CN103868889A - Micro cantilever beam array biochemical sensing device based on micro-mirror scanning and method - Google Patents
Micro cantilever beam array biochemical sensing device based on micro-mirror scanning and method Download PDFInfo
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- CN103868889A CN103868889A CN201210551559.4A CN201210551559A CN103868889A CN 103868889 A CN103868889 A CN 103868889A CN 201210551559 A CN201210551559 A CN 201210551559A CN 103868889 A CN103868889 A CN 103868889A
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Abstract
The invention relates to a micro cantilever beam array biochemical sensing device which comprises a reaction cell, a micro cantilever beam array, a semiconductor laser, a deflection micro-mirror, a signal generator, a photoelectric position sensitive detector (PSD) and a data processing device. The device utilizes deflection micro-mirror driving, achieves scanning detection of the micro cantilever beam array by a single focused laser beam, can achieve high-sensitivity, rapid and parallel detection of biochemical reaction information on the micro cantilever beam array, and can be applied in monitoring and detection of food safety, environmental pollution, biomedical sciences, scientific researches, production and manufacture and other fields. The invention also discloses a biochemical detection method utilizing the micro cantilever beam array biochemical sensing device.
Description
Technical field
The invention belongs to biochemical sensitive technical field, relate to particularly a kind of micro-cantilever array biochemical sensitive apparatus and method, these apparatus and method can be applicable to monitoring and the detection in the fields such as food security, environmental pollution, biomedicine, scientific research and the manufacturing.
Background technology
Micro-cantilever (later all claiming micro-beam) the biochemical sensitive technology detecting based on surface stress is the emerging sensing technology of one in recent years occurring, its principle is: probe (antigen or antibody) molecule is fixed to (modification) on the Gold plated Layer of micro-beam one side by direct or indirect mode, in the time of the target molecule in detected sample liquid and the probe molecule generation specific reaction in micro-beam gold surface, can make micro-beam surface stress change, thereby cause micro-beam deformed, detect the process of this distortion by optics or electrical method, can obtain the real-time information of biochemical reaction.Compared with traditional immuno-sensing method, the method is without using any enzyme mark, fluorescent material and radioactivity as reaction tracer agent, eliminate the impact of labeling process, highly sensitive (than the high several times of enzyme linked immune assay), can also carry out by monitoring micro-beam distortion the course of reaction of real-time, quantitative monitoring antigen-antibody, obtain the information of abundanter immune biochemical reaction.Through development these years, micro-cantilever sensing is used as a kind of emerging technology, carry out comparative study at the aspects such as bioengineering and environmental pollution monitoring technology and traditional method, as the rna transcription factor, enzyme, mercury emissions and volatile compound etc., because the gauge of micro-cantilever is only sub-micrometer scale, to micro-beam surface biochemical reaction (such as, the probe molecule of modifying is combined with target molecule) very sensitivity of the STRESS VARIATION that causes, make its detection limit reach even every milliliter of Ya Nake level of nanogram, be better than conventional enzyme-linked immunoassay method.
On single micro-cantilever detection system basis, affect, realize the fast parallel detection of multiple target molecules for further eliminating the ground unrests such as environment temperature is floated, solution variations in refractive index, micro-cantilever sensing technology is just progressively to many arrays development.Report that the method that realizes micro-beam array sensing Study of An mainly contains: (1) utilizes vertical cavity surface emitting laser device that sequential array light source is provided, micro-beam array is irradiated one by one, and recycling Optoelectronic Position Sensitive Detector (PSD) receives detection to the defection signal of each micro-beam; (2) utilize the area source after expanding to irradiate micro-beam array, record image before and after two-dimentional micro-beam array distortion and carry out the deformation detection of micro-beam with CCD.But because the neighbor distance of vertical cavity surface emitting laser device transmitting light beam can not regulate, it can only irradiate for the certain micro-beam array of spacing, and dirigibility is lower and price is also very expensive; In CCD area source detection method, produce disperse because the bending at micro-beam tip can make image, have a strong impact on the detection quality of spot displacement, cause its detection sensitivity not high, and speed is also slower when multi-channel detection.
How to utilize simple light path to design convenient and practical sensor-based system, realize micro-beam array high sensitivity, quick, parallel deformation detection, develop micro-beam array immune sensing device, and utilize array immunization sensor to carry out antibody affinity costant mensuration and be applied to how residual in food security and the parallel real-time in-situ detection of contents of many kinds of heavy metal ion, be the focus that biochemistry detection field is paid close attention to always.
Summary of the invention
For solving the above-mentioned problems in the prior art, make the present invention.
Principle of the present invention is to utilize deflection micro mirror to order about the laser beam generation periodic deflection that semiconductor laser emits, to scan micro-beam array; By optical lever principle, each micro-deflection of beam deformation signal in micro-beam array is amplified again, receive and detect by Optoelectronic Position Sensitive Detector (PSD) sequential, thus the biochemical reaction process information on the each micro-beam of Real-Time Monitoring.
Therefore, aspect first, the invention provides a kind of micro-cantilever array biochemical sensitive device (below sometimes referred to as " device of the present invention ") based on micro mirror scanning, this device comprises
Reaction tank, described reaction tank is used for holding damping fluid and testing sample;
Micro-beam array, micro-beam that described micro-beam array is arranged by more than two parallel interval forms and is removably fixed in airtight reaction tank, on wherein said each micro-beam, be fixed with detection of biological molecule or contrast molecule (in the present invention also referred to as " reference molecule ", fixing or micro-beam of being modified with reference molecule is also referred to as " with reference to beam ");
Semiconductor laser;
Deflection micro mirror, described deflection micro mirror is configured to change the light path of the laser beam of being launched by described semiconductor laser, and described laser beam is strafed on described each micro-beam;
Signal generator, input voltage size and the period of change of deflection micro mirror described in described signal generator control;
Optoelectronic Position Sensitive Detector (PSD), described Optoelectronic Position Sensitive Detector receives the laser spot being reflected by described micro-beam array, thereby produces and export the displacement signal about each micro-beam; With
Data processing equipment, described data processing equipment is processed the displacement signal of each micro-beam of being exported by described Optoelectronic Position Sensitive Detector, displacement data based on being fixed with the micro-beam that contrasts molecule is fixed with the data of each micro-beam deflection of detection of biological molecule about acquisition
Wherein in the time containing the target molecule of being combined with described detection of biological molecular specificity in testing sample, after reacting in reaction tank with described testing sample under the reaction conditions that is suitable for both, the micro-beam that is fixed with described detection of biological molecule can bend.
In a preferred embodiment, device of the present invention can also comprise the heating plate that is operatively connected with described reaction tank and the temperature controller being electrically connected with described heating plate are set, thereby the temperature of described temperature controller control heating plate is to regulate temperature in described reaction tank to be suitable for described detection of biological molecule and described target molecule reacts in described reaction tank.Described reaction can be any in receptors ligand combination, antigen-antibody reaction or molecular association reaction.
In another preferred embodiment, described device can also comprise analog/digital signal conversion device, described analog/digital signal conversion device converts the described displacement signal about each micro-beam to digital signal, described digital signal exports described data processing equipment to and processes, to obtain described micro-beam deflection data.
In another preferred embodiment, described signal generator can be by controlling voltage swing and the period of change of described deflection micro mirror, thereby make described deflection micro mirror change the sequential of described each micro-beam of laser beam flying with the light path of corresponding stepping amount deflection laser bundle, make it be suitable for the each micro-beam of scanning with different spacing interval thereby locate described laser beam.
Aspect second, the invention provides a kind of method (below sometimes referred to as " method of the present invention ") that uses device of the present invention to detect the target molecule in testing sample, said method comprising the steps of:
(1) by being fixed to respectively on the different micro-beam in micro-beam array from detection of biological molecule and contrast molecule that can described target molecule specific binding, on each micro-beam, fix a kind of molecule;
(2) by micro-beam parallel being fixed in reaction tank at intervals obtaining in step (1), and inject damping fluid in reaction tank, and damping fluid is flowed in reaction tank;
(3) start semiconductor laser Emission Lasers bundle;
(4) by signal generator control deflection micro mirror, make the light path of the laser beam that its deflection launched by described semiconductor laser, make it scan the each micro-beam in described micro-beam array with locating laser bundle;
(5) in reaction tank, add testing sample;
(6) receive by Optoelectronic Position Sensitive Detector the laser spot being reflected by described micro-beam array, thereby produce and export the displacement signal about each micro-beam;
(7) described data processing equipment receives and processes the displacement signal of each micro-beam of being exported by described Optoelectronic Position Sensitive Detector, the displacement data of micro-beam based on being fixed with contrast molecule is fixed with the data of each micro-beam deflection (in the time containing the target molecule of being combined with described detection of biological molecular specificity in testing sample, can bend after the micro-beam that is fixed with described detection of biological molecule reacts in reaction tank with described testing sample under the reaction conditions that is suitable for both) of detection of biological molecule about acquisition;
(8) according to whether comprising described target molecule in testing sample described in default amount of bow threshold decision.
In the method for the invention, thus can be by the temperature of operation temperature controller control heating plate to regulate temperature in described reaction tank to be suitable for described detection of biological molecule and described target molecule reacts in described reaction tank.Described reaction can be any in receptors ligand combination, antigen-antibody reaction or molecular association reaction.
In a preferred embodiment of method of the present invention, described signal generator can be according to input voltage and the period of change of user's setup control deflection micro mirror, thereby thereby the light path of controlling the laser beam that described deflection micro mirror launched by described semiconductor laser according to the default stepping amount deflection corresponding with the input voltage of setting and period of change changes the sequential of described each micro-beam of laser beam flying, makes it be suitable for the each micro-beam of scanning with different spacing interval to locate described laser beam.
In the present invention, described contrast molecule comprises at least one positive control molecule or at least one blank molecule, or the combination of at least one positive control molecule and at least one blank molecule.
In the present invention, described target molecule can be acceptor or part, and described detection of biological molecule is part or the acceptor that can be combined with described acceptor or ligand specificity.Described target molecule can also be antigen molecule, specific antibody or antibody fragment that described detection of biological molecule is described antigen molecule, and described specific antibody comprises monoclonal antibody or polyclonal antibody, preferably monoclonal antibody.
In a preferred embodiment of the invention, described antibody fragment can be Fab, Fab ' fragment or the F (ab ') of antibody
2fragment.
Concrete while implementing apparatus and method of the present invention, in practice, generally when the bending displacement amount of micro-cantilever reaches 10nm and when above, just can judge that testing result is positive.
Beneficial effect of the present invention
The present invention utilizes deflection micro mirror to drive, and has realized the single scanning probe of laser beam to micro-beam array that converge, and can realize the high sensitivity to biochemical reaction information on micro-beam array, quick, parallel detection.
The present invention is with respect to existing micro-beam array biochemical sensitive method and apparatus, and its advantage has:
(1) detection light channel structure is simple, easily realizes;
(2) what turntable driving parts used is a little micro mirror, and laser beam emitter only needs a little semiconductor laser, and cost is lower;
(3) scanning stepping amount can regulate arbitrarily, can conveniently position detection to the micro-beam array of various spacing;
(4) use same laser scans, ensured the consistance of radiation source on each micro-beam of micro-beam array;
(5) integrated micro-beam array biochemical sensor volume is little lightweight in this way for profit, conveniently moving.
Brief description of the drawings
In detailed description below in conjunction with accompanying drawing, above-mentioned feature and advantage of the present invention will be more obvious, wherein:
Fig. 1 is the global design schematic diagram of the micro-beam array biochemical sensitive device based on micro mirror scanning.
Fig. 2 is the schematic diagram that deflection micro mirror orders about laser beam flying micro-beam array.
Fig. 3 is the photo of experiment micro-beam array.
Fig. 4 is 250 μ m, 2 scanning shift curve maps of locating in interval in micro-beam array substrate, wherein: (a) directions X and (b) Y-direction.
Fig. 5 is the schematic diagram of two spot scan signals on micro-beam.
Fig. 6 is the displacement curve figure of two micro-beams under Temperature Excitation.
Fig. 7 utilizes kapillary to modify CLEN antibody to the photo on micro-beam array.
Fig. 8 shows the specific binding that utilizes micro-beam array to detect CLEN antigen-antibody.
Fig. 9 is the schematic diagram of biochemical reaction pool structure.
Embodiment
Definition
Detection of biological molecule: in the present invention, detection of biological molecule refer to can with the biomolecule of the studied target molecule specific binding that may exist in testing sample.This detection of biological molecule includes but not limited to antigen, antibody, acceptor or part etc., and its type can be the biomacromolecules such as protein, nucleic acid or carbohydrate.
Target molecule: in the present invention, target molecule is studied target molecule, it is the biomolecule that can be combined with detection of biological molecular specificity, and it includes but not limited to antigen, antibody, acceptor or part etc., and its type can be the biomacromolecules such as protein, nucleic acid or carbohydrate.
Antibody: antibody (antibody) refers to that the immune system of body is under antigenic stimulus, that the thick liquid cell being become by bone-marrow-derived lymphocyte or memory cell Proliferation, Differentiation produces, can with the immunoglobulin (Ig) of corresponding antigens generation specific binding.
Incomplete antibody fragment: by disulfide bond reducing agent, whole antibody is split into the symmetrical fragment that respectively carries sulfydryl, each incomplete antibody fragment comprises a complete light chain and complete heavy chain and Fc fragment.
F (ab ')
2: by pepsin hydrolysis, in hinge area, the heavy nearly C of interchain disulfide bond place cuts off Ig (immunoglobulin, immunoglobulin (Ig)), forms a bivalent Fab abbreviation F (ab ')
2fragment and some small fragment pFc '.Due to F (ab ')
2fragment has retained the biologic activity in conjunction with corresponding antigens, the spinoff of having avoided again the antigenicity of Fc fragment to cause, thereby be widely used as biological products.As diphtheria antimycin and lockjaw antimycin, after pepsin hydrolysis, reduce the generation of hypersensitivity because having removed the antigenicity of Fc fragment.PFc ' fragment is finally degraded, abiology activity.
Fab (fragment of antigen binding): papain makes Ig, and the heavy nearly N end of interchain disulfide bond place cuts off in hinge area, form two identical monovalent antigen binding fragments and be called for short Fab section (as shown in fig. 1), a crystallizable fragment is called for short Fc (fragment crystallizable) section.
Fab ': be monovalent antigen binding fragment with sulfydryl (F (ab ') 2 fragments that respectively carry sulfydryl that split by disulfide bond reducing agent).
Antigen binding site: the position that antibody molecule combines with antigen, CDR1, CDR2 and CDR3 light by Ig, heavy chain form.
Antigen: be a class energy induction of immunity system generation immune response, and the material of specific binding can occur with the product of immune response (antibody or effector cell).Antigen has immunogenicity and two kinds of character of reactionogenicity.Be divided into two classes according to antigenic property: comlete antigen and incomplete antigen.Comlete antigen (complete antigen) is the existing immunogenicity of a class, has again immunoreactive material.If most protein, bacterium, virus, bacterial exotoxin etc. are all comlete antigens.Incomplete antigen, haptens (hapten) is only to have immunoreactivity, and the material of non-immunogenicity, therefore claim again incomplete antigen.
Sulfhydrylization reagent: the difunctional cross-linking reagent that can connect antibody and gold with sulfydryl.
Disulfide bond reducing agent: disulfide bond claims again S-S key is the key between the sulphur atom of the oxidized and formation-S-S-form of 2 SH bases.Under the sulphur compound of mercaptoethylmaine (2-MEA), 2 mercapto ethanol, dithiothreitol (DTT) etc. exists, can have an effect with it, be reduced into sulfydryl (SH).These sulfide are exactly said disulfide bond reducing agent in the present invention.Disulfide bond in the case of micro-disulfide bond reducing agent exists between the heavy chain of antibody is reduced and other disulfide bond is not destroyed.
Micro-cantilever (micro-beam): typical micro-cantilever is made up of silicon nitride, as commercial triangle micro-cantilever (Veeco Instruments) (size: long 200um, the wide 20um of leg, thick 0.6um), the one-sided gold that is coated with 60nm; Antibody conventionally the sulfydryl (SH) by sulfhydrylization reagent and golden covalent bond and other one end of sulfhydrylization reagent (contain-COOH or-NH
2isoreactivity group) be combined with antibody and be secured to micro-beam surface.
The micro-cantilever sensor-based system detecting based on surface stress: micro-cantilever sensor-based system is mainly made up of laser instrument, micro-cantilever, photoelectric position sensor (PSD), temperature control system, peristaltic pump and data analysis treating apparatus.The step of typical micro-cantilever immunologic detection method is as follows: micro-cantilever is fixed in reaction tank, flows damping fluid by reaction tank with peristaltic pump control, in question response pond, after bubble emptying, the mobile damping fluid of speed with 0.1mL/min passes through reaction tank.The temperature of reaction tank is controlled at 37 ± 0.01 DEG C, and room temperature is controlled at 27 ± 0.01 DEG C.Laser instrument sends beam of laser and is radiated at the tip of micro-cantilever, after micro-cantilever reflection, impinges upon on the target surface of PSD.After the displacement signal of micro-cantilever is stablized, add the sample solution of damping fluid dilution, the most advanced and sophisticated displacement of PSD real time record micro-cantilever.
Sequential receives: the laser beam that laser instrument sends is after the reflection of deflection micro mirror, on order directive micro-cantilever array 1,2......n micro-cantilever, 1,2......n micro-cantilever reflects to PSD target surface irradiating the laser beam of coming successively again, records each laser spots position by PSD.
Stepping amount: the minimum angles that deflection micro mirror turns over while rotation at every turn.
Specific embodiments
In apparatus and method of the present invention, the biochemical reaction of utilization can be any in receptors ligand combination, antigen-antibody reaction or molecular association reaction, preferably conventional antigen-antibody reaction in this area.
In the time adopting antigen-antibody reaction, the antibody adopting is the antibody of being combined with detection of biological molecular specificity, can be polyclonal antibody or monoclonal antibody, it can, by any method preparation as known in the art, include but not limited to immunization, hybridoma method, chemical synthesis, gene engineering research etc.Described antibody can also be the hybrid antibody that genetic engineering is modified, and such as humanized antibody, camel source antibody etc. such as, for the antibody of certain mammal transformation, people-mouse hybrid antibody etc.
In the present invention, mentioned antigen includes but not limited to comlete antigen and haptens, and it can be the antigen of any type of dawn known in the art.
The testing sample of mentioning in the present invention can be biological sample, for example come from especially people's sample of mammal, comprise tissue sample (as histopathologic slide, biopsy, hair, swab etc.), cell sample (as cell smear, blood smear etc.), humoral sample (as blood, urine, cerebrospinal fluid, saliva etc.), excreta (such as vomitus, sweat, ight soil etc.).Described testing sample can also be environmental sample, such as pedotheque, water sample, floating dust etc.; The sample obtaining in other production fields, such as sewage sample, food samples etc.
The micro-beam using in the present invention can be prepared voluntarily according to the known method in this area, also can buy commercial goods, and this is not limited to them in the present invention.
As modifying or the method for fixed test biomolecule on micro-beam, in the present invention to this without any restriction, can adopt any known method in this area.
When using antibody or antibody fragment to divide the period of the day from 11 p.m. to 1 a.m as detection of biological, preferably use antibody fragment, described antibody fragment can be Fab, Fab ' fragment or F (ab ') 2 fragments of antibody.
As the example of the method for modified antibodies or antibody fragment on micro-beam, can first utilize sulfydryl self assembly that sulfhydrylization reagent carries to the gold-plated surface of micro-cantilever, then antibody or antibody fragment (for example, Fab, Fab ' fragment) are combined with sulfhydrylization reagent; Can be first antibody or antibody fragment (for example, Fab, Fab ' fragment) be bound up with sulfhydrylization reagent, then utilize sulfydryl self assembly that sulfhydrylization reagent carries to the gold-plated surface of micro-beam; Can also antibody be cracked into two haptens fragments with disulfide bond reducing agent, then utilize the sulfydryl self assembly of haptens fragment self to gold-plated surface; Also can be first antibody be hydrolyzed into F (ab ') with pepsin
2fragment, then with disulfide bond reducing agent by F (ab ')
2fragment is reduced into two Fab ' fragments, recycles the sulfydryl self assembly of each Fab ' fragment to the gold-plated surface of micro-beam.
The example of the disulfide bond reducing agent using in the present invention comprises mercaptoethylmaine (2-MEA), 2 mercapto ethanol, dithiothreitol (DTT) etc.Thereby the method and the condition that use disulfide bond reducing agent Reduction of Disulfide to split antibody are as known in the art, for example, can be according to the type of target antigen, size and character etc., select concrete disulfide bond reducing agent and concentration, antibody concentration in accordance with the instructions of the known method in this area or commercially available related kit, both consumptions and mass ratio, incubation conditions and incubative time etc.
The sulfhydrylization reagent using is in the present invention unrestricted, as long as it can connect with the Fab fragment of antibody, and can be fixed in the Gold plated Layer of micro-beam.
The sulfhydrylization reagent using in the present invention comprises mercapto functional group and carboxyl or amido functional group, wherein the sulfydryl of this reagent one end is for being fixed on gold surface by self assembly, and the carboxyl of the other end or amino (also can be exposed by activation) are for reacting with amino or the carboxyl terminal of antibody.The present invention can with sulfhydrylization reagent comprise sulfur alcohol compound, for example 11-thiol carboxylic acid, 2-aminoothyl mercaptan (AET) and 3-mercaptopropionic acid (MPA); Hydrochloric acid mercaptan imine; Sulfo group hydrocarbyl succinic imide-6-(3 ' 2-pyridine two sulphur-propionamide)-acetic acid esters (Sulfo-LC-SPDP); With 3, the two sulfosuccinimide propionic esters (DTSSP) of 3 '-bis-sulphur etc.
Explain the present invention below in conjunction with accompanying drawing.
In Fig. 1, illustrate the global design schematic diagram of the micro-beam array biochemical sensitive device based on micro mirror scanning.In Fig. 1, semiconductor laser (diameter 8mm, long 40mm, converge laser spots focal length adjustable) give off laser beam, change and strafe (reaction tank top is glass sheet) on the micro-beam being fixed in airtight biochemical reaction tank (volume 0.5ml) after light path through overshoot micro mirror, in pond environment temperature by temperature controller by heating plate control.Then use again Optoelectronic Position Sensitive Detector (PSD) to receive the laser spot position signalling being reflected by micro-beam array, input computing machine after optionally changing by A/D, just can realize the detection to the bending signal of micro-beam array, thereby obtain the corresponding biochemical reaction information on each beam in micro-beam array.
Fig. 2 is that deflection micro mirror orders about laser beam flying micro-beam array schematic diagram.In Fig. 2, the structure of deflection micro mirror and being configured to: the long 4mm of minute surface, wide 2mm, maximum input voltage 10V, 5 ° of maximum deflection angle.Its principle is as follows: by voltage swing and the period of change of signal generator control inputs deflection micro mirror, make micro mirror by each micro-beam (the micro-beam length 200-1000 μ m setting in stepping amount deflection laser Shu Shixu, location scanning micro-beam array, wide 90-100 μ m, thick 1 μ m, surface is coated with the gold layer that 0.02 μ m is thick, center distance between two micro-beams be 250 μ m), use again Optoelectronic Position Sensitive Detector (PSD) sequential to receive the laser spot position signalling being reflected by micro-beam array, realize the detection to the bending signal of micro-beam array.
Below in conjunction with specific embodiment, the present invention is being described further aspect micro-beam array sensor measuring, but is being not intended to limit the present invention.
Embodiment
The measurement to temperature variation of embodiment 1, micro-beam array method for sensing based on micro mirror scanning
1. by a commercialization micro-beam array (German micromotive company, as shown in Figure 3, wherein micro-beam length 500 μ m, wide 90 μ m, thick 1 μ m, surface is coated with the gold layer that 0.02 μ m is thick, and the center distance between two micro-beams is that 250 μ m) are fixed to system and (comprise laser instrument, deflection micro mirror, biochemical reaction tank, temperature controller, PSD, A/D converter, computing machine, in the biochemical reaction tank in as shown in Figure 1).
2. laser beam that what semiconductor laser sent converge is periodically strafed two fixed point 9 hours of spacing 250 μ m in micro-beam array substrate after the break-in of deflection micro mirror, scanning shift curve map is as shown in Figure 4: in directions X and Y-direction, all keeping parallelism is consistent in two scanning sites, do not occur larger skew, illustrative system scanning optical path is stable.
3. adjusting laser beam scanning site, accurately locates micro-beam 1,2 tips, periodically switches and gathers displacement signal, and schematic diagram as shown in Figure 5; After two beam displacement signals are stable, with high-precisive temperature controllers (0.01 DEG C of precision), the temperature of micro-beam array is progressively risen to 29 DEG C from 21 DEG C, gained corresponding data curve is as shown in Figure 6.As can be seen from Figure 6 heating up after 8 DEG C, the displacement response signal of two micro-beams has differed 21nm left and right, and error 3.6% (phase residual quantity 21nm is divided by total deflection 580nm) changes under excitation and is substantially consistent at same temperature.Because micro-cantilever sensing technology is mainly for intermolecular specific binding to the detection of biochemical reaction, as long as therefore can accurately measure this distinctive reaction information, the micro-beam deflection signal errors receiving on PSD target surface is not affect testing result in 10% left and right.
The detection to clenbuterol hydrochloride of embodiment 2, micro-beam array biochemical sensitive method based on micro mirror scanning
1. experimental provision and reagent
The concrete structure of detection system as shown in fig. 1, mainly comprise laser instrument (parameter: diameter 8mm, long 20mm, sending sharp light wavelength is 650nm, laser spot spot diameter be 200 μ m), deflection micro mirror (parameter: driving voltage 40V～50V, driving frequency 3KHz, 10 ° of scanning angle scopes), biochemical reaction tank (volume 0.5mL, hermetically sealed, by interior diameter 1mm injection port, interior diameter 1mm outlet, glass sheet, micro-beam fixed station composition, Fig. 9), temperature controller (0.01 DEG C of temperature-controlled precision, 0 DEG C～100 DEG C of temperature-control range), PSD (displacement resolution 1 μ m, target surface size 12mm × 12mm), A/D converter (12), computing machine.
Clenbuterol hydrochloride antibody, clenbuterol hydrochloride standard model CLEN, chloromycetin standard model CAP (above 3 kinds of samples are all taken from China Agricultural University's agronomy and Biotechnology Institute); Activator: 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC), N-hydroxy-succinamide (NHS); Mercaptan HS-CH
2-COOH (above 3 kinds of medicines are all purchased the company in SIGMA); PBS (4.0g NaCl+0.1g KH
2pO
4+ 1.48g Na
2hPO
4h
2o+500ml deionized water); TPBS (PBS+0.5%Tween-20); 98% concentrated sulphuric acid; 30% hydrogen peroxide, it is pure that mentioned reagent is analysis.
2. the modification of antibody on micro-beam array
Clean micro-beam array, the H that immersion ratio is 1: 3
2o
2and H
2sO
410min in mixed solution (room temperature), taking-up deionized water rinsing, put into orifice plate, after adding the 0.1mol/L mercaptan of 200 μ L, sealing leaves standstill 20h (room temperature), utilizes the sulfydryl (HS) that mercaptan carries to self-assemble on the gold-plated surface of micro-beam array one side.After having reacted, take out micro-beam array alcohol flushing, then use deionized water rinsing, put into new orifice plate, inject the 0.2mol/L EDC of 100 μ L and the 0.05mol/LNHS of 100 μ L and leave standstill 1.5h, the carboxyl of mercaptan on activation micro-beam array.Subsequently by micro-beam array taking-up deionized water rinsing, then be put into capillary array fit and modify on platform, No. 1 micro-beam is carried out to clenbuterol hydrochloride antibody modification, process as shown in Figure 7.No. 2 micro-beam is with reference to beam, does not modify any antibody, the signal deflection causing for detection of environmental factors such as temperature, solution variations in refractive index above.In the present embodiment, capillary inner diameter is designed to 200～230 μ m, and external diameter is 300～330 μ m, and one end is linked in Wei Liangshang, and the other end inserts and is equipped with in the orifice plate of clenbuterol hydrochloride antibody-solutions, leaves standstill 2h.Then take out micro-beam array TPBS and rinse, then be fixed in biochemical reaction tank, the PBS damping fluid that flows, debugging light path is tested.
3. specific experiment process
The laser beam directive deflection micro mirror that laser instrument sends, deflection micro mirror is by frequency 3KHz, magnitude of voltage 40V～50V (change in voltage cycle 1S, point 1000 steps complete) pumping signal drive deflection, the laser beam periodic scan micro-beam array after the reflection of deflection micro mirror.Regulate biochemical reaction tank position to make the tip of laser beam flying micro-beam 1 and micro-beam 2 in micro-beam array, then regulate PSD position that the laser beam of coming from micro-beam 1 and 2 reflections is beaten in PSD target surface central authorities, be convenient to the reception collection of signal.Debug after light path, Gather and input voltage signal and corresponding PSD target surface light intensity signal one minute, count in scanning process when light intensity signal is the strongest (illustrate laser beam flying on micro-beam 1 and micro-beam 2 and be reflected on PSD target surface caused response) two input voltage values, so while periodically inputting this two magnitudes of voltage, deflection micro mirror just orders about the micro-beam 1 of laser beam flying and micro-beam 2, property performance period location scanning.Micro-beam 1 of exporting on observation computing machine and the displacement signal curve of micro-beam 2, after two curves are all steady, input CAP standard specimen solution from injection port, wait for a period of time, until the displacement signal curve of micro-beam 1 and micro-beam 2 all steadily after, again from injection port input CLEN standard specimen solution, Deng all steady rear experiments of displacement signal curve of micro-beam 1 of a period of time and micro-beam 2, stop data acquisition.
4. micro-beam array testing result
Micro-beam array to the testing result of CLEN antigen and antibody specific reaction as shown in Figure 8, in Fig. 8, first add after the CAP standard specimen of 500ng/mL, two beam response amounts are consistent, and amplitude is less, illustrate: (1) this response signal may be to be caused by environmental perturbation (temperature is floated, solution refractive index and potential of hydrogen variation etc.); (2) clenbuterol hydrochloride antibody and the CAP standard specimen on beam 1, modified do not react.After signal is steady, add again the CLEN standard specimen of 10ng/mL, beam 1 response signal that is now modified with CLEN antibody is obviously greater than beam 2 response signals of unmodified CLEN antibody, illustrates: the specific reaction that CLEN antigen-antibody has occurred on (1) beam 1 has caused the variation of beam upper surface stress; (2) the response signal amplitude of beam 2 is less, may be to be caused by environmental perturbation.Finally, beam 1 response signal is deducted to beam 2 (with reference to beam) response signal, can obtain the only true micro-beam deformation signal (34nm) in conjunction with generation by CLEN antigen and antibody specific.
Claims (10)
1. a micro-beam array sensing device, comprises
Reaction tank, described reaction tank is used for holding damping fluid and testing sample;
Micro-beam array, micro-beam that described micro-beam array is arranged by more than two parallel interval forms and is removably fixed in airtight reaction tank, is fixed with detection of biological molecule or contrast molecule on wherein said each micro-beam;
Semiconductor laser;
Deflection micro mirror, described deflection micro mirror is configured to change the light path of the laser beam of being launched by described semiconductor laser, and described laser beam is strafed on described each micro-beam;
Signal generator, input voltage size and the period of change of deflection micro mirror described in described signal generator control;
Optoelectronic Position Sensitive Detector (PSD), described Optoelectronic Position Sensitive Detector receives the laser spot being reflected by described micro-beam array, thereby produces and export the displacement signal about each micro-beam; With
Data processing equipment, described data processing equipment is processed the displacement signal of each micro-beam of being exported by described Optoelectronic Position Sensitive Detector, displacement data based on being fixed with the micro-beam that contrasts molecule is to obtain the data of the each micro-beam deflection that is fixed with detection of biological molecule
Wherein in the time containing the target molecule of being combined with described detection of biological molecular specificity in testing sample, after reacting in reaction tank with described testing sample under the reaction conditions that is suitable for both, the micro-beam that is fixed with described detection of biological molecule can bend.
2. device claimed in claim 1, described device also comprises the heating plate that is operatively connected with described reaction tank and the temperature controller being electrically connected with described heating plate is set, thereby the temperature of described temperature controller control heating plate is to regulate temperature in described reaction tank to be suitable for described detection of biological molecule and described target molecule reacts in described reaction tank.
3. the device described in claim 1 or 2, is characterized in that described reaction is receptors ligand combination, antigen-antibody reaction or molecular association reaction.
4. the device described in any one in claim 1-3, described device also comprises analog/digital signal conversion device, described analog/digital signal conversion device converts the described displacement signal about each micro-beam to digital signal, described digital signal exports described data processing equipment to and processes, to obtain described micro-beam deflection data.
5. the device described in any one in claim 1-4, is characterized in that described contrast molecule comprises at least one positive control molecule.
6. the device described in any one in claim 1-5, is characterized in that described contrast molecule comprises at least one blank molecule.
7. the device described in any one in claim 1-6, it is characterized in that described signal generator is by controlling voltage swing and the period of change of described deflection micro mirror, thereby make described deflection micro mirror change the sequential of described each micro-beam of laser beam flying with the light path of corresponding stepping amount deflection laser bundle, make it be suitable for the each micro-beam of scanning with different spacing interval thereby locate described laser beam.
8. the device described in any one in claim 1-7, it is characterized in that described target molecule is antigen, specific antibody or antibody fragment that described detection of biological molecule is described antigen, described specific antibody comprises monoclonal antibody or polyclonal antibody, preferably monoclonal antibody.
9. device claimed in claim 8, is characterized in that described antibody fragment is Fab, Fab ' fragment or the F (ab ') of antibody
2fragment.
10. use micro-beam array sensing device to detect a method for the target molecule in testing sample, said method comprising the steps of:
(1) by being fixed to respectively on the different micro-beam in micro-beam array from detection of biological molecule and contrast molecule that can described target molecule specific binding, on each micro-beam, fix a kind of molecule;
(2) by micro-beam parallel being fixed in reaction tank at intervals obtaining in step (1), and inject damping fluid in reaction tank, and damping fluid is flowed in reaction tank;
(3) start semiconductor laser Emission Lasers bundle;
(4) by signal generator control deflection micro mirror, make the light path of the laser beam that its deflection launched by described semiconductor laser, make it scan the each micro-beam in described micro-beam array with locating laser bundle;
(5) in reaction tank, add testing sample;
(6) receive by Optoelectronic Position Sensitive Detector the laser spot being reflected by described micro-beam array, thereby produce and export the displacement signal about each micro-beam;
(7) described data processing equipment receives and processes the displacement signal of each micro-beam of being exported by described Optoelectronic Position Sensitive Detector, displacement data based on being fixed with the micro-beam that contrasts molecule is fixed with the data of each micro-beam deflection of detection of biological molecule about acquisition
(8) according to whether comprising described target molecule in testing sample described in default amount of bow threshold decision.
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