CN207096073U - Time-resolved fluorescence enzyme micro-plate reader testing standard plate - Google Patents
Time-resolved fluorescence enzyme micro-plate reader testing standard plate Download PDFInfo
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- CN207096073U CN207096073U CN201720760541.3U CN201720760541U CN207096073U CN 207096073 U CN207096073 U CN 207096073U CN 201720760541 U CN201720760541 U CN 201720760541U CN 207096073 U CN207096073 U CN 207096073U
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Abstract
The utility model discloses a kind of time-resolved fluorescence enzyme micro-plate reader testing standard plate.Time-resolved fluorescence enzyme micro-plate reader testing standard plate includes black ELISA Plate；Black ELISA Plate is provided with 96 enzyme mark holes；96 enzyme mark Kong Zhongjun contain rare-earth fluorescent solid matter.The utility model has filled up the blank of existing time-resolved fluorescence enzyme micro-plate reader measurement criteria, a kind of high stability, non-maintaining, easily operated, cheap time-resolved fluorescence enzyme micro-plate reader testing standard plate are provided, sensitivity that can be to time-resolved fluorescence ELIASA, repeatability and the metering performance index such as linear are examined and determine and calibrated.
Description
Technical field
Time-resolved fluorescence assay technical field is the utility model is related to, more particularly to a kind of evaluation time resolved fluorometric
The testing standard plate of enzyme micro-plate reader performance indications and meter characteristic.
Background technology
Fluorescence is a kind of luminescence phenomenon caused by material stimulated radiation.When material molecule is irradiated by the light source of specific wavelength
When, molecule absorption photon simultaneously causes the electronics induced transition in ground state the electronics in excitation state is unstable to excitation state,
Unnecessary energy is discharged in the form of photon during transition time ground state, so as to produce luminescence phenomenon.Pass through such a side
The light that formula is sent is referred to as fluorescence.
Time-resolved fluoroimmunoassay (TRFIA) is actually what is grown up on the basis of fluorescence analysis (FIA), it
It is a kind of special fluorescence analysis.Exciting light is generally in 90 ° with launching light not on the same line during fluorescence analysis, Er Qiefa
Ejected wave is long to have a displacement between excitation wavelength, therefore fluorescence analysis method can overcome general ultraviolet-vis spectroscopy
The influence of heterogeneous light in analytic approach, exciting light can not directly reach optoelectronic receiver, so as to which optical analysis be significantly increased
Sensitivity.But the veiling glare of exciting light still can turn into the bottleneck for improving analysis specificity and sensitivity.It is miscellaneous in order to eliminate
The influence of astigmatism, it is proposed that Time-resolved fluorescence assay method, this process employs rare earth metal fluorescence (Eu, Tb, Sm, Dy)
Fluorescence lifetime is longer and the characteristics of common fluorescent mark fluorescence lifetime is very short, when exciting light disappears after light irradiation is excited
When, the fluorescence of common fluorescent mark also disappears therewith, but the fluorescence lifetime of rare earth metal is longer, reachable 1~2ms, therefore
Receive the fluorescence signal in transmitting light path again after tens to hundreds of microseconds of wait after exciting light stopping, it is possible to overcoming sample
The influence of product background fluorescence, realize highly sensitive, high specific analysis.In various rare earth metals, the most frequently used rare earth metal
It is europium (Eu) and terbium (Tb), Eu fluorescence lifetimes are up to 1ms, but it is unstable in water, can overcome after adding reinforcing agent.
Time-resolved fluorescence enzyme micro-plate reader is to carry out material qualitative and quantitative analysis instrument using time-resolved fluorescence phenomenon
Instrument, it has the characteristics of small ambient interferences, high sensitivity.Time-resolved fluorescence enzyme micro-plate reader combines immune response
High specific, the high flux of enzyme micro-plate reader and the highly sensitive advantage of fluorescence signal, overcome sample background fluorescence signal
Interference, is widely used in fields such as food security, clinical examinations in recent years.Time-resolved fluorescence enzyme micro-plate reader leads to
A normal plate can carry out the detection of 96 or 384 samples, and can use third party's detection reagent.
Time-resolved fluorescence enzyme micro-plate reader detection process and principle are typically as follows：Resist first in ELISA Plate in coating
Body, then sample to be detected is added in enzyme mark hole, the testing compound in sample is just coated on the spy on enzyme mark hole
Heterogenetic antibody captures, and adds the detection antibody of rare earth metal mark again after washing, and detection antibody is anti-with being incorporated in capture
Object on body is further in conjunction with the structure of formation " sandwich hamburger type ".It is then passed through passage time resolved fluorometric enzyme after washing
The Eu marked on mark instrument detection antibody time-resolved fluorescence signal.The amount positive correlation of fluorescence intensity and product, the amount of product with
The content positive correlation of target compound in sample to be detected.Therefore, can be according to the intensity of fluorescence signal in enzyme mark hole after reaction
Judge the content of target compound in testing sample.
The degree of accuracy of time-resolved fluorescence enzyme micro-plate reader testing result and the metering performance of instrument are closely related, these meters
Measuring performance includes the index such as the launch wavelength degree of accuracy, the excitation wavelength degree of accuracy, sensitivity, repeatability, linear, these metering performances
The confirmation and calibration of index must be realized by measurement criteria.Time-resolved fluorescence enzyme micro-plate reader is from optical texture principle
More using optical filter as beam splitter, the wavelength accuracy of optical filter can remove optical filter to be closed by measurement verification
Testing calibration is carried out on the ultraviolet specrophotometer of lattice.But the metering mark of the metering performance index such as sensitivity, repeatability, linear
Standard is more short of, and also without corresponding calibration code or calibrating standard is formulated, can not carry out the calibrating or calibration of instrument, instrument
The quality of device analysis measurement result is difficult to ensure that the measurement criteria of development time resolved fluorometric enzyme micro-plate reader turns into when business
It is anxious.
Utility model content
The purpose of this utility model is to fill up the blank of existing time-resolved fluorescence enzyme micro-plate reader measurement criteria, there is provided
A kind of high stability, non-maintaining, easily operated, cheap time-resolved fluorescence enzyme micro-plate reader testing standard plate, pair when
Between the sensitivity of resolved fluorometric ELIASA, repeatability and the metering performance index such as linear examined and determine and calibrated.
Time-resolved fluorescence enzyme micro-plate reader testing standard plate, including black ELISA Plate；Black ELISA Plate is provided with 96
Enzyme mark hole；96 enzyme marks Kong Zhongjun contains rare-earth fluorescent solid matter.
Time-resolved fluorescence enzyme micro-plate reader testing standard plate described in the utility model, wherein, the rare-earth fluorescent is consolidated
Contain rare earth metal and reinforcing agent in state material.
Time-resolved fluorescence enzyme micro-plate reader testing standard plate described in the utility model, wherein, the rare-earth fluorescent is consolidated
State material is long-life rare earth Fluorescence solid-state material.
Time-resolved fluorescence enzyme micro-plate reader testing standard plate described in the utility model, wherein, the rare-earth fluorescent is consolidated
The matrix of state material is high molecular polymer, and using copolymerization method, direct polymerization forms in enzyme mark hole.
Time-resolved fluorescence enzyme micro-plate reader testing standard plate described in the utility model, wherein, 96 enzyme marks hole
The rectangular array arranged in 8 rows 12 is uniformly distributed.
Time-resolved fluorescence enzyme micro-plate reader testing standard plate of the present utility model and its preparation and application with it is existing
Technology is compared, and it protrudes effect and is：
(1) the utility model has filled up the blank of existing time-resolved fluorescence enzyme micro-plate reader measurement criteria, there is provided one
Kind of high stability, non-maintaining, easily operated, cheap time-resolved fluorescence enzyme micro-plate reader testing standard plate, can pair when
Between the sensitivity of resolved fluorometric ELIASA, repeatability and the metering performance index such as linear examined and determine and calibrated.
(2) this measurement criteria uses solid-state time-resolved fluorescence form, with using solution form and simulation of electronic circuits when
Between the calibrating mode of resolved fluorometric signal compare, time-resolved fluorescence signal repeatability is more preferable, and stability is higher.
(3) this measurement criteria can be repeatedly used, and the operation such as solution preparation, filling need not be carried out before use, is reduced
The uncertainty introduced using link.
Explanation and specific embodiment are tested time-resolved fluorescence enzyme micro-plate reader of the present utility model below in conjunction with the accompanying drawings
On-gauge plate is described further.
Brief description of the drawings
Fig. 1 is the structural representation of time-resolved fluorescence enzyme micro-plate reader testing standard plate；
Fig. 2 is the structural representation in enzyme mark hole.
Embodiment
Embodiment 1
With reference to shown in Fig. 1-2, time-resolved fluorescence enzyme micro-plate reader testing standard plate, including black ELISA Plate (1), material
For polystyrene；Black ELISA Plate (1) is provided with 96 enzyme mark holes (2)；Contain rare-earth fluorescent in 96 enzyme marks hole (2)
Solid matter (3).
Contain rare earth metal and reinforcing agent in rare-earth fluorescent solid matter (3), be long-life rare earth Fluorescence solid-state material.It is dilute
The matrix of native Fluorescence solid-state material (3) is high molecular polymer, and using copolymerization method, direct polymerization forms in enzyme mark hole (2).
96 enzyme mark holes (2) are uniformly distributed in the rectangular array of 8 rows 12 row.
Embodiment 2
The preparation technology of time-resolved fluorescence enzyme micro-plate reader testing standard plate, comprises the following steps：
(1) different amounts of rare earth metal solution is well mixed with reinforcing agent, crosslinking agent, monomer, solvent and initiator；
(2) by the isometric 96 enzyme mark holes of addition of mixed solution；
(3) by the way of thermal initiation or ultraviolet lighting trigger, the solid fluorescent material in enzyme mark hole is made to aggregate into tool
There is certain rigid rare-earth fluorescent solid matter.
When using thermal initiation, from azodiisobutyronitrile, peroxidating two acyl or persulfate as initiator, at 40 DEG C
~100 DEG C of temperature ranges trigger Raolical polymerizable.
When using ultraviolet lighting trigger when, from styrax, benzoin dimethylether, benzoin ethyl ether, benzoin isopropyl ether,
Benzoin isobutyl ether, diphenylethan or α, alpha, alpha-dimethyl epoxide-α-phenyl acetophenone initiated polymerization.
The rare earth metal solution is to include one or more of solution in Eu, Tb, Sm, Dy element；The enhancing
Agent is β-naphthoyltrifluoroacetone, trioctyl phosphine oxide and Triton X-100；The crosslinking agent is dimethacrylate second two
Alcohol ester；The monomer is methacrylic acid；The solvent is methanol or acetonitrile-water；The initiator is that thermal initiator or light draw
Send out agent；
Usage ratio is respectively 10mmol rare earth elements, 15mmol β-naphthoyltrifluoroacetone, 50mM trioctyl phosphine oxides
With 1mL Triton X-100,4mmol methacrylic acid, 10mL methanol or acetonitrile-water, 20mmol dimethacrylate
Glycol ester and 50mg azodiisobutyronitriles.
Embodiment 3
The application method of time-resolved fluorescence enzyme micro-plate reader testing standard plate：Using rare earth metal solution national standard thing
Matter is as standard, using the time-resolved fluorescence enzyme micro-plate reader by the qualified sepectrophotofluorometer of measurement verification to preparation
The fluorescence intensity of rare-earth fluorescent solid matter in each enzyme mark hole of testing standard plate is measured, and is calculated using calibration curve method
Go out the equivalent rare earth metal concentrations in each enzyme mark hole, the standard value as the testing standard plate.
When for repeatability index metrology and measurement, according to the rare earth gold used in time-resolved fluorescence enzyme micro-plate reader
Exciting for category is measured with launch wavelength, suitable detection gain, analysis time and the fluorescence time of integration is set, to the time point
The time-resolved fluorescence intensity replication 6 times in a certain enzyme mark hole of fluorescence enzyme micro-plate reader testing standard plate is distinguished, calculates relative mark
Sign of the quasi- deviation as repeatability.
When for linear index metrology and measurement, according to the rare earth metal used in time-resolved fluorescence enzyme micro-plate reader
Excite and be measured with launch wavelength, suitable detection gain, analysis time and the fluorescence time of integration are set, to time resolution
The time-resolved fluorescence intensity in serial different equivalent rare earth metal concentrations enzyme mark hole is entered on fluorescence enzyme micro-plate reader testing standard plate
Row measure, then the time-resolved fluorescence intensity measured is carried out most with the equivalent rare earth metal concentrations standard value in corresponding enzyme mark hole
A young waiter in a wineshop or an inn multiplies linear regression, calculates linearly dependent coefficient as linear sign.
When being tested for signal to noise ratio index computation, according to the rare earth metal used in time-resolved fluorescence enzyme analyser
Excite and be measured with launch wavelength, suitable detection gain, analysis time and the fluorescence time of integration are set, to time resolution
The time-resolved fluorescence intensity in a certain equivalent rare earth metal concentrations enzyme mark hole and be free of on fluorescence enzyme micro-plate reader testing standard plate
The time-resolved fluorescence intensity for having the enzyme mark hole of rare earth metal is determined respectively, calculates the ratio of two fluorescence intensities as letter
Make an uproar than sign.
For prominent the beneficial effects of the utility model, following comparative example experiment has also been carried out.
The time-resolved fluorescence enzyme micro-plate reader test board A of the prior art of comparative example 1
Concrete structure is：One or more in Eu, Tb, Sm, Dy rare earth element are hybridly prepared into solution, will be different dense
The earth solution of degree is added to after being mixed with reinforcing agent in black ELISA Plate, forms the test board of calibration.
The time-resolved fluorescence enzyme micro-plate reader test board B of the prior art of comparative example 2
Concrete structure is：Using electronic technology simulated time resolved fluorometric signal, under the control of single-chip microcomputer, LED is driven
It is luminous, at the same simulated time resolved fluorescence spectroscopy signal, the test signal of formation calibration after overdamping filters.
The time-resolved fluorescence enzyme micro-plate reader test board A of comparative example 3 application method and effect
Using time-resolved fluorescence enzyme micro-plate reader test board A and the utility model embodiment 1 to same time resolution
Fluorescence microplate reader carries out the test of repeated measuring index, the relative standard of continuous 6 calculating time-resolved fluorescence signals of measurement
Sign of the deviation as repeatability.Test board A relative standard deviation is used as 2.3%, using the phase of the utility model device
It is 0.7% to standard deviation.
The time-resolved fluorescence enzyme micro-plate reader test board B of comparative example 4 application method and effect
Using time-resolved fluorescence ELIASA test board B and the utility model embodiment 1 to same time-resolved fluorescence
ELIASA carries out the test of repdocutbility measuring index.Instrument and test board shut down after testing every time and opened again, when being repeated 6 times calculating
Between resolved fluorometric signal relative standard deviation as repeatability sign.Use test board B relative standard deviation for
5.6%, the relative standard deviation of the utility model device is used as 1.8%.
Contrasted from comparative example 1-4 and embodiment 1,
| Repeated % | Repdocutbility % | |
| The utility model | 0.7 | 1.8 |
| Test board A | 2.3 | / |
| Test board B | / | 5.6 |
Conclusion：The utility model embodiment 1 uses solid time-resolved fluorescence mode, in indexs such as repeatability, repdocutbilities
Upper better than solution and the calibrating installation of simulation of electronic circuits mode.
Embodiment described above is only that preferred embodiment of the present utility model is described, not to this practicality
New scope is defined, and on the premise of the utility model design spirit is not departed from, those of ordinary skill in the art are to this
The various modifications and improvement that the technical scheme of utility model is made, the protection of the utility model claims book determination all should be fallen into
In the range of.
Claims (5)
- A kind of 1. time-resolved fluorescence enzyme micro-plate reader testing standard plate, it is characterised in that：Including black ELISA Plate (1)；Black ELISA Plate (1) is provided with 96 enzyme mark holes (2)；Contain rare-earth fluorescent solid matter (3) in 96 enzyme marks hole (2).
- 2. time-resolved fluorescence enzyme micro-plate reader testing standard plate according to claim 1, it is characterised in that：The rare earth Contain rare earth metal and reinforcing agent in Fluorescence solid-state material (3).
- 3. time-resolved fluorescence enzyme micro-plate reader testing standard plate according to claim 2, it is characterised in that：The rare earth Fluorescence solid-state material (3) is long-life rare earth Fluorescence solid-state material.
- 4. time-resolved fluorescence enzyme micro-plate reader testing standard plate according to claim 3, it is characterised in that：The rare earth The matrix of Fluorescence solid-state material (3) is high molecular polymer, and using copolymerization method, direct polymerization forms in enzyme mark hole (2).
- 5. time-resolved fluorescence enzyme micro-plate reader testing standard plate according to claim 4, it is characterised in that：Described 96 Enzyme mark hole (2) is uniformly distributed in the rectangular array of 8 rows 12 row.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201720760541.3U CN207096073U (en) | 2017-06-27 | 2017-06-27 | Time-resolved fluorescence enzyme micro-plate reader testing standard plate |
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| CN201720760541.3U CN207096073U (en) | 2017-06-27 | 2017-06-27 | Time-resolved fluorescence enzyme micro-plate reader testing standard plate |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107064007A (en) * | 2017-06-27 | 2017-08-18 | 中国计量科学研究院 | Time-resolved fluorescence enzyme micro-plate reader testing standard plate and its preparation and application |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107064007A (en) * | 2017-06-27 | 2017-08-18 | 中国计量科学研究院 | Time-resolved fluorescence enzyme micro-plate reader testing standard plate and its preparation and application |
| CN107064007B (en) * | 2017-06-27 | 2024-04-02 | 中国计量科学研究院 | Time-resolved fluorogenic enzyme-labeled analyzer test standard plate and preparation and use methods thereof |
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