Based on computational studies, Lin-T2, Lin-C2, and Lin-C1 are stable at all three temperatures. Lin-T2 had a low increase in free energy with rising temperature, while Lin-C2 and Lin-C1 had a free energy of -0.00 kcal/mol, Table 3. The MFE and Centroid structures of all three sequences confirm their stability at the three temperatures, (Fig. 1). According to EBPP diagrams, all sequences had high stability and equilibrium probability at various oligonucleotide concentrations (0.5, 1, 1.5, 2 and 15 µM) and salt concentrations [Na+ (1 M), Mg+ (0.0 M)]. Lin-C2 and Lin-C1 suggest a lack of base pair formation, while Lin-T2 showed a low pairing probability of A and T bases but remained stable, (Supplementary Fig. S 1). The free energy of the Lin-T2 sequence increased slightly with rising temperature but did not impact its stability. Its secondary structure and high equilibrium probability of nucleotides indicate its stability, (Supplementary Fig. S 2).
The absorption spectra of designed structures indicate the formation of AgNCs and oligonucleotide sequence conjugation. Figure 2a exhibits a decrease (hypochromic shift) in the absorption peak of BiAu after conjugation with Lin-T2. After using Lin-T1 to form AgNC (NCLin-T1), a new peak appeared at 440 nm in the absorption spectrum of NCLin-T1, (Fig. 2c). Conjugation between NCLin-T1 and BiAu is displayed in (Fig. 2b). Both spectra showed an increase in absorption (hyperchromic shift). Figure 2d presents the conjugation between Lin-T1 and BiAu. Lin-T1 spectrum showed the increase in absorption. BiAu spectrum showed the decrease in absorption. Figure 2 (e) shows the absorption spectrum of BiAu@NCLin-T2. BiAu and NCLin-T2 spectra showed an increase in absorption following conjugation. A new peak at 433 nm indicates the formation of AgNC after creating AgNC using the Lin-T2 scaffold, as shown in (Fig. 2f). In Fig. 2 (g), a new peak at 445 nm confirms the formation of NCLin-C1. The hypochromic shift of Lin-C2 spectrum (at 260 nm) and emerging of new peak (430 nm) of NCLin-C2 spectrum confirm the formation of NCLin-C2, (Fig. 2h). Figure 2i exhibits absorption spectra of NCLin-T2, NCLin-C2, and NCLin-C1. NCLin-T2 has the highest absorption intensity due to the addition of A and T to 12 C in Lin-T2.
BiAu@NCLin-T1, BiAu@Lin-T1, BiAu@NCLin-T2, and BiAu@Lin-T2 possess elevated conjugation efficiency and density of oligonucleotides and NCs on BiAu, Table 4. Lin-T1 and Lin-T2 were conjugated with BiAu using the affinity of A to AuNPs and the salt-aging method. A high concentration of oligonucleotides was utilized to elevate the conjugation efficiency. Studies have shown that oligonucleotides react with BSA [20], so the presence of BSA in a part of the BiAu increases the percentage of conjugation.
In the FT-IR spectra of BiAu and BiAu @LinT2, (Fig. 3), appeared amide I bands of BSA at 1638 cm − 1 [11, 21]. Stretching vibrations band of O-H at 3433 cm − 1 [22] and band at 1033.74 in BiAu shifted to 3434 and 1074.78 cm − 1 bands in BiAu@LinT2, respectively, which showed the conjugation of LinT2 to BiAu. The new peak appeared in the BiAu@LinT2 spectrum at 1270 cm − 1 assigned to C–O stretching vibration [23]. These new peaks can be assigned to the interaction of oligonucleotides with hybrid nanostructures.
The XRD pattern of BiAu hybrid nanostructures is presented in (Fig. 4). The diffraction pattern corresponds to the Au2Bi3 standard XRD card of JCPDS 21–0099 and the Au standard card of JCPDS 04-0784 with hexagonal and cubic structures, respectively.
NPs have spherical shapes with a limited distribution size (Fig. 5). The composition of BiAu was confirmed with the TEM image. Due to high electron density, AuNPs are darker than Bi2S3 NPs (bright) in one hybrid nanostructure [21].
Based on the EDX spectrum from BiAu (Fig. 6), the presence of Bi and S (BiAu spectrum) indicates the hybridization of AuNPs with Bi2S3. The existence of C, S, N, and O can be due to the BSA-mediated biomineralization of Bi2S3, (Fig. 6a). Two other elements, Ag and P, were also found in the spectrum of BiAu @NCLin-T2, which signify the formation of NCLin-T2 and its conjugation on the BiAu (Fig. 6b).
Figure 7 shows the confirmation of the formation of NCs and designed fluorophores through the observed Stokes shifts. The structures of NCLin-T2 and NCLin-C2, (Fig. 7a,b) have large Stokes shifts (116 nm (NCLin-T2) and 106 nm (NCLin-C2)). NCLin-T1 and Lin-T1, (Fig. 7f,e) exhibit Stokes shifts of 43.5 nm and 31.5 nm, respectively. BiAu@NCLin-T1 and BiAu@Lin-T1, (Fig. 7h,g) show slight Stokes shifts (14 nm and 11 nm, respectively). However, the emission spectrum and the Stokes shift were not observed in BiAu and BiAu@NCLin-T2, (Fig. 7d,c). The properties of DNA-AgNCs are influenced by the bases, sequences, and structures of DNA templates. Bases C and G interact strongly with Ag+ and are influential in forming DNA-Ag NCs. However, A and T have weaker interactions, and DNA templates with only A and T sequences have difficulty forming fluorescent AgNCs [24]. The addition of A and T to 12 C in Lin-T2 increases fluorescence intensity and Stokes shift [25]. The distance of less than ten nm (see the supplementary material, the level 3 heading “The length of Lin-T2”) between 5'HEX and AgNC in BiAu@NCLin-T1, leads to the FRET (FÖrster resonance energy transfer) phenomenon [26]. The emission spectrum of NCLin-T1 exhibited hypochromic shift following conjugation with BiAu (Supplementary Fig. S 3e). In BiAu@NCLin-T2, (Supplementary Fig. S 3g), the BiAu has resulted in the quenching of NCLin-T2 emission spectrum. The use of AuNPs in fluorescence quenching systems results in high-efficiency quenchers [27]. The distance of less than ten nm between BiAu and NCs in conjugated nanostructures leads to creation of FRET, hypochromic shift and quenching with BiAu. The nanostructures investigated in this section can be presented as FRET biosensors [26].
The size of BiAu is larger than its crystalline size due to the portion consisting of BSA that can be hydrated in water. BiAu@LinT2 has a higher hydrodynamic size, indicating conjugation with Lin-T2. Both BiAu and BiAu@Lin-T2 are polydisperse and have a negative surface charge within the − 10 and + 10 mV range, which is nearly neutral. Zeta potential can affect NPs permeability in cell membranes, and cationic particles may cause toxicity related to cell wall disruption [28]. BiAu@Lin-T2 has a more zeta potential than BiAu, indicating that Lin-T2 is conjugated to BiAu. The zeta potential of BiAu is neutral, reducing toxicity associated with the cell wall and membrane. As a result, BiAu can be a low-toxicity nanocarrier that can penetrate the cell wall and membrane (Fig. 8).
The study investigated the antibacterial effect of BiAu@NCLin-T1 as a nano photosensitizer (NPS) in PDT conditions compared to non-PDT states. Under PDT states, the antibacterial effect of BiAu@NCLin-T1 on E.coli increased in all concentrations, while a slight effect was observed in non-PDT conditions. BiAu@NCLin-T1 had the most pronounced PDT effect on E.coli at concentrations 1.5 µM, (Fig. 10a). BiAu@NCLin-T2 had a greater PDT effect on gram-negative bacteria than gram-positive, with considerable effects observed against E.coli at 1.5 and 2 µM, (Fig. 10c). Among all nanostructures, BiAu@Lin-T2 had the most considerable PDT effect against gram-positive strains at concentration of 2 µM, (Fig. 9d).
BiAu has several functions. It has negligible antibacterial activity under non-PDT conditions, making it suitable for delivering PSs to eukaryotic cells. Though it didn't show a substantial antibacterial effect in the gram-positive strain under PDT conditions, it demonstrated considerable impact in the gram-negative strain at two concentrations of 0.28 and 0.56 nM, (Supplementary Fig. S 5). BiAu also showed an increase in antibacterial activity at all 4 mentioned concentrations (0.28, 0.56, 0.85, and 1.13 nM) compared to non-PDT and PDT mode in the PTT test. Bi2S3-based nanomaterials exhibit poor toxicity. They are employed as a theranostic agent in X-ray computed tomography and near-infrared light-induced PTT [11].
According to (Supplementary Fig. S 7), both NCLin-T1 and NCLin-T2 have antibacterial properties against both gram-positive and gram-negative strains in all concentrations under PDT and non-PDT conditions. However, the antibacterial effect (in non-PDT states) is more pronounced in gram-positive strains than gram-negative strain. A study in 2016 found that AgNCs stabilized with various oligonucleotide sequences exhibited remarkable antimicrobial effects against both gram-positive and gram-negative bacterial strains at low concentrations (such as 0.75, 1.5, and 2.25 µM) [29]. Under photo stimulation conditions, NCLin-T1 has a heightened antibacterial effect against E.coli, at 1.5 µM concentration, (Supplementary Fig. S 7a). In S.aureus, the PDT-induced antibacterial effect of NCLin-T1 has increased at concentrations of 1, 1.5, and 2 µM, (Supplementary Fig. S 7b). AgNCs generate Ag+, leading to an enhanced aPDTeffect [30]. According to the findings of antibacterial outcomes, the original nanosystem (BiAu@NCLin-T1) had the most antibacterial impact in PDT conditions. The highest %BR of BiAu@NCLin-T1 was observed in E.coli and S.aureus at concentrations of 1.5 and 2 µM, respectively, (Supplementary Fig. S 20).
BiAu@NCLin-T1 and BiAu@Lin-T1 had similar Stokes shifts and lower fluorescence intensity than NCLin-T1, NCLin-T2, NCLin-C2, and Lin-T1. BiAu@Lin-T1 showed great aPDT activity against gram-negative strains compared to Lin-T1 at all four oligonucleotide concentrations. BiAu@NCLin-T1 demonstrated aPDT at 1 and 1.5 µM concentrations against the gram-negative strains, (Fig. 10a), as well as against the gram-positive strains at the concentration of 2 µM, (Fig. 10b). BiAu@NCLin-T2 also had aPDT against gram-negative strains at certain concentrations (Fig. 10c) despite quenching. Modifications in fluorescence intensity and quenching did not affect ROS production. In 2020, Paula Caregnato et al synthesized porous silicon NPs with magnetic properties that quenched visible luminescence but retained the ability to generate oxygen ions and superoxide radicals [31].
Based on SEM analysis, E.coli, and S. aureus, which weren't treated with NCLin-T2 are presented in (Fig. 13a,c), respectively, and possess healthy and intact surfaces. E.coli treated with NCLin-T2 (at the oligonucleotide concentration of 1.5 µM) demonstrated alterations under photo stimulation conditions after 22 hours. Indentation, widening, crooked, and curved structures, as well as cell lysis, are also evident in (Fig. 13b). S.aureus treated with NCLin-T2 (at the oligonucleotide concentration of 2 µM) under photo stimulation conditions after 22 hours is illustrated in (Fig. 13d). The observed damages include dilatation and cell lysis. NCLin-T2 has the %BR of %37 against E.coli at the concentration of 1.5 µM and under photo stimulation conditions. This structure exhibits the %BR of 45.86% against S. aureus at the concentration of 2 µM (under photo-stimulation conditions). The mentioned %BR from NCLin-T2 confirm the damage and morphological alterations on gram-negative and gram-positive strains.