CN114178251B - Method for regenerating sample tube for multiparameter protein stability analyzer - Google Patents

Method for regenerating sample tube for multiparameter protein stability analyzer Download PDF

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CN114178251B
CN114178251B CN202111402629.5A CN202111402629A CN114178251B CN 114178251 B CN114178251 B CN 114178251B CN 202111402629 A CN202111402629 A CN 202111402629A CN 114178251 B CN114178251 B CN 114178251B
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CN114178251A (en
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范仕龙
王乐乐
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Tsinghua University
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B08—CLEANING
    • B08B—CLEANING IN GENERAL; PREVENTION OF FOULING IN GENERAL
    • B08B3/00—Cleaning by methods involving the use or presence of liquid or steam
    • B08B3/04—Cleaning involving contact with liquid
    • B08B3/10—Cleaning involving contact with liquid with additional treatment of the liquid or of the object being cleaned, e.g. by heat, by electricity or by vibration
    • B08B3/12—Cleaning involving contact with liquid with additional treatment of the liquid or of the object being cleaned, e.g. by heat, by electricity or by vibration by sonic or ultrasonic vibrations
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B08—CLEANING
    • B08B—CLEANING IN GENERAL; PREVENTION OF FOULING IN GENERAL
    • B08B13/00—Accessories or details of general applicability for machines or apparatus for cleaning
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B08—CLEANING
    • B08B—CLEANING IN GENERAL; PREVENTION OF FOULING IN GENERAL
    • B08B9/00—Cleaning hollow articles by methods or apparatus specially adapted thereto 
    • B08B9/02—Cleaning pipes or tubes or systems of pipes or tubes
    • B08B9/023—Cleaning the external surface
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B08—CLEANING
    • B08B—CLEANING IN GENERAL; PREVENTION OF FOULING IN GENERAL
    • B08B9/00—Cleaning hollow articles by methods or apparatus specially adapted thereto 
    • B08B9/02—Cleaning pipes or tubes or systems of pipes or tubes
    • B08B9/027—Cleaning the internal surfaces; Removal of blockages
    • B08B9/032—Cleaning the internal surfaces; Removal of blockages by the mechanical action of a moving fluid, e.g. by flushing
    • B08B9/0321—Cleaning the internal surfaces; Removal of blockages by the mechanical action of a moving fluid, e.g. by flushing using pressurised, pulsating or purging fluid
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    • F26—DRYING
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Abstract

The invention provides a regeneration method of a sample tube for a multi-parameter protein stability analyzer. After a regeneration experiment, the sample tube has excellent regeneration effect through data analysis, the coefficient of variation is less than 10%, and the optimal coefficient can be within 1%. Meanwhile, the regeneration times can reach 5 times, and the user cost in the final technical service process can be reduced to 15 percent. If the alkaline solution with the pH value of 9.0 to 11.0 or the acidic solution with the pH value of 1.5 to 3.0 is selected as the regenerant, the corrosion of strong acid and strong base to the sample tube can be effectively avoided, and the situation that the weak acid and weak base cannot achieve the regeneration effect can not occur. If the soaking time of the sample tube is 20 to 60 minutes or the ultra-sonic time of the ultrapure water is 30 to 120 seconds, the regeneration effect can be ensured, and meanwhile, too long experimental time cannot be wasted.

Description

Method for regenerating sample tube for multiparameter protein stability analyzer
Technical Field
The invention relates to sample tube regeneration, in particular to a method for regenerating a sample tube of a multi-parameter protein stability analyzer.
Background
The protein is a main executor of the physiological function of a living body, and the natural structure of the protein is the basis of the biological function, so that the stability research of the protein structure is widely applied to the fields of basic research of life science, the research and development of new drugs, the design and screening of drug molecules, the development of biological drug preparations, the optimization of biological molecular structures and the like. The existing protein/antibody/vaccine stability detection technology and method mainly comprise the following steps: the incubator method combines Size Exclusion Chromatography (SEC), PCR technology (dye method), DSC technology and DSF technology (dye method).
A multiparameter protein stability analyzer (model: uncle; specification: 54cm wide by 50cm deep by 58cm high by 50kg; manufacturing unit: uncariamed Labs company, USA) can realize the detection of various parameters of protein molecule conformation stability and solution colloid stability by adopting an unmarked endogenous fluorescence detection technology and a light scattering technology, and provides a series of protein stability related data such as Tm (thermal denaturation temperature), tagg (protein aggregation temperature), sample particle size and polydispersity, B22 (second dimensional coefficient), kD (diffusion interaction coefficient) and solution viscosity. In addition, the multi-parameter protein stability analyzer can be used for virus-like particle stability research, antibody drug coupling structure research, protein/small molecule combination/interaction research, biological agent stability research, wild species and mutant protein stability research, protein spatial structure and peptide chain folding mode and biological function correlation research, protein modification (such as glycosylation) influence research on structure stability, biological drug nanoparticle carrier stability research, structure biological research and the like. Through in-depth analysis of market demands and the existing technical method, the Uncle system of the protein stability analyzer has the characteristics of high flux (48 samples can be detected in one experiment and 144 samples can be analyzed in one day), multiple parameters (11 applications), trace quantity (only 9 mu l and 0.45 mu g of each sample are needed), four Tm analysis methods (freely selected according to protein denaturation fluorescence characteristics) and the like, so that the working efficiency is greatly improved, and the working cost is reduced.
Whether from the perspective of high efficiency, accuracy, or user operational experience, such multi-parameter protein stability analyzers are excellent protein stability research systems, but it is the cost of consumables that currently gives all users a concern. The sample tube of the multi-parameter protein stability analyzer is a capillary tube which is refined by quartz, is provided with an upper opening and a lower opening and is embedded in a magnetic material, each sample tube is provided with 16 sample adding holes, and the light transmittance and the heat conductivity are good. At present, the regeneration of quartz products adopts the procedures of ordinary acid soaking, spraying, drying and the like, but for the sample tube, the inner diameter and the outer diameter of the capillary tube are very small, so that the sample tube cannot be washed to a level capable of being reused by a conventional method. The cost of the sample tube is very high, the price of a single sample tube is more than 45 dollars, and if the sample tube is used once, the cost is very high for users, and the cost of a laboratory is greatly increased. Therefore, in order to reduce the use cost of users and practice "environmental protection" research, development of a new sample tube regeneration method is urgently needed.
Disclosure of Invention
The invention provides a regeneration method of a sample tube for a multi-parameter protein stability analyzer, which aims to improve the utilization rate of the sample tube and reduce the experiment cost.
A method for regenerating a sample tube for a multiparameter protein stability analyzer, comprising immersing the sample tube in a regenerant selected from the group consisting of an alkaline solution having a pH of 9.0 to 11.0 and/or an acidic solution having a pH of 1.5 to 3.0.
Optionally, the method further comprises the step of washing the sample tube with ultrapure water before soaking the sample tube in the regenerant.
Optionally, after the sample tube is soaked in the regenerant, the method further comprises the step of performing ultrasonic washing on the sample tube soaked in the regenerant in an ultrapure water bath, and optionally, the method further comprises rinsing the sample tube with ultrapure water after the ultrasonic washing.
Optionally, after rinsing the sample tube with ultrapure water, the method further comprises the step of blow-drying the sample tube with compressed air.
Optionally, wherein the acidic solution is selected from hydrochloric acid (HCl), sulfuric acid (H) 2 SO 4 ) Nitric acid (HNO) 3 ) And phosphoric acid (H) 3 PO 4 ) At least one of; the alkaline solution is selected from sodium hydroxide (NaOH), potassium hydroxide (KOH), and sodium silicate (Na) 2 SiO 3 ) And sodium carbonate (Na) 2 CO 3 ) At least one of (1).
Optionally, wherein the soaking time of the sample tube is in the range of 20 minutes to 60 minutes.
Optionally, wherein the time of the ultrasonic washing is in a range of 30 seconds to 120 seconds.
Optionally, wherein the steps of washing, soaking, sonicating, blow drying are repeated 2 to 5 times.
The technical scheme of the invention has the beneficial effects that:
after a regeneration experiment, the sample tube has excellent regeneration effect through data analysis, the coefficient of variation is less than 10%, and the optimal coefficient can reach within 1%. Meanwhile, the regeneration times can reach 5 times, and the user cost in the final technical service process can be reduced to 15 percent.
Optionally, an alkaline solution with a pH value of 9.0 to 11.0 is selected as a regenerant, so that the corrosion to the sample tube when the pH value of the alkaline solution is greater than 11.0 can be effectively avoided, and the regeneration effect cannot be achieved when the pH value is less than 9.0.
Optionally, an acidic solution with a pH value of 1.5 to 3.0 is selected as a regenerant, so that corrosion to the sample tube when the pH value of the acidic solution is less than 1.5 can be effectively avoided, and the regeneration effect cannot be achieved when the pH value is greater than 3.0.
Optionally, the soaking time of the sample tube is 20 to 60 minutes, so that the regeneration effect can be ensured, and meanwhile, too long experiment time is not wasted.
Optionally, the ultra-pure water ultrasonic time is 30 to 120 seconds, so that the regeneration effect can be ensured and meanwhile, too long experiment time is not wasted.
Drawings
FIG. 1 shows Tm and CV% of 4 protein solutions obtained after the sample tube is regenerated once by sodium hydroxide solutions with different pH values, wherein Tm represents the thermal denaturation temperature, and CV% represents the variation coefficient;
FIG. 2 shows a fluorescence signal curve and a static light scattering signal curve of a protein solution 7010 obtained after a sample tube is regenerated once by sodium hydroxide solutions with different pH values, wherein New Unis represents a brand New sample tube;
FIG. 3 shows Tm and CV% of 4 protein solutions obtained after the sample tube is regenerated once by hydrochloric acid solutions with different pH values;
FIG. 4 is a graph of fluorescence signal and static light scattering signal of IgG protein solution obtained after the sample tube is regenerated once by hydrochloric acid solutions of different pH values;
fig. 5 is Tm and CV% of 4 protein solutions obtained after five regenerations of the sample tube through a sodium hydroxide solution having a pH of 11 and an HCl solution having a pH of 3;
fig. 6 is a graph showing the fluorescence signal curve and the static light scattering signal curve of IgG protein solution obtained after five regenerations of the sample tube by a sodium hydroxide solution having a pH of 11 and an HCl solution having a pH of 3.
Fig. 7 shows Tm and CV% of 4 HSA solutions with different concentrations obtained after five regenerations of the sample tube with sodium hydroxide solutions with different pH values.
FIG. 8 is a graph showing fluorescence signal curves of 4 HSA solutions with different concentrations obtained after five regenerations of the sample tube with sodium hydroxide solutions with different pH values.
Fig. 9 shows Tm and CV% of 4 HSA solutions with different concentrations obtained after five regenerations of the sample tube with different pH values of hydrochloric acid solution.
FIG. 10 is a graph showing the fluorescent signal curves of 4 HSA solutions of different concentrations obtained after five regenerations of the sample tube in hydrochloric acid solutions of different pH values.
Detailed Description
As the sample tube belongs to the specially manufactured sample adding of various proteins for a multi-parameter protein stability analyzer, no related regeneration technology exists in China at present. The cost of the sample tube is very high, the price of a single sample tube is more than 45 dollars, and if the sample tube is used once, the sample tube is very expensive for protein users, so that the cost of a laboratory is greatly increased. In addition, at present, 30 multi-parameter protein stability analyzers Uncle in China are used with high efficiency, and the device is used by up to 150 units all over the world, so that the using amount of the sample tube is large.
Because the inner diameter and the outer diameter of the capillary tube of the sample tube are very small, the invention adopts a method combining the working procedures of soaking by a regenerant, ultrasonic treatment, compressed air blow-drying and the like, thereby achieving good regeneration effect. The regenerant adopted by the method is a reagent which is cheap and easy to obtain in a laboratory, the operation method is relatively simple, convenient and safe, and the method has wide application prospect in the actual experimental technology.
And (3) accurately calibrating the regeneration effect of the regenerated sample tube by using a multi-parameter protein stability analyzer Uncle, and determining the regeneration times of the sample tube.
The following embodiments will further explain the present invention by referring to the figures.
Example 1
A method for regenerating sample tubes using sodium hydroxide solutions of different pH values, comprising the steps of:
step 1, after the protein stability experiment is finished, washing off the protein solution in 3 sample tubes by using ultrapure water, and respectively soaking in 3 sodium hydroxide solution regenerants with pH values of 11.0, 10.0 and 9.0 for 30 minutes.
And 2, placing the sample tube soaked by the regenerant in an ultrasonic instrument, and performing ultrasonic treatment for 60 seconds by using ultrapure water.
And 3, washing the sample tube subjected to ultrasonic treatment with ultrapure water to flush residual regenerant.
And 4, finally, drying by using compressed air, and finishing the first regeneration experiment of the sample tube.
And 5, comparing Tm values of different water-soluble protein solutions before and after sample tube regeneration, coefficient of variation CV% calculated by formula 1 and coincidence degree of fluorescent signals or static light scattering signal curves of three repeated samples through testing and analysis.
Equation 1: CV% = SD/mean × 100%,
where SD refers to the standard deviation of all Tm before and after each regeneration, mean refers to the average of all Tm before and after each regeneration;
if CV% is less than 10%, the sample tube can be reused in the multi-parameter protein stability analyzer.
If the coincidence degree of the fluorescent signal curves or the static light scattering signal curves of the three repeated samples is good, the regeneration effect of the sample tube can be further explained to be good, and the regenerant can be preferably used.
And 6, selecting the sample tube soaked in the sodium hydroxide solution regenerant with the pH value of 11.0, and carrying out secondary regeneration according to the steps 1, 2, 3, 4 and 5.
And 7, carrying out third regeneration on the sample tube treated in the step 6 according to the same regeneration step.
And 8, carrying out fourth regeneration on the sample tube treated in the step 7 according to the same regeneration step.
And 9, performing fifth regeneration on the sample tubes treated in the step 8 according to the same regeneration step.
Wherein the protein stability experiment is as follows: placing 3 sample tubes loaded with 4 kinds of water-soluble protein solutions into a multi-parameter protein stability analyzer, and setting operation parameters as follows: the starting temperature of the temperature rising curve is 20 ℃; the heating rate is 0.3 ℃/min, the end temperature of the heating curve is 90 ℃, the running time is 4.5 hours, and the protein solution parameters under the New Unis condition are obtained and used as comparison data.
Furthermore, the samples in each sample tube were four different kinds of water-soluble protein solutions, which were named 7009 (Cas 13b protein, purity 90% or more, manufacturing unit: expression and purification of X-ray crystallography platform at university of Qinghua), 7010 (Cas 13a protein, purity 90% or more, manufacturing unit: expression and purification of X-ray crystallography platform at university of Qinghua), aux (Synpsin-1 protein, purity 90% or more, manufacturing unit: expression and purification of X-ray crystallography platform at university of Qinghua), and IgG (IgG from virus server, cat number: I5506-100MG, manufacturer: aldrich, co). The protein solution concentrations were 2.2mg/mL,1.8mg/mL,0.8mg/mL,1.5mg/mL, respectively. All protein solution concentrations were determined using a microspectrophotometer, model Nano Drop. The sequence of adding samples is 1-3 for 7009,4-6 for 7010,7-9 for Aux, and 10-12 for IgG. 3 replicates of each protein solution were made, for a total of 12 samples per tube. The number of sample tubes per experiment was 3, and the sample volume and sequence were the same for each.
Further, the data obtained in step 5 of example 1 are shown in fig. 1, and the coefficients of variation CV% of the four proteins obtained by regenerating the sodium hydroxide solution at the pH of 11.0 shown in fig. 1 are 7.76%,1.94%,2.69%, and 0.88%, respectively; the coefficient of variation CV% of four protein solutions obtained by regenerating the sodium hydroxide solution with the pH value of 10.0 are respectively 1.71%,2.07%,2.24% and 1.5%; the coefficient of variation CV% of the 4 protein solutions obtained by regenerating the sodium hydroxide solution with the pH value of 9.0 are respectively 1.0%,1.83%,5.52% and 1.05%. The CV percent of all the coefficient of variation is less than 10 percent, and the effect of the first regeneration experiment is good; as shown in FIG. 2, the fluorescence signal curve and the static light scattering signal curve of 7010 protein solution obtained by regenerating sodium hydroxide solution with pH values of 11.0, 10.0 and 9.0 respectively have the best coincidence ratio, the minimum fluctuation and the best regeneration effect of the three repeated samples under the condition of pH value of 11.0.
Preferably, the subsequent experiments were carried out with sodium hydroxide solution having a pH of 11.0 as regenerant.
Example 2
A method for regenerating sample tubes using hydrochloric acid solutions of different pH values, comprising the steps of:
step 1, after the protein stability experiment is finished, washing off the protein solution in 3 sample tubes by using ultrapure water, and respectively placing the sample tubes in 3 hydrochloric acid solution regenerants with the pH values of 3.0,2.0 and 1.5 for soaking for 30 minutes.
And 2, placing the sample tube soaked by the regenerant in an ultrasonic instrument, and performing ultrasonic treatment for 60 seconds by using ultrapure water.
And 3, washing the sample tube subjected to ultrasonic treatment with ultrapure water to flush residual regenerant.
And 4, finally, drying by using compressed air, and finishing the first regeneration experiment of the sample tube.
And 5, comparing Tm values of different water-soluble protein solutions before and after sample tube regeneration, coefficient of variation CV% calculated by formula 1 and coincidence degree of fluorescent signals or static light scattering signal curves of three repeated samples through testing and analysis.
Equation 1: CV% = SD/mean × 100%,
where SD refers to the standard deviation of all Tm before and after each regeneration, mean refers to the average of all Tm before and after each regeneration;
if CV% is less than 10%, the sample tube can be reused in the multi-parameter protein stability analyzer.
If the coincidence degree of the fluorescent signal curves or the static light scattering signal curves of the three repeated samples is good, the regeneration effect of the sample tube is good, and the regenerant can be preferred.
And 6, selecting the sample tube soaked in the regenerant of the hydrochloric acid solution with the pH value of 3.0, and performing secondary regeneration according to the steps 1, 2, 3, 4 and 5.
And 7, carrying out third regeneration on the sample tube treated in the step 6 according to the same regeneration step.
And 8, performing fourth regeneration on the sample tube treated in the step 7 according to the same regeneration step.
And 9, performing fifth regeneration on the sample tubes treated in the step 8 according to the same regeneration step.
Wherein the protein stability experiment is as follows: putting 3 sample tubes loaded with 4 water-soluble protein solutions into a multi-parameter protein stability analyzer, and setting operation parameters as follows: the starting temperature of the temperature rising curve is 20 ℃; the temperature rise rate is 0.3 ℃/min, the end point temperature of the temperature rise curve is 90 ℃, the running time is 4.5 hours, and the protein solution parameters under the New Unis condition are obtained and used as comparison data.
Further, the samples in each sample tube were four different kinds of water-soluble protein solutions, named 7009, 7010, aux and IgG. The protein solution concentrations were 2.2mg/mL,1.8mg/mL,0.8mg/mL,1.5mg/mL, respectively. The sample adding sequence is 1-3 to 7009,4-6 to 7010,7-9 to Aux and 10-12 to IgG in sequence. 3 replicates were made for each protein solution, for a total of 12 samples per tube. The number of sample tubes for each experiment was 3, and the volume and sequence of each sample was the same.
The data obtained in step 5 of example 2 are shown in fig. 3, and the hydrochloric acid solution with pH of 1.5 shown in fig. 3 is regenerated to obtain coefficients of variation CV% of the four proteins of 0.7%,0.38%,2.16%,0.81%; the coefficients of variation CV% of the four protein solutions obtained by regenerating the hydrochloric acid solution with the pH value of 2.0 are respectively 1.41%,1.27%,2.73% and 0.97%; the coefficients of variation CV% of the four protein solutions obtained by regenerating the hydrochloric acid solution with the pH value of 3.0 are respectively 0.56%,1.01%,1.13% and 0.42%. The CV percent of all the coefficient of variation is less than 10 percent, and the effect of the first regeneration experiment is good; as shown in fig. 4, the fluorescent signal curve and the static light scattering signal curve of the IgG protein solution obtained by regenerating hydrochloric acid solutions with pH values of 3.0,2.0, and 1.5, respectively, have the smoothest curve, the minimum fluctuation, and the best repeatability under the hydrochloric acid solution condition with pH value of 3.0.
Preferably, the subsequent experiments were performed with hydrochloric acid solution having a pH of 3.0 as regenerant.
Example 3
The data obtained in step 9 of examples 1 and 2 are shown in fig. 5, and the coefficients of variation CV% of the four proteins obtained after regenerating the sample tube with the sodium hydroxide solution having the pH of 11.0 shown in fig. 5 for 5 times were 0.31%,0.78%,1.87%, and 0.31%, respectively; after the hydrochloric acid solution with the pH value of 3.0 regenerates the sample tube for 5 times, the coefficient of variation of the four proteins is respectively 0.82%,0.77%,1.88% and 0.57% in coefficient of variation CV%. The Tm values of 4 protein solutions obtained by regenerating the sample tube for 5 times are very close, the variation coefficients are controlled within 10 percent, and the regeneration effect of the adopted regenerant is remarkable. FIG. 6 shows that after the sample tube was regenerated 5 times with sodium hydroxide solution at pH 11.0 and hydrochloric acid solution at pH 3.0, a fluorescence signal curve and a static light scattering signal curve of the IgG protein solution were obtained. The curve under the condition of the sodium hydroxide solution with the pH value of 11.0 is smoother, and the regeneration effect and the repeatability are optimal.
Further, according to the examples, a preferred regenerant is sodium hydroxide solution having a pH of 11.0.
Further, it can be seen from the examples that the sample tube can still be used in a multiparameter protein stability analyzer after 5 regenerations.
Further, according to the examples, it can be obtained that the coefficient of variation CV% is less than 2% under the conditions of sodium hydroxide at pH 11.0 and hydrochloric acid solution at pH 3.0 as regenerants.
Example 4
A method of selecting a regenerant comprising the steps of:
step 1, after the protein stability experiment is finished, washing off the protein solution in 3 sample tubes by using ultrapure water, and respectively soaking in 3 sodium hydroxide solution regenerants with pH values of 11.0, 10.0 and 9.0 for 30 minutes.
And 2, placing the sample tube soaked by the regenerant in an ultrasonic instrument, and performing ultrasonic treatment for 60 seconds by using ultrapure water.
And 3, washing the sample tube subjected to ultrasonic treatment with ultrapure water to wash residual regenerant.
And 4, finally, drying by using compressed air, and finishing the first regeneration experiment of the sample tube.
And 5, comparing Tm values of four protein solutions with different concentrations before and after the regeneration of the sample tube, coefficient of variation CV% calculated by formula 1 and the coincidence degree of the fluorescence signals of three repeated samples through testing and analysis (the characteristics of a static light scattering signal curve of the protein solution in the experiment are not typically referred to).
Equation 1: CV% = SD/mean × 100%,
where SD refers to the standard deviation of all Tm before and after each regeneration, mean refers to the average of all Tm before and after each regeneration;
if CV% is less than 10%, the sample tube can be reused in the multi-parameter protein stability analyzer.
If the coincidence degree of the fluorescence signal curves of the three repeated samples is good, the regeneration effect of the sample tube can be further explained to be good, and the regenerant can be preferably used.
And 6, carrying out second regeneration on the sample tube processed in the step 4 according to the steps 1, 2, 3, 4 and 5.
And 7, carrying out third regeneration on the sample tube treated in the step 6 according to the same regeneration step.
And 8, performing fourth regeneration on the sample tube treated in the step 7 according to the same regeneration step.
And 9, performing fifth regeneration on the sample tubes treated in the step 8 according to the same regeneration step.
And 10, changing the regenerant in the experimental step 1 into hydrochloric acid solutions with pH values of 3.0,2.0 and 1.5 respectively, and repeating the experimental steps 1 to 9.
Wherein the protein stability experiment is as follows: 3 sample tubes each carrying a solution of HAS (Albumin from human serum, cat # A1653-5G, manufacturer: sigma Aldrich, co) of 4 different concentrations were placed in a multiparameter protein stability analyzer, and the operating parameters were set as follows: the starting temperature of the temperature rising curve is 20 ℃; the temperature rise rate is 0.3 ℃/min, the end point temperature of the temperature rise curve is 90 ℃, the running time is 4.5 hours, and the protein solution parameters under the New Unis condition are obtained and used as comparison data.
Further, the sample in each sample tube was four different concentrations of HSA solution. The protein solution concentrations were: 150mg/mL (the highest detectable concentration of the apparatus is 150 mg/mL), 75mg/mL,37.5mg/mL,18.75mg/mL. The sample adding sequence is 1-3 of 150mg/mL,4-6 of 75mg/mL,7-9 of 37.5mg/mL and 10-12 of 18.75mg/mL in sequence. 3 replicates of each protein solution concentration were made, for a total of 12 samples per tube. The number of sample tubes for each experiment was 3, and the volume and sequence of each sample was the same.
Further, the data obtained in step 9 of example 4 are shown in fig. 7, and the CV% of the coefficients of variation of the four different concentrations of protein obtained after regenerating the sample tube with the sodium hydroxide solution at the pH of 11.0 shown in fig. 7 for five times are 3.6%,4.8%,4.6%, and 3.9%, respectively; after the sodium hydroxide solution with the pH value of 10.0 is used for regenerating the sample tube for five times, the coefficient of variation CV% of four protein solutions with different concentrations are respectively 3.6%,4%,3.3% and 2.9%; after the sample tube is regenerated for five times by using the sodium hydroxide solution with the pH value of 9.0, the coefficient of variation CV% of four protein solutions with different concentrations are respectively 4.6%,4.5%,2.9% and 3.1%. All the coefficient of variation CV% are less than 5%, and the data show that the regeneration experiment effect is good, and the regeneration effect of the adopted regenerant is remarkable. As shown in fig. 8, the fluorescence signal curves of four protein solutions with different concentrations obtained after the sodium hydroxide solution with pH values of 11.0, 10.0, and 9.0 respectively regenerates the sample tube five times have the colors of the curves sequentially from light to dark corresponding to four concentrations from low to high, which are respectively: 150mg/mL,75mg/mL,37.5mg/mL,18.75mg/mL. The coincidence degree of the curves shows that the fluorescence signal curves of four protein solutions with different concentrations and with the pH value of 11.0 are well coincided, the fluctuation is small, the repeatability is good, and when the concentration of HSA is less than 75mg/mL under the condition that the pH value is within the range of 9.0 to 10.0, the curves are not well coincided and the repeatability is poor. The data and curve analysis are combined to obtain the best coincidence degree of the fluorescent signal curves of the four protein solutions with different concentrations under the condition of the sodium hydroxide solution with the pH value of 11.0, and the best regeneration effect and repeatability are obtained.
Further, the data obtained in step 10 of example 4 is shown in fig. 9, and after the hydrochloric acid solution with pH value of 3.0 shown in fig. 9 regenerates the sample tube five times, the CV% of the variation coefficients of the four different concentrations of protein are 3.3%,4.6%,8.4%, and 4.3%, respectively; regenerating the sample tube by hydrochloric acid solution with the pH value of 2.0 for five times to obtain the coefficient of variation of four proteins with different concentrations, wherein the coefficient of variation CV% is respectively 3.8%,5.4%,7.3% and 6%; after the hydrochloric acid solution with the pH value of 1.5 is used for regenerating the sample tube for five times, the coefficient of variation of the four proteins with different concentrations is respectively 5.3%,5.8%,7.4% and 2.6% as coefficient of variation CV%. All the coefficient of variation CV% are controlled within 8%, and the data show that the regeneration experiment effect is good. Fig. 10 shows fluorescence signal curves of four protein solutions with different concentrations obtained after the hydrochloric acid solutions with pH values of 3.0,2.0, and 1.5 regenerate the sample tube for 5 times, wherein the colors of the curves are sequentially from light to dark, and the four concentrations are respectively: 150mg/mL,75mg/mL,37.5mg/mL,18.75mg/mL. The coincidence degree of the curves shows that the fluorescence signal curves of 4 protein solutions under the condition that the pH value is in the range of 1.5 to 2.0 are well coincided, the repeatability is good, when the pH value is 3.0 and the HSA concentration is less than 37.5mg/mL, the curves are not well coincided, and the repeatability is not good.
Further, it can be seen from the examples that at a protein solution concentration <37.5mg/mL, the acidic rejuvenating agent pH preferably ranges from 1.5 to 2.0, and the alkaline rejuvenating agent pH preferably ranges from 10.0 to 11.0.
Further, it can be seen from the examples that the pH of acidic rejuvenating agents is preferably in the range of 1.5 to 3.0 and the pH of alkaline rejuvenating agents is preferably in the range of 10.0 to 11.0 at protein solution concentrations <37.5mg/mL <75 mg/mL.
Further, it can be seen from the examples that the acidic rejuvenating agent pH preferably ranges from 1.5 to 3.0 and the alkaline rejuvenating agent pH preferably ranges from 9.0-11.0 at protein solution concentrations <75mg/mL <150 mg/mL.
Comparative example 1
In the prior art, various surfactants (such as soap water) are used as detergents for washing test tubes in chemical experiments, and the test tubes are brushed by dipping the detergents and then washed by water, and finally washed by distilled water.
The pickling method of the quartz tube is mainly realized by the procedures of putting the quartz tube in a horizontal type cleaning tank, soaking, spraying, drying and the like.
The residue in the quartz sample tube involved in the method is a protein solution, the protein is usually very difficult to wash due to the complex nature of the protein, and compared with the general residue, the reactant cannot be singly selected as a washing agent according to the acid-base property or the organic matter property. Meanwhile, the quartz sample tube is a capillary tube, the inner diameter and the outer diameter of the quartz sample tube are very small, and the quartz sample tube cannot be cleaned by a brush or a cleaning tank.
Comparative example 2
Washing the sample tubes described herein with a strong acid, such as aqua regia, which is typically regenerated by glassware washing, can cause damage to the sample tubes, making them unusable for protein stability analysis; meanwhile, unsafe factors of experiments are increased.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims (2)

1. A method for regenerating a sample tube for a multiparameter protein stability analyzer, comprising immersing the sample tube in a regenerant that will select an alkaline solution having a pH of 9.0 to 11.0 and an acidic solution having a pH of 1.5 to 3.0;
the sample tube is a capillary tube;
the acidic solution is selected from HCl and H 2 SO 4 、HNO 3 And H 3 PO 4 At least one of; the alkaline solution is selected from NaOH, KOH and Na 2 SiO 3 And Na 2 CO 3 At least one of; the soaking time of the sample tube is 20 minutesTo 60 minutes; before the sample tube is put into the regenerant for soaking, the method comprises the step of washing the sample tube with ultrapure water; after a sample tube is placed into a regenerant for soaking, the method comprises the step of carrying out ultrasonic washing on the sample tube soaked by the regenerant in an ultrapure water bath; the time of the ultrasonic washing is in the range of 30 seconds to 120 seconds; after the ultrasonic washing, rinsing the sample tube with ultrapure water; after the sample tube is washed by ultrapure water, the method comprises the step of blow-drying the sample tube by using compressed air.
2. The method for regenerating a sample tube for a multiparameter protein stability analyzer of claim 1, wherein the washing, soaking, sonicating, and blowing steps are repeated 2 to 5 times.
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5585436A (en) * 1978-12-19 1980-06-27 Fujitsu Ltd Surface treating method for quartz glass
CN101219429A (en) * 2007-01-10 2008-07-16 北京北方微电子基地设备工艺研究中心有限责任公司 Method for cleaning quartz parts surface in polycrystal etching cavity
CN101735904A (en) * 2008-11-20 2010-06-16 Axt公司 Cleaning solution and cleaning method adopting same
CN106824929A (en) * 2017-03-10 2017-06-13 北京北方华创微电子装备有限公司 A kind of vertical multi-stage quartz tube cleaner and cleaning method
CN107930550A (en) * 2017-12-27 2018-04-20 北京华福工程有限公司 Production aromatic hydrocarbons is prepared with the method for reaction tube and production aromatic hydrocarbons reaction tube
CN110465523A (en) * 2019-08-01 2019-11-19 上海至纯洁净系统科技股份有限公司 A kind of vertical quartz tube automatic rinser and cleaning method

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5585436A (en) * 1978-12-19 1980-06-27 Fujitsu Ltd Surface treating method for quartz glass
CN101219429A (en) * 2007-01-10 2008-07-16 北京北方微电子基地设备工艺研究中心有限责任公司 Method for cleaning quartz parts surface in polycrystal etching cavity
CN101735904A (en) * 2008-11-20 2010-06-16 Axt公司 Cleaning solution and cleaning method adopting same
CN106824929A (en) * 2017-03-10 2017-06-13 北京北方华创微电子装备有限公司 A kind of vertical multi-stage quartz tube cleaner and cleaning method
CN107930550A (en) * 2017-12-27 2018-04-20 北京华福工程有限公司 Production aromatic hydrocarbons is prepared with the method for reaction tube and production aromatic hydrocarbons reaction tube
CN110465523A (en) * 2019-08-01 2019-11-19 上海至纯洁净系统科技股份有限公司 A kind of vertical quartz tube automatic rinser and cleaning method

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