Bismuth-gold nanohybrid conjugated with a HEX-bound oligonucleotide; a novel nano photosensitizer to combat antimicrobial resistance

Bismuth-gold nanohybrid conjugated with a HEX-bound oligonucleotide; a novel nano photosensitizer to combat antimicrobial resistance | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Bismuth-gold nanohybrid conjugated with a HEX-bound oligonucleotide; a novel nano photosensitizer to combat antimicrobial resistance Atiyeh Nomani, Hamed Nosrati, Naser Faraji, Jalil Charmi, Siamak Javani This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4648298/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Sep, 2024 Read the published version in Scientific Reports → Version 1 posted 9 You are reading this latest preprint version Abstract Antimicrobial resistance (AMR) leads to a decrease in the adequacy of antimicrobial agents and an increase in the rate of adverse effects and mortality. The main objective of this project is to investigate the synergistic effect of BiAu@NCLin-T 1 and its substructures as an antimicrobial photodynamic therapy (aPDT) agent to combat microbial resistance. In addition, the effect of photothermal therapy (PTT) on some of the designed nanostructures at a temperature of 40°C was also tested. The antimicrobial test was carried out using the growth curve method against E. coli and S. aureus as Gram-negative and positive model bacteria. Computational methods were used to investigate the stability and entropy of oligonucleotide sequence structures. Various analyses were performed to identify the nanostructures, including Ultraviolet-visible (UV-vis) spectroscopy, Fourier-transform infrared spectroscopy (FTIR), dynamic light scattering (DLS), transmission electron microscopy (TEM), X-ray diffraction (XRD), scanning electron microscopy (SEM), energy-dispersive X-ray analysis (EDS) and fluorescence analysis. The BiAu@NCLin-T 1 appeared the significant aPDT impact against the gram-negative E.coli strain at two distinctive oligonucleotide concentrations (1, and 1.5 µM). Based on the results, the outlined nanostructures can act as a photosensitizer (PS), a photothermal treatment agent (PTT), and an antimicrobial agent to combat resistant bacteria. Biological sciences/Drug discovery Biological sciences/Microbiology antimicrobial drug resistance photosensitizing agents nanostructure phototherapy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Introduction Antimicrobial resistance (AMR) is a global public health threat that can hinder the control and treatment of infectious diseases [ 1 ]. It is related to wide mortality rates (around 700,000 deaths per year worldwide), and a remarkable impact on the effectiveness of antimicrobial agents [ 2 ]. Photodynamic therapy (PDT), as a new treatment method, is suggested to dominate AMR. PDT contains photosensitizers (PSs) and light to create reactive oxygen species (ROS) [ 3 , 4 ]. The currently approved PSs have poor water solubility, aggregation, low photostability, and phototoxicity issues [ 5 ]. Metal NPs can elevate the efficiency of PDT by increasing ROS generation and curing biocompatibility. LSPR (localized surface plasmon resonance), as a property of metal NPs, has been utilized to enhance the performance of aPDT by rectifying the efficiency of PSs. Gold nanoparticles (AuNPs) have exhibited LSPR, allowing them to absorb light at specific wavelengths and possess photoacoustic and photothermal properties [ 6 , 7 ]. DNA-AuNP conjugates are stable nanobioconjugates formed by linking single-strand DNA (ssDNA) to AuNPs [ 8 ]. Bismuth compounds have been used as medicines for gastrointestinal disorders and have recently been explored for viral infections, multidrug-resistant microbial infections, and cancer treatment [ 9 ]. Bismuth NPs exhibit long chemical stability, slight toxicity, controllable size and shape and simplicity of functionalization [ 10 ]. Nanomaterials predicated on Bi 2 S 3 exhibit negligible toxicity and have attracted widespread usage as a theranostic agent in X-ray computed tomography (CT) and near-infrared light-induced PTT [ 11 ]. Studies has indicated AgNPs have the antimicrobial activity through various molecular pathways, including the generation of ROS, disruption of biological membranes, and Trojan horse mechanism. AgNPs possess the capability to dominate AMR bacteria and counteract resistance mechanisms, such as preventing biofilm formation and regulating bacterial influx/efflux pumps [ 12 ]. Ag nanoclusters (AgNCs) are clusters of Ag atoms with a diameter of less than 2 nm, which demonstrate unique features such as well-defined structures and strong luminescence emission [ 13 ]. AgNCs can be synthesized using single-stranded nucleic acids as polydentate ligands, which provide biocompatibility and fluorescence properties [ 14 ]. A principal sight of planning oligonucleotide structures is to build structures that are thermodynamically stable and have the least energy state. Thermodynamic models are utilized to assess the equilibrium base-pairing probability of bases, intramolecular unfavorable interactions, the interaction of a nucleic acid chain with other chains, and the amount of free energy when the oligonucleotides are in the thermodynamic equilibrium [ 15 – 17 ]. Some of software such as ViennaRNA [ 16 ] and Nupack [ 17 ] are usually employed, for the thermodynamic examinations of oligonucleotide structures. The main current problem is the inefficiency of conventional PSs due to the mentioned disadvantages. Our hypothesis is designing a novel PS using AuNPs, AgNCs, and oligonucleotides to provide condition for better treatment of AMR. In the present work, Hex (Hexachlorofluorescein, the fluorescence dye) is employed as PS for aPDT of resistant bacteria. The aim of this project is to design a nanoplatform founded on the AgNCs and a hybrid NP, namely bismuth-gold (BiAu), to enhance the aPDT effect of Hex. This nano platform has been explored for its PDT (the wavelength of 520 nm) effect and as PSs on two model bacteria, namely E.coli and S.aureus , which are gram-negative and positive, respectively. BiAu hybrid NPs were functionalized with oligonucleotide sequences, namely Linker (LinT 1 − 2 ), and formed conjugated nanostructures named BiAu@Lin-T 1 and BiAu@Lin-T 2 . In Lin-T 1 , HEX has been linked to this sequence from the 5′ end. Lin-T 1 and Lin-T 2 are utilized as templates for AgNCs construction. Lin-T 1 and Lin-T 2 -mediated AgNCs conjugated with BiAu hybride nanostructures and created BiAu@NCLin-T 1 and BiAu@NCLin-T 2 , respectively. The PDT effect of nanostructures has been investigated in various concentrations. To explore the PTT effect, BiAu@NCLin-T 2 , BiAu, and Lin-T 2 have been selected. Material and methods Chemicals and instruments Bovine serum albumin (BSA), Tetrachloroauric (III) acid (HAuCl 4 ), Bismuth(III) nitrate (Bi(NO 3 ) 3 ), Sodium bromide (NaBr), Sodium hydroxide (NaOH), Silver nitrate (AgNO 3 ), Nitric acid (HNO 3 ), Sodium tetrahydridoborate (NaNH 4 ), Sodium citrate tribasic dihydrate (C 6 H 9 Na 3 O 9 ), Sodium dihydrogen phosphate (NaH 2 PO 4 ), Disodium hydrogen phosphate ((Na 2 HPO 4 ), Formaldehyde (CH 2 O), DeNovix DS-11 + UV-vis spectrophotometer, Perkin Elmer Spectrum RX1 FT-IR spectrophotometer, SZ-100z Dynamic Light Scattering & Zeta potential analyzer (company, Horiba Jobin Jyovin), TEM (Philips CM-120), XRD (Bruker D8 ADVANCE), SEM (AIS 2100 , Seron Technology, Korea), BioTek Cytation 3 Microplate Reader and EDS (TESCAN MIRA2). Synthesis of Bi 2 S 3 coated with BSA To synthesize Bi 2 S 3 , a biogenic mineralization approach is employed [ 11 ]. The synthesis process involves dissolving 250 mg of BSA in 8 mL of water. Then, while stirring vigorously, gradually add a solution of 50 mM Bi(NO 3 ) 3 in 1 mL of HNO 3 to the BSA solution. After stirring for 30 minutes, the pH is modified to a value of 12 by adding a solution of 2 M NaOH. The resulting mixture is then subjected to stirring. The mixture was left at room temperature (25° C) for 12 hours, allowing the formation of NPs of Bi 2 S 3 . The change in color from transparent to black confirms the NP formation. Then, the nanoparticles undergo purification through dialysis against water for 24–48 hours and are stored at a temperature of 4° C. Preparation of hybrid dimer BiAu BiAu is a type of metal-semiconductor heterodimer NPs that is obtained by growing AuNPs on the surface of Bi 2 S 3 [ 11 ]. We dissolved 30 mg of Bi 2 S 3 and 34 mg of HAuCl 4 in the 100 ml Milli-Q water at 140°C. Then, we dissolved 118 mg of C 6 H 9 Na 3 O 9 in 10 ml of Milli-Q water. Then, it was added to the gold solution very quickly and was held for 15 minutes at 140°C. After cooling to reach ambient temperature, excess salts were removed by filtration. UV-vis spectroscopy was used to determine the absorption and concentration of AuNPs. The final solution is purified using dialysis for 24 hours. The concentration of the AuNPs was estimated to be 10.6 nM by Lambert-Beers Law (the extinction coefficient of 13 nm Au NPs is 2.7×108 M − 1 cm − 1 at 525 nm). Characterization of synthesized NPs The formation of Bi 2 S 3 was determined by UV-vis spectroscopy. The identification of chemical structure was investigated by FTIR and EDX. The evaluation of colloidal stability and zeta potential was investigated by DLS. The morphology and size were determined by TEM. The structural characteristics were examined by XRD. Design and synthesis of oligonucleotides Four designed oligonucleotide sequences synthesized by Pishgam company with OD 260 : 8.00 are presented in Table 1 . These hybrid NPs are functionalized by two oligonucleotide sequences called Linker (LinT 1 − 2 ). Lin-C 1 and Lin-C 2 sequences were used as control sequences. Table 1 Designed sequences Name Sequence Bases number Lin-T 1 3-AAAAAAAAACCCCCCCCCCCCTTT-HEX-5 24 Lin-T 2 3-AAAAAAAAACCCCCCCCCCCCTTT-5 24 Lin-C 1 3-AAAAAAAAA-5 9 Lin-C 2 3-CCCCCCCCCCCCTTT-5 15 Validation of sequences by computational methods The step was performed for investigation of the stability and entropy of selected oligonucleotide sequences, Lin-T 2 , Lin-C 2 , and Lin-C 1 . Two factors, thermodynamic stability, and structural entropy, are examined using the Vienna RNA secondary structure server software ( http://rna.tbi.univie.ac.at/ ). This software can predict the secondary structure of two sequences using two methods: MFE (Minimum Free Energy) and Centroid structure [ 16 ]. The secondary structure of the mentioned oligonucleotide sequences will be examined at temperatures of 25, 37, and 40 ° C using the software. Moreover, the NUPACK software was utilized to examine the impact of varying concentrations on the stability of the oligonucleotide sequences. This software examines the thermodynamic equilibrium of oligonucleotides against environmental changes such as temperature and different concentrations based on thermodynamic parameters such as changes in free energy ( https://www.nupack.org/ ). The factors examined in the thermodynamic equilibrium state of nucleic acids are the equilibrium base-pairing probability (EBPP) and the probable equilibrium structures (Equilibrium structure probability) that nucleic acid molecules take in equilibrium state. The mentioned factors were investigated with this following manner, ensemble (all stacking) and salt concentrations [Na + (1 M), Mg + (0.0 M)] [ 17 ]. The selected sequences were subjected to different temperature and concentration conditions. At 25 ° C, the process of AgNCs synthesis (via the mentioned sequences) was performed. Additionally, the conjugation of Lin-T 2 with BiAu was performed at this temperature. The oligonucleotide concentrations in these two processes were 15µM. The antibacterial test was evaluated at four oligonucleotide concentrations of 0.5, 1, 1.5, and 2 µM. The test was conducted at a temperature of 37 ° C. A temperature of 40 ° C was also used for PTT test. Among three oligonucleotides, Lin-T 2 was placed at 40 ° C. Synthesis of oligonucleotides-templated AgNCs For AgNCs synthesizing, the concentration ratio of 1:6:6 was used for the oligonucleotide sequences, AgNO 3 , and NaBH 4 , respectively, with concentrations of 15 µM, 90 µM, and 90 µM. The resulting NCs were incubated overnight at room temperature and then washed with an Amicon Ultra-0.5ML filter to remove excess Ag + , NaBH 4 , and oligonucleotides. Fluorimetry analysis was also performed to characterize the NC formation. Conjugation of oligonucleotides and NCs with the BiAu To achieve this conjugation, the previously reported method was used with some modifications. The technique involves using the affinity of A bases to AuNP (existent in BiAu) and the salt-aging method [ 18 ]. The study involved conjugating Lin-T 1 , Lin-T 2 , NCLin-T 1 , and NCLin-T 2 with BiAu at varying concentrations. The mixture was vortexed and incubated for three days. NaBr was added gradually over 48 hours. The conjugation was confirmed by UV-vis spectroscopy, FT-IR, EDX, and DLS analysis. For further clarity, Table 2 , provides a description of designed nanostructures. Figures of nanostructures have been presented in (Supplementary material). Table 2 The description of designed nanostructures BiAu hybrid dimer (BSA- coated Bi 2 S 3 @ Au) NCLin-T 1 Lin-T 1 oligonucleotide- templated AgNCs NCLin-T 2 Lin-T 2 oligonucleotide- templated AgNCs BiAu@NCLin-T 1 NCLin-T 1 -BiAu conjugates BiAu@NCLin-T 2 NCLin-T 2 -BiAu conjugates BiAu@Lin-T 1 Lin-T 1 -BiAu conjugates BiAu@Lin-T 2 Lin-T 2 -BiAu conjugates NCLin-C 1 Lin-C 1 oligonucleotide- templated AgNCs NCLin-C 2 Lin-C 2 oligonucleotide- templated AgNCs Investigation of the antibacterial properties To investigate the antibacterial activity, E.coli (ATCC 43894) and S.aureus COL are employed as gram-negative and gram-positive models, respectively. The growth curve method will be utilized to conduct the antibacterial test. In this method, the bacteria will first be cultured in Laurie Bertani (LB) medium and then incubated at 37° C until they reach total growth. Then, the growth rate of bacteria is adjusted at OD: 0.1 inside the microplate. Then the bacteria are treated with four concentrations of 0.5, 1, 1.5, and 2 µM of oligonucleotides Lin 1 − 4 , nanostructures NCLin-T 1 , NCLin-T 2 , NCLin-C 2 , NCLin-C 1 , BiAu@Lin-T 1 , BiAu@Lin-T 2 , BiAu@NCLin-T 1 , and BiAu@NCLin-T 2 and four concentrations of 0.28, 0.56, 0.85 and 1.13 nM of BiAu. They are placed under photostimulation (PDT) and non-photo stimulation (non-PDT) conditions, followed by incubation at 37° C for 24 hours. The incident light was irradiated for 2 hours, using a light source of diod laser (with irradiation of 520 nm and 4.75 milli watts intensity). The OD of the bacteria will be measured at 600 nm after the end of the incubation period (22 hours). Additionally, to investigate the effect of PTT, nanostructures LinT 2 , BiAu, and BiAu@NCLin-T 2 were selected. The bacteria were treated with these compounds at 40° C for 2 hours. The bacteria will then be incubated at 37°C (non-PTT) and 40° C (PTT) for 24 hours inside an incubator, and their OD 600 will be measured after 22 hours. SEM analysis In this study, SEM analysis was performed on treated bacteria after 22 hours. To prepare the bacterial samples, fixation was performed. Fixation involves centrifugation of bacterial broth, and washing the resulting pellet with a phosphate buffer solution. Phosphate buffer and formaldehyde are added to the pellet to achieve the concentration of formaldehyde in the final volume equivalent to %2 (V/V). Then, dehydration was performed using different concentrations of ethanol (%30, %50, %70, % 80, % 90, and % 100). Then, a smear of the fixed bacterial sample was prepared on a slide for SEM analysis. Data analysis method The acquired data is presented using mean, standard deviation, percentage of reduction, and graph. Antibacterial test data analysis was done through the student’s t-test using Graph Pad Prism 9 software. The tests were performed with n value of 4 and significant differences were identified with * (P < 0.05), ** (P < 0.01), *** (P < 0.001), and ns (non-significant). Results Validation of sequences by computational tools Figure 1 displays details on the entropy, stability, and secondary structure of Lin-T 2 , Lin-C 2 , and Lin-C 1 oligonucleotides. Mountain plots are also presented at temperatures of 25, 37, and 40° C. The diagrams indicate the entropy of the nucleotide bases, with red indicating the lowest entropy and purple indicating the highest. Table 3 shows the thermodynamic free energy values of the sequences in kcal/mol, NUPACK and Vienna RNA results. Supplementary Fig. S 1 and S 2 show EBPP diagrams of Lin-T 2 , Lin-C 2 , and Lin-C 1 oligonucleotide sequences. Supplementary Fig. S 1 also possesses a histogram of the oligonucleotide concentrations of 0.5, 1, 1.5, 2, and 15 μM. The Equilibrium probability band, presented in both figures, displays the sequence's structural equilibrium. It highlights areas with more excellent stability, gradually increasing in probability from blue to brown. Supplementary Fig. S 2 presents the EBPP diagram (independent of concentration) featuring the oligonucleotides of Lin-T 2 , Lin-C 2 , and Lin-C 1 , along with the free energy of the complex at temperatures of 25, 37 °C (for all three sequences), and 40 °C (only for Lin-T 2 ). Table 3. The thermodynamic free energy of oligonucleotide at temperatures: 25, 37, and 40° C. Oligonucleotides The free energy of thermodynamic ensemble/ complex based on kcal/mol 25°C 37°C 40°C Lin-T 2 Vienna RNA -0.41 -0.15 -0.12 NUPACK -0.62 -0.42 -0.39 Lin-C 2 Vienna RNA -0.00 -0.00 --- NUPACK -0.00 -0.00 --- Lin-C 1 Vienna RNA -0.00 -0.00 --- NUPACK -0.00 -0.00 --- UV-visible spectroscopic analysis To confirm the formation of BiAu hybrid NPs, the conjugation of oligonucleotide sequences with BiAu, and the formation of AgNCs, the UV-visible absorption of the engineered nanostructures was investigated in the wavelength range of 220–700 nm. Figure 2 presents the absorption diagrams of the designed nanostructures along with the λ max of each one. The maximum absorption wavelength (λ max ) of the BiAu, (Lin-T 1 and BiAu@NCLin-T 1 ) and NCLin-T 1 are 535, 540 (Fig. 2b,c), and 548 nm, respectively. Estimation of the conjugation rate The concentration of oligonucleotides in conjugated samples and the concentration of primary oligonucleotides were calculated based on their absorbance at OD 260 nm and using the Beer-Lambert equation (A = εdc) [ 19 ]. In this equation (Absorbance, A) is the absorption (OD 260 nm) of Lin-T 2 and Lin-T 1 sequences in two unconjugated and conjugated states, ε is the molar absorption coefficient of Lin-T 2 and Lin-T 1 sequences with unit M − 1 cm − 1 , d is the length of the optical path trough the solution in cm, c is sample concentration in molar. The data regarding the percentage of conjugated oligonucleotides is presented in Table 4 . FT-IR analysis The formation of BiAu is determined through the appearance of bands 3433 and 1638 cm − 1 , in the FTIR spectrum of BiAu. The appearance of new bands such as 1270 cm − 1 confirmed the conjugation of oligonucleotides on BiAu, (Fig. 3) . Table 4 The percentage of conjugation rate for the Lin-T 2 and Lin-T 1 Samples Percentage of conjugation BiAu@NCLin-T 1 % 77.246 BiAu@Lin-T 1 % 83.48 BiAu@NCLin-T 2 % 83.55 BiAu@Lin-T 2 % 71.08 XRD analysis The XRD analysis was employed to investigate the structural and crystalline characteristics of the BiAu. Figure 4 shows the BiAu XRD spectrum, which confirms its crystalline structure. TEM analysis Figure 5 displays the morphology of BiAu and the corresponding size distribution histogram. The average size of the BiAu is 11.38 ± 4.38 nm (mean ± SD [n=70]), as depicted in the histogram. EDS analysis Figure 6 displays the EDX spectra of BiAu and BiAu@NCLin-T 2 and the corresponding SEM images for each configuration. The designated elements in the spectrum of each nanostructure, coupled with the difference observed in the intensity of the peaks and SEM images for each nanostructure, indicate the formation of the BiAu and the conjugation of the oligonucleotide sequence with the BiAu. Fluorimetric analysis of NCs Figure 7 presents the excitation (Ex) and emission (Em) spectra of the synthesized NCs, as well as the maximum excitation (λ max , Ex) and emission (λ max , Em) wavelengths. Lin-T 1 and BiAu were provided as control samples. In the spectra of Lin-T 1 , NCLin-T 1 , BiAu@Lin-T 1 , BiAu@NCLin-T 1 , BiAu@NCLin-T 2 , NCLin-T 2 and NCLin-C 2 , a fluorescence-induced Stokes shift was observed. The maximum emission wavelength was not observed in BiAu and BiAu@NCLin-T 2 . In Supplementary Fig. S 3, the emission diagrams of the synthesized fluorophores are compared. The shifts observed in the fluorescence intensity and wavelength peaks indicate the formation of NCs. DLS analysis The hydrodynamic size and zeta potential of BiAu and BiAu@Lin-T 2 are presented in (Fig. 8). The hydrodynamic sizes of BiAu and BiAu@Lin-T 2 are reported as 142.8±3.96 and 148.1±4.3 nm, respectively (Mean±SD [n=3]). Moreover, the dispersion index (PDI) has been demonstrated on each column in the chart. The zeta potential of BiAu and BiAu@Lin-T 2 are -5.45±0.64 and -0.35±0.35 mV, respectively (Mean±SD [n=3]). Antibacterial tests results Antibacterial tests were performed under different conditions: PDT, non-PDT, PTT, and non-PTT conditions. These tests was performed based on the concentration of the Lin-T 1 . The results have been reported as graphs based on the percentage of bacterial reduction (%Bacterial reduction, %BR) in each concentration. The reduction percentage has been specified on each column and has been calculated through the following formula: OD 600 Ctrl + and OD 600 Sample are the absorbance of untreated and treated bacteria at a wavelength of 600 nm, respectively. Furthermore, the effect of light and temperature on the positive control (Ctrl + ) was investigated. Supplementary Fig. S 4 shows the results based on the OD of the bacteria. It was found that a noteworthy distinction wasn't seen within the growth rate of E.coli and S. aureus beneath the conditions of PDT and non-PDT. Moreover, substantial distinction wasn't observed within the growth rates of both bacterial strains at temperatures of 37 and 40° C. The PDT effect of BiAu on two bacteria is depicted in (Supplementary Fig. S 5). In S. aureus , the considerable PDT effect wasn’t observed at all four concentrations. The effect of PDT was observed in E.coli at two concentrations, specifically 0.28 and 0.56 nM. BiAu showed a negligible PDT effect at 0.85 and 1.13 nM against E.coli . PDT-induced antibacterial effects BiAu@Lin-T 1 and BiAu@Lin-T 2 showed PDT effects against E.coli at all four concentrations. The PDT effect of both nanostructures against S.aureus was observed at 3 concentrations of 1, 1.5, and 2 µM, (Fig. 9). BiAu@NCLin-T 1 exhibited the PDT effect at all four concentrations against E.coli. While, BiAu@NCLin-T 2 had PDT effect at 3 concentrations of 0.5, 1.5, and 2 µM. In S. aureus , the PDT effect of BiAu@NCLin-T 1 was observed at two concentrations of 1.5 and 2 µM. BiAu@NCLin-T 2 demonstrated the PDT effect at a concentration of 1 µM, (Fig. 10). Supplementary Fig. S 6–9 present PDT and non-PDT effect of nanostructures Lin-T 1 , Lin-T 2 , NCLin-T 1 , NCLin-T 2 , NCLin-C 1 , NCLin-C 2 , Lin-C 1 and Lin-C 2 . Comparison of the antibacterial effect in PDT condition In E.coli , the BiAu@Lin-T 1 illustrated a significant enhancement of the PDT effect at concentrations of 0.5 and 2 µM compared to BiAu@NCLin-T 1 . BiAu@Lin-T 2 exhibited a substantial increase in the PDT effect at concentrations of 0.5, 1, and 1.5 compared to BiAu@NCLin-T 2 . At a concentration of 2 µM, the PDT effect of BiAu@NCLin-T 2 showed a substantial augmentation compared to BiAu@Lin-T 2 , (Fig. 11a,c). In S. aureus , the antibacterial effect of BiAu@NCLin-T 1 at concentrations of 1.5 and 2 µM has increased compared to BiAu@Lin-T 1 . BiAu@Lin-T 2 has exhibited a considerable increase at 3 concentrations of 1, 1.5, and 2 µM compared to BiAu@NCLin-T 2 , (Fig. 11b,d).' Supplementary Fig. S 10-13 present the antibacterial performance of BiAu@Lin-T 1 , BiAu@Lin-T 2 , Lin-T 1 , Lin-T 2 , BiAu, NCLin-T 1 , NCLin-T 2 , NCLin-C 2 , NCLin-C 1 , Lin-C 1 , and Lin-C 2 . The antibacterial effect of nanostructures in non-PDT conditions is presented in the supplementary material with the level 3 heading “Antibacterial comparison under non-PDT conditions”. PTT-induced antibacterial effect In Fig. 12, the antibacterial effect of the nanostructures at a temperature of 40°C was compared with the antibacterial effect under the condition of non-photo stimulation (temperature of 37°C). All three mentioned structures demonstrated a substantial PTT effect on E.coli . BiAu@NCLin-T 2 and BiAu in S. aureus showed a substantial PTT effect in all four concentrations. Supplementary Fig. S 19 shows the comparison between the antibacterial effect of nanostructures at a temperature of 40°C and the antibacterial efficacy under light stimulation conditions (temperature of 37°C). SEM analysis The morphological effects of aPDT were investigated by choosing the NCLin-T 2 (with concentrations of 1.5 µM for E.coli and 2 µM for S.aureus ). Images (a) and (b) in (Fig. 13) exhibit the untreated and treated E.coli , respectively. The untreated S.aureus bacteria are presented in (Fig. 13c) and the treated one is displayed in (Fig. 13d). Discussion Based on computational studies, Lin-T 2 , Lin-C 2 , and Lin-C 1 are stable at all three temperatures. Lin-T 2 had a low increase in free energy with rising temperature, while Lin-C 2 and Lin-C 1 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-C 2 and Lin-C 1 suggest a lack of base pair formation, while Lin-T 2 showed a low pairing probability of A and T bases but remained stable, (Supplementary Fig. S 1). The free energy of the Lin-T 2 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-T 2 . After using Lin-T 1 to form AgNC (NCLin-T 1 ), a new peak appeared at 440 nm in the absorption spectrum of NCLin-T 1 , (Fig. 2c). Conjugation between NCLin-T 1 and BiAu is displayed in (Fig. 2b). Both spectra showed an increase in absorption (hyperchromic shift). Figure 2d presents the conjugation between Lin-T 1 and BiAu. Lin-T 1 spectrum showed the increase in absorption. BiAu spectrum showed the decrease in absorption. Figure 2 (e) shows the absorption spectrum of BiAu@NCLin-T 2 . BiAu and NCLin-T 2 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-T 2 scaffold, as shown in (Fig. 2f). In Fig. 2 (g), a new peak at 445 nm confirms the formation of NCLin-C 1 . The hypochromic shift of Lin-C 2 spectrum (at 260 nm) and emerging of new peak (430 nm) of NCLin-C 2 spectrum confirm the formation of NCLin-C 2 , (Fig. 2h). Figure 2i exhibits absorption spectra of NCLin-T 2 , NCLin-C 2 , and NCLin-C 1 . NCLin-T 2 has the highest absorption intensity due to the addition of A and T to 12 C in Lin-T 2 . BiAu@NCLin-T 1 , BiAu@Lin-T 1 , BiAu@NCLin-T 2 , and BiAu@Lin-T 2 possess elevated conjugation efficiency and density of oligonucleotides and NCs on BiAu, Table 4 . Lin-T 1 and Lin-T 2 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 @LinT 2 , (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@LinT 2 , respectively, which showed the conjugation of LinT 2 to BiAu. The new peak appeared in the BiAu@LinT 2 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 Au 2 Bi 3 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 Bi 2 S 3 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 Bi 2 S 3 . The existence of C, S, N, and O can be due to the BSA-mediated biomineralization of Bi 2 S 3 , (Fig. 6 a). Two other elements, Ag and P, were also found in the spectrum of BiAu @NCLin-T 2 , which signify the formation of NCLin-T 2 and its conjugation on the BiAu (Fig. 6 b). Figure 7 shows the confirmation of the formation of NCs and designed fluorophores through the observed Stokes shifts. The structures of NCLin-T 2 and NCLin-C 2 , (Fig. 7a,b) have large Stokes shifts (116 nm (NCLin-T 2 ) and 106 nm (NCLin-C 2 )). NCLin-T 1 and Lin-T 1 , (Fig. 7f,e) exhibit Stokes shifts of 43.5 nm and 31.5 nm, respectively. BiAu@NCLin-T 1 and BiAu@Lin-T 1 , (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-T 2 , (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-T 2 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-T 2 ”) between 5'HEX and AgNC in BiAu@NCLin-T 1 , leads to the FRET (FÖrster resonance energy transfer) phenomenon [ 26 ]. The emission spectrum of NCLin-T 1 exhibited hypochromic shift following conjugation with BiAu (Supplementary Fig. S 3e). In BiAu@NCLin-T 2 , (Supplementary Fig. S 3g), the BiAu has resulted in the quenching of NCLin-T 2 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@LinT 2 has a higher hydrodynamic size, indicating conjugation with Lin-T 2 . Both BiAu and BiAu@Lin-T 2 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-T 2 has a more zeta potential than BiAu, indicating that Lin-T 2 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-T 1 as a nano photosensitizer (NPS) in PDT conditions compared to non-PDT states. Under PDT states, the antibacterial effect of BiAu@NCLin-T 1 on E.coli increased in all concentrations, while a slight effect was observed in non-PDT conditions. BiAu@NCLin-T 1 had the most pronounced PDT effect on E.coli at concentrations 1.5 µM, (Fig. 10a). BiAu@NCLin-T 2 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-T 2 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. Bi 2 S 3 -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-T 1 and NCLin-T 2 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-T 1 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-T 1 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-T 1 ) had the most antibacterial impact in PDT conditions. The highest %BR of BiAu@NCLin-T 1 was observed in E.coli and S.aureus at concentrations of 1.5 and 2 µM, respectively, (Supplementary Fig. S 20). BiAu@NCLin-T 1 and BiAu@Lin-T 1 had similar Stokes shifts and lower fluorescence intensity than NCLin-T 1 , NCLin-T 2 , NCLin-C 2 , and Lin-T 1 . BiAu@Lin-T 1 showed great aPDT activity against gram-negative strains compared to Lin-T 1 at all four oligonucleotide concentrations. BiAu@NCLin-T 1 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-T 2 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-T 2 are presented in (Fig. 13a,c), respectively, and possess healthy and intact surfaces. E.coli treated with NCLin-T 2 (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-T 2 (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-T 2 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-T 2 confirm the damage and morphological alterations on gram-negative and gram-positive strains. Conclusion AMR is a global threat to public health, affecting the control and treatment of infectious diseases. Inappropriate use of antimicrobial agents and lack of access to new drugs contribute to the crisis of AMR. We designed nanoplatforms to combat AMR. BiAu can have various functions, including PTT-agent for infectious and non-infectious diseases. It applies to drug delivery and diagnostics. The sequence of Lin-T 1 and Lin-T 2 can be used as templates for the formation of AgNCs. They can also be used to design new nanoplatforms with BiAu. According to the fluorimetric analysis of BiAu@NCLin-T 1 , BiAu@Lin-T 1 , BiAu@NCLin-T 2 , NCLin-T 1 , NCLin-T 2 , and NCLin-C 2 , they can be used as FRET biosensors and fluorescence quenching systems in colorimetric sensors. Different sequences and nanostructures, except Lin-C 1 and Lin-C 2 , can act as PSs, PTT, or antibacterial agents under various conditions. For example, BiAu@NCLin-T 1 and BiAu@NCLin-T 2 can be used as new PS for the removal of gram-negative bacteria. BiAu@Lin-T 2 can be used to combat gram-positive bacteria-induced infections. Declarations Data availability All data generated or analysed during this study are included in this published article (and its Supplementary Information files). Author Contributions statement Siamak Javani: Supervision, Conceptualization, Editing, Methodology, Investigation, Validation Hamed Nosrati: Validation, Methodology Naser Faraji: Software, Editing Atiyeh Nomani: Methodology, Conceptualization, Data curation, Writing, Original draft preparation, Software, Visualization, Editing, Writing- Reviewing, Formal analyses Investigation Jalil Charmi: Methodology. 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Supplementary Files Supplementarydata.docx Cite Share Download PDF Status: Published Journal Publication published 30 Sep, 2024 Read the published version in Scientific Reports → Version 1 posted Reviews received at journal 13 Jul, 2024 Reviews received at journal 04 Jul, 2024 Reviewers agreed at journal 03 Jul, 2024 Reviewers agreed at journal 03 Jul, 2024 Reviewers invited by journal 02 Jul, 2024 Editor assigned by journal 02 Jul, 2024 Editor invited by journal 02 Jul, 2024 Submission checks completed at journal 01 Jul, 2024 First submitted to journal 27 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4648298","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":326441459,"identity":"bba868bc-b569-4a0a-8205-8258753bdda6","order_by":0,"name":"Atiyeh Nomani","email":"","orcid":"","institution":"School of Advanced Technologies in Medicine, Golestan University of Medical Sciences, Iran","correspondingAuthor":false,"prefix":"","firstName":"Atiyeh","middleName":"","lastName":"Nomani","suffix":""},{"id":326441461,"identity":"85b23356-7327-4c99-abc3-b8dd046cc276","order_by":1,"name":"Hamed Nosrati","email":"","orcid":"","institution":"Zanjan Pharmaceutical Nanotechnology Research Center, Zanjan University of Medical Sciences, Zanjan, Iran","correspondingAuthor":false,"prefix":"","firstName":"Hamed","middleName":"","lastName":"Nosrati","suffix":""},{"id":326441464,"identity":"28349c5d-aa4d-462d-a73d-3f54209f4186","order_by":2,"name":"Naser Faraji","email":"","orcid":"","institution":"Department of Medical Nanotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran","correspondingAuthor":false,"prefix":"","firstName":"Naser","middleName":"","lastName":"Faraji","suffix":""},{"id":326441465,"identity":"f7f80c55-b01d-4594-b6ba-4c4e31d13f8b","order_by":3,"name":"Jalil Charmi","email":"","orcid":"","institution":"4Department of Physics, Faculty of Science, University of Zanjan, Zanjan 45371-38791, Iran","correspondingAuthor":false,"prefix":"","firstName":"Jalil","middleName":"","lastName":"Charmi","suffix":""},{"id":326441466,"identity":"8bbdff97-1b60-4b21-aeb2-85af8b4487a8","order_by":4,"name":"Siamak Javani","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/klEQVRIiWNgGAWjYDACZhBxAEIxJFRIgDgkaTlDjBYGiBYIYGxjIKxFvp356YYfZxjY+WckP3vwcJ5FNN/x5gcMH/fU4tRicJjN7GbPDQZmiRtp5gaJ2yRyZ545ZsA449lx3FqYGcxu8HwA+uXMATMJkJYNNxIMmHkOHMPtsGb2bzf/ALXInzn+TSJxDlDL/ecf8GphOMxjdpsH6DCD4z1AWxpAtvCAbKnB4xeestsyZySYDY/3lEkkHAP5Jafg4IwDB3A7rP/4tptvjtkkyx1m3yb5o6Yut+/48Y0PPhyow+0wCJBIRuECrThMSAuDHboAQVtGwSgYBaNg5AAA/H1ePAXnnVUAAAAASUVORK5CYII=","orcid":"","institution":"School of Advanced Technologies in Medicine, Golestan University of Medical Sciences, Iran","correspondingAuthor":true,"prefix":"","firstName":"Siamak","middleName":"","lastName":"Javani","suffix":""}],"badges":[],"createdAt":"2024-06-27 11:47:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4648298/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4648298/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-74273-z","type":"published","date":"2024-09-30T15:57:09+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":60944093,"identity":"bab08e42-8d27-402b-a516-749245555c3d","added_by":"auto","created_at":"2024-07-23 22:06:51","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1764654,"visible":true,"origin":"","legend":"\u003cp\u003ethe thermodynamic stability and entropy of oligonucleotide sequences Lin-T\u003csub\u003e2\u003c/sub\u003e, Lin-C\u003csub\u003e2\u003c/sub\u003e, and Lin-C\u003csub\u003e1\u003c/sub\u003e at three different temperatures: 25, 37, and 40° C, along with their secondary structures using both the MFE and Centroid structure methods; a) secondary structures of Lin-T\u003csub\u003e2\u003c/sub\u003e at 25° C; b) Lin-T\u003csub\u003e2\u003c/sub\u003e secondary structures at 37° C; c) Lin-T\u003csub\u003e2\u003c/sub\u003e secondary structures at 40° C; d) the secondary structure of Lin-C\u003csub\u003e2\u003c/sub\u003e at two temperatures of 25 and 37° C; e) the secondary structure of Lin-C\u003csub\u003e1\u003c/sub\u003e at two temperatures of 25 and 37° C.\u003c/p\u003e","description":"","filename":"Picture1.png","url":"https://assets-eu.researchsquare.com/files/rs-4648298/v1/fbe99c766a989104b6f0ba69.png"},{"id":60944092,"identity":"2afbe728-1e85-4901-8deb-e5c1a7a30ac3","added_by":"auto","created_at":"2024-07-23 22:06:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":125912,"visible":true,"origin":"","legend":"\u003cp\u003eabsorption graphs of designed nanostructures along with λ\u003csub\u003emax\u003c/sub\u003e of each and their comparison with each other.\u003c/p\u003e","description":"","filename":"Picture2.png","url":"https://assets-eu.researchsquare.com/files/rs-4648298/v1/311d6417873bcce895c52d8e.png"},{"id":60943038,"identity":"d46ecfb0-4863-46c4-ae4c-16ee6e14a24c","added_by":"auto","created_at":"2024-07-23 21:58:51","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":318402,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR spectrum of BiAu and BiAu@Lin-T\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e","description":"","filename":"Picture3.png","url":"https://assets-eu.researchsquare.com/files/rs-4648298/v1/90f0ae5571d229c0f0dc0bca.png"},{"id":60943041,"identity":"40a8eeee-c0c2-4832-bd7a-0ff2ecfee57d","added_by":"auto","created_at":"2024-07-23 21:58:51","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":529392,"visible":true,"origin":"","legend":"\u003cp\u003eX-ray diffraction pattern of BiAu\u003c/p\u003e","description":"","filename":"Picture4.png","url":"https://assets-eu.researchsquare.com/files/rs-4648298/v1/95589c16ab81c75dedcc0cf1.png"},{"id":60943043,"identity":"20409a5d-c181-4e90-a9d7-85ca0ce1dac4","added_by":"auto","created_at":"2024-07-23 21:58:51","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1689564,"visible":true,"origin":"","legend":"\u003cp\u003ea) TEM image of BiAu; b) size distribution diagram corresponding to TEM image. This diagram was obtained using Image J software.\u003c/p\u003e","description":"","filename":"Picture5.png","url":"https://assets-eu.researchsquare.com/files/rs-4648298/v1/f01e2be45e86fce2aed4e5bd.png"},{"id":60944095,"identity":"b366dc85-aa71-406e-9c41-605bba86dc1f","added_by":"auto","created_at":"2024-07-23 22:06:52","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1875838,"visible":true,"origin":"","legend":"\u003cp\u003ea) SEM image of BiAu and its corresponding EDX spectrum; b) SEM image of BiAu@NCLin-T\u003csub\u003e2\u003c/sub\u003e and its corresponding EDX spectrum.\u003c/p\u003e","description":"","filename":"Picture6.png","url":"https://assets-eu.researchsquare.com/files/rs-4648298/v1/8f0877b92fe808c95b649ab9.png"},{"id":60943046,"identity":"da83927f-1a14-45e3-80d6-8e839b98cf7a","added_by":"auto","created_at":"2024-07-23 21:58:52","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2005024,"visible":true,"origin":"","legend":"\u003cp\u003eexcitation and emission spectra of Lin-T\u003csub\u003e1\u003c/sub\u003e, BiAu and synthesized AgNCs. The vertical axis is the normalized fluorescence/absorbance intensity, based on arbitrary units (a.u.) and the horizontal axis is the wavelength in nanometers (nm).\u003c/p\u003e","description":"","filename":"Picture7.png","url":"https://assets-eu.researchsquare.com/files/rs-4648298/v1/b435a12dd4416cd49a25ea9f.png"},{"id":60943047,"identity":"fad864f7-9297-4eee-8d1f-f423ecf664ab","added_by":"auto","created_at":"2024-07-23 21:58:52","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":365157,"visible":true,"origin":"","legend":"\u003cp\u003ehydrodynamic size and zeta potential of BiAu and BiAu@Lin-T\u003csub\u003e2\u003c/sub\u003e: a) hydrodynamic size; b) zeta potential.\u003c/p\u003e","description":"","filename":"Picture8.png","url":"https://assets-eu.researchsquare.com/files/rs-4648298/v1/d585598681ec59ddd8bc1394.png"},{"id":60943049,"identity":"18614db7-222c-4889-8396-0b51b095cdaa","added_by":"auto","created_at":"2024-07-23 21:58:52","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1190991,"visible":true,"origin":"","legend":"\u003cp\u003eantibacterial performance of BiAu@Lin-T\u003csub\u003e1\u003c/sub\u003e and BiAu@Lin-T\u003csub\u003e2 \u003c/sub\u003eunder PDT and non-PDT conditions; a) antibacterial performance of BiAu@Lin-T\u003csub\u003e1\u003c/sub\u003e against \u003cem\u003eE.coli\u003c/em\u003e; b) antibacterial performance of BiAu@Lin-T\u003csub\u003e1\u003c/sub\u003e against \u003cem\u003eS.aureus\u003c/em\u003e; c) antibacterial performance of BiAu@Lin-T\u003csub\u003e2\u003c/sub\u003e against \u003cem\u003eE.coli\u003c/em\u003e; d) antibacterial performance of BiAu@Lin-T\u003csub\u003e2\u003c/sub\u003e against \u003cem\u003eS.aureus\u003c/em\u003e. Error bar indicates standard deviation.\u003c/p\u003e","description":"","filename":"Picture9.png","url":"https://assets-eu.researchsquare.com/files/rs-4648298/v1/d22fd0ca9c102fab6f504159.png"},{"id":60943051,"identity":"4f601319-feaf-4182-844b-84343e22ea84","added_by":"auto","created_at":"2024-07-23 21:58:52","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":1521281,"visible":true,"origin":"","legend":"\u003cp\u003eantibacterial performance of BiAu@NCLin-T\u003csub\u003e1\u003c/sub\u003e and BiAu@NCLin-T\u003csub\u003e2\u003c/sub\u003e under PDT and non-PDT conditions; a) antibacterial performance of BiAu@NCLin-T\u003csub\u003e1\u003c/sub\u003e against \u003cem\u003eE.coli\u003c/em\u003e; b) antibacterial activity of BiAu@NCLin-T\u003csub\u003e1\u003c/sub\u003e against \u003cem\u003eS.aureus\u003c/em\u003e; c) antibacterial performance of BiAu@NCLin-T\u003csub\u003e2\u003c/sub\u003e against \u003cem\u003eE.coli\u003c/em\u003e; d) antibacterial performance of BiAu@NCLin-T\u003csub\u003e2\u003c/sub\u003e against \u003cem\u003eS.aureus\u003c/em\u003e. Error bar indicates standard deviation.\u003c/p\u003e","description":"","filename":"Picture10.png","url":"https://assets-eu.researchsquare.com/files/rs-4648298/v1/3e0fe892a9ddc110b8813988.png"},{"id":60943044,"identity":"d9d0a261-b687-4804-972a-8bd688d2fcec","added_by":"auto","created_at":"2024-07-23 21:58:52","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":1647124,"visible":true,"origin":"","legend":"\u003cp\u003eantibacterial performance of BiAu@NCLin-T\u003csub\u003e1\u003c/sub\u003e and BiAu@NCLin-T\u003csub\u003e2\u003c/sub\u003e and their comparison with BiAu@Lin-T\u003csub\u003e1\u003c/sub\u003e and BiAu@Lin-T\u003csub\u003e2\u003c/sub\u003e under PDT conditions; a) antibacterial performance of BiAu@NCLin-T\u003csub\u003e1\u003c/sub\u003e and BiAu@Lin-T\u003csub\u003e1\u003c/sub\u003e in \u003cem\u003eE.coli\u003c/em\u003e; b) antibacterial performance of BiAu@NCLin-T\u003csub\u003e1\u003c/sub\u003e and BiAu@Lin-T\u003csub\u003e1\u003c/sub\u003e in \u003cem\u003eS.aureus\u003c/em\u003e; c) antibacterial performance of BiAu@ NCLin-T\u003csub\u003e2\u003c/sub\u003e and BiAu@Lin-T\u003csub\u003e2\u003c/sub\u003e in \u003cem\u003eE.coli\u003c/em\u003e; d) antibacterial performance of BiAu@ NCLin-T\u003csub\u003e2\u003c/sub\u003e and BiAu@Lin-T\u003csub\u003e2 \u003c/sub\u003eon \u003cem\u003eS.aureus\u003c/em\u003e. Error bar indicates standard deviation.\u003c/p\u003e","description":"","filename":"Picture11.png","url":"https://assets-eu.researchsquare.com/files/rs-4648298/v1/960282ee118ad6dc15480b6c.png"},{"id":60943045,"identity":"5639fbe2-0e34-40bb-9006-37aa8c22d6c0","added_by":"auto","created_at":"2024-07-23 21:58:52","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":584219,"visible":true,"origin":"","legend":"\u003cp\u003ecomparison of antibacterial performance of BiAu@NCLin-T\u003csub\u003e2\u003c/sub\u003e, BiAu and Lin-T\u003csub\u003e2\u003c/sub\u003e nanostructures under PTT and non-PDT conditions; a) antibacterial performance of BiAu@NCLin-T\u003csub\u003e2\u003c/sub\u003e against \u003cem\u003eE.coli\u003c/em\u003e; b) antibacterial activity of BiAu against \u003cem\u003eE.coli\u003c/em\u003e; c) antibacterial activity of Lin-T\u003csub\u003e2\u003c/sub\u003e against \u003cem\u003eE.coli\u003c/em\u003e; d) antibacterial performance of BiAu@NCLin-T\u003csub\u003e2\u003c/sub\u003e against \u003cem\u003eS.aureus\u003c/em\u003e; e) antibacterial activity of BiAu against \u003cem\u003eS.aureus\u003c/em\u003e; f) antibacterial activity of Lin-T\u003csub\u003e2\u003c/sub\u003e against \u003cem\u003eS.aureus\u003c/em\u003e. Error bar indicates standard deviation.\u003c/p\u003e","description":"","filename":"Picture12.png","url":"https://assets-eu.researchsquare.com/files/rs-4648298/v1/22b3d53fd8e2541a0f2961c7.png"},{"id":60944094,"identity":"36154003-6b88-477d-8525-dc8508d17cc2","added_by":"auto","created_at":"2024-07-23 22:06:52","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":971367,"visible":true,"origin":"","legend":"\u003cp\u003eSEM images of \u003cem\u003eE.coli\u003c/em\u003e and \u003cem\u003eS.aureus\u003c/em\u003e bacteria treated with NCLin-T\u003csub\u003e2\u003c/sub\u003e; a) untreated \u003cem\u003eE.coli\u003c/em\u003e bacteria; b) \u003cem\u003eE.coli\u003c/em\u003e bacteria treated with NCLin-T\u003csub\u003e2\u003c/sub\u003e; c) untreated\u003cem\u003e S.aureus\u003c/em\u003e bacteria; d)\u003cem\u003eS.aureus\u003c/em\u003e bacteria treated with NCLin-T\u003csub\u003e2\u003c/sub\u003e. The red arrows indicate the damage done to the bacterial strains.\u003c/p\u003e","description":"","filename":"Picture13.png","url":"https://assets-eu.researchsquare.com/files/rs-4648298/v1/affc18b88ef5e8afaba0157b.png"},{"id":66097868,"identity":"3e7ffc33-9800-4681-9383-0e4838687135","added_by":"auto","created_at":"2024-10-07 16:15:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":20550861,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4648298/v1/252a313c-0264-4a16-b31e-56ad4d3218cc.pdf"},{"id":60943052,"identity":"91b4d97f-e7a1-4cb9-9be7-3343233cffc1","added_by":"auto","created_at":"2024-07-23 21:58:52","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":20627186,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarydata.docx","url":"https://assets-eu.researchsquare.com/files/rs-4648298/v1/0013106f6fb8ce9265a15b3c.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Bismuth-gold nanohybrid conjugated with a HEX-bound oligonucleotide; a novel nano photosensitizer to combat antimicrobial resistance","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAntimicrobial resistance (AMR) is a global public health threat that can hinder the control and treatment of infectious diseases [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. It is related to wide mortality rates (around 700,000 deaths per year worldwide), and a remarkable impact on the effectiveness of antimicrobial agents [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Photodynamic therapy (PDT), as a new treatment method, is suggested to dominate AMR. PDT contains photosensitizers (PSs) and light to create reactive oxygen species (ROS) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The currently approved PSs have poor water solubility, aggregation, low photostability, and phototoxicity issues [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Metal NPs can elevate the efficiency of PDT by increasing ROS generation and curing biocompatibility. LSPR (localized surface plasmon resonance), as a property of metal NPs, has been utilized to enhance the performance of aPDT by rectifying the efficiency of PSs. Gold nanoparticles (AuNPs) have exhibited LSPR, allowing them to absorb light at specific wavelengths and possess photoacoustic and photothermal properties [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. DNA-AuNP conjugates are stable nanobioconjugates formed by linking single-strand DNA (ssDNA) to AuNPs [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBismuth compounds have been used as medicines for gastrointestinal disorders and have recently been explored for viral infections, multidrug-resistant microbial infections, and cancer treatment [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Bismuth NPs exhibit long chemical stability, slight toxicity, controllable size and shape and simplicity of functionalization [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Nanomaterials predicated on Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e exhibit negligible toxicity and have attracted widespread usage as a theranostic agent in X-ray computed tomography (CT) and near-infrared light-induced PTT [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eStudies has indicated AgNPs have the antimicrobial activity through various molecular pathways, including the generation of ROS, disruption of biological membranes, and Trojan horse mechanism. AgNPs possess the capability to dominate AMR bacteria and counteract resistance mechanisms, such as preventing biofilm formation and regulating bacterial influx/efflux pumps [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Ag nanoclusters (AgNCs) are clusters of Ag atoms with a diameter of less than 2 nm, which demonstrate unique features such as well-defined structures and strong luminescence emission [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. AgNCs can be synthesized using single-stranded nucleic acids as polydentate ligands, which provide biocompatibility and fluorescence properties [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. A principal sight of planning oligonucleotide structures is to build structures that are thermodynamically stable and have the least energy state. Thermodynamic models are utilized to assess the equilibrium base-pairing probability of bases, intramolecular unfavorable interactions, the interaction of a nucleic acid chain with other chains, and the amount of free energy when the oligonucleotides are in the thermodynamic equilibrium [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Some of software such as ViennaRNA [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] and Nupack [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] are usually employed, for the thermodynamic examinations of oligonucleotide structures. The main current problem is the inefficiency of conventional PSs due to the mentioned disadvantages. Our hypothesis is designing a novel PS using AuNPs, AgNCs, and oligonucleotides to provide condition for better treatment of AMR.\u003c/p\u003e \u003cp\u003eIn the present work, Hex (Hexachlorofluorescein, the fluorescence dye) is employed as PS for aPDT of resistant bacteria. The aim of this project is to design a nanoplatform founded on the AgNCs and a hybrid NP, namely bismuth-gold (BiAu), to enhance the aPDT effect of Hex. This nano platform has been explored for its PDT (the wavelength of 520 nm) effect and as PSs on two model bacteria, namely \u003cem\u003eE.coli\u003c/em\u003e and \u003cem\u003eS.aureus\u003c/em\u003e, which are gram-negative and positive, respectively. BiAu hybrid NPs were functionalized with oligonucleotide sequences, namely Linker (LinT\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;2\u003c/sub\u003e), and formed conjugated nanostructures named BiAu@Lin-T\u003csub\u003e1\u003c/sub\u003e and BiAu@Lin-T\u003csub\u003e2\u003c/sub\u003e. In Lin-T\u003csub\u003e1\u003c/sub\u003e, HEX has been linked to this sequence from the 5\u0026prime; end. Lin-T\u003csub\u003e1\u003c/sub\u003e and Lin-T\u003csub\u003e2\u003c/sub\u003e are utilized as templates for AgNCs construction. Lin-T\u003csub\u003e1\u003c/sub\u003e and Lin-T\u003csub\u003e2\u003c/sub\u003e-mediated AgNCs conjugated with BiAu hybride nanostructures and created BiAu@NCLin-T\u003csub\u003e1\u003c/sub\u003e and BiAu@NCLin-T\u003csub\u003e2\u003c/sub\u003e, respectively. The PDT effect of nanostructures has been investigated in various concentrations. To explore the PTT effect, BiAu@NCLin-T\u003csub\u003e2\u003c/sub\u003e, BiAu, and Lin-T\u003csub\u003e2\u003c/sub\u003e have been selected.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eChemicals and instruments\u003c/h2\u003e \u003cp\u003eBovine serum albumin (BSA), Tetrachloroauric (III) acid (HAuCl\u003csub\u003e4\u003c/sub\u003e), Bismuth(III) nitrate (Bi(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e3\u003c/sub\u003e), Sodium bromide (NaBr), Sodium hydroxide (NaOH), Silver nitrate (AgNO\u003csub\u003e3\u003c/sub\u003e), Nitric acid (HNO\u003csub\u003e3\u003c/sub\u003e), Sodium tetrahydridoborate (NaNH\u003csub\u003e4\u003c/sub\u003e), Sodium citrate tribasic dihydrate (C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e9\u003c/sub\u003eNa\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e), Sodium dihydrogen phosphate (NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e), Disodium hydrogen phosphate ((Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e), Formaldehyde (CH\u003csub\u003e2\u003c/sub\u003eO), DeNovix DS-11\u0026thinsp;+\u0026thinsp;UV-vis spectrophotometer, Perkin Elmer Spectrum RX1 FT-IR spectrophotometer, SZ-100z Dynamic Light Scattering \u0026amp; Zeta potential analyzer (company, Horiba Jobin Jyovin), TEM (Philips CM-120), XRD (Bruker D8 ADVANCE), SEM \u003cem\u003e(AIS 2100\u003c/em\u003e, Seron Technology, Korea), BioTek Cytation 3 Microplate Reader and EDS (TESCAN MIRA2).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eSynthesis of Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e coated with BSA\u003c/h2\u003e \u003cp\u003eTo synthesize Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e, a biogenic mineralization approach is employed [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The synthesis process involves dissolving 250 mg of BSA in 8 mL of water. Then, while stirring vigorously, gradually add a solution of 50 mM Bi(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e3\u003c/sub\u003e in 1 mL of HNO\u003csub\u003e3\u003c/sub\u003e to the BSA solution. After stirring for 30 minutes, the pH is modified to a value of 12 by adding a solution of 2 M NaOH. The resulting mixture is then subjected to stirring. The mixture was left at room temperature (25\u0026deg; C) for 12 hours, allowing the formation of NPs of Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e. The change in color from transparent to black confirms the NP formation. Then, the nanoparticles undergo purification through dialysis against water for 24\u0026ndash;48 hours and are stored at a temperature of 4\u0026deg; C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of hybrid dimer BiAu\u003c/h2\u003e \u003cp\u003eBiAu is a type of metal-semiconductor heterodimer NPs that is obtained by growing AuNPs on the surface of Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. We dissolved 30 mg of Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e and 34 mg of HAuCl\u003csub\u003e4\u003c/sub\u003e in the 100 ml Milli-Q water at 140\u0026deg;C. Then, we dissolved 118 mg of C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e9\u003c/sub\u003eNa\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e in 10 ml of Milli-Q water. Then, it was added to the gold solution very quickly and was held for 15 minutes at 140\u0026deg;C. After cooling to reach ambient temperature, excess salts were removed by filtration. UV-vis spectroscopy was used to determine the absorption and concentration of AuNPs. The final solution is purified using dialysis for 24 hours. The concentration of the AuNPs was estimated to be 10.6 nM by Lambert-Beers Law (the extinction coefficient of 13 nm Au NPs is 2.7\u0026times;108 M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at 525 nm).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization of synthesized NPs\u003c/h2\u003e \u003cp\u003eThe formation of Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e was determined by UV-vis spectroscopy. The identification of chemical structure was investigated by FTIR and EDX. The evaluation of colloidal stability and zeta potential was investigated by DLS. The morphology and size were determined by TEM. The structural characteristics were examined by XRD.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eDesign and synthesis of oligonucleotides\u003c/h2\u003e \u003cp\u003eFour designed oligonucleotide sequences synthesized by Pishgam company with OD\u003csub\u003e260\u003c/sub\u003e: 8.00 are presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. These hybrid NPs are functionalized by two oligonucleotide sequences called Linker (LinT\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;2\u003c/sub\u003e). Lin-C\u003csub\u003e1\u003c/sub\u003e and Lin-C\u003csub\u003e2\u003c/sub\u003e sequences were used as control sequences.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDesigned sequences\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eName\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSequence\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBases number\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLin-T\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3-AAAAAAAAACCCCCCCCCCCCTTT-HEX-5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLin-T\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3-AAAAAAAAACCCCCCCCCCCCTTT-5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLin-C\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3-AAAAAAAAA-5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLin-C\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3-CCCCCCCCCCCCTTT-5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eValidation of sequences by computational methods\u003c/h2\u003e \u003cp\u003eThe step was performed for investigation of the stability and entropy of selected oligonucleotide sequences, Lin-T\u003csub\u003e2\u003c/sub\u003e, Lin-C\u003csub\u003e2\u003c/sub\u003e, and Lin-C\u003csub\u003e1\u003c/sub\u003e. Two factors, thermodynamic stability, and structural entropy, are examined using the Vienna RNA secondary structure server software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://rna.tbi.univie.ac.at/\u003c/span\u003e\u003cspan address=\"http://rna.tbi.univie.ac.at/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). This software can predict the secondary structure of two sequences using two methods: MFE (Minimum Free Energy) and Centroid structure [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The secondary structure of the mentioned oligonucleotide sequences will be examined at temperatures of 25, 37, and 40\u003csup\u003e\u0026deg;\u003c/sup\u003e C using the software. Moreover, the NUPACK software was utilized to examine the impact of varying concentrations on the stability of the oligonucleotide sequences.\u003c/p\u003e \u003cp\u003eThis software examines the thermodynamic equilibrium of oligonucleotides against environmental changes such as temperature and different concentrations based on thermodynamic parameters such as changes in free energy (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.nupack.org/\u003c/span\u003e\u003cspan address=\"https://www.nupack.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The factors examined in the thermodynamic equilibrium state of nucleic acids are the equilibrium base-pairing probability (EBPP) and the probable equilibrium structures (Equilibrium structure probability) that nucleic acid molecules take in equilibrium state. The mentioned factors were investigated with this following manner, ensemble (all stacking) and salt concentrations [Na\u003csup\u003e+\u003c/sup\u003e (1 M), Mg\u003csup\u003e+\u003c/sup\u003e (0.0 M)] [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe selected sequences were subjected to different temperature and concentration conditions. At 25\u003csup\u003e\u0026deg;\u003c/sup\u003e C, the process of AgNCs synthesis (via the mentioned sequences) was performed. Additionally, the conjugation of Lin-T\u003csub\u003e2\u003c/sub\u003e with BiAu was performed at this temperature. The oligonucleotide concentrations in these two processes were 15\u0026micro;M. The antibacterial test was evaluated at four oligonucleotide concentrations of 0.5, 1, 1.5, and 2 \u0026micro;M. The test was conducted at a temperature of 37\u003csup\u003e\u0026deg;\u003c/sup\u003e C. A temperature of 40\u003csup\u003e\u0026deg;\u003c/sup\u003e C was also used for PTT test. Among three oligonucleotides, Lin-T\u003csub\u003e2\u003c/sub\u003e was placed at 40\u003csup\u003e\u0026deg;\u003c/sup\u003e C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eSynthesis of oligonucleotides-templated AgNCs\u003c/h2\u003e \u003cp\u003eFor AgNCs synthesizing, the concentration ratio of 1:6:6 was used for the oligonucleotide sequences, AgNO\u003csub\u003e3\u003c/sub\u003e, and NaBH\u003csub\u003e4\u003c/sub\u003e, respectively, with concentrations of 15 \u0026micro;M, 90 \u0026micro;M, and 90 \u0026micro;M. The resulting NCs were incubated overnight at room temperature and then washed with an Amicon Ultra-0.5ML filter to remove excess Ag\u003csup\u003e+\u003c/sup\u003e, NaBH\u003csub\u003e4\u003c/sub\u003e, and oligonucleotides. Fluorimetry analysis was also performed to characterize the NC formation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eConjugation of oligonucleotides and NCs with the BiAu\u003c/h2\u003e \u003cp\u003eTo achieve this conjugation, the previously reported method was used with some modifications. The technique involves using the affinity of A bases to AuNP (existent in BiAu) and the salt-aging method [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The study involved conjugating Lin-T\u003csub\u003e1\u003c/sub\u003e, Lin-T\u003csub\u003e2\u003c/sub\u003e, NCLin-T\u003csub\u003e1\u003c/sub\u003e, and NCLin-T\u003csub\u003e2\u003c/sub\u003e with BiAu at varying concentrations. The mixture was vortexed and incubated for three days. NaBr was added gradually over 48 hours. The conjugation was confirmed by UV-vis spectroscopy, FT-IR, EDX, and DLS analysis. For further clarity, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, provides a description of designed nanostructures. Figures of nanostructures have been presented in (Supplementary material).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe description of designed nanostructures\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBiAu\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ehybrid dimer (BSA- coated Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e @ Au)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNCLin-T\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLin-T\u003csub\u003e1\u003c/sub\u003e oligonucleotide- templated AgNCs\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNCLin-T\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLin-T\u003csub\u003e2\u003c/sub\u003e oligonucleotide- templated AgNCs\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBiAu@NCLin-T\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNCLin-T\u003csub\u003e1\u003c/sub\u003e-BiAu conjugates\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBiAu@NCLin-T\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNCLin-T\u003csub\u003e2\u003c/sub\u003e-BiAu conjugates\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBiAu@Lin-T\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLin-T\u003csub\u003e1\u003c/sub\u003e-BiAu conjugates\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBiAu@Lin-T\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLin-T\u003csub\u003e2\u003c/sub\u003e-BiAu conjugates\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNCLin-C\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLin-C\u003csub\u003e1\u003c/sub\u003e oligonucleotide- templated AgNCs\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNCLin-C\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLin-C\u003csub\u003e2\u003c/sub\u003e oligonucleotide- templated AgNCs\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eInvestigation of the antibacterial properties\u003c/h2\u003e \u003cp\u003eTo investigate the antibacterial activity, \u003cem\u003eE.coli\u003c/em\u003e (ATCC 43894) and \u003cem\u003eS.aureus\u003c/em\u003e COL are employed as gram-negative and gram-positive models, respectively. The growth curve method will be utilized to conduct the antibacterial test. In this method, the bacteria will first be cultured in Laurie Bertani (LB) medium and then incubated at 37\u0026deg; C until they reach total growth. Then, the growth rate of bacteria is adjusted at OD: 0.1 inside the microplate. Then the bacteria are treated with four concentrations of 0.5, 1, 1.5, and 2 \u0026micro;M of oligonucleotides Lin\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;4\u003c/sub\u003e, nanostructures NCLin-T\u003csub\u003e1\u003c/sub\u003e, NCLin-T\u003csub\u003e2\u003c/sub\u003e, NCLin-C\u003csub\u003e2\u003c/sub\u003e, NCLin-C\u003csub\u003e1\u003c/sub\u003e, BiAu@Lin-T\u003csub\u003e1\u003c/sub\u003e, BiAu@Lin-T\u003csub\u003e2\u003c/sub\u003e, BiAu@NCLin-T\u003csub\u003e1\u003c/sub\u003e, and BiAu@NCLin-T\u003csub\u003e2\u003c/sub\u003e and four concentrations of 0.28, 0.56, 0.85 and 1.13 nM of BiAu. They are placed under photostimulation (PDT) and non-photo stimulation (non-PDT) conditions, followed by incubation at 37\u0026deg; C for 24 hours. The incident light was irradiated for 2 hours, using a light source of diod laser (with irradiation of 520 nm and 4.75 milli watts intensity). The OD of the bacteria will be measured at 600 nm after the end of the incubation period (22 hours). Additionally, to investigate the effect of PTT, nanostructures LinT\u003csub\u003e2\u003c/sub\u003e, BiAu, and BiAu@NCLin-T\u003csub\u003e2\u003c/sub\u003e were selected. The bacteria were treated with these compounds at 40\u0026deg; C for 2 hours. The bacteria will then be incubated at 37\u0026deg;C (non-PTT) and 40\u0026deg; C (PTT) for 24 hours inside an incubator, and their OD\u003csub\u003e600\u003c/sub\u003e will be measured after 22 hours.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSEM analysis\u003c/h2\u003e \u003cp\u003eIn this study, SEM analysis was performed on treated bacteria after 22 hours. To prepare the bacterial samples, fixation was performed. Fixation involves centrifugation of bacterial broth, and washing the resulting pellet with a phosphate buffer solution. Phosphate buffer and formaldehyde are added to the pellet to achieve the concentration of formaldehyde in the final volume equivalent to %2 (V/V). Then, dehydration was performed using different concentrations of ethanol (%30, %50, %70, % 80, % 90, and % 100). Then, a smear of the fixed bacterial sample was prepared on a slide for SEM analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eData analysis method\u003c/h2\u003e \u003cp\u003eThe acquired data is presented using mean, standard deviation, percentage of reduction, and graph. Antibacterial test data analysis was done through the student\u0026rsquo;s t-test using Graph Pad Prism 9 software. The tests were performed with n value of 4 and significant differences were identified with * (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), ** (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), *** (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and ns (non-significant).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eValidation of sequences by computational tools\u003c/h2\u003e\n \u003cp\u003eFigure 1 displays details on the entropy, stability, and secondary structure of Lin-T\u003csub\u003e2\u003c/sub\u003e, Lin-C\u003csub\u003e2\u003c/sub\u003e, and Lin-C\u003csub\u003e1\u003c/sub\u003e oligonucleotides. Mountain plots are also presented at temperatures of 25, 37, and 40\u0026deg; C. The diagrams indicate the entropy of the nucleotide bases, with red indicating the lowest entropy and purple indicating the highest. Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e shows the thermodynamic free energy values of the sequences in kcal/mol, NUPACK and Vienna RNA results.\u003c/p\u003e\n \u003cp\u003eSupplementary Fig. S 1 and S 2\u0026nbsp;show EBPP diagrams of Lin-T\u003csub\u003e2\u003c/sub\u003e, Lin-C\u003csub\u003e2\u003c/sub\u003e, and Lin-C\u003csub\u003e1\u0026nbsp;\u003c/sub\u003eoligonucleotide sequences. Supplementary Fig. S 1 also possesses a histogram of the oligonucleotide concentrations of 0.5, 1, 1.5, 2, and 15 \u0026mu;M. The Equilibrium probability band, presented in both figures, displays the sequence\u0026apos;s structural equilibrium. It highlights areas with more excellent stability, gradually increasing in probability from blue to brown.\u0026nbsp;Supplementary Fig. S 2\u0026nbsp;presents the EBPP diagram (independent of concentration) featuring the oligonucleotides of Lin-T\u003csub\u003e2\u003c/sub\u003e, Lin-C\u003csub\u003e2\u003c/sub\u003e, and Lin-C\u003csub\u003e1\u003c/sub\u003e, along with the free energy of the complex at temperatures of 25, 37 \u0026deg;C (for all three sequences), and 40 \u0026deg;C (only for Lin-T\u003csub\u003e2\u003c/sub\u003e).\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eTable 3. The thermodynamic free energy of oligonucleotide at temperatures: 25, 37, and 40\u0026deg; C. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eOligonucleotides\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eThe free energy of thermodynamic ensemble/ complex based on kcal/mol\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e25\u0026deg;C\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e37\u0026deg;C\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e40\u0026deg;C\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eLin-T\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVienna RNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNUPACK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.39\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eLin-C\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVienna RNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e---\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNUPACK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e---\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eLin-C\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVienna RNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e---\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNUPACK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e---\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003eUV-visible spectroscopic analysis\u003c/h2\u003e\n \u003cp\u003eTo confirm the formation of BiAu hybrid NPs, the conjugation of oligonucleotide sequences with BiAu, and the formation of AgNCs, the UV-visible absorption of the engineered nanostructures was investigated in the wavelength range of 220\u0026ndash;700 nm. Figure\u0026nbsp;2 presents the absorption diagrams of the designed nanostructures along with the \u0026lambda;\u003csub\u003emax\u003c/sub\u003e of each one. The maximum absorption wavelength (\u0026lambda;\u003csub\u003emax\u003c/sub\u003e) of the BiAu, (Lin-T\u003csub\u003e1\u003c/sub\u003e and BiAu@NCLin-T\u003csub\u003e1\u003c/sub\u003e) and NCLin-T\u003csub\u003e1\u003c/sub\u003e are 535, 540 (Fig. 2b,c), and 548 nm, respectively.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003eEstimation of the conjugation rate\u003c/h2\u003e\n \u003cp\u003eThe concentration of oligonucleotides in conjugated samples and the concentration of primary oligonucleotides were calculated based on their absorbance at OD\u003csub\u003e260\u003c/sub\u003enm and using the Beer-Lambert equation (A\u0026thinsp;=\u0026thinsp;\u0026epsilon;dc) [\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\n \u003cp\u003eIn this equation (Absorbance, A) is the absorption (OD\u003csub\u003e260\u003c/sub\u003enm) of Lin-T\u003csub\u003e2\u003c/sub\u003e and Lin-T\u003csub\u003e1\u003c/sub\u003e sequences in two unconjugated and conjugated states, \u0026epsilon; is the molar absorption coefficient of Lin-T\u003csub\u003e2\u003c/sub\u003e and Lin-T\u003csub\u003e1\u003c/sub\u003e sequences with unit M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003ecm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, d is the length of the optical path trough the solution in cm, c is sample concentration in molar. The data regarding the percentage of conjugated oligonucleotides is presented in Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003eFT-IR analysis\u003c/h2\u003e\n \u003cp\u003eThe formation of BiAu is determined through the appearance of bands 3433 and 1638 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, in the FTIR spectrum of BiAu. The appearance of new bands such as 1270 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e confirmed the conjugation of oligonucleotides on BiAu, (Fig. 3) .\u003c/p\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003ctable id=\"Tab4\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eThe percentage of conjugation rate for the Lin-T\u003csub\u003e2\u003c/sub\u003e and Lin-T\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSamples\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePercentage of conjugation\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBiAu@NCLin-T\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e% 77.246\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBiAu@Lin-T\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e% 83.48\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBiAu@NCLin-T\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e% 83.55\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBiAu@Lin-T\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e% 71.08\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003eXRD analysis\u003c/h2\u003e\n \u003cp\u003eThe XRD analysis was employed to investigate the structural and crystalline characteristics of the BiAu. Figure 4 shows the BiAu XRD spectrum, which confirms its crystalline structure.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eTEM analysis\u003c/strong\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003cp\u003eFigure 5 displays the morphology of BiAu and the corresponding size distribution histogram. The average size of the BiAu is 11.38 \u0026plusmn; 4.38 nm (mean \u0026plusmn; SD [n=70]), as depicted in the histogram.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n \u003ch2\u003eEDS analysis\u003c/h2\u003e\n \u003cp\u003eFigure 6 displays the EDX spectra of BiAu and BiAu@NCLin-T\u003csub\u003e2\u003c/sub\u003e and the corresponding SEM images for each configuration. The designated elements in the spectrum of each nanostructure, coupled with the difference observed in the intensity of the peaks and SEM images for each nanostructure, indicate the formation of the BiAu and the conjugation of the oligonucleotide sequence with the BiAu.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n \u003ch2\u003eFluorimetric analysis of NCs\u003c/h2\u003e\n \u003cp\u003eFigure 7 presents the excitation (Ex) and emission (Em) spectra of the synthesized NCs, as well as the maximum excitation (\u0026lambda;\u003csub\u003emax\u003c/sub\u003e, Ex) and emission (\u0026lambda;\u003csub\u003emax\u003c/sub\u003e, Em) wavelengths. Lin-T\u003csub\u003e1\u003c/sub\u003e and BiAu were provided as control samples. In the spectra of Lin-T\u003csub\u003e1\u003c/sub\u003e, NCLin-T\u003csub\u003e1\u003c/sub\u003e, BiAu@Lin-T\u003csub\u003e1\u003c/sub\u003e, BiAu@NCLin-T\u003csub\u003e1\u003c/sub\u003e, BiAu@NCLin-T\u003csub\u003e2\u003c/sub\u003e, NCLin-T\u003csub\u003e2\u003c/sub\u003e and NCLin-C\u003csub\u003e2\u003c/sub\u003e, a fluorescence-induced Stokes shift was observed. The maximum emission wavelength was not observed in BiAu and BiAu@NCLin-T\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n \u003cp\u003eIn Supplementary Fig. S 3, the emission diagrams of the synthesized fluorophores are compared. The shifts observed in the fluorescence intensity and wavelength peaks indicate the formation of NCs.\u003c/p\u003e\n \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\n \u003ch2\u003eDLS analysis\u003c/h2\u003e\n \u003cp\u003eThe hydrodynamic size and zeta potential of BiAu and BiAu@Lin-T\u003csub\u003e2\u003c/sub\u003e are presented in (Fig. 8). The hydrodynamic sizes of BiAu and BiAu@Lin-T\u003csub\u003e2\u003c/sub\u003e are reported as 142.8\u0026plusmn;3.96 and 148.1\u0026plusmn;4.3 nm, respectively (Mean\u0026plusmn;SD [n=3]). Moreover, the dispersion index (PDI) has been demonstrated on each column in the chart. The zeta potential of BiAu and BiAu@Lin-T\u003csub\u003e2\u003c/sub\u003e are -5.45\u0026plusmn;0.64 and -0.35\u0026plusmn;0.35 mV, respectively (Mean\u0026plusmn;SD [n=3]).\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\n \u003ch2\u003eAntibacterial tests results\u003c/h2\u003e\n \u003cp\u003eAntibacterial tests were performed under different conditions: PDT, non-PDT, PTT, and non-PTT conditions. These tests was performed based on the concentration of the Lin-T\u003csub\u003e1\u003c/sub\u003e. The results have been reported as graphs based on the percentage of bacterial reduction (%Bacterial reduction, %BR) in each concentration. The reduction percentage has been specified on each column and has been calculated through the following formula:\u003c/p\u003e\n \u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" width=\"373\" height=\"63\"\u003e\u003c/p\u003e\n \u003cp\u003eOD\u003csub\u003e600\u003c/sub\u003e Ctrl\u003csup\u003e+\u003c/sup\u003e and OD\u003csub\u003e600\u003c/sub\u003e Sample are the absorbance of untreated and treated bacteria at a wavelength of 600 nm, respectively. Furthermore, the effect of light and temperature on the positive control (Ctrl\u003csup\u003e+\u003c/sup\u003e) was investigated. Supplementary Fig. S 4 shows the results based on the OD of the bacteria. It was found that a noteworthy distinction wasn\u0026apos;t seen within the growth rate of \u003cem\u003eE.coli\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e beneath the conditions of PDT and non-PDT. Moreover, substantial distinction wasn\u0026apos;t observed within the growth rates of both bacterial strains at temperatures of 37 and 40\u0026deg; C. The PDT effect of BiAu on two bacteria is depicted in (Supplementary Fig. S 5). In \u003cem\u003eS. aureus\u003c/em\u003e, the considerable PDT effect wasn\u0026rsquo;t observed at all four concentrations. The effect of PDT was observed in \u003cem\u003eE.coli\u003c/em\u003e at two concentrations, specifically 0.28 and 0.56 nM. BiAu showed a negligible PDT effect at 0.85 and 1.13 nM against \u003cem\u003eE.coli\u003c/em\u003e.\u003c/p\u003e\n \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\n \u003ch2\u003ePDT-induced antibacterial effects\u003c/h2\u003e\n \u003cp\u003eBiAu@Lin-T\u003csub\u003e1\u003c/sub\u003e and BiAu@Lin-T\u003csub\u003e2\u003c/sub\u003e showed PDT effects against \u003cem\u003eE.coli\u003c/em\u003e at all four concentrations. The PDT effect of both nanostructures against \u003cem\u003eS.aureus\u003c/em\u003e was observed at 3 concentrations of 1, 1.5, and 2 \u0026micro;M, (Fig. 9). BiAu@NCLin-T\u003csub\u003e1\u003c/sub\u003e exhibited the PDT effect at all four concentrations against \u003cem\u003eE.coli.\u003c/em\u003e While, BiAu@NCLin-T\u003csub\u003e2\u003c/sub\u003e had PDT effect at 3 concentrations of 0.5, 1.5, and 2 \u0026micro;M. In \u003cem\u003eS. aureus\u003c/em\u003e, the PDT effect of BiAu@NCLin-T\u003csub\u003e1\u003c/sub\u003e was observed at two concentrations of 1.5 and 2 \u0026micro;M. BiAu@NCLin-T\u003csub\u003e2\u003c/sub\u003e demonstrated the PDT effect at a concentration of 1 \u0026micro;M, (Fig. 10). Supplementary Fig. S 6\u0026ndash;9 present PDT and non-PDT effect of nanostructures Lin-T\u003csub\u003e1\u003c/sub\u003e, Lin-T\u003csub\u003e2\u003c/sub\u003e, NCLin-T\u003csub\u003e1\u003c/sub\u003e, NCLin-T\u003csub\u003e2\u003c/sub\u003e, NCLin-C\u003csub\u003e1\u003c/sub\u003e, NCLin-C\u003csub\u003e2\u003c/sub\u003e, Lin-C\u003csub\u003e1\u003c/sub\u003e and Lin-C\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\n \u003ch2\u003eComparison of the antibacterial effect in PDT condition\u003c/h2\u003e\n \u003cp\u003eIn \u003cem\u003eE.coli\u003c/em\u003e, the BiAu@Lin-T\u003csub\u003e1\u003c/sub\u003e illustrated a significant enhancement of the PDT effect at concentrations of 0.5 and 2 \u0026micro;M compared to BiAu@NCLin-T\u003csub\u003e1\u003c/sub\u003e. BiAu@Lin-T\u003csub\u003e2\u003c/sub\u003e exhibited a substantial increase in the PDT effect at concentrations of 0.5, 1, and 1.5 compared to BiAu@NCLin-T\u003csub\u003e2\u003c/sub\u003e. At a concentration of 2 \u0026micro;M, the PDT effect of BiAu@NCLin-T\u003csub\u003e2\u003c/sub\u003e showed a substantial augmentation compared to BiAu@Lin-T\u003csub\u003e2\u003c/sub\u003e, (Fig. 11a,c). In \u003cem\u003eS. aureus\u003c/em\u003e, the antibacterial effect of BiAu@NCLin-T\u003csub\u003e1\u003c/sub\u003e at concentrations of 1.5 and 2 \u0026micro;M has increased compared to BiAu@Lin-T\u003csub\u003e1\u003c/sub\u003e. BiAu@Lin-T\u003csub\u003e2\u003c/sub\u003e has exhibited a considerable increase at 3 concentrations of 1, 1.5, and 2 \u0026micro;M compared to BiAu@NCLin-T\u003csub\u003e2\u003c/sub\u003e, (Fig. 11b,d).\u0026apos;\u003c/p\u003e\n \u003cp\u003eSupplementary Fig. S 10-13\u0026nbsp;present the antibacterial performance of BiAu@Lin-T\u003csub\u003e1\u003c/sub\u003e, BiAu@Lin-T\u003csub\u003e2\u003c/sub\u003e, Lin-T\u003csub\u003e1\u003c/sub\u003e, Lin-T\u003csub\u003e2\u003c/sub\u003e, BiAu, NCLin-T\u003csub\u003e1\u003c/sub\u003e, NCLin-T\u003csub\u003e2\u003c/sub\u003e, NCLin-C\u003csub\u003e2\u003c/sub\u003e, NCLin-C\u003csub\u003e1\u003c/sub\u003e, Lin-C\u003csub\u003e1\u003c/sub\u003e,\u003csub\u003e\u0026nbsp;\u003c/sub\u003eand Lin-C\u003csub\u003e2\u003c/sub\u003e. The antibacterial effect of nanostructures in non-PDT conditions is presented in the supplementary material with the level 3 heading \u0026ldquo;Antibacterial comparison under non-PDT conditions\u0026rdquo;.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\n \u003ch2\u003ePTT-induced antibacterial effect\u003c/h2\u003e\n \u003cp\u003eIn Fig. 12, the antibacterial effect of the nanostructures at a temperature of 40\u0026deg;C was compared with the antibacterial effect under the condition of non-photo stimulation (temperature of 37\u0026deg;C). All three mentioned structures demonstrated a substantial PTT effect on \u003cem\u003eE.coli\u003c/em\u003e. BiAu@NCLin-T\u003csub\u003e2\u003c/sub\u003e and BiAu in \u003cem\u003eS. aureus\u003c/em\u003e showed a substantial PTT effect in all four concentrations.\u003c/p\u003e\n \u003cp\u003eSupplementary Fig. S 19 shows the comparison between the antibacterial effect of nanostructures at a temperature of 40\u0026deg;C and the antibacterial efficacy under light stimulation conditions (temperature of 37\u0026deg;C).\u0026nbsp;\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\n \u003ch2\u003eSEM analysis\u003c/h2\u003e\n \u003cp\u003eThe morphological effects of aPDT were investigated by choosing the NCLin-T\u003csub\u003e2\u003c/sub\u003e (with concentrations of 1.5 \u0026micro;M for \u003cem\u003eE.coli\u003c/em\u003e and 2 \u0026micro;M for \u003cem\u003eS.aureus\u003c/em\u003e). Images (a) and (b) in (Fig. 13) exhibit the untreated and treated \u003cem\u003eE.coli\u003c/em\u003e, respectively. The untreated \u003cem\u003eS.aureus\u003c/em\u003e bacteria are presented in (Fig. 13c) and the treated one is displayed in (Fig. 13d).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eBased on computational studies, Lin-T\u003csub\u003e2\u003c/sub\u003e, Lin-C\u003csub\u003e2\u003c/sub\u003e, and Lin-C\u003csub\u003e1\u003c/sub\u003e are stable at all three temperatures. Lin-T\u003csub\u003e2\u003c/sub\u003e had a low increase in free energy with rising temperature, while Lin-C\u003csub\u003e2\u003c/sub\u003e and Lin-C\u003csub\u003e1\u003c/sub\u003e had a free energy of -0.00 kcal/mol, Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The MFE and Centroid structures of all three sequences confirm their stability at the three temperatures, (Fig.\u0026nbsp;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 \u0026micro;M) and salt concentrations [Na\u003csup\u003e+\u003c/sup\u003e (1 M), Mg\u003csup\u003e+\u003c/sup\u003e (0.0 M)]. Lin-C\u003csub\u003e2\u003c/sub\u003e and Lin-C\u003csub\u003e1\u003c/sub\u003e suggest a lack of base pair formation, while Lin-T\u003csub\u003e2\u003c/sub\u003e showed a low pairing probability of A and T bases but remained stable, (Supplementary Fig. S 1). The free energy of the Lin-T\u003csub\u003e2\u003c/sub\u003e 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).\u003c/p\u003e \u003cp\u003eThe absorption spectra of designed structures indicate the formation of AgNCs and oligonucleotide sequence conjugation. Figure\u0026nbsp;2a exhibits a decrease (hypochromic shift) in the absorption peak of BiAu after conjugation with Lin-T\u003csub\u003e2\u003c/sub\u003e. After using Lin-T\u003csub\u003e1\u003c/sub\u003e to form AgNC (NCLin-T\u003csub\u003e1\u003c/sub\u003e), a new peak appeared at 440 nm in the absorption spectrum of NCLin-T\u003csub\u003e1\u003c/sub\u003e, (Fig.\u0026nbsp;2c). Conjugation between NCLin-T\u003csub\u003e1\u003c/sub\u003e and BiAu is displayed in (Fig.\u0026nbsp;2b). Both spectra showed an increase in absorption (hyperchromic shift). Figure\u0026nbsp;2d presents the conjugation between Lin-T\u003csub\u003e1\u003c/sub\u003e and BiAu. Lin-T\u003csub\u003e1\u003c/sub\u003e spectrum showed the increase in absorption. BiAu spectrum showed the decrease in absorption. Figure\u0026nbsp;2 (e) shows the absorption spectrum of BiAu@NCLin-T\u003csub\u003e2\u003c/sub\u003e. BiAu and NCLin-T\u003csub\u003e2\u003c/sub\u003e 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-T\u003csub\u003e2\u003c/sub\u003e scaffold, as shown in (Fig.\u0026nbsp;2f). In Fig.\u0026nbsp;2 (g), a new peak at 445 nm confirms the formation of NCLin-C\u003csub\u003e1\u003c/sub\u003e. The hypochromic shift of Lin-C\u003csub\u003e2\u003c/sub\u003e spectrum (at 260 nm) and emerging of new peak (430 nm) of NCLin-C\u003csub\u003e2\u003c/sub\u003e spectrum confirm the formation of NCLin-C\u003csub\u003e2\u003c/sub\u003e, (Fig.\u0026nbsp;2h). Figure\u0026nbsp;2i exhibits absorption spectra of NCLin-T\u003csub\u003e2\u003c/sub\u003e, NCLin-C\u003csub\u003e2\u003c/sub\u003e, and NCLin-C\u003csub\u003e1\u003c/sub\u003e. NCLin-T\u003csub\u003e2\u003c/sub\u003e has the highest absorption intensity due to the addition of A and T to 12 C in Lin-T\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eBiAu@NCLin-T\u003csub\u003e1\u003c/sub\u003e, BiAu@Lin-T\u003csub\u003e1\u003c/sub\u003e, BiAu@NCLin-T\u003csub\u003e2\u003c/sub\u003e, and BiAu@Lin-T\u003csub\u003e2\u003c/sub\u003e possess elevated conjugation efficiency and density of oligonucleotides and NCs on BiAu, Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Lin-T\u003csub\u003e1\u003c/sub\u003e and Lin-T\u003csub\u003e2\u003c/sub\u003e 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 [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], so the presence of BSA in a part of the BiAu increases the percentage of conjugation.\u003c/p\u003e \u003cp\u003eIn the FT-IR spectra of BiAu and BiAu @LinT\u003csub\u003e2\u003c/sub\u003e, (Fig.\u0026nbsp;3), appeared amide I bands of BSA at 1638 cm \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Stretching vibrations band of O-H at 3433 cm \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] and band at 1033.74 in BiAu shifted to 3434 and 1074.78 cm \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e bands in BiAu@LinT\u003csub\u003e2\u003c/sub\u003e, respectively, which showed the conjugation of LinT\u003csub\u003e2\u003c/sub\u003e to BiAu. The new peak appeared in the BiAu@LinT\u003csub\u003e2\u003c/sub\u003e spectrum at 1270 cm \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e assigned to C\u0026ndash;O stretching vibration [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. These new peaks can be assigned to the interaction of oligonucleotides with hybrid nanostructures.\u003c/p\u003e \u003cp\u003eThe XRD pattern of BiAu hybrid nanostructures is presented in (Fig.\u0026nbsp;4). The diffraction pattern corresponds to the Au\u003csub\u003e2\u003c/sub\u003eBi\u003csub\u003e3\u003c/sub\u003e standard XRD card of JCPDS 21\u0026ndash;0099 and the Au standard card of JCPDS 04-0784 with hexagonal and cubic structures, respectively.\u003c/p\u003e \u003cp\u003eNPs have spherical shapes with a limited distribution size (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The composition of BiAu was confirmed with the TEM image. Due to high electron density, AuNPs are darker than Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e NPs (bright) in one hybrid nanostructure [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBased on the EDX spectrum from BiAu (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e6\u003c/span\u003e), the presence of Bi and S (BiAu spectrum) indicates the hybridization of AuNPs with Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e. The existence of C, S, N, and O can be due to the BSA-mediated biomineralization of Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e, (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Two other elements, Ag and P, were also found in the spectrum of BiAu @NCLin-T\u003csub\u003e2\u003c/sub\u003e, which signify the formation of NCLin-T\u003csub\u003e2\u003c/sub\u003e and its conjugation on the BiAu (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e6\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eFigure 7 shows the confirmation of the formation of NCs and designed fluorophores through the observed Stokes shifts. The structures of NCLin-T\u003csub\u003e2\u003c/sub\u003e and NCLin-C\u003csub\u003e2\u003c/sub\u003e, (Fig.\u0026nbsp;7a,b) have large Stokes shifts (116 nm (NCLin-T\u003csub\u003e2\u003c/sub\u003e) and 106 nm (NCLin-C\u003csub\u003e2\u003c/sub\u003e)). NCLin-T\u003csub\u003e1\u003c/sub\u003e and Lin-T\u003csub\u003e1\u003c/sub\u003e, (Fig.\u0026nbsp;7f,e) exhibit Stokes shifts of 43.5 nm and 31.5 nm, respectively. BiAu@NCLin-T\u003csub\u003e1\u003c/sub\u003e and BiAu@Lin-T\u003csub\u003e1\u003c/sub\u003e, (Fig.\u0026nbsp;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-T\u003csub\u003e2\u003c/sub\u003e, (Fig.\u0026nbsp;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\u003csup\u003e+\u003c/sup\u003e 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 [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The addition of A and T to 12 C in Lin-T\u003csub\u003e2\u003c/sub\u003e increases fluorescence intensity and Stokes shift [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The distance of less than ten nm (see the supplementary material, the level 3 heading \u0026ldquo;The length of Lin-T\u003csub\u003e2\u003c/sub\u003e\u0026rdquo;) between 5'HEX and AgNC in BiAu@NCLin-T\u003csub\u003e1\u003c/sub\u003e, leads to the FRET (F\u0026Ouml;rster resonance energy transfer) phenomenon [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The emission spectrum of NCLin-T\u003csub\u003e1\u003c/sub\u003e exhibited hypochromic shift following conjugation with BiAu (Supplementary Fig. S 3e). In BiAu@NCLin-T\u003csub\u003e2\u003c/sub\u003e, (Supplementary Fig. S 3g), the BiAu has resulted in the quenching of NCLin-T\u003csub\u003e2\u003c/sub\u003e emission spectrum. The use of AuNPs in fluorescence quenching systems results in high-efficiency quenchers [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe size of BiAu is larger than its crystalline size due to the portion consisting of BSA that can be hydrated in water. BiAu@LinT\u003csub\u003e2\u003c/sub\u003e has a higher hydrodynamic size, indicating conjugation with Lin-T\u003csub\u003e2\u003c/sub\u003e. Both BiAu and BiAu@Lin-T\u003csub\u003e2\u003c/sub\u003e are polydisperse and have a negative surface charge within the \u0026minus;\u0026thinsp;10 and +\u0026thinsp;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 [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. BiAu@Lin-T\u003csub\u003e2\u003c/sub\u003e has a more zeta potential than BiAu, indicating that Lin-T\u003csub\u003e2\u003c/sub\u003e 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.\u0026nbsp;8).\u003c/p\u003e \u003cp\u003eThe study investigated the antibacterial effect of BiAu@NCLin-T\u003csub\u003e1\u003c/sub\u003e as a nano photosensitizer (NPS) in PDT conditions compared to non-PDT states. Under PDT states, the antibacterial effect of BiAu@NCLin-T\u003csub\u003e1\u003c/sub\u003e on \u003cem\u003eE.coli\u003c/em\u003e increased in all concentrations, while a slight effect was observed in non-PDT conditions. BiAu@NCLin-T\u003csub\u003e1\u003c/sub\u003e had the most pronounced PDT effect on \u003cem\u003eE.coli\u003c/em\u003e at concentrations 1.5 \u0026micro;M, (Fig.\u0026nbsp;10a). BiAu@NCLin-T\u003csub\u003e2\u003c/sub\u003e had a greater PDT effect on gram-negative bacteria than gram-positive, with considerable effects observed against \u003cem\u003eE.coli\u003c/em\u003e at 1.5 and 2 \u0026micro;M, (Fig.\u0026nbsp;10c). Among all nanostructures, BiAu@Lin-T\u003csub\u003e2\u003c/sub\u003e had the most considerable PDT effect against gram-positive strains at concentration of 2 \u0026micro;M, (Fig.\u0026nbsp;9d).\u003c/p\u003e \u003cp\u003eBiAu 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. Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e-based nanomaterials exhibit poor toxicity. They are employed as a theranostic agent in X-ray computed tomography and near-infrared light-induced PTT [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAccording to (Supplementary Fig. S 7), both NCLin-T\u003csub\u003e1\u003c/sub\u003e and NCLin-T\u003csub\u003e2\u003c/sub\u003e 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 \u0026micro;M) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Under photo stimulation conditions, NCLin-T\u003csub\u003e1\u003c/sub\u003e has a heightened antibacterial effect against \u003cem\u003eE.coli\u003c/em\u003e, at 1.5 \u0026micro;M concentration, (Supplementary Fig. S 7a). In \u003cem\u003eS.aureus\u003c/em\u003e, the PDT-induced antibacterial effect of NCLin-T\u003csub\u003e1\u003c/sub\u003e has increased at concentrations of 1, 1.5, and 2 \u0026micro;M, (Supplementary Fig. S 7b). AgNCs generate Ag\u003csup\u003e+\u003c/sup\u003e, leading to an enhanced aPDTeffect [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. According to the findings of antibacterial outcomes, the original nanosystem (BiAu@NCLin-T\u003csub\u003e1\u003c/sub\u003e) had the most antibacterial impact in PDT conditions. The highest %BR of BiAu@NCLin-T\u003csub\u003e1\u003c/sub\u003e was observed in \u003cem\u003eE.coli\u003c/em\u003e and \u003cem\u003eS.aureus\u003c/em\u003e at concentrations of 1.5 and 2 \u0026micro;M, respectively, (Supplementary Fig. S 20).\u003c/p\u003e \u003cp\u003eBiAu@NCLin-T\u003csub\u003e1\u003c/sub\u003e and BiAu@Lin-T\u003csub\u003e1\u003c/sub\u003e had similar Stokes shifts and lower fluorescence intensity than NCLin-T\u003csub\u003e1\u003c/sub\u003e, NCLin-T\u003csub\u003e2\u003c/sub\u003e, NCLin-C\u003csub\u003e2\u003c/sub\u003e, and Lin-T\u003csub\u003e1\u003c/sub\u003e. BiAu@Lin-T\u003csub\u003e1\u003c/sub\u003e showed great aPDT activity against gram-negative strains compared to Lin-T\u003csub\u003e1\u003c/sub\u003e at all four oligonucleotide concentrations. BiAu@NCLin-T\u003csub\u003e1\u003c/sub\u003e demonstrated aPDT at 1 and 1.5 \u0026micro;M concentrations against the gram-negative strains, (Fig.\u0026nbsp;10a), as well as against the gram-positive strains at the concentration of 2 \u0026micro;M, (Fig.\u0026nbsp;10b). BiAu@NCLin-T\u003csub\u003e2\u003c/sub\u003e also had aPDT against gram-negative strains at certain concentrations (Fig.\u0026nbsp;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 [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBased on SEM analysis, \u003cem\u003eE.coli\u003c/em\u003e, and \u003cem\u003eS. aureus\u003c/em\u003e, which weren't treated with NCLin-T\u003csub\u003e2\u003c/sub\u003e are presented in (Fig.\u0026nbsp;13a,c), respectively, and possess healthy and intact surfaces. \u003cem\u003eE.coli\u003c/em\u003e treated with NCLin-T\u003csub\u003e2\u003c/sub\u003e (at the oligonucleotide concentration of 1.5 \u0026micro;M) 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.\u0026nbsp;13b). \u003cem\u003eS.aureus\u003c/em\u003e treated with NCLin-T\u003csub\u003e2\u003c/sub\u003e (at the oligonucleotide concentration of 2 \u0026micro;M) under photo stimulation conditions after 22 hours is illustrated in (Fig.\u0026nbsp;13d). The observed damages include dilatation and cell lysis. NCLin-T\u003csub\u003e2\u003c/sub\u003e has the %BR of %37 against \u003cem\u003eE.coli\u003c/em\u003e at the concentration of 1.5 \u0026micro;M and under photo stimulation conditions. This structure exhibits the %BR of 45.86% against \u003cem\u003eS. aureus\u003c/em\u003e at the concentration of 2 \u0026micro;M (under photo-stimulation conditions). The mentioned %BR from NCLin-T\u003csub\u003e2\u003c/sub\u003e confirm the damage and morphological alterations on gram-negative and gram-positive strains.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eAMR is a global threat to public health, affecting the control and treatment of infectious diseases. Inappropriate use of antimicrobial agents and lack of access to new drugs contribute to the crisis of AMR. We designed nanoplatforms to combat AMR. BiAu can have various functions, including PTT-agent for infectious and non-infectious diseases. It applies to drug delivery and diagnostics. The sequence of Lin-T\u003csub\u003e1\u003c/sub\u003e and Lin-T\u003csub\u003e2\u003c/sub\u003e can be used as templates for the formation of AgNCs. They can also be used to design new nanoplatforms with BiAu. According to the fluorimetric analysis of BiAu@NCLin-T\u003csub\u003e1\u003c/sub\u003e, BiAu@Lin-T\u003csub\u003e1\u003c/sub\u003e, BiAu@NCLin-T\u003csub\u003e2\u003c/sub\u003e, NCLin-T\u003csub\u003e1\u003c/sub\u003e, NCLin-T\u003csub\u003e2\u003c/sub\u003e, and NCLin-C\u003csub\u003e2\u003c/sub\u003e, they can be used as FRET biosensors and fluorescence quenching systems in colorimetric sensors. Different sequences and nanostructures, except Lin-C\u003csub\u003e1\u003c/sub\u003e and Lin-C\u003csub\u003e2\u003c/sub\u003e, can act as PSs, PTT, or antibacterial agents under various conditions. For example, BiAu@NCLin-T\u003csub\u003e1\u003c/sub\u003e and BiAu@NCLin-T\u003csub\u003e2\u003c/sub\u003e can be used as new PS for the removal of gram-negative bacteria. BiAu@Lin-T\u003csub\u003e2\u0026nbsp;\u003c/sub\u003ecan be used to combat gram-positive bacteria-induced infections.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article (and its Supplementary Information files).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Siamak Javani: Supervision, Conceptualization, Editing, Methodology, Investigation, Validation Hamed Nosrati: Validation, Methodology Naser Faraji: Software, Editing Atiyeh Nomani: Methodology, Conceptualization, Data curation, Writing, Original draft preparation, Software, Visualization, Editing, Writing- Reviewing, Formal analyses Investigation Jalil Charmi: Methodology.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest\u0026hellip;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported financially by the Golestan University of Medical Sciences financial support (Grant number, 31-111679).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSteinman, A. \u0026amp; Navon-Venezia, S. 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Colloids and Surfaces A: Physicochem Eng Asp. 592(4):124577; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.colsurfa.2020.124577\u003c/span\u003e\u003cspan address=\"10.1016/j.colsurfa.2020.124577\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2020)\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"antimicrobial drug resistance, photosensitizing agents, nanostructure, phototherapy","lastPublishedDoi":"10.21203/rs.3.rs-4648298/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4648298/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAntimicrobial resistance (AMR) leads to a decrease in the adequacy of antimicrobial agents and an increase in the rate of adverse effects and mortality. The main objective of this project is to investigate the synergistic effect of BiAu@NCLin-T\u003csub\u003e1\u003c/sub\u003e and its substructures as an antimicrobial photodynamic therapy (aPDT) agent to combat microbial resistance. In addition, the effect of photothermal therapy (PTT) on some of the designed nanostructures at a temperature of 40\u0026deg;C was also tested. The antimicrobial test was carried out using the growth curve method against \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e as Gram-negative and positive model bacteria. Computational methods were used to investigate the stability and entropy of oligonucleotide sequence structures. Various analyses were performed to identify the nanostructures, including Ultraviolet-visible (UV-vis) spectroscopy, Fourier-transform infrared spectroscopy (FTIR), dynamic light scattering (DLS), transmission electron microscopy (TEM), X-ray diffraction (XRD), scanning electron microscopy (SEM), energy-dispersive X-ray analysis (EDS) and fluorescence analysis. The BiAu@NCLin-T\u003csub\u003e1\u003c/sub\u003e appeared the significant aPDT impact against the gram-negative \u003cem\u003eE.coli\u003c/em\u003e strain at two distinctive oligonucleotide concentrations (1, and 1.5 \u0026micro;M). Based on the results, the outlined nanostructures can act as a photosensitizer (PS), a photothermal treatment agent (PTT), and an antimicrobial agent to combat resistant bacteria.\u003c/p\u003e","manuscriptTitle":"Bismuth-gold nanohybrid conjugated with a HEX-bound oligonucleotide; a novel nano photosensitizer to combat antimicrobial resistance","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-23 21:58:47","doi":"10.21203/rs.3.rs-4648298/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2024-07-13T08:33:06+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-04T10:38:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"314893677893956929973141163369814508275","date":"2024-07-03T06:37:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"192462436642223698031073393957135532580","date":"2024-07-03T04:50:38+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-03T00:17:19+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-03T00:05:01+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-07-02T20:22:38+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-01T05:06:07+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-06-27T11:45:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9e3feedc-1dad-49d7-8884-f5159ffe4d25","owner":[],"postedDate":"July 23rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":34543324,"name":"Biological sciences/Drug discovery"},{"id":34543325,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2024-10-07T16:12:45+00:00","versionOfRecord":{"articleIdentity":"rs-4648298","link":"https://doi.org/10.1038/s41598-024-74273-z","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-09-30 15:57:09","publishedOnDateReadable":"September 30th, 2024"},"versionCreatedAt":"2024-07-23 21:58:47","video":"","vorDoi":"10.1038/s41598-024-74273-z","vorDoiUrl":"https://doi.org/10.1038/s41598-024-74273-z","workflowStages":[]},"version":"v1","identity":"rs-4648298","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4648298","identity":"rs-4648298","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}