CN117030645B - Optical system and detection method for single-channel pipettor precision on-site detection - Google Patents
Optical system and detection method for single-channel pipettor precision on-site detection Download PDFInfo
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- 238000001514 detection method Methods 0.000 title claims abstract description 72
- 230000003287 optical effect Effects 0.000 title claims abstract description 55
- 238000012544 monitoring process Methods 0.000 claims abstract description 26
- 239000007788 liquid Substances 0.000 claims description 134
- 239000000243 solution Substances 0.000 claims description 52
- 238000002835 absorbance Methods 0.000 claims description 34
- 239000012482 calibration solution Substances 0.000 claims description 23
- 239000000758 substrate Substances 0.000 claims description 23
- 238000000034 method Methods 0.000 claims description 21
- 230000000087 stabilizing effect Effects 0.000 claims description 20
- 239000011259 mixed solution Substances 0.000 claims description 17
- 238000002156 mixing Methods 0.000 claims description 16
- 238000002360 preparation method Methods 0.000 claims description 16
- 238000003756 stirring Methods 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- 238000012937 correction Methods 0.000 claims description 9
- 239000000203 mixture Substances 0.000 claims description 8
- 238000012546 transfer Methods 0.000 claims description 8
- 238000012360 testing method Methods 0.000 claims description 6
- KSYNLCYTMRMCGG-UHFFFAOYSA-J tetrasodium;2-[2-[bis(carboxylatomethyl)amino]ethyl-(carboxylatomethyl)amino]acetate;dihydrate Chemical compound O.O.[Na+].[Na+].[Na+].[Na+].[O-]C(=O)CN(CC([O-])=O)CCN(CC([O-])=O)CC([O-])=O KSYNLCYTMRMCGG-UHFFFAOYSA-J 0.000 claims description 6
- MPTQRFCYZCXJFQ-UHFFFAOYSA-L copper(II) chloride dihydrate Chemical compound O.O.[Cl-].[Cl-].[Cu+2] MPTQRFCYZCXJFQ-UHFFFAOYSA-L 0.000 claims description 5
- 239000012530 fluid Substances 0.000 claims description 5
- 239000012085 test solution Substances 0.000 claims description 5
- ORTQZVOHEJQUHG-UHFFFAOYSA-L copper(II) chloride Chemical compound Cl[Cu]Cl ORTQZVOHEJQUHG-UHFFFAOYSA-L 0.000 claims description 4
- IWZKICVEHNUQTL-UHFFFAOYSA-M potassium hydrogen phthalate Chemical compound [K+].OC(=O)C1=CC=CC=C1C([O-])=O IWZKICVEHNUQTL-UHFFFAOYSA-M 0.000 claims description 4
- 239000012895 dilution Substances 0.000 claims description 3
- 238000010790 dilution Methods 0.000 claims description 3
- XNGIFLGASWRNHJ-UHFFFAOYSA-L phthalate(2-) Chemical compound [O-]C(=O)C1=CC=CC=C1C([O-])=O XNGIFLGASWRNHJ-UHFFFAOYSA-L 0.000 claims description 3
- 229960003280 cupric chloride Drugs 0.000 claims description 2
- 238000001914 filtration Methods 0.000 claims description 2
- 230000001105 regulatory effect Effects 0.000 claims description 2
- 230000006641 stabilisation Effects 0.000 claims description 2
- 238000011105 stabilization Methods 0.000 claims description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims 2
- 230000007547 defect Effects 0.000 abstract description 6
- 230000007613 environmental effect Effects 0.000 abstract description 4
- 239000002585 base Substances 0.000 description 28
- 238000011065 in-situ storage Methods 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 3
- 238000000691 measurement method Methods 0.000 description 3
- 230000003068 static effect Effects 0.000 description 3
- 238000005303 weighing Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000011481 absorbance measurement Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000003637 basic solution Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 229910052736 halogen Inorganic materials 0.000 description 1
- 150000002367 halogens Chemical class 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- QFUMGPMULRUDNL-UHFFFAOYSA-J tetrasodium;ethane-1,2-diamine;tetraacetate;dihydrate Chemical compound O.O.[Na+].[Na+].[Na+].[Na+].CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O.NCCN QFUMGPMULRUDNL-UHFFFAOYSA-J 0.000 description 1
Classifications
-
- 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
- G01F—MEASURING VOLUME, VOLUME FLOW, MASS FLOW OR LIQUID LEVEL; METERING BY VOLUME
- G01F22/00—Methods or apparatus for measuring volume of fluids or fluent solid material, not otherwise provided for
Abstract
The optical system and the detection method for the single-channel liquid-transfering device precision on-site detection can accurately measure the single-channel liquid-transfering volume of the single-channel liquid-transfering device on site, one path of monitoring light path is added, the detection repeatability is improved, the detection efficiency is obviously improved, and the defects of heavy and high environmental requirements of the current single-channel liquid-transfering device detection equipment are overcome. The system comprises: the device comprises a point light source (1), a slit component (2), a first collimating lens (3), a second collimating lens (4), a beam shrinking lens (5), a small aperture diaphragm (6), an optical filter (7), a half-reflecting half-lens (8), a first spherical mirror (9), a first photoelectric sensor (10), a cuvette (11), a magnetic stirrer (12), a second spherical mirror (13) and a second photoelectric sensor (14).
Description
Technical Field
The invention relates to the technical field of optical detection of precise instruments, in particular to an optical system for on-site detection of single-channel pipettes and a detection method adopted by the optical system for on-site detection of the single-channel pipettes.
Background
Micropipettes are important links of experimental operation and field detection in the fields of medicine, chemistry, life science and the like, and the accuracy of the micropipettes plays a vital role in aspects of project research and development, result detection, performance stability and the like. Particularly, the rapid development of modern biology and medical technology makes the accuracy of micropipetting have higher requirements. In the traditional research work, a piston type handheld pipettor is a commonly used precise micro pipetting tool, and the pipetting accuracy can reach sub-micro upgrading at the highest. However, in actual situations, the product accuracy of different manufacturers and lots is different, even the same pipettor, and the accuracy of the same pipettor can be changed due to different operation habits of operators and different storage environments of the pipettor. Therefore, it is significant to detect the accuracy of the pipettes in various fields.
The detection methods of the pipettor commonly used at present comprise a static weighing method, a fluorescence measurement method, a laser measurement technology and the like. The static weighing method is the most common method for the current domestic detection mechanism, but is limited by high detection cost and difficult movement of detection equipment, so that the on-site detection of the pipettor is difficult to realize; the fluorescence measurement method has quite high requirements on the detection environment like a static weighing method, and does not meet the conditions required by field detection; the equipment required for laser measurement techniques is expensive, requires considerable expertise for operation, and is not suitable for such large-scale pipette detection.
Disclosure of Invention
In order to overcome the defects of the prior art, the technical problem to be solved by the invention is to provide an optical system for single-channel pipette precision on-site detection, which can accurately measure the single-channel pipette volume on site, increase one path of monitoring light path, improve the detection repeatability, remarkably increase the detection efficiency and overcome the defects of heavy weight and high environmental requirements of the current single-channel pipette detection equipment.
The technical scheme of the invention is as follows: the optical system for on-site detection of the precision of the single-channel pipettor comprises: a point light source (1), a slit component (2), a first collimating lens (3), a second collimating lens (4), a beam shrinking lens (5), a small aperture diaphragm (6), an optical filter (7), a half-reflecting half-lens (8), a first spherical mirror (9), a first photoelectric sensor (10), a cuvette (11), a magnetic stirrer (12), a second spherical mirror (13) and a second photoelectric sensor (14);
light generated by the point light source sequentially passes through the slit component, the first collimating lens, the second collimating lens, the beam shrinking lens and the aperture diaphragm to realize beam collimation and beam shrinking, and then the light is divided into a monitoring light path and a measuring light path through the optical filter and the half-reflecting half-lens, wherein the monitoring light path comprises a first spherical lens and a first photoelectric sensor, the light sequentially passes through the first spherical lens and the first photoelectric sensor, and the measuring light path comprises a cuvette, a second spherical lens and a second photoelectric sensor, the light sequentially passes through the cuvette, the second spherical lens and the second photoelectric sensor; the cuvette is used for placing liquid to be detected, and is divided into a stable liquid cuvette, a calibration liquid cuvette, a base liquid cuvette and a mixed liquid cuvette according to the difference of the liquid to be detected, and the cuvette is placed on the magnetic stirrer to realize uniform mixing of internal liquid through the magnetic stirrer.
According to the invention, liquid to be detected is placed in the cuvette, the cuvette is divided into the stable liquid cuvette, the calibration liquid cuvette, the base liquid cuvette and the mixed liquid cuvette according to different liquids to be detected, the cuvette is placed on the magnetic stirrer, internal liquid mixing is realized through the magnetic stirrer, light sequentially penetrates through the cuvette, the second spherical mirror and the second photoelectric sensor to obtain the light intensity of a measuring light path, meanwhile, the light intensity of a monitoring light path is obtained through the first spherical mirror and the first photoelectric sensor, the absorbance of the stable liquid cuvette, the calibration liquid cuvette, the base liquid cuvette and the mixed liquid is obtained by utilizing the Lambert-Beer photometric absorption principle, so that the detection constant can be calculated, the single-channel volume of the single-channel liquid pipette can be finally obtained, and the single-channel liquid pipette precision can be obtained by comparing with the nominal volume of the single-channel liquid pipette, therefore, the single-channel monitoring light path can be increased in situ, the detection repeatability is improved, the detection efficiency is remarkably increased, and the defects of high thickness of single-channel current single-channel liquid pipette detection equipment and environmental requirements are overcome.
The detection method of the optical system for detecting the precision of the single-channel pipettor on site is also provided, and comprises the following steps:
(1) Recording the temperature of the current test solution and the indoor temperature, air pressure and humidity;
(2) Confirming the volume VS to be detected of the single-channel pipettor to be detected;
(3) In order to determine the detection error of the volumes to be detected of the single-channel pipettor, repeatedly measuring each volume to be detected for at least n=10 times, and sequentially sucking 10 times of liquid to be detected through the single-channel pipettor to be detected to prepare a mixed liquid cuvette;
(4) And (3) measuring a correction coefficient S: the filter is switched to the wavelength of 520nm, the cuvette of the stabilizing solution is placed on the magnetic stirrer, after being stirred and mixed uniformly by the magnetic stirrer, the light intensity Ibuf,520, m under the measuring light path and the light intensity Ibuf,520, r under the monitoring light path are measured, the correction coefficient S is that the cuvette of the stabilizing solution is placed in the light splitting system, and the ratio of the light intensity Ibuf,520, m under the measuring light path and the light intensity Ibuf,520, r under the monitoring light path is calculated by the following formula:
(2)；
(5) Absorbance A at 520nm of calibration solution Cal520 And (3) measuring: switching the optical filter to 520nm wavelength, changing the cuvette into a calibration solution cuvette, stirring with a magnetic stirrer, and measuring the light intensity I under the measuring light path std,520,m And monitor the light intensity I under the light path std,520,r Absorbance A Cal520 The formula is:
(3)；
(6) Absorbance A at 520nm of base solution C520 And (3) measuring: switching the optical filter to 520nm wavelength, placing the substrate liquid cuvette on a magnetic stirrer, stirring with the magnetic stirrer, and measuring the light intensity I under the measuring light path dil,520,m And monitor the light intensity I under the light path dil,520,r Absorbance A C520 The formula is:
(4)；
(7) Absorbance A at 730nm of base solution C730 And (3) measuring: switching the optical filter to 730nm wavelength, placing the cuvette of the stabilizing solution on a magnetic stirrer, stirring and mixing uniformly by the magnetic stirrer, and measuring the light intensity I under the measuring light path buf,730,m And monitor the light intensity I under the light path buf,730,r The cuvette is replaced by a substrate liquid cuvette, and after being stirred and mixed uniformly by a magnetic stirrer, the light intensity I under a measuring light path is measured dil,730,m And monitor the light intensity I under the light path dil,730,r Absorbance A C730 The formula is:
(5)；
(8) Absorbance A at 520nm of the mixed solution M520 And (3) measuring: the optical filter is switched to the wavelength of 520nm, a cuvette of the mixed solution is arranged on a magnetic stirrer, and after being stirred and mixed uniformly by the magnetic stirrer, the light intensity I under a measuring light path is measured mix,520,m And monitor the light intensity I under the light path mix,520,r The absorbance A of the corresponding mixed solution after the ith delivery of the liquid to be measured M520 (i) The formula is:
(6)；
(9) Calculating a detection constant K j : detection constant K j Is the test volume V S The detection constant of the corresponding calibration solution is expressed as:
(7)；
(10) Calculating the pipetting volume: the pipetting volume is the transport volume when the liquid to be measured is transported for the ith time through the single channel pipettor to be detected, and the formula is:
(8)，
wherein V is CO Cuvette for substrate liquidInternally measured volume of base fluid, V T (i) The volume of the liquid to be measured which is transported for the ith time by the single-channel liquid transfer device.
Drawings
Fig. 1 is a schematic structural diagram of an optical system for single channel pipette precision field detection according to the present invention.
FIG. 2 is a flow chart of one particular embodiment of a method of detection of an optical system for single channel pipette precision field detection in accordance with the present invention.
Fig. 3 is an overall flow chart of a method of detection of an optical system for single channel pipette precision field detection in accordance with the present invention.
Detailed Description
In order that those skilled in the art will better understand the present invention, a technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in which it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.
It should be noted that the term "comprising" and any variations thereof in the description of the invention and the claims and in the above-described figures is intended to cover a non-exclusive inclusion, such that a process, method, apparatus, article, or device comprising a list of steps or elements is not necessarily limited to those steps or elements that are expressly listed or inherent to such process, method, article, or device, but may include other steps or elements not expressly listed.
As shown in fig. 1, the optical system for in-situ detection of precision of the single-channel pipette comprises: a point light source 1, a slit part 2, a first collimating lens 3, a second collimating lens 4, a beam shrinking lens 5, an aperture diaphragm 6, an optical filter 7, a half-reflecting half-lens 8, a first spherical lens 9, a first photoelectric sensor 10, a cuvette 11, a magnetic stirrer 12, a second spherical lens 13 and a second photoelectric sensor 14;
light generated by the point light source sequentially passes through the slit component, the first collimating lens, the second collimating lens, the beam shrinking lens and the aperture diaphragm to realize beam collimation and beam shrinking, and then the light is divided into a monitoring light path and a measuring light path through the optical filter and the half-reflecting half-lens, wherein the monitoring light path comprises a first spherical lens and a first photoelectric sensor, the light sequentially passes through the first spherical lens and the first photoelectric sensor, and the measuring light path comprises a cuvette, a second spherical lens and a second photoelectric sensor, the light sequentially passes through the cuvette, the second spherical lens and the second photoelectric sensor; the cuvette is used for placing liquid to be detected, and is divided into a stable liquid cuvette, a calibration liquid cuvette, a base liquid cuvette and a mixed liquid cuvette according to the difference of the liquid to be detected, and the cuvette is placed on the magnetic stirrer to realize uniform mixing of internal liquid through the magnetic stirrer.
According to the invention, liquid to be detected is placed in the cuvette, the cuvette is divided into the stable liquid cuvette, the calibration liquid cuvette, the base liquid cuvette and the mixed liquid cuvette according to different liquids to be detected, the cuvette is placed on the magnetic stirrer, internal liquid mixing is realized through the magnetic stirrer, light sequentially penetrates through the cuvette, the second spherical mirror and the second photoelectric sensor to obtain the light intensity of a measuring light path, meanwhile, the light intensity of a monitoring light path is obtained through the first spherical mirror and the first photoelectric sensor, the absorbance of the stable liquid cuvette, the calibration liquid cuvette, the base liquid cuvette and the mixed liquid is obtained by utilizing the Lambert-Beer photometric absorption principle, so that the detection constant can be calculated, the single-channel volume of the single-channel liquid pipette can be finally obtained, and the single-channel liquid pipette precision can be obtained by comparing with the nominal volume of the single-channel liquid pipette, therefore, the single-channel monitoring light path can be increased in situ, the detection repeatability is improved, the detection efficiency is remarkably increased, and the defects of high thickness of single-channel current single-channel liquid pipette detection equipment and environmental requirements are overcome.
Preferably, the optical filter comprises optical filters with two wavelengths of 520nm and 730nm, and the switching of the two optical filters is realized through motor driving.
Preferably, the cuvette is a cylinder, a cuboid or a cube.
Preferably, the light beam of the measuring light path is incident from the side surface of the cuvette, is emitted from the other side surface of the cuvette, and irradiates the second spherical mirror.
As shown in fig. 3, there is also provided a method for detecting an optical system for detecting accuracy of a single channel pipette in situ, comprising the steps of:
(1) Recording the temperature of the current test solution and the indoor temperature, air pressure and humidity;
(2) Confirming the volume VS to be detected of the single-channel pipettor to be detected;
(3) In order to determine the detection error of the volumes to be detected of the single-channel pipettor, repeatedly measuring each volume to be detected for at least n=10 times, and sequentially sucking 10 times of liquid to be detected through the single-channel pipettor to be detected to prepare a mixed liquid cuvette;
(4) And (3) measuring a correction coefficient S: the filter is switched to the wavelength of 520nm, the cuvette of the stabilizing solution is placed on the magnetic stirrer, after being stirred and mixed uniformly by the magnetic stirrer, the light intensity Ibuf,520, m under the measuring light path and the light intensity Ibuf,520, r under the monitoring light path are measured, the correction coefficient S is that the cuvette of the stabilizing solution is placed in the light splitting system, and the ratio of the light intensity Ibuf,520, m under the measuring light path and the light intensity Ibuf,520, r under the monitoring light path is calculated by the following formula:
(2)；
(5) Absorbance A at 520nm of calibration solution Cal520 And (3) measuring: switching the optical filter to 520nm wavelength, changing the cuvette into a calibration solution cuvette, stirring with a magnetic stirrer, and measuring the light intensity I under the measuring light path std,520,m And monitor the light intensity I under the light path std,520,r Absorbance A Cal520 The formula is:
(3)；
(6) Absorbance A at 520nm of base solution C520 And (3) measuring: switching the optical filter to 520nm wavelength, placing the substrate liquid cuvette on a magnetic stirrer, stirring with the magnetic stirrer, and measuring the light intensity I under the measuring light path dil,520,m And monitor the light intensity I under the light path dil,520,r Absorbance A C520 The formula is:
(4)；
(7) Absorbance A at 730nm of base solution C730 And (3) measuring: switching the optical filter to 730nm wavelength, placing the cuvette of the stabilizing solution on a magnetic stirrer, stirring and mixing uniformly by the magnetic stirrer, and measuring the light intensity I under the measuring light path buf,730,m And monitor the light intensity I under the light path buf,730,r The cuvette is replaced by a substrate liquid cuvette, and after being stirred and mixed uniformly by a magnetic stirrer, the light intensity I under a measuring light path is measured dil,730,m And monitor the light intensity I under the light path dil,730,r Absorbance A C730 The formula is:
(5)；
(8) Absorbance A at 520nm of the mixed solution M520 And (3) measuring: the optical filter is switched to the wavelength of 520nm, a cuvette of the mixed solution is arranged on a magnetic stirrer, and after being stirred and mixed uniformly by the magnetic stirrer, the light intensity I under a measuring light path is measured mix,520,m And monitor the light intensity I under the light path mix,520,r The absorbance A of the corresponding mixed solution after the ith delivery of the liquid to be measured M520 (i) The formula is:
(6)；
(9) Calculating a detection constant K j : detection constant K j Is the test volume V S The detection constant of the corresponding calibration solution is expressed as:
(7)；
(10) Calculating the pipetting volume: the pipetting volume is the transport volume when the liquid to be measured is transported for the ith time through the single channel pipettor to be detected, and the formula is:
(8)，
wherein V is CO For the volume of the substrate liquid actually measured in the substrate liquid cuvette, V T (i) The volume of the liquid to be measured which is transported for the ith time by the single-channel liquid transfer device.
Preferably, in the step (3), the preparation method of the liquid to be measured comprises: and (3) in the ponceau water solution, regulating the pH to 6.0+/-0.1, filtering through a 0.2 mu m filter, and determining the quality of ponceau required by preparing each liter of water of the ponceau water solution according to the volume to be detected.
Preferably, in the step (4), the preparation method of the stabilizing solution comprises the following steps: 4.08 g potassium hydrogen phthalate and 3.81g EDTA (ethylenediamine tetraacetic acid tetrasodium salt dihydrate) were dissolved per liter of water, the pH was adjusted to 6.0.+ -. 0.1, and filtered through a 0.2 μm filter.
Preferably, in the step (5), the preparation method of the calibration solution comprises the following steps: corresponding to the volume to be detected of single pipetting of the single-channel pipettor to be detected, mixing the liquid to be detected with the known volume with the base liquid with the known volume according to a certain proportion; wherein the volume V to be measured is used for the liquid to be measured S Is multiplied by the preparation factor of table 2; wherein the base liquid is multiplied by the volume V of the cupric chloride solution in the cuvette by the preparation factor CO ；
The dilution ratio R is calculated according to formula (1):
(1)，
wherein V is PS To actually measure the volume of the liquid to be measured, V C Is the volume of the measured base fluid.
Preferably, in the steps (6) and (7), the substrate liquid preparation method comprises the following steps: 1.12 g/l copper chloride dihydrate was dissolved in phthalate/EDTA stabilization and the pH was adjusted to 6.0.+ -. 0.1 and the resulting solution was filtered through a 0.2 μm filter.
Preferably, the stabilizing solution cuvette is filled with a stabilizing solution of a volume V CO Stabilizing the liquid in a clean and dry cuvette to obtain the liquid; the ratio of the calibration liquidColor dish with a delivery volume of V CO The calibration solution is obtained in a clean and dry cuvette; the substrate liquid cuvette is transported by a volume V CO The substrate liquid is obtained in a clean and dry cuvette; the mixed solution cuvette is prepared by sucking the liquid to be detected through a single-channel liquid transfer device to be detected and transferring the liquid to be detected into the substrate liquid cuvette; when preparing a plurality of cuvettes, each actual delivery volume error is V CO Within + -0.03% of the total volume at the test temperature is recorded.
The beneficial technical effects of the invention are as follows:
1. the single-channel liquid-transfering device on-site detection method with the monitoring light path is provided, the single-channel liquid-transfering volume of the single-channel liquid-transfering device is measured, and a new thought is provided for constructing and measuring a detection device of the micro liquid capacity;
2. according to the Lambert-Beer luminosity absorption principle, a path of monitoring light path is innovatively added according to the internal light path structure of the light splitting system, the monitoring light path improves the repeatability of detection, and the detection efficiency is remarkably improved;
3. the single-channel liquid transfer device on-site detection method overcomes the defects of heavy weight, high requirement on environment and the like of the existing single-channel liquid transfer device detection equipment.
The invention is further illustrated below with reference to examples.
Example 1
Step one: and (5) constructing a light splitting system.
The optical path diagram of the light splitting system in the embodiment of the invention is shown in fig. 1, and the light splitting system is used for collimating and beam shrinking of the point light source 1, and the generated collimated light beam is output in a certain wavelength and is divided into two paths through the half-reflecting half-lens 8. The point light source 1 is collimated and condensed, the point light source comprises a point light source 1, a slit component 2, a first collimating lens 3, a second collimating lens 4, a condensed beam lens 5 and a small aperture diaphragm 6, wherein the light beam sequentially penetrates through the slit component, the condensed beam is generated by the point light source 1, the light beam is collimated and condensed, the collimated light beam is output with a certain wavelength through a light filter 7, two paths of light paths are actually a monitoring light path and a measuring light path, the monitoring light path comprises a first spherical mirror 9 and a first photoelectric sensor 10, the light path sequentially penetrates through the first spherical mirror 9, and the measuring light path comprises a cuvette 11, a second spherical mirror 13 and a second photoelectric sensor 14, and the light path sequentially penetrates through the cuvette, the second spherical mirror 13 and the second photoelectric sensor. The cuvette is placed above the magnetic stirrer 12, and internal liquid mixing is achieved through the magnetic stirrer 12.
In one embodiment of the present invention, the point light source is a halogen lamp, and the light emitted from the point light source is irradiated onto the first collimating lens through a slit in the slit member.
In one embodiment of the invention, the optical filter comprises optical filters with two wavelengths of 520nm and 730nm, and the switching of the two optical filters is realized through motor driving.
In one embodiment of the invention, the monitoring light path is used for monitoring the fluctuation or attenuation influence of the external environment light and the point light source, and the correction coefficient in the fifth step is used for measuring to compensate the error caused by the disturbance of the external environment light, the shake or attenuation of the point light source, thereby improving the accuracy of the subsequent absorbance calculation and reducing the influence of the fluctuation problem of the light source on the photoelectric sensor at different moments.
In one embodiment of the invention, the cuvette is a container for holding a liquid to be tested, including but not limited to a cylinder, a cuboid, and a cube. The liquid to be detected comprises a base solution such as a stabilizing solution, a liquid to be detected and a base solution, and a calibration solution and a mixed solution prepared from the base solution according to certain conditions, wherein the specific preparation steps are shown in the second, third and fourth steps, and meanwhile, under the light path, the cuvette is easy to manually replace.
In one embodiment of the invention, the cuvette is placed in the measuring light path, the measuring light beam is incident on the side surface of the cuvette, and is emitted from the other side surface of the cuvette to irradiate on the second spherical mirror.
Step two: and (5) preparing a base solution.
And the base solution is used for preparing a stable solution cuvette, a base solution cuvette, a calibration solution cuvette and a mixed solution cuvette. The basic solution comprises a stabilizing solution, a solution to be tested and a base solution, and the specific preparation process is as follows:
stabilizing solution: each liter of water was dissolved with 4.08 g potassium hydrogen phthalate (CAS No. 877-24-7) and 3.81g EDTA (CAS No. 10378-23-1), the pH was adjusted to 6.0.+ -. 0.1, and filtered through a 0.2 μm filter.
Liquid to be measured: in ponceau aqueous solution, the pH is adjusted to 6.0.+ -. 0.1 and then filtered through a 0.2 μm filter. The required quality of ponceau per liter of water for the ponceau solution is different for different volumes to be measured, as shown in table 1. The liquid to be detected is used for preparing a calibration solution and is used as a coloring agent for a single-channel pipette to be detected.
TABLE 1
Base solution: 1.12. 1.12 g/l copper chloride dihydrate (CuCl2.2H2O) was dissolved in the phthalate/EDTA stabilizer and the pH was adjusted to 6.0.+ -. 0.1. The resulting solution was filtered through a 0.2 μm filter.
Step three: and (5) preparing a calibration solution.
The calibration solution is prepared and the volume V to be measured of single pipetting of the single-channel pipettor to be detected S Corresponding to each other. A known volume of the test solution was mixed with a known volume of the base solution according to table 2. The above solutions were prepared at the following doses:
liquid to be measured: using the desired volume to be measured V S Is multiplied by the preparation factor of table 2 (n=10 replicates).
Base solution: the preparation factors given in Table 2 were multiplied by the volume V of the copper chloride solution in the cuvette CO 。
TABLE 2
Calculating a dilution ratio R:
(1)，
wherein V is PS To actually measure the volume of the liquid to be measured, V C Is the volume of the measured base fluid.
Step four: preparing a stabilizing solution, a calibration solution, a base solution and a mixed solution cuvette.
The cuvette is configured to be placed in the spectroscopic system of step one for subsequent absorbance measurements. The stable liquid cuvette is transported by a volume V CO (between 4.5 mL and 5.5 mL) into a clean and dry cuvette by delivering a volume of V CO (between 4.5 mL and 5.5 mL) into a clean and dry cuvette, said cuvette being filled with a transfer volume V CO (4.5 mL-5.5 mL) into a clean and dry cuvette, wherein the cuvette is prepared by sucking the liquid to be detected into the cuvette by a single channel pipette to be detected, transferring the liquid to be detected into the cuvette, and when preparing a plurality of cuvettes, each actual delivery volume error is V CO Within + -0.03% of the total volume at the test temperature is recorded.
Step five: experimental procedure.
(1) Before the experiment starts, recording the temperature of the current test solution, the indoor temperature, the indoor air pressure and the indoor humidity;
(2) Confirm to be detected single channel pipettor volume V that awaits measuring S For a fixed-volume single-channel pipette, the volume to be measured is the nominal volume; for a variable volume single channel pipette, at least three volumes are determined, including the nominal volume, 50% or closest possible value of the nominal volume, and the lower limit of the usable volume range or 10% of the nominal volume (whichever is larger);
(3) In order to determine the detection error of the volumes to be detected of the single-channel pipettor, repeatedly measuring each volume to be detected for at least n=10 times, and sequentially sucking 10 times of liquid to be detected through the single-channel pipettor to be detected to prepare a mixed liquid cuvette;
(4) Measuring correction coefficient S, switching the optical filter of the light-splitting system to the wavelength of 520nm, placing the cuvette of the stable liquid in the light-splitting system, stirring and mixing uniformly by a magnetic stirrer, and measuring the light intensity I under a measuring light path buf,520,m And monitor the light intensity I under the light path buf,520,r The correction coefficient S is that a cuvette of the stable liquid is arranged in a light splitting system, and the light intensity I of a light path is measured buf,520,m The same monitoring light intensity I buf,520,r The specific formula of the ratio is as follows:
(2)；
(5) Absorbance A at 520nm of calibration solution Cal520 Measuring, namely switching the optical filter of the light splitting system to the wavelength of 520nm, replacing the cuvette into a cuvette of a calibration solution, stirring and mixing uniformly by a magnetic stirrer, and measuring the light intensity I under a measuring light path std,520,m And monitor the light intensity I under the light path std,520,r Absorbance A Cal520 The specific formula is as follows:
(3)；
(6) Absorbance A at 520nm of base solution C520 Measuring, namely switching the optical filter of the light-splitting system to the wavelength of 520nm, placing the substrate liquid cuvette in the light-splitting system, stirring and mixing uniformly by a magnetic stirrer, and measuring the light intensity I under a measuring light path dil,520,m And monitor the light intensity I under the light path dil,520,r Absorbance A C520 The specific formula is as follows:
(4)；
(7) Absorbance A at 730nm of base solution C730 Measuring, namely switching the optical filter of the light-splitting system to the wavelength of 730nm, placing the cuvette of the stabilizing solution in the light-splitting system, stirring and mixing uniformly by a magnetic stirrer, and measuring the light intensity I under a measuring light path buf,730,m And monitor the light intensity I under the light path buf,730,r The cuvette is replaced by a substrate liquid cuvette, and after being stirred and mixed uniformly by a magnetic stirrer, the light intensity I under a measuring light path is measured dil,730,m And monitor the light intensity I under the light path dil,730,r Absorbance A C730 The specific formula is as follows:
(5)；
(8) Absorbance A at 520nm of the mixed solution M520 Measuring, switching the optical filter of the light-splitting system to the wavelength of 520nm, placing the cuvette of the mixed solution in the light-splitting system, stirring and mixing uniformly by a magnetic stirrer, and measuring the light intensity I under the measuring light path mix,520,m And monitor the light intensity I under the light path mix,520,r The absorbance A of the corresponding mixed solution after the ith delivery of the liquid to be measured M520 (i) The specific formula is as follows:
(6)；
(9) Detection constant K j Calculating the detection constant K j Is the test volume V S The specific formula of the detection constant corresponding to the calibration solution is as follows:
(7)；
(10) And calculating a pipetting volume, wherein the pipetting volume is the transport volume when the to-be-detected liquid is transported for the ith time through the single-channel pipettor to be detected, and the specific formula is as follows:
(8)，
wherein V is CO For the volume of the substrate liquid actually measured in the substrate liquid cuvette, V T (i) The volume of the liquid to be measured which is transported for the ith time by the single-channel liquid transfer device.
The present invention is not limited to the preferred embodiments, but can be modified in any way according to the technical principles of the present invention, and all such modifications, equivalent variations and modifications are included in the scope of the present invention.
Claims (6)
1. The detection method of the optical system for detecting the precision of the single-channel pipettor on site is characterized by comprising the following steps of: the optical system for single channel pipettor precision field detection includes: a point light source (1), a slit component (2), a first collimating lens (3), a second collimating lens (4), a beam shrinking lens (5), a small aperture diaphragm (6), an optical filter (7), a half-reflecting half-lens (8), a first spherical mirror (9), a first photoelectric sensor (10), a cuvette (11), a magnetic stirrer (12), a second spherical mirror (13) and a second photoelectric sensor (14);
light generated by the point light source sequentially passes through the slit component, the first collimating lens, the second collimating lens, the beam shrinking lens and the aperture diaphragm to realize beam collimation and beam shrinking, and then the light is divided into a monitoring light path and a measuring light path through the optical filter and the half-reflecting half-lens, wherein the monitoring light path comprises a first spherical lens and a first photoelectric sensor, the light sequentially passes through the first spherical lens and the first photoelectric sensor, and the measuring light path comprises a cuvette, a second spherical lens and a second photoelectric sensor, the light sequentially passes through the cuvette, the second spherical lens and the second photoelectric sensor; the cuvette is placed with liquid to be detected, and is divided into a stable liquid cuvette, a calibration liquid cuvette, a base liquid cuvette and a mixed liquid cuvette according to the difference of the liquid to be detected, the cuvette is placed on a magnetic stirrer, and internal liquid mixing is realized through the magnetic stirrer;
the optical filter comprises optical filters with two wavelengths of 520nm and 730nm, and the two optical filters are switched by motor driving;
the cuvette is a cylinder, a cuboid or a cube;
the light beam of the measuring light path is incident from the side surface of the cuvette, is emitted from the other side surface of the cuvette and irradiates on the second spherical mirror;
the method comprises the following steps:
(1) Recording the temperature of the current test solution and the indoor temperature, air pressure and humidity;
(2) Confirm to be detected single channel pipettor volume V that awaits measuring s ；
(3) In order to determine the detection error of the volumes to be detected of the single-channel pipettor, repeatedly measuring each volume to be detected for at least n=10 times, and sequentially sucking 10 times of liquid to be detected through the single-channel pipettor to be detected to prepare a mixed liquid cuvette;
(4) And (3) measuring a correction coefficient S: the filter is switched to the wavelength of 520nm, the cuvette of the stabilizing solution is placed on the magnetic stirrer, after being stirred and mixed uniformly by the magnetic stirrer, the light intensity Ibuf,520, m under the measuring light path and the light intensity Ibuf,520, r under the monitoring light path are measured, the correction coefficient S is that the cuvette of the stabilizing solution is placed in the light splitting system, and the ratio of the light intensity Ibuf,520, m under the measuring light path and the light intensity Ibuf,520, r under the monitoring light path is calculated by the following formula:
（2）；
(5) Absorbance A at 520nm of calibration solution Cal520 And (3) measuring: switching the optical filter to 520nm wavelength, changing the cuvette into a calibration solution cuvette, stirring with a magnetic stirrer, and measuring the light intensity I under the measuring light path std,520,m And monitor the light intensity I under the light path std,520,r Absorbance A Cal520 The formula is:
（3）；
(6) Absorbance A at 520nm of base solution C520 And (3) measuring: switching the optical filter to 520nm wavelength, placing the substrate liquid cuvette on a magnetic stirrer, stirring with the magnetic stirrer, and measuring the light intensity I under the measuring light path dil,520,m And monitor the light intensity I under the light path dil,520,r Absorbance A C520 The formula is:
（4）；
(7) Absorbance A at 730nm of base solution C730 And (3) measuring: switching the optical filter to 730nm wavelength, placing the cuvette of the stabilizing solution on a magnetic stirrer, stirring and mixing uniformly by the magnetic stirrer, and measuring the light intensity I under the measuring light path buf,730,m And monitor the light intensity I under the light path buf,730,r The cuvette is replaced by a substrate liquid cuvette, and after being stirred and mixed uniformly by a magnetic stirrer, the light intensity I under a measuring light path is measured dil,730,m And monitor the light intensity I under the light path dil,730,r Suction pipeLuminosity A C730 The formula is:
（5）；
(8) Absorbance A at 520nm of the mixed solution M520 And (3) measuring: the optical filter is switched to the wavelength of 520nm, a cuvette of the mixed solution is arranged on a magnetic stirrer, and after being stirred and mixed uniformly by the magnetic stirrer, the light intensity I under a measuring light path is measured mix,520,m And monitor the light intensity I under the light path mix,520,r The absorbance A of the corresponding mixed solution after the ith delivery of the liquid to be measured M520 (i) The formula is:
（6）；
(9) Calculating a detection constant K j : detection constant K j Is the volume V to be measured s The detection constant of the corresponding calibration solution is expressed as:
（7），
r is the dilution ratio, calculated according to formula (1):
（1），
wherein V is PS To actually measure the volume of the liquid to be measured, V C Is the volume of the measured base fluid;
(10) Calculating the pipetting volume: the pipetting volume is the transport volume when the liquid to be measured is transported for the ith time through the single channel pipettor to be detected, and the formula is:
（8），
wherein V is CO For the volume of the substrate liquid actually measured in the substrate liquid cuvette, V T (i) For the ith transport of single-channel pipettesThe volume of the liquid to be measured.
2. The method for detecting the optical system for the on-site detection of the precision of the single-channel pipette according to claim 1, wherein the method comprises the following steps of: in the step (3), the preparation method of the liquid to be tested comprises the following steps: and (3) in the ponceau water solution, regulating the pH to 6.0+/-0.1, filtering through a 0.2 mu m filter, and determining the quality of ponceau required by preparing each liter of water of the ponceau water solution according to the volume to be detected.
3. The method for detecting the optical system for the on-site detection of the precision of the single-channel pipette according to claim 2, wherein the method comprises the following steps of: in the step (4), the preparation method of the stabilizing solution comprises the following steps: 4.08 g potassium hydrogen phthalate and 3.81g ethylene diamine tetraacetic acid tetrasodium salt dihydrate EDTA are dissolved per liter of water, the pH is adjusted to 6.0.+ -. 0.1 and filtered through a 0.2 μm filter.
4. The method for detecting an optical system for on-site detection of precision of a single-channel pipette according to claim 3, wherein: in the step (5), the preparation method of the calibration solution comprises the following steps: corresponding to the volume to be detected of single pipetting of the single-channel pipettor to be detected, mixing the liquid to be detected with the known volume with the substrate liquid with the known volume according to the proportion; wherein the volume V to be measured is used for the liquid to be measured s Is multiplied by a preparation factor; wherein the base liquid is multiplied by the volume V of the cupric chloride solution in the cuvette by the preparation factor CO 。
5. The method for detecting the optical system for the on-site detection of the precision of the single-channel pipette according to claim 4, wherein the method comprises the following steps of: in the steps (6) and (7), the preparation method of the base fluid comprises the following steps: 1.12 g/l copper chloride dihydrate was dissolved in phthalate/EDTA stabilization and the pH was adjusted to 6.0.+ -. 0.1 and the resulting solution was filtered through a 0.2 μm filter.
6. The method for detecting an optical system for single-channel pipette precision field detection according to claim 5,the method is characterized in that: the stable liquid cuvette is transported by a volume V CO Stabilizing the liquid in a clean and dry cuvette to obtain the liquid; the volume of the calibration solution cuvette is V CO The calibration solution is obtained in a clean and dry cuvette; the substrate liquid cuvette is transported by a volume V CO The substrate liquid is obtained in a clean and dry cuvette; the mixed solution cuvette is prepared by sucking the liquid to be detected through a single-channel liquid transfer device to be detected and transferring the liquid to be detected into the substrate liquid cuvette; when preparing a plurality of cuvettes, each actual delivery volume error is V CO Within + -0.03% of the total volume at the test temperature is recorded.
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| US5298978A (en) * | 1992-02-28 | 1994-03-29 | Artel, Inc. | Pipette calibration system |
| CN112041076A (en) * | 2018-04-23 | 2020-12-04 | 迈恩医疗解决方案有限公司 | Automatic analyzer and optical measuring method for obtaining a measuring signal from a liquid medium |
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| US6741365B2 (en) * | 2001-12-12 | 2004-05-25 | Artel, Inc. | Photometric calibration of liquid volumes |
| US7791716B2 (en) * | 2008-04-07 | 2010-09-07 | Artel, Inc. | System and method for liquid delivery evaluation using solutions with multiple light absorbance spectral features |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5298978A (en) * | 1992-02-28 | 1994-03-29 | Artel, Inc. | Pipette calibration system |
| CN112041076A (en) * | 2018-04-23 | 2020-12-04 | 迈恩医疗解决方案有限公司 | Automatic analyzer and optical measuring method for obtaining a measuring signal from a liquid medium |
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| Title |
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| 基于光度法的移液器在线计量系统研制;钟家栋;中国优秀硕士学位论文全文数据库工程科技Ⅱ辑(第05期);第C030-7页 * |
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