CN114231594A - Method for detecting thrombin activity and screening thrombin inhibitor in plasma by polypeptide microarray chip - Google Patents

Abstract

The invention discloses a method for detecting thrombin activity and screening thrombin inhibitors in plasma by using a polypeptide microarray chip, and relates to the technical field of polypeptide microarray chips. The polypeptide microarray chip detects thrombin activity in plasma, S1: placing the polypeptide microarray chip and activated plasma together for reaction, wherein the reaction temperature is kept between thirty and thirty-six degrees, the reaction time is kept over one hour, and S2: inhibitor detection inhibitors with different concentrations are respectively mixed with thrombin solution (30U/mL), standard serum (containing 30U/mL thrombin) or plasma, the using concentration of the serum or the plasma is 33 percent, the mixed solution reacts with a chip at 37 ℃ for 1h, and the chip is washed 3 times by washing solution-I, washing solution-II and ultrapure water containing 0.1 percent (V/V) Triton X-100 in sequence and is dried by spinning.

Description

Method for detecting thrombin activity and screening thrombin inhibitor in plasma by polypeptide microarray chip

Technical Field

The invention relates to the technical field of polypeptide microarray chips, in particular to a method for detecting thrombin activity and screening thrombin inhibitors in plasma by using the polypeptide microarray chip.

Background

The microarray biochip technology can integrate multiple biochemical reactions on one chip, and can realize efficient and rapid testing and analysis of bioactive substances. The microarray chip for immobilizing protease or a protease-specific substrate can simultaneously analyze the action mechanism and the action efficiency between various inhibitors and the protease 8.01. Ottamchandri et al simultaneously tested the inhibition efficiency of 3 compounds against 4 cysteine proteases using a protein microarray chip 10; neumann et al screened 6 compounds that bind to the thrombin active site using a small molecule microarray chip from nearly 10 million organic small molecules.

The existing method for screening thrombin inhibitors is generally only available, and the method can be used for calculating in an enzyme solution, so that a method for detecting thrombin activity and screening thrombin inhibitors in plasma by using a polypeptide microarray chip is provided.

Disclosure of Invention

The present invention is directed to at least one of the technical problems of the prior art, and provides a method for detecting thrombin activity and screening thrombin inhibitors in plasma using a polypeptide microarray chip.

In order to achieve the purpose, the invention provides the following technical scheme: polypeptide microarray chip for detecting thrombin activity in plasma

S1: placing the polypeptide microarray chip and activated plasma together for reaction, wherein the reaction temperature is kept between thirty and thirty-six degrees, and the reaction time is kept over one hour;

s2: inhibitor detection is carried out by mixing inhibitors with different concentrations with thrombin solution (30U/mL), spiked serum (containing 30U/mL thrombin) or plasma with the use concentration of 33%, reacting the mixed solution with chip at 37 deg.C for 1h, washing the chip with washing solution-I, washing solution-II and ultrapure water containing 0.1% (V/V) Triton X-100 for 3 times, and spin-drying;

s3: the enzyme hydrolysis process identifies that avidin marked by 3 mu mol/L fluorescein reacts with the chip at 37 ℃ for 1h, the chip is washed for 3 times by 1% (V/V) Tween-20-containing BSA washing solution-1, BSA-containing washing solution-I and ultrapure water in sequence, the chip is dried by spinning, a scanner is used for scanning, and gold nano particles with 0.3nmol/L react with the chip at 37 ℃ for 1 h;

s4: a thrombin solution (30U/ml) was prepared with an enzyme reaction buffer (pH 7.35,20mmol/L HEPES,0.14mol/L NaCl,2mmol/L CaCI2), reacted with the chip at 37 ℃ for 1 hour, and the chip was washed 3 times with wash solution-II (pH7.5, 20mmol/L LTris,2mmol/L EDTA,0.15mol/L NaCI) containing 0.1% (V/V). Triton X-100, and then spin-dried.

Method for screening thrombin inhibitor in plasma by polypeptide microarray chip

S1: blood sample preparation blood was collected by centrifugation within 6h, and frozen to fresh frozen plasma, stored at-80 ℃, 40. mu.L of plasma was diluted 1-fold with plasma dilution buffer (pH 7.35,20mmol/L HEPES,21.7mmol/L sodium citrate 60g/L BSA), 20. mu.L of activator (30pmol/L recombinant human tissue factor and 24. mu. mol/L phospholipid mixture (phosphatidylserine: phosphatidylethanolamine: phosphatidylcholine: 1:3, molar ratio), incubated at 37 ℃ for 5min, 20. mu.L HEPES buffer (pH 7.35,20mmol/L HEPES,60g/L HEPES BSA,0.1mol/L CaCl2) was added to prepare activated plasma, 1300g of activated plasma was centrifuged for 10min, and serum was isolated.

S2: preparing a gold nanoparticle probe, adding 2mL of 1% chloroauric acid solution into 100mL of ultrapure water, heating to boil, adding 2mL of 2% sodium citrate solution, continuously heating and stirring for 30min, synthesizing gold nanoparticles with the particle size of 30nm, taking 120 mu L of 1G/L CALNNNGK (biotin) G mixed solution (9:1, V/V), adding into 10mL of.4nmol/L gold nanoparticle solution, standing at room temperature for 1h, centrifuging to remove supernatant, suspending in probe buffer solution (pH7.5,50 mmol/L phosphate solution, 0.15mol/L NaCI, 0.1% (V/V) Tween-20, 1% (w0/V) BSA, and storing at 4 ℃;

s3: preparing a polypeptide microarray chip and preparing an enzyme hydrolysis process, preparing 0.5g/L polypeptide (CAEGGfPRRVVK (biotin)) solution by using spotting buffer solution (pH 8.5,0.3mol/L phosphate, 0.2mol/L NaCl, 35% (V/V) glycerol and 0.002% (20/V) BSA), spotting the solution on an optical-grade three-dimensional polymer D substrate by using a chip microarray chip spotting system, reacting for 14h at 30 ℃, covalently reacting aldehyde groups on the surface of the chip with amino and sulfhydryl groups on N-terminal cysteine residues of the polypeptide to form five-membered ring structures, fixing the polypeptide on the surface of the chip, washing for 2 times by using washing solution-1 (pH7.5,50mmol/I phosphate and 1% (10/V) BSA containing 0.1% (V/V) 20, placing blocking solution (pH7.5,50 mmol/L phosphate, 0.15mol/L NaCl, 1% (w/V) BSA,0.1mol/L ethanolamine) 309C for 1h, washed 3 times with ultrapure water, spun dry (480g,1min), and formulated with enzyme reaction buffer (pH 7.35,20mmol/L HEPES,0.14mol/L NaCl,2mmol/L CaCI2) to give 30U/mL gels

Reacting the hemozyme solution with the chip at 37 ℃ for 1 h; the chip was washed 3 times with washing solution-II (pH7.5, 20mmol/LTris,2mmol/LEDTA,0.15mol/L NaCI) containing 0.1% (V/V). Triton X-100, followed by spin-drying.

S4: respectively mixing the inhibitors with different concentrations with a thrombin solution (30U/mL), a standard serum (containing 30U/mL thrombin) or plasma, wherein the using concentration of the serum or the plasma is 33%, reacting the mixed solution with the chip at 37 ℃ for 1h, washing the chip for 3 times by using a washing solution-I, a washing solution-II and ultra-pure water which respectively contain 0.1% (V/V) Triton X-100, and spin-drying;

s5, extracting resonance light scattering signals by a chip scanner for signal detection and data analysis, wherein all data are signal values after background subtraction, calculating average values and standard deviations by using signal values of 6 identical points, and in inhibitor detection, a subarray without an inhibitor is used as a negative control and is defined as a minimum signal value of 1 mm; subarrays without thrombin served as positive controls and were defined as the maximum signal value mn. The relative signal intensity of the subarray was calculated using equation (1) where Δ I ═ I-Imin)/(Imax-Imin) x 100% where I is the signal value of the subarray to which both thrombin and inhibitor were added.

Compared with the prior art, the invention has the beneficial effects that:

(1) the method can quantitatively calculate the inhibition efficiency of the inhibitor in an enzyme solution and serum, and investigate the influence of a complex serum environment on the inhibitor, thereby solving the problem that the conventional method for screening the thrombin inhibitor can only be generally used, and the method can not only be used for calculating in the enzyme solution.

Detailed Description

In the description of the present invention, greater than, less than, exceeding, etc. are understood as excluding the present numbers, and the above, below, inside, etc. are understood as including the present numbers. If the first and second are described for the purpose of distinguishing technical features, they are not to be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated or implicitly indicating the precedence of the technical features indicated.

In the description of the present invention, unless otherwise explicitly limited, terms such as arrangement, installation, connection and the like should be understood in a broad sense, and those skilled in the art can reasonably determine the specific meanings of the above terms in the present invention in combination with the specific contents of the technical solutions.

Polypeptide microarray chip for detecting thrombin activity in plasma

S1: placing the polypeptide microarray chip and activated plasma together for reaction, wherein the reaction temperature is kept between thirty and thirty-six degrees, and the reaction time is kept over one hour;

s2: inhibitor detection is carried out by mixing inhibitors with different concentrations with thrombin solution (30U/mL), spiked serum (containing 30U/mL thrombin) or plasma with the use concentration of 33%, reacting the mixed solution with chip at 37 deg.C for 1h, washing the chip with washing solution-I, washing solution-II and ultrapure water containing 0.1% (V/V) Triton X-100 for 3 times, and spin-drying;

s3: the enzyme hydrolysis process identifies that avidin marked by 3 mu mol/L fluorescein reacts with the chip at 37 ℃ for 1h, the chip is washed for 3 times by 1% (V/V) Tween-20-containing BSA washing solution-1, BSA-containing washing solution-I and ultrapure water in sequence, the chip is dried by spinning, a scanner is used for scanning, and gold nano particles with 0.3nmol/L react with the chip at 37 ℃ for 1 h;

s4: a thrombin solution (30U/ml) was prepared with an enzyme reaction buffer (pH 7.35,20mmol/L HEPES,0.14mol/L NaCl,2mmol/L CaCI2), reacted with the chip at 37 ℃ for 1 hour, and the chip was washed 3 times with wash solution-II (pH7.5, 20mmol/L LTris,2mmol/L EDTA,0.15mol/L NaCI) containing 0.1% (V/V). Triton X-100, and then spin-dried.

Method for screening thrombin inhibitor in plasma by polypeptide microarray chip

S1: blood sample preparation blood was collected by centrifugation within 6h, and frozen to fresh frozen plasma, stored at-80 ℃, 40. mu.L of plasma was diluted 1-fold with plasma dilution buffer (pH 7.35,20mmol/L HEPES,21.7mmol/L sodium citrate 60g/L BSA), 20. mu.L of activator (30pmol/L recombinant human tissue factor and 24. mu. mol/L phospholipid mixture (phosphatidylserine: phosphatidylethanolamine: phosphatidylcholine: 1:3, molar ratio), incubated at 37 ℃ for 5min, 20. mu.L HEPES buffer (pH 7.35,20mmol/L HEPES,60g/L HEPES BSA,0.1mol/L CaCl2) was added to prepare activated plasma, 1300g of activated plasma was centrifuged for 10min, and serum was isolated.

S2: preparing a gold nanoparticle probe, adding 2mL of 1% chloroauric acid solution into 100mL of ultrapure water, heating to boil, adding 2mL of 2% sodium citrate solution, continuously heating and stirring for 30min, synthesizing gold nanoparticles with the particle size of 30nm, taking 120 mu L of 1G/L CALNNNGK (biotin) G mixed solution (9:1, V/V), adding into 10mL of.4nmol/L gold nanoparticle solution, standing at room temperature for 1h, centrifuging to remove supernatant, suspending in probe buffer solution (pH7.5,50 mmol/L phosphate solution, 0.15mol/L NaCI, 0.1% (V/V) Tween-20, 1% (w0/V) BSA, and storing at 4 ℃;

s3: preparing a polypeptide microarray chip and preparing an enzyme hydrolysis process, preparing 0.5g/L polypeptide (CAEGGfPRRVVK (biotin)) solution by using spotting buffer solution (pH 8.5,0.3mol/L phosphate, 0.2mol/L NaCl, 35% (V/V) glycerol and 0.002% (20/V) BSA), spotting the solution on an optical-grade three-dimensional polymer D substrate by using a chip microarray chip spotting system, reacting for 14h at 30 ℃, covalently reacting aldehyde groups on the surface of the chip with amino and sulfhydryl groups on N-terminal cysteine residues of the polypeptide to form five-membered ring structures, fixing the polypeptide on the surface of the chip, washing for 2 times by using washing solution-1 (pH7.5,50mmol/I phosphate and 1% (10/V) BSA containing 0.1% (V/V) 20, placing blocking solution (pH7.5,50 mmol/L phosphate, 0.15mol/L NaCl, 1% (w/V) BSA,0.1mol/L ethanolamine) 309C for 1h, washed 3 times with ultrapure water, spun dry (480g,1min), and formulated with enzyme reaction buffer (pH 7.35,20mmol/L HEPES,0.14mol/L NaCl,2mmol/L CaCI2) to give 30U/mL gels

Reacting the hemozyme solution with the chip at 37 ℃ for 1 h; the chip was washed 3 times with washing solution-II (pH7.5, 20mmol/LTris,2mmol/LEDTA,0.15mol/L NaCI) containing 0.1% (V/V). Triton X-100, followed by spin-drying.

S4: respectively mixing the inhibitors with different concentrations with a thrombin solution (30U/mL), a standard serum (containing 30U/mL thrombin) or plasma, wherein the using concentration of the serum or the plasma is 33%, reacting the mixed solution with the chip at 37 ℃ for 1h, washing the chip for 3 times by using a washing solution-I, a washing solution-II and ultra-pure water which respectively contain 0.1% (V/V) Triton X-100, and spin-drying;

s5, extracting resonance light scattering signals by a chip scanner for signal detection and data analysis, wherein all data are signal values after background subtraction, calculating average values and standard deviations by using signal values of 6 identical points, and in inhibitor detection, a subarray without an inhibitor is used as a negative control and is defined as a minimum signal value of 1 mm; subarrays without thrombin served as positive controls and were defined as the maximum signal value mn. The relative signal intensity of the subarray was calculated using equation (1) where Δ I ═ I-Imin)/(Imax-Imin) x 100% where I is the signal value of the subarray to which both thrombin and inhibitor were added.

The present invention is not limited to the above-described embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the spirit of the present invention.

Claims (2)

1. The polypeptide microarray chip for detecting thrombin activity in plasma is characterized by comprising the following steps:

s1: placing the polypeptide microarray chip and activated plasma together for reaction, wherein the reaction temperature is kept between thirty and thirty-six degrees, and the reaction time is kept over one hour;

s2: inhibitor detection is carried out by mixing inhibitors with different concentrations with thrombin solution (30U/mL), spiked serum (containing 30U/mL thrombin) or plasma with the use concentration of 33%, reacting the mixed solution with chip at 37 deg.C for 1h, washing the chip with washing solution-I, washing solution-II and ultrapure water containing 0.1% (V/V) Triton X-100 for 3 times, and spin-drying;

s3: the enzyme hydrolysis process identifies that avidin marked by 3 mu mol/L fluorescein reacts with the chip at 37 ℃ for 1h, the chip is washed for 3 times by 1% (V/V) Tween-20-containing BSA washing solution-1, BSA-containing washing solution-I and ultrapure water in sequence, the chip is dried by spinning, a scanner is used for scanning, and gold nano particles with 0.3nmol/L react with the chip at 37 ℃ for 1 h;

s4: a thrombin solution (30U/ml) was prepared with an enzyme reaction buffer (pH 7.35,20mmol/L HEPES,0.14mol/L NaCl,2mmol/L CaCI2), reacted with the chip at 37 ℃ for 1 hour, and the chip was washed 3 times with Wash-II (pH7.5, 20mmol/L Tris,2mmol/L LEDTA,0.15mol/L NaCI) containing 0.1% (V/V). Triton X-100, and then spin-dried.

2. The method for screening thrombin inhibitors in blood plasma by using the polypeptide microarray chip is characterized by comprising the following steps of:

s1: blood sample preparation blood was collected by centrifugation within 6h, and frozen to fresh frozen plasma, stored at-80 ℃, 40. mu.L of plasma was diluted 1-fold with plasma dilution buffer (pH 7.35,20mmol/L HEPES,21.7mmol/L sodium citrate 60g/L BSA), 20. mu.L of activator (30pmol/L recombinant human tissue factor and 24. mu. mol/L phospholipid mixture (phosphatidylserine: phosphatidylethanolamine: phosphatidylcholine: 1:3, molar ratio), incubated at 37 ℃ for 5min, 20. mu.L HEPES buffer (pH 7.35,20mmol/L HEPES,60g/L HEPES BSA,0.1mol/L CaCl2) was added to prepare activated plasma, 1300g of activated plasma was centrifuged for 10min, and serum was isolated.

S2: preparing a gold nanoparticle probe, adding 2mL of 1% chloroauric acid solution into 100mL of ultrapure water, heating to boil, adding 2mL of 2% sodium citrate solution, continuously heating and stirring for 30min, synthesizing gold nanoparticles with the particle size of 30nm, taking 120 mu L of 1G/L CALNNNGK (biotin) G mixed solution (9:1, V/V), adding into 10mL of.4nmol/L gold nanoparticle solution, standing at room temperature for 1h, centrifuging to remove supernatant, suspending in probe buffer solution (pH7.5,50 mmol/L phosphate solution, 0.15mol/L NaCI, 0.1% (V/V) Tween-20, 1% (w0/V) BSA, and storing at 4 ℃;

s3: preparing a polypeptide microarray chip and preparing an enzyme hydrolysis process, preparing 0.5g/L polypeptide (CAEGGfPRRVVK (biotin)) solution by using spotting buffer solution (pH 8.5,0.3mol/L phosphate, 0.2mol/L NaCl, 35% (V/V) glycerol and 0.002% (20/V) BSA), spotting the solution on an optical-grade three-dimensional polymer D substrate by using a chip microarray chip spotting system, reacting for 14h at 30 ℃, covalently reacting aldehyde groups on the surface of the chip with amino and sulfhydryl groups on N-terminal cysteine residues of the polypeptide to form five-membered ring structures, fixing the polypeptide on the surface of the chip, washing for 2 times by using washing solution-1 (pH7.5,50mmol/I phosphate and 1% (10/V) BSA containing 0.1% (V/V) 20, placing blocking solution (pH7.5,50 mmol/L phosphate, 0.15mol/L NaCl, 1% (w/V) BSA,0.1mol/L ethanolamine) 309C for 1h, washed 3 times with ultrapure water, spun dry (480g,1min), and formulated with enzyme reaction buffer (pH 7.35,20mmol/L HEPES,0.14mol/L NaCl,2mmol/L CaCI2) to give 30U/mL gels

Reacting the hemozyme solution with the chip at 37 ℃ for 1 h; the chip was washed 3 times with washing solution-II (pH7.5, 20mmol/LTris,2mmol/LEDTA,0.15mol/L NaCI) containing 0.1% (V/V). Triton X-100, followed by spin-drying.

S4: respectively mixing the inhibitors with different concentrations with a thrombin solution (30U/mL), a standard serum (containing 30U/mL thrombin) or plasma, wherein the using concentration of the serum or the plasma is 33%, reacting the mixed solution with the chip at 37 ℃ for 1h, washing the chip for 3 times by using a washing solution-I, a washing solution-II and ultra-pure water which respectively contain 0.1% (V/V) Triton X-100, and spin-drying;

s5, extracting resonance light scattering signals by a chip scanner for signal detection and data analysis, wherein all data are signal values after background subtraction, calculating average values and standard deviations by using signal values of 6 identical points, and in inhibitor detection, a subarray without an inhibitor is used as a negative control and is defined as a minimum signal value of 1 mm; subarrays without thrombin served as positive controls and were defined as the maximum signal value mn. The relative signal intensity of the subarray was calculated using equation (1) where Δ I ═ I-Imin)/(Imax-Imin) x 100% where I is the signal value of the subarray to which both thrombin and inhibitor were added.