A kind of high stable enhanced chemiluminescence substrateTechnical field
The present invention relates to chemiluminescence analytical technique, be a kind of ultramicro-analysis method, belong to International Classification of Patents GO1N21/17 " optical instrument test analysis " technical field.
Background technology
Chemiluminescence analytical technique is one of modern microanalysis method because it is highly sensitive, easy fast, good linearity, the term of validity is long, safe and harmless characteristics such as ("dead" and teratogen pollute) and becoming replace to put exempts from the important technology that joins with enzyme.Chemical analysis with horseradish peroxidase-labeled is after immune response is finished, and adds HRP substrate H2O2, and under alkali condition, HRP catalysis H2O2 makes luminous substrate luminol generation chemical reaction and luminous.But it is destroyed gradually with the prolongation of standing time that existing this technology one is H2O2, the 2nd, and the light signal instability that produces causes a deviation to testing result.
Summary of the invention
Purpose of the present invention is to overcome above-mentioned deficiency, and a kind of enhanced chemiluminescence substrate and detection method thereof of high stability is provided.
Technical scheme of the present invention is as follows.
A kind of high stable enhanced chemiluminescence substrate, this substrate by the luminol of special ratios, reinforcing agent to iodophenol, collaborative reinforcing agent tetraphenylboron sodium, oxygenant urea peroxide and H2O2, A liquid that is mixed with respectively and B liquid are formed.The A liquid of equivalent and B liquid contact with catalyzer (as horseradish peroxidase) label after the immune response, generate a kind of product of excited state by chemical reaction, and it produces luminescence phenomenon thereby the energy that discharges is transformed into photon in getting back to the process of ground state.Its advantage be have luminous sensitivity height, good stability, the result is accurate, easy to use and cost is low etc.Be applicable to full-automatic, semi-automatic and manual analysis.Can be widely used in clinical examination, biological study, legal medical expert identifies, in farming and animal husbandry and the environmental science to the detections of biomacromolecules such as nucleic acid and antigen-antibody.
Chemiluminescence immune analysis method becomes the outstanding person in the present external diagnosis reagent detection method because of environmentally safe, to comprehensive advantage and potentiality such as human body is nontoxic.
The objective of the invention is further to improve the stability of chemiluminescence immune assay substrate, for the suitability for industrialized production of product is brought bigger convenience and dirigibility with using.
Description of drawings
Accompanying drawing is to measure the synoptic diagram with a series of calibration object dose-response curve stability in 0～4 hour.
The time value and the result of calculation of accompanying drawing are as follows:
0min：y＝-0.9755x+1.2635???150min：y＝-0.9055x+1.1964
R2＝0.998???????????????????R2＝0.9972
30min：y＝-0.9205x+1.2034??180min：y＝-0.909x+1.2062
R2＝0.9977??????????????????R2＝0.9977
60min：y＝-0.9151x+1.2052??240min：y＝-0.9094x+1.2093
R2＝0.9972?????????????????R2＝0.9978
90min：y＝-0.9068x+1.198
R2＝0.9972
Embodiment
The substrate solution preparation: the borate buffer of 0.5mol/L pH8.6 is prepared A liquid and B liquid respectively: A liquid contains luminol 3.0mg/ml, and reinforcing agent is worked in coordination with reinforcing agent tetraphenylboron sodium 0.5mg/ml to iodophenol 0.25mg/ml; B liquid contains urea peroxide 0.4g/ml, H2O20.1g/ml.
The bin stability height.A liquid and B liquid are stored respectively.Preserved the term of validity 2 years for 2～8 ℃.
The reaction stability height.The A liquid and the B liquid that add mixed in equal amounts in the sample hose after the immune response of HRP mark (hole), same a series of calibration object dose-response curves of measuring in 0～4 hour stable (accompanying drawing); 0～4 hour with the sample measured value coefficient of variation less than 5%, its corresponding luminous value coefficient of variation (CV%) is less than 15%, referring to subordinate list: same sample measured value and the corresponding luminous value stability thereof measured in 0～4 hour.
Subordinate list
| Measured object 1 (5.3ng/ml) | Measured object 1 (112ng/ml) |
| Luminous value | Measured value | Luminous value | Measured value |
| ??0min | ??661112 | ????5.27 | ??129228 | ??113.88 |
| ??30min | ??670237 | ????5.33 | ??145643 | ??115.26 |
| ??60min | ??644213 | ????5.28 | ??147287 | ??110.57 |
| ??120min | ??598965 | ????5.22 | ??135181 | ??1115.10 |
| ??150min | ??571145 | ????5.16 | ??127741 | ??116.51 |
| ??180min | ??530768 | ????5.42 | ??127344 | ??107.61 |
| ??240min | ??489021 | ????5.65 | ??119057 | ??107.17 |
| Average | ??595066 | ????5.33 | ??133069 | ??112.30 |
| ??SD | ??68794 | ????0.16 | ??10303 | ??3.83 |
| ??CV％ | ??11.56％ | ????3.06％ | ??7.74％ | ??3.41％ |