WO2018000420A1 - 化学发光增强剂及化学发光免疫检测试剂盒 - Google Patents

化学发光增强剂及化学发光免疫检测试剂盒 Download PDF

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WO2018000420A1
WO2018000420A1 PCT/CN2016/088126 CN2016088126W WO2018000420A1 WO 2018000420 A1 WO2018000420 A1 WO 2018000420A1 CN 2016088126 W CN2016088126 W CN 2016088126W WO 2018000420 A1 WO2018000420 A1 WO 2018000420A1
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group
alkyl
chemiluminescence
alkynyl
alkenyl
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French (fr)
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胡鹍辉
王刚
夏福臻
钱纯亘
祝亮
邹定标
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深圳市亚辉龙生物科技股份有限公司
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Priority to PCT/CN2016/088126 priority Critical patent/WO2018000420A1/zh
Priority to US16/314,388 priority patent/US10745425B2/en
Publication of WO2018000420A1 publication Critical patent/WO2018000420A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/06—Phosphorus compounds without P—C bonds
    • C07F9/08—Esters of oxyacids of phosphorus
    • C07F9/141—Esters of phosphorous acids
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
    • C09K11/07—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials having chemically interreactive components, e.g. reactive chemiluminescent compositions
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/06—Phosphorus compounds without P—C bonds
    • C07F9/08—Esters of oxyacids of phosphorus
    • C07F9/09—Esters of phosphoric acids
    • C07F9/091—Esters of phosphoric acids with hydroxyalkyl compounds with further substituents on alkyl
    • 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

Definitions

  • the invention relates to the field of analytical detection technology, in particular to a chemiluminescence enhancer and a chemiluminescence immunoassay kit.
  • Chemiluminescence Immunoassay is a novel marker immunoassay that combines a chemiluminescent or bioluminescent system with an immune response for the detection of trace antigens or antibodies.
  • Chemiluminescence immunoassay is currently the most mainstream immunoassay technology in the world. It has the advantages of high sensitivity, wide detection range, short reaction time, fully automatic operation, good reproducibility and no pollution. It is a technology of release and exemption.
  • An ultra-sensitive micro-detection technique developed after fluorescent immunoassay.
  • the principle of chemiluminescence is that when the energy released by the ground state molecule absorption chemical reaction transitions to the excited state, the molecule in the excited state returns to the ground state in the form of light radiation.
  • Chemiluminescence immunoassay combines chemiluminescence with immunoassay methods to combine the high sensitivity of chemiluminescence with the high selectivity of immunoassays.
  • luminescent materials include luminol, isoluminol, 3-(2-helixadamantane)-4-methoxy-4-(3-phosphoryl)-phenyl- 1,2-dioxetane disodium salt (abbreviated as AMPPD), terpyridine pyridinium and acridine luminescent material.
  • AMPPD 1,2-dioxetane disodium salt
  • isoluminol, terpyridine pyridinium and acridine luminescent materials can be directly labeled as tracer molecules, belonging to flash type chemiluminescent substances.
  • Luminol and AMPPD rely on peroxidase and alkaline phosphatase as tracer molecular markers, respectively, and belong to enzymatic luminescent chemiluminescent substances.
  • the luminescence reaction of the acridine-based luminescent substance has no catalyst, and can be carried out in an alkaline environment, and the luminescence reaction is rapid, the background luminescence is low, and the signal-to-noise ratio is high.
  • the luminescent reaction has few interference factors, good reagent stability, simple chemiluminescence system and low cost of the excitation liquid, and is an ideal chemiluminescent substance.
  • the traditional acridine-based fully automatic chemiluminescence system mainly uses hydrogen peroxide in combination with an acidic solution and an alkaline solution as a luminescent starting reagent, and some of the luminescent starting reagent is added with a reinforcing agent polyethylene glycol octyl phenyl ether (Triton X- 100) and Tween 20 (Tween 20) to enhance luminescence, but the effect of enhancing the luminescence after the addition of the conventional enhancer is not obvious.
  • the conventional acridine luminescent material has a weak luminescence intensity.
  • a chemiluminescence enhancer for enhancing the luminescence intensity of an acridine-based luminescent material which comprises a betaine-based compound.
  • a chemiluminescence immunoassay kit comprising the above chemiluminescence enhancer and an acridine luminescent substance.
  • the betaine compound is a compound having a quaternary ammonium inner salt structure or an ammonium hydrazine structure, and is often used as an osmotic pressure regulator, a detergent or a bactericide.
  • the inventors have unexpectedly found that the betaine compound has an effect of enhancing the luminescence intensity of the acridine luminescent substance, and can be used for preparing a chemiluminescence enhancer.
  • the betaine compound can significantly enhance the luminescence intensity of acridine luminescent materials, and the enhancement ratio is higher than the traditional enhancers Triton X-100 and Tween 20.
  • Figure 1 is a graph showing the luminescence intensity of an acridinium ester after addition of a chemiluminescence enhancer containing tetradecamidopropyl hydroxypropyl sulfobetaine by an iFlash 3000 fully automated chemiluminometer;
  • Figure 2 is a graph showing the luminescence intensity of an acridine salt in the absence of an enhancer and the addition of a chemiluminescence enhancer and an Architect Trigger Solution (Abbott) commercial luminescent solution using an iFlash 3000 fully automated chemiluminometer.
  • the chemiluminescence enhancer comprising a betaine-based compound, which is useful for enhancing the luminescence intensity of an acridine-based luminescent substance.
  • the chemiluminescence enhancer may further include a solvent for dissolving the betaine compound or an acceptable impurity or the like.
  • the betaine compound is a compound having a quaternary ammonium internal salt structure or an ammonium hydrazine structure.
  • Acridine luminescent materials are a class of chemicals that can be used as chemiluminescent labels, such as acridinium esters or acridine salts.
  • R - is an anionic group
  • R 1 is an alkyl amide group of the C 5 ⁇ C 20 alkyl, C alkenyl or C 2 ⁇ C 10 alkynyl group of C 5 ⁇ C 20 of 2 ⁇ C
  • R 2 is a C 1 - C 10 alkyl group, a C 1 - C 10 alkoxy group, a C 2 - C 10 alkenyl group or a C 2 - C 10 alkynyl group
  • R 3 is a C 1 - C 10 alkane. a group, a C 1 -C 10 alkoxy group, a C 2 -C 10 alkenyl group or a C 2 -C 10 alkynyl group.
  • R - is an anionic group which is complexed with a quaternary ammonium internal salt to form a betaine compound.
  • the betaine compound is Compound I, and the structural formula of Compound I is:
  • R 1 is a C 5 - C 20 alkyl amide group, a C 5 - C 20 alkyl group, a C 2 - C 10 alkenyl group or a C 2 - C 10 alkynyl group
  • R 2 is a C 1 - C group
  • R 3 is C 1 -C 10 alkyl, C 1 -C 10 Alkoxy group, C 2 -C 10 alkenyl group or C 2 -C 10 alkynyl group. That is, in the present embodiment, -R - is -COO - .
  • betaine compound is Compound II, and the structural formula of Compound II is:
  • R 1 is a C 5 - C 20 alkyl amide group, a C 5 - C 20 alkyl group, a C 2 - C 10 alkenyl group or a C 2 - C 10 alkynyl group
  • R 2 is a C 1 - C group
  • R 3 is C 1 -C 10 alkyl, C 1 -C 10 Alkoxy group, C 2 -C 10 alkenyl group or C 2 -C 10 alkynyl group.
  • -R - is
  • betaine compound is Compound III
  • structural formula of Compound III is:
  • R 1 is a C 5 - C 20 alkyl amide group, a C 5 - C 20 alkyl group, a C 2 - C 10 alkenyl group or a C 2 - C 10 alkynyl group
  • R 2 is a C 1 - C group
  • R 3 is C 1 -C 10 alkyl, C 1 -C 10 Alkoxy group, C 2 -C 10 alkenyl group or C 2 -C 10 alkynyl group. That is, in the present embodiment, -R - is -CH 2 -SO 3 - .
  • betaine compound is compound IV, and the structural formula of compound IV is:
  • R 1 is a C 5 - C 20 alkyl amide group, a C 5 - C 20 alkyl group, a C 2 - C 10 alkenyl group or a C 2 - C 10 alkynyl group
  • R 2 is a C 1 - C group
  • R 3 is C 1 -C 10 alkyl, C 1 -C 10 Alkoxy group, C 2 -C 10 alkenyl group or C 2 -C 10 alkynyl group. That is, in the present embodiment, -R - is -CH 2 -CH 2 -SO 3 - .
  • betaine compound is compound V, and the structural formula of compound V is:
  • R 1 is a C 5 - C 20 alkyl amide group, a C 5 - C 20 alkyl group, a C 2 - C 10 alkenyl group or a C 2 - C 10 alkynyl group
  • R 2 is a C 1 - C group
  • R 3 is C 1 -C 10 alkyl, C 1 -C 10 Alkoxy group, C 2 -C 10 alkenyl group or C 2 -C 10 alkynyl group. That is, in the present embodiment, -R - is -CH(OH)-CH 2 -SO 3 - .
  • the C 5 - C 20 alkyl group in each structural formula may be a linear alkyl group or a branched alkyl group.
  • the C 2 -C 10 alkenyl group may be a linear alkenyl group or a branched alkenyl group.
  • the alkynyl group of C 2 to C 10 may be a linear alkynyl group or a branched alkynyl group.
  • the alkyl group of C 1 to C 10 may be a linear alkyl group or a branched alkyl group.
  • the alkoxy group of C 1 to C 10 may be a linear alkyl group or a branched alkyl group.
  • R 1 is a C 5 - C 20 alkyl amide group, a C 10 - C 20 alkyl group
  • R 2 is a C 1 - C 5 alkyl group, a C 1 - C 5 alkoxy group, or C 2 ⁇ C 5 alkenyl or C 2 ⁇ C 5 alkynyl group of.
  • R 3 is a C 1 - C 5 alkyl group, a C 1 - C 5 alkoxy group, a C 2 - C 5 alkenyl group or a C 2 - C 5 alkynyl group.
  • the alkylamide group refers to a group in which an alkyl group is bonded to an amide group, and its structural formula can be, for example, C 5 H 11 -CONH-, C 11 H 24 -CONH-, C 13 H 27 -CONH- or C 20 H 27 -CONH- and so on.
  • the amide group on the alkylamide group may also be bonded to a C 1 -C 10 alkyl group, and the structural formula thereof may be, for example, C 13 H 27 -CONH-(CH 2 ) 3 - or C 11 H 24 -CONH- (CH 2 ) 10 -etc.
  • R 1 may be C 5 , C 6 , C 7 , C 8 , C 9 , C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 or C 20 alkyl.
  • R 1 may also be an alkenyl group of C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , C 8 , C 9 or C 10 .
  • R 1 may also be a C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , C 8 , C 9 or C 10 alkynyl group.
  • R 2 may be an alkyl group of C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , C 8 , C 9 or C 10 .
  • R 2 may be a C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , C 8 , C 9 or C 10 alkoxy group.
  • R 2 may also be an alkenyl group of C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , C 8 , C 9 , C 10 .
  • R 2 may also be a C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , C 8 , C 9 or C 10 alkynyl group.
  • R 3 may be an alkyl group of C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , C 8 , C 9 or C 10 .
  • R 3 may be a C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , C 8 , C 9 or C 10 alkoxy group.
  • R 3 may also be an alkenyl group of C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , C 8 , C 9 , C 10 .
  • R 3 may also be a C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , C 8 , C 9 or C 10 alkynyl group.
  • R 1 , R 2 and R 3 may be the same or different.
  • R 1 , R 2 and R 3 are each selected from an alkenyl group or an alkynyl group, the alkenyl group or alkynyl group may be at any position in the corresponding group.
  • the betaine compound has an effect of enhancing the luminescence intensity of the acridine luminescent substance, and can be used for preparing a chemiluminescence enhancer.
  • Betaine compounds have the properties of amphoteric surfactants, simple structure, single composition, easy to control batch-to-batch, and effectively reduce the different effects of chemiluminescence enhancement effects due to batch differences.
  • the chemiluminescence enhancer can effectively enhance the luminescence intensity of the acridine luminescent material, the luminescence signal is obviously enhanced, and the reproducibility is good, and can be widely used in clinical diagnosis, scientific research, environmental sanitation detection and the like.
  • the compound having the above general structure can enhance the luminescence intensity of the acridine-based luminescent substance by at least two times, and the enhancement ratio is higher than the conventional enhancers Triton X-100 and Tween 20.
  • a chemiluminescence immunoassay kit comprising the above chemiluminescence enhancer and an acridine luminescent substance.
  • a betaine compound in general, can be dissolved in a solvent to form a chemiluminescence enhancer.
  • the solvent may be water, an acidic solution or an alkaline solution or the like.
  • a betaine compound is dissolved in water to prepare a chemiluminescence enhancer.
  • the acridine luminescent substance may be an acridinium ester.
  • an acridinium ester for example, NSP-DMAE-NHS acridinium ester, NSP-DMAE-HEG-NHS acridinium ester and the like.
  • the R in the formulae 1 to 6 may be H, an alkyl group or an alkylsulfo group, and Ar may be a group containing a phenyl group.
  • the specific luminescence process is as follows: an acridine compound represented by Formula 1 is added to a 9-position carbon atom of an acridine ring by hydrogen peroxide under acidic conditions to obtain a compound represented by Formula 2, and then a peroxygen is formed under alkaline conditions. Negative ions give a compound of the formula 3.
  • the chemiluminescence enhancer and the acridine-based luminescent material are separately placed and mixed at the time of use.
  • the chemiluminescence immunoassay kit further comprises a chemiluminescence pre-excitation liquid and an excitation liquid, and the chemiluminescence pre-excitation liquid comprises a HNO 3 and H 2 O 2 solution, and the excitation liquid is a NaOH solution.
  • the concentration of the HNO 3 solution is 0.05 mol/L to 0.3 mol/L, and the volume percentage of the H 2 O 2 solution is 0.1% to 1%.
  • the concentration of the NaOH solution is from 0.05 mol/L to 0.5 mol/L.
  • the concentration of the HNO 3 solution is 0.1 mol/L, and the volume percentage of the H 2 O 2 solution is 0.5%.
  • the concentration of the NaOH solution was 0.25 mol/L.
  • the chemiluminescence pre-excitation liquid and the basic excitation liquid act as a starting reagent for the luminescence, and cooperate with the chemiluminescence enhancer to further improve the luminescence intensity of the pyridine luminescent material.
  • a chemiluminescence enhancer can be added to the base excitation solution to prepare a chemical excitation solution, thereby improving the luminescence intensity of the pyridine luminescent material.
  • the above chemiluminescence immunoassay kit includes a chemiluminescence enhancer and an acridine luminescent substance.
  • the use of betaine compounds as chemiluminescence enhancers can effectively enhance the luminescence intensity of acridine luminescent materials, and the enhancement ratio is higher than that of the conventional enhancers Triton X-100 and Tween 20, and the reproducibility is good.
  • the betaine compound has the properties of an amphoteric surfactant, the structure is simple, the composition is single, the batch difference is very easy to control, the effect of the chemiluminescence enhancement effect caused by the batch difference is effectively reduced, and the chemiluminescence immunoassay is improved. With the sensitivity of the kit, the kit can be widely used in clinical diagnostics, scientific research, environmental sanitation testing and other fields.
  • the experimental method without specifying the specific conditions is usually purchased from Henan Provincial Road Purification Technology Co., Ltd. according to the conventional conditions. Tween 20 (sigma, article number V900548) and Triton X-100 (sigma, article number V900502).
  • Acridinium ester (NSP-DMAE-NHS) and acridine salt (NSP-SA-NHS) were purchased from Shanghai Maxtor Chemicals.
  • the instrument for detecting chemiluminescence is the iFlash 3000 fully automatic chemiluminometer.
  • a pre-excitation liquid containing HNO 3 and H 2 O 2 is disposed in ultrapure water, wherein the final concentration of HNO 3 in the pre-excitation liquid is 0.1 mol/L, and the volume percentage of the H 2 O 2 solution in the pre-excitation liquid It is 0.5%.
  • the base excitation solution containing NaOH was configured with ultrapure water, and the final concentration of NaOH in the base excitation solution was 0.25 mol/L.
  • a chemiluminescence enhancer is separately added to the base excitation solution, and the chemiluminescence enhancer includes a betaine compound Z1 to Z13, respectively, to obtain a chemical excitation solution.
  • the original concentration of each of the betaine compounds Z1 to Z13 in the chemiluminescence enhancer was 10 g/L, and the amount of the chemiluminescence enhancer was 2 ⁇ L to 30 ⁇ L.
  • the structural formula of the betaine compounds Z1 to Z13 included in the chemiluminescence enhancer is as follows:
  • R - is an anionic group, specifically the following four types of Y1 to Y5, and the specific structures of Y1 to Y5 are as follows:
  • R 1 is a C 5 - C 20 alkyl amide group, a C 5 - C 20 alkyl group, a C 2 - C 10 alkenyl group or a C 2 - C 10 alkynyl group
  • R 2 is a C 1 - C 10 group
  • R 3 is a C 1 -C 10 alkyl group or a C 1 -C 10 alkane
  • the specific structures corresponding to the betaine compounds Z1 to Z13 are shown in Table 1.
  • the acridine solution in this example is NSP-DMAE-HEG-NHS acridinium ester, and its structural formula is as follows:
  • the excitation solution contains a base excitation solution and a betaine compound Z1 to Z13, respectively.
  • the chemiluminescence signal is measured on a chemiluminescence detector, and the enhancement factor of each luminescent liquid signal value and the base luminescent liquid signal value is calculated.
  • the corresponding luminescence curve of Example 5 is shown in FIG.
  • the results of the luminous intensity of Examples 1 to 13 are shown in Table 2.
  • the experimental control group was the same as Examples 1 to 13 except that no chemiluminescence enhancer was added.
  • the addition of a chemiluminescence enhancer containing the betaine-based compounds Z1 to Z13 can effectively enhance the luminescence intensity of the acridinium ester.
  • the enhancement factor is more than 4 times.
  • Tween 20 and Triton X-100 were used as comparative examples.
  • the steps of disposing the pre-excitation liquid and disposing the base excitation liquid in the comparative examples were the same as in Examples 1 to 13. The difference is that when the chemical excitation solution is configured, 10 ⁇ L of Tween 20 and Triton X-100 having a concentration of 10 g/L, respectively, are added to the base excitation solution to obtain a comparative chemical excitation solution.
  • the chemiluminescence signal of the comparative example was measured on a chemiluminescence detector, and the enhancement factor of the signal value of each luminescent liquid and the signal value of the base luminescent liquid was calculated, and the results are shown in Table 3.
  • Architect Trigger Solution (Abbott, Cat. No. 6C55-60) commercial luminescent liquid was used as a control.
  • the sample solution of this example was 10 ⁇ L of a solution of acridine salt (NSP-SA-NHS) at a concentration of 1 pg/mL.
  • the pre-excitation liquid and the basic excitation liquid were the same as in Examples 1 to 13.
  • 10 ⁇ L of a Z5-containing chemiluminescence enhancer was added to the base excitation solution to obtain a chemical excitation solution.

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Abstract

提供一种化学发光增强剂及化学发光免疫检测试剂盒，化学发光增强剂具体为甜菜碱类化合物，可用于增强吖啶类发光物质的发光强度。包括该化学发光增强剂和吖啶类发光物质的化学发光免疫检测试剂盒，发光信号大大增强、检测灵敏度高，可广泛应用于临床诊断、科学研究、环境卫生检测等领域。

Description

化学发光增强剂及化学发光免疫检测试剂盒 技术领域：
本发明涉及分析检测技术领域，尤其涉及一种化学发光增强剂及化学发光免疫检测试剂盒。
背景技术：
化学发光免疫分析(Chemiluminescence Immunoassay，CLIA)是将化学发光或生物发光体系与免疫反应相结合，用于检测微量抗原或抗体的一种新型标记免疫测定技术。化学发光免疫分析是目前世界范围内最主流的免疫检测技术，具有灵敏度高、检测范围宽、反应时间短、全自动操作、重现性好、无污染等优点，是继放免技术、酶免技术、荧光免疫技术之后发展起来的一种超高灵敏度的微量检测技术。化学发光的原理是当基态分子吸收化学反应中释放的能量跃迁到激发态，处于激发态的分子以光辐射的形式返回到基态时产生的光。化学发光免疫分析将化学发光与免疫分析方法相结合，综合了化学发光的高灵敏度和免疫分析的高选择性。
在化学发光免疫分析中，常用的发光物质包括鲁米诺、异鲁米诺、3-(2-螺旋金刚烷)-4-甲氧基-4-(3-磷氧酰)-苯基-1，2-二氧环乙烷二钠盐(缩写为AMPPD)、三联吡啶钌和吖啶类发光物质。其中，异鲁米诺、三联吡啶钌和吖啶类发光物质可作为示踪分子直接标记，属于闪光型化学发光物质。鲁米诺和AMPPD分别依靠过氧化物酶和碱性磷酸酶作为示踪分子标记，属于酶促发光型化学发光物质。吖啶类类发光物质的发光反应具有不需催化剂，只要碱性环境中就可以进行，并且发光反应迅速，背景发光低，信噪比高。发光反应干扰因素少，试剂稳定性好，化学发光体系简单，激发液成本低等优点，是理想的化学发光物质。
传统的吖啶类全自动化学发光系统主要使用过氧化氢与酸性溶液以及碱性溶液配合作为发光启动试剂，有些在发光启动试剂中加入增强剂聚乙二醇辛基苯基醚(Triton X-100)和吐温-20(Tween 20)以增强发光，但是传统的增强剂加入后增强发光的效果并不明显。综上所述，传统的吖啶类发光物质的发光强度较弱。
发明内容：
基于此，有必要提供一种可以增强吖啶类发光物质的发光强度的化学发光增强剂及化学发光免疫检测试剂盒。
一种化学发光增强剂，用于增强吖啶类发光物质的发光强度，所述化学发光增强剂包括甜菜碱类化合物。
一种化学发光免疫检测试剂盒，包括上述化学发光增强剂和吖啶类发光物质。
甜菜碱类化合物为具有季铵内盐结构或称作铵鎓结构的化合物，常用作渗透压调节剂、清洁剂或杀菌剂等。发明人意外发现甜菜碱类化合物具有增强吖啶类发光物质的发光强度的作用，可用于制备化学发光增强剂。实验表明，该甜菜碱类化合物可明显增强吖啶类发光物质的发光强度，并且增强的倍数高于传统的增强剂Triton X-100和Tween 20。
附图说明
图1为采用iFlash 3000全自动化学发光仪测定加入含有十四烷酰胺丙基羟丙基磺基甜菜碱的化学发光增强剂后吖啶酯的发光强度曲线图；
图2为采用采用iFlash 3000全自动化学发光仪测定在无增强剂和分别加入化学发光增强剂以及Architect Trigger Solution(Abbott)商品发光液条件下吖啶盐的发光强度曲线图。
具体实施方式
为使本发明的上述目的、特征和优点能够更加明显易懂，下面结合附图和具体实施例对本发明的具体实施方式做详细的说明。在下面的描述中阐述了很多具体细节以便于充分理解本发明。但是本发明能够以很多不同于在此描述的其它方式来实施，本领域技术人员可以在不违背本发明内涵的情况下做类似改进，因此本发明不受下面公开的具体实施的限制。
一实施方式的化学发光增强剂，该化学发光增强剂包括甜菜碱类化合物，该化学发光增强剂可用于增强吖啶类发光物质的发光强度。当然，化学发光增强剂还可以包括溶解甜菜碱类化合物的溶剂或可接受的杂质等。
甜菜碱类化合物为具有季铵内盐结构或称作铵鎓结构的化合物。吖啶类发光物质是一类可用作化学发光标记物的化学物质，例如吖啶酯或吖啶盐等。
具体的，甜菜碱类化合物的结构式为：
Figure PCTCN2016088126-appb-000001
其中，R-为阴离子基团，R1为C5～C20的烷基酰胺基、C5～C20的烷基、C2～C10的烯基或C2～C10的炔基，R2为C1～C10的烷基、C1～C10的烷氧基、C2～C10的烯基或C2～C10的炔基，R3为C1～C10的烷基、C1～C10的烷氧基、C2～C10的烯基或C2～C10的炔基。
R-为阴离子基团，与季铵内盐结构配合形成甜菜碱类化合物。
在一个实施方式中，甜菜碱类化合物为化合物I，化合物I的结构式为：
Figure PCTCN2016088126-appb-000002
其中，R1为C5～C20的烷基酰胺基、C5～C20的烷基、C2～C10的烯基或C2～C10的炔基，R2为C1～C10的烷基、C1～C10的烷氧基、C2～C10的烯基或C2～C10的炔基，R3为C1～C10的烷基、C1～C10的烷氧基、C2～C10的烯基或C2～C10的炔基。即本实施方式中，-R-为-COO-。
另一实施方式中，甜菜碱类化合物为化合物II，化合物II的结构式为：
Figure PCTCN2016088126-appb-000003
其中，R1为C5～C20的烷基酰胺基、C5～C20的烷基、C2～C10的烯基或C2～C10的炔基，R2为C1～C10的烷基、C1～C10的烷氧基、C2～C10的烯基或C2～C10的炔基，R3为C1～C10的烷基、C1～C10的烷氧基、C2～C10的烯基或C2～C10的炔基。即本实施方式中，-R-为
Figure PCTCN2016088126-appb-000004
另一实施方式中，甜菜碱类化合物为化合物III，化合物III的结构式为：
Figure PCTCN2016088126-appb-000005
其中，R1为C5～C20的烷基酰胺基、C5～C20的烷基、C2～C10的烯基或C2～C10的炔基，R2为C1～C10的烷基、C1～C10的烷氧基、C2～C10的烯基或C2～C10的炔基，R3为C1～C10的烷基、C1～C10的烷氧基、C2～C10的烯基或C2～C10的炔基。即本实施方式中，-R-为-CH2-SO3 -。
再一实施方式中，甜菜碱类化合物为化合物IV，化合物IV的结构式为：
Figure PCTCN2016088126-appb-000006
其中，R1为C5～C20的烷基酰胺基、C5～C20的烷基、C2～C10的烯基或C2～C10的炔基，R2为C1～C10的烷基、C1～C10的烷氧基、C2～C10的烯基或C2～C10的炔基，R3为C1～C10的烷基、C1～C10的烷氧基、C2～C10的烯基或C2～C10的炔基。即本实施方式中，-R-为-CH2-CH2-SO3 -。
再一实施方式中，甜菜碱类化合物为化合物V，化合物V的结构式为：
Figure PCTCN2016088126-appb-000007
其中，R1为C5～C20的烷基酰胺基、C5～C20的烷基、C2～C10的烯基或C2～C10的炔基，R2为C1～C10的烷基、C1～C10的烷氧基、C2～C10的烯基或C2～C10的炔基，R3为C1～C10的烷基、C1～C10的烷氧基、C2～C10的烯基或C2～C10的炔基。即本实施方式中，-R-为-CH(OH)-CH2-SO3 -。
在本文中，为特别说明，各结构式中的C5～C20的烷基可以为直链烷基或支链烷基。C2～C10的烯基可以为直链烯基或支链烯基。C2～C10的炔基可以为直链炔基或支链炔基。C1～C10的烷基可以为直链烷基或支链烷基。C1～C10的烷氧基可以为直链烷基或支链烷基。
进一步的，R1为C5～C20的烷基酰胺基、C10～C20的烷基，R2为C1～C5的烷基、C1～C5的烷氧基、C2～C5的烯基或C2～C5的炔基。R3为C1～C5的烷基、C1～C5的烷氧基、C2～C5的烯基或C2～C5的炔基。
具体的，烷基酰胺基是指烷基与酰胺基连接的基团，其结构式例如可以为C5H11-CONH-、C11H24-CONH-、C13H27-CONH-或C20H27-CONH-等。更进一步的，烷基酰胺基上的酰胺基还可以连接C1～C10的烷基，其结构式例如可以为C13H27-CONH-(CH2)3-或C11H24-CONH-(CH2)10-等。
具体的，R1可以为C5、C6、C7、C8、C9、C10、C11、C12、C13、C14、C15、C16、C17、C18、C19或C20的烷基。R1还可以为C2、C3、C4、C5、C6、C7、C8、C9或C10的烯基。或者，R1还可以为C2、C3、C4、C5、C6、C7、C8、C9或C10的炔基。
具体的，R2可以为C1、C2、C3、C4、C5、C6、C7、C8、C9或C10的烷基。R2可以为C1、C2、C3、C4、C5、C6、C7、C8、C9或C10的烷氧基。R2还可以为C2、C3、C4、C5、C6、C7、C8、C9、C10的烯基。或者，R2还可以为C2、C3、C4、C5、C6、C7、C8、C9或C10的炔基。
具体的，R3可以为C1、C2、C3、C4、C5、C6、C7、C8、C9或C10的烷基。 R3可以为C1、C2、C3、C4、C5、C6、C7、C8、C9或C10的烷氧基。R3还可以为C2、C3、C4、C5、C6、C7、C8、C9、C10的烯基。或者，R3还可以为C2、C3、C4、C5、C6、C7、C8、C9或C10的炔基。
进一步的，R1、R2和R3可以相同也可以不同。当R1、R2及R3分别选自烯基或炔基时，烯基或炔基可以在相应的基团中的任意位置。
发明人意外发现甜菜碱类化合物具有增强吖啶类发光物质的发光强度的作用，可用于制备化学发光增强剂。甜菜碱类化合物具有两性表面活性剂的性质，结构简单、成分单一，批间差非常容易控制，有效减少了由于批次差异带来的化学发光增强效果的不同的影响。通过该化学发光增强剂，可有效增强吖啶类发光物质的发光强度，发光信号明显增强，而且重现性好，可广泛应用于临床诊断、科学研究、环境卫生检测等领域。进一步的，具有上述通式结构的化合物可以将吖啶类发光物质的发光强度增强至少两倍以上，增强的倍数高于传统的增强剂Triton X-100和Tween 20。
一种化学发光免疫检测试剂盒，包括上述化学发光增强剂和吖啶类发光物质。
一般的，可将甜菜碱类化合物溶解在溶剂中制成化学发光增强剂。溶剂可以为水、酸性溶液或碱性溶液等。本实施方式中，将甜菜碱类化合物溶解在水中制成化学发光增强剂。
具体的，吖啶类发光物质可以为吖啶酯。例如NSP-DMAE-NHS吖啶酯、NSP-DMAE-HEG-NHS吖啶酯等。
一实施方式的吖啶类发光物质的化学发光反应流程图如下所示：
Figure PCTCN2016088126-appb-000008
其中，式1所示结构表示的吖啶类化合物，式1～式6中的R可以为H、烷基或烷基磺基等，Ar可以为含有苯基的基团。具体发光过程为：式1所示的吖啶类化合物在酸性条件下通过过氧化氢加成吖啶环的9位碳原子，得到式2所示的化合物，然后在碱性条件下生成过氧负离子，得到式3所示的化合物。之后通过分子内进攻羰基，离去基团离去，生成不稳定中间体1，2-二氧杂环丁烷酮(式6所示的化合物)，然后1，2-二氧杂环丁烷酮开环生成吖啶酮，同时释放光子。
一般的，在化学发光免疫检测试剂盒中，化学发光增强剂和吖啶类发光物质是分开放置的，使用时再混合。
本实施方式中，化学发光免疫检测试剂盒还包括化学发光预激发液和激发液，化学发光预激发液包括HNO3和H2O2溶液，激发液为NaOH溶液。
具体的，HNO3溶液的浓度为0.05mol/L～0.3mol/L，H2O2溶液的体积百分含量为0.1％～1％。NaOH溶液的浓度为0.05mol/L～0.5mol/L。
本实施方式中，HNO3溶液的浓度为0.1mol/L，H2O2溶液的体积百分含量为0.5％。NaOH溶液的浓度为0.25mol/L。
化学发光预激发液和基础激发液作为发光的启动试剂，与化学发光增强剂配合，进一步提高啶类发光物质的发光强度。
具体的，可将化学发光增强剂加入基础激发液中制得化学激发液，从而提高啶类发光物质的发光强度。
上述化学发光免疫检测试剂盒，包括化学发光增强剂和吖啶类发光物质。将甜菜碱类化合物作为化学发光增强剂，可有效增强吖啶类发光物质的发光强度，增强的倍数高于传统的增强剂Triton X-100和Tween 20，而且重现性好。并且甜菜碱类化合物具有两性表面活性剂的性质，结构简单、成分单一，批间差非常容易控制，有效减少了由于批次差异带来的化学发光增强效果的不同的影响，提高化学发光免疫检测试剂盒检测灵敏度，该试剂盒可广泛应用于临床诊断、科学研究、环境卫生检测等领域。
以下为实施例部分。
以下实施例中，如无特别说明，未注明具体条件的实验方法，通常按照常规条件，所用实验材料甜菜碱类化合物购于河南省道纯化工技术有限公司。Tween20(sigma公司，货号V900548)和Triton X-100(sigma公司，货号V900502)。吖啶酯(NSP-DMAE-NHS)和吖啶盐(NSP-SA-NHS)采购于上海迈拓崴化工。检测化学发光的仪器为iFlash 3000全自动化学发光仪。
实施例1～实施例13
甜菜碱类化合物对吖啶类发光物质的发光强度的影响
1)配置预激发液
用超纯水配置含有HNO3和H2O2的预激发液，其中HNO3在预激发液中 的终浓度为0.1mol/L，H2O2溶液在预激发液中的体积百分含量为0.5％。
2)配置基础激发液
用超纯水配置含有NaOH的基础激发液，NaOH在基础激发液中的终浓度为0.25mol/L。
3)配置化学激发液
向基础激发液中分别加入化学发光增强剂，化学发光增强剂分别包括甜菜碱类化合物Z1～Z13，得到化学激发液。化学发光增强剂的中各甜菜碱类化合物Z1～Z13的原浓度均为10g/L，化学发光增强剂加入量为2μL～30μL。本实施例中分别加入10μL的分别含有Z1～Z13的化学发光增强剂到基础激发液中得到化学激发液。化学发光增强剂包括的甜菜碱类化合物Z1～Z13的结构式如下：
Figure PCTCN2016088126-appb-000009
其中，R-为阴离子基团，具体有以下Y1～Y5四类，Y1～Y5具体结构如下：
Figure PCTCN2016088126-appb-000010
R1为C5～C20的烷基酰胺基、C5～C20的烷基、C2～C10的烯基或C2～C10的炔基，R2为C1～C10的烷基、C1～C10的烷氧基、C2～C10的烯基或C2～C10的炔基，R3为C1～C10的烷基、C1～C10的烷氧基、C2～C10的烯基或C2～C10的炔基。甜菜碱类化合物Z1～Z13对应的具体的结构如表1所示。
表1：甜菜碱类化合物Z1～Z13的名称和结构式
Figure PCTCN2016088126-appb-000011
Figure PCTCN2016088126-appb-000012
4)化学发光信号的测定
以10μL、浓度为1pg/mL的吖啶溶液为样品，本实施例中吖啶溶液为NSP-DMAE-HEG-NHS吖啶酯，其结构式如下：
Figure PCTCN2016088126-appb-000013
向样品中加入100μL预激发液，再分别加入100μL各化学激发液。化学 激发液中分别含有基础激发液中以及甜菜碱类化合物Z1～Z13。在化学发光检测仪上测量化学发光信号，并计算各发光液信号值与基础发光液信号值的增强倍数。实施例5对应的发光曲线如图1所示。实施例1～13的发光强度结果如表2所示。
表2：实验对照组以及实施例1～13的发光强度
Figure PCTCN2016088126-appb-000014
其中，实验对照组除了不加化学发光增强剂，其余与实施例1～实施例13相同。从表2可以看出，加入含有甜菜碱类化合物Z1～Z13的化学发光增强剂可以有效的增强吖啶酯的发光强度。增强倍数在4倍以上。
对比例1～对比例2
为进一步验证甜菜碱类化合物对吖啶酯的发光强度的影响。采用Tween20和Triton X-100作为对比例。对比例中配置预激发液和配置基础激发液的步骤与实施例1～13相同。不同的是配置化学激发液时，采用分别将10μL的原浓度为10g/L的Tween 20和Triton X-100加入基础激发液中得到对比例的化学激发液。采用化学发光检测仪上测量对比例的化学发光信号，并计算各发光液信号值与基础发光液信号值的增强倍数，结果如表3所示。
表3：实验对照组以及对比例1和2的发光强度
Figure PCTCN2016088126-appb-000015
对比表2和表3的结果可知，使用甜菜碱类化合物能起到增强吖啶类化合物化学发光作用，且增强效果优于Tween 20和Triton X-100。并且本实施例1～13的化学发光也发光信号稳定，发光效率高。
实施例14
为进一步比较甜菜碱类化合物对吖啶类化合物化学发光增强作用，选用Architect Trigger Solution(Abbott公司，货号：6C55-60)商品发光液作为对照。本实施例的样品液为10μL浓度为1pg/mL的吖啶盐(NSP-SA-NHS)溶液。预激发液和基础激发液与实施例1～13相同。将10μL的含有Z5的化学发光增强剂加到基础激发液中得到化学激发液。向样品中加入100μL预激发液，再分别加入100μL的含有增强剂Z5对应的化学激发液或100μL的Architect Trigger Solution(Abbott)商品发光液。分别测定在无增强剂、有含有Z5的化学发光增强剂以及Architect Trigger Solution(Abbott)商品发光液 条件下吖啶盐的发光强度。结果如图2所示，与商品发光液Architect Trigger Solution(Abbott)发光信号进行对比。在本发明增强剂作用下，吖啶的发光信号大大增强，增强发光液性能接近Architect Trigger Solution(Abbott)。
从实施例1～14以及对比1的结果可知，甜菜碱类化合物对吖啶类化合物有很好的增强化学发光的效果，可提高诊断试剂盒的检测灵敏度。
本文中的所有数据、图像、仪器、试剂和步骤应理解为说明性而非限制性的。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (10)

  1. 一种化学发光增强剂，用于增强吖啶类发光物质的发光强度，其特征在于，所述化学发光增强剂包括甜菜碱类化合物。
  2. 根据权利要求1所述的化学发光增强剂，其特征在于，所述甜菜碱类化合物的结构式为：
    Figure PCTCN2016088126-appb-100001
    其中，R-为阴离子基团，R1为C5～C20的烷基酰胺基、C5～C20的烷基、C2～C10的烯基或C2～C10的炔基，R2为C1～C10的烷基、C1～C10的烷氧基、C2～C10的烯基或C2～C10的炔基，R3为C1～C10的烷基、C1～C10的烷氧基、C2～C10的烯基或C2～C10的炔基。
  3. 根据权利要求2所述的化学发光增强剂，其特征在于，所述甜菜碱类化合物的结构式为：
    Figure PCTCN2016088126-appb-100002
    其中，R1为C5～C20的烷基酰胺基、C5～C20的烷基、C2～C10的烯基或C2～C10的炔基，R2为C1～C10的烷基、C1～C10的烷氧基、C2～C10的烯基或C2～C10的炔基，R3为C1～C10的烷基、C1～C10的烷氧基、C2～C10的烯基或C2～C10的炔基。
  4. 根据权利要求2所述的化学发光增强剂，其特征在于，所述甜菜碱类化合物的结构式为：
    Figure PCTCN2016088126-appb-100003
    其中，R1为C5～C20的烷基酰胺基、C5～C20的烷基、C2～C10的烯基或C2～C10 的炔基，R2为C1～C10的烷基、C1～C10的烷氧基、C2～C10的烯基或C2～C10的炔基，R3为C1～C10的烷基、C1～C10的烷氧基、C2～C10的烯基或C2～C10的炔基。
  5. 根据权利要求2所述的化学发光增强剂，其特征在于，所述甜菜碱类化合物的结构式为：
    Figure PCTCN2016088126-appb-100004
    其中，R1为C5～C20的烷基酰胺基、C5～C20的烷基、C2～C10的烯基或C2～C10的炔基，R2为C1～C10的烷基、C1～C10的烷氧基、C2～C10的烯基或C2～C10的炔基，R3为C1～C10的烷基、C1～C10的烷氧基、C2～C10的烯基或C2～C10的炔基。
  6. 根据权利要求2所述的化学发光增强剂，其特征在于，所述甜菜碱类化合物的结构式为：
    Figure PCTCN2016088126-appb-100005
    其中，R1为C5～C20的烷基酰胺基、C5～C20的烷基、C2～C10的烯基或C2～C10的炔基，R2为C1～C10的烷基、C1～C10的烷氧基、C2～C10的烯基或C2～C10的炔基，R3为C1～C10的烷基、C1～C10的烷氧基、C2～C10的烯基或C2～C10的炔基。
  7. 根据权利要求2所述的化学发光增强剂，其特征在于，所述甜菜碱类化合物的结构式为：
    Figure PCTCN2016088126-appb-100006
    其中，R1为C5～C20的烷基酰胺基、C5～C20的烷基、C2～C10的烯基或C2～C10的炔基，R2为C1～C10的烷基、C1～C10的烷氧基、C2～C10的烯基或C2～C10的炔基，R3为C1～C10的烷基、C1～C10的烷氧基、C2～C10的烯基或C2～C10的炔基。
  8. 一种化学发光免疫检测试剂盒，其特征在于，包括如权利要求1～7任 一项所述的化学发光增强剂和吖啶类发光物质。
  9. 根据权利要求8所述的化学发光免疫检测试剂盒，其特征在于，所述吖啶类发光物质为吖啶酯或吖啶盐。
  10. 根据权利要求8所述的化学发光免疫检测试剂盒，其特征在于，还包括化学发光预激发液和激发液，所述化学发光预激发液包括HNO3和H2O2溶液，激发液包括NaOH溶液。
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