Chemiluminescence intensifier and chemiluminescence immune detection reagent kit
Technical field
The present invention relates to technical field of analysis and detection more particularly to a kind of chemiluminescence intensifier and chemiluminescence immunoassay to examine
Test agent box.
Background technique
Chemiluminescence immune assay (Chemiluminescence Immunoassay, CLIA) is by chemiluminescence or life
Object luminescence system is combined with immune response, for detecting a kind of novel markings immunoassay of trace antigen or antibody.
Chemiluminescence immune assay is the immunoassay technology of most mainstream in current world wide, have high sensitivity, detection range it is wide,
Reaction time is short, full automatic working, favorable reproducibility, it is pollution-free the advantages that, be after putting immune technology, enzyme immune technology, fluorescence immunoassay skill
A kind of Micrometry of the hypersensitivity to grow up after art.Chemiluminescent principle is when ground state molecule absorptionization
The energy jump discharged in reaction is learned to excitation state, the molecule in excitation state generates when returning to ground state in the form of light radiation
Light.Chemiluminescence immune assay combines chemiluminescence with immunoassay method, combines chemiluminescent high sensitivity
It is highly selective with immunoassay.
In chemiluminescence immune assay, common luminescent substance includes luminol, different luminol, 3- (2- spiral Buddha's warrior attendant
Alkane) -4- methoxyl group -4- (3- phosphorus oxygen acyl)-phenyl -1,2- dioxane disodium salt (being abbreviated as AMPPD), tris (bipyridine) ruthenium and
Acridine luminescent substance.Wherein, different luminol, tris (bipyridine) ruthenium and acridine luminescent substance can be used as tracer molecule and directly mark
Note, belongs to flash type chemiluminescent substance.Luminol and AMPPD rely on peroxidase and alkaline phosphatase as tracer respectively
Molecular labeling belongs to enzymatic light emitting-type chemiluminescent substance.The luminescence-producing reaction of acridine class luminescent substance, which has, is not required to catalyst,
As long as can be carried out in alkaline environment, and luminescence-producing reaction is rapid, background luminescence is low, and signal-to-noise ratio is high.Luminescence-producing reaction interference because
The advantages that element is few, and reagent stability is good, and chemical luminous system is simple, and exciting liquid is at low cost, is ideal chemiluminescent substance.
Traditional acridine Full-automatic chemiluminescence system mainly uses hydrogen peroxide and acid solution and alkaline solution
As the starting reagent that shines, reinforcing agent Triton X-100 (Triton is added in the starting reagent that shines in some for cooperation
X-100 it) is shone with Tween-20 (Tween 20) with enhancing, but traditional reinforcing agent enhances luminous effect not after being added
Obviously.In conclusion the luminous intensity of traditional acridine luminescent substance is weaker.
Summary of the invention
Based on this, it is necessary to provide a kind of chemiluminescence intensifier of luminous intensity that acridine luminescent substance can be enhanced
And chemiluminescence immune detection reagent kit.
A kind of chemiluminescence intensifier, for enhancing the luminous intensity of acridine luminescent substance, the chemiluminescent enhancement
Agent includes betaines compound.
In one embodiment, the structural formula of the betaines compound are as follows:
Wherein, R-For anionic group, R1For C5~C20Alkylamidoalkyl, C5~C20Alkyl, C2~C10Alkenyl or
C2~C10Alkynyl, R2For C1~C10Alkyl, C1~C10Alkoxy, C2~C10Alkenyl or C2~C10Alkynyl, R3For C1
~C10Alkyl, C1~C10Alkoxy, C2~C10Alkenyl or C2~C10Alkynyl.
In one embodiment, the structural formula of the betaines compound are as follows:
Wherein, R1For C5~C20Alkylamidoalkyl, C5~C20Alkyl, C2~C10Alkenyl or C2~C10Alkynyl, R2
For C1~C10Alkyl, C1~C10Alkoxy, C2~C10Alkenyl or C2~C10Alkynyl, R3For C1~C10Alkyl, C1~
C10Alkoxy, C2~C10Alkenyl or C2~C10Alkynyl.
In one embodiment, the structural formula of the betaines compound are as follows:
Wherein, R1For C5~C20Alkylamidoalkyl, C5~C20Alkyl, C2~C10Alkenyl or C2~C10Alkynyl, R2
For C1~C10Alkyl, C1~C10Alkoxy, C2~C10Alkenyl or C2~C10Alkynyl, R3For C1~C10Alkyl, C1~
C10Alkoxy, C2~C10Alkenyl or C2~C10Alkynyl.
In one embodiment, the structural formula of the betaines compound are as follows:
Wherein, R1For C5~C20Alkylamidoalkyl, C5~C20Alkyl, C2~C10Alkenyl or C2~C10Alkynyl, R2
For C1~C10Alkyl, C1~C10Alkoxy, C2~C10Alkenyl or C2~C10Alkynyl, R3For C1~C10Alkyl, C1~
C10Alkoxy, C2~C10Alkenyl or C2~C10Alkynyl.
In one embodiment, the structural formula of the betaines compound are as follows:
Wherein, R1For C5~C20Alkylamidoalkyl, C5~C20Alkyl, C2~C10Alkenyl or C2~C10Alkynyl, R2
For C1~C10Alkyl, C1~C10Alkoxy, C2~C10Alkenyl or C2~C10Alkynyl, R3For C1~C10Alkyl, C1~
C10Alkoxy, C2~C10Alkenyl or C2~C10Alkynyl.
In one embodiment, the structural formula of the betaines compound are as follows:
Wherein, R1For C5~C20Alkylamidoalkyl, C5~C20Alkyl, C2~C10Alkenyl or C2~C10Alkynyl, R2
For C1~C10Alkyl, C1~C10Alkoxy, C2~C10Alkenyl or C2~C10Alkynyl, R3For C1~C10Alkyl, C1~
C10Alkoxy, C2~C10Alkenyl or C2~C10Alkynyl.
A kind of chemiluminescence immune detection reagent kit, including chemiluminescence intensifier described in any of the above embodiments and acridine
Class luminescent substance.
In one embodiment, the acridine luminescent substance is acridinium ester or acridinium salt.
It in one embodiment, further include chemiluminescence preexciting liquid and exciting liquid, the chemiluminescence preexciting liquid
Including HNO3And H2O2Solution, exciting liquid include NaOH solution.
Betaines compound is with quatemay ammonium inner salt structure or to make the compound of ammonium structure, is commonly used for osmotic pressure tune
Save agent, detergent or fungicide etc..Inventors be surprised to learn that betaines compound has the hair of enhancing acridine luminescent substance
The effect of luminous intensity can be used for preparing chemiluminescence intensifier.Experiment shows that acridine can be remarkably reinforced in the betaines compound
The luminous intensity of class luminescent substance, and the multiple enhanced is higher than traditional reinforcing agent Triton X-100 and Tween 20.
Detailed description of the invention
Fig. 1 is to be added to contain myristamide propyl hydroxypropyl using the measurement of 3000 Full-automatic chemiluminescence apparatus of iFlash
The intensity profile of light emission figure of acridinium ester after the chemiluminescence intensifier of sulfobetaines;
Fig. 2 is to measure in no reinforcing agent using using 3000 Full-automatic chemiluminescence apparatus of iFlash and be separately added into chemistry
Acridinium salt shines under the conditions of luminescence enhancer and Architect Trigger Solution (Abbott) commodity luminescent solution
Intensity.
Specific embodiment
In order to make the foregoing objectives, features and advantages of the present invention clearer and more comprehensible, with reference to the accompanying drawing and specific real
Applying example, specific embodiments of the present invention will be described in detail.Be explained in the following description many details in order to
Fully understand the present invention.But the invention can be embodied in many other ways as described herein, art technology
Personnel can do similar improvement without violating the connotation of the present invention, therefore the present invention is not by following public specific implementation
Limitation.
The chemiluminescence intensifier of one embodiment, the chemiluminescence intensifier include betaines compound, the chemistry
Luminescence enhancer can be used for enhancing the luminous intensity of acridine luminescent substance.Certainly, chemiluminescence intensifier can also include molten
Solvent or acceptable impurity of solution betaines compound etc..
Betaines compound is with quatemay ammonium inner salt structure or to make the compound of ammonium structure.Acridine luminescent substance
It is a kind of chemical substance that can be used as chemiluminescent labels, such as acridinium ester or acridinium salt etc..
Specifically, the structural formula of betaines compound are as follows:
Wherein, R-For anionic group, R1For C5~C20Alkylamidoalkyl, C5~C20Alkyl, C2~C10Alkenyl or
C2~C10Alkynyl, R2For C1~C10Alkyl, C1~C10Alkoxy, C2~C10Alkenyl or C2~C10Alkynyl, R3For C1
~C10Alkyl, C1~C10Alkoxy, C2~C10Alkenyl or C2~C10Alkynyl.
R-For anionic group, betaines compound is cooperatively formed with quatemay ammonium inner salt structure.
In one embodiment, betaines compound is chemical compounds I, the structural formula of chemical compounds I are as follows:
Wherein, R1For C5~C20Alkylamidoalkyl, C5~C20Alkyl, C2~C10Alkenyl or C2~C10Alkynyl, R2
For C1~C10Alkyl, C1~C10Alkoxy, C2~C10Alkenyl or C2~C10Alkynyl, R3For C1~C10Alkyl, C1~
C10Alkoxy, C2~C10Alkenyl or C2~C10Alkynyl.I.e. in present embodiment ,-R-For-COO-。
In another embodiment, betaines compound is compound ii, the structural formula of compound ii are as follows:
Wherein, R1For C5~C20Alkylamidoalkyl, C5~C20Alkyl, C2~C10Alkenyl or C2~C10Alkynyl, R2
For C1~C10Alkyl, C1~C10Alkoxy, C2~C10Alkenyl or C2~C10Alkynyl, R3For C1~C10Alkyl, C1~
C10Alkoxy, C2~C10Alkenyl or C2~C10Alkynyl.I.e. in present embodiment ,-R-For
In another embodiment, betaines compound is compound III, the structural formula of compound III are as follows:
Wherein, R1For C5~C20Alkylamidoalkyl, C5~C20Alkyl, C2~C10Alkenyl or C2~C10Alkynyl, R2
For C1~C10Alkyl, C1~C10Alkoxy, C2~C10Alkenyl or C2~C10Alkynyl, R3For C1~C10Alkyl, C1~
C10Alkoxy, C2~C10Alkenyl or C2~C10Alkynyl.I.e. in present embodiment ,-R-For-CH2-SO3 -。
In a further embodiment, betaines compound is compounds Ⅳ, the structural formula of compounds Ⅳ are as follows:
Wherein, R1For C5~C20Alkylamidoalkyl, C5~C20Alkyl, C2~C10Alkenyl or C2~C10Alkynyl, R2
For C1~C10Alkyl, C1~C10Alkoxy, C2~C10Alkenyl or C2~C10Alkynyl, R3For C1~C10Alkyl, C1~
C10Alkoxy, C2~C10Alkenyl or C2~C10Alkynyl.I.e. in present embodiment ,-R-For-CH2-CH2-SO3 -。
In a further embodiment, betaines compound is compound V, the structural formula of compound V are as follows:
Wherein, R1For C5~C20Alkylamidoalkyl, C5~C20Alkyl, C2~C10Alkenyl or C2~C10Alkynyl, R2
For C1~C10Alkyl, C1~C10Alkoxy, C2~C10Alkenyl or C2~C10Alkynyl, R3For C1~C10Alkyl, C1~
C10Alkoxy, C2~C10Alkenyl or C2~C10Alkynyl.I.e. in present embodiment ,-R-For-CH (OH)-CH2-SO3 -。
Herein, the C to illustrate, in each structural formula5~C20Alkyl can be straight chained alkyl or branched alkyl.
C2~C10Alkenyl can be straight-chain alkenyl or branched-chain alkenyl.C2~C10Alkynyl can be straight-chain alkynyl or branch alkynyl.C1
~C10Alkyl can be straight chained alkyl or branched alkyl.C1~C10Alkoxy can be straight chained alkyl or branched alkyl.
Further, R1For C5~C20Alkylamidoalkyl, C10~C20Alkyl, R2For C1~C5Alkyl, C1~C5's
Alkoxy, C2~C5Alkenyl or C2~C5Alkynyl.R3For C1~C5Alkyl, C1~C5Alkoxy, C2~C5Alkenyl or
C2~C5Alkynyl.
Specifically, alkylamidoalkyl refers to that the group that alkyl is connect with amide groups, structural formula for example can be C5H11-
CONH-、C11H24-CONH-、C13H27- CONH- or C20H27- CONH- etc..Further, the amide groups in alkylamidoalkyl is also
It can connect C1~C10Alkyl, structural formula for example can be C13H27-CONH-(CH2)3Or C11H24-CONH-(CH2)10-
Deng.
Specifically, R1It can be C5、C6、C7、C8、C9、C10、C11、C12、C13、C14、C15、C16、C17、C18、C19Or C20Alkane
Base.R1It can also be C2、C3、C4、C5、C6、C7、C8、C9Or C10Alkenyl.Alternatively, R1It can also be C2、C3、C4、C5、C6、C7、
C8、C9Or C10Alkynyl.
Specifically, R2It can be C1、C2、C3、C4、C5、C6、C7、C8、C9Or C10Alkyl.R2It can be C1、C2、C3、C4、
C5、C6、C7、C8、C9Or C10Alkoxy.R2It can also be C2、C3、C4、C5、C6、C7、C8、C9、C10Alkenyl.Alternatively, R2May be used also
Think C2、C3、C4、C5、C6、C7、C8、C9Or C10Alkynyl.
Specifically, R3It can be C1、C2、C3、C4、C5、C6、C7、C8、C9Or C10Alkyl.R3It can be C1、C2、C3、C4、
C5、C6、C7、C8、C9Or C10Alkoxy.R3It can also be C2、C3、C4、C5、C6、C7、C8、C9、C10Alkenyl.Alternatively, R3May be used also
Think C2、C3、C4、C5、C6、C7、C8、C9Or C10Alkynyl.
Further, R1、R2And R3It can be the same or different.Work as R1、R2And R3When being respectively selected from alkenyl or alkynyl, alkene
Base or alkynyl can be in any positions in corresponding group.
Inventors be surprised to learn that betaines compound has the function of enhancing the luminous intensity of acridine luminescent substance, it can
It is used to prepare chemiluminescence intensifier.Betaines compound has the property of amphoteric surfactant, and structure is simple, ingredient list
One, difference between batch is very easy to control, effectively reduces the different shadows due to batch wise differences bring chemiluminescent enhancement effect
It rings.By the chemiluminescence intensifier, it can effectively enhance the luminous intensity of acridine luminescent substance, luminous signal is remarkably reinforced,
And favorable reproducibility, it can be widely applied to the fields such as clinical diagnosis, scientific research, environmental sanitation detection.Further, have upper
The luminous intensity of acridine luminescent substance can be enhanced at least twice or more by the compound for stating general formula structure, and the multiple of enhancing is high
In traditional reinforcing agent Triton X-100 and Tween 20.
A kind of chemiluminescence immune detection reagent kit, including above-mentioned chemiluminescence intensifier and acridine luminescent substance.
Chemiluminescence intensifier is made in a solvent in general, betaines compound can be dissolved.Solvent can for water,
Acid solution or alkaline solution etc..In present embodiment, betaines compound is dissolved in water, chemiluminescent enhancement is made
Agent.
Specifically, acridine luminescent substance can be acridinium ester.Such as NSP-DMAE-NHS acridinium ester, NSP-DMAE-
HEG-NHS acridinium ester etc..
The chemiluminescence reaction procedural style of the acridine luminescent substance of one embodiment is as follows:
Wherein, the acridine compound of representation shown in formula 1, the R in 1~formula of formula 6 can be H, alkyl or alkyl sulphur
Base etc., Ar can be the group containing phenyl.Specific luminescence process are as follows: acridine compound shown in formula 1 is in acid condition
By 9 carbon atoms of hydrogen peroxide addition acridine ring, 2 compound represented of formula is obtained, was then generated under alkaline condition
Negative oxygen ion obtains 3 compound represented of formula.Later by intramolecular attack carbonyl, leaving group is left away, and is generated in unstable
Mesosome 1,2- dioxetanes alkanone (6 compound represented of formula), then the open loop of 1,2- dioxetanes alkanone generates acridone,
Photon is discharged simultaneously.
In general, chemiluminescence intensifier and acridine luminescent substance are point in chemiluminescence immune detection reagent kit
Open to set, when use, remixes.
In present embodiment, chemiluminescence immune detection reagent kit further includes chemiluminescence preexciting liquid and exciting liquid, is changed
Learning the preexciting liquid that shines includes HNO3And H2O2Solution, exciting liquid are NaOH solution.
Specifically, HNO3The concentration of solution is 0.05mol/L~0.3mol/L, H2O2The volumn concentration of solution is
0.1%~1%.The concentration of NaOH solution is 0.05mol/L~0.5mol/L.
In present embodiment, HNO3The concentration of solution is 0.1mol/L, H2O2The volumn concentration of solution is 0.5%.
The concentration of NaOH solution is 0.25mol/L.
Chemiluminescence preexciting liquid and basic exciting liquid cooperate as luminous starting reagent with chemiluminescence intensifier,
Further increase the luminous intensity of pyridine class luminescent substance.
Chemi-excitation liquid is made specifically, chemiluminescence intensifier can be added in basic exciting liquid, to improve pyridine class
The luminous intensity of luminescent substance.
Above-mentioned chemiluminescence immune detection reagent kit, including chemiluminescence intensifier and acridine luminescent substance.By beet
Alkaloid compound can effectively enhance the luminous intensity of acridine luminescent substance as chemiluminescence intensifier, and the multiple of enhancing is high
In traditional reinforcing agent Triton X-100 and Tween 20, and favorable reproducibility.And betaines compound has both sexes
The property of surfactant, structure is simple, ingredient is single, and difference between batch is very easy to control, effectively reduces due to batch wise differences
The different influences of bring chemiluminescent enhancement effect improve chemiluminescence immune detection reagent kit detection sensitivity, the examination
Agent box can be widely applied to the fields such as clinical diagnosis, scientific research, environmental sanitation detection.
The following are embodiment parts.
In following embodiment, unless otherwise instructed, test method without specific conditions, usually according to normal condition,
Experimental material betaines compound used is purchased from Henan Titaning Chemical Technology Co., Ltd..Tween20 (sigma company, goods
Number V900548) and Triton X-100 (sigma company, article No. V900502).Acridinium ester (NSP-DMAE-NHS) and acridinium salt
(NSP-SA-NHS) buying steps in Shanghai and opens up chemical industry of rivering bend.Chemiluminescent instrument is detected as 3000 full-automatic chemical of iFlash hair
Light instrument.
1~embodiment of embodiment 13
Influence of the betaines compound to the luminous intensity of acridine luminescent substance
1) preexciting liquid is configured
Contain HNO with ultrapure water configuration3And H2O2Preexciting liquid, wherein HNO3It is final concentration of in preexciting liquid
0.1mol/L, H2O2Volumn concentration of the solution in preexciting liquid is 0.5%.
2) basic exciting liquid is configured
The basic exciting liquid containing NaOH, final concentration of 0.25mol/ of the NaOH in basic exciting liquid are configured with ultrapure water
L。
3) chemi-excitation liquid is configured
Chemiluminescence intensifier is separately added into basic exciting liquid, chemiluminescence intensifier respectively includes betaines
Object Z1~Z13 is closed, chemi-excitation liquid is obtained.The original content of each betaines compound Z1~Z13 in chemiluminescence intensifier
It is 10g/L, chemiluminescence intensifier additional amount is 2 μ of μ L~30 L.10 μ L are separately added into the present embodiment contains Z1 respectively
The chemiluminescence intensifier of~Z13 obtains chemi-excitation liquid into basic exciting liquid.The glycine betaine that chemiluminescence intensifier includes
The structural formula of class compound Z1~Z13 is as follows:
Wherein, R-For anionic group, specifically there is following tetra- class of Y1~Y5, Y1~Y5 specific structure is as follows:
R1For C5~C20Alkylamidoalkyl, C5~C20Alkyl, C2~C10Alkenyl or C2~C10Alkynyl, R2For C1
~C10Alkyl, C1~C10Alkoxy, C2~C10Alkenyl or C2~C10Alkynyl, R3For C1~C10Alkyl, C1~C10
Alkoxy, C2~C10Alkenyl or C2~C10Alkynyl.The corresponding specific structure such as table of betaines compound Z1~Z13
Shown in 1.
Table 1: the title and structural formula of betaines compound Z1~Z13
4) measurement of chemiluminescence signal
Using 10 μ L, concentration for 1pg/mL acridine solution as sample, in the present embodiment acridine solution be NSP-DMAE-HEG-
NHS acridinium ester, structural formula are as follows:
100 μ L preexciting liquid are added into sample, then are separately added into each chemi-excitation liquid of 100 μ L.Divide in chemi-excitation liquid
It Han You not be in basic exciting liquid and betaines compound Z1~Z13.Chemiluminescence letter is measured on chemiluminescence detector
Number, and calculate the intensification factor of each luminescent solution signal value Yu basic luminescence liquid signal value.The corresponding luminosity curve of embodiment 5 is as schemed
Shown in 1.The results are shown in Table 2 for the luminous intensity of embodiment 1~13.
Table 2: the luminous intensity of experimental comparison group and embodiment 1~13
Wherein, for experimental comparison group in addition to chemiluminescence intensifier is not added, remaining is identical as embodiment 1~13.It can be with from table 2
Find out, the chemiluminescence intensifier containing betaines compound Z1~Z13, which is added, can effectively enhance shining for acridinium ester
Intensity.Intensification factor is at 4 times or more.
1~comparative example of comparative example 2
Further to verify influence of the betaines compound to the luminous intensity of acridinium ester.Using 20 He of Tween
Triton X-100 is as a comparison case.The step of configuring preexciting liquid in comparative example and configuring basic exciting liquid and embodiment 1~
13 is identical.The difference is that using when configuration chemi-excitation liquid respectively by the original content of 10 μ L for 20 He of Tween of 10g/L
Triton X-100 is added in basic exciting liquid and obtains the chemi-excitation liquid of comparative example.Using measurement pair on chemiluminescence detector
The chemiluminescence signal of ratio, and the intensification factor of each luminescent solution signal value Yu basic luminescence liquid signal value is calculated, as a result such as table 3
It is shown.
Table 3: the luminous intensity of experimental comparison group and comparative example 1 and 2
Contrast table 2 and the result of table 3 are sent out it is found that can play enhancing acridine compound chemistry using betaines compound
Light action, and reinforcing effect is better than Tween 20 and Triton X-100.And the chemiluminescence of the present embodiment 1~13 also shines
Signal stabilization, luminous efficiency are high.
Embodiment 14
Further to compare betaines compound to acridine compound the enhancement of chemiluminescence, select
Architect Trigger Solution (Abbott company, article No.: 6C55-60) commodity luminescent solution is as control.This implementation
The sample liquid of example is acridinium salt (NSP-SA-NHS) solution that 10 μ L concentration are 1pg/mL.Preexciting liquid and basic exciting liquid and reality
It is identical to apply example 1~13.The chemiluminescence intensifier containing Z5 of 10 μ L is added in basic exciting liquid and obtains chemi-excitation liquid.To
100 μ L preexciting liquid are added in sample, then be separately added into 100 μ L containing the corresponding chemi-excitation liquid of reinforcing agent Z5 or 100 μ L
Architect Trigger Solution (Abbott) commodity luminescent solution.It measured respectively in no reinforcing agent, had containing Z5's
Acridinium salt under the conditions of chemiluminescence intensifier and Architect Trigger Solution (Abbott) commodity luminescent solution
Luminous intensity.As a result as shown in Fig. 2, with commodity luminescent solution Architect Trigger Solution (Abbott) luminous signal
It compares.Under reinforcing agent of the present invention effect, the luminous signal of acridine is greatly enhanced, and enhancing luminescent solution performance is close
Architect Trigger Solution(Abbott)。
From embodiment 1~14 and the result of comparison 1 it is found that betaines compound has well acridine compound
The detection sensitivity of diagnostic kit can be improved in the effect of enhanced chemiluminescence.
All data, image, instrument, reagent and step herein is interpreted as illustrative and not restrictive.Although
Combined above-mentioned specific embodiment to describe the present invention, many modifications and other variations for those skilled in the art be it is aobvious and
It is clear to.All this modifications and other variations can fall within the spirit and scope of the present inventions.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
Limitations on the scope of the patent of the present invention therefore cannot be interpreted as.It should be pointed out that for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to guarantor of the invention
Protect range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.