Use of TPE-ketoalkyne probes in the preparation of products for identifying urate and hydroxyapatite crystals
By combining TPE-Ketoalkyne probes with laser confocal fluorescence imaging technology, the problem of distinguishing between urate and hydroxyapatite crystals in the diagnosis of gouty arthritis in existing technologies has been solved, enabling rapid, specific identification and a simplified diagnostic process.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- AFFILIATED HUSN HOSPITAL OF FUDAN UNIV
- Filing Date
- 2023-06-07
- Publication Date
- 2026-06-02
AI Technical Summary
Existing diagnostic methods for gouty arthritis, such as ultrasound scanning, DECT, and CPLM, suffer from problems such as long processing time, low specificity, or high cost. They are difficult to quickly and accurately distinguish between urate and hydroxyapatite crystals, resulting in high false positive and false negative rates and affecting diagnostic efficiency.
Using a TPE-Ketoalkyne probe combined with laser confocal fluorescence imaging technology, urate and hydroxyapatite crystals are rapidly identified by aggregation-induced emission (AIE) through the specific binding of urate crystals, forming an aggregation-enhanced fluorescence signal.
It enables rapid and specific identification of urate and hydroxyapatite crystals, simplifies the diagnostic process, reduces reliance on professionals, and provides a detection method with comparable performance to CPLM imaging, suitable for basic research and clinical testing.
Smart Images
Figure CN116718576B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of sodium urate crystal identification technology, specifically to the application of the TPE-Ketoalkyne probe in the preparation of products that specifically identify urate and hydroxyapatite crystals. Background Technology
[0002] Gouty arthritis is one of the most common types of arthritis, characterized by the deposition of urate (MSU) crystals in the joints and soft tissues, leading to joint destruction. Furthermore, MSU crystal deposition increases the risk of cardiovascular and kidney disease, impairing patients' quality of life. The main treatment strategy for gouty arthritis is to reduce MSU crystal deposition through medication or surgery; therefore, timely and accurate detection of MSU crystals is crucial for the diagnosis, treatment, and prevention of recurrence of gouty arthritis.
[0003] Gouty arthritis is usually diagnosed by a specialist physician based on the patient's clinical presentation and relevant examinations. Because the clinical manifestations of acute arthritis caused by different types of crystal deposition are similar, clinical practice requires not only the detection of MSU crystals but also the specific identification of crystals similar to MSU crystals. In recent years, advanced imaging techniques have been continuously updated in the diagnosis of gout, especially ultrasound and dual-energy computed tomography (DECT), which have become indispensable techniques for the clinical detection of MSU crystals.
[0004] However, ultrasound scans are time-consuming and have low specificity, requiring specialized technicians for successful diagnosis. DECT (Disseminated Electroconvulsive Therapy) involves ionizing radiation, is relatively expensive, and has low sensitivity in the early stages of gout. Traditionally, compensated polarized light microscopy (CPLM) for MSU (metastatic urealyticum) crystals has been the gold standard for diagnosing gout. However, CPLM testing without proper training increases the error rate in identifying MSU crystals, leading to false negatives and false positives. For example, distinguishing MSU crystals from other crystals such as hydroxyapatite (HAP) can be challenging. Furthermore, the time-consuming process reduces clinical efficiency and increases stress. Stimulated Raman scattering (SRS) microscopy is a relatively new technique that enables high-speed imaging and has successfully diagnosed fresh gout tissue, identifying crystals in synovial fluid based on quantitative chemical analysis. However, laboratory-grade instruments are bulky and expensive, posing inconvenience for clinical use. Given the current situation, this technology remains limited to basic research applications.
[0005] Therefore, there is a need for a rapid and specific method or product for labeling MSU crystals to aid in the diagnosis of gout. Summary of the Invention
[0006] To overcome the shortcomings of the prior art, the present invention provides the application of the TPE-Ketoalkyne probe in the preparation of products that specifically identify urate and hydroxyapatite crystals.
[0007] To achieve the above objectives, the present invention adopts the following technical solution:
[0008] A first aspect of the present invention is to provide the use of a TPE-Ketoalkyne probe in the preparation of a product specifically distinguishing urate and hydroxyapatite crystals, the product comprising a TPE-Ketoalkyne probe that specifically binds to urate crystals.
[0009] Furthermore, the use of the above products is based on laser confocal fluorescence imaging technology.
[0010] Furthermore, the TPE-Ketoalkyne probe is used at a concentration of 5-80 μM.
[0011] A second aspect of the invention is to provide the use of a TPE-Ketoalkyne probe in the preparation of a product for the auxiliary diagnosis of gouty arthritis, the product comprising a TPE-Ketoalkyne probe that specifically binds to urate crystals.
[0012] Furthermore, the use of the above products is based on laser confocal fluorescence imaging technology.
[0013] Furthermore, the TPE-Ketoalkyne probe is used at a concentration of 5-80 μM.
[0014] A third aspect of the present invention is to provide a product for specifically identifying urate and hydroxyapatite crystals or for assisting in the diagnosis of gouty arthritis, the product comprising a TPE-Ketoalkyne probe that specifically binds to urate crystals.
[0015] Furthermore, the use of the above products is based on laser confocal fluorescence imaging technology.
[0016] Furthermore, the TPE-Ketoalkyne probe is used at a concentration of 5-80 μM.
[0017] The fourth aspect of the present invention is to provide a method for specifically identifying urate crystals and hydroxyapatite crystals, wherein the crystal sample to be tested is added to a phosphate buffer solution to prepare a solution, and then a TPE-Ketoalkyne probe is added and mixed, and observed by laser confocal fluorescence imaging.
[0018] The present invention adopts the above technical solution and has the following technical effects compared with the prior art:
[0019] This invention optimizes the rapid imaging technique for MSU crystals in in vitro studies, employing a TPE-Ketoalkyne probe. Its imaging results are comparable to the clinical gold standard method, CPLM, especially in differentiating between different types of crystalline arthritis caused by MSU and HAP crystals. Furthermore, this procedure is convenient and rapid, eliminating the need for professional assessment of crystal refractive properties and morphology; MSU and HAP crystals can be distinguished solely by the presence or absence of enhanced fluorescence signals. Moreover, the fluorescence produced by the TPE-Ketoalkyne probe differs from ordinary fluorescence; it is non-quenchable, allowing for long-term monitoring. This provides an excellent platform for basic research related to gout and offers a new auxiliary method for clinical gout detection. Attached Figure Description
[0020] Figure 1 These are schematic diagrams of the chemical structures of seven TPE probes; Figures A and B show the chemical structures of TPE-NH2, TPE-2NH2, TPE-4NH2, TPE-COOH, TPE-2COOH, TPE-4COOH and TPE-Ketoalkyne, respectively.
[0021] Figure 2 The fluorescence imaging results of the standard MSU crystal solution with seven TPE probes (TPE-NH2, TPE-2NH2, TPE-4NH2, TPE-COOH, TPE-2COOH, TPE-4COOH, and TPE-Ketoalkyne) are shown. Figure A shows the fluorescence imaging results of MSU crystal solution with TPE-NH2, TPE-2NH2, and TPE-4NH2 probes; Figure B shows the fluorescence imaging results of MSU crystal solution with TPE-COOH, TPE-2COOH, and TPE-4COOH probes; Figure C shows the fluorescence imaging results of MSU crystal solution with the TPE-Ketoalkyne probe; and Figure D shows the relative fluorescence intensities of the solutions after mixing the above seven TPE probes with MSU crystals.
[0022] Figure 3 The confocal fluorescence imaging results of MSU crystal standard with different concentrations of TPE-Ketoalkyne probe are shown; Figure A shows the fluorescence imaging results of MSU crystal solution mixed with different concentrations of TPE-Ketoalkyne probe; Figure B shows the relative fluorescence intensity of the solution after mixing different concentrations of TPE-Ketoalkyne probe with MSU crystal; Figure C shows the 3D fluorescence imaging results of 10 μM TPE-Ketoalkyne probe mixed with MSU crystal.
[0023] Figure 4The TPE-Ketoalkyne probe was used to specifically identify MSU and HAP standard crystals using laser confocal fluorescence imaging and CPLM imaging. Figure A shows the confocal fluorescence imaging results of the TPE-Ketoalkyne probe with MSU and HAP standard crystals; Figure B shows the confocal fluorescence imaging results of the TPE-Ketoalkyne probe with a equiproportional mixture of MSU and HAP standard crystals; and Figure C shows the CPLM imaging results of the TPE-Ketoalkyne probe with MSU, HAP, and a equiproportional mixture of MSU and HAP standard crystals.
[0024] Figure 5 The rapid and efficient labeling of MSU crystals in clinical samples by the TPE-Ketoalkyne probe was demonstrated using laser confocal fluorescence imaging and CPLM imaging. Figures A and B show the laser confocal fluorescence imaging results of TPE-Ketoalkyne probe-labeled MSU crystals in synovial fluid and tophi, respectively. Figures C and D show the CPLM imaging results of TPE-Ketoalkyne probe-labeled MSU crystals in synovial fluid and tophi, respectively. Figure E shows the fluorescence imaging results of TPE-Ketoalkyne and MSU crystals after mixing for different times (0, 60, 120 s). Figure F demonstrates the effectiveness of the TPE-Ketoalkyne probe in binding to MSU crystals using 3D imaging. Detailed Implementation
[0025] This invention provides the application of the TPE-Ketoalkyne probe in the preparation of products that specifically identify urate and hydroxyapatite crystals. The invention is described in detail below with reference to specific embodiments and accompanying drawings to provide a better understanding; however, these embodiments do not limit the scope of the invention.
[0026] Unless otherwise specified, the methods used in the embodiments are conventional methods, and the reagents used are commercially available reagents or reagents prepared according to conventional methods, unless otherwise specified.
[0027] Example 1
[0028] This embodiment uses 7 types of TPE probes (TPE-NH2). [1] TPE-2NH2 [2] TPE-4NH2 [3] TPE-COOH [4,5] TPE-2COOH [6] TPE-4COOH [7] TPE-Ketoalkyne [8,9] ; Specific chemical structures such as Figure 1(As shown) MSU crystals were labeled using fluorescence imaging technology. The specific experimental steps and results are as follows:
[0029] 1. Screening labeled standard MSU crystals for the most effective AIE probes
[0030] The standard MSU crystals were added to phosphate buffered saline (PBS) to prepare a solution with a concentration of 50 mg / ml. The same concentration (80 μM) of TPE probes TPE-NH2, TPE-2NH2, TPE-4NH2, TPE-COOH, TPE-2COOH, TPE-4COOH and TPE-Ketoalkyne were added to perform laser confocal fluorescence imaging.
[0031] like Figure 2 As shown, the TPE-NH2, TPE-2NH2, and TPE-4NH2 probes were scattered throughout the MSU crystal solution, and there was no statistically significant difference in fluorescence intensity. Figure 2 A); TPE-4COOH, TPE-2COOH, and TPE-COOH probes can bind to standard MSU crystals to varying degrees, and the fluorescence intensity shows a gradually increasing trend. Among them, the TPE-COOH probe shows relatively better fluorescence effect on the labeled MSU crystals. Figure 2 B); Compared with the previous 6 TPE probes, MSU crystals significantly enhanced the AIE effect of TPE-Ketoalkyne, resulting in the strongest relative fluorescence intensity in solution. Therefore, TPE-Ketoalkyne is the most effective probe molecule for labeling the standard MSU crystals. Figure 2 CD).
[0032] 2. Confocal fluorescence imaging of TPE-Ketoalkyne probes at different concentrations with standard MSU crystals
[0033] The standard MSU crystals were added to phosphate buffered saline (PBS) to prepare a solution with a concentration of 50 mg / ml. Different concentrations (5, 10, 20, 40, 60 and 80 μM) of TPE-Ketoalkyn probes were added for laser confocal fluorescence imaging.
[0034] like Figure 3 As shown, compared with the lowest concentration of 5 μM TPE-Ketoalkyne probe, the fluorescence intensity in the standard MSU crystal solution gradually increased with increasing probe concentration (10-80 μM), and this increase was statistically significant. Figure 3 AB). 3D fluorescence imaging results of the 10 μM TPE-Ketoalkyne probe and MSU crystal further demonstrate the effectiveness of this probe in binding to the standard MSU crystal. Figure 3 C).
[0035] 3. Group the samples according to the following categories, and perform CPLM imaging and laser confocal fluorescence imaging simultaneously.
[0036] The first set of samples consisted of standard MSU crystals, standard HAP crystals, and a mixed standard system (a mixture of standard MSU crystals and HAP crystals in equal proportions). The preparation of standard samples is well-established and commercially available.
[0037] The second set of samples consisted of MSU crystals in the patients' synovial fluid and tophi.
[0038] Each sample collected from both groups was prepared into a solution by adding it to phosphate-buffered saline (PBS): the concentrations of standard MSU crystals and standard HAP crystals in the first group were 50 mg / ml, and the concentration of the mixed system was 25 mg / ml; the concentration of joint liquid crystals in the second group was 15 mg / ml, and the concentration of tophi crystals was 100 mg / ml. Each sample solution was evenly distributed into two centrifuge tubes. One tube of sample solution was imaged using CPLM, and the other tube of sample solution was imaged using laser confocal fluorescence imaging after adding a TPE-Ketoalkyne probe (the final concentration was 10 μM for all samples except the mixed sample, which was 40 μM).
[0039] The results show that the TPE-Ketoalkyne probe can specifically label the MSU crystal standard in fluorescence imaging. Conversely, the same probe is uniformly distributed in the HAP crystal standard solution, meaning that the HAP crystals cannot be effectively labeled. Figure 4 A). When the TPE-Ketoalkyne probe was added to a solution containing equal proportions of standard MSU and HAP crystals, the probe specifically bound to the standard MSU crystals for imaging within the same system, but did not bind to the standard HAP crystals for imaging. Figure 4 B). Although CPLM can effectively image MSU crystals ( Figure 4 C), however, the imaging of mixed samples of standard MSU and HAP crystals requires the identification of an experienced physician, while the confocal fluorescence imaging process after adding the TPE-Ketoalkyne probe only requires judgment based on the presence or absence of aggregated enhanced fluorescence signals.
[0040] TPE-Ketoalkyne probes can effectively label MSU crystals in synovial fluid using fluorescence imaging. Figure 5 A) and MSU crystals in gouty tophi ( Figure 5 B), and the fluorescence imaging effect can achieve the effect of CPLM imaging. Figure 5 CD). More importantly, the TPE-Ketoalkyne probe can be rapidly imaged within 60 seconds after being mixed with the MSU crystal. Figure 5E). Similarly, 3D imaging demonstrated the effectiveness of the TPE-Ketoalkyne probe in binding to the MSU crystal ( Figure 5 F).
[0041] In summary, fluorescence imaging of TPE-Ketoalkyne-labeled MSU crystals can be a rapid detection method, especially in the identification of HAP crystals, where this AIE probe has a greater advantage.
[0042] The specific embodiments of the present invention have been described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, all equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.
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Claims
1. The application of TPE-Ketoalkyne probe in the preparation of products specifically distinguishing urate and hydroxyapatite crystals, characterized in that, The product includes a TPE-Ketoalkyne probe that specifically binds to urate crystals. The TPE-Ketoalkyne probe is used at a concentration of 5-80 μM. The chemical structure of the TPE-Ketoalkyne probe is shown below: 。 2. The application according to claim 1, characterized in that, The use of this product is based on laser confocal fluorescence imaging technology.
3. The application of TPE-Ketoalkyne probes in the preparation of products for the auxiliary diagnosis of gouty arthritis, characterized in that... The product includes a TPE-Ketoalkyne probe that specifically binds to urate crystals. The TPE-Ketoalkyne probe is used at a concentration of 5-80 μM. The chemical structure of the TPE-Ketoalkyne probe is shown below: 。 4. The application according to claim 3, characterized in that, The use of this product is based on laser confocal fluorescence imaging technology.
5. A method for specifically identifying urate crystals and hydroxyapatite crystals, characterized in that, The crystal sample to be tested was added to a phosphate buffer solution to prepare a solution, and then the TPE-Ketoalkyne probe was added dropwise and mixed. The mixture was then observed by laser confocal fluorescence imaging. The concentration of the TPE-Ketoalkyne probe used was 5-80 μM. The chemical structure of the TPE-Ketoalkyne probe is shown below: 。