Reactive fluorogenic compound and nanocomposite for sensing hydrogen sulfide comprising the same

Abstract

The present invention relates to a nanocomposite for detecting hydrogen sulfide; a method for preparing the same; a novel reactive fluorogenic compound to be used in the method; a kit for detecting hydrogen sulfide comprising the nanocomposite; and a method for providing information for the diagnosis of a disease, which causes abnormal secretion of hydrogen sulfide, by using the nanocomposite.

Claims

1. A nanocomposite for detecting hydrogen sulfide (H.sub.2S) comprising a C.sub.10-25 alkane or haloalkane, a neutral first surfactant, a cationic second surfactant, and a reactive fluorophore, wherein: the nanocomposite is a self-assembly formed by co-assembly of the first surfactant and the second surfactant, in which the self-assembly comprises a hydrophobic core containing the C.sub.10-25 alkane or haloalkane and the hydrophobic core comprises a reactive fluorophore, wherein the reactive fluorophore itself is non-fluorescent but exhibits fluorescence by a reaction with hydrogen sulfide, and wherein the C.sub.10-25 alkane or haloalkane is 1-iodooctadecane.

2. The nanocomposite of claim 1, wherein the reactive fluorophore is a molecule that is cleaved by a reduction reaction with HS.sup.− ions and decomposed into a fluorescent molecule and an aryl azide linker.

3. The nanocomposite of claim 1, wherein the reactive fluorophore is azidobenzylresorufin (ABR) in which a hydroxy (—OH) position, the 7.sup.th position of resorufin, is substituted with azidobenzyl; or azidobenzylfluorescein in which a hydroxy (—OH) position, the 7.sup.th position of fluorescein, is substituted with azidobenzyl.

4. The nanocomposite of claim 1, comprising the first surfactant and the second surfactant in a weight ratio of 10:90 to 50:50.

5. The nanocomposite of claim 1, comprising the reactive fluorophore in an amount of 1 to 30 parts by weight based on 100 parts by weight of the first surfactant.

6. The nanocomposite of claim 1, comprising the C.sub.10-25 alkane or haloalkane in an amount of 20 wt % to 50 wt % based on the weight of the reactive fluorophore.

7. A method for preparing the nanocomposite of claim 1, comprising a first step of mixing a mixture of 1-iodooctadeance and a reactive fluorophore dissolved in an organic solvent with a mixed aqueous solution comprising a neutral first surfactant and a cationic second surfactant.

8. The method of claim 7, wherein the organic solvent is used in an amount of 0.2 vol % (v/v) to 5 vol % (v/v) relative to the aqueous solution.

9. The method of claim 7, wherein the nanocomposite is provided in the form of an aqueous dispersion solution.

10. A kit for detecting hydrogen sulfide comprising the nanocomposite of claim 1.

11. The kit of claim 10, wherein the kit is used for the diagnosis of a disease causing abnormal secretion of hydrogen sulfide, which is selected from the group consisting of chronic kidney disease, cirrhosis, Down's syndrome, Alzheimer's disease, and diabetes.

12. A method for providing information for the diagnosis of a disease causing abnormal secretion of hydrogen sulfide, comprising: a first step of contacting the nanocomposite of claim 1 with a specimen isolated from a subject suspected of having a disease; a second step of measuring a fluorescence spectrum of a sample obtained from the first step; and a third step of deriving the concentration of hydrogen sulfide in the specimen from the fluorescence spectrum obtained from the second step.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIGS. 1a and 1b are graphs showing the absorption of resorufin (red curve) and ABR (orange curve) dissolved in DMSO and the fluorescence spectra at an excitation wavelength of 480 nm, respectively.

(2) FIG. 2a is a graph showing fluorescence spectral changes of ABR in an environment where NaHS is absent (orange curve) or present (red curve).

(3) FIG. 2b is a graph showing the fluorescence intensity of ABR in the presence of various reactive species (100 μM unless specified otherwise): (1) ABR only; (2) NaHS; (3) SCN.sup.−; (4) GSNO; (5) GSH (1 mM); (6) SO.sub.3.sup.2−; (7) S.sub.2O.sub.3.sup.2−; (8) L-cysteine; (9) homo-cysteine; (10) H.sub.2O.sub.2; (11) TBHP; (12) O.sub.2.sup.−; (13) OCl.sup.−; (14) NO.; (15) ClO.sub.4.sup.−; (16) OH.; and (17) t-BuO.

(4) FIG. 3a is a graph showing .sup.1H-NMR spectra of ABR in CDCl.sub.3.

(5) FIG. 3b is a graph showing .sup.1H-NMR spectra of ABR after reaction with H.sub.2S in DMSO-d.sub.6.

(6) FIG. 3c is a graph showing .sup.1H-NMR spectra of resorufin sodium salt in DMSO-d.sub.6.

(7) FIG. 4a is a diagram schematically showing the shape and operation principle of nanoABR.

(8) FIG. 4b is a graph showing the change in fluorescence intensity of nanoABR depending on the co-assembled surfactants before (black) and after (red) the addition of NaHS in a PBS buffer.

(9) FIGS. 4c and 4d are graphs showing the fluorescence intensity of nanoABR over time in the presence of NaHS (100 μM).

(10) FIG. 4e is a graph showing the intensity of recovered nanoABR fluorescence depending on the concentration of NaHS.

(11) FIGS. 5a, 5b, and 5c are graphs showing number-averaged hydrodynamic size distributions of nanoABR co-assembled with each of (a) SKC, (b) SDS, and (c) F127.

(12) FIG. 6 is a graph showing the fluorescence intensity of nanoABR in a PBS buffer in the presence of various reactive species (100 μM unless specified otherwise): (1) ABR only; (2) NaHS; (3) SCN.sup.−; (4) GSNO; (5) GSH (1 mM); (6) SO.sub.3.sup.2−; (7) S.sub.2O.sub.3.sup.2−; (8) L-cysteine; (9) homo-cysteine; (10) H.sub.2O.sub.2; (11) TBHP; (12) O.sub.2.sup.−; (13) OCl.sup.−; (14) NO.; (15) ClO.sub.4.sup.−; (16) OH.; and (17) t-BuO.

(13) FIG. 7 is graphs showing the absorption (a) and fluorescence (b, excited at 480 nm) spectra of resorufin. The red curves indicate the spectra of resorufin in water and the orange curves indicate the spectra of nanoABR after reaction with H.sub.2S in a PBS buffer.

(14) FIG. 8 is a graph showing the normalized florescence spectra of resorufin in various solvents, (red) H.sub.2O; (green) methanol; (sky blue) acetonitrile; (blue) dimethylsulfoxide. The observed polarity-dependent peak shift indicates that resorufin has negative solvatochromism.

(15) FIG. 9 is a graph showing the temporal fluorescence bleaching of free resorufin in water (black dot) and nanoABR (red dot) after reaction with H.sub.2S in a PBS buffer, under laser irradiation at 532 nm for 5 minutes.

(16) FIG. 10 is a graph showing the fluorescence intensity of nanoABR depending on the concentration of NaHS.

(17) FIG. 11 is graphs showing the spectra of (a) the methylene blue absorption and (b) the nanoABR fluorescence (excited at 480 nm) in the presence of NaHS at various concentrations in PBS.

(18) FIG. 12a is bright field/fluorescence images of HeLa cells treated with nanoABR (4×10.sup.−5 M) for 1 hour: (i) probe alone, (ii) probe+inhibitor (PAG), and (iii) probe+inducer (SNP).

(19) FIG. 12b is a graph showing the spectral profiles (left) and the relative intensities (right) of cytosolic fluorescence in FIG. 12a.

(20) FIG. 13 is a graph showing the cytotoxicity of nanoABR against HeLa cells.

(21) FIG. 14a is a diagram showing the fluorescence from nanoABR taken 30 minutes after mixing with sera from diabetes or normal mice (Ex 535 nm/Em 600 nm). Sera without mixing with nanoABR were only used as a control group.

(22) FIG. 14b is a graph showing the absolute values of the fluorescence intensities observed in FIG. 14a.

(23) FIG. 14c is a graph showing the control-normalized relative values for the fluorescence intensities observed in FIG. 14a.

(24) FIG. 15 is a graph showing the blood glucose levels of normal (blue) and diabetes (green) mouse models at selected time points after injection of streptozotocin (STZ) for inducing diabetes.

DETAILED DESCRIPTION OF THE INVENTION

(25) Hereinbelow, the present invention will be described in detail with accompanying exemplary embodiments. However, the exemplary embodiments disclosed herein are only for illustrative purposes and should not be construed as limiting the scope of the present invention.

(26) <Materials and Instrumentation>

(27) All chemical reagents were purchased from Aldrich and TCI and used without purification. DSPE-PEG-2000 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylenegly-col)]) was purchased from Avanti Polar Lipids, Inc. 4-Azidobenzyl bromide was synthesized by a method known in the art (H. Zhang et al., Talanta, 2015, 135: 149-154). .sup.1H NMR and .sup.13C NMR spectra of the synthesized compounds were recorded on a Bruker AVANCE 400 spectrometer. Elemental analysis was carried out using a FLASH 2000 (Thermo SCIENTIFIC, England) CHNS analyzer. Absorption and photoluminescence spectra were recorded on a UV-visible spectrometer (Agilent 8453) and the F-7000 fluorescence spectrophotometer (Hitachi, wavelength calibrated for excitation and emission), respectively. The nanoparticle size distribution was determined by a dynamic light scattering (DLS) method using a particle sizer (90Plus, Brookhaven Instruments Corporation)) at 25° C.

Example 1: Synthesis of ABR

(28) Resorufin (50 mg, 0.145 mmol) and K.sub.2CO.sub.3 (40 mg, 0.29 mmol) were dissolved in DMF (4 mL) and stirred at room temperature under an argon atmosphere. After the solution color changed to dark purple, 4-azidobenzyl bromide (61.5 mg, 0.29 mmol) dissolved in DMF (1 mL) was added dropwise to the stirred solution. The reaction mixture was stirred at 50° C. for 3 hours. After cooling to room temperature, the reaction mixture was poured into brine and extracted with ethyl acetate two times. The organic layer was separated and dried over anhydrous MgSO.sub.4. The solvent was evaporated at reduced pressure, and the residue was purified by column chromatography on silica gel with ethyl acetate/n-hexane 1:1 (v/v). ABR (35 mg) was obtained as an orange solid in 70.3% yield.

(29) .sup.1H NMR (400 MHz, CDCl.sub.3, δ): 7.72-7.70 (d, J=8.8 Hz, 1H), 7.42-7.39 (m, J=8.4 Hz and 10.0 Hz, 3H), 7.07-7.05 (d, J=8.4 Hz, 2H), 6.99-6.97 (dd, J=8.8 Hz and 2.8 Hz, 1H), 6.86-6.85 (d, J=2.4 Hz, 1H), 6.84-6.81 (dd, J=10.0 Hz and 2.0 Hz, 1H), 6.30 (d, J=2.0 Hz, 1H), 5.12 (s, 2H);

(30) .sup.13C NMR (150 MHz, CDCl.sub.3, δ): 186.34, 162.47, 149.82, 145.76, 145.64, 140.47, 134.74, 134.34, 132.03, 131.70, 129.19, 128.59, 119.44, 114.24, 106.80, 101.07, 70.32;

(31) Anal. calcd for C.sub.19H.sub.12N.sub.4O.sub.3: C 66.28, H 3.51, N 16.27; found: C 66.10, H 4.13, N 15.47.

(32) Specifically, in order to devise a H.sub.2S-responsive fluorogenic molecular probe, the present inventors have adopted a reaction strategy based on the chemoselective reduction of aryl azides to amines, which is triggered through nucleophilic attack by a hydrosulfide anion (HS.sup.−), which is the main active form of H.sub.2S under physiological conditions. In the probe design, the 4-azidobenzyl group was selected as a self-immolative aryl azide linker, and it was linked to a fluorescent emitter, resorufin, at its 7-hydroxy position. Since the 7-hydroxy substituent is known to efficiently quench the fluorescence of resorufin, the designed molecular probe, azidobenzylresorufin (ABR), is anticipated to be nonfluorescent and able to undergo self-immolative cleavage upon reaction with HS.sup.−, to release a 1,6-elimination product (azaquinone methide) and resorufin with fluorescence recovery. Indeed, the obtained ABR probe that is water-soluble was shown to be virtually nonfluorescent with a hypsochromic absorption shift compared to resorufin in organic media (FIG. 1). As expected from the design, it presented a fluorescence turn-on response toward H.sub.2S (FIG. 2a). Along the resulting fluorescence spectrum, the .sup.1H-NMR analysis confirmed that the fluorescent product is resorufin recovered through the self-immolative release from ABR after reduction by H.sub.2S (FIG. 3). ABR exhibited high chemoselectivity in spectroscopic response; when treated with various biologically relevant reaction sulfur, oxygen, and nitrogen species in acetonitrile, ABR responded only to H.sub.2S with substantial recovery of the resorufin fluorescence (FIG. 2b; not even responsive to excess GSH at a physiologically relevant concentration of 1 mM).

Example 2: Preparation of nanoABR for H.SUB.2.S Detection Using ABR as Fluorescent Molecule Nanoprobe

(33) ABR (0.014 mg) was homogeneously mixed with 1-iodooctadecane (0.005 mg) in a THF (0.2 mL) solvent. After the solvent was evaporated by air flow, the dried mixture was homogeneously dissolved in DMSO (10 μL) and mixed with Milli-Q water (990 μL) containing DSPE-PEG (0.1 mg) with or without stearalkonium chloride (SKC, 0.4 mg) with vigorous shaking to obtain an aqueous dispersion solution of self-assembled nanoABR. The other nanoABR probes co-assembled with differently charged co-surfactant molecules were prepared by following the same procedure with F127 (5 mg) or sodium dodecyl sulfate (0.5 mg) instead of SKC.

Comparative Example 1: Preparation of nanoABR Including Neutral Pluronic as Co-Surfactant

(34) NanoABR was prepared in the same manner as in Example 2 except that neutral Pluronic (F127) was used instead of SKC as the co-surfactant.

Comparative Example 2: Preparation of nanoABR Including Anionic SDS as Co-Surfactant

(35) NanoABR was prepared in the same manner as in Example 2 except that anionic sodium dodecyl sulfate (SDS) was used instead of SKC as the co-surfactant.

Example 3: Effect of Co-Surfactant on Polarity of Composite

(36) In order to apply the water-insoluble ABR to aqueous physiological media, it was formulated into a water-dispersed nanoreactor probe (nanoABR), as depicted in Example 1 and Comparative Example 1 or 2. As shown in FIG. 4a, nanoABR is a nano-scale molecular composite that is composed of DSPE-PEG and 1-iodooctadecane with or without a co-surfactant, and loaded inside with ABR. Prior to optimization of the nanoreactor probe, the nanoscopic polarity effect on the sensing reactivity of the embedded ABR was evaluated. Specifically, as in Example 2, and Comparative Examples 1 and 2, surfactants for co-assembly were selected among differently charged materials, such as cationic SKC (Example 2), neutral F127, and anionic SDS. Through such additional surfactants, i.e., co-assembly including co-surfactants, the interface between the hydrophobic core surface of nanoABR and the surrounding medium was allowed to be differently charged to impose different polarity influences on the nearby core area where ABR is embedded. Nanoassembly with each co-surfactant yielded a stable aqueous dispersion solution of nanoABR probes, all with similar colloidal sizes (50 nm to 60 nm in hydrodynamic diameter, FIG. 5).

(37) FIG. 4b shows the surfactant-dependent fluorescence reactivity of nanoABR to H.sub.2S in a PBS buffer. The parent nanoABR probe without additional co-surfactant assembly showed a very poor response, as opposed to the evident sensing behavior shown by ABR itself in organic media. Similarly, co-assembly with F127 that is electrically as neutral as DSPE-PEG in the parent nanoprobe only showed about 2.3-fold improvement in the fluorescence reactivity to H.sub.2S. Considering the fact that the medium effects induced within micelles retard the nucleophilic reaction therein, the poor reactivity of ABR in the nanoABR probe is attributable to the low polarity originating from 1-iodooctadecane in the inner hydrophobic core that may be lower than those of the polar organic media used in FIG. 2. Additionally, it can be considered that the resulting less polar medium within the nanoreactor may prevent the access of a polar sensing analyte from the surrounding aqueous medium to the ABR molecules entrapped inside. When anionic SDS was co-assembled, no notable alteration in the reactivity was observed, with negligibly increased fluorescence intensity (2.6-fold). Form these results, it can be speculated that the interfacial negative charges given by the anionic head group of SDS would favorably increase the internal medium polarity of nanoABR near the surface, but at the same time, they could electrostatically repel an anionic active form of H.sub.2S (HS.sup.−), and thus apparently result in no net influence on the reactivity due to these opposite effects. In contrast, it was shown that the co-assembly with cationic SKC greatly enhances the H.sub.2S-responsive fluorescence recovery from nanoABR (47-fold). Such greatly enhanced reactivity may contribute to the following two favorable effects from SKC: (1) the electrostatic attraction by which the positively charged surface can actively recruit the oppositely charged monoanionic HS.sup.− into the nanoreactor to increase the analyte concentration inside, and (2) the cationic charge-induced polarity in the near-surface nanoreactor medium that can stabilize the anionic polar transition state [ABR-SH].sup.− by the favorable electrostatic interaction.

Example 4: H.SUB.2.S Selectivity Evaluation of nanoABR

(38) For selectivity studies, nanoABR was prepared for the titration of biological reactive species in a PBS buffer at pH 7.4. The stock solution (100 μM) of reactive sulfur species (RSS) was also prepared using a PBS buffer. The stock solutions (100 μM) of reactive oxygen species (ROS) such as H.sub.2O.sub.2, tert-butyl hydroperoxide (TBHP), and OCl.sup.− were provided as 30 wt %, 70 wt %, and 5 wt % aqueous solutions, respectively. NO. was produced by adding stock solution of 3-(aminopropyl)-1-hydroxy-3-isopropyl-2-oxo-1-triazene (NOC-5) dissolved in DMSO. O.sub.2.sup.−′ was obtained from KO.sub.2 in DMSO containing 0.2 M 18-crown-6 ether for increasing solubility of KO.sub.2. OH. and t-BuO. were produced by Fenton reaction of Fe.sup.2+ (1 mM) with H.sub.2O.sub.2 (100 μM) and TBHP (100 μM), respectively.

(39) Specifically, based on the enhanced reactivity of nanoABR induced by SKC in a PBS buffer, its fluorogenic sensing characteristics were evaluated. As shown in the results on ABR in organic media, nanoABR also showed highly selective fluorescence reactivity to H.sub.2S among various biologically relevant reactive chemical species (FIG. 6). The nanoABR reacted with H.sub.2S showed evolution of absorption and fluorescence bands (λ.sub.max.abs=575 nm, λ.sub.max.fl=600 nm) that are typical of the monomer-like spectral profiles of resorufin (FIG. 7). However, these were all notably red-shifted compared to typical monomeric spectra of free resorufin in water (λ.sub.max.abs=555 nm, λ.sub.max.fl=585 nm), indicating that the negatively solvatochromic resorufin molecules recovered after the sensing reaction are kept in the less polar nanoreactor medium instead of released (FIG. 8). In addition, the recovered fluorescence of the nanoABR reacted with H.sub.2S is more photostable than free resorufin under laser irradiation at 532 nm (FIG. 9). As such, the improved photostability can be ascribed to the oxygen-shielding effect by nanoparticle encapsulation, thereby further confirming the retention of the recovered resorufin in the nanoreactor.

(40) FIG. 4c shows the temporal evolution of fluorescence recovery when nanoABR was treated with NaHS (100 μM) in a PBS buffer. As shown by the curve fitting in FIG. 4d, the fluorescence intensity at 600 nm shows a mono-exponential rise profile with a kinetic rate (k.sub.r) of 1.96×10.sup.−3 s.sup.−1. Although the completion of the reaction took about 60 minutes, the initial fluorescence reactivity was intense enough for the signal to be notably detected within 1 minute (the inset of FIG. 4c), and thus it can be applied to the real-time monitoring of H.sub.2S in biological systems. FIG. 4e shows the spectroscopic response depending on the concentration of NaHS in a PBS buffer, and the fluorogenic signal shows a linear correlation in the examined concentration range (5 μM to 150 μM). The detection limit of nanoABR was calculated to be 18 nM (FIG. 10), which is superior to the conventional colorimetric methylene blue formation assay (FIG. 11). Considering the physiological concentrations of H.sub.2S (serum: 30 μM to 100 μM, brain: 150 μM), the achieved detection sensitivity of nanoABR may allow for suitable monitoring of disease-related abnormal H.sub.2S levels in in vivo or in vitro biological samples.

Example 5: In Vitro Cell Labeling and Imaging

(41) A human cervical epitheloid carcinoma (HeLa) cell line was maintained in DEAM with 10% FBS, L-glutamine (5×10.sup.−3 M), and gentamicin (5 μg mL.sup.−1), in a humidified 5% CO.sub.2 incubator at 37° C. The cells were seeded onto 35 mm culture dishes and allowed to grow until 70% confluence. Prior to the experiment, cells were washed twice with the PBS buffer (pH 7.4) and then incubated in serum-free medium (1.9 mL) containing a nanoABR dispersion solution (100 μL). For endogenous sulfide imaging, cells were pretreated for 30 minutes in a serum-free medium containing sodium nitroprusside (SNP, 100 μM). For an inhibition test, cells were pretreated with DL-propargylglycine (PAG, 100 μM) for 30 minutes. The pretreated cells were washed twice with the PBS buffer (pH 7.4) to remove free nanoparticles just before the data acquisition, and subjected to microscopic imaging with a LEICA DMI3000B microscope equipped with a Nuance FX multispectral imaging system (CRI, USA).

(42) In order to demonstrate the feasibility of using nanoABR as a bioprobe, cellular internalization and fluorescence imaging of endogenous H.sub.2S by using HeLa cells were studied in the presence of chemical agents that stimulate or inhibit cellular generation of H.sub.2S (FIG. 12). When intact HeLa cells without chemical treatment were used as a control group, one-hour incubation with nanoABR at 37° C. was enough for reliable imaging of cytosolic fluorescence reactivity (FIG. 12a), suggesting that nanoABR is sufficiently taken up by cells, and also that its reactivity is sensitive enough to detect normal levels of endogenous H.sub.2S produced by intracellular enzymes under a physiological condition. In order to induce abnormal overproduction of H.sub.2S, cells were pretreated with sodium nitroprusside (SNP) that can upregulate H.sub.2S-producing enzymes, cystathionine β-synthase (CBS), and cystathionine γ-lyase (CSE) by NO. In such a cellular model overproducing H.sub.2S, nanoABR exhibited a more vivid cytosolic fluorescence reactivity that is two-fold more intense than that from the SNP-untreated control cells, reflecting the increased level of endogenous H.sub.2S. Upon pretreating cells with propargylglycine (PAG, 100 μM), a selective inhibitor of CBS and CSE, the cytosolic fluorescence reactivity of nanoABR became remarkably weak compared to that in PAG-untreated control cells, confirming that all of the imaged signals are in fact a response to cellular H.sub.2S. The cytosolic fluorescence spectra from cells, which are in accordance with that of resorufin along with their intensities depending on the cellular conditions (FIG. 12b), demonstrate that fluorogenic reactivity of nanoABR is operative to sensitively detect the level changes of endogenous H.sub.2S implicated in the cellular processes. The cell viability assay shows minimal toxic effects on live cells under the conditions adopted for imaging experiments (FIG. 13), suggesting the potential of nanoABR for biosensing applications.

Example 6: In Vitro Diagnostic Imaging of Diabetes

(43) The animal studies have been approved by the animal care and use committee of Korea Institute of Science and Technology, and all handling of mice was performed in accordance with the institutional regulations. A type 2 diabetes mouse model was prepared using CD-1 mice (male, 10 weeks of age, Orient Bio Inc., Korea) by anaesthetizing with intraperitoneal injection of 0.5% pentobarbital sodium (0.01 mL/g). Diabetes was induced by intraperitoneal injection of streptozotocin (STZ, 100 μL, 40 mg/mL in a PBS buffer), and the injection was repeated 4 times for 1 month. Mice with blood glucose levels between 250 mg/dL and 450 mg/dL were selected for the study. Mice were sacrificed and blood was collected heparinized capillary tubes. The capillary tubes were centrifuged, and the separated plasma was collected in microfuge tubes. Fluorescence imaging of H.sub.2S in plasma was carried out with an IVIS spectrum imaging system (Caliper, USA).

(44) In order to evaluate the practical biomedical utility of nanoABR, the possibility of diagnosing diabetes in vitro by detecting the H.sub.2S level change in serum extracted from a mouse model with chemically induced type 2 diabetes was tested. The levels of H.sub.2S in diabetes patients are known to drop in blood but increase in organs such as the pancreas or liver, and thus can be used as a potential blood biomarker having clinical importance for in vitro diagnosis of diabetes. FIG. 14a shows the fluorescence reactivity of nanoABR to plasma H.sub.2S, measured at 30 minutes after mixing with sera from normal or diabetic mice. It was observed that the fluorescence intensity recovered in sera collected from the diabetes model is statistically lower than that from normal mouse sera (FIG. 14b), suggesting a reduced blood concentration level of H.sub.2S due to diabetes. When normalized against the autofluorescence background from serum (FIG. 14c), this tendency is inversely correlated with an increase in the blood glucose level due to diabetes (FIG. 15). These results indicate that the molecularly assembled nanoABR probe retains structural integrity and high selectivity/sensitivity in biological fluids, and thus can suggest practical applicability for biomedical uses.

(45) In conclusion, the present inventors prepared a novel self-immolative azidobenzyl-substituted resorufin-based H.sub.2S-selective molecular probe (ABR) and studied its sensing reactivity within a molecularly assembled nanoreactor system (nanoABR) whose internal medium was elaborately engineered in terms of the electrical polarity on its surface. It was confirmed that the positively charged polar environment of the nanoABR interior established by co-assembly with a cationic co-surfactant (SKC) remarkably facilitates the nucleophilic sensing reaction of the embedded ABR in an electrostatic manner, through active recruitment of an anionic analyte (HS.sup.−) and stabilization of the anionic transition state of the sensing reaction. The sensing characteristics shown in physiological media having minimal cytotoxicity allowed for practical bioapplications to microscopic imaging of cellular processes and in vitro diagnostics of diabetes with blood samples from animal models.

(46) From the foregoing, one of ordinary skill in the art to which the present invention pertains will be able to understand that the present invention may be embodied in other specific forms without modifying the technical concepts or essential characteristics of the present invention. In this regard, the exemplary embodiments disclosed herein are only for illustrative purposes and should not be construed as limiting the scope of the present invention. On the contrary, the present invention is intended to cover not only the exemplary embodiments but also various alternatives, modifications, equivalents, and other embodiments that may be included within the spirit and scope of the present invention as defined by the appended claims.