CN109777794A - The luminescence system of a kind of ribalgilase and its inhibitor screening, preparation method and applications - Google Patents
The luminescence system of a kind of ribalgilase and its inhibitor screening, preparation method and applications Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 21
- 238000012216 screening Methods 0.000 title claims abstract description 21
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Abstract
The present invention relates to a kind of ribalgilase and its luminescence system of inhibitor screening, preparation method and applications.It is a kind of gold nanoclusters for glowing good based on bioaffinity, this gold nanoclusters are in a mild condition, using ribonucleic acid (RNA) and mercaptoundecylic acid as microenvironment, using ascorbic acid as mild reducing agent, by gold chloride (HAuCl4) be prepared.Issue the fluorescence of shiny red, 630 nm of emission wavelength or so.In this system, enzyme (such as RNase A) is sheared if there is ribonucleic acid, rna structure is destroyed, gold nanoclusters can not be formed, or luminous intensity is weak, thus achievees the purpose that qualitative, quantitative measures ribonuclease activity, this system can be also used for the screening of ribonuclease inhibitor.The preparation condition of gold nanoclusters is the water-bath at 30-40 DEG C, gold chloride, ribonucleic acid, ascorbic acid, mercaptoundecylic acid volume ratio be 0.8-1.2:6-10:8-15:0.8-1.2.Can be with qualitative and quantitative detection ribalgilase by the luminous intensity of luminescence system, quantitative detection range is 0 ~ 2.5 × 10‑4 U/mL。
Description
Technical field
Invention is related to the photochemistry side of a kind of specific detection ribalgilase (such as RNase A) and its inhibitor screening
Method belongs to large biological molecule Enzyme assay and inhibitor sifting field.
Background technique
The novel nano luminescent material that gold nanoclusters (AuNCs) are formed by tens to several hundred a gold atoms, in recent years, gold
Nano-cluster obtains the favor of many scientific research personnel because of its unique physics, chemical property, leads in bio-imaging, chemical sensitisation etc.
Domain shows the advantages such as preferable water solubility, hypotoxicity, biocompatibility, big stocks displacement, has wide application
Prospect.
In early days, gold nanoclusters preparation is real in the case where mercaptan compound, polyethyleneimine, polyamide-amine etc. are protected or are assisted
Existing, but the gold nanoclusters transmitting blue-fluorescence that these methods obtain, the tissue penetration in biologic applications can not show a candle to
Red or near infrared emission gold nanoclusters.The gold nanoclusters for sending out red fluorescence in order to obtain, would generally select in the preparation
Large biological molecules such as albumen, polypeptide, DNA etc. are used as template.The combination of large biological molecule and gold nanoclusters is not only that nano-cluster mentions
Ligand has been supplied, the photostability and biocompatibility of system are more improved.Xie in 2009 etc. is successfully closed using BSA as ligand for the first time
At gold nanoclusters, wherein BSA is simultaneously as stabilizer and reducing agent.This BSA-AuNCs is in addition to having good biofacies
Except capacitive and environment/cost advantage, moreover it is possible to the surface modification of promotion functions ligand.Later, BSA-AuNCs answering in all respects
Used in by wide coverage, such as detection pyrophosphate, dopamine, malachite green, alkaline phosphatase.Song and Gu etc. is with polypeptide
Template and reducing agent have synthesized polypeptide-AuNCs, and apply to proteinase activity detection.But Jenner is carried out by template of nucleic acid
When rice cluster preparation, nucleic acid can not make Au3+It is directly reduced, generally requires by reducing agents such as light, ascorbic acid.Tseng etc.
With A30Oligonucleotides is single-stranded with the method that ultraviolet light assists to be prepared for gold nanoclusters for template, Qing etc. using single stranded DNA as template,
HEPES has obtained gold nanoclusters as reducing agent, and is applied to Hg2+Detection.
Up to the present, there has been no relevant reports for the gold nanoclusters prepared using RNA as template and its application.
Summary of the invention
Technical solution of the present invention is using RNA as template, and for ascorbic acid as reducing agent, MUA is that adjuvant is prepared for stablizing
Gold nanoclusters solution.A large amount of base and Au in RNA molecule3+There is preferable interaction, can adsorb and in stablizing solution
Au3+, can be used as the template for preparing gold nanoclusters.This RNA-AuNCs solution issues very bright feux rouges in the excitation of 315 nm light.
And RNase A can quench the fluorescence of RNA-AuNCs, therefore realize RNase A Activity determination.
The invention belongs to large biological molecule probe fields, in particular to measurement life system ribonucleic acid shearing enzyme (such as
RNase A) activity and inhibitor screening fluorescence probe and method.Using a kind of gold for glowing good based on bioaffinity
Nano-cluster, this gold nanoclusters are in a mild condition, using ribonucleic acid (RNA) and mercaptoundecylic acid as microenvironment, with anti-bad
Hematic acid is as mild reducing agent, by gold chloride (HAuCl4) be prepared.
Mild condition provided by the present invention, easy to operate, high sensitivity and low-cost specific detection ribose core
The shiny red luminescence system of sour enzyme (such as RNase A) and its inhibitor screening, it is carrier system which, which is by ribonucleic acid (RNA),
Standby obtained gold nanoclusters are come what is realized, and emission wavelength 625-635 nm, stablizing storage life is 10 days or more.
The technical scheme is that be achieved through the following technical solutions:
Gold chloride and ribonucleic acid are added in distilled water, is uniformly mixed, at 30-40 DEG C after water-bath 8-15 minutes, slowly adds
Enter ascorbic acid, stirs reduction reaction 3-5h, obtain mixed solution；
Then mercaptoundecylic acid is added into the mixed solution, continues to stir 25-35min, centrifugation, obtaining supernatant is Jenner
Rice cluster.
The gold chloride, ribonucleic acid, ascorbic acid, mercaptoundecylic acid volume ratio be 0.8-1.2:6-10:8-15:
0.8-1.2。
Gold chloride in preferred embodiment, ribonucleic acid, ascorbic acid, mercaptoundecylic acid volume ratio be 1:8:10:1
The rna solution preparation method: weighing RNA, and PB buffer is added, and pH 7.2-7.6 is adjusted, in 3-4 DEG C of temperature
It is stand-by after the lower sealing avoid light place 10-12h of degree.
The mercaptoundecylic acid lays in liquid and preparation method thereof: weighing MUA, is dissolved in dehydrated alcohol, being configured to concentration is 0.1-
After being sealed against, it is stand-by to be stored in 3-4 DEG C of temperature for the stock solution of 0.15 M.
Another technical solution of the invention is by the luminescence system of the ribalgilase and its inhibitor screening special
Property detection ribalgilase RNase A reagent on application.
Qualitative recognition ribalgilase (such as RNase A)
In system to be measured implement gold nanoclusters preparation process, according to obtained product luminous situation in the presence of no RNase
Gold nanoclusters luminous intensity control, whether can qualitatively judge with the presence of RNase.
Quantitative test ribalgilase (such as RNase A)
1.0 mg RNase A are accurately weighed, is dissolved with 5.0 mL PB buffers (0.2 mM, pH 7.4), obtains 10 U/mL's
Stock solution continues to be diluted to 1 U/mL with PB buffer, and the stock solution prepared is sealed avoid light place at a temperature of 4 DEG C
It is stand-by after 12h.
By the RNase A of 400 μ L RNA(0.16 mg/mL) and various concentration at 37 DEG C co-incubation 1h, until RNA is complete
It is sheared entirely, 50 μ L HAuCl is added4(1 mM) is stirred after ten minutes, is slowly added to the AA solution (1 of the 500 fresh configurations of μ L
MM), it is vigorously stirred 4h.50 μ L MUA stock solutions (1 mM) are added after 4h to be restored into mixed solution, continue 30 points of stirring
Clock.Obtained solution 2000r is centrifuged 20 minutes, free gold atom is removed, obtains prepare liquid.Prepare liquid fluorescence is tested, is drawn
Fluorescence intensity (I/I processed0) with the standard curve of RNase A concentration relationship.
The preparation process for implementing gold nanoclusters in system to be measured, according to the luminous intensity of the gold nanoclusters of measurement, foundation
The concentration of RNase A in system to be measured is calculated in standard curve.
Another technical solution of the invention is to sieve the luminescence system of the ribalgilase and its inhibitor screening
Select the application on the active reagent of ribonuclease inhibitor.
Ribalgilase (such as RNase A) inhibitor screening
The RNase A for taking 2.5 μ L to contain different inhibitor is added to 400 μ L RNA(0.16 mg/mL) in, at 37 DEG C altogether
With culture 1h, until RNA reacts completely with RNase A.50 μ L HAuCl are added4(1 mM) is stirred after ten minutes, is slowly added to
The AA solution (1 mM) of the 500 fresh configurations of μ L, is vigorously stirred 4h.50 μ L MUA storage is added after 4h to be restored into mixed solution
Standby liquid (1 mM) continues stirring 30 minutes.By obtained solution 2000r be centrifuged 20 minutes, remove free gold atom, obtain to
Liquid is surveyed, is measured luminous intensity (I).The check experiment without inhibitor is done according to same step simultaneously, is shone according to what is obtained
Intensity (Im).Inhibiting rate is calculated by following formula:
(I-Im)/(I0-Im) ×100%
The inhibitory activity of RNase A inhibitor is judged according to inhibiting rate size.
It is that the gold nanoclusters system sending shiny red that carrier is prepared is glimmering that the system, which is by ribonucleic acid (RNA),
Light, 630 nm of emission wavelength or so, and save 10 days or more, luminous intensity does not obviously weaken phenomenon.
Solution prepares the water that water used in buffer is secondary distilled water or higher purity when the described fluoremetry.
The neutral buffered liquid is by KH2PO40.24 g, Na2HPO41.44 g(are if it is Na2HPO4·12H2O,
Then 3.63 g) are dissolved in 1000 mL water and being formulated.
The luminous intensity can be obtained by directly observing or testing in the UV lamp on sepectrophotofluorometer.
The present invention will both can be used for specific detection ribalgilase (such as RNase A), can be used for ribonucleic acid
Inhibitor activity screening.In the presence of no ribalgilase (such as RNase A), the gold that is prepared using RNA as template
Nano-cluster sends out very bright red fluorescence, but the presence of ribalgilase (such as RNase A) can destroy RNA structure, if there is
Ribonucleic acid shears enzyme (such as RNase A), and rna structure is destroyed, and gold nanoclusters can not be formed or luminous intensity is weak,
Thus the content of qualitative recognition ribalgilase or quantitative determination ribalgilase, this system can be also used for ribalgilase suppression
The screening of preparation.
In this system, the preparation condition of gold nanoclusters is the water-bath at 30-40 DEG C, gold chloride, ribonucleic acid, Vitamin C
Sour, mercaptoundecylic acid volume ratio is 0.8-1.2:6-10:8-15:0.8-1.2.It can be qualitative by the luminous intensity of luminescence system
Quantitative detection ribalgilase, quantitative detection range is 0 ~ 2.5 × 10-4 U/mL。
Such discrimination method is more intuitive, have it is easy to operate, high sensitivity and it is low in cost the features such as, be worthy to be popularized.
Detailed description of the invention
Fig. 1 is that the fluorescence photo of the gold nanoclusters that prepare by template of RNA under the ultraviolet light irradiation of 310 nm is (red glimmering
Light).
Fig. 2 is absorption (left side) and fluorescent emission (right side) spectrum of gold nanoclusters.
Fig. 3 is the fluorescence spectrum of gold nanoclusters placement after a week.
Fig. 4 is influence of the pH to gold nanoclusters luminous intensity.
The fluorescence spectrum variation of gold nanoclusters before and after RNase A is added in Fig. 5.Illustration: RNase is added under 310 nm ultraviolet lamps
Fluorescence photo (the Zuo Liang: before addition of the forward and backward gold nanoclusters of A；It is right dark: after addition).
Fig. 6 is the fluorescence spectrum of gold nanoclusters when various concentration RNase A is added.
Fig. 7 is gold nanoclusters fluorescence intensity (I/I0) with the relationship of RNase A concentration；Illustration: I/I0With RNase A concentration
Between quantitative relationship curve.
Fig. 8 is the fluorescence spectrum of gold nanoclusters in the presence of various concentration inhibitor.
Specific embodiment
Following tests and example are for further illustrating but being not limited to the present invention.
(1) gold nanoclusters are prepared by template of RNA
50 μ L gold chloride (HAuCl are added in 4.0 mL distilled water4, 1 mM) and 400 μ L ribonucleic acid (RNA, 0.16 mg/
ML), it is uniformly mixed, water-bath after ten minutes, is slowly added to the ascorbic acid (AA, 1 mM) of the 500 fresh configurations of μ L at 37 DEG C,
It is vigorously stirred 4h.50 μ L mercaptoundecylic acids (MUA, 1 mM) is added after 4h to be restored into mixed solution, continues 30 points of stirring
Clock.Obtained system is centrifuged 20 minutes with 2000 revs/min of speed, the gold atom to dissociate in solution is removed, is clarified
Transparent pale pink solution is gold nanoclusters, photo such as Fig. 1 under 310 nm ultraviolet lights, measures absorption spectrum and glimmering
Optical emission spectroscopy such as Fig. 2, luminous intensity I0。
(2) gold nanoclusters Stability and adaptability is tested
In the dark, 4 DEG C of refrigerations select different time to measure luminous intensity to the gold nanoclusters that (1) is obtained, to judge that system shines
Stability.Result of implementation such as Fig. 3.
The gold nanoclusters that (1) obtains are placed in the buffer of different pH value, measure its luminous intensity, judges it in difference
Luminous adaptability under physiological environment.Result of implementation such as Fig. 4.
(3) qualitative recognition ribalgilase (such as RNase A)
The preparation process for implementing gold nanoclusters according to the method for above (1) in system to be measured, according to the luminous feelings of obtained product
Condition is compareed with gold nanoclusters luminous intensity obtained in (1), as luminous intensity is decreased obviously, or is not shone, then qualitatively judging has
RNase exists.Result of implementation such as Fig. 5.
(4) quantitative test ribalgilase (such as RNase A):
1.0 mg RNase A are accurately weighed, is dissolved with 5.0 mL PB buffers (0.2 mM, pH 7.4), obtains 10 U/mL's
Stock solution continues to be diluted to 1 U/mL with PB buffer, and the stock solution prepared is sealed avoid light place at a temperature of 4 DEG C
It is stand-by after 12h.
By the RNase A of 400 μ L RNA(0.16 mg/mL) and various concentration at 37 DEG C co-incubation 1h, until RNA is complete
It is sheared entirely, 50 μ L HAuCl is added4(1 mM) is stirred after ten minutes, is slowly added to the AA solution (1 of the 500 fresh configurations of μ L
MM), it is vigorously stirred 4h.50 μ L MUA stock solutions (1 mM) are added after 4h to be restored into mixed solution, continue 30 points of stirring
Clock.Obtained solution 2000r is centrifuged 20 minutes, free gold atom is removed, obtains prepare liquid.Prepare liquid fluorescence is tested, is drawn
Fluorescence intensity (I/I processed0) with the standard curve of RNase A concentration relationship.Result of implementation such as Fig. 6, Fig. 7.
The preparation process for implementing gold nanoclusters in system to be measured according to the method in claim 3, according to the Jenner of measurement
The concentration of RNase A in system to be measured is calculated in the luminous intensity of rice cluster, establishing criteria curve.
(5) ribalgilase (such as RNase A) inhibitor screening
The RNase A for taking 2.5 μ L to contain different inhibitor is added to 400 μ L RNA(0.16 mg/mL) in, at 37 DEG C altogether
With culture 1h, until RNA reacts completely with RNase A.50 μ L HAuCl are added4(1 mM) is stirred after ten minutes, is slowly added to
The AA solution (1 mM) of the 500 fresh configurations of μ L, is vigorously stirred 4h.50 μ L MUA storage is added after 4h to be restored into mixed solution
Standby liquid (1 mM) continues stirring 30 minutes.By obtained solution 2000r be centrifuged 20 minutes, remove free gold atom, obtain to
Liquid is surveyed, is measured luminous intensity (I).The check experiment without inhibitor is done according to same step simultaneously, is shone according to what is obtained
Intensity (Im).Inhibiting rate is calculated by following formula:
(I-Im)/(I0-Im) ×100%
The inhibitory activity of RNase A inhibitor is judged according to inhibiting rate size.Result of implementation such as Fig. 8.
Claims (7)
1. the luminescence system of a kind of ribalgilase and its inhibitor screening, which is characterized in that the system is by Ribonucleic RNA
For the gold nanoclusters that carrier is prepared, system issues shiny red fluorescence, emission wavelength 625-635 nm, storage life be 10 days with
On.
2. the preparation method of the luminescence system of ribalgilase according to claim 1 and its inhibitor screening, feature
It is, includes the following steps:
Gold chloride and ribonucleic acid are added in distilled water, is uniformly mixed, at 30-40 DEG C after water-bath 8-15 minutes, slowly adds
Enter ascorbic acid, stirs reduction reaction 3-5h, obtain mixed solution；
Then mercaptoundecylic acid is added into the mixed solution, continues to stir 25-35min, centrifugation, obtaining supernatant is Jenner
Rice cluster.
3. the preparation method of the luminescence system of ribalgilase according to claim 2 and its inhibitor screening, feature
Be, gold chloride, ribonucleic acid, ascorbic acid, mercaptoundecylic acid volume ratio be 0.8-1.2:6-10:8-15:0.8-1.2.
4. the preparation method of the luminescence system of ribalgilase according to claim 2 and its inhibitor screening, feature
It is, the rna solution preparation method: weighs RNA, PB buffer is added, pH 7.2-7.6 is adjusted, at 3-4 DEG C
At a temperature of seal it is stand-by after avoid light place 10-12h.
5. the preparation method of the luminescence system of ribalgilase according to claim 2 and its inhibitor screening, feature
It is, the mercaptoundecylic acid lays in liquid and preparation method thereof: weighs MUA, be dissolved in dehydrated alcohol, being configured to concentration is 0.1-0.15
After being sealed against, it is stand-by to be stored in 3-4 DEG C of temperature for the stock solution of M.
6. the luminescence system of ribalgilase described in claim 1 and its inhibitor screening is in specific detection ribalgilase
Application on the reagent of RNase A.
7. the luminescence system of ribalgilase described in claim 1 and its inhibitor screening is in screening ribonuclease inhibitor
Active reagent on application.
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| CN114384053A (en) * | 2022-01-13 | 2022-04-22 | 山东大学 | Flash and glow adjustable near-infrared chemiluminescence system using nano-gold cluster as luminophor |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070249063A1 (en) * | 2004-08-30 | 2007-10-25 | Deshong Philip R | Biosensors |
| WO2010002949A2 (en) * | 2008-06-30 | 2010-01-07 | The Regents Of The University Of California | Remote optical switch for localized control of gene interference |
| CN106955360A (en) * | 2008-11-24 | 2017-07-18 | 西北大学 | Polyvalent RNA-nanoparticle compositions |
| CN108273056A (en) * | 2018-02-01 | 2018-07-13 | 中国科学院长春应用化学研究所 | A kind of modified gold nano-material/nucleic acid probe nanometer system and preparation method thereof, application |
| CN108410949A (en) * | 2018-03-05 | 2018-08-17 | 湖北大学 | Active probes of a kind of detection exonuclease I and its preparation method and application |
| CN109097029A (en) * | 2018-09-16 | 2018-12-28 | 吉林化工学院 | A kind of application of silicon nano/gold nanoclusters ratio fluorescent probe synthesized and its rifampin ratio fluorescent is detected |
-
2019
- 2019-01-04 CN CN201910008821.2A patent/CN109777794A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070249063A1 (en) * | 2004-08-30 | 2007-10-25 | Deshong Philip R | Biosensors |
| WO2010002949A2 (en) * | 2008-06-30 | 2010-01-07 | The Regents Of The University Of California | Remote optical switch for localized control of gene interference |
| CN106955360A (en) * | 2008-11-24 | 2017-07-18 | 西北大学 | Polyvalent RNA-nanoparticle compositions |
| CN108273056A (en) * | 2018-02-01 | 2018-07-13 | 中国科学院长春应用化学研究所 | A kind of modified gold nano-material/nucleic acid probe nanometer system and preparation method thereof, application |
| CN108410949A (en) * | 2018-03-05 | 2018-08-17 | 湖北大学 | Active probes of a kind of detection exonuclease I and its preparation method and application |
| CN109097029A (en) * | 2018-09-16 | 2018-12-28 | 吉林化工学院 | A kind of application of silicon nano/gold nanoclusters ratio fluorescent probe synthesized and its rifampin ratio fluorescent is detected |
Non-Patent Citations (2)
| Title |
|---|
| SHERIF M. SHAWKYA: "Gold aggregating gold: A novel nanoparticle biosensor approach for the", 《BIOSENSORS AND BIOELECTRONICS》 * |
| 金征宇等, 天津科学技术出版社 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114384053A (en) * | 2022-01-13 | 2022-04-22 | 山东大学 | Flash and glow adjustable near-infrared chemiluminescence system using nano-gold cluster as luminophor |
| CN114384053B (en) * | 2022-01-13 | 2023-09-08 | 山东大学 | Flash and glow adjustable near infrared chemiluminescence system with nano gold clusters as luminophors |
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