CN1824797A - DNA fluorescent capillary biosensor and preparing process thereof - Google Patents
DNA fluorescent capillary biosensor and preparing process thereof Download PDFInfo
- Publication number
- CN1824797A CN1824797A CN 200510022413 CN200510022413A CN1824797A CN 1824797 A CN1824797 A CN 1824797A CN 200510022413 CN200510022413 CN 200510022413 CN 200510022413 A CN200510022413 A CN 200510022413A CN 1824797 A CN1824797 A CN 1824797A
- Authority
- CN
- China
- Prior art keywords
- dna
- fcbs
- probe
- kapillary
- fluorescence
- 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
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
This invention discloses a DNA fluorescence capillary biologic sensor (DNA-FCBS) and its preparation method, it belongs to biochemical field. DNA-FCBS is made by using crosslinking agent fixing DNA probe in capillary tube, it includes capillary tube, poly lysine and DNA probe. It can realize the classification of DNA and quantitative determination. DNA sample solution with butt is inhaled by DNA-FCBS for hybridization and coloration reaction, and then the butt DNA is quantitative determined in fluorescence device according to the fluorescence strength. Different butt nucleotides can be determined by changing DNA-FCBS inner probe type, multiple probes are fixed in DNA-FCBS to determine multiple butt nucleotides once time. The DNA-FCBS also can be made into all kinds of DNA fluorescence capillary biologic testing cassette. The reproducibility of this invention is good, operation is simple, and trace quick determination can be realized, so gene detection can be popular and practical.
Description
Technical field
The present invention relates to DNA fluorescent capillary biosensor (DNA-FCBS) of a kind of DNA of being used for fluorescence capillary analysis (DNA-FCA) and preparation method thereof, belong to biological chemistry and molecular biological analysis field.The present invention is applicable to the gene test of biological samples such as medicine, health, agricultural.
Background technology
The DNA detection method mainly is to utilize DNA chip and DNA transmitter at present.The essence of DNA chip analysis is to carry out orderly dot matrix at the substrate surface of small area very, arranges a series of addressable identification molecules, according to the principle of base complementrity, with accurate scanner or CCD camera technique target DNA is discerned.The characteristics of DNA chip are a plurality of target DNAs of energy measurement, but the possibility of non-specific hybridization is arranged, length consuming time, and the cost of manufacture height, the detecting instrument costliness is difficult to popularize.
Optical fiber DNA biosensor is a state-of-the-art technology in the DNA biosensor, mainly be on the silica fibre surface with chemical method or physics method fixing DNA probe or cDNA, optical fiber one end that will be fixed with dna probe then is immersed in the solution and fluorescently-labeled target DNA hybridization; Also dna probe can be fixed on the fibre-optic end, then that some the photoconductive fibers that are fixed with dna probe are synthetic a branch of, form an optical fiber DNA sensor microarray, the other end of optical fiber is coupled in the fluorescent microscope, by fiber optic conduction, make fluorescent marker on the target DNA generation fluorescence that is excited from the laser of fluorescent microscope, the fluorescent signal that is produced still turns back in the fluorescent microscope through optical fiber, by the CCD imaging, obtain the collection of illustrative plates of DNA hybridization.But the deficiency of optical fiber DNA transmitter is, the exposed part frangibility of an end, and the fixed amount of dna probe and less in conjunction with the target DNA amount, a little less than the fluorescent signal of generation, detection sensitivity is low, less stable, detecting instrument complexity, costliness.
Summary of the invention
The purpose of this invention is to provide DNA fluorescent capillary biosensor (DNA-FCBS) of a kind of DNA of being used for fluorescence capillary analysis (DNA-FCA) and preparation method thereof, solve the existing in prior technology problem.
Measuring principle of the present invention is the principle according to two dna single chain base complementrities, dna probe molecule with known array detects complementary target DNA molecule, according to the quantitative and qualitative target DNA molecule of the fluorescence intensity of fluorescent mark material on the target DNA molecule, perhaps utilize the complementary double chain DNA molecule that combine formation of embedded type fluorescence dye pair and dna probe molecule to carry out mark, carry out fluoroscopic examination then, reach the qualitative and quantitative purpose of target DNA molecule.
The DNA-FCBS that the present invention is used for DNA-FCA is characterized in that it comprises kapillary, poly-lysine, dna probe as shown in Figure 1; During use, substitute test solution groove in the conventional fluorometric analysis, be placed in the fluorescence analyser light path with DNA-FCBS and permanent seat.Utilize DNA-FCBS can implement DNA-FCA, implement the mensuration and the research of gene.
DNA-FCBS material of the present invention can be silica glass, opticglass or transparent plastics, and its length is 3～5cm, and capacity is 2～10 μ L, and internal diameter is 0.14～0.90mm, and external diameter is 0.55～1.20mm, and wall thickness is 0.10～0.25mm; Its endoporus can be circular, also can be rectangle, the length of side is 0.25～0.45mm during rectangle, wall thickness is 0.10～0.25mm; Immobilized DNA concentration is 10～100 μ mol/L.
Can change the dna probe kind among the DNA-FCBS of the present invention, make the DNA-FCBS and the DNA fluorescent capillary bioassay box of different purposes.
A kind of dna probe can be fixed among the DNA-FCBS of the present invention, also two or more dna probes can be fixed simultaneously.
DNA-FCBS of the present invention can be directly used in commercial various fluorescence analysers.
DNA-FCBS preparation method of the present invention may further comprise the steps:
1) chemically modified capillaceous
Utilize capillarity that 0.2% poly-lysine is sucked in the kapillary, at room temperature, place to make its reaction 1h on the rotation mixed instrument, poly-lysine is covalently bind on the inwall capillaceous.
2) dna probe immobilization
Water-wash away in the kapillary after the free poly-lysine with aseptic second distillation, suck the dna probe solution 10 μ L of 10～100 μ mol/L, placing under 42 ℃ on the rotation mixed instrument, make it carry out covalent attachment reaction 30min; At 80 ℃ of dry 1h of following kapillary, suck the stationary liquid (methyl alcohol: acetate=3: 1), place 5min of precooling more rapidly then; Wash out kapillary internal fixing liquid with aseptic redistilled water, after the emptying, repeat heating (80 ℃) step and precooling fixing step 3 times, every step 5min uses 0.2% sodium lauryl sulphate (SDS) and aseptic redistilled water respectively to clean at last 1 time.
3) sealing of chemically modified group
The kapillary that is fixed with dna probe is immersed in 6～12h in the prehybridization solution that contains tortoise milt DNA, seals unconjugated poly-lysine, make DNA-FCBS; At last, DNA-FCBS is kept in-20 ℃ the refrigerator standby.
Basic fundamental parameter of the present invention is: the target DNA measurement range of Cy5 mark detects and is limited to 1.7pmol at 0.1～1.0 μ mol/L, and relative standard deviation RSD＜4.60%, dna probe fixed concentration are 10-100 μ mol/L; Hybridization time 1～2h, 45 ℃ of hybridization temperature, melting temperature(Tm) are 95～97 ℃, reusable 6 times of DNA-FCBS.
Advantage of the present invention and positively effect are:
1) available DNA-FCBS substitutes conventional fluorescence test solution groove, realizes the DNA-FCA method;
2) can fix a kind of dna probe separately among the DNA-FCBS, also can fix two or more dna probes simultaneously, measure when carrying out two above target DNAs;
3) can change dna probe kind among the DNA-FCBS, make the DNA-FCBS and the DNA fluorescent capillary bioassay box of different purposes;
4) DNA-FCBS can be directly used in commercial various fluorescence analysers;
5) dna probe can be reused after the immobilization in DNA-FCBS, can save a large amount of valuable reagent, reduces experimental cost;
6) easy and simple to handle, be easy to carry and preserve, can be used for that DNA measures in the various biological samples, can produce huge economic benefit.
Description of drawings
Fig. 1 DNA-FCBS structural representation of the present invention
S sample, 1 kapillary, 2 poly-lysines, 3DNA probe among the figure
Fig. 2 embodiment 2 measures the canonical plotting that DNA obtains with DNA-FCBS of the present invention
Specific embodiment
In conjunction with the accompanying drawings embodiments of the invention are further described:
Present embodiment be with synthetic 100 μ mol/L oligonucleotide sequences 5 '-GAAA CCTG TTTG TTGG ATAC-3 ' is as dna probe, is fixed in the kapillary internal surface as stated above, makes DNA-FCBS.
During mensuration, with DNA-FCBS suck 33 μ mol/L target nucleotide sequences 5 '-GTATCC AACAAACA GGTTTC-3 ' solution 10 μ L, under 45 ℃, make its hybridization 90min; Afterwards the double-stranded DNA that forms is dyeed with bromine second pyridine (EB); Wash out free EB staining fluid in the DNA-FCBS with 0.2%SDS and diethyl ester coke acid (DEPC); With RF-5000 fluorophotometric instrument at excitation wavelength 526nm, emission wavelength 584nm place measures, and the result who obtains is: the mean value of fluorescence intensity is 74.01 ± 14.12 before and after the hybridization, results of statistical analysis t=4.1106, there is significant difference p＜0.05 before and after the hybridization.
Present embodiment be with DNA-FCBS of the present invention suck the target nucleotide sequences 5 of 1 μ mol/L Cy5 mark '-GTATCCAACAAACA GGTTTC-3 solution 10 μ L, under 45 ℃, make its hybridization 90min; Wash out free target nucleotide in the DNA-FCBS with 0.2%SDS and DEPC; At excitation wavelength 646nm, emission wavelength 664nm place measures with RF-5000 fluorophotometric instrument, and the result is as shown in table 1; The mean value of fluorescence intensity is 61.66 ± 18.22 before and after the hybridization, results of statistical analysis t=6.7684, and there is highly significant difference 0.005＜P＜0.01 before and after the hybridization.
Table 1.
| The DNA-FCBS sequence number | Fluorescence intensity before the hybridization | Hybridization back fluorescence intensity | The |
| 1 2 3 4 | 158.75 199.25 172.50 188.75 | 247.25 266.13 223.88 228.63 | 88.5 66.88 51.38 39.88 |
| The mean value standard deviation | 179.81 —— | 241.47 —— | 61.66 18.22 |
Present embodiment is to be that 90min, hybridization temperature are under 45 ℃ the experiment condition in hybridization time, suck the Cy5 marker target nucleotide solution of a series of different concns with DNA-FCBS of the present invention, with RF-5000 fluorophotometric instrument at excitation wavelength 646nm, emission wavelength 664nm place measures, and the result as shown in Figure 2.The target nucleotide concentration of Cy5 mark has good linear relationship with fluorescence intensity in the scope of 0.1～1 μ mol/L: y=139.73x+39.613, relation conefficient is 0.9985, detection limit 1.7pmol.
Embodiment 4
Present embodiment is precision and a reproducibility of investigating DNA-FCBS of the present invention.95 ℃ of 45 ℃ of hybridization temperatures, hybridization time 90min, melting temperature(Tm), unwind under the experiment condition of time 4min, suck 1 μ mol/LCy5 labeled target dna sample repeatedly with DNA-FCBS of the present invention, with RF-5000 fluorophotometric instrument at excitation wavelength 646nm, emission wavelength 664nm repeatedly measures at the place, and its result is as shown in table 2.
As can be seen, DNA-FCBS can reuse 6 times, and fluorescence intensity obviously reduces after the 7th time; The fluorescence intensity mean value of 6 replications is 172.93, and standard deviation is 9.47, and relative standard deviation is 5.5%.
Table 2.
| The replication number of | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| Fluorescence intensity | 188.58 | 168.82 | 175.11 | 177.11 | 162.75 | 165.18 | 58.00 | 39.25 | 19.88 | 17.69 |
Claims (6)
1. the DNA capillary biosensor (DNA-FCBS) that is used for DNA fluorescence capillary analysis (DNA-FCA), it is characterized in that it comprises kapillary (1), poly-lysine (2), dna probe (3), utilize DNA-FCBS can implement DNA-FCA, implement the mensuration and the research of gene.
2. DNA-FCBS as claimed in claim 1, the material that it is characterized in that kapillary (1) can be silica glass, opticglass or transparent plastics, length is 3～5cm, capacity is 2～10 μ L, internal diameter is 0.14～0.90mm, and external diameter is 0.55～1.20mm, and wall thickness is 0.10～0.25mm; The endoporus of kapillary (1) can be circular, also can be rectangle, the length of side is 0.25～0.45mm during rectangle, wall thickness is 0.10～0.25mm; The immobilized DNA concentration and probe concentration is 10～100 μ mol/L.
3. DNA-FCBS as claimed in claim 1 is characterized in that changing dna probe (3) kind among the DNA-FCBS, makes the DNA-FCBS and the DNA fluorescent capillary bioassay box of different purposes.
4. DNA-FCBS as claimed in claim 1 is characterized in that fixing a kind of dna probe (3) in DNA-FCBS, also can fix two or more dna probe (3) simultaneously.
5. DNA-FCBS as claimed in claim 1 is characterized in that being directly used in commercial various fluorescence analysers.
6. DNA-FCBS preparation method as claimed in claim 1 is characterized in that may further comprise the steps:
1) chemically modified capillaceous
Utilize capillarity that 0.2% poly-lysine is sucked in the kapillary (1), at room temperature, place to make its reaction 1h on the rotation mixed instrument, poly-lysine is covalently bind on the inwall of kapillary (1).
2) dna probe immobilization
Water-wash away in the kapillary free poly-lysine (2) afterwards with aseptic second distillation, suck dna probe (3) the solution 10 μ L of 10～100 μ mol/L, placing under 42 ℃ on the rotation mixed instrument, make it carry out covalent attachment reaction 30min; At the dry 1h of 80 ℃ of following kapillaries (1), suck the stationary liquid (methyl alcohol: acetate=3: 1), place 5min of precooling more rapidly then; Wash out kapillary (1) internal fixing liquid with aseptic redistilled water, after the emptying, repeat heating (80 ℃) step and precooling fixing step 3 times, every step 5min respectively cleans 1 time with 0.2% sodium lauryl sulphate and aseptic redistilled water at last.
3) sealing of chemically modified group
The kapillary (1) that will be fixed with dna probe (3) is immersed in 6～12h in the prehybridization solution that contains tortoise milt DNA, seals unconjugated poly-lysine (2), makes DNA-FCBS; At last, DNA-FCBS is kept in-20 ℃ the refrigerator standby.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510022413 CN1824797A (en) | 2005-12-28 | 2005-12-28 | DNA fluorescent capillary biosensor and preparing process thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510022413 CN1824797A (en) | 2005-12-28 | 2005-12-28 | DNA fluorescent capillary biosensor and preparing process thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1824797A true CN1824797A (en) | 2006-08-30 |
Family
ID=36935638
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200510022413 Pending CN1824797A (en) | 2005-12-28 | 2005-12-28 | DNA fluorescent capillary biosensor and preparing process thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1824797A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104849247A (en) * | 2015-04-15 | 2015-08-19 | 刘骁勇 | Method for detecting heavy metal ion based on DNA and heavy metal ion mismatch principle |
| CN112903593A (en) * | 2021-01-11 | 2021-06-04 | 电子科技大学 | Rapid biochemical analyzer based on sequence combination |
-
2005
- 2005-12-28 CN CN 200510022413 patent/CN1824797A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104849247A (en) * | 2015-04-15 | 2015-08-19 | 刘骁勇 | Method for detecting heavy metal ion based on DNA and heavy metal ion mismatch principle |
| CN112903593A (en) * | 2021-01-11 | 2021-06-04 | 电子科技大学 | Rapid biochemical analyzer based on sequence combination |
| CN112903593B (en) * | 2021-01-11 | 2022-06-03 | 电子科技大学 | Rapid biochemical analyzer based on sequence combination |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Piunno et al. | Fiber optic biosensor for fluorimetric detection of DNA hybridization | |
| JP4091437B2 (en) | Fluorescent signal reference device | |
| Danz et al. | Surface plasmon resonance platform technology for multi‐parameter analyses on polymer chips | |
| US20020055111A1 (en) | Three-dimensional probe carriers | |
| CN1117284C (en) | Microfluid biochip detection-analysis board and its detection method | |
| US20230111586A1 (en) | Consumable for analyte detection | |
| CN111812075A (en) | SERS-SPR dual-mode sensor and preparation method and application thereof | |
| US20070160998A1 (en) | Device for analyzing an interaction between target and probe molecules | |
| JP2004529349A (en) | Waveguides and assays | |
| CN1824797A (en) | DNA fluorescent capillary biosensor and preparing process thereof | |
| CN107858403B (en) | A kind of trace target object detecting method based on single molecular fluorescence sensing | |
| WO2008072209A2 (en) | Method and microdevice to identify and/or quantify analytes in biological samples | |
| CN1605861A (en) | Preparation and detection method for electrochemical quantitative polymerase chain reaction detecting chip | |
| EP1570271A2 (en) | Waveguide system for detection of fluorescently labeled nucleic acid sequences | |
| CN1166954C (en) | Reverse protein chip | |
| JP2003232791A (en) | Probe solid-phase reaction array | |
| KR100848636B1 (en) | Selectively hybridizable substance immobilization fiber, fiber array comprising bundle of such fibers, selective hybridizing method, device therefor, and base | |
| Muhammad et al. | Novel fluorescent membrane for metronidazole sensing prepared by covalent immobilization of a pyrenebutyric acid derivative | |
| JP4217318B2 (en) | Method and apparatus for quantitative analysis of target nucleic acid | |
| US20020086325A1 (en) | Affinity detecting/analytical chip, method for production thereof, detection method and detection system using same | |
| Kuswandi et al. | Recent advances in optical DNA biosensors technology | |
| JP2002340900A (en) | Biosensor, biosensor array, and detection method | |
| Mogalisetti et al. | Enzyme kinetics in femtoliter arrays | |
| US20240165626A1 (en) | Methods and systems for dispensing and extracting reagents | |
| Herron et al. | Planar waveguide biosensors for nucleic acid hybridization reactions |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |