CN106680344B - A kind of production method of triple strand dna biosensor - Google Patents

A kind of production method of triple strand dna biosensor Download PDF

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Publication number
CN106680344B
CN106680344B CN201611126555.6A CN201611126555A CN106680344B CN 106680344 B CN106680344 B CN 106680344B CN 201611126555 A CN201611126555 A CN 201611126555A CN 106680344 B CN106680344 B CN 106680344B
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ssdna
strand dna
triple strand
concentration
hybridization
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CN106680344A (en
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鲁理平
杨冰洁
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Beijing University of Technology
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Beijing University of Technology
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/308—Electrodes, e.g. test electrodes; Half-cells at least partially made of carbon

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Abstract

A kind of production method of triple strand dna sensor, belongs to biosensor preparation field.The following steps are included: 1) determine the ssDNA-1 in triple strand dna composition, ssDNA-2 and ssDNA-3, wherein first ssDNA-1 sequence and Article 2 ssDNA-2 partial complementarity match, and Article 3 ssDNA-3 sequence is identical with Article 2 sequence but 5 ' and 3 ' directions are completely opposite；2) by ssDNA-1 and ssDNA-2 Complementary hybridization, dsDNA is obtained；3) dsDNA hybridized and ssDNA-3 is subjected to bulk crossing, obtains stablizing triple strand dna；4) triple strand dna hybridized is assembled into glassy carbon electrode surface by nitrogen-doped graphene (NGs), completes the production of triple strand dna sensor.The stable formation and the connectivity problem between triple strand dna and electrode that the present invention solves triple strand dna.

Description

A kind of production method of triple strand dna biosensor
Technical field
The present invention relates to a kind of production methods of triple strand dna biosensor, belong to biosensor preparation field.
Background technique
Biosensor, which refers to, utilizes biological substance (such as enzyme, protein, DNA, antibody, antigen, biomembrane, microorganism, thin Born of the same parents etc.) it is used as recognition component, biochemical reaction is transformed into physics, the chemical signal that can be quantified, thus allows for living matter The device for detecting and monitoring with chemical substance.The main functional elements of biosensor are bioactive substances, this substance Interaction can be generated with target target molecule, and the result of this interaction is converted to physics or change for detection Learn information.Biosensor has the characteristics that highly selective, small in size, response is fast, instrument cost is low.
The working principle of DNA sensor is, single stranded DNA and target fixed on the electrode using single stranded DNA as recognition component The bio signal that DNA hydridization generates, converted element are converted into discernible signal.DNA electrochemica biological sensor has selection Property it is strong, preparation is simple, rapid reaction the advantages that.When not having object, the form of the single-chain nucleic acid chain in solution is uncertain , it can form various space conformations.After object is added, due to the complementary pairing between nucleic acid base, electrostatic force, model De Huali, Hyarogen-bonding etc., nucleic acid chains are folded into a variety of space structures, such as hair clip type (hairpin), false knot (pseudoknot), G- tetrad (G-quartet), the structures such as stem ring (stem-loop).
What is applied in the present invention is triple strand structure, is to be formed on the basis of DNA double helical structure, although this structure It is already found, but yet there are no applied to sensor field.3 chains for forming three chains are homopurine or homologous phonetic Pyridine is generally divided into two types: purine-purine-pyrimidine (Pu-Pu-Py) type, pyrimidine-purine-pyrimidine (Py-Pu-Py) type；Py- The stability of three spiral of Pu-Py type is by its internal factor (such as length and sequence composition etc.), external factor (such as pH, temperature With counter ion counterionsl gegenions etc.) influence.The stability of three spiral of Pu-Pu-Py type is not influenced substantially by pH, the monovalence of metal and two Valence ion can increase its thermal stability.Three chains used in this experiment are Pu-Pu-Py types, pass through and adjust MgCl2Concentration adjust The ratio that the stability and triple strand dna for saving triple strand dna are formed.
Another two-dimensional material in this experiment: nitrogen-doped graphene has the performances such as unique electricity, optics, machinery, no Only there is designability, Modulatory character in configuration aspects, and its huge specific surface area and the features such as being easy to functionalization make its Wide application prospect is shown in terms of as biosensor material.Using nitrogen-doped graphene to single stranded DNA in this experiment The characteristic stronger than double-stranded DNA suction-operated, carries out the assembling of DNA.
Summary of the invention
Technical problem to be solved by the present invention lies in stablizing between formation and triple strand dna and electrode for triple strand dna Connectivity problem.The present invention forms stable triple strand structure by adjusting the condition of DNA hybridization.Using nitrogen-doped graphene conduct Substrate is adsorbed onto electrode surface, and nitrogen-doped graphene has good suction-operated to single stranded DNA, adsorbs and makees to double-stranded DNA With very weak, so that triple strand dna can stand well in electrode surface, three sections of single-stranded formation three-legged structures make DNA exist Electrode surface is more stable.
In order to solve the above technical problems, the present invention adopts the following technical scheme:
A kind of production method of triple strand dna biosensor, comprising the following steps:
1) three single stranded DNAs: ssDNA-1, ssDNA-2, ssDNA-3 are determined.Wherein the part ssDNA-1 and ssDNA-2 is mutual It recruits pair；Three companies meet Pu-Pu-Py type structure；Remaining unpaired base is identical.
2) designed ssDNA-1 and ssDNA-2 hybridization, ratio is 1:1, forms dsDNA, hybridization conditions: hybridization conditions: 95 DEG C of denaturation 5min, temperature are down to 20 DEG C.
3) dsDNA and ssDNA-3 hybridization, hybridization conditions: MgCl2Concentration be 5mM, in 4 DEG C of hybridization 12h of refrigerator, hybridization It freezes after the completion spare.
4) prepare glass-carbon electrode: polishing glass-carbon electrode is successively cleaned by ultrasonic with secondary water, ethyl alcohol, secondary water, uses nitrogen The dry electrode surface of air-blowing, is added dropwise 3 μ L nitrogen-doped graphene solution, and standing is dried, rinsed with secondary water, is dried with nitrogen.
5) 15 μ L triple strand dnas are added dropwise to electrode surface, places 1h under wet condition, obtains triple strand dna sensor.
Preferably, the ssDNA of design itself will not hybridize winding, not will form hair fastener or other structures, structure is most Freedom in minor affairs can be zero, have quite stable single-stranded structure.
Preferably, the stock solution of DNA is PBS buffer solution, concentration 5mM, pH=7.4.
Preferably, the concentration ratio of the hybridization of the ssDNA-1 and ssDNA-2 is 1:1.
Preferably, it is 1:1 that dsDNA, which hybridizes concentration ratio with ssDNA-3, and buffer is PBS, the MgCl containing 5mM2, PBS concentration is 5mM, pH=7.4.
Preferably, nitrogen-doped graphene solvent used is ethyl alcohol, and concentration is 1 μ g/mL, and volume used is 3 μ L.
Preferably, the concentration of triple strand dna used is 15 μM, and volume is 15 μ L.
The invention has the following advantages that
1) novel: the present invention uses triple strand dna, is that other patents of invention are not used.
2) stablize: the single stranded portion that this triple strand structure is adsorbed on electrode surface forms triangle in electrode surface, has Good stability.
3) pollution-free: entire detection process does not need to use organic solvent or toxic reagent.
Detailed description of the invention
Fig. 1 is the building schematic diagram of triple strand dna sensor of the present invention.
The impedance diagram (A) and cyclic voltammogram (B) of Fig. 2 different modifying process
Specific embodiment
In order to which the contents of the present invention, objects, technical solutions and advantages are more clearly understood, below in conjunction with Figure of description And embodiment, the present invention will be described in further detail.It should be appreciated that specific embodiment described herein is only to explain The present invention is not intended to limit the present invention.General replacement known to those skilled in the art is also covered by of the invention In protection scope.
Embodiment 1
1. the design of single stranded DNA in three stranded DNA structure
Selected triple strand dna is
SsDNA-1:5 ' AAAAAAAAAATCTCTCTCTCTCT 3 '
SsDNA-2:5 ' AGAGAGAGAGAGAAAAAAAAAAA3 '
SsDNA-3:5 ' AAAAAAAAAAAGAGAGAGAGAGA3 '
Wherein, it should be noted that the ssDNA of design itself will not be intertwined, not will form hair fastener or other Structure, structure minimum free energy are zero, have quite stable single-stranded structure.Unpaired single-stranded base sequence can be adsorbed on Nitrogen-doped graphene surface, so that DNA and electrode assembling are to together.
2. experimental section
1) the single stranded DNA dry powder of order is centrifuged 5min, revolving speed is set as 6000 revs/min, and it is slow that appropriate 5mM PBS is added Fliud flushing (pH=7.4), is made into 50 μM of concentration, and concussion 3min is full and uniform to solution.
2) ssDNA-1 and ssDNA-2 concentration 1:1 hybridizes, and hybridization conditions: 95 DEG C of denaturation 5min, temperature are dropped with constant speed To 20 DEG C, dsDNA is formed.
3) dsDNA and ssDNA-3 concentration 1:1 hybridizes, and final triple strand dna concentration is 15 μM, hybridization conditions: MgCl2It is dense Degree is overregulated optimization, and ultimate density is 5mM, is freezed after the completion of 4 DEG C of hybridization 12h of refrigerator, hybridization spare.
4) with 0.05 μm of Al2O3Powder polishing glass-carbon electrode, is successively cleaned by ultrasonic with secondary water, ethyl alcohol, secondary water, It is dried with nitrogen the nitrogen-doped graphene solution that 3 μ L, 1 μ g/mL is added dropwise after electrode surface immediately, standing is dried, is rinsed with secondary water, It is dried with nitrogen.
5) 15 μ L, 15 μM of triple strand dnas are added dropwise to electrode surface, places 1h under wet condition, obtains triple strand dna sensor.
6) for the assembling of verificating sensor, each step is all verified (attached drawing 2) with impedance and cyclic voltammetric.
Although the present invention has been described by way of example and in terms of the preferred embodiments, institute not only in the description and the implementation Column use, and the embodiment is only for the purposes of explanation, for those skilled in the art, are not departing from essence of the invention Can make under the premise of mind and range it is several change and retouch, the protection scope advocated of the present invention should be described in claims Subject to.

Claims (7)

1. a kind of production method of triple strand dna sensor, which comprises the following steps:
1) composition sequence of triple strand dna, including ssDNA-1, the sequence of ssDNA-2 and ssDNA-3 are determined；
Selected triple strand dna is
SsDNA-1:5 ' AAAAAAAAAATCTCTCTCTCTCT 3 '
SsDNA-2:5 ' AGAGAGAGAGAGAAAAAAAAAAA3 '
SsDNA-3:5 ' AAAAAAAAAAAGAGAGAGAGAGA3 '
2) ssDNA-1 hybridizes with ssDNA-2, forms dsDNA, hybridization conditions: then temperature is down to 20 DEG C to 95 DEG C of denaturation 5min；
3) dsDNA and ssDNA-3 hybridization, hybridization conditions: MgCl2Concentration be 5mM, in 4 DEG C of hybridized overnights of refrigerator, hybridization is completed After freeze it is spare；
4) preparation of glass-carbon electrode: nitrogen-doped graphene is added dropwise in polishing glass-carbon electrode, ultrasonic cleaning, and N doping content is: 3.0wt%~5.0wt%, standing are dried；
5) triple strand dna is added dropwise in electrode surface, places 1h at room temperature, obtains triple strand dna sensor.
2. according to method of claim 1, which is characterized in that ssDNA-1, ssDNA-2, ssDNA-3 are dissolved in PBS buffer solution, PBS concentration is 5mM, pH=7.4.
3. according to method of claim 1, which is characterized in that the concentration ratio of the hybridization of the ssDNA-1 and ssDNA-2 is 1: 1。
4. according to method of claim 1, which is characterized in that it is 1:1 that dsDNA, which hybridizes concentration ratio with ssDNA-3, and buffer is Contain 5mM MgCl2PBS, PBS concentration be 5mM, pH=7.4.
5. according to method of claim 1, which is characterized in that the diameter of glass-carbon electrode used is 3mm, and polishing glass-carbon electrode is to use 0.05μm Al2O3Powder.
6. according to method of claim 1, which is characterized in that nitrogen-doped graphene solvent used is ethyl alcohol, and concentration is 1 μ g/mL, Volume is 3 μ L.
7. according to method of claim 1, which is characterized in that the concentration of triple strand dna used is 15 μM, and volume is 15 μ L.
CN201611126555.6A 2016-12-09 2016-12-09 A kind of production method of triple strand dna biosensor Expired - Fee Related CN106680344B (en)

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Citations (4)

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Publication number Priority date Publication date Assignee Title
CN101846648A (en) * 2010-04-20 2010-09-29 上海大学 Electrochemical biosensor modified by graphene quantum dot and preparation method thereof
CN104326468A (en) * 2014-08-29 2015-02-04 河南赛沃思生物科技有限公司 Three dimensional graphene with functionalized amino, biosensor, preparation method and applications
CN105021680A (en) * 2015-06-30 2015-11-04 上海集成电路研发中心有限公司 Graphene sensor based method for detecting MicroRNA
CN105806909A (en) * 2016-03-22 2016-07-27 南京邮电大学 DNA biosensor based on AuNPs@MoS2 and construction and application thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101846648A (en) * 2010-04-20 2010-09-29 上海大学 Electrochemical biosensor modified by graphene quantum dot and preparation method thereof
CN104326468A (en) * 2014-08-29 2015-02-04 河南赛沃思生物科技有限公司 Three dimensional graphene with functionalized amino, biosensor, preparation method and applications
CN105021680A (en) * 2015-06-30 2015-11-04 上海集成电路研发中心有限公司 Graphene sensor based method for detecting MicroRNA
CN105806909A (en) * 2016-03-22 2016-07-27 南京邮电大学 DNA biosensor based on AuNPs@MoS2 and construction and application thereof

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