Triphenylamine-pyridinium salt fluorescent probe for rapid detection of white urine eggs and preparation method of triphenylamine-pyridinium salt fluorescent probe

Abstract

The invention relates to a triphenylamine-pyridinium salt fluorescent probe for rapid detection of white urine eggs, the triphenylamine-pyridinium salt fluorescent probe comprises anions and cations, the cations have a structural formula shown as a formula I, and at least one of R1, R2 and R3 is a group shown as a formula II; in the group shown in the formula II, R of the # imgabs0 # is linear alkyl with 1-20 carbons; and when two or three substitution sites in R1, R2 and R3 are connected with the group shown in the formula II, R in the groups shown in the formula II connected with different substitution sites are the same or different. The probe provided by the invention has ultrahigh sensitivity and a fluorescent colorimetric detection effect, and is beneficial to early prevention of chronic diseases and timely intervention of related diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of bedside rapid detection, and in particular relates to a triphenylamine-pyridinium salt fluorescent probe for rapid detection of urine albumin and a preparation method thereof. Background Art

[0002] Human serum albumin (HSA) is the most abundant protein in plasma and plays a vital role in various physiological processes. Abnormally low levels of HSA in plasma (<35 g / L) and elevated levels of albumin in urine (>30 mg / L) are thought to be associated with specific diseases, including cirrhosis, malnutrition, gastrointestinal cancer, systemic lupus erythematosus, and glomerulonephritis. The levels of albumin (ALB) in blood and urine can be accurately measured during formal physical examinations based on medical technology. However, current detection methods do not meet the needs of rapid, on-site, real-time monitoring of ALB or even home testing due to time-consuming pretreatment and high proficiency requirements for operators, which is not conducive to early prevention of chronic diseases and timely intervention of related diseases.

[0003] In the past five years, the development of fluorescent probes has promoted the rapid on-site detection of ALB due to its advantages such as fast response, simple use and signal visualization. Designing new fluorescent probes with high sensitivity (S) is the basic goal to achieve the final application. The most commonly used design strategy for ALB fluorescent probes is based on the sensing mechanism, that is, the probe with intramolecular charge transfer (ICT) properties is kept in the "fluorescence completely off" state in water to produce an ideal initial signal (I 0 ), during the detection process, the detection environment encapsulates the environmentally sensitive probe in the hydrophobic cavity, resulting in fluorescence recovery and generating a detection signal (I).

[0004] Hydrophilic AIEgens (Aggregation-Induced Emission luminogens) are a class of organic luminescent materials that emit almost no light in a dispersed state but significantly enhance their luminescence in an aggregated state. Hydrophilic AIEgens have been shown to be a promising alternative to urinary protein fluorescent probes. Currently, most studies focus on improving S by making full use of the molecular rotor structure. However, the enhancement of S depends on two factors, namely, reducing the initial fluorescence (I 0 ) and the amplification factor (I). However, the molecular rotor structure affects these two factors in a conflicting manner. As has been widely demonstrated, the stacking of molecular rotors in water can suppress non-radiative deactivation to aggregation-induced emission (AIE), which competes with the quenching effect of ICT solvent relaxation, leading to partial recovery of I. 0 (I 0This intrinsic property of molecular rotors makes it very difficult to increase S for most dye-based probes; to complicate the problem, the size distribution of aggregates on the large nanoscale may exacerbate I 0 This may reduce the accuracy of the test, especially for healthy individuals and patients with early CKD (early chronic kidney disease), because the concentration of ALB in urine is relatively low at this time, which will further affect the accuracy of the test.

[0005] Although some ALB fluorescent probes have been reported to use multi-ionic substituents to hinder their aggregation in water, thus obtaining extremely low initial fluorescence I 0 From the results, although these hydrophilic probes of ALB may also be suitable for bedside urine analysis and auxiliary judgment of kidney disease, their initial fluorescence I 0 There are still different degrees of increase, which leads to a decrease in the growth multiple (I). In addition, the POCT detection effect of the above-mentioned ALB fluorescent probe molecular structure in detecting urine albumin has not been reported yet, and this type of fluorescent probe still lacks a clear molecular design solution. Summary of the invention

[0006] 1. Technical issues to be resolved

[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 urine albumin, the main structural feature of which is a triphenylamine-pyridinium salt structure modified with a long alkyl chain, with ultra-high sensitivity and fluorescent colorimetric detection effect, and is suitable for fluorescent colorimetric bedside rapid detection, especially for healthy individuals and early CKD patients. The present invention is conducive to 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 urine albumin, which comprises an anion and a cation, wherein the cation has a structural formula shown in Formula I:

[0010]

[0011] At least one of the above 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 of 1 to 20 carbon atoms;

[0014] When two or three substitution positions among R1, R2 and R3 are connected to the group shown in formula II, R in the group shown in formula II connected to different substitution positions are the same or different; preferably, they are the same.

[0015] Among them, when R1, R2 and R3 are connected to the same R in the group shown in formula II, a centrally symmetrical fluorescent probe molecule is obtained; when R1, R2 and R3 are connected to different R in the group shown in formula II, an asymmetric fluorescent probe molecule is obtained.

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

[0017]

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

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

[0020] S1, using 4-methylpyridine and halogenated alkane as raw materials, reacting in acetonitrile to obtain N-alkyl substituted 4-methylpyridine salt;

[0021] S2. Using N-alkyl substituted 4-methylpyridinium salt and aldehyde triphenylamine as raw materials, reacting in ethanol to obtain a triphenylamine-pyridinium salt fluorescent probe, wherein the aldehyde triphenylamine is 4-aldehyde triphenylamine, 4,4'-diadehyd triphenylamine or 4,4',4"-trialdehyde triphenylamine; the reaction process is expressed as follows:

[0022]

[0023] Wherein, Ra and Rb are independently H or an aldehyde group; X is chlorine or bromine; R2 and R3 are independently H or a group represented by formula II;

[0024]

[0025] In the above formula, R is a straight chain alkyl group having 1 to 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 16-24h, preferably 18h at 80°C; wherein the halogenated alkane is a brominated alkane or a chloroalkane; and the molar ratio thereof to 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 of ethyl acetate is added to the reaction system, stirred at room temperature, cooled and filtered to obtain 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 the N-alkyl-substituted 4-methylpyridinium salt prepared in S1 and the catalyst tetrahydropyrrole are added, and the mixture is heated to 75-85°C for reaction for 20-30h (preferably 80°C for reaction for 25h), and the obtained reaction solution is concentrated by vacuum evaporation, separated and purified by silica gel column chromatography, and then recrystallized from ethyl acetate to obtain the red target product.

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

[0030] In a third aspect, the present invention also provides a test paper for rapid detection of urine protein, which comprises an adsorption test paper and a probe molecule loaded on the test paper substrate, wherein the probe molecule is the triphenylamine-pyridinium salt fluorescent probe. The test paper can realize bedside rapid detection of urine protein.

[0031] (III) Beneficial effects

[0032] The triphenylamine-pyridinium salt fluorescent probe of the present invention is an ALB fluorescent probe having 1-3 ion-substituted hydrophilic pyridinium salts and symmetrically connected hydrophobic alkyl chains of different lengths, which can be used for accurate detection of urine protein and rapid bedside detection; the hydrophilicity of the probe is mainly increased by different numbers of ion substituents of the pyridinium salt, so that the probe has good solubility in aqueous solution, and the hydrophobicity of the probe is increased by the hydrophobic alkyl chain, so that the probe can interact with a hydrophobic environment (such as a hydrophobic pocket of a protein). Hydrophobic alkyl chains of different lengths can adjust the hydrophobicity of the probe, thereby affecting its binding ability with a target molecule.

[0033] The triphenylamine-pyridinium salt fluorescent probe of the present invention is a hydrophilic AIEgenss, which can remain monodisperse in water and obtain an extremely low initial fluorescence (I 0 ), and the fluorescence response signal of the probe obtained an extremely high growth factor (I). It is worth noting that the probe unexpectedly red-shifted emission during the detection of real urine samples, thereby enabling the acquisition of rapid detection devices such as test strips or kits for ALB fluorescent colorimetric point-of-care testing (POCT).

[0034] Experimental verification shows that the fluorescent probe provided by the present invention has the following advantages:

[0035] (1) Ultra-high sensitivity: With extremely low initial fluorescence (I 0) and an extremely high growth multiple (I). The fluorescent probe C-3 prepared in Example 3 exhibits an ultra-high sensitivity of >3000 times in its fluorescence response. In an experiment simulating the urine of patients with chronic kidney disease (CKD), the fluorescence signal and the spiked ALB concentration show a ratio change, and have high correlation, high accuracy, and low detection limit.

[0036] (2) Hospital urine tests always require morning urine. As a new type of probe with high sensitivity, the experiment evaluated the fluorescent probe C-3's ability to maintain a gradually enhanced spectral response to ALB in six series of urine samples collected under different conditions, such as before lunch, after dinner, after staying up late, after taking medicine, after drinking beer, and after drinking a lot of water. The experimental results show that compared with routine health examinations in hospitals, fluorescent probe C-3 can be used to detect urinary albumin with fewer restrictions on urine collection, providing flexibility for on-site testing or 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 by the naked eye. Therefore, a fluorescent colorimetric point-of-care test (POCT) kit can be developed to facilitate the qualitative or semi-quantitative bedside rapid detection of urine protein. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0039] Figure 2 is the fitted correlation curve between ALB concentration and fluorescence intensity.

[0040] Figure 3 A curve showing the relationship between the fluorescence intensity and the ALB concentration when the concentration of the fluorescent probe C-3 was 1 μM and 10 μM was drawn using fluorescence titration.

[0041] Figure 4 When the ALB concentration is low, the fluorescence is "off"; as the ALB concentration increases, the fluorescence shows a significant 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 respond to 10 μM ALB.

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

[0044] Figure 7The fluorescence intensity of the blank solution, AMY, SOD, GAL, LYS, APP, LPS, GLB, 0.1μM ALB, 0.45μM ALB, 4.5μM ALB, and 10μM ALB solutions was added with equal amounts of C-3 probe.

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

[0046] Fig. 9 After albumin was spiked into the urine of three healthy adult volunteers, the fluorescence intensity of the fluorescent probe C-3 showed a trend of increasing with the increase of albumin concentration in the urine.

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

[0048] Fig.11 The spectral responsiveness of volunteer 1 to ALB was gradually enhanced in six series of urine samples obtained under different conditions, including before lunch, after dinner, after staying up late, after taking medicine, after drinking beer, and after drinking a lot of water.

[0049] Fig.12 This is a picture of a portable test strip prepared by impregnating filter paper with the fluorescent probe C-3.

[0050] Fig.13 This is a photo of the test strip showing four color changes in fluorescence that are visible to the naked eye after three urine samples of healthy adult volunteers spiked with different concentrations of ALB were added to the test strip.

[0051] Fig.14 A 24-color Hue ring for rapid detection of urine protein based on the fluorescent probe C-3.

[0052] Fig.15 This is a statistical graph showing that the color coordinate ratio R / B is positively correlated with the ALB concentration. DETAILED DESCRIPTION

[0053] In order to better explain the present invention and facilitate understanding, the present invention is described in detail below through specific implementation modes in conjunction with the accompanying drawings.

[0054] Examples 1-5

[0055] Example 1-5: Five fluorescent probes C-1, C-2, C-3, C-4 and C-5 were prepared in sequence. The preparation process is as follows:

[0056] The first step: take 4-methylpyridine (reactant 1) and halogenated alkanes A-1, A-2, A-3, A-4, and A-5 as reaction raw materials, and the molar ratio of 4-methylpyridine to halogenated alkanes is 1:1; dissolve the two reaction raw materials in acetonitrile, heat to 80°C and react for 18 hours, add ethyl acetate 3 times the volume of the reaction system, and stir at room temperature to remove excess raw materials, 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: Take 4-formyl triphenylamine (reactant 2) and dissolve it in ethanol, add the N-alkyl substituted 4-methylpyridinium salts B-1, B-2, B-3, B-4, B-5 ​​prepared in the first step and the catalyst tetrahydropyrrole (accounting for 5% of the mass of the N-alkyl substituted 4-methylpyridinium salt), wherein the molar ratio of the N-alkyl substituted 4-methylpyridinium salt to the 4-formyl triphenylamine is 1:1.

[0058] The reaction system was heated to 80°C for 25 hours. The mixed solution was concentrated to 1 / 3 of the original volume by evaporation under vacuum. After separation and purification by silica gel column chromatography, it was recrystallized from ethyl acetate to obtain red compounds C-1, C-2, C-3, C-4, and C-5; mass spectrum 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 process of Examples 1-5 is shown as follows. The names of the halogenated alkanes A-1, A-2, A-3, A-4, and A-5 are ethyl bromide, butyl chloride, hexyl chloride, octane chloride, and decane chloride, respectively; the names of the intermediates B-1, B-2, B-3, B-4, and B-5 are N-ethyl-4-methylpyridinium bromide, N-butyl-4-methylpyridinium chloride, N-hexyl-4-methylpyridinium chloride, N-octyl-4-methylpyridinium chloride, and N-decyl-4-methylpyridinium chloride, respectively.

[0060]

[0061] Embodiment 6-10

[0062] Examples 6-10: Five fluorescent probes C-6, C-7, C-8, C-9 and C-10 were prepared in sequence. The preparation process is as follows:

[0063] The first step: take 4-methylpyridine (reactant 1) and halogenated alkanes A-1, A-2, A-3, A-4, and A-5 as reaction raw materials, and the molar ratio of 4-methylpyridine to halogenated alkanes is 1:1; dissolve the two reaction raw materials in acetonitrile, heat to 80°C and react for 18 hours, add ethyl acetate 3 times the volume of the reaction system, and stir at room temperature to remove excess raw materials, 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: Take 4,4'-dialdehyde triphenylamine (reactant 3) and dissolve it in ethanol, add the N-alkyl substituted 4-methylpyridinium salts B-1, B-2, B-3, B-4, B-5 ​​prepared in the first step and the catalyst tetrahydropyrrole (accounting for 5% of the mass of the N-alkyl substituted 4-methylpyridinium salt), wherein the molar ratio of the N-alkyl substituted 4-methylpyridinium salt to the 4,4'-dialdehyde triphenylamine is 2:1.

[0065] The reaction system was heated to 80°C for 25 hours. The mixed solution was concentrated to 1 / 3 of the original volume by evaporation under vacuum. After separation and purification by silica gel column chromatography, it was recrystallized from ethyl acetate to obtain red compounds C-6, C-7, C-8, C-9 and C-10; mass spectrum 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 process of Examples 6-10 is shown as follows. The names of the halogenated alkanes A-1, A-2, A-3, A-4, and A-5 are ethyl bromide, butyl chloride, hexyl chloride, octane chloride, and decane chloride, respectively; the names of the intermediates B-1, B-2, B-3, B-4, and B-5 are N-ethyl-4-methylpyridinium bromide, N-butyl-4-methylpyridinium chloride, N-hexyl-4-methylpyridinium chloride, N-octyl-4-methylpyridinium chloride, and N-decyl-4-methylpyridinium chloride, respectively.

[0067]

[0068] The following is combined with application examples to further study the spectral responsiveness, sensing performance, selectivity and anti-interference characteristics of the fluorescent probe.

[0069] Application Example 1

[0070] Albumin ALB was added to PBS buffer in the following amounts: 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, 4.0 μM to obtain 13 samples, and fluorescent probe C-3 was added to each sample. The concentration of the fluorescent probe in the 13 samples was equal and 1 μM. Figure 1 The fluorescence spectra of these 13 samples are shown in Figure 2. According to the 3σ / k rule, the detection limit (LOD) of ALB is 14nM (0.93mg / L) (Figure 2 Figure 2 ), which is far below the threshold of microalbuminuria (30 mg / L).

[0071] Application Example 2

[0072] Fluorescence titration experiments were used to monitor the changes in fluorescence intensity and analyze the interaction between the fluorescent probe and ALB. Titration samples containing 1 μM fluorescent probe C-3 and 10 μM fluorescent probe C-3 were prepared, and their initial fluorescence intensities I0 were tested respectively. Then, ALB was gradually added dropwise by titration. During the addition process, the required volume of ALB mother solution was added at a time. After each addition, the solution was vortexed at 2000 rpm for 1 minute and allowed to stand for 1 minute. The fluorescence intensity of the solution was measured and recorded. The recorded values ​​were used to draw a curve showing the relationship between fluorescence intensity and ALB concentration. Figure 3 As shown, the fluorescence titration spectra of the fluorescent probe C-3 at 10 μM showed a similar "off-on" trend compared to 1 μM fluorescent probe C-3, but the sensitive range (0-60 μM) was changed compared to 1 μM. The C50 values ​​from the two experiments were 0.55 μM and 4.42 μM (see Figure 3 ), which are comparable to the thresholds for microalbuminuria (0.45 μM) and macroalbuminuria (4.5 μM), respectively.

[0073] like Figure 4 As shown in the figure, when the concentration of fluorescent probe C-3 is 10 μM, when the concentration of ALB is lower than 0.45 μM, the fluorescence is "dark" or "off state". When ALB is continuously added to increase its concentration to 3 μM or above, the fluorescence shows a clear change from orange to bright yellow. Therefore, benefiting from the initial "fluorescence off" state, the concentration of ALB can be reflected by the fluorescence from dark to yellow to bright during the titration process (such as Figure 4 The color change during this process is significant, so it can be observed directly by the naked eye, or by constructing a positive correlation between color coordinates and ALB concentration on a smartphone APP, thereby easily achieving semi-quantitative real-time detection of ALB.

[0074] Application Example 3

[0075] Prepare two solutions of fluorescent probes C-1, C-6, and C-8 in PBS, A and B, with the probe concentration of each solution being 10 μM. Add 10 μM ALB to group B, shake and let stand, and then test the fluorescence spectra of groups A and B. Figure 5 As shown, the fluorescence intensity in group A is extremely low, and the fluorescence in group B is restored. Based on the spectral response results, it is proved that compounds C-1, C-6, and C-8 can all be used as fluorescent probes for ALB to achieve high-sensitivity detection of ALB.

[0076] Application Example 4

[0077] Prepare 7 10 μM ALB solutions in parallel, add 100 U / mL of amylase (AMY), 500 U / mL of superoxide dismutase (SOD), 500 U / mL of galactosidase (GAL), 1000 μg / mL of lysozyme (LYS), 1000 μg / mL of aminopeptidase (APP), 1000 U / mL of lipase (LPS) and 500 μg / mL of globulin (GLB) as interfering molecules to each solution, add the same concentration of fluorescent probe C-3 (10 μM) to each solution; finally, prepare a solution containing only the same concentration of fluorescent probe C-3 as a reference. Irradiate the mixed solution with excitation light in the range of 500-850 nm for the above 8 solution samples, test the fluorescence intensity, and draw the excitation wavelength-fluorescence intensity change curve. 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 appears in the presence of the experimental concentration of biomacromolecules at the scanning wavelength in the remaining 7 solutions.

[0078] Further, a solution containing only fluorescent probe C-3 was prepared, with a concentration of 10 μM (solution number 1: probe), solutions containing the above experimental concentrations of biomacromolecules and fluorescent probe C-3 (10 μM) (solution numbers 2-8: probe-AMY, probe-SOD, probe-GAL, probe-LYS, probe-APP, probe-LPS and probe-GLB), a solution containing 0.1 μM ALB and fluorescent probe C-3 (solution number 9), a solution containing 0.45 μM ALB and fluorescent probe C-3 (solution number 10), a solution containing 4.5 μM ALB and fluorescent probe C-3 (solution number 11), and a solution containing 10 μM ALB and fluorescent probe C-3 (solution number 12). The fluorescence intensity was tested at an excitation light wavelength of 470 nm, and the test results are as follows. Figure 7As shown, compared with solution No. 1 containing only the fluorescent probe, the fluorescence intensity increased slightly to varying degrees after adding biomacromolecules such as AMY, SOD, GAL, LYS, APP, lipase LPS and GLB. Although the increase in fluorescence intensity induced by AMY is close to the induction signal of 0.1 μM ALB, the increase in fluorescence intensity caused by these biomacromolecules is much smaller than the increase induced by ALB at high concentrations, such as solutions No. 10 and No. 11, which have much higher fluorescence intensities than other solutions at the albuminuria threshold concentration (0.45 and 4.5 μM), respectively. These results show that the fluorescent probe C-3 exhibits excellent selectivity for ALB.

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

[0080] Through 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), it can be seen that the fluorescent probe C-3 of the present invention has excellent selectivity and anti-interference. When ALB is present, the "off→on" fluorescence effect of the probe on ALB can still occur in a solution environment interfered by complex biological macromolecules, which is beneficial to improve the detection sensitivity.

[0081] Application Example 5

[0082] Albumin was spiked into the urine of three healthy adult male volunteers, among which the amounts of ALB added in the urine of volunteer 1 were 0, 0.1μM, 0.2μM, 0.3μM, 0.45μM, 1μM, 2μM, 3μM, 4.5μM, 6μM, 8μM, 10μM, 15μM, and 20μM, respectively; the amounts of ALB added in the urine of volunteer 2 were 0, 0.1μM, 0.3μM, 0.4μM, 0.8μM, 2μM, 4μM, 6μM, 8μM, 9μM, 10μM, and 20μM, respectively; the amounts of ALB added in the urine of volunteer 3 were 0, 0.1μM, 0.2μM, 0.4μM, 0.8μM, 1μM, 2μM, 4μM, 6μM, 8μM, 9μM, 10μM, 20μM, and 30μM, respectively. The concentration of fluorescent probe C-3 in urine is 10 μM. Fig. 9 As shown, the fluorescence intensity of the fluorescent probe C-3 showed a trend of increasing with the increase of albumin concentration in urine, and the detection limit (LOD) value calculated according to the 3σ / k rule was much lower than the clinical judgment threshold of proteinuria (30 mg / L).

[0083] Application Example 6

[0084] The response performance of fluorescent probe C-5 to spiked albumin was tested in the urine of adult healthy male volunteers. 0, 0.1μM, 0.2μM, 0.3μM, 0.5μM, 0.7μM, 1μM, 2μM, 4μM, 6μM, 8μM, 10μM, 15μM, and 20μM ALB were spiked into the urine sample in sequence, and then 10μM C-5 probe was added in sequence. After shaking and standing, the fluorescence spectrum was tested, as shown in Figure 2. Fig.10 As shown in the figure, C-5 exhibits long-wavelength spontaneous luminescence in urine, and the fluorescence peak blue-shifts and increases with the increase of ALB concentration. This process can produce two fluorescence color changes under 470nm and 365nm flashlight irradiation, which is very conducive to naked eye observation and can be used for rapid bedside detection of urine protein by fluorescence colorimetry.

[0085] Application Example 7

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

[0087] In order to verify the high sensitivity of the novel probe of the present invention, 6 series of urine samples were collected from volunteer 1 under different conditions, such as before lunch, after dinner, after staying up late, after taking medicine, after drinking beer, and after drinking a lot of water. Different concentrations of ALB were added to each series of urine samples, and then the fluorescent probe C-3 was added to the urine at a concentration of 10 μM. Fig.11 As shown, the six serial urine samples maintained a gradually increasing spectral response to ALB. These results indicate that the fluorescent probe C-3 can be used to detect urinary albumin with high sensitivity and with less restrictions on the timing of urine collection compared to routine health examinations in hospitals, providing flexibility for on-site testing or even home testing.

[0088] Application Example 8

[0089] The probe fluorescent C-3 is soaked in filter paper to prepare a portable test paper (the test paper can be as Fig.12 As shown in Figure 2), rapid bedside detection of urine protein can be achieved. Fig.13 As shown in the figure, after adding urine samples of three healthy adult volunteers spiked with different concentrations of ALB to the test strip, the fluorescent color of the test strip showed four color changes (blue-red-orange-yellow) that can be discerned by the naked eye. Furthermore, a mobile phone APP can be used to quickly analyze the color difference (ΔE*) and color coordinate ratio (R / B), thereby realizing semi-quantitative bedside rapid detection of urine protein.

[0090] like Fig.12 As shown, the Hue value of each pattern column is recorded on the 24-color Hue ring, where the color change caused by ALB spans 180° ( Fig.14 ). For a more intuitive look, these colors are divided into three groups ( Fig.11 The white dashed box in the figure was used to specify the risk of chronic kidney disease (CKD). The Hue values ​​of Group 1 were 225° to 270°, corresponding to normal u-ALB concentrations (0-0.2 μM). The Hue values ​​of Group 2 were concentrated between 348° and 4°, and the corresponding u-ALB concentrations (0.45, 0.8 μM) may be caused by physiological or pathological reasons. The Hue values ​​of Group 3 ranged from 25° to 60°, corresponding to abnormal u-ALB levels, representing a high risk of CKD. In addition, after analysis and statistics, such as Fig.15 As shown, the color coordinate ratio (R / B) is also positively correlated with the ALB concentration, which provides another rapid detection signal mode for the quantitative detection of urine albumin.

[0091] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions described in the above embodiments may still be modified, or some or all of the technical features therein may be replaced by equivalents. These modifications or replacements, or the technical features in the above embodiments may be combined in the manner described in the embodiments if they do not conflict with each other, and these modifications, replacements 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 urine albumin, characterized in that: It includes anions and cations, and the cations have the structural formula shown in Formula I: At least one of the above R1, R2 and R3 is a group represented by formula II; In the group shown in formula II, R is a straight chain alkyl group of 1 to 20 carbon atoms; When two or three substitution positions among R1, R2 and R3 are connected to the group shown in formula II, R in the group shown in formula II connected to different substitution positions may be the same or different.

2. The triphenylamine-pyridinium salt fluorescent probe according to claim 1, characterized in that: R1, R2 and R3 are independently selected from H and one of the following groups:

3. The triphenylamine-pyridinium salt fluorescent probe according to claim 1 or 2, characterized in that: The anion is chloride or bromide.

4. A method for preparing a triphenylamine-pyridinium salt fluorescent probe for rapid detection of urine albumin, characterized in that: It includes the following steps: S1, using 4-methylpyridine and halogenated alkane as raw materials, reacting in acetonitrile to obtain N-alkyl substituted 4-methylpyridine salt; S2. Using N-alkyl substituted 4-methylpyridinium salt and aldehyde triphenylamine as raw materials, reacting in ethanol to obtain a triphenylamine-pyridinium salt fluorescent probe, wherein the aldehyde triphenylamine is 4-aldehyde triphenylamine, 4,4'-diadehyd triphenylamine or 4,4',4"-trialdehyde triphenylamine; the reaction process is expressed as follows: Wherein, Ra and Rb are independently H or an aldehyde group; X is chlorine or bromine; R2 and R3 are independently H or a group represented by formula II; In the above formula, R is a straight chain alkyl group having 1 to 20 carbon atoms.

5. The preparation method according to claim 4, characterized in that: In S1, the reaction temperature is 75-85°C and the reaction time is 16-24h.

6. The preparation method according to claim 4, characterized in that: In S1, the halogenated alkane is a brominated alkane or a chloroalkane; and the molar ratio of the halogenated alkane to 4-methylpyridine is 1:

1.

7. The preparation method according to claim 4, characterized in that: In S1, after the reaction is completed, ethyl acetate is added in an amount of 1.5 to 5 times the reaction system, stirred at room temperature, cooled and filtered to obtain N-alkyl substituted 4-methylpyridinium salt.

8. The preparation method according to claim 4, characterized in that: In S2, aldehyde triphenylamine is dissolved in ethanol, and the N-alkyl substituted 4-methylpyridinium salt prepared in S1 and the catalyst tetrahydropyrrole are added, and the mixture is heated to 75-85°C for reaction for 20-30 hours. The obtained reaction solution is concentrated by vacuum evaporation, separated and purified by silica gel column chromatography, and then recrystallized using ethyl acetate to obtain the red target product.

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

10. A test paper for rapid detection of urine albumin, characterized in that: The invention comprises an adsorption test paper and a probe molecule loaded on a substrate of the test paper, wherein the probe molecule is the triphenylamine-pyridinium salt fluorescent probe according to any one of claims 1 to 3.

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