CN1304598C - ATP and NAD and method for analysis of associated enzyme and substrate - Google Patents

Abstract

The invention relates to an analysis technology of coenzymes and coenzyme-related enzymes and substrates. The nucleic acid ligation system used in the analysis is composed of molecular beacon nucleic acid probes, nucleic acid fragments and nucleic acid ligase with NAD as a coenzyme in the corresponding buffer solution; the analysis of NAD, related enzymes and substrates is carried out in NAD-free buffer , The analysis of ATP is carried out in ATP-free buffer. For the analysis, molecular beacons, ligase and two or more paired nucleic acid strands are added to the corresponding connection system buffer, incubated at 37°C, and the fluorescence intensity is monitored. After the fluorescence is stabilized, add NAD, ATP, related enzymes or substrates to be tested, and record the change of fluorescence intensity. The detection and analysis of the coenzymes ATP, NAD, related enzymes and substrates in the present invention is fast, sensitive, accurate, simple in operation, low in investment, has important scientific value and broad market prospect, and has great social and economic benefits.

Description

A method for analyzing ATP, NAD and related enzymes and substrates

technical field

The invention relates to a detection method in the field of biochemistry, in particular to an analysis method for coenzymes, related enzymes and substrates.

Background technique

As the coenzyme of more than 300 known dehydrogenases, NAD exists in the cells of all animals, plants and microorganisms, and is an important substance involved in energy metabolism in biological fermentation, life and physiological processes. The establishment of fast, sensitive and accurate analytical methods not only helps to promote the study of life processes, but also has important practical significance for the fields of medical clinical diagnosis and drug analysis.

At present, most of the detection methods of NAD and NADH, including the analysis of related enzymes, use some enzymes to convert NAD into NADH, and then use NADH to have the maximum absorption in the 360nm ultraviolet region to establish its ultraviolet absorption spectrophotometry or use the fluorescence of NADH for direct fluorescence measurement. , there are also electrochemical methods, high-efficiency capillary electrophoresis and high-efficiency capillary electrophoresis-electrochemical methods, but the detection limit of NAD in the methods reported in the literature is generally only 10 ^-7 M-10 ^-8 M. Recently, it has been reported that the development of NAD ( H) The flow injection analysis method has a detection limit of 1 pmol, and the detection linear range of the above method is generally about two orders of magnitude. The sensitivity of the above method is not enough, so that the analysis of micro system and extremely micro system is limited; at the same time, it is easily interfered by other substances, which is not conducive to the analysis of living organisms.

Adenosine triphosphate (ATP) is a high-energy compound in organisms. It stores energy for the needs of cell life activities. It is a transfer station for energy transfer in biological cells; the amount of ATP can directly reflect cell activity. In the presence of enzymes, ATP is rapidly hydrolyzed, so measuring the content of endogenous ATP can reflect the number of living cells; measuring the ATP content in cultured tumor cells directly killed by different concentrations of chemotherapy drugs in vitro can judge the tumor cell’s ability to chemotherapy. drug sensitivity. In terms of environmental analysis, the amount of ATP content can also reflect the number of microorganisms, that is, the activity of sludge. The detection of ATP produced by microbial metabolism has become the focus of manufacturers in many industries such as cosmetics, food, and pharmaceuticals; As a signaling molecule of epithelial cells, it plays a very important role in the discrimination of cell death mode (apoptosis or necrosis). At present, ATP is mostly detected by the fluorescein method, which can detect as low as 10 ^-12 -10 ^-14 M, but at the same time, there are disadvantages such as large initial investment (need to purchase related instruments) and complicated operation and result analysis compared with the traditional plate culture method.

Therefore, establishing a fast, sensitive and accurate analysis method for ATP and NAD not only helps to promote the research of life process, but also has important practical significance for clinical diagnosis, drug analysis and other fields.

Contents of the invention

The present invention aims to develop a fast and highly sensitive analysis method for ATP, NAD and related enzymes and substrates, so as to solve the problems of low sensitivity and complicated operation in the current analysis method. Provide a new way for their analysis and application.

The present invention realizes the purpose of the invention through the following technical solutions. The nucleic acid connection system used in the analysis method of the present invention consists of a molecular beacon nucleic acid probe whose ring part matches the nucleic acid fragment sequence, has a fluorescent label at one end of the tail, and a fluorescent label or a fluorescent quenching group at the other end, and a molecular beacon nucleic acid probe with a molecular beacon ring The partial pair of two or more nucleic acid fragments and the DNA or RNA nucleic acid ligase with NAD as the coenzyme are composed in the corresponding NAD-free or ATP-free connection system buffer solution; the NAD analysis method is: in the NAD-free connection system Add molecular beacons, ligase, and two or more paired nucleic acid strands to the buffer, incubate at 37°C, and monitor the fluorescence intensity at the same time. After the fluorescence is stable, add different concentrations of NAD, observe and record the fluorescence intensity. Change; the ATP analysis method is: add molecular beacons, ligase and two or more paired nucleic acid strands to the ATP-free connection system buffer, incubate at 37°C, monitor the fluorescence intensity at the same time, and wait for the fluorescence to stabilize Add ATP, observe and record the change of fluorescence intensity; the relevant enzyme analysis method is: add molecular beacon, ligase, two or more paired nucleic acid strands, enzyme reaction substrate and NADH, incubated at 37°C, while monitoring the fluorescence intensity, after the fluorescence is stable, add relevant enzymes, observe and record the change of fluorescence intensity; the analysis method of the substrate is: add molecular beacons to the linker system without NAD, ligase , two or more paired nucleic acid strands, NADH and related enzymes, incubate at 37°C while monitoring the fluorescence intensity, add the enzyme reaction substrate after the fluorescence is stable, observe and record the changes in the fluorescence intensity of the sample.

The wavelength used for the fluorescence detection in the above analysis method is selected according to the fluorescent dye modified by the molecular beacon, the excitation wavelength is 338-560mm, and the emission wavelength is 505-660mm.

The present invention is described in detail below in conjunction with accompanying drawing.

Description of drawings

Fig. 1 is a schematic diagram of the analytical method of the present invention

Figure 2 shows the experimental results of NAD analysis

Figure 3 shows the experimental results of NAD analysis selectivity

Figure 4 is the experimental results of ATP analysis

Figure 5 is the experimental results of the analysis of related enzymes (GLDH)

Figure 6 is the experimental results of the analysis of related substrates (α-KG)

As shown in Figure 1 (above), the detection system of the present invention consists of molecular beacons, nucleic acid fragments 1 and 2, and nucleic acid ligase, wherein nucleic acid fragments 1 and 2 hybridize to half of the circular part of the molecular beacon respectively. When NAD or ATP exists, the nucleic acid ligase will connect the nucleic acid fragments 1 and 2 to form a long-chain ligation product, and the hybridization of the ligation product with the molecular beacon ring will open the molecular beacon and produce an obvious fluorescent signal, so that Highly sensitive detection and analysis of NAD or ATP is realized.

In the system shown in Figure 1 (bottom), if NAD is produced by a reaction catalyzed by glutamate dehydrogenase (GLDH), then the activity of GLDH can be analyzed in real time. Similarly, if NAD is produced by reactions catalyzed by other enzymes, other enzyme activities can also be analyzed in real time. Similarly, the method for analyzing the enzyme substrate (α-ketoglutarate) can be established by using the present invention. This method can quickly, sensitively and accurately measure NAD, ATP, related enzymes and substrates, with simple operation and low investment. This new analysis method has wide applications in the fields of biological analysis, medical clinical diagnosis, pathological research, etc. prospect.

Detailed ways

Embodiment 1 (NAD analysis)

Add 300 nM molecular beacon 5'-(TAMRA)-CCTCTC CGT to 100 μL reaction buffer (30 mM Tris-HCl (PH 8.0), 2.5 mM CaCl ₂ , 5 mM DTT, 10 mM MgCl ₂ , 1.2 mM EDTA, and 0.05% BSA) GTC TG TACTTC CCG TCA GAGAGG-(DABCYL)-3', 6.0 U of E.coli DNA ligase, 300nM nucleic acid fragment 5'-GAC GGG AAG-3' and 300nM another nucleic acid fragment 5'-TAC AAG ACA C- 3', incubate at 37°C, and monitor the fluorescence intensity with a fluorescence instrument F-2500 at the same time, the excitation wavelength is 521nm, and the emission wavelength is 578nm. After the fluorescence is stable, add different concentrations of NAD, and the real-time scanning curve of the fluorescence intensity is obtained as shown in Figure 2 (left) shown.

Figure 2 (left) is the real-time monitoring curve of nucleic acid connection corresponding to different concentrations of NAD. It can be seen that as the concentration of NAD increases, the initial velocity of the reaction increases. The initial velocity of the connection is plotted against the concentration of NAD to obtain Figure 2 (right). From the data in the figure, the minimum detection concentration of NAD is 3×10 ^-10 M (S/N=3), which is 2-3 orders of magnitude higher than the literature; the minimum detection amount is 3×10 ^-14 mol, and there are two detection linearities The ranges are 0.3-40nM and 40-300nM, respectively.

Add 300nM NAD, NADH, NADP, NADPH, dATP, ATP, ADP and AMP to the above buffer solution respectively, record the change of the fluorescence intensity of the sample, obtain the initial fluorescence enhancement velocity of each sample respectively, and then take the NAD sample as the benchmark After normalization processing, the results are shown in Figure 3. Except for a small amount of fluorescence enhancement by adding NADH, NADP, NADPH, dATP, ATP, ADP and AMP have no obvious fluorescence enhancement signals, indicating that the method has high selectivity.

The wavelength used for fluorescence detection is selected according to the fluorescent dye modified by the molecular beacon (see Table 1).

Table 1. Commonly used fluorescent dyes and their maximum absorption and emission wavelengths Fluorescent dye name Maximum absorption wavelength (nm) Maximum emission wavelength (nm) Tetramethylrhodamine (TAMRA) 555 580 Fluorescein 495 525 Fluorescein isothiocyanate (FITC) 492 519 Carboxyfluorescein (FAM) 492 518 Rhodamine 123 (Rhodamine 123) 560 540-660 Acridine orange 405 585 Acridine yellow 455 620 Propidium iodide 488 620 Ethidium bromide 488 610 Mithramycin 457 570 Pyronin Y (Pyronin Y) 488 580 Hoechst 33258 (Hoechst 33258) 338 505 Fluorescent yellow Lucifer yellow 428 544 Eosin 525 546 SYBR Green I 498 522 SYBR Gold 495 540 SYPRO Orange 475 580 SYPRO Red 545 635

Note: In order to avoid the influence of the excitation light on the emission light, it should be adjusted appropriately according to the actual situation during the specific measurement.

Embodiment 2 (ATP analysis)

Add 400 nM molecular beacon 5'-(TAMRA)-CCTCTC CGT GTC TG TAC TTC CCG TCA GAGAGG-(DABCYL)- to 100 μL reaction buffer (66 mM Tris-HCl (PH 8.0), 6.6 mM MgCl ₂ , 10 mM DTT) 3', 1.4U of T4 DNA ligase, 400nM nucleic acid fragment 5'-GAC GGG AAG-3' and 400nM another nucleic acid fragment 5'-TAC AAG ACA C-3', incubated at 37°C, while using a fluorometer F -2500 monitors the fluorescence intensity, the excitation wavelength is 521nm, and the emission wavelength is 578nm. After the fluorescence is stabilized, different concentrations of ATP are added to obtain the real-time scanning curve of the fluorescence intensity, as shown in Figure 4 (left).

Figure 4 (left) is the real-time monitoring curve of nucleic acid connection corresponding to different concentrations of ATP. It can be seen that with the increase of ATP concentration, the initial speed of the reaction increases. Figure 4 (right) was obtained by plotting the initial connection velocity against the ATP concentration, the lower limit of detection was 2nM, and the linear analysis range was 2-300nM.

Embodiment 3 (glutamate dehydrogenase analysis)

Add 5 mM NH ₄ Cl _{, 0.5} mM α-Ketoglutarate (α- KG), 300μM NADH, 300nM molecular beacon 5'-(TAMRA)-CCTCTC CGT GTC TG TAC TTC CCG TCA GAGAGG-(DABCYL)-3', 0.48U E.coli DNA ligase, 300nM nucleic acid fragment 5'-GAC GGG AAG-3' and 300nM another nucleic acid fragment 5'-TAC AAG ACA C-3', incubated at 37°C, while monitoring the fluorescence intensity with a fluorescence instrument F-2500, the excitation wavelength is 521nm, the emission wavelength is 578nm, and the fluorescence is stable Then add 0.34U glutamate dehydrogenase, observe and record the change of fluorescence intensity.

The experimental results are shown in Figure 5. It can be seen that after the addition of glutamate dehydrogenase, the fluorescence intensity of the sample increases rapidly, and the experimental results prove that this method can realize the detection of glutamate dehydrogenase.

Embodiment 4 (alpha-ketoglutarate analysis)

Add 5 mM NH ₄ Cl, 0.34 Unit of glutamate dehydrogenase to 100 μL of reaction buffer (30 mM Tris-HCl (PH 8.0), 5 mM DTT, 2.5 mM CaCl ₂ , 10 mM MgCl ₂ , 1.2 mM EDTA and 0.05% BSA) , 300μM NADH, 300nM molecular beacon 5'-(TAMRA)-CCTCTC CGT GTC TG TAC TTC CCG TCA GAGAGG-(DABCYL)-3', 0.48U E.coli DNA ligase, 300nM nucleic acid fragment 5'-GAC GGG AAG-3' and 300nM another nucleic acid fragment 5'-TAC AAG ACA C-3', incubated at 37°C, while monitoring the fluorescence intensity with a fluorescence instrument F-2500, the excitation wavelength is 521nm, the emission wavelength is 578nm, after the fluorescence is stable Add 0.5mM α-Ketoglutarate (α-KG), observe and record the change of fluorescence intensity.

The experimental results are shown in Figure 6. It can be seen that the fluorescence intensity of the sample increases rapidly after the addition of α-Ketoglutarate, which proves that this method can realize the detection of α-Ketoglutarate.

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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