CN107643251A - The method of gold nanorods probe in detecting polyadenosine diphosphate ribose polymerase 1 - Google Patents
The method of gold nanorods probe in detecting polyadenosine diphosphate ribose polymerase 1 Download PDFInfo
- Publication number
- CN107643251A CN107643251A CN201710669001.9A CN201710669001A CN107643251A CN 107643251 A CN107643251 A CN 107643251A CN 201710669001 A CN201710669001 A CN 201710669001A CN 107643251 A CN107643251 A CN 107643251A
- Authority
- CN
- China
- Prior art keywords
- gold nanorods
- solution
- probe
- parp
- diphosphate ribose
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 title claims abstract description 74
- 239000000523 sample Substances 0.000 title claims abstract description 50
- 238000000034 method Methods 0.000 title claims abstract description 24
- 229920000776 Poly(Adenosine diphosphate-ribose) polymerase Polymers 0.000 title claims abstract description 17
- 108010064218 Poly (ADP-Ribose) Polymerase-1 Proteins 0.000 claims abstract description 60
- 238000006243 chemical reaction Methods 0.000 claims abstract description 36
- 108020004414 DNA Proteins 0.000 claims abstract description 35
- 102000053602 DNA Human genes 0.000 claims abstract description 28
- 244000197975 Solidago virgaurea Species 0.000 claims abstract description 20
- 235000000914 Solidago virgaurea Nutrition 0.000 claims abstract description 20
- 238000009396 hybridization Methods 0.000 claims abstract description 14
- 230000005540 biological transmission Effects 0.000 claims abstract description 13
- 238000001514 detection method Methods 0.000 claims abstract description 13
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 claims abstract description 12
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 claims abstract description 12
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 claims abstract description 12
- 239000001177 diphosphate Substances 0.000 claims abstract description 12
- XPPKVPWEQAFLFU-UHFFFAOYSA-J diphosphate(4-) Chemical compound [O-]P([O-])(=O)OP([O-])([O-])=O XPPKVPWEQAFLFU-UHFFFAOYSA-J 0.000 claims abstract description 12
- 235000011180 diphosphates Nutrition 0.000 claims abstract description 12
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 10
- 229920000642 polymer Polymers 0.000 claims abstract description 10
- 238000003786 synthesis reaction Methods 0.000 claims abstract description 10
- 230000000694 effects Effects 0.000 claims abstract description 9
- 238000000149 argon plasma sintering Methods 0.000 claims abstract description 5
- 238000002360 preparation method Methods 0.000 claims abstract 2
- 238000000870 ultraviolet spectroscopy Methods 0.000 claims abstract 2
- 102100023712 Poly [ADP-ribose] polymerase 1 Human genes 0.000 claims description 57
- 239000007788 liquid Substances 0.000 claims description 15
- BAWFJGJZGIEFAR-NNYOXOHSSA-O NAD(+) Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-O 0.000 claims description 14
- 230000004913 activation Effects 0.000 claims description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 10
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical compound [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 claims description 10
- 239000010931 gold Substances 0.000 claims description 9
- 229910052737 gold Inorganic materials 0.000 claims description 9
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 8
- 239000008367 deionised water Substances 0.000 claims description 7
- 229910021641 deionized water Inorganic materials 0.000 claims description 7
- 230000003213 activating effect Effects 0.000 claims description 6
- 238000002371 ultraviolet--visible spectrum Methods 0.000 claims description 6
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 claims description 5
- QIGBRXMKCJKVMJ-UHFFFAOYSA-N Hydroquinone Chemical compound OC1=CC=C(O)C=C1 QIGBRXMKCJKVMJ-UHFFFAOYSA-N 0.000 claims description 5
- 108020004682 Single-Stranded DNA Proteins 0.000 claims description 5
- 239000005457 ice water Substances 0.000 claims description 5
- 238000004321 preservation Methods 0.000 claims description 5
- 229910001961 silver nitrate Inorganic materials 0.000 claims description 5
- 229910000033 sodium borohydride Inorganic materials 0.000 claims description 5
- 239000012279 sodium borohydride Substances 0.000 claims description 5
- 239000011780 sodium chloride Substances 0.000 claims description 5
- BAWFJGJZGIEFAR-NNYOXOHSSA-N NAD zwitterion Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-N 0.000 claims description 4
- 229950006238 nadide Drugs 0.000 claims description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 2
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 claims description 2
- 229910052799 carbon Inorganic materials 0.000 claims description 2
- 229910052802 copper Inorganic materials 0.000 claims description 2
- 239000010949 copper Substances 0.000 claims description 2
- FDWREHZXQUYJFJ-UHFFFAOYSA-M gold monochloride Chemical compound [Cl-].[Au+] FDWREHZXQUYJFJ-UHFFFAOYSA-M 0.000 claims description 2
- 230000008859 change Effects 0.000 abstract description 9
- 102000015087 Poly (ADP-Ribose) Polymerase-1 Human genes 0.000 abstract 3
- 238000010521 absorption reaction Methods 0.000 description 7
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 6
- 238000010586 diagram Methods 0.000 description 6
- 238000000862 absorption spectrum Methods 0.000 description 5
- 230000009881 electrostatic interaction Effects 0.000 description 5
- 230000003247 decreasing effect Effects 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 238000006555 catalytic reaction Methods 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 230000001186 cumulative effect Effects 0.000 description 3
- RJHLTVSLYWWTEF-UHFFFAOYSA-K gold trichloride Chemical class Cl[Au](Cl)Cl RJHLTVSLYWWTEF-UHFFFAOYSA-K 0.000 description 3
- 238000012482 interaction analysis Methods 0.000 description 3
- 229910001629 magnesium chloride Inorganic materials 0.000 description 3
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 3
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 3
- 230000002776 aggregation Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 238000006116 polymerization reaction Methods 0.000 description 2
- 238000002211 ultraviolet spectrum Methods 0.000 description 2
- 238000005054 agglomeration Methods 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention discloses a kind of method of gold nanorods probe colorimetric determination polyadenosine diphosphate ribose polymerase 1 (PARP 1), comprise the following steps：1) selection and hybridization of double-stranded DNA are activated；2) synthesis of gold nanorods probe；3) double-stranded DNA, PARP 1, NAD are activated+Hybrid reaction, carry out the preparation of polyadenosine diphosphate ribose polymer (PAR)；4) gold nanorods probe and polyadenosine diphosphate ribose polymer (PAR) effect, and reaction solution is detected using ultraviolet-visible spectrophotometer observation；5) golden rod solution before and after reunion is characterized using transmission electron microscope and details in a play not acted out on stage, but told through dialogues light scattering.The present invention is using electropositive gold nanorods after electronegative PAR surface self-organizations, and solution is changed into almost colourless from reddish brown, and color change is obvious, therefore using colorimetric determination PARP 1.The present invention is with operating cost is low, detection is quick, simplicity, sensitive height, good selective.
Description
Technical field
The technology of polyadenosine diphosphate ribose polymerase -1 is quantitatively detected the present invention relates to a kind of, belongs to biosensor technique
Field.
Background technology
The main method of traditional PARP-1 detections has labelled with radioisotope method, protein immunoblotting method, enzyme-linked
Immunoabsorption etc..Labelled with radioisotope method needs prior labeled substrate NAD+, testing result is more reliable, sensitive, can
To detect very small amount PARP-1, but have that detection range is narrow, operation sequence is numerous and diverse, costly, it is necessary to very accurate measurement
Instrument, it is time-consuming longer, exist in a large amount of actual sample context of detection compared with Multiple Constraints.Colorimetric method is not only cheap, is not required to
Complicated instrument and equipment is wanted, and measurement is easy, quick, along with visually observable color change, thus has obtained very fast
Development.
The content of the invention
Technical problem：It is an object of the invention to provide a kind of polymerization of gold nanorods probe in detecting polyadenosine diphosphate ribose
The method of enzyme -1, it is the gold nanorods probe colorimetric determination polyadenosine diphosphate ribose polymerization based on electrostatic interaction
The analysis method of enzyme -1 (PARP-1).
Technical scheme：It is optical after reuniting for electropositive gold nanorods probe in electronegative PAR environment
Significant change occurs for matter, so as to establish colorimetric determination PARP-1.Gold nanorods reunite after, between aggregation intercouple with
And the change of golden rod surrounding environment dielectric properties makes color be changed into light brown red finally by dark brown red as colourless while golden
The UV absorption at rod longitudinal direction peak has apparent decline.Quantitative detection can be carried out to PARP-1 by UV absorption decreasing value.
The method of the gold nanorods probe in detecting polyadenosine diphosphate ribose polymerase -1 of the present invention comprises the following steps：
1) selection and hybridization of double-stranded DNA are activated；
2) synthesis of gold nanorods probe；
3) double-stranded DNA, PARP-1, NAD are activated+Hybrid reaction, carry out polyadenosine diphosphate ribose polymer P AR system
It is standby；
4) gold nanorods probe and polyadenosine diphosphate ribose polymer (PAR) effect, and utilize ultraviolet-visible spectrum
Instrument observation detects to reaction solution；
5) golden rod solution before and after reunion is characterized using transmission electron microscope and details in a play not acted out on stage, but told through dialogues light scattering.
Wherein：
Step 1) the activation double-stranded DNA chooses the single stranded DNA of two particular sequences, the 95-100 DEG C of water in cushioning liquid
Room temperature is cooled to after bath, forms the activation double-stranded DNA of hybridization, is reacted with PARP-1, for activating PARP-1.
The cushioning liquid is:10mM Tris-HCl, 0.1M NaCl, pH 7.2~7.4.
Step 2) the gold nanorods probe synthesis comprises the following steps that：Gold nanorods are closed using one kettle way
Into taking a mouth bottle, CTAB solution added into deionized water, then fast drop gold chloride, be then slowly added to successively
Silver nitrate solution quinol solution reacts, and adds sodium borohydride ice water solution, shaken well, after finally probe solution is centrifuged
Redissolve in deionized water, room temperature preservation.
Prepared by step 3) the polyadenosine diphosphate ribose polymer P AR comprises the following steps that：A centrifuge tube is taken, first
PARP-1 is configured to various concentrations with reaction cushioning liquid, is then respectively adding activation double-stranded DNA and nicotinamide adenine two
Nucleotides NAD+In 35~40 DEG C of water-baths.
The activation Double stranded DNA concentration is 500~1000nM.
The NAD+Concentration is 100~500 μM.
What step 4) gold nanorods probe and polyadenosine diphosphate ribose polymer P AR were acted on comprises the following steps that：Will
By activation double-stranded DNA, NAD+The PAR and gold nanorods solution being prepared with various concentrations PARP-1 are abundant at 20~30 DEG C
Reaction, record is carried out to its reaction solution and ultraviolet-visible spectrum detects, and observes gold nanorods color and reunion situation.
Step 5) transmission electron microscope and details in a play not acted out on stage, but told through dialogues light scattering comprise the following steps that to golden rod solution sign before and after reunion：Take golden rod
Solution is added drop-wise on the copper mesh of carbon film support respectively before and after reunion, is placed at room temperature, overnight naturally dry, carries out transmission electron microscope inspection
Survey, in addition, solution is added drop-wise on slide respectively before and after taking golden rod reunion, dries at room temperature, carry out details in a play not acted out on stage, but told through dialogues scattering measuring.
Beneficial effect：Principle is simple, experimental period is short, raw materials used cost is relatively low, and experimental result is with the naked eye
Directly observation is without any large-scale instrument.The present invention is using electropositive gold nanorods in electronegative PAR surface self-organizations
Afterwards, solution is changed into almost colourless from reddish brown, and color change is obvious, therefore using colorimetric determination PARP-1.The present invention
It is effectively utilized the characteristic of metal nano material, it is not necessary to detected by expensive precision instrument, simplify detection method, greatly
Reduce PARP-1 testing costs, the present invention have operating cost is low, detection is quick, it is easy, sensitive it is high, that selectivity is good etc. is excellent
Point.
Brief description of the drawings
Figure 1A shows the schematic diagram that PAR is generated under PARP-1 catalytic action；Ratios of the Figure 1B using gold nanorods as probe
Color method detects PARP-1 flow chart；
Fig. 2A shows a column diagrams：Fig. 2 B show a lines；
Fig. 3 A show the transmission electron microscope picture of gold nanorods probe, and Fig. 3 B show golden rod with a small amount of PAR (by 0.1U
PARP-1 catalysis form) reaction after solution transmission electron microscope picture；Fig. 3 C show that golden rod (is urged with enough PAR by 0.8U PARP-1
Change form) reaction after solution transmission electron microscope picture；
Fig. 4 A show the dark-field imaging figure of gold nanorods probe, and Fig. 4 B show the details in a play not acted out on stage, but told through dialogues of gold nanorods after addition PAR
Image；
Fig. 5 shows quantitative detection PARP-1 ultraviolet spectra variation diagram.A：Under different amounts of PAR effects, obtain
Ultraviolet spectrogram (PARP-1 concentration：B：UV absorption decreasing value (Δ A745) with the matched curve of PARP-1 concentration；
Embodiment
Embodiment 1：
Gold nanorods probe colorimetric determination polyadenosine diphosphate ribose polymerase -1 based on electrostatic interaction
Analysis method, detecting step are：
Gold nanorods probe synthesis step：Gold nanorods are synthesized using one kettle way.A mouth bottle is taken, 38mL is dense
Spend and added for 0.2mol/L CTAB solution into 30mL deionized water, then fast drop 3mL gold chlorides, then delay successively
Slow add after the quinol solution that 0.02mol/L silver nitrate solutiones 346 μ L and 2mL contain 0.044g reacts 5 minutes adds
2.6mL concentration is the freshly prepared sodium borohydride ice water solutions of 0.5mmol/L, shaken well, is incubated more than 12 hours at 30 DEG C,
Cumulative volume is 76mL.Finally probe solution is centrifuged 20~30 minutes at 13000rpm, 20 DEG C, centrifugation is redissolved afterwards twice is going
In ionized water, room temperature preservation.This solution is as probe solution.
Activate DNA hybridization step：The single stranded DNA of two particular sequences is chosen, 95 DEG C in hybridization buffer environment
(10mM Tris-HCl, 0.1M NaCl, pH 7.4) water-bath is cooled to room temperature after 5 minutes, forms the double-stranded DNA of hybridization, with
PARP-1 reacts, for activating PARP-1.
PARP-1 catalyzes and synthesizes PAR specific steps：A centrifuge tube is taken, is first configured to PARP-1 with reaction cushioning liquid
Various concentrations, then to containing 10 μ L, 1 μM of activation double-stranded DNA and 10 μ L, 500 μM of NADH
(NAD+) reaction cushioning liquid in (50mM Tris-HCl, 50mM KCl, 2mM MgCl2, 50 μM of Zn (OAc)2, pH 7.4)
0.1U PARP-1 is added, is reacted 1 hour in 37 DEG C of water-baths.
PARP-1 Activity determinations：The gold nanorods that 80 μ L brand-news are added in the PAR solution being prepared to above-mentioned reaction are molten
Liquid mixes, fully reaction 15 minutes.After reaction terminates, ultraviolet-visible spectrum is carried out to its reaction solution and detected, observes and records gold
Nanometer rods color and reunion situation.Experimental result such as Fig. 5 shows that PARP-1 is in good linear relationship in 0.05~1U, detection
Limit is 0.006U.
Embodiment 2：
Gold nanorods probe colorimetric determination polyadenosine diphosphate ribose polymerase -1 based on electrostatic interaction
Analysis method, detecting step are：
Gold nanorods probe synthesis step：Gold nanorods are synthesized using one kettle way.A mouth bottle is taken, 38mL is dense
Spend and added for 0.2mol/L CTAB solution into 30mL deionized water, then fast drop 3mL gold chlorides, then delay successively
Slow add after the quinol solution that 0.02mol/L silver nitrate solutiones 346 μ L and 2mL contain 0.044g reacts 5 minutes adds
2.6mL concentration is the freshly prepared sodium borohydride ice water solutions of 0.5mmol/L, shaken well, is incubated more than 12 hours at 30 DEG C,
Cumulative volume is 76mL.Finally probe solution is centrifuged 20~30 minutes at 13000rpm, 20 DEG C, centrifugation is redissolved afterwards twice is going
In ionized water, room temperature preservation.This solution is as probe solution.
Activate DNA hybridization step：The single stranded DNA of two particular sequences is chosen, 95 DEG C in hybridization buffer environment
(10mM Tris-HCl, 0.1M NaCl, pH 7.4) water-bath is cooled to room temperature after 5 minutes, forms the double-stranded DNA of hybridization, with
PARP-1 reacts, for activating PARP-1.
PARP-1 catalyzes and synthesizes PAR specific steps：A centrifuge tube is taken, is first configured to PARP-1 with reaction cushioning liquid
Various concentrations, then to containing 10 μ L, 1 μM of activation double-stranded DNA and 10 μ L, 500 μM of NADH
(NAD+) reaction cushioning liquid in (50mM Tris-HCl, 50mM KCl, 2mM MgCl2, 50 μM of Zn (OAc)2, pH 7.4)
0.5U PARP-1 is added, is reacted 1 hour in 37 DEG C of water-baths.
PARP-1 Activity determinations：The gold nanorods that 80 μ L brand-news are added in the PAR solution being prepared to above-mentioned reaction are molten
Liquid mixes, fully reaction 15 minutes.After reaction terminates, ultraviolet-visible spectrum is carried out to its reaction solution and detected, observes and records gold
Nanometer rods color and reunion situation.Experimental result such as Fig. 5 shows that PARP-1 is in good linear relationship in 0.05~1U, detection
Limit is 0.006U.
Embodiment 3：
Gold nanorods probe colorimetric determination polyadenosine diphosphate ribose polymerase -1 based on electrostatic interaction
Analysis method, detecting step are：
Gold nanorods probe synthesis step：Gold nanorods are synthesized using one kettle way.A mouth bottle is taken, 38mL is dense
Spend and added for 0.2mol/L CTAB solution into 30mL deionized water, then fast drop 3mL gold chlorides, then delay successively
Slow add after the quinol solution that 0.02mol/L silver nitrate solutiones 346 μ L and 2mL contain 0.044g reacts 5 minutes adds
2.6mL concentration is the freshly prepared sodium borohydride ice water solutions of 0.5mmol/L, shaken well, is incubated more than 12 hours at 30 DEG C,
Cumulative volume is 76mL.Finally probe solution is centrifuged 20~30 minutes at 13000rpm, 20 DEG C, centrifugation is redissolved afterwards twice is going
In ionized water, room temperature preservation.This solution is as probe solution.
Activate DNA hybridization step：The single stranded DNA of two particular sequences is chosen, 95 DEG C in hybridization buffer environment
(10mM Tris-HCl, 0.1M NaCl, pH 7.4) water-bath is cooled to room temperature after 5 minutes, forms the double-stranded DNA of hybridization, with
PARP-1 reacts, for activating PARP-1.
PARP-1 catalyzes and synthesizes PAR specific steps：A centrifuge tube is taken, is first configured to PARP-1 with reaction cushioning liquid
Various concentrations, then to containing 10 μ L, 1 μM of activation double-stranded DNA and 10 μ L, 500 μM of NADH
(NAD+) reaction cushioning liquid in (50mM Tris-HCl, 50mM KCl, 2mM MgCl2, 50 μM of Zn (OAc)2, pH 7.4)
1.0U PARP-1 is added, is reacted 1 hour in 37 DEG C of water-baths.
PARP-1 Activity determinations：The gold nanorods that 80 μ L brand-news are added in the PAR solution being prepared to above-mentioned reaction are molten
Liquid mixes, fully reaction 15 minutes.After reaction terminates, ultraviolet-visible spectrum is carried out to its reaction solution and detected, observes and records gold
Nanometer rods color and reunion situation.Experimental result such as Fig. 5 shows that PARP-1 is in good linear relationship in 0.05~1U, detection
Limit is 0.006U.
Fig. 2A shows a column diagrams：The Zeta potential of the gold nanorods newly synthesized, it can be seen that gold nanorods surface is certain
With a large amount of positive charges；B column diagrams：PAR substantially diminishes with the Zeta potential after gold nanorods reaction a period of time, potential value,
Show that electronegative PAR makes gold nanorods reunite；Fig. 2 B show a lines：The ultra-violet absorption spectrum of gold nanorods；B/c/d lines：Gold
Nanometer rods probe is respectively in PARP-1, NAD+, PARP-1/NAD+In ultra-violet absorption spectrum；It can be seen that gold nano
The ultra-violet absorption spectrum of rod probe does not almost change, and illustrates that they can not all cause the reunion of golden rod；E/f/g lines：Gold
Nanometer rods probe is respectively in PARP-1/activated DNA, NAD+In/activated DNA and activated DNA
Ultra-violet absorption spectrum；As can be seen from the figure the UV absorption intensity of golden rod has slight reduction, and corresponding color
There occurs slight change, illustrates that activating double-stranded DNA can also lure that slight reunion occurs for golden rod into；H lines：Gold nanorods exist
activated DNA/
PARP-1/NAD+In ultra-violet absorption spectrum；As can be seen from the figure there occurs bright for the UV absorption intensity of golden rod
Aobvious change, illustration show that solution colour also becomes colourless by dark brown red, and gold nanorods group can be made by illustrating the PAR of generation
It is poly-.
Fig. 3 A show the transmission electron microscope picture of gold nanorods probe, and scale is 100 nanometers；As can be seen from the figure new synthesis
Gold nanorods probe there is good monodispersity in water；Fig. 3 B show golden rod with a small amount of PAR (by 0.1U PARP-1
Catalysis form) reaction after solution transmission electron microscope picture：As can be seen from the figure the distance between gold nanorods substantially diminish, explanation
Electronegative PAR makes mutually to further between golden rod by electrostatic interaction；Fig. 3 C show golden rod with enough PAR (by 0.8U
PARP-1 catalysis form) reaction after solution transmission electron microscope picture：As can be seen from the figure gold nanorods and enough PAR are in reaction solution
In almost completely reunite.
Fig. 4 A show the dark-field imaging figure of gold nanorods probe, and scale is 60 microns；As can be seen from the figure new synthesis
Gold nanorods dispersiveness very well and show consistent color, illustrate that golden rod possesses good colloidal stability；Fig. 4 B show
Shown add PAR after gold nanorods darkfield image；As can be seen from the figure originally scattered point is reunited together and bright spot
Also bright gold color is become by kermesinus, illustrates that there occurs obvious agglomeration for golden rod after adding PAR.
Fig. 5 shows quantitative detection PARP-1 ultraviolet spectra variation diagram.A：Under different amounts of PAR effects, obtain
Ultraviolet spectrogram (PARP-1 concentration：0th, 0.01,0.05,0.1,0.25,0.5,0.75,1,3U (from top to bottom)；Illustration：Gold
Relation between nanometer rods solution colour and PARP-1 concentration；B：UV absorption decreasing value (Δ A745) with the plan of PARP-1 concentration
Close curve；Illustration：UV absorption decreasing value (Δ A745) linear relationship between PARP-1 concentration；As can be seen from the figure
PARP-1 is in good linear relationship in 0.05U to 1U.
Claims (9)
- A kind of 1. method of gold nanorods probe in detecting polyadenosine diphosphate ribose polymerase -1, it is characterised in that this method Comprise the following steps：1) selection and hybridization of double-stranded DNA are activated；2) synthesis of gold nanorods probe；3) double-stranded DNA, PARP-1, NAD are activated+Hybrid reaction, carry out polyadenosine diphosphate ribose polymer P AR preparation；4) gold nanorods probe and polyadenosine diphosphate ribose polymer (PAR) effect, and seen using ultraviolet-visible spectrophotometer Examine and reaction solution is detected；5) golden rod solution before and after reunion is characterized using transmission electron microscope and details in a play not acted out on stage, but told through dialogues light scattering.
- 2. the method for gold nanorods probe in detecting polyadenosine diphosphate ribose polymerase -1 according to claim 1, its It is characterised by, step 1) the activation double-stranded DNA chooses the single stranded DNA of two particular sequences, 95-100 DEG C in cushioning liquid Room temperature is cooled to after water-bath, forms the activation double-stranded DNA of hybridization, is reacted with PARP-1, for activating PARP-1.
- 3. the method for gold nanorods probe in detecting polyadenosine diphosphate ribose polymerase -1 according to claim 1, its It is characterised by, step 2) the gold nanorods probe synthesis comprises the following steps that：Gold nanorods are closed using one kettle way Into taking a mouth bottle, CTAB solution added into deionized water, then fast drop gold chloride, be then slowly added to successively Silver nitrate solution quinol solution reacts, and adds sodium borohydride ice water solution, shaken well, after finally probe solution is centrifuged Redissolve in deionized water, room temperature preservation.
- 4. the method for gold nanorods probe in detecting polyadenosine diphosphate ribose polymerase -1 according to claim 2, its It is characterised by, the cushioning liquid is:10mM Tris-HCl, 0.1M NaCl, pH 7.2~7.4.
- 5. the method for gold nanorods probe in detecting polyadenosine diphosphate ribose polymerase -1 according to claim 1, its It is characterised by, prepared by step 3) the polyadenosine diphosphate ribose polymer P AR comprises the following steps that：A centrifuge tube is taken, PARP-1 is first configured to various concentrations with reaction cushioning liquid, is then respectively adding activation double-stranded DNA and nicotinamide adenine Dinucleotides NAD+In 35~40 DEG C of water-baths.
- 6. the method for gold nanorods probe in detecting polyadenosine diphosphate ribose polymerase -1 according to claim 2, its It is characterised by, the activation Double stranded DNA concentration is 500~1000nM.
- 7. the method for gold nanorods probe in detecting polyadenosine diphosphate ribose polymerase -1 according to claim 3, its It is characterised by, the NAD+Concentration is 100~500 μM.
- 8. the method for gold nanorods probe in detecting polyadenosine diphosphate ribose polymerase -1 according to claim 1, its Be characterised by, step 4) gold nanorods probe and polyadenosine diphosphate ribose polymer P AR effect comprise the following steps that：Will By activation double-stranded DNA, NAD+The PAR and gold nanorods solution being prepared with various concentrations PARP-1 are abundant at 20~30 DEG C Reaction, record is carried out to its reaction solution and ultraviolet-visible spectrum detects, and observes gold nanorods color and reunion situation.
- 9. the method for gold nanorods probe in detecting polyadenosine diphosphate ribose polymerase -1 according to claim 1, its It is characterised by, step 5) transmission electron microscope and details in a play not acted out on stage, but told through dialogues light scattering comprise the following steps that to golden rod solution sign before and after reunion：Take gold Solution is added drop-wise on the copper mesh of carbon film support respectively before and after rod is reunited, and is placed at room temperature, overnight naturally dry, carries out transmission electron microscope Detection, in addition, solution is added drop-wise on slide respectively before and after taking golden rod reunion, dries at room temperature, carry out details in a play not acted out on stage, but told through dialogues scattering measuring.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710669001.9A CN107643251B (en) | 2017-08-08 | 2017-08-08 | Method for detecting poly (adenosine diphosphate ribose) polymerase-1 by gold nanorod probe |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710669001.9A CN107643251B (en) | 2017-08-08 | 2017-08-08 | Method for detecting poly (adenosine diphosphate ribose) polymerase-1 by gold nanorod probe |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107643251A true CN107643251A (en) | 2018-01-30 |
| CN107643251B CN107643251B (en) | 2020-06-02 |
Family
ID=61110078
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710669001.9A Expired - Fee Related CN107643251B (en) | 2017-08-08 | 2017-08-08 | Method for detecting poly (adenosine diphosphate ribose) polymerase-1 by gold nanorod probe |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107643251B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111521602A (en) * | 2020-05-20 | 2020-08-11 | 河南大学 | Detection method of PARP-1 concentration and application thereof |
| CN116441555A (en) * | 2023-06-16 | 2023-07-18 | 北京建工环境修复股份有限公司 | Functionalized gold nanorod probe, preparation method and application thereof in pentachlorophenol dark field microscopic imaging detection |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004026820A2 (en) * | 2002-09-19 | 2004-04-01 | Aventis Pharmaceuticals Inc. | Method for assaying compounds that decrease the activity of poly(adp-ribose)-polymerase (parp) |
| CN101818198A (en) * | 2010-03-05 | 2010-09-01 | 中国科学院上海微系统与信息技术研究所 | Method of colorimetric detection of target DNA by combining nanometer gold with polythiophene ramification |
| CN104865216A (en) * | 2015-06-10 | 2015-08-26 | 常熟理工学院 | Method for colorimetrically detecting chloramphenicol based on nano-gold nucleic acid aptamer |
| CN105606671A (en) * | 2016-01-18 | 2016-05-25 | 南京农业大学 | Detection method of poly(adenosine diphosphate-ribose) polymerase |
| CN105842181A (en) * | 2016-06-03 | 2016-08-10 | 盐城工学院 | Method for detecting cyanide ions based on gold nanorods |
| CN106442514A (en) * | 2016-11-24 | 2017-02-22 | 桂林理工大学 | Simple ultrasensitive colorimetric detection method for bivalent copper ions |
-
2017
- 2017-08-08 CN CN201710669001.9A patent/CN107643251B/en not_active Expired - Fee Related
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004026820A2 (en) * | 2002-09-19 | 2004-04-01 | Aventis Pharmaceuticals Inc. | Method for assaying compounds that decrease the activity of poly(adp-ribose)-polymerase (parp) |
| CN101818198A (en) * | 2010-03-05 | 2010-09-01 | 中国科学院上海微系统与信息技术研究所 | Method of colorimetric detection of target DNA by combining nanometer gold with polythiophene ramification |
| CN104865216A (en) * | 2015-06-10 | 2015-08-26 | 常熟理工学院 | Method for colorimetrically detecting chloramphenicol based on nano-gold nucleic acid aptamer |
| CN105606671A (en) * | 2016-01-18 | 2016-05-25 | 南京农业大学 | Detection method of poly(adenosine diphosphate-ribose) polymerase |
| CN105842181A (en) * | 2016-06-03 | 2016-08-10 | 盐城工学院 | Method for detecting cyanide ions based on gold nanorods |
| CN106442514A (en) * | 2016-11-24 | 2017-02-22 | 桂林理工大学 | Simple ultrasensitive colorimetric detection method for bivalent copper ions |
Non-Patent Citations (1)
| Title |
|---|
| 聂怀军: "DNA损伤识别与修复酶活性新分析方法研究", 《中国优秀硕士学位论文全文数据库 基础科学辑》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111521602A (en) * | 2020-05-20 | 2020-08-11 | 河南大学 | Detection method of PARP-1 concentration and application thereof |
| CN116441555A (en) * | 2023-06-16 | 2023-07-18 | 北京建工环境修复股份有限公司 | Functionalized gold nanorod probe, preparation method and application thereof in pentachlorophenol dark field microscopic imaging detection |
| CN116441555B (en) * | 2023-06-16 | 2023-08-22 | 北京建工环境修复股份有限公司 | Functionalized gold nanorod probe, preparation method and application thereof in pentachlorophenol dark field microscopic imaging detection |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107643251B (en) | 2020-06-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Shao et al. | A multiple signal amplification sandwich-type SERS biosensor for femtomolar detection of miRNA | |
| Liu et al. | A simple and convenient fluorescent strategy for the highly sensitive detection of dopamine and ascorbic acid based on graphene quantum dots | |
| Yang et al. | Development of a lateral flow strip biosensor based on copper oxide nanoparticles for rapid and sensitive detection of HPV16 DNA | |
| Ma et al. | A single quantum dot-based nanosensor for the signal-on detection of DNA methyltransferase | |
| Gao et al. | Ultrasensitive and specific microRNA detection via dynamic light scattering of DNA network based on rolling circle amplification | |
| Chen et al. | Homogeneous two-dimensional visual and fluorescence analysis of circulating tumor cells in clinical samples via steric hindrance regulated enzymes recognition cleavage and elongation | |
| CN108315421A (en) | Method of the constant-temperature amplification with the combination of quantum dot fluorescence Resonance energy transfer for detecting a variety of MicroRNAs simultaneously | |
| CN110982900B (en) | Biosensor based on bismuth alkene nanosheet fluorescence quenching, miRNA detection kit and application | |
| Fu et al. | A LAMP-based ratiometric electrochemical sensing for ultrasensitive detection of Group B Streptococci with improved stability and accuracy | |
| CN103290132A (en) | Nucleic acid nano-gold biosensor for detecting mercury ions and kit | |
| Xiong et al. | Optical analysis of biological hydrogen sulphide: an overview of recent advancements | |
| Wu et al. | Ultrasensitive electrochemiluminescence detection of p53 gene by a novel cloth-based microfluidic biosensor with luminol-gold nanoparticles and hybridization chain reaction amplification | |
| CN107607515A (en) | A kind of method based on Au@AgNCs detection sulphions | |
| Huang et al. | Electrochemiluminescent sensor based on Ru (bpy) 32+-doped silica nanoprobe by incorporating a new co-reactant NBD-amine for selective detection of hydrogen sulfide | |
| CN107643251A (en) | The method of gold nanorods probe in detecting polyadenosine diphosphate ribose polymerase 1 | |
| Li et al. | DNAzyme-catalyzed etching process of Au/Ag nanocages visualized via dark-field imaging with time elapse for ultrasensitive detection of microRNA | |
| CN111748608B (en) | Nano cage probe, application thereof and nucleic acid detection method | |
| Wang et al. | Cu 2+ modulated DNA-templated silver nanoclusters as a turn-on fluorescence probe for the detection of quinolones | |
| CN104059976A (en) | Preparation method and application of non-sulfydryl nucleic acid-nanogold conjugate | |
| Zhang et al. | Label-free detection of DNA methylation by surface-enhanced Raman spectroscopy using zirconium-modified silver nanoparticles | |
| Ge et al. | A novel DNA biosensor for the ultrasensitive detection of DNA methyltransferase activity based on a high-density “hot spot” SERS substrate and rolling circle amplification strategy | |
| Wang et al. | A dual-fluorescence biosensor assembled by quantum dots and phenazinium dyes: a comparative study for DNA detection | |
| Kong et al. | Dual-responsive ratiometric fluorescence detection of Ce4+ and ascorbic acid by regulating oxidase-mimicking activity of Ce4+-based nanocomplex | |
| Liu et al. | A comparison of PMT-based and CCD-based sensors for electrochemiluminescence detection of sunset yellow in soft drinks | |
| Zhu et al. | In-situ generation of potassium ferricyanide for label-free and enzyme-free chemiluminescence detection of telomerase activity |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20200602 |