CN1731180A - Sensing method of protein chip and detection system therefor - Google Patents
Sensing method of protein chip and detection system therefor Download PDFInfo
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Abstract
The invention relates to a sensing method of a protein array chip and its detecting system in the field of biology technique. The method comprises the following steps: letting the light reflected by the protein array chip to get through a one-dimensional magnifying lens array and inject into a time are phase modulator, changing the voltage to let the ejected s light and p light to get through the polarizing prism to generate intervention, changing the image into the electric signal by CCD after imaging lens and inputting it into the computer; when changing a phase of p light, it collects an image; using the collected five images as a circulation to obtain the time phase changing information aroused by the reaction of each unit of the array chip.
Description
Technical field
The invention belongs to biological technical field, particularly be used for realizing the method and the protein chip detecting system thereof of bio-molecular interactions such as high precision, high flux, real-time sensor matter-protein, protein-DNA, protein-effector molecules, Ag-Ab, ligand-receptor, medicine-target.
Background technology
Life science develops into the structure-function relationship of illustrating gene from gene sequencing, promptly changes proteomics over to from genomics.Gene is a kind of inhereditary material, by A, T, four base compositions of C, G, and strict pairing, structure is more stable.Being based upon the genetic chip of hybridizing on the principle basis has become the important technological platform of genomics.Protein is the expression of gene product, is the citation form of vital movement, has space-time characterisation, and promptly different space structures has different functional characteristics, and different time has different expression activities.Therefore, protein detection and genetic test difference is bigger.At present, protein detection mainly is to use ripe gene chip detecting technique, and proteomics research has been played certain impetus.But this method has two deficiencies: the one, need carry out fluorescence or isotope labeling, and can influence activity of proteins or binding site, thereby influence accuracy of detection; The 2nd, can't quantize to detect protein-protein interaction, and this just is the important content of proteomics research.
Surface plasma resonance sensing is based on a kind of optical detecting method of the variations in refractive index that detects sensing surface, sample need not mark, highly sensitive, and can detect in real time, thereby in detecting, protein-chip has special advantages (see Chinese patent ZL99107780.6, the applying date is on May 28th, 1999).Surface plasma resonance sensing has 2 kinds of detection methods, and a kind of is the light intensity detection method, and another kind is the phase-detection method, and the latter's is highly sensitive in the former.At the beginning of 2005, Sweden BIAcore company adopts the light intensity detection method based on the surface plasma resonance sensing principle, has released BIAcore T100 bio-molecular interaction array detection systems.This system is based on the four-way detection technique of BIAcore 1000 products such as grade, and four sample passages are promptly arranged, and sensing chip correspondence with it is prepared into four lines, fixes 96 probes on every row, once can detect 384 kinds of samples, sees Fig. 1.Among the figure, 1 is four microjet passages, and 2 is sensing chip.The physical dimension that 4 row probes are arranged is consistent with 4 microchannels, and sensing chip is corresponding one by one with the microjet passage during use, when guaranteeing that sample solution flows through the microchannel can with the probe reaction of being fixed.Company of u.s.a. applied biosystem company (Applied Biosystem) has designed 20 * 20 battle arrays based on grating coupled surface plasmons resonance sensing method, once can detect 400 samples, sees Fig. 2.Collimate in parallel light beam 3 sees through the surface that windowpane 4 incides gold-plated film grating 5, at sample solution and grated interface place excitating surface plasma ripple.Resonate when the reflecting light level wave vector equates with the surface plasma wave vector, thereby cause reflective light intensity significantly to weaken, CCD (charge-coupled image sensor) 6 receives light intensity to be changed.The former characteristics are to detect to use the sample solution consumption few, but are difficult to further improve flux; The latter can further improve flux, must be transparent but need to guarantee detect sample, be restricted in actual applications, and remolding sensitivity the former is low.
The inventor once detected based on light intensity, protein microarray surface plasma resonance image-forming detection system and detection method have been invented, this invention energy detection arrays sensing chip (see Chinese patent 03147877.8, the applying date is on Dec 10th, 2003), sensing principle as shown in Figure 3.Rectangle parallel lines light beam 300 sees through on the interface that prism 307 and refractive index oil reservoir 308 be mapped to the substrate of sensing chip 309 and golden film.When incident angle is suitable, produce plasma resonance, significantly die down from the substrate of sensing chip 309 light intensity with the boundary reflection of golden film, behind imaging len 310, be converted to electric signal, through 316 processing of interface circuit 315 input computing machines by CCD 314.Probe array fixing on the sensing chip 309 distributes, and CCD 314 receives also is the variation of the light that causes of corresponding array biological respinse.But the sensitivity of light intensity detection method is than low 1-2 the order of magnitude of phase-detection method.For this reason, the inventor has invented the method and system of space phase interferometric modulator array detection of biological chip again and (has seen Chinese patent, publication number CN1588064A, the applying date is on August 27th, 2004), this method is based on phase-detection, highly sensitive, and detect the product that flux can be higher than BIAcore T100 and Applied Biosystems, Inc., see Fig. 4.Rectangle parallel lines light beam 400 sees through on the interface that prism 407 and refractive index oil reservoir 408 be mapped to the substrate of sensing chip 409 and golden film.When incident angle is suitable, produce plasma resonance, from the phase place generation acute variation of the light of the boundary reflection of the substrate of array sensing chip 409 and golden film.Reflected light produces space interference behind one dimension amplifying lens group 410, wollaston prism 411, polarizing prism 412, be imaged on through imaging lens group 413 on the target surface of CCD 414.Like this, the phase change of the light that causes during biological respinse changes into the phase change of interference fringe, and CCD 414 is converted to electric signal with interference image, and input computing machine 416 is handled behind interface circuit 415.Because space phase interferometric modulator array detection method is based on the phase change of measuring interference fringe, in order to measure phase change delicately, be poised for battle each unit on the biographies sense chip, at least to there be its 3 adjacent interference fringes to be incident upon on 12 CCD pixels that are close to delegation, the row of CCD and the pixel of row are limited, thereby the detection flux of this method also is very limited.
Protein more than 100,000 kinds is arranged in the human body, thousands of kinds of protein are just arranged in the cell, just more in the tissue.Proteomics research is once to obtain the express spectra of all proteins in a cell, tissue and even the body, thereby discloses essence, disease generation and development and the pharmaceutically-active mechanism of vital movement.Meanwhile, in the face of human various difficult diseases, press for from the numerous materials of occurring in nature and filter out specific drug, promptly find new drug and develop.Undoubtedly, the two all needs the method for sensing of high precision, high flux, detection of biological interaction of molecules in real time, and obviously four kinds of above-mentioned methods all can not meet the demands.
Summary of the invention
The objective of the invention is to overcome the deficiency of above-mentioned technology, a kind of new bio-molecular interaction method for sensing and system be provided, can detect the protein-chip of different capabilities array, obtain in real time between the protein and with the interactional information of effector molecules.
The invention provides a kind of method for sensing of protein-chip, it is characterized in that, this method specifically comprises the steps: based on time domain phase modulation (PM) interference surface plasma resonance array bio-sensing principle
1) laser that sends of frequency stabilized laser then allows light beam obtain rectangle parallel lines polarized light by the polarizer and rectangular aperture through laggard row space filtering of attenuator and collimation;
2) allow described rectangle parallel lines light beam inject the bio-sensing unit that is used for detecting protein-chip, enter one dimension amplifying lens group by its light beam that reflects, adjust the enlargement factor of described one dimension amplifying lens group, shape and size when making the hot spot of ejaculation return to the described bio-sensing of incident unit;
3) light that allows described one dimension amplifying lens group penetrate is injected the time domain phase-modulator, s light in the incident light is parallel with y ' axle with the x ' axle of described time domain phase-modulator respectively with p polarisation of light direction, change the voltage be applied on the described time domain phase-modulator then successively, guarantee that the phase change of the p light that penetrates is followed successively by-π ,-pi/2,0, pi/2 and π; The s light and the p light that allow described time domain phase-modulator penetrate produce interference by polarizing prism, through being imaged on behind the imaging len on the CCD target surface, convert interference image to electric signal by CCD, deposit computing machine after treatment in; The phase place of a p light of the every change of described time domain phase-modulator, computing machine is just gathered piece image, connects to gather 5 width of cloth images, finishes a cycling;
4) continuously change the voltage that is applied on the described time domain phase-modulator, continuous acquisition image thereupon according to the described method of step 3); And utilization is stored in the coordinate of the specific program centering institute images acquired in the computing machine, then utilize the Hariharan algorithm that image is resolved, the phase change that produces when in real time obtaining light by described bio-sensing unit promptly obtains the information of bio-molecular interaction in real time.
In step 4), select earlier one of them unit on the CCD that interference image covered of a sensing unit in the described biologic array sensing chip, the react phase change of caused light of the probe that utilizes the Hariharan algorithm to resolve again to be fixed on this sensing unit and other biomolecule.
In step 4), the plurality of units that is close in delegation on the CCD that interference image covered of a sensing unit in the described biologic array sensing chip of selection or a few row is carried out the average processing earlier, the react phase change of caused light of the probe that utilizes the Hariharan algorithm to resolve again to be fixed on this sensing unit and other biomolecule is to obtain more high precision.
The present invention also provides a kind of time domain phase modulation (PM) interference surface plasma resonance array detection protein-chip system of above-mentioned method for sensing, comprise the incident arm that produces the rectangle parallel lines polarized light that detects usefulness, detect the bio-sensing unit of protein-chip, produce the reflection arm of interference image, and obtain the interference image signal and carry out the signal processing unit of subsequent treatment, it is characterized in that: described reflection arm is positioned at the side that the bio-sensing unit reflected light penetrates, and comprises the one dimension amplifying lens group that places successively on the reflection optical axis, the time domain phase-modulator that has driving circuit, polarizing prism and imaging len; Computing machine in the described signal processing unit links to each other with the driving circuit of described time domain phase-modulator, to realize the control to the time domain phase-modulator.
In the present invention, described one dimension amplifying lens group can be made up of the flat-protruding cylindrical mirror peace-recessed cylindrical mirror of common optical axis, the concave surface of described putting down-recessed cylindrical mirror is a plane of light incidence, the convex surface of described putting down-protruding cylindrical mirror is the light outgoing plane, and the enlargement factor of described one dimension amplifying lens group is 2～4 times.
In the present invention, described time domain phase-modulator is an electro-optic crystal, and material is KDP, KD
*P, ADP, LiTaO
3Or LiNbO
3
In the present invention, the described time domain phase-modulator light wavelength of being modulated is 0.4 μ m～2 μ m;
In the present invention, the clear aperture of described time domain phase-modulator is 4 * 4-20 * 20mm
2, length is 10-120mm.
In the present invention, described bio-sensing unit is a surface plasma resonance biosensor, comprises right angle or Dove prism, refractive index oil reservoir, array sensing chip and places sample pool under the described array sensing chip; Described right angle or Dove prism, refractive index oil reservoir are identical with the refractive index of the optical glass substrate of array sensing chip; The material of the optical glass substrate of described right angle or Dove prism and array sensing chip is a flint glass.
In the present invention, described incident arm is that collimated light source comprises frequency-stabilized He-Ne or the semiconductor laser that places bio-sensing unit one side, and is arranged on attenuator, spatial filter, collimating apparatus, the polarizer and rectangular aperture on the described laser optical axis; Described attenuator metal-plated membrane, attenuation ratio is 0-50%, and the aperture of the diaphragm of described spatial filter is 5-50 μ m, and the clear aperture of described rectangular aperture is 4 * 4-20 * 20mm
2
This time domain phase modulation (PM) interference surface plasma resonance array bio-sensing method provided by the invention and the time domain phase modulation (PM) interference surface plasma resonance array detection protein-chip system that utilizes this method, can parallel (simultaneously promptly) detect the catoptrical phase change that causes when occurring on the array sensing chip bio-molecular interaction on each sensing unit, offer biologist and physician parsing.The present invention can realize high precision, high flux, need not mark and detect in real time, once can detect the interaction of biomolecule such as several ten thousand protein-protein, protein-DNA, DNA-DNA, antibody-antigen, ligand-receptor, medicine-target, obtain dynamics, specificity, locus, steric effect and information such as structure and function.
Description of drawings
Fig. 1 is array detection sensing chip and the microjet access diagram of existing a kind of BIAcore T100.
Fig. 2 be existing a kind of Applied Biosystem company detect principle and sensing chip synoptic diagram based on grating coupling SPR.
Fig. 3 is the principle schematic of existing protein microarray surface plasma resonance image-forming detection system and detection method.
Fig. 4 interferes the principle schematic of table to plasma resonance array detection biochip system for the existing space phase modulation (PM).
Fig. 5 is the structural representation of time domain phase modulation (PM) interference surface plasma resonance array detection protein-chip of the present invention system.
Fig. 6 is the comparison synoptic diagram of time domain phase modulation (PM) interference of the present invention and existing space phase modulation (PM) principle of interference.
Fig. 7 is the structural representation of array sensing chip of the present invention.
Fig. 8 is the optical schematic diagram of one dimension amplifying lens group of the present invention.
Fig. 9 is the principle of work synoptic diagram of time domain phase-modulator of the present invention.
Figure 10 is the driving circuit block diagram of time domain phase-modulator of the present invention.
Figure 11 is the block scheme of signal processing unit of the present invention.
Figure 12 is an array sensing chip image centering program flow diagram of the present invention.
Figure 13 carries out the program flow diagram that phase place is resolved for the Hariharan of utilization algorithm of the present invention.
Embodiment
Below in conjunction with accompanying drawing time domain phase modulation (PM) interference surface plasma resonance array bio-sensing method of the present invention and protein chip detecting system thereof are illustrated.
The present invention is made up of bio-sensing unit, incident arm, reflection arm and signal processing unit four major parts based on array detection, as shown in Figure 5.Wherein, the bio-sensing unit is a surface plasma resonance biosensor, comprises right angle or Dove prism 507, places the refractive index oil reservoir 508 and the array sensing chip 509 of right angle or Dove prism 507 bottom surfaces and places sample pool 510 below the array sensing chip 509; Described incident arm is positioned at a side of bio-sensing unit, is made of frequency-stabilized He-Ne or semiconductor laser 501, attenuator 502, aperture 503, collimating apparatus 504, the polarizer 505 and rectangular aperture 506 etc.; Described reflection arm is positioned at the opposite side with the corresponding bio-sensing of incident arm unit, comprise one dimension amplifying lens group 511 successively, have time domain phase-modulator 512, polarizing prism 513 and the imaging len 514 of driving circuit 518, time domain phase-modulator driving circuit 518 selects circuit, amplification control circuit, output control circuit, computer interface etc. partly to form by normal signal generator, signal high-voltage amplifier, high-voltage signal output circuit, signal type; Described signal processing unit adopts prior art, comprise the CCD (charge-coupled image sensor) 515, signal processing circuit board 516 and the computing machine 517 that connect successively, described signal processing circuit board 516 is inserted on the mainboard of computing machine 517, can handle the electric signal of CCD 515 outputs, be converted into digital signal, input computing machine 517.Signal processing circuit board 516 comprises conventional computer interface, drives sequential circuit, memory bank, A/D change-over circuit and correlated double sampling circuit etc.Computing machine 517 in the described signal processing unit links to each other with the driving circuit 518 of described time domain phase-modulator, to realize the control to the time domain phase-modulator.
Time domain phase modulation (PM) interference surface plasma resonance array bio-sensing method of the present invention adopts time domain phase modulation (PM) interference surface plasma resonance array detection protein-chip of the present invention system, and concrete steps are as follows:
The laser that frequency-stabilized He-Ne or semiconductor laser 501 send decays to the light beam of light intensity suitable (promptly guarantee the back produced interference image gray scale suitable) behind attenuator 502, the light beam that allows attenuator 502 penetrate again carries out spatial filtering and collimation by aperture 503 and collimating apparatus 504, and then the light behind the collimation becomes rectangle parallel lines light beam behind the polarizer 505 and rectangular aperture 506.
Adjust rectangle parallel lines polarized light and inject the incident angle of prism 507, guarantee that light beam is by prism 507 and refractive index oil reservoir 508, be mapped on the interface of the glass substrate of described array sensing chip 509 and golden film, make the light wave total reflection and produce evanescent wave, excite the plasma wave on the golden film surface on the described array sensing chip 509, promptly produce surface plasma body resonant vibration, incident angle at this moment is called resonance angle.
Allow described rectangle parallel lines light beam reflect, see through glass substrate, refractive index oil reservoir 508 and the prism 507 of array sensing chip 509, inject one dimension amplifying lens group 511 from the glass substrate of described array sensing chip 509 and the interface of golden film; Adjust the enlargement factor of described one dimension amplifying lens group, using rectangle parallel polarized light from the rectangular aperture 506 of described collimation incides on described prism 508 and the array sensing chip 509 and reflection back and cause the hot spot of compression, shape and size when being restored to the described bio-sensing of incident unit after its folded light beam is by one dimension amplifying lens group 511.
Rectangle parallel lines polarized light after the recovery of one dimension amplifying lens group ejaculation is injected time domain phase-modulator 512 again, just can change the voltage that is applied on the time domain phase-modulator 512 by the output signal amplitude of regulating time domain phase-modulator driving circuit 518, thereby regulate the phase differential between s light and the p light, make its successively according to-π ,-pi/2,0, pi/2 and π equiphase circulation change.The s light and the p light that penetrate from the time domain phase-modulator interfere behind polarizing prism 513, be imaged on by imaging len 514 on the target surface of CCD 515, adjust imaging len 514, can guarantee that the complete correspondence of image is imaged on the target surface of CCD 515, neither exceed target surface, be not less than target surface again.Described signal processing unit converts interference image to electric signal by CCD 515 earlier, becomes digital signal through signal processing circuit board 516 treatment conversion again, then deposits computing machine 517 in; The phase place of a p light of the every change of described time domain phase-modulator, computing machine is just gathered piece image, connects to gather 5 width of cloth images, finishes a cycling.
After computing machine utilizes the coordinate of array sensing unit interference image on the software program centering array sensing chip 509 earlier, utilize the Hariharan algorithm that every group 5 width of cloth image that CCD 515 obtains handled again, specifically can adopt following dual mode: first, select earlier one of them unit on the CCD that interference image covered of a sensing unit in the described biologic array sensing chip, the react phase change of caused light of the probe that utilizes the Hariharan algorithm to resolve again to be fixed on this sensing unit and other biomolecule; Second, the plurality of units that is close in delegation on the CCD that interference image covered of a sensing unit in the described biologic array sensing chip of selection or a few row is carried out the average processing earlier, the react phase change of caused light of the probe that utilizes the Hariharan algorithm to resolve again to be fixed on this sensing unit and other biomolecule is to obtain more high precision.Continuously change the voltage that is applied on the time domain phase-modulator, the continuous acquisition image, resolve continuously, the phase change that causes by biochemical reaction in the time of just can obtaining light in real time by the bio-sensing unit, promptly obtain the information of bio-molecular interaction in real time, offer the biologist or the physician resolves, obtain the biology or the medical information of being correlated with.
Sample solution is imported described sample pool 510, " part " in " acceptor " on each unit that is fixed on array sensing chip 509 and sample solution is when combining, phase change just takes place in the p light component in the rectangle parallel lines polarized light, produce interference through time domain phase-modulator 512 and polarizing prism 513 backs, because this is the time domain phase modulation (PM), the phase change of the interference fringe that produces only is presented in the time domain, and to present on the plane that striped moves be that the position changes and be different from interference fringe phase change that the space phase modulation produces.This interference fringe only shows as the light intensity of following modulation on the same unit of CCD 515 and changes, and the CCD515 real time record is got off, and deposits computing machine 517 in after signal processing circuit board 516 is handled; Computing machine 517 is handled the data that deposit in real time, obtains the information that reacts on each sensing unit on the array sensing chip 509 simultaneously.The reaction of each unit is constantly carried out on the array sensing chip 509, and computing machine 517 promptly provides corresponding information in real time.
Fig. 6 is the comparison synoptic diagram of time domain phase modulation (PM) interference of the present invention and existing space phase modulation (PM) principle of interference.As previously mentioned, the phase change of the interference fringe that the time domain phase modulation (PM) produces only is presented in the time domain, to present on the plane that striped moves be that the position changes and be different from interference fringe phase change that space phase modulation produces, and the light intensity of following modulation that only shows as on the same unit of CCD 515 changes.With the CCD target surface is object of reference, and space phase interferometric modulator waveform is to be parallel to target surface, and the waveform that the time domain phase modulation (PM) is interfered is perpendicular to target surface, thereby the time domain phase modulation (PM) interferes the detection flux can be much higher than space phase interferometric modulator detection flux.
As shown in Figure 7, array sensing chip of the present invention can adopt following structure: it is by glass substrate 19, and golden film 20, coupling connection layer 21 and sensor array unit 22 are formed.Sensor array unit 22 is probe biomolecule (also can claim " acceptor "), can combine (also can claim and " part " coupling connection) with the biomolecule of being analyzed, and joins on the layer 21 forming array at coupling with some model machine point; Different molecular probes can be fixed in each unit in the sensor array unit 22, thereby once can analysis of various biomolecules.
As shown in Figure 8, one dimension amplifying lens group of the present invention can adopt following structure: it is made up of a flat protruding cylindrical mirror peace-recessed cylindrical mirror of common optical axis.The concave surface of flat-recessed cylindrical lens 23 is plane of light incidence facing to the light that is reflected by prism 507, and the plane of flat-protruding cylindrical mirror 24 is facing to the plane of flat-recessed cylindrical lens 23, and the convex surface of flat-protruding cylindrical mirror 24 is the light outgoing plane.
As shown in Figure 9, time domain phase-modulator principle of work of the present invention is to utilize the electrooptical effect of electro-optic crystal that the light (light of surface plasma body resonant vibration promptly takes place) from 509 reflections of array sensing chip is carried out the time domain phase modulation (PM).Described time domain phase-modulator can be lithium niobate, and (LiNbO3, LN) crystal use laterally modulation, and extra electric field is along x (or along y) direction, and the linearly polarized light transmission direction of incident is parallel to main shaft z.After adding electric field, the main shaft x ' of new index ellipsoid and y ' but have rotated 45 ° around the z axle still in the x-y plane.Be L, be the crystal of d that it is that phase differential between x ', the y ' component is that electric light postpones along direction of an electric field thickness for length
Adjust the placement angle of modulator, make the s light of reflection, p polarisation of light direction parallel with x ', the y ' axle of electro-optic crystal respectively.Behind the light process polarizing prism by the electro-optic crystal ejaculation, received by CCD, its light intensity is
Wherein, I
s, I
pBe respectively the light intensity of s light and p light, Γ is an electric light phase delay, and δ is owing to the phase change that SPR causes takes place.As can be seen from the above equation, the light intensity that CCD receives is an interference fringe, and is irrelevant with volume coordinate x, y, only is subjected to the influence of SPR and electro-optic crystal.That is to say that for more specific on the CCD target surface (x, y are unique to be determined), the light intensity that CCD receives only changes in time.If Γ is known, also only relevant by the δ that this light intensity calculates, and irrelevant with pixel state on every side with light intensity and time, this with regard to fundamental difference in sampling and the phase calculation of CCD to the space interference striped.For example, in the phase place of space interference striped is resolved, sample frequency (promptly the pixel number that occupies of phase space interference striped) is weekly resolved precision to last phase place fundamental influence, and in the phase calculation of above-mentioned interference fringe, sample frequency has no impact basically to δ.Therefore, the sample point of each pixel of CCD on just can corresponding array sensing chip in theory, the detection flux of total system is determined that by the pixel sum of CCD and the processing power of electronic signaling system this has just improved the detection flux of whole sensor-based system greatly.That measure is phase differential δ between s light and the p light, utilizes electrooptic modulator to introduce additional phase differential at interval successively by certain hour between s light and p light
Obtain light intensity signal I respectively
1, I
2, I
3, I
4, I
5These five points can be determined the time dependent sinusoidal signal of row, and the phase differential δ that measure just transforms the initial phase of sinusoidal signal for this reason.Use the Hariharan algorithm, ask
As long as make computing machine send instruction, the driving voltage value of an electro-optic crystal of every change is promptly adopted piece image thereupon, and continuously changing 5 times is a circulation, and 5 width of cloth images are adopted in a circulation altogether; Utilization utilizes the Hariharan algorithm, this 5 width of cloth image is handled just can obtain a result.
As shown in figure 10, the driving circuit 518 of time domain phase-modulator of the present invention is a prior art, mainly selects circuit, amplification control circuit, output control circuit, computer interface etc. to form by signal generator, signal high-voltage amplifier, high-voltage signal output circuit, signal type.Computing machine sends by interface circuit and selects signal type and enlargement ratio instruction, after signal type selects circuit to receive the instruction that computing machine sends, control-signals generator produces needed signal, and it is selective that signal generator can produce sine wave, triangular wave or step signal.Simultaneously, after amplification control circuit received the enlargement ratio instruction that computing machine sends, the enlargement ratio of control signal high-voltage amplifier guaranteed to reach needed voltage.Then, computing machine sends the signal output order by computer interface, after output control circuit receives instruction, by the high-voltage signal output circuit high-voltage signal is applied on the time domain phase-modulator 512, makes its action, the light signal that will pass through modulation.
As shown in figure 11, signal processing unit of the present invention comprises CCD 515, signal processing circuit board 516 and computing machine 517, and described signal processing circuit board 516 can adopt following structure: it partly is made up of computer interface, driving sequential circuit, memory bank, A/D change-over circuit and correlated double sampling circuit etc.After computing machine sent the sampling instruction, the machine interface started the driving sequential as calculated, produces various time sequential pulses.At first, start CCD, convert picture signal to electric signal.Then, start correlated-double-sampling work, gather successively and keep electric signal in each unit of CCD, for analog to digital conversion creates conditions.Correlated-double-sampling is that double sampling is carried out in black level reference interval and the signal level interval in same pixel period, and obtains the true composition of the electric signal of correlation interference striped after double sampling value simulation subtracted each other, and reaches the high-fidelity sampling.The input of A/D change-over circuit links to each other with the output of correlated double sampling circuit, and the analog electrical signal with each unit of CCD is converted to digital signal successively.Driving under the time sequence control, the digital signal with the output of A/D change-over circuit deposits memory bank in successively, and memory bank can be deposited the data that the CCD of delegation goes up each unit, also can deposit 1 frame to 5 frame even the view data that receives of multiframe CCD more.Computing machine can once read in all images data in the memory bank by interface.
As shown in figure 12, the software flow that carries out centering operation in computing machine is as follows: at first, read first width of cloth image of collection, find with chip on the corresponding basic fixed position point of point of fixity; Then, by this basic fixed position point beginning, find the origin coordinates of each sample point; Then, determine the regional extent of each sample point, just finished the work of array sensing chip centering, just can utilize the Hariharan algorithm in the zone of determining, each sample point to be carried out resolving of phase place according to the size of sample point.
As shown in figure 13, to carry out the process that phase place resolves as follows for the Hariharan of utilization algorithm of the present invention: start computing machine, send instruction and give signal processing unit; Signal processing unit is started working, and carries out reception, collection and the analog to digital conversion of image, and the view data of A/D change-over circuit output deposits memory bank in.After data acquisition was finished, computing machine is reads image data from the memory bank of signal processing unit, carried out the phase place of the first five width of cloth image and resolved.Then, read following five width of cloth images, ask its PHASE DISTRIBUTION value, and compare, obtain the phase differential of corresponding point and draw out the phase change curve with first width of cloth image corresponding point.
Claims (10)
1. the method for sensing of a protein-chip is characterized in that, this method specifically comprises the steps: based on time domain phase modulation (PM) interference surface plasma resonance array bio-sensing principle
1) laser that sends of frequency stabilized laser then allows light beam obtain rectangle parallel lines polarized light by the polarizer and rectangular aperture through laggard row space filtering of attenuator and collimation;
2) allow described rectangle parallel lines light beam inject the bio-sensing unit that is used for detecting protein-chip, enter one dimension amplifying lens group by its light beam that reflects, adjust the enlargement factor of described one dimension amplifying lens group, shape and size when making the hot spot of ejaculation return to the described bio-sensing of incident unit;
3) light that allows described one dimension amplifying lens group penetrate is injected the time domain phase-modulator, s light in the incident light is parallel with y ' axle with the x ' axle of described time domain phase-modulator respectively with p polarisation of light direction, change the voltage be applied on the described time domain phase-modulator then successively, guarantee that the phase change of the p light that penetrates is followed successively by-π ,-pi/2,0, pi/2 and π; The s light and the p light that allow described time domain phase-modulator penetrate produce interference by polarizing prism, through being imaged on behind the imaging len on the CCD target surface, convert interference image to electric signal by CCD, deposit computing machine after treatment in; The phase place of a p light of the every change of described time domain phase-modulator, computing machine is just gathered piece image, connects to gather 5 width of cloth images, finishes a cycling;
4) continuously change the voltage that is applied on the described time domain phase-modulator, continuous acquisition image thereupon according to the described method of step 3); And utilization is stored in the coordinate of the specific program centering institute images acquired in the computing machine, then utilize the Hariharan algorithm that image is resolved, the phase change of being drawn by biochemical reaction when in real time obtaining light by described bio-sensing unit promptly obtains the information of bio-molecular interaction in real time.
2. the method for sensing of protein-chip according to claim 1, it is characterized in that: in step 4), select earlier one of them unit on the CCD that interference image covered of a sensing unit in the described biologic array sensing chip, the react phase change of caused light of the probe that utilizes the Hariharan algorithm to resolve again to be fixed on this sensing unit and other biomolecule.
3. the method for sensing of protein-chip according to claim 1, it is characterized in that: in step 4), the plurality of units that is close in delegation on the CCD that interference image covered of a sensing unit in the described biologic array sensing chip of selection or a few row is carried out the average processing earlier, the react phase change of caused light of the probe that utilizes the Hariharan algorithm to resolve again to be fixed on this sensing unit and other biomolecule is to obtain more high precision.
4. time domain phase modulation (PM) interference surface plasma resonance array detection protein-chip system that adopts the described method for sensing of claim 1, comprise the incident arm that produces the rectangle parallel lines polarized light that detects usefulness, detect the bio-sensing unit of protein-chip, produce the reflection arm of interference image, and obtain the interference image signal and carry out the signal processing unit of subsequent treatment, it is characterized in that: described reflection arm is positioned at the side that the bio-sensing unit reflected light penetrates, and comprises the one dimension amplifying lens group that places successively on the reflection optical axis, the time domain phase-modulator that has driving circuit, polarizing prism and imaging len; Computing machine in the described signal processing unit links to each other with the driving circuit of described time domain phase-modulator, to realize the control to the time domain phase-modulator.
5. time domain phase modulation (PM) interference surface plasma resonance array detection protein-chip according to claim 4 system, it is characterized in that: described one dimension amplifying lens group is made up of the flat-protruding cylindrical mirror peace-recessed cylindrical mirror of common optical axis, the concave surface of described putting down-recessed cylindrical mirror is a plane of light incidence, the convex surface of described putting down-protruding cylindrical mirror is the light outgoing plane, and the enlargement factor of described one dimension amplifying lens group is 2～4 times.
6. time domain phase modulation (PM) interference surface plasma resonance array detection protein-chip according to claim 4 system, it is characterized in that: described time domain phase-modulator is an electro-optic crystal, material is KDP, KD*P, ADP, LiTaO
3Or LiNbO
3
7. time domain phase modulation (PM) interference surface plasma resonance array detection protein-chip according to claim 4 system, it is characterized in that: the light wavelength that described time domain phase-modulator is modulated is 0.4 μ m～2 μ m;
8. time domain phase modulation (PM) interference surface plasma resonance array detection protein-chip according to claim 4 system, it is characterized in that: the clear aperture of described time domain phase-modulator is 4 * 4-20 * 20mm
2, length is 10-120mm.
9. time domain phase modulation (PM) interference surface plasma resonance array detection protein-chip according to claim 4 system, it is characterized in that: described bio-sensing unit is a surface plasma resonance biosensor, comprises right angle or Dove prism, refractive index oil reservoir, array sensing chip and places sample pool under the described array sensing chip; Described right angle or Dove prism, refractive index oil reservoir are identical with the refractive index of the optical glass substrate of array sensing chip; The material of the optical glass substrate of described right angle or Dove prism and array sensing chip is a flint glass.
10. time domain phase modulation (PM) interference surface plasma resonance array detection protein-chip according to claim 4 system, it is characterized in that: described incident arm is that collimated light source comprises frequency-stabilized He-Ne or the semiconductor laser that places bio-sensing unit one side, and is arranged on attenuator, spatial filter, collimating apparatus, the polarizer and rectangular aperture on the described laser optical axis; Described attenuator metal-plated membrane, attenuation ratio is 0-50%, and the aperture of the diaphragm of described spatial filter is 5-50 μ m, and the clear aperture of described rectangular aperture is 4 * 4-20 * 20mm
2
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