CN101093220A - Detetion method in vitro, kit, and instrument based on magnetic grains and microelectrodes - Google Patents
Detetion method in vitro, kit, and instrument based on magnetic grains and microelectrodes Download PDFInfo
- Publication number
- CN101093220A CN101093220A CN 200610086617 CN200610086617A CN101093220A CN 101093220 A CN101093220 A CN 101093220A CN 200610086617 CN200610086617 CN 200610086617 CN 200610086617 A CN200610086617 A CN 200610086617A CN 101093220 A CN101093220 A CN 101093220A
- Authority
- CN
- China
- Prior art keywords
- microelectrode
- magnetic particle
- detection method
- method based
- external detection
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Abstract
An external detecting-method based on magnetic particle and microelectrode includes fixing specific labeled object with magnetic particle, neutralizing material to be tested in liquid phase, hybridizing specific labeled object with gold particle to form magnetic-gold particle composite body, separating material to be tested out after composite body is silver-dyed, transferring separated material to be tested on to microelectrode and using electric detector to detect impedance of said material after drying for carrying out quantitative or qualitative analysis on material to be tested.
Description
[technical field]
The present invention relates to a kind of external detection method, kit and the instrument of biomedical sector, more precisely, be a kind ofly to carry out the technology, related kit of vitro detection and a kind of robotization detecting instrument of technological development thus at the biomacromolecule material based on magnetic nano particle and gold nano grain.
[background technology]
At present biological detection adopts optical means usually, and by means such as fluorescence labeling, chemiluminescence, enzyme connection, but optical means exists sensitivity lower, poor stability, and the equipment needed thereby costliness such as is not easy to carry at shortcoming.
In recent years, along with the fusion of nanometer technology, microelectric technique and biotechnology, multidisciplinary intersection infiltration has produced the electrical detection method thereupon.Professor Mirkin of U.S. Northwest University has taken the lead in realizing need not the anthrax DNA electrical detection of pcr amplification, and (Science 295,1503-1506,2002), the following (see figure 1) of its concise and to the point process: 1) Chang Gui photoetching process prepares microelectrode, with dna probe molecule (P1) Covalent Immobilization in the microelectrode gap; 2) dna probe molecule (P2) of dna molecular to be measured, nanogold particle mark and the hybridization of P1 probe molecule, nanogold particle will be assembled in the microelectrode gap; 3) silver-colored dye liquor is handled, and at deposition one deck silver of gold grain surface selectivity, changes interelectrode resistance (it also is the step that signal amplifies that silver dyes); 4) the while monitoring current is surveyed the dna molecular identifying.This based on the biomolecule detection method of microelectrode need not complicated optical system, be convenient to the integrated of detecting instrument, can make the detecting instrument of the formula of taking; For the user, simple to operate.
But this detection method also has its limitation, 1) because the interaction of probe molecule and substrate, interaction between probe molecule, the absorption of liquid intermediate ion on probe molecule, the pattern of substrate surface complexity is so probe molecule is difficult to the distribution of homogeneous, directed being arranged on the substrate; 2) have only near the testing molecule of substrate could be fixed on on-chip probe molecule hybridization, most detection molecules is free on solution, makes detection sensitivity lower (see figure 2) greatly; 3) silver dyes part Ag in the process
+Can form the Ag particle, non-specific adsorption is on electrode base sheet, and ground unrest increases (Nano Letters 5,1475-1482 2005); 4) little current detecting is carried out in liquid phase, be difficult to avoid the interference of effects of ion to electric current, so signal to noise ratio (S/N ratio) is lower.
[summary of the invention]
At the above-mentioned shortcoming of prior art, the technical purpose that the present invention will reach provides a kind of highly sensitive, detection method that specificity is good, and develops a kind of detector, has apparatus expensive heaviness, the deficiency that sensitivity is not high at present thereby solve.
Technical scheme, the present invention proposes solution hybridization, silver dyes, the route (see figure 4) that the solid phase microelectrode detects, step is as follows: 1) enrichment, magnetic nano particle enrichment in the liquid phase material to be detected that utilizes specific marker thing 1 (L1) to modify, test substance can be nucleic acid, albumen, comprises DNA, RNA, antigen, antibody etc.; 2) form magnetic-gold grain complex, can add specific marker thing 2 (L2), gold nano grain, or directly add the L2 of gold nano grain mark; 3) silver dyes, and adds silver-colored dye liquor in magnetic-gold grain complex surfaces selective deposition one deck silver, 4) separate the complex after the externally-applied magnetic field separation of Silver is dyed; 5) electrical detection is dyed the back complex with silver and is transferred on the microelectrode, surveys its impedance after the dried.
Solution hybridization has following advantage: 1) adopt glucosan or agarose or chitosan-modified magnetic particle, can reduce hybridization, silver dyes the non-specific adsorption in the process; 2) the magnetic particle diameter is less than 1 μ m, and specific marker thing L1 can modify the magnetic nano particle surface more evenly, in an orderly manner; 3) magnetic nano particle has increased the reaction interface of specific marker thing molecule and testing sample, enrichment test substance (see figure 3) effectively greatly; 4) the magnetic nano particle homogeneous is dispersed in the reactant liquor, and reaction is more near physiological environment.Uniform sequential modification, high reaction interface, homogeneous disperses to make that the reaction time is faster, sensitivity is higher, specificity is better in the liquid phase.
Solid phase detects, and following advantage is arranged: after 1) silver dyed, magnetic-gold grain complex was separated in magnetic field, removed the Ag particle that produces without Au catalysis, and complex is distributed in the solution again, transferred on the microelectrode, had reduced Ag
+The ground unrest (Nano Letters 5,1475-1482 2005) that the Ag particle that spontaneous reduction produces in solution causes; 2) electrical signal is reliable, and repeatability improves; 3) though the detection material difference, silver dye and the electrical measurement process the same, can realize measuring simultaneously on the micro-electrode chip a plurality of samples of multiple material or a kind of material.Therefore, our scheme, sensitivity, quick, stable, the big molecule of detection of biological specifically more.
The specific marker thing, according to nucleic acid hybridization and antigen and antibody specific combination principle, the specific marker thing is DNA, RNA, antigen or antibody, its common feature is that L1, L2 can while and test substance combinations.
Gold grain, silver-colored particle can catalysis Ag
+Both become Ag at its surface reduction, so all can be used for binding specificity label L2 (Science 295,1503-1506,2002).But also catalysis Ag of some enzymes of bibliographical information is arranged recently
+Become Ag at its surface reduction, generate the Ag particle, so these enzymes also can be used in conjunction with L2 (Nano Letters5,1475-1482 2005).
Silver dyes the separation method of back complex, dyes the back complex by the externally-applied magnetic field separation of Silver and has quick, easy advantage, also can separate complex by traditional centrifuge method in addition.
The electrical detection instrument is made up of signal generator, amplifier and display three parts.Signal generator produces the voltage signal of-1～1V, puts on the microelectrode.Amplify by amplifier, draw current-voltage curve through display.Can be in conjunction with automation equipment, the robotization detector that hybridization, the silver that makes up a kind of biomacromolecule dyes, all finishes by an integrated robotization pick-up unit need not manual step-by-step operation.
[description of drawings]
Fig. 1 is the electrical detection method route figure of professor's Mirkin initiative, and its hybridization, silver dye and little current detecting is all carried out on substrate,
1 is electrode, and 2 is that probe 1,3 is a test substance, and 4 is that the probe 2,5 of gold grain mark is Ag
+
Fig. 2 is on-chip hybridization, have only near the testing molecule of substrate could be fixed on on-chip probe molecule hybridization, most detection molecules is free on solution, makes detection sensitivity lower greatly.
1 is can be in the gold grain of probe molecule combination, and 2 is the specific probe molecule, and 3 is substrate.
Fig. 3 is the solution hybridization based on the magnetic particle, has both increased reaction interface, again near physiological environment, combines molecule to be checked to greatest extent, has improved sensitivity.
1 for being distributed to the magnetic particle in the solution, and 2 is can be in conjunction with the specific marker thing of magnetic particle, and 3 is test substance.
Fig. 4 is solution hybridization, the solid phase detection of biological molecule electrical detection route map that the present invention innovated
1 is the magnetic particle, and 2 is can be in conjunction with the specific marker thing L1 of magnetic particle, and 3 is test substance, 4 is specific marker thing L2, and 5 is the gold grain of energy and L2 combination, and 6 silver for formation after silver-colored the dying dye magnetic-gold grain complex, 7 is externally-applied magnetic field, and 8 is the microelectrode detector.
Fig. 5 is that detection method of the present invention detects the anthrax nucleic acid fragment.
Fig. 6 is that detection method of the present invention detects mouse AA98 monoclonal antibody.
[embodiment]
Detection of biological samples example 1
Utilize experimental technique of the present invention to carry out the detection of anthrax DNA, used 50nm magnetic particle is bought by Dynal company, mark is with reference to instructions on the magnetic particle for probe 1, and silver-colored transfection reagent box Sigma company buys, and silver dyes process with reference to instructions, the probe 2 of gold grain mark is seen document, and (Science 295,1503-1506,2002), the anthrax nucleic acid fragment is bought in match Parkson, Beijing company, the gap is that the microelectrode of 50 μ m prepares by photoetching method, and concrete implementation step is as follows:
1) enrichment: the magnetic nano particle that the anthrax dna probe (P1) of 50 μ L is modified, the anthrax DNA sample mix of 50 μ L, hybridization is 20 minutes under the room temperature, externally-applied magnetic field absorption magnetic particle, PBS is distributed in the 100 μ L hybridization solutions after cleaning three times again.
2) form magnetic particle-gold nano grain complex: add under gold grain label probe 2 (P2) 10 μ L, the room temperature and hybridized 20 minutes, form complex, the externally-applied magnetic field absorbing complex, PBS cleans three times.
3) silver dyes: add 20 μ L silver dye liquor A and 20 μ L silver dye liquor B, reacted 5 minutes, the externally-applied magnetic field separation obtains black precipitate, and PBST cleans once, and distilled water is distributed to 20 μ L solution after cleaning twice again.
4) electrical detection: sediment is transferred on the microelectrode, and 55 ℃ of heating made water evaporates in 10 minutes, and its impedance is surveyed in dry back.For detecting this sensitivity based on nano particle and microelectrode electrical detection method, the anthrax dna segment is diluted, contain 10 respectively in the 50 μ L solution
4, 10
5, 10
6With 10
7Individual anthrax dna segment.Pure water is made blank.The result shows that the logarithm of anthrax dna segment number and the logarithm that records corresponding resistor are linear approximate relationship (as Fig. 5), can be used for quantitative test.The resistance that records of blank group is 10
6, contain 10
4It is 10 that individual anthrax dna segment group records resistance
3, signal noise ratio about 10
3Be fixed on on-chip detection between electrode with probe molecule, signal noise ratio has improved two orders of magnitude above (Science295,1503-1506,2002).
Detection of biological samples example 2
Utilize experimental technique of the present invention to carry out the detection of mouse AA98 monoclonal antibody, used 50nm magnetic particle is bought by Dynal company, A albumen on the magnetic particle mark with reference to instructions, two anti-Sigma companies of silver transfection reagent box, gold grain mark buy, silver dyes process with reference to instructions, the gap is that the microelectrode of 50 μ m prepares by photoetching method, and concrete implementation step is as follows:
1) enrichment: the magnetic nano particle that the A of 100 μ L is protein modified, the mouse AA98 monoclonal antibody of 100 μ L is mixed, following 120 minutes of room temperature, externally-applied magnetic field absorption magnetic particle, PBS is distributed in the 100 μ L hybridization solutions after cleaning three times again.
2) form magnetic particle-gold nano grain complex: hybridization is 20 minutes under two anti-20 μ L of adding gold grain mark, the room temperature, forms complex, the externally-applied magnetic field absorbing complex, and PBS cleans three times.
3) silver dyes: add 20 μ L silver dye liquor A and 20 μ L silver dye liquor B, react after 5 minutes externally-applied magnetic field and separate and obtain black precipitate, and externally-applied magnetic field absorption, PBST cleans once, is distributed to 20 μ L solution again after twice of the distilled water cleaning.
4) electrical detection: sediment is transferred on the microelectrode, and 55 ℃ of heating made water evaporates in 10 minutes, and its impedance is surveyed in dry back.For detecting this sensitivity based on nano particle and microelectrode electrical detection method, antibody is diluted, contain 10pg respectively in the 100 μ L solution, 100pg, 1ng, 10ng antibody.Pure water is made blank.The result shows that the logarithm of antibody concentration and the logarithm that records corresponding resistor are linear approximate relationship, can be used for quantitative test (as Fig. 6).
Claims (18)
1. external detection method based on magnetic particle and microelectrode, it is characterized in that comprising the following steps 1) design specific marker thing 1 (L1), specific marker thing 2 (L2), make L1 and L2 energy simultaneously in conjunction with test substance, L1 is fixed on the magnetic particle, L2 energy joining gold particle; 2) in solution, be fixed with magnetic particle, L2, gold grain and the test substance of L1 in conjunction with forming magnetic-gold grain complex; 3) add silver-colored dye liquor at complex surfaces selective deposition silver; 4) separation of Silver is dyed complex and is transferred on the microelectrode, surveys its impedance by detector after the dried.
2. the external detection method based on magnetic particle and microelectrode according to claim 1 is characterized in that described test substance is nucleic acid and protein.
3. the external detection method based on magnetic particle and microelectrode according to claim 2 is characterized in that described nucleic acid is DNA and RNA.
4. the external detection method based on magnetic particle and microelectrode according to claim 2 is characterized in that described nucleic acid comprises RNA, cDNA and the anthrax DNA of bird flu.
5. the external detection method based on magnetic particle and microelectrode according to claim 2 is characterized in that described protein is antigen or antibody.
6. the external detection method based on magnetic particle and microelectrode according to claim 2 is characterized in that described protein comprises tumor marker.
7. the external detection method based on magnetic particle and microelectrode according to claim 6 is characterized in that described tumor marker comprises alpha-fetoprotein, carcinomebryonic antigen, prostate specific antigen, CA-199, CA-125, CA-153.
8. the external detection method based on magnetic particle and microelectrode according to claim 1 is characterized in that, with silver-colored particle or energy catalysis Ag
+The enzyme that is reduced into silver at its surface selectivity is replaced the gold grain in the claim 1.
9. the external detection method based on magnetic particle and microelectrode according to claim 1 is characterized in that, it is externally-applied magnetic field or centrifugation that described silver dyes the complex separation method.
10. the external detection method based on magnetic particle and microelectrode according to claim 1 is characterized in that described magnetic particle surface is modified with glucosan or agarose or shitosan.
11. the external detection method based on magnetic particle and microelectrode according to claim 1 is characterized in that one or more electrodes are arranged on the described electrode chip, the microelectrode gap is 1-200 μ m.
12. the external detection method based on magnetic particle and microelectrode according to claim 1, it is characterized in that by manual operation finish hybridization, silver dyes and detect step.
13. the external detection method based on magnetic particle and microelectrode according to claim 1, it is characterized in that by full-automatic detector finish hybridization automatically, silver dyes and detect step.
14., it is characterized in that being used for the qualitative detection or the detection by quantitative of test substance according to the purposes of the described external detection method based on magnetic particle and microelectrode of claim 1-13.
15. be used for the kit of the described external detection method based on magnetic particle and microelectrode of claim 1, it is characterized in that comprising hybridization kit, silver-colored transfection reagent box.
16. kit according to claim 15 is characterized in that the magnetic grain size in the kit is 8-800nm, finishing has glucosan or agarose or shitosan.
17. be used for the detection system of the described external detection method based on magnetic particle and microelectrode of claim 1, it is characterized in that comprising the described kit of claim 15, described microelectrode of claim 11 and detector, and the described full-automatic detector of claim 13.
18. the detection system based on magnetic particle and microelectrode according to claim 17, its microelectrode is characterized as interdigital electrode.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200610086617 CN101093220A (en) | 2006-06-23 | 2006-06-23 | Detetion method in vitro, kit, and instrument based on magnetic grains and microelectrodes |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200610086617 CN101093220A (en) | 2006-06-23 | 2006-06-23 | Detetion method in vitro, kit, and instrument based on magnetic grains and microelectrodes |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101093220A true CN101093220A (en) | 2007-12-26 |
Family
ID=38991583
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200610086617 Pending CN101093220A (en) | 2006-06-23 | 2006-06-23 | Detetion method in vitro, kit, and instrument based on magnetic grains and microelectrodes |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101093220A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101923092A (en) * | 2010-06-28 | 2010-12-22 | 宁波大学 | Method for preparing carcinoembryonic antigen working electrode for screen printing electrode |
| CN102089661A (en) * | 2008-07-10 | 2011-06-08 | 英国政府创新、大学与技能部 | Sample carrier for effecting chemical assays |
| CN102435736A (en) * | 2011-11-29 | 2012-05-02 | 桂林理工大学 | Method for measuring antigen of ovarian cancer embryo by electrochemical luminescence (ECL) immunosensor |
| CN115646562A (en) * | 2022-09-27 | 2023-01-31 | 郑州大学 | Control chip based on micro-magnetic action, detection assembly, detection system and method |
-
2006
- 2006-06-23 CN CN 200610086617 patent/CN101093220A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102089661A (en) * | 2008-07-10 | 2011-06-08 | 英国政府创新、大学与技能部 | Sample carrier for effecting chemical assays |
| CN102089661B (en) * | 2008-07-10 | 2014-09-24 | 英国政府创新、大学与技能部 | Sample carrier for effecting chemical assays |
| CN101923092A (en) * | 2010-06-28 | 2010-12-22 | 宁波大学 | Method for preparing carcinoembryonic antigen working electrode for screen printing electrode |
| CN102435736A (en) * | 2011-11-29 | 2012-05-02 | 桂林理工大学 | Method for measuring antigen of ovarian cancer embryo by electrochemical luminescence (ECL) immunosensor |
| CN115646562A (en) * | 2022-09-27 | 2023-01-31 | 郑州大学 | Control chip based on micro-magnetic action, detection assembly, detection system and method |
| CN115646562B (en) * | 2022-09-27 | 2023-08-11 | 郑州大学 | Micro-magnetic effect-based control chip, detection assembly, detection system and method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Wei et al. | Graphene nanocomposites modified electrochemical aptamer sensor for rapid and highly sensitive detection of prostate specific antigen | |
| Wang et al. | Low sample volume origami-paper-based graphene-modified aptasensors for label-free electrochemical detection of cancer biomarker-EGFR | |
| Wang et al. | A silver–palladium alloy nanoparticle-based electrochemical biosensor for simultaneous detection of ractopamine, clenbuterol and salbutamol | |
| JP2020126068A (en) | Co-binder assisted assay methods | |
| Xu et al. | Electrochemical biosensors based on magnetic micro/nano particles | |
| kholafazad Kordasht et al. | Biosensing of microcystins in water samples; recent advances | |
| CN101231287B (en) | Method for preparing biosensor by arraying nanometer particles in external field evoked electrode | |
| Wu et al. | Digital single virus electrochemical enzyme-linked immunoassay for ultrasensitive H7N9 avian influenza virus counting | |
| Ding et al. | Electrochemical and electrochemiluminescence determination of cancer cells based on aptamers and magnetic beads | |
| Ming et al. | Folding paper-based aptasensor platform coated with novel nanoassemblies for instant and highly sensitive detection of 17β-estradiol | |
| Liu et al. | Sensitive chemiluminescence immunoassay by capillary electrophoresis with gold nanoparticles | |
| Liu et al. | Versatile apoferritin nanoparticle labels for assay of protein | |
| Baldo et al. | Carbon nanotube-based sensing devices for human Arginase-1 detection | |
| Nur Topkaya et al. | Electrochemical aptasensors for biological and chemical analyte detection | |
| Liu et al. | Recent advances in chemiluminescence detection coupled with capillary electrophoresis and microchip capillary electrophoresis | |
| Yao et al. | Enzyme-free surface plasmon resonance aptasensor for amplified detection of adenosine via target-triggering strand displacement cycle and Au nanoparticles | |
| Fan et al. | Smartphone-based electrochemical system with multi-walled carbon nanotubes/thionine/gold nanoparticles modified screen-printed immunosensor for cancer antigen 125 detection | |
| Zitka et al. | Microfluidic tool based on the antibody‐modified paramagnetic particles for detection of 8‐hydroxy‐2′‐deoxyguanosine in urine of prostate cancer patients | |
| Truong et al. | Development of label-free impedimetric Hcg-immunosensor using screen-printed electrode | |
| Mao et al. | Accelerated and signal amplified nanozyme-based lateral flow assay of acetamiprid based on bivalent triple helix aptamer | |
| CN101093220A (en) | Detetion method in vitro, kit, and instrument based on magnetic grains and microelectrodes | |
| Wang et al. | Multivalent aptamer nanoscaffold cytosensor for glioma circulating tumor cells during Epithelial–Mesenchymal transition | |
| JP2009520983A (en) | Sensing device and method for determining the amount of target molecule in an analyte | |
| Zhang et al. | Universal and high-speed zeptomolar protein serum assay with unprecedented sensitivity | |
| Yan et al. | Utilizing dual carriers assisted by enzyme digestion chemiluminescence signal enhancement strategy simultaneously detect tumor markers CEA and AFP |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C57 | Notification of unclear or unknown address | ||
| DD01 | Delivery of document by public notice |
Addressee: Wen Jingwei Document name: Written notice of preliminary examination of application for patent for invention |
|
| C06 | Publication | ||
| PB01 | Publication | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |