A triphenylamine-pyridinium salt fluorescent probe for rapid detection of urinary white and a preparation method thereof

Abstract

This invention relates to a triphenylamine-pyridinium salt fluorescent probe for rapid detection of urinary albumin, comprising an anion and a cation. The cation has the structural formula shown in Formula I: at least one of R1, R2, and R3 is a group shown in Formula II; in the group shown in Formula II, R is a straight-chain alkyl group with 1-20 carbons; and when two or three substitution sites of R1, R2, and R3 are connected to the group shown in Formula II, R in the groups shown in Formula II connected to different substitution sites may be the same or different. The probe of this invention has ultra-high sensitivity and fluorescence colorimetric detection effect, which is beneficial for the early prevention of chronic diseases and timely intervention of related diseases.

Description

Technical Field

[0001] This invention belongs to the field of point-of-care rapid detection technology, specifically, it relates to a triphenylamine-pyridinium salt fluorescent probe for rapid detection of urinary albumin and its preparation method. Background Technology

[0002] Human serum albumin (HSA) is the most abundant protein in blood plasma and plays a vital role in various physiological processes. Abnormally low plasma HSA levels (<35 g / L) and elevated albumin concentrations in urine (>30 mg / L) are considered associated with specific diseases, including cirrhosis, malnutrition, gastrointestinal cancers, systemic lupus erythematosus, and glomerulonephritis. The levels of albumin (ALB) in blood and urine can be accurately measured in formal physical examinations using medical technology. However, time-consuming pretreatment and the high level of operator skill required prevent current testing methods from meeting the needs for rapid, on-site, real-time monitoring of ALB, or even home testing. This hinders early prevention of chronic diseases and timely intervention in related conditions.

[0003] In the past five years, the development of fluorescent probes has facilitated rapid on-site detection of ALB due to their advantages such as fast response speed, ease of use, and signal visualization. Designing novel fluorescent probes with high sensitivity (S) is a fundamental goal for achieving the final application. The most common design strategy for ALB fluorescent probes is based on the sensing mechanism, which involves keeping the probe with intramolecular charge transfer (ICT) properties in a "completely fluorescence-off" state in water to generate an ideal initial signal (I0). During the detection process, the detection environment encapsulates the environmentally sensitive probe in a hydrophobic cavity, causing fluorescence recovery and generating a detection signal (I).

[0004] Hydrophilic AIEgens (Aggregation-Induced Emission luminogens) are a class of organic luminescent materials that exhibit almost no emission in the dispersed state but significantly enhanced emission in the aggregated state. Hydrophilic AIEgens have proven to be promising alternatives to urinary protein fluorescent probes. Currently, most research focuses on improving S by fully utilizing molecular rotor structures; however, the enhancement of S depends on two factors: reducing initial fluorescence (I0) and increasing the growth fold (I). However, the molecular rotor structure affects these two factors in conflicting ways. As has been widely demonstrated, the stacking of molecular rotors in water can suppress nonradiative inactivation to induce aggregation-induced emission (AIE), which competes with the quenching effect of ICT solvent relaxation, leading to partial recovery of I0 (I0 is increased). This inherent property of molecular rotors makes it very difficult to improve S for most dye-based probes; complicating matters further, the particle size distribution of aggregates at the large nanoscale can exacerbate fluctuations in I0, which may reduce detection accuracy, especially for healthy individuals and patients with early CKD (early CKD), as the concentration of ALB in urine is relatively low at these times, which will further affect the accuracy of the detection.

[0005] Although some ALB fluorescent probes have been reported to use multi-ionic substituents to inhibit their aggregation in water, resulting in extremely low initial fluorescence I0, the results suggest that while these hydrophilic ALB probes may be suitable for point-of-care urine analysis and auxiliary diagnosis of kidney disease, their initial fluorescence I0 still increases to varying degrees, leading to a decrease in the fold increase (I). Furthermore, the POCT detection efficacy of the aforementioned ALB fluorescent probe molecular structures in detecting urinary albumin has not yet been reported, and a well-defined molecular design scheme for such fluorescent probes remains lacking. Summary of the Invention

[0006] (a) Technical problems to be solved

[0007] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a triphenylamine-pyridinium salt fluorescent probe for rapid detection of urinary albumin. Its main structural feature is a triphenylamine-pyridinium salt structure modified with a long alkyl chain. It has ultra-high sensitivity and fluorescence colorimetric detection effect, and is suitable for rapid point-of-care fluorescence colorimetric detection. In particular, for healthy individuals and early CKD patients, the present invention is beneficial for the early prevention of chronic diseases and timely intervention of related diseases.

[0008] (II) Technical Solution

[0009] In a first aspect, the present invention provides a triphenylamine-pyridinium salt fluorescent probe for rapid detection of urinary albumin, comprising an anion and a cation, wherein the cation has the structural formula shown in Formula I:

[0010]

[0011] At least one of R1, R2 and R3 is a group represented by Formula II;

[0012]

[0013] In the group shown in Formula II, R is a straight-chain alkyl group with 1-20 carbon atoms;

[0014] Furthermore, when two or three of R1, R2, and R3 are connected to the group shown in Formula II at different substitution sites, R in the group shown in Formula II at different substitution sites may be the same or different; preferably the same.

[0015] When the R in the groups of Formula II connected to R1, R2, and R3 are all the same, a centrosymmetric fluorescent probe molecule is obtained; when the R in the groups of Formula II connected to R1, R2, and R3 are different, an asymmetric fluorescent probe molecule is obtained.

[0016] According to a preferred embodiment of the present invention, R1, R2 and R3 are each independently selected from H and one of the following groups:

[0017]

[0018] According to a preferred embodiment of the present invention, the anion is chlorine or bromine.

[0019] Secondly, the present invention provides a method for preparing a triphenylamine-pyridinium salt fluorescent probe for rapid detection of urinary albumin, comprising the following steps:

[0020] S1. Using 4-methylpyridine and haloalkanes as raw materials, react in acetonitrile to obtain N-alkyl-substituted 4-methylpyridine salts;

[0021] S2. Using N-alkyl-substituted 4-methylpyridinium salt and aldehyde triphenylamine as raw materials, a triphenylamine-pyridinium salt fluorescent probe is obtained by reacting in ethanol. The aldehyde triphenylamine is 4-aldehyde triphenylamine, 4,4'-dialdehyde triphenylamine, or 4,4',4”-trialdehyde triphenylamine. The reaction process is described below:

[0022]

[0023] Where Ra and Rb are independently H or aldehyde groups; X is chlorine or bromine; R2 and R3 are independently H or groups shown in Formula II;

[0024]

[0025] In the above formula, R is a straight-chain alkyl group with 1-20 carbon atoms.

[0026] According to a preferred embodiment of the present invention, in S1, the reaction temperature is 75-85°C, the reaction time is 16-24 h, preferably 18 h at 80°C; wherein, the haloalkane is a bromoalkane or a chloroalkane; and its molar ratio with 4-methylpyridine is 1:1.

[0027] According to a preferred embodiment of the present invention, in S1, after the reaction is completed, 1.5-5 times the amount of ethyl acetate in the reaction system is added, the mixture is stirred at room temperature, cooled and filtered to obtain an N-alkyl-substituted 4-methylpyridinium salt.

[0028] According to a preferred embodiment of the present invention, in S2, aldehyde triphenylamine is dissolved in ethanol, and N-alkyl-substituted 4-methylpyridinium salt prepared in S1 and the catalyst tetrahydropyrrole are added. The mixture is heated to 75-85°C and reacted for 20-30 h (preferably at 80°C for 25 h). The resulting reaction solution is concentrated by vacuum evaporation, purified by silica gel column chromatography, and then recrystallized with ethyl acetate to obtain the red target product.

[0029] According to a preferred embodiment of the present invention, in S2, the amount of tetrahydropyrrole catalyst added is 4-6% of the mass of the N-alkyl-substituted 4-methylpyridinium salt.

[0030] Thirdly, the present invention also provides a test strip for rapid detection of urinary protein, comprising an adsorption test strip and probe molecules loaded on the test strip substrate, wherein the probe molecules are the aforementioned triphenylamine-pyridinium salt fluorescent probe. The test strip enables rapid point-of-care detection of urinary protein.

[0031] (III) Beneficial Effects

[0032] This invention relates to a triphenylamine-pyridinium salt fluorescent probe, an ALB fluorescent probe symmetrically linked by a hydrophilic pyridinium salt with 1-3 ion-substituted groups and hydrophobic alkyl chains of varying lengths. It can be used for precise and rapid point-of-care detection of urinary proteins. Its main advantages lie in increasing the probe's hydrophilicity through varying numbers of ion-substituents in the pyridinium salt, resulting in good solubility in aqueous solutions. The hydrophobic alkyl chains enhance the probe's hydrophobicity, enabling it to interact with hydrophobic environments (such as the hydrophobic pockets of proteins). The hydrophobicity of the probe can be modulated by varying the length of the hydrophobic alkyl chains, thereby affecting its binding affinity to target molecules.

[0033] The triphenylamine-pyridinium salt fluorescent probe of this invention is a hydrophilic AIEgenss that remains monodisperse in water, exhibiting extremely low initial fluorescence (I0), and its fluorescence response signal shows an extremely high growth fold (I). Notably, the probe unexpectedly exhibits redshift emission during the detection of real urine samples, thereby enabling rapid testing devices such as test strips or kits for point-of-care testing (POCT) of ALB fluorescence.

[0034] Experiments have verified that the fluorescent probe provided by this invention has the following advantages:

[0035] (1) Ultra-high sensitivity: It has extremely low initial fluorescence (I0) and extremely high growth factor (I). The fluorescent probe C-3 prepared in Example 3 showed an ultra-high sensitivity of >3000 times in fluorescence response. In the experiment simulating urine of patients with chronic kidney disease (CKD), the fluorescence signal showed a ratio change with the concentration of spiked ALB, and had high correlation, high accuracy and low detection limit.

[0036] (2) Hospital urinalysis always requires morning urine. As a novel probe with high sensitivity, the experiment evaluated the gradually increasing spectral response of fluorescent probe C-3 to ALB in six series of urine samples collected under different conditions, including before lunch, after dinner, after staying up late, after taking medication, after drinking beer, and after drinking large amounts of water. The experimental results show that compared with routine hospital health checkups, fluorescent probe C-3 can be used to detect urinary albumin with fewer restrictions on urine collection, providing flexibility for on-site testing and even home testing.

[0037] (3) The fluorescent probe provided by the present invention has a good colorimetric effect in urine samples, making it easier to identify directly with the naked eye. This enables the development of a point-of-care testing (POCT) kit to facilitate rapid bedside detection of urine protein qualitative or semi-quantitative results. Attached Figure Description

[0038] Figure 1 The fluorescence spectra of ALB and 1 μM fluorescent probe C-3 were obtained by adding different concentrations of ALB and 1 μM to the PBS buffer of Application Example 1.

[0039] Figure 2 The curve showing the correlation between ALB concentration and fluorescence intensity is used for fitting.

[0040] Figure 3 To plot the relationship between fluorescence intensity and ALB concentration at fluorescent probe C-3 concentrations of 1 μM and 10 μM, a fluorescence titration method was used.

[0041] Figure 4 When the ALB concentration is low, the fluorescence is "off"; as the ALB concentration increases, the fluorescence shows a clear change from orange-yellow to bright yellow.

[0042] Figure 5 The fluorescence spectra of fluorescent probes C-1, C-6, and C-8 at 10 μM are shown in response to 10 μM ALB.

[0043] Figure 6 Fluorescence intensity curves under excitation light of 550-850 nm were obtained by adding equal amounts of C-3 probe to blank solutions and solutions of ALB+AMY, ALB+SOD, ALB+GAL, ALB+LYS, ALB+APP, ALB+LPS and ALB+GLB respectively.

[0044] Figure 7 The fluorescence intensity of the C-3 probe was measured by adding equal amounts of it to blank solutions and solutions containing AMY, SOD, GAL, LYS, APP, LPS, GLB, 0.1μm ALB, 0.45μm ALB, 4.5μm ALB, and 10μm ALB.

[0045] Figure 8 To investigate the fluorescence reaction of fluorescent probe C-3 with ALB in LAB under the influence of ions, amino acids, glucose, urine components, and exogenous drugs.

[0046] Figure 9 The fluorescence intensity of fluorescent probe C-3 increased with increasing albumin concentration in the urine of three healthy adult volunteers after albumin was added to their urine.

[0047] Figure 10 To collect the fluorescence spectrum and color response of fluorescent probe C-5 after adding albumin to the urine of volunteer 1.

[0048] Figure 11 To investigate the gradually increasing spectral response of six series of urine samples from volunteer 1 under different conditions, including before lunch, after dinner, after staying up late, after taking medication, after drinking beer, and after drinking large amounts of water, the study aimed to obtain data on the response of urine to ALB.

[0049] Figure 12 Image of a portable test strip prepared by impregnating the probe fluorescent C-3 with filter paper.

[0050] Figure 13 The photo shows four color changes of fluorescence visible to the naked eye after urine samples from three healthy adult volunteers were spiked with different concentrations of ALB and dropped onto the test strip.

[0051] Figure 14 A 24-color Hue ring for rapid detection of urinary protein based on probe fluorescence C-3.

[0052] Figure 15 This is a statistical graph showing a positive correlation between the color coordinate ratio R / B and ALB concentration. Detailed Implementation

[0053] To better explain and facilitate understanding of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0054] Examples 1-5

[0055] Examples 1-5 show the sequential preparation of five fluorescent probes C-1, C-2, C-3, C-4, and C-5, with the following preparation process:

[0056] Step 1: Take 4-methylpyridine (reactant 1) and haloalkanes A-1, A-2, A-3, A-4, and A-5 as reactants, with a molar ratio of 4-methylpyridine to haloalkanes of 1:1; dissolve the two reactants in acetonitrile, heat to 80°C and react for 18 hours, add 3 times the volume of ethyl acetate to the reaction system, and stir at room temperature to remove excess reactants, cool and filter to obtain N-alkyl-substituted 4-methylpyridine salts B-1, B-2, B-3, B-4, and B-5.

[0057] Step 2: Dissolve 4-aldehyde triphenylamine (reactant 2) in ethanol, add the N-alkyl-substituted 4-methylpyridinium salts B-1, B-2, B-3, B-4, and B-5 prepared in Step 1 and the catalyst tetrahydropyrrole (accounting for 5% of the mass of the N-alkyl-substituted 4-methylpyridinium salts), wherein the molar ratio of the N-alkyl-substituted 4-methylpyridinium salts to 4-aldehyde triphenylamine is 1:1.

[0058] The reaction system was heated to 80℃ and reacted for 25 hours. The mixture was evaporated and concentrated to 1 / 3 of its original volume under vacuum. After purification by silica gel column chromatography, it was added and recrystallized with ethyl acetate to give red compounds C-1, C-2, C-3, C-4, and C-5. Mass spectrometry data: C-1: MS(ESI, m / z): 377.20; C-2: MS(ESI, m / z): 405.23; C-3: MS(ESI, m / z): 433.26; C-4: MS(ESI, m / z): 461.29; C-5: MS(ESI, m / z): 489.32.

[0059] The reaction processes in Examples 1-5 are shown below. The names of the haloalkanes A-1, A-2, A-3, A-4, and A-5 are, in order, bromoethane, chlorobutane, chlorohexane, chlorooctane, and chlorodecane; the names of the intermediates B-1, B-2, B-3, B-4, and B-5 are, in order, N-ethyl-4-methylpyridine bromide, N-butyl-4-methylpyridine chloride, N-hexyl-4-methylpyridine chloride, N-octyl-4-methylpyridine chloride, and N-decyl-4-methylpyridine chloride.

[0060]

[0061] Examples 6-10

[0062] Examples 6-10 show the sequential preparation of five fluorescent probes C-6, C-7, C-8, C-9, and C-10, with the preparation process as follows:

[0063] Step 1: Take 4-methylpyridine (reactant 1) and haloalkanes A-1, A-2, A-3, A-4, and A-5 as reactants, with a molar ratio of 4-methylpyridine to haloalkanes of 1:1; dissolve the two reactants in acetonitrile, heat to 80°C and react for 18 hours, add 3 times the volume of ethyl acetate to the reaction system, and stir at room temperature to remove excess reactants, cool and filter to obtain N-alkyl-substituted 4-methylpyridine salts B-1, B-2, B-3, B-4, and B-5.

[0064] Step 2: Dissolve 4,4'-dialdehyde triphenylamine (reactant 3) in ethanol, add the N-alkyl-substituted 4-methylpyridinium salts B-1, B-2, B-3, B-4, and B-5 prepared in Step 1, and the catalyst tetrahydropyrrole (accounting for 5% of the mass of the N-alkyl-substituted 4-methylpyridinium salts), wherein the molar ratio of the N-alkyl-substituted 4-methylpyridinium salts to 4,4'-dialdehyde triphenylamine is 2:1.

[0065] The reaction system was heated to 80℃ and reacted for 25 hours. The mixture was evaporated and concentrated to 1 / 3 of its original volume under vacuum. After purification by silica gel column chromatography, it was added and recrystallized with ethyl acetate to give red compounds C-6, C-7, C-8, C-9 and C-10. Mass spectrometry data: C-6: MS(ESI, m / z): 254.64; C-7: MS(ESI, m / z): 282.67; C-8: MS(ESI, m / z): 310.70; C-9: MS(ESI, m / z): 338.73; C-10: MS(ESI, m / z): 366.76; C-6: MS(ESI, m / z): 355.15.

[0066] The reaction processes in Examples 6-10 are shown below. The names of the haloalkanes A-1, A-2, A-3, A-4, and A-5 are, in order, bromoethane, chlorobutane, chlorohexane, chlorooctane, and chlorodecane; the names of the intermediates B-1, B-2, B-3, B-4, and B-5 are, in order, N-ethyl-4-methylpyridine bromide, N-butyl-4-methylpyridine chloride, N-hexyl-4-methylpyridine chloride, N-octyl-4-methylpyridine chloride, and N-decyl-4-methylpyridine chloride.

[0067]

[0068] The following application examples further investigate the spectral response, sensing performance, selectivity, and anti-interference properties of the fluorescent probe.

[0069] Application Example 1

[0070] Albumin (ALB) was added to PBS buffer at concentrations of 0, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 1.0 μM, 2.0 μM, 3.0 μM, and 4.0 μM, resulting in 13 samples. Fluorescent probe C-3 was added to each sample, with the concentration of the fluorescent probe being equal at 1 μM in all 13 samples. Figure 1 The fluorescence spectra of these 13 samples are shown below. Based on the 3σ / k rule, the limit of detection (LOD) for ALB is calculated to be 14 nM (0.93 mg / L). Figure 2 As shown in the figure, it is far below the threshold for microalbuminuria (30 mg / L).

[0071] Application Example 2

[0072] Fluorescent titration was used to monitor changes in fluorescence intensity and analyze the interaction between the fluorescent probe and ALB. Titration samples containing 1 μM and 10 μM of fluorescent probe C-3 were prepared, and their initial fluorescence intensities (I0) were measured. Then, ALB was added gradually via titration. During the addition, the required volume of ALB stock solution was added one at a time, followed by vortexing at 2000 rpm for 1 minute and allowing the solution to stand for 1 minute after each addition. The fluorescence intensity of the solution was measured and recorded, and the recorded values ​​were used to plot a curve showing the relationship between fluorescence intensity and ALB concentration. Figure 3 As shown, compared to the 1 μM fluorescent probe C-3, the fluorescence titration spectrum of fluorescent probe C-3 at 10 μM exhibited a similar "off-on" trend, but the sensitivity range (0-60 μM) changed compared to 1 μM. The C50 values ​​from the two experiments were 0.55 μM and 4.42 μM (see...). Figure 3 These values ​​are comparable to the thresholds for microalbuminuria (0.45 μM) and macroalbuminuria (4.5 μM), respectively.

[0073] like Figure 4 As shown, when the concentration of fluorescent probe C-3 is 10 μM, the fluorescence is "dark" or "off" when the ALB concentration is below 0.45 μM. Continuing to add ALB to increase its concentration to 3 μM or higher, the fluorescence shows a clear change from orange-yellow to bright yellow. Therefore, benefiting from the initial "fluorescence off" state, the ALB concentration can be reflected by the change in fluorescence from dark to yellow to bright during the titration process (e.g., ...). Figure 4As shown in the figure, the color change during this process is significant, so it can be observed directly with the naked eye, or it can be easily detected in real time by constructing a positive correlation between color coordinates and ALB concentration on a smartphone APP.

[0074] Application Example 3

[0075] Prepare two solutions, A and B, containing fluorescent probes C-1, C-6, and C-8 in PBS, with a probe concentration of 10 μM in each solution. Add 10 μM ALB to group B, shake and let stand, then measure the fluorescence spectra of groups A and B. Figure 5 As shown, the fluorescence intensity in group A is extremely low, while the fluorescence in group B recovers. Based on this spectral response result, it is demonstrated that compounds C-1, C-6, and C-8 can all be used as fluorescent probes for ALB to achieve highly sensitive detection of ALB.

[0076] Application Example 4

[0077] Seven parallel 10 μM ALB solutions were prepared. Each solution contained one of the following biomolecules as interfering molecules: amylase (AMY) 100 U / mL, superoxide dismutase (SOD) 500 U / mL, galactosidase (GAL) 500 U / mL, lysozyme (LYS) 1000 μg / mL, aminopeptidase (APP) 1000 μg / mL, lipase (LPS) 1000 U / mL, and globulin (GLB) 500 μg / mL. The same concentration of fluorescent probe C-3 (10 μM) was also added to each solution. Finally, a control solution containing only the same concentration of fluorescent probe C-3 was prepared. The eight solutions were irradiated with excitation light in the range of 500-850 nm, and the fluorescence intensity was measured. Excitation wavelength-fluorescence intensity curves were plotted. Figure 6 As shown, compared with the reference solution containing only the probe, the "off-on" fluorescence effect of the fluorescent probe C-3 on ALB still appeared in the other 7 solutions at the experimental concentration of biomacromolecules at the scanning wavelength.

[0078] Further, solutions containing only fluorescent probe C-3 at a concentration of 10 μM were prepared (solution number 1: probe); solutions containing the above-mentioned experimental concentrations of biomacromolecules and fluorescent probe C-3 (10 μM) were prepared (solution numbers 2-8: probe-AMY, probe-SOD, probe-GAL, probe-LYS, probe-APP, probe-LPS, and probe-GLB); solutions containing 0.1 μM ALB and fluorescent probe C-3 were prepared (solution number 9); solutions containing 0.45 μM ALB and fluorescent probe C-3 were prepared (solution number 10); solutions containing 4.5 μM ALB and fluorescent probe C-3 were prepared (solution number 11); and solutions containing 10 μM ALB and fluorescent probe C-3 were prepared (solution number 12). Fluorescence intensity was measured at an excitation wavelength of 470 nm. The test results are as follows: Figure 7 As shown, compared to solution 1 containing only the fluorescent probe, the addition of biomolecules such as AMY, SOD, GAL, LYS, APP, lipase LPS, and GLB resulted in slight increases in fluorescence intensity to varying degrees. Although the fluorescence intensity increase induced by AMY was close to the sensing signal of 0.1 μM ALB, the increase in fluorescence intensity induced by these biomolecules was much smaller than that induced by ALB at high concentrations, such as solutions 10 and 11, where the fluorescence intensity was much higher than other solutions at albuminuria threshold concentrations (0.45 and 4.5 μM), respectively. These results indicate that the fluorescent probe C-3 exhibits excellent selectivity for ALB.

[0079] In addition, such as Figure 8 As shown, the fluorescence response of probe C-3 to ALB is not affected by most of the test ions (ions: Na+). + ,K + Ca 2+ Mg 2+ NH4 + SO4 2- HCO3 - ,Cl -The effects of various substances (concentration 0.5 mM), amino acids (AA: Cys (40 μM), Trp (125 μM), Val (100 μM), Ala (100 μM), Arg (50 μM), Leu (75 μM), Cystine (50 μM)), glucose (Glu, 0.6 mM), urine components (u-species: uric acid (0.2 mM), urea (5 M), Creatine (50 μM), 4-pyridoxic acid (4 μM), 3-hydroxyanthranlic acid (50 μM)), and exogenous drugs (Drugs & metabolites, concentration 10 μM)) on ALB concentration were investigated, indicating that the probe can be used to monitor ALB in complex samples. The test involved preparing a C-3 solution (10 μM) containing the above substances and a C-3 solution (10 μM) containing the above substances and 10 μM ALB, and then measuring the fluorescence spectra of each sample.

[0080] The above comparison of the spectral intensity changes caused by ALB and amylase (AMY), superoxide dismutase (SOD), galactosidase (GAL), lysozyme (LYS), aminopeptidase (APP), lipase (LPS), and globulin (GLB) shows that the fluorescent probe C-3 of the present invention has excellent selectivity and anti-interference properties. When ALB is present, the probe can still exhibit an "off-on" fluorescence effect on ALB in a solution environment with complex biomolecular interference, which is beneficial to improving detection sensitivity.

[0081] Application Example 5

[0082] Albumin was added to the urine of three healthy adult male volunteers. The ALB levels added to the urine of volunteer 1 were 0, 0.1 μM, 0.2 μM, 0.3 μM, 0.45 μM, 1 μM, 2 μM, 3 μM, 4.5 μM, 6 μM, 8 μM, 10 μM, 15 μM, and 20 μM, respectively. The ALB levels added to the urine of volunteer 2 were 0, 0.1 μM, 0.3 μM, 0.4 μM, 0.8 μM, 2 μM, 4 μM, 6 μM, 8 μM, 9 μM, 10 μM, and 20 μM, respectively. The ALB levels added to the urine of volunteer 3 were 0, 0.1 μM, 0.2 μM, 0.4 μM, 0.8 μM, 1 μM, 2 μM, 4 μM, 6 μM, 8 μM, 9 μM, 10 μM, 20 μM, and 30 μM, respectively. The concentration of fluorescent probe C-3 in urine was 10 μM. For example... Figure 9 As shown, the fluorescence intensity of fluorescent probe C-3 increased with increasing albumin concentration in urine, and the detection limit (LOD) calculated according to the 3σ / k rule was much lower than the clinically determined proteinuria threshold (30 mg / L).

[0083] Application Example 6

[0084] The response of the fluorescent probe C-5 to spiked albumin was tested in the urine of healthy adult male volunteers. ALB was added sequentially at concentrations of 0, 0.1 μM, 0.2 μM, 0.3 μM, 0.5 μM, 0.7 μM, 1 μM, 2 μM, 4 μM, 6 μM, 8 μM, 10 μM, 15 μM, and 20 μM, followed by the addition of 10 μM of the C-5 probe. After shaking and incubation, the fluorescence spectrum was measured, as shown below. Figure 10 As shown, C-5 exhibits long-wavelength autoluminescence in urine, and the fluorescence peak shifts and intensifies with increasing ALB concentration. This process produces two fluorescence color changes under both 470nm and 365nm flashlight illumination, making it highly suitable for naked-eye observation and enabling rapid bedside detection of urinary protein using fluorescence colorimetry.

[0085] Application Example 7

[0086] Currently, existing detection methods and probes have high requirements for the concentration of albumin in urine. Therefore, hospitals always require morning urine for urine tests because the concentration of albumin is higher in morning urine, making it easier to detect and identify abnormal albumin concentrations.

[0087] To verify the high sensitivity of the novel probe of this invention, six series of urine samples were collected from volunteer 1 under different conditions: before lunch, after dinner, after staying up late, after taking medication, after drinking beer, and after drinking large amounts of water. Different concentrations of ALB were added to each series of urine samples, and then fluorescent probe C-3 at a concentration of 10 μM was added to each sample. Figure 11 As shown, the six series of urine samples maintained a progressively increasing spectral response to ALB. These results demonstrate that the fluorescent probe C-3 can be used for highly sensitive detection of urinary albumin compared to routine hospital health checks, with fewer restrictions on the timing of urine collection, providing flexibility for on-site and even home testing.

[0088] Application Example 8

[0089] Portable test strips were prepared by impregnating filter paper with the probe-loaded fluorescent C-3. (The test strips can be prepared as follows) Figure 12 As shown in the image, this technology enables rapid bedside detection of urine protein. Figure 13 As shown, after adding urine samples from three healthy adult volunteers, each spiked with different concentrations of ALB, to the test strip, the fluorescent color of the strip exhibited four color changes visible to the naked eye (blue-red-orange-yellow). Furthermore, a mobile app can be used to quickly analyze the color difference (ΔE*) and color coordinate ratio (R / B), thereby enabling rapid, semi-quantitative point-of-care detection of urinary protein.

[0090] like Figure 12As shown, the 24-color Hue ring records the Hue value for each pattern column, where the color change caused by ALB spans 180°. Figure 14 To make it more intuitive, these colors are divided into three groups ( Figure 11 The white dashed boxes in the figure indicate the risk of chronic kidney disease (CKD). Group 1 has Hue values ​​ranging from 225° to 270°, corresponding to normal u-ALB concentrations (0-0.2 μM). Group 2 has Hue values ​​concentrated between 348° and 4°, with corresponding u-ALB concentrations (0.45, 0.8 μM), possibly due to physiological or pathological causes. Group 3 has Hue values ​​ranging from 25° to 60°, corresponding to abnormal u-ALB levels, representing a high risk of CKD. Furthermore, statistical analysis, such as... Figure 15 As shown, the color coordinate ratio (R / B) is also positively correlated with ALB concentration, which provides another rapid detection signal mode for the quantitative detection of urinary albumin.

[0091] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features. These modifications or substitutions, or combinations of technical features in the above embodiments that do not conflict with each other, can be made in accordance with the manner described in the embodiments. These modifications, substitutions or combinations do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A triphenylamine-pyridinium salt fluorescent probe for rapid detection of urinary albumin, characterized in that, The structural formula is as follows: 。 2. The application of the triphenylamine-pyridinium salt represented by the following structural formula in the preparation of rapid detection fluorescent probes, urine albumin fluorescent colorimetric instant test strips or kits; 。 3. The application according to claim 2, characterized in that, The preparation method of the triphenylamine-pyridinium salt is as follows: S1. Using 4-methylpyridine and chlorohexane as raw materials, react in acetonitrile to obtain N-alkyl-substituted 4-methylpyridine salt; S2. Using N-alkyl-substituted 4-methylpyridinium salt and aldehyde triphenylamine as raw materials, react in ethanol to obtain triphenylamine-pyridinium salt fluorescent probe, wherein the aldehyde triphenylamine is 4-aldehyde triphenylamine.

4. The application according to claim 3, characterized in that, In S1, the reaction temperature is 75-85℃ and the reaction time is 16-24h.

5. The application according to claim 3, characterized in that, In S1, the haloalkane is a bromoalkane or a chloroalkane; its molar ratio with 4-methylpyridine is 1:

1.

6. The application according to claim 3, characterized in that, In S1, after the reaction is complete, 1.5-5 times the volume of ethyl acetate is added to the reaction system, stirred at room temperature, cooled and filtered to obtain N-alkyl-substituted 4-methylpyridinium salt.

7. The application according to claim 3, characterized in that, In S2, aldehyde triphenylamine is dissolved in ethanol, and N-alkyl-substituted 4-methylpyridinium salt prepared in S1 and the catalyst tetrahydropyrrole are added. The mixture is heated to 75-85℃ and reacted for 20-30 hours. The resulting reaction solution is concentrated by vacuum evaporation, purified by silica gel column chromatography, and then recrystallized with ethyl acetate to obtain the red target product.

8. The application according to claim 7, characterized in that, In S2, the amount of catalyst tetrahydropyrrole added is 4-6% of the mass of the N-alkyl-substituted 4-methylpyridinium salt.

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