CN102321759B - Fluorescence method for detecting S1 nuclease and inhibitor thereof - Google Patents
Fluorescence method for detecting S1 nuclease and inhibitor thereof Download PDFInfo
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- CN102321759B CN102321759B CN 201110245341 CN201110245341A CN102321759B CN 102321759 B CN102321759 B CN 102321759B CN 201110245341 CN201110245341 CN 201110245341 CN 201110245341 A CN201110245341 A CN 201110245341A CN 102321759 B CN102321759 B CN 102321759B
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Abstract
The invention is a fluorescence method for detecting S1 nuclease and inhibitors thereof by using oligonucleotide and graphene oxide. The method uses graphene oxide as a solid carrier, allows a dye-labelled oligonucleotide probe to adhere to the surface of graphene oxide by the interaction between graphene oxide and nucleic acid bases, and performs fluorescence quenching. Undegraded complementary strands can form a double-spiral structure with the probe by hybridization, and are detached from the surface of graphene oxide, and thus the system fluorescence is recovered; in the presence of S1 nuclease, the complementary strands are degraded into small base gragments, and can not form a double-spiral structure with the probe, and the probe still adheres to the surface of graphene oxide, and the fluorescence is in a quenching state; adenosine triphosphate can effectively inhibit the degradation effect of nuclease on the complementary strands, and thus the system fluorescence is recovered. Therefore, by detecting the change of the system fluorescence intensity, the detection of S1 nuclease and inhibitors thereof can be realized. The method of the invention is simple and rapid, has high sensitivity and selectivity, and has important meaning with respect to nuclease detection.
Description
Technical field
The invention belongs to bio-sensing and analysis field, particularly a kind of fluorescence detection method that utilizes oligonucleotide and graphene oxide to detect nuclease and inhibitor thereof.
Background technology
Nuclease refers to act on the enzyme of the phosphodiester bond of nucleic acid.According to the different classification of substrate, can be divided into DNA enzyme and RNA enzyme; Different according to cutting part, can be divided into endonuclease, restriction enzyme and exonuclease again.Nuclease in organism is responsible for the degraded of the inside and outside catalytic nucleic acid of cell, and vital process is played very important effect.The purposes of nuclease comprises that the important gene such as shearing, modification and degraded after synthetic and reparation and the rna transcription that participates in DNA copy and the genetic expression process; Be responsible for removing unnecessary, nucleic acid that structure and function is abnormal, also can remove the Exogenous Nucleic Acid of invading cell simultaneously; Nucleic acid in Digestive system in degraded food is in order to absorption; It is the important tool enzyme in the extracorporeal recombinant DNA technology.
Due to the significance of nuclease physiological function, be subject in recent years scientists and more and more pay close attention to, therefore quick in the urgent need to seeking, easy, highly sensitive method detects nuclease.Thereupon, the test material of nuclease and the development research of method are become a focus.The novel analytical procedure that is directed at present the nuclease detection is developed rapidly in fields such as life science and disease molecules diagnosis.To deepening continuously of nuclease research, will make larger contribution to exploration and the future of humanity of life quintessence.
Graphene oxide is learned and electrical properties with its special mechanics, quantum in recent years, paid attention to by physics and material educational circles.Due to advantages such as it have that excellent electric property, thermal conductivity are good, good water solubility, specific surface area are large, also the detection for biomolecules provides new thinking.The present successful Application in bio-sensing along with Single Walled Carbon Nanotube, graphene oxide also becomes the object of sensory field favor gradually.Due to the special property of graphene oxide, make that testing process is simple to operate, reaction fast, greatly reduced like this step of experiment, can improve significantly sensitivity and the specificity of detection simultaneously.
Summary of the invention
Technical problem: the technical problem to be solved in the present invention is the deficiency that exists for existing nuclease detection method, a kind of fluorescent method that utilizes oligonucleotide and graphene oxide to detect S1 nuclease and inhibitor thereof is provided, and it has higher specificity and sensitivity.
Technical scheme: the present invention detects the fluorescent method of S1 nuclease and inhibitor thereof, it is characterized in that the method comprises the following steps:
A. the oligonucleotide fluorescent probe with dye marker joins in buffered soln, take 480nm as excitation wavelength, carries out fluoroscopic examination, records the fluorescent emission bands of a spectrum of this probe;
B. graphene oxide is joined in mentioned solution, graphene oxide forms oligonucleotide/graphene oxide mixture as the oligonucleotide probe of the free rolled state of solid phase carrier absorption, at this moment the oxidized Graphene quencher of fluorescence of dyestuff;
C. through the oligonucleotide complementary strand of S1 nuclease cutting, join in oligonucleotide/graphene complex and react;
D. the oligonucleotide complementary strand forms little base fragment through the specificity Degradation of S1 nuclease, can't form double-spiral structure with the oligonucleotide chain probe on graphene oxide surface, thereby oligonucleotide probe can not break away from the surface of graphene oxide, and the fluorescent signal of dyestuff can not recover; The degree of recovering according to fluorescent signal realizes the qualitative and detection by quantitative to the S1 nuclease;
E. the oligonucleotide complementary strand through the effect of S1 nuclease degradation, adds in oligonucleotide/graphene oxide mixture and reacts under the inhibitor adenosine triphosphate exists;
F. the oligonucleotide complementary strand can not formed double-spiral structure with oligonucleotide probe by the S1 nuclease degradation under the inhibitor adenosine triphosphate exists, and oligonucleotide probe breaks away from the surface of graphene oxide, and the fluorescent signal of dyestuff is recovered; The degree of recovering according to fluorescent signal realizes the qualitative and detection by quantitative to the inhibitor adenosine triphosphate.
Wherein:
Step a) described oligonucleotide fluorescent probe with dye marker joins in buffered soln, detects with spectrophotofluorometer, and dyestuff used is fluorescein; Dye marker is at 5 ' end of oligonucleotide probe; The oligonucleotide probe based composition is: 5 '-FAM-ATCTTGACTATGTGGGTGCT-3 '; Buffered soln used is 20mM Tris-HCl, 100mM NaCl, 5mM KCl, 5mM MgCl
2, pH=7.4; Fluorescence detection method used is: probe solution is joined the scanning of carrying out fluorescence emission spectrum in 1.6mL buffered soln, excitation wavelength is 480nm, and the emission wavelength sweep limit is 490-650nm.
Step b) described oligonucleotide/graphene oxide complex formation is to make oligonucleotide probe be adsorbed onto the graphene oxide surface by non-covalent interaction; After oligonucleotide was adsorbed onto the graphene oxide surface, the oxidized Graphene quencher of the fluorescence of dyestuff was after joining graphene oxide in the buffered soln that contains oligonucleotide probe, room temperature was placed 5 minutes, then carry out fluoroscopic examination, excitation wavelength is 480nm, and sweep limit is 490-650nm.
Step c) through the oligonucleotide complementary strand of S1 nuclease cutting, join in oligonucleotide/graphene complex and react, oligonucleotide complementary strand probe based composition used is: 5 '-AGCACCCACATAGTCAAGAT-3 '; Adding after the oligonucleotide complementary strand solution that contains S 1 enzyme or other isopyknic contrast solution fluorescent signal to recover the time used is room temperature, about 25 minutes.
Described fluorescent signal detects with spectrophotofluorometer, and excitation wavelength is 480nm, and the emission wavelength sweep limit is 490-650nm.Described fluorescent probe is any one section single stranded oligonucleotide, and dye marker is at 5 ' end, or 3 ' end.Described dye marker is fluorescein or fluorescein isothiocyanate.
Steps d) described oligonucleotide complementary strand is through the effect of S1 nuclease degradation, be by the S1 nuclease to single stranded DNA or the specific Degradation of RNA, form 5 ' phosphoric acid structure.
The S1 nuclease is cut process enzyme used to the enzyme of oligonucleotide complementary strand, and to cut solution be 2mM CH
3COONa, 15mMNaCl, 0.1mMZnSO
4, pH=4.6; Enzyme Qie Wendu is 37 ℃, and the time is 30 minutes.
Step e) described oligonucleotide complementary strand through the effect of S1 nuclease degradation, is that adenosine triphosphate can suppress the S1 nuclease to the specificity Degradation of single stranded DNA or RNA cutting under the inhibitor adenosine triphosphate exists; When the inhibitor adenosine triphosphate existed, the S1 nuclease was identical with above-mentioned enzyme tangent condition to the action condition of oligonucleotide complementary strand.
Beneficial effect: according to fluorescence detection method of the present invention, can realize simultaneously the detection to S1 nuclease and inhibitor adenosine triphosphate thereof.When containing other nuclease and inhibitor in system, still can realize its high-sensitivity detection.Therefore, the inventive method is expected to detection for actual clinical sample amplifying nucleic acid enzyme provides a kind of easy, method fast.
Description of drawings
Fig. 1 is that the present invention utilizes oligonucleotide and graphene oxide to detect the fundamental diagram of the fluorescence detection method of nuclease and inhibitor thereof.
Fig. 2 is that the present invention utilizes oligonucleotide and graphene oxide to detect without the fluoroscopic examination of the DNA chain of S1 enzyme liberating figure as a result.A is oligonucleotide probe; B is oligonucleotide probe+graphene oxide; C is oligonucleotide probe+complementary strand+graphene oxide; D is oligonucleotide probe+incomplementarity chain+graphene oxide.
Fig. 3 is differential responses system fluoroscopic examination comparison diagram as a result in S1 enzyme and inhibitor testing process thereof.A is oligonucleotide probe; B is oligonucleotide probe+graphene oxide; C is oligonucleotide probe+S1 nuclease+complementary strand+graphene oxide; D is oligonucleotide probe+S1 nuclease+adenosine triphosphate+complementary strand+graphene oxide.
Embodiment
A kind of fluorescence detection method that utilizes oligonucleotide and graphene oxide to detect S1 nuclease and inhibitor thereof comprises the following steps:
1) oligonucleotide probe with dye marker joins in buffered soln, and solution is carried out fluoroscopic examination, records the fluorescent emission bands of a spectrum of this probe; This probe is one section single-chain nucleic acid of arbitrary sequence, and an end is marked with fluorescein.
2) graphene oxide is joined in buffered soln as solid phase carrier, because the graphene oxide surface has and can carry out the chemical group that non covalent bond is combined with oligonucleotide probe, can make the oligonucleotide probe of dye marker be adsorbed onto the surface of graphene oxide, thereby form oligonucleotide/graphene oxide mixture, between dyestuff and graphene oxide, efficient energy occurs and shift this moment, the effective quencher of the oxidized Graphene of the fluorescence of dyestuff.
3) add the oligonucleotide complementary strand to react in oligonucleotide/graphene oxide mixture, by base complementrity pairing effect, two strand Nucleotide hybridization form double-spiral structure.
4) the double-stranded formation of complementary strand mediation makes it from graphene oxide surface disengaging, distant between this moment dye groups and graphene oxide, effective energy can not occur to be shifted, the fluorescent signal that is marked at the dyestuff of probe end is restored, and stronger fluorescent signal can be detected.
5) when the oligonucleotide complementary strand after the S1 nuclease degradation forms little base fragment, join in oligonucleotide/graphene complex and react, the base fragment after degraded can not with oligonucleotide chain formation double-spiral structure.
6) the oligonucleotide complementary strand can't form double-spiral structure with oligonucleotide owing to being subject to the effect of S1 nuclease degradation, thereby oligonucleotide probe can not break away from from the surface of graphene oxide, and the fluorescent signal of dyestuff can not recover.
7) when the oligonucleotide complementary strand under the inhibitor adenosine triphosphate exists after the effect of S1 nuclease, join in oligonucleotide/graphene complex and react, complementary strand can by the S1 nuclease degradation, therefore can not form double-spiral structure with oligonucleotide under the inhibitor adenosine triphosphate exists.
8) the oligonucleotide complementary strand due under the effect of inhibitor adenosine triphosphate not by the S1 nuclease degradation, can form double-spiral structure with oligonucleotide probe, thereby make oligonucleotide probe break away from the surface of graphene oxide, the fluorescent signal of dyestuff is recovered.
According to the present invention, step 1) be: the oligonucleotide probe of dye marker is joined in buffered soln, solution is carried out fluoroscopic examination; Wherein, oligonucleotide probe of the present invention is one section single-chain nucleic acid of arbitrary sequence.
The temperature that graphene oxide is joined in buffered soln as solid phase carrier according to the present invention, step 2), dye marker oligonucleotide probe are adsorbed onto the graphene oxide surface can be room temperature, preferably 25 ℃.
According to the present invention, step 2) it is very fast that the oligonucleotide probe described in is adsorbed onto the speed on graphene oxide surface, and the time of observing quenching of fluorescence is about 2 minutes.
According to the present invention, step 3) described in to add the temperature that complementary strand reacts in oligonucleotide/graphene oxide mixture can be room temperature, preferably 25 ℃.By base complementrity pairing effect, form double-spiral structure.
According to the present invention, step 4) after the oligonucleotide described in and its complementary strand formed double-spiral structure, the time that breaks away from from the graphene oxide surface was about 25 minutes.
According to the present invention, step 5) the oligonucleotide complementary strand described in can be 29 ℃～39 ℃ through the temperature of reaction of the Degradation of S1 nuclease, preferably 37 ℃.
According to the present invention, step 5) the oligonucleotide complementary strand described in is 30 minutes through the time of the Degradation of S1 nuclease.
According to the present invention, step 6) described in through the oligonucleotide complementary strand after the S1 nuclease degradation, adding the time of reacting in oligonucleotide/graphene complex is 25 minutes.
According to the present invention, step 7) described in when the inhibitor adenosine triphosphate exists, add the effect of S1 nuclease in the oligonucleotide complementary strand, same step 5) of temperature of reaction and reaction times.
According to the present invention, step 8) described in when the inhibitor adenosine triphosphate exists, the oligonucleotide complementary strand through after the effect of S1 nuclease adds the time of reacting in oligonucleotide/graphene complex to be about 25 minutes.
Below in conjunction with accompanying drawing, further illustrate the present invention with the detection embodiment of S1 nuclease.But the present invention is not limited in and detects nuclease and inhibitor thereof, also is not subjected to the restriction of other conditions in embodiment.
Fluoroscopic examination instrument used is fluorescent sub-photometer (RF-5301, Japan).The fluorescence spectral measuring condition: xenon lamp excites, and exciting and launching slit width is 5.0nm and 10.0nm, and voltage is 950V, time of response 2S, and excitation wavelength is 480nm, emission wavelength sweep limit 490～650nm; Measure sample volume 1.60mL with the 3mL quartz colorimetric utensil; Room temperature.
In the embodiment of the present invention graphene oxide used be according to document (W.S.Hummers, R.E.Offeman, J.Am.Chem.Soc.1958,80,1339-1339) in the method for Hummers synthetic.
In the embodiment of the present invention oligonucleotide probe used and complementary strand be all available from Shanghai biotechnology company limited, and sequence is respectively (5 '-FAM-ATCTTGACTATGTGGGTGCT-3 ') and (5 '-AGCACCCACATAGTCAAGAT-3 '); S1 nuclease and adenosine triphosphate are available from Shanghai biotechnology company limited; Enzyme is cut the liquid composition: (2mM CH
3COONa, 15mM NaCl, 0.1mMZnSO
4, pH 4.6; Buffered soln composition used is: 20mM Tris-HCl, 100mM NaCl, 5mMKCl, 5mM MgCl
2, pH 7.4.
Embodiment 1 preparation oligonucleotide/graphene oxide mixture
The fluorescein-labeled few core nucleotide probe that is 2 μ M with 1 μ L concentration joins in the Tris-HCl buffered soln of 1mL, then adds 0.6mL water to carry out fluoroscopic examination; And then to add 1.8 μ L concentration be the graphene oxide solution of 1mg/mL, and this probe can be adsorbed onto rapidly the graphene oxide surface, and with the quench of fluorescein.
The detection of embodiment 2 oligonucleotide complementary strands
Detect 1: the oligonucleotide complementary strand that is 30 μ M with 1 μ L concentration joins in oligonucleotide/graphene oxide mixture, complementary strand and oligonucleotide probe form double-spiral structure, from graphene oxide surface disengaging, carry out fluoroscopic examination (excitation wavelength 480nm, emission wavelength 490～650nm) can be observed the recovery phenomenon of fluorescence.The incomplementarity chain that is 50 μ M with 1 μ L concentration simultaneously adds in contrast, and other condition is identical.
The detection of embodiment 3 nucleases and inhibitor thereof
Detect 2: the fluorescein-labeled few core nucleotide probe that is 1.6 μ M with 1 μ L concentration joins in the Tris-HCl buffered soln of 1mL, then adds the water of 0.6mL to carry out fluoroscopic examination; And then to add 1.5 μ L concentration be the graphene oxide solution of 1mg/mL, and this probe can be adsorbed onto rapidly the graphene oxide surface, and with the quench of fluorescein.
Be that the S1 nuclease of 0.01U and oligonucleotide complementary strand that 1 μ L concentration is 30 μ M join the 3uL enzyme and cut liquid (2mM CH with 3uL concentration
3COONa, 15mM NaCl, 0.1mM ZnSO
4, pH 4.6) in, reaction is 30 minutes under 37 ℃; Again reacted mixed solution is joined in oligonucleotide/graphene oxide mixture, in contrast, other condition with detect in 1 identical.
Simultaneously, do following test as a comparison.
Detect 3: be that the adenosine triphosphate of 150mM and oligonucleotide complementary strand that 1 μ L concentration is 30 μ M join the 3uL enzyme and cut that in liquid, (pH 4.6,2mM CH with 2.5 μ L concentration
3COONa, 15mM NaCl, 0.1mM ZnSO
4), then to add 3uL concentration be the S1 nuclease of 0.01U, reaction is 30 minutes under 37 ℃; Reacted mixed solution is joined in oligonucleotide/graphene oxide mixture, in contrast, other condition with detect in 1 identical.
Result:
In detection 1, the fluorescent signal recovery strength of oligonucleotide complementary strand and incomplementarity chain is compared and is differed greatly.As seen the selectivity of experiment is very strong.
In detection 2 and 3, the oligonucleotide complementary strand is compared through the effect of S1 nuclease degradation and the Degradation when adenosine triphosphate exists, and fluorescence intensity differs greatly.As seen the present invention has the specificity of height to the detection of nuclease and inhibitor thereof.
Claims (10)
1. fluorescent method that detects S1 nuclease and inhibitor thereof is characterized in that the method comprises the following steps:
A. the oligonucleotide fluorescent probe with dye marker joins in buffered soln, take 480 nm as excitation wavelength, carries out fluoroscopic examination, records the fluorescent emission bands of a spectrum of this probe;
B. graphene oxide is joined in mentioned solution, graphene oxide forms oligonucleotide/graphene oxide mixture as the oligonucleotide probe of the free rolled state of solid phase carrier absorption, at this moment the oxidized Graphene quencher of fluorescence of dyestuff;
C. through the oligonucleotide complementary strand of S1 nuclease cutting, join in oligonucleotide/graphene complex and react;
D. the oligonucleotide complementary strand forms little base fragment through the specificity Degradation of S1 nuclease, can't form double-spiral structure with the oligonucleotide chain probe on graphene oxide surface, thereby oligonucleotide probe can not break away from the surface of graphene oxide, and the fluorescent signal of dyestuff can not recover; The degree of recovering according to fluorescent signal realizes the qualitative and detection by quantitative to the S1 nuclease;
E. the oligonucleotide complementary strand through the effect of S1 nuclease degradation, adds in oligonucleotide/graphene oxide mixture and reacts under the inhibitor adenosine triphosphate exists;
F. the oligonucleotide complementary strand can not formed double-spiral structure with oligonucleotide probe by the S1 nuclease degradation under the inhibitor adenosine triphosphate exists, and oligonucleotide probe breaks away from the surface of graphene oxide, and the fluorescent signal of dyestuff is recovered; The degree of recovering according to fluorescent signal realizes the qualitative and detection by quantitative to the inhibitor adenosine triphosphate.
2. the fluorescent method of detection according to claim 1 S1 nuclease and inhibitor thereof, it is characterized in that the described oligonucleotide fluorescent probe with dye marker of step a) joins in buffered soln, detect with spectrophotofluorometer, dyestuff used is fluorescein; Dye marker is at 5 ' end of oligonucleotide probe; The oligonucleotide probe based composition is: 5 '-FAM-ATCTTGACTATGTGGGTGCT-3 '; Buffered soln used is 20 mM Tris-HCl, 100 mM NaCl, 5 mM KCl, 5 mM MgCl
2, pH=7.4; Fluorescence detection method used is: probe solution is joined the scanning of carrying out fluorescence emission spectrum in 1.6 mL buffered soln, excitation wavelength is 480nm, and the emission wavelength sweep limit is 490-650nm.
3. the fluorescent method of detection according to claim 1 S1 nuclease and inhibitor thereof, it is characterized in that the described oligonucleotide of step b)/graphene oxide complex formation, is to make oligonucleotide probe be adsorbed onto the graphene oxide surface by non-covalent interaction; After oligonucleotide was adsorbed onto the graphene oxide surface, the oxidized Graphene quencher of the fluorescence of dyestuff was after joining graphene oxide in the buffered soln that contains oligonucleotide probe, room temperature was placed 5 minutes, then carry out fluoroscopic examination, excitation wavelength is 480nm, and sweep limit is 490-650nm.
4. the fluorescent method of detection according to claim 1 S1 nuclease and inhibitor thereof, it is characterized in that step c) is through the oligonucleotide complementary strand of S1 nuclease cutting, join in oligonucleotide/graphene complex and react, oligonucleotide complementary strand probe based composition used is: 5 '-AGCACCCACATAGTCAAGAT-3 '; Add fluorescent signal after the oligonucleotide complementary strand solution that contains the S1 enzyme or other isopyknic contrast solution to recover about 25 minutes time used.
5. the fluorescent method of detection S1 nuclease according to claim 1 and inhibitor thereof, is characterized in that described fluorescent signal detects with spectrophotofluorometer, and excitation wavelength is 480nm, and the emission wavelength sweep limit is 490-650nm.
6. the fluorescent method of detection S1 nuclease according to claim 1 and inhibitor thereof, is characterized in that described fluorescent probe is any one section single stranded oligonucleotide, and dye marker is at 5 ' end, or 3 ' end.
7. the fluorescent method of detection S1 nuclease according to claim 1 and inhibitor thereof, is characterized in that described dye marker is fluorescein.
8. the fluorescent method of detection according to claim 1 S1 nuclease and inhibitor thereof, it is characterized in that the described oligonucleotide complementary strand of step d) is through the effect of S1 nuclease degradation, be by the S1 nuclease to single stranded DNA or the specific Degradation of RNA, form 5 ' phosphoric acid structure.
9. the fluorescent method of detection according to claim 8 S1 nuclease and inhibitor thereof, it is characterized in that the S1 nuclease enzyme of oligonucleotide complementary strand being cut process enzyme used, to cut solution be 2 mM CH
3COONa, 15 mM NaCl, 0.1 mM ZnSO
4, pH=4.6; Enzyme Qie Wendu is 37 ℃, and the time is 30 minutes.
10. the fluorescent method of detection according to claim 1 S1 nuclease and inhibitor thereof, it is characterized in that the described oligonucleotide complementary strand of step e) under the inhibitor adenosine triphosphate exists through the effect of S1 nuclease degradation, be that adenosine triphosphate can suppress the S1 nuclease to the specificity Degradation of single stranded DNA or RNA cutting.
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| CN102788824B (en) * | 2012-06-06 | 2014-07-02 | 河南赛沃思生物科技有限公司 | Preparation method of DNA biosensor |
| CN102998292B (en) * | 2012-12-13 | 2014-10-29 | 湖北中烟工业有限责任公司 | Fluorescence detection method for detecting ***e by use of oligonucleotide and graphene oxide |
| WO2014207515A1 (en) | 2013-06-25 | 2014-12-31 | Tubitak (Turkiye Bilimsel Ve Teknolojik Arastirma Kurumu) | Fluorescence method for detecting nuclease activity |
| EP3596228B1 (en) | 2017-03-14 | 2022-09-28 | Tubitak | Method for rapid identification of microorganisms producing nuclease enzymes |
| CN107557431A (en) * | 2017-07-18 | 2018-01-09 | 天津大学 | A kind of kit of Visual retrieval S1 nucleases |
| CN111665351B (en) * | 2020-06-20 | 2021-12-03 | 江南大学 | Method for quickly and specifically determining RNA content |
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