阅读说明：本技术 一种生物传感器及其应用 (A kind of biosensor and its application ) 是由 宋建军 焦政 李重阳 陈立勇 刘仁源 李建霖 于 2019-07-04 设计创作，主要内容包括：本发明提供了一种生物传感器及其应用,所述生物传感器包括2-10种探头；所述探头包被有不同抗原的单克隆抗体；所述抗原包括肝肿瘤标志物。本发明通过在生物传感器上设置针对不同抗原的多个探头,且各探头的检测过程不会相互影响,不仅实现了同一样本中不同抗原的同时检测,而且实现了多种样本的同时检测。(The present invention provides a kind of biosensor and its application, the biosensor includes 2-10 kind probe；Described pop one's head in is coated with the monoclonal antibody of not synantigen；The antigen includes liver tumour marker.The present invention is by setting on a biosensor for multiple probes of not synantigen, and the detection process respectively popped one's head in will not influence each other, detection while not only realizing in same sample not synantigen, but also detection while realize a variety of samples.)

1. a kind of biosensor, which is characterized in that the biosensor includes 2-10 kind probe (1)；

The probe (1) is coated with the monoclonal antibody of not synantigen；

The antigen includes liver tumour marker.

2. biosensor according to claim 1, which is characterized in that the biosensor includes 3-8 kind probe (1), preferably 5-6 kind pops one's head in (1).

3. biosensor according to claim 1 or 2, which is characterized in that the monoclonal antibody includes alpha-fetoprotein Monoclonal antibody, 3 monoclonal antibody of human a-fetoprotein heteroplasmon, Cea Monoclonal Antibodies, carbohydrate antigen 125 monoclonal are anti- In body or Carbohydrate Antigen 19-9 monoclonal antibody any one or at least two combination.

4. a kind of formulating method of the standard curve of the biosensor as described in claim any one of 1-3, which is characterized in that institute State method the following steps are included:

(1) it will be mixed after at least two antigen standard gradient dilutions, configuration obtains the antigen standard of at least 7 parts various concentrations Liquid；

(2) colloidal gold labeled monoclonal antibody of antigen is added into step (1) the antigen titer, mixing is incubated for, and obtains detection liquid；

(3) probe is successively immersed in step (2) described detection liquid, mixing is incubated for, and is carried out optical detection, is obtained thickness Value；

(4) using thickness value as ordinate, the concentration of antigen is abscissa, and drafting obtains the standard curve of not synantigen.

5. according to the method described in claim 4, it is characterized in that, step (2) and the temperature of step (3) described incubation are 15- 40 DEG C, preferably 37-38 DEG C；

Preferably, the time of step (2) and step (3) described incubation is 60-100s, preferably 70-90s.

6. a kind of associated detecting method of antigen, which is characterized in that the method uses raw as described in claim any one of 1-3 Object sensor.

7. according to the method described in claim 6, it is characterized in that, the described method comprises the following steps:

Sample to be tested is mixed incubation with colloidal gold labeled monoclonal antibody by (1 '), obtains solution to be measured；

(2 '), which will pop one's head in, successively to be immersed in step (1 ') described solution to be measured, and mixing is incubated for, and is carried out optical detection, is obtained thickness Value；

The concentration of antigen in the sample to be tested is calculated according to standard curve in (3 ').

8. the method according to the description of claim 7 is characterized in that step (the 1 ') colloidal gold labeled monoclonal antibody includes colloidal gold Labelled AFP monoclonal antibody, 3 monoclonal antibody of colloid gold label human a-fetoprotein heteroplasmon, colloid gold label cancer embryo are anti- Former monoclonal antibody, colloid gold label carbohydrate antigen 125 monoclonal antibody and colloid gold label Carbohydrate Antigen 19-9 monoclonal are anti- The combination of body.

9. method according to claim 7 or 8, which is characterized in that the temperature of step (1 ') and step (the 2 ') incubation It is 15-40 DEG C, preferably 37-38 DEG C；

Preferably, the time of step (1 ') and step (the 2 ') incubation is 60-100s, preferably 70-90s.

10. a kind of application of biosensor as described in any one of claims 1-3 in joint-detection liver tumour marker.

Technical field

The invention belongs to field of molecular detection, it is related to a kind of biosensor and its application.

Background technique

Clinically used liver tumour marker includes alpha-fetoprotein (AFP), human a-fetoprotein heteroplasmon 3 (AFP-L3), cancer Embryonal antigen (CEA), carbohydrate antigen 125 (CA125), Carbohydrate Antigen 19-9 (CA19-9) etc..However single tumor markers Specificity and sensitivity are lower, it usually needs joint Diagnostic Value of Several Serum Tumor Markers, to improve the specificity and sensitivity of lesion detection. The detection method of tumor markers mainly includes colloidal gold paper chromatography, enzyme-linked immunization and chemiluminescence immunoassay at present Deng, however different tumor markers are detected one by one it is usually necessary to use kit and special instrument, operation repeats high, consumption Time-consuming length, higher cost.Traditional kit detection method is difficult to efficiently carry out the quick multi objective joint-detection of multisample.

107345966 A of CN discloses a kind of methadone detection method based on biomembrane interference technique, the method inspection Survey that the time is short, and entire detection process only needs 5min or so, and detection sensitivity is minimum can reach 10ng/mL, can be criticized simultaneously The detection for measuring sample at most can once carry out the detection of 80 samples, and the entire time only has 30min, realizes high-throughput sample Quickly detection.However the Testing index of this method is only limitted to methadone, can not achieve multi objective joint-detection.

Therefore it provides a kind of biosensor of high throughput multi objective is led for the joint-detection of antigen in Molecular Detection Domain is of great significance.

Summary of the invention

In view of the deficiencies of the prior art, the present invention provides a kind of biosensor and its application, the biosensors By loading the probe of the different monoclonal antibodies of multiple coatings, cooperates microwell plate, realize the multisample multi objective joint of antigen Detection, high degree of automation, detection efficiency are high, the time is short.

To achieve this purpose, the present invention adopts the following technical scheme:

In a first aspect, the biosensor includes 2-10 kind probe 1 the present invention provides a kind of biosensor；

The probe 1 is coated with the monoclonal antibody of not synantigen；

The antigen includes liver tumour marker.

In the present invention, a variety of probes of not synantigen, and the detection respectively popped one's head in are directed to by being arranged on a biosensor Process will not influence each other, detection while not only realizing in same sample not synantigen, but also realize a variety of samples It detects simultaneously.

Preferably, the biosensor includes 2-10 kind probe 1, such as can be 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 Kind, 8 kinds, 9 kinds or 10 kinds, preferably 3-8 kind, further preferably 5-6 kind.

Preferably, the monoclonal antibody includes that alpha-fetoprotein monoclonal antibody, 3 monoclonal of human a-fetoprotein heteroplasmon are anti- It is any one in body, Cea Monoclonal Antibodies, carbohydrate antigen 125 monoclonal antibody or Carbohydrate Antigen 19-9 monoclonal antibody Kind or at least two combination.

Second aspect, the present invention provides a kind of formulation sides of standard curve of biosensor as described in relation to the first aspect Method the described method comprises the following steps:

(1) it will be mixed after at least two antigen standard gradient dilutions, configuration obtains the antigen mark of at least 7 parts various concentrations Quasi- liquid；

(2) colloidal gold labeled monoclonal antibody of antigen is added into step (1) the antigen titer, mixing is incubated for, is examined Survey liquid；

(3) probe is successively immersed in step (2) described detection liquid, mixing is incubated for, and is carried out optical detection, is obtained thickness Angle value；

(4) using thickness value as ordinate, the concentration of antigen is abscissa, and drafting obtains the standard curve of not synantigen.

Preferably, step (2) and the temperature of step (3) described incubation are 15-40 DEG C, such as can be 15 DEG C, 16 DEG C, 17 ℃、18℃、19℃、20℃、21℃、22℃、23℃、24℃、25℃、26℃、27℃、28℃、29℃、30℃、31℃、32 DEG C, 33 DEG C, 34 DEG C, 35 DEG C, 36 DEG C, 37 DEG C, 38 DEG C, 39 DEG C or 40 DEG C, preferably 37-38 DEG C.

Preferably, the time of step (2) and step (3) described incubation be 60-100s, such as can be 60s, 61s, 62s, 63s、64s、65s、66s、67s、68s、69s、70s、71s、72s、73s、74s、75s、76s、77s、78s、79s、80s、81s、 82s, 83s, 84s, 85s, 86s, 87s, 88s, 89s, 90s, 91s, 92s, 93s, 94s, 95s, 96s, 97s, 98s, 99s or 100s, preferably 70-90s.

The third aspect, the present invention provides a kind of associated detecting method of antigen, the method is used such as first aspect institute State biosensor.

Preferably, it the described method comprises the following steps:

Sample to be tested is mixed incubation with colloidal gold labeled monoclonal antibody by (1 '), obtains solution to be measured；

(2 '), which will pop one's head in, successively to be immersed in step (1 ') described solution to be measured, and mixing is incubated for, and is carried out optical detection, is obtained thickness Angle value；

The concentration of antigen in the sample to be tested is calculated according to standard curve in (3 ').

Preferably, step (the 1 ') colloidal gold labeled monoclonal antibody includes colloid gold label alpha-fetoprotein monoclonal antibody, glue Body gold marks 3 monoclonal antibody of human a-fetoprotein heteroplasmon, colloid gold label Cea Monoclonal Antibodies, colloid gold label sugar The combination of class antigen 125 monoclonal antibody and colloid gold label Carbohydrate Antigen 19-9 monoclonal antibody.

Preferably, step (1 ') and the temperature of step (the 2 ') incubation are 15-40 DEG C, for example, can be 15 DEG C, 16 DEG C, 17℃、18℃、19℃、20℃、21℃、22℃、23℃、24℃、25℃、26℃、27℃、28℃、29℃、30℃、31℃、32 DEG C, 33 DEG C, 34 DEG C, 35 DEG C, 36 DEG C, 37 DEG C, 38 DEG C, 39 DEG C or 40 DEG C, preferably 37-38 DEG C.

Preferably, the time of step (1 ') and step (the 2 ') incubation be 60-100s, such as can be 60s, 61s, 62s、63s、64s、65s、66s、67s、68s、69s、70s、71s、72s、73s、74s、75s、76s、77s、78s、79s、80s、 81s, 82s, 83s, 84s, 85s, 86s, 87s, 88s, 89s, 90s, 91s, 92s, 93s, 94s, 95s, 96s, 97s, 98s, 99s or 100s, preferably 70-90s.

Fourth aspect, the present invention provides a kind of biosensors as described in relation to the first aspect in joint-detection liver tumour mark Application in will object.

Compared with prior art, the invention has the following beneficial effects:

(1) biosensor of the invention can carry out multiple determination to multiple samples, and detection efficiency is high, sample consumption It is few；

(2) biosensor of the invention can detect crude samples, and sample preprocessing requires low；

(3) biosensor of the invention directly detects the concentration of antigen, the inspection of indices by double antibody sandwich method Survey carries out in an enzyme mark hole, and detection flux is big；

(4) biosensor of the invention only need to be once loaded, remaining step is automatically performed by instrument, the degree of automation Height, it is easy to operate, it is time saving and energy saving.

Detailed description of the invention

Fig. 1 is the structural schematic diagram of biosensor；

Fig. 2 is 96 microwell plate reagent pipetting volume figures.

Fig. 3 A is AFP standard curve, and Fig. 3 B is AFP-L3 standard curve, and Fig. 3 C is CEA standard curve, and Fig. 3 D is CA125 Standard curve, Fig. 3 E are CA19-9 standard curve.

Specific embodiment

The technological means and its effect taken for the present invention is further explained, with reference to embodiments with attached drawing to this hair It is bright to be further described.It is understood that the specific embodiments described herein are used only for explaining the present invention, rather than Limitation of the invention.