CN106018371B - Enzyme coupling nucleic acid-argentum nanometer probe - Google Patents

Enzyme coupling nucleic acid-argentum nanometer probe Download PDF

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Publication number
CN106018371B
CN106018371B CN201610526250.8A CN201610526250A CN106018371B CN 106018371 B CN106018371 B CN 106018371B CN 201610526250 A CN201610526250 A CN 201610526250A CN 106018371 B CN106018371 B CN 106018371B
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solution
added
nucleic acid
sodium borohydride
acid
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CN106018371A (en
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仰大勇
张志昆
刘阳
杨璐
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Tianjin University
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Tianjin University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy

Abstract

The invention discloses a kind of enzyme coupling nucleic acid-argentum nanometer probes, are made of following methods: (1) with phosphate buffer solution being respectively that solvent prepares oligonucleotides acid solution, silver nitrate solution and sodium borohydride solution；(2) oligonucleotides acid solution and silver nitrate solution are taken, is uniformly mixed, phosphate buffer solution is added, sodium borohydride solution is added in stirring, stirs, and FeSO is added in the liquid that (3) are obtained to step (2)4Aqueous solution, is added D-AAO aqueous solution, and concussion mixes；The oligonucleotide is -3 ˊ of 5 ˊ-CCCTTAATCCCC, and probe of the invention is used for fluorescence analysis, detects to the D- amino acid in food, and detection is offline to can reach 10nM；Have many advantages, such as that sensitivity for analysis is high, selectivity is strong and uses easy；Compared with traditional detection method, sample is easy to operate, at low cost without performing the derivatization, and realizes the quick and easy detection of D- amino acid.

Description

Enzyme coupling nucleic acid-argentum nanometer probe
Technical field
The invention belongs to technical field of biological, and in particular to a kind of identification chiral D-amino acid enzyme coupling nucleic acid- Argentum nanometer probe.
Background technique
Amino acid is the basic component units of protein, closely related with vital movement.There is hand in Most amino-acids Property enantiomter, l-amino acid and D- amino acid (except glycine).The overwhelming majority be present in tellurian life entity with L-amino acid synthetic proteins matter, only there are the peptide chains of l-amino acid in a small number of bacterial cells.Although D- amino acid is not involved in human body The synthesis of protein, but it with special physiological function, suitable D- amino acid is necessary to life less.
Excessive D- amino acid can be metabolized by D-AAO, can generate hydrogen peroxide, be caused to the body of people Certain damage, in addition, D- amino acid largely enters body, it would be possible to cause the exception or even death of vital movement.For example, The higher D-Ser containing concentration in the Deiter's cells of patients with amyotrophic lateral sclerosis.Therefore, chiral Recognition is in life It is particularly important in the process, if containing excessive D- amino acid in food, once being absorbed into human body, toxicity can be generated to human body Property.
Currently, the analysis of chiral isomer, the detection of especially small molecule isomer amino acids is still relatively complicated.D- ammonia Base acid detection method mainly includes high pressure lipuid chromatography (HPLC), gas chromatography, circular dichroism detector, electrochemical assay, enzyme process and Micro-fluidic chip method etc..But above method complex steps, it is complicated for operation, at high cost, need large scale equipment and professional etc. no Foot.Currently, being had not been reported using the work that nano silver detects D- amino acid as fluorescence probe.
Summary of the invention
The purpose of the present invention is overcome the deficiencies of the prior art and provide a kind of probe of enzyme coupling nucleic acid/silver nanoparticle.
Technical solution of the present invention is summarized as follows:
A kind of enzyme coupling nucleic acid-argentum nanometer probe, is made of following methods:
(1) respectively with phosphate buffer solution be solvent prepare 0.1-0.5mM oligonucleotides acid solution, prepare 0.7- The silver nitrate solution of 3.5mM, and prepare 0.7-3.5mM sodium borohydride solution；
(2) oligonucleotides acid solution described in 50 μ L and 50 μ L silver nitrate solutions are taken, is uniformly mixed, the phosphoric acid of 4.85mL is added Salt buffer solution stirs 15-25min, and 50 μ L sodium borohydride solutions are added, and stirs 50-90min；Wherein oligonucleotide, nitric acid The molar ratio of silver and sodium borohydride are as follows: 1:7:7；
(3) FeSO of the 0.5-5.0mM of 0.56mL is added in the liquid obtained to step (2)425-40 μ is added in aqueous solution The 100000U/L D-AAO aqueous solution of L, concussion mix；The concentration of the phosphate buffer solution is 20mM, pH It is 6.8.
The sequence of oligonucleotide is preferably -3 ˊ of 5 ˊ-CCCTTAATCCCC.
Advantages of the present invention:
(1) enzyme coupling nucleic acid-argentum nanometer probe is used for fluorescence analysis, detects to the D- amino acid in food, examines It surveys and offline can reach 10nM；Have many advantages, such as that sensitivity for analysis is high, selectivity is strong and uses easy；
(2) compared with traditional detection method, sample is easy to operate, at low cost without performing the derivatization, and realizes D- amino The quick and easy detection of acid.
Detailed description of the invention
Fig. 1 is enzyme coupling nucleic acid-argentum nanometer probe fluorescence photo；
Fig. 2 is enzyme coupling nucleic acid-argentum nanometer probe fluorescence spectra；
Fig. 3 is the fluorescence spectra that enzyme coupling nucleic acid-argentum nanometer probe identifies D-alanine；
Fig. 4 is the fluorescence spectra that enzyme coupling nucleic acid-argentum nanometer probe identifies D-Arg；
Fig. 5 is the fluorescence spectra that enzyme coupling nucleic acid-argentum nanometer probe identifies D-phenylalanine.
Specific embodiment
The present invention is further illustrated combined with specific embodiments below.
The D-AAO that various embodiments of the present invention are acted on is commercially available pig kidney D-AAO.
The embodiment of the present invention is in order to enable those skilled in the art to more fully understand the present invention, if in the application It is improved on the basis of -3 ˊ of 5 ˊ-CCCTTAATCCCC of oligonucleotide, and is used to prepare enzyme in conjunction with D-AAO Coupling nucleic acid-argentum nanometer probe, also belongs to the scope of the present invention.
Embodiment 1
A kind of enzyme coupling nucleic acid-argentum nanometer probe, is made of following methods:
(1) respectively with phosphate buffer solution be solvent prepare 0.3mM oligonucleotides acid solution, prepare the nitric acid of 2.1mM Silver-colored solution, and prepare 2.1mM sodium borohydride solution；
(2) oligonucleotides acid solution described in 50 μ L and 50 μ L silver nitrate solutions are taken, is uniformly mixed, 4.85mL phosphate is added Buffer solution stirs 20min, and 50 μ L sodium borohydride solutions are added, and stirs 70min；
(3) FeSO of the 1.0mM of 0.56mL is added in the liquid obtained to step (2)4Aqueous solution is added 35 μ L's 100000U/L D-AAO aqueous solution, concussion mix；The oligonucleotide is -3 ˊ of 5 ˊ-CCCTTAATCCCC.
The concentration of the phosphate buffer solution is 20mM, pH 6.8.
The fluorescence photo of enzyme coupling nucleic acid/silver nanoparticle probe is shown in Fig. 1.
Fluorogram is Fig. 2.
Embodiment 2
A kind of enzyme coupling nucleic acid/argentum nanometer probe, is made of following methods:
(1) respectively with phosphate buffer solution be solvent prepare 0.1mM oligonucleotides acid solution, prepare the nitric acid of 0.7mM Silver-colored solution, and prepare 0.7mM sodium borohydride solution；
(2) oligonucleotides acid solution described in 50 μ L and 50 μ L silver nitrate solutions are taken, is uniformly mixed, the phosphoric acid of 4.85mL is added Salt buffer solution stirs 15min, and 50 μ L sodium borohydride solutions are added, and stirs 50min；
(3) FeSO of the 0.5mM of 0.56mL is added in the liquid obtained to step (2)4Aqueous solution is added 25 μ L's 100000U/L D-AAO aqueous solution, concussion mix；The oligonucleotide is -3 ˊ of 5 ˊ-CCCTTAATCCCC.
The concentration of the phosphate buffer solution is 20mM, pH 6.8.
Fluorogram is Fig. 2.
Embodiment 3
A kind of enzyme coupling nucleic acid-argentum nanometer probe, is made of following methods:
(1) respectively with phosphate buffer solution be solvent prepare 0.5mM oligonucleotides acid solution, prepare the nitric acid of 3.5mM Silver-colored solution, and prepare 3.5mM sodium borohydride solution；
(2) oligonucleotides acid solution described in 50 μ L and 50 μ L silver nitrate solutions are taken, is uniformly mixed, the phosphoric acid of 4.85mL is added Salt buffer solution stirs 25min, and 50 μ L sodium borohydride solutions are added, and stirs 90min；
(3) FeSO of the 5.0mM of 0.56mL is added in the liquid obtained to step (2)4Aqueous solution is added 40 μ L's 100000U/L D-AAO aqueous solution, concussion mix；The oligonucleotide is -3 ˊ of 5 ˊ-CCCTTAATCCCC.
The concentration of the phosphate buffer solution is 20mM, pH 6.8.
Fluorogram is Fig. 2.
Embodiment 4
Enzyme coupling nucleic acid-the argentum nanometer probe prepared with embodiment 1 detects the chiral Recognition of D-alanine:
In three centrifuge tubes, enzyme coupling nucleic acid-argentum nanometer probe of 90 μ L is added, then is separately added into the ultrapure of 10 μ L Water (blank), the 10 of 10 μ L–2The 10 of the l-Alanine of M and 10 μ L–2The D-alanine of M, 37 DEG C of 30min of water-bath use fluorescence point Light photometer is excited in 568nm, 620nm transmitting measurement.See Fig. 3.
Embodiment 5
Enzyme coupling nucleic acid-the argentum nanometer probe prepared with embodiment 2 detects the chiral Recognition of D- amino acid:
In three centrifuge tubes, enzyme coupling nucleic acid-argentum nanometer probe of 90 μ L is added, then is separately added into the ultrapure of 10 μ L Water (blank), the 10 of 10 μ L–2The 10 of the L-arginine of M and 10 μ L–2The D-Arg of M, 37 DEG C of 30min of water-bath use fluorescence point Light photometer is excited in 568nm, 620nm transmitting measurement.See Fig. 4.
Embodiment 6
Enzyme coupling nucleic acid-argentum nanometer probe prepared with embodiment 3 detects the chiral Recognition of D- amino acid, fragmented three In heart pipe, enzyme coupling nucleic acid-argentum nanometer probe of 90 μ L is added, then is separately added into the ultrapure water (blank) of 10 μ L, 10–2M's The 10 of L-phenylalanine and 10 μ L–2The D-phenylalanine of M, 37 DEG C of 30min of water-bath are swashed using sepectrophotofluorometer in 568nm Hair, 620nm transmitting measurement.See Fig. 5.
Experiments have shown that: it can be to D-alanine, D-Arg, D- benzene with enzyme coupling nucleic acid-argentum nanometer probe of the invention Alanine, D-His, D-Lys, D-Ser, D-Thr etc. are identified.

Claims (1)

1. a kind of enzyme coupling nucleic acid-argentum nanometer probe, it is characterized in that being made of following methods:
(1) respectively with phosphate buffer solution be solvent prepare 0.1-0.5mM oligonucleotides acid solution, prepare 0.7-3.5mM's Silver nitrate solution and preparation 0.7-3.5mM sodium borohydride solution；
(2) oligonucleotides acid solution described in 50 μ L and 50 μ L silver nitrate solutions are taken, is uniformly mixed, the phosphate that 4.85mL is added is slow Solution is rushed, 15-25min is stirred, 50 μ L sodium borohydride solutions are added, stirs 50-90min；The oligonucleotide, silver nitrate and The molar ratio of sodium borohydride are as follows: 1:7:7；
(3) FeSO of the 0.5-5.0mM of 0.56mL is added in the liquid obtained to step (2)4Aqueous solution is added 25-40 μ L's 100000U/L D-AAO aqueous solution, concussion mix；The concentration of the phosphate buffer solution is 20mM, and pH is 6.8；
The sequence of the oligonucleotide is -3 ˊ of 5 ˊ-CCCTTAATCCCC.
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Citations (3)

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EP1889907A1 (en) * 2005-05-23 2008-02-20 Kaneka Corporation Novel amino group transferase, gene encoding the same and method of using the same
CN102692409A (en) * 2012-06-25 2012-09-26 华东理工大学 Method and kit for detecting concentration of chiral amino acid
CN105618783A (en) * 2015-01-28 2016-06-01 江苏科技大学 Preparation method for silver nanoparticles with optical activity

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US7521427B2 (en) * 2004-11-09 2009-04-21 Georgia Tech Research Corporation Peptidyl allyl sulfones

Patent Citations (3)

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Publication number Priority date Publication date Assignee Title
EP1889907A1 (en) * 2005-05-23 2008-02-20 Kaneka Corporation Novel amino group transferase, gene encoding the same and method of using the same
CN102692409A (en) * 2012-06-25 2012-09-26 华东理工大学 Method and kit for detecting concentration of chiral amino acid
CN105618783A (en) * 2015-01-28 2016-06-01 江苏科技大学 Preparation method for silver nanoparticles with optical activity

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Min Duan et al..DNA-stabilized silver nanoclusters with guanine-enhanced fluorescence as a novel indicator for enzymatic detection of cholesterol†.《Analytical Methods》.2013,第5卷
生物大分子包被的金、银纳米簇在药物和生物分子分析中的应用;常勇;《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》;20140915(第09期);第8页倒数第2段、第18页第1段

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