Singlet oxygen generation by hybrid rhodamine B-gold nanostructures in chitosan biomolecule environment: an EPR study

Singlet oxygen generation by hybrid rhodamine B-gold nanostructures in chitosan biomolecule environment: an EPR study | 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 Research Article Singlet oxygen generation by hybrid rhodamine B-gold nanostructures in chitosan biomolecule environment: an EPR study Jelena Pajović, Radovan Dojčilović, Dušan Milivojević, Dušan K. Božanić, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6595834/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Gold nanoparticles of various shapes were synthesized by reduction of gold salts in the presence of chitosan biomolecules as stabilizing agents. Fluorescence and photosensitizing properties of the rhodamine B dye were studied after its mixing with pure chitosan and chitosan-gold nanoparticle solutions. It was found that gold nanoparticles significantly affect the fluorescence intensity and the singlet oxygen production of the photosensitizer. Metal-enhanced fluorescence and metal-enhanced singlet oxygen generation effects were observed, probably as a direct consequence of the activation of the surface plasmon of the nanoparticles upon irradiation. Photosensitizing activity of the rhodamine B dye was investigated by using electron paramagnetic resonance (EPR) spectroscopy with TEMP as spin-trap molecules. The singlet oxygen generation was followed via changes in the intensity of EPR signal of the radical adduct, TEMPO. It was found that gold nanoparticles facilitate the production of singlet oxygen, while the chitosan molecules influence TEMPO stability and tend reduce the intensity of the EPR signal, especially at prolonged times following the irradiation. chitosan gold nanoparticles photosenzitizer singlet oxygen rhodamine B electron-paramagnetic resonance Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Rhodamine B (RhB) belongs to the class of hydrophilic xanthene derivatives and it is widely used as a dye in various industries. It exhibits high absorption in the visible region, coupled with moderate to high fluorescence quantum yield [ 1 ]. Also, it is very sensitive to the polarity of the solvent and undergoes spirolactam/ring-opening structural transformations as acidity increases. Since the open form is fluorescent and the spirolactam form is not, RhB, and rhodamine derivatives in general, are extensively studied as pH fluorescence sensors [ 2 – 4 ]. Rhodamine B shows high photosensitizing activity [ 5 , 6 ] and it has been studied as an active molecule for photodynamic therapy (PDT) of cancer [ 6 , 7 ]. Ngen et al . [ 7 ] conjugated rhodamine B to porphyrin in order to create mitochondria-targeting photosensitizers that can absorb light in near-infrared region, where its penetration through tissues is the most efficient. RhB was introduced as an additional moiety component to the well-known porphyrin photosensitizer due to its large two-photon absorption cross section at 800 nm. The two-photon excitation of RhB and the resonant energy transfer (RET) between the components enabled efficient singlet oxygen production at low near-infrared incident energies. Recently, surface modification of inorganic nanoparticles with functional molecules emerged as route for fabrication of hybrid materials for therapeutic and diagnostic applications [ 8 , 9 ]. We used this approach in our previous studies, where we functionalized noble-metal nanoparticles with fluorescent biomolecules to influence their optical properties and test them as probes for fluorescent bioimaging [ 10 – 14 ]. The molecules from the rhodamine-family proved to be good capping agents for inorganic particles providing functionalities suitable for imaging and photodynamic therapy (PDT) applications [ 15 , 16 ]. Pallavi et al [ 16 ] showed that photosensitizing potency of rhodamine 6G can be improved by its attachment to the surface of gold nanoparticles (Au NPs). This method also enables an increase in the uptake of photosensitizer molecules by the cells, which is a necessary condition for the efficient PDT treatment. Also, rhodamine capped gold nanoparticles were successfully used for the detection of Cr 3+ ion in living cells and contaminated waters [ 17 ]. Finally, it is important to emphasize that in vitro and in vivo cytotoxicity tests on zebrafish embryos revealed that these hybrid particles were fairly safe i.e. non-toxic [ 16 ]. In a series of preceding studies [ 18 – 24 ], we have shown that polysaccharide biopolymers such as starch, chitosan and alginate can be used as controlled environments for the synthesis of inorganic nanoparticles. In the present paper, we used polysaccharide chitosan, derived by deacetylation from the highly abundant biopolymer chitin, to control the growth of the gold the nanoparticles. Chitosan biomolecules possess both amino and hydroxyl groups exhibiting excellent chelating properties and enable fabrication of the nanoparticles with relatively narrow size distribution [ 25 ]. The obtained gold nanoparticles in chitosan were further functionalized with rhodamine B photosensitizers. Electron paramagnetic resonance (EPR) spectroscopy was used to study the changes in singlet oxygen production after the RhB molecules were added to the gold-chitosan colloid. Experimental Materials Gold(III) chloride trihydrate (HAuCl 4 ·3H 2 O), chitosan, D-glucose, rhodamine B and 2,2,6,6-tetramethylpiperidine (TEMP, a spin trap molecule) were purchased from Sigma–Aldrich and used as received. High-purity water was used in all procedures. Sample preparation The gold nanoparticles in chitosan were prepared by using the D-glucose as reducing agent for tetrachloroaurate ions. Briefly, an aqueous solution of HauCl 4 was mixed with chitosan dissolved in 1% acetic acid solution. The concentration of chitosan in the solution was kept constant at 0.5 wt./vol.%, while the concentrations of the gold ions varied between 0.15 mM and 0.375 mM. An appropriate amount of 0.1 M D-glucose solution was added to the gold-chitosan mixture and stirred for 24 h in the dark at room temperature. The mixture gradually changed the colour to dark red. The list of the samples with corresponding preparation conditions are given in the Table 1 . Table 1 The list of samples and the amount of chemicals used in fabrication. Label [Au] (10 − 5 M) Chitosan (wt./vol. %) 0.1 M D-glucose (ml) Chitosan 0 0.5 0 ChAu 3.75 0.5 0.25 ChAu2 7.50 0.5 0.5 ChAu4 15 0.5 1 The obtained gold-chitosan samples were further mixed with rhodamine B dye solution [5·10 − 4 M]. The final RhB concentration in each of the samples was 5 µM. For comparison, the same set of the samples was also prepared by mixing RhB with the solution of the pure chitosan instead of the colloid. The four-step procedure for the sample preparation is schematically presented in Fig. 1 . Methods Absorbance spectra were recorded on a Thermo Evolution 600 UV–vis spectrophotometer in the range from 350 to 900 nm. Photoluminescence (PL) spectroscopy studies were carried out on PerkinElmer LS45 fluorescence spectrophotometer. The excitation wavelength was 530 nm. For PL measurements, each sample containing rhodamine B was diluted by factor 1000 due to extremely strong fluorescence intensity signal coming from the dye. EPR Spectrometer MiniScope 300, Magnettech, Berlin, Germany, operating at a nominal frequency of 9.5 GHz, was used for electron paramagnetic resonance measurements. The spectra were obtained at room temperature. The microwave power was 1 mW (microwave attenuation of 20 dB) with a modulation amplitude of 0.2 mT. The Hamamatsu spotlight LC8, λ > 300 nm, was used as a UV light source. The production of the singlet oxygen ( 1 O 2 ) by the samples was followed via spin trap method. The formation of 1 O 2 was detected through its specific reaction with spin trap TEMP (2,2,6,6-tetramethylpiperidine) molecule. The reaction produces a radical adduct, TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl) with a stable paramagnetic response. Results and discussion Figure 2 a shows TEM micrographs of ChAu samples prepared with various initial concentrations of gold salts. The reduction of the gold ions in the presence of chitosan molecules results in formation of nanoparticles with various shapes. Besides spherical particles, the particles in form of discs, rectangles and triangles can be observed (Fig. 2 a). The corresponding particle size distributions obtained from TEM analysis are shown in Fig. 2 b. The average size of the particles increases with increasing in concentration of gold salts, but the changes are within the standard deviation of the particle distributions. The solution of the pure chitosan and the obtained gold-chitosan colloid samples were further mixed with fixed amount of rhodamine B aqueous solution. The UV-vis absorption spectra of the Ch-RhB, ChAu and ChAu-RhB samples are shown in Fig. 3 (a). The ChAu sample exhibits strong absorption at ~ 540 nm, which originates from surface plasmon resonance (SPR) of gold nanoparticles. Also, SPR peak of Au NPs and the RhB absorption peak partially overlap and the absorption spectrum of the ChAu-RhB is the result of the superposition of the spectra of the components. It should be noted that the UV-vis absorption spectra (not shown) of the ChAu-RhB, ChAu2-RhB and ChAu4-RhB samples showed that the change in the initial concentration of the gold ions from 3.75·10 − 5 M to 15·10 − 5 M did not affect the position of the absorption peak, only its intensity. Figure 3 b shows photoluminescent spectra of the pure Ch-solution, chitosan-gold colloid and their corresponding mixtures with rhodamine B. It can be noticed that there is a strong increase in fluorescence intensity of rhodamine B after its interaction with gold nanoparticles. This is a consequence of plasmon-enhanced fluorescence effect [ 26 – 28 ]. Namely, metallic particles can affect both emission efficiency (e.g. quantum yield) and fluorescence lifetime of the fluorophore placed in the vicinity of their surfaces [ 26 ]. As can be seen in Fig. 3 a, the position of the absorption SPR peak of gold nanoparticles at ~ 540 nm is close to the excitation wavelength used in the PL measurements. The excitation of the surface plasmon leads to the enhanced near-field intensity of the electric field surrounding the particle. This in turn increases the rate of excitation of the rhodamine B and, consequently, results in overall higher fluorescence. Another effect that should also be taken into account is that the nanoparticles influence the deexcitation dynamics of the nearby excited fluorophore. The coupling can result in appearance of new nonradiative deexcitation pathways, which in turn decreases the lifetime of the excited states of the molecule. This effect also leads to an increased molecular photostability and prolonged photophysical activity of the molecule [ 29 ]. Both effects can be responsible for the enhanced photosensitizing activity of the rhodamine B in the presence of nanoparticles [ 30 – 32 ], as the results of EPR spectroscopy presented below show. Formation of singlet oxygen via UV-illumination of RhB (in water), Ch-RhB and ChAuRhB samples were studied by EPR spectroscopy. EPR spectra of the TEMPO radicals formed through singlet oxygen interaction with TEMP spin trap were shown in Fig. 4 . As can be seen, photosensitization activity of rhodamine B strongly depends on the physicochemical environment surrounding the photosensitizer. The intensity of the EPR signal is the lowest in the case when the rhodamine B is in the chitosan solution. This might be due to the fact that chitosan molecules can generate other reactive radicals [33] that can interact with TEMPO and decrease its EPR signal [ 34 – 35 ]. In contrast, the signal intensity increases when the rhodamine B is in the vicinity of metallic surface (Fig. 4 ). The sensitizing activity is stronger than the activity in water, even if the photosensitizer is surrounded by chitosan molecules. The mechanism of the metal-enhanced singlet oxygen generation is of the same nature as in the case of the metalenhanced fluorescence discussed above [36]. The enhancement of the electric field around the Au NP increases the number of photosensitizer molecules in the singlet excited state, which, due to intersystem crossing transitions, leads to a higher population of rhodamine B molecules in their triplet states. This, in turn, increases the rate of singlet oxygen generation. The enhanced generation of singlet oxygen can also be the effect of the increased photostability of photosensitizer, which increases the probability of its interaction with surrounding oxygen molecules. Although singlet oxygen generation and fluorescence are competing processes, they can be both enhanced via the interaction of a fluorophore/photosensitizer with plasmonic nanostructure. However, the contribution of the metal-enhanced electric field surrounding the particle to the each of the processes might be different. Metallic particle might also quench the fluorescence of the fluorophore in its vicinity, without influencing the singlet oxygen generation process [36]. On the other hand, chitosan biomolecules can also affect stability of the EPR signal. Figure 5 a shows the EPR spectra of RhB, as Ch-RhB and ChAu-RhB samples recorded in the moment of irradiation and, successively, every five minutes after the irradiation. The dependence of the normalized signal intensities from time are presented in Fig. 5 b. The EPR signal intensity of the RhB sample, where photosensitizer is surrounded solely with water molecules, is fairly stable in time (Fig. 5 b). In contrast, when RhB is in chitosan solution, the relative intensity of the signal gradually decreases. This behaviour was also noticed in the case of ChAu-RhB sample, but the decrease in relative intensity of the signal is less pronounced and starts with a delay. Obviously, the instability of the EPR signal with time in photosensitizer’s environments other than water should be taken into account if this method is used for the detection of the singlet oxygen generation. Conclusions Fluorescence and photosensitizing activity of rhodamine B strongly depends on its physicochemical environment. Both, metal-enhanced fluorescence and metal-enhanced singlet oxygen generation was observed when rhodamine B was introduced into gold nanoparticle-chitosan solution. The obtained results were discussed in terms of an increase in the near-field intensity of electrical filed surrounding the gold nanoparticles due to their metallic nature. When rhodamine B is in the proximity of the particle surfaces, this effect can increase the rate of the excitation and consequently enhance either the fluorescence or the generation of singlet oxygen via interaction of oxygen molecules with its triplet states. The generation of singlet oxygen by rhodamine B was studied in various environments (water, chitosan and chitosan-gold nanoparticle solutions) by using spin-trap method and EPR spectroscopy. The chitosan proved to be a good agent for the control of the growth of gold nanoparticles, but chitosan biomolecules may influence the TEMPO radicals formed after the interaction of singlet oxygen with TEMP spin-trap molecules. For this reason, they reduced the intensity of the EPR signal obtained. Nevertheless, due to metal-enhanced singlet oxygen generation effect, an excess of the single oxygen was observed after illumination of the ChAu-RhB sample, with respect to that obtained with illumination of rhodamine B water solution. Declarations Acknowledgments Funding The research was funded by the Ministry of Science, Technological development and Innovation of the Republic of Serbia under Grant Agreement No 451-03-66/2024-03/200017 and 451-03-66/2024-03/200162. Ethical statement: No statement required Author contribution statement J.P. and R.D. preparation of the samples; J.P. and R.D. D.K.B. fluorescent and UV Vis absorption spectroscopy measurements, data analysis and visualization; D.M. EPR spectroscopy measurements, data analysis; D.K.B. and V. Dj. conceived the project and the methodology; V.Dj. writing, reviewing and editing the paper. 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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-6595834","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":476388669,"identity":"10b171bc-1b89-4a68-aff2-16b5e138ffcd","order_by":0,"name":"Jelena Pajović","email":"","orcid":"","institution":"Faculty of Physics, University of Belgrade","correspondingAuthor":false,"prefix":"","firstName":"Jelena","middleName":"","lastName":"Pajović","suffix":""},{"id":476388670,"identity":"e8002b48-e7c7-4628-82e5-b87ffb249317","order_by":1,"name":"Radovan Dojčilović","email":"","orcid":"","institution":"Vinča Institute of Nuclear Sciences, National Institute of the Republic of Serbia, University of Belgrade","correspondingAuthor":false,"prefix":"","firstName":"Radovan","middleName":"","lastName":"Dojčilović","suffix":""},{"id":476388671,"identity":"9416316b-bc4d-440c-9454-5b7f4f28ebe2","order_by":2,"name":"Dušan Milivojević","email":"","orcid":"","institution":"Vinča Institute of Nuclear Sciences, National Institute of the Republic of Serbia, University of Belgrade","correspondingAuthor":false,"prefix":"","firstName":"Dušan","middleName":"","lastName":"Milivojević","suffix":""},{"id":476388672,"identity":"706d8bc9-eb7b-45cd-9b95-53804feead51","order_by":3,"name":"Dušan K. Božanić","email":"","orcid":"","institution":"Vinča Institute of Nuclear Sciences, National Institute of the Republic of Serbia, University of Belgrade","correspondingAuthor":false,"prefix":"","firstName":"Dušan","middleName":"K.","lastName":"Božanić","suffix":""},{"id":476388673,"identity":"3039fb45-6c43-445d-af7f-9c5ef844063a","order_by":4,"name":"Vladimir Djoković","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4klEQVRIie2RsQrCMBCGLwTsEux6XdpXSCmIj9PSl+igRRHi4gMovod0TAm0Sx6go5Oz3RxNRUSXqJtgPsId4fLBfwTA4fhFpKSmIvhAFsP5QgkWnytAb51LUz5SxrVSvBfTKOlycSTVHPy1HPUXixLIJs92AuNDl6050S2gTumeWRQudaKYxnSia4FENAAdUGoL9lCSzV2JjEJswQYlZwWm3FsOygy4UcAWbNgl3hYYb1ujZFqyWGcralPGnVJ45mXkr7wT9lUZhq1S1mCA8umSgmLvf8d/nZf21w6Hw/GXXAEOCk9hYrgo3wAAAABJRU5ErkJggg==","orcid":"","institution":"Vinča Institute of Nuclear Sciences, National Institute of the Republic of Serbia, University of Belgrade","correspondingAuthor":true,"prefix":"","firstName":"Vladimir","middleName":"","lastName":"Djoković","suffix":""}],"badges":[],"createdAt":"2025-05-05 15:38:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6595834/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6595834/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":85487368,"identity":"fb1f15d1-fa2b-401b-ad22-439adcfefcf8","added_by":"auto","created_at":"2025-06-26 12:22:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":127581,"visible":true,"origin":"","legend":"\u003cp\u003eThe schematic diagram of the sample preparation procedure\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6595834/v1/f4417ab440816728733280ba.png"},{"id":85487374,"identity":"80693677-2adf-40c9-86d2-d3bfa15f22bf","added_by":"auto","created_at":"2025-06-26 12:22:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":174582,"visible":true,"origin":"","legend":"\u003cp\u003ea) TEM micrographs of chitosan-gold samples obtained with various initial concentration of gold salts; b) The corresponding particle size distributions obtained from analysis of TEM images\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6595834/v1/669287860f878f487524ba9b.png"},{"id":85487369,"identity":"6cc11a82-e42e-4f35-b244-efdcdfb3031b","added_by":"auto","created_at":"2025-06-26 12:22:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":77958,"visible":true,"origin":"","legend":"\u003cp\u003ea) UV-vis spectra of the pure chitosan (Ch), chitosan-rhodamine B (Ch-RhB), chitosan-gold colloid (ChAu) and chitosan-gold-rhodamine B (ChAu‑RhB) samples; b) Photoluminescence spectra of ChM, Ch-RhB, ChAu and ChAu‑RhB samples (λ\u003csub\u003eexc\u003c/sub\u003e = 530 nm)\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6595834/v1/80576a3f349014f1a3c40c90.png"},{"id":85487373,"identity":"7ab3bcc9-eede-4fbd-b9b9-45a7e4bb8b13","added_by":"auto","created_at":"2025-06-26 12:22:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":34584,"visible":true,"origin":"","legend":"\u003cp\u003eEPR spectra of TEMPO radicals recorded after UV-illumination of rhodamine B (in water) as well as Ch‑RhB and ChAu-RhB samples in the presence of TEMP spin trap; The reported spectra were collected 5 min after the illumination\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6595834/v1/50539f0d9e2ca8bba1d81cb5.png"},{"id":85487372,"identity":"c76c5068-c211-4a59-a206-b49073241780","added_by":"auto","created_at":"2025-06-26 12:22:03","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":89315,"visible":true,"origin":"","legend":"\u003cp\u003ea) EPR TEMPO signals of RhB, Ch-RhB and ChAu-RhB recorded at various times (0, 5, 10, 15 and 20 min) after light excitation of the photosensitizer; b) Time dependence of the relative EPR signal intensity\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6595834/v1/1aeb64fe692d620399fe9746.png"},{"id":85488980,"identity":"9e3b6a54-46cc-4803-bf12-2d9d31f0e5f7","added_by":"auto","created_at":"2025-06-26 12:38:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":871285,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6595834/v1/303297fc-3863-403f-b5f3-275b9d4680d8.pdf"},{"id":85488394,"identity":"2d9bcbf2-5767-43ef-ab27-d65dfbab6191","added_by":"auto","created_at":"2025-06-26 12:30:03","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":298142,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.docx","url":"https://assets-eu.researchsquare.com/files/rs-6595834/v1/f17498e8004e53e0d56e9ab5.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Singlet oxygen generation by hybrid rhodamine B-gold nanostructures in chitosan biomolecule environment: an EPR study","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRhodamine B (RhB) belongs to the class of hydrophilic xanthene derivatives and it is widely used as a dye in various industries. It exhibits high absorption in the visible region, coupled with moderate to high fluorescence quantum yield [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Also, it is very sensitive to the polarity of the solvent and undergoes spirolactam/ring-opening structural transformations as acidity increases. Since the open form is fluorescent and the spirolactam form is not, RhB, and rhodamine derivatives in general, are extensively studied as \u003cem\u003epH\u003c/em\u003e fluorescence sensors [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Rhodamine B shows high photosensitizing activity [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] and it has been studied as an active molecule for photodynamic therapy (PDT) of cancer [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Ngen \u003cem\u003eet al\u003c/em\u003e. [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] conjugated rhodamine B to porphyrin in order to create mitochondria-targeting photosensitizers that can absorb light in near-infrared region, where its penetration through tissues is the most efficient. RhB was introduced as an additional moiety component to the well-known porphyrin photosensitizer due to its large two-photon absorption cross section at 800 nm. The two-photon excitation of RhB and the resonant energy transfer (RET) between the components enabled efficient singlet oxygen production at low near-infrared incident energies. Recently, surface modification of inorganic nanoparticles with functional molecules emerged as route for fabrication of hybrid materials for therapeutic and diagnostic applications [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. We used this approach in our previous studies, where we functionalized noble-metal nanoparticles with fluorescent biomolecules to influence their optical properties and test them as probes for fluorescent bioimaging [\u003cspan additionalcitationids=\"CR11 CR12 CR13\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The molecules from the rhodamine-family proved to be good capping agents for inorganic particles providing functionalities suitable for imaging and photodynamic therapy (PDT) applications [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Pallavi et al [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] showed that photosensitizing potency of rhodamine 6G can be improved by its attachment to the surface of gold nanoparticles (Au NPs). This method also enables an increase in the uptake of photosensitizer molecules by the cells, which is a necessary condition for the efficient PDT treatment. Also, rhodamine capped gold nanoparticles were successfully used for the detection of Cr\u003csup\u003e3+\u003c/sup\u003e ion in living cells and contaminated waters [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Finally, it is important to emphasize that \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e cytotoxicity tests on zebrafish embryos revealed that these hybrid particles were fairly safe i.e. non-toxic [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn a series of preceding studies [\u003cspan additionalcitationids=\"CR19 CR20 CR21 CR22 CR23\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], we have shown that polysaccharide biopolymers such as starch, chitosan and alginate can be used as controlled environments for the synthesis of inorganic nanoparticles. In the present paper, we used polysaccharide chitosan, derived by deacetylation from the highly abundant biopolymer chitin, to control the growth of the gold the nanoparticles. Chitosan biomolecules possess both amino and hydroxyl groups exhibiting excellent chelating properties and enable fabrication of the nanoparticles with relatively narrow size distribution [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The obtained gold nanoparticles in chitosan were further functionalized with rhodamine B photosensitizers. Electron paramagnetic resonance (EPR) spectroscopy was used to study the changes in singlet oxygen production after the RhB molecules were added to the gold-chitosan colloid.\u003c/p\u003e"},{"header":"Experimental","content":"\u003cp\u003eMaterials\u003c/p\u003e \u003cp\u003eGold(III) chloride trihydrate (HAuCl\u003csub\u003e4\u003c/sub\u003e\u0026middot;3H\u003csub\u003e2\u003c/sub\u003eO), chitosan, D-glucose, rhodamine B and 2,2,6,6-tetramethylpiperidine (TEMP, a spin trap molecule) were purchased from Sigma\u0026ndash;Aldrich and used as received. High-purity water was used in all procedures.\u003c/p\u003e \u003cp\u003eSample preparation\u003c/p\u003e \u003cp\u003eThe gold nanoparticles in chitosan were prepared by using the D-glucose as reducing agent for tetrachloroaurate ions. Briefly, an aqueous solution of HauCl\u003csub\u003e4\u003c/sub\u003e was mixed with chitosan dissolved in 1% acetic acid solution. The concentration of chitosan in the solution was kept constant at 0.5 wt./vol.%, while the concentrations of the gold ions varied between 0.15 mM and 0.375 mM. An appropriate amount of 0.1 M D-glucose solution was added to the gold-chitosan mixture and stirred for 24 h in the dark at room temperature. The mixture gradually changed the colour to dark red. The list of the samples with corresponding preparation conditions are given in the Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\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\u003e The list of samples and the amount of chemicals used in fabrication.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\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 \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLabel\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e[Au] (10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e M)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChitosan (wt./vol. %)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.1 M D-glucose (ml)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChitosan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChAu\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChAu2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChAu4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe obtained gold-chitosan samples were further mixed with rhodamine B dye solution [5\u0026middot;10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e M]. The final RhB concentration in each of the samples was 5 \u0026micro;M. For comparison, the same set of the samples was also prepared by mixing RhB with the solution of the pure chitosan instead of the colloid. The four-step procedure for the sample preparation is schematically presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMethods\u003c/p\u003e \u003cp\u003eAbsorbance spectra were recorded on a Thermo Evolution 600 UV\u0026ndash;vis spectrophotometer in the range from 350 to 900 nm.\u003c/p\u003e \u003cp\u003ePhotoluminescence (PL) spectroscopy studies were carried out on PerkinElmer LS45 fluorescence spectrophotometer. The excitation wavelength was 530 nm. For PL measurements, each sample containing rhodamine B was diluted by factor 1000 due to extremely strong fluorescence intensity signal coming from the dye.\u003c/p\u003e \u003cp\u003eEPR Spectrometer MiniScope 300, Magnettech, Berlin, Germany, operating at a nominal frequency of 9.5 GHz, was used for electron paramagnetic resonance measurements. The spectra were obtained at room temperature. The microwave power was 1 mW (microwave attenuation of 20 dB) with a modulation amplitude of 0.2 mT. The Hamamatsu spotlight LC8, λ\u0026thinsp;\u0026gt;\u0026thinsp;300 nm, was used as a UV light source. The production of the singlet oxygen (\u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e) by the samples was followed \u003cem\u003evia\u003c/em\u003e spin trap method. The formation of \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e was detected through its specific reaction with spin trap TEMP (2,2,6,6-tetramethylpiperidine) molecule. The reaction produces a radical adduct, TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl) with a stable paramagnetic response.\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea shows TEM micrographs of ChAu samples prepared with various initial concentrations of gold salts. The reduction of the gold ions in the presence of chitosan molecules results in formation of nanoparticles with various shapes. Besides spherical particles, the particles in form of discs, rectangles and triangles can be observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The corresponding particle size distributions obtained from TEM analysis are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb. The average size of the particles increases with increasing in concentration of gold salts, but the changes are within the standard deviation of the particle distributions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe solution of the pure chitosan and the obtained gold-chitosan colloid samples were further mixed with fixed amount of rhodamine B aqueous solution. The UV-vis absorption spectra of the Ch-RhB, ChAu and ChAu-RhB samples are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(a).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe ChAu sample exhibits strong absorption at ~\u0026thinsp;540 nm, which originates from surface plasmon resonance (SPR) of gold nanoparticles. Also, SPR peak of Au NPs and the RhB absorption peak partially overlap and the absorption spectrum of the ChAu-RhB is the result of the superposition of the spectra of the components. It should be noted that the UV-vis absorption spectra (not shown) of the ChAu-RhB, ChAu2-RhB and ChAu4-RhB samples showed that the change in the initial concentration of the gold ions from 3.75\u0026middot;10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e M to 15\u0026middot;10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e M did not affect the position of the absorption peak, only its intensity. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb shows photoluminescent spectra of the pure Ch-solution, chitosan-gold colloid and their corresponding mixtures with rhodamine B. It can be noticed that there is a strong increase in fluorescence intensity of rhodamine B after its interaction with gold nanoparticles. This is a consequence of plasmon-enhanced fluorescence effect [\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Namely, metallic particles can affect both emission efficiency (e.g. quantum yield) and fluorescence lifetime of the fluorophore placed in the vicinity of their surfaces [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. As can be seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, the position of the absorption SPR peak of gold nanoparticles at ~\u0026thinsp;540 nm is close to the excitation wavelength used in the PL measurements. The excitation of the surface plasmon leads to the enhanced near-field intensity of the electric field surrounding the particle. This in turn increases the rate of excitation of the rhodamine B and, consequently, results in overall higher fluorescence. Another effect that should also be taken into account is that the nanoparticles influence the deexcitation dynamics of the nearby excited fluorophore. The coupling can result in appearance of new nonradiative deexcitation pathways, which in turn decreases the lifetime of the excited states of the molecule. This effect also leads to an increased molecular photostability and prolonged photophysical activity of the molecule [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Both effects can be responsible for the enhanced photosensitizing activity of the rhodamine B in the presence of nanoparticles [\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], as the results of EPR spectroscopy presented below show.\u003c/p\u003e \u003cp\u003eFormation of singlet oxygen \u003cem\u003evia\u003c/em\u003e UV-illumination of RhB (in water), Ch-RhB and ChAuRhB samples were studied by EPR spectroscopy. EPR spectra of the TEMPO radicals formed through singlet oxygen interaction with TEMP spin trap were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. As can be seen, photosensitization activity of rhodamine B strongly depends on the physicochemical environment surrounding the photosensitizer. The intensity of the EPR signal is the lowest in the case when the rhodamine B is in the chitosan solution. This might be due to the fact that chitosan molecules can generate other reactive radicals [33] that can interact with TEMPO and decrease its EPR signal [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In contrast, the signal intensity increases when the rhodamine B is in the vicinity of metallic surface (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The sensitizing activity is stronger than the activity in water, even if the photosensitizer is surrounded by chitosan molecules.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe mechanism of the metal-enhanced singlet oxygen generation is of the same nature as in the case of the metalenhanced fluorescence discussed above [36]. The enhancement of the electric field around the Au NP increases the number of photosensitizer molecules in the singlet excited state, which, due to intersystem crossing transitions, leads to a higher population of rhodamine B molecules in their triplet states. This, in turn, increases the rate of singlet oxygen generation. The enhanced generation of singlet oxygen can also be the effect of the increased photostability of photosensitizer, which increases the probability of its interaction with surrounding oxygen molecules. Although singlet oxygen generation and fluorescence are competing processes, they can be both enhanced \u003cem\u003evia\u003c/em\u003e the interaction of a fluorophore/photosensitizer with plasmonic nanostructure. However, the contribution of the metal-enhanced electric field surrounding the particle to the each of the processes might be different. Metallic particle might also quench the fluorescence of the fluorophore in its vicinity, without influencing the singlet oxygen generation process [36].\u003c/p\u003e \u003cp\u003eOn the other hand, chitosan biomolecules can also affect stability of the EPR signal. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea shows the EPR spectra of RhB, as Ch-RhB and ChAu-RhB samples recorded in the moment of irradiation and, successively, every five minutes after the irradiation. The dependence of the normalized signal intensities from time are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe EPR signal intensity of the RhB sample, where photosensitizer is surrounded solely with water molecules, is fairly stable in time (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). In contrast, when RhB is in chitosan solution, the relative intensity of the signal gradually decreases. This behaviour was also noticed in the case of ChAu-RhB sample, but the decrease in relative intensity of the signal is less pronounced and starts with a delay. Obviously, the instability of the EPR signal with time in photosensitizer\u0026rsquo;s environments other than water should be taken into account if this method is used for the detection of the singlet oxygen generation.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eFluorescence and photosensitizing activity of rhodamine B strongly depends on its physicochemical environment. Both, metal-enhanced fluorescence and metal-enhanced singlet oxygen generation was observed when rhodamine B was introduced into gold nanoparticle-chitosan solution. The obtained results were discussed in terms of an increase in the near-field intensity of electrical filed surrounding the gold nanoparticles due to their metallic nature. When rhodamine B is in the proximity of the particle surfaces, this effect can increase the rate of the excitation and consequently enhance either the fluorescence or the generation of singlet oxygen \u003cem\u003evia\u003c/em\u003e interaction of oxygen molecules with its triplet states. The generation of singlet oxygen by rhodamine B was studied in various environments (water, chitosan and chitosan-gold nanoparticle solutions) by using spin-trap method and EPR spectroscopy. The chitosan proved to be a good agent for the control of the growth of gold nanoparticles, but chitosan biomolecules may influence the TEMPO radicals formed after the interaction of singlet oxygen with TEMP spin-trap molecules. For this reason, they reduced the intensity of the EPR signal obtained. Nevertheless, due to metal-enhanced singlet oxygen generation effect, an excess of the single oxygen was observed after illumination of the ChAu-RhB sample, with respect to that obtained with illumination of rhodamine B water solution.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e The research was funded by the Ministry of Science, Technological development and Innovation of the Republic of Serbia under Grant Agreement No 451-03-66/2024-03/200017 and 451-03-66/2024-03/200162.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical statement:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo statement required\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJ.P. and R.D. preparation of the samples; J.P. and R.D. D.K.B. fluorescent and UV Vis absorption spectroscopy measurements, data analysis and visualization; D.M. EPR spectroscopy measurements, data analysis; D.K.B. and V. Dj. conceived the project and the methodology; V.Dj. writing, reviewing and editing the paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no competing interests to declare that are relevant to the content of this article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eM. Beija, C. A. M. Afonso, J. M. G. Martinho, Chem. Soc. Rev. (2009) https://doi.org/10.1039/B901612K\u003c/li\u003e\n \u003cli\u003eY. Zhou, K. Chu, H. Zhen, Y. Fang and C. 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(2022) https://doi.org/10.1007/s40820-022-00856-y\u003c/li\u003e\n\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":"colloid-and-polymer-science","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":" Learn more about [Colloid and Polymer Science](https://www.springer.com/journal/396) ","snPcode":"396","submissionUrl":"https://mc.manuscriptcentral.com/cps","title":"Colloid and Polymer Science","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"chitosan, gold nanoparticles, photosenzitizer, singlet oxygen, rhodamine B, electron-paramagnetic resonance","lastPublishedDoi":"10.21203/rs.3.rs-6595834/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6595834/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGold nanoparticles of various shapes were synthesized by reduction of gold salts in the presence of chitosan biomolecules as stabilizing agents. Fluorescence and photosensitizing properties of the rhodamine B dye were studied after its mixing with pure chitosan and chitosan-gold nanoparticle solutions. It was found that gold nanoparticles significantly affect the fluorescence intensity and the singlet oxygen production of the photosensitizer. Metal-enhanced fluorescence and metal-enhanced singlet oxygen generation effects were observed, probably as a direct consequence of the activation of the surface plasmon of the nanoparticles upon irradiation. Photosensitizing activity of the rhodamine B dye was investigated by using electron paramagnetic resonance (EPR) spectroscopy with TEMP as spin-trap molecules. The singlet oxygen generation was followed \u003cem\u003evia\u003c/em\u003e changes in the intensity of EPR signal of the radical adduct, TEMPO. It was found that gold nanoparticles facilitate the production of singlet oxygen, while the chitosan molecules influence TEMPO stability and tend reduce the intensity of the EPR signal, especially at prolonged times following the irradiation.\u003c/p\u003e","manuscriptTitle":"Singlet oxygen generation by hybrid rhodamine B-gold nanostructures in chitosan biomolecule environment: an EPR study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-26 12:21:58","doi":"10.21203/rs.3.rs-6595834/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-31T05:35:22+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-30T15:02:59+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-14T16:54:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"253104188053883041107708249128523142197","date":"2025-07-09T01:09:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"146733568639511549483196937237522068519","date":"2025-07-08T11:31:25+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-23T08:53:51+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-15T13:59:18+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-15T13:57:42+00:00","index":"","fulltext":""},{"type":"submitted","content":"Colloid and Polymer Science","date":"2025-05-05T15:33:23+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"colloid-and-polymer-science","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":" Learn more about [Colloid and Polymer Science](https://www.springer.com/journal/396) ","snPcode":"396","submissionUrl":"https://mc.manuscriptcentral.com/cps","title":"Colloid and Polymer Science","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b3933c91-331a-4be8-87cc-42f80633958d","owner":[],"postedDate":"June 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-10-17T09:53:06+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-26 12:21:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6595834","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6595834","identity":"rs-6595834","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}