CN101038202A - Method for contrasting and correcting accuracy of liquid transfering device volume with europium atom labeling method - Google Patents
Method for contrasting and correcting accuracy of liquid transfering device volume with europium atom labeling method Download PDFInfo
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- CN101038202A CN101038202A CN 200710067193 CN200710067193A CN101038202A CN 101038202 A CN101038202 A CN 101038202A CN 200710067193 CN200710067193 CN 200710067193 CN 200710067193 A CN200710067193 A CN 200710067193A CN 101038202 A CN101038202 A CN 101038202A
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- pipettor
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Abstract
The present invention discloses a capacity precision comparing and adjusting method of the liquid transfer vessel by using a europium atom labelling method. The method includes the following steps: 1. the liquid is sucked by a standard liquid transfer vessel: the drop head of the standard liquid vessel is put into the europium labeled solution to make the drop head full of liquid; 2. the liquid is discharged by the standard liquid transfer vessel: the liquid transfer vessel is moved to the bottom of the inner wall of the polyethylene lath and all the europium labeled solution is discharged; 3. said processes are repeated and the standard liquid transfer vessel is used for adding a certain of holes; 4. a parallel test: the same solution labelled with europium is sucked by the liquid transfer vessel to be detected and then the steps of 1-3 are repeated and the liquid transfer vessel adds holes have a same number; 5. the result testing: at the same time, said added polyethylene lath is placed on the time resolution detecting meter for measuring the fluorescence value, and the number of the fluorescent substance grain is reflected by the fluorescence value (CPS value) thus, the capacity of the standard liquid transfer vessel and the capacity of the liquid transfer vessel to be detected can be compared and adjusted. The present invention has a high sensitivity, a simple operation and the method processes are easy to be repeated.
Description
Technical field
The present invention relates to a kind of precision comparing and Calibration Method to the pipettor capacity, specifically a kind of europium atom labeling method that adopts is to the precision comparing of pipettor capacity and the authentication method of calibration.
Background technology
Pipettor is the formula that the measures sample injector that adopts air displacement principle, is widely used in technical fields such as medical and health, environment measuring, food security, fine chemistry industry, is the common tool of each scientific research institution, laboratory quantitative test.The degree of accuracy of pipettor capacity is directly connected to the reliability of experimental result.At present, both at home and abroad the calibration steps of pipettor capacity precision has two kinds: 1. gravimetry." People's Republic of China's national metrological verification regulations " JJG 646-90 regulation, with of the standard method of distilled water weight method as the calibrating of pipettor capacity precision, but this complex operation, experiment is subjected to influences such as temperature, humidity, air pressure, detection sensitivity is lower simultaneously, is 0.1mg only, and experimental error is bigger, and 3 weighing results are difficult to differentiate the reason that error causes, this method is applicable to that capacity is the calibration of the above pipettor of 20 μ l.2. spectrophotometry.As potassium dichromate method, eosin method etc., and the method application " Lang Bai-than ear " colorimetric principle, defective is that high concentration, low concentration solution are existed deviation from linearity, detection sensitivity is 10
-3-10
-4G/L.In addition,, do not propose as yet so far both at home and abroad or set up effective comparison and calibration steps the micropipettor of capacity less than 20ul, and the low capacity pipettor main application of sample instrument of molecular biology, genetic engineering, protein engineering experiment just.
Summary of the invention
Technical matters to be solved by this invention be to propose a kind of highly sensitive, good reproducibility, easy and simple to handle with europium atom labeling method to pipettor capacity precision comparing and Calibration Method.
For solving the problems of the technologies described above, the present invention comprises following job step with europium atom labeling method to pipettor capacity precision comparing and Calibration Method: 1, standard pipettor imbibition: the water dropper of standard pipettor is immersed in the europium label solution, and water dropper is filled fully; 2, standard pipettor tapping: the standard pipettor is moved to tygon lath inwall bottom and the emptying europium label solution in it; 3, repeat said process, utilize the some holes of standard pipettor application of sample; 4, parallel experiment: draw same europium label solution with pipettor to be checked, repeat above-mentioned 1-3 step, with pipettor to be checked also application of sample equal number hole; 5, the result measures: said added polyethylene lath is put into simultaneously surveys the fluorescence reading on the time resolution detector, reflect fluorescent material amounts of particles in the solution by recording fluorescent value (CPS value), standard pipettor and pipettor to be checked are carried out volume ratio pair and calibration.
Above-mentioned with europium atom labeling method to pipettor capacity precision comparing and Calibration Method, utilize each application of sample 20 hole of standard pipettor and pipettor to be checked.
Above-mentioned with europium atom labeling method to pipettor capacity precision comparing and Calibration Method, before imbibition, earlier with standard pipettor and pipettor to be checked and imbibition nozzle and europium label solution equilibrium at room temperature 30 minutes.
Above-mentioned with europium atom labeling method to pipettor capacity precision comparing and Calibration Method, before imbibition, earlier with europium atom solution with strengthening the liquid dilution, to be controlled at CPS value be between the 100000-200000/100 μ l to concentration the most at last, and solution fully is mixed.
Above-mentioned with europium atom labeling method to pipettor capacity precision comparing and Calibration Method, before imbibition, carry out pre-imbibition earlier, promptly select cleaning, suitable imbibition nozzle to be placed on the standard pipettor sleeve, compress that imbibition nozzle makes it and sleeve between do not have the clearance, pre-suction europium label solution also flows back in the former container, and imbibition, discharge opeing repeat repeatedly.
Above-mentioned with europium atom labeling method to pipettor capacity precision comparing and Calibration Method, during imbibition, the water dropper of standard pipettor and pipettor to be checked is immersed 2-3mm place under the europium label solution liquid level, slowly loosen button then reposefully, after treating that 1-2 water dropper in second is filled fully, water dropper leaves liquid level, adherent stop 2-3 second, drops down excess liquid.
Above-mentioned with europium atom labeling method to pipettor capacity precision comparing and Calibration Method, during tapping, standard pipettor and pipettor to be checked are moved to numbered tygon lath inwall bottom, keep plumbness, 1-2 second is waited in press push button to the first rest point position gently, mobile water dropper pastes inwall, press knob down fully,, then water dropper is upwards removed along inwall to drain the europium label solution in the water dropper.
Above-mentioned with europium atom labeling method to pipettor capacity precision comparing and Calibration Method, the excitation wavelength of described time-resolved fluorescence instrument is 336～337nm, the mensuration wavelength is 610～613nm.
Above-mentioned with europium atom labeling method to pipettor capacity precision comparing and Calibration Method, mensuration obtains calculating after the CPS value CPS average of two pipettors, utilizes formula CV=SD/CPS average * 100% and R=[pipettor CPS average to be checked-standard pipettor CPS average]/standard pipettor CPS average * 100% relative error calculates CV value (precision of expression pipettor--reflection stochastic error) and relative error R (represent the accuracy of pipettor to be checked---reflection systematic error) respectively; The be above standard pipettor of specification error scope of CV value is abandoned; Up to specification but when unequal, regulate pipettor top knob as the CV value of pipettor to be checked with the CPS average of standard pipettor, adjust its amount of capacity; Duplicate detection step 1-5 and aforementioned calculation set-up procedure equate that with the CPS average of standard pipettor promptly R trends towards till 0 until pipettor CPS average to be checked once more.
The present invention is owing to adopted said method, and it calculates related data separately by bioassay standard pipettor and the fluorescent value of pipettor to be checked to same europium label solution suction sample.Since under same europium label concentration, the size that how much reflects capacity of granule number, thus can compare to the capacity of two pipettors, and calculate relative error.The CV value of measurement result should be within the permissible error scope of " quantitatively, adjustable pipette measurement verification regulations " JJG 646-90 regulation.The pipettor that the CV value is surpassed JJG 646-90 specification error scope should be abandoned; Up to specification as CV, and the CPS average of pipettor to be checked equates with the CPS average of standard pipettor, represents that then both capacity are identical.CV as pipettor to be checked is up to specification, but when unequal, represents that both capacity are inequality with the CPS average of standard pipettor.At this moment, just need calibrate the capacity of pipettor to be checked: regulate pipettor top knob with special spanner, turn clockwise, capacity is transferred big; Be rotated counterclockwise, capacity is turned down, carry out 20 mark solution application of samples subsequently again and measure, until pipettor to be checked with till CPS average between the standard pipettor equates.It utilizes time-resolved fluorescence detection technique (time-resolvedfluoroimmunoassay, TR-FIA) high sensitivity that is had and high stability characteristics, with group of the lanthanides europium atom (Eu) as tracer material (atomic number 63, atomic radius 2.56, atomic volume 28.9cm
3/ mol), (β-NTA) is a fluorescence enhancement solution with β-naphthoyltrifluoroacetone, with 336～337nm is excitation wavelength, with 610～613nm serves as to measure wavelength, differentiate luminoscope with the time and detect Luhrmann's purple fluorescence spectrum, JJG 646-90 is a criterion with " People's Republic of China's national metrological verification regulations ", sets up accurately effectively, uses simply and rapidly europium atom labelling technique to pipettor capacity precision comparing and Calibration Method.It has background low (200～400cps), it is highly sensitive that (detection sensitivity reaches 10
-12～10
-14G/L), high specificity (exciting light and radiative STOKES displacement are greater than 200nm), good reproducibility (CV＜1%), tracer stable (the europium half life period reaches up to ten thousand years), the linear Fan Kuan of typical curve etc. detect advantage.Utilize the inventive method, can carry out the relevant regulations of recognizing each other about medical test, medical imaging between medical institutions better, be the specific requirement of each laboratory internal soundness control, medical test, check result " single-pass " provide technical guarantee between medical institutions in order to advance again.
Description of drawings
Fig. 1 is the process flow diagram of comparison of the present invention and calibration steps.
Embodiment:
Comparison and calibration steps with 100 μ l pipettor to be checked and 100 μ l standard pipettors is that example is specifically described below.This method comprises following job step:
1, the pipettor that settles the standard: select the standard pipettor of 100 μ l pipettors of 1 national metrological service calibrating for use as this experiment.
2, capacity is set: rotate pipettor top adjusting knob, with the capacity regulating of standard pipettor and pipettor to be checked to 100 μ l scales.
3, with europium atom solution with strengthening liquid (β-naphthoyltrifluoroacetone (β-NTA)) dilution, to be controlled at CPS value be that solution fully is mixed between the 100000-200000/100 μ l to concentration the most at last.Like this, the linear ratio of CPS value is better, also can reduce because the error that occurring in nature radiation background produces.
4, temperature balance: earlier with standard pipettor, pipettor to be checked and imbibition nozzle and europium label solution equilibrium at room temperature 30 minutes.Like this, can make degree of the expanding with heat and contract with cold unanimity of imbibition nozzle, solute and solvent can fully mix in the solution simultaneously, guarantee consistance.
5, pre-imbibition: select clean, suitable imbibition nozzle to be placed on the standard pipettor sleeve, reverse a little compress that imbibition nozzle makes it and sleeve between do not have the clearance.The vertical pipettor that grips sucks the europium label solution in advance and flows back in the former container, and imbibition, discharge opeing repeat 5 times altogether, and the whole operation action is soft, avoids button lively.Because water has adsorbability when touching the plastics imbibition nozzle for the first time, just can make imbibition nozzle keep wettability by 5 pre-backs of inhaling, the adsorbability of imbibition and tapping is consistent after promptly guaranteeing.
6, imbibition: vertically grip the standard pipettor, button is depressed into first rest point, water dropper immerses 2-3mm place under the europium label solution liquid level, slowly loosen button then reposefully, wait 1-2 water dropper in second to be filled fully after, water dropper leaves liquid level, adherent stop 2-3 second, drop down excess liquid.
7, tapping: pipettor moved to number tygon lath inwall bottom, keep plumbness, press push button to the first rest point position gently, wait for 1-2 second, mobile water dropper pastes inwall, presses knob down fully, to drain the europium label solution in the water dropper, then water dropper is upwards removed along inwall, be total to application of sample 20 holes with the standard pipettor.
8, parallel experiment: draw same europium label solution with pipettor to be checked, repeat above-mentioned 5-8 step, with pipettor to be checked also application of sample 20 holes.
9, the result measures: the tygon lath in above-mentioned application of sample 40 holes is put into survey fluorescence reading on the time resolution detector simultaneously, reflects the fluorescent material amounts of particles that holds in the liquid by recording the CPS value.Under same concentration, the numerical value of CPS has reflected the size of capacity, thereby can the capacity of two pipettors be compared mutually.
10, computing method: by above-mentioned standard pipettor and each 20 application of sample of pipettor to be checked, use the time-resolved fluorescence method measured value, calculate CPS average, CV and the relative error R of two pipettors.Computing formula is: CV=SD/CPS average * 100%; Relative error R=[pipettor CPS average to be checked-standard pipettor CPS average]/standard pipettor CPS average * 100%.
11, volume ratio is right: the CV value of pipettor to be checked should be within the scope of " quantitatively, adjustable pipette measurement verification regulations " J JG 646-90 regulation.The pipettor that the CV value is surpassed JJG 646-90 specification error scope should be abandoned; CV as pipettor to be checked is up to specification, and both CPS averages are equal, represent that then both capacity are identical.
12, capacity calibration: up to specification as pipettor CV to be checked, but when unequal, represent that both capacity are unequal with the CPS average of standard pipettor.At this moment, just need calibrate the capacity of pipettor to be checked: regulate pipettor top knob with special spanner, turn clockwise, capacity is transferred big; Be rotated counterclockwise, capacity is turned down.Repeat the 5-12 step subsequently once more, until pipettor CPS average (X to be checked
R) with the CPS average (X of standard pipettor
S) equate that promptly R trends towards till 0.
In the inventive method, the application of sample hole count of pipettor can not be 20 times, and as 10 times, 25 times or the like, the above accuracy in application of sample 20 holes can be higher.
Claims (9)
1, with europium atom labeling method to pipettor capacity precision comparing and Calibration Method, it is characterized in that it comprises following job step: 1, standard pipettor imbibition: the water dropper of standard pipettor is immersed in the europium label solution, and water dropper is filled fully; 2, standard pipettor tapping: the standard pipettor is moved to tygon lath inwall bottom and the emptying europium label solution in it; 3, repeat said process, utilize the some holes of standard pipettor application of sample; 4, parallel experiment: draw same europium label solution with pipettor to be checked, repeat above-mentioned 1-3 step, with pipettor to be checked also application of sample equal number hole; 5, the result measures: said added polyethylene lath is put into simultaneously surveys the fluorescence reading on the time resolution detector, reflect fluorescent material amounts of particles in the solution by recording fluorescent value CPS value, standard pipettor and pipettor to be checked are carried out volume ratio pair and calibration.
2, as claimed in claim 1 with europium atom labeling method to pipettor capacity precision comparing and Calibration Method, it is characterized in that, utilize each application of sample 20 hole of standard pipettor and pipettor to be checked.
3, as claimed in claim 1 or 2 with europium atom labeling method to pipettor capacity precision comparing and Calibration Method, it is characterized in that, before imbibition, earlier with standard pipettor and pipettor to be checked and imbibition nozzle and europium label solution equilibrium at room temperature 30 minutes.
4, as claimed in claim 1 or 2 with europium atom labeling method to pipettor capacity precision comparing and Calibration Method, it is characterized in that, before imbibition, earlier europium atom solution is diluted with strengthening liquid, concentration is controlled at the CPS value between the 100000-200000/100 μ l the most at last, and solution fully is mixed.
5, as claimed in claim 1 or 2 with europium atom labeling method to pipettor capacity precision comparing and Calibration Method, it is characterized in that, before imbibition, carry out pre-imbibition earlier, promptly select cleaning, suitable imbibition nozzle to be placed on the standard pipettor sleeve, compress that imbibition nozzle makes it and sleeve between do not have the clearance, pre-suction europium label solution also flows back in the former container, and imbibition, discharge opeing repeat repeatedly.
6, as claimed in claim 1 or 2 with europium atom labeling method to pipettor capacity precision comparing and Calibration Method, it is characterized in that, during imbibition, the water dropper of standard pipettor and pipettor to be checked is immersed 2-3mm place under the europium label solution liquid level, slowly loosen button then reposefully, treat that 1-2 water dropper in second is filled fully after, water dropper leaves liquid level, adherent stop 2-3 second, drop down excess liquid.
7, as claimed in claim 1 or 2 with europium atom labeling method to pipettor capacity precision comparing and Calibration Method, it is characterized in that, during tapping, standard pipettor and pipettor to be checked are moved to numbered tygon lath inwall bottom, keep plumbness, press push button to the first rest point position gently, wait for 1-2 second, mobile water dropper pastes inwall, presses knob down fully, to drain the europium label solution in the water dropper, then water dropper is upwards removed along inwall.
8, as claimed in claim 1 or 2 with europium atom labeling method to pipettor capacity precision comparing and Calibration Method, it is characterized in that the excitation wavelength of described time-resolved fluorescence instrument is 336～337nm, the mensuration wavelength is 610～613nm.
9, as claimed in claim 1 or 2 with europium atom labeling method to pipettor capacity precision comparing and Calibration Method, it is characterized in that, mensuration obtains calculating after the CPS value CPS average of two pipettors, utilizes formula CV=SD/CPS average * 100% and R=[pipettor CPS average to be checked-standard pipettor CPS average]/standard pipettor CPS average * 100% relative error calculates CV value and relative error R respectively; The be above standard pipettor of specification error scope of CV value is abandoned; Up to specification but when unequal, regulate pipettor top knob as the CV value of pipettor to be checked with the CPS average of standard pipettor, adjust its amount of capacity; Duplicate detection step 1-5 and aforementioned calculation set-up procedure equate that with the CPS average of standard pipettor promptly R trends towards till 0 until pipettor CPS average to be checked once more.
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| CNB2007100671932A CN100422696C (en) | 2007-02-06 | 2007-02-06 | Method for contrasting and correcting accuracy of liquid transfering device volume with europium atom labeling method |
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| CNB2007100671932A CN100422696C (en) | 2007-02-06 | 2007-02-06 | Method for contrasting and correcting accuracy of liquid transfering device volume with europium atom labeling method |
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| CN100422696C CN100422696C (en) | 2008-10-01 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103175953A (en) * | 2011-12-26 | 2013-06-26 | 苏州新波生物技术有限公司 | Europium nanoparticle fluorescence quality control serum as well as preparation method and application thereof |
| CN107131929A (en) * | 2017-04-01 | 2017-09-05 | 合肥迪安医学检验所有限公司 | A kind of self-calibrating method of sample loading gun |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN2068418U (en) * | 1989-12-21 | 1991-01-02 | 西南石油学院 | Batch liquid-removing apparatus for chemical test |
| US5492673A (en) * | 1992-02-28 | 1996-02-20 | Artel, Inc. | Reagent system for calibration of pipettes and other volumetric measuring devices |
| US20060096349A1 (en) * | 2002-08-27 | 2006-05-11 | Andrzej Czernecki | Method of pipette calibration |
-
2007
- 2007-02-06 CN CNB2007100671932A patent/CN100422696C/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103175953A (en) * | 2011-12-26 | 2013-06-26 | 苏州新波生物技术有限公司 | Europium nanoparticle fluorescence quality control serum as well as preparation method and application thereof |
| CN103175953B (en) * | 2011-12-26 | 2015-04-15 | 苏州新波生物技术有限公司 | Europium nanoparticle fluorescence quality control serum as well as preparation method and application thereof |
| CN107131929A (en) * | 2017-04-01 | 2017-09-05 | 合肥迪安医学检验所有限公司 | A kind of self-calibrating method of sample loading gun |
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| Publication number | Publication date |
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| CN100422696C (en) | 2008-10-01 |
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Granted publication date: 20081001 Termination date: 20120206 |