JFNF: preparation and characterization
Preparation of the JFNF was done using previously published protocol . In short, locally collected jellyfish specimens (Rhopilema nomadica) were processed by mechanical cutting while the needed proteins collagen and Q-mucin were extracted by solvent precipitation and centrifugation. Next, JF proteins were dissolved and added to a PCL solution to prepare the ES solution used in a home-built electrospinning setup (Fig. 1A). Scaffold structure optimization was performed by varying the process parameters such as JF/PCL ratio, the voltage applied (13–17 kV), and tip-sample distance. Figure 1B shows the scaffold’s environmental scanning electron microscopy (ESEM), highlighting its fibrous nature. For additional analysis of the scaffold (See also Additional file 1: Figures S1 and S2).
In situ synthesis of AuNP and Bimetallic NPs
As previously shown, this scaffold can be used to reduce Ag ions into AgNPs . We attribute this phenomenon to the chemical reduction properties of Q-mucin glycoproteins’ presence on the fiber surface [30, 31]. Notably, as Silver’s electronegativity value is + 1.93 eV, it can be concluded that it is possible to synthesize other noble metal NPs that exhibit higher electronegativity values, including Au (+ 2.54 eV)  and bimetallic NPs.
Control over the synthesis products can be achieved via the pH of the reaction  because the latter affects the proteins’ ternary structure, which, in turn, determines the number of sites available for the reaction. To test this effect in our case, we synthesized AuNPs and bimetallic (Au–Ag) NPs complexes on the JFNF in pivotal acidic (pH 3) and alkaline (pH 9) environments. Figure 1C–F Shows Environmental Scanning Electron Microscopy (ESEM) images and corresponding X-ray diffraction (XRD) spectra of the scaffold. At pH 3, large spherical particles and hexagonal and triangular crystals were found (Fig. 1D, Additional file 1: Figure S3; Table S1), while small particles were generated at alkaline conditions. The phenomenon is attributed to the disulfide bonds protonation allowing the formation of a large area available for the synthesis at pH 3, resulting in large crystalline structures. In contrast, the disulfide bonds are deprotonated and tightly packed under alkaline conditions, limiting the volume and area of nucleation, resulting in small spherical AuNPs (Fig. 1E, Additional file 1: Figure S4; Table S1).
Next, we synthesized the bimetallic NPs. In this study, Ag and Au ions were added simultaneously to the scaffold in alkaline pH. Figure 1C depicts the ESEM images showing the synthesized particles. With the limitation of the characterization techniques, we hypothesize that the obtained structures are in the form of Au–Ag solid-solution as often obtained in the steady-state phase diagram . XRD analysis (Fig. 1D and Additional file 1: Figure S5) of the bimetallic NPs clearly indicates the formation of Au, Ag, and AgCl crystals.
Correlation between the chemical reducing substrate (JFNF) properties and the size and structures of NPs reveals that larger fibers facilitated the growth of larger spherical particles than smaller-diameter ones (Table S1). The effect of fiber diameter on triangular and hexagonal Au particles’ growth is inconclusive due to the diverse particle population obtained. No correlation between the content of the solution and particle size was found (Additional file 1: Figure S6). Changing the pH didn’t affect the morphological structure of the JFNF and they retained their original structure.
Photothermal properties of AuNPs and Ag-Au NPs
While it is evident that the pristine scaffold did not show PT characteristics, the NPs-doped scaffolds exhibited pronounced PT properties: AuNPs synthesized at pH 3 reached an elevated temperature of ~ 45 °C, while the ones synthesized at pH 9 reached a higher temperature of 80 °C. Notably, the bimetallic NPs produced at this pH showed a similar PT profile, indicating that these can function both as antibacterial and PT materials.
The time-dependent characteristics can be understood in light of thermogravimetric analysis (TGA, Additional file 1: Figure S8) of these materials, which shows a distinct melting point of the host scaffold around 60 °C, corresponding to the sharp increase in the PT profiles. This transition was not observed in AuNPs produced at pH 3 since the maximum heating temperature did not reach the melting point. The difference between the PT characteristics found in pH 3 and pH 9 can be attributed to the different size and dispersion of the particles produced in the syntheses (see also Additional file 1: Figures S3, S4; Table S1): It is known that light localization at hot spots between the dense population of particles, increases the amount of non-radiative processes which are transferred to heat . Therefore, heat is generated more efficiently at the small and dense population (pH 9) than at the large and scattered ones (pH 3).
Antibacterial and antibiofilm activity
The PT experiments allowed us to optimize and tune the conditions needed to be applied in the PT-induced antibacterial and antibiofilm experiments. In this respect, we choose irradiation times of ~ 60 s at moderate laser power (1 W/Cm2), which allows heating the scaffold to desired temperatures without damaging it.
To evaluate the scaffold’s antibacterial (“passive”) and PT-induced (“active”) properties, we performed disk diffusion antibacterial qualitative assays against gram-positive Bacillus subtilis bacteria known for their resistance to the harsh environment . Additional file 1: Figure S9 shows the results of the assay tested with and without laser irradiation. Clear evidence of antibacterial activity is shown in scaffolds with Au–Ag NPs. ESEM scan of the inhibition zone highlights the borderline between the treated area, which clearly became bacteria-free, and the untreated one (Fig. 3A).
Next, the antibiofilm properties of scaffolds with Au–Ag NPs were evaluated. In this case, due to the high resistance properties of the matured biofilms, we also applied PT treatment.
It is evident that upon irradiation (Fig. 3 and Additional file 1: Figure S9), mature biofilms located directly underneath the NP-decorated scaffolds were denatured. The corresponding ESEM images indicated that the produced thermal shock had caused massive damage to the bacteria’s membrane, causing membrane collapse (Fig. 3B, C). Due to that short lasing time, it can be concluded that the destruction of the biofilm takes place solely because of the high-temperature increment and not due to the passive antibacterial effect, which occurs at longer time scales. However, we have found that the irradiated bimetallic-based scaffolds kept the treated surface clean of biofilms for at least 24 h after removal in contrast to the AuNPs-based scaffold in which regrowth of colonies was found (Fig. 4 and Additional file 1: Figure S10). We attribute this observation to the diffusion of the antibacterial silver ions from the NPs to the surface, making this surface resistant to bacterial regrowth.
Laser irradiation applied to a reference sample composed of a pristine scaffold did not affect the bacterial colony. Interestingly, this type of scaffold exhibited strong adhesion to the biofilm, causing partial biofilm removal from the growth medium to the scaffold without the need for heating (Additional file 1: Figure S10).
Quantitative antibacterial growth inhibition assay
To check the validity of our methodology to different types of bacteria, we performed a quantitative antibacterial growth inhibition assay in aqueous media. This was done using three different bacterial strains: E. coli (Gram-negative) (Fig. 5A), S. epidermidis (Gram-positive) (Fig. 5B), and P. aeruginosa (Gram-negative, Fig. 5C), a pathogenic bacterium capable of forming biofilm layers, as a model bacterial strain.
The analyses indicate that the growth of all three tested bacterial strains was utterly inhibited by scaffolds decorated with Au–Ag NP and AuNPs (pH 3), and this, with and without application of laser. These observations suggest that the scaffold’s passive antibacterial activity exhibits high antibacterial properties, and laser irradiation is not required to suppress bacterial growth in these cases, as the difference between the two in each scaffold was not significant.
Surprisingly, despite their excellent PT properties, AuNPs (pH 9)-based scaffold showed no significant inhibition with or without the laser irradiation except in the case of S. epidermidis (Fig. 5B). This can be attributed to the slow diffusion of metal ions and short irradiation time, which was inefficient in damaging the bacteria in this particular case. In all cases, pristine fibers scaffold without the Np did not show any antibacterial effect with and without laser irradiation.
Quantitative antibiofilm inhibition assay
Finally, we performed quantitative analysis on mature P. aeruginosa BFs (Fig. 5D). It is evident that scaffolds that contain NPs successfully devastated the BF. The heat generated by the irradiated Au–Ag NP and AuNP (pH 3 & pH 9) was sufficient to completely denaturate the biofilm beneath the scaffolds even in a short irradiation time of 30 s by this “sanitizing” the solid growth medium. In this case, the passive antibacterial function of the scaffolds is not necessary for bacterial removal but is nevertheless very useful for the prevention of new contaminations as demonstrated in the qualitative assay which was performed on Bacillus subtilis biofilm (Fig. 3, Additional file 1: Figure S11).
Reference experiments performed on pristine JFNF scaffold (Fig. 5A–C “pristine scaffold”) showed no significant antibacterial activity, with or without laser treatment. In addition, direct irradiation by laser on the bacteria or biofilm did not show any inhibition growth or biofilm removal.