CN1376917A - Method for testing ultratrace DNA by dual-amplifying technique and electrochemical quartz crystal microbalance - Google Patents
Method for testing ultratrace DNA by dual-amplifying technique and electrochemical quartz crystal microbalance Download PDFInfo
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- CN1376917A CN1376917A CN 02115772 CN02115772A CN1376917A CN 1376917 A CN1376917 A CN 1376917A CN 02115772 CN02115772 CN 02115772 CN 02115772 A CN02115772 A CN 02115772A CN 1376917 A CN1376917 A CN 1376917A
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Abstract
A method for detecting ultratrace DNA by electrochemical quartz crystal microbalance and double amplifying technique includes such steps as plating a thin Au or Ag film or quartz crystal, selectively adsorbing DNA by quartz crystal oscillator, molecular hybridizing of DNA to obtain dual-chain DNa, amplifying the detected signals in mass and electrochemical areas by the nano microbead marked by chemical or biologic reagent which can selectively act on the dual chains of DNA and has electrochemical activity, and measuring the mass signal and electrochemical signal. Its advantages are high selectivity and high sensitivity.
Description
Technical field
The present invention relates to the method that a kind of dual amplifying technique electrochemical quartz crystal microbalance detects ultratrace DNA.
Background technology
QCM (Quartz Crystal Microbalance) (QCM) claim piezoelectric sensor again, and its theoretical foundation is based on piezoelectric effect.The principle of piezoelectric DNA biosensor is: plate skim gold film or silverskin earlier on quartz wafer, manage to allow optionally adsorption of DNA of quartz crystal oscillator then, pass through dna molecule hybridize, another DNA that contains complementary base sequence is discerned, be combined into double-stranded DNA, utilize frequency change reflection hybridisation events.Because QCM can have frequency response to the quality that is increased in surperficial nanogram level, thereby is a kind of highstrung quality detection device, is suitable for detecting the change of single stranded DNA (ssDNA) quality before and after hybridization, thereby can uses the Study on Biosensor with DNA.QCM and electrochemical techniques coupling have constituted electrochemical quartz crystal microbalance (EQCM).It had both had the high-quality sensitivity of QCM, can carry out the quality determination of nanogram level; Simultaneously can obtain galvanochemistry information again, the subtle change of on-site measurement electrode surface.
The Applied Electrochemistry QCM (Quartz Crystal Microbalance) as the biology sensor development rapidly in recent years, demonstrating great potential aspect making nucleic acid molecular hybridization identification and the mensuration, have broad application prospects, often only utilize quality aspect signal for the EQCM Study on Biosensor at present, be the QCM biology sensor.Though QCM is a kind of mass detector of sensitivity, can record Ya Nake level mass change, but in actual detected, the scope that the concentration of DNA directly detects well below QCM, particularly as detecting the biology sensor that ultratrace changes, its sensitivity can't reach requirement.
Summary of the invention
The present invention provides a kind of dual amplifying technique electrochemical quartz crystal microbalance to detect the method for ultratrace DNA at the problems referred to above, and this method can detect the target dna of ultratrace, and highly sensitive.
Technical scheme provided by the invention is: a kind of dual amplifying technique electrochemical quartz crystal microbalance detects the method for ultratrace DNA, plating skim gold film or silverskin on quartz wafer, allow optionally adsorption of DNA of quartz crystal oscillator then, pass through dna molecule hybridize, be combined into double-stranded DNA, its special character is: will be marked with and can do quality and the dual amplification of galvanochemistry to detection signal with the selection effect of dna double chain and chemicals or biological reagent marking nano microballon with electrochemical activity, and carry out then that quality signal will detect and electrochemical signals mensuration.
Above-mentioned nano microsphere is magnetic micro-beads or albumin microballon.
Above-mentioned can be adriamycin class medicine with selection effect of dna double chain and the chemicals with electrochemical activity.
Above-mentioned can be actinomycin D with selection effect of dna double chain and the biological reagent with electrochemical activity.
Above-mentioned quartz crystal oscillator was immersed in absolute methanol solution earlier 10～60 minutes before using, and ultrasonic cleaning is 1～3 minute then, with redistilled water washing 3～5 times, dried again.
The present invention is being modified on the nano microsphere as guide molecule with double-stranded DNA selectively acting and medicine or biological reagent with electrochemical activity, the nano microsphere of guide molecule mark interacts with the ds-DNA that is modified on the EQCM electrode, because the big quality of nano microsphere, the ds-DNA on medicine or bio-guide molecule and the EQCM electrode interacts and causes frequency change significantly.This method can make the lowest detection lower limit from 10
-8The mol/L order of magnitude drops to 10
-13The mol/L order of magnitude.Simultaneously because the big surface area of nano microsphere, increased the quantity of guide molecule on electrode, electrochemical signals strengthens widely when electrochemical gaging, thereby reflect card, identification mutually and amplify the mensuration target dna from quality and electrochemical signals two aspects by electrochemical quartz crystal microbalance (EQCM) biosensor technology, and then improve sensitivity on both side.Compare with the piezoelectric DNA biosensor of routine, the present invention can make lowest detectable limit improve 2～5 orders of magnitude, thereby not only can detect the target dna of ultratrace, and selectivity is strong, highly sensitive.
Embodiment
Embodiment 1: the nano-magnetic microsphere of modifying with adriamycin (ADM) forms the target double chain DNA molecule and proves conclusively and do quality and amplify, and detects the target dna of ultratrace.
One, the preparation of the magnetic microsphere of adriamycin modification: preparation 2.0 * 10
-6The aqueous solution of mol/L adriamycin is used to prepare the cross-linking reaction between amino-aldehyde radical, behind the 0.02g aldehyde radical magnetic microsphere to be added (available commercially available prod), place 4 ℃ to stir down, reacted 4 hours in this potpourri, microballoon cleans 3 times with double distilled water on the magnetic force frame, add the redistilled water of 0.5mL, 4 ℃ of preservations are standby.
Two, self assembly alpha-lipoic acid monofilm: quartz wafer was immersed in absolute methanol solution earlier 30 minutes before using, and ultrasonic cleaning is 1 minute then, with redistilled water washing 3 times, dried in the air again.The quartz wafer of washes clean is placed the Teflon reaction tank of QCM rapidly, add the ethanolic solution of 0.5 milliliter of 0.3mol/L alpha-lipoic acid, with the redistilled water washing, dry in the air after 2 hours.
Three, the curing of probe: from the Teflon reaction tank of QCM, take out chip electrode, the N-hydroxy-succinamide (NHS) that drips hydrochloric acid 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) of 10 microlitre 100mg/mL and 10 microlitre 100mg/Ml was placed 20 minutes to the film modified quartzy electrode slice surface of self-assembled monolayer.The redistilled water washing is dried in air.' end has-NH to add 10 microlitres 5 on the wafer gold electrode surfaces
2SsDNA (5 '-XATGGGTATTCAACATTTCCG, X=-NH
2) probe solution (10
-5Mol/L), reaction is 30 minutes.(PBS, pH=7.0) washing is dried standby in air with the 1/15mol/L phosphate buffer solution.
Four, hybridization and chimeric: will be encased in the Teflon reaction tank of QCM through the chip electrode after the above-mentioned processing, with 0.2mol/L phosphate buffer solution (pH=7.0, contain 50% formamide) as hybridization buffer solution, join in the Teflon reaction tank of QCM, after treating the wafer frequency stabilization (Δ f≤± 1Hz/min), add the not complementary target dnas of 10 microlitres (ncDNA) (5 ' GACGTCAGCA), start QCM, recording frequency changes as blank, adds its complementary target dna (cDNA) (5 ' CGGAAATGTTGAATACC) of 10 microlitres after about 5 minutes.After frequency change is stable, add 5-20 microlitre Act D again or through the nano-magnetic microsphere of the 0.5mg/mL Act D modified of step 1 preparation in the Teflon reaction tank, make it with ds-DNA chimeric 15 minutes, recording frequency changes.
Five, electrochemical gaging: Xiao Chi adds the 2.0mol/L hac buffer in the Teflon reaction, (the EQCM gold electrode through modification is a working electrode with three-electrode system, the Ag/AgCl contrast electrode, platinum is to electrode) carry out differential pulse voltammetry (DPV) electrochemical gaging, sweeping journey is 0-1.0V.After test was finished, the crystal oscillator electrode was immersed in 30min in the methanol solution, ultrasonic cleaning 1min, and again with redistilled water washing 3 times, each 1min, crystal oscillator is promptly reusable.
Its result shows, compares with the piezoelectric DNA biosensor of routine, and the present invention has greatly improved the sensitivity of QCM, makes lowest detectable limit drop to 10
-12～10
-13Mol/L.
Embodiment 2: the nano-magnetic microsphere of modifying with actinomycin D (ActD) forms the target double chain DNA molecule and proves conclusively and do quality and amplify, and detects the target dna of ultratrace.
The preparation of the nano-magnetic microsphere that actinomycin D is modified: preparation 1.0 * 10
-6The acetone soln of mol/L actinomycin D is used to prepare the cross-linking reaction between amino-aldehyde radical, behind the 0.02g aldehyde radical magnetic microsphere to be added, place 4 ℃ to stir down, reacted 4 hours in this potpourri, microballoon cleans 3 times with acetone on the magnetic force frame, add the acetone of 0.5mL, 4 ℃ of preservations are standby.All the other steps are with embodiment 1.
Embodiment 3: the albumin nanometer microballoon of modifying with mitoxantrone forms the target double chain DNA molecule and proves conclusively and do quality and amplify, and detects the target dna of ultratrace.
The preparation of the albumin nanometer microballoon that mitoxantrone is modified: preparation 2.0 * 10
-6The aqueous solution of mol/L mitoxantrone is used to prepare the cross-linking reaction between amino-aldehyde radical, behind the 0.02g aldehyde radical albumin microsphere to be added, place 4 ℃ to stir down, reacted 4 hours in this potpourri, 12000 rev/mins centrifugal, clean 3 times with double distilled water, add the redistilled water of 0.5mL, 4 ℃ of preservations are standby.All the other steps are with embodiment 1.
Other anthracene ring antitumor medicinals such as adriamycin class medicines such as the also available adriamycin epirubicin of the present invention, zorubicin and bisantrene or other can with the Nano microsphere of the medicine of double-stranded DNA selectively acting or biological reagent mark as the magnifying tags thing.
Claims (5)
1. a dual amplifying technique electrochemical quartz crystal microbalance detects the method for ultratrace DNA, plating skim gold film or silverskin on quartz wafer, allow optionally adsorption of DNA of quartz crystal oscillator then, pass through dna molecule hybridize, be combined into double-stranded DNA, it is characterized in that: will be marked with and to do quality and the dual amplification of galvanochemistry with the selection effect of dna double chain and chemicals or biological reagent marking nano microballon with electrochemical activity to detection signal, and carry out then that quality signal will detect and electrochemical signals mensuration.
2. method according to claim 1 is characterized in that: described nano microsphere is magnetic micro-beads or albumin microballon.
3. method according to claim 1 and 2 is characterized in that: above-mentioned can be adriamycin class medicine with selection effect of dna double chain and the chemicals with electrochemical activity.
4. method according to claim 1 and 2 is characterized in that: above-mentioned can be actinomycin D with selection effect of dna double chain and the biological reagent with electrochemical activity.
5. method according to claim 1 and 2 is characterized in that: quartz crystal oscillator was immersed in absolute methanol solution earlier 10～60 minutes before using, and ultrasonic cleaning is 1～3 minute then, with redistilled water washing 3～5 times, dried again.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021157723A CN1180251C (en) | 2002-04-28 | 2002-04-28 | Method for testing ultratrace DNA by dual-amplifying technique and electrochemical quartz crystal microbalance |
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|---|---|---|---|
| CNB021157723A CN1180251C (en) | 2002-04-28 | 2002-04-28 | Method for testing ultratrace DNA by dual-amplifying technique and electrochemical quartz crystal microbalance |
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| Publication Number | Publication Date |
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| CN1376917A true CN1376917A (en) | 2002-10-30 |
| CN1180251C CN1180251C (en) | 2004-12-15 |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101852707A (en) * | 2010-05-20 | 2010-10-06 | 中国科学院化学研究所 | Quartz crystal microbalance signal amplification method taking polystyrene spheres as template |
| CN101871872A (en) * | 2010-06-11 | 2010-10-27 | 东华大学 | Method for detecting formaldehyde by quartz crystal microbalance sensor |
| CN102778408A (en) * | 2012-06-15 | 2012-11-14 | 天津大学 | Device of quartz crystal monitor (QCM) chip with surface modified by zinc oxide nanometer chain and preparation method of QCM chip |
| CN103954521A (en) * | 2014-04-28 | 2014-07-30 | 国家电网公司 | Method for testing content of corrosive sulphur in insulating oil |
| CN104165819A (en) * | 2013-08-29 | 2014-11-26 | 北京至感科技有限公司 | Online real-time magnetic particle monitoring system |
| CN104198323A (en) * | 2013-09-03 | 2014-12-10 | 北京至感科技有限公司 | Highly sensitive acid sensor |
| CN104388570A (en) * | 2014-12-04 | 2015-03-04 | 中国科学院苏州生物医学工程技术研究所 | Piezoelectric thin film technology-based nucleic acid single-gene mutation detection method |
| CN111537603A (en) * | 2019-07-26 | 2020-08-14 | 临沂大学 | QCM-based cell detection method |
| CN111534572A (en) * | 2019-07-26 | 2020-08-14 | 临沂大学 | QCM-based nucleic acid sequence detection method |
| CN111812319A (en) * | 2020-07-21 | 2020-10-23 | 中南大学 | Microfluidic quartz wafer array sensing detection system and detection method thereof |
Families Citing this family (1)
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| CA2469116C (en) * | 2003-06-25 | 2009-08-18 | Asf-Keystone, Inc. | Three-piece motion control truck system |
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2002
- 2002-04-28 CN CNB021157723A patent/CN1180251C/en not_active Expired - Fee Related
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101852707A (en) * | 2010-05-20 | 2010-10-06 | 中国科学院化学研究所 | Quartz crystal microbalance signal amplification method taking polystyrene spheres as template |
| CN101852707B (en) * | 2010-05-20 | 2011-10-19 | 中国科学院化学研究所 | Quartz crystal microbalance signal amplification method taking polystyrene spheres as template |
| CN101871872A (en) * | 2010-06-11 | 2010-10-27 | 东华大学 | Method for detecting formaldehyde by quartz crystal microbalance sensor |
| CN101871872B (en) * | 2010-06-11 | 2011-11-09 | 东华大学 | Method for detecting formaldehyde by quartz crystal microbalance sensor |
| CN102778408B (en) * | 2012-06-15 | 2014-11-26 | 天津大学 | Device of quartz crystal monitor (QCM) chip with surface modified by zinc oxide nanometer chain and preparation method of QCM chip |
| CN102778408A (en) * | 2012-06-15 | 2012-11-14 | 天津大学 | Device of quartz crystal monitor (QCM) chip with surface modified by zinc oxide nanometer chain and preparation method of QCM chip |
| CN104165819A (en) * | 2013-08-29 | 2014-11-26 | 北京至感科技有限公司 | Online real-time magnetic particle monitoring system |
| CN104198323A (en) * | 2013-09-03 | 2014-12-10 | 北京至感科技有限公司 | Highly sensitive acid sensor |
| CN103954521A (en) * | 2014-04-28 | 2014-07-30 | 国家电网公司 | Method for testing content of corrosive sulphur in insulating oil |
| CN103954521B (en) * | 2014-04-28 | 2016-04-20 | 国家电网公司 | Detect the method for active sulfur content in insulating oil |
| CN104388570A (en) * | 2014-12-04 | 2015-03-04 | 中国科学院苏州生物医学工程技术研究所 | Piezoelectric thin film technology-based nucleic acid single-gene mutation detection method |
| CN111537603A (en) * | 2019-07-26 | 2020-08-14 | 临沂大学 | QCM-based cell detection method |
| CN111534572A (en) * | 2019-07-26 | 2020-08-14 | 临沂大学 | QCM-based nucleic acid sequence detection method |
| CN111812319A (en) * | 2020-07-21 | 2020-10-23 | 中南大学 | Microfluidic quartz wafer array sensing detection system and detection method thereof |
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|---|---|
| CN1180251C (en) | 2004-12-15 |
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