CN105300938B - Based on molecular dynamics detection method associated with acousto-optic - Google Patents
Based on molecular dynamics detection method associated with acousto-optic Download PDFInfo
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Abstract
This case is related to based on molecular dynamics detection method associated with acousto-optic, it goes to capture biomolecule to be measured and forms interlayer structure by the first antibody for being combined with magnetic bead and the secondary antibody for being combined with optical markings molecule, and magnetic bead is quickly adsorbed to the acoustic wave piezoelectric sensor surface of the body of Molecular Adsorption containing optical markings under magnetic fields together with the interlayer structure, molecular dynamics information is obtained by the change of sensor signal, at the same time by the deployable accurate optical detection of optical markings molecule, and the result obtained with this is corrected kinetic parameter.This case can quickly capture the biological tests molecule of ultramicron without can be directly detected to sample pre-treatments from complex samples；The dynamic process test to biomolecule is realized, obtains the information such as testing molecule quality, viscoplasticity, concentration, and the kinetic parameter such as association reaction performance graph measuring and calculating reaction rate, binding constant or dissociation constant.
Description
Technical field
It is used to test the molecular dynamics detection methods of target biological molecules in testing liquid the present invention relates to a kind of, especially
It is related to a kind of molecular dynamics detection method based on the compound joint technology of acousto-optic.
Background technology
Acoustic wave piezoelectric sensing technology is a kind of non-optical, highly sensitive quantitative measurement technology of rapid rising in recent years.Pass through
Special sex modification is carried out to the sensor interface, is hardly influenced by sample translucency and viscosity, can be in reaction system
In rapid capture target molecule, these molecules can change piezoelectric material vibration resonant frequency after being combined with sensor surface believes
Number, the reacting condition of the signal amplitude and the phase quality of binding molecule, the information of viscoplasticity equimolecular content, and combine anti-
Performance graph is answered to be parsed to kinetic parameters such as reaction rate, binding constant or dissociation constants.Shortcoming is exactly,
Since acoustics piezo technology is in itself without the ability for distinguishing labeled molecule, can only detection molecules be combined with the surface or depart from
Cause the increase of quality, belong to markless detection, thus the non-specific adhesion of some non-targeted molecules and sensor easily causes
False positive results.Non-specific adhesion refers to that other foreign proteins and molecule can be adsorbed onto piezoelectric sensor surface in sample, cause
Mass adsorption and frequency shifts, so as to cause false positive results, cause error, although can be reduced by strengthening surface modification
The generation of false positive results, but there is small part to adsorb.
The content of the invention
In view of the deficiencies of the prior art, the present invention intends to be examined by building a kind of brand-new molecular dynamics
Survey method so that it can suppress the generation of false positive results to greatest extent, improve the essence of its kinetic parameter obtained
Exactness.
Due to the presence of false positive results so that piezoelectric detection technology is in dynamic parameter and terminal the concentration inspection to determinand
Application in terms of survey is very restricted, and error is larger.Optical detecting method high sensitivity, detection it is semi-automatic, complete
Automatic molecule diagnosis is based on optical instrument；And if optical detective technology can be combined with piezoelectric detection technology, it will
Improve the precision and application range of piezoelectric detection technology.However, both combinations are there is many technology barriers, it is two first
Person's detection apparatus structure used is totally different, and how to be integrated out in a detection cell can be to the detection device knot of both parameters
Structure, the problem of becoming very headache；Also, more troubling, conventional art is with ELISA, Western Blot
Deng the most ripe of optical detecting method development, but " direct " detection of the complex fluid sample to high viscosity, translucency difference
Very difficult, they are required to carry out pre-treatment to sample, such as centrifuge, filtering, dilution etc..In addition, optical detective technology easily by
Background interference, such as viscosity, density factor, its test limit is relatively low, i.e., can not accurately test extremely low concentration content.
Therefore, it is another object of the present invention to by the improvement to the molecular dynamics detection method, can be based on acousto-optic
Associated with angle go to coordinate to integrate optical detection and piezoelectric detection technology, both is formed an organic whole, take long benefit each other
It is short.
To achieve the above object, the present invention is achieved through the following technical solutions：
One kind is based on molecular dynamics detection method associated with acousto-optic, including：
Step 1) sputters one layer of layer gold in acoustic wave piezoelectric sensor surface, and can capture optics in the layer gold surface modification
The optical markings Molecular Adsorption body of mark molecule；The acoustic wave piezoelectric sensor is then placed in the bottom of detection cell, the detection
At least there is one to be capable of the horizontal cavity for housing acoustic wave piezoelectric sensor, the runner that an accommodating fluid frees in and out, one in pond
A top surface for being capable of printing opacity, also, the fluid in the runner can directly be contacted with the optical markings Molecular Adsorption body；
Step 2) can produce the first antibody of specific binding in magnetic bead surfaces modification with testing molecule；
Step 3) by optical markings molecular modification to secondary antibody, wherein, which can produce with testing molecule
Raw specific binding, and the site specifically bound is different from first antibody；
The magnetic bead for being modified with first antibody is placed in runner by step 4) with the secondary antibody for being modified with optical markings molecule
In fluid, acoustic wave piezoelectric sensor gathered data is opened, the prepare liquid containing testing molecule is injected into runner, at this time control stream
Body does not flow, and first antibody, secondary antibody are combined with testing molecule and testing molecule are clamped in the middle sandwich knot of formation respectively
Structure, introduces magnetic field so that optical markings molecule and optical markings Molecular Adsorption body are fast in the lower section of the acoustic wave piezoelectric sensor
Speed combines, and testing molecule is fixed on the layer gold surface, so as to cause the signal intensity of acoustic wave piezoelectric sensor, treats sound wave
After the data stabilization of piezoelectric transducer collection, runner is controlled fluid flow out, and with new fluid scouring runner, treat that acoustic wave piezoelectric passes
After the signal of sensor is stablized again, that is, complete single detection process；
Step 5) sets an optical detector circuitry in the detection cell top face, by gathering optical markings molecule
Optical parameter obtains the ultimate density of testing molecule, and goes to correct by acoustic wave piezoelectric sensor institute on the basis of the ultimate density
The data error brought, so as to obtain the kinetic parameter of more accurately testing molecule.Optical markings molecule can be divided into fluorescence mark
Score son and visible ray mark molecule, correspondingly, corresponding optical markings Molecular Adsorption body is also classified into fluorescent tag molecule
Adsorbent and visible ray mark molecule adsorbent, fluorescent tag molecule adsorbent are mostly antibody class, can be with corresponding fluorescent marker
Molecule produces specific adsorption, it is seen that signal Molecular Adsorption body is mostly active chemistry, can be with corresponding visible signal
Molecule produces Covalent bonding together.Common fluorescent tag molecule has rhodamine, cyanine dye cy3, cy5 and FITC points of cyanine dye
Son, common visible ray mark molecule have nanogold and nano silver.Mark according to fluorescent tag molecule, then optical detection electricity
The optical parameter that road is collected is to excite fluorescent tag molecule by laser light source, and fluorescent tag molecule is excited caused fluorescence
Light intensity；Mark according to visible ray mark molecule, using the nanometer enhancement effect of metallic, enhance interface to visible
Beam divergence signal, the optical parameter that optical detector circuitry collects are luminous intensities after the nano metal particles of light source activation strengthen
Or angular rate signal.Different optical markings molecules is chosen, optical detector circuitry should just choose the spy of corresponding testing principle
Survey module.
Preferably, it is described based on molecular dynamics detection method associated with acousto-optic, wherein, the acoustic wave piezoelectric sensing
The material of device is selected from piezoelectric ceramics, quartz, lithium niobate, zinc oxide, aluminium nitride or its combination.
Preferably, it is described based on molecular dynamics detection method associated with acousto-optic, wherein, passed in the acoustic wave piezoelectric
One side of the sensor away from the layer gold is additionally provided with basalis.
Preferably, it is described based on molecular dynamics detection method associated with acousto-optic, wherein, the temperature of the fluid is
37±0.5℃。
Preferably, it is described based on molecular dynamics detection method associated with acousto-optic, wherein, the thickness of the layer gold is
10~20nm.
Preferably, it is described based on molecular dynamics detection method associated with acousto-optic, wherein, the bore of the runner is
10~100 μm.
Preferably, it is described based on molecular dynamics detection method associated with acousto-optic, wherein, the magnetic bead it is a diameter of
20nm~1.5 μm.
Preferably, it is described based on molecular dynamics detection method associated with acousto-optic, wherein, the optical markings molecule
Selected from nanogold, nano silver, rhodamine, cyanine dye cy3, cyanine dye cy5 or FITC molecule (FITC：fluorescein
Isothiocyanate, fluorescein isothiocynate) in one kind.
The beneficial effects of the invention are as follows：
1) without can be directly detected to sample pre-treatments, the life of ultramicron can be quickly captured from complex samples
Thing testing molecule, by means of the optical markings carried out to molecule, it is possible to achieve the accurate detection to molecular concentration to be measured；And with
This surveys concentration and the relevant parameter that acoustic wave piezoelectric sensor collects effectively is corrected；
2) realize and the dynamic process of biomolecule is tested, obtain the information such as testing molecule quality, viscoplasticity, concentration, and
The kinetic parameters such as association reaction performance graph measuring and calculating reaction rate, binding constant or dissociation constant, error is small, and precision is high.
Brief description of the drawings
Fig. 1 is the principle schematic based on molecular dynamics detection method associated with acousto-optic.
Fig. 2 is the principle schematic using detection cell as the test system of core.
Fig. 3 is the piezoelectric transducer kinetic test result figure measured by embodiment 1.
Fig. 4 is the AFP concentration fluorometric investigation result figures measured by embodiment 1.
Fig. 5 is the AFP concentration piezoelectricity test result figures measured by embodiment 1.
Embodiment
The present invention is described in further detail below in conjunction with the accompanying drawings, to make those skilled in the art with reference to specification text
Word can be implemented according to this.
This case provide an embodiment based on molecular dynamics detection method associated with acousto-optic, wherein, with FITC molecules make
For optical markings molecule, optical markings Molecular Adsorption body is used as using FITC antibody；Specifically include following steps：
Step 1) sputters one layer of layer gold in acoustic wave piezoelectric sensor surface, and can capture FITC in the layer gold surface modification
The FITC antibody of molecule；The acoustic wave piezoelectric sensor is then placed in the bottom of detection cell, which at least has one
It is capable of runner, the top for being capable of printing opacity that cavity, the accommodating fluid of horizontal accommodating acoustic wave piezoelectric sensor free in and out
Face, also, the fluid in runner can directly be contacted with FITC antibody；Acoustic wave piezoelectric sensor is really one layer by piezoelectric
The layer structure of composition, both sides are equipped with lead end, its structure and principle belongs to the prior art, and details are not described herein for this case,
And the purpose in its surface sputtering layer gold is intended to improve the biocompatibility of sensor by the characteristic of gold, and repaiied in gold surface
Decorations FITC antibody falls within the prior art, such as can utilize the Covalent bonding together of sulfydryl and gold, select cysteine, mercaptan or
The probe biomolecules such as nucleic acid probe or antibody are fixed on sensor surface by the molecule of other functional groups Han-SH.FITC antibody
Concrete form or type need not be defined, it only need to meet can with FITC molecules produce specific reaction it is in combination i.e.
Can, because the effect of FITC antibody is exactly to identify and capture in a fluid FITC molecules and clenched.Here " inspection
Survey pond " it is a component for being used for piezoelectric detection and optical detection actually, it collects reaction and is detected on one, the bottom of detection cell
For acoustic wave piezoelectric sensor, centre is runner, and runner is used to be connected with other such as sampling devices, temperature control device, and top is
Transparent surface, optical detection are mainly located at the FITC molecules of sensor surface from transparent surface irradiation excitation.Therefore, it is referred to here
" detection cell " in fact without being defined to a specific structure, only need to meet above-mentioned requirement；And in runner
Fluid can directly be contacted with FITC antibody, the fluid that actually also show in runner is directly contacted with layer gold, this
In the complete pipeline in border of described " runner " actually also and in the narrow sense, it is jaggy, and piezoelectricity in bottom surface
Sensor has filled up this notch just so that runner becomes a complete closed pipe that can be accommodated fluids free flow and walk
Road, certainly, pipeline here are also not necessarily that the shape such as round pipeline in the narrow sense, square all may be used.
Step 2) can produce the first antibody of specific binding in magnetic bead surfaces modification with testing molecule；Magnetic bead is positioned at stream
In the fluid in road, first antibody is used for the good compatibility for capturing biomolecule to be measured, magnetic bead and fluid reagent, and specific surface area is big,
The contact probability of reagent and testing molecule is added, beneficial to acceleration reaction process.
Step 3) by FITC molecular modifications to secondary antibody, wherein, which can also produce with testing molecule
Specific binding, and the site specifically bound is different from first antibody；If identical with the binding site of first antibody, easily make
Into competitive reaction, and this will deviate from the original intention that this case sets double antibody.The purpose that first antibody and secondary antibody are used in combination is just
It is to make them form interlayer structure after being combined with biomolecule to be measured, the magnetic bead combined on first antibody can be used to accelerate whole
The process of a reaction, the FITC molecules in secondary antibody can be fixed by the FITC antibody modified in layer gold, and FITC molecules from
Body can be used for optical detection, what the ratio of the biomolecule to be measured in FITC molecules and interlayer structure was to determine, by means of
FITC molecules directly can accurately measure the ultimate density of biomolecule to be measured, but in the prior art, this is nothing in fact
Method realize, reason be exactly in the prior art to by optical detection concentration just must to sample progress pre-treatment, and
In most cases, since the complexity of sample differs, the effect of pre-treatment can not absolute guarantee, and the technical solution of this case
Preceding processing when also solving the problems, such as optical detection while piezoelectric detection, the i.e. generation of interlayer structure are virtually realizing
To the purification ＆ isolation of sample to be tested, and this purification ＆ isolation is efficient, and effect is better than the conventional macroscopic processing such as centrifugation, dilution
Means.After testing molecule is fixed on sensor surface by interlayer structure, repeatedly washed away by fluid convection road, can
Impurity is effectively removed, only leaves biomolecule to be measured in itself, therefore, the refining effect of this specific adsorption mode is very high
Effect.First antibody and secondary antibody can not be defined as a clear and definite structural formula, because their selection is according to be measured
It is fixed that biomolecule is come, and different biomolecule to be measured has the different antibody that can be specifically bound from it, and each combination
The antibody structure formula in site is different from, and therefore, this case need not provide the concrete structure formula of first antibody and secondary antibody.
The magnetic bead for being modified with first antibody is placed in the fluid of runner by step 4) with the secondary antibody for being modified with FITC molecules
In, acoustic wave piezoelectric sensor gathered data is opened, the prepare liquid containing testing molecule is injected into runner, controls fluid not at this time
Flowing, first antibody, secondary antibody are combined with testing molecule and testing molecule are clamped in middle formation interlayer structure respectively,
The purpose of lower section introducing magnetic field of acoustic wave piezoelectric sensor, the magnetic field of introducing is to make magnetic bead rapid aggregation around sensor surface,
So that FITC molecules are quickly combined with FITC antibody, testing molecule is fixed in layer gold surface with the pattern of interlayer structure,
So as to cause the signal intensity of acoustic wave piezoelectric sensor, after the data stabilization of acoustic wave piezoelectric sensor collection, fluid stream is controlled
Go out runner, and with new fluid scouring runner, after the signal of acoustic wave piezoelectric sensor is stablized again, that is, complete single and detected
Journey；The incorporation way in magnetic field is also unrestricted, can manually pipette a block permanent magnet to realize, can also set one block of electromagnetism
Iron, the switch in magnetic field is controlled by power on/off.The flow rates of fluid are preferably 50~5000 μ L/min.
Step 5) sets an optical detector circuitry in detection cell top face, excites FITC molecules by irradiating, collection
The fluorescence intensity that FITC molecules are sent after being excited obtains the ultimate density of testing molecule, and goes to repair on the basis of the ultimate density
The data error just as caused by acoustic wave piezoelectric sensor, so as to obtain the kinetic parameter of more accurately testing molecule.And arrive
How to be detected in optical detector circuitry, then belong to the prior art, this case think without the concrete structure to optical detector circuitry into
Row limits, but this case can provide a simply example, for example, optical detector circuitry can include laser light source, lens,
Reflective mirror, grating, photomultiplier etc., light source irradiate excitation FITC molecules after adjusting position by speculum and lens,
FITC molecules are excited to produce fluorescence, after lens, grating filters, are received by photomultiplier, can be obtained after processor is handled
Go out corresponding data.
In the above-described embodiments, the material of acoustic wave piezoelectric sensor is selected from piezoelectric ceramics, quartz, lithium niobate, zinc oxide, nitrogen
Change aluminium or its combination.The preferably combination of lithium niobate, zinc oxide and aluminium nitride three, this combines the sensitivity that can obtain higher.
In the above-described embodiments, basalis, substrate are further preferably provided with one side of the acoustic wave piezoelectric sensor away from layer gold
Layer is preferably silicon layer, and the effect of basalis is to increase the antijamming capability of sensor, improves its measuring accuracy.
In the above-described embodiments, the temperature of fluid is 37 ± 0.5 DEG C.
In the above-described embodiments, the thickness of layer gold is preferably 10~20nm, if the thickness of layer gold is less than 10nm, is easily reduced
The reactivity that FITC antibody is combined with layer gold；If the thickness of layer gold is more than 20nm, the sensitivity for causing sensor is declined,
Error rate rises.
In the above-described embodiments, the bore of runner is preferably 10~100 μm.The size of bore will influence fluid in runner
The smoothness of flow velocity and fluid, so as to change testing molecule and the speed and efficiency of antibody binding reaction, therefore, bore value
It should be restricted.
In the above-described embodiments, the diameter of magnetic bead is preferably 20nm~1.5 μm.The size of magnetic bead can also influence magnetic bead to anti-
The acceleration degree answered, if bead diameter is too small, will cause acceleration effect unobvious；If diameter is excessive, and is easily sent out by gravity
Raw sedimentation is reunited, so as to lose the acceleration effect as caused by its larger ratio surface.Therefore, the diameter of magnetic bead should be limited
System.
Detection cell is a part for whole detecting system, it needs to combine sampling device, temperature control device, piezoelectric signal inspection
Slowdown monitoring circuit and optical signalling detection circuit complete whole detection process jointly, but, due to sampling device, temperature control device, piezoelectricity
Signal deteching circuit and optical signalling detection circuit belong to the prior art, and details are not described herein for this case.For example, in piezoelectric sensing
The both ends of device are equipped with lead end, and lead end is connected with piezoelectric signal detection circuit, which is used to gather, analyze, record
The real time data collected with output piezoelectric transducer, but this case omits these details herein, is exactly because these are thin
Section belongs to the prior art, also has many commercially available piezoelectric transducer detecting instruments to integrate these functions well, and make
With conveniently.This case below will describe the detection process of molecular dynamics with reference to Fig. 1 and Fig. 2：Prepare liquid containing testing molecule 5
Entered by sampling device in the runner 3 of detection cell, according to the characteristics of testing molecule 5, first antibody 10 and secondary antibody 8 are right
Testing molecule 5 carries out specific adsorption, so that testing molecule 5 be wrapped, forms interlayer structure, 2 surface of layer gold
FITC antibody 9 carries out specific adsorption to FITC molecules 6 at the same time, introduces magnetic by permanent magnet or electromagnet 11 in this process
, under magnetic fields, entrainment of accelerated 2 table of layer gold being fixed on acoustic wave piezoelectric sensor 1 of magnetic bead 7 of testing molecule 5
Face, after cancelling magnetic field, the other impurities in uncombined magnetic bead 7, first antibody 10, secondary antibody 8 and prepare liquid are with fluid quilt
Runner 3 is washed out, testing molecule 5 is screened by the crawl of first antibody 10 and secondary antibody 8 is fixed on 2 surface of layer gold, and this
One dynamic process all have recorded by acoustic wave piezoelectric sensor 1, so as to obtain the quality of testing molecule 5, viscoplasticity, concentration
Etc. information, and association reaction performance graph calculates the kinetic parameters such as reaction rate, binding constant or dissociation constant, is solved obtaining
During from constant, the material that FITC molecules 6 can be made to be dissociated with FITC antibody 9 need to be only added in a fluid；Due to second anti-
Be connected with FITC molecules 6 on body 8, secondary antibody 8 is in again that the ratio that determines is combined with testing molecule 5, therefore, FITC molecules 6 it is dense
Degree just can directly react the concentration of testing molecule 5, and testing molecule 5 is virtually just having already passed through after the above process
One separation, purification, diluted process, this not carrying out testing molecule by optical detective technology originally " straight
Connect " detection become reality；By the top surface 4 for being capable of printing opacity on detection cell, optical signalling detection circuit can be made to obtain to be measured point
The ultimate density of son 5, the concentration is also accurate more than the data that piezoelectric detection technology is obtained, although optical signalling and piezoelectric signal
Unit it is different, but they can be converted into the concentration of testing molecule 5.Optical signalling converts when endpoint concentration is calculated
Concentration results it is with a high credibility, so select optics conversion as a result, and with this concentration results to correct piezoelectric detection when
Dynamic Signal.Such as：The concentration that piezoelectricity is surveyed is 100ng/mL, and the only 90ng/mL that optics is surveyed, that just illustrates that piezoelectricity has 10%
Signal be as caused by non-specific adsorption, it is necessary to which the performance graph of piezoelectricity is modified.Adopt in such a way, can be with
Piezo technology and optical technology are integrated well so that both sides can learn from other's strong points to offset one's weaknesses, with the absorption in piezo technology
Journey solves the pretreatment process in optical technology, and the false positive in piezo technology is corrected with high-precision result in optical technology
As a result, the combination of two kinds of technologies can bring qualitative leap to the effect of detection.
Specific embodiment is listed below：
Embodiment 1 (selects alpha-fetoprotein Alpha fetoprotein (AFP) to be used as testing molecule)
1) acoustic wave piezoelectric sensor is fixed on runner bottom surface, runner bore is 10~100 μm；Piezoelectric transducer resonance frequency
Rate scope is 5~500MHz.
2) to obtain more preferably biocompatibility, one layer of gold is sputtered or evaporates in piezoelectric sensor surface, thickness for 10~
20nm, then combines FITC antibody on the surface of gold.
3) the epoxy group modified magnetic bead (quantity about 5 × 10 that diameter is about 1.1 μm is selected9/ mL), with PBST buffer solution for cleaning
(1mol/L PBS+0.05%tween20).
4) appropriate magnetic bead, the PBSB buffer solutions (PBS+0.1%BSA) of addition, with saturated ammonium sulfate solution and AFP antibody are taken
One (AFP antibody can produce specific reaction with AFP), piping and druming mixes, and is incubated overnight at room temperature.
5) the 0.4 μ L of DMSO solution (3mg/mL) of FITC are taken, with AFP antibody two, piping and druming mixes.By FITC and AFP antibody
Two mixed solution adds desalination centrifugal column (cutoff 7KDa), and 1000G is centrifuged 1~2 minute, elutes uncombined small
Molecule, collects the two-FITC solution of AFP antibody that has marked, put 4 DEG C be incubated overnight it is spare.
6) AFP molecules are made into 100,50,25,12.5,6.25,3.125,1.56,0.78ng/ with PBS buffer dilution
mL。
7) AFP molecules to be measured and AFP molecular criterias liquid are mixed with one coated magnetic bead of AFP antibody respectively and is incubated and adds
Enter the AFP antibody two of suitable FITC marks, 40min is incubated at 37 DEG C.
8) can be under magnetic fields, when AFP molecular complexes (i.e. interlayer structure) flow through acoustic wave piezoelectric sensor region
(＜ 1min) just reaches surface and FITC antibody bindings in layer gold in short period.
9) after the revocation of magnetic field, uncombined magnetic bead is eluted first, is deposited in sample between AFP concentration and frequency of sound wave movement
In linear relationship.AFP concentration is higher, and the AFP complex molecule numbers of formation are more, and then combined with sonic sensor surface
Molecular number increase, causes the variable quantity of frequency also to increase.
10) while photomultiplier can also be utilized to detect fluorescence intensities of the FITC in 488nm, with obtained by AFP titers
As a result corresponding standard curve is made, and the ultimate density of AFP molecules to be measured is analyzed with this.
11) resonance peak is analyzed and corrected to the ultimate density of the AFP molecules to be measured obtained using step 10) as correction reference
Change is so as to obtain magnetic bead from the kinetic curve of film combination-desorption.
Fig. 3 show dynamic overall process of the piezoelectric transducer to sample test：System stablize added in backward runner it is to be measured
Sample, under magnetic fields, testing molecule is combined after forming interlayer structure with magnetic bead with sensor surface, and quality increase, causes
Sensor resonant frequency declines；After the revocation of magnetic field during buffer solution for cleaning, impurity and uncombined molecule are eluted, sensor surface knot
Mass lost is closed, causes resonant frequency to raise.Corresponding quality can be calculated by the amount of movement of frequency in the dynamic process to become
Change amount, and then calculate the concentration of testing molecule in sample.And the molecular weight by combining and eluting in the unit interval can then calculate
Go out the dynamical phase related parameter such as reaction rate, binding constant or dissociation constant.Sensor surface is cleaned after reaction,
It is ready for sample detection next time.
Fig. 4 shows that the fluorescence intensity of magnetic bead complex (i.e. interlayer structure) is with the increase of AFP molecular concentrations to be measured
Increase, and FITC average fluorescent strengths value and AFP concentration are proportionate functional relation in runner, goodness of fit coefficients R2For
0.99967(R2Maximum is 1, represents optimum linearity regression fit).AFP fluorometric investigation concentration linearly interval for 6.25~
100ng/mL。
Fig. 5 is embodied to be become using normalized frequency of the piezoelectric transducer test AFP concentration in 0.78~100ng/mL sections
Change response, the regression fit linearity preferably (R between frequency shifts amount and AFP concentration2=0.99256).With fluorescence detection result
Compare, (fluoroscopic examination is limited to 6.25ng/mL, such as Fig. 4 to the lower 0.78ng/mL of detectable limit of sonic sensor under equal conditions
It is shown), sensitivity higher, especially also has 0~1ng/mL extremely low concentration AFP molecules significantly response (see square frame enlarging section in Fig. 5
Point).But degree of fitting higher of the fluorescence results in the range of 6.25~100ng/mL, thus its confidence level is higher than piezoelectric transducer
Test result, it is possible thereby to correct the piezoelectric detection result of response with the result of optical detection.
Although embodiment of the present invention is disclosed as above, it is not restricted in specification and embodiment listed
With it can be applied to various suitable the field of the invention completely, can be easily for those skilled in the art
Realize other modification, therefore under the universal limited without departing substantially from claim and equivalency range, it is of the invention and unlimited
In specific details and shown here as the legend with description.
Claims (8)
1. one kind is based on molecular dynamics detection method associated with acousto-optic, it is characterised in that including：
Step 1) sputters one layer of layer gold in acoustic wave piezoelectric sensor surface, and can capture optical markings in the layer gold surface modification
The optical markings Molecular Adsorption body of molecule；The acoustic wave piezoelectric sensor is then placed in the bottom of detection cell, the detection cell is extremely
There is runner, the energy that cavity, an accommodating fluid for being capable of horizontal accommodating acoustic wave piezoelectric sensor frees in and out less
The top surface of enough printing opacities, also, the fluid in the runner can directly be contacted with the optical markings Molecular Adsorption body；
Step 2) can produce the first antibody of specific binding in magnetic bead surfaces modification with testing molecule；
Step 3) by optical markings molecular modification to secondary antibody, wherein, which can produce special with testing molecule
The opposite sex combines, and the site specifically bound is different from first antibody；
The magnetic bead for being modified with first antibody is placed in the fluid of runner by step 4) with the secondary antibody for being modified with optical markings molecule
In, acoustic wave piezoelectric sensor gathered data is opened, the prepare liquid containing testing molecule is injected into runner, controls fluid not at this time
Flowing, first antibody, secondary antibody are combined with testing molecule and testing molecule are clamped in middle formation interlayer structure respectively,
The lower section of the acoustic wave piezoelectric sensor introduces magnetic field so that optical markings molecule is quickly tied with optical markings Molecular Adsorption body
Close, testing molecule is fixed on the layer gold surface, so as to cause the signal intensity of acoustic wave piezoelectric sensor, treats acoustic wave piezoelectric
After the data stabilization of sensor collection, runner is controlled fluid flow out, and with new fluid scouring runner, treat acoustic wave piezoelectric sensor
Signal again stablize after, that is, complete single detection process；
Step 5) sets an optical detector circuitry in the detection cell top face, by the optics for gathering optical markings molecule
Parameter obtains the ultimate density of testing molecule, and goes to correct on the basis of the ultimate density and brought by acoustic wave piezoelectric sensor
Data error, so as to obtain the kinetic parameter of more accurately testing molecule.
2. according to claim 1 be based on molecular dynamics detection method associated with acousto-optic, it is characterised in that the sound wave
The material of piezoelectric transducer is selected from piezoelectric ceramics, quartz, lithium niobate, zinc oxide, aluminium nitride or its combination.
3. according to claim 1 be based on molecular dynamics detection method associated with acousto-optic, it is characterised in that in the sound
One side of the wave pressure electric transducer away from the layer gold is additionally provided with basalis.
4. according to claim 1 be based on molecular dynamics detection method associated with acousto-optic, it is characterised in that the fluid
Temperature be 37 ± 0.5 DEG C.
5. according to claim 1 be based on molecular dynamics detection method associated with acousto-optic, it is characterised in that the layer gold
Thickness be 10~20nm.
6. according to claim 1 be based on molecular dynamics detection method associated with acousto-optic, it is characterised in that the runner
Bore be 10~100 μm.
7. according to claim 1 be based on molecular dynamics detection method associated with acousto-optic, it is characterised in that the magnetic bead
A diameter of 20nm~1.5 μm.
8. according to claim 1 be based on molecular dynamics detection method associated with acousto-optic, it is characterised in that the optics
The one kind of mark molecule in nanogold, nano silver, rhodamine, cyanine dye cy3, cyanine dye cy5 or FITC molecule.
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