CN112143178B

CN112143178B - Electrostatic composite fluorescent array sensor and application

Info

Publication number: CN112143178B
Application number: CN202011019700.7A
Authority: CN
Inventors: 韩进松; 王浩; 李飞; 马宗辉; 黄慧
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2020-09-24
Filing date: 2020-09-24
Publication date: 2022-06-28
Anticipated expiration: 2040-09-24
Also published as: CN112143178A

Abstract

The invention discloses an electrostatic compound fluorescence array sensor and application thereof. Is compounded by the interaction of water-soluble fluorescent conjugated polymer (PPE) with different side chain substitutions and graphene oxide. The method is used for detecting various proteins and has the characteristics of high accuracy and sensitivity, short detection time, low cost, high repeatability and the like.

Description

Electrostatic composite fluorescent array sensor and application

Technical Field

The invention relates to a fluorescence detection sensor and application, in particular to an electrostatic compound fluorescence array sensor and application.

Background

Proteins play an important role in organisms. The detection of proteins is crucial for clinical diagnosis and the analysis of specific proteins can provide valuable information for identifying specific physiological and pathological states. On the one hand, excessive abnormal proteins in vivo may cause some diseases, such as Alzheimer's disease, caused by amyloid aggregation in brain tissue. On the other hand, the occurrence and development of certain diseases (such as cancer, prostatitis, hypoproteinemia and the like) are often accompanied by the over-expression of various proteins, and the proteins can be used as biomarkers for clinical disease diagnosis. Due to the structural diversity of proteins and the complexity of clinical sample systems, accurate detection of proteins remains a very challenging task.

Traditional protein detection methods, such as enzyme-linked immunosorbent assay (ELISA), Polymerase Chain Reaction (PCR), polyacrylamide gel electrophoresis (PAGE), electrochemistry, fluorescence, etc., all have high sensitivity. However, these methods are expensive, risk of sample contamination, and time consuming to operate. These methods employ a "lock-and-key" binding sensing mode, requiring highly selective receptors for each analyte, which limits their widespread use in the detection and identification of various proteins.

The above-described deficiencies and inadequacies of conventional protein detection methods are complemented and remedied by methods for detecting protein sequences based on mimicking the olfactory system of a mammal. Compared with the traditional detection system based on the 'lock-key' combination mode, the detection aim can be achieved by low selective combination between the sensor unit and the analyte, and the complexity of the design of the sensor unit is avoided. Fluorescent array sensors differentiate between different analytes by binding multiple receptors to different analytes to generate multiple sets of detection signals. In recent years, array sensor technology has developed rapidly, and in particular, has played a unique advantage in complex samples. The method has been applied in a number of detection fields, such as the detection of bacterial microorganisms, volatile gases, food products, etc. Many array sensors are currently disclosed for the detection of proteins, including colorimetric and fluorescent array sensors. Wang et al (Wang et al. Langmuir 2019, 35, 5599-. Sun et al (Sun et al. Sensors & actors: B.chemical 2019, 296, 126677) designed a double-nano plasma colorimetric sensor array for protein identification, with glutathione as a regulatory factor, and with carbon nanodots (CDs) to induce reversible aggregation of gold nanoparticles (AuNPs) to construct a fluorescence array sensing unit, thereby realizing differentiation of multiple proteins.

The array sensor reported in the literature usually needs a plurality of sensing elements, and has the disadvantages of complicated operation, high production cost, narrow range of protein detection, low sensitivity and poor reproducibility. Therefore, there is still a need in the art to develop a fluorescence array sensor with simple operation, high sensitivity and wide detection range for detecting proteins.

Disclosure of Invention

The invention aims to: the invention aims to provide the electrostatic compound fluorescent array sensor which has the characteristics of capability of detecting various protein types, high accuracy and sensitivity, quick detection time and the like.

The invention also aims to provide application of the electrostatic composite fluorescence array sensor.

The technical scheme is as follows: the invention provides an electrostatic compound fluorescence array sensor, which is compounded by the interaction of water-soluble fluorescence conjugated polymer (PPE) with different side chain substitutions and graphene oxide.

Further, the water-soluble fluorescent conjugated polymer (PPE) with different side chain substitutions has the following structural general formula:

wherein:

R₁each independently selected from: hydrogen, substituted or unsubstituted C1-10 straight or branched chain alkyl and cycloalkyl, C1-10 alkenyl, C1-10 alkynyl, C1-10 alkoxy, aromatic ring, saturated or aromatic heterocycle, hydroxyl, carboxyl, ester;

R₂Each independently selected from: hydrogen, substituted or unsubstituted C1-10 linear or branched alkyl and cycloalkyl, C1-10 alkenyl, C1-10 alkoxy, aromatic ring, saturated heterocycle or aromatic heterocycle, halogen, nitrile group, nitro, hydroxyl, carboxyl, ester;

n is an integer of 8 to 200.

Further, said R₁The groups are:

n is an integer of 8 to 200; m is an integer of 1 to 10.

Further, said R₂The groups are:

n is an integer of 8 to 200; m is an integer of 1 to 10.

Further, the structural formula of the electrostatic compound fluorescence array sensor is shown as formula I, formula II and formula III:

wherein n is an integer from 8 to 200; m is an integer of 1 to 10.

Further, the structural formula of the fluorescent conjugated polymer of the three sensing units in the fluorescent sensor is shown as PPE 1, PPE 2 and PPE 3:

further, the preparation method of the water-soluble fluorescent conjugated polymer (PPE) with different side chain substitutions comprises the following steps: the diiodobenzene containing different substituents is used as a raw material, and is coupled with 1, 4-diacetylene benzene compounds to generate quaternary ammonium salt with trimethylamine, so that a target product is obtained.

Further, the water-soluble fluorescent conjugated polymer (PPE) with different side chain substitutions is diluted by PBS buffer solution, different amounts of negatively charged graphene oxide are respectively added and uniformly mixed to obtain the fluorescence titration curves of the graphene oxide and the fluorescent polymer, and the electrostatic compound is used for constructing the sensing unit of the array sensor when the graphene oxide quenches the fluorescence intensity of the polymer to thirty percent.

The electrostatic compound fluorescence array sensor is applied to protein detection.

Furthermore, different sensing units of the electrostatic composite fluorescence array sensor are mixed with protein respectively to determine the fluorescence intensity of each sensing unit.

Further, the fluorescence data were processed and analyzed using statistical analysis software SYSTAT (version 13.0). Through Linear Discriminant Analysis (LDA), the data matrix is classified, data are visualized, and the distinguishing and detection of various proteins are realized.

As a preferable scheme:

the construction method of the fluorescence array sensor comprises the following steps: chemically synthesized water-soluble fluorescent polymers (PPE 1, PPE 2 and PPE 3) with different substituents are diluted to a final concentration of 2 mu M by 0.1mM PBS (pH 7.0), different concentrations of negatively charged graphene oxide (the final concentrations of graphene oxide are 0.0002, 0.0004, 0.0006, 0.0008, 0.001, 0.0012, 0.0014, 0.0016, 0.0018, 0.002, 0.0025, 0.003, 0.004 and 0.005g/L in sequence) are added to obtain fluorescence titration curves of the graphene oxide and the fluorescent polymers, and the electrostatic compound is selected to be used for constructing a sensing unit of the array sensor, wherein the ratio of the graphene oxide to quench the fluorescence intensity of the polymer to thirty percent.

The methods for distinguishing different kinds of proteins as described above are: the polymer-graphene oxide complex array sensor unit obtained by the above construction was diluted with 0.1mM PBS (pH 7.0) buffer solution to a final concentration of 2 μ M, and a novel fluorescence array sensor was prepared. Adding 100 mu L of different protein (the final concentration of the protein is 5 mu M) including Human Serum Albumin (HSA), Bovine Serum Albumin (BSA), pepsin (Pep), trypsin (Try), lipase (Lip), papain (Pap), cytochrome C (Cyt), lysozyme (Lys), myoglobin (Myo), canavalin (Con), fibrinogen (Fib), beta-lactoglobulin (beta-lac) and other samples, shaking for 10 seconds, repeatedly testing each protein group for six times, and recording fluorescence intensity data.

And processing the fluorescence data by using various algorithms such as linear discriminant analysis and the like, classifying the data matrix to obtain a two-dimensional or three-dimensional array distinguishing fingerprint of the protein, and realizing visual identification. And detecting unknown samples by using the obtained two-dimensional or three-dimensional array distinguishing fingerprint as a model, and calculating the prediction accuracy of the unknown samples to realize the distinguishing and detection of the proteins.

Has the beneficial effects that: the invention can simultaneously distinguish and detect dozens of even hundreds of protein types, has high accuracy and sensitivity, low cost and high repeatability, and the detection time is less than one minute without professional technicians.

Drawings

FIG. 1 shows the change of the interaction fluorescence intensity of graphene oxide with different concentrations and three water-soluble fluorescent polymers;

FIG. 2 is a non-linear fitting of the fluorescence intensity changes of graphene oxide with different concentrations and three water-soluble fluorescent polymers;

FIG. 3 shows the fluorescence response signals of 12 proteins to three constructed sensing units respectively;

fig. 4 is a visualization (LDA) of the rapid recognition of proteins by a fluorescent array sensor.

Detailed Description

Example 1 Synthesis of fluorescent high molecular Polymer PPE1

The fluorescent high molecular polymer used in the following examples was prepared according to the synthesis scheme shown in the following figure by the following steps:

monomer 1(1.04g, 2.35mmol) and monomer 2(2.09g, 2.35mmol) were dissolved in toluene/DIEA (7.2mL/4.9mL), and Pd (PPh) was added₃)₂Cl₂(4.95mg, 7.05. mu. mol) and CuI (2.69mg, 14.10. mu. mol) were stirred at room temperature for 24 h. Adding saturated NH to the mixture₄Cl solution and CHCl₃The aqueous layer was separated and washed with CHCl₃And (4) extracting. Anhydrous MgSO (MgSO)₄Drying, filtering and removing the solvent under reduced pressure. The crude product was dissolved in a small amount of CHCl₃In (1), an excess of n-hexane was slowly added for precipitation, and the precipitation process was repeated three times to obtain compound 3 as a yellow-brown solid (2.63g, 90%). Molecular weight M _nThe test was about 1.4X10⁴The PDI was 3.9.¹H NMR(300MHz，CDCl₃) δ ═ 7.12-7.24(m, 2H), 6.94-7.10(m, 2H), 4.40-4.64(m, 2H), 4.02-4.34(m, 4H), 3.48-3.87(m, 60H), 3.30-3.40(m, 12H), 2.25-2.47(m, 4H). Infrared IR (cm)^-1)：v 2912，2870，1508，1489，1469，1420，1389，1351，1271，1200，1096，1026，943，849，719，650。

Compound 3(100mg, 0.083mmol, calculated on repeating monomer structure) is dissolved in degassed THF/EtOH (10mL/5mL) and N (CH) is added slowly₃)₃(2mL) in N₂Stir at ambient room temperature for 2 days. Then adding N (CH)₃)₃(2mL) was stirred for 6 days. The solvent was removed under reduced pressure, redissolved in distilled water and dialyzed against deionized water for 7 days. Polymer 4 after freeze-drying was a yellow solid (90mg, 83%) which was the polymer shown in PPE 1. Hydrogen spectrum¹H NMR (300MHz, MeOD) δ 7.21-7.45(m, 4H), 4.54-4.65(m, 2H), 4.16-4.38(m, 4H), 3.47-3.89(m, 60H), 3.32-3.37(m, 12H), 3.12-3.25(m, 18H), 2.31-2.46(m, 4H). Infrared IR (cm)^-1)：v 3421，2871，2359，1649，1600，1508，1489，1419，1350，1272，1200，1091，1049，944，849。

Example 2 Synthesis of fluorescent Polymer PPE 2

polymer 4(25mg, 0.082mmol) was dissolved in N, N-dimethylethylenediamine (25mL) and stirred at 50 ℃ for 24 h. The solvent was removed under reduced pressure and the crude product was washed with n-hexane. Drying in vacuo gave an orange solid as polymer 5(52mg, 0.077mmol, 94%). ¹H NMR(300MHz，CDCl3)：δ7.14(s，2H)，4.52(s，4H)，3.31(s，4H)，2.81(s，4H)，2.17(s，12H).¹³C NMR(300MHz，CDCl3)：δ168.97，157.57，118.45，113.49，92.94，90.67，68.57，58.10，44.97，36.44.

Polymer 5(40mg, 0.1mmol) was dissolved in dichloromethane (25mL) and iodomethane (15mL) was added. Stir at room temperature overnight. The solvent was removed under reduced pressure, washed with hexanes, and dried in vacuo to afford PPE2(63mg, 0.094mmol, 94%) as a dark orange solid.¹H NMR(300MHz，D₂O)：δ7.09(s，2H)，4.50(s，4H)，3.74(s，4H)，3.52(s，4H)，3.27(s，18H).¹³C NMR(300MHz，D₂O)：δ167.51，156.13，118.10，113.54，93.03，90.86，66.21，65.33，54.16，36.38.

Example 3 Synthesis of fluorescent Polymer PPE 3

The fluorescent high molecular polymer used in the following examples was prepared according to the synthetic scheme shown in the following figure by the following steps:

monomer 6(820mg, 1.00mmol) and monomer 7(347mg, 1.05mmol) were dissolved in dichloromethane/DIEA/triethylamine (2mL/2mL/1mL) and Pd (PPh) was added₃)₂Cl₂(1.4mg, 2. mu. mol) and CuI (0.4mg, 2. mu. mol) were stirred at ambient temperature for 24 h. Polymer 8(70mg, 0.082mmol) was dissolved in N, N-dimethylethylenediamine (25mL) and stirred at 50 ℃ for 24 h. The solvent was removed under reduced pressure and the crude product was washed with n-hexane. Drying in vacuo afforded polymer 9 as an orange solid (89.4mg, 0.077mmol, 94%). Polymer 9(40mg, 0.0344mmol) was dissolved in dichloromethane (25mL) and iodomethane (15mL) was added. Stir at room temperature overnight. The solvent was removed under reduced pressure, washed with hexane and dried under vacuum to give PPE 3 as a dark orange solid (116mg, 0.094mmol, 94%).

Example 4 fluorescence quenching titration experiment of Polymer and graphene oxide

The synthesized polymer was used to prepare a stock solution having a concentration of 0.1 mM. 48. mu.L of each of PPE1, PPE2 and PPE3 was diluted to 12mL (4. mu.M) for use, and graphene oxide solutions of different concentrations were prepared. 500 mu L of standby polymer solution is added into a fluorescence cuvette, and then 500 mu L of graphene oxide with different concentrations are respectively added. The final concentration of the fluorescent polymer PPE1-PPE3 was 2. mu.M, and the final concentrations of graphene oxide were 0.0002, 0.0004, 0.0006, 0.0008, 0.001, 0.0012, 0.0014, 0.0016, 0.0018, 0.002, 0.0025, 0.003, 0.004, and 0.005g/L, respectively. And (3) testing by using a fluorescence spectrophotometer, wherein the excitation wavelength of the three macromolecules is 410nm, the emission wavelengths are 465nm, 460nm and 460nm respectively, and recording fluorescence data to obtain a fluorescence spectrum (shown in figure 1). The graphene oxide has a strong fluorescence quenching effect on fluorescent polymers, and the fluorescence intensity is remarkably reduced along with the increase of the concentration of the graphene oxide. According to the fluorescence titration curve shown in the attached figure 2, the quenching abilities of graphene oxide to fluorescent polymers PPE 1-3 are different, and the concentration and the proportion of the graphene oxide and the fluorescent polymers are selected to construct a sensor unit when the fluorescence intensity of the PPE1-PPE3 is quenched to 30%. Wherein when the PPE1 is quenched to 30%, the concentration of the graphene oxide is 0.001 g/L; when the PPE2 was quenched to 30%, the concentration of graphene oxide was 0.0008 g/L; when the PPE3 is quenched to 30%, the concentration of the graphene oxide is 0.0025 g/L;

Example 5 architecture of fluorescent array sensor

mu.L of each stock solution of PPE1, PPE2 and PPE3 was transferred, diluted to 5mL with PBS buffer at a concentration of 8. mu.M and shaken well. 200. mu.L of graphene oxide stock solution (1mg/mL) was diluted to 10mL, and 1000. mu.L, 800. mu.L and 2500. mu.L were respectively diluted to 5 mL. Mixing the high molecular polymer PPE 1-PPE 3 diluent with the graphene oxide diluent with the corresponding concentration in a ratio of 1: 1, and shaking up to obtain three composite systems of PPE1-GO1, PPE2-GO2 and PPE3-GO3 for later use.

The method for distinguishing different kinds of proteins is as follows: 100 mu L of three compounds of PPE1-GO1, PPE2-GO2 and PPE3-GO3 are added into different protein stock solutions (with the concentration of 10 mu M) with the final concentration of 5 mu M, and the mixture is shaken for 10 seconds. The excitation wavelength is 410nm, the fluorescence intensity of the complex combined with the protein is measured at the emission wavelengths of 465nm, 460nm and 460nm respectively, and the change of the relative fluorescence intensity is taken as a detection signal (I-I)₀/I₀). Each protein and each complex were tested 6 times for 12 proteins, 3 complexes, and 6 × 12 × 3 ═ 216 fluorescence signals were obtained, and then the 216 fluorescence signals were used to form a data matrix (table 1), and 3 sensor units had different fluorescence responses to different proteins (fig. 3).

Example 6 analysis and processing of fluorescence array sensor data

The fluorescence data were processed and analyzed using the statistical analysis software SYSTAT (version 13.0). The fluorescence response pattern was converted to canonical pattern using the method of Linear Discriminant Analysis (LDA). All variables were used for the model (full mode) with a tolerance set to 0.001. The mahalanobis distance of each individual mode in the multidimensional space to each group centroid is calculated and the assignment of all classes is based on the shortest mahalanobis distance. According to the LDA chart, even if the structure of the protein is not greatly different, the concentration is low, the variety is large, different proteins can be finally distinguished (figure 4), and the Jackknifed Classification Matrix shows that the accuracy of the protein distinguishing reaches 99 percent (Table 2).

Table 1 shows the data matrix of the fluorescent response of the array sensor to different proteins (12 protein concentrations of 5. mu.M)

TABLE 2 accuracy verification of classification matrices

Ability of array sensor to discriminate unknown samples: the 12 proteins were blinded in random order for a total of 48 unknown samples. The test was performed according to the above procedure and the relative fluorescence intensity change was recorded (Table 3). And then, linear discriminant analysis is carried out to verify the capability of the model for testing unknown samples and distinguish the types of the proteins. 48 unknown samples are tested, 48 samples are correctly detected, and the accuracy is 100%.

Table 3 shows a data matrix of the fluorescence response of the array sensor to the unknown sample

In the invention, diiodobenzene and 1, 4-diacetylene benzene compounds with different substituents are used for synthesizing a polymer, and the polymer is prepared into different types of quaternary ammonium salts in order to increase the water solubility of the polymer and be suitable for complex environmental detection of biological fluids and the like. The graphene oxide is used as an ideal sensor construction material and has very wide application. After the negatively charged graphene oxide is combined with the positively charged fluorescent molecules, fluorescence is remarkably quenched, and amplification of detection signals in sensor application is facilitated. And forming an electrostatic compound by the three fluorescent macromolecules and the graphene oxide with different concentrations to construct a fluorescent sensor array. Common proteins with similar structures, including human serum protein, bovine serum albumin, pepsin, trypsin, lipase, papain, cytochrome, lysozyme, myoglobin, sword bean protein, fibrinogen and beta-lactoglobulin, are selected as analytes, different kinds of proteins are respectively added into a fluorescence array sensor, the concentration of each protein is 5 mu M, each protein and each sensing unit are tested for 6 times, and an enzyme-labeling instrument is used for testing the fluorescence intensity. A data model of 12 (protein) × 3 (compound) × 6 (repetition times) is obtained, and a data set is analyzed and processed by Linear Discriminant Analysis (LDA), so that the fluorescent array sensor has 99% correct distinguishing capability for 12 proteins and 100% correct distinguishing capability for unknown samples. The invention provides an electrostatic compound array sensor which is simple in construction, simple and convenient to operate, easy to practically apply, and capable of distinguishing various test proteins and further popularizing the distinguishing of more proteins.

The advantages of this embodiment: the array sensor has the advantages of simple preparation method, low cost, high sensitivity, short detection time, capability of accurately distinguishing various proteins, accuracy rate of unknown samples of nearly 100 percent, good reproducibility and capability of being used for detecting actual unknown samples. The sensor array operates without the need for specialized technicians.

Claims

1. An electrostatic composite fluorescent array sensor, comprising: is compounded by the interaction of water-soluble fluorescent conjugated polymers with different side chain substitutions and graphene oxide,

the structural formula of the fluorescent conjugated polymer of the three sensing units in the fluorescent sensor is shown as formula I, formula II and formula III:

wherein n is an integer of 8 to 200; m is an integer of 1 to 10.

2. An electrostatic complex fluorescent array sensor according to claim 1, characterized in that: the structural formula of the fluorescent conjugated polymer of the three sensing units in the fluorescent sensor is shown as PPE 1, PPE 2 and PPE 3:

3. an electrostatic complex fluorescent array sensor according to claim 1, characterized in that: the preparation method of the water-soluble fluorescent conjugated polymer with different side chain substitutions comprises the following steps: the diiodobenzene containing different substituents is used as a raw material, and is coupled with 1, 4-diacetylene benzene compounds to generate quaternary ammonium salt with trimethylamine, so that a target product is obtained.

4. An electrostatic composite fluorescent array sensor according to claim 1, wherein: diluting water-soluble fluorescent conjugated polymers with different side chain substitutions by using PBS buffer solution, respectively adding different amounts of negatively charged graphene oxide, uniformly mixing to obtain fluorescence titration curves of the graphene oxide and the fluorescent polymers, and selecting an electrostatic compound for constructing a sensing unit of the array sensor, wherein the electrostatic compound quenches the fluorescence intensity of the polymers to thirty percent by using the graphene oxide.

5. Use of the electrostatic complex fluorescent array sensor of claim 1 in protein detection for non-diagnostic and therapeutic purposes.

6. Use according to claim 5, characterized in that: the detection method comprises the following steps: the method comprises the steps of respectively mixing different sensing units of the electrostatic composite fluorescent array sensor with protein, measuring the fluorescence intensity of each sensing unit, processing fluorescence data, classifying data matrixes, and distinguishing and detecting various proteins.