CN1304598C - ATP and NAD and method for analysis of associated enzyme and substrate - Google Patents
ATP and NAD and method for analysis of associated enzyme and substrate Download PDFInfo
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- CN1304598C CN1304598C CNB2004100466597A CN200410046659A CN1304598C CN 1304598 C CN1304598 C CN 1304598C CN B2004100466597 A CNB2004100466597 A CN B2004100466597A CN 200410046659 A CN200410046659 A CN 200410046659A CN 1304598 C CN1304598 C CN 1304598C
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Abstract
The present invention relates to an analysis technology for a coenzyme, a relative enzyme and a substrate thereof. A nucleic acid connecting system used for analysisi is prepared froom a molecular beacon nucleic acid probe, a nucleic acid segment and nucleic acid ligase using NAD as the coenzyme in a corresponding buffer solution; NAD, the relative enzyme and the substrate are analyzed in an NAD-free buffer solution, ATP is analyzed in an ATP-free buffer solution, molecular beacons, the ligase and two or more than two paired nucleic acid chains are added in a corresponding connection system buffer solution for the analysis at the temperature of 37 DEG C, the fluorescence intensity is monitored, NAD, ATP, the relative enzyme or the substrate to be measured are added until the fluorescent is stabilized, and the variation of the fluorescence intensity is recorded. The analysis technology has the advantages of fast, sensitive and accurate measurement and analysis of the coenzyme ATP and NAD, the relative enzyme and the substrate, simple operation and less input, has significant scientific value and broad market prospects and has larger social benefits and economic benefits.
Description
Technical field
The present invention relates to the detection method in the biochemical field, be specifically related to the analytical procedure of a kind of coenzyme and relevant enzyme and substrate.
Background technology
NAD is present in all animals, plant and the microbial cell as the coenzyme of present known more than 300 kinds of desaturases, is a kind of important substance that biological fermentation, life and physiological process participate in energy metabolism.The research that set up fast, sensitivity and accurate analytical method not only helps to promote vital process also has important and practical meanings for fields such as medical clinic applications, pharmaceutical analysiss simultaneously.
The detection of NAD, NADH at present comprises that the analytical procedure of its relevant enzyme mostly utilizes some enzymes that NAD is converted into NADH, utilize NADH to have maximum absorption to set up its ultra-violet absorption spectrometry or utilize the fluorescence of NADH to carry out direct fluorometric assay then at the 360nm ultraviolet region, also have electrochemical method, high performance capillary electrophoresis and HPCE-electrochemical method coupling, but the detectability of the method NAD of bibliographical information generally only is 10
-7M-10
-8M, report has developed NAD (H) flow injection analysis method recently, and detectability just reaches 1pmol, and the detection linearity range of aforesaid method is generally about two orders of magnitude.The aforesaid method insufficient sensitivity, feasible analysis to small system and denier system is restricted; Be subjected to simultaneously the interference of other material easily, be unfavorable for the living organisms analysis.
Triphosaden (ATP) is a macroergic compound in the organism, and its storage power needs for cell activities, is the transhipment station that the biomass cells self-energy shifts; What of ATP content can directly be reflected cytoactive, after the necrocytosis because the existence of ATP enzyme is arranged in the cell, the rapid hydrolysis of ATP, the content of therefore measuring Endogenous ATP can reflect the quantity of viable cell; At the ATP content of external test in the culture of tumor cell of different concns chemotherapeutics direct killing, can judge the sensitivity of this tumour cell to chemotherapeutics.Aspect environmental analysis, what of ATP content can reflect that also microbial numbers is a sludge activity, and detecting the ATP that microbial metabolism produces has become the focus that the manufacturer pays close attention in many industries such as makeup, food, medicine; Intracellular in addition ATP also can be used as an epithelial signaling molecule, plays important effect in the process distinguishing of necrocytosis pattern (apoptosis or necrosis).At present ATP detects the fluorescein method that mostly adopts, and minimumly detects 10
-12-10
-14M, but have downpayment having high input (needs are bought pertinent instruments) and operation and the more traditional shortcomings such as plating method complexity of interpretation of result simultaneously.
Therefore, set up fast, sensitive and accurately ATP and NAD analytical procedure not only help to promote the research of vital process, also there is important and practical meanings the while for fields such as clinical diagnosis, pharmaceutical analysiss.
Summary of the invention
The present invention is intended to develop a kind of analytical procedure of ATP, NAD and relevant enzyme and substrate of fast high-sensitive, to solve problem such as the not high and complicated operation of sensitivity that the present analysis method exists.For their analysis and application provides a kind of brand-brand-new way.
The present invention is achieved through the following technical solutions goal of the invention.The nucleic acid connected system that analytical procedure of the present invention is used is by the molecular beacon nucleic acid probe that ring portion and nucleic acid fragment sequence are complementary, afterbody one end has fluorescent mark, the other end has fluorescent mark or fluorescence quenching group, is the DNA of coenzyme or RNA nucleic acid ligase with two or more nucleic acid fragments of molecular beacon ring portion paired and with NAD at no NAD accordingly or do not have in the connected system buffered soln of ATP and form; The NAD analytical procedure is: add molecular beacon, ligase enzyme and two or more paired nucleic acid chains in the linked system damping fluid of no NAD, at 37 ℃ of incubations, monitor fluorescence intensity simultaneously, treat to add respectively after fluorescence is stablized the NAD of different concns, the variation of observing and writing down fluorescence intensity; The ATP analytical procedure is: add molecular beacon, ligase enzyme and two or more paired nucleic acid chains in the linked system damping fluid of no ATP, at 37 ℃ of incubations, monitor fluorescence intensity simultaneously, treat that the stable back of fluorescence adds ATP, observes and write down the variation of fluorescence intensity; The relevant enzyme analytical procedure is: add molecular beacon, ligase enzyme, two or more paired nucleic acid chains, enzyme reaction substrate and NADH in the linked system damping fluid of no NAD, at 37 ℃ of incubations, monitor fluorescence intensity simultaneously, treat that the stable back of fluorescence adds relevant enzyme, observes and write down the variation of fluorescence intensity; The analytical procedure of substrate is: the linker system at no NAD adds molecular beacon, ligase enzyme, two or more paired nucleic acid chains, NADH and relevant enzyme, at 37 ℃ of incubations, monitor fluorescence intensity simultaneously, add enzyme reaction substrate Deng the stable back of fluorescence, observe and note down the variation of fluorescent intensity.
The fluorescence dye that the used wavelength of fluoroscopic examination in the above-mentioned analytical procedure is modified according to molecular beacon is selected, and excitation wavelength is 338-560mm, and emission wavelength is 505-660mm.
Below in conjunction with accompanying drawing in detail the present invention is described in detail.
Description of drawings
Fig. 1 is an analytical procedure schematic diagram of the present invention
The experimental result that Fig. 2 analyzes for NAD
Fig. 3 analyzes optionally experimental result for NAD
The experimental result that Fig. 4 analyzes for ATP
Fig. 5 is the experimental result that relevant enzyme (GLDH) is analyzed
Fig. 6 is to the related substrates (experimental result that α-KG) analyzes
Such as Fig. 1 (on) signal, detection architecture of the present invention by molecular beacon, nucleic acid fragment 1,2 and nucleic acid ligase formed, wherein nucleic acid fragment 1,2 respectively with half hybridization of molecular beacon annulus. When NAD or ATP exist, nucleic acid ligase is connected to form nucleic acid fragment 1,2 the connection product of a long-chain, molecular beacon is opened in the hybridization of this connection product and molecular beacon ring portion, produces obvious fluorescence signal, has so just realized NAD or the high-sensitive detection analysis of ATP.
In the system shown in Fig. 1 (descending), if NAD is produced by the reaction of glutamte dehydrogenase (Glutamate Dehydrogenase, GLDH) catalysis, just can carry out real-time analysis to the activity of GLDH this moment. In like manner, if NAD is produced by other enzymatic reaction, also can carry out real-time analysis to other enzymatic activity. In like manner utilize the present invention can set up analytical method to zymolyte (KG). This method can carry out fast NAD, ATP, relevant enzyme and substrate, sensitive and measure accurately, simple to operate, less investment, this brand-new analysis approach has broad application prospects in fields such as bioanalysis, medical clinic applications, pathological studies.
Embodiment
Embodiment 1 (NAD analysis)
At 100 μ L reaction buffers (30mM Tris-HCl (PH 8.0), 2.5mM CaCl
2, 5mM DTT, 10mM MgCl
21.2mM EDTA and 0.05%BSA) middle 300nM molecular beacon 5 '-(the TAMRA)-CCTCTC CGT GTC TG TACTTC CCG TCA GAGAGG-(DABCYL)-3 ' that adds, 6.0U the E.coli dna ligase, 300nM nucleic acid fragment 5 '-GAC GGG AAG-3 ' and another nucleic acid fragment 5 ' of 300nM-TAC AAG ACA C-3 ', at 37 ℃ of incubations, simultaneously with luminoscope F-2500 monitoring fluorescence intensity, excitation wavelength 521nm, emission wavelength 578nm, treat that the stable back of fluorescence adds the NAD of different concns, obtains fluorescence intensity real time scan curve shown in Fig. 2 (left side).
Fig. 2 (left side) be different concns NAD the real-time monitoring curve that connects of corresponding nucleic acid, as can be seen, along with the raising of NAD concentration, the initial velocity of reaction is increasing, will connect initial velocity and NAD concentration is mapped obtain Fig. 2 (right side).To obtain the NAD minimal detectable concentration be 3 * 10 to data from figure
-10M (S/N=3) relatively improves 2-3 the order of magnitude with document; The lowest detection amount is 3 * 10
-14Mol has two to detect linearity range, is respectively 0.3-40nM and 40-300nM.
NAD, the NADH, NADP, NADPH, dATP, ATP, ADP and the AMP that in above-mentioned damping fluid, add 300nM respectively, the fluorescence intensity of record sample changes, the fluorescence of trying to achieve each sample respectively strengthens initial velocity, be that benchmark is made normalized with the NAD sample again, the result as shown in Figure 3, have a small amount of fluorescence enhancing except that adding NADH, NADP, NADPH, dATP, ATP, ADP and AMP etc. do not have obvious fluorescence enhancing signal, illustrate that this method has very high selectivity.
The fluorescence dye that the used wavelength of fluoroscopic examination is modified according to molecular beacon is selected (seeing Table 1).
Table 1. fluorescence dye commonly used and maximum absorption and emission wavelength
| The fluorescence dye title | Maximum absorption wavelength (nm) | Maximum emission wavelength (nm) |
| Tetramethyl-rhodamine (TAMRA) | 555 | 580 |
| Fluorescein (Fluorescein) | 495 | 525 |
| Fluorescein isothiocyanate (FITC) | 492 | 519 |
| Fluoresceincarboxylic acid (FAM) | 492 | 518 |
| Rhodamine 123 (Rhodamine 123) | 560 | 540-660 |
| Acridine orange (Acridine orange) | 405 | 585 |
| Trypaflavine (Acridine yellow) | 455 | 620 |
| Propidium iodide (Propidium iodide) | 488 | 620 |
| Bromination second pyridine (Ethidium bromide) | 488 | 610 |
| Mithramycin (Mithramycin) | 457 | 570 |
| Pyronine Y (Pyronin Y) | 488 | 580 |
| Hirst 33258 (Hoechst 33258) | 338 | 505 |
| Fluorescent yellow Lucifer yellow | 428 | 544 |
| Yihong Eosin | 525 | 546 |
| SYBR Green I | 498 | 522 |
| SYBR Gold | 495 | 540 |
| SYPRO Orange | 475 | 580 |
| SYPRO Red | 545 | 635 |
Annotate:, when concrete the measurement, suitably adjust according to practical situation for avoiding exciting light to radiative influence.
Embodiment 2 (ATP analysis)
At 100 μ L reaction buffers (66mM Tris-HCl (PH 8.0), 6.6mM MgCl
2, 10mM DTT) in add the T4DNA ligase enzyme of 400nM molecular beacon 5 '-(TAMRA)-CCTCTC CGT GTC TG TAC TTC CCG TCA GAGAGG-(DABCYL)-3 ', 1.4U, 400nM nucleic acid fragment 5 '-GAC GGG AAG-3 ' and another nucleic acid fragment 5 ' of 400nM-TAC AAG ACA C-3 ', at 37 ℃ of incubations, simultaneously with luminoscope F-2500 monitoring fluorescence intensity, excitation wavelength 521nm, emission wavelength 578nm, treat that the stable back of fluorescence adds the ATP of different concns, obtains fluorescence intensity real time scan curve shown in Fig. 4 (left side).
Fig. 4 (left side) be different concns ATP the real-time monitoring curve that connects of corresponding nucleic acid, as can be seen, along with the raising of ATP concentration, the initial velocity of reaction is increasing.Mapping obtains Fig. 4 (right side) to ATP concentration will to connect initial velocity, and it is limited to 2nM under detecting, and the linear analysis scope is 2-300nM.
Embodiment 3 (glutamate dehydrogenase analysis)
At 100 μ L reaction buffers (30mM Tris-HCl (PH 8.0), 5mM DTT, 2.5mM CaCl
2, 10mM MgCl
2, 1.2mM EDTA and 0.05%BSA) in add 5mM NH
4Cl, 0.5mM α-Ketoglutarate (α-KG), 300 μ MNADH, 300nM molecular beacon 5 '-(TAMRA)-CCTCTC CGT GTC TG TAC TTC CCG TCA GAGAGG-(DABCYL)-3 ', 0.48U the E.coli dna ligase, 300nM nucleic acid fragment 5 '-GAC GGG AAG-3 ' and another nucleic acid fragment 5 ' of 300nM-TAC AAG ACA C-3 ', at 37 ℃ of incubations, simultaneously with luminoscope F-2500 monitoring fluorescence intensity, excitation wavelength 521nm, emission wavelength 578nm, treat that the stable back of fluorescence adds the 0.34U glutamate dehydrogenase, observes and write down the variation of fluorescence intensity.
Experimental result can see that the fluorescence intensity of sample strengthens rapidly after glutamate dehydrogenase adds as shown in Figure 5, and experimental result proves that this method can realize the detection to glutamate dehydrogenase.
Embodiment 4 (α-Tong Wuersuan analysis)
At 100 μ L reaction buffers (30mM Tris-HCl (PH 8.0), 5mM DTT, 2.5mM CaCl
2, 10mM MgCl
2, 1.2mM EDTA and 0.05%BSA) in add 5mM NH
4Cl, 0.34Unit glutamate dehydrogenase, 300 μ M NADH, 300nM molecular beacon 5 '-(TAMRA)-CCTCTC CGT GTC TG TAC TTC CCG TCA GAGAGG-(DABCYL)-3 ', 0.48U the E.coli dna ligase, 300nM nucleic acid fragment 5 '-GAC GGG AAG-3 ' and another nucleic acid fragment 5 ' of 300nM-TAC AAG ACA C-3 ', at 37 ℃ of incubations, simultaneously with luminoscope F-2500 monitoring fluorescence intensity, excitation wavelength 521nm, emission wavelength 578nm, treat that the stable back of fluorescence adds 0.5mM α-Ketoglutarate (α-KG), observe and write down the variation of fluorescence intensity.
Experimental result can see that the fluorescence intensity of sample strengthens rapidly, proves that this method can realize the detection to α-Ketoglutarate after α-Ketoglutarate adds as shown in Figure 6.
Claims (1)
1, the analytical procedure of a kind of ATP, NAD and relevant enzyme and substrate, it is characterized in that nucleic acid connected system that this method uses by ring portion and nucleic acid fragment sequence be complementary, afterbody one end has the molecular beacon nucleic acid probe that the fluorescent mark the other end has fluorescent mark or fluorescence quenching group, form with two or more nucleic acid chains of molecular beacon ring portion paired, DNA or RNA nucleic acid ligase and the linked system buffered soln that do not have NAD accordingly or do not have an ATP
(1) the NAD analytical procedure is: add molecular beacon in the linked system damping fluid of no NAD, be the DNA of coenzyme or RNA nucleic acid ligase and two or more and molecular beacon ring portion paired nucleic acid chains with NAD, at 37 ℃ of incubations, monitor fluorescence intensity simultaneously, treat that fluorescence adds the NAD sample respectively after stable, observe and write down the variation of fluorescence intensity
(2) the ATP analytical procedure is: add molecular beacon, T4 dna ligase and two or more and molecular beacon ring portion paired nucleic acid chains in the linked system damping fluid of no ATP, at 37 ℃ of incubations, monitor fluorescence intensity simultaneously, treat that the stable back of fluorescence adds ATP, observe and write down the variation of fluorescence intensity
(3) the relevant enzyme analytical procedure is: add molecular beacon in the linked system damping fluid of no NAD, be the DNA of coenzyme or RNA nucleic acid ligase, two or more and molecular beacon ring portion paired nucleic acid chains, NADH and enzyme reaction substrate with NAD, at 37 ℃ of incubations, monitor fluorescence intensity simultaneously, treat that the stable back of fluorescence adds relevant enzyme, observe and write down the variation of fluorescent intensity
(4) analytical procedure of substrate is: add molecular beacon in the linked system damping fluid of no NAD, be the DNA of coenzyme or RNA nucleic acid ligase, two or more and molecular beacon ring portion paired nucleic acid chains, NADH and relevant enzyme with NAD, at 37 ℃ of incubations, monitor fluorescence intensity simultaneously, add enzyme reaction substrate Deng the stable back of fluorescence, observe and write down the variation of fluorescent intensity.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101644679B (en) * | 2008-08-05 | 2011-08-31 | 国家纳米科学中心 | Kit and method for using same to detect ATP content, enzyme and medicament |
| CN103207167B (en) * | 2012-12-25 | 2015-04-15 | 西安交通大学 | Preparation method of fluorescence resonance system for rapid detection of ATP in mitochondria |
| CN103276096A (en) * | 2013-06-08 | 2013-09-04 | 中南大学 | Nicking endonuclease constant-temperature amplification technology based ATP or NAD system and detection method thereof |
| CN106018387A (en) * | 2016-05-13 | 2016-10-12 | 孙晓晖 | Adenosine triphosphate (ATP ) fluorescence detection method |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1148870A (en) * | 1994-04-06 | 1997-04-30 | 布拉福国际公司 | Method for calibrating chemical analysis |
| US6355421B1 (en) * | 1997-10-27 | 2002-03-12 | Boston Probes, Inc. | Methods, kits and compositions pertaining to PNA molecular beacons |
| WO2003054223A2 (en) * | 2001-12-20 | 2003-07-03 | Mcmaster University | Tripartite molecular beacons |
| CN1488763A (en) * | 2002-10-09 | 2004-04-14 | 英科新创(厦门)科技有限公司 | Improved molecular beacon probe and its use |
| US6743582B2 (en) * | 2000-06-08 | 2004-06-01 | Becton, Dickinson And Company | Probes and methods for detection of nucleic acids |
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2004
- 2004-08-13 CN CNB2004100466597A patent/CN1304598C/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1148870A (en) * | 1994-04-06 | 1997-04-30 | 布拉福国际公司 | Method for calibrating chemical analysis |
| US6355421B1 (en) * | 1997-10-27 | 2002-03-12 | Boston Probes, Inc. | Methods, kits and compositions pertaining to PNA molecular beacons |
| US6743582B2 (en) * | 2000-06-08 | 2004-06-01 | Becton, Dickinson And Company | Probes and methods for detection of nucleic acids |
| WO2003054223A2 (en) * | 2001-12-20 | 2003-07-03 | Mcmaster University | Tripartite molecular beacons |
| CN1488763A (en) * | 2002-10-09 | 2004-04-14 | 英科新创(厦门)科技有限公司 | Improved molecular beacon probe and its use |
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