CN101382517B

CN101382517B - Method for detecting nucleic acid by electrochemical activity switch molecule beacon

Info

Publication number: CN101382517B
Application number: CN2008102012296A
Authority: CN
Inventors: 程圭芳; 何品刚; 黄翠华; 张帆; 林莉; 吴继魁; 方禹之
Original assignee: East China Normal University
Current assignee: East China Normal University
Priority date: 2008-10-15
Filing date: 2008-10-15
Publication date: 2012-05-23
Anticipated expiration: 2028-10-15
Also published as: CN101382517A

Abstract

The invention discloses a method for detecting nucleic acid by using electrochemical activity switch molecular beacon, which belongs to the technical field of chemical analysis methods and comprises two steps of the synthesis of the electrochemical activity switch molecular beacon and the detection of nucleic acid molecules with specific sequences of a sample. The molecular beacon has no electrochemical activity, but when combined with target molecules, the molecular beacon has electrochemical activity due to the change of the stem-loop structure thereof, and generates oxidation peak current which has linear relationship with the concentration of the nucleic acid in the solution, therefore, the content of the target molecules of the nucleic acid can be calculated. The method takes the electrochemical activity switch molecular beacon as a detection probe without a reagent and being fixed, has the advantages of good selectivity, sensitivity, simpleness and convenience, and can quickly and directly carry out homogeneous detection on such biomolecules as nucleic acid with wide linear detection range, thereby particularly being applicable to such humble situations as fields and the like, and detecting the sample with large objective parameter change.

Description

A kind of method that detects nucleic acid with electrochemical activity switch molecule beacon

Technical field

The present invention relates to a kind of method, belong to the technical field of chemical analysis method with electrochemical activity switch molecule beacon detection nucleic acid.

Background technology

Molecular beacons technology is the emphasis of probe biomolecule research.It can: pcr amplification product is carried out detection by quantitative and pcr amplification process (real-time PCR) is done real-time online detection [document: Tyagi, F.R.Kramer, Nat Biotechnol, 1996,14,303-308; (b) Alsmadi OA, Al-Kayal F, Al-Hamed M, and Meyer BF, BMC Med Genet2006,7,43-49.]; Be used for gene quantitatively, qualitative detection and be used for the analysis of point mutation etc. and the detection [document: Du H, Disney MD, Miller BL, Krauss TD, J.Am.Chem.Soc., 2003,125,4012-4013 of double-stranded DNA; (d) Marras, S.A.E., Kramer, F.R.and Tyagi, S.Genet.Anal.Biomol.E, 1999,14,151-156; Carry out basic medical research and the detection and diagnosis [document: Fan, C., the Plaxco that directly apply in the clinical medicine various diseases such as tumour, SARS, hepatitis B and AIDS viruses; K.W., Heeger, A.; J.Proc.Natl.Acad.Sci.U.S.A.2003,100,9134-9137; (f) Douglas Horejsh, Federico Martini, Fabrizio Poccia; Giuseppe Ippolito, Nucleic Acids Res, 2005; 33, e13 has very important meaning in the development of numerous areas such as medical science, molecular biology, environmental science.

Most molecular beacons are fluorescent molecular bacon.Because huger, the valuable detecting instrument of fluorescent molecular bacon Technology Need, be not suitable for some simpler and cruder yards solution of emergent event in the open air in one's power: need the occasion of field work etc. like biological and chemical attack etc.And that electrochemical measuring technique has is highly sensitive, selectivity good, easy advantage such as inexpensive and portable.The galvanochemistry molecular beacon adopts the galvanochemistry molecular beacon that is fixed in electrode surface with an end usually at present.Its method mainly is to realize conversion [document: Fan, C.et al.Proc.Natl.Acad.Sci., 2003,100, the 9134-9137 of biomolecule/electrochemical signals through the distance that changes electrochemical activity molecule and electrode surface; Wang, J., Li, J., Baca, A.J., Hu, J., Zhou, F., Yan, W., Pang, D.-W., Anal.Chem., 2003,75,3941-3945; S.S.W.Yeung, T.M.H.Lee., I.M.Hsing., J.Am.Chem.Soc., 2006,128,13374-13375; A.E.Radi, J.L.A.Sa ', E.Baldrich, C.K.O ' Sullivan, J.Am.Chem.Soc., 2006,128,117-124.], having the background current height, detection sensitivity is low and need the fixing mode of employing, jumbo sample detection is had any problem etc.

Summary of the invention

To the deficiency that existing detection technique exists, the objective of the invention is to release a kind of method that detects nucleic acid with electrochemical activity switch molecule beacon.This method is detector probe with the electrochemical activity switch molecule beacon, and no reagent need not fixed, biomolecule such as selectivity is good, sensitive, easy, fast direct homogeneous determination nucleic acid.

The object of the invention is realized through following technical scheme.This technical scheme comprises two steps of the nucleic acid molecules of particular sequence in synthesizing of electrochemical activity switch molecule beacon and the test sample.Described molecular beacon does not have electrochemical activity, but with after target molecule combines, has electrochemical activity because of its loop-stem structure changes, produce with solution in the linear oxidation peak current of concentration of nucleic acid.Also can calculate the content of target set nucleic acid molecule thus.

Describe technical scheme of the present invention in detail at present.A kind of method with electrochemical activity switch molecule beacon detection nucleic acid is characterized in that the concrete operations step:

Synthesizing of first step electrochemical activity switch molecule beacon

Get 30 μ L 3.3 * 10 when every 1.0OD nucleic acid amount is synthetic ^-3MolL ^-1Carminic acid solution with add the 20mg imidazoles, the pH value to 6.5 of regulating this solution, activated carboxylic is 30 minutes under the room temperature; In the presence of coupling agent 12mgEDC and 27mgNHS; Add and contain the PBS solution with loop-stem structure particular sequence nucleic acid that the 1.0OD two ends are modified with amino, the pH value to 7.3 of regulating this PBS solution is 400 μ L with liquor capacity, under ice-water bath, stirs 24 hours; Bag filter was dialysed 48 hours in PBS solution; Remove not the carminic acid with the nucleic acid coupling,, obtain concentration and be not less than 1 * 10 with the purification of HPLC method ^-5MolL ^-1Electrochemical activity switch molecule beacon, preserve subsequent use in 4 ℃ of refrigerators;

The nucleic acid molecules of particular sequence in the second step test sample

In the sample that contains particular sequence nucleic acid solution to be measured, add 50 μ L 1 * 10 ^-5MolL ^-1The electrochemical activity switch molecule beacon that obtains of the first step, the hybridization conditions of control solution is the pH=7.3 and [Na of PBS solution ⁺]=1.8mol/L, 50 ℃ of following hybridization reactions 30 minutes adopt the carboxylic carbon nano-tube modified electrode to sweep DPV in 0.2～0.9V scope, the oxidation peak current value at record 0.7V place, the particular sequence nucleic acid concentration is linear in this oxidation peak current value and the sample.

Advantage of the present invention:

1, adopt electrochemical activity switch molecule beacon as detector probe, need not other reagent, unfixing, method is simple, convenient.

2, adopt Electrochemical Detection, have sensitivity, selectivity is good, background signal is low, and sample is not had the turbidity requirement, and required instrument is simple, and low price is applicable to that simple and crude occasion such as field uses.

3, can in the WS, detect by the nucleic acid to particular sequence, and the range of linearity that detects is wide, is applicable to that aim parameter changes sample detection greatly.

Embodiment

Combine embodiment to further specify technical scheme of the present invention and principle of work at present.Embodiment operates according to the concrete operations step of the method for above-mentioned detection nucleic acid fully.

Embodiment: the nucleic acid determination of human body growth factor promoter in the solution (porpholilinogen deaminase from 170to 142) sequence

The first step: press the synthetic of 3.0OD nucleic acid amount, get 90 μ L 3.3 * 10 ^-3MolL ^-1Carminic acid solution, add the 60mg imidazoles, the pH value to 6.5 of regulating this solution; Activated carboxylic is 30 minutes under the room temperature, and in the presence of coupling agent 12mgEDC and 27mgNHS, the two ends that add 3.0OD are modified with the amino specific nucleic acid fragment PBS solution (pH=7.3) that contains porpholilinogen deaminase from 170to 142 complementary seriess; Liquor capacity is 1200 μ L; Mixed liquor stirred 24 hours under ice-water bath, and bag filter (molecular weight is 7000) was dialysed 48 hours in PBS solution, remove excessive not with the carminic acid of nucleic acid coupling; Purify with the HPLC method, making 1100 μ L concentration is 1.5 * 10 ^-5MolL ^-1Electrochemical activity switch molecule beacon, preserve subsequent use in 4 ℃ of refrigerators.

Second step: containing 0.1,0.3,0.6,0.9,1.5 μ molL respectively ^-1In the particular sequence target nucleic acid solution of human body growth factor promoter (porpholilinogen deaminase from 170to 142), add 50 μ L 1 * 10 respectively ^-5MolL ^-1The electrochemical activity switch molecule beacon that obtains of the first step, the hybridization conditions of the solution of control is pH=7.3PBS and [Na ⁺]=1.8mol/L, 50 ℃ of following hybridization reactions 30 minutes adopt the carboxylic carbon nano-tube modified electrode to sweep DPV in 0.2～0.9V scope, the oxidation peak current value at record 0.7V place, this oxidation peak current value is respectively 0.32,0.64,1.16,1.62,2.75 μ A.Through calculating, its linear equation is: Y=0.122+1.83C, and Y is peak current μ A in the formula, C is target nucleic acid concentration μ molL ^-1, related coefficient is: 0.9986.

Principle of work:

Carminic acid is the hydroxy-anthraquione compounds, and electrochemical response is all arranged under positive and negative current potential.Be respectively: be positioned at-near the 0.4V a pair of redox peak, the anthraquinone radicals of corresponding carminic acid is reduced into quinhydrones and reversed reaction thereof, and is positioned at+oxidation peak of 0.720V, and corresponding quinhydrones group is oxidized to the process of two quinoid structures.

When electrochemical activity switch molecule beacon exists with ring stem structure form; When it is in closure state; Because two terminal carminic acid molecules of its stem lean on very closely, form stable intermolecular ydrogen bonding between its quinhydrones group, thereby make two carminic acid molecular associations; Carminic acid molecule after the association is in stable status more, does not show electrochemical activity; And when electrochemical activity switch molecule beacon with complementary particular sequence nucleic acid chains effect and open after, the variation of its configuration is separated terminal carminic acid molecule, this moment, the association state of carminic acid molecule was destroyed, so can show electrochemical activity.With the signaling molecule of carminic acid as electrochemical activity switch molecule beacon; Utilize the configuration that takes place after the nucleic acid chains effect of electrochemical activity switch molecule beacon and complementary particular sequence to change; The carminic acid molecule can and be dissociated at the association state changes between state, thus make its have galvanochemistry nonactive/ability changed between activity.

Claims

1. A method for detecting nucleic acid with an electrochemically active switch molecular beacon, characterized in that, the specific steps:

The first step Synthesis of electrochemically active switch molecular beacon

Take 30μL 3.3× ^10-3 molL ^-1 carminic acid solution and add 20mg imidazole when synthesizing every 1.0OD nucleic acid amount, adjust the pH value of the solution to 6.5, activate the carboxyl group at room temperature for 30 minutes, and add 12mgEDC and 27mgNHS as the coupling agent In the presence of presence, add a PBS solution containing a 1.0OD nucleic acid with a specific sequence of stem-loop structure modified with amino groups at both ends, adjust the pH value of the PBS solution to 7.3 and the solution volume to 400 μL, stir in an ice-water bath for 24 hours, and the dialysis bag was placed in Dialyze in PBS solution for 48 hours to remove carminic acid that is not coupled to nucleic acid, purify by HPLC, and obtain an electrochemically active switch molecular beacon with a concentration of not less than 1×10 ^-5 molL ^-1 , and store in a refrigerator at 4°C spare;

The second step is to detect nucleic acid molecules of a specific sequence in the sample

Add 50 μL of 1×10 ^-5 molL ^-1 electrochemically active switch molecular beacon obtained in the first step to the sample containing the nucleic acid solution of the specific sequence to be tested. The hybridization conditions of the control solution are the pH of the PBS solution = 7.3 and [Na ⁺ ]=1.8mol/L, hybridization reaction at 50°C for 30 minutes, use carboxylated carbon nanotube modified electrode to scan DPV in the range of 0.2-0.9V, record the oxidation peak current value at 0.7V, the oxidation peak current value There is a linear relationship with the concentration of specific sequence nucleic acids in the sample.