA/CuO to 2.94 × 10⁻⁶ S·cm⁻¹ at 7.5 × 10¹⁷ ions·cm⁻². The chain scissioning, which increases the speed of ionic movement, was the reason for this rise in σ_ac due to rising ion beam impacts. Additionally, as the ion beam effects increase, the values of σ_ac conductivity is increased due to the induced free carriers by ion irradiation.

Furthermore, the complex impedance, Z*, is given by.⁴²

\[ Z^* = Z' + iZ'' \] [13]

The real impedance is represented by Z', which provides information on the resistive fraction, and the reactance portion is

The highest achievable potential barrier, W_M, was estimated by utilizing the subsequent Eq. 16.⁴⁹

1. The temperature is T and m is estimated by the sloping curves of Ln (ε'') against Ln (ω), which is illustrated in Fig. 14, according to the Eq. 17.⁵⁰

2. Wₘ = -4k_B T / m    [16]

3. ε'' = Aω^m    [17]

4. The derived Wₘ decreased from 0.24 eV for PVA/CuO, to 0.21 eV, 0.16 eV, and 0.15 eV, correspondingly for the 2.5 × 10¹⁷ ions.cm⁻², 5 × 10¹⁷ ions.cm⁻² and 7.5 × 10¹⁷ ions.cm⁻² samples. Therefore, the treatment of the films improved their electrical conductivity, as seen by the reduction in the Wₘ value.

5. The handmade ion beam source was applied to effectively irradiate the films. PVA/CuO composite sheet manufacturing was confirmed by XRD and FTIR. The XRD examined the amorphicity of the bombarded PVA/CuO, which rose with ion exposure as a result of variations in dislocation density and crystallite size. Exposure to various ion beam fluencies increased the conductivity. The impedance characteristics of the bombarded PVA/CuO film were revealed by the energy density, which increased from 1.77 × 10⁻⁴ J/m³ for the pure composite to 15.7 × 10⁻⁴ J/m³ for the irradiated samples. The relaxation time reduced from 9.36 × 10⁻⁸ sec for PVA/CuO to 7.96 × 10⁻⁸ sec, 7.23 × 10⁻⁸ sec, and to 6.58 × 10⁻⁸ sec for 2.5 × 10¹⁷ ions.cm⁻², 5 × 10¹⁷ ions.cm⁻² and 7.5 × 10¹⁷ ions.cm⁻². In contrast, the dielectric loss rose with increasing irradiation from 19 for PVA/CuO to 1042 for the irradiated film. The findings of this work open up new possibilities for diverse dielectric devices, such as microelectronic devices, batteries, and supercapacitors.

6. Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2024R399), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia.

7. M. M. Abdelhamied https://orcid.org/0009-0003-3439-1791

8. graphene oxide (PVA-CuO-GO) nanocomposites: synthesis, structural and optical properties." Sci. of Advan. Mat., 15, 412 (2023).

ECS Journal of Solid State Science and Technology, 2024 13 063003

28. Z. Zare-Akbari, H. Farhadnejad, B. Furughi-Nia, S. Abedin, M. Yadollahi, and M. Khorsand-Ghayeni, "PH-sensitive bionanocomposite hydrogel beads based on carboxymethyl cellulose/ZnO nanoparticle as drug carrier." Int. J. Biol. Macromol., 93, 1317 (2016).

29. M. T. Khorasani, A. Joorabloo, A. Moghaddam, H. Shamsi, and Z. MansooriMoghadam, "Incorporation of ZnO nanoparticles into heparinised polyvinyl alcohol/chitosan hydrogels for wound dressing application." Int. J. Biol. Macromol., 114, 1203 (2018).

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[System]
You are a **scientific‐rigor auditor**. You will receive the parsed contents of a research paper. Your job is to identify **only** those errors or flaws that directly undermine the **scientific validity** of the paper’s methods, analyses, or conclusions.

Your sole focus is to identify flaws—such as errors in experimental design, data integrity, calculations, statistical inference, or reproducibility—that directly call into question the validity of a specific claim, paragraph, or the paper as a whole.

**Do not** report issues that are purely presentational, rhetorical, stylistic, or related to citation practices.

---

After you’ve done a **detailed walkthrough** of the paper, output exactly in this format—no extra keys or commentary:

```
<analysis>
{detailed walk‑through of how you checked each section/figure and why you flagged (or did not flag) any flaw}
</analysis>

<response>
{
  "has_error": <true|false>,
  "errors": [
    {
      "location": "Section 2.1",
      "description": "Claim that ‘all X are Y’ is contradicted by citation [5], which shows examples of non‑Y X."
    },
    {
      "location": "Figure 3",
      "description": "XAxis labeled ‘Time (s)’ but units appear to be milliseconds; scale bar mismatches caption."
    }
    // …more entries…
  ]
}
</response>
```

- **Do not** include any other keys or prose outside these two tagged blocks.
- **Do not** report stylistic or citation issues.
- Be as precise as possible about **where** (section ID or figure/table) and **what** the scientific flaw is.
- Each description for the errors must be rooted in a scientific rationale explaining why it is 'wrong' (not how it could be improved).

Begin your analysis now.

[User]
Liu et al. Journal of Nanobiotechnology (2024) 22:746
https://doi.org/10.1186/s12951-024-03026-x

Journal of Nanobiotechnology

RESEARCH Open Access

A green, versatile, and facile strategy for anti-biofouling surface with ultra-high graft density polyethylene glycol

Wenjie Liu1,2, Suqin He1*, Hao Liu1, Zeyu Shou2,3, Kaiyuan Huo2, Hongping Xiang4, Aihan Feng4, Wei Lu5* and Na Li2*

Abstract

Implantable catheters are susceptible to severe complications due to non-specific protein adhesion on their surfaces. Polyethylene glycol (PEG) coatings, the gold standard for resistance to non-specific protein adhesion, present a challenge in achieving high-density grafting, which significantly restricts their use as anti-biofouling coatings. Herein, we exploited the strong interaction between polyphenols (PCs) and polycations (K6-PEG) to graft PEG onto the surface of PC-Cu (A network of metal polyphenols composed of proanthocyanidins and metal copper ions, with expectation for the coating with excellent resistance to non-specific protein adhesion (PC-Cu@K6-PEG). The introduction of K6-PEG resulted in enhanced stability and modulus of PC-Cu, as well as a reduction in the surface adhesion energy and contact angle of PC-Cu. In contrast to previously reported PEG coatings, PC-Cu@K6-PEG exhibited a markedly elevated grafting density of PEG (4.06 chains/nm²), which was more than double the highest value previously reported (1.9 chains/nm²), due to the diffusing ability of K6-PEG throughout the PC-Cu networks. PC-Cu@K6-PEG displays robust resistance to a variety of proteins, microbials, and platelet attachment, thereby preventing thrombosis. The coating ability of PC-Cu onto diverse substrates, combined with the simple, straightforward and environmentally benign process of fabricating PC-Cu@K6-PEG, suggests that this strategy has significant potential for use in anti-biofouling surfaces.

Keywords Non-specific adsorption, Anti-biofouling coating, Peptide, Polyethylene glycol (PEG), Metal-phenol networks (MPNs)

Introduction

In the medical field, the need for antifouling coatings is increasing as many devices require direct contact with human tissue, such as implants, catheters, sensors, due to the anti-biofouling coatings not only enhancing the protective performance of medical devices, but also significantly improving the clinical experience and safety and security of patients by improving biocompatibility [1]. For example, catheters as one of the most important medical product categories and currently used for a wide range of medical conditions, the incidence of bacterial infections and thrombosis is as high as 5–15% and 10–20%, respectively [2–4]. These can lead to serious catheter-related

*Correspondence:
Suqin He hesq@zzu.edu.cn
Wei Lu luwei@wmu.edu.cn
Na Li lina0701@ucas.ac.cn

Full list of author information is available at the end of the article

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consequences, such as sepsis and deep vein thrombosis [5]. The main cause is attributed to non-specific adhesion of proteins. Non-specific adhesion of proteins induces bacteria easily adhere to the surface of catheter materials, secreting extracellular matrix and multiply, and further forming a difficult-to-remove bacterial biofilm, leading to a variety of infections and inflammatory responses [6, 7]. When exposed to the bloodstream, non-specific protein adhesion results in platelet adhesion and activation, fibrin formation, and eventual thrombus formation through interaction with fibrinogen and thrombin, causing death of patient in some serious cases [8]. Over the past decades, the mainstay of systemic therapy has long relied on the systemic use of anticoagulants and antibiotics. However, overdosing of these drugs may be plagued by several problems, such as heparin-induced postoperative bleeding and hyperkalemia, and bacterial resistance caused by long-term antibiotic use [9]. The development of implantable devices with a high level of resistance to protein adsorption (anti-biofouling) is therefore of great importance for public health.

Engineering the surface of catheters that resist non-specific protein adsorption is considered an effective means. Various coating materials, such as polyethylene glycol (PEG), poly-2-oxazoline, poly(hydroxyethylmethacrylate) and poly[N-(2-hydroxypropyl) methacrylamide] have been coated on different substrate materials and shown to reduce the adsorption of non-specific proteins [10]. Among these, polyethylene glycol (PEG) is considered to be a most effective coating material due to its strong resistance to hydration and spatial location and has been widely used in the manufacture of anti-biofouling surface materials [11, 12]. There are generally two main categories, graft from (GF) and graft to (GT) [13, 14], in the preparation of anti-biofouling coatings. The GF method is a surface initiated (SI) polymerisation process, in which the polymer chains are grown in situ from initiator molecules attached onto the surface. The generation of polymer brushes on the material surface is usually achieved by controlled radical polymerisation such as atom transfer radical polymerisation (ATRP) and reversible addition chain transfer polymerisation (RAFT) [15]. For example, Nagasaki [16–18] et al. designed a series of PEGs capped with different groups (which contained N6 (pentaethylenehexamine), PAMA (poly[2-(N, N-dimethylamino) ethylmethacrylate]); -SH (thiols), etc.) to bind to gold surfaces via the groups capped at the end of the PEG chain, resulting in a nonspecific antiprotein adsorption effect on the gold surface sensor. Later, mussel-inspired bioadhesion techniques were widely used to adapt to different material surfaces. For example, Meagher [19] et al. generated poly(ethylene glycol methyl ether methacrylate) (MW∼475) (PEGMA) polymers in situ on various substrates by surface-initiated ATRP (SI-ATRP) and showed

good resistance to static fouling. In the GT process, pre-synthesised polymer chains from a melt or polymer solution are attached to either physically or chemically a surface through a process known as 'tethering'. For instance, Messersmith et al. [20, 21] employed 3,4-dihydroxyphenylalanine (DOPA), derived from mussel adhesion protein (MAP), to affix polyethylene glycol (PEG) to a titanium dioxide (TiO₂) substrate, thereby imparting the material with resistance to protein adsorption.

Previous studies have shown that the length and grafting density of the PEG chains on the surface have a close relationship with the resistance to protein adsorption. Among these, the graft density has been the focus of research because a high graft density can form a denser hydration layer and fill in the voids or the defects on the surface that the brushes do not cover. GT is simple and straightforward, but limiting the density of grafted PEGs by preventing further attachment when existing PEGs begin to adhere to the surface [22]. In contrast, GF is a bottom-up approach, which in principle makes it easier to achieve high-density grafted brush layers. However, due to the self-termination effect of the free radical reaction and the reduced reactive grafting activity caused by the spatial resistance of the PEG molecular brushes, the preparation of high-density grafted brush by GF is still a big challenge. The reported maximum grafting density of PEG was 0.79 chains/nm² and 1.9 chains/nm² for the GF and GT strategy, respectively [12, 23]. Besides, the GF method requires the maintenance of radical activity over a long period of time and harsh reaction conditions, which makes its industrial application challenging [24].

Both for GT and GF methods, the use of organic reagents is usually involved, which leads to concerns about biosafety. In addition, introducing anti-biofouling coatings to implant surfaces in a rational and effective manner is a major challenge due to the diversity of catheter materials. Therefore, a general, simple and effective green strategy for high-density surface polymer brush grafting is urgently needed.

Metal-polyphenol networks (MPNs) formed by the coordinative interaction between metal ions and phenolic ligands in polyphenol MPNs have received increasing attention due to their fast and simple deposition process on different shapes and a wide range of substrates (silicon, quartz, gold and polypropylene sheets, etc.), since the report by Caruso et al. [25]. It has been shown that MPNs have a certain degree of inherent anti-biofouling ability, attributed to a certain degree of hydrophilicity on their surfaces [26]. However, polyphenols are prone to non-specific adsorption of proteins and polysaccharides through multiple interactions, which can lead to unexpected biological events. In addition, the long-term stability of polyphenols is difficult to ensure due to their susceptibility to oxidation, which also limits the potential of MPNs for anti-biofouling applications.

There are strong and diverse interactions between polyphenols and polycations, including hydrogen bonding, hydrophobicity and electrostatic interactions, which can be used to separate polyphenols and create functional coatings. For example, Xu, F. et al. [27] describe the co-substitution of tannic acid with alkyl and quaternary ammonium groups to construct a cationic coating material (AQTA), which was then used to functionalise a multifunctional medical device for antimicrobial and haemostatic applications. In our group’s previous studies, functional peptides were introduced into polyphenol-based coatings via positively charged K6 (hexameric lysine) in order to achieve enhanced osseointegration of implants [28, 29]. Herein, by linking PEG to short positively charged peptide, hexameric lysine (K6), we fabricated an anti-biofouling coating on pre-constructed PC-Cu MPNs coating onto diverse substrates through a green process (Fig. 1a). Our results demonstrated that the introduction of polyethylene glycol (PEG) was not a classical adsorption process that occurs only on the surface, but a process of penetration into the whole PC-Cu MPNs coating, which leads to PEG layer with a markedly elevated grafting density (4.06 chains/nm²) and long-lasting anti-biofouling properties and resistance to the attachment of proteins, microorganisms and platelet, and avoidance of thrombosis (Fig. 1b).

Proanthocyanidins (PC, 95%), anhydrous copper chloride (CuCl₂, 98%), and tris(hydroxymethyl)aminomethane (Tris) (≥99%), sodium chloride (NaCl, 99.99%), Urea (99.99%), Tween 20, Sodium hydroxide (NaOH) (AR), ammonia (AR), dexamethasone and sodium β-glycerophosphate (β-GP, 13408-09-8) were purchased from Macklin. K6-PEG was ordered from Hangzhou Specific Peptide Biologicals Ltd. with purity >95%, MW=3095.37). All chemicals were used without any purification. FITC-labeled bovine serum proteins and lysozyme were synthesized by coupling FITC to the free ε-amino group of the proteins to form a thiourea bond, according to reported method [30]. Hemoglobin was purchased from Te Ai (Shanghai) Chemical Industry Development Co. Ltd, Mucins and Fibronectin were purchased from Shanghai Yuan Ye Biotechnology Co. Ltd, Escherichia coli (DH5α) and Staphylococcus aureus (SH1000). LIVE/DEAD BacLight Bacterial Viability Kit was purchased from ThermoFisher Co. Silicon wafers, titanium (Ti) plates, and circular glass coverslips (14 mm diameter, nested) were cleaned with piranha solution (70% H₂SO₄ and 30% H₂O₂, V/V) for 2 h at 98 ℃, then rinsed with MILI-Q water and dried in a gentle air stream before use.

Figure 1

Fig. 1 (a) Preparation process of the PC-Cu@K6-PEG coating; (b) Schematic representation of the PC-Cu@K6-PEG coating anti-biofouling (proteins; bacteria; platelets)

(Note: piranha solution is highly oxidizing and corrosive and should be prepared and used with extreme caution.) Experiments with deionized (DI) water purified using the MILI-Q system have a resistivity in excess of 18.25 MΩ·cm.

## Preparation of PC-Cu@K6-PEG coating

The components were assembled on clean, treated wafers (including PC-Cu MPNs substrates and K6-PEG surface layers). The silicon substrate was immersed in 500 µl of PC solution (concentration of 3 mM, dissolved in deionised water, pH 5.5). The pH was then adjusted to 5.5 with 0.1 M sodium hydroxide. Finally, 500 µl of Subsequently, a solution of CuCl₂ (concentration of 5 mM, dissolved in deionised water, without adjusting the pH) was added, and the silicon wafer was then shaken on the shaking table (at 800 rpm) for 30 s. Following this, the wafer was left to stand for 10 min. Following 30 s shaking period (at 200 rpm) and a 10-minute resting period, 500 µl of Tris-HCl buffer (100 mM, pH 8) was added and mixed for 30 s. The silicon wafer was then removed and the surface was rinsed slowly with deionised water, and then blown dry with nitrogen to obtain a layer of MPN. The aforementioned steps were repeated in order to obtain MPNs with varying numbers of layers per cycle, which were recorded as MPNn (n = 5, 10, 20) (The following defaults to n = 10 unless explicitly stated). For subsequent grafting of K6-PEG, the aforementioned procedure is typically repeated at least five times. Prior to commencing the subsequent step, the coatings were immersed in PC solution, removed and rinsed, and then immersed in K6-PEG (1 mg/mL) (dissolved in Tris-HCl (10 mM, pH 9)) and placed on a shaker (at 70 rpm) for 24 h. Subsequently, the coatings were removed and rinsed thoroughly with deionised water, and then blown dry with nitrogen gas to obtain the PC-Cu@K6-PEG coatings (All the above experimental procedures were carried out at room temperature). All the pH adjustments mentioned above were made using 1 mM salt. Specifically, 1 mM hydrochloric acid and 1 mM sodium hydroxide were used for the above pH adjustments. In order to meet the requirements of subsequent experiments, the coatings were also applied to glass substrates with a diameter of 14 mm and to gold substrates. The procedure was identical to that previously described.

## Thickness test

The ellipsometric spectroscopy model (SE) can accurately monitor the changes in polarization state of incident light waves at material interfaces upon reflection by measuring the polarizing angle (ψ) and phase difference (Δ), and also can calculate the refractive index, thickness, and quality of the information interface coating. The Woollam M2000UI ellipsometric spectrometer (J.A. Woollam Co.,

1. Inc., Lincoln, NE) was utilized, with WVASE32 serving as the analysis software, employing two incident angles, 65° and 70°. The values of ψ (polarization angle) and Δ (phase) were measured between 245 and 1000 nm. These two parameters (ψ and Δ) are related to the thickness and optical properties of the sample and can be calculated using the following equations:

2. \(\tan \psi e^{i\Delta} \equiv \frac{r_p}{r_s} \equiv \rho(\theta_0, h_1, \ldots, h_j, N_a, N_S, N_1, \ldots, N_j)\)

3. where \(r_p\) and \(r_s\) are the reflection coefficients parallel and perpendicular to the incident surface, respectively, \(\rho\) is the complex reflectance, \(\theta_0\) is the angle of incidence, \(h_j\) is the thickness of the layer, and \(N_a\), \(N_S\), and \(N_j\) are the complex refractive indices of the environment, substrate, and layer, respectively.

4. The assembled PC-Cu coatings and PC-Cu@K6-PEG coatings were characterized using X-ray photoelectron spectroscopy (ESCALAB 250) to obtain peak data under the conditions of a take-off angle of 90° and an energy of 20 eV. In this case, the PC-Cu@K6-PEG layer was etched twice, each time with a thickness of 15 nm and a cumulative etch depth of 30 nm.

5. The contact angle is the angle between the solid-liquid interface passing through the liquid interior and the gas-liquid interface. In research, the WCA is commonly used to measure the hydrophilicity of materials. This experiment used the static contact angle drop method using the KRUSS DSA1 1.80 version contact angle analyzer. Deionized water was used as the probing droplet to measure the WCA of the samples. Specifically, 5 μL of ultrapure water was precisely dropped onto the sample surface, and the moment of contact between the ultrapure water and the sample surface was captured using a high-speed camera. Then use OneAttension software to analyze the size of the WCA.

6. Surface mechanical properties, adhesion, and roughness of the coatings were measured using an atomic force microscope (AFM, Dimension Icon, Bruker). Firstly, the samples were placed on the AFM sample stage and securely fixed using vacuum adsorption. Subsequently, the surface of the coating samples was characterized using the tapping mode of MikroMash silicon cantilevers, and roughness was calculated using accompanying software. To calculate the Young's modulus, the Derjaguin-Muller-Toporov model was employed to fit the retraction curves of force versus separation obtained from the AFM measurements. The principle of adhesion energy testing involves applying appropriate force to the coating using an AFM probe. As the probe approaches the coating, it experiences attractive forces. Upon contact with the coating, these attractive forces transform into repulsive forces, and the measured force corresponds to the adhesion energy of the coating.

7. The stability of the hydrophilicity of the PC-Cu@K6-PEG coatings was evaluated by immersing the PC-Cu@K6-PEG coatings in PBS (pH 7.3) for 0–7 days or irradiating them continuously with UV for 24 h, and then measuring the WCA on the surface of the blown-dry samples by the static solid-drop method. The ability of the samples to withstand extreme environments was evaluated by immersing the PC-Cu MPNs coatings with the PC-Cu@K6-PEG coatings in pH = 2 and pH = 12 solutions, and measuring the thickness of the immersed thicknesses by optical ellipsometry (M-2000UI, J.A. Woollam).

8. The grafting density (δ) of K6-PEG on the PC-Cu surface was calculated using the following equation:

9. \(\delta = \frac{\rho S \Delta L N_A}{M S}\)

10. In this context, the symbol S denotes the grafted area. It is assumed that the density of K6-PEG, denoted by the symbol \(\rho\), is 1 g/cm³. The symbol \(\Delta L\) denotes the thickness of the K6-PEG contribution. The symbol \(N_A\) denotes Avogadro's constant. The symbol M denotes the molecular weight of K6-PEG. Finally, the symbol δ denotes the grafting density.

11. The antisorption process of the coating on the surface of the gold base against proteins was monitored using a quartz crystal microbalance (QCM-D) with a dissipation function. The gold-plated quartz crystal sensors were subjected to a cleaning process involving a solution of ammonia (25%), hydrogen peroxide (30%), and water (1:1:5) at a temperature of 75 °C for one hour. This was followed by a comprehensive rinsing with deionized water and drying under nitrogen. Subsequently, the gold-plated quartz crystal sensor was mounted in the flow chamber of the Q-Sense instrument and a phosphate-buffered saline (PBS) buffer solution was flowed through the chip until the system reached equilibrium. Subsequently, a variety of proteins (dissolved in PBS, pH = 7) were introduced into the system in a sequential manner, resulting in a discernible shift in the resonant frequency.

before reaching equilibrium once more. Finally, the PBS solution was circulated through the chip to remove any residual proteins that had not adhered to the surface. The injection solution was injected at a flow rate of 100 µL/min, and the change in resonance frequency (Δf) was monitored in real time at different harmonics (ν = 3, 5, 7, 9, etc.). The ν = 3 data were used for image plotting.

The ability to non-specifically passivate the adsorption of a wide range of proteins is key to achieving biofouling. We measured the magnetic changes of different proteins (bovine serum proteins, lysozyme, fibrinogen, mucin, and hemoglobin) during the flow over the surface of the chips coated with PC-Cu MPNs and PC-Cu@K6-PEG coatings, respectively using a quartz crystal microbalance with dissipation (QCM-D). In addition, the amounts of proteins adsorbed on the surface of the gold sensor was further obtained by Sauerbrey formula fitting. In order to visualize the effect, we incubated FITC-labeled bovine serum proteins as well as lysozyme with the materials at 37 °C for 24 h. After removal, we blocked the slices under light-avoidance conditions, captured the images with a laser confocal microscope, and semi-quantitatively analyzed the fluorescence using the software image J.

Antimicrobial adhesion activity of the surfaces was tested using Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria; firstly, E. coli and S. aureus epidermidis were incubated for 24 h on agar solids at 37 ℃, and then passaged twice to ensure monoclonality. Subsequently, a set of fresh colonies was selected and dispersed in LB medium and shaken overnight in a 37 ℃ incubator. The next day, 100 µl of the bacterial solution was aspirated in a 96 wells plate for counting, and then the bacteria were resuspended in a solution containing 0.2% liquid medium and 99.8% physiological saline (maintaining a bacterial concentration of 10^7 cfu/ml). Apply 500 µl of bacterial solution to the sample surface. After incubation at 37 ℃ for 8 h, the surface bacteria were slightly rinsed with saline and then fixed with 2.5% glutaraldehyde solution overnight. After further dehydration, the bacterial attachment to the sample surface was observed by scanning electron microscopy (SEM). Furthermore, following the 24 h incubation of the bacteria on the sample surface, the bacteria were removed by washing with saline. A bacterial live-dead dye was then added to the samples, which were incubated at 37 °C for 20 min. The samples were subsequently blocked in a closed environment and observed under a laser confocal microscope. The bacterial adherence on the coated surfaces was analysed fluorescently and semi-quantitatively using the software Image J.

Fresh whole blood from rabbits (purchased from Zhejiang Laboratory Animal Centre) was centrifuged at 4 °C for 15 min (1500 rpm) to obtain an erythrocyte suspension. The suspension was then washed with saline and centrifuged at 1500 rpm for 3 min. This process was repeated at least three times until the supernatant was clear. The collected erythrocyte suspension was then diluted to 4% by volume with saline. Place the sample in a 1.5 ml test tube. The diluted suspension (500 µL) and saline (500 µL) were added to each well and incubated with the samples at 37 °C for 1 h. Meanwhile, the cell suspensions of the negative and positive control groups were incubated with saline and 0.2% Triton solution without sample, respectively. The cell suspension was then centrifuged at 2000 rpm for 3 min to obtain the supernatant. Finally, 150 µL of supernatant from each group was transferred to a 96 wells plate and OD 540 was read using an enzyme-linked immunosorbent assay (Biotek, USA):

Hemolysis ratio (%) = (OD samples – OD negative) / (OD positive – OD negative)

Samples were placed in 12 wells plates and equilibrated with PBS overnight. All samples were then immersed in 2 mL of PRP (obtained by centrifuging fresh blood at 1500 rpm/min for 15 min) and incubated at 37 °C for 3 h. After incubation, samples were washed with PBS to remove nonadherent platelets and then fixed with 2.5% glutaraldehyde solution at 4 °C for 4 h. The samples were subjected to a series of grades of ethanol (25%, 50%, 75%, 95%, and 100%, v/v) at room temperature for 30 min per step and dried under vacuum. The morphology and number of platelets adhering to the surface of the samples were observed using field emission scanning electron microscopy.

Nine New Zealand White rabbits (purchased from the Zhejiang Animal Experiment Centre) were randomly divided into three groups (3 rabbits in each group) were anaesthetised with sodium pentobarbital at a concentration of 30 mg/ml and a dose of 1 ml/kg. The left carotid artery and right external jugular vein were surgically exposed and cannulated. PU catheters (unmodified, PC-Cu MPNs-modified and PC-Cu@K6-PEG-modified) were then attached at one end to the rabbit jugular vein and at the other end to the rabbit carotid artery to create a closed circulatory system. After 2 h of in vitro circulation, the unmodified, PC-Cu MPNs-modified and PC-Cu@K6-PEG-modified PU tubes were removed and rinsed three times with saline. The thrombus weight of

the tubes was determined by weighing the tubes before and after the experiment, and the catheter occlusion rate was quantified by calculating the percentage of lumen area occlusion using computerized image analysis. The rabbits were anaesthetized using gas and subsequently euthanized by cervical dislocation.

Statistical significance was tested by ANOVA, with p<0.05 considered significant: *p<0.05, **p<0.01, ***p<0.001. Error bars represent the standard deviation of the mean for each type of experiment, which was repeated at least three times for each experimental group.

The protocol for all animal implantation surgeries was approved by the Wenzhou Institute, University of Chinese Academy of Sciences, with ethics number WIUCAS24022602. All procedures were conducted by following the standards described in the guidelines for the care and use of laboratory animals. All procedures were conducted by following the standards described in the guidelines for the care and use of laboratory animals.

The PC-Cu MPNs coatings were prepared by self-assembly in the aqueous phase, as previously reported [31]. Subsequently, K6-PEG was deposited onto the MPNs coatings by immersing MPNs into K6-PEG solution

was measured. The WCA of various substrates with the PC-Cu MPNs coating reduced to 20–30°, and further significant reduction of the PC-Cu@K6-PEG coated surfaces were observed (<10° and even <5° for some substrates).

biomedical applications, the coatings were subjected to UV irradiation. The results demonstrated that there was no notable alteration in the WCA on the coating surface following 24 h of UV irradiation (Fig. 3g). Additionally, no considerable change in the WCA on the coating surface was observed after the coating was immersed in PBS for 7 days (Fig. 3h). These findings suggest that the coating retains its initial superhydrophilicity, which is conducive to long-lasting antifouling performance.

The fabrication of anti-biofouling polymer brushes represents the primary method of mitigating non-specific adhesion to medical device surfaces. The material composition and structural properties of polymer brushes are of significant relevance to the prevention of biofouling. One of the most crucial aspects is the grafting density of polymer brushes. It has been demonstrated

that a high graft density enhances the structural stability, hydration strength (a stronger hydration layer), and resistance to bio-contamination of polymer brushes [36]. A comparison of the grafting densities of the polymer brushes obtained from the aforementioned methods and the present work was illustrated in Fig. 4a [37–42]. The maximum grafting density reported in other documents was 0.79 chains/nm² for GF strategy and 1.9 chains/nm² for GT strategy. In this work, the calculated graft density of K6-PEG on PC-Cu MPNs was found to be 4.06 chains/nm². Given these observations, an investigation was undertaken into the adsorption mechanism of K6-PEG in MPN coatings with a view to elucidating the rationale behind the elevated graft density observed on the coating surface. By blocking one or more of these interactions, it is possible to compare the change in layer thickness of PC-Cu@K6-PEG with that of the MPNs control group, thereby enabling an investigation of the

interaction between K6-PEG and MPNs. As illustrated in Fig. 4b, the coatings were immersed in 100 mM Tween 20 (shielded from hydrophobic interaction), 100 mM NaCl (shielded from electrostatic interaction), and 100 mM urea (shielded from hydrogen bonding interaction), respectively. There was essentially no reduction in MPNs in the NaCl solution. In contrast, the desorption rate of PC-Cu@K6-PEG was 30%, suggesting that the deposition process is primarily electrostatically driven. This result was consistent with the dependent deposition of K6-PEG on pH, which was an indication on electrostatic interaction [31]. Furthermore, the deposited thickness of K6-PEG on MPNs is strongly relied on the immersing time (Fig. 4c). It showed very slow depositing process with time, there

was no plateau even after the tested 48 h. It was further found that the deposition process was also related to the thickness of the initial MPN. As illustrated in Fig. 4d, for the MPNs with different cycle numbers and thickness of 29.54 ± 1.10 nm, 65.53 ± 2.35 nm, and 119.71 ± 0.57 nm, the increased thicknesses were 9.34 ± 1.61 nm, 15.12 ± 2.09 nm, and 21.58 ± 1.19 nm, respectively, after soaking into K6-PEG solution at pH 9 for 24 h (Fig. 4d). The increased thicknesses attributed to K6-PEG deposition were obtained as 9.34 ± 1.61 nm, 15.12 ± 2.09 nm, and 21.58 ± 1.19 nm for the MPNs respectively. To better observe the contribution of precoated MPNs to the increased thickness, the increased thickness was normalized by the thickness of MPNs, and the data was showed

in (Fig. 4e). It’s found that the ratio of increased thickness to the thickness of precoated MPNs was very close to 0.25, meaning per 100 nm precoated MPNs contributed to a 25 nm increase of K6-PEG coating.

The elemental distribution of PC-Cu@K6-PEG at varying depths was subsequently examined through XPS etching. Specifically, the elemental distribution on the surface of PC-Cu@K6-PEG was initially analyzed by XPS. Subsequently, the layer was etched downward once (through a thickness of 15 nm) by XPS, maintaining the original position. Ultimately, the layer was etched downward once more, also through a thickness of 15 nm, by XPS. The results demonstrate that the surface of PC-Cu@K6-PEG films was comprised of 57.59% carbon (C), 8.15% nitrogen (N), and 33.72% oxygen (O) atoms, respectively. Following the initial 15 s of etching, a decrease in the percentage of detected N and O atoms was observed, while the percentage of detected C atoms increased. Following an additional 15 s of etching, the percentage of C, N, and O atoms in the film remained at 76.89%, 7.09%, and 15.20%, respectively (Fig. 4f). As the etching process continues, the proportions of C, N, and O atoms tend to stabilize, with the atomic proportion of the N element remaining at 6.77%. These results strongly indicated that the deposition of K6-PEG occurred not only on the surface but also within the MPN. The deposition process of K6-PEG is proposed in Fig. 4g. Initially, the free K6-PEG in solution is attracted to the MPN by electrostatic and hydrogen bonding interactions, resulting in the adsorption of K6-PEG on the MPN. The positive charge of K6 changed the zeta potential on the MPN surface, thereby creating an electrostatic potential energy difference (ΔE) between the top and bottom of the MPN. In the presence of this negative electric field, the positively charged K6 is further attracted deeper into the MPN until an even distribution of K6-PEG throughout the MPN layer is achieved.

Contact of the interventional catheter with blood results in the rapid adsorption of plasma proteins (especially fibrinogen) to the surface of the material, which then undergoes a conceptual transformation to form fibrin. The adsorption and denaturation of proteins can give rise to a number of complications, including acute thrombosis, acute inflammation, and a reduction or loss of the biological function of the surface. Consequently, it is of paramount importance to assess the adsorption properties of proteins on the surface of implant materials [43].

The non-fouling properties of hydrophilic materials are closely related to the hydration layer near the surface. This tightly bound water layer forms a physical and energetic barrier that prevents protein adsorption to the surface [44, 45]. In order to determine the dynamic passivation adsorption capacity of the coatings for different proteins, a series of tests were carried out using a quartz crystal microbalance (Q-CMD) to assess the adsorption of PC-Cu MPNs and PC-Cu@K6-PEG coatings for a range of proteins. Proteins with disparate physicochemical properties (including molecular weight (Mw) and isoelectric point (IP)) were selected for investigation (BSA has a molecular weight of 68 KD and IP 4.8; Lys has a molecular weight of 14–15 KD and IP 10.5–11.0; fibrinogen has a molecular weight of 330–340 KD and an IP 5.5; haemoglobin has a molecular weight of 67 KD and IP 4.5–5.0; and mucin has a molecular weight of 110 KD and IP 3.0–5.0). Specifically, 1 mg/ml BSA, Lys, fibrinogen, haemoglobin and mucin were dissolved in 1× PBS (pH=7.4), the gold-coated sensor was mounted in a Q-CMD chamber, the peristaltic pump speed was 90 rpm and the system temperature was set at 25℃. PBS was then passed through until the curves became smoother and then the PBS was changed to the protein solutions above. As illustrated in Fig. 5a–e, the time-dependent resonance frequencies on the gold sensor surface demonstrated that proteins were adsorbed by flowing on the material surface. In comparison to the PC-Cu MPNs group, the PC-Cu@K6-PEG coating group exhibited a significantly reduced change in frequency (6–9 times lower). In addition, the mass of proteins adsorbed on the surface of the gold sensor was calculated using the equation under the viscoelastic model, and the blank control group adsorbed the most amount of proteins on the surface up to 1,235.32 ng/cm², while the amount of proteins adsorbed on the PC-Cu@K6-PEG was significantly reduced down to a minimum of 4.09 ng/cm² (Fig. 5f), and the anti-adsorption efficiency against proteins was 99.67%. These results indicate that the PC-Cu@K6-PEG coating has good anti-adsorption properties for proteins with a wide spectral range.

Furthermore, in order to more effectively illustrate the coating’s capacity to resist protein adsorption, the samples were incubated with 1 mg/mL of fluorescein isothiocyanate-labeled bovine serum proteins (FITC-BSA) and lysozyme (FITC-Lys) for a period of 24 h. This allowed for a qualitative and semi-quantitative examination of the static adsorption behaviours of proteins on the surface of the materials. As shown in Fig. 5g, a large amount of fluorescent protein adsorbed on the blank glass slice (control), resulting in high surface fluorescence intensity. In contrast, there was slight decrease [in] fluorescent signal on the PC-Cu MPN coatings and almost no visible fluorescent signal on PC-Cu@K6-PEG coating. From the quantitative analysis in the mean fluorescence (Fig. 5h), the amount of fluorescence adsorbed on the surface of the PC-Cu@K6-PEG coating was significantly reduced, with a fluorescence intensity of 0.5 a.u., which was reduced by a factor of ~150 compared to that of the PC-Cu MPNs and by a factor of ~200 compared to the blank glass.

Previous studies have demonstrated that a significant proportion of bacteria contain a variety of protein appendages that play a direct or indirect role in adhesion [46], such as flagella, hyphae, and coils [47–49]. Bacterial adhesion is typically divided into three stages, commencing with reversible initial adhesion. This is followed by a transition to enhanced irreversible adhesion, and finally the development of a mature biofilm [50, 51]. Consequently, the inhibition of initial bacterial adhesion represents a pivotal strategy for the prevention of biofilm formation. The initial 8 h following bacterial contact with the surface is regarded as the “decisive period” for the prevention of bacterial adhesion. Consequently, the samples were incubated with Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) for 8 h in order to investigate their resistance to bacteria. Following an eight hours incubation period, the bacteria were stained with a LIVE/DEAD BacLight bacterial viability kit (Steps provided by supplier) and observed under a fluorescence microscope (Fig. 6a). It was observed that severe E. coli and S. aureus adhesion occurred on blank glass slice group (control) and the PC-Cu MPNs group. In contrast, only a few E. coli and S. aureus adhesion were observed on the surface of the PC-Cu@K6-PEG sample. A semi-quantitative fluorescence analysis (Fig. 6b) showed that the fluorescence intensity of the PC-Cu MPNs group was slightly reduced (by about two-fold) compared to

(control). Figure 5i illustrated the relative fluorescence intensities by normalizing the samples' intensity with the control's, in which a reduced relative fluorescence intensity on PC-Cu MPN coating and significant reduction on PC-Cu@K6-PEG coating was observed. These results suggested that the coating had a strong passivation effect on protein adsorption.

the blank glass slides, whereas the average fluorescence intensity of the PC-Cu@K6-PEG group was reduced by about 50 folds compared to that of the blank glass slides group, and by about 25 folds compared to that of the PC-Cu MPNs. To provide a more visual representation of the coating's resistance to bacterial adhesion, the incubated bacteria were dehydrated and cured, and then observed for bacterial adhesion on the sample surface by SEM. As illustrated in Fig. 6c, a considerable number of bacteria adhered to the wafer substrate (control group). In contrast, the number of bacteria on the surface of PC-Cu MPNs was relatively reduced, while a significant reduction in bacterial adherence was observed on the surface of PC-Cu@K6-PEG. Furthermore, the quantitative analysis results (Fig. 6d) demonstrated the number of bacterial adhesions was slightly reduced in the PC-Cu MPNs group compared to the control (by about twofold), whereas the number of bacterial adhesions on the PC-Cu@K6-PEG group was reduced by about 100 folds compared to the control group and by about 50 folds compared to the PC-Cu MPNs. The results were attributed to the strong hydrophilicity of PEG in PC-Cu@K6-PEG, which formed a tenacious hydration layer that could strongly repel bacteria when they approached the surface for adhesion [52]. Furthermore, Tanaka et al. [53] proposed the "intermediate water concept" to elucidate the water-mediated repulsion. This concept posits that the physical barrier of intermediate water plays a pivotal role in preventing the adsorption of proteins onto the substrate [50]. The dense intermediate hydration layer formed by the high density of PEG chains on the surface of the coatings successfully prevented the adhesion of bacteria and proteins on the surface. Together, these results demonstrate that the surface-modified PC-Cu@K6-PEG coatings are highly resistant to the adhesion of proteins and bacteria.

One of the most crucial criteria for the successful application of blood-contacting materials and devices is their blood compatibility. This is primarily assessed through in vitro analyses that examine the adhesion of blood

adhesion and aggregation with a value of less than 50/cm², and the reduction of platelet adhesion was about 99.84%. Normalised to the number of platelets adhering to the surface of the control samples in Fig. 7b, as shown in Fig. 7c, we can see that the number of platelets adhering to the surface of the PC-Cu MPNs coatings differs from that of the control group by only one star, while the PC-Cu@K6-PEG group shows a significantly smaller difference than the latter group. These data suggest PC-Cu@K6-PEG coating exhibited considerable antithrombotic potential.

In addition, in order to illustrate more the performance of PEG brushes with high graft density in anti-biofouling, the results of this study were compared with those of other studies (Table S2). It can be seen that in the GT’s strategy, the highest graft density PEG brushes achieved 98.93% anti-sorption of bovine serum proteins and 99% anti-adhesion of platelets. In the strategy of GF, the PEG brush with the highest graft density achieved 90% anti-sorption of human serum proteins and more than 99.5% antiadhesion to platelets. In the present study, antisorption of lysozyme, bovine serum proteins, fibrinogen, mucin, and hemoglobin were studied, with a maximum antisorption effect of 99.7% and an antimutagenic effect

of more than 99.8% on platelets (see Table S2 for more comparisons).

In order to further simulate clinical applications, in vitro catheter blood circulation experiments were conducted [60]. Rabbit arteriovenous shunt circuits were connected using commercially available medical polyurethane catheters with different surface treatments. (Fig. 8a) Following 2 h of ex vivo flow circulation without any systemic heparin anticoagulation, thrombus formation was found to be slightly reduced in the PC-Cu MPNs group in comparison with the catheter without coatings (Fig. 8b). Almost no obvious thrombus formation was observed in the PC-Cu@K6-PEG group. SEM analysis additionally demonstrated that the grafting of K6-PEG effectively inhibited thrombus formation (Fig. 8c). The blood-contact surfaces of bare PU and PU-MPNs tubes showed severe thrombosis with properties of densely interconnected polymerized fibrin networks, red blood cells, and activated platelets, in the PU-PC-Cu@K6-PEG group, only a very small number of haemocytes were seen on the surface (Fig. 8c). Quantitative analysis of these results further showed that thrombosis was significantly reduced in the PC-Cu@K6-PEG group compared to the other groups, as evidenced by the assessment of thrombus weight, occlusion rate (Fig. 8d-e). Specifically, PC-Cu@K6-PEG showed an approximately 30 folds and 21 folds reduction in mean clot weight compared with the PU tubing group and the PC-Cu MPNs group, respectively (Fig. 8d). Furthermore, the catheter occlusion rate was quantified by calculating the percentage of lumen area occlusion using computerised image analysis (Fig. 8e). The PC-Cu@K6-PEG modified catheters exhibited a significant reduction in thrombus occlusion rate compared to the PU tube group and PC-Cu MPNs group, with an occlusion rate of approximately 5%. These results clearly demonstrated that the K6-PEG modification imparts excellent anti-thrombotic properties to the material surface.

The two main factors involved in thrombosis on blood contact implants are fibrinogen (Fib) adsorption and activation and platelet adhesion and activation [61]. Preliminary experiments confirmed the anti-absorptive capacity of the K6-PEG-modified surface against a wide range of proteins (including Fib, hemoglobin, etc.), as well as its resistance to platelet adhesion, suggesting a potential anti-thrombotic capacity. This was subsequently confirmed by the results of in vitro blood circulation experiments in rabbits. Therefore, the PC-Cu@K6-PEG coating has good prospects for use in medical antithrombotic catheters.

In summary, the coating with strong resistance to nonspecific protein adsorption, PC-Cu@K6-PEG, was successfully engineered through direct depositing K6-PEG onto pre-constructed PC-Cu MPNs. The introduction of

K6-PEG into MPN has been demonstrated to improve both the stability and the mechanical properties of PC-Cu MPN, while simultaneously reducing the contact angle and adhesion energy of PC-Cu. K6-PEG is able to penetrate throughout PC-Cu MPNs, resulting in the formation of PC-Cu@K6-PEG with an ultrahigh PEG grafting density (4.06 chains/nm²). The deposition of K6-PEG to PC-Cu showed significantly reducing the adsorption of a variety of proteins (Lys; hemoglobin; Fib; mucoprotein; BSA) and bacteria (E. coli and S. aureus). Ex vivo blood circulation test demonstrated that PC-Cu@K6-PEG significantly reduced platelet adhesion on the surface of implanted catheters and prevented thrombus formation. It should be highlighted that the preparation of PC-Cu@K6-PEG coatings is a straightforward and environmentally friendly process that can be applied to a wide variety of substrates without any limitations in terms of material or shape, we believe this strategy offers a promising avenue for the development of anti-biofouling coating applications. In future studies, we envision that by changing the molecular weight of the PEG or by changing the structure of the PEG chain to obtain a defect-free surface of the PEG brushes, a complete anti-biofouling effect can be achieved.

Wenjie Liu: Experimental work, data Formal analysis, manuscript writing; Hao Liu: Experimental work, data Formal analysis; Zeyu Shou: Experimental work, data Formal analysis; Kaiyuan Huo, Hongping Xiang, Aihan Feng: data Formal analysis; Suqin He, Wei Lu: Conception and design; Na Li: Conception and design, data Formal analysis, manuscript writing.

Thanks for the Wenzhou Municipal Bureau of Science and Technology (Y20240088) and the Wenzhou Institute of the Chinese Academy of Sciences (WIUCASQD2021043) for funding this work. Thank you to the following contributors: Wenjie Liu: Experimental work, data Formal analysis, manuscript writing; Hao Liu: Experimental work, data Formal analysis; Zeyu Shou: Experimental work, data Formal analysis; Kaiyuan Huo, Hongping Xiang, Aihan Feng: data Formal analysis; Suqin He, Wei Lu: Conception and design; Na Li: Conception and design, data Formal analysis, manuscript writing.

The online version contains supplementary material available at https://doi.org/10.1186/s12951-024-03026-x.

No datasets were generated or analysed during the current study.

The authors declare no competing interests.

All authors declare full consent for publication.

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Supplementary Material

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1School of Materials Science and Engineering, Zhengzhou University, Zhengzhou 450001, Henan, China

Received: 27 September 2024 / Accepted: 19 November 2024
Published online: 02 December 2024

2Wenzhou Institute, University of Chinese Academy of Sciences, Wenzhou Key Laboratory of Perioperative Medicine, Wenzhou 325001, Zhejiang, China

5Department of Cardiovascular Surgery, The Quzhou Affiliated Hospital of Wenzhou Medical University, Quzhou People’s Hospital, Quzhou 32400, Zhejiang, China

3Department of Orthopedics, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, Zhejiang, China

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