CN110455732B - Biological sample treatment method, ferrous ion or total ferric ion determination method of biological sample and detection kit - Google Patents

Biological sample treatment method, ferrous ion or total ferric ion determination method of biological sample and detection kit Download PDF

Info

Publication number
CN110455732B
CN110455732B CN201910801327.1A CN201910801327A CN110455732B CN 110455732 B CN110455732 B CN 110455732B CN 201910801327 A CN201910801327 A CN 201910801327A CN 110455732 B CN110455732 B CN 110455732B
Authority
CN
China
Prior art keywords
solution
biological sample
sample
ions
ferrous
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201910801327.1A
Other languages
Chinese (zh)
Other versions
CN110455732A (en
Inventor
周昊函
徐巍
田佳怡
梁琳
李乐
梁婷
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Jilin University
Original Assignee
Jilin University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Jilin University filed Critical Jilin University
Priority to CN201910801327.1A priority Critical patent/CN110455732B/en
Publication of CN110455732A publication Critical patent/CN110455732A/en
Application granted granted Critical
Publication of CN110455732B publication Critical patent/CN110455732B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/38—Diluting, dispersing or mixing samples
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
    • G01N2001/2866—Grinding or homogeneising
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/38—Diluting, dispersing or mixing samples
    • G01N2001/386—Other diluting or mixing processes

Landscapes

  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

The application provides a biological sample processing method, a ferrous ion or total ferric ion determination method of a biological sample and a detection kit. According to the biological sample processing method, the acidic solution can fully expand and crack cells by virtue of a low-permeability principle, and meanwhile, iron ions in the cells can be fully released under the action of acid, and do not react with a color developing agent, so that the measurement of the iron ions is not influenced. According to the method for measuring the ferrous ions of the biological sample and the method for measuring the total ferric ions of the biological sample, the concentration of the reducing agent is increased, so that the ferric ions are fully reduced, and the alkaline liquor is added, so that the tripyridyl triazine and the ferrous ions in the solution generate a stable bluish purple complex, and the method can be used for detecting the ferrous ions, and is simple, easy and accurate. The ferrous ion and total iron ion detection kit can be used for measuring ferrous ions and total iron ions in a biological sample.

Description

Biological sample treatment method, ferrous ion or total ferric ion determination method of biological sample and detection kit
Technical Field
The invention relates to the technical field of biological sample detection, in particular to a biological sample processing method, a biological sample ferrous ion determination method, a biological sample total ferric ion determination method, a biological sample ferrous ion detection kit and a biological sample total ferric ion detection kit.
Background
In the organism, a plurality of trace metal elements participate in different physiological metabolic activities. Iron is a mineral and plays a vital role in many biological processes, such as DNA synthesis, electron transfer, and oxygen transport. Iron is a transition element that can form a range of oxidation states, most commonly ferrous iron (Fe)2+Or ferrous iron) and ferric iron (Fe)3+). Ferritin participates in many reactions, usually chemical reactions that utilize the transient changes in the oxidation state of iron. Both deficiency and excess of iron may cause serious human dysfunction. Therefore, finding a suitable iron ion quantitative detection method in clinical, medicine and environmental aspects is always the direction of efforts of researchers.
At present, the quantitative analysis of iron element is mainly focused on soil, water quality, atmosphere and the like, and the common methods include an atomic absorption method, an inductively coupled plasma emission spectrometry, a fluorescence probe method and a spectrophotometry. The former two methods, although accurate in measurement, have high requirements for equipment, expensive measuring instruments, cumbersome in operation and difficult in application to common laboratories. The fluorescent probe method is still in the research and exploration stage and is not popularized and applied. The spectrophotometry has relatively simple and mature requirements on equipment, reagents and operation, and is widely applied at present. Commonly used spectrophotometry methods include 2,4, 6-tripyridyltriazine chromogenic spectrophotometry, sulfosalicylic acid spectrophotometry, phenanthroline spectrophotometry.
At present, the three spectrophotometric methods are mainly used for measuring environmental samples such as water samples, soil and the like. The required amount of samples is large, the experimental system is large (at least 50mL samples), biological experimental samples mainly comprise cells, tissues, blood samples and the like, the sample amount is limited, the pH value of the biological samples is different from the pH value range of environmental samples, and the pH value requirement applied to the experimental system of the environmental samples is not suitable for biological life science experiments. In addition, the reaction between the cell and tissue lysate and the color reagent of the method interferes with the experimental result. At present, iron ion detection kits for biological samples in biological research are imported abroad, are expensive and have confidential kit components. If the simple and feasible biological sample iron ion detection kit with the independent intellectual property rights in China can be invented, the blank of the market is filled, and the kit makes a contribution to the scientific research field in China.
In the conventional biological sample processing method, the method for releasing cell contents is usually cell lysis, and currently, a general cell lysate and tripyridyl triazine on the market react to generate an opalescent precipitate, so that the method cannot be used for measuring iron ions. The biological sample is placed in double distilled water by using osmotic pressure gradient to expand and crack cells, but most of iron ions are combined with protein, DNA or RNA and are difficult to release, and the contents released by the cells expanded and cracked by the double distilled water cannot react with the tripyridyltriazine to generate a complex. Therefore, how to properly process the biological sample so that the iron element in the sample can be accurately detected and how to effectively measure the iron element in the biological sample are all problems to be overcome.
Disclosure of Invention
In view of the above, it is necessary to provide a biological sample processing method, a biological sample ferrous ion measuring method, a biological sample total ferric ion measuring method, a biological sample ferrous ion detecting kit and a biological sample total ferric ion detecting kit, which can accurately detect the iron element in the biological sample.
A method of processing a biological sample, comprising the steps of:
adding a biological sample into an acidic solution to obtain a biological sample solution;
treating the biological sample solution by adopting an ultrasonic oscillation or ultrasonic crushing method to obtain a mixed solution;
and centrifuging the mixed solution, discarding the precipitate, and reserving and taking supernatant to obtain the treated biological sample solution.
In one embodiment, the operation of adding the biological sample to the acidic solution to obtain the biological sample solution is as follows:
adding the biological sample into an acid solution with the molar concentration of 8-10%, and standing for 10-15 hours at the temperature of 0-4 ℃ to obtain a biological sample solution;
treating the biological sample solution by adopting an ultrasonic oscillation or ultrasonic crushing method to obtain a mixed solution, wherein the operation of obtaining the mixed solution is as follows:
and treating the biological sample solution for 15-30 minutes by adopting an ultrasonic oscillation or ultrasonic crushing method to obtain the mixed solution, wherein the ultrasonic oscillation condition is 20-25 ℃, 40-50 kHz and high frequency, the ultrasonic crushing condition is 300-400W, the working time is 10s, the interval is 15s, and the working frequency is 180 times.
In one embodiment, the operation of adding the biological sample to the acidic solution to obtain the biological sample solution is as follows:
adding the biological sample into an acid solution with the molar concentration of 8% -10% to obtain a biological sample solution;
treating the biological sample solution by adopting an ultrasonic oscillation or ultrasonic crushing method to obtain a mixed solution, wherein the operation of obtaining the mixed solution is as follows:
and treating the biological sample solution for 45 minutes to 1.5 hours by adopting an ultrasonic oscillation or ultrasonic crushing method to obtain the mixed solution, wherein the ultrasonic oscillation condition is 20-25 ℃, 40kHz-50kHz and high frequency, the ultrasonic crushing condition is 300-400W, the working time is 10s, the interval is 15s, and the working frequency is 180 times.
A ferrous ion determination method of a biological sample comprises the following steps:
sequentially adding a solution D, a solution A and a solution C into a biological sample solution prepared by the biological sample treatment method to obtain a first sample, wherein the solution D is a saturated alkaline solution, the solution A is a buffer solution, and the solution C is a color developing agent solution;
reacting the first sample at 37 ℃ for 30 minutes to obtain a second sample;
measuring the second sample at a wavelength of 595nm to obtain an absorbance value;
preparing a standard solution of ferrous ions, and preparing a standard curve to obtain a standard curve equation;
and substituting the absorbance value of the second sample into the standard curve equation to calculate the ferrous ion concentration of the biological sample solution.
In one embodiment, solution a is an acetic acid-sodium acetate buffer with pH 4.7, solution C is a solution of tripyridazine acetic acid, and solution D is a saturated solution of sodium hydroxide.
A method for measuring total iron ions in a biological sample comprises the following steps:
adding the solution B into the biological sample solution prepared by the biological sample treatment method, standing for 10 minutes, and then sequentially adding the solution D, the solution A and the solution C to obtain a first sample, wherein the solution B is a reducing agent solution, the solution D is a saturated alkaline solution, the solution A is a buffer solution, and the solution C is a color developing agent solution;
reacting the first sample at 37 ℃ for 30 minutes to obtain a second sample;
measuring the second sample at a wavelength of 595nm to obtain an absorbance value;
preparing a standard solution of ferrous ions, and preparing a standard curve to obtain a standard curve equation;
and substituting the absorbance value of the second sample into the standard curve equation to calculate the total iron ion concentration of the biological sample solution.
In one embodiment, the pH of the first sample is 4-5.
In one embodiment, the solution a is an acetic acid-sodium acetate buffer solution with pH 4.7, the solution B is a hydroxylamine hydrochloride or ascorbic acid solution, the solution C is a tris-pyridine triazine acetic acid solution, and the solution D is a saturated sodium hydroxide solution.
A ferrous ion detection kit for a biological sample comprises a solution A, a solution C and a solution D, wherein the solution A is a buffer solution, the solution C is a color developing agent solution, and the solution D is a saturated alkaline solution.
In one embodiment, the solution a is an acetic acid-sodium acetate buffer solution with pH 4.7, the solution C is a tris-pyridine triazine acetic acid solution, and the solution D is a saturated sodium hydroxide solution.
A total iron ion detection kit for a biological sample comprises a solution A, a solution B, a solution C and a solution D, wherein the solution A is a buffer solution, the solution B is a reducing agent solution, the solution C is a color developing agent solution, and the solution D is a saturated alkaline solution.
In one embodiment, the solution a is an acetic acid-sodium acetate buffer solution with pH 4.7, the solution B is a hydroxylamine hydrochloride or ascorbic acid solution, the solution C is a tris-pyridine triazine acetic acid solution, and the solution D is a saturated sodium hydroxide solution.
In one embodiment, the concentration of the acetic acid-sodium acetate buffer solution is 0.1moL/L, and the concentration of the hydroxylamine hydrochloride solution is 100 g/L; the concentration of the terpyridyl triazine acetic acid solution is 1 g/L.
According to the biological sample processing method, the acidic solution can fully expand and crack cells by virtue of a low-permeability principle, and simultaneously, iron ions in the cells can be fully released under the action of acid, and are not subjected to other reactions with a color developing agent, so that the measurement of the iron ions is not influenced.
According to the method for measuring the ferrous ions of the biological sample and the method for measuring the total ferric ions of the biological sample, the cells and tissues are fully cracked and the ferric ions are released at the same time by the method for treating the biological sample, so that the subsequent measurement is ensured to be carried out smoothly. Meanwhile, the liquid D is added in the testing process, the pH value of a measuring system is adjusted, the detection capability of ferrous ions is effectively improved, and the capability of accurately measuring the ferrous ions or total ferric ions in the biological sample can be realized.
According to the ferrous ion detection kit for the biological sample, after the solution D, the solution A and the solution C are added into the solution of the biological sample, the absorbance value can be tested by a spectrophotometer, and then the concentration of ferrous ions in the biological sample is calculated according to a standard curve. The method for detecting the iron element of the organism is simple and universal, saves manpower and material resources, and ensures the accuracy of the experiment.
According to the total ferric ion detection kit for the biological sample, after the solution B, the solution D, the solution A and the solution C are added into the solution of the biological sample, the absorbance value can be tested by a spectrophotometer, and then the concentration of ferrous ions in the biological sample is calculated according to a standard curve. The method for detecting the iron element of the organism is simple and universal, saves manpower and material resources, and ensures the accuracy of the experiment.
Drawings
FIG. 1 is a schematic flow diagram of a biological sample processing method according to one embodiment;
FIG. 2 is a schematic flow chart of a biological sample processing method according to an embodiment;
FIG. 3 is a schematic flow chart of a biological sample processing method according to another embodiment;
FIG. 4 is a schematic flow chart of a ferrous ion determination method for a biological sample according to an embodiment;
FIG. 5 is a schematic flow chart of a method for measuring total iron ions in a biological sample according to an embodiment;
FIG. 6 is the full-wavelength absorbance detection, from which it can be seen that the optimum wavelength of the present invention is 595 nm;
FIG. 7 is a standard curve for measuring ferrous ion concentration of biological samples according to examples 1 and 2 of the present invention;
fig. 8 is a standard curve for measuring the ferrous ion concentration of a biological sample according to examples 3 and 4 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
As shown in fig. 1, a biological sample processing method according to an embodiment includes the steps of:
and S110, adding the biological sample into the acidic solution to obtain a biological sample solution.
Specifically, the biological sample is obtained by freezing cultured cells or extracted tissues with liquid nitrogen and then grinding the frozen cells or extracted tissues. Alternatively, the biological sample is obtained by disrupting the cultured cells or the extracted tissue by French press. It is understood that the biological sample may also be cultured cells or extracted tissue directly without being subjected to liquid nitrogen freezing followed by grinding or French press disruption.
The acidic solution can be HCL solution, H2SO4Solution or HNO3Solutions, and the like.
In S110, the acidic solution ruptures cells by the low permeability principle, and promotes the release of iron ions by the acidic environment.
And S112, treating the biological sample solution by adopting an ultrasonic oscillation or ultrasonic crushing method to obtain a mixed solution.
In S112, the ultrasonic vibration can promote the cell expansion and the release of iron ions through mechanical action.
And S114, centrifuging the mixed solution, discarding the precipitate, and reserving the supernatant to obtain the treated biological sample solution.
In S114, the mixed liquid is centrifuged at 0-5 deg.C (4 deg.C) and 10000-12000 g for 10 min.
In S114, impurities such as excess cell debris can be discarded by high-speed centrifugal sedimentation.
According to the biological sample processing method, the acidic solution can fully expand and crack cells by virtue of a low-permeability principle, and simultaneously, iron ions in the cells can be fully released under the action of acid, and are not subjected to other reactions with a color developing agent, so that the measurement of the iron ions is not influenced.
Specifically, referring to fig. 2, in an embodiment, the method for processing a biological sample includes the following steps:
s120, adding the biological sample into an acid solution with the molar concentration of 8% -10%, and standing for 10-15 hours at the temperature of 0-4 ℃ to obtain a biological sample solution.
Wherein the biological sample is obtained by freezing cultured cells or extracted tissue with liquid nitrogen and grinding. Alternatively, the biological sample is obtained by disrupting the cultured cells or the extracted tissue by French press. It is understood that the biological sample may also be cultured cells or extracted tissue directly without being subjected to liquid nitrogen freezing followed by grinding or French press disruption.
Wherein the acidic solution can be HCL solution or H2SO4Solution or HNO3Solutions, and the like.
S122, treating the biological sample solution for 15-30 minutes by adopting an ultrasonic oscillation or ultrasonic crushing method to obtain a mixed solution, wherein the ultrasonic oscillation condition is 20-25 ℃, 40-50 kHz, high frequency, the ultrasonic crushing condition is 300-400W, the working time is 10S, the interval is 15S, and the working times are 180 times.
And S124, centrifuging the mixed solution, discarding the precipitate, and reserving the supernatant to obtain the treated biological sample solution.
In S124, the mixed solution is centrifuged at 0-4 deg.C at 10000-12000 g for 10-15 min.
Specifically, referring to fig. 3, in another embodiment, the method for processing a biological sample includes the following steps:
s130, adding the biological sample into an acid solution with the molar concentration of 8% -10% to obtain a biological sample solution.
Wherein the biological sample is obtained by freezing cultured cells or extracted tissue with liquid nitrogen and grinding. Alternatively, the biological sample is obtained by disrupting the cultured cells or the extracted tissue by French press. It is understood that the biological sample may also be cultured cells or extracted tissue directly without being subjected to liquid nitrogen freezing followed by grinding or French press disruption.
Wherein the acidic solution can be HCL solution or H2SO4Solution or HNO3Solutions, and the like.
S132, treating the biological sample solution for 45 minutes to 1.5 hours by adopting an ultrasonic oscillation or ultrasonic crushing method to obtain a mixed solution, wherein the ultrasonic oscillation condition is 20-25 ℃, the frequency is high, the ultrasonic crushing condition is 300-400W, the working time is 10S, the interval is 15S, and the working frequency is 180 times.
S134, centrifuging the mixed solution, discarding the precipitate, and reserving the supernatant to obtain the treated biological sample solution.
In S134, the mixed solution is centrifuged at 0-4 deg.C at 10000-12000 g for 10-15 min.
In addition, referring to fig. 4, a method for measuring ferrous ions of a biological sample according to an embodiment is further provided, including the following steps:
s210, adding liquid D, liquid A and liquid C into the prepared biological sample solution in sequence to obtain a first sample, wherein the liquid D is a saturated alkaline solution, the liquid A is a buffer solution, and the liquid C is a color developing agent solution.
Wherein, due to the difference of the pH value of the biological sample, the amount of the added reagent can be adjusted according to the proportion, so that the pH of the first sample is between 4 and 5.
Wherein the biological sample solution is prepared using the method described above. That is, the biological sample solution may be prepared by the biological sample treatment method described in S110 to S114, may be prepared by the biological sample treatment method described in S120 to S124, or may be prepared by the biological sample treatment method described in S130 to S134.
Wherein, the solution A can be acetic acid-sodium acetate, acetic acid, oxalate, phosphate and other buffer solutions. The solution C is a solution of tripyridyl triazine acetic acid. The solution D can be saturated sodium hydroxide solution or saturated potassium hydroxide solution.
Preferably, the solution a is an acetic acid-sodium acetate buffer solution with pH 4.7, the solution C is a tris-pyridine triazine acetic acid solution, and the solution D is a saturated sodium hydroxide solution.
More preferably, the concentration of the acetic acid-sodium acetate buffer solution is 0.1moL/L, and the concentration of the tris-pyridine triazine acetic acid solution is 1 g/L.
And S220, reacting the first sample at 37 ℃ for 30 minutes to obtain a second sample.
The reaction of S220 can be performed in an incubator. The reaction can be allowed to cool to room temperature.
And S230, measuring the second sample at the wavelength of 595nm to obtain an absorbance value.
S240, preparing a standard solution of ferrous ions, and preparing a standard curve to obtain a standard curve equation.
The specific operation of S240 is:
Fe2+standard solution (100 mg/L): 0.1755g (NH) was weighed4)2Fe(SO4)2·6H 20, dissolving with 30mL sulfuric acid (1moL/L), and transferring to a constant volume of 250 mL. Diluted to 10mg/L before use.
And (3) working curve configuration: 10mg/L Fe was added to each of 50mL volumetric flasks numbered in advance2+0.0mL, 0.20mL, 0.50mL, 0.80mL, 1.00mL, 2.00mL, 3.00mL, 4.00mL, 5.00mL, 6.00mL, 7.00mL, 8.00mL, 9.00mL, and 10.0mL of the solution were added, followed by 2.0mL of solution C, 2.0mL of solution B, and 5.0mL of solution A, and mixed to a constant volume.
On a spectrophotometer, recording absorbance values of different concentrations by taking 598nm as a test wavelength. A standard curve was prepared.
And S250, substituting the absorbance value of the second sample into a standard curve equation, and calculating the ferrous ion concentration of the solution of the biological sample.
The method for measuring the ferrous ions of the biological sample can be directly used for measuring the concentration of the ferrous ions in the biological sample solution.
Referring to fig. 5, a total iron ion measuring method of a biological sample according to another embodiment includes the following steps:
s310, adding the solution B into the prepared biological sample solution, standing for 10 minutes, and then sequentially adding the solution D, the solution A and the solution C to obtain a first sample, wherein the solution B is a reducing agent solution, the solution D is a saturated alkaline solution, the solution A is a buffer solution, and the solution C is a color developing agent solution.
Wherein, due to the difference of the pH value of the biological sample, the amount of the added reagent can be adjusted according to the proportion, so that the pH of the first sample is between 4 and 5.
Wherein the biological sample solution is prepared using the method described above. That is, the biological sample solution may be prepared by the biological sample treatment method described in S110 to S114, may be prepared by the biological sample treatment method described in S120 to S124, or may be prepared by the biological sample treatment method described in S130 to S134.
Wherein, the solution A can be acetic acid-sodium acetate, acetic acid, oxalate, phosphate and other buffer solutions. The solution B may be hydroxylamine hydrochloride, ascorbic acid, etc. The solution C is a solution of tripyridyl triazine acetic acid. The solution D can be saturated sodium hydroxide solution or saturated potassium hydroxide solution.
Preferably, the solution a is an acetic acid-sodium acetate buffer solution with pH 4.7, the solution B is a hydroxylamine hydrochloride solution, the solution C is a tris-pyridine triazine acetic acid solution, and the solution D is a saturated sodium hydroxide solution.
More preferably, the concentration of the acetic acid-sodium acetate buffer solution is 0.1moL/L, and the concentration of the hydroxylamine hydrochloride solution is 100 g/L; the concentration of the terpyridyl triazine acetic acid solution is 1 g/L.
The preparation method of the solution A, the solution B and the solution C is as follows.
Solution A: acetic acid-sodium acetate buffer at pH 4.7 (0.1 moL/L): 0.82g of sodium acetate was weighed, dissolved in 100mL of water, and 0.6mL of glacial acetic acid was added to the solution and mixed well. And B, liquid B: hydroxylamine hydrochloride solution (100 g/L): 10.0g of hydroxylamine hydrochloride was weighed and dissolved in 100mL of water. And C, liquid C: tripyridyltriazine acetic acid solution (1 g/L): 0.25g of tripyridyltriazine is weighed out and dissolved in 250mL of a 0.01moL/L acetic acid solution.
And S320, reacting the first sample at 37 ℃ for 30 minutes to obtain a second sample.
The reaction of S320 can be performed in an incubator. The reaction can be allowed to cool to room temperature.
S330, measuring the second sample at the wavelength of 595nm to obtain an absorbance value.
S340, preparing a standard solution of ferrous ions, and preparing a standard curve to obtain a standard curve equation.
The specific operation of S340 is:
Fe2+standard solution (100 mg/L): 0.1755g (NH) was weighed4)2Fe(SO4)2·6H 20, dissolving with 30mL sulfuric acid (1moL/L), and transferring to a constant volume of 250 mL. Diluted to 10mg/L before use.
And (3) working curve configuration: 10mg/L Fe was added to each of 50mL volumetric flasks numbered in advance2+0.0mL, 0.20mL, 0.50mL, 0.80mL, 1.00mL, 2.00mL, 3.00mL, 4.00mL, 5.00mL, 6.00mL, 7.00mL, 8.00mL, 9.00mL, and 10.0mL of the solution were added, followed by 2.0mL of solution C, 2.0mL of solution B, and 5.0mL of solution A, and mixed to a constant volume.
On a spectrophotometer, recording absorbance values of different concentrations by taking 598nm as a test wavelength. A standard curve was prepared.
And S350, substituting the absorbance value of the second sample into a standard curve equation, and calculating the total iron ion concentration of the solution of the biological sample.
In addition, the concentration of ferric ions in the biological sample can be tested. The concentration of ferrous ions measured when the B solution is not added is subtracted from the total concentration of ferric ions, and the result is the concentration of ferric ions in the biological sample. According to the method for measuring the ferrous ions of the biological sample and the method for measuring the total ferric ions of the biological sample, the cells and tissues are fully cracked and the ferric ions (including the ferrous ions and the ferric ions) are released at the same time by the method for treating the biological sample, so that the subsequent measurement is ensured to be carried out smoothly. Meanwhile, the D liquid is added in the testing process, strong acid added in the biological sample treatment process is neutralized, the pH value of a detection system is adjusted, the detection capability of ferrous ions is effectively improved, and the capability of accurately detecting the ferrous ions or total ferric ions in the biological sample can be realized.
According to the method for measuring the ferrous ions of the biological sample and the method for measuring the total ferric ions of the biological sample, as TPTZ can only react with the ferrous ions, the concentration of the ferrous ions in the solution is measured firstly, then the ferric ions in the solution are reduced into the ferrous ions by the reducing agents such as ascorbic acid, and the concentration of the total ferric ions in the solution is measured, and the concentration of the ferric ions in the solution can be measured by subtracting the concentrations of the ferrous ions and the ferric ions. And adding the biological sample solution to be detected into the solution D, the solution A and the solution C, placing the solution into a spectrophotometer to measure an OD value, bringing the obtained light absorption value into a standard curve, and calculating a value to be the corresponding ferrous ion concentration a in the solution. And adding the biological sample solution to be detected into the solution B, the solution D, the solution A and the solution C in sequence, reducing all ferric iron in the solution into ferrous iron, and measuring to obtain the total iron element concentration B in the solution. The ferric iron concentration in the solution can be obtained as b-a through calculation.
According to the method for measuring the ferrous ions of the biological sample and the method for measuring the total ferric ions of the biological sample, the cells and tissues are fully cracked and the ferric ions (including the ferrous ions and the ferric ions) are released at the same time by the method for treating the biological sample, so that the subsequent measurement is ensured to be carried out smoothly. Compared with the traditional 2,4, 6-tripyridyl triazine color development spectrophotometry, the method for measuring the ferrous ions of the biological samples and the method for measuring the total ferric ions of the biological samples increase the liquid D in the test process, neutralize the strong acid added in the sample treatment process, and adjust the pH value of the system. The concentration of the reducing agent is improved by about 10 times, so that trace iron ions in a measurement system are fully reduced. Because the biological sample is precious and has a small amount, the concentration of the ferric ions is low, so that trace ferric ions cannot be fully reduced, the numerical values before and after the reducing agent is added are similar, and the exact ferric ions cannot be fully measured. In the method for measuring ferrous ions in the biological sample and the method for measuring total ferric ions in the biological sample, the concentration of the reducing agent is increased, so that trace ferric ions in the sample are fully reduced, and accurate measurement is performed. And the absorption wavelength of the spectrophotometer is adjusted to 595nm, the pH of the experimental final system is adjusted to 4-5, so that the tripyridyl triazine and ferrous ions in the solution generate stable blue-violet complex, and the stable blue-violet complex can be placed in an enzyme labeling instrument, the spectrophotometer and other instruments for detection. The method for measuring the ferrous ions of the biological sample and the method for measuring the total ferric ions of the biological sample are simple, feasible and accurate, so that the method for detecting the iron element of the organism is simple and universal, the manpower and material resources are saved, and the accuracy of the experiment is ensured. The invention improves the 2,4, 6-tripyridyltriazine color development spectrophotometry, so that the total volume of an experimental system is 100uL, and the method is suitable for biological sample experiments.
In addition, the ferrous ion detection kit for the biological sample of an embodiment is also provided, and comprises a solution A, a solution C and a solution D, wherein the solution A is a buffer solution, the solution C is a color developing agent solution, and the solution D is a saturated alkaline solution.
Wherein, the solution A can be acetic acid-sodium acetate, acetic acid, oxalate, phosphate and other buffer solutions. The solution C can be a solution of tripyridyl triazine acetic acid. The solution D can be saturated sodium hydroxide solution or saturated potassium hydroxide solution.
Preferably, the solution a is an acetic acid-sodium acetate buffer solution with pH 4.7, the solution C is a tris-pyridine triazine acetic acid solution, and the solution D is a saturated sodium hydroxide solution.
More preferably, the concentration of the acetic acid-sodium acetate buffer solution is 0.1moL/L, and the concentration of the hydroxylamine hydrochloride solution is 100 g/L; the concentration of the terpyridyl triazine acetic acid solution is 1 g/L.
According to the ferrous ion detection kit for the biological sample, after the solution D, the solution A and the solution C are added into the solution of the biological sample, the absorbance value can be tested by a spectrophotometer, and then the concentration of ferrous ions in the biological sample is calculated according to a standard curve. The method for detecting the iron element of the organism is simple and universal, saves manpower and material resources, and ensures the accuracy of the experiment.
In addition, the total iron ion detection kit for the biological sample of an embodiment is also provided, and comprises solution A, solution B, solution C and solution D, wherein the solution A is a buffer solution, the solution B is a reducing agent solution, the solution C is a color developing agent solution, and the solution D is a saturated alkaline solution.
Wherein, the solution A can be acetic acid-sodium acetate, acetic acid, oxalate, phosphate and other buffer solutions. The solution B can be hydroxylamine hydrochloride and ascorbic acid solution. The solution C can be a solution of tripyridyl triazine acetic acid. The solution D can be saturated sodium hydroxide solution or saturated potassium hydroxide solution.
Preferably, the solution a is an acetic acid-sodium acetate buffer solution with pH 4.7, the solution B is a hydroxylamine hydrochloride solution, the solution C is a tris-pyridine triazine acetic acid solution, and the solution D is a saturated sodium hydroxide solution.
More preferably, the concentration of the acetic acid-sodium acetate buffer solution is 0.1moL/L, and the concentration of the hydroxylamine hydrochloride solution is 100 g/L; the concentration of the terpyridyl triazine acetic acid solution is 1 g/L.
According to the total iron ion detection kit for the biological sample, after the solution B, the solution D, the solution A and the solution C are added into the solution of the biological sample, the absorbance value can be tested by a spectrophotometer, and then the concentration of total iron ions in the biological sample is calculated according to a standard curve. The method for detecting the iron element of the organism is simple and universal, saves manpower and material resources, and ensures the accuracy of the experiment.
The biological sample treatment method has the advantages that the reagents are common, the method is simple and effective, and most importantly, the biological samples such as cells and the like can fully release iron ions while expanding and cracking, and the method can be used for detecting the subsequent iron ions. However, the currently used cell lysis method is that the commercial lysis solution lyses cells, and although the time consumption is low, the introduced chemical components react with TPTZ to generate milky white precipitates, which cannot be used in subsequent experiments. However, after a biological sample is cracked by hypotonic solution such as common double distilled water and the like, complex formation is not seen in the reaction with TPTN, and iron ions are difficult to be sufficiently released because most iron ions exist in cells in a mode of being combined with protein, DNA and RNA, and the iron ions can be sufficiently released by damaging a combined hydrogen bond by adding hydrochloric acid with a proper proportion.
The experimental system of the method for measuring the ferrous ions of the biological sample and the method for measuring the total ferric ions of the biological sample is reduced to hundreds of microliter, the requirement on the instrument is lower, and both a spectrophotometer and an enzyme-labeling instrument can be used. Although the atomic absorption method is accurate, the steps are complex, the requirement on equipment is high, the equipment and experiment cost is greatly reduced, and manpower, material resources and time are saved.
The total ferric ions referred to hereinabove include ferrous ions and ferric ions.
The following is a detailed description of the embodiments.
Example 1
Processing of the biological sample:
step 11, culturing the ovarian cancer Skov3 cell line for 24 hours to obtain 5X 106And (4) collecting the cultured cells, centrifuging, and removing the supernatant to obtain cell masses.
Step 12, freezing the collected cell mass in liquid nitrogen overnight and grinding into powder.
And step 13, placing the biological sample powder obtained in the step 12 in 9% HCL, and carrying out ultrasonic oscillation on the sample for 20 minutes after overnight standing at 4 ℃, wherein the oscillation condition is 25 ℃, 40kHz and high frequency.
And step 14, placing the mixed solution after shaking in the step 13 in a high-speed centrifuge at 4 ℃ for 12000g, centrifuging for 10 minutes, discarding the precipitate, and reserving and taking supernatant.
Determination of optimum wavelength by improved TPTZ spectrophotometry:
after being treated by the above determination scheme, the standard ferrous chloride stock solution of 1 ng/microliter is placed in a microplate reader for full-wavelength scanning. Double distilled water was used as a blank control. As a result, as shown in FIG. 6, it can be seen that the optimum detection wavelength of the method of the present invention is 595 nm.
And (3) measuring ferrous ions of the biological sample:
experimental reagent: ascorbic acid, ammonium ferrous sulfate hexahydrate, acetic acid, anhydrous sodium acetate, terpyridyl triazine and secondary distilled water.
Step 21, reagent preparation:
solution A: 20mg of sodium acetate and 800 mu L of acetic acid are mixed and then the volume is increased to 50mL by using double distilled water.
And C, liquid C: 200mg of 2,4, 6-tripyridyl triazine is dissolved by adding 100 mu L of hydrochloric acid, and double distilled water is added to reach the volume of 50 mL.
And (3) liquid D: saturated sodium hydroxide solution.
Step 22, preparation of a standard substance:
ferrous chloride (FeCl) was prepared at a concentration of 4 ng/. mu.L, 2 ng/. mu.L, 1 ng/. mu.L, 0.5 ng/. mu.L, 0.25 ng/. mu.L, 0.125 ng/. mu.L, 0.0625 ng/. mu.L, 0.03125 ng/. mu.L, 0.015625 ng/. mu.L, 0 ng/. mu.L2) The solvent of the solution is double distilled water.
Step 23, standard curve configuration:
231, putting 20 mu L of the standard substance into the cells of a 384-hole plate, and simultaneously setting a blank control and 20 mu L/hole of double distilled water;
232, adding 5 mu L of D liquid into each standard substance and each blank control hole;
step 233, adding 67.5 mul/hole A solution;
234, adding 7.5 mu L/hole C solution;
235, placing the 384-hole plate in a 37 ℃ incubator, fully reacting for 30 minutes, and cooling to room temperature;
and 236, placing the pore plate in an enzyme labeling instrument, measuring the absorbance value of each pore at the wavelength of 595nm, and making a standard curve. The absorbance was measured and a standard curve was plotted as shown in FIG. 7, where R is shown2The linear correlation is better when the value is 0.9997.
The detection operation of the ferrous ions of the sample comprises the following steps:
31, putting 20 mu L of a sample into a cell of a 384-pore plate, and simultaneously setting a blank control, namely 20 mu L/hole of double distilled water;
step 32, adding 5 mu L of D liquid per hole;
step 33, adding 63.75 mu L/hole A solution;
step 34, adding 7.5 mu L/hole C solution;
step 35, placing the 384-hole plate in a 37 ℃ incubator, fully reacting for 30 minutes, and cooling to room temperature;
step 36, placing the pore plate in an enzyme labeling instrument, and measuring the absorbance value of each pore at the wavelength of 595 nm;
and step 37, substituting the absorbance value of the measured sample into a standard curve equation, and calculating the ferrous ion concentration of the sample to be 0.11 ng/mu L.
And (3) recovery rate inspection:
an iron standard solution of parallel 3 groups is selected to be subjected to an adding standard recovery experiment at a concentration of 1 ng/. mu.L, and the adding standard recovery rate is (adding standard sample determination value-sample determination value)/adding standard amount multiplied by 100 percent through a recovery rate calculation formula, so that the average adding standard recovery rate is 99.6 percent, the S is 0.4, and the RSD is 0.37 percent. It can be seen that the recovery rate of the present invention is better.
Example 2
Processing of the biological sample:
step 11, Skov3 cell line, after 24 hours of culture, 5X 10 cells were obtained6And (4) collecting the cultured cells, centrifuging, and removing the supernatant to obtain cell masses.
Step 12, freezing the collected cell mass in liquid nitrogen overnight and grinding into powder.
And step 13, placing the biological sample powder obtained in the step 12 in 9% HCL, and carrying out ultrasonic oscillation on the sample for 20 minutes after overnight standing at 4 ℃, wherein the oscillation condition is 25 ℃, 40kHz and high frequency.
And step 14, placing the mixed solution after shaking in the step 13 in a high-speed centrifuge at 4 ℃ for 12000g, centrifuging for 10 minutes, discarding the precipitate, and reserving and taking supernatant.
Determination of optimum wavelength by improved TPTZ spectrophotometry:
after being treated by the above determination scheme, the standard ferrous chloride stock solution of 1 ng/microliter is placed in a microplate reader for full-wavelength scanning. Double distilled water was used as a blank control. As a result, as shown in FIG. 6, it can be seen that the optimum detection wavelength of the method of the present invention is 595 nm.
Biological sample total iron ion determination:
experimental reagent: ascorbic acid, ammonium ferrous sulfate hexahydrate, acetic acid, anhydrous sodium acetate, terpyridyl triazine and secondary distilled water.
Step 21, reagent preparation:
solution A: 20mg of sodium acetate and 800 mu L of acetic acid are mixed and then the volume is increased to 50mL by using double distilled water.
And B, liquid B: ascorbic acid (vitamin C)0.264g was dissolved in 5mL double distilled water.
And C, liquid C: 200mg of 2,4, 6-tripyridyl triazine is dissolved by adding 100 mu L of hydrochloric acid, and double distilled water is added to reach the volume of 50 mL.
And (3) liquid D: saturated sodium hydroxide solution.
Step 22, preparation of a standard substance:
ferrous chloride (FeCl) was prepared at a concentration of 4 ng/. mu.L, 2 ng/. mu.L, 1 ng/. mu.L, 0.5 ng/. mu.L, 0.25 ng/. mu.L, 0.125 ng/. mu.L, 0.0625 ng/. mu.L, 0.03125 ng/. mu.L, 0.015625 ng/. mu.L, 0 ng/. mu.L2) The solvent of the solution is double distilled water.
Step 23, standard curve configuration:
231, putting 20 mu L of the standard substance into the cells of a 384-hole plate, and simultaneously arranging a blank control, namely 20 mu L/hole of double distilled water;
232, adding 5 mu L of D liquid into each standard substance and each blank control hole;
step 233, adding 67.5 mul/hole A solution;
234, adding 7.5 mu L/hole C solution;
235, placing the 384-hole plate in a 37 ℃ incubator, fully reacting for 30 minutes, and cooling to room temperature;
and 236, placing the pore plate in an enzyme labeling instrument, measuring the absorbance value of each pore at the wavelength of 595nm, and making a standard curve. The absorbance was measured and a standard curve was plotted as shown in FIG. 7, where R is shown2The linear correlation is better when the value is 0.9997.
The detection operation of total iron ions of the sample comprises the following steps:
31, putting 20 mu L of a sample into a cell of a 384-pore plate, and simultaneously setting a blank control, namely 20 mu L/hole of double distilled water;
32, adding 3.75 mu L of the solution B into each sample and each blank control hole, and standing for 10 minutes;
step 33, adding 5 mu L of D liquid per hole;
step 34, adding 63.75 mu L/hole A solution;
step 35, adding 7.5 mu L/hole C solution;
step 36, placing the 384-hole plate in a 37 ℃ incubator, fully reacting for 30 minutes, and cooling to room temperature;
step 37, placing the pore plate in an enzyme labeling instrument, and measuring the absorbance value of each pore at the wavelength of 595 nm;
and step 38, substituting the absorbance value of the measured sample into a standard curve equation, and calculating the total iron ion concentration of the sample to be 0.19 ng/mu L.
And (3) recovery rate inspection:
an iron standard solution of 3 parallel groups is selected to be 1 ng/. mu.L for an adding standard recovery experiment, and the adding standard recovery rate is (adding standard sample determination value-sample determination value)/adding standard amount multiplied by 100 percent through a recovery rate calculation formula, so that the average adding standard recovery rate is 101.3 percent, S is 0.5, and RSD is 0.55 percent. It can be seen that the recovery rate of the present invention is better.
Example 3
Processing of the biological sample:
step 11, culturing the ovarian cancer Skov3 cell line for 24 hours to obtain 5X 106And (4) collecting the cultured cells, centrifuging, and removing the supernatant to obtain cell masses.
And step 12, placing the cell mass block in 10% HCL to directly perform ultrasonic oscillation for 1 hour, wherein the oscillation condition is 25 ℃, 50kHz and high frequency.
And step 13, placing the mixed solution after shaking in the step 12 in a 0 ℃ high-speed centrifuge of 10000g, centrifuging for 10 minutes, discarding the precipitate, and reserving and taking the supernatant.
Determination of optimum wavelength by improved TPTZ spectrophotometry:
after being treated by the above determination scheme, the standard ferrous chloride stock solution of 1 ng/microliter is placed in a microplate reader for full-wavelength scanning. Double distilled water was used as a blank control. As a result, as shown in FIG. 6, it can be seen that the optimum detection wavelength of the method of the present invention is 595 nm.
And (3) measuring ferrous ions of the biological sample:
step 21, reagent preparation:
solution A: phosphate buffer.
And C, liquid C: 200mg of 2,4, 6-tripyridyl triazine is dissolved by adding 100 mu L of hydrochloric acid, and double distilled water is added to reach the volume of 50 mL.
And (3) liquid D: saturated potassium hydroxide solution.
Step 22, preparation of a standard substance:
preparation of 4 ng/. mu.L, 2 ng/. mu.L, 1 ng/. mu.L, 0.5 ng/. mu.L, 0.25 ng/. mu.L, 0.125 n/. mu.LFerrous chloride (FeCl) at a concentration of g/. mu.L, 0.0625 ng/. mu.L, 0.03125 ng/. mu.L, 0.015625 ng/. mu.L, 0 ng/. mu.L2) The solvent of the solution is double distilled water.
Step 23, standard curve configuration:
231, putting 20 mu L of the standard substance into the cells of a 384-hole plate, and simultaneously arranging a blank control, namely 20 mu L/hole of double distilled water;
232, adding 5 mu L of D liquid into each standard substance and each blank control hole;
step 233, adding 67.5 mul/hole A solution;
234, adding 7.5 mu L/hole C solution;
235, placing the 384-hole plate in a 37 ℃ incubator, fully reacting for 30 minutes, and cooling to room temperature;
and 236, placing the pore plate in an enzyme labeling instrument, measuring the absorbance value of each pore at the wavelength of 595nm, and making a standard curve. Measuring absorbance and plotting a standard curve as shown in FIG. 8, R can be seen2The linear correlation is better when the value is 0.9997.
The detection operation of the ferrous ions of the sample comprises the following steps:
31, putting 20 mu L of a sample into a cell of a 384-pore plate, and simultaneously setting a blank control, namely 20 mu L/hole of double distilled water;
step 32, adding 5 mu L of D liquid per hole;
step 33, adding 63.75 mu L/hole A solution;
step 34, adding 7.5 mu L/hole C solution;
step 35, placing the 384-hole plate in a 37 ℃ incubator, fully reacting for 30 minutes, and cooling to room temperature;
step 36, placing the pore plate in an enzyme labeling instrument, and measuring the absorbance value of each pore at the wavelength of 595 nm;
and step 37, substituting the absorbance value of the measured sample into a standard curve equation, and calculating the ferrous ion concentration of the sample to be 0.108 ng/mu L.
And (3) recovery rate inspection:
an iron standard solution of parallel 3 groups is selected to be subjected to an adding standard recovery experiment at a concentration of 1 ng/. mu.L, and the adding standard recovery rate is (adding standard sample determination value-sample determination value)/adding standard amount multiplied by 100% through a recovery rate calculation formula, so that the average adding standard recovery rate is 100.8%, S is 0.4, and RSD is 0.86%. It can be seen that the recovery rate of the present invention is better.
Example 4
Processing of the biological sample:
step 11, culturing the ovarian cancer Skov3 cell line for 24 hours to obtain 5X 106And (4) collecting the cultured cells, centrifuging, and removing the supernatant to obtain cell masses.
And step 12, placing the cell mass block in 10% HCL to directly perform ultrasonic oscillation for 1 hour, wherein the oscillation condition is 25 ℃, 50kHz and high frequency.
And step 13, placing the mixed solution after shaking in the step 12 in a 0 ℃ high-speed centrifuge of 10000g, centrifuging for 10 minutes, discarding the precipitate, and reserving and taking the supernatant.
Determination of optimum wavelength by improved TPTZ spectrophotometry:
after being treated by the above determination scheme, the standard ferrous chloride stock solution of 1 ng/microliter is placed in a microplate reader for full-wavelength scanning. Double distilled water was used as a blank control. As a result, as shown in FIG. 6, it can be seen that the optimum detection wavelength of the method of the present invention is 595 nm.
Biological sample total iron ion determination:
step 21, reagent preparation:
solution A: phosphate buffer.
And B, liquid B: 5g of hydroxylamine hydrochloride was dissolved in 5mL of double distilled water.
And C, liquid C: 200mg of 2,4, 6-tripyridyl triazine is dissolved by adding 100 mu L of hydrochloric acid, and double distilled water is added to reach the volume of 50 mL.
And (3) liquid D: saturated potassium hydroxide solution.
Step 22, preparation of a standard substance:
ferrous chloride (FeCl) was prepared at a concentration of 4 ng/. mu.L, 2 ng/. mu.L, 1 ng/. mu.L, 0.5 ng/. mu.L, 0.25 ng/. mu.L, 0.125 ng/. mu.L, 0.0625 ng/. mu.L, 0.03125 ng/. mu.L, 0.015625 ng/. mu.L, 0 ng/. mu.L2) The solvent of the solution is double distilled water.
Step 23, standard curve configuration:
231, putting 20 mu L of the standard substance into the cells of a 384-hole plate, and simultaneously arranging a blank control, namely 20 mu L/hole of double distilled water;
232, adding 5 mu L of D liquid into each standard substance and each blank control hole;
step 233, adding 67.5 mul/hole A solution;
234, adding 7.5 mu L/hole C solution;
235, placing the 384-hole plate in a 37 ℃ incubator, fully reacting for 30 minutes, and cooling to room temperature;
and 236, placing the pore plate in an enzyme labeling instrument, measuring the absorbance value of each pore at the wavelength of 595nm, and making a standard curve. Measuring absorbance and plotting a standard curve as shown in FIG. 8, R can be seen2The linear correlation is better when the value is 0.9997.
The detection operation of total iron ions of the sample comprises the following steps:
31, putting 20 mu L of a sample into a cell of a 384-pore plate, and simultaneously setting a blank control, namely 20 mu L/hole of double distilled water;
32, adding 3.75 mu L of the solution B into each sample and each blank control hole, and standing for 10 minutes;
step 33, adding 5 mu L of D liquid per hole;
step 34, adding 63.75 mu L/hole A solution;
step 35, adding 7.5 mu L/hole C solution;
step 36, placing the 384-hole plate in a 37 ℃ incubator, fully reacting for 30 minutes, and cooling to room temperature;
step 37, placing the pore plate in an enzyme labeling instrument, and measuring the absorbance value of each pore at the wavelength of 595 nm;
and step 38, substituting the absorbance value of the measured sample into a standard curve equation, and calculating the total iron ion concentration of the sample to be 0.201 ng/mu L.
And (3) recovery rate inspection:
an iron standard solution of parallel 3 groups is selected to be 1 ng/. mu.L for an adding standard recovery experiment, and the adding standard recovery rate is (adding standard sample determination value-sample determination value)/adding standard amount multiplied by 100 percent through a recovery rate calculation formula, so that the average adding standard recovery rate is 103 percent, S is 0.6, and RSD is 1.2 percent. It can be seen that the recovery rate of the present invention is better.
Comparative example 1
Cell lysis solution commonly used in the market is used for cell lysis, and milky white suspension is formed after the tripyridyltriazine is added, and no bluish purple complex is formed. Among them, the tissue/cell lysate commonly used in the market is RIPA tissue/cell lysate, and the major manufacturers (and the goods numbers) include: sigma (R0278), Thermo Scientific (78430), Dingguo (WB-0072), Baiolaibo (BL1357), etc., whose components were mainly 50mmol/L Tris-HCl,150mmol/L NaCl, 0.1% SDS, 1% NP-40, 2. mu.g/ml Aprotinin, 2. mu.g/ml Leuteptin, 1mM PMSF, 1.5mM EDTA, 1mM NaVanadate (phosphatase inhibitor, candidate).
Comparative example 2
Comparative example 2 is substantially the same as example 2 except that comparative example 2 does not prepare liquid D, nor does it have step 232 and step 33.
In comparative example 2, in the absence of solution D, the biological sample was treated with a strong acid and the pH was low, so that the measurement system could not be maintained at the optimum pH for the stable complex formed by tripyridyltriazine and ferrous ion, i.e., the optimum pH was 3-5, resulting in an unstable complex, which was very small and could not be measured at all.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (2)

1. A method for measuring ferrous ions in a biological sample is characterized by comprising the following steps:
culturing an ovarian cancer Skov3 cell line for 24 hours to obtain 5 × 106 cells, collecting the cultured cells, centrifuging, and removing supernatant to obtain cell masses;
freezing the collected cell mass in liquid nitrogen overnight, and grinding into powder;
placing the obtained biological sample powder in 9% HCL, carrying out ultrasonic oscillation on the sample for 20 minutes after overnight standing at 4 ℃, wherein the oscillation condition is 25 ℃ and 40 kHz;
placing the mixed solution after the oscillation in a high-speed centrifuge at 4 ℃ for 12000g, centrifuging for 10 minutes, removing the precipitate, and reserving and taking supernatant to obtain a treated biological sample solution;
sequentially adding a solution D, a solution A and a solution C into the biological sample solution to obtain a first sample, wherein the solution D is a saturated sodium hydroxide solution, the solution A is a buffer solution, and the solution C is a color developing agent solution;
reacting the first sample at 37 ℃ for 30 minutes to obtain a second sample;
measuring the second sample at a wavelength of 595nm to obtain an absorbance value;
preparing a standard solution of ferrous ions, and preparing a standard curve to obtain a standard curve equation;
substituting the absorbance value of the second sample into the standard curve equation to calculate the ferrous ion concentration of the biological sample solution;
wherein the preparation method of the solution A comprises mixing 20mg of sodium acetate and 800 μ L of acetic acid, and diluting to 50mL with double distilled water;
the preparation method of the solution C comprises the steps of adding 100 mu L hydrochloric acid into 200mg of 2,4, 6-tripyridyltriazine for dissolution, and adding double distilled water to reach the constant volume of 50 mL.
2. A method for measuring total iron ions in a biological sample is characterized by comprising the following steps:
culturing an ovarian cancer Skov3 cell line for 24 hours to obtain 5 × 106 cells, collecting the cultured cells, centrifuging, and removing supernatant to obtain cell masses;
freezing the collected cell mass in liquid nitrogen overnight, and grinding into powder;
placing the obtained biological sample powder in 9% HCL, carrying out ultrasonic oscillation on the sample for 20 minutes after overnight standing at 4 ℃, wherein the oscillation condition is 25 ℃ and 40 kHz;
placing the mixed solution after the oscillation in a high-speed centrifuge at 4 ℃ for 12000g, centrifuging for 10 minutes, removing the precipitate, and reserving and taking supernatant to obtain a treated biological sample solution;
adding the solution B into the biological sample solution, standing for 10 minutes, and then sequentially adding the solution D, the solution A and the solution C to obtain a first sample, wherein the solution B is a reducing agent solution, the solution D is a saturated alkaline solution, the solution A is a buffer solution, and the solution C is a color developing agent solution;
reacting the first sample at 37 ℃ for 30 minutes to obtain a second sample;
measuring the second sample at a wavelength of 595nm to obtain an absorbance value;
preparing a standard solution of ferrous ions, and preparing a standard curve to obtain a standard curve equation;
substituting the absorbance value of the second sample into the standard curve equation to calculate the total iron ion concentration of the biological sample solution;
the preparation method of the solution A comprises the following steps: mixing 20mg of sodium acetate and 800 mu L of acetic acid, and then using double distilled water to fix the volume to 50 mL;
the preparation method of the solution B comprises dissolving 0.264g ascorbic acid in 5mL double distilled water;
the preparation method of the solution C comprises the steps of adding 100 mu L hydrochloric acid into 200mg of 2,4, 6-tripyridyltriazine for dissolution, and adding double distilled water to reach the constant volume of 50 mL;
the solution D is saturated sodium hydroxide solution.
CN201910801327.1A 2019-08-28 2019-08-28 Biological sample treatment method, ferrous ion or total ferric ion determination method of biological sample and detection kit Active CN110455732B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201910801327.1A CN110455732B (en) 2019-08-28 2019-08-28 Biological sample treatment method, ferrous ion or total ferric ion determination method of biological sample and detection kit

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201910801327.1A CN110455732B (en) 2019-08-28 2019-08-28 Biological sample treatment method, ferrous ion or total ferric ion determination method of biological sample and detection kit

Publications (2)

Publication Number Publication Date
CN110455732A CN110455732A (en) 2019-11-15
CN110455732B true CN110455732B (en) 2021-09-28

Family

ID=68489567

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201910801327.1A Active CN110455732B (en) 2019-08-28 2019-08-28 Biological sample treatment method, ferrous ion or total ferric ion determination method of biological sample and detection kit

Country Status (1)

Country Link
CN (1) CN110455732B (en)

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105384769B (en) * 2015-07-20 2018-02-02 北京师范大学 The preparation and application of the nano-silicon dioxide particle of terpyridine moieties modification
CN108445231A (en) * 2018-03-21 2018-08-24 浙江经贸职业技术学院 Detect kit, the method and its application of lactoferrin and beta lactoglobulin
CN108801954B (en) * 2018-06-28 2021-05-14 湖北兴瑞硅材料有限公司 Method for measuring content of iron ions in organic silicon product

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
天然活性多糖研究进展;张淑杰 等;《食品工业科技》;20160926;第38卷(第2期);第379-382、389页 *
张淑杰 等.天然活性多糖研究进展.《食品工业科技》.2016,第38卷(第2期),第379-382、389页. *

Also Published As

Publication number Publication date
CN110455732A (en) 2019-11-15

Similar Documents

Publication Publication Date Title
Cao et al. Naked-eye sensitive detection of nuclease activity using positively-charged gold nanoparticles as colorimetric probes
Goodwin Quantification of serum inorganic phosphorus, phosphatase, and urinary phosphate without preliminary treatment
Meng et al. A versatile electrochemical biosensor for the detection of circulating microRNA toward non‐small cell lung cancer diagnosis
US9222885B2 (en) Method for assaying nucleic acids by fluorescence
CN103033619B (en) A kind of protein chip kit of comprehensive detection lung cancer marker and method
CN108484622A (en) The synthesis of multi signal fluorescence probe and its application for distinguishing detection Hcy, Cys and GSH simultaneously
CN109142710B (en) Method for rapidly and sensitively detecting tetrodotoxin TTX
CN107502339B (en) A kind of ratio fluorescent probe identifying nilotinib and its preparation and recognition methods
CN107449759B (en) Efficient mercury ion detection method, probe molecule and kit
US20150037805A1 (en) Double-stranded nucleic acid, use and kit thereof in ribonuclease detection
CN112129736B (en) Homogeneous phase visualization/fluorescence POCT detection method for mucin and circulating tumor cells
CN105928917B (en) A kind of silver nanoclusters sensor and its preparation method and application
CN110455732B (en) Biological sample treatment method, ferrous ion or total ferric ion determination method of biological sample and detection kit
CN111855625B (en) CA125 detection kit based on Cu-MOF and application thereof
CN103630518B (en) A kind of new method of the fluorescence probe detection hydrogen sulfide synthase activity of use hydrogen sulfide and its application
Sun et al. An amplified fluorescence detection of T4 polynucleotide kinase activity based on coupled exonuclease III reaction and a graphene oxide platform
CN110592186B (en) AND molecular logic gate sensing system AND preparation method AND application thereof
US20230313272A1 (en) Rna quality assay
CN113960153A (en) ICP-MS (inductively coupled plasma-mass spectrometry) detection method for 12 elements in serum
CN110468182A (en) It is a kind of detect Platelet-derived growth factor BB homogeneous bioanalytical method and its application
CN107356577B (en) Universal sulfotransferase activity analysis method
Chen et al. A sequential injection fluorometric procedure for rapid determination of total protein in human serum
Subashchandrabose et al. Trace element analysis of urine by ICP-MS/MS to identify urinary tract infection
CN110398589B (en) Kit for quantitatively detecting content of anti-mullerian hormone and detection method thereof for non-disease diagnosis purpose
CN113267475A (en) Method for fluorescence detection of cadmium ions by one-step quenching method

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant