Background technology
Bioanalysis and biomedical information acquisition are the bases of biomedical research and disease clinical diagnosis; How to set up one highly sensitive, selectivity good, cost is low, analysis speed is fast, robotization and can be in complex system the detection method of advantage such as on-line continuous detection, prevention of disease, diagnosis and treatment have important significance for theories and using value.According to the detection quantity of information what, usually detection technique is divided into several aspects such as single biology sensor, bio-sensing chip and biochip.Its essence all is to utilize the biomolecule recognition principle, converts the various variations that produce in its process physical signallings such as to light, electricity, magnetic, piezoelectricity, sound and carries out check and analysis.In various detection meanss, Electrochemical Detection and optical detection are two maximum major types detection techniques of research.Particularly earlier biomolecule is carried out fluorescent material or chemical luminous substrate enzyme labeling, the route that detects through optical signalling then remains present main flow detection means.But also there are various limitation simultaneously in above classic method; As the former easily poor the and latter of duplicating property often have the shortcomings such as optical system that photobleaching phenomenon, the dynamics range of fluorescence molecule are narrow and needs are accurate, so constantly seek the dream that new detection means is still scientist.
In recent years, nano material has become the most active research direction in bioanalysis field at the mark of biomolecule and the application in the detection.Nano material has characteristics such as specific surface area is big, high activity, utmost point smallness usually, and these characteristics require corresponding with the desired multifunction of sensor, microminiaturization, high speed etc.In addition, nano particle also shows some specific performances at aspects such as optics, magnetic, catalysis.Such as; Because semi-conductor nano particles has that fluorescent yield is high, emission peak is narrow, the Stocks displacement is with advantages such as adjustable grain; Since Science report in 1998 is used for the mark of biomacromolecule; Quantum dot just is widely used in the luminescent material mark of various biomolecule, maybe for opening up that high sensitivity and the multichannel optical biosensor that detects simultaneously supplied.The surface plasma body resonant vibration that utilizes various metal nanoparticles (Au, the Ag) characteristic of optical effect is grown up various marks and detection method one of them bright spot especially, wherein typical is exactly a series of activities of being carried out around nanometer Au.Wait the characteristics utilize nanometer Au under the guiding of dna fragmentation, can form supramolecular structure like professor Mirkin, set up with sulfhydrylation oligonucleotide probe marking nano Au and detect the new method of specific polynucleotide sequence as the assembling molecule.1997, they utilized nanometer Au particle to change and produce redness at the accumulation process middle distance and arrive the yellow color variation, detect the polynucleotide of 10fmol/L.2000, their combining nano Au mark and silver dyed developing technology, have amplified hybridization signal effectively, than sensitive 100 times of the burnt induced fluorescence detection method of the sensitiveest existing copolymerization.2002, they improve make just join/signal intensity ratio of single base mismatch improves greatly, reached 10
5: 1, and detection sensitivity is brought up to 500fmol.In addition, in patent ZL2113147.3, people such as Liu also propose to utilize a kind of nano gold mark silver dyeing detection method of genetic chip.
In the present invention; We have proposed a kind of novel nanometer Pd that utilizes and have carried out biomolecular labeling; And the catalytic performance of combining nano Pd; Utilize electroless plating to carry out signal and amplify, a kind of new bio Molecular Detection route that finally detects by the performance that is shown after the electroless plating such as characteristics such as optics, magnetic, as shown in Figure 1.Compare with present various documents and patent institute reported method, this route has the following advantages: (1) is seen from the reaction kinetics angle of target molecule and probe molecule, is more conducive to the carrying out that reacts during nanometer Pd mark.Generally be inversely proportional to particle grain size because be connected with the target molecule of nano particle to the rate of propagation of detecting probe surface, promptly the diameter of particle is more little, and its reaction velocity is fast more.In addition; Because nanometer Pd particle diameter surface is less to the non-specific adsorption of biomolecule; Thereby the target biological molecules that is more conducive to mark disperses in solution, preserves, mutual agglomeration between the particle that has been occurred when effectively having avoided some other nanoparticle label target biological molecules; (2) with respect to other detection means, carry out more simple, the convenience of chemical deposition technique route through the catalytic action of Pd, do not need expensive detecting instrument.This point is similar to patent ZL2113147.3, but seeing that the price of present Pd will be lower than Au, so this technology path will be more cheap on cost.Simultaneously, do not lose detection sensitivity again, only need a spot of target molecule to be attached on the probe molecule in the solution, just can detect through the catalytic action of Pd.(3) compare with the used relative nano gold mark silver dyeing method of patent ZL2113147.3,, be widely used in the various catalytic reactions, thereby have wider catalytic performance because nanometer Pd is traditional catalysis material, can not only catalysis Ag
+, simultaneously can also catalysis Cu
2+, CO
2+, Ni
2+Wait each metal ion species.Therefore, reach (or the institute dyes) metalline of plating subsequently from the catalysis enlarge-effect, route that we carry will more be superior to traditional Au standard silver dyeing technique.Such as, though traditional Au standard silver dyeing technique also is to Ag through nanometer Au
+Also original catalytic action, and Ag is deposited on the Au particle surface.But it is to observe the black splotch that simple substance silver is appeared through naked eyes that final signal detects, or with the gray scale of coming analytic signal after the common scanner scanning again with related software.Obviously, sort signal intensity depends on the size and the color depth of deposition of silver spot.But when being mark with nanometer Pd; Not only can detect (like the deposition of Cu) through the color of deposition; Carry out magnetics through magnetic materials such as catalyzing N i or Co and detect as utilize the magneto sensor of giant magnetoresistance effect to carry out signal and read but also can utilize; At this moment, its detection sensitivity will be higher than top color detection far away.In addition, electrochemical means also capable of using such as cyclic voltammetry etc. detect above-mentioned Pd catalytic deposition result.
Summary of the invention
Technical matters:The objective of the invention is to propose a kind of biological detecting method based on nanometer palladium mark and catalytic deposition amplification thereof; One of purpose is to propose a kind of method of biomolecule being carried out nanometer Pd mark like (DNA or protein etc.); Promptly earlier through functional group and the surperficial ligand complex effects of Pd such as sulfydryl or amino; Specified stabiliser is connected to nano grain surface, utilizes simultaneously other functional groups such as carboxyl etc. on the stabilizing agent further biomolecule to be connected to its surface like (DNA or protein etc.) again.Made particle is in buffer solution more than final the requirement
Good dispersiveness is arranged.
Two of the object of the invention is the catalytic performances that are conceived to Pd, and through chemical plating method, the signal that realize to detect thing amplifies, and finally detects by means of post-depositional various physical propertys such as light, magnetic, electricity etc.
Technical scheme:According to above-mentioned target; At first to search out a kind of suitable nanometer Pd granulate preparation and finishing path; Some basic demands that require the gained particle can satisfy to carry out biomolecular labeling and detection as: particle diameter is less, in the homogeneous, ability stable existence buffer solution like PBS and making things convenient for when biomolecule is connected, can reduce non-specific adsorption to the other biological molecule.Secondly, find a kind of easy catalytic deposition process, require this process that the interaction between the biomolecule is not had obvious influence, can have tangible signal simultaneously again and amplify and convenient detection based on the Pd particle.
Biological detecting method based on nanometer palladium mark and catalytic deposition amplification thereof of the present invention utilizes nanometer palladium particle right
Biomolecule is carried out mark, and the catalytic performance of combining nano palladium, utilizes electroless plating to carry out signal and amplifies, and finally detects by the optics that is shown after the electroless plating, magnetic properties.
Described nanometer palladium particle, its particle diameter are reductive agent with ethanol or hydrazine hydrate between 0-100nm, are that the stabilizing agent reduction obtains with sodium citrate, PVP, PEG-SH, CTAB or SDS.
Described biomolecule comprises dna fragmentation, protein.
Before to nanometer palladium finishing biomolecule, utilize two step method, with HS-(CH
2)
n-COOH or H
2N-(CH
2)
n-COOH replaces the stabilizing agent molecule and is connected to the palladium particle surface; So-called two-step approach is meant that the first step is replaced the stabilizing agent of nanometer palladium particle surface earlier by TA, utilizes for second step then, again with the TA part of nanometer palladium particle surface by HS-(CH
2)
n-COOH replaces; Wherein, n is between 4-20;
Directly the Pd nano particle that obtains and said biomolecule are directly hatched and make nanometer Pd particle mark biomolecule.
With the HS-(CH that is obtained
2)
n-COOH modification palladium nano-particles and biomolecule are hatched and are made nanometer Pd particle mark biomolecule.
After the biomolecule and probe molecule generation biological agent of nanometer palladium particle mark, utilize the electroless plating process, by the catalytic action of Pd, around nanometer palladium particle, further deposit metallic particles and carry out the signal amplification.
The electroless plating process refers under cold situation, utilizes redox reaction around nanometer Pd, to obtain layer of metal simple substance or alloy; Wherein, the metal that relates to comprises nickel, copper, zinc, cobalt, silver, gold, iron or the alloy that is combined between them.
Described detection is based on optics, magnetic and other characteristic that is shown after the above-mentioned electroless plating; Optics refers to post-depositional change color, can judge according to naked eyes, applies flexibly the gray scale scanning appearance and carries out; Magnetic refers to the magnetism characteristic that coating shows, and carries out input by giant magnetoresistance effect and superconducting quantum interference device (SQUID); Other characteristics comprise after the above coating dissolving, relend and help electrochemical techniques, detect the concentration of metal ions after dissolving.
This testing process is not only applicable to biochip test, carries out high-throughout screening, can be used for single biology sensor simultaneously yet, carries out the detection of single biomolecule.
Beneficial effect:Compare with document or patent that the front has been reported; Although the technical scheme of being stated among the present invention is from see above-mentioned route somewhat similar " the Au standard silver dyes " technology to a certain degree; Equally also be to utilize the metal nanoparticle mark; Utilize its catalytic action to carry out signal again and amplify, but see that from the sensitivity angle of input this route is superior to " the Au standard silver dyes " again.Because the former catalytic process is wider, price is also more cheap.See from nano material and biological detection subject combining development trend; The extended nanometer Pd mark biomolecule technology of this patent will enlarge the biomolecular labeling technology; And the certain effect of performance in other biological detection technique such as Electrochemical Detection, catered to subject development.
Embodiment
Doing further to explain in the face of top technology path down, is stabilizing agent with the sodium citrate at first, utilizes coprecipitation to prepare the Pd nano particle of mean grain size about 10nm.When carrying out dna marker, can utilize two kinds of approach of direct method or indirect method to be connected to nanometer Pd surface.The former refer to before made nanometer Pd particle directly and sulfydryl or amido modified dna fragmentation hatch and obtain.The latter refers to before connecting dna molecular, use two-step approach earlier, and is as shown in Figure 2, with H
2N-(CH
2)
n-COOH (wherein; N is between 4-20) replace the stabilizing agent that the front prepares the Pd particle, and then under the effect of EDC and NHS, with amido modified DNA; Schiff key (Schiff bond) through forming is connected to the Pd nano grain surface, thereby accomplishes the nanometer Pd mark of target molecule.The Pd nanoparticle label biomolecule that said process obtains will be more more stable than direct method in buffer solution.After accomplishing nanometer palladium mark, through being connected to the biochip surface of known array on the target molecule of the interaction between the biomolecule (like crossover process) with nanometer Pd particle mark.After washing the target molecule of non-specific adsorption in microarray surface; According to process shown in Figure 1; The gained chip is soaked the people in the chemical plating fluid of cupric, carry out the signal amplification; Take out the chip water after several minutes and simply clean, finally around the Pd of linking objective molecule particle, deposit a large amount of copper particles.The result shows; Utilize the extraordinary dna sequence dna of having distinguished complete complementation (the copper color is very dark), base of mispairing (copper lighter color) and two bases of mispairing (copper lighter color) of electroless plating; Its sensitivity is suitable with Au standard silver dyeing technique, can reach about 500fmol.
Need to prove, above said in, the preparation of nanometer Pd is to be that stabilizing agent, ethanol are that reductive agent prepares in the WS with the sodium citrate.When with PEG
2000-SH, polyvinylpyrrolidone (PVP), cetyl trimethyl ammonium bromide (CTAB) or sodium dodecyl (SDS) etc. are stabilizing agent; Or when being reductive agent with the hydrazine hydrate; Prepared nanometer Pd particle is applicable to top mentioned two-step approach finishing process too, finally is translated into 5-sulfydryl undecanoic acid and modifies the Pd nano particle.In addition, see, not only comprised amido modified dna molecular chain, also can expand to protein molecule simultaneously from the biomolecule type of modifying, all can utilize the Schiff key with these molecular modifications to the Pd particle surface.See that from the process of electroless plating nanometer Pd not only can be to Cu
2+Carry out catalytic deposition, simultaneously can also be to Ni
2+, Co
2+Plasma deposits, thereby realizes that detection signal amplifies.And work as with Ni
2+, Co
2+When plasma is plating bath, can also carry out quantitative test to it according to the magnetic property of institute's coating.It is the detection synoptic diagram that detects magnetic head that Fig. 3 has provided with the giant magnetoresistance induction pick-up, and when in the site that does not have coating, its detection signal is zero, only in the site of fully just joining, the detection electric current is arranged, thereby has improved detection sensitivity, is convenient to carry out high flux screening.In addition, see from the detection technique angle, above be to detect in the cited process through probe stationary made microarray on glass baseplate, in fact this process also is fit to scopes such as single biology sensor, bio-sensing chip and biochip.For the biology sensor of single biology sensor, also can carry out signal and amplify through catalytic deposition process based on nanometer Pd such as piezoelectric quartz crystal.
Embodiment one:With ethanol is that reductive agent, sodium citrate are that stabilizing agent prepares nanometer Pd particle
With c (sodium citrate)/c (H
2PdCl
4)=10, c (H
2PdCl
4)=0.5mM is an example, in the round-bottomed flask of 100mL, adds 15mL H earlier
2PdCl
4Solution (2mM), 0.0882g sodium citrate, 2mL ethanol and 43ml ddH
2O, mechanical raking reflux 3h.Solution becomes sepia by pale brown look, promptly obtains the stable Pd nano particle of sodium citrate.Then, (12000rmin 30min), removes supernatant, is dissolved in ddH more again with made particle high speed centrifugation
2Among the O, so repeatable operation is 3 times, keeps next step use.Can find out from characterization result, gained grain size homogeneous, rounded, mean grain size is about 10 nm.
Embodiment two:With ethanol is that reductive agent, PVP are that stabilizing agent prepares nanometer Pd particle
With 106.4 mg PdCl
2, 6.0 mL concentration are that the HCl WS of 0.2 M adds the people and steams in the water to 294 mL two, ultrasonic 30 minutes, are made into 2.0 mM H
2PdCl
4Solution.Get wherein 15 mL, and in the round-bottomed flask that mixed solution (content of ethanol is between 10-70 vol %) and a certain amount of PVP (between the 0.333-133 mg) of 35 mL water and ethanol joins 100 mL reflux 3h.After the reaction, (12000rmin 30min), removes supernatant, is dissolved in ddH more again with made particle high speed centrifugation
2Among the O, so repeatable operation is 3 times, keeps next step use.
Embodiment three:With PEG
2k-SH be stabilizing agent, hydrazine hydrate be reductive agent prepare nanometer Pd particle,
With 0.500 g PdCl
2, 0.33 g NaCl adds the people and steams in the water to 250 mL two, ultrasonic 30 minutes, is made into 0.011 M Na
2PdCl
4The WS.Then with 0.55 mmol PEG
2k-SH is dissolved in the Na of the above preparation of 10 mL
2PdCl
4In the WS.Under vigorous stirring, in this mixed solution, dropwise add the WS that 10 mL contain 0.22 mmol hydrazine hydrate and 0.22 mmol NaOH.Along with the carrying out of reaction, the color of solution becomes black rapidly.After dripping, be reflected at and continue to stir 30 minutes.After the reaction, (12000rmin 30min), removes supernatant, is dissolved in ddH more again with made particle high speed centrifugation
2Among the O, so repeatable operation is 3 times, keeps next step use.
Embodiment four:With CTAB is that stabilizing agent, ethanol are that reductive agent prepares nanometer Pd particle
Under 30 ° of C, it is in toluene/tetrahydrofuran mixed solution of 5/1 that 4 g palladium acetylacetonates, 5 g CTAB are dissolved in 200 mL volume ratios, and to wherein adding 25 mL absolute ethyl alcohols.Then, under 65 ° of C, with top gained mixed solution reflux 12 h.Along with the carrying out of reaction, the color of solution transfers brownish black gradually to.After reaction finishes, stir adding 100mL absolute ethyl alcohol in the gained mixed solution down, from solution, deposit to promote the Pd nano particle.After removing supernatant, the gained deposition is cleaned three times with ethanolic solution, so that remove unnecessary stabilizing agent and residual toluene solution.At last, water cleans twice, thereby made Pd nano particle is transferred in the WS, is made into the pd nano particle WS of 1mg/ml, keeps next step use.
Embodiment five:The HS-DNA of Pd nano particle directly modifies
:
Pd colloidal solution that 2 mL embodiment one to four are prepared and 1 OD sulfydryl probe P1 (Pd-5 '-HS-AGC TCT CAA ACT TTT-3 ', its ultimate density is greatly about 3.51 μ M) mix, 16 h hatched under the room temperature.Add 3 ml phosphate buffers (pH 7 for 10 mM PB, 0.1 M NaCl), continue to hatch 48 h.Centrifugal 25 min under 14000rpm then are to separate unlabelled DNA.Remove supernatant liquor after centrifugal, 5 ml phosphate buffers (10 mM PB, 0.1 M NaCl are used in the black precipitate of bottom again; PH 7) dilution, centrifugal 25 min under 14000rpm remove supernatant once more; The lower black deposition adds 1ml 2 * PBS liquid (10 mM PB; 0.3 M NaCl, pH 7), mother liquor be stored in 4 ℃ subsequent use.
Embodiment six:HS-(the CH that utilizes two-step approach that the Pd nano grain surface is carried out
2) the n-COOH modification
:
Can also utilize two-step approach (its process is as shown in Figure 2) to part front gained Pd nano particle, HS-(CH is carried out on its surface
2) n-COOH, reach non-specific adsorption so that further improve the stability of gained Pd nano particle in buffer solution to biomolecule.
The first step, colloid Pd solution that 2 ml embodiment one, embodiment two and embodiment four is prepared and the TA aqueous solution of 1 ml 1mol/L, ultrasonic 2 minutes, and then hatch 8 h under the room temperature.Then, centrifugal 25 min under 14000rpm remove TA that does not exchange to the Pd surface and the sodium citrate stabilizing agent (or other stabilizing agents) that exchanges.Repeat above process twice, each remove supernatant liquor after centrifugal, the black precipitate of bottom is again with 5 ml WS dilution dispersion.In second step, above solution is dispersed in the 2 ml water, and adds the HS-(CH of 1 ml, 0.5 mol/L
2)
11-COOH aqueous solution was hatched 24 hours.Afterwards, centrifugal 30 min under 14000rpm remove the HS-(CH that does not exchange to the Pd surface
2)
11-COOH and the TA that exchanges.Buffering is followed towards the each DNA of liquid, adds 3 ml phosphate buffers (10 mM PB, 0.1 M NaCl; PH 7); Repeat above process twice, each remove supernatant liquor after centrifugal, 2 ml (10 mM PB are used in the black precipitate of bottom again; 0.1 M NaCl, pH 7) buffer solution dilution dispersion.At last, lower black deposition is added 1ml 2 * PBS liquid (pH 7 for 10 mM PB, 0.3 M NaCl), mother liquor be stored in 4 ℃ subsequent use.
Embodiment seven:With Pd-S-(CH
2)
5-COOH nano particle and H
2N-DNA (H
2N AGC TCT CAA ACT TTT-3 ') directly modifies.
With above embodiment six gained 1mlPd-S-(CH2) 5-COOH solution; Under 14000rpm, centrifugal 30min removes supernatant; Black precipitate with bottom is diluted to 1ml with the MES damping fluid then, adds 0.4mg EDC (about 2mM) and 0.6mg NHS room temperature reaction 15 minutes.Then, the H2N AGC TCT CAA ACT TTT-3 ' solution of 1 OD is added in the above-mentioned solution, continue to hatch 24h.After the reaction, with above mixed solution centrifugal 30min under 14000rpm.After removing supernatant, the lower black deposition is added 1ml 2 * PBS liquid (pH 7 for 10 mM PB, 0.3 M NaCl), mother liquor be stored in 4 ℃ subsequent use.
Embodiment eight:The preparation of dna microarray and and the nano-particle modified target dna of Pd between hybridization
It is surperficial through the glass slide of aldehyde radical modification to utilize point sample instrument that the oligonucleotide probe (the living worker in Shanghai is synthetic again) of following four 16 bases is selected in advance, and hatches fixing.
5 '-AGC TCT CAA ACT TTT C-3 ' just joins
1 base of 5 '-AGC TCT CAC ACT TTT C-3 ' mispairing
2 bases of 5 '-AGC TCT CCC ACT TTT C-3 ' mispairing
3 bases of 5 '-AGC TCT GCC ACT TTT C-3 ' mispairing
The Pd-5 '-HN AGC TCT CAA ACT TTT-3 ' that obtains with embodiment five or embodiment seven is then hybridized, and hybridization solution is MicroHyb, and hybridization conditions is identical with fluorescence method.After the hybridization, use 2 * SSC/0.1%SDS and 0.1 * SSC/ 0.1%SDS solution respectively to clean 10 min respectively, remove the Pd nano particle of non-specific adsorption on glass sheet.
Embodiment nine:Microarray after the hybridization is carried out the copper electroless plating
Amplify and development carrying out signal in the sheet base immersion Tongran plating bath after the hybridization of embodiment eight gained.Wherein, solution composition is made up of following A and two kinds of solution of B, before carrying out electroless plating, and on-the-spot preparation.A solution 3g CuSO
4, 4g NaOH, 14g sodium-potassium tartrate, 100ml water, the formalin solution of B solution 37.2%.Before the electroless plating, directly above A, two kinds of solution of B are mixed, above acquisition microarray is immersed react then.Reaction 10 min under the room temperature, washing dries up.The result shows, utilizes the extraordinary dna sequence dna of having distinguished complete complementation (the copper color is very dark), base of mispairing (copper lighter color) and two bases of mispairing (copper lighter color) of electroless plating.Its sensitivity is suitable with Au standard silver dyeing technique, can reach about 500fmol.
Embodiment ten:Microarray after the hybridization is carried out the nickel electroless plating
Sheet base after the embodiment eight gained hybridization is immersed nickel to be dyed and carries out signal in the plating bath and amplify and develop.Wherein, solution composition is made up of following A and two kinds of solution of B, before carrying out electroless plating, and on-the-spot preparation.A solution is reducing solution, amido borine (DMAB) 0. 39/ L.In addition, reductive agent can also be NaBH
4, KBH
4Deng amido borines such as alkali metal borohydride, trimethylamine groups borine, triethyamino borine and hydrazine etc., be good with DMAB.In the B solution NiSO
4(with Ni
2+Meter)/gL
-14~7, NaH
2PO
2H
2O/ gL
-120~40 malic acid/gL
-110~30 succinic acids/gL
-15~15.Before the electroless plating, directly above A, two kinds of solution of B are mixed, and its pH value is transferred to 4~5.Then above acquisition microarray is immersed and react, 70~93 ℃ are reacted 10 min down, and washing dries up.
Embodiment 11:Microarray after the hybridization is carried out nickel, cobalt-base alloy electroless plating
Amplify and development carrying out signal in the sheet base immersion nickel after the hybridization of embodiment eight gained, the cobalt mix plating.Wherein, solution composition is made up of following A and two kinds of solution of B, before carrying out electroless plating, and on-the-spot preparation.A solution is reducing solution, amido borine (DMAB) 0. 39/ L.In addition, reductive agent can also be NaBH
4, KBH
4Deng amido borines such as alkali metal borohydride, trimethylamine groups borine, triethyamino borine and hydrazine etc., be good with DMAB.In the B solution NiSO
430 g/L, CoSO
48-14 g/L, NaH
2PO
220 g/L, C
6H
5O
7Na
364 g/L, complexing agent 0. 020 g/L, stabilizing agent 0. 003 g/L, surfactant 0. 5 g/L.Before the electroless plating, directly above A, two kinds of solution of B are mixed, and its pH value is transferred to 8~8.5.Then above acquisition microarray is immersed and react, 30~40 ℃ are reacted 10 min down, and washing dries up.
Embodiment 12:Seeing that the input that the magnetic properties of coating is carried out
When being the electroless plating material with Co, Ni or its potpourri; Because formed coating not only has the change color that similar front Cu coating is shown; They self also have magnetic properties simultaneously; And these characteristics can carry out signal and read through different biological magnetic field checkout equipments, like spin-valve sensor (Spin Valves), giant magnetoresistance induction pick-up (GMR Sensors), superconducting quantum interference device (SQUID) (SQUIDs), anisotropic magnetoresistive (AMR) ring type sensor, the small-scale combination sensor of ear suddenly (Hall Crosses) and tunnel junction (TMR) sensor etc.
Be example explanation its application (its principle process is asked for an interview Fig. 3) with the giant magnetoresistance induction pick-up below.After microarray after embodiment ten and embodiment 11 will hybridizes carries out electroless plating, first with washed with de-ionized water chip 3 times, remove free Ni
2+, Co
2+Ion and other solution compositions, and then with PBS buffer solution cleaning 2 times, clean once with distilled water more at last.After chip being blown over, place in the vacuum drying chamber and spend the night with argon gas.Then, placing the homemade GMR sensor device in laboratory to carry out signal chip reads.Wherein, the GMR read head is made up of spin valve, and sensitivity is 1.3 mV/V*Oe, the range of linearity ± 20Oe.Reference literature adopts Wheatstone bridge to change resistance signal into voltage signal output.Because the magnetic-field component to along the surface is very responsive.When externally-applied magnetic field tilted, the reference signal of GMR sensor can change, and effective GMR signal of generation also can change.Directly multiply each other the average magnetic field of sensor surface and transducer sensitivity, the resistance signal that just obtains the GMR sensor changes.Find in the experiment, the variation of above-mentioned signal can extraordinary reflection microarray in the variation of dna sequence dna, electric signal when sequence is matched fully and during one of mispairing can produce the electric signal difference of about 20 μ V.The gray scale test result of this and front matches, and the feasibility of this route is described fully.