CN103234961A - Micro-cantilever beam array biochemical sensing apparatus and biochemical detection method thereof - Google Patents
Micro-cantilever beam array biochemical sensing apparatus and biochemical detection method thereof Download PDFInfo
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Abstract
The present invention relates to a micro-cantilever beam array biochemical sensing apparatus and a biochemical detection method. The micro-cantilever beam array biochemical sensing apparatus comprises a biochemical reaction cell, a micro-cantilever beam array, a vertical cavity surface emitting laser (VCSEL) array, a signal generator, a photoelectric position sensitive detector (PSD) and a data treatment device. According to the apparatus, a laser beam emitted by the VCSEL array is adopted to periodically scan the micro-cantilever beam array, an optical lever principle is adopted to amplify bending deformation signals of various micro-cantilever beams in the micro-cantilever beam array, and the PSD is adopted to perform time program detection receiving so as to real-timely monitor biochemical reaction process information on various micro-cantilever beams. With the present invention, highly sensitive, rapid and parallel detection on the biochemical reaction information of the micro-cantilever beam array can be achieved, and the apparatus can be used for monitoring and detection in food safety, environmental pollution, bio-medicine, scientific researches, production manufacturing and other fields. The invention further discloses a biochemical detection method of the micro-cantilever beam array biochemical sensing apparatus.
Description
Technical field
The invention belongs to the biochemical sensitive technical field, relate to the biochemistry detection method of a kind of micro-cantilever array biochemical sensitive device and described micro-cantilever array biochemical sensitive device particularly, 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
The micro-cantilever biochemical sensing technology that detects based on surface stress is a kind of emerging sensing technology that occurs in recent years, its principle is: probe (antigen or antibody) molecule is fixed (modification) on the Gold plated Layer of micro-cantilever one side with direct or indirect mode, when the target molecule in the detected sample liquid and the probe molecule generation specific reaction on the micro-cantilever gold surface, the micro-cantilever surface stress is changed, thereby cause micro-cantilever flexural deformation, detect the process of this distortion by optics or electrical method, can obtain the real-time information of biochemical reaction.Compare with traditional immuno-sensing method, this method need not to use any enzyme mark, fluorescent material and radioactivity as the reaction tracer agent, eliminated the influence of labeling process, highly sensitive (than the high several times of enzyme linked immune assay), can also come the course of reaction of real-time, quantitative monitoring antigen-antibody by the distortion of monitoring micro-cantilever, obtain abundanter immune biochemical reaction information.Through development these years, the micro-cantilever sensing is used as a kind of emerging technology, compare research at 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 only is sub-micrometer scale, to micro-cantilever surface biochemical reaction (such as, the probe molecule of modifying is combined with target molecule) STRESS VARIATION that causes sensitivity very, make its detection limit reach every milliliter of nanogram even Ya Nake level, be better than conventional enzyme-linked immunoassay method.
On single micro-cantilever detection system basis, for further eliminating the fast parallel detection of ground unrest influences such as the environment temperature is floated, solution variations in refractive index, the multiple target molecules of realization, the micro-cantilever sensing technology is just progressively to many arrays development.Reported that the method that realizes the micro-cantilever array sensing Study of An mainly contains: (1) utilization fiber array side by side is as array light source, micro-cantilever array is shone one by one, and recycling optoelectronic position sensing detector (PSD) receives detection to the defection signal of each root micro-cantilever; (2) utilize area source irradiation micro-cantilever array after expanding, record the deformation detection that image before and after the two-dimentional micro-cantilever array distortion carries out micro-cantilever with CCD.
Because fiber array is that the rear end has connected a plurality of semiconductor lasers in essence, cause its wiring complicated, auxiliary equipment is more, and it is big that volume becomes, and dirigibility is lower; The flatness of fiber end face is also very big to the angle of divergence and the light spot shape influence of outgoing laser beam simultaneously, and this will greatly influence consistance and effective detection range that fiber array gives off laser beam; In the CCD area source detection method, because the bending at micro-cantilever tip can make image produce disperse, have a strong impact on the detection quality of spot displacement, cause its detection sensitivity not high.
How to utilize simple light path to design convenient and practical sensor-based system, realize the micro-cantilever array high sensitivity, fast, parallel deformation detection, develop micro-cantilever array immune sensing device, and utilize the array immunization sensor to carry out the antibody affinity costant and measure and be applied to that the how residual and parallel real-time in-situ of contents of many kinds of heavy metal ion detects in the food security, be the focus of biochemistry detection field concern always.
Summary of the invention
For solving the above-mentioned problems in the prior art, the invention provides the biochemistry detection method of a kind of micro-cantilever array biochemical sensitive device and described micro-cantilever array biochemical sensitive device.
The principle of micro-cantilever array biochemical sensitive device of the present invention is that the laser beam of utilizing vertical cavity surface emitting laser arrays to launch periodically scans micro-cantilever array; Amplify by the flexural deformation signal of optical lever principle with each micro-cantilever in the micro-cantilever array again, receive detection with optoelectronic position sensing detector (PSD) sequential, thereby monitor the biochemical reaction process information on each micro-cantilever in real time.
Therefore, aspect first, the invention provides a kind of micro-cantilever array biochemical sensitive device based on vertical cavity surface emitting laser arrays (following abbreviate as sometimes " device of the present invention ").Described micro-cantilever array biochemical sensitive device comprises:
Biochemical reaction tank, it is used for holding damping fluid and testing sample;
Micro-cantilever array, it comprises the micro-cantilever that two above parallel interval are arranged, and removably be fixed in the airtight biochemical reaction tank, detection of biological molecule or contrast molecule (be also referred to as " reference molecule " in the present invention, the micro-cantilever of fixing or be modified with reference molecule is also referred to as " with reference to beam ") are fixedly arranged on wherein said each micro-cantilever;
Vertical cavity surface emitting laser arrays (VCSELs), it comprises the laser instrument that two above parallel interval are arranged;
Signal generator, it controls the input voltage period of change of described vertical cavity surface emitting laser arrays, to control the periodicity light on and off of each laser instrument;
Optoelectronic position sensing detector (PSD), it receives by described micro-cantilever array laser light reflected luminous point, thereby produces and export the displacement signal about each micro-cantilever;
Data processing equipment, it handles the displacement signal by each micro-cantilever of described optoelectronic position sensing detector output, based on the data of the displacement data that the micro-cantilever that contrasts molecule fixedly is arranged about each micro-cantilever bending of obtaining fixedly to have the detection of biological molecule;
Wherein, when containing the target molecule of being combined with described detection of biological molecular specificity in the testing sample, fixedly there are the micro-cantilever of described detection of biological molecule and described testing sample in biochemical reaction tank, can bend after the reaction under both reaction conditionss being suitable for.
In a preferred embodiment, micro-cantilever array biochemical sensitive device of the present invention can also comprise the heating plate that is operatively connected with described biochemical reaction tank and the temperature controller that is electrically connected with described heating plate are set, thereby the temperature that described temperature controller is controlled heating plate is to regulate temperature in the described biochemical reaction tank and be suitable for described detection of biological molecule and described target molecule reacts in described biochemical reaction tank.
In another preferred embodiment, described reaction is receptors ligand combination, antigen-antibody reaction or molecular association reaction.
In another preferred embodiment, described micro-cantilever array biochemical sensitive device can also comprise the analog/digital signal conversion device, described analog/digital signal conversion device converts described displacement signal about each micro-cantilever to digital signal, described digital signal exports described data processing equipment to and handles, to obtain the crooked data of described micro-cantilever.
In another preferred embodiment, described contrast molecule comprises at least a positive control molecule.
In another preferred embodiment, described contrast molecule comprises at least a blank molecule.
In another preferred embodiment, described signal generator can be by the described Vcsel array input voltage period of change of control, makes each laser instrument in the described vertical cavity surface emitting laser arrays light every micro-cantilever with the periodic scan correspondence one by one.
In another preferred embodiment, described target molecule is antigen, specific antibody or antibody fragment that described detection of biological molecule is described antigen, and described specific antibody comprises monoclonal antibody or polyclonal antibody.
In another preferred embodiment, described target molecule is antigen, and described detection of biological molecule is the antibody fragment of described antigen, and described antibody fragment is Fab, Fab ' fragment or the F (ab ') of antibody
2Fragment.
Aspect second, the invention provides the biochemistry detection method of above-mentioned any one micro-cantilever array biochemical sensitive device, described biochemistry detection method uses micro-cantilever array biochemical sensitive device of the present invention to detect the method (following abbreviate as sometimes " method of the present invention ") of the target molecule in the testing sample, and described biochemistry detection method may further comprise the steps:
(1) will be fixed to respectively on the different micro-cantilevers in the micro-cantilever array with detection of biological molecule that can the combination of described target molecule specificity and contrast molecule, fix a kind of molecule on each micro-cantilever;
(2) micro-cantilever array that obtains in the step (1) is fixed in the biochemical reaction tank, and in biochemical reaction tank, injects damping fluid, and damping fluid is flowed in biochemical reaction tank;
(3) periodically light one by one by each laser instrument in the signal generator control vertical cavity surface emitting laser arrays, with the emission laser beam;
(4) fine setting micro-cantilever array position makes laser beam that described vertical cavity surface emitting laser arrays sends each micro-cantilever in can the described micro-cantilever array of location scanning;
(5) in biochemical reaction tank, add testing sample;
(6) receive by described micro-cantilever array laser light reflected luminous point by the optoelectronic position sensing detector, thereby generation and output are about the displacement signal of each micro-cantilever;
(7) described data processing equipment receives and handles the displacement signal by each micro-cantilever of described optoelectronic position sensing detector output, based on the displacement data of the micro-cantilever that the contrast molecule is fixedly the arranged data about each micro-cantilever bending of obtaining fixedly to have the detection of biological molecule (when containing the target molecule of being combined with described detection of biological molecular specificity in the testing sample, fixedly having the micro-cantilever of described detection of biological molecule and described testing sample in biochemical reaction tank, after being suitable for reacting under both reaction conditionss, can bend);
(8) according to whether comprising described target molecule in the default described testing sample of amount of bow threshold decision.
Concrete when implementing micro-cantilever array biochemical sensitive device of the present invention and biochemistry detection method thereof, in practice, generally reach 10nm and when above, just can judge that testing result is positive when the bending displacement amount of micro-cantilever.
Beneficial effect of the present invention: the present invention utilizes vertical cavity surface emitting laser arrays to realize scanning probe to micro-cantilever array, can realize to biochemical reaction information on the micro-cantilever array high sensitivity, fast, parallel detection.
The present invention is with respect to existing micro-cantilever array biochemical sensitive method and apparatus, and its advantage has:
(1) the detection light channel structure is simple, realizes easily;
(2) laser signal that sends of vertical cavity surface emitting laser arrays is more stable, consistance is better, has increased substantially the biochemistry detection precision;
(3) compact conformation of vertical cavity surface emitting laser arrays, evenly, make its to the location of micro-cantilever array more quick and precisely, scan efficiency is higher;
(4) utilize the integrated micro-cantilever array biochemical sensor volume of this method little, in light weight, conveniently moving.
Description of drawings
From the 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 based on the global design synoptic diagram of the micro-cantilever array biochemical sensitive device of vertical cavity surface emitting laser arrays.
Fig. 2 is the schematic diagram that vertical cavity surface emitting laser arrays is launched the laser beam flying micro-cantilever array one by one.
Fig. 3 is the photo that micro-cantilever array is used in experiment.
Fig. 4 a and Fig. 4 b are the scanning shift curve maps that 2 of the 250 μ m in interval locate in the micro-cantilever array substrate, and wherein: Fig. 4 a is directions X, and Fig. 4 b is Y-direction.
Fig. 5 is the synoptic diagram of two spot scan signals on the micro-cantilever.
Fig. 6 is the displacement curve figure of two micro-cantilevers under the temperature excitation.
Fig. 7 is the synoptic diagram of biochemical reaction pool structure.
Fig. 8 utilizes kapillary to modify the photo of CLEN antibody to the micro-cantilever array.
Fig. 9 shows the specificity combination that utilizes micro-cantilever array to detect the CLEN antigen-antibody.
Embodiment
Definition
The detection of biological molecule: in the present invention, the detection of biological molecule refers to the biomolecule that can be combined with the target molecule specificity that is studied 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 biomacromolecules such as protein, nucleic acid or carbohydrate.
Target molecule: in the present invention, the target molecule that target molecule namely is studied, it is the biomolecule that can be combined with the detection of biological molecular specificity, and it includes but not limited to antigen, antibody, acceptor or part etc., and its type can be biomacromolecules such as protein, nucleic acid or carbohydrate.
Antibody: antibody (antibody) refers to the immune system of body under antigenic stimulus, breeds the thick liquid cell immunoglobulin (Ig) that produce, that can be combined with corresponding antigens generation specificity that is divided into by bone-marrow-derived lymphocyte or memory cell.
The incomplete antibody fragment: by the disulfide bond reduction agent whole antibody is split into the fragment of the symmetry that respectively carries sulfydryl, each incomplete antibody fragment comprises a complete light chain and complete heavy chain and Fc fragment.
F (ab ')
2: Ig(immunoglobulin, immunoglobulin (Ig)) cut off by the pepsin hydrolysis nearly C of disulfide bond place between the hinge area heavy chain, forms two valency Fabs abbreviation F (ab ')
2Fragment and some small fragment pFc'.Because F (ab ')
2Fragment has kept the biologic activity in conjunction with corresponding antigens, the spinoff of having avoided the antigenicity of Fc fragment to cause again, thereby be widely used as biological products.As diphtheria antimycin and lockjaw antimycin, behind pepsin hydrolysis, reduced the generation of hypersensitivity because of the antigenicity of having removed the Fc fragment.The pFc' fragment finally is degraded, the abiology activity.
Fab (fragment of antigen binding): papain cuts off the Ig nearly N end of disulfide bond place between the hinge area heavy chain, 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 ': the monovalent antigen binding fragment (F (ab ') that is the band sulfydryl
2The fragment that respectively carries sulfydryl that is split by the disulfide bond reduction agent).
Antigen binding site: the position that antibody molecule combines with antigen, CDR1, CDR2 and CDR3 light by Ig, heavy chain form.
Antigen: be class energy induction of immunity system generation immune response, and can the material that specificity is combined take place with the product (antibody or effector cell) of immune response.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, and immunoreactive material is arranged again.All be comlete antigen as most protein, bacterium, virus, bacterial exotoxin etc.Incomplete antigen, namely haptens (hapten) is only to have immunoreactivity, and the material of non-immunogenicity, so claim incomplete antigen again.
Sulfhydrylization reagent: the difunctional cross-linking reagent that can connect antibody and gold with sulfydryl.
The disulfide bond reduction agent: disulfide bond claims the S-S key again, is the key between the sulphur atom of the oxidized and formation-S-S-form of 2 SH base.In the presence of the sulphur compound of mercaptoethylmaine (2-MEA), 2 mercapto ethanol, dithiothreitol (DTT) etc., can have an effect with it, be reduced into sulfydryl (SH).These sulfide are exactly said disulfide bond reduction agent among the present invention.Disulfide bond between the heavy chain of antibody under the situation that the disulfide bond reduction agent of trace exists is reduced and other disulfide bond is not destroyed.
Micro-cantilever: typical micro-cantilever is made by 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 usually the sulfydryl by sulfhydrylization reagent (SH) with the covalent bond of gold and an other end of sulfhydrylization reagent (contain-COOH or-NH
2The isoreactivity group) is combined with antibody and is secured to the micro-cantilever surface.
Micro-cantilever sensor-based system based on the surface stress detection: the micro-cantilever sensor-based system mainly is 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 the biochemical reaction tank, flow damping fluid by biochemical reaction tank with peristaltic pump control, treat to pass through biochemical reaction tank with the mobile damping fluid of the speed of 0.1mL/min after the bubble emptying in the biochemical reaction tank.The temperature control of biochemical reaction tank is at 37 ± 0.01oC, and room temperature is controlled at 27 ± 0.01oC.Laser instrument sends the tip that beam of laser is radiated at micro-cantilever, impinges upon on the target surface of PSD after the micro-cantilever reflection.After the displacement signal of micro-cantilever is stable, add the sample solution of damping fluid dilution, the most advanced and sophisticated displacement of PSD real time record micro-cantilever.
Sequential receives: 1 on the vertical cavity surface emitting laser arrays, 2 ... on the n laser instrument order directive micro-cantilever array 1,2 ... the n micro-cantilever, 1,2 ... the n micro-cantilever will shine the laser beam of coming again successively and reflect to the PSD target surface, record each laser spots position by PSD.
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 the molecular association reaction, antigen-antibody reaction commonly used in preferred this area.
When adopting antigen-antibody reaction, the antibody that adopts is the antibody of being combined with the detection of biological molecular specificity, can be polyclonal antibody or monoclonal antibody, it can include but not limited to immunization, hybridoma method, chemical synthesis, gene engineering research etc. by any method preparation as known in the art.Described antibody can also be the hybrid antibody that genetic engineering is modified, and such as humanized antibody, camel source antibody etc. are at the antibody of certain mammal transformation, for example people-mouse hybrid antibody etc.
Mentioned antigen includes but not limited to comlete antigen and haptens among the present invention, and it can be the antigen of any kind of dawn known in the art.
The testing sample of mentioning among 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 (for example vomitus, sweat, ight soil etc.).Described testing sample can also be environmental sample, such as pedotheque, water sample, floating dust etc.; The sample that obtains in other production fields is such as sewage sample, food samples etc.
The micro-cantilever of Shi Yonging can prepare voluntarily according to the known method in this area in the present invention, also can buy the commercial goods, and this is not limited to them in the present invention.
As modifying at micro-cantilever or the method for fixed test biomolecule, 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 the F (ab ') of antibody
2Fragment.
Example as the method for modified antibodies on micro-cantilever or antibody fragment, can utilize sulfydryl self assembly that sulfhydrylization reagent carries earlier 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 earlier antibody or antibody fragment (for example, Fab, Fab ' fragment) be bound up with sulfhydrylization reagent, utilize sulfydryl self assembly that sulfhydrylization reagent carries then to the gold-plated surface of micro-cantilever; Can also antibody be cracked into two haptens fragments with the disulfide bond reduction agent, utilize the sulfydryl self assembly of haptens fragment self then to gold-plated surface; Also can be earlier with pepsin antibody be hydrolyzed into F (ab ')
2Fragment uses the disulfide bond reduction agent with F (ab ') then
2Fragment is reduced into two Fab ' fragments, and the sulfydryl self assembly of each Fab ' fragment of recycling is to the gold-plated surface of micro-cantilever.
The example of the disulfide bond reduction agent of using among the present invention comprises mercaptoethylmaine (2-MEA), 2 mercapto ethanol, dithiothreitol (DTT) etc.Thereby method and the condition of using disulfide bond reduction agent reduction disulfide bond to split antibody are as known in the art, for example, can be according to type, size and the character etc. of target antigen, the instructions of abideing by known method in this area or commercially available related kit is selected concrete disulfide bond reduction agent and concentration, antibody concentration, both consumptions and mass ratio, incubation conditions and incubation time etc.
The sulfhydrylization reagent of Shi Yonging is unrestricted in the present invention, as long as it can connect with the Fab fragment of antibody, and can be fixed on the Gold plated Layer of micro-cantilever.
The sulfhydrylization reagent that uses among the present invention comprises mercapto functional group and carboxyl or amido functional group, wherein the sulfydryl of this reagent one end is used 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 used for reacting with amino or the carboxyl terminal of antibody.The present invention can with sulfhydrylization reagent comprise sulfur alcohol compound, 11-thiol carboxylic acid for example, 2-aminoothyl mercaptan (AET) and 3-mercaptopropionic acid (MPA); The 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 '-two sulphur etc.
Explain the present invention below in conjunction with accompanying drawing.
Illustrate the global design synoptic diagram based on the micro-cantilever array biochemical sensitive device of vertical cavity surface emitting laser arrays among Fig. 1.In Fig. 1, the laser beam that vertical cavity surface emitting laser arrays (VCSELs) 1 sends shines (biochemical reaction tank 1 top is glass sheet) on the micro-cantilever array 4 that is fixed in the airtight biochemical reaction tank 3 one by one by reflective mirror 2, environment temperature is controlled by heating plate 6 by temperature controller 5 in the biochemical reaction tank 3.Then use optoelectronic position sensing detector (PSD) 7 to receive by micro-cantilever array 4 laser light reflected light spot position signals again, randomly by A/D 8 conversion back input computing machines 9, just can realize the detection to micro-cantilever array 4 crooked signals, thereby obtain the corresponding biochemical reaction information on each beam in the micro-cantilever array 4.
Fig. 2 is vertical cavity surface emitting laser arrays emission laser beam flying micro-cantilever array schematic diagram.In Fig. 2, the structure of vertical cavity surface emitting laser arrays 1 and being configured to: the laser array number is 2, high 160 μ m, wide 250 μ m, and length overall 0.5mm, the center distance of adjacent laser instrument is 250 μ m, maximum input voltage 5V.Its principle is as follows: by the change in voltage cycle of signal generator control input vertical cavity surface emitting laser arrays 1, make vertical cavity surface emitting laser arrays 1 give off laser beam to scan one by one every micro-cantilever 10(micro-cantilever beam length 500 μ m in the micro-cantilever array 4 by the cycle of setting, wide 90 μ m, thick 1 μ m, the surface is coated with the thick gold layer of 0.02 μ m, center distance between two micro-cantilevers 10 is 250 μ m), use optoelectronic position sensing detector 7(PSD again, 22mm * 22mm) sequential receives by micro-cantilever array 4 laser light reflected light spot position signals, realizes the detection to micro-cantilever array 4 crooked signals.
Below in conjunction with specific embodiment the present invention is being described further aspect the micro-cantilever array sensor measuring, but is being not intended to restriction the present invention.
Embodiment
1. with a commercialization micro-cantilever array (German micromotive company, as shown in Figure 3, micro-cantilever beam length 500 μ m wherein, wide 90 μ m, thick 1 μ m, the surface is coated with the thick gold layer of 0.02 μ m, and the center distance between two micro-cantilevers is 250 μ m) system that is fixed to (comprises VCSELs, biochemical reaction tank, temperature controller, PSD, A/D converter, computing machine, as shown in Figure 1) in the biochemical reaction tank in.
2. the laser beam sent of vertical cavity surface emitting laser arrays is periodically strafed two fixed points 9 hours of spacing 250 μ m in the micro-cantilever array substrate, the scanning shift curve map is shown in Fig. 4 a and Fig. 4 b: on directions X and Y-direction, two scanning sites are the keeping parallelism unanimity all, bigger skew do not occur, illustrative system scanning light path is stable.
3. regulate the laser beam flying site, accurately locate micro-cantilever 1,2 tips, periodic scan is also gathered displacement signal, and synoptic diagram is (being two scanning pictures among Fig. 5) as shown in Figure 5; After two beam displacement signals are stable, with high precision temperature control device (0.01 ℃ of precision) temperature of micro-cantilever array is progressively risen to 29 ℃ from 21 ℃, gained corresponding data curve is as shown in Figure 6.As can be seen from Figure 6 after 8 ℃ of intensifications, the displacement response signal of two micro-cantilevers has differed about 21nm, and error 3.59% (phase residual quantity 21nm is divided by total deflection 585nm) is consistent under same temperature variation excitation substantially.Because the micro-cantilever sensing technology mainly is at intermolecular specificity combination to the detection of biochemical reaction, as long as therefore can accurately measure this distinctive reaction information, the crooked signal errors of the micro-cantilever that receives on the PSD target surface is not influence testing result about 10%.
1. experimental provision and reagent
The concrete structure of detection system as shown in fig. 1, mainly comprise vertical cavity surface emitting laser arrays (parameter: 2 arrays, high 160 μ m, wide 250 μ m, length overall 500 μ m, adjacent laser instrument center distance 250 μ m, maximum input voltage 5V, sending Wavelength of Laser is 650nm), biochemical reaction tank (volume 0.5mL, hermetically sealed, mainly by interior diameter 1mm injection port 11, outlet 12, glass sheet 13, micro-cantilever array fixed station 14 is formed, Fig. 7), temperature controller (0.01 ℃ of temperature-controlled precision, 0 ℃～100 ℃ of temperature-control range), PSD(displacement resolution 1 μ m, target surface size 22mm * 22mm), A/D converter (16), computing machine.
Clenbuterol hydrochloride antibody, clenbuterol hydrochloride standard model CLEN, chloromycetin standard model CAP; Activator: 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC), N-hydroxy-succinamide (NHS); Mercaptan HS-CH
2-COOH (above medicine is all purchased the company in SIGMA); PBS (4.0g NaCl+0.1g KH
2PO
4+ 1.48g Na
2HPO
4H
2O+500 ml deionized waters); TPBS (PBS+0.5% Tween-20); 98% concentrated sulphuric acid; It is pure that 30% hydrogen peroxide, mentioned reagent are analysis.
2. the modification of antibody on the micro-cantilever array
Clean micro-cantilever array, the immersion ratio is the H of 1:3
2O
2And H
2SO
410 min (room temperature) in the mixed solution, take out and use deionized water rinsing, put into orifice plate, add to seal behind the 0.1 mol/L mercaptan of 200 μ L and leave standstill 20 h (room temperature), the sulfydryl that utilizes mercaptan to carry (HS) self-assembles on the gold-plated surface of micro-cantilever array one side.After reaction is finished, take out the micro-cantilever array alcohol flushing, use deionized water rinsing again, put into new orifice plate, inject the 0.2 mol/L EDC of 100 μ L and the 0.05 mol/L NHS of 100 μ L and leave standstill 1.5 h, the carboxyl of mercaptan on the activation micro-cantilever array.Subsequently micro-cantilever array is taken out and use deionized water rinsing, the capillary array fit that is put into kapillary 15 compositions is again modified on the platform, and No. 1 micro-cantilever is carried out the clenbuterol hydrochloride antibody modification, and process as shown in Figure 8.No. 2 micro-cantilever is with reference to beam, does not modify any antibody above, the signal deflection that causes for detection of environmental factors such as temperature, solution variations in refractive index.In the present embodiment, capillary inner diameter is designed to 200 ~ 230 μ m, and external diameter is 300 ~ 330 μ m, and an end is linked on the micro-cantilever, and the other end inserts and is equipped with in the orifice plate of clenbuterol hydrochloride antibody-solutions, leaves standstill 2 h.Take out micro-cantilever array then and wash with TPBS, be fixed on again in the biochemical reaction tank, the PBS damping fluid that flows, the debugging light path experimentizes.
3. concrete experimentation
The laser beam that vertical cavity surface emitting laser arrays sends is (being set to 1s) scanning micro-cantilever array periodically.Regulate the tip that the biochemical reaction tank position makes laser beam flying micro-cantilever 1 and micro-cantilever 2 in micro-cantilever array, regulate the PSD position again the laser beam of coming from micro-cantilever 1 and 2 reflections are beaten in PSD target surface central authorities, be convenient to the reception collection of signal.The micro-cantilever of exporting on the observation computing machine 1 and the displacement signal curve of micro-cantilever 2, treat that two curves are all steadily back from injection port input CAP standard specimen solution, wait for a period of time, the displacement signal curve for the treatment of micro-cantilever 1 and micro-cantilever 2 all steadily after, again from injection port input CLEN standard specimen solution, wait for a period of time, the displacement signal curve for the treatment of micro-cantilever 1 and micro-cantilever 2 all steadily the back experiment finish, stop data acquisition.
4. micro-cantilever array testing result
Micro-cantilever array to the testing result of CLEN antigen and antibody specific reaction as shown in Figure 9, after adding the CAP standard specimen of 500 ng/mL earlier among Fig. 9, two beam response amount unanimities, and amplitude is less, illustrates: (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 of modifying on the beam 1 do not react.After treating that signal steadily, the CLEN standard specimen that adds 10 ng/mL again, beam 1 response signal that be modified with CLEN antibody this moment is obviously greater than beam 2 response signals of unmodified CLEN antibody, and illustrate: the specific reaction that the CLEN antigen-antibody has taken place 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.At last, beam 1 response signal is deducted beam 2(with reference to beam) response signal, can obtain only by the true micro-cantilever deformation signal (33 nm) of CLEN antigen and antibody specific in conjunction with generation.
The above only is preferred embodiment of the present invention, not in order to limiting the present invention, all any modifications of doing within the spirit and principles in the present invention, is equal to and replaces and improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1. micro-cantilever array biochemical sensitive device, it is characterized in that: it comprises:
Biochemical reaction tank, it is used for holding damping fluid and testing sample;
Micro-cantilever array, it comprises the micro-cantilever that two above parallel interval are arranged, and removably is fixed in the airtight biochemical reaction tank, wherein, detection of biological molecule or contrast molecule is arranged fixedly on described each micro-cantilever;
Vertical cavity surface emitting laser arrays (VCSELs), it comprises the laser instrument that two above parallel interval are arranged;
Signal generator, it controls the input voltage period of change of described vertical cavity surface emitting laser arrays, to control the periodicity light on and off of each laser instrument;
Optoelectronic position sensing detector (PSD), it receives by described micro-cantilever array laser light reflected luminous point, thereby produces and export the displacement signal about each micro-cantilever;
Data processing equipment, it handles the displacement signal by each micro-cantilever of described optoelectronic position sensing detector output, based on the data of the displacement data that the micro-cantilever that contrasts molecule fixedly is arranged with each micro-cantilever bending of obtaining fixedly to have the detection of biological molecule;
Wherein, when containing the target molecule of being combined with described detection of biological molecular specificity in the testing sample, fixedly there are the micro-cantilever of described detection of biological molecule and described testing sample in biochemical reaction tank, can bend after the reaction under both reaction conditionss being suitable for.
2. micro-cantilever array biochemical sensitive device as claimed in claim 1, it is characterized in that: described micro-cantilever array biochemical sensitive device also comprises the heating plate that is operatively connected with described biochemical reaction tank and the temperature controller that is electrically connected with described heating plate is set, thereby the temperature that described temperature controller is controlled heating plate is to regulate temperature in the described biochemical reaction tank and be suitable for described detection of biological molecule and described target molecule reacts in described biochemical reaction tank.
3. micro-cantilever array biochemical sensitive device as claimed in claim 1 or 2 is characterized in that: described reaction is receptors ligand combination, antigen-antibody reaction or molecular association reaction.
4. micro-cantilever array biochemical sensitive device as claimed in claim 1 or 2, it is characterized in that: described micro-cantilever array biochemical sensitive device also comprises the analog/digital signal conversion device, described analog/digital signal conversion device converts described displacement signal about each micro-cantilever to digital signal, described digital signal exports described data processing equipment to and handles, to obtain the crooked data of described micro-cantilever.
5. micro-cantilever array biochemical sensitive device as claimed in claim 1 or 2, it is characterized in that: described contrast molecule comprises at least a positive control molecule.
6. micro-cantilever array biochemical sensitive device as claimed in claim 1 or 2, it is characterized in that: described contrast molecule comprises at least a blank molecule.
7. micro-cantilever array biochemical sensitive device as claimed in claim 1 or 2, it is characterized in that: described signal generator is by the input voltage period of change of the described vertical cavity surface emitting laser arrays of control, makes each laser instrument in the described vertical cavity surface emitting laser arrays light every micro-cantilever with the periodic scan correspondence one by one.
8. micro-cantilever array biochemical sensitive device as claimed in claim 1 or 2, 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.
9. micro-cantilever array biochemical sensitive device as claimed in claim 1 or 2, it is characterized in that: described target molecule is antigen, and described detection of biological molecule is the antibody fragment of described antigen, and described antibody fragment is Fab, Fab ' fragment or the F (ab ') of antibody
2Fragment.
10. biochemistry detection method as any described micro-cantilever array biochemical sensitive device in the claim 1 to 9, it uses the target molecule in the described micro-cantilever array sensing device detection testing sample, and it is characterized in that: described biochemistry detection method may further comprise the steps:
To be fixed to respectively on the different micro-cantilevers in the micro-cantilever array with detection of biological molecule that can the combination of described target molecule specificity and contrast molecule, fix a kind of molecule on each micro-cantilever;
Described micro-cantilever array is fixed in the biochemical reaction tank, and in biochemical reaction tank, injects damping fluid, and damping fluid is flowed in biochemical reaction tank;
Periodically light one by one by each laser instrument in the signal generator control vertical cavity surface emitting laser arrays, with the emission laser beam;
Fine setting micro-cantilever array position makes laser beam that described vertical cavity surface emitting laser arrays sends each micro-cantilever in can the described micro-cantilever array of location scanning;
In biochemical reaction tank, add testing sample;
Receive by described micro-cantilever array laser light reflected luminous point by the optoelectronic position sensing detector, thereby generation and output are about the displacement signal of each micro-cantilever;
Described data processing equipment receives and handles the displacement signal by each micro-cantilever of described optoelectronic position sensing detector output, based on the data of the displacement data that the micro-cantilever that contrasts molecule fixedly is arranged about each micro-cantilever bending of obtaining fixedly to have the detection of biological molecule;
According to whether comprising described target molecule in the default described testing sample of amount of bow threshold decision.
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103837472A (en) * | 2014-03-03 | 2014-06-04 | 安徽理工大学 | Micro cantilever beam deflection scanning system and method for micro cantilever beam array sensor based on multi-angle plane transmitting mirrors |
| CN104215607A (en) * | 2014-09-18 | 2014-12-17 | 中国科学院合肥物质科学研究院 | Optical fiber cantilever beam sensor for food pathogenic bacteria and detection method |
| CN105548011A (en) * | 2016-01-15 | 2016-05-04 | 中国科学技术大学 | Micro-cantilever array biochemical sensing device and method based on optical fiber array |
| CN108181459A (en) * | 2017-11-29 | 2018-06-19 | 中国科学技术大学 | The micro-cantilever array detection method of microcysin LR based on aptamer |
| CN108181488A (en) * | 2017-11-14 | 2018-06-19 | 中国科学技术大学 | A kind of micro-cantilever array detection method of the organophosphorus pesticide based on aptamer |
| CN108225203A (en) * | 2017-11-29 | 2018-06-29 | 全球能源互联网研究院有限公司 | A kind of MEMS sensor of bidimensional physical quantity parallel detection |
| CN108474724A (en) * | 2015-09-30 | 2018-08-31 | Koc 大学 | Use the sensor device of the cantilever based on fiber of embedded sleeve |
| CN108956587A (en) * | 2018-07-11 | 2018-12-07 | 安徽理工大学 | A method of ionic adsorption is monitored based on micro-cantilever sensing technology |
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| CN113295321A (en) * | 2021-05-26 | 2021-08-24 | 江苏大学 | Embedded runner type micro-cantilever sensor and detection method |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1700879A (en) * | 2002-11-26 | 2005-11-23 | 金伯利-克拉克环球有限公司 | Healthcare monitoring system |
| CN101135624A (en) * | 2007-04-03 | 2008-03-05 | 中国科学技术大学 | Monitoring molecule conformation transition and biochemical reaction method and device thereof |
| EP2202519A1 (en) * | 2008-12-29 | 2010-06-30 | Danmarks Tekniske Universitet - DTU | A detector system for identifying substances in a sample |
| CN102645529A (en) * | 2012-04-27 | 2012-08-22 | 南京中迅微传感技术有限公司 | Micro-cantilever array biochemical sensing method based on acousto-optic modulation and device |
-
2013
- 2013-04-16 CN CN2013101323176A patent/CN103234961A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1700879A (en) * | 2002-11-26 | 2005-11-23 | 金伯利-克拉克环球有限公司 | Healthcare monitoring system |
| CN101135624A (en) * | 2007-04-03 | 2008-03-05 | 中国科学技术大学 | Monitoring molecule conformation transition and biochemical reaction method and device thereof |
| EP2202519A1 (en) * | 2008-12-29 | 2010-06-30 | Danmarks Tekniske Universitet - DTU | A detector system for identifying substances in a sample |
| WO2010076310A1 (en) * | 2008-12-29 | 2010-07-08 | Danmarks Tekniske Universitet - Dtu | A detector system for identifying substances in a sample |
| CN102645529A (en) * | 2012-04-27 | 2012-08-22 | 南京中迅微传感技术有限公司 | Micro-cantilever array biochemical sensing method based on acousto-optic modulation and device |
Non-Patent Citations (1)
| Title |
|---|
| HANS PETER LANG ET AL: "Cantilever array sensors", 《MATERIALS TODAY》 * |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103837472A (en) * | 2014-03-03 | 2014-06-04 | 安徽理工大学 | Micro cantilever beam deflection scanning system and method for micro cantilever beam array sensor based on multi-angle plane transmitting mirrors |
| CN104215607A (en) * | 2014-09-18 | 2014-12-17 | 中国科学院合肥物质科学研究院 | Optical fiber cantilever beam sensor for food pathogenic bacteria and detection method |
| CN108474724A (en) * | 2015-09-30 | 2018-08-31 | Koc 大学 | Use the sensor device of the cantilever based on fiber of embedded sleeve |
| CN105548011A (en) * | 2016-01-15 | 2016-05-04 | 中国科学技术大学 | Micro-cantilever array biochemical sensing device and method based on optical fiber array |
| CN108181488A (en) * | 2017-11-14 | 2018-06-19 | 中国科学技术大学 | A kind of micro-cantilever array detection method of the organophosphorus pesticide based on aptamer |
| CN108181459A (en) * | 2017-11-29 | 2018-06-19 | 中国科学技术大学 | The micro-cantilever array detection method of microcysin LR based on aptamer |
| CN108225203A (en) * | 2017-11-29 | 2018-06-29 | 全球能源互联网研究院有限公司 | A kind of MEMS sensor of bidimensional physical quantity parallel detection |
| CN108225203B (en) * | 2017-11-29 | 2022-07-22 | 全球能源互联网研究院有限公司 | MEMS sensor for two-dimensional physical quantity parallel detection |
| CN108956587A (en) * | 2018-07-11 | 2018-12-07 | 安徽理工大学 | A method of ionic adsorption is monitored based on micro-cantilever sensing technology |
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