CN1222773C - An optical circulation amplification type biological chip detecting process - Google Patents
An optical circulation amplification type biological chip detecting process Download PDFInfo
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- CN1222773C CN1222773C CN 02139105 CN02139105A CN1222773C CN 1222773 C CN1222773 C CN 1222773C CN 02139105 CN02139105 CN 02139105 CN 02139105 A CN02139105 A CN 02139105A CN 1222773 C CN1222773 C CN 1222773C
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- adenine dinucleotide
- reduced
- biological sample
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- nicotinamide adenine
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Abstract
The present invention discloses a light circulation amplifying type biological chip detecting method which comprises: enzyme circulation and biological luminescence are combined, the FMNH2 accumulation of circulation on electrodes is realized through NADH/NAD<+> oxidation reduction, and highly cumulated FMNH2 reacts with luciferase as another luminous substrate, aldehyde with long chains and oxygen gas to emit fluorescent light with the wave length of 490 nm. The light has long hold time and large light density. The method of the present invention has the advantages of high sensitivity, low cost and simple operation and is suitable for the analysis of various biologic samples, and the method has a favorable prospect in the aspects of developing high density biological chips and protein chips.
Description
Technical field
The present invention relates to bioassay technique, more specifically relate to a kind of method of light circulation scale-up version biochip test, be applicable to the analysis of all kinds of biological samples on the biochip.
Background technology
At present, bio-chip test method mainly is based on radioactive isotope and optical detection, but because isotopic pollution and harm, people are in the use of avoiding it as far as possible, so fluorescence has become the main detection means of biochip with chemiluminescence.Yet because the extremely low concentration of biological sample to be measured has limited the concentration of label, so can only detect very faint optical signalling, this just requires to use very sensitive pick-up unit, for example: cold light coupling device (cooled CCD), remove to catch sort signal, the price of these devices is also very high at present, and obviously this has just caused the costliness that detects cost.In highdensity biochip, the reaction chamber space is very narrow and small, and the reaction substrate that can add is very a small amount of, and this has limited the sensitivity of detection means equally, so detection means commonly used is difficult to use in the superchip development.Can come the concentration of amplification of nucleic acid by PCR method in the DNA chip, yet protein can't resemble the amplification of carrying out concentration the nucleic acid at present, this is a key obstacle of exploitation protein-chip.About bioluminescence, refer to that at present the wavelength that firefly and extra large endophytic bacteria are sent out is the cold light of 490nm more.Luminous may be a kind of strategy of going after profits and advoiding disadvantages concerning these biologies, such as: remove harmful O
2Perhaps escape the hunting of natural enemy.The substrate of luminescence-producing reaction has related generally to fluorescein, luciferase and O
2Deng.Though it is human to after deliberation comparatively thorough of bioluminescence, but up to now, the mankind also only are confined to two aspects to noctilcent application: the one, and the monitoring of hygiene, whether for example: exceed standard by the quantitative measurement bacterial population to ATP or NADH, the range of linearity to ATP or NADH detection can reach 10 at present
-12-10
-6M; The 2nd, bioluminescence gene lux is applied to gene expression analysis and immunoassay as reporter gene.Exquisite mechanism in this biosome of bioluminescence is applied to biochip does not still have report and application.
Summary of the invention
The object of the invention is to provide a kind of method of light circulation scale-up version biochip test, galvanochemistry and bioluminescence are combined, by galvanochemistry circulation accumulation target product, sensitivity is improved one to two order of magnitude, reduce the detection cost, be mainly used in protein-chip and high-density biochip.
In order to achieve the above object, the present invention is by the following technical solutions:
The reaction substrate that relates to has: specific oxidation reductase (NADH-FMN Oxidereductase), flavin mononucleotide (FMN) (FMN), the flavin mononucleotide reduced (FMNH of nicotinamide adenine dinucleotide reduced (NADH) or NADPH (NADPH) and flavin mononucleotide (FMN) (FMN)
2), nicotinamide adenine dinucleotide (NAD) or nicotinamide-adenine dinucleotide phosphate (NADP), nicotinamide adenine dinucleotide reduced (NADH) or NADPH (NADPH),, oxygen (O
2), above aldehyde (R-CHO), marine bacteria luciferase (Luciferase) and the electron mediator ferrocene etc. of seven carbon atoms.
Its step is as follows:
1, biological sample to be detected (acceptor) is fixed in the reaction chamber of chip,, is fixed in the reaction chamber of chip as antibody.
2, nicotinamide adenine dinucleotide reduced (NADH) or NADPH (NADPH) and the specific oxidation reductase (NADH-FMNOxidereductase) of flavin mononucleotide (FMN) (FMN) are marked on another kind can with above-mentioned acceptor identification and the biological sample that combines, this biological sample can be discerned the biological sample (part) that also combines with above-mentioned acceptor, as: antigen.
3, step 2 acceptance of the bid the is recorded a demerit biological sample respective reaction that biological sample (part) point sample of enzyme fixes with step 1 on chip is indoor.
4, with 20 to the 50 ℃ of following incubations of reaction of two kinds of biological samples in step 1 and 2 20 to 50 minutes.
5, with the biological sample of a series of buffer solution and the non-specific bond of elute soln wash-out and then use deionized water wash.Can two biological samples be discerned mutually and detect in conjunction with can using following luminescence-producing reaction, can discern mutually and the situation of combination under just have light signal and occur, reach the purpose of biological detection with this.
6, the nicotinamide adenine dinucleotide reduced (NADH) of oxygen or the solution of NADPH (NADPH), flavin mononucleotide (FMN) (FMN) and any one electron mediator are removed in adding in the said chip reaction chamber; Nicotinamide adenine dinucleotide reduced (NADH) or NADPH (NADPH), flavin mononucleotide (FMN) (FMN) generate nicotinamide adenine dinucleotide (NAD) or nicotinamide-adenine dinucleotide phosphate (NADP) and flavin mononucleotide reduced (FMNH at once under the catalysis of enzyme
2); Nicotinamide adenine dinucleotide that is generated in enzymatic reaction (NAD) or nicotinamide-adenine dinucleotide phosphate (NADP) are reduced into nicotinamide adenine dinucleotide reduced (NADH) or reduced form niacinamide urea purine dinucleotide phosphoric acid (NADPH) again by the electron mediator ferrocene under the effect of chip electrode.
7, flavin mononucleotide reduced (FMNH after above-mentioned circulation is carried out 100 to 10000
2) a large amount of accumulation, what at this moment add oxygenation contains the above aldehyde (R-CHO) of seven carbon atoms and the reaction solution of luciferase (Luciferase), and luminescence-producing reaction begins and stable lasting 0.01 to 8 second; When reaction is carried out, use the optical analyzer sensed light signal.
8, signal is analyzed, biological sample to be measured is carried out qualitative and quantitative.
The present invention has the following advantages for the detection method that present biochip is used always: do not have radiological hazard; Can use the very low optical instrument of detection sensitivity to detect, detect with low cost; The detection sensitivity height exceeds 2 to 4 orders of magnitude than detection method at present commonly used; Solved the analysis difficult problem that can not increase and cause because of protein; Use amount of reagent to be specially adapted to high-density biochip less.
Detection method of the present invention has following effect: cheap detecting instrument will play a significant role in the detection of biochip, solve the too high difficult problem of current chip testing cost; Because it has solved the difficulty that protein-chip can not increase from another angle, it will promote the development of protein-chip greatly; It is cold light that this detection method is used amount of reagent fluorescence few, highly sensitive, easy to operate, that sent, and these advantages are undoubtedly huge power concerning the development of high-density biochip.
Embodiment
The screening of type hepatitis antibody medicine:
1, will show the various bacteriophage separation and purification of all kinds of possible hepatitis antibodies of type by display technique of bacteriophage, be individually fixed on the chip in each reaction chamber with the acetaldehyde method then, and be numbered for various antibody.
2, use the Hepatitis virus of specific oxidation reductase (NADH-FMNOxidereductase) the mark deactivation of nicotinamide adenine dinucleotide reduced (NADH) or NADPH (NADPH) and flavin mononucleotide (FMN) (FMN).
3, in each reaction chamber, add deactivation that the enzyme labeling of 0.005 μ l crosses Hepatitis virus with the chip point sample instrument, and 37 ℃ of incubations 30 minutes.
4, at first mark deactivation Hepatitis virus and other non-specific junction mixture with the enzyme of buffer solution of sodium phosphate washing non-specific binding.
5, the phosphate solution that in the said chip reaction chamber, adds nicotinamide adenine dinucleotide reduced (NADH), flavin mononucleotide (FMN) (FMN) and the electron mediator ferrocene of removing oxygen.Nicotinamide adenine dinucleotide reduced (NADH), flavin mononucleotide (FMN) (FMN) generate nicotinamide adenine dinucleotide (NAD) and flavin mononucleotide reduced (FMNH under the catalysis of enzyme
2).
6, in enzymatic reaction, on chip electrode, apply-voltage of 1.0v, make the nicotinamide adenine dinucleotide (NAD) that is generated be reduced into nicotinamide adenine dinucleotide reduced (NADH) again at once by the electron mediator ferrocene.
7, flavin mononucleotide reduced (FMNH is carried out after 1000 to 10000 in above-mentioned circulation
2) a large amount of accumulation, at this moment add the reaction solution that contains aldehyde C-9 and marine bacteria luciferase (Luciferase) of oxygenation.
8, use the photomultiplier sensed light signal, and record the numbering of the reaction chamber of light signal.
9, by photomultiplier bundled software signal is analyzed, further quantitative after qualitative to biological sample to be measured.
10,, and can further analyze the pairing gene situation of these antibody according to gained result can judge which kind of phage display antibody of Hepatitis virus and the amount of displaying.
Claims (1)
1, a kind of method of light circulation scale-up version biochip test comprises the following steps:
A, biological sample to be detected is fixed in the reaction chamber of chip;
B, with nicotinamide adenine dinucleotide reduced or NADPH and the specific oxidation reductase of flavin mononucleotide (FMN) be marked on another kind can with above-mentioned biological sample identification and the biological sample that combines;
C, the step B acceptance of the bid biological sample respective reaction that the biological sample point sample of enzyme fixes with steps A on chip of recording a demerit is indoor;
D, with 20 to the 50 ℃ of following incubations of reaction of two kinds of biological samples among steps A and the step B 20 to 50 minutes;
E, with the biological sample of buffer solution and the non-specific bond of elute soln wash-out and then use deionized water wash;
F, in the said chip reaction chamber, add nicotinamide adenine dinucleotide reduced or the NADPH of removing oxygen, the solution of flavin mononucleotide (FMN) and electron mediator, nicotinamide adenine dinucleotide reduced or NADPH, flavin mononucleotide (FMN) generates nicotinamide adenine dinucleotide or nicotinamide-adenine dinucleotide phosphate and flavin mononucleotide reduced at once under the catalysis of enzyme, nicotinamide adenine dinucleotide that is generated in enzymatic reaction or nicotinamide-adenine dinucleotide phosphate directly or by the electron mediator ferrocene are reduced into nicotinamide adenine dinucleotide reduced or NADPH again under the effect of chip electrode;
Flavin mononucleotide reduced accumulation after G, above-mentioned circulation are carried out 100 to 10000, what at this moment add oxygenation contains the above aldehyde of seven carbon atoms and the reaction solution of luciferase, luminescence-producing reaction begins and stable lasting 0.01 to 8 second, when reaction is carried out, use the optical analyzer sensed light signal;
H, signal is analyzed, biological sample to be measured is carried out qualitative and quantitative.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02139105 CN1222773C (en) | 2002-09-26 | 2002-09-26 | An optical circulation amplification type biological chip detecting process |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02139105 CN1222773C (en) | 2002-09-26 | 2002-09-26 | An optical circulation amplification type biological chip detecting process |
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| Publication Number | Publication Date |
|---|---|
| CN1485617A CN1485617A (en) | 2004-03-31 |
| CN1222773C true CN1222773C (en) | 2005-10-12 |
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|---|---|---|---|
| CN 02139105 Expired - Fee Related CN1222773C (en) | 2002-09-26 | 2002-09-26 | An optical circulation amplification type biological chip detecting process |
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Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100425973C (en) * | 2006-03-30 | 2008-10-15 | 湖南大学 | Nano biological sensor for detecting NADH concentration and its detecting method |
| CN106018387A (en) * | 2016-05-13 | 2016-10-12 | 孙晓晖 | Adenosine triphosphate (ATP ) fluorescence detection method |
| EP3379252B1 (en) * | 2017-03-20 | 2022-10-26 | National Cheng Kung University | Molecular probe for signal amplification and assay using the same |
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Owner name: XIE BIN Free format text: FORMER OWNER: WUHAN VIROLOGY INSTITUTE,CHINAN ACADEMY OF SCIENCES Effective date: 20041231 |
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Effective date of registration: 20041231 Address after: 430071 small Hongshan, Wuhan, Hubei, Wuchang Applicant after: Xie Bin Address before: 430071 small Hongshan, Wuhan, Hubei, Wuchang Applicant before: Wuhan Institute of Virology, Chinese Academy of Sciences |
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