Multi-channel fluorescence array sensor and its construction and use method

Abstract

The present invention discloses a multi-channel fluorescence array sensor and a method for constructing and using the same, which belongs to the field of biosensor technology. The multi-channel fluorescence array sensor is composed of negatively charged conjugated polymer nanoparticles Pdots and positively charged porphyrin compounds PPB and PPC with different side chain substitutions through electrostatic action, and is used to distinguish and detect different glycosaminoglycans. The preparation process of the array sensor is simple and the number of sensing elements is small; Pdots has excellent properties such as high light stability, signal amplification ability, and two-photon performance; porphyrin has stable chemical properties and many excellent photophysical properties; at the same time, the characteristics of multi-signal channel output can reduce the interference of background signal noise, effectively improving the practical application value of the array sensor.

Description

Technical Field

[0001] The present invention belongs to the field of biosensor technology, and in particular relates to a multi-channel fluorescence array sensor and a construction and use method thereof. Background Art

[0002] Glycosaminoglycans (GAGs), also known as mucopolysaccharides, are linear polyanionic polysaccharides composed of repeating disaccharide units, mainly including hyaluronic acid (HA), chondroitin sulfate (CS), dermatan sulfate (DS), heparin (Hep), etc. The disaccharide units of the above GAGs are composed of hexuronic acid (D-glucuronic acid / L-iduronic acid) and hexosamine (N-acetylglucosamine / N-acetylgalactosamine). They exhibit different biological activities and are widely used in various scenarios. Among them, heparin is the natural polyanion with the highest charge density known in biological systems and is commonly used as an anticoagulant; hyaluronic acid exists in our epithelial and conjunctival tissues and is used in ophthalmic surgery and skin care products; chondroitin sulfate is composed of 1,4-β-D-glucuronic acid and 1,3-N-acetyl-β-D-galactosamine repeating units, which can relieve pain and inflammation caused by osteoarthritis. The disaccharide repeating units that make up these polysaccharides have similar chemical structures, and their biosynthesis process is very complicated, which brings great difficulties to the characterization and detection of glycosaminoglycans.

[0003] Traditional heparin quality control methods mainly include nuclear magnetic resonance, high performance liquid chromatography and mass spectrometry. Although these methods have high sensitivity, the detection equipment they rely on is relatively expensive, and the data analysis process needs to be completed by experienced spectrum analysts. When a small amount of other glycosaminoglycan analog impurities exist in a certain polysaccharide, these methods are difficult to make rapid and accurate identification, which poses a serious potential threat to the clinical safety application of such drugs. The above factors limit the widespread application of such methods in heparin detection and quality control.

[0004] Considering the high heterogeneity of the chemical structure of glycosaminoglycans such as heparin, it is difficult to design specific fluorescent probes based on the traditional "lock-key" model. However, array sensors can simulate the olfactory and taste systems of mammals to perform pattern recognition on analogs. Compared with the detection system of the "lock-key" binding mode, the low-selective binding between the cross-reactive sensor unit and the analyte can achieve the detection purpose, avoiding the complexity of the sensor unit design. Fluorescent array sensors generate multiple groups of detection signals by binding to different analytes through multiple receptors, thereby distinguishing different analytes. The generated pattern response provides fingerprint characteristics for each analyte, which can realize the parallel detection of multiple analytes and avoid the complexity of the sensor unit design.

[0005] In his master's thesis "Construction of supramolecular fluorescence sensor array based on tetraphenylethylene and porphyrin and its application in glycosaminoglycan detection", Yang Zhiyu mentioned that he constructed an AIE fluorescence sensor array based on tetraphenylethylene-polydiallyldimethylammonium chloride. By adjusting the loading amount of tetraphenylethylene formic acid (TPE) on the cationic polymer polydiallyldimethylammonium chloride (PDDA), PDDA-TPE was synthesized. 100 、PDDA-TPE 75 、PDDA-TPE 50 and PDDA-TPE 25 These four AIE supramolecular nanoprobes were used to construct an AIE sensor array that can distinguish a variety of glycosaminoglycans. In order to avoid the problem that short-wavelength fluorescence is easily interfered by biological background signals, five porphyrins with different numbers and positions of positively charged units were synthesized and assembled on the surface of graphene oxide to form five nanocomposites to construct a porphyrin-graphene oxide sensor array PP-GO. Both types of sensors constructed by them can distinguish a variety of glycosaminoglycans, but both PDDA-TPE arrays and PP-GO arrays have a problem in actual use, that is, both types of sensors rely on changes in signal intensity of a single wavelength to identify glycosaminoglycans, which may cause the test results to be affected by external factors such as sensor concentration and instrument parameter settings, and cannot well reduce or even eliminate background noise.

[0006] Based on the above considerations, if a new type of array sensor can be designed, and this sensor can use the ratio of fluorescence intensity changes at two or even more different wavelengths as a sensing signal, it is expected to eliminate the negative effects caused by sensor concentration and instrument parameter settings. Summary of the invention

[0007] The purpose of the present invention is to solve the problems existing in the prior art, provide a new multi-channel fluorescence array sensor, and disclose its construction method and use method. The array sensor includes two sensing units formed by conjugated polymer nanoparticles and porphyrin compounds with different side chain substitutions through electrostatic compound action, and can amplify signal response through energy resonance transfer effect; and because six groups of signal channels are generated, the background noise can be reduced through multi-signal output of double emission peaks, and then linear discriminant analysis is used for data processing, so that small molecule heparin and glycosaminoglycan analogs such as dermatan sulfate and oversulfated chondroitin sulfate can be recognized and distinguished.

[0008] The present invention is implemented as follows: a multi-channel fluorescence array sensor, which is formed by compounding negatively charged conjugated polymer nanoparticles Pdots and positively charged porphyrin compounds PPB and PPC with different side chain substitutions through electrostatic action, and there is a fluorescence resonance energy transfer (FRET) effect between the conjugated polymer nanoparticles and the porphyrin compounds with different side chain substitutions, further amplifying the fluorescence response signal; the conjugated polymer nanoparticles are carboxyl functionalized polymer nanoparticles prepared by nano coprecipitation method based on the hydrophobic interaction between fluorene polymer and PSMA, and the structural formula of fluorene polymer is as follows:

[0009]

[0010] Wherein, n is a natural number between 8 and 200;

[0011] The structural formulas of two porphyrin compounds with different side chain substitutions are as follows:

[0012]

[0013] Furthermore, it includes two sensing units that generate six groups of signal channels. The two sensing units refer to the Pdots+PPB system and the Pdots+PPC system. The excitation wavelengths of the two systems are both 410nm. Given a single fixed excitation wavelength, multi-channel signals can be output, wherein the emission wavelengths of the Pdots+PPB system are 490nm, 680nm and 725nm; the emission wavelengths of the Pdots+PPC system are 490nm, 680nm and 720nm.

[0014] The construction method of the above multi-channel fluorescence array sensor is as follows:

[0015] 1) 5,10,15,20-tetrapyridin-4-ylporphyrin is used as a raw material to undergo electrophilic substitution reaction with two electrophilic reagents, 4-bromomethylphenylboronic acid and iodomethane, to generate pyridinium quaternary ammonium salts PPB and PPC;

[0016] 2) using 2,2'-(9-nonyl-9-octyl-9H-fluorene-2,7-diyl)bis(4,4,5,5-tetramethyl-1,3,2-dioxaborane) as a raw material, coupling with 5,8-dibromoquinoxaline to obtain a quinoxaline-substituted fluorene polymer;

[0017] 3) Carboxyl-functionalized conjugated polymer nanoparticles Pdots were prepared by coprecipitation of quinoxaline-substituted fluorene polymers and amphiphilic PMSA;

[0018] 4) The conjugated polymer nanoparticle mother solution was diluted with PBS buffer, and different concentrations of positively charged porphyrin compounds PPB and PPC with different side chain substitutions were added respectively to obtain titration curves of the conjugated polymer nanoparticles and the porphyrin compounds. The electrostatic complex when PPB quenched the fluorescence intensity of the conjugated polymer nanoparticles to 60% and the electrostatic complex when PPC quenched the fluorescence intensity of the conjugated polymer nanoparticles to 50% were selected to construct the sensing units of the array sensor, namely, the Pdots+PPB system and the Pdots+PPC system.

[0019] Furthermore, the Pdots+PPB and Pdots+PPC systems were obtained by mixing a PBS (10 mM, pH = 7.4) solution of conjugated polymer nanoparticles with a mass concentration of 0.01 mg / mL and a PBS solution of porphyrin compounds PPB and PPC with a mass concentration of 0.04 mg / mL in a volume ratio of 1:1.

[0020] The multi-channel fluorescence array sensor can be used in glycosaminoglycan identification and heparin quality control. The specific method of use is as follows:

[0021] 1) Take 100 μL of the sensor unit solution in a 96-well plate, add 100 μL of the test solution, use 10 mM PBS buffered saline as a blank, shake, scan the 96-well plate with an ELISA reader, repeat the test 7 times for each group of glycosaminoglycan test sample solutions, and record the fluorescence intensity data; glycosaminoglycans can produce electrostatic and π-π stacking effects with PPB and PPC. The FRET effect formed by the conjugated polymer nanoparticles and porphyrin compounds is destroyed, resulting in changes in the fluorescence values ​​of the multiple channels, and the generated signal response represents the fingerprint signals of different glycosaminoglycans;

[0022] 2) The fluorescence data were processed and analyzed using the statistical analysis software SYSTAT (version 13.0), and the fluorescence response pattern was converted into a canonical pattern using linear discriminant analysis (LDA), and the data matrix was classified to obtain a two-dimensional array fingerprint of glycosaminoglycans for visual identification;

[0023] 3) The obtained two-dimensional array distinguishing fingerprint map is used as a model to detect unknown samples, and the accuracy of the prediction of unknown samples is calculated to achieve the distinction and detection of glycosaminoglycans.

[0024] Furthermore, the glycosaminoglycan analog test sample solution includes sample solutions of hyaluronic acid (HA), chondroitin sulfate sodium salt (LSA), chondroitin sulfate C sodium salt (LSC), dermatan sulfate (DS), chondroitin sulfate (CS), persulfated chondroitin sulfate (OSCS), and small molecular weight heparin (LMWH).

[0025] Furthermore, the final concentration of the measured heparin and other glycosaminoglycan analogs sample solutions was 25 μg / mL.

[0026] Beneficial effects:

[0027] 1. The present application utilizes carboxyl-functionalized conjugated polymer nanoparticles and two porphyrin compounds (PPB and PPC) substituted with different side chains to bind through electrostatic interaction. The two have a FRET effect, which can further amplify the fluorescence response signal. After adding the glycosaminoglycan solution, the fluorescence spectrum undergoes obvious signal changes. Through double emission peaks and multi-signal channel output, and then with the help of linear discriminant analysis for data processing, it can realize the recognition and differentiation of small molecular heparin and structural analogs such as dermatan sulfate and oversulfated chondroitin sulfate, and thus realize the detection and differentiation of a variety of glycosaminoglycan analogs;

[0028] 2. The multi-channel sensor array constructed in this application obtains multiple detection signals through a single measurement, which not only effectively reduces the number of sensor elements, but also eliminates the negative effects caused by factors such as sensor concentration and instrument parameter settings, and reduces background noise, compared with the sensor unit that relies on the signal intensity change of a single wavelength to identify glycosaminoglycans, which is of great significance to improving the practical application ability of the sensor;

[0029] 3. The present application provides a multi-channel fluorescence array sensor based on the FRET effect, which is simple to construct, easy to operate, does not require the assistance of professional technicians, is easy to apply in practice, can correctly identify and distinguish a variety of glycosaminoglycans, and has the potential to be applied to the quality control of small molecule heparin;

[0030] 4. The preparation method of the array sensor constructed in this application is simple, low-cost, highly sensitive, and has a short detection time. It can accurately distinguish a variety of glycosaminoglycans and assist in the quality control of small molecule heparin. The accuracy rate for unknown samples is nearly 100%, and the reproducibility is good. It can be used for the detection of actual unknown samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 is the mass spectrum of PPB prepared in Example 1;

[0032] Figure 2 is the H NMR spectrum of PPB prepared in Example 1;

[0033] Figure 3 is the mass spectrum of PPC prepared in Example 1;

[0034] Figure 4 is the H NMR spectrum of PPC prepared in Example 1;

[0035] Figure 5 is the H NMR spectrum of compound 6 prepared in Example 1;

[0036] Figure 6 is the H NMR spectrum of compound 8 prepared in Example 1;

[0037] Figure 7 The fluorescence intensity changes of the interaction between different concentrations of PPB and conjugated polymer nanoparticles;

[0038] Figure 8 is the change in fluorescence intensity of the interaction between different concentrations of PPC and conjugated polymer nanoparticles;

[0039] Fig. 9 It is the fluorescence response signal of six groups of signal channels output by the two constructed sensor units to the seven glycosaminoglycan analogs;

[0040] Fig.10 It is a visualization diagram (LDA) of the rapid recognition of glycosaminoglycans by a multi-channel fluorescence array sensor. DETAILED DESCRIPTION

[0041] The preferred embodiments of the present invention are described in detail below so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more definite definition of the protection scope of the present invention.

[0042] Embodiment 1

[0043] 1) Synthesis of the porphyrin compound 4,4,4,4-porphyrin-5,10,15,20-tetrayltetrakis(1-(4-borylbenzyl)pyridin-1-ium (PPB)

[0044] The specific synthetic route is as follows

[0045]

[0046] 4-Bromomethylphenylboronic acid (compound 2, 105 mg, 0.4849 mmol) was dissolved in N,N-dimethylformamide (3 mL), and 5,10,15,20-tetrakis(pyridin-4-yl)porphyrin (compound 1, 50 mg, 0.0808 mmol) was added, and the reaction was stirred at 65°C for 48 h. After the reaction, the mixture was transferred to a 50 mL centrifuge tube, acetone was added to the mixture, a solid was precipitated, and the supernatant was removed by centrifugation. The solid was washed three times, and the solvent was removed under reduced pressure to obtain a brown solid (89 mg, 95%).

[0047] 1H NMR (300MHz, DMSO-d6) δ9.67(d,J=6.0Hz,8H),9.23(s,8H),9.05(d,J=5.0Hz,8H),8.29(s, 8H),8.03(d,J=7.5Hz,8H),7.86(d,J=7.7Hz,8H),6.24(s,8H),2.09(s,2H),-3.07(s,2H).

[0048] 2) Synthesis of the porphyrin compound 4,4,4,4-porphyrin-5,10,15,20-tetrayltetrakis(1-methylpyridin-1-ium) (PPC)

[0049] The specific synthetic route is as follows

[0050]

[0051] 5,10,15,20-tetrapyridin-4-ylporphyrin (compound 1, 200 mg, 0.3232 mmol) was dissolved in N,N-dimethylformamide (25 mL), iodomethane (compound 3, 500 μL) was added thereto, and the mixture was stirred overnight at 90°C. After the reaction was completed, the mixture was transferred to a 50 mL centrifuge tube, and ether was added to the mixture to precipitate a solid. The supernatant was removed by centrifugation, and the solid was washed with ether three times. The solvent was removed under reduced pressure to obtain a brown solid (364 mg, 94.91%).

[0052] 1 H NMR (400MHz, DMSO-d6) δ9.50(d,J=6.3Hz,8H),9.22(s,8H),9.01(d,J=6.7Hz,8H),4.74(s,12H),-3.09(s,2H).

[0053] 3) Synthesis of polyfluorene

[0054] The specific synthetic route and synthetic steps are as follows

[0055]

[0056] Weigh a certain amount of compound 4 (4.985 g, 8.9 mmol), compound 5 (5.4172 g, 0.01948 mmol), Pd(ddpf)Cl2 (600 mg, 0.712 mmol) and potassium acetate (3.9250 g, 0.04 mol) in a 250 mL reaction tube; evacuate, replace with argon three times, add ultra-dry 1,4-dioxane, stir, evacuate the system, and replace with argon three times. Stir and react at 90 ° C for 16 hours. After the reaction is completed, add 150 mL of deionized water to the reaction system, extract with dichloromethane three times, remove the solvent under reduced pressure, and wash the crude product with saturated sodium chloride three times. Dry with anhydrous sodium sulfate, filter, collect the liquid, remove the solvent under reduced pressure, and dry in vacuum to obtain product 6.

[0057] 1 H NMR (400MHz, Chloroform-d) δ7.88–7.61(m,6H),2.10–1.92(m,4H),1.46–0.93(m,44H),0.81(t,J=7.1Hz,6H),0.62–0.46(m,4H).

[0058]

[0059] Compound 7 (2 g, 6.8 mmol) was dissolved in ethanol, stirred for 5 min under N2 protection, the reaction system was cooled to 0°C, NaBH4 (2.57 g, 68 mmol) was slowly added, and stirred overnight at room temperature. After the reaction was completed, the reaction solution was cooled to 0°C, 20 mL of deionized water was added, stirred for 10 min, the solvent was removed under reduced pressure, extracted with ether three times, the ether was removed under reduced pressure, washed with saturated sodium chloride, dried over anhydrous Na2SO4, filtered, the filtrate was collected, and the solvent was removed under reduced pressure. Impurities were removed by column chromatography using PE:EA=10:1 to obtain solid 8.

[0060] 1 H NMR(300MHz,Chloroform-d)δ9.01(s,2H),8.04(s,2H).

[0061] Compound 8 (1.2 mg, 4.5 mmol) was dissolved in anhydrous ethanol, and glyoxal (900 μL, 6.3 mmol) and triethylamine (1.138 g, 1.125 mmol) were added. Under N2 protection, the reaction was allowed to react at room temperature overnight. The reaction was stopped and the reaction solution was filtered to obtain a white solid, which was recrystallized from ethanol to obtain needle-shaped crystals 9.

[0062]

[0063] Monomer 6 (100 mg, 0.155 mmol) and monomer 9 (45 mg, 0.155 mmol) were dissolved in toluene / potassium carbonate (8 mL / 1.5 mL), Pd(PPh3)2Cl2 (4 mg) was added, the reaction flask was replaced with argon three times, and the reaction was stirred at 100 ° C for 48 hours. The reaction was stopped, and the reaction solution was cooled to room temperature and transferred to 120 mL of methanol. Filtered to obtain a yellow-green solid, which was washed with methanol three times. The product was dissolved in dichloromethane, washed with saturated sodium chloride solution, and dried over anhydrous sodium sulfate. Filtered and the liquid was collected. The solvent was removed under reduced pressure. After the chloroform was dissolved, the chloroform solution was slowly dripped into 150 mL of methanol to precipitate. The precipitate was filtered and vacuum dried to obtain a green solid polymer 10.

[0064] 4) Preparation of polymer nanoparticles

[0065] Weigh 5 mg of the polyfluorene prepared in step 3) (i.e., solid polymer 12), add 5 mL of anhydrous tetrahydrofuran to dissolve, and prepare a polymer solution with a mass concentration of 1 mg / mL;

[0066] Weigh 5 mg of poly(styrene-maleic anhydride) copolymer, add 5 mL of anhydrous tetrahydrofuran to dissolve, and prepare a PMSA stock solution with a mass concentration of 1 mg / mL;

[0067] Take 250 μL of 1 mg / mL polymer solution and 100 μL of PSMA stock solution, add them to 5 mL of anhydrous THF, dissolve them by ultrasonication, and mix them evenly. Under ultrasonic conditions, quickly inject the mixed solution into 10 mL of deionized water, remove THF in the solution under reduced pressure at 80°C, and make the remaining solution constant to 5 mL. Filter through a 0.22 μm filter to obtain conjugated polymer nanoparticles (Pdots).

[0068] Conjugated polymers (CPs) have a large delocalized π-electron backbone, thus possessing strong optoelectronic advantages. In addition, CPs can effectively coordinate electronic coupling, which can enhance light harvesting and light amplification performance through energy transfer mechanisms. Pdots, which combine the properties of conjugated polymers and nanoparticles, are a novel class of fluorescent materials with advantages such as high brightness, excellent photostability, low cytotoxicity, and large absorption cross-sections.

[0069] 5) Fluorescence quenching titration experiment of polymer nanoparticles and porphyrin compounds

[0070] The prepared polymer nanoparticle mother solution was diluted to 0.005 mg / mL with PBS, and the porphyrin compound was then diluted to different concentration gradients with PBS.

[0071] 100 μL of 0.005 mg / mL conjugated polymer nanoparticle solution and 100 μL of different concentrations of porphyrin compounds were placed in a 96-well plate (the final concentrations of porphyrin compounds were 0.005 mg / mL, 0.01 mg / mL, 0.02 mg / mL, 0.025 mg / mL, 0.03 mg / mL, 0.04 mg / mL, 0.05 mg / mL, 0.1 mg / mL, and 0.2 mg / mL, respectively). The Pdots were tested using an ELISA reader. The excitation wavelength of the Pdots was 410 nm. The fluorescence intensity of the Pdots changed after interacting with different concentrations of PPB and PPC (see Figure 7 and Figure 8 ).

[0072] Porphyrin compounds have a strong fluorescence quenching effect on conjugated polymer nanoparticles. As the concentration of porphyrin compounds increases, the fluorescence intensity of conjugated polymer nanoparticles decreases significantly, while the fluorescence intensity of porphyrin compounds increases. There is an obvious fluorescence resonance energy transfer (FRET) effect between the two.

[0073] Observation of the fluorescence titration curve shows that different concentrations of PPB and PPC have different quenching abilities for conjugated polymer nanoparticles Pdots, and the efficiency of the FRET effect is also different. The electrostatic complexes when PPB quenches the fluorescence intensity of conjugated polymer nanoparticles to 60% and PPC quenches the fluorescence intensity of conjugated polymer nanoparticles to 50% are selected to construct the sensing unit of the multi-channel array sensor.

[0074] The specific construction method is: dilute the 0.05 mg / mL conjugated polymer nanoparticle mother solution with 10 mM PBS (pH = 7.4) buffer to 0.01 mg / mL, dissolve PPB and PPC in PBS to prepare 1 mg / mL stock solutions for standby use. Dilute the stock solutions of the two porphyrin compounds with PBS to 0.04 mg / mL. Mix the conjugated polymer nanoparticle dilutions with the dilutions of the two porphyrin compounds in a volume ratio of 1:1, shake well, and obtain two sensing units of Pdots+PPB and Pdots+PPC. The final concentration of PPB is 0.01 mg / mL, and the final concentration of PPC is also 0.01 mg / mL.

[0075] The two sensing units generate six signal channels. The Pdots+PPB sensing unit generates three signal channels, Channel 1, Channel 2 and Channel 3. The excitation wavelength of the three channels is 410nm, and the emission wavelengths are 490nm, 680nm and 725nm respectively. The Pdots+PPC sensing unit generates three signal channels, Channel 4, Channel 5 and Channel 6. The excitation wavelength of the three channels is 410nm, and the emission wavelengths are 490nm, 680nm and 720nm respectively. That is to say, a total of six signal channels can be generated under the same excitation wavelength.

[0076] 6) Differentiate between different types of glycosaminoglycans

[0077] Take 100 μL of the two sensor units Pdots+PPB and Pdots+PPC in a 96-well plate, add 100 μL of different types of glycosaminoglycan stock solution, and the final concentration of glycosaminoglycan is 25 μg / mL. Shake for 10 seconds and test the fluorescence data with an enzyme-labeled instrument. For the Pdots+PPB system, the multi-channel fluorescence intensity output by the reaction of the electrostatic complex and glycosaminoglycan can be measured at emission wavelengths of 490nm, 680nm and 725nm respectively; for the Pdots+PPC system, the multi-channel fluorescence intensity output by the reaction of the electrostatic complex and glycosaminoglycan can be measured at emission wavelengths of 490nm, 680nm and 720nm respectively; using the relative fluorescence intensity change as the detection signal (I-I0 / I0), each glycosaminoglycan and each sensor unit were tested 7 times, with a total of 7 glycosaminoglycans, 2 sensor units, and 6 signal channels, 7×7×6=294 fluorescence signals can be obtained, and then these 294 fluorescence signals are used to form a data matrix as shown below

[0078]

[0079]

[0080] The two sensor units have different fluorescence responses to different glycosaminoglycans (see Fig. 9 ).

[0081] 7) Analysis and processing of fluorescence array sensor data

[0082] The fluorescence data were processed and analyzed using statistical analysis software. The linear discriminant analysis (LDA) statistical method was used to convert the fluorescence response pattern into a canonical pattern. The Mahalanobis distance from each single pattern to the centroid of each group in the multidimensional space was calculated, and the assignment of all species was based on the shortest Mahalanobis distance. LDA generated multiple factors representing the linear combination of the data matrix. Since the cumulative percentage of the first two factors was high, the 2D spectrum was drawn using the first two factors (see Fig.10 ), it can be seen from the LDA graph that even though the seven glycosaminoglycans have high structural similarity and low concentrations, they can be clearly divided into seven different groups, and there is no obvious intersection and overlap between all the groups, indicating that the cross-array sensor we designed is very effective in distinguishing different types of glycosaminoglycans.

[0083] The accuracy verification results of the jackknife discriminant classification matrix are as follows

[0084]

[0085]

[0086] It can be seen from the verification data that the sensor unit constructed in the embodiment has a 100% accuracy rate in distinguishing seven types of glycosaminoglycans.

[0087] 8) Detect the ability of array sensors to distinguish unknown samples

[0088] Seven glycosaminoglycans were blindly tested in random order, with a total of 35 unknown samples. The test was performed according to the above steps, and the relative fluorescence intensity changes of the unknown samples were recorded as follows:

[0089]

[0090]

[0091] Linear discriminant analysis was used to verify the model's ability to test unknown samples and distinguish the types of glycosaminoglycans. 35 unknown samples were tested and 35 samples were correctly detected, with an accuracy rate of 100%.

[0092] This application uses a nano-coprecipitation method to prepare carboxyl-functionalized Pdots based on the hydrophobic interaction between conjugated polymers and poly(styrene-comaleic anhydride) (PSMA). A multi-channel fluorescence array sensor is constructed using the fluorescence resonance energy transfer effect between Pdots and porphyrin compounds, which is applied to the detection and identification of glycosaminoglycans and the quality control of heparin.

[0093] Negatively charged polymer nanoparticles and positively charged porphyrin molecules can bind to each other through electrostatic interactions, shortening the distance between the molecules. At the same time, since the emission spectrum of polymer nanoparticles overlaps with the absorption spectrum of porphyrin molecules, there is a FRET effect between the two, which is beneficial to the amplification of detection signals and the reduction of signal noise in sensor applications.

[0094] Common glycosaminoglycans with similar structures including hyaluronic acid, chondroitin sulfate sodium salt, chondroitin sulfate C sodium salt, dermatan sulfate, chondroitin sulfate, chondroitin persulfate, and small molecule heparin were selected as analytes, and different types of glycosaminoglycans were added to the fluorescence array sensor, respectively. The final concentration of each glycosaminoglycan was 25 μg / mL, and 7 groups of repeated experiments were tested for each glycosaminoglycan and sensor unit. The fluorescence intensity of the multi-channel output was tested by an ELISA instrument, and PBS solution was used as a blank control to obtain a data model of 7 groups of glycosaminoglycans × 2 groups of sensor units × 7 repetitions.

[0095] The training data set was analyzed and processed using SYSTAT13.0 software through a linear discriminant analysis algorithm. The fluorescence array sensor had 100% correct discrimination ability for the seven glycosaminoglycans and 100% correct prediction accuracy for unknown samples.

[0096] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A multi-channel fluorescence array sensor, characterized in that: It is composed of negatively charged conjugated polymer nanoparticles Pdots and positively charged porphyrin compounds PPB and PPC with different side chain substitutions through electrostatic interaction. The conjugated polymer nanoparticles are carboxyl functionalized polymer nanoparticles prepared by nano-coprecipitation method based on the hydrophobic interaction between fluorene polymer and PSMA. The structural formula of fluorene polymer is as follows: Wherein, n is a natural number between 8 and 200; The structural formulas of two porphyrin compounds with different side chain substitutions are as follows: The multi-channel fluorescence array sensor includes two sensing units and six groups of signal channels. The two sensing units are a Pdots+PPB system and a Pdots+PPC system. The excitation wavelengths of the two systems are both 410 nm. The emission wavelengths of the Pdots+PPB system are 490 nm, 680 nm and 725 nm; the emission wavelengths of the Pdots+PPC system are 490 nm, 680 nm and 720 nm.

2. The method for constructing a multi-channel fluorescence array sensor according to claim 1, characterized in that: The corresponding process is as follows: 1) 5,10,15,20-tetrapyridin-4-ylporphyrin is used as a raw material to undergo electrophilic substitution reaction with two electrophilic reagents, 4-bromomethylphenylboronic acid and iodomethane, to generate pyridinium quaternary ammonium salts PPB and PPC; 2) using 2,2'-(9-nonyl-9-octyl-9H-fluorene-2,7-diyl)bis(4,4,5,5-tetramethyl-1,3,2-dioxaborane) as a raw material, coupling with 5,8-dibromoquinoxaline to obtain a quinoxaline-substituted fluorene polymer; 3) Carboxyl-functionalized conjugated polymer nanoparticles Pdots were prepared by coprecipitation of quinoxaline-substituted fluorene polymers and amphiphilic PMSA; 4) The conjugated polymer nanoparticle mother solution was diluted with PBS buffer, and different concentrations of positively charged porphyrin compounds PPB and PPC with different side chain substitutions were added respectively to obtain titration curves of the conjugated polymer nanoparticles and the porphyrin compounds. The electrostatic complex when PPB quenched the fluorescence intensity of the conjugated polymer nanoparticles to 60% and the electrostatic complex when PPC quenched the fluorescence intensity of the conjugated polymer nanoparticles to 50% were selected to construct the sensing units of the array sensor, namely, the Pdots+PPB system and the Pdots+PPC system.

3. The method for constructing a multi-channel fluorescence array sensor as claimed in claim 2, characterized in that: The Pdots+PPB and Pdots+PPC systems were obtained by mixing a PBS solution of conjugated polymer nanoparticles with a mass concentration of 0.01 mg / mL and a PBS solution of porphyrin compounds PPB and PPC with a mass concentration of 0.04 mg / mL in a volume ratio of 1:

1.

4. The method for using the multi-channel fluorescence array sensor according to claim 1, characterized in that: The multi-channel fluorescence array sensor can be used in glycosaminoglycan recognition and heparin quality control. The process is as follows: 1) Take 100 μL of the two sensor units Pdots+PPB and Pdots+PPC respectively in a 96-well plate, add 100 μL of the sample solution to be tested, use 10 mM PBS buffer solution as a blank, shake, scan the 96-well plate with an ELISA reader, repeat the test 7 times for each group of glycosaminoglycan sample solution to be tested, and record the fluorescence intensity data; 2) Use statistical analysis software to process and analyze fluorescence data, use linear discriminant analysis to convert fluorescence response patterns into canonical patterns, classify data matrices, and obtain two-dimensional array fingerprints of glycosaminoglycans to achieve visual identification; 3) The obtained two-dimensional array distinguishing fingerprint map is used as a model to detect unknown samples, and the accuracy of the prediction of unknown samples is calculated to achieve the distinction and detection of glycosaminoglycans.

5. The method for using the multi-channel fluorescence array sensor as claimed in claim 4, characterized in that: The glycosaminoglycan sample solution to be tested includes sample solutions of hyaluronic acid, chondroitin sulfate sodium salt, chondroitin sulfate C sodium salt, dermatan sulfate, chondroitin sulfate, persulfated chondroitin and small molecule heparin.

6. The method for using the multi-channel fluorescence array sensor as claimed in claim 5, characterized in that: The final concentration of the measured heparin and other glycosaminoglycan analogs sample solutions was 25 μg / mL.

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