Carbonic anhydrase IX activated and disassembled nano-probe as well as construction method and application thereof

Abstract

The invention belongs to the technical field of biomedical detection and molecular imaging, and particularly discloses a carbonic anhydrase IX activated response type nanoprobe and a construction method and application thereof. The probe SQ-H-SA is a self-detachable fluorescent nanoprobe based on squaric acid dye, has a synergistic mechanism of self-assembly complete background quenching, target-triggered dual signal amplification and whole blood autofluorescence inhibition, and realizes high sensitivity, background interference resistance and accurate imaging capability for CA detection. Not only is the high-throughput analysis capability of clinical samples achieved, but also the diagnosis precision of liquid biopsy is improved, and first-time selective and cell membrane specific fluorescence imaging of CAIX in cancer cells and clinical lung cancer tissues is achieved. The invention also provides a preparation method and application of the probe SQ-H-SA.

Description

Technical Field

[0001] This invention relates to the fields of biomedical detection and molecular imaging technology, and in particular to carbonic anhydrase IX activated disassembly nanoprobes, their construction methods, and applications. Background Technology

[0002] Carbonic anhydrases (CAs) are a class of zinc-containing metalloenzymes that catalyze the reversible hydration of carbon dioxide. It plays a crucial role in maintaining physiological pH balance and tumor microenvironment acidification. Among them, CAIX is an isoenzyme in the CA family. As a transmembrane glycoprotein, it is highly expressed in a variety of solid tumors (such as lung cancer, kidney cancer, and breast cancer), but almost not expressed in normal tissues. It is a highly promising biomarker for cancer diagnosis and prognosis.

[0003] Although CAIX is widely recognized as a key biomarker for tumor diagnosis and treatment monitoring, quantitative detection and accurate imaging of CAIX still face technical challenges. Current detection technologies for CAIX suffer from the following bottlenecks: 1) Insufficient sensitivity and signal-to-noise ratio: Traditional small molecule probes have high background fluorescence (e.g., rhodamine, BODIPY) or limited signal amplification (usually <30-fold), making it difficult to detect low concentrations of CA; 2) Severe interference from whole blood testing: Blood autofluorescence and albumin (~50 mg / mL) interference prevent direct application to clinical samples, requiring plasma separation (which may result in the loss of soluble CAIX fragments); 3) Inability to achieve in situ visualization: Existing probes cannot specifically target CAIX on cancer cell membranes, leading to activation of endocytosis in normal cells (false positive signals) and low tissue imaging contrast (T / N ratio <2).

[0004] Given the shortcomings of current CA detection technology, there is an urgent need to develop a new CA detection technology that combines high sensitivity, resistance to background interference, and accurate imaging capabilities. Summary of the Invention

[0005] This invention aims to address at least one of the technical problems existing in the prior art. To this end, this invention provides a carbonic anhydrase IX activated and disassembled nanoprobe, its construction method, and its applications. The probe SQ-H-SA of this invention achieves a novel carbonic anhydrase (CA) detection technology with high sensitivity, resistance to background interference, and accurate imaging capabilities through a synergistic mechanism of self-assembly-complete background quenching, target-triggered dual signal amplification, and whole blood autofluorescence inhibition. Furthermore, this probe also possesses cancer cell membrane-specific imaging and high-throughput detection and analysis capabilities, effectively improving diagnostic accuracy in liquid biopsies.

[0006] The present invention also provides a method for preparing the probe SQ-H-SA.

[0007] This invention also proposes the application of probe SQ-H-SA in the preparation of tumor detection reagents and in tumor detection.

[0008] The present invention also proposes a tumor diagnostic kit containing the probe SQ-H-SA.

[0009] In a first aspect, the present invention provides a fluorescent nanoprobe SQ-H-SA, the structural formula of which is shown below:

[0010]

[0011] According to a specific embodiment of the present invention, a self-disassembling fluorescent nanoprobe (SQ-H-SA) based on squareaine (SQ) dye is proposed, achieving a technological breakthrough through the following synergistic mechanism:

[0012] Self-assembly completely quenches the background: SQ molecules form stable aggregates through π-π stacking and antiparallel dipole interactions, with a quantum yield as low as 1.16% in the aqueous phase. Target-triggered dual signal amplification: (1) CA binding induces probe depolymerization, eliminating the aggregation quenching effect (ACQ); (2) The monomer SQ is shielded by the hydrophobic chamber of CA, increasing the quantum yield to 34.36% and enhancing the total signal by 45 times (3μM CAIX, within 7min). Whole blood autofluorescence inhibition: The probe has a molar extinction coefficient of 3.24×105M-1cm-1 at 600nm, and inhibits 70% of the blood background fluorescence through the internal filtration effect (IFE), achieving high-sensitivity detection of CA in 50-fold diluted whole blood (LOD=3.5nM). Cancer cell membrane-specific imaging: The synergistic hydrogen bonding and hydrophobic interaction between the probe and CAIX (ΔG=-8.145kcal / mol) achieves cancer cell membrane targeting, avoiding activation by endocytosis of normal cells, and the T / N ratio in clinical lung cancer tissue imaging reaches 4:1. Whole blood testing method: Mix 40 μM SQ-H-SA with 50-fold diluted whole blood, incubate at 37°C for 10 min, and directly read the signal using a fluorescence spectrometer (λex / λem = 600 / 640 nm) without centrifugation or lysis of red blood cells. Cancer tissue imaging: Immerse fresh tumor tissue in 5 μM SQ-H-SA solution. Fluorescence signal saturation occurs within 10 minutes, and high-contrast images are obtained using a confocal microscope (λex = 640 nm).

[0013] A second aspect of the present invention provides a method for preparing the fluorescent nanoprobe SQ-H-SA described in the first aspect of the present invention, comprising the following steps:

[0014] S1. 3,3-Dimethyl-3H-indole and 6-bromohexanoic acid were mixed in a solvent and reacted. After the reaction was completed, the mixture was poured into a petroleum ether / ethyl acetate solution, cooled, and the crude product was precipitated. The purified compound was 6-(3,3-dimethyl-3H-indole)hexanoic acid.

[0015] S2. Dissolve 6-(3,3-dimethyl-3H-indole)hexanoic acid and 3,4-dihydroxy-3-cyclobutene-1,2-dione in a solvent, heat to carry out the reaction, and purify to obtain the dye Dye-COOH after the reaction is completed.

[0016] S3. Dissolve Dye-COOH and 4-(2-aminoethyl)benzenesulfonamide in a solvent, and add 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate and N,N-diisopropylethylamine in sequence. Stir the mixture under a nitrogen atmosphere to carry out the reaction. After the reaction is completed, purify the solution to obtain the fluorescent nanoprobe SQ-H-SA.

[0017] According to some embodiments of the present invention, the molar ratio of 3,3-dimethyl-3h-indole and 6-bromohexanoic acid in step S1 is 1:(0.5-2).

[0018] According to some embodiments of the present invention, the molar ratio of 3,3-dimethyl-3h-indole and 6-bromohexanoic acid in step S1 is 1:(0.7-1.5).

[0019] According to some embodiments of the present invention, the solvent in step S1 includes anhydrous acetonitrile, and the concentration of 3,3-dimethyl-3h-indole in the reaction solution is 1 to 5 mmol / mL.

[0020] According to some embodiments of the present invention, the reaction described in step S1 is carried out at 70-80°C for 4-6 hours under an inert atmosphere.

[0021] According to some embodiments of the present invention, the volume ratio of petroleum ether to ethyl acetate in the petroleum ether / ethyl acetate solution in step S1 is 3:(0.5-2).

[0022] According to some embodiments of the present invention, the molar ratio of 6-(3,3-dimethyl-3H-indole)hexanoic acid and 3,4-dihydroxy-3-cyclobutene-1,2-dione in step S2 is (2-5):1.

[0023] According to some embodiments of the present invention, the molar ratio of 6-(3,3-dimethyl-3H-indole)hexanoic acid and 3,4-dihydroxy-3-cyclobutene-1,2-dione in step S2 is (3-4):1.

[0024] According to some embodiments of the present invention, the heating reaction in step S2 is a reflux reaction at 100-130°C for 6-10 hours.

[0025] According to some embodiments of the present invention, the solvent in step S2 is a mixed solvent of toluene, pyridine and n-butanol.

[0026] According to some embodiments of the present invention, the volume ratio of toluene, pyridine and n-butanol in the mixed solvent in step S2 is (2-2.4):(1-1.2):(1-1.2).

[0027] According to some embodiments of the present invention, the purification in step S2 is performed by silica gel column separation and purification, using a mixed solution of dichloromethane and methanol as the eluent, wherein the volume ratio of dichloromethane to methanol in the eluent is 20:(0.5-2).

[0028] According to some embodiments of the present invention, the molar ratio of Dye-COOH, 4-(2-aminoethyl)benzenesulfonamide, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate and N,N-diisopropylethylamine in step S3 is 1:(2-10):(0.2-1):(1-5).

[0029] According to some embodiments of the present invention, the molar ratio of Dye-COOH, 4-(2-aminoethyl)benzenesulfonamide, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate and N,N-diisopropylethylamine in step S3 is 1:(4-6):(0.4-0.6):(2-4).

[0030] According to some embodiments of the present invention, the solvent in step S3 includes anhydrous dichloromethane, and the concentration of Dye-COOH in the reaction solution is 0.05 to 0.1 mmol / mL.

[0031] According to some embodiments of the present invention, the reaction in step S3 is a stirred reaction at room temperature for 1 to 3 hours.

[0032] According to some embodiments of the present invention, the purification in step S3 is performed by silica gel column separation and purification, using a mixed solution of dichloromethane and methanol as the eluent, wherein the volume ratio of dichloromethane to methanol in the eluent is 30:(0.5-2).

[0033] According to some embodiments of the present invention, the preparation of the fluorescent nanoprobe SQ-H-SA further includes a self-assembly process, the specific steps of which are as follows:

[0034] The fluorescent nanoprobe SQ-H-SA was dissolved in DMSO to obtain a stock solution. The stock solution was then added to a DPBS:DMSO mixed buffer and mixed to obtain a self-assembly-disassembly driven fluorescent nanoprobe SQ-H-SA with a final concentration of 0.5–50 μM.

[0035] According to some embodiments of the present invention, the final concentration of the self-assembly-disassembly driven fluorescent nanoprobe SQ-H-SA is 1–20 μM.

[0036] According to some embodiments of the present invention, the final concentration of the self-assembly-disassembly driven fluorescent nanoprobe SQ-H-SA is 2–10 μM.

[0037] According to some embodiments of the present invention, the volume ratio of DPBS to DMSO in the DPBS:DMSO mixed buffer is (18-20):1.

[0038] According to some embodiments of the present invention, the mixing method is to use a vortex mixer to mix at 200-300 rpm for 4-6 minutes.

[0039] A third aspect of the present invention provides the application of the fluorescent nanoprobe SQ-H-SA described in the first aspect in the preparation of tumor detection reagents.

[0040] A fourth aspect of the present invention provides the application of the fluorescent nanoprobe SQ-H-SA described in the first aspect in tumor detection.

[0041] According to some embodiments of the present invention, the fluorescent nanoprobe SQ-H-SA is used for the quantitative or qualitative detection of carbonic anhydrase in tumor detection.

[0042] According to some embodiments of the present invention, the fluorescent nanoprobe SQ-H-SA is used for the quantitative or qualitative detection of carbonic anhydrase IX (CAIX) in tumor detection.

[0043] According to some embodiments of the present invention, the fluorescent nanoprobe SQ-H-SA is used in tumor detection for whole blood detection, monitoring of the efficacy of tumor treatment, and fluorescence imaging of tumor tissue.

[0044] In a fifth aspect, the present invention provides a tumor diagnostic kit comprising the fluorescent nanoprobe SQ-H-SA as described in claim 1.

[0045] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0046] 1) The fluorescent nanoprobe SQ-H-SA of this invention forms a completely fluorescence-quenched nanoaggregate through rigid self-assembly, and its quantum yield (Φ) F As low as 1.16% (excitation wavelength λ) ex =600nm), compared to the BODIPY-based probe (baseline Φ) reported in the literature. F The background was reduced by 4.6 times (~5.3%); combined with CA recognition-triggered disassembly and protein hydrophobic cavity shielding effect, Φ FThe fluorescence intensity was increased by 30-fold (to 34.36%), and the total fluorescence enhancement reached 45-fold (within 7 min under 3 μM CA IX stimulation), significantly outperforming existing self-assembled probes (maximum enhancement of 38-fold). The two-stage amplification mechanism (disassembly to eliminate aggregation quenching + hydrophobic environment enhancement) resulted in a signal-to-noise ratio (S / N) as high as 162, which is 10.8-fold higher than that of traditional ligand-based probes (S / B = 15).

[0047] 2) The fluorescent nanoprobe SQ-H-SA of this invention has a detection limit as low as 3.50 nM (S / N = 3) for CA IX, and a dynamic range spanning 3.5 nM-3 μM (linear RL). 2 >0.99), which improves sensitivity by 5900 times compared to the rhodamine-based competitive method (LOD = 20.6 μM); the probe's absorption spectrum (638 nm, ε = 3.24 × 10⁻⁶) is also improved. 5 M -1 cm -1 The ligand probe precisely overlaps with the autofluorescence spectrum of blood (550-650nm), and suppresses 70% of background interference based on the internal filtering effect (IFE), achieving accurate detection of CA IX in 50-fold diluted whole blood (recovery rate 98.2±1.4%). This overcomes the false negative problem caused by centrifugation loss in traditional ligand probes in plasma / serum detection (such as ELISA method underreporting content by 23%).

[0048] 3) The fluorescent nanoprobe SQ-H-SA of this invention enables high-throughput analysis of clinical samples and membrane-specific imaging. In a 96-well plate system, it can complete the parallel detection of 21 clinical blood samples in just 10 minutes. The CAIX positive signal and the healthy group have a discrimination of ****P<0.0001 (F / F0=5.2vs.1.1), which is highly correlated with the ELISA results. At the same time, the probe has achieved specific in vivo imaging of cancer cell membrane CAIX for the first time. The fluorescence intensity ratio of tumor to adjacent tissue is 4:1 (clinical lung cancer sample, n=9). Moreover, it avoids the internalization and activation of normal cells through the size exclusion effect (Dh~190nm). The smaller molecular probe reduces the false positive rate.

[0049] 4) The fluorescent nanoprobe SQ-H-SA of this invention integrates treatment monitoring and multimodal diagnosis and treatment, and can quantitatively track the dynamic changes in postoperative blood CAIX levels in lung cancer patients (post-treatment concentration decreased to 64.2±8.7μM, a decrease of 50.6%), providing real-time feedback for personalized treatment. Combined with fluorescence imaging and flow cytometry, it achieves precise intraoperative tissue boundary delineation (fluorescence intensity gradient ≥3.5×10⁻⁶). 8 The radiance) improves efficiency by 90% compared to traditional H&E pathological slide analysis (reducing the detection cycle from 3 days to 30 minutes).

[0050] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

[0051] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0052] Figure 1 This is a schematic diagram of the synthesis process of the probe SQ-H-SA in Example 1 of the present invention.

[0053] Figure 2 The diagram shows the design and screening results of the fluorescent probes in Example 2 of this invention; (a) molecular structures of the reference dye and four candidate probes; (b) screening results of photophysical properties; (c) signal-to-background ratio (SBR) of each dye and probe at λex / λem = 600 / 640 nm; (d) and (e): the lowest energy conformation and key interaction diagram of docking probe 6 molecules to the CAⅡ and CAⅨ active sites.

[0054] Figure 3 Figure 3 shows the stability analysis results of probe SQ-H-SA in Example 3 of this invention; (a) absorption spectrum of probe SQ-H-SA in DPBS containing different concentrations of DMSO (0-50% v / v); (b) dynamic light scattering (DLS) spectrum of probe SQ-H-SA before and after activation; (c) transmission electron microscopy (TEM) images of probe SQ-H-SA before and after activation; (d) time-dependent fluorescence monitoring results of probe SQ-H-SA; (e) concentration-dependent assembly stability monitoring results of probe SQ-H-SA; (f) DLS spectrum of probe SQ-H-SA after concentration cycling and dilution cycling.

[0055] Figure 4 The following figures show the test results of the response performance of the probe SQ-H-SA to carbonic anhydrase in Example 4 of this invention; wherein, (a) normalized absorption spectra of assembled SQ-H-SA, disassembled probe and monomeric SQ-H-SA; (b) fluorescence titration curves with increasing CAⅡ concentration; (c) fluorescence titration curves with increasing CAIX concentration; (d) dose-response curves of activated fluorescence intensity versus CAⅡ / IX concentration; (e) time-resolved fluorescence decay curves of SQ-H-SA before and after CAⅡ treatment; and (f) absolute fluorescence quantum yield (Φ) before and after CAⅡ activation. F (g) Circular dichroism chromatograms of SQ-H-SA and CAⅡ; (h) Zeta potential chromatograms of SQ-H-SA nanoprobe, free CAⅡ and its complex; (i) Real-time fluorescence kinetics at different CAⅡ concentrations.

[0056] Figure 5The following are the results of quantitative analysis of endogenous CA in clinical whole blood samples according to Example 5 of the present invention: (a) Schematic diagram of clinical testing process and accuracy verification; (b) Fluorescence response results of SQ-H-SA to blood samples with different endogenous CA levels; (c) Quenching effect of SQ-H-SA on autofluorescence of 2% whole blood; (d) Time-dependent fluorescence change of SQ-H-SA at 645nm in 2% whole blood; (e) Comparison of fluorescence spectra of each group; (f) Fluorescence intensity results of SQ-H-SA when CAⅡ is added to 50-fold diluted blood; (g) Relationship between emission intensity and CAⅡ concentration; (h) Comparison of CA levels in lung cancer patients, healthy donors and lung cancer patients after treatment; (i) Correlation between SQ-H-SA fluorescence detection and ELISA results.

[0057] Figure 6 The following are high-throughput quantitative detection results of clinical whole blood samples according to Embodiment 6 of the present invention: (a) a schematic diagram of the whole blood quantitative analysis workflow; (b) a graph of spiked blood quantitative detection results; (c) a graph of dose-response correlation results; (d) a graph of microplate fluorescence analysis results; (e) a graph of linear calibration and regression analysis results; (f) a graph of multimodal imaging high-throughput clinical screening results; (g) a graph of CA level comparison results; (h) a graph of parallel fluorescence verification results; and (i) a graph of CA level comparison results.

[0058] Figure 7 The images shown are rapid targeted imaging results of SQ-H-SA in normoxic and cancer cells in Example 7 of this invention; (a) a schematic diagram of the mechanism of hypoxia-induced CAⅨ upregulation under CoCl2 treatment; (b) real-time membrane imaging results; (c) hypoxia-dependent targeted imaging results; (d) flow cytometry analysis results of LA795 cells; (e) kinetic quantitative results of image b; and (f) quantitative analysis results of image c.

[0059] Figure 8 The following are the results of tissue fluorescence detection in Example 7 of the present invention: (a) a schematic diagram of fluorescence imaging of lung cancer and adjacent normal tissue; (b) a time-delayed fluorescence imaging result of human lung cancer tissue and adjacent normal tissue; (c) a normalized fluorescence intensity result of Figure b; (d) a clinical tissue fluorescence imaging result after 30 min of culture; (e) a quantitative analysis result of fluorescence intensity in Figure c; (f) a confocal imaging result of LCT / PCT sections stained with probe; and (g) a pathological H&E staining result of the tissue.

[0060] Figure 9 This is a schematic diagram illustrating the response mechanism of the SQ-H-SA nanofluorescent probe of the present invention and its application in the quantitative detection of CA enzymes in whole blood and precise imaging of clinical cancer tissues. Detailed Implementation

[0061] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0062] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0063] Example 1: Synthesis method of probe SQ-H-SA

[0064] This embodiment provides a method for synthesizing the probe SQ-H-SA, and the specific steps are as follows:

[0065] 1) 3,3-Dimethyl-3H-indole (5 g, 34.43 mmol) and 6-bromohexanoic acid (6.72 g, 34.32 mmol) were mixed in anhydrous acetonitrile (15 mL) and heated in an inert atmosphere in a sealed reaction vessel at 75 °C for 5 h; the mixture was poured into a petroleum ether / ethyl acetate (PE / EA, 3:1, v / v) solution and cooled to 0 °C to precipitate the crude product; the crude product was filtered and rotary evaporated under reduced pressure to obtain the compound: 6-(3,3-dimethyl-3H-indole)hexanoic acid (2 g, yield 22.1%).

[0066] 2) The 6-(3,3-dimethyl-3H-indole)hexanoic acid (1 g, 3.81 mmol) and 3,4-dihydroxy-3-cyclobutene-1,2-dione (150 mg, 1.15 mmol) obtained in step 1) were dissolved in a mixed solvent system of toluene / pyridine / n-butanol (2:1:1, v / v / v); the reaction was heated to 115 °C, refluxed for 8 h, and stirred vigorously; after vacuum concentration, the solution was purified by column chromatography (gradient: DCM / MeOH = 20:1, v / v) to obtain the dark green solid dye Dye-COOH (100 mg, yield 58%).

[0067] The structure of the dye Dye-COOH:

[0068]

[0069] NMR and mass spectrometry characterization of the dye:

[0070] 1H NMR (500MHz, DMSO-d6) δ11.95(s,2H),7.44(d,J=9.4Hz,2H),7.28-7.23(m,4H),7.09(d,J=8.5Hz,2H),5.72(d, J=14.8Hz,2H),4.00(s,4H),2.13(q,J=7.7,6.6Hz,4H),1.69-1.55(m,10H),1.49(q,J=7.4Hz,5H),1.33(s,5H).

[0071] MS(MALDI-TOF-MS):m / z=624.320,calcd for(C 38 H 44 N₂O₆) = 624.778.

[0072] 3) Dye-COOH (50 mg, 0.4 mmol) and 4-(2-aminoethyl)benzenesulfonamide (200 mg, 2.1 mmol) were dissolved in anhydrous dichloromethane (DCM, 5 mL), and then 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU, 80 mg, 0.21 mmol) and N,N-diisopropylethylamine (DIPEA, 150 mg, 1.2 mmol) were added sequentially. The mixture was stirred at room temperature under a nitrogen atmosphere for 2 h; after concentration under reduced pressure, the crude product was directly purified by silica gel chromatography (DCM / MeOH = 30:1, v / v) to obtain the target probe SQ-H-SA (30 mg, yield 76.0%).

[0073] The schematic diagram of the synthesis process of the probe SQ-H-SA is shown below. Figure 1 As shown.

[0074] Structure of probe SQ-H-SA:

[0075]

[0076] Mass spectrometry and NMR characterization of probe SQ-H-SA:

[0077] 1H NMR (500MHz, DMSO-d6) δ8.05-7.92(m,2H),7.76-7.62(m,6H),7.53(d,J=7.4Hz,2H),7.44-7.25(m,10H),7.18(t,J=7.1Hz,2H),5.82(s ,2H),4.07(s,3H),3.31-3.20(m,9H),2.75(t,J=7.7Hz,4H),2.07(t,J=7.4Hz,4H),1.70(s,12H),1.59-1.54(m,4H),1.41-1.31(m,4H).

[0078] 13 C NMR (176MHz, DMSO) δ159.53,147.53,139.88,134.92,129.60,127.62,126.36,126.13,125.69,122.58,119.25,1 05.20,91.69,78.33,76.52,74.71,63.86,62.37,61.33,53.85,53.28,49.07,35.33,32.97,27.00,25.06,22.53.

[0079] MS(MALDI-TOF-MS):m / z=989.420,calcd for(C 54 H 64 N6O8S2)=989.260.

[0080] Example 2: Rational Design and Screening of CA Enzyme-Responsive Self-Assembled Fluorescent Probes

[0081] Based on the principles of self-assembly stability, CA-specific activation, and collaborative optimization of connector arm length, such as Figure 2 As shown in Figure a, molecules containing sulfonamide (SA) ligands and SQ dyes were designed as reference dyes (1-2) and candidate probes (3-6).

[0082] The photophysical properties of each dye and probe were screened, and the results are as follows: Figure 2Figure b shows the photophysical screening criteria in DPBS / DMSO (19:1, v / v): absolute fluorescence quantum yield (ΦF) and lifetime (τ) before and after 210 μM CA treatment, including the maximum absorption wavelength and molar extinction coefficient, and the absolute ΦF values ​​of dye 2 and probe 6 monomers in DMSO. The results show that CA activation of probe 6 increased the fluorescence quantum yield by 30-fold (ΦF: 1.16% → 34.36%); the fluorescence lifetime was extended to 3.13 ns (before activation: 2.41 ns), indicating that CA induced probe disassembly. The signal-to-background ratio (SBR) results for each dye and probe at λex / λem = 600 / 640 nm are shown below. Figure 2 As shown in Figure c, the 638nm absorption peak overlaps with the blood autofluorescence spectrum. By utilizing the internal filtration effect (IFE) to suppress background interference, probe 6 achieves an ultra-high signal-to-background ratio (SBR = 162 ± 3), which is more than 10 times higher than existing probes, and achieves excellent anti-interference ability.

[0083] The probe 6 molecule was docked to the lowest energy conformation and key interactions of the CAII and CAIX active sites, respectively. Figure 2 As shown in Figures d and e, molecular docking reveals that the binding affinity for CAIX is significantly higher than that for CAII (ΔG = -8.145 vs. -6.776 kcal / mol), and the specificity is mediated by the Leu9 / Gly42 / Lys147 hydrogen bond and the Thr93 / Lys108 hydrophobic interaction.

[0084] Through the above systematic screening, the SQ-H-SA (probe 6) with a six-carbon linker was determined to be the optimal structure, which balances self-assembly stability and CA activation efficiency.

[0085] Example 3: Self-assembly characteristics and stability test of probe SQ-H-SA

[0086] 4.49 mg of probe SQ-H-SA prepared in Example 1 was dissolved in 5 mL of DMSO to prepare a 1 mM stock solution, which was stored at -20 °C. The absorption spectra of the probe in DPBS containing different concentrations of DMSO (0-50% v / v) were tested, and the results are as follows: Figure 3 As shown in Figure a.

[0087] Self-assembly of probe SQ-H-SA: 5 μL of DMSO stock solution (1 mM) of SQ-H-SA was injected into 995 μL of preheated DPBS buffer (containing 5% DMSO, pH 7.4), and vortexed for 5 min (250 rpm) to prepare a 5 μM nanoprobe solution, which self-assembled into stable fluorescence quenching aggregates.

[0088] Dynamic light scattering (DLS) spectra of SQ-H-SA before and after activation with 210 μM CA ( Figure 3 Figure b) and transmission electron microscopy (TEM) images ( Figure 3 The results in Figure c were used for characterization; the DLS test results showed that the particle size distribution peak was located at 190±10nm, indicating the formation of uniform nano-aggregates; the TEM image showed that the average diameter of the probe spherical nanoparticles was 180nm.

[0089] The long-term stability of the probe was tested, and the results are as follows: Figure 3 As shown in Figure d, after 21 days of storage at 25℃ under light-protected conditions, the fluorescence intensity showed little fluctuation, demonstrating its stability. Subsequently, the concentration-dependent assembly stability of the probe was tested. The UV-Vis absorption spectra of the SQ-H-SA nanofluorescent probe (×1:5μM) under continuous concentration cycling and dilution cycling are shown in Figure d. Figure 3 As shown in Figure e, the DLS spectrum results are as follows: Figure 3 As shown in Figure f, it also proves that the device assembly can maintain stability under changing external conditions.

[0090] The combined results of the above tests indicate that the probe SQ-H-SA possesses both good self-assembly characteristics and stability.

[0091] Example 4: Fluorescence response and sensitivity detection of CA by probe SQ-H-SA in a buffer system

[0092] Dissolve 4.49 mg of the probe in 5 mL of DMSO to prepare a 1 mM stock solution, which was stored at -20 °C. The detection system was DPBS buffer solution (10 mM, pH 7.4, containing 5% DMSO).

[0093] The reaction system of probe SQ-H-SA and carbonic anhydrase was shaken at room temperature for 30 min, and its fluorescence emission and UV-Vis absorption spectra were measured. The excitation wavelength of the fluorescence instrument was set to 600 nm, the emission wavelength receiving range was 610-800 nm, the slit width was 5 nm / 5 nm, and the absorption spectrum measurement range was set to 550-750 nm. The test results are as follows. Figure 4 As shown.

[0094] in, Figure 4Figure a shows the normalized absorption spectra, illustrating the assembled SQ-H-SA (5 μM) nanoprobe (black curve in the figure), the disassembled probe after incubation with 210 μM CA for 30 min (red curve in the figure), and the monomeric SQ-H-SA in DPBS / DMSO (1:1 v / v) (blue curve in the figure), demonstrating the occurrence of assembly and disassembly. Figures b and c show the fluorescence titration curves with increasing concentrations of CAⅡ (0-210 μM) and CAⅨ (0-3 μM), respectively. The results show that under 3 μM CAIX stimulation, the fluorescence intensity increases 45-fold at 645 nm, and at 210 μM... Under CAⅡ stimulation, the fluorescence intensity increased 160-fold at 645 nm, demonstrating the probe's specific recognition response to CAⅡ / Ⅸ; Figure d shows the dose-response curve of activated fluorescence intensity (645 nm) versus CAⅡ / Ⅸ concentration (0-3 μM), demonstrating its linear quantification of CAⅡ / Ⅸ; Figure e shows the time-resolved fluorescence decay curves of SQ-H-SA (5 μM) before (τ = 2.41 ns) and after (τ = 3.13 ns) treatment with 210 μM CAⅡ, demonstrating that the probe is encapsulated by the enzyme, increasing its fluorescence lifetime; Figure f shows the absolute fluorescence quantum yield (Φ) of CAⅡ (210 μM) before activation (1.16%) and after activation (34.36%). F The graph further demonstrates its responsiveness; Figure g shows the circular dichroism (CD) spectra of the nanoprobes SQ-H-SA (1-5 μM) and CAⅡ (20 μM), proving the binding of the enzyme to the probe; Figure h shows the Zeta potential of the SQ-H-SA (5 μM) nanoprobe, free CAⅡ (210 μM) and their complex, further proving the binding of the enzyme to the probe; Figure i shows the real-time fluorescence kinetics (fluorescence intensity F at 645 nm) results at different CAⅡ concentrations (0, 5, 14, 140, 210 μM), showing the response time: t 1 / 2 =2.3 min, and reached the plateau phase within 7 min. Detection limit (LOD): calculated at 3σ / S as 3.50 nM (n=3), proving that the probe not only has a rapid response but also extremely high sensitivity.

[0095] The combined results of the above tests demonstrate the qualitative and quantitative analytical capabilities of the probe SQ-H-SA for CAⅡ / Ⅸ.

[0096] Example 5: Quantitative detection of endogenous CAIX in whole blood using probe SQ-H-SA.

[0097] Dissolve 4.49 mg of the probe in 5 mL of DMSO to prepare a 1 mM stock solution, which was stored at -20 °C. The detection system was PBS buffer solution (10 mM, pH 7.4, containing 50% DMSO).

[0098] like Figure 5The clinical testing procedure and accuracy verification diagram shown in Figure a illustrates the sample processing: Whole blood (EDTA anticoagulated) from healthy donors and lung cancer patients was diluted 50-fold (DPBS buffer) and pretreated with 1 mM ABS for 30 min to quench endogenous CA activity. Then, 40 μM SQ-H-SA (final concentration 5 μM) was added, and the mixture was incubated at 37°C for 10 min. Fluorescence intensity was measured (λex = 600 nm, λem = 645 nm). The fluorescence emission and UV-Vis absorption spectra were then measured. The excitation wavelength of the fluorometer was set to 600 nm, the emission wavelength receiving range was 610-800 nm, and the slit width was 5 nm / 5 nm. The results are as follows: Figure 5 As shown.

[0099] in, Figure 5 Figure b shows the fluorescence response of 40 μM SQ-H-SA to blood samples with different endogenous CA levels. F and F0 represent the fluorescence intensity at 654 nm in the absence of 1 mM ABS, respectively, used to screen for the optimal dilution factor. The result showed that a 50-fold dilution yielded the best effect. Figure c shows the effect of SQ-H-SA (0-40 μM) on 2% whole blood in DPBS. ex The quenching effect of autofluorescence at 570 nm was used to screen for the optimal quenching concentration, and the result showed that 40 μM SQ-H-SA was the most effective. Figure d shows the time-dependent fluorescence change of 40 μM SQ-H-SA at 645 nm in 2% whole blood from 0 to 10 min, demonstrating its rapid detection response. Figure e shows the fluorescence spectra comparison of baseline (pink area), ABS-induced quenching (black curve), endogenous CA in healthy blood (red curve), blood from lung cancer patients (blue curve), and post-treatment blood (green curve), demonstrating that the probe can be applied to whole blood detection. Figure f shows the effect of adding 0-2.8 μM CAⅡ to 50-fold diluted blood with 40 μM SQ-H-SA. The fluorescence intensity of SQ-H-SA is shown, where "0" represents blood treated with ABS to remove CA, and "X" represents endogenous CA signal. Figure g shows the relationship between emission intensity and CAII concentration (0, X, X+0.7, X+1.05, X+1.4, X+2.1, X+2.8), used to determine whole blood CA concentration. Data are mean ± standard deviation (n=3), where the standard curve is a linear equation established based on recovery experiments with added exogenous CAII, R0. 2 =0.994, demonstrating the quantitative analysis of whole blood detection by the probe; the h-plot shows the comparison of CA levels, showing that the CA signal in lung cancer patients (Disease) is significantly higher than that in healthy donors (Normal), and significantly reduced after treatment (Treated) (****P<0.0001), further highlighting the probe's ability to be applied to whole blood detection through case comparison; the i-plot shows the correlation between the probe SQ-H-SA fluorescence detection and ELISA results, using the gold standard to verify the reliability of the method data.

[0100] The combined results of the above tests indicate that the probe SQ-H-SA has promising applications in whole blood testing.

[0101] Example 6: High-throughput analysis of whole blood samples using the SQ-H-SA probe.

[0102] Dissolve 4.49 mg of the probe in 5 mL of DMSO to prepare a 1 mM stock solution, which was stored at -20 °C. The detection system was PBS buffer solution (10 mM, pH 7.4, containing 50% DMSO).

[0103] Figure 6 Figure a in the diagram illustrates the workflow for quantitative analysis of whole blood using a multimodal imaging system and a microplate fluorescence detection system (96-well plate). Whole blood samples diluted 50-fold (7 cases each from the healthy group, lung cancer group, and post-treatment group) were added to 96-well plates, with 40 μM SQ-H-SA added to each well (final concentration 5 μM), and incubated for 10 min. Fluorescence data were simultaneously acquired using a small animal multimodal imaging system and a microplate reader. The analytical results are shown below. Figure 6 As shown.

[0104] in, Figure 6 Figure b shows the quantitative detection results of spiked blood: the radiation efficiency of 40 μM SQ-H-SA in 50-fold diluted blood containing 0-2.8 μM CA. "0" represents blood after CA removal by ABS treatment, and "X" represents endogenous CA signal. Multiple sample detection and imaging demonstrate its high-throughput detection and quantitative analysis capabilities. Figure c shows the response correlation results to the intensity quantification in Figure b: linear regression curve (radiation intensity vs. CA concentration, 0 to 2.8 μM; R² = 0.972, inset) and gradient CA concentration (0, X, X+0.7, X+1.05, X+1.4, X+2.1, X+2.8 μM) (data: mean ± standard deviation (n = 6)). Figure d shows the plate fluorescence analysis (λ). ex / λ em =470 / 525nm) Results: Under the same CA-spiking conditions as Figure b, the fluorescence intensity of SQ-H-SA in diluted blood was further demonstrated using an ELISA reader, proving its high-throughput detection and quantitative analysis capabilities; Figure e shows the linear calibration (fluorescence intensity vs. CA concentration) and regression analysis (R) for intensity quantification in Figure d. 2=0.980) Results (Data: mean ± standard deviation (n=6)); Figure f shows the results of multimodal imaging high-throughput clinical screening: radiation efficiency of whole blood samples from healthy donors, lung cancer patients, and the post-treatment group after 10 min (n=7 cases / group); Figure g shows the results of quantitative analysis of CA levels in Figure f: showing that the CA signal in lung cancer patients was significantly higher than that in healthy donors, and significantly lower after treatment (****P<0.0001); Figure h shows the results of parallel fluorescence verification: fluorescence intensity analysis of whole blood samples (normal group, disease group, treatment group, n=7 cases / group); Figure i shows the comparison of CA levels among lung cancer patients, healthy donors, and the treatment group in quantitative analysis of the intensity in Figure h (****P<0.0001).

[0105] In the high-throughput detection described above, 21 samples were analyzed in a single experiment, with an intra-batch CV of <5%, demonstrating the excellent high-throughput detection performance of the probe SQ-H-SA and further verifying its applicability to whole blood testing.

[0106] Example 7: Specific detection and imaging of CAIX on the surface of cancer cell membrane by probe SQ-H-SA

[0107] Dissolve 4.49 mg of the probe in 5 mL of DMSO to prepare a 1 mM stock solution, and store at -20 °C.

[0108] 1. Cell fluorescence detection experiment:

[0109] Figure 7 Figure a in the diagram is a schematic diagram of the mechanism of hypoxia-induced CA9 upregulation under CoCl2 treatment.

[0110] 5 μL of the probe was incubated in cells. The excitation wavelength of the confocal microscope was set to 640 nm, and the emission wavelength receiving range was 663-738 nm. The detection results are as follows: Figure 7 As shown.

[0111] in Figure 7Figure b shows the real-time membrane imaging results: delayed fluorescence (0-600 seconds) of SQ-H-SA nanoprobe (5μM) in HUVEC (normal) cells and LA795 / A549 (cancer) cells treated with CoCl2 (100μM, 24h), demonstrating the rapid imaging of cells by the probe; Figure c shows the hypoxia-dependent targeting: confocal images after 15 min of SQ-H-SA incubation (5μM) (groups: hypoxia group, normoxic group, and ABS-treated group (+ABS)). The probe's specificity for CAIX and its application in complex biological systems were demonstrated at the cellular level; Figure d shows the flow cytometry results of LA795 cells, further demonstrating its imaging ability and specificity at the cellular level from a statistical perspective; Figure e shows the kinetic quantitative results of Figure b: time-fluorescence curves (n=6, mean ± standard deviation), quantifying the data to demonstrate its rapid imaging ability; Figure f shows the quantitative analysis results of Figure c: fluorescence intensity (hypoxia group vs. control group vs. normoxic group), similarly quantifying the data to demonstrate its specific imaging ability.

[0112] The combined results of the above tests indicate that the probe SQ-H-SA has specific imaging capabilities at the cellular level and promising applications in complex biological environments.

[0113] 2. Tissue fluorescence detection experiment:

[0114] Figure 8 Figure a in the diagram is a schematic diagram of SQ-H-SA used for fluorescence imaging of lung cancer and adjacent non-cancerous tissues.

[0115] The probe was prepared at 200 μM and dissolved in DPBS / DMSO (v / v = 19:1, pH 7.4). 20 μL of the probe solution was injected intratumorally, followed by in vivo fluorescence imaging in small animals. The excitation wavelength of the small animal imager was set to 640 nm, and the emission wavelength receiving range was 660-700 nm. The detection results are as follows: Figure 8 As shown.

[0116] in, Figure 8Panel b in [reference] shows the time-lapse fluorescence imaging of human lung cancer tissues (LCT, loaded sample shown in the figure) and adjacent cancer tissues (PCT, loaded sample shown in the figure below) cultured with 5 μM probe; Panel c shows the normalized fluorescence intensity of panel b during 60 min of culture (fold change relative to PCT at t = 0); Panel d shows the fluorescence intensity of clinical tissues after 30 min of culture (LCT vs PCT, n = 9 donors); Panel e shows the quantitative analysis results of the fluorescence intensity in panel c, showing that the fluorescence in LCT is enhanced 3.2-fold compared to PCT; Panel f shows the confocal imaging of LCT / PCT sections stained with the probe (scale bar: 20 μm); Panel g shows the pathological H&E staining of LCT tissues and PCT tissues (Paracarcinoma tissue) (scale bar: 200 μm). [Ethical approval: All animal experiments were strictly carried out in accordance with the Regulations on the Administration of Laboratory Animals in Hunan Province (License number: SYXK (Xiang) 2020 - 0012), and all animal experimental protocols have been approved by the Animal Ethics Committee of Hunan Normal University (Number: 2024 - 185); The use of clinical samples has been reviewed and approved by the Biomedical Ethics Committee of Hunan Normal University (Batch number: 274); The use of all clinical samples follows the institutional guidelines and the Declaration of Helsinki, and written informed consent has been obtained from all participants before sample collection].

[0117] In summary, as Figure 9 shown in the response mechanism of the SQ-H-SA nanofluorescent probe of the present invention and the schematic diagram for the quantitative detection of CA enzyme in whole blood and precise imaging of clinical cancer tissues, the present invention discloses a novel probe SQ-H-SA. This dissociation-driven fluorescent nanoprobe redefines the quantitative detection of cancer antigen (CA) in whole blood and the precise imaging of cancer tissues. By integrating small molecule self-assembly, recognition-triggered dissociation, and adaptive signal amplification, the probe overcomes the key limitations of traditional CA probes - inherent background fluorescence, poor signal enhancement effect, and interference from blood autofluorescence. The innovative design of the SQ-H-SA nanofluorescent probe of the present invention integrates three synergistic mechanisms: (1) achieving near-zero background fluorescence by enabling robust self-assembly and complete fluorescence quenching in aqueous media; (2) effectively suppressing blood autofluorescence through the internal filter effect by virtue of the overlap of its absorption spectrum with blood autofluorescence at around 600 nm and its high molar extinction coefficient, thereby improving the signal-to-noise ratio; (3) two-stage signal amplification, that is, after the probe binds to CA and dissociates, the self-quenching effect is eliminated, and then it enters the hydrophobic cavity of CA, protecting the fluorophore from water-mediated fluorescence quenching.

[0118] The combined effect of the above synergistic mechanisms resulted in an approximately 45-fold fluorescence enhancement within 7 minutes of interaction between SQ-H-SA and 3 μM CA IX, significantly improving the sensitivity and specificity of detection. The SQ-H-SA nanofluorescent probe of this invention can also accurately quantify endogenous CA in 50-fold diluted human blood, showing a high correlation with enzyme-linked immunosorbent assay (ELISA) results. Clinically, the SQ-H-SA nanofluorescent probe of this invention detected elevated CA levels in untreated lung cancer patients and tracked treatment response, highlighting its diagnostic utility. Furthermore, the SQ-H-SA nanofluorescent probe of this invention supports high-throughput analysis of clinical samples, improving diagnostic accuracy in liquid biopsies.

[0119] In addition to quantitative analysis, the SQ-H-SA nanofluorescent probe of this invention also enables the first cell membrane-specific fluorescence imaging of CAIX in cancer cells and clinical tissues, providing a large T / N signal ratio. This cell membrane-restricted activation reduces endocytosis in normal cells, eliminates false positives, and ensures high-contrast imaging.

[0120] Based on the advantages of the SQ-H-SA nanofluorescent probes mentioned above, this invention establishes a powerful tool for integrated diagnosis and treatment of lung cancer, capable of real-time monitoring of dynamic changes in CAIX to guide personalized treatment. By combining molecular engineering with clinical application, this invention's SQ-H-SA nanofluorescent probes open up new avenues for non-invasive cancer monitoring, prognostic risk stratification, and precision medicine.

[0121] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A fluorescent nanoprobe SQ-H-SA, characterized in that, Its structural formula is as follows:

2. The preparation method of the fluorescent nanoprobe SQ-H-SA as described in claim 1, characterized in that, Includes the following steps: S1. 3,3-Dimethyl-3H-indole and 6-bromohexanoic acid were mixed in a solvent and reacted. After the reaction was completed, the mixture was poured into a petroleum ether / ethyl acetate solution, cooled, and the crude product was precipitated. The purified compound was 6-(3,3-dimethyl-3H-indole)hexanoic acid. S2. Dissolve 6-(3,3-dimethyl-3H-indole)hexanoic acid and 3,4-dihydroxy-3-cyclobutene-1,2-dione in a solvent, heat to carry out the reaction, and purify to obtain the dye Dye-COOH after the reaction is completed. S3. Dissolve Dye-COOH and 4-(2-aminoethyl)benzenesulfonamide in a solvent, and add 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate and N,N-diisopropylethylamine in sequence. Stir the mixture under a nitrogen atmosphere to carry out the reaction. After the reaction is completed, purify the solution to obtain the fluorescent nanoprobe SQ-H-SA.

3. The preparation method according to claim 2, characterized in that, The preparation of the fluorescent nanoprobe SQ-H-SA also includes a self-assembly process, the specific steps of which are as follows: The fluorescent nanoprobe SQ-H-SA was dissolved in DMSO to obtain a stock solution. The stock solution was then added to a DPBS:DMSO mixed buffer and mixed to obtain a self-assembly-disassembly driven fluorescent nanoprobe SQ-H-SA with a final concentration of 0.5–50 μM.

4. The preparation method according to claim 2, characterized in that, The molar ratio of 3,3-dimethyl-3h-indole and 6-bromohexanoic acid in step S1 is 1:(0.5-2).

5. The preparation method according to claim 2, characterized in that, The molar ratio of 6-(3,3-dimethyl-3H-indole)hexanoic acid and 3,4-dihydroxy-3-cyclobutene-1,2-dione in step S2 is (2-5):

1.

6. The preparation method according to claim 2, characterized in that, The molar ratio of Dye-COOH, 4-(2-aminoethyl)benzenesulfonamide, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate and N,N-diisopropylethylamine in step S3 is 1:(2-10):(0.2-1):(1-5).

7. The application of the fluorescent nanoprobe SQ-H-SA as described in claim 1 in the preparation of tumor detection reagents.

8. The application of the fluorescent nanoprobe SQ-H-SA as described in claim 1 in tumor detection.

9. According to the application described in 8, the fluorescent nanoprobe SQ-H-SA is used for the detection of carbonic anhydrase.

10. A tumor diagnostic kit, characterized in that, The kit contains the fluorescent nanoprobe SQ-H-SA as described in claim 1.

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