CN110514648A - A kind of chemiluminescence analysis POCT detection device and its application - Google Patents
A kind of chemiluminescence analysis POCT detection device and its application Download PDFInfo
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- CN110514648A CN110514648A CN201810806198.0A CN201810806198A CN110514648A CN 110514648 A CN110514648 A CN 110514648A CN 201810806198 A CN201810806198 A CN 201810806198A CN 110514648 A CN110514648 A CN 110514648A
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- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 235000019154 vitamin C Nutrition 0.000 description 1
- 239000011718 vitamin C Substances 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- 230000010148 water-pollination Effects 0.000 description 1
- 229920001285 xanthan gum Polymers 0.000 description 1
- 229940082509 xanthan gum Drugs 0.000 description 1
- 235000010493 xanthan gum Nutrition 0.000 description 1
- 239000000230 xanthan gum Substances 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
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
Abstract
The present invention relates to a kind of chemiluminescence analysis POCT detection device in chemiluminescence field and its applications.The chemiluminescence analysis POCT detection device separately designs reagent card and POCT detection device, integrates and uses, and acquires sample to be tested using reagent card, easy to carry.Meanwhile the detection method of chemiluminescence immune assay POCT is carried out using the device, under the premise of not interrupting reaction, after repeatedly, choosing reading twice, to widen detection range, and in the detection process, simplicity rapidly calculates testing concentration.In addition, method of the invention can 100% correctly identify double-antibody method detection in HD-HOOK effect sample, the method can significantly improve the accuracy of double antibody sandwich method immunoassays, and reduce the false negative rate of double antibody sandwich method immunoassays.
Description
Technical field
The invention belongs to chemiluminescence fields, and in particular to a kind of chemiluminescence analysis POCT detection device and its answer
With.
Background technique
Chemiluminescence analysis is then to develop relatively rapid on-radiation immunoassay technology in recent years, and principle is to utilize change
The amplification that luminescent substance carries out signal is learned, and by its luminous intensity, immune cohesive process is directly measured, which has become
For one of the important directions of immunology detection.Light-induced chemiluminescent method is one of common method of chemiluminescence analytical technique, can
For studying the intermolecular interaction of biology, clinically it is mainly used for the detection of disease.The technology incorporates high molecular particle
The research of the related fieldss such as technology, organic synthesis, protein chemistry and clinical detection.With traditional enzyme-linked immune analytic method phase
Than it has the characteristics that homogeneous, high sensitivity and easy to operate is easy to automate.Therefore, application prospect is very wide.
It, can be because when material concentration height to be detected is to a certain concentration in the detection pattern of double antibodies sandwich in chemiluminescence
For double antibodies sandwich compound cannot be formed to the relatively low phenomenon of signal value, referred to as high dose-hook effect (HD-HOOK effect
It answers).That is, high dose-hook effect refers in two-site sandwich immunization experiment, the high dose of dose-effect curve
Section, linear trend instead of in platform-like it is unlimited after prolong, be turned under curved, like a hook, cause to generate false negative
Phenomenon.In immune detection frequent occurrence, incidence accounts for positive sample 30% or so to HD-HOOK effect.Since HD-HOOK is imitated
It is since its concentration exceeds the range of linearity of detection kit also that the presence answered, which causes to be detected sample and cannot correctly be divided into,
It is concentration itself is exactly the value, so that misdiagnosis of the experiment, especially causes false negative rate to rise.
Meanwhile existing chemoluminescence method has the disadvantage in that A, instrument system are bulky, takes up a large area, meanwhile,
Since test flux is big, reagent card is used with 100 tests as whole unit, is required to laboratory sample size；B, instrument system
And reagent price is expensive, maintenance cost is high, is not suitable for basic medical unit；C, equipment instrument is big, cannot carry entrance
Diagnosis and treatment scene；D, chemiluminescence system mainly uses serum and blood plasma as sample, cannot generally use whole blood, limiting it makes
Use range.
Newly rise in recent years a kind of clinical detection (bedside detect bedside testing) carried out beside patient i.e.
When examine (point-of-care testing) technology, abbreviation POCT detection technique, POCT detection technique mainstream is fluorescent quantitation
Chromatography or colloidal gold, the mainly fluorescent microsphere of package fluorescent material or colloidal gold carry out immune inspection by the method that film layer is analysed
That surveys examines technology fastly.But since both technologies mainly carry out release detection on NC film, since the CV of film itself just has
5% or more, therefore will be 10% or more as the POCT detection CV-of solid phase embrane method, detection precision is very poor, for sensitivity
Exigent project such as cTnI, it is quantitative just to become extremely difficult.
Therefore, it is urgent to provide a kind of wide scopes for avoiding HD-HOOK effect, while having the characteristics that quick, portable change
Learn luminesceence analysis POCT detection device and its application.
Summary of the invention
In view of the deficiencies of the prior art, the present invention provides a kind of chemiluminescence analysis POCT detection devices, by reagent
Card and POCT detection device separately design, and integrate and use, and acquire sample to be tested using reagent card, easy to carry.Meanwhile utilizing this
Device carries out the detection method of chemiluminescence analysis POCT, under the premise of not interrupting reaction, after repeatedly reading, chooses two
For secondary reading to widen detection range, and in the detection process, simplicity rapidly calculates testing concentration.In addition, of the invention
Method 100% can correctly identify HD-HOOK effect sample in double-antibody method detection, and the method can significantly improve double
The accuracy of antibody sandwich immunoassays, and reduce the false negative rate of double antibody sandwich method immunoassays.
For this purpose, first aspect present invention provides a kind of chemiluminescence analysis POCT detection device comprising:
Reagent pipetting volume module is used for the sample to be tested of the doubtful molecule containing object to be measured and chemiluminescence reaction institute occurs
Reaction forms testing mixture after the reagent mixing needed；
Reaction module is used to that chemiluminescence reaction to occur for testing mixture obtained in reagent pipetting volume module to provide conjunction
Suitable temperature environment；
Detection module is used to record chemiluminescent signal value described in n times；Wherein, the chemiluminescence letter of n-th record
Number value is denoted as reading RLUn；And any signal value twice in the chemiluminescence signal value of the n times record is chosen, it is denoted as respectively
RLUm and reading RLUk are read, and the difference amplification of RLUm and RLUk is denoted as A, amplification A=(RLUm/RLUk-1) × 100%；
With
Processor module is used for according to a series of standard substance of the molecule containing object to be measured of known concentrations and any
The difference amplification A ' of the reading RLUm ' and RLUk ' of two secondary responses do standard curve；The amplification A is compared with standard curve
Compared with to determine the concentration of sample；
Wherein, n, m and k are the natural number greater than 0, and k < m≤n, n >=2.
In certain embodiments of the present invention, described device further includes light excitation module, is used for successive t excitation institute
It states testing mixture and chemiluminescence occurs, wherein t is natural number greater than 0, and n≤t.
In other embodiments of the invention, described device further includes the reagent card being used cooperatively, the reagent card
On offer sample to be tested hole, donating agent hole and receptor agents hole；The sample to be tested hole is used to contain sample to be tested, described
Donating agent hole is used to contain donating agent, and the receptor agents hole is used to contain receptor agents；
When detecting, the reagent card is arranged in the POCT detection device, under the control of circuit control module, institute
Reaction module is stated for adjusting the temperature of substance in the reagent card and reagent card, the reagent pipetting volume module is described for shifting
Substance in reagent card, the smooth excitation module are used for successive t transmitting laser, and the detection module is for recording described in n times
Chemiluminescent signal value；Wherein, the chemiluminescence signal value of n-th record is denoted as reading RLUn；And choose the n times record
Chemiluminescence signal value in any signal value twice, be denoted as reading RLUm and reading RLUk respectively, and by RLUm and RLUk
Difference amplification be denoted as A；The processor module according to a series of standard substance of the molecule containing object to be measured of known concentrations with
And the difference amplification A ' of the reading RLUm ' and RLUk ' of any two secondary response do standard curve；By the amplification A and standard curve
It is compared, to determine the concentration of sample；
Wherein, t, n, m and k are the natural number greater than 0, and k < m≤n≤t, n >=2.
In certain embodiments of the present invention, dilution fluid apertures is also provided on the reagent card, the dilution fluid apertures is used
To contain dilution；The sample to be tested hole, donating agent hole, receptor agents hole and the dilution equal overlay film closed orifices of fluid apertures.
In certain embodiments of the present invention, it is additionally provided with bar code area on the reagent card, is set in the bar code area
There is bar code.
In certain embodiments of the present invention, described device further includes bar code scanning module, the bar code scanning module
The information in bar code is read for identification.
Second aspect of the present invention, which provides, a kind of utilizes device as described in the first aspect of the invention to carry out chemiluminescence point
The method for analysing POCT detection comprising following steps:
(1) anti-after mixing the sample to be tested of the doubtful molecule containing object to be measured with reagent needed for generation chemiluminescence reaction
Testing mixture should be formed；
(2) successively chemiluminescence occurs for the t excitation testing mixture, and n times record the chemiluminescent signal value；
Wherein, the chemiluminescence signal value of n-th record is denoted as reading RLUn；
(3) any signal value twice in the chemiluminescence signal value of the n times record is chosen, is denoted as reading RLUm respectively
With reading RLUk, and the difference amplification of RLUm and RLUk is denoted as A；
(4) according to a series of standard substance of the molecule containing object to be measured of known concentrations and its in step (2) and step (3)
In the difference amplification A ' of reading RLUm ' and RLUk ' of any two secondary response do standard curve；
(5) it is compared by the A value with standard curve, to determine the concentration of sample；
Wherein, t, n, m and k are the natural number greater than 0, and k < m≤n≤t, n >=2.
In certain embodiments of the present invention, the amplification A=(RLUm/RLUk-1) × 100%.
In other embodiments of the invention, the n is greater than 2.
In certain embodiments of the present invention, in step (1), the chemiluminescence reaction is that homogeneous chemistry shines instead
It answers.
In other embodiments of the invention, in step (1), reagent packet needed for the generation chemiluminescence reaction
Include receptor agents and donating agent；Wherein:
It include donor in the donating agent, the donor can generate singlet oxygen under excited state；
It include receptor in the receptor agents, the receptor can be reacted with singlet oxygen generates detectable chemiluminescence
Signal value.
In certain embodiments of the present invention, the receptor is filled with luminophor and lanthanide compound
High molecular particle.
In other embodiments of the invention, the luminophor is selected from olefin(e) compound, is preferably selected from diformazan
Base thiophene, compound of di-butanedione, dioxine, enol ether, enamine, 9- alkylidene xanthane, 9- alkylidene-N-9,10
Acridan, aryl ether alkene, Aryimidazole and lucigenin and their derivative, be more preferably selected from thioxene and
Its derivative.
In certain embodiments of the present invention, the lanthanide compound is europium complex.
In other embodiments of the invention, it is non-that the receptor, which includes olefin(e) compound and metallo-chelate,
Particulate forms, and it is solvable in water-bearing media.
In certain embodiments of the present invention, the receptor and the first specific junction mixture of object to be measured molecule directly or
It combines indirectly.
In other embodiments of the invention, the donor is filled with the high molecular particle of Photoactive compounds,
It can produce singlet oxygen under red laser excitation.
In certain embodiments of the present invention, the Photoactive compounds are selected from methylene blue, rose-red, Bu Lin and phthalocyanine
One of.
In other embodiments of the invention, the donor is either directly or indirectly combined with marker.
In certain embodiments of the present invention, in step (1), reagent needed for the generation chemiluminescence reaction is also wrapped
Include object to be measured molecule the second specific junction mixture reagent；Preferably, second specific junction mixture of object to be measured molecule and label
Object specific junction mixture either directly or indirectly combines.
In other embodiments of the invention, in step (1), first by the sample to be tested of the molecule containing object to be measured with by
Body reagent and the mixing of object to be measured molecule the second specific junction mixture reagent, then again mix it with donating agent.
In certain embodiments of the present invention, in step (2), using energy and/or reactive compound excite it is described to
It surveys mixture and chemiluminescence occurs；Preferably, its generation is excited with the red exciting light irradiation testing mixture of 600~700nm
Chemiluminescence.
In other embodiments of the invention, in step (2), the Detection wavelength of the chemiluminescence signal value is recorded
For 520~620nm.
In certain embodiments of the present invention, the object to be measured molecule is antigen or antibody；Wherein, the antigen is
Refer to that the substance with immunogenicity, the antibody refer to the immunoglobulin that can identify specific exotic that body generates.
In other embodiments of the invention, the standard substance is positive control.
In some preferred embodiments of the invention, the method specifically includes the following steps:
(a1) first time incubation is carried out after mixing the doubtful sample to be tested containing determined antigen (or antibody) with receptor agents；
The first step is incubated gained mixed liquor again to mix with donating agent, forms testing mixture after second of incubation；
(a2) chemiluminescence, n times are occurred with the red exciting light of the 600~700nm successive t excitation testing mixture
The chemiluminescent signal value is recorded, Detection wavelength is 520~620nm；Wherein, the chemiluminescence signal value of n-th record
Just it is denoted as reading RLUn；
(a3) any signal value twice in the chemiluminescence signal value of the n times record is chosen, is denoted as reading respectively
RLUm and reading RLUk, and the difference amplification of RLUm and RLUk is denoted as A, amplification A=(RLUm/RLUk-1) × 100%；
(a4) according to a series of positive reference substance of the molecule containing object to be measured of known concentrations and its in step (a2) and
The difference amplification A ' of the reading RLUm ' and RLUk ' of any two secondary response do standard curve in step (a3)；
(a5) according to the A value determine object to be measured molecular concentration be standard curve first transition or declining
Section, then the RLU1 of object to be measured molecule is substituted into its corresponding standard curve and calculates concentration；
Wherein, t, n, m and k are the natural number greater than 0, and k < m≤n≤t, n >=2.
The invention has the benefit that of the present invention kind of chemiluminescence analysis POCT detection device, by reagent card and
POCT detection device separately designs, and integrates and uses, and acquires sample to be tested using reagent card, easy to carry.Meanwhile utilizing the device
The detection method for carrying out chemiluminescence analysis POCT, under the premise of not interrupting reaction, after repeatedly reading, selection is read twice
For number to widen detection range, and in the detection process, simplicity rapidly calculates testing concentration.In addition, method of the invention
100% it can correctly identify HD-HOOK effect sample in double-antibody method detection, the method can significantly improve double antibody
The accuracy of sandwich method immunoassays, and reduce the false negative rate of double antibody sandwich method immunoassays.
Detailed description of the invention
Below in conjunction with attached drawing, the present invention is further described.
Fig. 1: AFP using signal value obtained by the method for the present invention and A respectively with concentration of specimens graph of relation.
Fig. 2: HCG+ β using signal value obtained by the method for the present invention and A respectively with concentration of specimens graph of relation.
Fig. 3: Ferr using signal value obtained by the method for the present invention and A respectively with concentration of specimens graph of relation.
Fig. 4: anti-HIV using signal value obtained by the method for the present invention and A respectively with concentration of specimens graph of relation.
Fig. 5: MYO using signal value obtained by the method for the present invention and A respectively with concentration of specimens graph of relation.
Fig. 6: NT-proBNP using signal value obtained by the method for the present invention and A respectively with concentration of specimens graph of relation.
Fig. 7: PCT using signal value obtained by the method for the present invention and A respectively with concentration of specimens graph of relation.
Fig. 8: cTnI using signal value obtained by the method for the present invention and A respectively with concentration of specimens graph of relation.
Specific embodiment
To be readily appreciated that the present invention, the present invention is described more detail below.But before describing the present invention in detail, it should be understood that
The present invention is not limited to the specific embodiments of description.It is also understood that term used herein is only for description specific implementation
Mode, and be not offered as restrictive.
In the case where providing numberical range, it should be understood that in the upper and lower bound of the range and the prescribed limit
Any other regulation or between two parties each of between numerical value numerical value is encompassed by the present invention between two parties.These small range of upper limits
It can independently be included in lesser range with lower limit, and be also covered by the present invention, obey any clear in prescribed limit
The limit of exclusion.Defined range include one or two limit in the case where, exclude any of the limit that those include or
The range of the two is also included in the present invention.
Unless otherwise defined, the usual reason of all terms used herein and those skilled in the art
Solve meaning having the same.Although similar or equivalent any method and material can also be with method described herein and material
It is used in implementation or test of the invention, but preferred method and material will now be described.
Unless otherwise stated, disclosed in this invention experimental method, detection method, preparation method be all made of this technology neck
Molecular biology, biochemistry, chromatin Structure and the analysis of domain routine, analytical chemistry, cell culture, recombinant DNA technology and
The routine techniques of related fields.These technologies have perfect explanation in the prior art, and for details, reference can be made to Sambrook etc.
MOLECULAR CLONING:A LABORATORY MANUAL, Second edition, Cold Spring Harbor
Laboratory Press, 1989and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY, John Wiley&Sons, New York, 1987and periodic updates；the
Series METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe, CHROMATIN
STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；METHODS IN
ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic
Press, San Diego, 1999；With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin
Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..
I, term
Term " chemiluminescence analysis measurement " of the present invention refers to that chemical reaction can generate certain and be in excited electronic state
Product, when radiation transistion or energy, which is transferred to other irradiative molecules, occurs that the molecule again occurs for this product molecule
When radiation transistion, luminescence phenomenon is just generated.It is this due to absorbing chemical energy, so that molecule is generated electron excitation and luminous phenomenon
Referred to as chemiluminescence.The method for carrying out chemical analysis using chemiluminescence and measuring determinand is known as chemiluminescence analysis measurement side
Method.
It can be liquid chemiluminescence analysis determining method, and can be gas phase chemiluminescence analysis determining method, also
It can be solid state chemistry luminesceence analysis measuring method；Preferably liquid chemiluminescence analysis determining method
It can be general chemistry luminesceence analysis measuring method (energy supply reaction is general chemical reaction), be also possible to biology
(energy supply reaction is biochemical reaction to chemiluminescence analysis measuring method；Abbreviation BCL), it can also be that Electrochemiluminescprocess process is surveyed
Determine method (energy supply reaction is electrochemical reaction, abbreviation ECL) etc.；Preferably general chemistry luminesceence analysis measuring method.
It not only can be heterogeneous phase chemistry luminesceence analysis measuring method, but also can also be that heterogeneous phase chemistry luminesceence analysis is surveyed
Determine method, preferably heterogeneous phase chemistry luminesceence analysis measuring method.
Term " object to be measured molecule " of the present invention is either immune molecule, such as antigen or antibody；It can be again
Inorganic compound, such as metal ion, hydrogen peroxide, CN-Or NO2-；It is also possible to organic compound, such as: oxalic acid, Vitamin C
Acid, imines, acetylcholine etc.；It can also be carbohydrate, such as: glucose or lactose；It can also be amino acid, hormone, enzyme, fat
Acid, vitamin and drug；It is preferred that immune molecule.It includes object to be measured molecules for term " sample to be examined " of the present invention.This hair
It includes samples to be tested for the bright term " mixed liquor to be measured ".
Term " reagent needed for chemiluminescence analysis measurement " of the present invention, refers to that a chemical reaction will generate chemistry
Luminescence phenomenon, it is necessary to which meet the following conditions: first is that the reaction must provide enough excitation energy, and individually be mentioned by a certain step
For because the energy of back reaction release will cannot shine because vibration relaxation disappears in the solution；Second is advantageous
Reaction process, make chemical reaction energy can at least be received by a kind of substance and generate excitation state；Third is excitation state point
Son must have certain chemiluminescence quantum efficiency to release photon, or can shift its energy and make to another molecule
Entrance excitation state and release photon.
Reagent needed for chemiluminescence analysis measurement is including but not limited to following substance: (1) anti-in chemiluminescence reaction
Answer object；(2) catalyst in chemiluminescence reaction, sensitizer or inhibitor；(3) reactant in coupling reaction, catalyst, increasing
Quick dose etc..
Term " successively " of the present invention is a temporal characteristics, indicates that the number of repeatedly " excitation " is according to chronomere
Come what is distinguished.
Term " antibody " of the present invention is used with most wide meaning, and the antibody including any isotype retains to antigen
The antibody fragment of specific binding, including but not limited to Fab, Fv, scFv and Fd segment, chimeric antibody, humanized antibody, list
The fusion protein of chain antibody, bispecific antibody and antigen-binding portion thereof and non-antibody protein comprising antibody.In any need
In the case where, antibody can be further with other parts, and biotin or Streptavidin etc. are conjugated.
Term " antigen " of the present invention refers to the substance with immunogenicity, such as protein, polypeptide.It is representative anti-
Original includes but is not limited to: cell factor, tumor markers, Matrix metalloprotease-9, cardiovascular diabetes related protein etc..Term
" tumor markers " refer in the generation and breeding of tumour, either by body pair caused by tumour cell itself
Tumour cell reaction and generate, reaction tumour exist and growth a substance.The representative tumor markers packet in this field
It includes (but being not limited to): alpha-fetoprotein (AFP), cancer antigen 125 (CA125) etc..
Term " in conjunction with " of the present invention refer to due to for example covalently, the interaction such as electrostatic, hydrophobic, ion and/or hydrogen bond,
Including but not limited to two intermolecular direct joints caused by such as salt bridge and the interaction of water bridge.
Term " specific binding " of the present invention, refer between two kinds of substances it is mutual discrimination and selective binding it is anti-
Answer, said from stereochemical structure angle be exactly conformation between corresponding reactant correspondence.
Term " biotin " of the present invention is widely present in animal vegetable tissue, there are two cyclic structure on molecule,
Respectively imidazolone ring and thiphene ring, wherein imidazolone ring is the main portions in conjunction with Streptavidin.The biotin of activation
It can be coupled under the mediation of protein cross agent with known almost all creatures macromolecular, including protein, nucleic acid, more
Sugar and lipid etc.；And " Streptavidin " is a kind of protein secreted by streptomycete, molecular weight 65kD." Streptavidin "
Molecule is made of 4 identical peptide chains, wherein every peptide chain can combine a biotin.Therefore each antigen or antibody can be same
When be coupled multiple biotin molecules, thus generate " tentacle effect " improve sensitivity for analysis.
In the case where any need, any reagent used in the present invention, including antigen, antibody, receptor or donor, it can
With biotin-conjugated according to actual needs or Streptavidin etc..
Term " donor " of the present invention can generate after referring to the activation by energy or reactive compound and receptor
The sensitizer of the reactive intermediate of such as singlet oxygen of reaction.Donor can be (such as dyestuff and aromatic compound) of photoactivation
Or (such as enzyme, metal salt) of chemical activation.
In particular embodiments of the invention, the donor is photosensitizer, and the photosensitizer can be known in the art
Photosensitizer, it is preferably stable with respect to light and the compound with singlet oxygen effecting reaction, non-limiting example do not include example
Methylene blue, rose-red, porphyrin, phthalein as disclosed in United States Patent (USP) US5709994 (patent document is hereby incorporated by reference)
The derivative with 1-50 replacing group of the compounds such as cyanines and chlorophyll and these compounds, the substituent group are used
In make these compounds with more lipophilicity or with more hydrophily, and/or as being connected to specific binding pair member
Linking group.The example of other photosensitizers well known by persons skilled in the art can also be used in the present invention, such as the U.S.
The content recorded in patent US6406913, the patent document are hereby expressly incorporated by reference.
In other specific embodiments of the invention, the donor is other sensitizers of chemical activation, non-limiting
Example be certain compounds, their catalyzing hydrogen peroxides are converted into singlet oxygen and water.The example of some other donor includes:
Isosorbide-5-Nitrae-dicarboxyethyl-Isosorbide-5-Nitrae-naphthalene endoperoxides object, 9,10- diphenylanthrancene -9,10- endoperoxides object etc., heat these chemical combination
Object or these compounds, which directly absorb light, can discharge singlet oxygen.
Term " receptor " of the present invention is to refer to react the compound that can produce detectable signal with singlet oxygen.
For donor by energy or reactive compound induced activation and the singlet oxygen for discharging upper state, the singlet oxygen of the upper state is close
The receptor of distance is captured, to transmit energy to activate the receptor.
In some embodiments of the invention, the receptor is such substance: it undergoes the change with singlet oxygen
Reaction is learned to form unstable metastable state intermediate, the metastable state intermediate can be decomposed, subsequently or simultaneously be shone.These
The typical example of substance includes but is not limited to: enol ether, enamine, 9- alkylidene xanthan gum, 9- alkylidene-N- alkyl Acridane, fragrant second
Alkene ether, bicyclic ethylene oxide, thioxene, armaticity imidazoles or lucigenin.
In other specific embodiments of the invention, the receptor can be reacted with singlet oxygen to be formed and can be divided
Solution at the hydroperoxides or dioxy cyclobutane of ketone or carboxylic acid derivates olefines；It can be decomposed by the effect of light steady
Determine dioxy cyclobutane；It can be reacted with singlet oxygen to form the acetylene class of diones；Azo-compound or azo can be formed
The hydrazone class or hydrazides of carbonyls, such as luminol；With the aromatic compounds that can form endoperoxides species.It can root
Specific, the non-limiting example of the receptor utilized according to the disclosure and claimed invention are recorded in U.S. Patent number
US5340716 (patent document is hereby incorporated by reference).
In other specific embodiments of the invention, " donor " and/or " receptor " can be wrapped by functional group
" donor microballoon " and/or " receptor microballoon " is formed on matrix." matrix " of the present invention is that those skilled in the art institute is public
The microballoon or particle known, can be any size, can be organic or inorganic, can be inflatable or not
It is expandable, can be it is porous or non-porous, with any density, but preferably have and the close density of water, it is excellent
Choosing can float in water, and be made of transparent, partially transparent or opaque material.Described matrix can be with or without charge,
When having charge, preferably negative electrical charge.Described matrix can be solid (such as polymer, metal, glass, organic and inorganic matter
Such as mineral, salt and diatom), small oil droplet (such as hydrocarbon, fluorocarbon, silicic fluid), vesica (as synthesis such as
Phosphatide or natural such as cell and organelle).Matrix can be latex particle or contain organic or inorganic polymer
Other particles, lipid bilayer such as liposome, phospholipid capsule bubble, small oil droplet, silicon particle, metal-sol, cell and crystallite dyestuff.Matrix
Usually have multifunctionality, or can be integrated to by special or non-specific covalently or non-covalently interaction donor or
On receptor.It is available or is merged in there are many functional group.Typical functional group includes carboxylic acid, acetaldehyde, amino, cyanogen
Base, vinyl, hydroxyl, sulfydryl etc..A unrestricted example for being suitable for the invention matrix is carboxy-modified latex
Particle.The details of this matrix can be found in United States Patent (USP) US5709994, and (this two patents document is herein with US5780646
Full text is incorporated by reference).
II, specific embodiment
The basic principle of double-antibody method:
The basic principle of double antibody sandwich method is well-known to those skilled in the art.Conventional way is by first antibody
It is fixed on solid phase carrier, then by first antibody and antigen-reactive, then is reacted with the secondary antibody of label, chemical hair is finally carried out
Light or enzyme-linked chromogenic reaction detect signal.
The present invention is described more detail below.
For this purpose, chemiluminescence analysis POCT detection device involved in first aspect present invention comprising:
Reagent pipetting volume module is used for the sample to be tested of the doubtful molecule containing object to be measured and chemiluminescence reaction institute occurs
Reaction forms testing mixture after the reagent mixing needed；
Reaction module is used to that chemiluminescence reaction to occur for testing mixture obtained in reagent pipetting volume module to provide conjunction
Suitable temperature environment；
Detection module is used to record chemiluminescent signal value described in n times；Wherein, the chemiluminescence letter of n-th record
Number value is denoted as reading RLUn；And any signal value twice in the chemiluminescence signal value of the n times record is chosen, it is denoted as respectively
RLUm and reading RLUk are read, and the difference amplification of RLUm and RLUk is denoted as A, amplification A=(RLUm/RLUk-1) × 100%；
With
Processor module is used for according to a series of standard substance of the molecule containing object to be measured of known concentrations and any
The difference amplification A ' of the reading RLUm ' and RLUk ' of two secondary responses do standard curve；The amplification A is compared with standard curve
Compared with to determine the concentration of sample；
Wherein, n, m and k are the natural number greater than 0, and k < m≤n, n >=2.
In certain embodiments of the present invention, described device further includes light excitation module, is used for successive t excitation institute
It states testing mixture and chemiluminescence occurs, wherein t is natural number greater than 0, and n≤t.
In other embodiments of the invention, described device further includes the reagent card being used cooperatively, the reagent card
On offer sample to be tested hole, donating agent hole and receptor agents hole；The sample to be tested hole is used to contain sample to be tested, described
Donating agent hole is used to contain donating agent, and the receptor agents hole is used to contain receptor agents；
When detecting, the reagent card is arranged in the POCT detection device, under the control of circuit control module, institute
Reaction module is stated for adjusting the temperature of substance in the reagent card and reagent card, the reagent pipetting volume module is described for shifting
Substance in reagent card, the smooth excitation module are used for successive t transmitting laser, and the detection module is for recording described in n times
Chemiluminescent signal value；Wherein, the chemiluminescence signal value of n-th record is denoted as reading RLUn；And choose the n times record
Chemiluminescence signal value in any signal value twice, be denoted as reading RLUm and reading RLUk respectively, and by RLUm and RLUk
Difference amplification be denoted as A；The processor module according to a series of standard substance of the molecule containing object to be measured of known concentrations with
And the difference amplification A ' of the reading RLUm ' and RLUk ' of any two secondary response do standard curve；By the amplification A and standard curve
It is compared, to determine the concentration of sample；
Wherein, t, n, m and k are the natural number greater than 0, and k < m≤n≤t, n >=2.
In certain embodiments of the present invention, dilution fluid apertures is also provided on the reagent card, the dilution fluid apertures is used
To contain dilution；The sample to be tested hole, donating agent hole, receptor agents hole and the dilution equal overlay film closed orifices of fluid apertures.
In certain embodiments of the present invention, it is additionally provided with bar code area on the reagent card, is set in the bar code area
There is bar code.
In certain embodiments of the present invention, described device further includes bar code scanning module, the bar code scanning module
The information in bar code is read for identification.
Chemiluminescence analysis is carried out using device as described in the first aspect of the invention involved in second aspect of the present invention
The method of POCT detection comprising following steps:
(1) anti-after mixing the sample to be tested of the doubtful molecule containing object to be measured with reagent needed for generation chemiluminescence reaction
Testing mixture should be formed；
(2) successively chemiluminescence occurs for the t excitation testing mixture, and n times record the chemiluminescent signal value；
Wherein, the chemiluminescence signal value of n-th record is denoted as reading RLUn；
(3) any signal value twice in the chemiluminescence signal value of the n times record is chosen, is denoted as reading RLUm respectively
With reading RLUk, and the difference amplification of RLUm and RLUk is denoted as A；
(4) according to a series of standard substance of the molecule containing object to be measured of known concentrations and its in step (2) and step (3)
In the difference amplification A ' of reading RLUm ' and RLUk ' of any two secondary response do standard curve；
(5) it is compared by the A value with standard curve, to determine the concentration of sample；
Wherein, t, n, m and k are the natural number greater than 0, and k < m≤n≤t, n >=2.
In certain embodiments of the present invention, the amplification A=(RLUm/RLUk-1) × 100%.
In other embodiments of the invention, the n is greater than 2.For example, n can be 3,4 or 5 etc..In the present invention,
When the n is greater than 2, the sensitivity of the method detection is higher, and the ability of anti-HD-HOOK effect is stronger.
In certain embodiments of the present invention, in step (1), the chemiluminescence reaction is that homogeneous chemistry shines instead
It answers.
In other embodiments of the invention, in step (1), reagent packet needed for the generation chemiluminescence reaction
Include receptor agents and donating agent；Wherein:
It include donor in the donating agent, the donor can generate singlet oxygen under excited state；
It include receptor in the receptor agents, the receptor can be reacted with singlet oxygen generates detectable chemiluminescence
Signal value.
In certain embodiments of the present invention, the receptor is filled with luminophor and lanthanide compound
High molecular particle.
In other embodiments of the invention, the luminophor is selected from olefin(e) compound, is preferably selected from diformazan
Base thiophene, compound of di-butanedione, dioxine, enol ether, enamine, 9- alkylidene xanthane, 9- alkylidene-N-9,10
Acridan, aryl ether alkene, Aryimidazole and lucigenin and their derivative, be more preferably selected from thioxene and
Its derivative.
In certain embodiments of the present invention, the lanthanide compound is europium complex.
In other embodiments of the invention, it is non-that the receptor, which includes olefin(e) compound and metallo-chelate,
Particulate forms, and it is solvable in water-bearing media.
In certain embodiments of the present invention, the receptor and the first specific junction mixture of object to be measured molecule directly or
It combines indirectly.
In other embodiments of the invention, the donor is filled with the high molecular particle of Photoactive compounds,
It can produce singlet oxygen under red laser excitation.
In certain embodiments of the present invention, the Photoactive compounds are selected from methylene blue, rose-red, Bu Lin and phthalocyanine
One of.
In other embodiments of the invention, the donor is either directly or indirectly combined with marker.
In certain embodiments of the present invention, in step (1), reagent needed for the generation chemiluminescence reaction is also wrapped
Include object to be measured molecule the second specific junction mixture reagent；Preferably, second specific junction mixture of object to be measured molecule and label
Object specific junction mixture either directly or indirectly combines.
In other embodiments of the invention, in step (1), first by the sample to be tested of the molecule containing object to be measured with by
Body reagent and the mixing of object to be measured molecule the second specific junction mixture reagent, then again mix it with donating agent.
In certain embodiments of the present invention, in step (2), using energy and/or reactive compound excite it is described to
It surveys mixture and chemiluminescence occurs；Preferably, its generation is excited with the red exciting light irradiation testing mixture of 600~700nm
Chemiluminescence.
In other embodiments of the invention, in step (2), the Detection wavelength of the chemiluminescence signal value is recorded
For 520~620nm.
In certain embodiments of the present invention, the object to be measured molecule is antigen or antibody；Wherein, the antigen is
Refer to that the substance with immunogenicity, the antibody refer to the immunoglobulin that can identify specific exotic that body generates.
In other embodiments of the invention, the standard substance is positive control.
In some preferred embodiments of the invention, the method specifically includes the following steps:
(a1) first time incubation is carried out after mixing the doubtful sample to be tested containing determined antigen (or antibody) with receptor agents；
The first step is incubated gained mixed liquor again to mix with donating agent, forms testing mixture after second of incubation；
(a2) chemiluminescence, n times are occurred with the red exciting light of the 600~700nm successive t excitation testing mixture
The chemiluminescent signal value is recorded, Detection wavelength is 520~620nm；Wherein, the chemiluminescence signal value of n-th record
Just it is denoted as reading RLUn；
(a3) any signal value twice in the chemiluminescence signal value of the n times record is chosen, is denoted as reading respectively
RLUm and reading RLUk, and the difference amplification of RLUm and RLUk is denoted as A, amplification A=(RLUm/RLUk-1) × 100%；
(a4) according to a series of positive reference substance of the molecule containing object to be measured of known concentrations and its in step (a2) and
The difference amplification A ' of the reading RLUm ' and RLUk ' of any two secondary response do standard curve in step (a3)；
(a5) according to the A value determine object to be measured molecular concentration be standard curve first transition or declining
Section, then the RLU1 of object to be measured molecule is substituted into its corresponding standard curve and calculates concentration；
Wherein, t, n, m and k are the natural number greater than 0, and k < m≤n≤t, n >=2.
Here, the method is used for double it should be strongly noted that the above method is the method for non-disease diagnostic purpose
In antibody sandwich immunization or double antigens sandwich immunization detection process, widen detection range by reading twice, with
In detection process, the amplification A read twice is made comparisons with critical value, to judge whether sample to be tested needs to carry out again after diluting
Measurement.
Preferably, the antigen refers to the substance with immunogenicity.Such as protein, polypeptide.Representative antigen packet
It includes (but being not limited to): cell factor, tumor markers, Matrix metalloprotease-9, cardiovascular diabetes related protein etc..
The antibody refers to the immunoglobulin that can identify specific exotic that body generates.
In the embodiment of the present invention, the antigen or antibody are selected from from alpha-fetoprotein (AFP), HBsAb hepatitis type B virus table
Face antibody (HBsAb), human chorionic gonadotropin's gland swashs and β subunit (HCG+ β), hepatitis B surface antigen (HBsAg), cancer antigen 125
(CA125), C peptide (CP), ferritin (Ferr) and Anti-HCV etc..
It can be not particularly limited, be can be any (or anti-containing object to be measured antigen with the sample that the method for the present invention detects
Body) sample, representative example may include serum sample, urine specimen, saliva sample etc..Currently preferred sample is blood
Final proof sheet.
Preferably, it can be specifically bound between the marker and marker specific junction mixture.
It is furthermore preferred that the marker is biotin, the marker specific junction mixture is Streptavidin.
Preferably, the receptor refers to the high molecular particle filled with luminophor and lanthanide compound.It shines
Compound can be the derivative etc. of Dioxene (dioxine) or thioxene (thioxene), lanthanide series
Closing object can be Eu (TTA)3/ TOPO or Eu (TTA)3/ Phen, the particle can be by buying in the market.The surface functional group of receptor can
Be it is any can connexin matter group, such as carboxyl, aldehyde radical, amido, epoxy ethyl or halogenated alkyl it is various it is known can
Connect the functional group of protein.
Preferably, the donor is filled with the high molecular particle of Photoactive compounds, under red laser excitation, can produce
Raw singlet oxygen ion.In the case that when it, distance is close enough with receptor, single line oxonium ion is transmitted to receptor, with the hair in receptor
Optical compounds reaction, generates ultraviolet light, and ultraviolet light further excites lanthanide compound, generates the photon of certain wavelength.
Photoactive compounds can be phthalocyanine dye etc., which can also be by buying in the market.
In detection range, the concentration of object to be measured antigen shows as the quantity of double-antibody sandwich compound, and and photon
Number is directly proportional；But when object to be measured antigen concentration is excessively high, part determined antigen in conjunction with single antibody, leads to dual anti-folder respectively
Heart compound is reduced, and optical signal is relatively low, cannot reflect the actual concentration of object to be measured antigen.
Similarly, in detection range, the concentration of object to be measured antibody shows as the quantity of double antigens sandwich compound, and with
Number of photons is directly proportional；But when object to be measured antibody concentration is excessively high, part test antibodies with single antigen binding, cause double respectively
Antigen sandwich compound is reduced, and optical signal is relatively low, cannot reflect the actual concentration of object to be measured antibody.
Method of the invention is optionally wherein read twice after repeatedly reading, and reading gained signal value increases more twice
Relationship between width, so as to play the role of widening detection range and distinguish HD-HOOK effect sample.The difference read twice
It is different to be determined by following three aspects:
In a first aspect, after donor is irradiated by red laser (600~700nm), releasing singlet oxygen when reading for the first time
Ion.After a part of singlet oxygen ion transport to receptor, by a series of chemical reaction, launch 520~620nm high energy
The light of grade；And a part of singlet oxygen ion is then anti-with the object to be measured antigen (or antibody) that is not combined by antibody (or antigen)
It answers, so that the concentration of object to be measured antigen (or antibody) reduces.For the sample of low concentration, object to be measured antigen (or antibody) is dense
After degree decline, double antibodies sandwich compound is reduced, and second of read signal value can reduce；And for high concentration sample, object to be measured
After antigen (or antibody) concentration reduces, double antibodies sandwich compound increases, and second of read signal value increases instead.
Second aspect, for low concentration sample, donor in first time reading process by red laser (600~
It 700nm) irradiates, after discharging singlet oxygen ion, energy is lost, and second of read signal can reduce.
The third aspect, for HD-HOOK effect, when reading for the first time, balance, In is had not yet been reached in antigen-antibody reaction
The interval time read twice, reaction can still be carried out towards positive direction, and second of read signal can increase.
III, embodiment
To keep the present invention easier to understand, below in conjunction with embodiment, present invention be described in more detail, these realities
Apply example only serve it is illustrative, it is not limited to application range of the invention.If raw material used in the present invention or component nothing
Specified otherwise can be made by commercial sources or conventional method.
Embodiment 1: chemiluminescence analysis POCT detection device
Chemiluminescence immune assay POCT detection device is developed by Bo Yang biotechnology (Shanghai) Co., Ltd. comprising:
Reagent pipetting volume module is used for the sample to be tested of the doubtful molecule containing object to be measured and chemiluminescence reaction institute occurs
Reaction forms testing mixture after the reagent mixing needed；
Reaction module is used to that chemiluminescence reaction to occur for testing mixture obtained in reagent pipetting volume module to provide conjunction
Suitable temperature environment；
Detection module is used to record chemiluminescent signal value described in n times；Wherein, the chemiluminescence letter of n-th record
Number value is denoted as reading RLUn；And any signal value twice in the chemiluminescence signal value of the n times record is chosen, it is denoted as respectively
RLUm and reading RLUk are read, and the difference amplification of RLUm and RLUk is denoted as A, amplification A=(RLUm/RLUk-1) × 100%；
With
Processor module is used for according to a series of standard substance of the molecule containing object to be measured of known concentrations and any
The difference amplification A ' of the reading RLUm ' and RLUk ' of two secondary responses do standard curve；The amplification A is compared with standard curve
Compared with to determine the concentration of sample；
Wherein, n, m and k are the natural number greater than 0, and k < m≤n, n >=2.
Described device further includes light excitation module, is used for the successive t excitation testing mixture and chemiluminescence occurs,
Wherein, t is natural number greater than 0, and n≤t.
Described device further includes the reagent card being used cooperatively, and sample to be tested hole, donating agent are offered on the reagent card
Hole and receptor agents hole；The sample to be tested hole is used to contain sample to be tested, and the donating agent hole is used to contain donating agent,
The receptor agents hole is used to contain receptor agents；
When detecting, the reagent card is arranged in the POCT detection device, under the control of circuit control module, institute
Reaction module is stated for adjusting the temperature of substance in the reagent card and reagent card, the reagent pipetting volume module is described for shifting
Substance in reagent card, the smooth excitation module are used for successive t transmitting laser, and the detection module is for recording described in n times
Chemiluminescent signal value；Wherein, the chemiluminescence signal value of n-th record is denoted as reading RLUn；And choose the n times record
Chemiluminescence signal value in any signal value twice, be denoted as reading RLUm and reading RLUk respectively, and by RLUm and RLUk
Difference amplification be denoted as A；The processor module according to a series of standard substance of the molecule containing object to be measured of known concentrations with
And the difference amplification A ' of the reading RLUm ' and RLUk ' of any two secondary response do standard curve；By the amplification A and standard curve
It is compared, to determine the concentration of sample；
Wherein, t, n, m and k are the natural number greater than 0, and k < m≤n≤t, n >=2.
Dilution fluid apertures is also provided on the reagent card, the dilution fluid apertures is used to contain dilution；The sample to be tested
Hole, donating agent hole, receptor agents hole and the dilution equal overlay film closed orifices of fluid apertures.
It is additionally provided with bar code area on the reagent card, bar code is equipped in the bar code area, the bar code is one-dimensional
Code.
Described device further includes bar code scanning module, and the bar code scanning module reads the letter in bar code for identification
Breath.The bar code scanning module supports IC card scanning, printed bar code medium (paper or reagent card) scanning, and information, which is read in, to be used
Contact type scanning either non-contact scanning, mode can be the modes such as infrared or radio frequency；The information includes but unlimited
In detection and analysis project name, standard curve, reagent component, lot number, effect phase, manufacturer's information.
The shape of the reagent card is circular single part reagent card.
Embodiment 2: conventional method and the method for the present invention detect alpha-fetoprotein (AFP) sample respectively
The present embodiment uses alpha-fetoprotein (AFP) detection kit of Bo Yang biotechnology (Shanghai) Co., Ltd. production
(chemoluminescence method) detects the content of alpha-fetoprotein in sample.POCT detection device is by rich positive biotechnology (Shanghai) limited public affairs
Department's exploitation.
The alpha-fetoprotein antigen of high concentration is subjected to gradient dilution, common detection methods and detection side of the invention are respectively adopted
Method measures the signal value of the sample of the alpha-fetoprotein containing various concentration.
Common detection methods the following steps are included:
A, by the determinand sample of known concentration with reagent 1 (receptor solution in conjunction with mouse monoclonal antibody) and reagent 2
(the mouse monoclonal antibody solution in conjunction with biotin) mixing, 37 DEG C of incubation 10min；Then reagent 3 is added (with Streptavidin
In conjunction with donor solution), 37 DEG C of incubation 2.5min obtain reaction solution；
B, laser irradiation is carried out to the reaction solution in step A, photon counter reading reads RLU, as a result such as 1 institute of table
Show.
The step of using method of reading twice of the invention, is as follows:
A, it will know the determinand sample of concentration, reagent 1 (receptor solution in conjunction with mouse monoclonal antibody) and reagent 2 (with
The mouse monoclonal antibody solution that biotin combines), then reagent 3 is added (in conjunction with Streptavidin in 37 DEG C of incubation 10min
Donor solution), 37 DEG C of incubation 2.5min obtain reaction solution；
B, laser irradiation is carried out to the reaction solution in step A, photon counter is read for the first time, is as a result denoted as RLU1；
Then again 37 DEG C continue to incubate 7min, photon counter second is read, and is as a result denoted as RLU2, and calculate the increasing of second of signal value
Width A=(RLU2/RLU1-1) × 100%, testing result is as shown in table 1 and Fig. 1:
Table 1:
As shown in Table 1, from 50ng/ml to 51,200ng/ml, signal value increases with concentration and increases concentration, and concentration is after of continuing rising
Height, signal value are increased with alpha-fetoprotein concentration and are reduced, i.e., concentration is greater than 51,200ng/ml and (defines this concentration to turn for HD-HOOK
Point, defining its amplification is A0) then HD-HOOK, in conventional detection, antigen concentration is higher than the sample report concentration of this detection range
It will be relatively low (reported concentrations are respectively less than 51,200ng/ml).
The method of the present invention widens detection range, instruction HD-HOOK sample or super detection range sample by reading twice.
Each sample to be tested successively detects signal value result RLU1, RLU2, by the RLU amplification A=(RLU2/RLU1- of second of reading
1) × 100% one of the index as judgement sample concentration ranges.By table 1 and Fig. 1 it is found that signal value is increased to concentration is continuous
51,200ng/ml (being defined as first transition), signal value starts to increase with concentration and decline (being defined as last transition) later, but
It is amplification A is persistently to rise with concentration.It is detected to obtain RLU1, RLU2 and A of sample to be tested with the method for the present invention.
RLU2 the and A standard curve (such as Fig. 1) for making gamut (such as 0-3,276,800ng/ml), first passes through determinand
A value determines that its concentration is to substitute into its corresponding standard curve in first transition or last transition, then by the RLU2 of test substance
Calculate exact concentration.
Embodiment 3: conventional method and the method for the present invention detect human chorionic gonadotrophin and β subunit (HCG+ respectively
β) sample
The human chorionic gonadotrophin and β subunit (HCG+ produced using Bo Yang biotechnology (Shanghai) Co., Ltd.
β) detection kit (chemoluminescence method) detects the content of human chorionic gonadotrophin and β subunit in sample.POCT inspection
Device is surveyed to be developed by Bo Yang biotechnology (Shanghai) Co., Ltd..
The human chorionic gonadotrophin of high concentration and β subunit antigen are subjected to gradient dilution, conventional inspection is respectively adopted
The signal of survey method and detection method measurement human chorionic gonadotrophin containing various concentration and the sample of β subunit
Value.
Common detection methods include the following steps
A, by the determinand sample of known concentration with reagent 1 (receptor solution in conjunction with mouse monoclonal antibody) and reagent 2
(the mouse monoclonal antibody solution in conjunction with biotin) mixing, 37 DEG C of incubation 10min；Then reagent 3 is added (with Streptavidin
In conjunction with donor solution), 37 DEG C of incubation 2.5min obtain reaction solution；
B, laser irradiation is carried out to the reaction solution in step A, photon counter reading reads RLU, as a result such as 2 institute of table
Show.
The step of using method of reading twice of the invention, is as follows:
A, it will know the determinand sample of concentration, reagent 1 (receptor solution in conjunction with mouse monoclonal antibody) and reagent 2 (with
The mouse monoclonal antibody solution that biotin combines), then reagent 3 is added (in conjunction with Streptavidin in 37 DEG C of incubation 10min
Donor solution), 37 DEG C of incubation 2.5min obtain reaction solution；
B, laser irradiation is carried out to the reaction solution in step A, photon counter is read for the first time, is as a result denoted as RLU1；
Then again 37 DEG C continue to incubate 7min, photon counter second is read, and is as a result denoted as RLU2, and calculate the increasing of second of signal value
Width A=(RLU2/RLU1-1) × 100%, testing result as shown in table 2 and figure 2:
Table 2:
As shown in Table 2, concentration from 100mIU/ml to 102,400mIU/ml signal value with concentration increase and increase, concentration after
Height of continuing rising, signal value are increased with human chorionic gonadotrophin and β subunit concentration and are reduced, i.e., concentration is greater than 102,
(define this concentration is HD-HOOK inflection point to 400mIU/ml, and defining its amplification is A0) then HD-HOOK, in conventional detection, antigen
The sample report concentration that concentration is higher than this detection range will be relatively low (reported concentrations are respectively less than 102,400mIU/ml).
The method of the present invention widens detection range, instruction HD-HOOK sample or super detection range sample by reading twice.
Each sample to be tested successively detects signal value result RLU1, RLU2, by the RLU amplification A=(RLU2/RLU1- of second of reading
1) × 100% one of the index as judgement sample concentration ranges.By table 2 and Fig. 2 it is found that signal value is increased to concentration is continuous
51,200mIU/ml (being defined as first transition), signal value starts to increase with concentration and decline (being defined as last transition) later,
But amplification A is persistently to rise with concentration.It is detected to obtain RLU1, RLU2 and A of sample to be tested with the method for the present invention.
RLU2 the and A standard curve (such as Fig. 2) for making gamut (such as 0-6,553,600mIU/ml), first passes through determinand A
Value determines that its concentration is to substitute into its corresponding standard curve meter in first transition or last transition, then by the RLU2 of test substance
Calculate exact concentration.
Embodiment 4: conventional method and the method for the present invention detect ferritin (Ferr) sample respectively
Ferritin (Ferr) detection kit (chemiluminescence produced using Bo Yang biotechnology (Shanghai) Co., Ltd.
Method) detect the content of ferritin in sample.POCT detection device is developed by Bo Yang biotechnology (Shanghai) Co., Ltd..
The ferritin antigen of high concentration is subjected to gradient dilution, common detection methods and detection method are respectively adopted
Measure the signal value of the sample of the ferritin containing various concentration.
Common detection methods the following steps are included:
A, by the determinand sample of known concentration with reagent 1 (receptor solution in conjunction with mouse monoclonal antibody) and reagent 2
(the mouse monoclonal antibody solution in conjunction with biotin) mixing, 37 DEG C of incubation 10min；Then reagent 3 is added (with Streptavidin
In conjunction with donor solution), 37 DEG C of incubation 2.5min obtain reaction solution；
B, laser irradiation is carried out to the reaction solution in step A, photon counter reading reads RLU, as a result such as 3 institute of table
Show.
The step of using method of reading twice of the invention, is as follows:
A, it will know the determinand sample of concentration, reagent 1 (receptor solution in conjunction with mouse monoclonal antibody) and reagent 2 (with
The mouse monoclonal antibody solution that biotin combines), then reagent 3 is added (in conjunction with Streptavidin in 37 DEG C of incubation 10min
Donor solution), 37 DEG C of incubation 2.5min obtain reaction solution；
B, laser irradiation is carried out to the reaction solution in step A, photon counter is read for the first time, is as a result denoted as RLU1；
Then again 37 DEG C continue to incubate 7min, photon counter second is read, and is as a result denoted as RLU2, and calculate the increasing of second of signal value
Width A=(RLU2/RLU1-1) × 100%, testing result as shown in table 3 and figure 3:
Table 3:
As shown in Table 3, from 50ng/ml to 51,200ng/ml, signal value increases with concentration and increases concentration, and concentration is after of continuing rising
Height, signal value are increased with ferritin levels and are reduced, i.e., concentration is greater than 51,200ng/ml and (defines this concentration to turn for HD-HOOK
Point, defining its amplification is A0) then HD-HOOK, in conventional detection, antigen concentration is higher than the sample report concentration of this detection range
It will be relatively low (reported concentrations are respectively less than 51,200ng/ml).
The method of the present invention widens detection range, instruction HD-HOOK sample or super detection range sample by reading twice.
Each sample to be tested successively detects signal value result RLU1, RLU2, by the RLU amplification A=(RLU2/RLU1- of second of reading
1) × 100% one of the index as judgement sample concentration ranges.By table 3 and Fig. 3 it is found that signal value is increased to concentration is continuous
51,200ng/ml (being defined as first transition), signal value starts to increase with concentration and decline (being defined as last transition) later, but
It is amplification A is persistently to rise with concentration.It is detected to obtain RLU1, RLU2 and A of sample to be tested with the method for the present invention.
RLU2 the and A standard curve (such as Fig. 3) for making gamut (such as 0-3,276,800ng/ml), first passes through determinand A
Value determines that its concentration is to substitute into its corresponding standard curve meter in first transition or last transition, then by the RLU2 of test substance
Calculate exact concentration.
Embodiment 5: conventional method and the method for the present invention detect human immune defect virus antibody (anti-HIV) sample respectively
This
Using human immune defect virus antibody (anti-HIV) inspection of Bo Yang biotechnology (Shanghai) Co., Ltd. production
Test agent box (chemoluminescence method) detects the content of human immune defect virus antibody in sample.POCT detection device is by rich sun
The exploitation of biotechnology (Shanghai) Co., Ltd..
The human immune defect virus antibody of high concentration is subjected to gradient dilution, common detection methods and Ben Fa are respectively adopted
The signal value of the bright detection method measurement sample of human immune defect virus antibody containing various concentration.
Common detection methods the following steps are included:
A, by the determinand sample of known concentration with reagent 1 (receptor solution with HIV antigen binding) and reagent 2 (with life
The HIV antigenic solution that object element combines) mixing, 37 DEG C of incubation 10min；Then (the donor in conjunction with Streptavidin of reagent 3 is added
Solution), 37 DEG C of incubation 2.5min obtain reaction solution；
B, laser irradiation is carried out to the reaction solution in step A, photon counter reading reads RLU, as a result such as 4 institute of table
Show.
The step of using method of reading twice of the invention, is as follows:
A, it will know the determinand sample of concentration, reagent 1 (receptor solution with HIV antigen binding) and reagent 2 are (with biotin
In conjunction with HIV antigenic solution), 37 DEG C of incubation 10min, then be added reagent 3 (donor solution in conjunction with Streptavidin), 37
DEG C incubate 2.5min obtain reaction solution；
B, laser irradiation is carried out to the reaction solution in step A, photon counter is read for the first time, is as a result denoted as RLU1；
Then again 37 DEG C continue to incubate 7min, photon counter second is read, and is as a result denoted as RLU2, and calculate the increasing of second of signal value
Width A=(RLU2/RLU1-1) × 100%, testing result as shown in table 4 and figure 4:
Table 4:
As shown in Table 4, from 25ng/ml to 25600ng/ml, signal value increases with concentration and increases concentration, and concentration is after of continuing rising
Height, signal value are increased with human immune defect virus antibody concentration and are reduced, i.e., concentration (defines this concentration greater than 25600ng/ml
For HD-HOOK inflection point, defining its amplification is A0) then HD-HOOK, in conventional detection, antigen concentration is higher than this detection range
Sample report concentration will be relatively low (reported concentrations are respectively less than 25600ng/ml).
The method of the present invention widens detection range, instruction HD-HOOK sample or super detection range sample by reading twice.
Each sample to be tested successively detects signal value result RLU1, RLU2, by the RLU amplification A=(RLU2/RLU1- of second of reading
1) × 100% one of the index as judgement sample concentration ranges.By table 4 and Fig. 4 it is found that signal value is increased to concentration is continuous
25600ng/ml (is defined as first transition), and signal value starts to increase with concentration and decline (being defined as last transition) later, but
It is amplification A is persistently to rise with concentration.It is detected to obtain RLU1, RLU2 and A of sample to be tested with the method for the present invention.
RLU2 the and A standard curve (such as Fig. 4) for making gamut (such as 0-1638400ng/ml), first passes through determinand A value
Determine that its concentration is to substitute into its corresponding standard curve calculating in first transition or last transition, then by the RLU2 of test substance
Exact concentration.
Embodiment 6: conventional method and the method for the present invention detect myoglobins (MYO) sample respectively
Myoglobins (MYO) detection kit (chemiluminescence produced using Bo Yang biotechnology (Shanghai) Co., Ltd.
Method) detect the content of myoglobins in sample.POCT detection device is developed by Bo Yang biotechnology (Shanghai) Co., Ltd..
The myoglobins antigen of high concentration is subjected to gradient dilution, common detection methods and detection side of the invention are respectively adopted
Method measures the signal value of the sample of the myoglobins containing various concentration.
Common detection methods the following steps are included:
A, by the determinand sample of known concentration with reagent 1 (receptor solution in conjunction with mouse monoclonal antibody) and reagent 2
(the mouse monoclonal antibody solution in conjunction with biotin) mixing, 37 DEG C of incubation 10min；Then reagent 3 is added (with Streptavidin
In conjunction with donor solution), 37 DEG C of incubation 2.5min obtain reaction solution；
B, laser irradiation is carried out to the reaction solution in step A, photon counter reading reads RLU, as a result such as 5 institute of table
Show.
The step of using method of reading twice of the invention, is as follows:
A, it will know the determinand sample of concentration, reagent 1 (receptor solution in conjunction with mouse monoclonal antibody) and reagent 2 (with
The mouse monoclonal antibody solution that biotin combines), then reagent 3 is added (in conjunction with Streptavidin in 37 DEG C of incubation 10min
Donor solution), 37 DEG C of incubation 2.5min obtain reaction solution；
B, laser irradiation is carried out to the reaction solution in step A, photon counter is read for the first time, is as a result denoted as RLU1；
Then again 37 DEG C continue to incubate 7min, photon counter second is read, and is as a result denoted as RLU2, and calculate the increasing of second of signal value
Width A=(RLU2/RLU1-1) × 100%, testing result is as shown in table 5 and Fig. 5:
Table 5:
As shown in Table 5, from 6ng/ml to 25600ng/ml, signal value increases with concentration and increases concentration, and concentration is after of continuing rising
Height, signal value are increased with human immune defect virus antibody concentration and are reduced, i.e., concentration (defines this concentration greater than 25600ng/ml
For HD-HOOK inflection point, defining its amplification is A0) then HD-HOOK, in conventional detection, antigen concentration is higher than this detection range
Sample report concentration will be relatively low (reported concentrations are respectively less than 25600ng/ml).
The method of the present invention widens detection range, instruction HD-HOOK sample or super detection range sample by reading twice.
Each sample to be tested successively detects signal value result RLU1, RLU2, by the RLU amplification A=(RLU2/RLU1- of second of reading
1) × 100% one of the index as judgement sample concentration ranges.By table 5 and Fig. 5 it is found that signal value is increased to concentration is continuous
25600ng/ml (is defined as first transition), and signal value starts to increase with concentration and decline (being defined as last transition) later, but
It is amplification A is persistently to rise with concentration.It is detected to obtain RLU1, RLU2 and A of sample to be tested with the method for the present invention.
RLU2 the and A standard curve (such as Fig. 5) for making gamut (such as 0-409,600ng/ml), first passes through determinand A value
Determine that its concentration is to substitute into its corresponding standard curve calculating in first transition or last transition, then by the RLU2 of test substance
Exact concentration.
Embodiment 7: conventional method and the method for the present invention detect N-terminal atrial natriuretic peptide (NT-proBNP) sample respectively
Examination is detected using the N-terminal atrial natriuretic peptide (NT-proBNP) of Bo Yang biotechnology (Shanghai) Co., Ltd. production
Agent box (chemoluminescence method) detects the content of N-terminal atrial natriuretic peptide in sample.POCT detection device is by rich positive biotechnology
's exploitation.
The N-terminal atrial natriuretic peptide antigen of high concentration is subjected to gradient dilution, common detection methods and Ben Fa are respectively adopted
The signal value of the sample of the bright detection method measurement atrial natriuretic peptide of N-terminal containing various concentration.
Common detection methods the following steps are included:
A, by the determinand sample of known concentration with reagent 1 (receptor solution in conjunction with mouse monoclonal antibody) and reagent 2
(the mouse monoclonal antibody solution in conjunction with biotin) mixing, 37 DEG C of incubation 10min；Then reagent 3 is added (with Streptavidin
In conjunction with donor solution), 37 DEG C of incubation 2.5min obtain reaction solution；
B, laser irradiation is carried out to the reaction solution in step A, photon counter reading reads RLU, as a result such as 6 institute of table
Show.
The step of using method of reading twice of the invention, is as follows:
A, it will know the determinand sample of concentration, reagent 1 (receptor solution in conjunction with mouse monoclonal antibody) and reagent 2 (with
The mouse monoclonal antibody solution that biotin combines), then reagent 3 is added (in conjunction with Streptavidin in 37 DEG C of incubation 10min
Donor solution), 37 DEG C of incubation 2.5min obtain reaction solution；
B, laser irradiation is carried out to the reaction solution in step A, photon counter is read for the first time, is as a result denoted as RLU1；
Then again 37 DEG C continue to incubate 7min, photon counter second is read, and is as a result denoted as RLU2, and calculate the increasing of second of signal value
Width A=(RLU2/RLU1-1) × 100%, testing result is as shown in table 6 and Fig. 6:
Table 6:
As shown in Table 6, from 62.5pg/ml to 256000pg/ml, signal value increases with concentration and increases concentration, and concentration continues
It increases, signal value is increased with N-terminal atrial natriuretic peptide concentration and reduced, i.e., greater than 256000pg/ml, (define this concentration is concentration
HD-HOOK inflection point, defining its amplification is A0) then HD-HOOK, in conventional detection, antigen concentration is higher than the sample of this detection range
This report concentration will be relatively low (reported concentrations are respectively less than 256000pg/ml).
The method of the present invention widens detection range, instruction HD-HOOK sample or super detection range sample by reading twice.
Each sample to be tested successively detects signal value result RLU1, RLU2, by the RLU amplification A=(RLU2/RLU1- of second of reading
1) × 100% one of the index as judgement sample concentration ranges.By table 6 and Fig. 6 it is found that signal value is increased to concentration is continuous
256000pg/ml (is defined as first transition), and signal value starts to increase with concentration and decline (being defined as last transition) later, but
It is amplification A is persistently to rise with concentration.It is detected to obtain RLU1, RLU2 and A of sample to be tested with the method for the present invention.
RLU2 the and A standard curve (such as Fig. 6) for making gamut (such as 0-4096000pg/ml), first passes through determinand A value
Determine that its concentration is to substitute into its corresponding standard curve calculating in first transition or last transition, then by the RLU2 of test substance
Exact concentration.
Embodiment 8: conventional method and the method for the present invention detect Procalcitonin (PCT) sample respectively
Procalcitonin (PCT) detection kit (chemiluminescence produced using Bo Yang biotechnology (Shanghai) Co., Ltd.
Method) detect the content of Procalcitonin in sample.POCT detection device is developed by Bo Yang biotechnology (Shanghai) Co., Ltd..
The Procalcitonin antigen of high concentration is subjected to gradient dilution, common detection methods and detection side of the invention are respectively adopted
Method measures the signal value of the sample of the Procalcitonin containing various concentration.
Common detection methods the following steps are included:
A, by the determinand sample of known concentration with reagent 1 (receptor solution in conjunction with mouse monoclonal antibody) and reagent 2
(the mouse monoclonal antibody solution in conjunction with biotin) mixing, 37 DEG C of incubation 10min；Then reagent 3 is added (with Streptavidin
In conjunction with donor solution), 37 DEG C of incubation 2.5min obtain reaction solution；
B, laser irradiation is carried out to the reaction solution in step A, photon counter reading reads RLU, as a result such as 7 institute of table
Show.
The step of using method of reading twice of the invention, is as follows:
A, it will know the determinand sample of concentration, reagent 1 (receptor solution in conjunction with mouse monoclonal antibody) and reagent 2 (with
The mouse monoclonal antibody solution that biotin combines), then reagent 3 is added (in conjunction with Streptavidin in 37 DEG C of incubation 10min
Donor solution), 37 DEG C of incubation 2.5min obtain reaction solution；
B, laser irradiation is carried out to the reaction solution in step A, photon counter is read for the first time, is as a result denoted as RLU1；
Then again 37 DEG C continue to incubate 7min, photon counter second is read, and is as a result denoted as RLU2, and calculate the increasing of second of signal value
Width A=(RLU2/RLU1-1) × 100%, testing result is as shown in table 7 and Fig. 7:
Table 7:
As shown in Table 7, from 1ng/ml to 12,500ng/ml, signal value increases with concentration and increases concentration, and concentration is after of continuing rising
Height, signal value are increased with Procalcitonin concentration and are reduced, i.e., concentration is greater than 12,500ng/ml and (defines this concentration to turn for HD-HOOK
Point, defining its amplification is A0) then HD-HOOK, in conventional detection, antigen concentration is higher than the sample report concentration of this detection range
It will be relatively low (reported concentrations are respectively less than 12,500ng/ml).
The method of the present invention widens detection range, instruction HD-HOOK sample or super detection range sample by reading twice.
Each sample to be tested successively detects signal value result RLU1, RLU2, by the RLU amplification A=(RLU2/RLU1- of second of reading
1) × 100% one of the index as judgement sample concentration ranges.By table 7 and Fig. 7 it is found that signal value is increased to concentration is continuous
12,500ng/ml (being defined as first transition), signal value starts to increase with concentration and decline (being defined as last transition) later, but
It is amplification A is persistently to rise with concentration.It is detected to obtain RLU1, RLU2 and A of sample to be tested with the method for the present invention.
RLU2 the and A standard curve (such as Fig. 7) for making gamut (such as 0-312,500ng/ml), first passes through determinand A value
Determine that its concentration is to substitute into its corresponding standard curve calculating in first transition or last transition, then by the RLU2 of test substance
Exact concentration.
Embodiment 9: conventional method and the method for the present invention detect Troponin I (cTnI) sample respectively
Using Troponin I (cTnI) detection kit (chemistry hair of Bo Yang biotechnology (Shanghai) Co., Ltd. production
Light method) detect the content of Troponin I in sample.POCT detection device is opened by Bo Yang biotechnology (Shanghai) Co., Ltd.
Hair.
The Troponin I antigen of high concentration is subjected to gradient dilution, common detection methods and present invention detection are respectively adopted
Method measures the signal value of the sample of the Troponin I containing various concentration.
Common detection methods the following steps are included:
A, by the determinand sample of known concentration with reagent 1 (receptor solution in conjunction with mouse monoclonal antibody) and reagent 2
(the mouse monoclonal antibody solution in conjunction with biotin) mixing, 37 DEG C of incubation 10min；Then reagent 3 is added (with Streptavidin
In conjunction with donor solution), 37 DEG C of incubation 2.5min obtain reaction solution；
B, laser irradiation is carried out to the reaction solution in step A, photon counter reading reads RLU, as a result such as 8 institute of table
Show.
The step of using method of reading twice of the invention, is as follows:
A, it will know the determinand sample of concentration, reagent 1 (receptor solution in conjunction with mouse monoclonal antibody) and reagent 2 (with
The mouse monoclonal antibody solution that biotin combines), then reagent 3 is added (in conjunction with Streptavidin in 37 DEG C of incubation 10min
Donor solution), 37 DEG C of incubation 2.5min obtain reaction solution；
B, laser irradiation is carried out to the reaction solution in step A, photon counter is read for the first time, is as a result denoted as RLU1；
Then again 37 DEG C continue to incubate 7min, photon counter second is read, and is as a result denoted as RLU2, and calculate the increasing of second of signal value
Width A=(RLU2/RLU1-1) × 100%, testing result is as shown in table 8 and Fig. 8:
Table 8:
As shown in Table 8, from 0.2ng/ml to 500ng/ml, signal value increases with concentration and increases concentration, and concentration is after of continuing rising
Height, signal value are increased with Troponin I concentration and are reduced, i.e., concentration is greater than 500ng/ml and (defines this concentration to turn for HD-HOOK
Point, defining its amplification is A0) then HD-HOOK, in conventional detection, antigen concentration is higher than the sample report concentration of this detection range
It will be relatively low (reported concentrations are respectively less than 500ng/ml).
The method of the present invention widens detection range, instruction HD-HOOK sample or super detection range sample by reading twice.
Each sample to be tested successively detects signal value result RLU1, RLU2, by the RLU amplification A=(RLU2/RLU1- of second of reading
1) × 100% one of the index as judgement sample concentration ranges.By table 8 and Fig. 8 it is found that signal value is increased to concentration is continuous
500ng/ml (is defined as first transition), and signal value starts to increase with concentration and decline (being defined as last transition) later, still
Amplification A is persistently to rise with concentration.It is detected to obtain RLU1, RLU2 and A of sample to be tested with the method for the present invention.
RLU2 the and A standard curve (such as Fig. 8) for making gamut (such as 0-62,500ng/ml), it is true to first pass through determinand A value
Its fixed concentration is to substitute into its corresponding standard curve calculating really in first transition or last transition, then by the RLU2 of test substance
Cut concentration.
It should be noted that embodiment described above for explaining only the invention, is not constituted to of the invention any
Limitation.By referring to exemplary embodiments, invention has been described, it should be appreciated that word used in it is descriptive
With explanatory vocabulary, rather than limited vocabulary.The present invention can be made within the scope of the claims by regulation
Modification, and the present invention is revised in without departing substantially from scope and spirit of the present invention.Although the present invention described in it relates to
And specific method, material and embodiment, it is not intended that the present invention is limited to particular case disclosed in it, on the contrary, this hair
It is bright to can be extended to other all methods and applications with the same function.
Claims (26)
1. a kind of chemiluminescence analysis POCT detection device comprising:
Reagent pipetting volume module is used for the sample to be tested of the doubtful molecule containing object to be measured and occurs needed for chemiluminescence reaction
Reaction forms testing mixture after reagent mixing；
Reaction module is used to that chemiluminescence reaction to occur for testing mixture obtained in reagent pipetting volume module to provide suitably
Temperature environment；
Detection module is used to record chemiluminescent signal value described in n times；Wherein, the chemiluminescence signal value of n-th record
It is denoted as reading RLUn；And any signal value twice in the chemiluminescence signal value of the n times record is chosen, it is denoted as reading respectively
RLUm and reading RLUk, and the difference amplification of RLUm and RLUk is denoted as A, amplification A=(RLUm/RLUk-1) × 100%；With
Processor module, be used for according to the standard substance of the molecule containing object to be measured of known concentrations a series of and it is any twice
The difference amplification A ' of the reading RLUm ' and RLUk ' of reaction do standard curve；The amplification A is compared with standard curve, is come
Determine the concentration of sample；
Wherein, n, m and k are the natural number greater than 0, and k < m≤n, n >=2.
2. the apparatus according to claim 1, which is characterized in that described device further includes light excitation module, is used for successive t
Chemiluminescence occurs for the secondary excitation testing mixture, wherein t is the natural number greater than 0, and n≤t.
3. device according to claim 1 or 2, which is characterized in that described device further includes the reagent card being used cooperatively, institute
It states and offers sample to be tested hole, donating agent hole and receptor agents hole on reagent card；The sample to be tested hole is used to contain to be measured
Sample, the donating agent hole are used to contain donating agent, and the receptor agents hole is used to contain receptor agents；
When detecting, the reagent card is arranged in the POCT detection device, described anti-under the control of circuit control module
Answer module for adjusting the temperature of substance in the reagent card and reagent card, the reagent pipetting volume module is for shifting the reagent
Substance in card, the smooth excitation module are used for successive t transmitting laser, and the detection module is for recording chemistry described in n times
Luminous signal value；Wherein, the chemiluminescence signal value of n-th record is denoted as reading RLUn；And choose the change of the n times record
Any signal value twice in luminous signal value is learned, is denoted as reading RLUm and reading RLUk respectively, and by the difference of RLUm and RLUk
Value amplification is denoted as A；The processor module is according to the standard substance of the molecule containing object to be measured of known concentrations a series of and appoints
The difference amplification A ' of reading RLUm ' and RLUk ' of two secondary responses of anticipating does standard curve；The amplification A and standard curve are carried out
Compare, to determine the concentration of sample；
Wherein, t, n, m and k are the natural number greater than 0, and k < m≤n≤t, n >=2.
4. device according to claim 3, which is characterized in that dilution fluid apertures is also provided on the reagent card, it is described dilute
Fluid apertures is released to be used to contain dilution；The sample to be tested hole, donating agent hole, receptor agents hole and the equal overlay film closing of dilution fluid apertures
Aperture.
5. device according to claim 3 or 4, which is characterized in that be additionally provided with bar code area, the item on the reagent card
Bar code is equipped in shape code area.
6. device according to claim 5, which is characterized in that described device further includes bar code scanning module, the bar code
Scan module reads the information in bar code for identification.
7. the side that a kind of device using as described in any one of claim 1-6 carries out chemiluminescence analysis POCT detection
Method comprising following steps:
(1) shape is reacted after mixing the sample to be tested of the doubtful molecule containing object to be measured with reagent needed for generation chemiluminescence reaction
At testing mixture；
(2) successively chemiluminescence occurs for the t excitation testing mixture, and n times record the chemiluminescent signal value；Its
In, the chemiluminescence signal value of n-th record is denoted as reading RLUn；
(3) any signal value twice in the chemiluminescence signal value of the n times record is chosen, is denoted as reading RLUm and reading respectively
Number RLUk, and the difference amplification of RLUm and RLUk is denoted as A；
(4) appoint according to a series of standard substance of the molecule containing object to be measured of known concentrations and its in step (2) and step (3)
The difference amplification A ' of reading RLUm ' and RLUk ' of two secondary responses of anticipating does standard curve；
(5) it is compared by the A value with standard curve, to determine the concentration of sample；
Wherein, t, n, m and k are the natural number greater than 0, and k < m≤n≤t, n >=2.
8. the method according to the description of claim 7 is characterized in that the amplification A=(RLUm/RLUk-1) × 100%.
9. method according to claim 7 or 8, which is characterized in that the n is greater than 2.
10. the method according to any one of claim 7-9, which is characterized in that in step (1), the chemiluminescence
Reaction is homogeneous chemistry luminescence-producing reaction.
11. method according to any one of claims of claim 7-10, which is characterized in that in step (1), the generation chemistry
Reagent needed for luminescence-producing reaction includes receptor agents and donating agent；Wherein:
It include donor in the donating agent, the donor can generate singlet oxygen under excited state；
It include receptor in the receptor agents, the receptor can be reacted with singlet oxygen generates detectable chemiluminescence signal
Value.
12. according to the method for claim 11, which is characterized in that the receptor is filled with luminophor and group of the lanthanides member
The high molecular particle of plain compound.
13. according to the method for claim 12, which is characterized in that the luminophor is selected from olefin(e) compound, preferably
Selected from thioxene, compound of di-butanedione, dioxine, enol ether, enamine, 9- alkylidene xanthane, 9- alkylene
Base-N-9,10 acridans, aryl ether alkene, Aryimidazole and lucigenin and their derivative, are more preferably selected from two
Methylthiophene and its derivative.
14. method according to claim 12 or 13, which is characterized in that the lanthanide compound is europium complex.
15. according to the method for claim 11, which is characterized in that the receptor includes olefin(e) compound and metal-chelating
Object is non-particulate forms, and solvable in water-bearing media.
16. method described in any one of 1-15 according to claim 1, which is characterized in that the receptor and object to be measured point
Sub first specific junction mixture either directly or indirectly combines.
17. method described in any one of 1-16 according to claim 1, which is characterized in that the donor is filled with photosensitive
The high molecular particle of compound can produce singlet oxygen under red laser excitation.
18. according to the method for claim 17, which is characterized in that the Photoactive compounds be selected from methylene blue, rose-red,
One of Bu Lin and phthalocyanine.
19. method described in any one of 1-18 according to claim 1, which is characterized in that the donor and marker are direct
Ground combines indirectly.
20. the method according to any one of claim 7-19, which is characterized in that in step (1), the generation chemistry
Reagent needed for luminescence-producing reaction further includes object to be measured molecule the second specific junction mixture reagent；Preferably, the object to be measured point
Sub second specific junction mixture is either directly or indirectly combined with marker specific junction mixture.
21. according to the method for claim 20, which is characterized in that in step (1), first by the to be measured of the molecule containing object to be measured
Sample is mixed with receptor agents and object to be measured molecule the second specific junction mixture reagent, then again mixes it with donating agent
It closes.
22. the method according to any one of claim 7-21, which is characterized in that in step (2), using energy and/
Or reactive compound excites the testing mixture that chemiluminescence occurs；Preferably, it is shone with the red exciting light of 600~700nm
Penetrating testing mixture excites it that chemiluminescence occurs.
23. the method according to any one of claim 7-22, which is characterized in that in step (2), record the chemistry
The Detection wavelength of luminous signal value is 520~620nm.
24. the method according to any one of claim 7-23, which is characterized in that the object to be measured molecule is antigen
Or antibody；Wherein, the antigen refers to that the substance with immunogenicity, the antibody refer to that capable of identifying for body generation is specific outer
Carry out the immunoglobulin of object.
25. the method according to any one of claim 7-24, which is characterized in that the standard substance is positive right
According to.
26. the method according to any one of claim 7-21, which is characterized in that the method specifically includes following step
It is rapid:
(a1) first time incubation is carried out after mixing the doubtful sample to be tested containing determined antigen (or antibody) with receptor agents；Again will
The first step incubates gained mixed liquor and mixes with donating agent, forms testing mixture after second of incubation；
(a2) chemiluminescence, n times record are occurred with the red exciting light of the 600~700nm successive t excitation testing mixture
The chemiluminescent signal value, Detection wavelength are 520~620nm；Wherein, the chemiluminescence signal value of n-th record is just remembered
To read RLUn；
(a3) choose any signal value twice in the chemiluminescence signal value of n times record, be denoted as respectively reading RLUm and
RLUk is read, and the difference amplification of RLUm and RLUk is denoted as A, amplification A=(RLUm/RLUk-1) × 100%；
(a4) according to a series of positive reference substance of the molecule containing object to be measured of known concentrations and its in step (a2) and step
(a3) the difference amplification A ' of the reading RLUm ' and RLUk ' of any two secondary response do standard curve in；
(a5) determine that object to be measured molecular concentration is the first transition in standard curve either in descending area according to the A value
Between, then the RLU1 of object to be measured molecule is substituted into its corresponding standard curve and calculates concentration；
Wherein, t, n, m and k are the natural number greater than 0, and k < m≤n≤t, n >=2.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116148459A (en) * | 2023-01-09 | 2023-05-23 | 浙江宝太智能科技有限公司 | Reading compensation method for chemiluminescent reader |
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| CN116148459B (en) * | 2023-01-09 | 2024-02-23 | 浙江宝太智能科技有限公司 | Reading compensation method for chemiluminescent reader |
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| CB02 | Change of applicant information | ||
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Address after: 201210 floor 3 and 5, building 1, No. 88, Cailun Road, free trade zone, Pudong New Area, Shanghai Applicant after: Boyang Biotechnology (Shanghai) Co., Ltd. Applicant after: Kemei Diagnostic Technology Co., Ltd Address before: 201210 Pudong New Area, Shanghai, Zhangjiang Cai Lun Road, building 2, 3 floor, east 2. Applicant before: Boyang Biotechnology (Shanghai) Co., Ltd. Applicant before: Beijing Kemei Biological Technology Co., Ltd. |
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| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB02 | Change of applicant information | ||
| CB02 | Change of applicant information |
Address after: 200131 3rd and 5th floors, building 1, No.88 Cailun Road, Pudong New Area pilot Free Trade Zone, Shanghai Applicant after: Kemei Boyang diagnostic technology (Shanghai) Co., Ltd Applicant after: Kemei Diagnostic Technology Co., Ltd Address before: 201210 the third and fifth floors of Building 1, No. 88, Cailun Road, Pudong New Area, Shanghai Applicant before: Boyang Biotechnology (Shanghai) Co., Ltd Applicant before: Kemei Diagnostic Technology Co., Ltd |