A kind of method of test sample pH valueTechnical 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, needsIt 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 pHThe precision of test paper (card) detection is low.And pH meter is mainly surveyed by electrode to the instrument for accurately measuring solution phThe 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 batchQuickly 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 5When~6, usually indicate to suffer from trichomonas or bacterial vaginitis after exclusive PCR factor, therefore the pH value measurement of leukorrhea can be madeFor 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, inspectionTime 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 examinePerson'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 itselfThe 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 strandedIt 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 tableThe 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. problemAnd the accurate of result interpretation is influenced, and result is simulation value, it is not accurate quantitative analysis.In addition, these test paper (card) interpretationsInstrument 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 potentialPhysical 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 depositThe 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 inspectionThe 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 analysisIt 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 indicatorIn 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 solutionThe 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 thatThe 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 usedThere is versatility, to realize that the batch of clinically leukorrhea pH value quickly measures, solves at present clinically leukorrhea pH value detection and existThe 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 followsAdditional 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 measuredThe 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 550~700nmValue.
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 descriptionObviously, 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, answerWork 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 realitiesIt 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 fieldArt personnel.Particular technique or condition are not specified in embodiment, according to the literature in the art described technology or conditions orPerson carries out according to product description.Reagents or instruments used without specified manufacturer, being can be by the normal of commercially available acquisitionAdvise 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 markInstrument analysis principle carries out a quasi-instrument of analysis test, such as certain spectrophotometers, micro-spectrophotometer, Biochemical AnalyzerAlso often with the analytic function for having microplate reader, some microplate reader detection modules are placed in self-reacting device, or on this basisImprovement 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 inventionDevice, 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 toolThe 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.OperationPerson can adjust the amount of reagent or test fluid according to the micropore board type of selection to obtain ideal effect, and for such tuneIt 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 rangeSample can realize test by replacing the suitable mating method of the invention of acid-base indicator, but the adjustment still belongs to this hairThe 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 equipmentDegree, 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 instrumentDegree 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 thisThe 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 sampleMultiple 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 40It 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 optimizationIt 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, thenAdd 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 differenceEnter 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 preparedWhen standard solution:
It prepares 0.1mol/L citric acid solution: taking citric acid (C6H8O7·H2O: molecular weight 210.14) 21.01g is dissolved in950mL 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) 29.41gIt 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 10μL。
(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 intoSolution 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, toSample 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 pHThe 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 acquisitionDirectrix 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 (sufferersGroup) 12, healthy person (Normal group) 24.It is computed Normal group and sufferer group vaginal fluid measurement result is classifiedStatistics 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 justOften 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 detectingThe 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 realNow 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 showThe description of example " or " some examples " etc. means specific features, structure, material or spy described in conjunction with this embodiment or examplePoint is included at least one embodiment or example of the invention.In the present specification, schematic expression of the above terms are notIt is interpreted as that identical embodiment or example must be directed to.Moreover, particular features, structures, materials, or characteristics describedIt can be combined in any suitable manner in any one or more of the embodiments or examples.In addition, those skilled in the art canDifferent 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 exampleProperty, it is not considered as limiting the invention, those skilled in the art within the scope of the invention can be to above-mentionedEmbodiment is changed, modifies, replacement and variant.