CN111948185B

CN111948185B - Sensor for instantly identifying development process of acute kidney injury based on two-dimensional amplification array mode, preparation method, application and use method

Info

Publication number: CN111948185B
Application number: CN202010854955.9A
Authority: CN
Inventors: 余伯阳; 田蒋为; 张然; 喻谢安
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2020-08-21
Filing date: 2020-08-21
Publication date: 2023-03-21
Anticipated expiration: 2040-08-21
Also published as: CN111948185A

Abstract

The invention discloses a sensor for instantly identifying the development process of acute kidney injury based on a two-dimensional amplification array mode, a preparation method, application and a use method. The sensor comprises polydopamine-polyethyleneimine (PDA-PEI) copolymer and DNA chains modified with fluorophores of three different emission wavelengths. Adding dopamine hydrochloride and polyethyleneimine into a buffer solution, stirring, filtering and dialyzing to obtain a polydopamine-polyethyleneimine copolymer carrier with excellent quenching effect; the sensor can adsorb three kinds of DNA for modifying different fluorophores through electrostatic action to form a multi-channel sensor. Based on the charge difference of proteins with different particle diameters, DNA fluorescent signal molecules of the sensor can be induced to dissociate to form fluorescent fingerprints of different degrees of kidney injury, the kidney injury with different degrees can be rapidly identified by means of a multivariate statistical method, and the sensor is high in sensitivity and strong in specificity.

Description

Sensor for instantly identifying development process of acute kidney injury based on two-dimensional amplification array mode, preparation method, application and use method

Technical Field

The invention relates to a biosensor, a preparation method and application thereof, in particular to a sensor for instantly identifying the development process of acute kidney injury based on a two-dimensional amplification array mode, a preparation method, application and application thereof.

Background

Acute kidney injury is a clinical syndrome of acute decline or loss of kidney filtration function in a short period of time due to different causes, can cause multiple organ failure, and is an important disease affecting human health. Acute kidney injury encompasses the entire process of reduced renal function, covering the entire progression of reduced renal filtration function from mild decline to the need for renal replacement therapy and even end-stage renal disease. Therefore, differentiating the occurrence and progression of renal injury is a prerequisite for clinical diagnosis and treatment of acute renal injury.

In 2004, the acute dialysis quality guide group proposed a diagnostic criteria (RIFLE) that classified acute kidney injury into 3 grades-Risk (Risk), injury (initial), failure (Failure) and two outcomes-Loss of renal function (Loss) and end-stage renal disease (ESKD) based on the relative changes in serum creatinine and urine volume; in 2007, the acute kidney injury network expert group proposed a modified version based on the RIFLE standard; in 2012, the global kidney disease improvement prognosis organization proposed the latest diagnosis and staging standard based on the first two standards, namely, serum creatinine and urine volume were used as the main indicators. However, creatinine and urine volume are susceptible to factors such as sex, age, diet, etc., and creatinine is only significantly increased after a 50% reduction in renal filtration function. The kidneys have a strong compensatory capacity, and after acute kidney injury, the renal structure may not be completely repaired even if serum creatinine is restored to the pre-injury baseline level. Therefore, the integrity of the kidney structure and function cannot be fully reflected only by the change of creatinine and urine volume, and the degree of kidney injury development cannot be accurately determined. During the filtration process of the kidney, the glomerular filtration system plays a crucial role. The filtering barrier of the glomerulus comprises endothelial cells, podocytes and glomerular basement membrane, and under the combined action of the endothelial cells, the podocytes and the glomerular basement membrane, the protein with the particle size of less than 8nm and positive charge can be freely filtered under a normal physiological state. With the aggravation of kidney injury and the destruction of glomerular filtration system, proteins with particle size of more than 8nm and negatively charged proteins can be discharged via urine along with the filtration system. Therefore, by depending on the pathological characteristics of the occurrence and development of the renal injury and by means of two core indexes of a glomerular filtration system, namely charge and particle size, a novel rapid identification method of the development process of the renal injury can bring a new opportunity for the accurate identification of the acute renal injury.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to provide a sensor for instantaneously identifying the development process of acute renal injury based on a two-dimensional amplification array mode, which has the characteristics of high sensitivity, strong specificity and the like.

Another object of the invention is to provide a method for the preparation, use and method of use of said sensor.

The technical scheme is as follows: the invention provides a sensor for instantaneously identifying the development process of acute kidney injury based on a two-dimensional amplification array mode, which comprises polydopamine-polyethyleneimine (PDA-PEI) copolymer and a DNA chain modified with three fluorophores with different emission wavelengths.

The preparation method of the sensor for instantaneously identifying the development process of the acute kidney injury based on the two-dimensional amplification array mode comprises the following steps:

(1) Preparation of polydopamine-polyethyleneimine (PDA-PEI) copolymer carrier: adding dopamine hydrochloride and polyethyleneimine into a buffer solution, stirring at room temperature in a dark place, filtering, and dialyzing to remove unreacted dopamine hydrochloride and polyethyleneimine to obtain a polydopamine-polyethyleneimine (PDA-PEI) copolymer carrier;

(2) And (3) constructing a sensor: and mixing the polydopamine-polyethyleneimine (PDA-PEI) copolymer carrier with the DNA chains modified with three fluorophores with different emission wavelengths to obtain the PDA-PEI/DNAs sensor, wherein the DNA chains modified with the three fluorophores with different emission wavelengths are respectively AAAAA-Cy7, AAAAAAAAAA-Texas Red and AAAAAAAAAAAAAAAAAA-VIC.

Further, the air conditioner is characterized in that,

the excitation wavelength and the emission wavelength of the AAAAA-Cy7, the AAAAAAAAAA-Texas Red and the AAAAAAAAAAAAAAAAAAAA-VIC are 735nm and 755nm respectively; 590nm and 615nm;535nm,555nm.

Further, the weight average molecular weight of the polyethyleneimine is 600Da-10000Da.

The sensor for instantaneously identifying the development process of the acute kidney injury based on the two-dimensional amplification array mode is used for monitoring the development process of kidney injuries of different models.

The sensor for instantly identifying the development process of acute kidney injury based on the two-dimensional amplification array mode is used for evaluating the development process of kidney injury of different models and the action mechanism and the reagent of drugs for protecting the kidney injury at different stages.

The use method of the sensor for instantly identifying the development process of the acute kidney injury based on the two-dimensional amplification array mode comprises the steps of collecting urine of kidney injuries of different models, adding the sensor, instantly measuring fluorescence spectra of three signal molecules, forming characteristic fluorescence fingerprints and identifying the development process of the acute kidney injury.

Further, passing the collected urine through a 50kDa ultrafiltration tube, performing ultrafiltration centrifugation to obtain urine samples with different molecular weights, respectively adding the urine samples into sensors, instantaneously determining three signal molecules induced by the urine with the molecular weight lower than 50kDa and three signal molecules induced by the urine with the molecular weight higher than 50kDa, establishing a linear discriminant function based on six signal molecules induced by two dimensions of the molecular weight and the charged charge, and quantitatively identifying the development process of acute kidney injury of different models by means of a multivariate statistical analysis method.

The application of the transient identification sensor in rapid identification of kidney injury of different models comprises a cisplatin-induced kidney injury model and a unilateral ureter ligation model.

The principle of the invention is illustrated as follows:

the preparation of the PDA-PEI copolymer carrier is that DA is oxidized into dopaquinone under the condition of normal oxygen, and then cyclization, oxidation and rearrangement reactions are carried out to form o-dihydroxyindole, and indole ring self-polymerization is carried out to form PDA; the added PEI is grafted to the PDA through the reaction of Michael addition and Schiff base; in addition, PEI can also promote homogeneous polymerization of DA into PDA, and a PDA-PEI copolymer with a good quenching effect is formed.

The construction principle of the sensor is that the positive charge PDA-PEI is combined with the three DNAs with negative charges through electrostatic adsorption to form the PDA-PEI/DNAs sensor. Based on the Fluorescence Resonance Energy Transfer (FRET) effect, the fluorescence of the three DNAs is substantially quenched.

Urine samples with different damage degrees are collected and added into the constructed sensor, and the protein charge in the urine can induce different fluorescent signal molecules of the sensor to dissociate so as to instantly generate a fluorescent signal. Due to the charge difference of the proteins in the urine samples with different damage degrees, the sensor can be induced to generate different fluorescent fingerprint spectrums, and further the damage degree is identified. In order to accurately identify the occurrence and development of renal injury, urine is filtered through a 50kDa ultrafiltration tube to obtain urine samples with different molecular weights, the samples with different molecular weights are respectively added into the constructed sensors, and fluorescent signal molecules for identifying the development process of renal injury can be added from two dimensions of protein particle size and charged charge, so that the development process of renal injury can be quickly and accurately identified (figure 1).

The DNAs signal molecule fluorescence spectrum is collected by a fluorescence enzyme labeling instrument.

Specifically, the sensor disclosed by the invention is simple to prepare, mild in reaction condition, low in cost and easy to prepare in batches. The constructed sensor is added into the urine sample to obtain a response result instantly: in the process of occurrence and development of renal injury, due to the filtration barrier of glomeruli, the difference of the particle size and the charge of the protein in urine can induce the dissociation of a fluorescence signal molecule of a sensor from two dimensions, so that a unique fluorescence fingerprint is generated, the development process of renal injury can be rapidly identified, and a new strategy is provided for rapid detection of renal injury and evaluation of potential renal protection effect of a medicament. The invention has wide application range, not only can be used for quickly detecting different types of kidney injuries, but also can quickly screen potential kidney protection medicaments according to the established model.

The action mechanism of the invention is based on that the particle size and the charged charge of protein in urine are different to reflect the process of renal injury due to the difference of glomerular filtration barrier in the occurrence and development process of renal injury, and the two dimensions of the particle size and the charged charge of the protein can be reflected according to the change of fluorescent molecules of the constructed sensor. The instantaneous identification is to add the sensor into the urine and read the fluorescence spectrum immediately to obtain the result, and the sensor and the urine do not need to be incubated for a long time to realize rapid detection.

Has the advantages that: compared with the prior art, the invention has the following advantages:

the sensor provided by the invention is simple in preparation method, mild in reaction condition, low in cost and easy to prepare in batches, and three signal channels are constructed, so that the development process of renal injury can be preliminarily evaluated; in order to further accurately identify the occurrence and development states of renal injury, urine passes through a 50kDa ultrafiltration tube to obtain urine samples with different molecular weights, and the urine samples react with the sensors respectively to amplify fluorescent signal molecular variables from two dimensions of particle size and charged difference, so that the accurate identification of the occurrence and development states of renal injury is realized. More importantly, the invention has wide application range, can be used for quickly identifying the renal injury and can also be used for screening potential renal protection drugs. In addition, the sensor constructed by the invention has the characteristics of transient response, simple preparation and the like, and provides a new tool and a new method for screening potential kidney protection medicaments.

The invention prepares a polydopamine-polyethyleneimine (PDA-PEI) copolymer carrier with good quenching effect by a one-step method, adsorbs DNAs by electrostatic action and quenches the fluorescence of the DNAs to form the PDA-PEI/DNAs sensor. The interaction of the sensor with urine frees the DNAs, producing a fluorescent signal instantaneously. After renal injury occurs, the difference of glomerular filtration systems caused by renal injury of different degrees can cause the difference of the particle size and the charge of protein in urine, so as to induce the dissociation of fluorescence signal molecules of the sensor, generate different fluorescence signals and reflect different states of renal injury. The sensor prepared by the invention has wide application prospect in the aspects of rapid identification of kidney injury, screening of potential protective drugs for kidney injury and the like.

Drawings

FIG. 1 is a schematic diagram of the detection principle of the present invention;

FIG. 2 is a transmission electron microscope image of the PDA-PEI carrier prepared in example 1;

FIG. 3 is a graph showing the particle size of the PDA-PEI carrier prepared in example 1;

FIG. 4 is a UV spectrum of the PDA-PEI carrier prepared in example 1;

FIG. 5 is an infrared spectrum of the PDA-PEI carrier prepared in example 1;

FIG. 6 is a graph showing fluorescence spectra of DNAs prepared in example 1;

FIG. 7 is a graph showing fluorescence titration of three DNAs with different concentrations of PDA-PEI vector prepared in example 1;

FIG. 8 is a graph of the score of the Principal Component Analysis (PCA) of the cisplatin-induced renal injury model and the partial least squares analysis (PLS-DA) of the sensor obtained in example 1 (Panel B);

FIG. 9 is a Linear Discriminant Analysis (LDA) score plot for the cisplatin-induced renal injury model for the sensor obtained in example 1;

FIG. 10 is a Principal Component Analysis (PCA) score chart and partial minimum discriminant analysis (PLS-DA) chart (B) of the UUO model based on the protein charge induction three variable sensors obtained in example 1;

FIG. 11 is a Linear Discriminant Analysis (LDA) score plot of the sensor obtained in example 1 for three variables of the UUO model;

FIG. 12 is a graph of Principal Component Analysis (PCA) of a UUO model based on protein particle size and charge two-dimensional pattern induced six variable sensors obtained in example 1 (Panel A) and a partial minimum discriminatory analysis (PLS-DA) (Panel B);

fig. 13 is a Linear Discriminant Analysis (LDA) score plot of six variables of the UUO model for the sensor obtained in example 1.

Detailed Description

In the following examples, the type of a fluorescence microplate reader used for reading the fluorescence spectrum is Thermo Fisher Scientific Oy 3001; the type of an enzyme-labeling instrument used for reading the ultraviolet absorption numerical value is American BioTek company; the instrument model used for the determination of the infrared spectrum is BRUKER-MPA of BRUKER company, switzerland; the Zeta potential is measured by a Malvern Zeta sizer-Nano Z instrument; the transmission electron microscope image of the PDA-PEI is measured by adopting a JEOL JEM-200CX instrument at an acceleration voltage of 200 kV; dopamine hydrochloride was purchased from solibao technologies ltd; the polyethyleneimine is available from McLin Biochemical technology, inc. (M.W.600Da, 1800Da, 10000 Da); DNAs (AAAAA-Cy 7, AAAAAAAAAA-Texas Red and AAAAAAAAAAAAAAAAAAAA-VIC) were synthesized by Shanghai Shichen technology effective Co., ltd; tris-HCl (1M, pH 7.4) was purchased from Solebao technologies, inc.

Example 1

Construction of PDA-PEI/DNAs sensor and verification of successful synthesis

1. Preparation of PDA-PEI vector

(1) Diluting 100. Mu.L of Tris-HCl (1M, pH 7.4) buffer to 10mL with ultrapure water for later use;

(2) Respectively weighing 10mg of dopamine hydrochloride (DA & HCl) and 10mg of polyethyleneimine (PEI, M.W.600Da), adding the two into 10mL of Tris-HCl buffer solution, and stirring on a magnetic stirrer at room temperature in a dark place for 2-8h;

(3) The solution is filtered by a 0.22 mu m cellulose ester membrane and then is placed in a dialysis bag (molecular weight cut-off 1000 Da) for dialysis for 24h to remove unreacted DA and PEI, and the PDA-PEI copolymer carrier is obtained.

FIG. 2 shows a schematic representation of the transmission electron microscope of the PDA-PEI carrier prepared in example 1.

FIG. 3 is a schematic diagram showing the dynamic light scattering particle size characterization of the PDA-PEI carrier prepared in example 1.

As can be seen from FIGS. 2 and 3, the TEM indicates that the prepared PDA-PEI carrier is spherical, uniform in size and uniform in dispersion, and Dynamic Light Scattering (DLS) indicates that the average particle size of the synthesized PDA-PEI carrier is 290nm, which indicates that the PDA-PEI carrier, i.e. the polydopamine-polyethyleneimine (PDA-PEI) copolymer carrier, is successfully prepared.

FIG. 4 shows UV spectra of DA, PEI and PDA-PEI copolymer carriers, respectively. The PDA-PEI copolymer carrier has characteristic peaks at 280nm and 420nm, the peak observed at 280nm is characteristic of DA, and the peak near 420nm is characteristic peak of dopamine pigment obtained by intramolecular cyclization of DOPAquinone which is an oxidation product of DA, thereby further verifying the successful preparation of the PDA-PEI carrier. FIG. 5 shows the IR spectra of PDA-PEI copolymer carrier, DA and PEI, respectively. Appeared at about 1650cm in the IR spectrum of PDA-PEI ^-1 The peak of (a), was caused by C = N vibration, further verifying the success of the synthesis of PDA-PEI.

2. A sensor for instantaneously identifying the development process of acute renal injury by a two-dimensional amplification array mode comprises the following steps: construction of PDA-PEI/DNAs sensor

And (3) placing the prepared PDA-PEI copolymer carrier and DNAs on an oscillator to be fully mixed to obtain the PDA-PEI/DNAs sensor. The sensor was constructed with PBS (pH = 7.4) and ultrapure water = 1: 1 (v/v) as the total reaction system, 100nM DNAs and 250ng/mL PDA-PEI.

FIG. 6 shows the fluorescence spectra of three DNAs, respectively. The optimal excitation and emission wavelength of 5A modified Cy7 was 735, 755nm; the optimal excitation and emission wavelength of the 10A modified Texas Red is 590, 615nm; the optimal excitation and emission wavelength for the 20A modified VIC was 535, 555nm. The 3 signal molecule emission spectra are not overlapped and have no mutual influence, so that the method can be applied to a multi-channel fluorescence array sensor and realizes multi-channel simultaneous detection.

FIG. 7 shows a graph of the fluorescence titration of the PDA-PEI copolymer vector against three DNAs signal molecules. Different concentrations of PDA-PEI were added to a single DNA, the fluorescence intensity of the optimal emission wavelength of the DNA was measured, and an optimal curve of a set of models of the same binding sites was fitted. The fluorescence intensity is continuously reduced along with the increase of the concentration of the PDA-PEI carrier, and when the concentration of the PDA-PEI is 250ng mL ^-1 In the process, the quenching efficiency of 3 fluorescent signal molecules is over 90 percent, and finally the platform period is reached, which shows that the PDA-PEI copolymer carrier has good quenching performance.

Table 1 shows the association constants of the PDA-PEI copolymer support with different DNAs signal molecules, respectively, as determined by fitting a fluorescence titration curve. By Scatchard equation: log [ (IoF-IF)/IF]＝logKa+nlog[Q]The ratio of the association constants (Ka) of the three signal molecules 5A-Cy7, 10A-Texas Red,20A-VIC was calculated to be 3.68X 10 ⁵ ，3.20×10 ⁶ ，2.47×10 ⁷ (M ^-1 ) The difference of orders of magnitude is shown, and the binding force of the 20A-VIC and the PDA-PEI is strongest.

TABLE 1

Fluorophores	Association constant (Ka), (M) ^-1	R ²
			5A-Cy7	3.68×10 ⁵	0.997
10A-Texas Red	3.20×10 ⁶	0.997
			20A-VIC	2.47×10 ⁷	0.992

Example 2

And (3) inspecting the application of the PDA-PEI/DNAs fluorescent array sensor in identifying the development process of the cisplatin model kidney injury.

1. The sensor was prepared as in example 1;

2. identifying cisplatin model kidney progression injury progression. The method comprises the following steps:

(1) Cisplatin molding: cisplatin was injected intraperitoneally at a dose of 5mg/kg for 6 days.

(2) Collecting urine: collecting blank urine of mice and urine of 1-6 days after administration by bladder squeezing method every day, and storing at-20 deg.C for use.

(3) And (3) feeding a sensor: mu.L of each 10. Mu.L of collected mouse urine on different days of administration was transferred to a 96-well plate, and 190. Mu.L of the constructed array sensor (final concentration of PDA-PEI is 250ng mL) ^-1 And the final concentration of 3 DNA signal molecules is 100 nM), and mixing.

(4) The fluorescence values were read and calculated: after a sensor is added, the mixture is placed in a fluorescence microplate reader, the reading values are optimally excited and emitted by 3 signal molecules respectively, the excitation is carried out at 535nm, and fluorescence signals are collected at 540-600 nm; excitation at 590nm, and collecting fluorescence signals at 600-700 nm; 735nm excitation, 740-800nm collection of fluorescence signals, the bandwidth is 5nm, and the fluorescence signals are used as the unique fluorescence fingerprint of mouse urine of cisplatin with different administration days.

FIG. 8 shows the Principal Component Analysis (PCA) score plot and partial least squares analysis (PLS-DA) plot of the sensor versus cisplatin-induced renal injury model. 1 represents 0 days of administration, 2 represents 1 day of administration, 3 represents 2 days of administration, 4 represents 3 days of administration, 5 represents 4 days of administration, 6 represents 5 days of administration, and 7 represents 6 days of administration. The principal component analysis approach, processed by SIMCA-P software, can reduce the dimensionality of the data to locate patterns in the data. Under the PCA unsupervised mode, the urine of mice dosed with cisplatin on different days was significantly aggregated into 6 groups, wherein one group was given on day 0, one group was given on day 1, one group was given on day 2, one group was given on day 3, one group was given on day 4, and one group was given on day 5-6. The analysis result of the PCA can provide a main basis for constructing the discriminant function in the later period. Based on the grouping result of PCA, the PLS-DA is verified again, and the result shows that the 6 groups can be obviously separated in 0 day, 1 day, 2 days, 3 days, 4 days and 5-6 days, which shows that the constructed method can be used for rapidly identifying the development process of the cisplatin-induced kidney injury.

FIG. 9 is a Linear Discriminant Analysis (LDA) score plot of the sensor versus cisplatin-induced renal injury model. 1 represents blank group, 2 represents administration for 1 day, 3 represents administration for 2 days, 4 represents administration for 3 days, 5 represents 4 days, and 6 represents administration for 5-6 days. The graph is obtained by processing SPSS software, and LDA can maximally improve the ratio of the inter-class difference to the intra-class difference, so that the LDA can be used for quantitatively distinguishing the fluorescence response modes of the urine samples of the sensor and the cisplatin on different administration days. In this figure, each point represents the response pattern of urine to sensor for different days of cisplatin administration, and urine from cisplatin model mice was successfully divided into 6 groups based on the number of administrations.

According to the LDA analysis result, grouping conditions of the LDA are quantized, three variables are used as indexes, and a constructed linear discriminant function is as follows:

Y1＝4.398X1+11.464X2+0.338X3-634.26；Y2＝3.488X1+12.083X2+0.365X3-655.456；

Y3＝4.884X1+13.214X2+0.494X3-851.46；Y4＝5.345X1+13.917X2+0.453X3-942.266；

y5=6.310X1+13.227X2+0.579X3-950.20; y6=3.843X1+13.042X2+0.532X3-791.283. Wherein Y1-6 represents the cisplatin-induced renal injury grouping, and X1, X2 and X3 represent the fluorescence intensities of 3 signaling molecules. The value of the urine sample induced fluorescence signal with unknown injury degree is directly brought into the constructed discriminant function, the grouping condition is attributed, and the development process of renal injury can be rapidly identified.

Example 3

Based on the size of charges carried by urine protein, the application of the PDA-PEI/DNAs fluorescent array sensor in identifying the development process of the kidney injury of the UUO model.

1. The preparation process of the sensor is the same as that of example 1;

2. identifying the UUO model kidney development injury course. The method comprises the following steps:

(1) UUO molding: 4% chloral hydrate 10mL kg ^-1 After the mice are anesthetized by intraperitoneal injection, the mice are placed on an operating table, and after the abdomen is shaved, conventional disinfection is carried out by iodophor; making a longitudinal incision about 1cm in the lower abdomen of the left side, incising the skin, subcutaneous tissues and muscle layer by layer, and exposing the kidney of the left side; the ureter is doubly ligated with No. 4 suture at the position close to the free ureter of the renal pelvis; seam by seamClosing the incision and disinfecting by iodophor.

(2) Collecting urine: collecting urine of blank mice by bladder squeezing method every day for a fixed time, ligating the urine of mice for 1-14 days, and storing at-20 deg.C for use.

(3) And (3) feeding a sensor: mu.L of each 10. Mu.L of collected mouse urine on different ligation days was transferred to a 96-well plate, and 190. Mu.L of the constructed array sensor (final concentration of PDA-PEI is 250ng mL) ^-1 And the final concentration of 3 DNA signal molecules is 100 nM), and mixing.

(4) The fluorescence values were read and calculated: after a sensor is added, the mixture is placed in a fluorescence microplate reader, the reading values are optimally excited and emitted by 3 signal molecules respectively, the excitation is carried out at 535nm, and fluorescence signals are collected at 540-600 nm; excitation at 590nm, and collecting fluorescence signals at 600-700 nm; 735nm excitation, 740-800nm collection of fluorescence signals, the bandwidth is 5nm, and the fluorescence signals are used as the fluorescence fingerprint spectra of the urine of the mouse on different ligation days.

FIG. 10 is a Principal Component Analysis (PCA) score plot and partial least squares analysis (PLS-DA) plot of a UUO model based on protein charge induction three variable sensors. 1 for blank group, 2 for ligation for 1 day, 3 for ligation for 2 days, 4 for ligation for 3 days, 5 for ligation for 4 days, 6 for ligation for 5 days, 7 for ligation for 6 days, 8 for ligation for 7 days, 9 for ligation for 8 days, 10 for ligation for 9 days, 11 for ligation for 10 days, 12 for ligation for 11 days, 13 for ligation for 12 days, 14 for ligation for 13 days, and 15 for ligation for 14 days. The principal component analysis method can reduce the dimensionality of the data to find patterns in the data, as processed by the SIMCA-P software. Under PCA unsupervised mode, urine of mice ligated by UUO model on different days was significantly gathered into 3 groups, urine was significantly gathered together after ligation for 1-3 days, urine was gathered into one group after ligation for 4-7 days, and urine was gathered into another group after ligation for 8-14 days. Therefore, the PCA result can provide basis for late recognition of the UUO model urine grouping. And then, carrying out PLS-DA analysis on the basis of PCA grouping results, wherein the results show that blank group samples are divided into one group, the blank group samples are divided into one group in 1-3 days, the blank group samples are divided into one group in 4-7 days and the blank group samples are divided into one group in 8-14 days, urine of the UUO model mouse can be divided into 4 groups according to different ligation days, the four stages of the renal injury development process are represented, and the development process of the UUO model renal injury can be effectively identified.

FIG. 11 is a plot of Linear Discriminant Analysis (LDA) scores of the fluorescence response of the sensor to mouse urine from UUO model for different ligation days; 1 represents blank, 2 represents ligation for 1-3 days, 3 represents ligation for 4-7 days, and 4 represents ligation for 8-14 days. The graph is obtained by processing through SPSS software, and LDA can improve the ratio of the inter-class difference to the intra-class difference to the maximum extent, so that the LDA can be used for quantitatively distinguishing the fluorescence signal difference induced by urine samples after different ligation days of mice. In this figure, each point represents the response pattern of urine to sensors for different ligation days for the mice, and the UUO model mice were successfully divided into 4 groups according to the ligation days.

According to the LDA analysis result, grouping conditions of the LDA are quantified, and a linear discriminant function is constructed by taking 3 signal molecules as variables as follows:

Y1＝5.517X1+0.498X2+2.952X3-342.782；Y2＝5.078X1+0.409X2+3.035X3-323.389；

y3=5.589X1+0.442X2+3.404X3-399.586; y4=6.146X1+0.553X2+3.718X3-485.485. Wherein Y represents a grouping of the progression of renal injury; x1, X2 and X3 represent the fluorescence intensities of 3 signal molecules. And (3) substituting the specific numerical values of the 3 fluorescence signals induced by the unknown sample into the constructed linear discriminant function, and accurately identifying the attribution of the development stage of the renal injury so as to judge the injury degree of the renal injury.

Example 4

Based on the charge of urine protein and the particle size, the PDA-PEI/DNAs fluorescent array sensor is applied to accurately identify the development process of the UUO model kidney injury.

1. The sensor was prepared as in example 1;

(1) UUO molding: 4% chloral hydrate 10mL kg ^-1 After the anesthetized mouse is injected into the abdominal cavity, the mouse is placed on an operating table, and after the abdomen is shaved, iodophor is used for routine disinfection; making a longitudinal incision about 1cm in the lower abdomen of the left side, incising the skin, subcutaneous tissues and muscle layer by layer, and exposing the kidney of the left side; the ureter is doubly ligated with No. 4 suture at the position close to the free ureter of the renal pelvis; suture incision layer by layerIodophor is routinely used for disinfection.

(2) Collecting and treating urine: the urine of a blank mouse is collected by a bladder squeezing method for a fixed time every day, and the urine of the mouse is ligated for 1 to 14 days. And (3) passing the collected urine through a 50kDa ultrafiltration tube, performing ultrafiltration and centrifugation to obtain a urine sample with the molecular weight of less than 50kDa and a urine sample with the molecular weight of more than 50kDa, and storing the urine samples at-20 ℃ for later use.

(3) And (3) feeding a sensor: taking 10 mu L of urine samples with different molecular weights obtained by ultrafiltration to a 96-well plate respectively, and adding 190 mu L of the constructed array sensor (the final concentration of the PDA-PEI is 250ng mL) ^-1 And the final concentration of 3 DNA signal molecules is 100 nM), and mixing.

FIG. 12 is a Principal Component Analysis (PCA) score plot and partial least squares discriminant analysis (PLS-DA) plot of six variable sensor pairs UUO model induced based on protein particle size and charge two-dimensional patterns. 1 for blank group, 2 for ligation for 1 day, 3 for ligation for 2 days, 4 for ligation for 3 days, 5 for ligation for 4 days, 6 for ligation for 5 days, 7 for ligation for 6 days, 8 for ligation for 7 days, 9 for ligation for 8 days, 10 for ligation for 9 days, 11 for ligation for 10 days, 12 for ligation for 11 days, 13 for ligation for 12 days, 14 for ligation for 13 days, and 15 for ligation for 14 days. As can be seen in FIG. 12, after increasing the fluorescence signal variation, the urine of UUO model mice was subdivided into 7 groups of 0 days, 1-2 days, 3-4 days, 5-7 days, 8-9 days, 10-11 days, and 12-14 days, respectively, under the PCA unsupervised mode. Compared with the grouping result only depending on 3 fluorescence signal variables, the grouping of 8-14 days is more finely distinguished, the original group which belongs to 1 group is divided into 3 groups, and the whole development process of the renal injury is refined. Subsequently, based on the PCA grouping results, PLS-DA analysis was performed to verify the rationality of the constructed renal injury development process grouping, and the PLS-DA successfully divided 14-day urine of the UUO model into 7 groups according to the injury degree. The method for expanding the variable can realize accurate identification of the development process of the kidney injury of the UUO model.

Figure 13 is a Linear Discriminant Analysis (LDA) score plot of the fluorescence response of the sensor to mouse urine from different days of ligation for the UUO model for six variables. 1 represents blank, 2 represents ligation for 1-2 days, 3 represents ligation for 3-4 days, 4 represents ligation for 5-7 days, 5 represents 8-9 days, 6 represents 10-11 days, and 7 represents 12-14 days. The graph is obtained by processing SPSS software, and LDA can improve the ratio of the inter-class difference to the intra-class difference to the maximum extent, so that the graph can be used for quantitatively distinguishing the fluorescence reaction mode of urine samples of sensors and UUO model mice after different ligation days. In the graph, each point represents the response mode of urine of a mouse to a sensor on different ligation days, the urine of the UUO model for 14 days is successfully divided into 7 groups according to the injury degree, and the development process of kidney injury of the UUO model can be accurately identified by a variable lifting method.

According to the LDA analysis result, grouping conditions of the LDA are quantified, and a linear discriminant function is constructed by taking 6 signal molecules as variables as follows:

Y1＝0.912X1+0.805X2-1.118X3+6.271X4+12.471X5+5.435X6-1860.994；

Y2＝1.130X1+0.945X2-1.313X3+4.949X4+13.317X5+5.489X6-1822.502；

Y3＝0.809X1+1.050X2-1.270X3+6.410X4+12.615X5+6.410X6-2031.469；

Y4＝1.000X1+0.800X2-1.166X3+6.867X4+12.512X5+7.121X6-2200.629；

Y5＝1.057X1+0.860X2-1.207X3+6.320X4+13.131X5+7.170X6-2231.456；

Y6＝1.165X1+0.790X2-1.169X3+5.315X4+13.485X5+6.667X6-2064.167；

Y7＝1.506X1+0.636X2-1.191X3+5.323X4+14.047X5+6.933X6-2226.907；

wherein Y represents the grouping of UUO model renal lesions; x1, X2 and X3 represent the fluorescence intensity of three signal molecules induced by the urine sample with molecular weight higher than 50 kDa; x5, X6 and X7 represent the fluorescence intensities induced by 3 signal molecules in urine samples with molecular weights below 50 kDa. According to the specific numerical values of 6 fluorescence signals induced by an unknown sample, the constructed linear discriminant function is brought in, the grouping condition of the linear discriminant function is attributed, and the development process of the renal injury can be rapidly identified.

In conclusion, the sensor disclosed by the invention is simple in preparation method, mild in reaction conditions, low in cost and easy to prepare in batches, three DNAs (deoxyribonucleic acids) for modifying different fluorophores are used as fluorescent signal molecules to construct three signal channels, and the development process of renal injury can be preliminarily evaluated; in order to further accurately identify the occurrence and development states of renal injury, urine passes through a 50kDa ultrafiltration tube to obtain urine samples with different molecular weights, and the urine samples react with the sensors respectively to amplify fluorescent signal molecular variables from two dimensions of particle size and charged difference, so that the accurate identification of the occurrence and development states of renal injury is realized. More importantly, the invention has wide application range, can be used for quickly identifying the renal injury and can also be used for screening potential renal protection drugs. In addition, the sensor constructed by the invention has the characteristics of transient response, simple preparation and the like, and provides a new tool and a new method for screening potential kidney protection medicaments.

Claims

1. A sensor for instantaneously identifying the development progress of acute renal injury based on a two-dimensional amplification array pattern, characterized in that: comprises polydopamine-polyethyleneimine (PDA-PEI) copolymer and DNA chains modified with fluorophores of three different emission wavelengths,

(2) And (3) constructing a sensor: mixing a polydopamine-polyethyleneimine (PDA-PEI) copolymer carrier with DNA chains modified with three fluorophores with different emission wavelengths to obtain the PDA-PEI/DNAs sensor, wherein the DNA chains modified with the three fluorophores with different emission wavelengths are respectively AAAAA-Cy7, AAAAAAAAAA-Texas Red and AAAAAAAAAAAAAAAAAAAA-VIC,

the excitation wavelength and the emission wavelength of the AAAAA-Cy7, the AAAAAAAAAA-Texas Red and the AAAAAAAAAAAAAAAAAAAA-VIC are 735nm and 755nm respectively; 590nm,615nm;535nm,555nm,

the weight average molecular weight of the polyethyleneimine is 600Da-10000Da.

2. Use of the sensor for transiently identifying the progression of acute kidney injury according to claim 1 based on a two-dimensional amplification array pattern for evaluating the progression of kidney injury in different models and the mechanism of action and reagents of drugs for protection of kidney injury at different stages.

3. The method for using the sensor for instantly identifying the development process of acute kidney injury based on the two-dimensional amplification array mode as claimed in claim 1, collecting urine of kidney injury of different models, adding the sensor, instantly measuring the fluorescence spectra of three signal molecules, forming characteristic fluorescence fingerprints, and identifying the development process of acute kidney injury.

4. The use method of claim 3, wherein the collected urine is filtered through a 50kDa ultrafiltration tube, and subjected to ultrafiltration and centrifugation to obtain urine samples with different molecular weights, the urine samples are respectively added into a sensor, three signal molecules induced by urine with the molecular weight lower than 50kDa and three signal molecules induced by urine with the molecular weight higher than 50kDa are instantaneously measured, and based on six signal molecules induced by two dimensions of the molecular weight and the charged size, a linear discriminant function is established by means of a multivariate statistical analysis method to quantitatively identify the development process of acute kidney injury of different models.