Journal Pre-proofs Aggregation-based fluorescence amplification strategy: “turn-on” sensing of aminoglycosides using near-IR carbocyanine dyes and pre-micellar surfac‐ tants Sofia A. Zakharenkova, Ekaterina A. Katkova, Irina A. Doroshenko, Anna S. Kriveleva, Aleksandra N. Lebedeva, Tatyana A. Vidinchuk, Anna V. Shik, Sergei S. Abramchuk, Tatyana A. Podrugina, Mikhail K. Beklemishev PII: DOI: Reference: S1386-1425(20)31088-X https://doi.org/10.1016/j.saa.2020.119109 SAA 119109 To appear in: Spectrochimica Acta Part A: Molecular and Bio‐ molecular Spectroscopy Received Date: Revised Date: Accepted Date: 2 June 2020 2 October 2020 14 October 2020 Please cite this article as: S.A. Zakharenkova, E.A. Katkova, I.A. Doroshenko, A.S. Kriveleva, A.N. Lebedeva, T.A. Vidinchuk, A.V. Shik, S.S. Abramchuk, T.A. Podrugina, M.K. Beklemishev, Aggregation-based fluorescence amplification strategy: “turn-on” sensing of aminoglycosides using near-IR carbocyanine dyes and pre-micellar surfactants, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2020), doi: https://doi.org/10.1016/j.saa.2020.119109 This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Elsevier B.V. All rights reserved. Aggregation-based fluorescence amplification strategy: "turn-on" sensing of aminoglycosides using near-IR carbocyanine dyes and pre-micellar surfactants Sofia A. Zakharenkovaa, Ekaterina A. Katkovaa, Irina A. Doroshenkoa, Anna S. Krivelevaa, Aleksandra N. Lebedevaa, Tatyana A. Vidinchuka, Anna V. Shika, Sergei S. Abramchukb, Tatyana A. Podruginaa, Mikhail K. Beklemisheva* aM.V.Lomonosov Moscow State University, Department of Chemistry, 119991 GSP-1 Moscow, Russia bNesmeyanov Institute of Organoelement Compounds (INEOS), Russian Academy of Sciences, Vavilova 28, Moscow, 119991, Russia *e-mail: [email protected] Abstract. This study is aimed at developing sensing schemes without obtaining selective receptors. A series of simple carbocyanine dyes was synthesized, whose emission was quenched in water with formation of nanoparticles in the range of 20–100 nm. Fluorescence in near-IR region is “turned on” in the presence of a drug cation of middle molecular weight (400–700 Da) and sodium dodecyl sulfate (SDS), as well as anionic drugs and a cationic surfactant (cetyltrimethylammonium bromide, CTAB). Aggregates (clusters) up to 100–200 nm in size were detected using dynamic light scattering (DLS) and Rayleigh light scattering (RLS) techniques in the systems: cationic analyte – SDS, carbocyanine dye – CTAB, and in all brightly fluorescent ternary systems dye – surfactant – analyte. Small ions (<200 Da) incapable of multi-point binding do not form the aggregates or cause the emission enhancement. The “turn-on” signal is only observed at the surfactant submicellar concentrations insufficient to solubilize the dye nanoparticles. Based on these findings, we suggest a rapid and simple method for the detection of ≥ 4·10–5 mol/L of neomycin in urine. The proposed strategy paves the way for developing more selective methods. Keywords: fluorescence spectroscopy, self-assembly, aggregation, ion pair, carbocyanine, aminoglycoside Introduction Fluorescence-based methods for detection of small molecules are inexpensive easy to operate, and often exhibit low detection limits. Molecular fluorescence sensors are based on different types of interactions, including supramolecular ones [1, 2]. A challenging problem is sensing of small molecules without a considerable synthetic effort, which invokes interest to detection strategies based on aggregation of amphiphilic species [3] and ionic self-assembly [4–6] (several authors prefer to use term “clusters” instead of “aggregates” [7]). Fluorescent aggregation-based methods, which are sometimes highly sensitive and selective, are realized using various principles: displacement processes (AB + С, AC + B) [8], formation of surfactant micelles to encapsulate a hydrophobic fluorophore [9–11] or joint micelles of the fluorophore and analyte [12], and aggregation of the fluorophore-containing micelles by a polymeric analyte [13]. Aggregation-based technique includes a group of detection methods in which the signal of analyte does not result from a direct analyte–fluorophore interaction – indirect signalling methods, which widen the range of possible analytes and expand the opportunities of tuning the sensing selectivity [14, 15]. A number of indirect signalling-based fluorescent methods were developed using aggregation [3], including formation of the ternary aggregates of a target analyte with a pre-micellar surfactant and a fluorescent dye. Such aggregates can be formed when the analyte lowers the critical micelle concentration (CMC) of the surfactant. The process is visualized upon involvement of a dye, leading to optical changes [16–18]. In many cases analytes cause emission quenching [17, 18]; however, more practical is signal enhancement that can be achieved by pre-quenching the fluorophores by a number of ways [15, 19–21], including placement of the dye in water, which results in its aggregation [22]. However, the concrete form of the dye often remains unknown. Aminoglycosides are widely used as antibiotics; the instrumental methods for their determination such as HPLC-MS [23] and other techniques [24, 25] have been developed. Optical sensing of aminoglycosides is a challenge, since their molecules do not have any chromophoric or fluorophoric groups. Fluorescent derivatives of aminoglycosides have been obtained by use of derivatization technique designed for HPLC methods [26]. However, reversed-phase HPLC faces difficulties in the determination of aminoglycosides because of their poor chromatographic retention [27]. Several fluorescent methods for aminoglycosides without derivatization were developed [28], including the methods using aptasensors [29, 30]; an aggregational pattern-based fluorescent method was used in the discrimination of an artificial mixture of aminoglycosides [12]. Although specific immune tests for aminoglycosides, which are based on enzyme-linked immunosorbent assay (ELISA) [31, 32] and requires aminoglycoside conjugation with proteins and animal immunization steps, are widely used, there is increasing interest in rapid techniques that do not involve the use of enzymes or antibodies. 2 This proof-of-concept study rests on two fruitful ideas of aggregation-based sensing: preliminary fluorophore quenching [15, 19, 20] and aggregation of the target compound with a surfactant [16–18]. Taking into account that the aggregation-based sensing events greatly depend on hydrophobicity of the fluorophores, we synthesized and studied a series of carbocyanines (I, II, IV– VI) that are different in their hydrophobicity following the principles described earlier [33]. A commercially available near-IR dye III was also studied. The purposes of this study are to reveal the origin of fluorescence enhancement of carbocyanine dyes observed with model analytes and surfactants and to evaluate the feasibility of non-covalent sensing of a selected analyte (neomycin). Neomycin is part of medicines for external use; when applied to damaged skin or granulation tissue, it can be quickly absorbed into the body and cause kidney problems and hearing difficulty [34]. Neomycin metabolism is negligible; hence, all absorbed fraction of neomycin is excreted by the kidneys [35]. Also, it can adversely affect the kidneys [36], which requires its determination in urine. Materials and Methods HOOC N 1 X R I-III N R2 N I IV-VI N R I: R1 = R2 = (CH2)10COOH, X = Br– IV: R = CH3 II: R1 = R2 = (CH2)5CH3, X = I– V: R = (CH2)5CH3 III (Cy5.5-COOH): R1 = CH3, R2 = (CH2)6COOH, X = Cl– VI: R = (CH2)10COOH Scheme 1. Dye structures. Compounds. Dyes I, II, and IV–VI were synthesized according to [33] as illustrated in Scheme 2. A more detailed protocol and characterization data are given in the ESI. Carbocyanine III (Cy5.5-COOH, CAS No 1449612-07-0) was purchased from Lumiprobe (Hannover, Germany). The structures of compounds used as model analytes are given in Scheme S1 in ESI. 3 NHPh PhN RX N X 1 Py, Ac2O N R1 X I-II 2b-c HOOC N R1 N R1 CH3I HOOC N N I 3 AcONa 4 HOOC Ac2O N I PhN NHPh N R X 2a-c X N R IV-VI NAcPh N R 5a-c Scheme 2. Synthetic route to dyes I, II (top) and IV–VI (bottom). RESULTS AND DISCUSSION 1. Fluorescent Properties of Carbocyanine Dyes Organic solution. In organic solvents (ethanol, THF) dyes I–VI show absorption maxima at ~350 nm and 650–680 nm and fluorescence bands at 400–500 nm and 650–750 nm (Fig. 1a, b), which correspond to their molecular forms. The quantum yields of the NIR emission do not exceed ~2% (Table S1 in ESI). Aqueous solutions without surfactants. The dilution of the ethanolic (or THF) solution of the new dyes I or II with ≥10-fold excess of water or an aqueous buffer produces a visually transparent solution, which exhibits weak red absorption (Fig. S2) and almost no NIR emission, while the fluorescence at 400—500 nm remains prominent (Fig. 1c). In this study we focused on NIR emission as it is more responsive to model analytes. 4 Fig. 1. Absorbance (a) and fluorescence (b) spectra of ethanolic solutions of dyes I – VI; fluorescence spectra of dyes I – III: (c) in an aqueous solution without surfactant (pH 7.4, 7 mM phosphate buffer) and (d) with submicellar (0.7 mM) or micellar (24 mM) SDS in the same buffer. Transmission electron microscopy (TEM) evidences (Fig. 2a) that in water the dye forms ballshaped nanoparticles of size 20–100 nm (Fig. 2a). No regular nanoparticles were found by TEM in the sample of the dried ethanolic solution of dye I (Fig. S3). As mentioned above, the nanoparticles of dye I demonstrate very low fluorescence intensity in the region higher than 600 nm (Fig. 1d). A similar aggregational form was observed in TEM for another fluorophore [37]. 5 Fig. 2. TEM images of the solutions of dye I: a – in water (a THF solution (1 g/L) was mixed with water (1 + 40, v/v) and dried on the copper grid); b – in water containing submicellar SDS (0.7 mM). Aqueous solutions with surfactants. Surfactants at submicellar concentrations (0.1–0.03 critical micelle concentration (CMC) value) do not considerably increase the dyes emission compared with the buffer solution (Fig. 1d). Presumably, in submicellar solutions the dyes also exist in the form of nanoparticles, as found for dye I in TEM (Fig. 2b). At micellar concentration of surfactants (3 CMC values) the dyes are strongly emitting (Fig. 1d), most probably, due to distribution of the dye molecules in the micelles. This process is supposed to be accompanied by de-aggregation and inhibition of radiationless decay, which in turn can be explained by the rigidization of the dye in the micelle environment [38]. 2. Fluorescence in the dye – analyte – surfactant systems The effect of model analytes on the emission intensity of dyes was studied at physiological pH 7.4. In the absence of surfactant, no effect of analytes was noticed with respect to the blank experiments. Analytes in submicellar surfactants. In the premicellar solutions of SDS or CTAB, the emission of dyes I–III is quenched (similar to pure water, section 1). The feasibility of fluorescence enhancement was studied by adding differently charged medicinal drugs. In the 500-nm emission range, no regular effect of the model analyte on the emission was found (data not shown). By contrast, the NIR fluorescence was enhanced for the combinations of an analyte with the oppositely charged surfactant: cationic species (aminoglycosides) with SDS (Fig. 3,a) and anionic species 6 (cephalosporins, penicillins) with CTAB (Fig. 3,b). Similar results were obtained with the other dyes (Fig. S4 in ESI). The signals could be also acquired by photographing 96-well plates (Fig. 3 insets) with a NIR photo camera (the setup is shown in Fig. S1). Studies of a wider range of compounds in the system dye I – analyte – submicellar surfactant revealed that the species of molecular weight less than 200 Da, even those charged oppositely to the surfactant, induced smaller signals compared to those of the drugs (Fig. 4a, b). Let us construct a conceivable scheme of the observed fluorescence enhancement for dye I and aminoglycosides that yield the highest signals. At the first stage, the cationic drugs (charge +3...+5 at pH 7.4, https://www.drugbank.ca/) form aggregates with the oppositely charged SDS. The high charge of these cations could therefore be considered as the main driving force for binding with SDS. However, anionic drugs (cephalosporins and penicillins) also yield a signal with an oppositely charged surfactant (CTAB, Fig. 4,b), though their physiological charge does not exceed –1 (the lower charge may be the reason for lower selectivity for the anionic species (Fig. 4,b) compared to aminoglycosides (Fig. 4,a). Therefore, it is probably important that the analyte could form multi-point bonds in order to be bound in the aggregates. The studied drugs do have several centers of binding (their structures are shown in Scheme S1 in ESI). The other party in this binding (Scheme S3) could be micelle-like structures of the surfactant formed upon aggregation (the micelles per se are absent in the initial submicellar solution). At the second stage, the SDS – analyte aggregates formed could solubilize the dye nanoparticles to enhance the fluorescent signal (the situation with CTAB slightly differs). This stage is considered in section 3 below. Fig. 3. Fluorescence spectra (and NIR images of the plates) with dye I: (a) in submicellar SDS: without added analytes (1) and with analytes: cationic (blue colors: 2–5) or anionic (red colors: 6, 7); (b) in submicellar CTAB: without analytes (1) and with cationic (blue colors: 2, 3) or anionic (red 7 colors: 6–9) analytes. Analytes: 2 – amikacin, 3 – neomycin, 4 – gentamicin, 5 – streptomycin, 6 – ceftriaxone, 7 – cefazoline, 8 – ceftazidime, 9 – cefotaxime. Conditions: pH 7.4 (7 mM phosphate buffer); 1.5 mM analyte, 5 M dye I, 0.7 mM SDS or 0.1 mM CTAB (here and below the concentrations are given for the reaction mixture). Fig. 4. NIR fluorescence intensity in the system dye I – submicellar SDS (0.7 mM, left) and dye I – submicellar CTAB (0.1 mM, right) in the presence of 1.5 mM of various species at pH 7.4. The molar weight of the cation or anion of the species, whichever is heavier, is shown beside its name. The charge at pH 7.4 (according to https://www.drugbank.ca/) is given in parentheses. * As sulfates. **As sodium salts. Analytes in micellar surfactants. In micellar solutions of surfactants the dyes exhibit high NIR emission (Fig. 1d); this is the maximum signal developed with the dyes under such conditions, for which reason it is not increased by the analytes (Fig. S5). 8 3. Aggregation in the systems dye – analyte – surfactant The aggregational phenomena were studied to understand the reasons behind the emission enhancement we observed (Figs. 3, 4). Analyte – surfactant aggregation. According to the DLS data, the submicellar (0.03– 0.1×CMC) SDS solutions with cationic drugs contained particles with an average size of 60 nm (Fig. 5). For the micellar concentrations of the surfactant (3×CMC), no similar aggregates were observed, probably, due to ability of the surfactant micelles to solubilize the surfactant – analyte AGs [39]. Fig. 5. Particle sizes according to DLS technique in micellar (3×CMC) and submicellar (0.03×CMC) solutions of SDS with and without streptomycin. Concentrations: 0.24 or 24 mM SDS, 1.5 mM analyte, pH 7.4. Dye – analyte – surfactant aggregation: cationic analytes with SDS. In studying aggregation in the presence of fluorophores, the DLS technique faces problems caused by the strong dye fluorescence under the laser light. Characterization of the dye – analyte – surfactant systems was performed using Rayleigh light scattering (RLS) spectra that qualitatively reflect the particle sizes. The analyte – SDS aggregates showed intense RLS spectra, not significantly changing upon adding a dye (Fig. 6a,b and Fig. S6 in ESI). This means that the particle size is not appreciably increased, which allows us to hypothesize that the dye enters already formed aggregates. As a result, the dye – analyte – SDS ternary aggregates are formed. However, not every large cationic species will give rise to emission enhancement. For example, amikacin does not form an aggregate with SDS, as evidenced by the RLS spectra, and consequently does not form a ternary aggregate in the presence of the dye. No considerable emission is observed with this drug (Fig. 6c; for detailed spectra and other examples, see Fig. S6 in ESI). 9 Thus, the RLS and fluorescence sudies demonstrate that emission is turned on only it there are aggregates of the (cationic) analyte and (anionic) surfactant. If the dye has an affinity for these binary aggregates, it is solubilized in them, transforming from an aggregated conditon (nanoparticles) to molecular state, thereby increasing its quantum yield. Dye – analyte – surfactant aggregation: anionic analytes with CTAB. A different pattern has been observed for the cationic surfactants. Judging by the RLS intensity (Fig. 6,b and Fig. S6) we observe (1) no formation of large particles in the system analyte – CTAB, but (2) presence of a large aggregate dye – CTAB. The dye – CTAB aggregates (observed for dyes I and II) may be formed due to CTAB cation interaction with the system of the dye (Scheme S3 in ESI), as it is typical of quaternary ammonium salts to give complexes with the -systems of aromatic compounds [1, 40]. Fluorescence in the ternary systems dye – CTAB – analyte is only observed for some anionic drugs (ceftriaxone, ceftazidime and cefotaxime) that form large ternary aggregates (Fig. 6b,c). There is no fluorescence with CTAB and other species giving large anions (cefazoline or penicillin); at the same time, these drugs do not form large aggregates dye – CTAB – analyte (judging by the RLS intensity that is far lower that for dye I – CTAB, Fig. S6 in ESI). Based on this data, we can suppose that fluorescence of anionic analytes appears only in the ternary aggregates dye – CTAB – analyte. It should be analyzed how the ternary aggregates can be formed. In case of cationic drugs and SDS (previous subsection), the SDS ensembles dissolved the dye nanoparticles. In case of anionic drugs, we can imagine two scenarios of ternary aggregates formation: (1) the dye – anionic drug aggregate reacts with the cationic surfactant, which entails solubilization of the dye in the surfactant micelle-like substructure of the ternary aggregate thus formed; (2) the ternary aggregate is formed during simultaneous interaction of the three components: dye nanoparticles, ions of the surfactant and the drug. The existing techniques do not allow for distinguishing between these two scenarios; however, both of them result in conversion of the dye into its molecular (fluorescent) form (Scheme 3). Not every analyte of the proper charge will cause fluorescent enhancement. The RLS spectra show that small species that are unable to give multiple non-covalent bonds (such as arginine or diethylamine in the presence of SDS, Fig. 4 or S7) do not form the aggregates with the oppositely charged submicellar surfactants. This is in agreement with the fact that only large ions (having multiple binding centers) can form the aggregates and selectively enhance fluorescence. Overall, fluorescence is turned on in the ternary aggregates dye – surfactant – analyte, independently of the the surfactant and analyte charges. (Scheme 3). This mechanism can be implemented if the analyte species is large and multifunctional (to allow for multi-point binding) and the dye fluorescence without the analyte is pre-quenched in a submicellar surfactant. 10 Fig. 6. Rayleigh light scattering and fluorescence (in the inserts) spectra of the systems: neomycin (Neo) – SDS – dye I (a) and ceftriaxone (Ceftr) – CTAB – dye I (b); correlation diagrams between the NIR fluorescence and RLS intensities of the ternary aggregates (analyte – surfactant – dye I): with SDS (c) and CTAB (d). The RLS and emission spectra for the other analytes are given in Fig. S6 in ESI. In the correlation diagrams (c, d) the RLS signals were calculated as the intergal intensities of the ternary aggregates spectra within 360–650 nm excluding the intensities of the binary aggregates (surfactant – dye I). 11 Scheme 3. Suggested mechanism for fluorescence enhancement: dye fluorescence quenching in water (process I); for cationic analytes: aggregation with SDS (process IIa) and solubilization of the dye nanoparticles in the formed aggregate (process IIb); for anionic analytes: aggregation of dye nanoparticles with CTAB (process IIIa) and inclusion of the anionic analyte into the aggregate with solubilization of the dye nanoparticles (process IIIb). 12 4. Effects of the nature of the dye and surfactant on fluorescence enhancement Above we considered the emission enhancement scheme for dye I and two surfactants (SDS and CTAB). Below we provide the data for some other surfactants and dyes. Nature of the surfactant. Studies of a number of surfactants with dyes I–III and four selected analytes (Fig. S8) showed that the most efficient surfactants for sensing cationic and anionic analytes are SDS and CTAB, respectively. All other surfactants used (including non-ionic ones) yield either a high blank signal or (more frequently) almost no signal with respect to the blank. To explore role of surfactant, two systems were examined in TEM (Fig. 7): dye I – ceftriaxone – CTAB (fluorescent) and dye I – cetriaxone – benzyldimethyltetradecylammonium chloride (BDTAC) (non-fluorescent). When CTAB is used, no dye nanoparticles were found over the whole TEM sample, which implies that the dye was completely solubilized in the aggregates, as can be clearly seen in the images (Fig. 7a, b). On the contrary, in the case of BDTAC (Fig. 7b, c), numerous dye nanoparticles, which are apparently unable to solubilize in this surfactant – analyte pair and thus remain non-fluorescent, were observed. These results imply that the dye nanoparticles can be converted into molecular form when fluorescent aggregates are formed; on the other hand, no dissolution is observed in the system without fluorescence. We believe these data are a strong argument in favor of the suggested mechanism for fluorescence enhancement (Scheme 3). The results (Fig. 7) confirm that not all surfactants of the proper charge will form ternary aggregates and cause emission enhancement: there should be certain affinity of the surfactant to the dye nanoparticles. Fig. 7. TEM images of dye I – ceftriaxone mixture with two surfactants: a, b – CTAB (NIR fluorescence), c, d – benzyldimethyltetradecylammonium chloride (no NIR fluorescence). Conditions: 1.5 mM analyte, 5 M dye I, 0.1 CMC of each surfactant, no buffer. The NIR images of the vials with the said solutions are also shown. 13 Effect of dye hydrophobicity. All dyes provide signals with anionic and cationic analytes (ceftriaxone and neomycin, respectively, are shown as examples in Fig. 8, see also Fig. S4). However, sensing is only feasible on a low background level (signal without analytes). The low backround in submicellar CTAB or SDS is observed for the most hydrophobic dyes, I and II. Emission of the less hydrophobic dyes is insufficiently quenched in water, and these dyes demonstrate a higher background signal with SDS (dye VI), CTAB (dyes III, V) or both surfactants (dye IV); the most hydrophilic dye IV is not useful for sensing at all under these conditions. The hydrophobic property of a carbocyanine dye is important for the suggested sensing scheme because the dye should (1) form quenched nanoparticles in aqueous solution (ensuring low background signal) and (2) be efficiently included in the ternary aggregates along with the analyte and the counter-ion (ensuring the high signal of interest). From this viewpoint, dye III (logP = 6.9) is inferior to dyes I and II (logP = 9.7 and 9.0) as it yields a higher blank signal in the submicellar CTAB (Fig. 8). Future development should be directed to synthesizing carbocyanine dyes with logP of 9 and higher. Fig. 8. NIR fluorescence image of the plate wells with surfactant – analyte mixtures with dyes I–VI of different lipophilicity (measured as logP*). The surfactants are added at concentrations of onetenth of their CMC (0.1 mM CTAB, 0.7 mM SDS), pH 7.4 (7 mM phosphate buffer), 1.5 mM analyte, 5 M dye. * Distribution coefficient of the molecular form in n-octanol–water system calculated using https://www.molinspiration.com/cgi-bin/properties 14 5. Sensing of neomycin with dye I and SDS The selective signal induced by the aminoglycoside antibiotics allowed us to develop a NIR test for neomycin using dye I and submicellar SDS. Testing was performed in a 96-well plate, the signal was recorded by using a NIR photo camera (Fig. S1) and digitized using ImageJ software. Selection of conditons. Dependence of the signal on the reactant concentrations is shown in Fig. 9. Excessive amount of dye results in a lower signal, probably, due to exceeding the solubilization capacity of the aggregates. The working pH range is 4–8, and the SDS concentration in the reaction solution should not exceed 2 mM (note that the CMC of SDS in pure water is 8 mM). The signal can be measured directly after mixing the solutions and does not change for hours thereafter. The chosen conditions are given in caption to Fig. 9. Fig. 9. NIR fluorescent signal of dye I in the presence of neomycin and without it for different concentrations: of the dye (a), SDS (b), and pH (c). General conditions: pH 7.4 (7 mM phosphate buffer), 0.7 mM SDS, 2.5 M dye I, 0.4 mM neomycin. In varying pH, the following buffers were used in 7 mM concentrations (by anion): borate (pH 9–10), phosphate (pH 5.5–8.1), acetate (pH 3.5– 5.0), and HCl (pH 1.8–2.8). Selectivity of neomycin sensing was characterized by cross-reactivity and interferences. To evaluate cross-reactivity, the system response to various organic species and inorganic salts was studied without neomycin. As it can be seen from Fig. 10a, signals were obtained from neomycin itself and other aminoglycosides (amikacin, gentamycin and streptomycin, wells No 3–5), while the compounds of other classes yielded virtually no emission. To study the interferences of the same species with neomycin detection, the signals of their mixtures with neomycin were measured (Fig. 10b; Fig. S9 in ESI contains the quantitative data for the images of Fig. 10). Neomycin response was only reduced by Cu2+ and Fe3+ salts due to their quenching effect; smaller amounts of these ions did not interfere (0.05 mM Cu2+ and 1.5 mM Fe3+ in the final solution). We can conclude that the 15 detection of neomycin is selective with respect to organic species, including large anionic molecules (cephalosporins, penicillins), as well as non-charged species, amino acids and inorganic salts. Fig. 10. Images of the plates for the system dye I – SDS without neomycin (a) and with it (b) in the presence of equal molar amounts of diverse species. Numbers of wells: 1a — blank, 2a, 2b – only neomycin, 3 — amikacin*, 4 — gentamicin, 5 — streptomycin*, 6 — arginine, 7 — diethylamine, 8 — cefazolin**, 9 — ceftazidime**, 10 — cefotaxime**, 11 — ceftriaxone**, 12 — ampicillin**, 13 — aspartic acid, 14 — trisodium citrate, 15 — pefloxacin, 16 — phenylalanine, 17 — glycine, 18 — proline, 19 — histidine, 20 — cysteine, 21 — glutamine, 22 — serine, 23 — aniline, 24 — glucose, 25 — sulfathiazole, 26 — urea, 27 — K4P2O7, 28 — chloride**, 29 — nitrate**, 30 — sulfate**, 31 — acetate**, 32 — KCl, 33 — CaCl2, 34 — KI, 35 — MgCl2, 36 — (NH4)2SO4, 37 — FeCl3, 38 — CuSO4. Conditions: 0.7 mM SDS, pH 7.4 (7 mM phosphate buffer), 4 M dye I, 1.5 mM neomycin. Note. The NIR light, as not perceived by eye, should be imaged in black and white, but in Figs. 10, 11 we are keeping the red hue yielded by the digital camera as more convenient for perception. * As sulfates **As sodium salts Determination of neomycin in urine. Urine samples collected from healthy volunteers of Moscow University were spiked with neomycin solution at different concentrations. Determination was performed in a 96-well plate with photographic recording of the NIR signal. The amount of urine was 1/7 (v:v) of the resulting solution, as higher amounts increased the background signal. The 16 calibration plots for the determination of neomycin in aqueous buffer practically coincided with those in urine, which confirmed the high selectivity of the method (Fig. 11). The fluorescent signal is proportional to the log concentration of neomycin within 0.05 – 2 mM (initial concentration in the aqueous solution or sample) with an average RSD of 0.07 (in water) and 0.09 (with urine) for the said concentration range and a limit of detection of 4×10–5 M (by 3s criterion). Fig. 11. Calibration plot for sensing neomycin in distilled water and in urine. Insert: images of the plates with the corresponding concentrations of neomycin in urine and water. The following solutions were added to each well: 30 L of 8 mM SDS, 30 L of 67 mM phosphate buffer, 30 L of 25 M dye I in water, 50 L of urine sample spiked with neomycin or an aqueous neomycin solution, and 220 L of water. Four parallel runs were performed for each concentration of neomycin. The exposure time was 0.2 s. Confidence intervals for the error bars were calculated as , where t is Student's coefficient (3.18), s is standard deviation, and n is number of runs (4). Detection of other aminoglycosides under the conditions of neomycin detection. Other aminoglycosides can also be detected in urine. The logarithmically linear relationships between fluorescence intensity and concentration in urine were observed for gentamycin and streptomycin (Table S2) with LODs for of 50 and 100 М, respectively. The interferences were similar to those found for neomycin (Fig. 10). 17 Conclusions This study reveals a number of aggregational effects that deserve interest. First is the formation of hydrophobic dye nanoparticles in water. Quenching in water is usually associated with the aggregation of the dye, but the information on the concrete structure of those aggregates is very scarce. Second is the ability of the dye nanoparticles to solubilize in the analyte – surfactant aggregates and the ability of anionic analytes to enter the dye – surfactant aggregates; both processes involve transfer of the dye into its fluorescent (probably, molecularly dispersed) form within the aggregates, resulting in emission enhancement. Lack of investigation of such processes possibly resulted in gap in the literature. The formation of the aggregates is governed (1) by the analyte charge (the aggregates only can be formed from species oppositely charged to the used surfactant), (2) the ability of analyte to form multiple bonds (small ions do not induce aggregation). Such requirements are necessary but insufficient, as the third component must have an affinity to the binary aggregate as follows: dye to the cationic analyte – surfactant, or anionic analyte to the dye – surfactant. These limitations will allow for tuning the selectivity by varying the nature of the surfactant and fluorophore. The third phenomenon to be emphasized is an unusual role of surfactant as not a micelleforming but an aggregate-forming agent. A cationic surfactant can aggregate the dye nanoparticles, presumably due to cation- interactions; an anionic surfactant aggregates with the highly charged aminoglycosides. However, it is likely that within those aggregates the surfactants exist in the form of micelle-like structures bound together (with the analyte ions or the dye nanoparticles) by cooperative bonding. It is essential, that these aggregational events only take place in submicellar solutions, as surfactant micelles solubilize all types of aggregates. The micellar solutions are not of interest from the sensing viewpoint, as the dyes are brightly fluorescent in the micelles without the analytes. Fourthly, the selective signal of aminoglycosides over other species is not caused by selective interaction with the dye (because the analytes have no direct contacts with the dye) or even with the surfactant (though aminoglycosides do form the aggregates with SDS). Rather, the selective signal is due to formation of the aggregate, which is cross-linked by multiple non-covalent bonds to yield a nanoparticle that, in turn, is capable of solubilizing the dye and converting it into the fluorescent form. From the practical viewpoint, the sensing scheme provides an approach for the detection of large species, multiple charged ions or molecules capable of multi-point binding. In comparison to 18 other instrumental techniques, the method is more rapid and simple; the signal can be registered by a modified digital camera without using a stationary instrument. As compared to immune-based tests, the suggested method does not require any enzymes or antibodies. In contrast to direct fluorescent methods, the described approach does not require a sophisticated synthesis of receptors; the synthesis of fluorophores is performed using standard methods, and even commercially available dyes can be used. The "turn-on" signal in the NIR region minimizes the interferences from a biological matrix. Such schemes may be used as an alternative to fluorophore–spacer–receptor (FSR) sensors. The selectivity and sensitivity of the fluorescent methods based on the suggested scheme can be further improved as described below. We suggest that a selective sensing method can be developed for a target analyte provided a complementary counter-ion (not a surfactant) could be found to form a stable ion pair with the analyte. If both particles can form multi-point bonds, the resulting ion pairs will be able to produce large aggregates (nanoparticles). 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Podrugina: methodology, investigation, Mikhail K. Beklemishev: conceptualization, supervision, funding acquisition, writing – review and editing 22 23 Formation of quenched carbocyanine nanoparticles in water Submicellar surfactants as aggregate-forming agents Fluorescence enhancement in dye–analyte–surfactant aggregates Selectivity to large organic ions capable of cooperative binding Rapid detection of neomycin in urine without using a spectrofluorimeter 24