CN1844897A - Method and apparatus for monitoring interaction of probe molecule and target molecule on micro-cantilever - Google Patents
Method and apparatus for monitoring interaction of probe molecule and target molecule on micro-cantilever Download PDFInfo
- Publication number
- CN1844897A CN1844897A CN 200610078270 CN200610078270A CN1844897A CN 1844897 A CN1844897 A CN 1844897A CN 200610078270 CN200610078270 CN 200610078270 CN 200610078270 A CN200610078270 A CN 200610078270A CN 1844897 A CN1844897 A CN 1844897A
- Authority
- CN
- China
- Prior art keywords
- micro
- cantilever
- subsystem
- optical
- light
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Investigating Or Analysing Materials By Optical Means (AREA)
Abstract
The invention relates to a method for method for detecting the interactive condition between the probe molecule and the target molecule of micro suspend beam, and relative device. Wherein, said device is formed by an optical light subsystem, an optical filter subsystem and an optical image subsystem; said optical light subsystem is at the front of optical filter subsystem and as the incidence of micro suspend beam; said optical filter subsystem is at the back of optical light subsystem and as the reflect of micro suspend beam; said optical image subsystem is at the back of optical filter subsystem to image the micro suspend beam; said optical filter subsystem is a straight limit filter system. The invention uses diffraction filter technique to transform the corner distortion signal of micro suspend beam into the optical intensity signal of micro suspend beam image received by optical receiver; and according to the change of optical intensity, it can find the interactive condition between the probe molecule and the target molecule of micro suspend beam.
Description
Technical field
The present invention relates to a kind of method and apparatus of monitoring biochemical reaction process, probe molecule and interactional method of target molecule and device on particularly a kind of monitoring micro-cantilever.
Background technology
Biochip technology is started the beginning of the nineties in last century, has obtained swift and violent development in recent years especially.It is by integrated thousands of the probe molecules relevant with life on the chip of centimeter square, various biochemical reaction process in life science and the medical science are detected, thereby realize bioactivators such as gene, part, antigen are carried out the test and the analysis of efficient quick.
Though present existing biochip is existing multiple, but its course of work is identical substantially: at first be to make biochip, mainly be to adopt the method for surface chemistry to handle the solid-phase matrix surface, as glass sheet or silicon chip, make the bioprobe molecule then, as DNA, RNA by specific order modify on the substrate (at present commercial reached with 400,000 kinds of different dna moleculars orderly be arranged in 1cm
2On).Next is the processing of detected sample, because biological sample to be detected usually is the potpourri of some biomolecule, can not be directly and chip react, so in general need sample is carried out specific biological treatment, obtain DNA wherein, target molecules such as protein.For the signal extraction behind the match reaction, existing following several method: as isotope labeling, fluorescence labeling, surface plasma body resonant vibration (SPR) and X-ray diffraction etc.Particularly,, also must need carry out mark to target molecule because the detection side's ratio juris that is subjected to being adopted in the existing chip technology limits, wherein general with fluorescein labeling method or isotope-labelling method.Be that ready detected sample is contacted with biochip to trigger the reaction of probe molecule and target molecule with that, such as certain probe molecule is a single stranded DNA sequence, target molecule is and its pairing dna single chain-ordering just, and the two will match hybridization reaction so.After reaction is finished biochip is washed, the target molecule that specific biochemical reaction has taken place with probe molecule has been stayed in the position of probe molecule time, according to before to the different mark modes of target molecule by use the laser scanning fluorescent microscope, or the signal of each differential responses point also passes through the related software analysis image and just can obtain relevant biological information on the method acquisition chip of isotope egative film exposure.
As mentioned above, the prior biological chip technology must carry out mark to sample in the course of the work, and this uses comparatively trouble, and mark itself may cause additional influence to sample, makes that the result who detects is difficult to analyze.In addition, the just whole biochemical reaction process that the existing chip technology obtains carry out object information, the not reflection of the information that course of reaction is carried out.
Biochemical sensor based on micro-cantilever is after atomic force microscope (AFM) occurs, a kind of new sensing technology that develops rapidly.Its principle is: one through having modified the micro-cantilever of probe molecule after the chemical conversion treatment, when biochemical reaction takes place on its surface, can biochemical reaction take place such as the probe molecule on the target molecule in the detected sample and the little beam time, the surface stress of little beam (surface energy just) thereby can change causes little beam to produce flexural deformation, just can know that by monitoring this distortion whole biochemical reaction carries out the information of process.
Summary of the invention
The purpose of this invention is to provide probe molecule and interactional method of target molecule and isolated plant on a kind of monitoring micro-cantilever.Caused micro-cantilever deflection of beam corner distortion when this method and apparatus between the probe molecule and target molecule biochemical reaction takes place by detecting on the micro-cantilever surface, multiple specific biochemical reaction process in real time, parallel change visible optical image information into, in real time, the parallel multiple biochemical reaction process of monitoring.
Probe molecule and the interactional device of target molecule are made up of illumination optical subsystem, optical filter subsystem and imaging optical subsystem on the monitoring micro-cantilever provided by the present invention;
Described illumination optical subsystem is positioned at the prime of described optical filter subsystem, and is positioned at the incident side of micro-cantilever;
Described optical filter subsystem is positioned at the back level of described illumination optical subsystem, and is positioned at the reflection side of micro-cantilever;
Described imaging optical subsystem is positioned at the back level of described optical filter subsystem, to the micro-cantilever imaging;
Described optical filter subsystem is the straight border filtering system.
Described straight border filtering system is made up of fourier transform lens and optically filtering unit, or directly is made up of the optically filtering unit.
Described optically filtering unit is the edge of a knife, slit or rectangular opening.
Described optically filtering unit is positioned on the optics spectrum plane of described micro-cantilever.
Probe molecule and the interactional device of target molecule can be monitored at least one micro-cantilever, particularly micro-cantilever array on this monitoring micro-cantilever probe molecule and target molecule interact.
The principle of work of this device is the irradiate light that the illumination optical subsystem sends (micro-cantilever) micro-cantilever array.Micro-cantilever array is in original state, and the reflector of the little beam of its each root is all got an identical initial angle.The diffracted ray that reflects from (micro-cantilever) micro-cantilever array enters optical filter subsystem, the identical position stack on the optics spectrum plane of (micro-cantilever) micro-cantilever array of this diffracted ray pools the optical diffraction spectrum, diffraction spectra behind optically filtering enters imaging optical subsystem, forms visible images.
The illumination optical subsystem that is adopted in the device is made up of light source, light source filtering hole, semi-transparent semi-reflecting lens and light lens, and light source is positioned at the place ahead in light source filtering hole, and light source filtering hole is positioned at the place ahead of the front focal plane or the front focal plane of light lens.Described light source can be common white light, laser etc., constitutes the parallel light scheme when light source filtering hole just in time is positioned at the front focal plane of light lens; When being positioned at the place ahead of front focal plane of light lens, light source filtering hole constitutes the optical illumination scheme that converges.
When the illumination optical subsystem shines (micro-cantilever) micro-cantilever array with directional light, optical filter subsystem is made up of fourier transform lens and optically filtering unit, at this moment, the optics of (micro-cantilever) micro-cantilever array spectrum plane is exactly the back focal plane of fourier transform lens; When the illumination optical subsystem shines (micro-cantilever) micro-cantilever array with converging light, optical filter subsystem is the optically filtering unit, at this moment, the optics spectrum plane of (micro-cantilever) micro-cantilever array is exactly directly to be converged by diffraction spectra to form, and the light lens in the illumination optical subsystem also plays fourier transform lens simultaneously at this moment.
Imaging optical subsystem is then the same with prior art to be made up of imaging len and optical receiver, and described optical receiver can be human eye, charge-coupled image sensor (being CCD) or other light-sensitive detector spares.
Probe molecule and the interactional method of target molecule on the monitoring micro-cantilever provided by the present invention, it is the incident light irradiation micro-cantilever that sends with the illumination optical subsystem in probe molecule on the above-mentioned monitoring micro-cantilever and the interactional device of target molecule, on the optics of micro-cantilever spectrum plane with above-mentioned monitoring micro-cantilever on the filter unit of straight border in probe molecule and the interactional device of target molecule the diffraction spectra that micro-cantilever reflects is carried out Filtering Processing, by the imaging optical subsystem in probe molecule on the above-mentioned monitoring micro-cantilever and the interactional device of target molecule micro-cantilever is imaged on the optical receiver again, make the micro-cantilever deformation signal that causes when taking place to interact change the light intensity signal of micro-cantilever picture corresponding on the optical receiver into, determine the interaction situation of probe molecule and target molecule on the micro-cantilever according to the variation of light intensity signal by the probe molecule on the target molecule in the detected sample and the micro-cantilever.
The illumination intensity of the incident light that described illumination optical subsystem sends should reach the saturation value of optical receiver as far as possible.
The principle of probe molecule and the interactional method of target molecule is that micro-cantilever deforms when probe molecule on the micro-cantilever and target molecule interaction on the monitoring micro-cantilever of the present invention, and its free-ended corner can change thereupon.This corner when being out of shape at micro-cantilever changes, with a branch of rayed micro-cantilever, the deflection of reflection ray promptly detects the light intensity of micro-cantilever imaging by representing through the diffraction spectra energy that receives behind the optically filtering unit, the corner in the time of just can detecting the micro-cantilever distortion.
For probe molecule on the above-mentioned monitoring micro-cantilever and the interactional device of target molecule, its detection sensitivity is determined by following formula:
D
R＝F1， (1)
D wherein
RBe the detection sensitivity of this device, F is the function that characterizes the optically filtering operation, and I is the illumination intensity of this device.
After micro-cantilever unit (micro-cantilever) is subjected to extraneous induction, it will produce corner.The diffraction spectra of micro-cantilever unit will produce a translation on the optics spectrum plane of micro-cantilever unit this moment.Because the energy of diffraction spectra is not equally distributed on the optics spectrum plane of micro-cantilever unit, so the light intensity that this translation will cause seeing through the optically filtering unit changes.As a result, the intensity of visible images also can change thereupon on the optical receiver, and this changes the corner that has just reflected the micro-cantilever unit.Extraneous in other words induction can be by the light intensity reflection of visible images.Formula (1) shows that reasonably the optically filtering operation can be optimized the F in the formula, makes F reach maximum, thereby makes the detection sensitivity of optical measuring device reach maximum.Be positioned at shape and the position that micro-cantilever unit optics is composed the optically filtering unit on the plane by adjusting, just can select to make the diffraction spectra of which level as required by optically filtering unit, the detection sensitivity that so just can regulate optical measuring device.
As the optically filtering unit of selecting the micro-cantilever diffraction spectra, the present invention has adopted the optically filtering unit of straight border (abbreviating straight flange as) form, is specially the edge of a knife, slit or rectangular opening, to optimize the F in the formula (1).The boundary definition of optically filtering unit on the micro-cantilever unit diffraction spectra moving direction " logical light " and " obstructed light " two states.After micro-cantilever produced corner, its diffraction spectra moves to the transparent zone territory from the opaque zone territory of this boundary definition, and was perhaps opposite; Correspondingly, the luminous energy that reflects on the micro-cantilever that receives on the optical receiver will increase or reduce.Diffraction spectra depends on the initial position and the shape on this border in this borderline energy gradient (variation of the luminous flux that the unit amount of movement of diffraction spectra is brought, i.e. detection sensitivity).The present invention learns that through experimental analysis repeatedly the optically filtering unit of straight flange can obtain maximum energy gradient, so the edge of a knife, slit or rectangular opening will be best optically filtering unit.And, because diffraction spectra everywhere intensity gradient on the back focal plane of fourier transform lens is different, the intensity gradient maximum at Zero-order diffractive peak, and then the initial position of optically filtering unit efficiency frontier should be positioned at the Zero-order diffractive peak near, optical measuring device could obtain maximum detection sensitivity like this.
In addition, the minimum-value aperture in the probe molecule and the interactional device of target molecule is the optically filtering unit on the micro-cantilever because the present invention monitors, and therefore, in order to receive distinct image, the logical light yardstick of optically filtering unit should have a lower limit.Prescribe a time limit down less than this when the logical light yardstick of optically filtering unit, the optical imagery that receives can not correctly reflect the distribution of extraneous induction.Formula (2) has provided the principle of design of the logical light yardstick of optically filtering unit:
D＝λf/T≤d， (2)
Wherein D is that the half-angle of Airy spot of optically filtering unit is wide, T is the logical light yardstick of optically filtering unit, λ is the wavelength of lighting source in probe molecule and the interactional device of target molecule on the monitoring micro-cantilever, f is the focal length of imaging optical subsystem, d is the smallest dimension of optical receiver sensing unit, if receive with charge-coupled image sensor (CCD), d is exactly effective size of charge-coupled device (CCD) pixel.
Formula (2) shows, has only when the wide smallest dimension less than the optical receiver sensing unit of the half-angle of the Airy of optically filtering unit spot, and the visible images that obtains could reflect correctly that extraneous induction distributes.The analysis showed that like this, in the optically filtering unit of straight flange, the optically filtering unit of edge of a knife form will be best selection, because its logical light yardstick is a half-plane, the half-angle of the Airy spot of optically filtering unit is wide the narrowest, the image that receives is subjected to the influence of optics aliasing smaller, and image is also the most clear.From angle of practical application, after logical light yardstick arrived certain degree greatly, the optics aliasing was very little, and therefore all right selection slit and rectangular opening are as the optically filtering unit.
When adopting the filter unit of straight flange form, what detection sensitivity can be more concrete is expressed as:
Wherein λ is the illumination wavelengths of probe molecule and the interactional device of target molecule on the monitoring micro-cantilever, and N is the quantification progression of optical receiver, and L is the length of micro-cantilever reflective surface on its yawing moment.Formula (3) shows that the quantification progression N that improves optical receiver also will effectively improve detection sensitivity.
Because the width of micro-cantilever diffraction spectra is that after improving illumination intensity, the peak width of diffraction spectra can not change by the geometric scale decision of the reflector in the micro-cantilever unit, and amplitude will improve thereupon.In other words, micro-cantilever produces identical corner and will cause bigger light intensity to change, and this will improve optical detection sensitivity.Because the detection sensitivity of probe molecule and the interactional device of target molecule is proportional on illumination intensity and the monitoring micro-cantilever, so increase the detection sensitivity that the light intensity of incident light will increase probe molecule and the interactional device of target molecule on the monitoring micro-cantilever effectively.It should be noted that, here the illumination intensity of probe molecule and the interactional device of target molecule is the comprehensive result of light source and optical receiver on the monitoring micro-cantilever of mentioning, it can directly be realized by the brightness of regulating light source, can also realize by the gain of regulating optical receiver, also can both regulate simultaneously.Range of adjustment is to make illumination intensity reach the saturation value of optical receiver as far as possible.
The present invention is with micro-cantilever corner deformation signal, by the diffraction spectra filtering technique, be converted on the optical receiver light intensity signal of corresponding micro-cantilever picture, can determine the interaction situation of probe molecule and target molecule on the micro-cantilever according to the variation of light intensity signal.Little beam (micro-beam array) image that collects on (probe molecule on little beam also not with the target molecule generation biochemical reaction of sample) optical receiver 10 before sample can also being injected is as the background image, subtract each other comparison with reacted image, highlight micro-beam array position and brightness that biochemical reaction has taken place, indicate the biochemical reaction of pairing probe molecule.After finishing, the biochemical reaction of probe molecule and target molecule do not need biochip is washed.Need not during detection the detected sample mark, having eliminated may be by the added influence of mark generation; The generating process of real-time, parallel monitoring whole biochemical reaction can be realized, thereby abundanter biological information can be obtained; Can detect the probe molecule on the micro-cantilever array and the interaction of target molecule, highly sensitive; Be specially adapted to be difficult to the mark tracer, as the detected target molecule of luciferin or tracer isotope; Optical measuring device wherein adopts non-interferometry mode, optics resistance to shock height.
Description of drawings
Fig. 1 is a micro-cantilever array chip and from the optical monitoring device synoptic diagram of top parallel light scheme.
Fig. 2 is optically filtering unit (edge of a knife form) and filtering principle thereof.
Fig. 3 is the influence of illumination intensity to the optical measuring device detection sensitivity.
Fig. 4 is micro-cantilever array chip and the optical monitoring device synoptic diagram that converges the optical illumination scheme.
Fig. 5 is the micro-cantilever array chip of common light source-fourier transform lens scheme and the optical monitoring device synoptic diagram of parallel light scheme.
Fig. 6 is a micro-cantilever array chip and from the optical monitoring device synoptic diagram of parallel beneath optical illumination scheme.
Fig. 7 is the micro-cantilever array synoptic diagram that has only a micro-cantilever in the cell.
Fig. 8 is the micro-cantilever array synoptic diagram that the complex root micro-cantilever is arranged in the cell.
Fig. 9 is a dna double chain hybridization reaction synoptic diagram.
Figure 10 is an antigen---antibody specificity hybridization reaction synoptic diagram.
Figure 11 is micro-cantilever array chip and enlarged drawing thereof.
Figure 12 is that the optical monitoring device of Fig. 1 is monitored goat-anti people IgM and human serum immunoglobulin M (IgM) results of interaction on the micro-cantilever array bio-chip.
Embodiment
Following experimental technique if no special instructions, is conventional method.
Fig. 1 has provided the optical monitoring device synoptic diagram of micro-cantilever array chip and parallel light scheme.Wherein, micro-cantilever array chip 7 is placed in the top light-transmitting reaction vessels 6, the temperature of the constant temperature system control reaction vessel by contacting with container bottom, and its temperature stability is ± 0.01K.Detected sample passes in and out reactors by peristaltic pump through flow system 12.
Probe molecule and the interactional device of target molecule are made up of illumination optical subsystem, optical filter subsystem and imaging optical subsystem on the monitoring micro-cantilever.The illumination optical subsystem is made up of light source 1, light source filtering hole 2, semi-transparent semi-reflecting lens 4 and light lens 5.Optical filter subsystem is made up of optically filtering unit 8 and fourier transform lens 13.Imaging optical subsystem is made up of imaging len 9 and optical receiver 10.
Light source filtering hole 2 (3 for light barrier) is placed on the rear (among the figure right-hand) of light source 1.Light source filtering hole 2 just in time is positioned at the front focus of light lens 5, and light is parallel beam by light lens 5 collimation, and the irradiate light that this parallel beam is reflected by semi-transparent semi-reflecting lens 4 and is reflected by micro-cantilever array 7 on micro-cantilever array 7.The diffracted ray that returns from micro-cantilever array 7 sees through semi-transparent semi-reflecting lens 4, is converged on its back focal plane by fourier transform lens 13, forms the optical diffraction spectrum of micro-cantilever array 7.Optically filtering unit 8 is placed on the back focal plane of fourier transform lens 13, and has set in advance transparent zone territory and opaque zone territory.The part that diffraction spectra only drops on 8 transparent zone territories, optically filtering unit could arrive optical receiver 10 through imaging len 9, and on optical receiver 10, form visible images, the part that drops on 8 opaque zone territories, optically filtering unit is blocked, and can not arrive optical receiver 10.When injecting, sample is placed with in the reaction vessel 6 of micro-cantilever array 7, target molecule in the sample and the probe molecule generation biochemical reaction on the micro-cantilever and when causing micro-cantilever to produce the corner distortion, correspondingly, the diffraction light that reflection from micro-cantilever array 7 is returned also produces a deflection, shows on fourier transform lens 13 back focal planes to be exactly the translation of a diffraction spectra.A part of light that the translation of diffraction spectra makes it drop on 8 transparent zone territories, optically filtering unit has originally moved into the opaque zone territory (perhaps opposite) of optically filtering unit 8, and is partly blocked (perhaps can pass through optically filtering unit 8) by the obstructed light of optically filtering unit 8.Therefore can will reduce (or increasing) by the light of optically filtering unit 8, the luminous energy that arrives optical receiver 10 reduces (or increasing).Being reflected on the optical receiver 10 is exactly weaken (or enhancing) of visible images light intensity.The visible light intensity variations that promptly receives has just reflected the information that biochemical reaction takes place.In the example of Fig. 1, though the optically filtering unit 8 usefulness edges of a knife realize that as top analysis as can be known, optically filtering unit 8 also can be realized by slit or rectangular opening.Just when the optically filtering unit 8 usefulness edges of a knife were realized, the visible images spatial resolution that receives was the highest.In fact, (can utilize the principle of design of formula (2) to calculate the size of slit or rectangular opening) after the transparent zone territory of slit or rectangular opening reaches certain yardstick, spatial resolution has not been subjected to the influence of transparent zone territory size substantially.As for physically how realizing optically filtering unit 8, can make the edge of a knife, slit and rectangular opening with the method for machinery.The canonical parameter of each parts is: the common white light of light source 1 usefulness, laser etc.; The aperture in light source filtering hole 2 is 0.02-1mm; The focal length of light lens 5 is 50-100mm (or littler); The focal length 50-100mm of fourier transform lens 13 (lens numerical aperture NA=1, or littler); The blade that the edge of a knife 8 can adopt light-proof material to make, the blade place should approach as far as possible, reaches micron dimension (as 5um); The focal length 5-100mm of imaging len 9 (lens numerical aperture NA=1, or littler).
Fig. 2 has provided among Fig. 1 the relative position relation of optics filter unit 8 and diffraction spectra on fourier transform lens 13 back focal planes.A represents among Fig. 2 is micro-cantilever when the corner distortion does not take place, the relative position relation of optically filtering unit 8 and diffraction spectra; What B represented among Fig. 2 is after micro-cantilever generation corner is out of shape, the relative position relation of optically filtering unit 8 and diffraction spectra.In Fig. 2, the opaque zone territory of optically filtering unit is by 8 expressions of optically filtering unit, and the filtering boundary of definition is by 701 expressions.1801,1802 and 1803 represent 0 grade, 1 grade and 2 grades of diffraction spectra 18 respectively.The more senior time diffraction light that do not draw among Fig. 2, be because luminous energy on the higher order of diffraction time quite a little less than, optically filtering has not been had too much influence.In micro-cantilever generation corner when distortion,, the initial position of optically filtering unit 8 is positioned at the zero level of diffraction spectra, i.e. the position of diffraction spectra intensity gradient maximum (see among Fig. 2 shown in the A).After the micro-cantilever generation corner distortion, its diffraction spectra mobile sees among Fig. 2 shown in the B.Because the translation of diffraction spectra, its part diffraction light has shifted out the transparent zone territory and has entered the opaque zone territory, and correspondingly, the light intensity that receives on the optical receiver 10 will die down.Because the distortion difference of each micro-cantilever, so corner is also different, the translational movement of its diffraction spectra is also inequality, the diffraction spectra of each micro-cantilever unit changes also just different by optically filtering unit 8 transparent zone territory light quantities, being reflected on the optical receiver 10 is exactly that light intensity changes with corner, and the intensity image that receive on the receiver this moment is exactly because the reflection that little beam that biochemical reaction causes is out of shape takes place on little beam.That is to say, real-time " seen " biochemical reaction that is carried out on the micro-cantilever array.In fact, the optically filtering unit not only can be realized with the edge of a knife in the diagram, also can realize with slit or rectangular opening.
Fig. 3 has provided the influence of illumination intensity to the optical measuring device detection sensitivity.Because the increase of illumination intensity will improve the diffraction peak height of micro-cantilever diffraction spectra, and can not influence the physical dimension of little beam diffraction spectra, in this case, the light quantity by the optically filtering unit will increase with the increase of the light intensity of throwing light on.Promptly produce under the situation of identical deflection angle, increase the illumination light good general and produce bigger intensity gradient variation, so the detection sensitivity of optical measuring device rises at micro-cantilever.Among Fig. 3 1901 is the sensitivity curves when the illumination light intensity is increased to 2 times of original illumination light intensity (corresponding 1902 shown in sensitivity curves).Compare with the sensitivity curve shown in 1902, after illumination light was forced doubly, the detection sensitivity of optical measuring device also doubled.In the utilization of reality, range of adjustment is to make the illumination intensity of incident light should reach the saturation value of optical receiver as far as possible, and after with digital quantization illumination light intensity, such as with the 8-bit optical receiver time, the gray level of its initial illumination intensity should be near 255; During with the optical receiver of 12-bit, the gray level of its initial illumination intensity should be near 4095; By that analogy, the quantification progression of optical receiver is high more, and correspondingly, the sensitivity of optical measuring device is also high more.
Fig. 4 has provided the structure key diagram of the optical monitoring device of micro-cantilever array chip and " converging the optical illumination scheme ".It is identical with the optical monitoring device of Fig. 1 that it implements thinking.But from the light of illumination subsystems outgoing is not directional light, but converges light.Converging ray can be realized by the position that light source filtering hole 2 is placed on light lens 5 front focal plane the place aheads (front focal plane is taken back a bit among the figure).The distance that light lens 5 front focus are departed from light source filtering hole 2 directly has influence on the enlargement factor of optical measuring device.Because be the caustic illumination of using foreign currency, the fourier transform lens 13 of optical filter subsystem can save.The plane that diffracted ray converges is exactly the optics spectrum plane of micro-beam array, optically filtering unit 8 is placed on this plane get final product.Light source filtering hole 2 is near more apart from light lens 5 front focus, and the enlargement ratio of optical measuring device is more little; Light source filtering pitch-row is far away more from light lens 5 front focus, the enlargement ratio of optical measuring device big more (light source filtering hole 2 can be regulated by the optical translation platform with respect to the distance of light lens 5 front focus).Than Fig. 1, this scheme is owing to having saved fourier transform lens 13, and is therefore simpler.
Fig. 5 has provided the structure explanation of the optical monitoring device of micro-cantilever array chip and " common light source-fourier transform lens scheme ".It is identical with the optical monitoring device of Fig. 1 that it implements thinking.Difference is light lens and fourier transform lens shared.Light source filtering hole 2 is placed on the front focal plane of lens 5.The light beam line that sends in light source filtering hole 2 arrives lens 5 (do this moment light lens with) through semi-transparent semi-reflecting lens 4 and is collimated into parallel beam, and this parallel beam is radiated on the micro-cantilever array 7, and is reflected by micro-cantilever array 7.The diffracted ray that returns from micro-cantilever array 7 passes through lens 5 (do this moment fourier transform lens with) once more and is converged on its back focal plane, and the optical diffraction that forms micro-cantilever array 7 is composed.Process afterwards is consistent with Fig. 1.Compared to Figure 1, this method is because shared light lens and fourier transform lens, and it is simpler that structure seems.
Fig. 6 has provided the micro-cantilever array chip identical with Fig. 1 and the optical monitoring device synoptic diagram of parallel light scheme.It is identical with the optical monitoring device of Fig. 1 that it implements thinking.Difference is that micro-cantilever array places the transparent isothermal reaction container in below, reads the distortion situation of light path micro-cantilever array from below illumination micro-cantilever array chip and after reading biochemical reaction.The benefit of doing like this is when the reaction vessel pond is put into or replaced in the top of reaction vessel with biochip, can avoid and read the light path conflict, and it is more convenient that structure seems.Fig. 4 and Fig. 5 also can adopt below illumination shown in Figure 6 to read light path.
What Fig. 7 showed is the micro-cantilever array synoptic diagram that has only a micro-cantilever in the cell.Its micro-cantilever unit is the micro-cantilever of rectangle, has foursquare reflector at its free end and is used for increasing effective reflective area.
Fig. 8 is little beam of making complex root in support frame lattice, modifies with a kind of bioprobe molecule, with the bio-target molecule in the mode test sample reagent of probability on the little beam of complex root in the same framework lattice.
What Fig. 9 showed is to modify the micro-joist unit of ssDNA probe and the pairing single stranded DNA target molecule generation hybridization reaction in the sample, causes the synoptic diagram of little beam distortion.
What Figure 10 showed is to be modified with the micro-joist unit of biological antibody molecule and the reaction of the pairing biological antigens molecule generation specific hybrid in the sample, causes the synoptic diagram of little beam distortion.
1, the preparation of micro-cantilever array chip
With the micro-cantilever array chip of MEMS technology etching making shown in Fig. 7 and 8.Its micro-cantilever is made of variable shaped beam 14, reflector 15 and chi frame 16.The reflector of rectangle is connected in the free-end of micro-cantilever, is used for increasing effective detection reflective area.
With SiNx material micro-cantilever array chip solution is example, production process is as follows: at first direct growth goes out to make the required SiNx film of device architecture on silicon chip substrate, utilize the pattern etching method on the SiNx film, to make the micro-cantilever array structure, comprise variable shaped beam, reflector and chi frame.Then from the upper surface of structure to variable shaped beam the micro-cantilever array and reflector gold evaporation film, golden thickness 20～50 nanometers.The silicon substrate of part forms micro-cantilever array bio-chip beyond the last removal devices structure middle frame.The distortion beam length 200um of micro-cantilever, wide 20um, thick 1um.
This micro-cantilever array bio-chip and enlarged drawing thereof as shown in figure 11, A is the micro-cantilever array chip, B and C are the enlarged drawing of this micro-cantilever array chip.The micro-joist unit that two kinds of different structures are arranged in this micro-cantilever array chip.Enlarged drawing among the B is the co-deflection girder construction micro-cantilever unit of different beam lengths, and the enlarged drawing among the C is the single variable shaped beam structure micro-cantilever unit of different beam lengths.Little beam number is (5 * 2) * (11 * 4)=440 on the array chip.
2, the preparation of human serum immunoglobulin M (IgM) micro-cantilever array sensing chip:
Reagent: freeze-drying goat-anti people IgM diagnostic serum (single 1:140 that tires that spreads), freeze-drying human serum immunoglobulin reference serum (containing IgM:1.43g/L).3 one mercaptopropionic acids (MPA); Hydrochloric acid 1 one ethyls 1 (3 one dimethylaminopropyl) carbodiimides (EDC); N monohydroxy succinimide (NHS).
Preparation method: the gold-plated micro-cantilever array chip of cleaning is put people MPA solution (99g/100mL) soak 1h, take out the water flushing, nitrogen drying.Place mixed solution (1: 1) the activation 1h of 100g/L EDC and 100g/L NHS again, take out with excessive EDC and the NHS of distilled water flush away, nitrogen drying.Get IgM antibody (freeze-drying goat-anti people IgM diagnostic serum) 10uL, be diluted to 500uL, get the antibody 20uL after the dilution in the experiment at every turn, drip on micro-cantilever array chip gilding incubation 45min under 37 ℃ of constant temperature with pH 6.1 PBS buffer solution.Taking-up is washed with PBS, BSA 20uL is dripped on micro-cantilever array chip gilding afterwards, incubation 1h under 37 ℃ of constant temperature again, with sealing bag by non-specific adsorption site on the micro-cantilever array chip gilding of antibody, take out the back and clean, make the micro-cantilever array bio-chip sensor that is used for detecting human serum immunoglobulin M (IgM) with distilled water.
3, the interaction of goat-anti people IgM and human serum immunoglobulin M (IgM) on the optical monitoring device monitoring micro-cantilever array bio-chip of usefulness Fig. 1
In the optical monitoring device of Fig. 1, light source 1 is common white light, and the aperture in light source filtering hole 2 is 0.02mm; The focal length of light lens 5 is 50mm; The focal length 50mm of fourier transform lens 13 (lens numerical aperture NA=1); The blade that the edge of a knife 8 adopts light-proof material to make, the blade place is 5 microns; The focal length 50mm of imaging len 9 (lens numerical aperture NA=1).
Concrete detection method is as follows:
1) this micro-cantilever array bio-chip is placed in (37 ℃) in the top light-transmitting reaction vessels 6, the temperature of the constant temperature system control reaction vessel by contacting with container bottom, its temperature stability is ± 0.01K.
2) flow through container by peristaltic pump constant speed control PBS buffer solution (pH 6.1), in optical monitoring device, by observing the brightness of micro-cantilever array reflector, chip monitoring micro-cantilever array distortion in real time.
3) treat that the stable back of micro-cantilever array adds the IgM solution (20 μ g/ml) that dilutes with PBS by peristaltic pump, monitors the micro-cantilever array distortion in real time.
4) treat that micro-cantilever array reaches and stop to flow after stable, finish experiment.
5) extract the deformation information of micro-beam array, analyze experimental data.
Testing result as shown in figure 12, among Figure 12, before A is antigen-antibody reaction, observed micro-cantilever array image in optical monitoring device; After B is antigen-antibody reaction, observed micro-cantilever array image in optical monitoring device.The oval marks zone is the micro-cantilever array position, and lastrow is the co-deflection girder construction micro-cantilever array of different beam lengths, the single girder construction micro-cantilever array of the different beam lengths of next behavior.
1 of embodiment has provided the results of interaction with goat-anti people IgM and human serum immunoglobulin M (IgM) on the optical monitoring device monitoring micro-cantilever array bio-chip of Fig. 1, Fig. 4,5 with 6 optical monitoring device monitoring micro-cantilever array bio-chip on goat-anti people IgM with the results of interaction of human serum immunoglobulin M (IgM) identical with Figure 12.
What embodiment 1 provided is to detect a kind of antigen-antibody with micro-cantilever array.For detecting multiple different antigen-antibody simultaneously, only need different antibody, utilize the method for pointwise spraying to modify on the different micro-cantilevers on the chip, with top method, detect different antigen.
What embodiment 1 provided is to utilize optical monitoring device of the present invention and method to detect the interaction result of antibody and antigen.Utilize the intermolecular interaction of optical monitoring device of the present invention and method monitoring other biological, identical with embodiment 1 as the interactional method between agglutinin and sugar, Avidin and biotin, acceptor and part and the dna molecular, monitoring effect is also identical with embodiment 1.
Claims (10)
1, probe molecule and the interactional device of target molecule on the monitoring micro-cantilever are made up of illumination optical subsystem, optical filter subsystem and imaging optical subsystem;
Described illumination optical subsystem is positioned at the prime of described optical filter subsystem, and is positioned at the incident side of micro-cantilever;
Described optical filter subsystem is positioned at the back level of described illumination optical subsystem, and is positioned at the reflection side of micro-cantilever;
Described imaging optical subsystem is positioned at the back level of described optical filter subsystem, to the micro-cantilever imaging;
Described optical filter subsystem is the straight border filtering system.
2, device according to claim 1 is characterized in that: described straight border filtering system is made up of fourier transform lens and optically filtering unit.
3, device according to claim 1 is characterized in that: described straight border filtering system is made up of the optically filtering unit.
4, according to claim 1 or 2 or 3 described devices, it is characterized in that: described illumination optical subsystem is made up of light source, light source filtering hole, semi-transparent semi-reflecting lens and light lens; Described light source is positioned at the place ahead in light source filtering hole, and described light source filtering hole is positioned at the place ahead of the front focal plane or the front focal plane of light lens.
5, device according to claim 4 is characterized in that: described light source filtering hole is positioned at the front focal plane of light lens; The light lens while of described illumination optical subsystem is as the fourier transform lens of described straight border filtering system.
6, according to claim 1 or 2 or 3 described devices, it is characterized in that: described optically filtering unit is the edge of a knife, slit or rectangular opening.
7, device according to claim 6 is characterized in that: described optically filtering unit is positioned on the optics spectrum plane of described micro-cantilever.
8, according to claim 1 or 2 or 3 described devices, it is characterized in that: described micro-cantilever at least one; Be preferably array.
9, probe molecule and the interactional method of target molecule on the monitoring micro-cantilever, it is the incident light irradiation micro-cantilever that sends with the illumination optical subsystem in arbitrary described device among the claim 1-8, on the optics of micro-cantilever spectrum plane with claim 1-8 in the filter unit of straight border in arbitrary described device the diffraction spectra that micro-cantilever reflects is carried out Filtering Processing, by the imaging optical subsystem in arbitrary described device among the claim 1-8 micro-cantilever is imaged on the optical receiver again, make the micro-cantilever deformation signal that causes when taking place to interact change the light intensity signal of micro-cantilever picture corresponding on the optical receiver into, determine the interaction situation of probe molecule and target molecule on the micro-cantilever according to the variation of light intensity signal by the probe molecule on the target molecule in the detected sample and the micro-cantilever.
10, method according to claim 9 is characterized in that: described micro-cantilever at least one; Be preferably array.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200610078270 CN1844897A (en) | 2006-05-18 | 2006-05-18 | Method and apparatus for monitoring interaction of probe molecule and target molecule on micro-cantilever |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200610078270 CN1844897A (en) | 2006-05-18 | 2006-05-18 | Method and apparatus for monitoring interaction of probe molecule and target molecule on micro-cantilever |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1844897A true CN1844897A (en) | 2006-10-11 |
Family
ID=37063841
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200610078270 Pending CN1844897A (en) | 2006-05-18 | 2006-05-18 | Method and apparatus for monitoring interaction of probe molecule and target molecule on micro-cantilever |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1844897A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009103197A1 (en) * | 2008-02-21 | 2009-08-27 | 天津市先石光学技术有限公司 | A sensor for measuring the amount of an analyte in human tissue fluid, a fluid channel unit and a detecting method thereof |
| CN103460039A (en) * | 2010-12-10 | 2013-12-18 | 巴塞尔大学 | Method for staging cancer progression by AFM |
| CN106092409A (en) * | 2016-06-13 | 2016-11-09 | 常州大学 | Utilize the method that optics quickly measures DNA molecular intermolecular forces |
| CN106124008A (en) * | 2016-06-13 | 2016-11-16 | 常州大学 | A kind of micro-simply supported beam device weighing DNA molecular quality |
-
2006
- 2006-05-18 CN CN 200610078270 patent/CN1844897A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009103197A1 (en) * | 2008-02-21 | 2009-08-27 | 天津市先石光学技术有限公司 | A sensor for measuring the amount of an analyte in human tissue fluid, a fluid channel unit and a detecting method thereof |
| US8349258B2 (en) | 2008-02-21 | 2013-01-08 | Tianjin Sunshine Optics Technologies Co., Ltd. | Sensor and method for measuring amount of analyte in human interstitial fluid, fluid channel unit |
| CN103460039A (en) * | 2010-12-10 | 2013-12-18 | 巴塞尔大学 | Method for staging cancer progression by AFM |
| CN106092409A (en) * | 2016-06-13 | 2016-11-09 | 常州大学 | Utilize the method that optics quickly measures DNA molecular intermolecular forces |
| CN106124008A (en) * | 2016-06-13 | 2016-11-16 | 常州大学 | A kind of micro-simply supported beam device weighing DNA molecular quality |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US7158224B2 (en) | Optically active substrates | |
| JP5816083B2 (en) | Microarray evaluation system and method | |
| JP6039717B2 (en) | Detection system and method | |
| CN105758834B (en) | A kind of biochip test method of induced with laser and CCD acquisition | |
| JP2006208294A (en) | Device and method for concurrently imaging plasmon resonance and fluorescence | |
| JP6291842B2 (en) | Detection method, microarray analysis method, and fluorescence reader | |
| US7126688B2 (en) | Microarray scanning | |
| CN101441175B (en) | Surface plasma resonance detection device based on laser scanning surface confocal microscopic system | |
| JP2008546997A (en) | Optical reader | |
| JP2000512744A (en) | System and method for detecting label material | |
| US20120267549A1 (en) | Methods and apparatus for fluorescence sensing employing fresnel zone plates | |
| CN1869689A (en) | Micro-cantilever array bio-chip | |
| CN1869659A (en) | Light-sensing system that uses light guides | |
| CN1954213A (en) | Analytical platform and method for generating protein expression profiles of cell populations | |
| JP2010256161A (en) | Plasmon excitation sensor, and assay method using the same | |
| CN1844897A (en) | Method and apparatus for monitoring interaction of probe molecule and target molecule on micro-cantilever | |
| US20210010920A1 (en) | Spectroscopic analysis device, spectroscopic analysis method, program, recording medium, and microscope | |
| US20030232427A1 (en) | Optically active substrates for examination of biological materials | |
| CN2758753Y (en) | Multi-channel surface plasma resonance image sensor | |
| CN101074928A (en) | Resonance imager of light-exciting color surface piasma | |
| WO2014132717A1 (en) | Interaction analysis device | |
| CN101939635B (en) | Molecular diagnostic system based on evanescent illumination and fluorescence | |
| JP2007085744A (en) | Measuring apparatus and method of surface plasmon resonance | |
| JP2010066212A (en) | Measurement apparatus, and method of measuring object to be observed | |
| CN110168353B (en) | Biochip, biochip unit, biochip reader, and biochip manufacturing method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |