CN101573618A

CN101573618A - Surface mapping by optical manipulation of particles in relation to a functionalized surface

Info

Publication number: CN101573618A
Application number: CNA2007800342613A
Authority: CN
Inventors: 克里斯多佛·克努森; 克里斯托·杜克; 加里·斯泰西; 丹·米特; 埃文·坦纳; 奥斯曼·阿克恰基尔; 傅浩军; 罗伯特·兰斯洛特; 塔尼亚·恰克拉巴蒂; 肯尼斯·布拉德利
Original assignee: Haemonetics Corp
Current assignee: Haemonetics Corp
Priority date: 2006-09-15
Filing date: 2007-09-14
Publication date: 2009-11-04

Abstract

Methods and apparatus for analyzing surface properties of particles are provided. A method for analyzing the surface properties of the particle includes a associating a first particle with a first capture zone having a specific binding affinity for a first chemical species, applying an optical force to the first particle, sensing a response of the first particle to the optical force, and using the sensed response to determine the presence, absence or quantity of the first chemical species on the first particle surface. This process may be repeated in parallel to test multiple particles. In addition to directly testing the surface properties of the particles, the method can be used in direct, indirect and competitive assays to determine the presence, absence or quantity of free or immobilized analytes. A fluidic cartridge with capture zones having avidities that are tuned for the use of optical forces is provided. A software routine for performing the method is also provided.

Description

Carry out the surface mapping by the optical manipulation particle relevant with functionalized surface

Cross reference with related application

Present patent application requires the U.S. Provisional Patent Application series number No.60/844 of submission on September 15th, 2006,911 " combination of measuring Ag-Ab by a plurality of objects of operation in the electromotive force that limits at light is identified (Determination ofantigen-antibody attachment for protein assay and blood typing bymanipulation of multiple bodies within an optically defined potential) to be used for analysis of protein and blood group ", the U.S. Provisional Patent Application series number No.60/844 that on September 15th, 2006 submitted to, 890 " being used for the substrate functionalization (Substrate Functionalization for BloodTyping) that blood group is identified ", the U.S. Provisional Patent Application series number No.60/844 that on September 15th, 2006 submitted to, 910 " the analysis boxes (Analytical cartridge for opticalmanipulation) that are used for optical manipulation ", the U.S. Provisional Patent Application series number No 60/844 that on September 15th, 2006 submitted to, 763 " integrating the compact Sample testing device (Compact sampletesting apparatus incorporating holographic optical trapping) of holographic optical trap ", and the U.S. Provisional Patent Application series number No.60/931 of submission on May 22nd, 2007, the right of priority of 451 " being used for the analysis boxes (Analytical cartridge for blood typing) that blood group is identified ".It is reference that all above-mentioned temporary patent applications draw with it in full at this.

Technical field

The present invention relates to make power Operations Analyst thing or the probe particle of using up generation,, comprise the cell surface antigen analysis to be used for analysis.

Background of invention

Application becomes the molecule of array with for example microwell plate with the biochip form, make the information about the growing amount in the natural world of can retrieving.Although known have a multiple version, the use of this array generally includes probe or analyte is fixed in the substrate, and uses various technology to measure the interaction of immobilized molecule and other solution molecule mutually.The object lesson of this array technique is included in the immunoassay that carries out in the microwell plate (enzyme-linked immuno assay for example, ELISAs), biological nucleic acid chip (for example by Affymetrix and Illumina commercial those) and protein bio-chip (Invitrogen ProtoArray for example ^TM).The most common substrate of glass of utilizing of biochip, and the microwell plate of ELISA is usually made from the polystyrene of γ-radiation.Although from immobilized molecule still reference feature and being arranged on this meaning in position of qualification relative to each other, array generally is orderly, so-called " solution phase array " is also by commercialization.Luminex X-Map ^TMCommercial measurement combining of analyte and pearl suspending liquid.Suspending liquid produces by many batches of particles are mixed, the described particle common mark of respective mixtures of bonding probes and two kinds of different ratios of fluorescence identifier molecule.Use flow cytometer to detect the fluorescence labeling analyte and the identifier ratio of combination.

Except detecting individual molecule, also used array technique analysing particulates (comprising biological cell).For example, at ELISA or the Flow cytometry cell surface antigen of passing through known in the art.The U.S. Patent No. 6,251,615 of Oberhardt discloses use microscopic examination cell, to detect the cell that one of a plurality of trapping regions with the antibody receptor that has coupling combine.

The major limitation of conventional arrays is, analyzes effectively allowing in order to reach enough near the bonding state of balance, needs the long incubation time usually.This may need to make sample or reagent many hours of incubation in ELISA microwell plate or biochip, and in some cases, surpasses typical 8 times each workday.Such delay causes additional expense, has hindered the use in emergency condition, and owing to reagent reduces the quality of data in the degraded of the incubation time durations of spinning out.Other restriction of conventional method is to need analyte markers step and tight cleaning step, and this is introducing additional cost aspect work force-summing device, and may influence the quality of data.Use the specimen and the reagent of relative large volume based on the method for microwell plate.

As known in the art, red blood cell (" RBCs ") from different patients can have different antigen in its surface, to may in the blood acceptor, cause harmful immune response from patient's whole blood to another patient's blood transfusion that lacks those antigens with some RBC antigen.Therefore, carrying out blood group by the aggegation analysis usually identifies.

Blood group is identified needs to identify blood group antigens or the antibody of presenting on the RBCs surface of object.Usually be accredited as A and Blood group antigen B, and the various antigen relevant with the Rh system, wherein D antigen is most important clinically.At present, blood group identifies it is (referring to, for example, the U.S. Patent No. 4,894,347 of Hillyard etc.) undertaken by the aggegation that detects erythrocyte (RBCs) after adding blood group antibody.The positive findings that the antibody of the aggegation of blood sample indication antigen and adding is complementary (for example Blood group antigen A, Rh blood group antigens).

Because antibody response is in interpersonal difference, and since in the blood of object antibody may be pre-existing in, therefore have the possibility of spurious results.It is incorrect that false results causes blood group to be identified, this produces the possibility of disadvantageous transfusion reaction again.In addition, a large amount of relatively blood of existing Technology Need (～3mL) be used for check.Therefore, the ability of carrying out the blood group evaluation with smaller size smaller will increase the comfort level of donor, and will be extremely useful when processing anaemia patient for example suffers from the neonate of erythroblastosis fetalis (HDN).The ability of carrying out somatotype from simple sample blood also will be at present employed blood sample to be input to remarkable improvement corresponding to the prior art a plurality of independent reaction volume of each check antigen respectively.

In addition, measure a small amount of RBCs or other cell or the organism and multiple reaction reagent of the broad sense parallel interactional ability that---has known special compatibility for specific analyte separately---more, great analysis and diagnostic value will be arranged.A kind of example application of such system is from the RBCs of the single blood sample parallel analysis for ABO and two kinds of system's antigens of Rh.

Holographic optical trap (HOT) provides useful instrument for the operation of micron and nano particle biological example composition.The system that is used to produce and use this ligh trap of the ultimate principle of holographic optical trap and structure is disclosed in U.S. Patent No. 6,055, and in 106, U. S. application series number Nos.10/701,324 and 09/886,802, it draws at this in full and is reference.

Research small sample particle need be placed on sample in the fluid box in based on microscopical system, for example on the slide, so that it can keep aliging with the optics of microscopic system.Such fluid box is also referred to as specimen slides, sample chip or cover plate.The specimen slides that can be used for the holographic optical trap technology is disclosed in U.S. Patent Application Serial No.10/294, and 599, among the U.S. Patent Publication No.20030119177, it draws with it in full at this in full is reference.

Summary of the invention

According to illustrative embodiment of the present invention, used the method for the surface nature of analyzing one or more particles that respectively have the surface.This method comprises that the suspending liquid that will contain a plurality of particles is incorporated into and has at least one and chemical species and have in the sampling receptacle of trapping region of given compatibility, at least a portion in a plurality of particles is contacted with at least one trapping region, to allow between at least one trapping region and at least a portion particle, binding interactions taking place, binding interactions is associated with the surface composition of particle, optical force is applied at least a portion particle two simultaneously at least, this power tends to cause the response of particle, wherein this response depends on the existence of binding interactions between particle and at least one trapping region, do not exist or degree, respond to of the response of at least two particles, and the composition of particle surface is determined in the required response of usability to optical force.

Apply optical force and can comprise use holographic optical tweezer (HOT).Contact can comprise a plurality of particles are contacted with the single trapping district, and respond to the response of a plurality of particles to optical force.Method can comprise makes first particle contact with first trapping region that first kind of chemical species is had the specificity binding affinity, to allow between first trapping region and first particle, binding interactions taking place, the existence of first kind of chemical species molecule on binding interactions and first particle surface is associated, second particle contacted with second trapping region that second kind of chemical species is had the specificity binding affinity, to allow between second trapping region and second particle, binding interactions taking place, the existence of second kind of chemical species molecule on binding interactions and second particle surface is associated, first and second particle are applied optical force, this power tends to cause the response of particle, respond to the response to optical force of first and second particle, the first kind of chemical species on first particle surface and the existence of second kind of chemical species on second particle surface are determined in the required response of usability, do not exist or quantity.

Method can be included in and identify particle before particle applies power.Method also can comprise the evaluation particle, selects first group to identify particle, applies optical force to first group of particle, selects second group of particle, and applies optical force to second group of particle.

Step that can repetition methods is obtained threshold value to reach data.

Power can have normal direction in trapping region and the component that leaves the trapping region orientation.Optionally or in addition, power can have the component that is parallel to the plane that trapping region determines.Power can tend to make unconjugated particle with the planar process that limits with trapping region to first direction and second direction in parallel move to the position that separates with trapping region.

Response can comprise from trapping region removes particle.

Particle can be a cell.Chemical species can be a cell surface antigen.

In embodiments, particle is a red blood cell, and at least one trapping region comprises the cell surface antigen specific probe.In addition, mensuration can be used for further determining blood group, comprises the blood group that relates to surface antigen more than three kinds, for example rare (minor) blood group.

Probe can be for example antibody, antibody fragment or aptamers.Contact procedure can comprise the permission particle from the solution sedimentation, for example uses gravity to make particles settling or use optical force make particles settling.Contact can also comprise with solution be incorporated into part by first surface and part by in the volume that is limited with first surperficial separated lid, at least one is optically transparent basically for surperficial and lid.The distance that lid and first surface are separated by can arriving less than its 1000 times more than 1 times for first particle diameter.The distance that lid and first surface are separated by can arriving less than its 20 times more than 2 times for first particle diameter.The distance that lid and first surface are separated by can be in 15 microns to 500 microns scope.The obstruction in the space that lid and the surface distance of being separated by be big must to be enough to be unfavorable for that first surface and lid are limited, but still little be enough to allow the sedimentation in of first particle less than 10 minutes.The step that applies optical force comprises uses the holographic optical tweezer.

First trapping region can comprise a plurality of first kind of target part being had specific probe molecule, the structure of probe molecule makes it have affinity to the particle that has a plurality of first kind of target part on it, selects the structure of probe molecule to be shifted out by the power in the particular range with permission.Specific scope can be for example 1 to 1000pN, 10 to 200pN or 20 to arrive 100pN.Method can also comprise second trapping region, contain and a plurality of second kind of target part had specific probe molecule, probe molecule is configured from the teeth outwards, so that the particle that has a plurality of second kind of target part on it is had affinity, select the structure of probe molecule to be shifted out by the power in the particular range with permission.Specific scope can be for example 1 to 1000pN, 10 to 200pN or 20 to arrive 100pN.The probe molecule of trapping region is formed in the particular range and shifts out by changing the length of the coupling part that connects surface and probe.The probe molecule of trapping region also can be formed in the particular range and shift out by changing the surface density of probe.Can use and utilize non-binding surface portion probe to be separated so that obtain the method for first kind and second kind affinity.Can select surface density by the expection density according to cell surface antigen on the cell surface, the pair cell surface characterizes.

Apply optical force and can comprise that the center with ligh trap is set in from the certain distance of particle, the energy of position of particle is high on the surface, in the center of ligh trap minimum, makes and shifted out the surface as fruit granule by the optical force of trap that particle will move to the center of ligh trap.

Contact can comprise by passive stream first particle importing solution.

Method can comprise that change is applied to the optics intensity of force on the particle, causes the value of power to increase in time, and record shifts out particle the amount of the required power in surface.Method can comprise that power and the reference power that will record compare, existing, not existing or measuring with first kind of chemical species on definite first particle surface.Induction can comprise the use automated microscope.The application of optical force can be robotization.

Sampling receptacle is used in the operation that is selected from diluted sample and filtration before the analysis.

According to embodiment of the present invention, method be used for determining patient's the multiple haemocyte surface antigen of blood sample existence, do not exist or measure.Method comprises dilutes blood sample, the blood sample of dilution is contacted with first and second trapping region, first trapping region is functionalized so that first kind of blood cell antigen had special compatibility, second trapping region is functionalized so that second kind of blood cell antigen had special compatibility, to have first optical force that shifts out component be applied to first the district at least one cell on, if the size of first power is adjusted to when being enough to not have first kind of blood cell antigen on cell, cell is shifted out from first district, but still be not enough to and exist the cell of first kind of blood cell antigen to shift out, to have second optical force that shifts out component is applied at least one cell in second district, if being adjusted to, the size of second power is enough to when there be not second kind of blood cell antigen in cell, cell is shifted out from second district, but still be not enough to exist the cell of second kind of blood cell antigen to shift out, and detect cell and from the district, shift out still and do not shift out.Contact may further include and allows cell sedimentation under gravity.

According to embodiment of the present invention, the device that is used to analyze the existence of the target part on one or more particles, do not exist or measure comprises substrate, be positioned at suprabasil first and second trapping region, each trapping region contains a plurality of probe portion, wherein the affinity of first and second trapping region is constructed such that the particle that trapping region is had than high-affinity tends to stay in the presence of the displacement optical force, tend to be transferred in the presence of the displacement optical force and trapping region is had the particle that hangs down compatibility, optical force is positioned at particular range.

Optional, device can comprise lid, and wherein at least one in bottom, trapping region and the lid is optically transparent; The entrance and exit of the volume that defines by trapping region and lid; Spacer is used to separate lid and trapping region, and its size makes in case after containing solution of mammalian cell and being imported into, and most of cell will deposit on the trapping region in 5 minutes or shorter time.The size of spacer also allows by passive stream solution to be imported in the volume that is defined by trapping region and lid.

Device can comprise the optically transparent lid of infrared light.The particular range that can be used for device can be for example 1 to 1000pN, 10 to 200pN or 20 to arrive 100pN.Device can be configured for blood group and identify, also can operate the sample preparation step that is selected from diluted sample and filtration with execution.

According to embodiment of the present invention, method comprises the solution that IgM solution is cracked into the fragment that contains a plurality of antigen binding structural domains, binding structural domain further is cracked into the antigen-binding subfragrnent, and subfragrnent is combined with substrate to produce at the trapping region of specificity in conjunction with first kind of antigen of identification.Method can also comprise second trapping region of generation specificity in conjunction with second kind of antigen of identification.Method can comprise for example particle is exposed to first and second trapping region, and the particle and the compatibility in zone are graded, to identify the common antigen that exists at particle surface.Particle can be a cell.Can comprise compatibility grading particle is applied optical force.This power can be applied simultaneously on second particle.This power can use HOT to apply.

According to embodiment, there is the existence that is used for determining a plurality of molecules of solution, the method that does not exist or measure, comprise providing and have first trapping region at least with first kind of immobilization probe and the substrate that has second trapping region of second kind of immobilized probe, trapping region and analyte solution are contacted under the condition of analyte molecule and probe combination being enough to allow, the analyte of combination is contacted with having with the particle of the immobilization general probe of the analyte molecule specificity combination of multiple combination, particle is applied optical force, and observe the response of particle to optical force, this response depends on the combination between general probe and the analyte that combines.

Optional or in addition, method can comprise that the analyte with combination contacts with a plurality of particles, and particle is applied optical force, and the response of observation particle.Particle is applied power can carry out simultaneously.Analyte solution can contact with a plurality of trapping regions, and its mode causes and cause setting up the fluid connection between trapping region.Optional or in addition, analyte solution can contact with a plurality of trapping regions and not allow that fluid is communicated with between the trapping region.Analyte solution can comprise patient's antibody.The antibody that general probe can comprise at least one class patient has wide optionally second antibody.The type of antibody can be selected from one of IgG, IgE, IgA and IgM.

According to embodiment of the present invention, computer program on the touched computer-readable medium that computer system is used comprises that computer code, the startup of identifying the multiple purpose particle that contacts with trapping region apply the computer code of displacement optical force and write down the computer code of observed particle to the response of optical force particle.

Optional or in addition, computer program can comprise the computer code of selecting a plurality of fields of microscope, and/or determine when that having accumulated threshold quantity data computing machine encodes.

According to embodiment of the present invention, the method of analyzing the surface nature of a plurality of particles comprises that the suspension that will contain a plurality of particles imports the fluid box that has a plurality of trapping regions in substrate, box has been determined the liquid column that lid defines, thereby allow particle deposition contact trapping region, particle is applied optical force; And the observation particle is to the response of optical force.The height of selecting liquid column had been less than 10 minutes or had been less than in period of 5 minutes deposition and contacting with trapping region to allow red blood cell.

According to embodiment of the present invention, the method that is used for competitive analysis comprises a plurality of sandwich structures of generation, each sandwich structure contains the set that is fixed on suprabasil first kind of probe molecule, set with first kind of reversible occupy-place molecule that combines of probe molecule, and have a particle a plurality of and the reversible second kind of probe molecule that combines of occupy-place molecule, add analyte solution, incubation is enough to allow the analyte molecule that first kind and second kind of probe molecule have compatibility was replaced to the time of small part occupy-place molecule, to change the bond strength between particle and the substrate, particle is applied optical force, observe the response of particle, and use this to respond to determine the inclusions of analyte solution optical force.

Has the zones of different that specific sandwich structure can be placed on substrate for different respective analyte molecules, so that report a plurality of analyte kinds.The occupy-place molecule can tend to produce the stronger adhesion than corresponding analyte molecule produced between particle and substrate.Optional or in addition, the occupy-place molecule can tend to produce the more weak adhesion than corresponding analyte molecule produced between particle and substrate.Optical force can be applied simultaneously on a plurality of particles.

According to embodiment of the present invention, the combined sample disposal system is used HOT performance and microscope, and comprise shell, the sample stage that has sample area, HOT subsystem, this subsystem comprise the HOT emission port of laser, diffraction optical element, sensing sample area, light source that at least one has the output source that points to sample area, can be with the object lens of sample area optical alignment, be arranged to focus on the camera of sample area.In shell, contain sample area, HOT emission port and output light source at least.

Optional or in addition, all parts are installed on the removable frame.System also can comprise and is selected from following at least a parts: the computer interface of integration, response lid are opened and are closed the security mechanism of laser lighting, are used to locate the anchor clamps of fluid sample box, and the base of belt wheel.

The accompanying drawing summary

With reference to following detailed, after with reference to the accompanying drawings, These characteristics of the present invention will be more readily understood, in these figure:

Fig. 1 is according to embodiment of the present invention, the process flow diagram of the method on analysing particulates surface;

Fig. 2 is the planimetric map that is used for the device with trapping region of Fig. 1 method;

Fig. 3 has shown Fig. 2 device that has analyzed haemocyte;

Fig. 4 is a synoptic diagram, has shown the cross-sectional view of Fig. 2-3 device, comprises the trapping region that has antibody probe;

Fig. 5 is a synoptic diagram, has shown Fig. 4 device after cell has been deposited on trapping region;

Fig. 6 is a synoptic diagram, has shown the xsect of Fig. 2-4 device, and the probe density that changes has been described;

Fig. 7 is a synoptic diagram, has shown the xsect of Fig. 2-4 device, and the use of biologically inert part has been described;

Fig. 8 is a synoptic diagram, has shown according to embodiments of the invention to contain the system that analyzes box and instrument;

Fig. 9 is a synoptic diagram, has shown the skeleton view of the box of embodiment of the present invention;

Figure 10 is according to embodiment of the present invention, by the exploded view of box among Fig. 9 of lamination assembling manufacturing;

Figure 11 is the box that has a plurality of analyses storehouse according to embodiments of the present invention;

Figure 12 is the box with a plurality of analyses storehouse according to another embodiment of the invention;

Figure 13 a shown according to embodiment of the present invention, by capillary action with the process of load fluids in the box in, the box of Figure 12;

Figure 13 b has shown the fluid sample box of integrating according to embodiments of the present invention;

Figure 13 c has shown another fluid sample box according to embodiments of the present invention;

Figure 14 schematically illustrates the HOT and the microscopy instrument of combination according to embodiments of the present invention;

Figure 15 schematically illustrates alternative according to embodiments of the present invention combination HOT and microscopy instrument;

Figure 16 is the microscopical skeleton view that is used for the embodiment of Figure 14-16;

Figure 17 has shown the skeleton view of the HOT subsystem of the embodiment that is used for Figure 14-16;

That Figure 18 has shown is compact according to embodiments of the present invention, movably, the synoptic diagram of combination HOT and microscopical instrument;

Figure 19 schematically illustrates the parts of Figure 19 instrument;

Figure 20 is the process flow diagram that is used for the software operation of execution graph 1 method on self-reacting device;

Figure 21 is to produce the reaction scheme of catching the surface of bag quilt with the IgM antibody fragmentization;

Figure 22 a has schematically shown analysis according to embodiments of the present invention, and wherein analyte antigen combines with substrate, and is discerned by the probe of particle mark;

Figure 22 b has schematically shown the analysis of Figure 22 a, and wherein antigen is not discerned by probe;

Figure 23 a schematically illustrates the analysis of embodiment of the present invention, and wherein analyte antigen combines with substrate, and is discerned by the compound of the probe of antibody and particle mark;

Figure 23 b is the synoptic diagram of the analysis of Figure 23 a, and wherein antigen is not discerned by compound;

Figure 24 is the reaction scheme that shows the competitive analysis of embodiment of the present invention, and wherein the adhesion between particle and the trapping region reduces after importing sample;

Figure 25 is the reaction scheme that shows the competitive analysis of embodiment of the present invention, and wherein the adhesion between particle and the trapping region increases after importing sample.

The detailed description of specific embodiments

Definition.When being used for this instructions and appending claims, unless context has other requirement, following term will have the meaning of indication:

" optical force " is meant that the illumination of using suitable orientation or pattern is applied to the power on the particle, causes to produce and tends to make the potential energy states of particle in the rising of assigned direction top offset.

" particle " is meant the object that has the surface and can be handled by optical force.Cell and virus are " particles ", and little polymerization and inorganic object, for example the microballoon or the microballon of various difformities and composition also are.

For particle, " surface " is meant the part that particle can be approaching with probe.

For fluid box or other sampling receptacle, " passive stream " is meant that sampling receptacle passes through capillary action or alternate manner and do not use the pump draw fluid.

When relating to the instrument that is used for grain size analysis, term " robotization microscopy " is meant that by suitable location and/or software program instrument can be located the particle that is used to analyze at least automatically and be write down their position.

During entity on relating to particle surface, " chemical species " is meant any molecule, divides sub-assemblies, big molecule, divide sub-assemblies or part greatly.Term " chemical species " clearly comprises biological and abiotic big molecule, for example peptide, albumen, carbohydrates, glycoprotein, antibody, nucleic acid, polymkeric substance, medicinal composition etc.

" probe " is meant for given target particle, molecule, assembly or part to have the assembly of specificity in conjunction with tendentious molecular entity, particle or molecular entity.

" trapping region " be meant comprise acceptor so that its can with the given interactional surf zone of analyte specificity.

Herein and " array " that use in any claims be meant a plurality of element groups that are placed on the discrete locus, no matter be in one or more time occasions, and though also be rule at interval, periodic intervals or other.

" blood group " comprises and measures any individuality or the combination that is selected from following antigen: A, B, Rh (all 53 kinds of Rh antigen Rh1 that comprise International Society of Blood Transfusion (International Society Blood Transfusion) definition are to Rh53), A ₃, A _x, A _End, A _m, A _y, A _El, B ₃, B _x, B _m, B _El, Duffy, Kell, Kidd, Lewis, MNSs, Lutheran, Diego, Cartwright, Xg, Scianna, Dombrock, Colton, Landsteiner-Weiner, Chido-Rodgers, H, K _x, Gerbich, Cromer, Knops, Indian, Ok, Raph, John Milton Hagen, I, Globoside, GIL, and other blood group antigens.

Illustrative embodiment of the present invention relates to the interactional method and apparatus of observing between particle surface and other surface, extracting and the relevant information of interactional tendentiousness takes place on the surface, and use this information acquisition about the composition on surface, with the conclusion of other character of the analyte of surface interaction or analyte sample.Interaction can be by comprising that at a plurality of particles applying the directional light educational level on the cell surveys, to test they and the interactional ability of molecular recognition elements.By using parallel ligh trap or optical tweezer technology, such test can be carried out in mode quick, the composition benefit, to produce a lot of high-quality measurements.Embodiment also relates to uses self-reacting device in a parallel manner particle to be applied power and the induction apparatus and method to the response of applied force.Various aspects of the present invention especially can be applicable to biology and biochemical analysis, comprise the analysis (for example diagnosis of blood group evaluation and cancer and other morbid state) of cell surface antigen, the analysis of antibody or distinctive other biomarker of morbid state, and use the array antibody on the protein chip to carry out the analysis of albumen.Specific embodiments of the present invention relates to uses thin and transparent bin analysing particulates.Because bin is thin, can make particle contact trapping region more faster than what reach usually in the conventional affine bioanalysis.

Fig. 1 is the process flow diagram according to the method for embodiment of the present invention characterizing particles surface nature.In the process of routine analyzer, a plurality of particles to be analyzed are with one or more trapping regions combinations and contact (step 100).Trapping region is designed to catch the particle with some surface nature.Particle can be by gravity settling with contacting of trapping region or the power that applies more on one's own initiative is for example centrifugal, magnetic-particle is applied magnetic field or makes with light tweezer (comprise use HOT) finish.Density as fruit granule is too low for gravity settling, and active method described later may be preferred.Then particle is applied optical force, the mode that applies is tended to the position (step 110) of disturbance particle.Optical force can have normal direction in and leave the component (for example when use plane trapping region time, towards out-of-plane power) of trapping region orientation.Optical force also can have the component that is parallel to surface or trapping region plane.Optical force also can be designed to the plane of trapping region planar quadrature in move particle (for example, attempting to quicken particle) along diagonal line so that it is moved in the horizontal and vertical directions.The value of power and orientation can be constant; move according to predetermined pattern; or according to moving by observing the feedback that particle obtains, and can tend to various mode disturbance particle, described mode depends on the bonding state of particle with respect to trapping region.Except with the rectilinear motion, the power or the power of form regularly can be induced particle to rotate back and forth, reverse or be out of shape (if flexible) motion, or be shown the combination of above-mentioned behavior.The induction module of the device (the robotization microscope of the Fig. 8 that for example describes below) that is fit to is used to determine masterpiece is gone out the existence and the degree (step 120) of the particle disturbance of response.For example, as the result who applies optical force, particle can be shifted out from trapping region or replaces.Particle is used to determine interactional character between particle surface and the trapping region to the sensed response of power.The character that can be determined comprise chemical species on particle or the trapping region surface existence, do not exist or measure.Perhaps, the response of sensing can be used for determining that the character of interactional environment (for example the existence of competitive bond and amount, temperature, allosteric effector, or SOLUTION PROPERTIES) takes place for particle and trapping region.

In specific embodiment, trapping region comprises the probe that particular chemical thing class is had the specificity binding affinity.Particle and the contact of such trapping region to be to resist the relative or absolute trend of optical force displacement, shown probe institute specificity at chemical species whether be present on the surface of particle.In embodiments, also can determine the amount or the concentration of chemical species on the particle.In other embodiment, comprise in those embodiments that are described with reference to figure 24-25, the interaction of particle and trapping region be used to analyte in the analytical solution existence, do not exist or measure.As what describe below, the embodiment of the specific illustrative of Fig. 1 method comprises by sample being imported the micro fluid box particle is combined with trapping region, and the cell sedimentation under gravity that exists in the permission fluid sample, with the trapping region of contact antibody sandwich.Then box is inserted in the aut.eq., celluar localization on trapping region, is applied power, and competent cell goes out the change in location of response to masterpiece,, and obtain blood group with the surface antigen of determining to exist on the cell.

Because many important application of embodiment of the present invention are biology or biochemical in itself, therefore in embodiment described herein, analyzed particle is a cell.But, the particle of many other types also can be characterized, include but not limited to virus, vesica, organelle, silicon dioxide microsphere, polystyrene or other polymer microballoon, be combined with the microstructure of the microballoon of sample analytes molecule, microstructure in irregular shape, fluorescence or other mark, the microstructure of tape font code (comprising the active identifier of identifier, fluorescence or Raman of nucleic acid coding etc.), and inorganic structure comprises semiconductor, core-shell nanoparticles, quantum dot, magnetic microsphere and metal construction.Particle can be in 0.1 to 100 micron magnitude range for example, and diameter is between 1 to 10 micron in specific embodiment.In general, say for the meaning of certain wavelength light educational level sensitivity that given medium, is used to analyze from particle, particle should be have optically active.

Fig. 2 has shown the planimetric map of the substrate 200 that has a plurality of trapping regions 210.Each trapping region has the surface of using at the probe functionalization of particular chemical thing class.For immunoassay and biochip mating surface, trapping region 210 can contain the set of immobilization probe in trapping region.Substrate 200 can be to various wavelength only transparent, with the optical imagery that allows particle with by from applying optical force with the probe opposition side through irradiation, although in optional embodiment, irradiation is from top or undertaken by these two kinds of paths.Probe can be covalently bound on the surface, for example, by use silane on substrate of glass 200 or other chemical species that is fit to, and can use epoxide, mercaptan, amine or other reactive group known in the art.A concrete scheme that connects antibody fragment is described below with reference to Figure 21.Trapping region 210 also can comprise the nonplanar structure that is connected with substrate, example gel pad (referring to for example disclosure of U.S. Patent No. 6,642,000), hole, kapillary or other structure.Shown 3 trapping regions 210, but can provide more or less.Substrate 200 can be motionless basically, and the optical force that can not be used to particle tested moves.Perhaps, trapping region 210 can be can with light tweezer operation repetitive, to form cubical array (for example, trapping region 210 is placed on the surface of optical activity particle).The U.S. Patent Application Serial No.10/1735 that on Dec 12nd, 2003 submitted to, 39 disclose the generation of cubical array, draw at this to be reference.

As above-mentioned, trapping region 210 contains the probe that given chemical species is had special compatibility.In other words, the compatibility of a kind of thing class of trapping region selective binding is higher than other and may be present in thing class in the analyte sample.Therefore, the entity in the specimen and the binding interactions of trapping region have indicated trapping region in this sample to be designed to it is had the existence of the chemical species of pathoklisis.The example that can be used for the probe of embodiment of the present invention comprises nucleotide, oligonucleotides (and chemical derivative), linearity or cyclic DNA (plasmid for example, clay, BACs, Acs), mRNA, cDNA, mitochondrial RNA (mt RNA), engineered rna, aptamers, PNA (peptide nucleic acid), polyclonal antibody, monoclonal antibody, the engineered antibody of reorganization, antibody fragment, antigen, haptens, antibody FAB subunit (words that need are modified), domain antibodies, the artificial antibody, albumen, the albumen of modifying, recombinant protein, recombinant antigen, enzyme, the co-factor of enzyme or inhibitor, albumen composition, agglutinin, the albumen of histidine mark, the albumen of tape label, molecular imprinting, plastics antibody (plastibodies), membrane receptor, full cell, cell fragment and cell substructure, cynapse, agonist/antagonist, cell, organelle (for example microsome), micromolecule (Benzodiazepine for example, prostaglandin, microbiotic, medicine, metabolin, drug metabolite, natural products), carbohydrates and derivant thereof (comprising immunodominant carbohydrate), steroids, hormone, peptide, natural or artificial polymkeric substance, natural and artificial receptors and chemical derivative thereof, chelating reagent, crown ether ligand, supramolecular assemblies, indicator (pH, electromotive force, membrane potential, and tissue sample (micro-array tissue) redox-potential).

Fig. 3 has shown a specific embodiment, wherein planar substrates 200 has trapping region 210, this trapping region is functionalized to comprise the antibody probe at the red blood cell surface antigen of embodiment of the present invention, and this embodiment can be used for blood group to be identified, comprises and determines rare and rare blood group.First trapping region 210 is with antibody (anti-A antibody) functionalization of one or more selective binding A antigen, second trapping region 211 be with antibody (anti-B antibody) functionalization of one or more selective binding B antigen, and the 3rd trapping region 212 is with functionalization antibody (the anti-Rhesus factor antibody) functionalization of one or more and Rh factor selective binding.When needs are checked other antigen (for example check relate to 4,5 or even the rare or rare blood group of antigen more than 10 kinds), other trapping region can be provided.In use, use lancet to obtain patient's one droplet blood (for example 0.1-10 microlitre), (for example the buffering of heparin, metal-chelator (for example EDTA, citric acid/ACDA) EDTA, neodicoumarin or other blood coagulation inhibitor) etc. is oozed salt solusion and is diluted with containing anti-agglomerating agent.The blood sample of dilution is assigned in the substrate, allows the cell settlement in the blood sample also to contact with lip-deep trapping region thus.Sedimentation will take place by the deposition process under the gravity, but centrifugal, magnetic separates or other method also can be used.Such just as will be discussed further below, when the solution on the sample of surperficial 200 tops is held suitably low height, (for example be less than 10 minutes, 5 minutes or 1 minute) will take place with immediate mode in the gravity settling of the particle of right quantity, exempt the needs to these other methods.Select the magnitude of dilution, make, allow to use a plurality of cell measurements to determine every kind of cell surface antigen at a plurality of red blood cells 220 of each trapping region sedimentation.For example, can use the thinning agent that constitutes by buffer saline and anti-agglomerating agent (for example phosphoric acid buffer or Tris buffered saline and EDTA) with 1000 times of whole blood dilutions.If sample is the whole blood of dilution, lymphocyte 230 also can be deposited on the trapping region 210, minority, and these cells can be left in the basket, although in optional embodiment, lymphocyte can replace RBCS or analyzed except that RBCS.Although be shown as zone separately, trapping region 210 also can be continuous, if each regional border be known (for example, by observe suprabasil border or with the relative position of reference surfaces).Optional, trapping region 210 can be separated by physical barriers, comprises the solid walls in bin or hole, or the surface energy by the operation substrate is to produce void area hydrophobic or lyophoby.

Image recognition program based on software is used to identify red blood cell 220.The evaluation of red blood cell 220 can be used micrometron and image recognition, opens so that it and lymphocyte 230 or other object are differentiated.Image recognition program can be identified red blood cell according to the identification to various different characteristics, and these characteristics include but not limited to: the continuity at redness, high-contrast, size, circularity and edge.Image recognition program can be considered the effect of figure image focus, and can utilize and do not have the image of object to come subtracting background in the visual field.The health of cell also can be analyzed; Contrast is an indication of cell health.Based on this pair cell type and healthy analysis, selected one or more cells 220, optical force is applied on the cell 220.Optical force can be the form of light tweezer or ligh trap, in embodiments, is to use the holographic optical tweezer to apply.Power has the components that leave surface 200 orientations, if so that the power value is enough, will tend to make cell 220 from surface displacement come out (optional further on same or other direction).Usually, possible displacement force component be with the direction of the planar quadrature of substrate 200 on, but also can be positioned at or near the plane of substrate, to produce lateral shift.Must be enough to make cell 220 displacements of the antigen that the antibody that do not contain given trapping region 210 discerns if power is strong, but also be not enough to strong must shifting out and contain the cell 220 of discerning antigen, the displacement of cell 220 so, show on the surface of cell 220, not have this antigen, do not have displacement then to show on cell 220 and have cell surface antigen.This process can repeat for a plurality of cells in each trapping region 210, to obtain the significance,statistical result.This process also can be carried out abreast, so that power is applied simultaneously on a plurality of cells 220, the displacement of cell 220 is also by parallel induction.In specific embodiment, parallel processing uses HOT to finish.

Therefore, use method and apparatus described herein to produce many benefits.Use multiple probe to make to carry out the particle that accepts inspection qualitative more completely in parallel or continuous mode, for example analyze RBCs to determine blood group or hypotype.This method can be carried out in mode fast, for example in 5 to 10 minutes, particularly by using parallel processing and with the liquid level sedimentation of particle from low clearance.As what describe below, comprise 22a-25 with reference to figure, above-mentioned technology can be used for various analysis type.

As above-mentioned, optical force can be the form of ligh trap, and it can be described to have the field of force of center and radial tilting zone, makes the particle that is arranged in tilting zone have the potential energy of rising, and does not have resistance, is tending towards towards the center displacement.The center of ligh trap can be positioned on the cell 220, and ligh trap is removed with respect to substrate 200 from its reference position, with the compatibility of 220 pairs of trapping regions 210 of test cell.In this manner, the cell that is moved out of will tend to along with move at the center of ligh trap.Perhaps, the center of ligh trap can be from the certain distance of cell 220, but near must being enough to still applies optical force on cell 220.In this structure, will tend to towards the middle cadion-acceleration of ligh trap and stay the there in conjunction with weak cell.Ligh trap also can be placed to such an extent that all with cell 220 certain distance is arranged on vertical and horizontal direction, so that power will tend to make cell 220 with cornerwise mode displacement.In this any configuration, shifting out property optical force can apply with constant level providing qualitative results, or increases gradually in time, and is quantitative with the compatibility of trapping region 210 with pair cell 220.In this quantitative embodiment, can compare with known or simultaneously-measured reference value shifting out power (the relevant parameter of optical force that is applied when being moved out of trapping region 210) with cell 220.For example, can be included in the dilution with antigen coated " standard " particle, the power that shifts out of cell 220 can compare with the power that shifts out of particle.These particles are stable (for example, the glass particle of the probe from envelope antigen, antigen mimicking thing or other embodiment makes up) in the time of can being designed to standing storage.The cell 220 in given trapping region 210 or the power that shifts out of other particle also can compare with the power that shifts out of contrast or reference trapping region 210.For the purpose of analyzing, if applied enough power make not in conjunction with or the particle displacement of non-specific binding, may not need to apply the big power that the specificity that the district that is hunted down is discerned is shifted out in conjunction with particle of must being enough to.But, in some cases, may wish from trapping region, " to isolate " such cell or other particle, with further analysis or use (for example in cell culture medium, grow, nucleic acid in the analysis of cells or albumen, or the like).

If trapping region 210 has low-down affinity for given particle, and this particle is applied in the optical force displacement, and so the most confirmable is that adhesion between particle and the trapping region 210 is less than displacement force.Similarly, if 210 pairs of given particles of trapping region have very high affinity, and this particle is not applied in the displacement of optical force institute, and so the most certainly the adhesion between particle and the trapping region 210 is greater than displacement force.Therefore, if desired, the surface nature of cell 220 or other particle can be come quantitative measurement by particle tested with the compatibility of the trapping region 210 that particle is had different affinities in the dynamic range that increases.

Fig. 4 has shown the schematic cross section of the substrate 200 with two trapping regions 210 (called after 214 and 215), and each trapping region has optionally binding affinity for the different blood group antigens 225 of red blood cell (" RBC ").The antibody 300 that produces at first kind of antigen is attached on the trapping region 214 of substrate 200 by connector 310.The antibody 300 that produces at second kind of antigen (different) ' be attached to by connector 310 on the trapping region 215 of substrate 200 with first kind of antigen.In the art, for much being known to the method on glass, plastics or the metal surface with antibodies.For example, various silane coupled chemical species and with functional group or assorted functional group crosslinking chemical can from Pierce Biotechnologies (Rockford, Il) and Gelest (Morrisville PA) obtains.But as will being explained in more detail below, probe-immobilized method and the structure that is suitable for the inventive method most may propose one group of new challenge.

Fig. 5 has shown the red blood cell 220 after the sedimentation under gravity, and they contact with substrate 200 lip-deep antibody now as a result.Although between the surface of antibody 300, connector 310 or substrate 200 some non-specific binding may take place, but 300 specificitys of the antibody on cell 220 in the given trapping region at the existence of cell surface antigen 225, to cause more secure adhesion of these cells 220 and substrate 200, to such an extent as to when applying the optical force of suitable size and Orientation, those cells 220 with identification antigen 22 5 will keep, and those cells 220 that do not have identification antigen 22 5 will be to leave trapping region (214 or 215 s') direction displacement.

The bond strength of cell 220 will be determined by Several Factors, comprise antibody 300,300 ' to antibody 300,300 in the compatibility of antigen 225, the substrate 200 ' surface density, and cell 220 surfaces go up antigens density (although many cell surface antigens can be in cell membrane " floating " and concentrate on a zone).Make a concerted effort be called as " affinity " of trapping region from multiple probe-summary that target interaction (being antibody-AI) produces here.

If red blood cell 220 be added in the thin liquid level (for example 1mm to 0.5mm or still less, perhaps in addition be thinned to about 1 to 2 occupied height of individual layer red blood cell), the red blood cell general is very fast to contact (for example being less than 10 minutes) with trapping region 214-215.In order making the best combination interaction to take place between cell 220 and the trapping region 214-215, may to need some extra incubation times, this is because transposition, the morphological change in the cell or other effect of cellular antigens.Yet, association reaction reach with the All Time of the enough approaching point of balance compare with traditional immuno analytical method very short, in traditional immuno analytical method, the incubation that may need many hours and make and carry out combination by the combination of convection current and diffusion.

Particle surface analytical approach described herein can be utilized the instrumentation with limited optical force strength range.May be favourable with the intersity limitation of optical force in a scope, because use too many luminous energy (for example because cell is sacrifices consisting of an ox, a sheep and a pig solid in conjunction with getting) possibility pair cell or antibody to cause damage, increase local temperature (this may cause that fluid flows), or otherwise damage the measurement result that obtains.On the contrary, use too little optical force (together with in conjunction with weak cell) may produce the with a low credibility of poor dynamic range and experimental data.Therefore, embodiment of the present invention utilization has the substrate of trapping

region

214 and 215, and described trapping region becomes with cell 220 and has affinity in particular range.For example, scope can be 1 to 1000pN.Yet, keep enough signal to noise ratio (S/N ratio)s simultaneously for fear of light heating effect improperly, scope can select 10 to 200pN, or 20 to 100pN.In embodiments, change compatibility, so that in order to the illumination of 1064nm and have the optics strength that the HOT of the about 500mW power of each trap produces.

Fig. 6 and 7 has schematically illustrated trapping region 214 with transformation and 215 substrate 200.In Fig. 6, the surface density of antagonist 300 is adjusted: trapping region 214 has lower density, and trapping region 215 has higher density.The manufacturing of this trapping region can be finished by the amount of restriction silane, crosslinking chemical or antibody, and can comprise the reactive and non-reacted blocking agent of use.Also can change affinity by the length (for example from 5 to 50 carbon atoms) that changes crosslinking chemical.

Fig. 7 has shown the

trapping region

214 and 215 that contains antibody 300 and biologically inert part 320.Biologically inert part 320 can be for example to be connected to hydrophilic polymer polyalkylene glycol moiety for example in the substrate 200 by the silane key.Biologically inert part 320 is used to reduce antibody density, and the non-specific binding between pair cell 220 and the trapping region interacts and has minimum influence or not influence simultaneously.Biologically inert part also can be included on the zone between the trapping region, if should exist in the zone.Connector 310 also can be composition biologically inert or that have biologically inert.The surface density of antibody 310 also can by with antibody with have reactive but the reagent of biologically inert merges and changes; The for example glycocoll when using the amine reactant cross-linker, the maybe halfcystine when use sulfydryl reactant cross-linker.

Flexible particle is cell 220 for example, also can be out of shape when combining strongly with trapping region 214,215 and flattens.As a result, it may be difficult using optical force to reclaim cell.Although do not need to reclaim cell for example blood group is identified for the simple analysis method, the user may wish to reclaim cell and is used for further experiment or use in other is used.If analyzed is for example glass microsphere of rigid particles, the geometry between the flat substrate 200 and the particle of curved surface does not match interactional effective affinity between restriction particle and the trapping region.Therefore, flexibly connect thing 310 significantly by selecting its length to say so on particle curvature scale, cell 220 or other particle can combine with trapping region 214, and keep curvature largely simultaneously.For example, the length of probe can be adjusted to greater than 10nm, 100nm or 1 μ m.Perhaps, having the substrate 200 (for example micropore or kapillary) that the curvature zone is arranged on the particle rank can be used for preferably and the particle complementation that is positioned at wherein.In this optional embodiment, the light tweezer can be used for particle is positioned in the crooked zone.

Fig. 8 is the synoptic diagram of the increasingly automated system of embodiment of the present invention execution graph 1 method.Fluid box 400 has a plurality of trapping regions 214,215 and 216.In use, box 400 is equipped with sample to be analyzed, and in the anchor clamps 510 of instrument 500 it is installed on the optical instrument 500 by box 400 is positioned.Box 400 can be by manually or for more full automatic operation, come loading or unloading from anchor clamps 510 by the robot manipulation.In embodiments, anchor clamps come approaching by the hinged lid that lifts instrument 500.Instrument comprises microscope assembly 520 and optical force assembly 530.Microscope assembly comprises the optical element that is used to locate the particle that combines with trapping region 214-216 and is used to respond to the response of the optical force that particle applies optical force assembly 530.530230 utilizations and the relevant information in the position of particle on trapping region 214-216 of optical force assembly, and particle applied power, the direction of power and value tend to make in conjunction with weak or unconjugated particle displacement, simultaneously its size can not shift out the district 214-216 that is hunted down go up to the chemical entities on the particle surface have that specific probe discerns by force in conjunction with particle.Perhaps, optical force assembly 530 applies the power of variation, and microscope assembly induction particle is to the response of those power, the level of the power that computation module 540 applies when being recorded in particle displacement.In embodiments, optical force produces by holographic optical tweezer (HOT).Use HOT has the imaging of permission and the force of labor optical element has common focussing plane (because HOT trap plane can optimisedly be adjusted, to work with specific micro-assembly) advantage, and allow simultaneously a plurality of particles to be applied power, even particle is arranged in a plurality of sites of three dimensions.Micro-assembly 520 and optical force assembly are by 540 controls of common computation module.Computation module 540 can comprise processor, operation circuit, data storage media and data I/O sub-component, and can comprise personal computer or special-purpose specific hardware.The parts of computation module 540 can separate physically, even can be positioned at the outside (for example on computer network) of instrument 500, but are communicated with data, so that carry out method described herein.Different with system of the prior art, instrument 500 can apply optical force simultaneously and respond to the displacement of a plurality of particles.Computation module 540 can execution analysis, and data are submitted to the user, and analysis result and machine-readable identification symbol (for example bar code, RFID label etc.) can be interrelated, these identifiers directly or indirectly (for example in company with packing) are associated with fluid box 400.

Fluid box 400 can be configured to use the very detected biological components of small sample quantities.Use only several cell polar the earth of indication biological components to reduce required sample size, this has quickened breadboard diagnostic test.Because sample size reduces, each sample of collecting can be used for carrying out more diagnostic test.In addition, the reduction of sample size has also reduced the pain and the discomfort of sample donor.

Show in the embodiment as Fig. 9-13 that fluid box 400 can be configured to have internal reservoir, kapillary and passage, to promote the importing and the movement of sample of fluid sample.Optional, fluid box will be easy to use various routines or microfluid principle is mixed with thinning agent or other material, as the professional understood of present technique field.Shown simply, cheaply in the structure, fluid box 400 has utilized capillary the principles of science, to realize the passive chamber of flowing through in the fluid box 400.Capillary action, fluid flow direction and speed are regulated by the moistened surface character and the geometric size that change inner passage and chamber.The fluid that fluid box 400 also can depend on other power generation flows, and for example gravity, aerodynamic force, waterpower, mechanical force or electric osmose driving force comprise optical drive mechanism.

In embodiments, fluid box 400 is built into has inlet, and the user can import the small amount of sample fluid wherein.Fluid sample is blood for example, if carry out the test of blood group identification diagnosis, imports in the sample reservoir by capillary action.Sample fluid is directed to be inquired after in the storehouse, carries out sample analysis therein.Under the situation of blood group evaluation program, inquire after storehouse (being also referred to as " sample bin " in this article) and can comprise trapping region.After sample and trapping region interacted, the sample that can use HOT to analyze to obtain interacted.In addition, fluid box 400 can be configured to have one or more exit points, allows the gas effusion or allows the user further to control the order that fluid flows through container.

Fig. 9-11 has shown the embodiment of fluid box 400.Fluid box 400 can be formed by the laminated substrates of a plurality of polymerizations.By cutting technique laser ablation for example, can in the laminated plastics plate, form various heteroid endo conformations and shape.Perhaps, parts can be made by machinery, injection moulding, 3D printing, photoetch or other technology of routine.Use bonding agent, ultra-sonic welded or other technology bonding the plate and the cover plate that obtain, formation has the laminated substrates of moulding inner chamber.Cut can produce point, passage, valve, reservoir and other fluid part in fluid box 400.

Fig. 9 is the explanation of the fluid box 400 of embodiment of the present invention.Fluid box 400 comprises the box body 612 that can be formed by a plurality of polymeric layers.Bottom 614 is formed by multiple layer polymer, and interlayer accompanies adhesive agent layer.When adding upper caldding layer 616 in the process of making box 400, the chamber in the bottom 614 has formed at least one kapillary 618 and storehouse 620 is analyzed in one or more analysis.In fluid box 400, also form at least one inlet 622, be used to import sample.Outlet 24 also can be provided, when using syringe, be used for discharging gas, take out flowing of sample or control container by port.Mating holes 626 is convenient to calibrate by being placed on the alignment pin on the stationary installation (for example 510 among Fig. 8), and is used in the assembling layer different with aligning in the adhesion process.In Fig. 9, inlet 622 (and outlet) cuts out in the bottom 612 of box 400, and kapillary 618 and analysis storehouse 620 cut out in the end face of bottom.When adding top layer with sealed hair tubule 618 and storehouse 620, box 400 can upset under inlet is open.Each port of inlet and fluid box 400 can seal with film, keeps the inner passage aseptic, and feasible being merely able to imports (and taking-up) sample by hypodermic needle.

Figure 10 has shown the exploded view of the box 400 of Fig. 9.Fluid box 400 can use based on the technology of laser ablation polymkeric substance laminate and make and assemble.The laminated plastics plate is also bonding with cut, and to form laminated chip, it comprises port, passage, valve, reservoir and other fluidic component.The layout of multilayer layer compressing tablet at first use CAD software for example AutoCAD prepare.Use general laser system CO then ₂Laser cutting system is by cutting every layer from the cad file conversion.Use optically transparent contact adhesive thin layer that the layer of bottom 614 is bonded together then, form three-dimensional structure.Various plastic material comprises polyester, acrylic acid and silicone elastomer, can be used as bottom layer.Each bottom plastic layer is peeled off the two-layer pressure-sensitive adhesive that back sheet protects by siliconeization and is clipped in the middle.After the cut, remove peel ply, the part that cuts out is alignd on the mark post of one group of calibration hole 626 that cuts out in being passed in every layer with agreeing with.Layer is piled up to produce thickness.After being pressed onto them together, can the structure of assembling be compressed by the roller of laminating machine.This process may be repeated to form sandwich construction.Behind the bottom 614 that has produced kapillary with required form and storehouse, add upper caldding layer 616.Specifically, add upper cover plate 628, must be enough to cover storehouse and the kapillary that cuts out just greatly, but be not enough to cover whole substrate.Around the cover plate 628 adhesive phase 630 (be applied to the bottom and go up but have a window, be used to pass through cover plate).On the top of cover plate, apply another layer bonding agent, on bonding agent and cover plate, apply transparent polymer covering then, with the fluid box 400 of sealed bottom 614 and generation sealing.Complete fluid box 400 can have the standard size that can place on the standard microscope platform; For example about 1 inch multiply by 3 inches.Be used for fluid box 400 will analyze the material of storehouse 620 boundary should be to optical transparency, so that fluid box 400 can be shone by the microscope illumination source, so that HOT laser can be able to interact with sample.Analyze storehouse 620 and comprise one or more trapping regions (for example 210 among Fig. 2-3).

Figure 11 has shown and has had a plurality of sample analysis storehouse 620 and the fluid box 400 that is connected kapillary 618.Box 400 has inlet 631 and imports in the reservoir 634 by the sample that capillary action will contain particle on a small quantity to allow the user.Sample enters kapillary 635 and analyzes in the storehouse 620 then, allows particle deposition (being sedimentation) and interacts with trapping region.Sample can import under pressure, perhaps talks about more easily, uses the kapillary 618 with suitable dimension to import by capillary action.In above-mentioned example, the bottom surface of cover plate 628 can have the trapping region of one or more functionalization, is used for being present in conjunction with hypothesis the specific antigen of sample.With after sample contacts, use the HOT system to survey trapping region, the sample particle that has been attached on the trapping region can " be pulled out " to detect adhesion.The adhesion of experiencing is compared with the expection adhesion matrix of having set up, can confirm specific chemical entities for example cell surface antigen whether exist.Apply in the zone of functionalization trapping region in employing, with other material bag by cover plate to help prevent the generation of non-specific binding, may be helpful.Silane is to have the material that helps this purpose, and as in U.S. Patent No. 5,620, disclosed in 857, drawing in full with it at this is reference.

Figure 12 and 13a have shown the optional structure of the fluid box of embodiment of the present invention.Figure 12 has shown and can be used for carrying out grain size analysis, comprising the planimetric map of the sample box 400 that blood group is identified according to embodiment of the present invention.Box 400 comprises bottom 712.Mating holes 626 allows to position in the anchor clamps of analytical instrument 500.Inlet 622 is accepted analytic sample.When sample is added to inlet 622 the time, box 400 will draw sample by a plurality of kapillaries 628 and enter into the sample analysis storehouse 620 that can be separated from each other.Figure 13 has shown that being in part fills the box 400 of state.Simultaneously, the air of holding back will be overflowed by outlet 724.Finally, sample bin is full of sample fully.Perhaps, can be by exporting 724 suctions or applying negative pressure, with sample traction passing through box.As mentioned above, be filled in case analyze storehouse 620, particle with sedimentation with the contact trapping region.The surface is optically transparent above and/or under the analysis storehouse 620, uses the light tweezer to allow imaging and to make, and makes particle and to stand optical force by imaging, with the potential binding interactions of test with the trapping region probe.Although have the trapping region 210 that is used to analyze main blood group antigens shown in Figure 12-13, box 400 goes for analyzing extensively different particle surface.

Because the sample bin 620 of box 400 is separated from one another, so trapping region can be easily with different probe functionalization.For example, can use cross-linker molecules to make trapping region have reactivity, different antibody-solutions can be assigned in several storehouses 620 simultaneously.Perhaps, be functionalized and the individual substrate (not shown) that produces trapping region 210 can place and be installed in several storehouses 620.For example, trapping region 210 can be printed on the glass sheet, then with its installation.Perhaps make a plurality of trapping regions 210 in (for example in the storehouse 620 at Fig. 9) in the single continuous substrate.Box 400 can use the laminating method of Figure 10 to make up.Perhaps, kapillary, storehouse and inlet can be on the base material piece machine manufacturing or etch, perhaps can moldedly have the material in these chambeies.Packing ring and the lid that is fit to can be fixed on the bottom then.Lid can comprise through hole, is used for sample and enters inlet 622.

Figure 13 b has shown the fluid box of highly integrating according to embodiments of the present invention 400 able to programme.Box 400 is dilute sample and delivery reagent automatically automatically before analysis.Fluid box 400 comprises built-in operation valve and pump and inline filtrator, and it can be by remote control and driving, for example by pneumatic or motor machine path.Optional, box 400 can have internal sensor or tensimeter, is used for feedback control loop to produce data.Various different valves and pump (for example flexible membrane valve and membrane pump) can be included in the box 400, are used for self fluid-operated and control.Before being used for analytical procedure, box has the damping fluid reservoir and the pearl solution reservoir of preloaded.By keeping transfer valve and vent valve open simultaneously, by capillary action with damping fluid or pearl solution its reservoir that leads.After loading reservoir, close transfer valve and vent valve.Now, before use, box can be preserved in controlled environment for a long time (for example make and be transported to the user) with the state that loads in advance.In use, sample fluid (for example whole blood in the blood group evaluation program) at first imports in the sample reservoir by inlet (inlet 2)., blood sample can be mixed with damping fluid before and dilutes entering into test storehouse (test section 1) to V6 and pump P1, P2 by activated valve V3 suitably.Filtrator (PI) can help to remove pollutant or the aggregation in the blood sample.The blood sample that finished sample for example dilutes also can be filtered (F2 and F3) before entering the test section, to remove undesired material (for example cell or some antibody).Also can import particle or pearl that (by control P3, P4 and valve 7 to 14) has specific surface nature, with the test section in finished sample and surperficial trapping region interact.

Figure 13 c has illustrated another fluid box according to embodiments of the present invention.Box can form from the multilayer polymeric substrate.Box contains fluid layer, and it comprises test section, damping fluid reservoir, whole blood reservoir and all fluid passages, and the pneumatic control layer, and it comprises pneumatic channel (not shown) all distributions and that be used to activate each valve and pump.Box has connectivity port (being presented at the bottom near box), and the pneumatic control passage is connected by integrated manifold with external pneumatic or mechanical sources.Valve normally cuts out, and can seal the normal fluid pressure with opposing box inside.Damping fluid at first imports in the reservoir by capillary action or pressure-driven program.By shut-off valve v3 and v4 reservoir is sealed then.After blood being imported in the blood reservoir, valve V1 and V2 open.By the speed and the volume of accurate control pump, damping fluid and blood sample are mixed, and are filled in the test section safely.

In the embodiment of Fig. 9-13c, can select the bottom surface in sample analysis storehouse and the distance between the end face, with the balance between the obstruction tendentiousness of optimizing required time of particles settling, the optical instrument 500 accessible depths of focus and very narrow storehouse.For example, the lid in storehouse can be apart between 15 to 100 microns of the bottoms in storehouse, make that when with respect to gravity direction vertically and when remaining on room temperature, cell or other particle were deposited to trapping region within 10 minutes.On the other hand, lid and bottom can be at a distance of between 2 to 10 times of the diameter of analyzed particle, perhaps between 3 of the diameter of analysing particulates to 5 times.In embodiments, spacing can be adjusted, the fluid level that make to keep allow major part in the solution, the overwhelming majority or basically all red blood cells be less than sedimentation within 10 minutes.Note having different sizes and will can the height in storehouse be adjusted accordingly with different velocity sedimentations with the particle of density.

Figure 14-19 has shown the microscopy instrument 500 of compact applications HOT, and it contains several compound systems, is connected to each other in portable packing.Except comprising all required parts of operation HOT optics force applying assembly and imaging function, device also contains user's interface element.For example, system can comprise monitor, keyboard, mouse and possible touch-screen user interface display in addition.

Term holographic optical trap used herein comprises the utilization holographic method and produce optical gradient forces in the fluid of designated volume, to allow to handle particle.Usually, monochromatic light (for example continuous wave laser) is by dynamic facies model optical element (dynamic phase patterning optical element), promptly the spatial light modulator (SLM) with reflective operation is disperseed, and is modulated in real time so that the phase place of folded light beam (and having only phase place) can be used as the function of position in the light beam.The essence of optical gradient forces is the principle of clearly understanding, and can carry out by using following technology in the present invention: HOT, light tweezer, ligh trap, light vortex, light bottle, optical gradient and the electromotive force that limits of the light of broad sense more.The method of holographic optical trap is described in the U.S. Patent No. of for example submitting on February 2nd, 1,998 6,055, the U.S. Patent Application Serial No.10/1735 that 106 (" Grier ") and on Dec 12nd, 2003 submit among 395 (" Gruber "), draws at this and to be reference.

Suitable laser instrument provided as any, and Dan Se light beam is used to produce the electromotive force that limits on volume of fluid basically.Available laser instrument comprises YLF Lasers device, diode pumping YAG laser instrument and the flashlamp-LD pumped YAG lasers of solid-state laser, diode-pumped laser, semiconductor laser, fibre laser, fibre substrate (fiber-hosted) laser instrument, gas laser, dye laser, alexandrite laser instrument, free electron laser, VCSEL laser instrument, diode laser, titanium-sapphire laser, doped YAG laser instrument, doping.The Nd:YAG laser instrument of the diode pumping of operating between 10mW and 20W is preferred.That the optimal wavelength of laser beam that is used to form the optics field of force of research biologic material comprises is infrared, near infrared, visible redness, green and visible blue wavelength, and wherein the wavelength from about 400nm to about 1200nm is most preferred.Simultaneously or the multiple laser that uses the different wave length of selecting in succession with the different interactions of optimization with probe or target, also be possible.

Instrument can be accommodated in size and shape all is fit to be placed in the removable framework or the shell on the trolley that can find usually in the medical environment.In general, the trolley that is used for mobile medical science and diagnostic device can be made enough for a short time, is used for being placed on the space on sick bed or the operating room limit.For example, the trolley that has the instrument that places can be about 26 inches, about 26 inches long, 18 inches wide and about 32 inches high (height of waist), have 4 wheels and are used to move, and is perhaps littler.HOT system and microscopical shell should carry out the daily servicing and the repairing of internal part easily.User's interface element can be used as annex and is fixed on the bottom enclosure rack system, and interconnects by flexible wire or use Radio Transmission Technology.Compact system has the microscope imaging ability, includes object lens, camera and essential tube lens.

System provides illumination capability for the sample that is studied.Can provide illumination for bright-field microscope, fluorescent microscope or these two.System can comprise bright visual field illumination capability, and wherein light source is positioned at the sample top to illuminate the whole relevant visual field.Such light source can be integrated directly in the shell carriage.For fluorescent microscope, fluorescence light source can be included in the shell carriage, and is arranged to from sample below illumination is provided, and perhaps can from the beginning go up, for example the diode that links to each other with the hinged lid of instrument.Other imaging mode is included in mode commonly used in the microscopy and also can uses.

The focusing of micro objective and HOT parts is controlled by central source, so that terminal user is operated easily.Sample stage is in case after sampling receptacle manually or is automatically loaded thereon, providing controlled mobile on X and the Y direction at least.The motion of platform can be motorized, and is controlled by central source.Pre-meter systems will hold 1 inch and multiply by 3 inches and the 2 inches sample slides that multiply by 3 inches.The thickness of slide can be at 1mm in the scope of 3mm.But, should be realized that system and platform can be configured to admit the specimen preparation thing of other size and profile, comprise the box 400 of Fig. 9-13.System is airtight for maintenance, and inlet port or sample gate can be provided, and they can be opened so that sample is installed on the platform by the user, close before system operation then.The z-axial adjustment mechanism of robotization can respond to the different-thickness of different sample panel automatically, then by adjusting distance between platform and object lens from the normal moveout correction difference in thickness.Where the surface that can use robotization z-axial adjustment mechanism roughly to discern box 400 is positioned at.Can use imaging processing program to analyze a series of bright field-of-view images that obtain at differing heights then,, and before activating HOT, set final platform height with the target particles in the evaluation given area.

All lasing light emitters that link to each other with system are closed in the box-type shell.Can imagine that at least a laser instrument will be used for the HOT system, but also can provide other laser instrument to be used to operate sample, illumination is provided or monitors focus.Laser instrument and any essential transfer wire all should be contained in the removable trolley, preferably in the closure carriage/cabinet of system.Instrument also comprises computer system, is configured to control the HOT system, drives focusing system, autofocus system and user's interface element.Computer system can be used easy-to-use software, for example the LabView software systems of MicrosoftWindows and National Instruments.The power requirement of the electric parts of all of system can by with particular geographic area on any wall of using the compatible single power supply line of electrical socket provide.For example, power lead can link to each other with 110 volts of power supplys with 15 or 20 amperes.Because instrument is compact, removable and wieldy, it can be placed in traditional hygiene care or the diagnostic test room environmental.

Figure 14 has shown the synoptic diagram of the HOT system that has microscope and camera component.Diagram is duplicated from U.S. Patent Application Serial No.10/701324, and the full text of this application draws at this and is reference.Because the size and the arrangement of parts, figure has shown static system.This device has used the HOT component system, has the YAG that produces laser beam 10, and laser beam 10 arrives spectroscope 51 by diffraction optical element (DOE) 13, transmission optical devices L1, so that it can act on the sample that is positioned at zone " B ".In addition, system comprises from the imaging of top illumination, and the position under video camera 64C, send operating personnel 65 to the image of catching sample and with them.Image can be sent on the screen.This system also comprises the computing machine 66 that links to each other with various electronic units, uses to operating personnel so that Control Software to be provided.The system of this existing technology has used conventional microscope, laser instrument, illumination and camera parts, makes it quite big.Each component subsystems needs metastable installation surface, thereby requires these systems to assemble in environment stable, that work is desk-top.

Another kind of HOT system and microscope combination are presented among Figure 15 to 17, and from U.S. Patent Application Serial No.091886,802 duplicate, and the full text of this application draws at this and is reference.As what illustrate among Fig. 2 A to show, system unit and the system similarity of describing in the past.HOT laser instrument 102 produces light beam 1100 to spectroscope 30, with beam direction optical element 22, and by the analyte in the dorsal pore 28 arrival vessel 10 of condenser lens 12.The major part of this system is the microscope that shows among Figure 16.Microscope 60 is to fix greatly, for providing carriage from light source 61, the object lens 56 of top and the HOT system that includes in the nosepiece 54.The HOT laser system links to each other with outside lasing light emitter by optical fiber 150.As shown in Fig. 2 C, HOT subsystem 50 provides carriage 52 loading the several times mirror, and one be two relay len TO1 and TO2 then, optical element 51, optical channel 53A and 53D, and spectroscope 58.

Linking to each other with microscope 60, among these figure is other parts but be not presented at, for example computing machine, camera, sample stage, move and focusing and laser generator.Must be around the microscope 60 of traditional scale the subsystem of interconnective a plurality of complexity, make total system not move.

Be displayed among Figure 18 according to the removable HOT microscopy instrument 500 of the compact of illustrative embodiment of the present invention, schematically illustrating of its building block is presented among Figure 19.Instrument 500 comprises the shell 902 of the service part that is used to hold instrument.Shell can be with any in light weight, durable and can prevent that the rigid material that the laser of wavelength that HOT uses sees through from making.The structure 904 that shell preferably can be positioned on belt wheel is for example on the trolley, or as its part.Trolley 904 should have at least two wheels 906, is preferably 4 wheels, so that stable.

Instrument 500 can be constructed such that bigger electric parts for example computing machine 908 and laser generator 910 and 912 and any other big electric parts power supply for example, can be placed in the base section 914, to keep lower center of gravity, increase the stability of removable trolley.Such parts if they outwards do not launch laser, can not need to be closed.The supply of the needs of printing opacity, limited locomotivity or laser and the luminous component that should be closed can be placed in the top 916 of shell 902 sealings for the purpose of safety.Shell can be configured to have closable opening, and for example lid 918, and the user can pass through its internal part near instrument 500, and places the sample box 400 of wanting instrument to handle.Perhaps, pallet can be stretched out automatically and regained by instrument 500, is used to accept box 400 and its is suitably placed be used for analyzing.Other mechanism that is used for sample box is loaded into instrument also can use, and as known in the art, comprise rotatable mechanism and adds the slit of spring.Opening 920 by movably lid 918 protections should be enough big, so that operating personnel can be placed on sample box 400 in the sample area 922 of sample stage 924.Lid 918 should comprise safety switch (kill-switch), and when opening with convenient lid, device can not be operated insecurely.Location, sample stage 924 adjacent lid below.Plateform system can be to have H117XY platform that is used for Nikon TE2000U and the Prior ProScan II controller that focuses on the button circle.Platform 924 and focusing knob can have scrambler.Place object lens 926 adjacent sample stage 924 belows, is used for the trapping region imaging on the box 400, and is used to form holographic optical trap.Object lens 926 can be Nikon CFl Plan Apo 40X Air, NA=0.95, WD=0.12-0.16.Rotating nosepiece can be Nikon TE 200 rotating disks.

HOT subsystem 928 is positioned at adjacent object lens 926 belows, can be from BioRyx 200 (Arryx, Inc.) transformation use.The parts of HOT subsystem, comprise diffraction optical element (DOE), luminous point block device (spot blocker), collimator and other relevant lens and relay optics, can make up according to the given monochromatic wavelength that is used to produce optical force (for example 1064nm).The DOE that uses can be spatial light modulator (SLM), for example from Boulder NonlinearSystems (model P512-1064).SLM can have dielectric coat, is used for high power operation.The central spot block device also can be used to block zero order reflection and the high-order district band from SLM.The HOT system has the emission port that points to sample stage.The ligh trap light that dichronic mirror 927 can be used for sending from HOT subsystem 928 imports box 400, allows other illumination and imaging wavelength to propagate by light path simultaneously.

The optical fiber 930 that is used for laser instrument is connected to lasing light emitter can be the Oz optical fiber.The HOT laser instrument can be the IPG 10W fibre laser that has the FC connector.In addition, collimator can be used for being connected to the HOT subsystem, for example the FC2O-IR-T/A of miniature laser system.The HOT subsystem also links to each other with computing machine 908 (not showing), is used to control diffraction optical element.

Other parts comprise light source, can be included in the shell 902.A kind of means of illumination that is used for compact system has used bright visual field light source 932.For the top from sample area 922 provides illumination, a kind of compact structure that is used for bright visual field illumination 932 is in order to place it in removable lid 918, as shown in Figure 18.Bright visual field illumination 932 like this can comprise the led light source that output wavelength is about 525nm, and it has and is used to guarantee to throw light on uniform scatterer 934.

Optional light source, or preferred additional light source can comprise the fluorescence excitation illuminator 936 that is placed on sample area 922 belows.Fluorescent exciting propagates into dichronic mirror 940 by exciter filter 938.Fluorescent illumination can be the part of sub-component, for example with fall to penetrating the Exfo Excite illuminator (for example DAPI-FITCTR (Nikon) in the TE2000U cube) that the fluorescence cube links to each other.Perhaps, the light source that is used for fluorescence excitation also can be incorporated into and be arranged in shell 902 lids 918 sample area top.

Another kind of optional component subsystems is a laser alignment guidance system (LAG) 942, is used for the automatic focus of secondary station 924 with respect to object lens 926.It can be the diode of operating at 635nm that LAG aims at laser instrument 942.Laser instrument can be by lasing light emitter 912 energy supplies that separate, at a distance, and laser can propagate in the main shell 902 by optical fiber 944.Perhaps, laser instrument 942 can get for a short time and enough directly be assembled in the shell 902.Aim at laser instrument 942 and directly or by photoconduction (for example optical device) laser beam 946 is shipped to spectroscope 948 for example on the dichronic mirror, then laser can be propagated by being positioned at the emission optical filter 950 in sample stage object area the place ahead.

The subsystem component of another kind of usability that can backup system is a camera 952, is used for collecting and the image of the sample that transmission is used to analyze by common computation module 540, or sends the operating personnel of device to.Camera can be charge-coupled device (CCD) (CCD) or equivalent.Camera is preferably mounted in the shell 902, so as with the position alignment of the sample area 922 of sample stage 924.The part optical device of camera can comprise tube lens 954, with image focusing on the camera surface.The example that is suitable for the camera of system is Qimaging RetigaExi color cooled.Video output can see on graphoscope 956 that it also can show other control, monitoring and data analysis characteristics.

System can be by user interface 960 control, and the user interface can comprise LCD user's interface screen 956, is used for text is passing the user, and touch screen capability can be provided.Perhaps, it is several by key control 962 that user interface 960 can provide, the automatic processing of start-up system.Optional in addition, but do not show, can be computer keyboard, be used for and the computing machine installed software direct interaction that can control various subsystems of the present invention.

All subsystems of compact HOT/ microscopic system can be connected with the structure of belt wheel by conventional mechanical securing member known in the art with individual component.In addition, can add conventional shock mitigation system.

Fig. 8 and 18 instrument 500 can increasingly automatedly be operated.Figure 20 is the process flow diagram of the illustrative controlling schemes of implementation on instrument 500.Although be described in a continuous manner, the many or all operations of this embodiment can be carried out on a plurality of particles in a parallel manner by instrument 500.Sample is loaded on the box 400 and with box according to after in same zone 922, and after allowing particles settling and contacting trapping region 210, can begin analysis.Analysis can start (step 1200) by manual button (physically or pass through GUI).Perhaps, microscope assembly (Fig. 8 520) can detect the existence of box 400 with computation module (Fig. 8 540), follows the tracks of particle and finishes their subsidence stage to determine when particle, begins the optical force analysis then.If desired, can apportioned amount the outer time, make particle and trapping region 210 further combined with.The temperature of box also can be controlled automatically, to optimize integration or to guarantee unanimity as a result.Instrument will be autofocusing at (step 1205) on the trapping region.Instrument is found one or more particles automatically then, therefrom selects one or more analyze immediately (steps 1210) then.Optional, whether instrument will meet certain criteria group (1220) to determine it to the particle imaging.For example, in the blood group identification and analysis, it is to lose time preferably the time that lymphocyte is applied optical force, when the worst, if lymphocyte can not be come out respectively, will make distortion as a result.Instrument can use Figure recognition program, size and shape data (for example to the object in red blood cell or the concrete outward appearance scope of other particle detection), absorption data (for example redness in the red blood cell), fluorescent microscope data (for example existence of dyestuff or labelled antibody), other spectroscopic data or other non-spectral measurement to carry out this mensuration.Instrument will start light field gradient (step 1230) automatically near selected particle then, particle is applied potential displacement power.By during applying power and/or, and respond to particle thus, can measure compatibility or the affinity of trapping region particle to any displacement (step 1240) that masterpiece goes out to respond afterwards to the particle continuous imaging.As mentioned above, because trapping region contains the probe of specificity in conjunction with target analyte thing class, therefore can determine the existence of these analytes on the particle surface.For follow-up analysis, instrument will write down the position of each analyzed particle with respect to the trapping region of its institute's combination in calculator memory.The feature of trapping region can be from machine-readable mark, from box is determined with respect to the understanding of the direction of reference mark (box also can with key so that insert with specific direction) or other method.Repeat selection and the analysis and the optional automatic focus step of particle, stop threshold value (step 1250) up to reaching.Threshold value can be, the giving determined number, elapsed time maximal value or surpassed the statistical measurement (for example having obtained to be lower than the standard deviation of particular value) of error of the particle of giving determined number, analyzing in each trapping region of for example analyzed altogether particle.In addition, can be to the sampling of a plurality of visuals field in single trapping district 210 or a plurality of trapping region 210.In order to compensate positioning error, each visual field changes can carry out automatic focusing program.Analyze data then and be and pass the user (step 1260).Under the situation of blood group evaluation or other clinical practice, analysis can comprise the value on blood group or other the clinical relevance.

Figure 21 schematically illustrates an embodiment, comprises using derived from the antigen-binding proteins fragment bag of IgM by the method for surface, for example trapping region 210.For example hydrochloric acid 2-mercaptoethylmaine, dithiothreitol (DTT) or 2 mercapto ethanol are handled with disulfide reducing agent will to contain the solution of IgM molecule.For example, (Rockford, ImmunoPur IgM fragmentation kit IL) comprises instructions and the reagent that is used for various cleavage methods from Pierce Biotechnology company.By the relative concentration of control recovery time, temperature and reductive agent, these reactions can produce the antibody fragment with specified molecular weight.These fragments can be fixed on matrix then for example on glass or quartz slide, particle, three-dimensional structure, resin or the gel.Afterwards, the matrix of acquisition will have special compatibility to the antigen of IgM identification.In one embodiment, matrix is the zone of glass slide or glass slide.Immobilization can use various connection chemical substance to carry out, wherein several can (Rockford IL) obtains from Pierce Biotechnologies company.For example, 4-[N-maleimide ylmethyl] cyclohexane-1-carboxylic acid sulfosuccinimide ester (Sulfo-SMCC) can be used for sulfydryl on the fragment and the amino silane on the glass matrix crosslinked.A plurality of such matrix (be positioned at the zone on the absolute construction, or be positioned at the zone in the adjacent or non-conterminous zone of continuous structure jointly) can be with fragment bag quilt, to produce the different zone of compatibility.Cell can be exposed to a plurality of zones.By interactional intensity or the relevant parameter of relative intensity between observation and cell and the surface, can identify lip-deep antigen.In one embodiment, cell is a red blood cell, antigentic specificity calmodulin binding domain CaM identification blood group.

As above-mentioned, above-mentioned method and apparatus not only can be used for blood group to be identified, and can be used for biology, biological chemistry and the chemical analysis program of many other types.For example, replace analyzing red blood cell, people can analyze lymphocyte, cultured cells, bacterium, biopsy samples etc.In each trapping region, can use the probe of one or more types; For example, trapping region can have the potpourri of three kinds of antibody, and they have produced at certain type the stem cell or the specificity of cancer cell when merging.It is contemplated that and use the protein combination ligand analysis of same-type to test syphilis, HIV or hepatitis B cell surface antigen.Analyzed cell can be purified (cell sorting that for example uses fluorescent activation) before use.In addition, in single substrate, can test many different samples, perhaps can in single substrate, carry out the many different tests of simple sample.

Although use embodiment of the present invention can carry out polytype analysis, they much belong to three types that describe below: directly analysis, indirect analysis and competitive analysis.

Directly analyze to comprise how the probe molecule that relates to direct mark is observed with the interactional parameter of target.In relating to the embodiment of observation to the response of optical force, label can be optically active substance, for example particle.For example, the diameter that has an immobilized antibody is that the glass of 1-100 micron or plastic bead can be used for surveying specificity or non-specific binding to suprabasil target analyte.Analyte can covalently or non-covalently be attached in the substrate.Probe and label also can be merged into the single creature entity; For example express natural or genetically engineered cell as the surface protein of probe.For the blood group identification and analysis, can use the general antigen of all red blood cells is adhered to red blood cell from the teeth outwards, the particle that has blood group antigens contacts with red blood cell, and the response of the optical force of combined particle is used as parameter, and the pair cell surface characterizes.Figure 22 a and 22b have shown the example of direct analysis.Analyte antigen 225 is attached to trapping region 210, and the particle 220 that has the probe 300 of sessile antibody contacts with trapping region 210 and combination.Binding interactions if any, its intensity uses the optical force that applies to measure as mentioned above.If antibody 300 identification antigen 22s 5 as showing among Figure 22 a, will need just can make particle 220 displacements than higher power required under the optional situation that shows among Figure 22 so, under latter event antibody 300 nonrecognition not associativity antigen 22 5 '.Perhaps, target can be an antibody, and the probe on the particle can be an antigen.

On the contrary, indirect analysis comprises the character that detects the second kind of probe that combines with first kind of probe.Similar to direct analysis, if second kind of suitable mark of probe quilt, parameter can be the optical force of second kind of probe.Second kind of probe can be or be specific to the label (particle or other) of first kind of probe the chemical part of first kind of probe.Among the embodiment that in Figure 23 a, shows, unlabelled monoclonal mouse antibody 310 can be used as first kind of probe, and contact with fixing target antigen 225 (for example directly be fixed in the substrate 200 or be incorporated on the immobilized cell), second kind of probe can be that probe portion 300 is the anti-mouse antibodies of polyclonal rabbit with the particle 220 of probe portion 300 marks of combination.Second kind of probe particle 220 can contact with first kind of probe antibody 310, makes its combination, and by observing particle the specificity binding interactions tested in the response of the optical force that applies.Shown in Figure 22 b, lack the specificity association reaction and make particle 220 than low optical force level bottom offset.In another embodiment, HIV (human immunodeficiency virus) antigen is incorporated in the substrate.Substrate is exposed to the patients serum.If HIV antibody exists, they will combine with suprabasil HIV antigen.By remove patient's serum with buffer solution for cleaning, stay the antibody of intact basically specificity combination.The particle that is marked with the antibody (for example rabbit anti-human igg antibody) of specificity at human antibodies is imported, and by applying the combination of optical force test specificity.Such analysis is particularly advantageous under the situation of frequency multiplexing technique, because can use particle only a kind of or the only probe mark of several types to come the much bigger analyte molecule of amount detection.For example, the various antigen of human pathogen can be fixed in the trapping region separately, and add the patient's sample (for example blood or serum) that contains antibody in the mode that can cause a plurality of interregional fluids to be communicated with.After being enough to allow the time of antibodies, can adding the solution of the particle that is marked with anti-human antibodies (antibody of for example anti-IgG, anti-IgM, anti-IgG and IgA or its combination), and allow it to be attached on the trapping region.Can use optical force (comprising parallel optical force) to survey the response of particle then, to obtain about the pathogen exposure in patient's past or the conclusion of immune state.By using the grain type of 1 to 4 kind of mark, such technology can parallelly be applied to several, tens kinds or even hundreds of kind antigen.Because the grain type that needs is few like this, it is simple relatively that every kind of grain type is carried out mark, for example uses to have different exciting or the fluorophore of emission wavelength, to understand the result.In another example, can determine patient's antibody distribution situation at a plurality of antigens, it is irritated to be used for diagnosis.

Competitive analysis comprises and detects with the analyte of combination or report the relevant incident of replacement of thing or other molecule.In this U.S. Patent No. 5620857 of drawing Weetall for reference etc., the method that makes with being at war with property of light tweezer immunoassay is disclosed.But disclosed analysis is preferably carried out in a continuous manner, produces less data point.Therefore, the information that can obtain in preset time is few, and the result's of acquisition corresponding confidence level is lower.On the contrary, competitive analysis embodiment described herein can be carried out in a parallel manner; For example by using HOT to test a plurality of particles simultaneously.As a result, can obtain the result that the more data point is used for higher confidence level, and/or can test more eurypalynous target.Figure 24 and 25 has illustrated the structure of use optical force with competitive some analyses that combine.

As shown in the reaction scheme of Figure 24, in trapping region 214 and 215, formed the ternary sandwich structure.The sandwich probe 300 that comprises the substrate combination ', Covalent Immobilization at occupy-place molecule 227 on the trapping region of substrate and Covalent Immobilization the particle bonding probes 300 on particle 220.Occupy-place molecule 227 can have binding ability to probe 300 and 300 ', and the target analytes in this binding ability and the target sample is same or similar at least.Trapping region 214 and 215 have probe 300,300 ' and specificity at the occupy-place molecule of different analytes 225.For example, the occupy-place thing can be the target antigen of recombinant forms.In addition, the probe 300 of substrate combination ' can have different compatibilities or specificity to all types of target analyte, and can become ternary complex with the suitable occupy-place molecule 227 and probe 300 assembled in advance of particle combination.When adding sample, if analyte molecule to probe 300 and 300 ' have compatibility, they will tend to replace occupy-place molecule 227, reach balance through reasonable time, the degree of balance depends on relative binding constant and mass action.Interact as fruit granule 220 and the mode of trapping region,, have only some occupy-place molecules will tend to be replaced by competitiveness the analyte of intermediate concentration 225 times with multivalence.It is detected that these intermediatenesses will can be used as the difference of the amount of replacing the required optical force of particle.

Figure 25 has shown the reaction scheme of competitive analysis, and wherein 227 pairs of probes 300 of occupy-place molecule and 300 ' compatibility are lower than analyte molecule 225.

Trapping region

214 and 215 have probe 300,300 ' with the occupy-place molecule of specificity at different analytes 225.As a result, if the occupy-place molecule is replaced by target analyte 225 competitiveness from the sample that adds, the binding interactions between trapping region 215 and the particle 220 will increase, and this can be detected as the increase of replacing the required optical force of particle.If the analyte of 214 pairs of uncrossed reactions of trapping region has compatibility, will not be subjected to disturbance to the adhesion of sandwich structure in this zone.In other analysis embodiment, this method can be carried out on a plurality of particles (for example 3,5,10,100 or more) in a parallel manner.

In the optional embodiment of the competitive analysis of Figure 24-25, can add the target sample, mode is to make it only contact with a target region or contact with a plurality of target regions a time.For example, trapping region can be arranged in the hole independent, that separate basically of sampling receptacle, the generation of sandwich structure is by suitable occupy-place molecule 227 being joined in the trapping region of the antibody 300 ' that has the substrate combination, clean, adding the probe 300 of particle combination and cleaning once more.Structure is prepared to use then.

But if trapping region is a single suprabasil simple region in the single fluid structure, it is obviously complicated more that situation will become.In this case, the fluid of adding will be communicated with a plurality of trapping regions.As a result, the occupy-place molecule may need the time period longer with the trapping region incubation, just can make them attempt a plurality of probe type and combine with the probe that is fit to.Because particle 220 tends to must be enough to sedimentation greatly, they will tend to not visit a plurality of trapping regions 210.In order to overcome this problem, the probe solution of particle combination directly can be assigned to the top of suitable trapping region 201.In order to prevent diffusion and cross reaction, can form temporary transient pore structure.Temporary transient pore structure can be to be pressed in the substrate time to look and be enough to the dropper of sedimentation head.Perhaps, the array of through hole can contact with substrate, distributes particle and make its sedimentation before via-hole array is removed.In the optional embodiment of another kind, trapping region is arranged in the dolly dimple of substrate, and adds the fluid that contains particle of certain volume, the volume that this volume comprises less than Kong Suoneng (illustrating that surperficial energy effect can allow the forward meniscus).The reagent and the sample that add larger volume will flood a plurality of depressions and enter all trapping regions.

Under any circumstance, the sandwich array structure that is communicated with of the fluid of acquisition can be used for multiple analytes in the competitive binding analysis solution (for example can measure 3,10,100 or even 1000 kinds or how different analytes).In specific embodiment, sandwich array is the protein group sensor, can be for diagnosis or research purpose detection of biological tens to several thousand kinds of different albumen in the product that imitate.Can increase dynamic range by using a plurality of sandwich structure types (for example mark or location in the trapping region that separates) with known location.A plurality of sandwich structures can be built in different analyte concentration scopes and respond.For example, the relative compatibility of occupy-place molecule and probe is different between a plurality of trapping regions of structure, is used to detect the same analyte in the variable concentrations scope.

Embodiment 1, use robotization microscope and optical force are carried out the blood group identification and analysis

The user opens sample gate, and sample strip is placed on inside.The user closes door and pushes " begin test " on the touch-screen.Object lens and imaging plane therefore are from the position far below (about 1mm) lid (cover plate) bottom.Microstat moves to its limit to find them, moves to then the center corresponding to first trapping region on the sample strip.Software is opened red automatic focus laser instrument, and guides the focus knob rotation, with the speed rising object lens more than the 100 μ m/s.The view camera image is sensed red flash of light up to system, and expression laser-bounce goes out the basal surface of cover plate.Use suitable algorithm to determine when that the reflection of laser reaches its peak value.The automatic focus laser instrument is closed then, opens the green LED illuminator, adjusts object lens, so that imaging plane approximately is positioned at the centre (based on the position of basal surface and the coverslip thickness of expectation) of cover plate.Digital camera obtains background image, and it will be deducted at any successive image that is used for the red blood cell evaluation.Guide focusing then, make imaging plane move to about 20 microns place at most, estimating position below, cover plate top.Then focus is raise more lentamente, camera images gets access to the image of variation.Algorithm will determine when that red blood cell has entered focus and left focus then once more.In case surpassed correct plane, focus will be returned this plane, and carry out Flame Image Process to determine where there is complete RBCs in the sample.According to size, circle and edge continuity, identify available RBCs.With the too approaching cell of other cell or be arranged in the cell in the inaccessiable zone of ligh trap or the cell approaching, be left in the basket with the edge peptide of camera images.Remaining RBCs once test 4 (by we can with the laser power restriction of forcing).Ligh trap is placed on the center of each cell, and is parallel to cover plate with the speed of about 7 μ m/s and moves about 20 μ m, but ligh trap is placed on several microns of cell tops, makes the normal direction on the common motion of experiencing of cell and surface slightly tilt.

Repeat same step, all testable RBCs are examined in the visual field.If RBC is not retained in the short distance in its original position after moving, it is considered to be bonded on the surface.Based on the binomial distribution statistical test, whether the software decision moves to another visual field on the identical trapping region, or does not accept the result and move to next trapping region.If the former selects the position in the next visual field according to the helical pattern around the initial visual field, so that minimize the degree of approach at mobile time and maximization and trapping region center.According to statistics, if desired, can detect many visuals field to the single trapping district.Each when platform is moved to the different visuals field, use automatic focus program usually based on image, even platform only is to move in same trapping region, automatic focus is estimated only 10 microns beginnings under the position from RBCs.In case trapping region is finished, platform moves to next trapping region center with the visual field, and reuses the automatic focus based on image.In case collect statistics from all trapping regions, screen displaying goes out the result, LED and laser instrument are closed, and platform moves down 1mm and turns back to first trapping region center, and the user can freely open sample bin and fetch sheet.

In optional embodiment, the disclosed method that is used for surface analysis can be used as computer program and carries out, and uses with computer system.Such execution can comprise and a series ofly is fixed on the computer instruction that can touch on the medium on the computer-readable medium (for example floppy disk, CD-ROM, ROM or hard disk) for example, or for example be transferred to the computer instruction of computer system by modulator-demodular unit or other interface device with the communication adapter that network is connected by media.But media can be a catalyst to be situated between by (for example optics or analog communication line), or the media of carrying out with wireless technology (for example microwave, infrared or other transmission technology).The instruction of series of computation machine has embodied all or part function that this paper front frame of reference is described.The professional in present technique field will recognize that these computer instructions can be write as with many program languages, are used for many computer organizations or operating system.

In addition, such instruction can be stored in any memory storage, for example semiconductor, magnetic, optics or other memory storage, and can use any mechanics of communication transmission, for example optics, infrared, microwave or other transmission technology.Estimate that such computer program can be used as removable media (for example hard-pressed bale dress software) distribution that has the printing of enclosing or e-file, pre-install (for example in the ROM or hard disk of system) with computer system, or distribute from server or BBBS (Bulletin Board System)BS by network (for example internet or WWW).Certainly, certain embodiments of the present invention can be used as software (for example computer program) and the combination of hardware realizes.Other embodiment of the present invention as hardware completely or completely software (for example computer program) realize.

Its purpose of described embodiment of the present invention only is exemplary, obviously can carry out a large amount of changes and modification for the professional in present technique field.Change that all are such and modification all are intended to be included in the scope of the invention of appended claims qualification.

Claims

1. analyze one or more methods that respectively have the surface nature of surperficial particle, this method comprises:

The suspending liquid that will have a plurality of particles is incorporated in the sampling receptacle with at least one trapping region, and described trapping region has given compatibility to chemical species;

At least a portion in a plurality of particles is contacted with at least one trapping region, and to allow between this at least one trapping region and this at least a portion particle binding interactions taking place, described binding interactions is relevant with the surface composition of particle;

Optical force is applied in this at least a portion particle two simultaneously at least, and this power tends to cause the response of particle, wherein said response depend on the binding interactions between particle and at least one trapping region existence, do not exist or degree;

Respond to of the response of at least two particles to optical force;

The required response of usability determine the chemical species between particle surface and trapping region existence, do not exist or measure.

2. the process of claim 1 wherein that applying optical force comprises use HOT.

3. the process of claim 1 wherein that contact comprises a plurality of particles are contacted with the single trapping district, and respond to of the response of a plurality of particles optical force.

4. aforesaid right requires each method, also comprises:

First particle is contacted with first trapping region that first kind of chemical species is had the specificity binding affinity, allowing between first trapping region and first particle binding interactions taking place, described binding interactions is relevant with the existence of the molecule of first kind of chemical species between first particle surface and first trapping region;

Second particle contacted with second trapping region that second kind of chemical species is had the specificity binding affinity, allowing between second trapping region and second particle binding interactions taking place, described binding interactions is relevant with the existence of the molecule of second kind of chemical species between second particle surface and second trapping region;

First and second particle are applied optical force, and this power tends to cause the response of particle;

Respond to first and of the response of second particle to optical force;

The required response of usability is determined in the existence of first kind of chemical species between first particle surface and first trapping region and second kind of chemical species between second particle surface and second trapping region, is not existed or measure.

5. aforesaid right requires each method, also is included in particle applied to identify particle before the power.

6. the method for claim 5 further comprises the evaluation particle, selects first group to identify particle, applies optical force to first group of particle, selects second group of particle, and applies optical force to second group of particle.

7. aforesaid right requires each method, comprises that also repetitive process obtains threshold value to reach data.

8. aforesaid right requires each method, and wherein said power has normal direction is left trapping region in trapping region and direction component.

9. aforesaid right requires each method, and wherein said power has the component on the plane that is parallel to the trapping region qualification.

10. aforesaid right requires each method, and wherein said power tends to make unconjugated particle to be displaced to the position that separates with trapping region with normal direction in first direction on the plane that trapping region limits and with second direction that is parallel to the plane that trapping region limits.

11. aforesaid right requires each method, wherein response comprises from trapping region and shifts out particle.

12. aforesaid right requires each method, wherein particle is a cell.

13. aforesaid right requires each method, wherein chemical species is a cell surface antigen.

14. the method for claim 13, wherein particle is a red blood cell, and at least one trapping region comprises the cell surface antigen specific probe.

15. the method for claim 14 is wherein measured and is used to further determine blood group.

16. the method for claim 15, wherein blood group comprises the cell surface antigen of measuring more than three kinds.

17. the method for claim 16, wherein blood group is rare blood group type.

18. the method for claim 14, its middle probe are one of antibody, antibody fragment, peptide part and aptamers.

19. aforesaid right requires each method, wherein contact comprises that the permission particle passes the solution sedimentation.

20. the method for claim 19, wherein contact comprises that also use gravity causes particles settling.

21. the method for claim 19, wherein contact comprises that also the use optical force causes particles settling.

22. the method for claim 19, wherein optical force is applied on first particle, and simultaneously first particle is coated over basically in the same solution with its institute's sedimentation.

23. aforesaid right requires each method, wherein contact also comprise with solution be incorporated into part by first surface and part by in the volume that is limited with first surperficial separated lid, at least one of surperficial and lid is optically transparent basically.

24. the method for claim 23, wherein lid and first surface distance of being separated by for first particle diameter more than 1 times and be less than 1000 times.

25. the method for claim 23, wherein lid and first surface distance of being separated by for first particle diameter more than 2 times and be less than 20 times.

26. the method for claim 23, wherein lid and first surface distance of being separated by is in 15 microns to 500 microns scope.

27. the method for claim 23, wherein lid stops up even as big as being unfavorable for the space that first surface and lid are limited with the distance that the surface is separated by, but is small enough to allow the sedimentation in less than 10 minutes of first particle.

28. aforesaid right requires each method, wherein applies optical force and comprises use holographic optical tweezer.

29. aforesaid right requires each method, wherein first trapping region comprises a plurality of first kind of target part being had specific probe molecule at least, the structure of described probe molecule makes that the particle that has a plurality of first kind of target part on it is had affinity, select the structure of probe molecule, shifted out by the power in the particular range with permission.

30. the method for claim 29, wherein particular range is 1 to 1000pN.

31. the method for claim 30, wherein particular range is 10 to 200pN.

32. the method for claim 31, wherein particular range is 20 to 100pN.

33. each method of claim 29 to 31 also comprises

Second trapping region, comprise and a plurality of second kind of target part had specific probe molecule, described probe molecule is configured from the teeth outwards, makes the particle that has a plurality of second kind of target part on it is had affinity, select the structure of probe molecule, shifted out by the power in the particular range with permission.

34. the method for claim 33, wherein particular range is 1 to 1000pN.

35. the method for claim 33, wherein particular range is 10 to 200pN.

36. the method for claim 33, wherein particular range is 20 to 100pN.

37. each method of claim 29 to 36, wherein the probe molecule of trapping region is configured to shift out in particular range by changing the length of the coupling part that connects surface and probe.

38. each method of claim 29 to 36, wherein the probe molecule of trapping region is configured to shift out in particular range by changing the surface density of probe.

39. the method for claim 38 also comprises and uses non-binding surface portion that probe is separated, thereby obtains first kind and second kind of density.

40. the method for claim 38 comprises that also the cell surface antigen of pair cell characterizes, wherein surface density is selected based on the estimated density of the cell surface antigen on the cell surface.

41. aforesaid right requires each method, wherein apply optical force and comprise that the center with ligh trap is set in the certain distance of particle, the energy of position of particle is remarkable in the surface, in the center of ligh trap minimum, so that shifted out the surface as fruit granule by the optical force of trap, particle will move to the center of ligh trap.

42. aforesaid right requires each method, wherein contact comprises by passive stream first particle is imported in the solution.

43. aforesaid right requires each method, also comprises changing the value that is applied to the optical force on the particle, and capable value is increased in time, and record shifts out particle the amount of the required power in surface.

44. the method for claim 43 comprises that also power and the reference power that will record compare, existing, not existing or measuring with first kind of chemical species on definite first particle surface.

45. aforesaid right requires each method, wherein induction comprises use robotization microscope.

46. the method for claim 45, wherein applying of optical force is robotization.

47. aforesaid right requires each method, also is included in to analyze to use sampling receptacle to be selected from the operation of diluted sample and filtration before.

48. aforesaid right requires each method, wherein applies optical force and comprises use HOT.

49. the existence of multiple haemocyte surface antigen, the method that does not exist or measure in definite patient's the blood sample, this method comprises:

The dilute blood sample;

The blood sample of dilution is contacted with first and second trapping region, and first trapping region is functionalized so that first kind of blood cell antigen had special compatibility, and second district is functionalized so that second kind of blood cell antigen had special compatibility;

To have first optical force that shifts out component be applied to first the district at least one cell on, the size of first power is adjusted to is enough to not exist on it cell of first kind of blood cell antigen to shift out from first district, but still is not enough to and will exists the cell of first kind of blood cell antigen to shift out on it;

To have second optical force that shifts out component is applied at least one cell in second district, the size of second power is adjusted to be enough to and will not to exist the cell of second kind of blood cell antigen to shift out from second district on it, but still is not enough to will exist on it cell of second kind of blood cell antigen to shift out; And

The detection cell shifts out still from the district and does not shift out.

50. the method for claim 49, wherein contact further comprises the sedimentation under gravity of permission cell.

51. analyze the device that the target part on one or more particles exists, do not exist or measure, this device comprises:

Substrate;

Be positioned at suprabasil first and second trapping region, each trapping region contains a plurality of probe portion, wherein construct the affinity of first and second trapping region, make the particle that trapping region is had than high-affinity in the presence of the displacement optical force, tend to stay, tend to by displacement in the presence of the displacement optical force and trapping region is had the particle that hangs down compatibility, optical force is positioned at particular range.

52. the device of claim 51 also comprises:

Lid, wherein at least one in bottom, trapping region and the lid is optically transparent;

The entrance and exit of the volume that limits by trapping region and lid;

Be used to separate the spacer of lid and trapping region, after its size makes in a single day the solution that contains mammalian cell is imported into, most of cell will deposit on the trapping region in 5 minutes or shorter time, and the size of spacer also allows by passive stream solution to be imported in the volume that is limited by trapping region and lid.

53. the device of claim 52, wherein lid is optically transparent to infrared light.

54. the device of claim 51 or 52, wherein particular range is 1 to 1000pN.

55. the device of claim 54, wherein particular range is 10 to 200pN.

56. the device of claim 55, wherein particular range is 20 to 100pN.

57. being configured to blood group, each device of claim 51 to 56, wherein said device identify.

58. each device of claim 51 to 57, wherein said device can be operated the sample preparation step that is selected from diluted sample and filtration with execution.

59. method comprises:

IgM solution is cracked into the solution of the fragment that contains a plurality of antigen binding structural domains;

Binding structural domain further is cracked into the antigen-binding subfragrnent; And

Subfragrnent is combined with substrate to produce the trapping region of specificity in conjunction with first kind of antigen of identification.

60. the method for claim 59 also comprises producing second trapping region of specificity in conjunction with second kind of antigen of identification.

61. the method for claim 60 further is exposed to particle first and second trapping region, and the particle and the compatibility in zone are graded, to identify the antigen that exists on particle surface.

62. the method for claim 61, wherein particle is a cell.

63. the method for claim 60, wherein the grading of compatibility comprises particle is applied optical force.

64. the method for claim 63, wherein power is applied simultaneously on second particle.

65. the method for claim 64, wherein power uses HOT to apply.

66. determine the existence of a plurality of molecules in the solution, the method that does not exist or measure, this method comprises:

Substrate is provided, and described substrate has first trapping region that has first kind of immobilization probe at least, and second trapping region that has second kind of immobilization probe;

Trapping region and analyte solution are contacted under the condition of analyte molecule and probe combination being enough to allow;

The analyte of combination is contacted the analyte molecule specificity combination that combines of this general probe and multiple thing class with the particle that has the immobilization general probe;

Particle is applied optical force; And

Observe the response of particle to optical force, this response depends on the combination between general probe and the analyte that combines.

67. the method for claim 66 comprises that also the analyte with combination contacts with a plurality of particles, and particle is applied optical force, and the response of observing particle.

68. the method for claim 67 wherein applies power to particle and carries out simultaneously.

69. the method for claim 67, wherein analyte solution is so that set up the mode that fluid is communicated with and contact with a plurality of trapping regions between the trapping region.

70. the method for claim 67, wherein analyte solution contacts with a plurality of trapping regions and does not allow fluid connection between the trapping region.

71. each method of claim 66-69, wherein analyte solution contains patient's antibody.

72. the method for claim 71, wherein patient's antibody of comprising at least for a kind of general probe has wide optionally second antibody.

73. the method for claim 72, wherein said classification is selected from one of IgG, IgE, IgA and IgM.

74. be used for the computer program on the touched computer-readable medium of computer system, this computer program comprises:

Identify the computer code of a plurality of purpose particles that contact with trapping region;

Actuating applies the computer code of displacement optical force to particle;

Write down the computer code of observed particle to the response of optical force.

75. the computer program of claim 74 also comprises the computer code of selecting a plurality of fields of microscope.

76. the computer program of claim 74 also comprises determining when that accumulating threshold quantity data computing machine encodes.

77. analyze the method for the surface nature of a plurality of particles, this method comprises:

The suspension that will contain a plurality of particles imports the fluid box that has a plurality of trapping regions in substrate, and described box limits the liquid column that is limited by lid;

Allow particles settling with the contact trapping region;

Particle is applied optical force; And

Observe the response of particle to optical force,

The height of wherein selecting liquid column contacts with trapping region to allow red blood cell being less than sedimentation in period of 10 minutes.

78. the method for claim 77, the wherein said period is less than 5 minutes.

79. be used for the method for competitive analysis, comprise:

Produce a plurality of sandwich structures, each sandwich structure comprises the set that is fixed on suprabasil first kind of probe molecule, with the set of first kind of reversible occupy-place molecule that combines of probe molecule, and have particle a plurality of and the reversible second kind of probe molecule that combines of occupy-place molecule;

Add analyte solution;

Incubation a period of time, the described time is enough to allow the analyte molecule that first kind and second kind of probe molecule have compatibility is replaced to small part occupy-place molecule, thereby changes the bond strength between particle and the substrate;

Particle is applied optical force;

Observe the response of particle to optical force;

Use response to determine the content of analyte solution.

80. the method for claim 79 wherein has the zones of different that specific sandwich structure is placed on substrate for different respective analyte molecules, so that report a plurality of analyte kinds.

81. claim 79 or 80 each methods, wherein to tend to the adhesion that produces between particle and substrate stronger than the adhesion that corresponding analyte molecule produces for the occupy-place molecule.

82. claim 79 or 80 each methods, it is more weak than the adhesion that corresponding analyte molecule produces that wherein the occupy-place molecule tends to the adhesion that produces between particle and substrate.

83. each method of claim 79 to 82, wherein optical force is applied simultaneously on a plurality of particles.

84. use HOT ability and microscopical combined sample disposal system, comprising:

Shell;

The sample stage that has sample area;

The HOT subsystem comprises:

Laser instrument,

Diffraction optical element,

Point to the HOT emission port of sample area,

At least one points to the light source that has output source of sample area,

The optional object lens that can aim at sample area,

Be arranged to focus on the camera of sample area,

Wherein hold sample area, HOT emission port and output light source in the shell at least.

85. the combined sample disposal system of claim 84, wherein said system and all parts are loaded on the removable frame.

86. the system of claim 84, also comprise and be selected from following at least a parts: the computer interface of integration, response lid are opened and are closed the release mechanism of laser lighting, the anchor clamps of location fluid sample box, the mounting of belt wheel, and be installed in wherein light source.

87. shown and describe HOT and microscopic system.

88. shown and describe fluid box.