CN112924422A

CN112924422A - Multi-channel array sensor and preparation method and application thereof

Info

Publication number: CN112924422A
Application number: CN202110080177.7A
Authority: CN
Inventors: 田蒋为; 余伯阳; 张然
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2021-01-21
Filing date: 2021-01-21
Publication date: 2021-06-08
Anticipated expiration: 2041-01-21
Also published as: CN112924422B

Abstract

The invention discloses a multichannel array sensor which comprises nanogold-polyethyleneimine (AuNPs-PEI) copolymer and protein modified with three fluorophores with different emission wavelengths. The invention also discloses a preparation method of the sensor, which comprises the steps of adding chloroauric acid and polyethyleneimine into buffer solution, stirring, filtering and dialyzing to obtain a nanogold-polyethyleneimine copolymer carrier with excellent quenching effect; the sensor can adsorb three proteins for modifying different fluorophores through electrostatic action to form a multi-channel sensor. The invention also discloses the application of the sensor, which can induce fluorescence signal molecules in the sensor to dissociate to form specific fluorescence fingerprints based on different action mechanisms of the nephrotoxic drugs and the difference of urine protein particle size and charge in the development process of renal injury, and can quickly identify the action mechanism of the nephrotoxic drugs and the development process of the renal injury by means of a multivariate statistical method.

Description

Multi-channel array sensor and preparation method and application thereof

Technical Field

The invention belongs to the biosensing technology, and particularly relates to a multi-channel array sensor and a preparation method and application thereof.

Background

Drug-induced renal injury refers to abnormal renal structure or function caused by drugs, and is mainly manifested by acute tubular necrosis, acute interstitial nephritis and permeable nephropathy. Many drugs are metabolized or excreted by the kidney and tend to accumulate in the kidney and damage the glomerulus, tubules or renal basement membrane. In addition, part of the medicines can cause the rapid decline of bilateral renal filtration function in a short time, so that toxic substances in vivo are accumulated, and acute renal failure can be caused seriously. Studies have shown that nephrotoxic drugs cause acute renal failure in 19% -25% of critically ill patients, with a high probability of causing chronic or end-stage renal disease. The action mechanism of kidney injury caused by different nephrotoxic drugs is complex, for example, aminoglycoside drugs can interfere renal tubular transport by damaging mitochondrial and lysosome functions to cause renal tubular cytotoxic reaction, and further cause renal tubular epithelial cell necrosis; the sulfonamide antibiotics or metabolites thereof are easy to form crystals in the intrarenal tissues and deposit in the lumen of the distal renal tubule to block the urine flow and stimulate the interstitial reaction, thereby causing the obstructive nephropathy; non-steroidal anti-inflammatory drugs affect the hemodynamic changes in renal ischemic injury by inhibiting cyclooxygenase-catalyzed production of prostaglandins, reducing the synthesis of vasodilating prostaglandins in arachidonic acid, and altering the activity balance between renal vasoconstrictors and vasodilators. Therefore, different drugs can cause renal function damage through different action routes, which brings great challenges to clinical diagnosis of renal injury, seriously affects preclinical safety assessment and clinical treatment, and further hinders accurate treatment of renal toxicity induced by different drugs.

The current clinical diagnosis method of renal injury still uses the evaluation of renal filtration function by using blood creatinine and urea nitrogen as 'gold indexes'. However, the development of the renal injury diagnosis method is severely limited by the defects of poor specificity, low accuracy and the like of the evaluation of the hysteresis of the renal injury by the blood creatinine and the urea nitrogen. Proteinuria is a typical symptom of kidney disease. Proteinuria refers to the comprehensive manifestation of impaired renal function in which the content of protein in urine is increased due to abnormal glomerular filtration function and renal tubular reabsorption function, but clinical determination of the total amount of urine protein does not indicate the development process of renal diseases. The filtration function of the kidney is explored, and the filtration barrier of the glomerulus is found to serve as a urine protein molecular sieve and better pass the protein with positive charges, so that the particle size and the charge difference of the urine protein bring a new breakthrough point for identifying the development process of the kidney injury.

The traditional method is limited due to the defects of expensive instrument, complex operation, time consumption, low sensitivity and the like, and the fluorescent fingerprint method is different from the specific identification of a lock and a key and can be used for identifying the selective combination between an analyte and a receptor. In this recognition mode, the differential signal response of the analyte to the receptor depends on the strength of the binding force, and a characteristic fingerprint is formed. Therefore, the fluorescence fingerprint can reflect the overall change of a complex mixture only through the combined action of a plurality of sensor units and a large amount of analytes, and provides a new tool and a new method for exploring drug-induced renal injury.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the technical problems in the prior art, the invention provides a multi-channel array sensor with the characteristics of high sensitivity, strong specificity and the like, and also provides a preparation method and application of the sensor.

The technical scheme is as follows: the multichannel array sensor comprises nanogold-polyethyleneimine (AuNPs-PEI) copolymer and protein modified with three fluorophores with different emission wavelengths.

Wherein, the proteins modified with three different emission wavelength fluorophores are bovine serum albumin-FITC, peanut agglutinin-RhB and beta-lactoglobulin-Cy 5.

Further, the bovine serum albumin-FITC (BSA-FITC), the peanut agglutinin-RhB (PNA-RhB) and the beta-lactoglobulin-Cy 5 (beta-Lac-Cy 5) have the excitation wavelength and the emission wavelength respectively and sequentially: 495nm, 520nm, 550nm, 575nm, 654nm and 667 nm.

The preparation method of the multichannel array sensor comprises the following steps:

(1) preparing a nanogold-polyethyleneimine (AuNPs-PEI) copolymer carrier: mixing chloroauric acid (HAuCl)₄·3H₂O) and Polyethyleneimine (PEI) are added into buffer solution, stirred at room temperature in a dark place and then filtered, and dialyzed to remove unreacted chloroauric acid and polyethyleneimine to obtain a nanogold-polyethyleneimine (AuNPs-PEI) copolymer carrier;

(2) and (3) constructing a sensor: mixing a nanogold-polyethyleneimine (AuNPs-PEI) copolymer carrier with Proteins modified with fluorophores with three different emission wavelengths to obtain the AuNPs-PEI/Proteins sensor.

In the step (1), the molecular weight of the polyethyleneimine is 600Da-10000Da (M.W.), and the mass ratio of the chloroauric acid to the polyethyleneimine is 1:1-1: 10. Preferably, the molecular weight of the polyethyleneimine is 1800Da, the mass ratio of the chloroauric acid to the polyethyleneimine is 1:1, and the concentrations of the chloroauric acid and the polyethyleneimine are both 0.01 g/mL.

In the step (2), the proteins modified with three fluorophores with different emission wavelengths are bovine serum albumin-FITC (BSA-FITC), peanut agglutinin-RhB (PNA-RhB) and beta-lactoglobulin-Cy 5 (beta-Lac-Cy 5).

Further, the excitation wavelength and the emission wavelength of the BSA-FITC, the PNA-RhB and the beta-Lac-Cy 5 are 495nm and 520nm respectively; 550nm, 575 nm; 654nm, 667 nm.

Wherein, in the step (2), the concentration of the AuNPs-PEI copolymer in the construction of the sensor is 0.01ng mL^-1-12.5ng mL^-1The concentration of the three proteins modified with fluorophores of different emission wavelengths was 0.0075 nmol/mL. Preferably, the concentration of the AuNPs-PEI copolymer is 10ng mL^-1。

The invention also discloses application of the multichannel array sensor in monitoring the development process of renal injury caused by different nephrotoxic drugs.

The invention also discloses application of the multichannel array sensor in identifying action mechanisms of different nephrotoxic drugs.

The use method of the multichannel array sensor for rapidly identifying the action mechanism of the nephrotoxic drug and the development process of renal injury comprises the following steps: collecting urine samples of different nephrotoxic drug-induced renal injuries, adding a sensor, and instantaneously measuring fluorescence spectra of three signal molecules to form characteristic fluorescence fingerprints so as to identify the development process of each drug-induced renal injury.

Further, based on the aging relation of drug-induced renal injury, urine samples of different drug administration days (1, 3, 5, 7, 9 and 14 days) are collected, sensors are respectively added, the fluorescence values of three signal molecules induced by different drug administration days are measured instantly, the variables are expanded, the 3 variables are changed into 18 variables, and a linear discriminant function is established by means of a multivariate statistical analysis method to quantitatively identify the action mechanism of the renal toxicity drug.

The nephrotoxic drugs of the invention include aminoglycoside antibiotics, non-steroidal anti-inflammatory drugs and proton pump inhibitors.

The principle of the invention is illustrated as follows:

the AuNPs-PEI copolymer carrier is prepared because the branched PEI has good water solubility, contains rich nitrogen elements in the branched PEI, can stabilize AuNPs, and contains rich amino groups which are easy to functionalize on the surface. PEI with the molecular weight of 1800Da is used as a template, Au (III) ions are reduced by utilizing the reducibility of amino groups on the surface of the PEI through a self-reduction method, and the AuNPs-PEI copolymer with good quenching effect is formed.

The construction principle of the sensor is that positive charge AuNPs-PEI is combined with three negatively charged Proteins through electrostatic adsorption to form the AuNPs-PEI/Proteins sensor. The fluorescence of the three Proteins is essentially quenched based on the Fluorescence Resonance Energy Transfer (FRET) effect.

Urine samples of different damage degrees of each medicine are collected and added into the constructed sensor, and the particle size and the charge of the protein 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 difference of the protein particle size and the charges in the urine samples with different damage degrees, the sensor can be induced to generate different fluorescence fingerprint spectrums, and then the damage degree of the kidney induced by each medicament is identified. Further, in order to accurately identify the action mechanism of the nephrotoxic drug, based on the time-dependent relationship of nephrotoxic drug-induced renal injury, urine samples of the drug for different administration days (1, 3, 5, 7, 9, 14 days) are collected, sensors are respectively added, the fluorescence values of three signal molecules induced by different administration days are measured instantaneously, the variables are expanded, and the 3 variables are changed into 18 variables, so that the action mechanisms of different nephrotoxic drugs are quickly and accurately identified (fig. 1).

The fluorescence spectra of the protein signal molecules are collected by a fluorescence microplate reader.

Specifically, the constructed sensor is added into a urine sample, and response results can be obtained instantly: in the process of developing drug-induced kidney injury, the dissociation of fluorescence signal molecules of the sensor is induced by the particle size of protein in urine and the difference of charges carried by the protein in urine, so that unique fluorescence fingerprints are generated, the development process of kidney injury induced by different drugs can be rapidly identified, the variable is expanded by utilizing the aging relation of kidney injury induced by different nephrotoxic drugs, the purpose of rapidly identifying the action mechanism of the nephrotoxic drug is further realized, and a new strategy is provided for rapidly detecting the development process of kidney injury and evaluating the potential action mechanism of the drug.

The action mechanism of the invention is based on that the difference of the glomerular filtration barrier in the development process of renal injury induced by nephrotoxic drugs causes the difference of the particle size of protein in urine and the charge carried by the urine to reflect the progress of renal injury, and simultaneously introduces the aging relationship of drug-induced renal injury, increases variables and accurately identifies the action mechanisms of different nephrotoxic drugs. The rapid identification is that the sensor is added into urine to immediately read the fluorescence spectrum to obtain the result, and the sensor does not need to be incubated with the urine for a long time.

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

the sensor provided by the invention has the advantages of simple preparation method, mild reaction conditions, low cost and easiness in batch preparation, and the three signal channels are constructed, so that the development process of each nephrotoxic drug-induced renal injury can be preliminarily evaluated, and the rapid identification of the action mechanism of the nephrotoxic drug can be realized. More importantly, the invention has wide application range, can be used for rapidly identifying the development process of the renal injury and evaluating the potential action mechanism of the medicament. In addition, the sensor constructed by the invention has the characteristics of quick response, simple preparation and the like, and provides a new tool and a new method for screening potential kidney protection medicaments.

The method prepares the nanogold-polyethyleneimine (AuNPs-PEI) copolymer carrier with good quenching effect through a one-step method, adsorbs Proteins through electrostatic action and quenches the fluorescence of the Proteins, and forms the AuNPs-PEI/Proteins sensor. The interaction of the sensor with urine frees the Proteins, which instantaneously generates a fluorescent signal. After the drug induces the kidney injury, the difference of glomerular filtration systems caused by the kidney injury with different degrees can cause the difference of the particle size and the charge of the protein in urine, so as to induce the dissociation of the fluorescence signal molecules of the sensor, and generate different fluorescence signals which can reflect different states of the kidney injury. The sensor prepared by the invention has wide application prospect in the aspects of rapid identification of the development process of the kidney injury, evaluation of the potential action mechanism of the drug, screening of potential protective drugs for the kidney injury and the like.

Drawings

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

FIG. 2 is a transmission electron microscope image of AuNPs-PEI vector prepared in example 1;

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

FIG. 4 is a UV spectrum of AuNPs-PEI vector prepared in example 1;

FIG. 5 is an infrared spectrum of AuNPs-PEI vector prepared in example 1;

FIG. 6 is a fluorescence spectrum of Proteins prepared in example 1;

FIG. 7 is a graph of the fluorescence titration of three Proteins by AuNPs-PEI vectors prepared in example 1 in different volumes;

FIG. 8 is a Principal Component Analysis (PCA) and partial minimum discriminant analysis (PLS-DA) of the sensor of example 1 for a model of aminoglycoside antibiotic (gentamicin, tobramycin, neomycin) induced renal injury (A is gentamicin, B is tobramycin, C is neomycin);

FIG. 9 is a Principal Component Analysis (PCA) and partial least squares discriminant analysis (PLS-DA) of the proton pump inhibitor (omeprazole, pantoprazole, lansoprazole, esomeprazole) induced kidney injury model (A is omeprazole, B is pantoprazole, C is pantoprazole, D is esomeprazole) with the sensor of example 1;

FIG. 10 is the Principal Component Analysis (PCA) and partial minimum discriminant analysis (PLS-DA) of the sensor of example 1 for the non-steroidal anti-inflammatory drug (ibuprofen, naproxen, diclofenac sodium) induced kidney injury model (A is ibuprofen, B is naproxen, C is diclofenac sodium);

FIG. 11 is a graph of a partial minimum second discriminant analysis (PLS-DA) of the sensor of example 1 at different dosing time points for three mechanisms of action of nephrotoxic drug-induced renal injury;

FIG. 12 is a plot of the Linear Discriminant Analysis (LDA) scores of the sensor of example 1 for nephrotoxic drug-induced renal injury for three mechanisms of action at different dosing time points.

Detailed Description

The present application will be described in detail with reference to specific examples.

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 picture of AuNPs-PEI is measured by adopting a JEOL JEM-200CX instrument at an acceleration voltage of 200 kV; chloroauric acid (HAuCl)₄·3H₂O) purchased from shanghai alatin gmbh; the polyethyleneimine is available from McLin Biochemical technology, Inc. (M.W.600Da, 1800Da, 10000 Da); proteins (BSA-FITC, PNA-RhB and. beta. -Lac-Cy5) were synthesized by Biliger Biotech, Inc.

Example 1

Construction of AuNPs-PEI/Proteins sensor and verification of successful synthesis

1. Preparation of AuNPs-PEI vector

(1) A certain mass of polyethyleneimine (PEI, M.W.1800Da) was accurately weighed and diluted with ultrapure water to a concentration of 0.01g/mL for use.

(2) Accurately weighing a certain mass of chloroauric acid (HAuCl)₄·3H₂O) powder was added to PBS buffer solution to a final concentration of 0.01g/mL according to the product instructions, and stored in a refrigerator at 4 ℃ in the dark for later use.

(3) Sucking 125 μ L of chloroauric acid (0.01g/mL) and adding into 2.5mL of ultrapure water, slowly dropwise adding 100 μ L of polyethyleneimine (0.01g/mL, M.W.1800Da), and stirring at room temperature on a magnetic stirrer at 750rpm in the dark for 8 h;

(4) 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 2000Da) for dialysis for 24h to remove unreacted chloroauric acid and PEI, and the AuNPs-PEI copolymer carrier is obtained.

FIG. 2 shows a schematic representation of AuNPs-PEI carrier transmission electron microscopy prepared in example 1.

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

As can be seen from FIGS. 2 and 3, the TEM indicates that the prepared AuNPs-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 AuNPs-PEI carrier is 35.45nm, which indicates that the AuNPs-PEI carrier, namely the nanogold-polyethyleneimine (AuNPs-PEI) copolymer carrier, is successfully prepared.

FIG. 4 shows UV spectra of AuNPs and AuNPs-PEI copolymer carriers, respectively. AuNPs have maximum absorption peaks at 521nm, and AuNPs-PEI has maximum absorption peaks at 525nm, the maximum absorption peaks are red-shifted, and the successful preparation of AuNPs-PEI copolymer is further verified. FIG. 5 shows the infrared spectra of PEI, AuNPs-PEI vectors, respectively. A peak of about 1650cm < -1 > appears in the infrared spectrum of AuNPs-PEI and is caused by C ═ N vibration, and the synthesis success of AuNPs-PEI is further verified.

2. A multi-channel array sensor for rapidly identifying the action mechanism of nephrotoxic drugs and the development process of renal injury thereof comprises: construction of AuNPs-PEI/Proteins sensor

And (3) fully mixing the prepared AuNPs-PEI copolymer carrier and Proteins on an oscillator to obtain the AuNPs-PEI/Proteins sensor. In the total reaction system of the construction method of the sensor, the molar concentration of each Protein is 0.075nmol/mL, and the final concentration of AuNPs-PEI is 10ng mL^-1。

FIG. 6 shows the fluorescence spectra of three Proteins, respectively. The optimal excitation and emission wavelength of FITC modified Bovine Serum Albumin (BSA) is 495, 520 nm; the optimal excitation and emission wavelength of the RhB modified peanut agglutinin (PNA) is 550, 575 nm; the optimal excitation and emission wavelength of Cy5 modified beta-lactoglobulin (beta-Lac) is 654, 667 nm. 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 the fluorescence titration of three Proteins signaling molecules by AuNPs-PEI copolymer carrier. AuNPs-PEI with different concentrations was added to a single Protein, the fluorescence intensity of the optimal emission wavelength of the Protein was measured, and the 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 AuNPs-PEI carrier, and when the final concentration of AuNPs-PEI is 10ng mL^-1In the process, the quenching efficiency of 3 fluorescent signal molecules is over 90 percent, and finally the plateau period is reached, which shows that the quenching performance of the AuNPs-PEI copolymer carrier is good.

Table 1 shows the association constants of AuNPs-PEI copolymer carriers with different Proteins signaling 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 three signal molecules, BSA-FITC, PNA-RhB, beta-Lac-Cy 5, was calculated to be 1.41X 10⁸，2.68×10⁶，7.43×10⁷(M^-1) The BSA-FITC has the strongest binding force with AuNPs-PEI.

TABLE 1

Fluorophores	Association constant (Ka), (M)^-1	R²
			BSA-FITC	1.41×10⁸	0.999
PNA-RhB	2.68×10⁶	0.993
			β-Lac-Cy5	7.43×10⁷	0.998

The detection principle of the sensor of the present invention is shown in fig. 1, and will be described below with reference to specific embodiments.

Example 2

And (3) investigating the application of the AuNPs-PEI/Proteins fluorescent array sensor in identifying the development process of the aminoglycoside antibiotics (gentamicin, tobramycin and neomycin) model kidney injury.

1. The sensor was prepared as in example 1;

2. identifying aminoglycoside antibiotic drug model kidney development injury process. The method comprises the following steps:

(1) molding the medicine: the mice are divided into 3 groups, each group comprises 21 mice, each group of mice is respectively administered with gentamicin, tobramycin and neomycin by intragastric administration, the clinical dose of the human is converted into the dose of the experimental mice, and the doses are 97.07mg/kg/d, 46.41mg/kg/d and 303mg/kg/d respectively and are continuously administered for 14 days.

(2) Collecting urine: the urine of mice was collected by bladder squeezing for a fixed period of time every day, and the urine was collected for 1, 3, 5, 7, 9, and 14 days after administration and stored at-20 ℃ for later use.

(3) And (3) feeding a sensor: 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 AuNPs-PEI is 10ng mL)^-1And the final concentration of 3 Protein signal molecules is 0.075nmol/mL), and mixing uniformly.

(4) The fluorescence values were read and calculated: after a sensor is added, the mixture is placed in a fluorescence microplate reader, reading values are optimally excited and emitted by 3 signal molecules respectively, excitation is carried out at 495nm, and fluorescence signals are collected at 520 nm; exciting at 550nm, and collecting fluorescence signals at 575 nm; 654nm excitation, 667nm collection fluorescence signal, bandwidth of 5nm, as each drug different days of administration mouse urine peculiar fluorescence fingerprint.

FIG. 8 shows the Principal Component Analysis (PCA) score plot and partial least squares analysis (PLS-DA) plot for the aminoglycoside antibiotic drug model for the sensor. 1 for 0 days, 2 for 1 day, 3 for 3 days, 4 for 5 days, 5 for 7 days, 6 for 9 days, and 7 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. In the PCA unsupervised mode, urine fractions of mice dosed with gentamicin (panel a) on different days were significantly aggregated into 6 groups, with a 0-day group, a 1-day group, a 3-day group, a 5-day group, a 7-9-day group, and a 14-day group. 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 verification of PLS-DA is carried out again, and the result shows that 6 groups can be obviously separated in 0 day, 1 day, 3 days, 5 days, 7-9 days and 14 days; by the same token, the results of the PCA analysis and PLS-DA verification of tobramycin administration (panel B) show that the 7 groups can be clearly separated by 0 day, 1 day, 3 days, 5 days, 7 days, 9 days and 14 days; the neomycin administration (panel C) is firstly subjected to PCA analysis and then is subjected to PLS-DA re-verification, and the results show that 5 groups can be obviously separated in 0 day, 1 day, 3 days, 5-9 days and 14 days, which indicates that the constructed method can be used for rapidly identifying the development process of the aminoglycoside antibiotic drug model induced kidney injury.

Example 3

And (3) inspecting the application of the AuNPs-PEI/Proteins fluorescent array sensor in identifying the development process of the proton pump inhibitor (omeprazole, pantoprazole, lansoprazole and esomeprazole) model kidney injury.

1. The sensor was prepared as in example 1;

2. identifying the progress of renal progression injury in a proton pump inhibitor drug model. The method comprises the following steps:

(1) molding the medicine: the mice are divided into 4 groups, each group comprises 21 mice, each group of mice is respectively administered with omeprazole, pantoprazole, lansoprazole and esomeprazole by intragastric administration, the clinical dosage of the human is converted into the dosage of the experimental mice, and the dosage is respectively 6.07mg/kg/d, 4.55mg/kg/d and 6.07mg/kg/d by continuous administration for 14 days.

FIG. 9 shows the Principal Component Analysis (PCA) score plot and partial least squares analysis (PLS-DA) plot of the sensor versus proton pump inhibitor drug model. 1 for 0 days, 2 for 1 day, 3 for 3 days, 4 for 5 days, 5 for 7 days, 6 for 9 days, and 7 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 the PCA unsupervised mode, the urine fractions of mice dosed with omeprazole (panel a) on different days were significantly aggregated into 6 groups, of which 0-day group, 1-day group, 3-5-day group, 7-day group, 9-day group, and 14-day group. 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 verification of PLS-DA is carried out again, and the result shows that 6 groups can be obviously separated in 0 day, 1 day, 3-5 days, 7 days, 9 days and 14 days; by the same principle, the pantoprazole administration (figure B) is firstly analyzed by PCA and then verified by PLS-DA, and the results show that 7 groups can be obviously separated in 0 day, 1 day, 3 days, 5 days, 7 days, 9 days and 14 days; the Lansoprazole administration (panel C) is firstly subjected to PCA analysis and then subjected to PLS-DA re-verification, and the results show that the 5 groups can be obviously separated in 0 day, 1-3 days, 5-7 days, 9 days and 14 days; the results of the PCA analysis of the esomeprazole administration (panel D) followed by the re-validation of PLS-DA showed that the 6 groups were clearly separated by 0, 1-3, 5, 7, 9 and 14 days; the constructed method is demonstrated to be used for the rapid identification of the development process of the proton pump inhibitor drug model induced kidney injury.

Example 4

And (3) inspecting the application of the AuNPs-PEI/Proteins fluorescent array sensor in identifying the development process of the non-steroidal anti-inflammatory drug (ibuprofen, naproxen and diclofenac sodium) model kidney injury.

1. The sensor was prepared as in example 1;

2. identifying a non-steroidal anti-inflammatory drug model of the kidney development injury process. The method comprises the following steps:

(1) molding the medicine: the mice are divided into 3 groups, each group comprises 21 mice, each group of mice is respectively administered with ibuprofen, naproxen and diclofenac sodium by intragastric administration, the dosage is converted into the dosage of experimental mice by the clinical dosage of human, and the dosage is respectively 91.0mg/kg/d, 121.3mg/kg/d and 15.2mg/kg/d for 14 days of continuous administration.

FIG. 10 shows a Principal Component Analysis (PCA) score plot and partial least squares analysis (PLS-DA) plot of the sensor versus NSAID drug model. 1 for 0 days, 2 for 1 day, 3 for 3 days, 4 for 5 days, 5 for 7 days, 6 for 9 days, and 7 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. In the PCA unsupervised mode, mice dosed with ibuprofen (panel a) on different days had a distinct urine pool of 6 groups, with a 0-day group, a 1-day group, a 3-5-day group, a 7-day group, a 9-day group, and a 14-day group. 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 verification of PLS-DA is carried out again, and the result shows that 6 groups can be obviously separated in 0 day, 1 day, 3-5 days, 7 days, 9 days and 14 days; on the same reason, the naproxen administration (B picture) is firstly analyzed by PCA and then verified by PLS-DA, and the results show that 6 groups can be obviously separated in 0 day, 1 day, 3 days, 5-7 days, 9 days and 14 days; the results of the PCA analysis of diclofenac sodium administration (figure C) and the subsequent validation of PLS-DA show that the 0 day, 1 day, 3-5 days, 7 days, 9 days and 14 days of 6 groups can be obviously separated, which indicates that the constructed method can be used for the rapid identification of the development process of the non-steroidal anti-inflammatory drug induced kidney injury.

Example 5

Three mechanisms of action were selected for nephrotoxic drugs: aminoglycoside antibiotics (gentamicin, tobramycin, neomycin), proton pump inhibitors (omeprazole, pantoprazole, lansoprazole, esomeprazole) and non-steroidal anti-inflammatory drugs (ibuprofen, naproxen, diclofenac sodium).

The administration dose is converted into the dose of a mouse experimental animal according to the clinical dose of a human, and the difference of the action mechanisms of sensors on the renal injury induced by the three major classes of nephrotoxic drugs at different administration time points (0 day, 1 day, 3 days, 5 days, 7 days, 9 days and 14 days) is considered.

1. The sensor was prepared as in example 1;

2. the mechanism of renal tissue injury by three classes of drugs (as exemplified by gentamicin) was differentiated at different time points. Comprises the following steps

(1) Molding the medicine: the mice were gavaged and administered with gentamicin, the dose was converted to the clinical dose of human, and the doses were 97.07mg/kg/d for 14 days.

(4) The fluorescence values were read and calculated: after a sensor is added, the mixture is placed in a fluorescence microplate reader, reading values are optimally excited and emitted by 3 signal molecules respectively, excitation is carried out at 495nm, and fluorescence signals are collected at 520 nm; exciting at 550nm, and collecting fluorescence signals at 575 nm; 654nm excitation, 667nm collection fluorescence signal, bandwidth of 5nm, as drug in different days of administration mouse urine peculiar fluorescence fingerprint.

FIG. 11 shows a partial minimum discriminant analysis (PLS-DA) of the renal injury induced by three mechanisms of action of a nephrotoxic drug at different time points of administration of the sensor. 1 represents an aminoglycoside antibiotic, 2 represents a proton pump inhibitor, and 3 represents a non-steroidal anti-inflammatory drug. PLS-DA analysis was first performed using 18 variables as indicators, processed by SIMCA-P software, and the results indicated that the three classes of drugs could be initially separated, whereas the aminoglycoside drug had some crossover with the proton pump inhibitor (panel A). Then, the results are further analyzed, and by using the VIP value (VIP value > 1) in the software, six signal molecules, namely, BSA-FITC signals of 3 days, 5 days and 7 days after administration, beta-Lac-Cy 5 of 5 days and 7 days after administration and PNA-RhB of 5 days after administration, play a main recognition role in 18 signal molecules, so that the fluorescence values of the six signal molecules are taken as variables to carry out further analysis, and the results in a B diagram show that the sensor can better distinguish three major classes of nephrotoxic drugs according to the action mechanisms of renal injury, thereby indicating that the constructed method can be used for quickly recognizing the action mechanisms of the nephrotoxic drugs.

Figure 12 presents a Linear Discriminant Analysis (LDA) score plot of the sensor for nephrotoxic drug-induced renal injury for the three mechanisms of action at different dosing time points. 1 represents an aminoglycoside antibiotic, 2 represents a proton pump inhibitor, and 3 represents a non-steroidal anti-inflammatory drug. 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 LDA can be used for quantitatively distinguishing the fluorescence reaction mode of a sensor and a nephrotoxic drug induced renal injury urine sample of three action mechanisms. In the figure, each point represents the response pattern of the nephrotoxic drug urine to the sensor for each mechanism, and the mouse urine was successfully divided into 3 groups according to the difference of the mechanism of action.

According to the LDA analysis result, grouping conditions of the LDA are quantified, six variables of the VIP value are taken as indexes, and a constructed linear discriminant function is as follows:

Y1＝1.186X1-0.353X2+7.049X3+1.514X4+2.491X5+0.909X6-177.657；

Y2＝0.411X1+0.017X2+7.264X3+2.039X4+2.210X5+0.615X6-205.042；

Y3-0.556X 1-0.191X2+7.916X3+1.965X4+3.477X5+1.440X 6-260.784. Wherein Y1-3 represents the renal injury mechanism group, X1, X2, X3, X4, X5 and X6 represent the fluorescence intensity of 6 signal molecules. The value of the urine sample induced fluorescence signal of unknown injury mechanism is directly brought into the constructed discriminant function, the grouping condition is attributed, and the action mechanism of renal injury can be rapidly identified.

Claims

1. A multi-channel array sensor is characterized by comprising nanogold-polyethyleneimine AuNPs-PEI copolymer and protein modified with fluorophores of three different emission wavelengths.

2. The multi-channel array sensor of claim 1, wherein the proteins modified with fluorophores of three different emission wavelengths are bovine serum albumin-FITC, peanut agglutinin-RhB, and β -lactoglobulin-Cy 5.

3. The multi-channel array sensor of claim 2, wherein the bovine serum albumin-FITC, peanut agglutinin-RhB and beta-lactoglobulin-Cy 5 excitation wavelengths and emission wavelengths are, respectively, in the following order: 495nm, 520nm, 550nm, 575nm, 654nm and 667 nm.

4. A method of manufacturing a multi-channel array sensor as claimed in any one of claims 1 to 3, comprising the steps of:

(1) preparing a nanogold-polyethyleneimine AuNPs-PEI copolymer carrier: adding chloroauric acid HAuCl4 & 3H2O and polyethyleneimine PEI into a buffer solution, stirring at room temperature in a dark place, filtering, dialyzing to remove unreacted chloroauric acid and polyethyleneimine, and obtaining a nanogold-polyethyleneimine AuNPs-PEI copolymer carrier;

(2) and (3) constructing a sensor: and fully mixing the nanogold-polyethyleneimine AuNPs-PEI copolymer carrier with the protein modified with three fluorophores with different emission wavelengths to obtain the AuNPs-PEI/Proteins sensor.

5. The method for preparing a multi-channel array sensor according to claim 4, wherein in the step (1), the mass ratio of the chloroauric acid to the polyethyleneimine is 1:1-1: 10.

6. The method for preparing a multi-channel array sensor according to claim 4, wherein in the step (1), the weight average molecular weight of the polyethyleneimine is 600Da-10000 Da.

7. The method for preparing a multi-channel array sensor according to claim 4, wherein in the step (2), the concentration of the nanogold-polyethyleneimine AuNPs-PEI copolymer is 0.01ng mL^-1-12.5ng mL^-1The concentration of the three proteins modified with fluorophores of different emission wavelengths was 0.0075 nmol/mL.

8. Use of the multi-channel array sensor of claim 1 for monitoring the progression of renal injury caused by different nephrotoxic drugs.

9. Use of a multi-channel array sensor according to claim 1 for identifying the mechanism of action of different nephrotoxic drugs.

10. The use of claim 8 or 9, wherein the nephrotoxic drug comprises an aminoglycoside antibiotic, a nonsteroidal anti-inflammatory drug, and a proton pump inhibitor.