CN1519329A - Reactor of biochip in micro array - Google Patents
Reactor of biochip in micro array Download PDFInfo
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- CN1519329A CN1519329A CNA03102954XA CN03102954A CN1519329A CN 1519329 A CN1519329 A CN 1519329A CN A03102954X A CNA03102954X A CN A03102954XA CN 03102954 A CN03102954 A CN 03102954A CN 1519329 A CN1519329 A CN 1519329A
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Abstract
A microarray biochip reactor is composed of the first member with the first recess for holding a sample solution and a microarray biochip, the second member able to be moved onto the first member and with the second recess relative to the first recess, and a group of electrodes in the second recess and in touch with the solution in the first recess.
Description
Technical field
The present invention relates to a kind of micro-array biochip reactor, particularly a kind of micro-array biochip reactor that comprises electrode.
Background technology
In the present stage scientific development, gene information is considerable to the whole process of life.The various information that comprised in the life are all relevant with the arrangement of gene fundamental, for example generation of the color of the formation of organ, hair and skin, disease, proteinic composition and expression, and height is fat or thin or the like, all is subjected to the control of gene.Therefore, the research of present stage for the life science aspect concentrates on large-scale gene expression mostly, and the gene information that obtains organism how fast, easily and accurately, to be used for the research in next stage, for example discovery of drug development, gene therapy, disease gene or the like accurately.
For obtaining in a large number of the fast development that adapts to life science and bioinformation, the correlation technique of biochip development is developed and improves in a large number and apace, detects correlation technique, biochip manufacturing technology, microfluid system correlation technique, information biology and molecular biological correlation techniques etc. such as spectroscopy such as surface chemistry, fluorescent.In other words, biochip is the Development Technology of a cross-cutting integration, and the development of each link all can affect biochip to obtain the important goal of bioinformation fast and correctly.
With molecular biology is example, relevant Development Technology comprise sample pre-treatments (samplepreparation), gene sequencing (gene sequence), probe design (probe design), polymerase chain reaction (polymerase chain reaction, PCR), electrophoretic separation (electrophoresis) and gene recombination reaction (hybridization) correlation technique or the like.Traditional molecular biotechnology is quite long for the time of the above required cost of relating operation, therefore at above-mentioned correlation technique many technical breakthroughs and improvement are arranged all, for example exploitation of sample pre-treatments reagent, multitube road electrocapillary phoresis sequencing instrument easily and fast, rapid gene comparison software are to the help of gene probe design, the development of microminiaturization pcr chip, the development of electrocapillary phoresis chip and the various kinds invention of acceleration hybridization of all kinds.
The birth of biochip, the time of obtaining of disease detection or bioinformation is significantly reduced, for example, originally detect disease with cell culture technology or judge that bacterial classification needs three to five days, even the resistance test that need make infection pathogen sometimes then must spend a couple of days again by tens of days, and utilization biochip correlation technique then can foreshorten in 6 hours and just can obtain relevant information.Yet, still can't adapt to the actual demand of some disease or medical procedure this detection time of 6 hours, for example, some disease is found to the death time from diagnosis and is no more than one to two.So still at developing biochip technology, the research staff puts focus on the subject under discussion that shortens in the detection reaction time that biochip is required one after another.
Shorten detection time, just must be from checkout procedure main technical setting about consuming time, wherein, in the middle of 6 hour above-mentioned proving time, round pcr needs 1.5 hours approximately, and nucleic acid hybridization needs 4 hours approximately, and these two technical processes have accounted for the time of whole biochip test process about 92%.So become to attach most importance to the detection time that how to shorten these two technology, at present, existing many research staff have done considerable effort.
As hybridize auxiliary (Hybridization Helper) technology (US Patent 5,030,557), its principle is for utilizing one section nucleotide sequence (being Helper), itself and corpse or other object for laboratory examination and chemical testing nucleic acid are complementary to going up or downstream part of probe (probe) nucleic acid and corpse or other object for laboratory examination and chemical testing nucleic acid hybridization zone, utilize the characteristic of this one section nucleic acid (Helper) and corpse or other object for laboratory examination and chemical testing nucleic acid hybridization, with corpse or other object for laboratory examination and chemical testing nucleic acid stretching (removing in disorder curling unfavorable hybridization configuration originally), so that carry out hybridization.
In addition, also having a kind of technology, is the patented invention that is proposed by Gen-Probe company, is called nucleic acid precipitation reagent (Nucleic acid precipitating reagents) (US patent5,132,207).Its principle is to utilize different salt damping fluids, makes corpse or other object for laboratory examination and chemical testing nucleic acid be easy to be deposited near the probe nucleic acid, increases the subregion concentration of corpse or other object for laboratory examination and chemical testing nucleic acid by this method, to promote the carrying out of hybridization.
Also having a kind of technology, is the patented invention that is proposed by Chiron company, is called dendritic nucleotide sequence (Branched oligonucleotide multimer) technology (US Patent 5,624,802 and 5,594,118).Its principle is for utilizing one section probe earlier, and this section probe stationary is used for catching corpse or other object for laboratory examination and chemical testing nucleic acid in chip surface.Utilize one section dendritic nucleotide sequence (Branched oligonucleotidemultimer, itself and the complementation of corpse or other object for laboratory examination and chemical testing nucleic acid) to receive on the corpse or other object for laboratory examination and chemical testing nucleic acid again; At last having the nucleic acid of fluorescent or radioactive rays to detect sub-complementation with sign again hybridizes on dendritic nucleotide sequence.Because a dendritic nucleotide sequence can have the hybridization of tens of hundreds of even detecting thereon, can strengthen detecting intensity greatly, so can shorten hybridization time.
In addition, a kind of volume that is called is got rid of the technology of preparation (Volume exclusion agents) (USPatent 4,886,741) be the patent application that is proposed by Microprobe company, it utilizes organic molecule to form reticulated structure, and but this reticulated structure exclusive segment hybridization buffer makes the subregion concentration of corpse or other object for laboratory examination and chemical testing nucleic acid heighten, and then promotes the carrying out of hybridization.
The patented invention that Becton Dickinson company is proposed, be called both sexes hydrocarbon polymer (Amphipathic hydrocarbon polymer, (the US Patent 5,853 of technology AHP), 986), it utilizes both sexes organic molecule (wetting ability and hydrophobic nature) to form reticulated structure.But this reticulated structure exclusive segment hybridization buffer heightens the subregion concentration of corpse or other object for laboratory examination and chemical testing nucleic acid, and then promotes the carrying out of hybridization.
Also has a kind of device (US Patent 6 that is called dynamic crossing system (Dynamic hybridization system), 255,050), the patented invention that is proposed for Lorne Park Research company, this system utilizes semipermeable partition that probe nucleic acid is fixed on the semipermeable partition, on the other hand, flow towards the semipermeable partition direction with mode actuating fluid (including corpse or other object for laboratory examination and chemical testing nucleic acid) such as pneumatic or vacuum compression.Because corpse or other object for laboratory examination and chemical testing nucleic acid has and postpones so corpse or other object for laboratory examination and chemical testing nucleic acid causes accumulation phenomena near semipermeable partition, so can improve the concentration of corpse or other object for laboratory examination and chemical testing nucleic acid by phenomenon during fluid process permeable membrane, to improve hybridization speed.Its principle is can not pass through from the hole of semipermeable partition with probe nucleic acid complementary corpse or other object for laboratory examination and chemical testing nucleic acid.
The patent of invention US Patent 5 that U.S. Nanogen company proposes, 728,532,5,849,486,6,017,696 and 6,099, in 803, utilize electrode application voltage to attract the biomolecules of electronegative property, cause near the partial concentration of electrode greatly to improve, and the collision probability of increase and surperficial fixed bioprobe molecule, to reach the purpose that improves biomolecules hybridization speed, simultaneously also utilize electrode to apply a reverse voltage, get rid of the biomolecules of electronegative property, utilize the control voltage swing to reach the assay of differentiating single base difference.
The patent of invention US Patent 6 that U.S. Genomic Solution company proposes, 238,910, utilize flowing of biological sample in the temperature control fluid channel, increase the probability of colliding between biomolecules, to reach the biomolecules liquid mixing and to quicken the purpose of hybridization.
The United States Patent (USP) 6 that Agilent company is proposed, 258,593 and 6, in 186,659, at biomolecules hybridization design slide glass stationary installation, required space when the device of this sealing can provide slide glass to carry out the biomolecules hybridization effectively, and the mode of utilizing bubbles entrain solution is quickened the mixing of sample, reaches the purpose of quickening the biomolecules hybridization, and this device has also increased the accessibility of biomolecules hybridization operation simultaneously.
In addition, according to United States Patent (USP) 6,162,400 invention utilizes centrifugal force to drive flowing of sample solution, quicken the mixing of sample molecule and increase with the biomolecules that is fixed on the slide glass between the collision probability, the carrying out of acceleration biomolecules hybridization.
The method that above-mentioned hybridization speed increases, mostly be to improve the mode of corpse or other object for laboratory examination and chemical testing nucleic acid concentration, or it is a corpse or other object for laboratory examination and chemical testing is stretching, or utilize dendritic structure, or utilize flowing of sample liquids, increase collision probability between biomolecules etc., but these methods that allow nucleic acid hybridization quicken, still have some restrictions, for example much need under big scale, could operate.
Summary of the invention
The object of the present invention is to provide a kind of micro-array biochip reactor, it comprises:
One first member, or be called carrier, it has one first groove, can hold a sample solution; And
One second member, or be called lid, it is positioned on first member, is provided with one group of above electrode, and this electrode can contact with this sample solution;
Between first member and second member, link, hold sample solution with the space that a fixed volume is provided in integrally formed mode.With micro-array biochip reactor of the present invention, utilize above-mentioned electrode to apply more than one electric field, drive molecule contained in the above-mentioned sample solution and change its travel direction with the change of electric field level, direction or frequency, molecule is in moving process, increase solution blended uniformity coefficient, quicken simultaneously to collide between biomolecules, thereby reach the purpose that increases biochemical reaction rate, shortens the reaction times.
Another order of the present invention is to provide a kind of micro-array biochip reactor, and it comprises:
One first member, or be called carrier, it has one first groove, can hold a micro-array biochip that contains a reaction zone; And
One second member, or be called lid movably is arranged on first member, have on this second member one corresponding to second groove of this reaction zone and one group with top electrode, it is arranged in second groove.
For purpose of the present invention, feature and advantage can be become apparent, below by specific embodiment, and conjunction with figs., elaborate.
Description of drawings
Figure 1A～1E is five kinds of arrangement of electrodes synoptic diagram of the embodiment of the invention;
Fig. 2 A～2E is the lid (2A-2B) and carrier (2C-2D) synoptic diagram of a kind of embodiment of micro-array biochip reactor of the present invention, and wherein Fig. 2 A, 2C figure are top view, and Fig. 2 B, 2D are front view, and Fig. 2 E is the A-A ' sectional view of Fig. 2 C;
Fig. 3 A～3D is the lid (3A-3B) and carrier (3C-3D) synoptic diagram of another embodiment of micro-array biochip reactor of the present invention, and wherein Fig. 3 A, 3C figure are top view, and Fig. 3 B, 3D are front view;
Fig. 4 A～4B is the hybridization figure as a result of different concns probe biomolecule (0.05,0.1, and 0.5 μ M), and the biological sample concentration with fluorescent molecule marker is 1 μ M.Fig. 4 A in voltage of alternating current ± 2.5V, under 30 minutes the condition of 60Hz continuous action, carries out hybridization for adopting micro-array biochip reactor of the present invention; And Fig. 4 B left standstill 30 minutes for adopting traditional molecular biology method, carried out hybridization.
Fig. 5 A～5B is the hybridization figure as a result of different concns probe biomolecule (0.01,0.05,0.1 and 0.5 μ M), and the biological sample concentration with fluorescent molecule marker is 0.5 μ M.Fig. 5 A in voltage of alternating current ± 10V, under 30 minutes the condition of 60Hz continuous action, carries out hybridization for adopting micro-array biochip reactor of the present invention; And Fig. 5 B left standstill 30 minutes for adopting traditional molecular biology method, carried out hybridization.
Fig. 6 A～6B is the hybridization figure as a result of different concns probe biomolecule (0.05,0.1 and 0.5 μ M), and the biological sample concentration with fluorescent molecule marker is 1 μ M.Fig. 6 A in voltage of alternating current ± 25V, under 30 minutes the condition of 60Hz continuous action, carries out hybridization for adopting micro-array biochip reactor of the present invention; And Fig. 6 B left standstill 30 minutes for adopting traditional molecular biology method, carried out hybridization.
Fig. 7 A～7D is the hybridization figure as a result of different concns probe biomolecule (0.01,0.05,0.1 and 0.5 μ M).Fig. 7 A～7D is for adopting traditional molecular biology method, leave standstill in 40 ℃ of constant temperature under 4 hours the condition, carry out hybridization, the biological sample concentration that wherein has the fluorescent molecule marker is respectively 0.5 μ M (Fig. 7 A), 1.0 μ M (Fig. 7 B), 5.0 μ M (Fig. 7 C) and 10 μ M (Fig. 7 D).Fig. 7 E is for adopting micro-array biochip reactor of the present invention, under room temperature, voltage of alternating current ± 10V, and 60H
zVoltage transitions frequency, 30 minutes condition of continuous action under, carry out hybridization, the biological sample concentration that wherein has fluorescent labelling is 0.5 μ M.
Fig. 8 A～8F is the hybridization figure as a result under concentration 10nM biological sample exists.Fig. 8 A adopts micro-array biochip reactor of the present invention, in voltage of alternating current ± 10V, and 60H
zElectric voltage frequency change, under 30 minutes the condition of continuous action, carry out hybridization; Fig. 8 B～8F carries out the hybridization of 1 hour (Fig. 8 B), 2 hours (Fig. 8 C), 4 hours (Fig. 8 D), 12 hours (Fig. 8 E) and 16 hours (Fig. 8 F) for adopting commercial goods hybridization instrument.
Fig. 9 A～9B is three kinds of different biological molecules probe AlO3 of concentration 0.5 μ M, and the hybridization of O3 and P3 is figure as a result, and the biological sample concentration with fluorescent molecule mark meter is 5 μ M.Fig. 9 A adopts micro-array biochip reactor of the present invention, in voltage of alternating current ± 10V, and 60H
zElectric voltage frequency change, under 30 minutes the condition of continuous action, carry out hybridization; Fig. 9 B left standstill 30 minutes for adopting traditional biological molecules method, carried out hybridization.
Embodiment
Micro-array biochip reactor of the present invention comprises lid 1, electrode 2, second groove 3, the second groove border 4, hole 5, binding site 6, carrier 7, the first grooves 8 and micro-array chip 9 are characterised in that to comprise one group of above electrode, utilize above-mentioned electrode that more than one electric field is provided, drive the contained molecule of sample solution in the biochip reaction device, make above-mentioned molecule can change its travel direction along with the change of direction of an electric field, molecule can increase the collision of molecules probability in moving process, thereby the speed of increase biochemical reaction, shorten the reaction times.
Arrangement of electrodes in the micro-array biochip reactor of the present invention is shown in Figure 1A～1E, on rectangle lid 1, with one group of electrode 2, with form as Figure 1A or 1B, or two groups of above electrodes 2, with form as Fig. 1 C, 1D or 1E, be arranged at long or wide both sides or the centre of lid 1, the variation of other form is easy to be pushed away by those skilled in the art.Above-mentioned electrode is made up of gold and silver, copper, nickel, platinum, stainless steel or other metal.Though above-mentioned electrode not is only to apply electric field with the interaction that electrode is set in groups for being provided with in groups when implementing, but can adjust the frequency that direction of an electric field, size or direction of an electric field change by manual or computer programing design.For example, adjust direction of an electric field and can select any two electrodes according to need, and between any two electrodes, anode and negative electrode can change arbitrarily; Also can adopt alternating-current as power supply, thereby can do various directions of an electric field variations.By adjusting interelectrode potential difference, can obtain different electric field level, for example between 0 to 200V; And adjust the frequency that direction of an electric field changes, can realize in the time of sample solution by adjusting electric field action.
The embodiment of micro-array biochip reactor of the present invention, with Fig. 2 A～2E and Fig. 3 A～3D is example, this reactor comprises first member, second member and one group of above electrode, first member is that carrier 7, the second members shown in Fig. 2 C and 2D or 3C and the 3D figure are the lid 1 shown in Fig. 2 A and 2B or Fig. 3 A and the 3B.This lid 1 is made up of the organic or inorganic material with carrier 7, and above-mentioned organic materials comprises, but is not limited to synthetic rubber, natural rubber, synthetic macromolecule, polyethylene, polystyrene, polypropylene and polyvinyl chloride.Above-mentioned inorganic materials comprises, but is not limited to metal, stupalith, silicon and glass.Below with Fig. 2 A～2E and Fig. 3 A～3D two kinds of embodiments are described respectively.
One embodiment of the present invention please refer to Fig. 2 A～2E, and Fig. 2 A～2B is the synoptic diagram of lid, and Fig. 2 C～2D is the synoptic diagram of carrier, and Fig. 2 A, 2C be top view, and Fig. 2 B, 2D are front view, and Fig. 2 E figure is the A-A ' sectional view of Fig. 2 C.In Fig. 2 C, 2D and 2E, carrier 7 is embedded with a micro-array biochip 9, on one first groove 8 is arranged, can directly carry sample solution, its border can be designed to O type ring, prevent the sample solution seepage, and first groove 8 purpose is set when carrying out biochemical hybridization, provide enough molecule mobile spaces to react by biomolecules.Among Fig. 2 A and the 2B, have one group of electrode 2 on the lid 1 on the corresponding carrier in the scope of first groove 8, if having more than two groups, or even increase by an independent electrode, corresponding to respectively organizing the electrode setting originally, can freely change design, also can be used in combination mutually.When combination lid 1 and carrier 7, first groove 8 becomes an enclosed space, so according to need one or more holes 5 can be set on the lid 1 corresponding to first groove 8, so that inject or the output sample solution, or after the reaction when needing to clean, can inject or export cleaning solution, present embodiment has two reflecting points on slide, and therefore two holes 5 are set.The purpose of this hole design is the turnover that makes things convenient for sample and cleaning solution, to finish the biomolecules hybridization.In addition, the mode that is one of the forming between lid 1 and the carrier 7 links, and holds sample solution with the space that a fixed volume is provided.
Another embodiment of the present invention please refer to Fig. 3 A～3D, and Fig. 3 A～3B is the synoptic diagram of lid, and Fig. 3 C～3D is the synoptic diagram of carrier, and Fig. 3 A, 3C be top view, and Fig. 3 B, 3D are front view.Among Fig. 3 C and the 3D, carrier 7 surfaces have first groove 8, are used to carry micro-array biochip, and first groove 8 can be put into the size of general slide glass in the present embodiment.Two ends are provided with the binding site 6 of lid 1 and carrier 7 on other carrier 7, can optionally increase and decrease its number or change its position.Please refer to Fig. 3 A～3B again, the contact surface with carrier 7 on the lid 1 has second groove 3 that holds sample solution, and its border 4 is identical with the material of lid 1, and perhaps, border 4 also can be designed as O type ring, to prevent the sample solution seepage.Second groove 3 purpose is set when carrying out biochemical hybridization, provide enough molecule mobile spaces to react by biomolecules.Hold on the lid 1 in the scope of second groove 3 of sample solution and have one group of electrode 2, as described above, electrode group number or need add the single electrode of a correspondence in addition all can change according to need, and the electrode 2 that present embodiment shows is arranged at second groove, 3 both sides.Lid 1 is arranged on the carrier 7 with packaged type, connects with binding site 6, and binding site 6 can be corresponding to carrier 7 appropriate changes.In addition, when combination lid 1 and carrier 7, second groove 3 becomes an enclosed space, so can a plurality of holes 5 be set at second groove 3 according to need, so that inject or the output sample solution, or when needing to clean after the reaction, can inject or export cleaning solution, present embodiment has two reflecting points on slide, therefore two holes 5 are set.The purpose of design of this hole is for making things convenient for the turnover of sample and cleaning solution, to finish the biomolecules hybridization.
In addition, no matter which kind of material above-mentioned lid 1, carrier 7 or electrode 2 adopt, all must not can react with sample solution or cleaning solution.
Adopt in the sample solution that micro-array biochip reactor of the present invention reacts, comprise organic and inorganic or biomolecules, charged ion or uncharged neutral molecule.Above-mentioned organic molecule comprises, but is not limited to, organic acid, organic bases or amino acid.Above-mentioned inorganic molecule comprises, but is not limited to, metal ion or inorganic salts.Above-mentioned biomolecules comprises, but is not limited to double-stranded DNA, single stranded DNA, RNA, protein or polypeptide.
When adopting micro-array biochip reactor of the present invention to carry out hybridization, the entire reaction system also comprises the power supply unit that can adjust strength of electric field, alternating-electric field switching frequency and can control direction of an electric field.One of feature of the present invention is for applying extra electric field and constantly changing direction of an electric field, allow the biomolecules or the charged particle that have electric charge in certain zone, move, even the drive neutral molecule is mobile, and reach the probability that increases molecular impact, but not allow solution flow or disturbance on a macro scale, need not improve the concentration of corpse or other object for laboratory examination and chemical testing nucleic acid, also need not accurate little processing, needn't improve temperature of reaction, be not to increase whole turnover rate yet, can increase the biomolecules hybridization.
It below is the Application Example that adopts micro-array biochip reactor of the present invention to be carried out.
Adopt micro-array biochip reactor of the present invention, cooperation one can be adjusted the power supply unit of strength of electric field, alternating-electric field switching frequency and may command direction of an electric field, in the result of biochip reaction result who applies gained under the alternating-electric field effect and traditional biological chip reaction (leaving standstill), shown in Fig. 4 A～4B to Fig. 9 A～9B.
At first, relatively under probe biomolecule concentration difference, sample concentration fixed situation, adopt micro-array biochip reactor of the present invention and traditional molecular biology method to carry out the situation of hybridization.Probe biomolecule concentration is respectively 0.05,0.1 and 0.5 μ M, and the sample concentration with fluorescent molecule marker is all 1 μ M.Adopt micro-array biochip reactor of the present invention, utilize the switching frequency of voltage of alternating current ± 2.5V, 60Hz, continuous action was carried out the biomolecules hybridization in 30 minutes, obtained result such as Fig. 4 A; Adopt traditional molecular biology method to carry out hybridization, left standstill 30 minutes, obtain result such as Fig. 4 B.Relatively two groups of results show, the present invention utilizes voltage of alternating current to carry out the method that sample mix and molecule film micro area move, and can increase the speed of biomolecules hybridization effectively.The present invention applies efficient and the speed that device that voltage of alternating current quickens hybridization speed can improve hybridization effectively, reaches more than 75%, even reaches 99%; And that tradition leaves standstill the resulting drop as a result of method is quite big, even result that can't its hybridization of identification.In other words, utilize alternating-electric field to continuingly act on the sample solution that contains biomolecules with fixed frequency, can increase the uniformity coefficient of sample solution effectively, make when biochip carries out the biomolecules hybridization, biomolecules collision probability each other is suitable, thereby obtains more consistent reaction result.
Then, the sample concentration that will have the fluorescent molecule marker is reduced to 0.5 μ M, with the probe biomolecule concentration of 0.1 and 0.5 μ M, relatively adopts micro-array biochip reactor of the present invention and traditional molecular biology method to carry out the situation of hybridization.Adopt micro-array biochip reactor of the present invention, voltage of alternating current is increased to ± 10V, the switching frequency of 60Hz, continuous action 30 minutes is carried out the biomolecules hybridization, and the result who obtains is shown in Fig. 5 A; Adopt the reaction of traditional biological molecular hybridization, left standstill 30 minutes, the result who obtains is shown in Fig. 5 B.Even under lower sample concentration effect, owing to improve voltage of alternating current,, utilize voltage of alternating current to carry out sample mix and the molecule film micro area moves still as can be seen with micro-array biochip reactor of the present invention, compare with traditional method, increased the speed of biomolecules hybridization effectively.
In addition, still with the different biological molecules concentration and probe concentration, under the fixed sample concentration, relatively adopt micro-array biochip reactor of the present invention and traditional molecular biology method to carry out the situation of hybridization.Probe biomolecule concentration still is respectively 0.05,0.1 and 0.5 μ M, and the sample concentration with fluorescent molecule marker then is 1 μ M.Adopt micro-array biochip reactor of the present invention, voltage of alternating current is increased to ± 25V, the switching frequency of 60Hz, continuous action 30 minutes is carried out the biomolecules hybridization, and the result who obtains as shown in Figure 6A; With the reaction of traditional biological molecular hybridization, left standstill 30 minutes, the result who obtains is shown in Fig. 6 B.Comparative result shows, with micro-array biochip reactor of the present invention, utilizes voltage of alternating current to carry out sample mix and the molecule film micro area moves, and can increase the speed of biomolecules hybridization than traditional method effectively.
In addition, with the different concns bioprobe, relatively under traditional biological molecular hybridization method under 0.5～10 μ M concentration biological sample and 0.5 μ M concentration biological sample, adopt the result of micro-array biochip reactor of the present invention.Bioprobe concentration is respectively 0.01,0.05,0.1 and 0.5 μ M, and the biological sample concentration with fluorescent molecule marker is respectively 0.5 μ M (Fig. 7 A), 1.0 μ M (Fig. 7 B), 5.0 μ M (Fig. 7 C), 10 μ M (Fig. 7 D) and 0.5 μ M (Fig. 7 E).Fig. 7 A～7D takes to react 4 hours in the mode that 40 ℃ of constant temperature leave standstill for utilizing the conventional hybridization reaction method, finishes the result of biomolecules hybridization.Fig. 7 E utilizes micro-array biochip reactor of the present invention, at room temperature utilizes voltage of alternating current 10V, 60Hz voltage transitions frequency to carry out the result of 30 minutes biomolecules hybridization gained.Can see significantly, adopt micro-array biochip reactor of the present invention can increase biomolecules hybridization speed effectively, shorten the hybridization time and reduce biological sample concentration.The reaction of traditional biological molecular hybridization is in order to obtain reaction result preferably, the concentration that often needs to prolong the hybridization time or improve biological sample is reached its purpose, thus, the cost of time and sample all can relatively improve, and is unfavorable for the application of biochip microarray.On the other hand, the result who obtains shows, the resulting experimental result of different biological sample concentration can't obtain crossbreeding effect preferably because biological sample concentration improves, and possible reason is because the mixing of biological sample might not evenly cause.Micro-array biochip reactor of the present invention utilizes alternating-electric field to reach the mixed uniformly purpose of biomolecules promptly in order to improve this phenomenon, makes the biomolecules hybridization can obtain more consistent experimental result.
Then, relatively adopt the result of micro-array biochip reactor of the present invention and commercial goods hybridization instrument.In the presence of biological sample concentration 10nM, adopt micro-array biochip reactor of the present invention, apply voltage of alternating current ± 10V, the variation of 60Hz electric voltage frequency, carried out the biomolecules hybridization 30 minutes, obtain the result shown in Fig. 8 A; Simultaneously, utilize commercial goods hybridization instrument, when identical biological sample concentration, carry out 1 hour (shown in Fig. 8 B), 2 hours (shown in Fig. 8 C), 4 hours (shown in Fig. 8 D), 12 hours (shown in Fig. 8 E) and 16 hours (shown in Fig. 8 F) biomolecules hybridizations respectively.The result shows that compared to the hybridization result of commercial goods hybridization instrument gained, micro-array biochip reactor of the present invention can be reached the purpose of quick bio molecular hybridization reaction effectively.
In addition, carry out the hybridization influence test of micro-array biochip reactor of the present invention for the probe biomolecule of different freedom of movement.The probe biomolecule of different freedom of movement is meant different binding between probe biomolecule and substrate, for instance, AlO3 structure of the present invention is 5 '-amino linker (amino linker)-TTTTTTTTTTTTTTT-(probe sequence)-3 ', the structure of O3 is 5 '-TTTTTTTTTTTTTTT-(probe sequence)-3 ', and the structure of P3 is 5 '-amino linker (amino linker)-(probe sequence)-3 '.Three kinds of different biological molecules concentration and probe concentration all are fixed as 0.5 μ M, and sample concentration also is fixed as 5 μ M.Adopt micro-array biochip reactor of the present invention, apply voltage of alternating current ± 10V, 60Hz switching frequency, continuous action 30 minutes is carried out the biomolecules hybridization, and the result who obtains is shown in Fig. 9 A; Adopt the reaction of traditional biological molecular hybridization, left standstill 30 minutes, the result who obtains is shown in Fig. 9 B.Comparative result adopts micro-array biochip reactor of the present invention as can be seen, applies that voltage of alternating current carries out sample mix and the molecule film micro area moves, and can improve biomolecules hybridization speed and specificity effectively.Because the effect of alternating-electric field makes the O3 probe can not be adsorbed in chip surface, and can reduce the anti-non-again specificity reaction of follow-up biological molecular hybridization effectively.For the influence of probe molecule freedom of movement, the reaction effect of the AlO3 probe molecule P3 probe molecule reaction result more relatively poor than freedom of movement is more even preferably for freedom of movement.
Above result proves, adopts micro-array biochip reactor of the present invention can quicken the carrying out of biochemical reaction effectively.And in process of the test, find, for few nucleic acid microarray (oligo microarray), when sample concentration is too high, the diffusional effect of sample molecule can reach balance at short notice, the effect that is alternating-electric field is also not obvious, and the result of resulting biomolecules hybridization there is no significant difference; And in low concentration sample, the biochip under the alternating-electric field effect, its hybridization efficient and result are better than the result that tradition leaves standstill the method gained significantly.In addition, carry out the biochip of hybridization with the tradition method of leaving standstill, hybridization result's reproducibility is relatively poor, the reliability that is this result still remains to be discussed, yet the biomolecules hybridization with the control of alternating-current place can obtain more consistent experimental result, promptly adopts micro-array biochip reactor of the present invention and voltage of alternating current, really can improve biochip gained result's exactness and reliability, solve the biochip problem that This is what people generally disapprove of the most all the time.
Analyze from another viewpoint, under the situation that does not have the external force effect, because the molecular weight of biomolecules is bigger, and to a certain degree interaction power arranged each other, make the rate of diffusion of biomolecules in solution greatly reduce, thereby cause biomolecules skewness in solution, and do not have reproducibility, cause the result of traditional biological hybridization that chip carries out to produce bigger drop.And utilize electric field action when biological sample solution, because the charged relation of biomolecules itself, therefore under electric field action, biomolecules can be advanced regularly along with direction of an electric field.Therefore, when the biomolecules that reactive force is arranged each other is present in the sample solution, can distinguish to some extent with the biomolecules that does not produce affinity interaction because of effect of electric field, even the more weak biomolecules of avidity to each other, also can separate, make sample solution more evenly help the carrying out of biochip hybridization reaction because of effect of electric field.According to more than, because of the effect of alternating-electric field hybridization result is preferably arranged in theory, also can be confirmed clearly from experimental result.
In addition, above result also shows, utilize under the electric field action, when less sample concentration, the concentration of bioprobe is high more, then the result of hybridization gained is good than the result of traditional method gained, because the probability of colliding between the molecule is relatively higher, but when concentration and probe concentration was above greater than finite concentration, then the resulting result of hybridization there is no difference significantly between the two, because when sample molecule concentration is high, and the collision probability between the probe molecule improves naturally.For example the result of 0.1 and 0.5 μ M concentration and probe concentration gained hybridization there is no significant difference in fact.This result means when concentration and probe concentration arrives finite concentration, hybridization promptly reaches capacity, in other words, though the high result that can guarantee hybridization of concentration and probe concentration, the hybridization efficiency of its probe is lower, and this is because when concentration and probe concentration is high, probe molecule closely is fixed in chip surface, steric hindrance when causing hybridization has reduced the reaction compartment of each probe molecule on the contrary, even the result who causes hybridization is than the low time difference of concentration and probe concentration.Therefore, suitable concentration and probe concentration except saving the cost of manufacture of biochip, also is one of important parameter that reaches the required consideration of best hybridization result simultaneously.
Though the present invention discloses as above with preferred embodiment, so it is not that any person skilled in the art is within the spirit and scope that do not break away from the present invention, when doing various changes and retouching in order to qualification the present invention.Therefore, protection scope of the present invention is as the criterion with the scope that claims were defined.
Claims (31)
1. micro-array biochip reactor, it comprises:
One first member, it has one first groove, and this groove can hold a sample solution;
One second member is arranged on first member; And
One group of electrode is arranged on second member, can contact with this sample solution.
2. micro-array biochip reactor as claimed in claim 1 is characterized in that, this first member and second member are made of organic materials.
3. micro-array biochip reactor as claimed in claim 2 is characterized in that this organic materials is selected from any one in synthetic rubber, natural rubber, the synthetic macromolecule.
4. micro-array biochip reactor as claimed in claim 2 is characterized in that this organic materials is selected from any one in polyethylene, polystyrene, polypropylene or the polyvinyl chloride.
5. micro-array biochip reactor as claimed in claim 1 is characterized in that, this first member and second member are made of inorganic materials.
6. micro-array biochip reactor as claimed in claim 5 is characterized in that this inorganic materials is selected from any one in metal, stupalith, silicon or the glass.
7. micro-array biochip reactor as claimed in claim 1 is characterized in that, this second member also comprises one or more holes, and this hole is in communication with the outside first member of combination and second member.
8. micro-array biochip reactor as claimed in claim 1 is characterized in that this sample solution comprises a part.
9. micro-array biochip reactor as claimed in claim 8 is characterized in that this molecule is selected from organic molecule, inorganic molecule or biomolecules, and is charged ion or uncharged neutral molecule.
10. micro-array biochip reactor as claimed in claim 9 is characterized in that this organic molecule is selected from any one in organic acid, organic bases or the amino acid.
11. micro-array biochip reactor as claimed in claim 9 is characterized in that, this inorganic molecule is metal ion or inorganic salts.
12. micro-array biochip reactor as claimed in claim 9 is characterized in that, this biomolecules is double-stranded DNA, single stranded DNA, RNA, protein or peptide.
13. micro-array biochip reactor as claimed in claim 1 is characterized in that, this micro-array biochip reactor also comprises one second group of electrode, and it is arranged on this second member.
14. micro-array biochip reactor as claimed in claim 1 is characterized in that, this micro-array biochip reactor also comprises an independent electrode, and this independent electrode is arranged on this second member corresponding to this group electrode.
15. as claim 1,13 or 14 described micro-array biochip reactors, it is characterized in that, but the described combination of interactions of respectively organizing between electrode is used.
16., it is characterized in that the material of described electrode is gold and silver, copper, nickel, platinum or stainless steel as claim 1,13 or 14 described micro-array biochip reactors.
17., it is characterized in that described first member, second member or electrode can not react with described sample solution as claim 1,13 or 14 described micro-array biochip reactors.
18. micro-array biochip reactor as claimed in claim 1 is characterized in that, is embedded with a micro-array biochip in described second member.
19. micro-array biochip reactor as claimed in claim 1 is characterized in that, links in integrally formed mode between described first member, second member.
20. a micro-array biochip reactor, it comprises:
One first member, it has one first groove, and this first groove holds a micro-array biochip that contains a reaction zone;
One second member is arranged on first member with packaged type, has second groove corresponding to this reaction zone on it; And
One group of electrode is arranged in second groove.
21. micro-array biochip reactor as claimed in claim 20 is characterized in that, this first member, second member or micro-array biochip are made of organic materials.
22. micro-array biochip reactor as claimed in claim 21 is characterized in that, this organic materials is synthetic rubber, natural rubber or synthetic macromolecule.
23. micro-array biochip reactor as claimed in claim 21 is characterized in that, this organic materials is polyethylene, polystyrene, polypropylene or polyvinyl chloride.
24. micro-array biochip reactor as claimed in claim 20 is characterized in that, this first member, second member or micro-array biochip are made of inorganic materials.
25. micro-array biochip reactor as claimed in claim 24 is characterized in that, this inorganic materials is metal, stupalith, silicon or glass.
26. micro-array biochip reactor as claimed in claim 20 is characterized in that, this second groove has one or more holes.
27. micro-array biochip reactor as claimed in claim 20 is characterized in that, this micro-array biochip reactor also comprises one second group of electrode, is arranged in this second groove.
28. micro-array biochip reactor as claimed in claim 20 is characterized in that, this micro-array biochip reactor also comprises an independent electrode, is arranged on this second member corresponding to this group electrode.
29. as claim 20,27 or 28 described micro-array biochip reactors, it is characterized in that, but this respectively organizes combination of interactions use between electrode.
30., it is characterized in that the material of this electrode is gold and silver, copper, nickel, platinum or stainless steel as claim 20,27 or 28 described micro-array biochip reactors.
31., it is characterized in that described first member, second member or electrode can not react with the sample solution in described second groove as claim 20,27 or 28 described micro-array biochip reactors.
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| CN104849114A (en) * | 2015-05-13 | 2015-08-19 | 安徽农业大学 | Special slide for comet experiment |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104849114A (en) * | 2015-05-13 | 2015-08-19 | 安徽农业大学 | Special slide for comet experiment |
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