CN109632674A - A kind of method of test sample pH value - Google Patents

A kind of method of test sample pH value Download PDF

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Publication number
CN109632674A
CN109632674A CN201910083444.9A CN201910083444A CN109632674A CN 109632674 A CN109632674 A CN 109632674A CN 201910083444 A CN201910083444 A CN 201910083444A CN 109632674 A CN109632674 A CN 109632674A
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China
Prior art keywords
value
sample
tested
test sample
acid
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Pending
Application number
CN201910083444.9A
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Chinese (zh)
Inventor
吴海苹
吴尚毅
叶庆新
陈淑妹
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Xiamen Limulus Reagent Biological Polytron Technologies Inc
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Xiamen Limulus Reagent Biological Polytron Technologies Inc
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Priority to CN201910083444.9A priority Critical patent/CN109632674A/en
Publication of CN109632674A publication Critical patent/CN109632674A/en
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/78Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
    • G01N21/80Indicating pH value
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N2021/775Indicator and selective membrane

Abstract

The present invention provides a kind of methods of test sample pH value comprising acid-base indicator is added in microwell plate;The pH standard solution of known different pH value and sample to be tested are respectively added in the microwell plate containing the acid-base indicator, every hole adds 10 ~ 500 μ L, and vibration plate mixes, and the OD value in each hole is surveyed with instrument;Using the pH value of pH standard solution as abscissa, OD value is ordinate, draws standard curve;Calculate the pH value of sample to be tested;To pass through the method test sample pH value of test absorbance value, so that the sample size that sample uses when detecting is few, can batch detection, detection speed it is fast, can accurate quantitative analysis, and the instrument used has versatility, to realize that the batch of clinically leukorrhea pH value quickly measures, solve the problems, such as that at present clinically the detection of leukorrhea pH value exists.

Description

A kind of method of test sample pH value
Technical field
The present invention relates to pH value fields of measurement, and in particular to a kind of method of test sample pH value.
Background technique
The measurement method of pH value mainly has indicator method, electrochemical process and optical sensor method etc.;Wherein, pH test paper (card) Measurement belongs to indicator method, and when with pH test paper (card) test sample, colour developing result usually will not be completely the same with standard color block, needs It wants proofer to estimate a numerical value between the two closest discontinuous standard color blocks that develop the color, error is easy to appear, so that pH The precision of test paper (card) detection is low.And pH meter is mainly surveyed by electrode to the instrument for accurately measuring solution ph The physical quantity variation of quantity of fluid current potential and pH value determination, but it is only used for monocyte sample test, and required sample size is more, can not batch Quickly measurement.
Therefore, the method for test sample pH value still has much room for improvement at present.
Summary of the invention
The present invention is directed to solve one of the technical problem in above-mentioned technology at least to a certain extent.
It should be noted that the present invention is the following discovery based on inventor and completes:
The measurement of leukorrhea pH value is basic vaginal fluid inspection item, and normal leukorrhea pH value is about 4.5, and pH value is greater than 5 When~6, usually indicate to suffer from trichomonas or bacterial vaginitis after exclusive PCR factor, therefore the pH value measurement of leukorrhea can be made For colpitis auxiliary diagnosis means.Traditional vaginal fluid pH value measurement is to contact secretion with common accurate pH test paper, PH value is read by developing the color to compare with standard color block.However following problems are often deposited in clinical application:
1, clinically leukorrhea sampling is usually the inspection in a manner of cotton swab, due to the semifluid character of secretion itself, In addition the water sorption of cotton balls, the wet degree of cotton swab generally can not infiltrate enough pH test paper and be allowed to sufficiently develop the color, inspection Time is particularly evident when extending.
2, by improving, diminution test paper area is made into test card for some producers, adds sample liquids from special well, It to reduce the required sample liquid measure of detection, but also reduces color lump and generates area, to influence the interpretation of vision.
3, when with pH test paper (card) test sample, colour developing result usually will not be completely the same with standard color block, needs to examine Person's one numerical value of estimation between the closest two discontinuous standard color blocks of developing the color, in view of the requirement to pH value accurate reading, Such as the exceptional value in normal vagina pH value 3.8~4.2 and Amsel standard (goldstandard of bacterial vaginitis clinical diagnosis) (pH > 4.5) are close, therefore all very high to the sensitivity and accuracy requirement of pH value measurement, but due to common precision test paper itself The colour vision error of sensitivity and human eye causes experimental error usually in 0.5 pH or more, to often result in testing result and determine to be stranded It is difficult;
4, some producers carry out auxiliary by test paper (card) interpretative instrument and distinguish color interpretation result at present, but often because of test paper table The interference of face degree of roughness difference, test paper remained on surface liquid reflective are interfered, dry and wet paper card reading differs greatly etc. problem And the accurate of result interpretation is influenced, and result is simulation value, it is not accurate quantitative analysis.In addition, these test paper (card) interpretations Instrument is typically only capable to mating special test paper (card) and uses, and different manufacturers are not generally general, and user is in replacement test paper (card) Instrument must be bought when producer again will cause waste.
5, pH meter is to commonly use at present accurately to measure the instrument of solution ph, mainly passes through electrode measurement liquid current potential Physical quantity variation and pH value determination are only used for monocyte sample test, need to calibrate and be easily affected by temperature before use, but also deposit The problem of in terms of electrode cleaning, disinfection, it is impossible to be used in the batch of leukorrhea etc micro-example pH value quickly measures.
In view of this, inventor makes sample develop the color using chemical reagent, then with the method for apparatus measures sample OD value, with inspection The pH value of sample, this method when detecting needed for sample size it is few, can measure batch, detection speed is fast, can be surveyed with accurate quantitative analysis It measures and instrument is versatile.
For this purpose, the invention proposes a kind of methods of test sample pH value comprising:
(1) acid-base indicator is added in microwell plate;
(2) the pH standard solution of known different pH value and sample to be tested are respectively added micro- containing the acid-base indicator In orifice plate, every hole adds 10~500 μ L, and vibration plate mixes, and the OD value in each hole is surveyed with instrument;
(3) using the pH value of pH standard solution as abscissa, the OD value of pH standard solution is ordinate, draws standard curve;
(4) pH value of sample to be tested is calculated.
The method of test sample pH value according to the present invention draws out pH by testing the absorbance value of pH standard solution The standard curve of standard solution, then pass through the absorbance value of standard curve and sample to be tested, obtain the pH value of sample to be tested, so that The sample size that sample uses when detecting is few, can batch detection, detection speed it is fast, can accurate quantitative analysis, and the instrument tool used There is versatility, to realize that the batch of clinically leukorrhea pH value quickly measures, solves at present clinically leukorrhea pH value detection and exist The problem of.
In addition, a kind of method of the test sample pH value proposed according to that above embodiment of the present invention, can also have as follows Additional technical characteristic:
According to embodiments of the present invention, the acid-base indicator in the step (1) by the dehydrated alcohol of 5~100mL and 0~ The bromocresol green mixed dissolution of 0.25g, then be settled to 100mL with water and be made.
It according to embodiments of the present invention, is the OD value phase that sample to be tested is found from the standard curve in the step (4) The pH value answered, to obtain the pH value of the sample to be tested;Or the regression equation of the standard curve is calculated, then will be to be measured The OD value of sample substitutes into the regression equation, the pH value of sample to be tested is calculated, to obtain the pH value of the sample to be tested.
According to embodiments of the present invention, the acid-base indicator of 5~20 μ L is added in the step (1) every hole in microwell plate.
According to embodiments of the present invention, the middle microplate reader of the step (2) surveys the OD in each hole under the wavelength of 550~700nm Value.
According to embodiments of the present invention, the OD value in each hole is surveyed under the wavelength of 620nm or 630nm in the step (2).
According to embodiments of the present invention, using the citric acid-sodium citrate pH standard of known different pH value in the step (2) Solution.
According to embodiments of the present invention, the pH value of the citric acid-sodium citrate pH standard solution is 3.8~5.8.
Additional aspect and advantage of the invention will be set forth in part in the description, and will partially become from the following description Obviously, or practice through the invention is recognized.
Detailed description of the invention:
Fig. 1 is the standard curve of pH standard solution of the invention.
Specific embodiment
In order to better understand the above technical scheme, exemplary embodiment of the present invention is described in more detail below, answer Work as understanding, may be realized in various forms the present invention and should not be limited by the embodiments set forth herein.On the contrary, providing these realities It applies example to be to be able to thoroughly understand the present invention, and the scope of the present invention can be communicated completely to the skill of this field Art personnel.Particular technique or condition are not specified in embodiment, according to the literature in the art described technology or conditions or Person carries out according to product description.Reagents or instruments used without specified manufacturer, being can be by the normal of commercially available acquisition Advise product.
It should be noted that microplate reader of the present invention does not refer in particular to the instrument of a certain model instead of, refer to using enzyme mark Instrument analysis principle carries out a quasi-instrument of analysis test, such as certain spectrophotometers, micro-spectrophotometer, Biochemical Analyzer Also often with the analytic function for having microplate reader, some microplate reader detection modules are placed in self-reacting device, or on this basis Improvement is done some, such as is made into work station, it is possible to have the detection function, these all belong to this kind of instrument of microplate reader of the present invention Device, in the protection scope of invention.The acquisition cost of user's instrument can be saved by being applicable in these instruments.
It is of the present invention micro, refer to the seldom scope of relatively measurable sample liquid volume, without referring to some tool The volume of body, being usually only necessary to 0.05-0.1mL prepare liquid can be used to this test, test for example, by using 96 half bore microwell plates.Operation Person can adjust the amount of reagent or test fluid according to the micropore board type of selection to obtain ideal effect, and for such tune It is whole, still belong to protection scope of the present invention.
The scheme of acid-base indicator of the invention is suitable for sample of the pH between 3.6-6.6, for being more than the pH range Sample can realize test by replacing the suitable mating method of the invention of acid-base indicator, but the adjustment still belongs to this hair The protection scope of bright method.
Quick detection of the present invention is not to refer in particular to some time, is specifically dependent upon the read plate speed of selected test equipment Degree, most microplate reader can be completed in 1 minute, such as uncle is selected to rise Elx808 series microplate reader.The inspection of different model instrument Degree of testing the speed slightly has difference, but for using different instruments by optimization, to obtain the improvement in detection required time, still belongs to this The protection scope of invention.
Batch of the present invention is not to refer in particular to some numerical value, specifically with the microwell plate model of operator's selection and to test sample Multiple holes number it is related, by taking 96 orifice plates as an example, if the multiple holes number of each test specimens be 2, the batch tested every time can be at 40 It is more than sample.And use 384 to wait high-throughput plate and its necessary instrument then and can get the batch of higher single test, for optimization It detects carrier and promotes batch, still belong to the scope of the present invention.
Embodiment
1, acid-base indicator: (by taking 100ml amount as an example) is prepared referring to following table 1:
1 acid-base indicator preparation program of table
Serial number Raw material Dosage Remarks
A Dehydrated alcohol 5~100ml As solvent, preferred volume 20ml
B Bromocresol green 0~0.25g It is uniformly dissolved with dehydrated alcohol, preferably 0.05g
C H2O 0~95ml Dissolved mixture constant volume to 100ml
Wherein, if there is precipitating in when above-mentioned A and B mixed dissolution, 0~10ml of 0.1M sodium hydroxide dissolution can be added dropwise, then Add water fixed molten to 100ml.The dissolved liquid color of A and B should be bluish-green (slightly biased indigo plant) color can add dropwise if there is larger color difference Enter 0.1M sodium hydroxide adjustment solution colour, stops being added when meeting.
Use the pH value linear working range of the acid-base indicator agent: 3.6-6.6.
2, pH standard solution:
PH standard solution can be prepared in the present embodiment, can also directly choose the standard solution of corresponding pH value.Wherein, pH is prepared When standard solution:
It prepares 0.1mol/L citric acid solution: taking citric acid (C6H8O7·H2O: molecular weight 210.14) 21.01g is dissolved in 950mL pure water is uniformly dissolved rear constant volume to 1L, to obtain 0.1mol/L citric acid solution.
It prepares 0.1mol/L sodium citrate solution: taking sodium citrate (Na3C6H5O7·2H2O: molecular weight 294.12) 29.41g It is dissolved in 950mL pure water, is uniformly dissolved rear constant volume to 1L, to obtain 0.1mol/L sodium citrate solution.
(citric acid-sodium citrate) pH standard solution is prepared according to the following table 2 scheme:
2 citric acid-sodium citrate pH standard solution of table is prepared
3, the method for using microplate reader to detect clinically leukorrhea sample pH: (by taking 150 μ L half bore, 96 microwell plate as an example)
(1) acid-base indicator by above-mentioned preparation is sub-packed in microwell plate, and every hole adds 5~20 μ L, it is preferable that every hole adds 10 μL。
(2) the 96 half bore microwell plates that 10 μ L acid-base indicators have been added are taken, the pH standard of above-mentioned six pH value is separately added into Solution or sample to be tested, every hole add 10~500 μ L, it is preferable that every hole adds 100 μ L;Wherein each 3 multiple holes of pH standard solution, to Sample each 2 or 3 multiple holes;Vibration plate mixes after the completion of sample-adding, rises 808 type microplate reader with uncle and surveys OD630Value.After tested, each pH The OD of titer630As shown in table 3 below.
The OD of table 3pH standard solution630
(3) using the pH value of pH standard solution as abscissa, OD630Value is ordinate, draws standard curve;The mark of acquisition Directrix curve is as shown in Figure 1, the regression equation for being computed acquisition is as shown in table 4.
Table 4pH calibration curve formula
Curve equation A B R value
Y=A × X+B 0.639 -2.33 0.9939
(4) pH value of sample to be tested is calculated.36 clinical samples being added are selected, wherein all kinds of vaginitis patient (sufferers Group) 12, healthy person (Normal group) 24.It is computed Normal group and sufferer group vaginal fluid measurement result is classified Statistics such as following table 5.
5 vaginitis patient of table and healthy person vaginal fluid pH result
Group Quantity Age PH value
Sufferer group 6 40.0±8.7 5.41±0.42
Normal group 30 28.6±6.4 4.18±0.47
In terms of the result of detection, range of this programme detection pH value in 3.8-5.8 is linearly good, can meet clinical leukorrhea just Often detection needs, and sufferer group and Normal group measurement result meet clinical expection.
The measurement of relatively existing sample pH as a result, the method for test sample pH value according to the present invention, when detecting The sample size used is few, can batch detection, detection speed it is fast, can accurate quantitative analysis, and the instrument used has versatility, can be real Now clinically the batch of leukorrhea pH value quickly measures, and solves the problems, such as that at present clinically the detection of leukorrhea pH value exists.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show The description of example " or " some examples " etc. means specific features, structure, material or spy described in conjunction with this embodiment or example Point is included at least one embodiment or example of the invention.In the present specification, schematic expression of the above terms are not It is interpreted as that identical embodiment or example must be directed to.Moreover, particular features, structures, materials, or characteristics described It can be combined in any suitable manner in any one or more of the embodiments or examples.In addition, those skilled in the art can Different embodiments or examples described in this specification are engaged and be combined.
Although the embodiments of the present invention has been shown and described above, it is to be understood that above-described embodiment is example Property, it is not considered as limiting the invention, those skilled in the art within the scope of the invention can be to above-mentioned Embodiment is changed, modifies, replacement and variant.

Claims (8)

1. a kind of method of test sample pH value characterized by comprising
(1) acid-base indicator is added in microwell plate;
(2) microwell plate containing the acid-base indicator is respectively added in the pH standard solution of known different pH value and sample to be tested In, every hole adds 10 ~ 500 μ L, and vibration plate mixes, and the OD value in each hole is surveyed with instrument;
(3) using the pH value of pH standard solution as abscissa, the OD value of pH standard solution is ordinate, draws standard curve;
(4) pH value of sample to be tested is calculated.
2. a kind of method of test sample pH value as described in claim 1, which is characterized in that the soda acid in the step (1) Indicator is settled to 100 mL systems by the dehydrated alcohol of 5 ~ 100 mL and the bromocresol green mixed dissolution of 0 ~ 0.25 g, then with water At.
3. a kind of method of test sample pH value as described in claim 1, it is characterised in that: be from institute in the step (4) The corresponding pH value of OD value that sample to be tested is found in standard curve is stated, to obtain the pH value of the sample to be tested;Or it calculates The regression equation of the standard curve, then the OD value of sample to be tested is substituted into the regression equation, calculate sample to be tested PH value, to obtain the pH value of the sample to be tested.
4. a kind of method of test sample pH value as claimed in claim 1 or 2, which is characterized in that the step (1) is in micropore The acid-base indicator of 5 ~ 20 μ L is added in every hole in plate.
5. a kind of method of test sample pH value as described in claim 1, which is characterized in that with enzyme mark in the step (2) Instrument surveys the OD value in each hole under the wavelength of 550 ~ 700nm.
6. a kind of method of test sample pH value as claimed in claim 5, which is characterized in that in 620nm in the step (2) Or under the wavelength of 630nm, the OD value in each hole is surveyed.
7. a kind of method of test sample pH value as described in claim 1, which is characterized in that using in the step (2) Know the citric acid-sodium citrate pH standard solution of different pH value.
8. a kind of method of test sample pH value as claimed in claim 7, which is characterized in that the citric acid-sodium citrate The pH value of pH standard solution is 3.8 ~ 5.8.
CN201910083444.9A 2019-01-29 2019-01-29 A kind of method of test sample pH value Pending CN109632674A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113218904A (en) * 2021-07-08 2021-08-06 北京矿冶研究总院 Sampling assembly, pH detection device and pH detection method

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