CN1834657B - Analyzer and analyzing method - Google Patents
Analyzer and analyzing method Download PDFInfo
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- CN1834657B CN1834657B CN2006100207534A CN200610020753A CN1834657B CN 1834657 B CN1834657 B CN 1834657B CN 2006100207534 A CN2006100207534 A CN 2006100207534A CN 200610020753 A CN200610020753 A CN 200610020753A CN 1834657 B CN1834657 B CN 1834657B
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- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
Abstract
This invention discloses a kind of analysis device; its constitution includes analysis reactor and separating system. The system includes nm separating system, the nm separating system is size separating system use nm size substance as separating object, and/or magnetic suspension absorbing system, the magnetic suspension absorbing system is that absorbing substance is suspended in appointed place in column under function of magnetic field to absorb separating goal substance. The device in this invention can separate sample object, and at least can comparing concentrate object, so detecting sensitivity is greatly improved.
Description
Technical field
The present invention relates to the method for a kind of analytical equipment, this analytical equipment of preparation and use the method that this analytical equipment is analyzed.
Background technology
With the biochip kit is example, we can say the subject matter that analytical equipment faces before making eye bright.
Among the present invention, term " biochip kit " is called for short " chip agent box ", includes but not limited to Biochip, Microarray, Bioarray in the English.Chip comprises microfluidic circuit chip (being equivalent to the Microchannel Biochip in the English), micro-array chip (being equivalent to Biochip, Microarray, Bioarray in the English) etc.Increasing biology sensor is also becoming biochip.Biochip is because it has high flux and microminiaturization, and range of application is very extensive, comprises gene expression detection, genescreen, drug screening, medical diagnosis on disease treatment, environmental monitoring and fields such as improvement, judicial expertise.
Sensitivity, specificity, stability, reproducibility are of paramount importance problems in the biochip kit research and development.Constantly improve owing to analyze mark Huaihe River, the sensitivity that therefore improves biochip kit just becomes the heat subject of continuous pursuit.Developing highly sensitive chip agent box, is that most researchers is devoted to the target innovating and develop.Other analytical equipment also has analogue.
Summary of the invention
The objective of the invention is: a kind of high-sensitivity analysis device, particularly biochip kit are provided, prepare the method for this kit, and use the method that this kit is analyzed.
The objective of the invention is to realize by the enforcement following technical proposals:
A kind of analytical equipment, its composition comprises analysis reactor and piece-rate system, it is characterized in that: described piece-rate system comprises: a. nanometer piece-rate system, b. is or/and the magnetic levitation adsorption system; The former a is that the material with nano-scale is the piece-rate system of separate targets thing; Latter b is meant that adsorbate under the action of a magnetic field, is suspended in the assigned address in the pillar, comes the adsorption system of adsorptive separation object.
In an embodiment of the present invention, discussed special geometric properties (for example nanofilter separation) in the time of in detail based on separate targets thing nano particle, or the nanometer piece-rate system of special mechanical property (for example nano particle centrifuging), and based on the magnetic levitation adsorption system of affine absorption.As the analytical equipment of one of preferred version of the present invention, be in order to analyze the analytical equipment of plurality of target thing.As the more preferably analytical equipment of one of scheme of the present invention, be in same analysis reactor, to be fixed with multiple probe, to have the analytical equipment that is used to separate plurality of target thing piece-rate system.
In analytical equipment of the present invention, its nanometer piece-rate system comprises containing the nanofiltration system of stablizing filter medium.In an embodiment of the present invention, contain the nanofiltration system of stablizing filter medium and comprise nanofilter (Nano-filter), its average pore size is at nano-scale, preferred 15-100nm, more preferably 15-80nm, and filter medium wherein comprises hollow fiber nanometer chimney filter, nanofiltration membrane and giant molecule ultra filtration membrane.
In analytical equipment of the present invention, its nanometer piece-rate system comprises containing the nanofiltration system that can cancel filter medium.In an embodiment of the present invention, can cancel filter medium and comprise the particle with magnetic that can change its accumulation shape by change magnetic field.
In the analytical equipment of invention, its nanometer piece-rate system comprises the nanometer centrifugation systems.In an embodiment of the present invention, the nanometer centrifugation systems comprises the separable centrifugation systems that goes out nano particle (for example high speed centrifugation, for example centrifugal force is greater than the centrifugation systems of 15000g), and it is different from other centrifugation systems commonly used in the analysis.
In analytical equipment of the present invention, its nanometer piece-rate system comprises with separation comprising that totivirus particulate and viral fragment particulate are the piece-rate system of object.A lot of viruses and viral fragment all have the form of particle.For example the hepatitis B grain diameter is between 40nm-50nm, the about 20nm of its fragment diameter of particle.
In analytical equipment of the present invention, its magnetic levitation adsorption system is the adsorption system that comprises the adsorbate that contains the magnetic particle.
In analytical equipment of the present invention, be the magnetic levitation adsorption system of adsorbate to contain the magnetic levitation adsorption system that the magnetic particle is an adsorbate, to comprise with the magnetic nanometer.
In analytical equipment of the present invention, its composition also includes one or more affine nano particles, and wherein said affine nano particle contains nano particle, and the aglucon that is fixed on the separate targets thing on the nano particle.In an embodiment of the present invention, aglucon comprises the polypeptide aglucon, and the polypeptide aglucon comprises antigen and antibody.In an embodiment of the present invention, nano particle comprises inorganic matrix nano particle and organic substrate nano particle, the nano particle bag of preferred chemical modification
Analytical equipment of the present invention, in one or more affine nano particles that its composition comprises, the free aglucon content that has a kind of affine nano particle at least is less than 50%, and the content of non-affine nano particle is less than 50%.In the part embodiment of the embodiment of the invention, described affine nano particle, contain at least nano particle, covalent bonding the coupling group on the nano particle, covalent bonding at activated group on the coupling group and covalent bonding the aglucon on activated group.Described activated group is selected from: amino, general formula is-RNH
2Group, do not contain NH
2Organic group.When described activated group is selected from amino or does not contain NH
2Organic group the time: described coupling group be evenly distributed density greater than 1.85 μ mol/m
2Nanoparticle surface or/and described activated group be evenly distributed density greater than 1.85 μ mol/m
2Nanoparticle surface.When described activated group is selected from general formula be-RNH
2Group the time, described activated group be evenly distributed density greater than 0.5 μ mol/m
2Nanoparticle surface.Described coupling group comprises the organosilicon coupling group.At least a described affine nano particle has the purity of affinity chromatography level.
Analytical equipment of the present invention in its at least one analysis reactor, both had been fixed with probe antibody, was fixed with antigen of probe again.In the part embodiment of embodiments of the invention, analytical equipment is the kit that can detect target antigen and target antibody in a reactor simultaneously.
Analytical equipment of the present invention, existing aglucon is the affine nano particle of antigen in its at least one analysis reactor, it is the affine nano particle of antibody that aglucon is arranged again.In the part embodiment of embodiments of the invention, analytical equipment is the kit that can detect target antigen and target antibody in a reactor simultaneously.
In analytical equipment of the present invention, constitute the affine nano particle of its constituent, comprise the affine nano particle that is used for mark.In the part embodiment of the embodiment of the invention, the affine nano particle of mark has magnetic, easily as the biology sensor kit.
Analytical equipment of the present invention is for having the biochip kit of above-mentioned piece-rate system and reactor.In the part embodiment of the embodiment of the invention, the biochip reaction device is a movable reactor, realizes the continuous operation between piece-rate system and the reactor easily.
A kind of analytical approach of the present invention the steps include: sampling and analytical equipment of the present invention is provided.
Analytical approach of the present invention also comprises the steps: with described nanometer piece-rate system, separates the evaluating objects thing particle that may exist in the acquisition sample; Again isolated evaluating objects thing particle is fixed in the reactor.
Analytical approach of the present invention also comprises the steps: with described affine nano particle, and the evaluating objects thing reaction with existing in the sample forms object/affine nano-particle compound; Use described nanometer piece-rate system then, separate targets thing/affine nano-particle compound; Again isolated object/affine nano-particle compound is fixed in the reactor.
Analytical approach of the present invention also comprises the steps: with described affine nano particle, and the evaluating objects thing reaction with existing in the sample forms object/affine nano-particle compound; Use described nanometer piece-rate system then, compartment analysis object particle and described object/affine nano-particle compound; Again isolated evaluating objects thing particle and object/affine nano-particle compound are fixed in the reactor.
In the part embodiment of the embodiment of the invention, in the formation of the above-mentioned object/affine nano-particle compound reaction, the concentration of affine nano particle (w/v) is 10,000/to 1%.
In the part embodiment of the embodiment of the invention, the maximum concentration (w/v) when above-mentioned object/affine nano-particle compound separates is less than 10%.
In the part embodiment of the embodiment of the invention, above-mentioned object/affine nano-particle compound concentration fixedly time the in reactor is 10,000/to 1%.
Analytical approach of the present invention also comprises the steps: the affine nano particle of magnetic is fixed on the evaluating objects thing in the reactor.
Analytical approach of the present invention also comprises the steps: with described magnetic levitation adsorption system adsorptive separation evaluating objects thing; Again isolated evaluating objects thing is fixed in the reactor.
A kind of method for preparing analytical equipment of the present invention, comprise the preparation of described affine nano particle, make its contain in the composition of affine nano particle: the content of on-fixed aglucon is less than 5% of the aglucon total amount, and the content of non-affine nano particle is less than 30% of nano particle total amount.The content of described on-fixed aglucon reduces by centrifuge method.The content of described non-affine nano particle reduces by affinity chromatography.
A kind of method for preparing analytical equipment of the present invention comprises that preparation has the kit of above-mentioned piece-rate system and reactor.
The invention has the advantages that:
Because analytical equipment of the present invention is by new forming, especially wherein Chuan Xin piece-rate system, so that analytical equipment of the present invention can the object in the sample be separated, at least can carry out concentrated (object concentration increases greatly with the ratio of other biomolecule concentration) relatively of object, thereby improve detection sensitivity greatly.
Embodiment
Term definition
In the present invention, term " probe " is meant the material of catching (comprising affinity interaction) object in the reactor, by interaction in order to be fixed on.Probe comprise polypeptide or/and with the interactional medicine of polypeptide.Known can be a lot of with the probe of target polypeptides effect, for example ion exchanger, medicine, polypeptide, polysaccharide, vitamin, microbiotic, function organism, antigen and virus, cell or their composition.Probe also comprise nucleic acid or/and with the medicine of nucleic acid interaction.
Term of the present invention " mark aglucon " is meant and is contained in the label, by itself and object interact (comprising affinity interaction) realize material to the object mark, comprise aglucon (being equivalent to the Ligand in the English), for example: the aptamer molecule of antigen, antibody, part, part index enhanced system evolution technology screening, aglucon, polypeptide, polysaccharide, common enzyme, co-factor, microbiotic, steroids, virus, cell etc.
Term of the present invention " polypeptide " is equivalent to " polypeptide " in the English, comprise natural or synthetic protein, protein fragments, synthetic peptide or the like, in the immune detection common object and detect in general aglucon, for example antigen, antibody, or the like all belong to polypeptide.
Term of the present invention " affine nano particle " or " aglucon/nano-particle compound " are meant the nano particle that is fixed with aglucon.Aglucon is fixed with a lot of modes on nano particle, a kind of example of fixed form is that covalence key closes in aglucon/nano-particle compound of preparing in the inventive method.It should be noted that, the prepared product that contains aglucon/nano-particle compound, be generally the potpourri (being called for short aglucon/nanoparticle mixture) that contains aglucon/nano-particle compound, because of it also contains aglucon that more or less is not fixed to nano particle and the nano particle that more or less is not fixed with aglucon basically.
Term of the present invention " nano particle " is meant in three dimensions to have the solid phase carrier particle of one dimension less than 1000nm at least; Term " activation nano particle " is meant the compound that activated group and nano particle form by covalent bond.
Term of the present invention " biological chip base " is called for short the sheet base, is meant in order to form the solid phase carrier of probe points thereon.Solid phase carrier of the present invention comprises conventional carrier and nanostructured carrier, and wherein said conventional carrier is the solid phase carrier of surperficial essentially no nanostructured, and described nanostructured carrier is the solid phase carrier that nanostructured is arranged on the surface.Usually have on the sheet base can with aglucon reactive activity group.
Term of the present invention " mark substance " is meant in order to form or to participate in forming the material of detecting signal, for example rhodamine, CY3, CY5 etc.
Term of the present invention " analysis reactor " is called for short " reactor ", is meant that the place of the probe and the reaction of object reaches the integral body of the entire infrastructure composition that is communicated with it.Reactor comprises border or isolation structure, sheet base, be fixed on the probe on the sheet base and other dependency structure of being connected (for example stream, feed liquor structure, fluid structure, immobilization label, or the like).In addition, among the present invention: according to the number n of reactor on the chip, chip is defined as single reactor chip (n=1) and multiple reactor chip (n is equal to or greater than 2); According to the liquid phase medium that is added in the testing process can be in reactor directed flow, reactor is defined as flow reactor and non-current reactor, and will be that the chip of feature is defined as flow chip and non-current chip respectively with flow reactor and non-current reactor
Below will the present invention be described in more detail by embodiment.
Embodiment 1. is used for the preparation of the nanometer piece-rate system of analytical equipment
The nanometer piece-rate system for preparing in the present embodiment comprises nanometer piece-rate system (prefilter+nanometer separation vessel) that contains pre-flock and the nanometer piece-rate system (nanometer separation vessel) that does not contain pre-flock.The purpose of pre-flock is the big component of separate targets thing of removing the ratio nano piece-rate system.In the present embodiment, used prefilter is all membrane filter, and the normal pore size is 0.2 μ m or 0.4 μ m.For example, aperture 0.2 a μ m (or 0.4 μ m), area 1cm
2The miniature displacer of pre-filter membrane.
The nanometer piece-rate system for preparing in the present embodiment except that said system, can also comprise parts such as connecting pipe.The nanometer piece-rate system of present embodiment preparation, wherein flowing of separating medium can be finished with micro pump (for example flow velocity 1 μ l-100 μ is l/ minute).
About the more detailed description of nanometer separation vessel, state embodiment as follows.
Embodiment 1.1. contains the preparation of the filtering system of stablizing filter medium
In the present embodiment, used filter medium is respectively: hollow fiber chimney filter, filter membrane and ultra filtration membrane, their average pore size are between 10-35nm, in order to the separate targets thing of size greater than average pore size that dam.Nanometer piece-rate system in the present embodiment, filter medium does not wherein have the variation of form at filter process, for example filter membrane filter membrane always.More detailed description is stated embodiment as follows.
Embodiment 1.1.1 contains the nanofilter of hollow fiber nanometer chimney filter
In the present embodiment, the nanometer piece-rate system contains hollow fiber nanometer chimney filter, for example takes from the hollow fiber nanometer chimney filter of the Planova series of products (hollow fiber chimney filter average pore size is respectively 15nm, 19nm, 35nm or the like) of Asahi company.Planova is a kind of important viral filter that removes.Wherein, the hollow fiber nanometer chimney filter of Panova20N, its average pore size is 19nm.In the present embodiment, " hollow fiber nanometer chimney filter " is meant that average pore size is at nano-scale, preferred 15-100nm, the more preferably hollow fiber chimney filter of 15-45nm.Hollow fiber nanometer chimney filter can be connected with other parts in a different manner, thereby makes different nanometer piece-rate systems.
A scheme in the present embodiment is a nanofilter, contains: 1). hollow fiber nanometer chimney filter; 2) feed tube and drain pipe, they link to each other with hollow fiber nanometer chimney filter liquid feeding end, outlet end respectively; 3). filter cylinder, it is used to hold hollow fiber chimney filter and filtered solution; 4). cartridge cover and bottom, they contain hollow fiber chimney filter fixed sturcture.In the present embodiment, filter cylinder is the stainless steel cylinder of homemade internal diameter 5mm, and its length is 10cm, has waste liquid outlet on it.Nanofilter in the present embodiment is the separate targets thing with the filter trapped substance.And the Planova filter then is the separate targets thing with the filtered solution.
The nanofilter of present embodiment preparation, its principle of work is: make sample (for example 30 times of dilute serums or contain 30 times of dilute serums of object/affine nano-particle compound) enter hollow fiber nanometer chimney filter through feed tube, thereby will be by its particle of the holding back (virus and the viral derivative particles of size more than 15nm for example, or/and object/affine nano-particle compound) is retained in the dialysis tubing, and the molecule of reduced size is gone out by the orifice flow on the dialysis tube wall.
The nanofilter of present embodiment preparation can be separated or/and concentrate the nano particle in the sample (for example virion is or/and object/affine nano-particle compound).
The nanometer piece-rate system of present embodiment preparation, brief note is Z111 in following examples.
Embodiment 1.1.2. contains the nanofilter of nanofiltration membrane
In the present embodiment, nanofilter contains nanofiltration membrane, for example is selected from the filter membrane in the Viresolve NFP series of filter membrane in the DV series of PULL company and Millipore company.DV, Viresolve NFP are the important viral filters that removes.For example, take from the filter membrane among the DV20, its average pore size is 20nm; Take from the filter membrane among the Viresolve NFP, its average pore size is 28nm.In the present embodiment, " nanofiltration membrane " be meant average pore size at nano-scale, average pore size at nano-scale, preferred 15-100nm, the more preferably filter membrane of 15-45nm.Filter membrane can be connected with other parts in a different manner, thereby makes different nanometer piece-rate systems.
A scheme in the present embodiment is a nanofilter: contain 1). nanofiltration membrane, 2). filter cylinder, to contain the top cover of feed tube and the bottom of filter membrane support portion and drain pipe is arranged, it is used to hold filter membrane.In the present embodiment, filter cylinder is the stainless steel cylinder of homemade internal diameter 5mm, and spiral marking connection between its top cover and the bottom can be dismantled, with civilian dress, remove filter membrane.
The nanometer piece-rate system of present embodiment preparation, its principle of work is: make sample (for example 30 times of dilute serums or contain 30 times of dilute serums of object/affine nano-particle compound) enter filter cylinder through feed tube, will be by its particle of holding back (virus and the viral derivative particles of size more than 15nm for example, or/and object/affine nano-particle compound) is retained in the nanofiltration membrane top, and the molecule of reduced size is flowed out by filter membrane.
A scheme in the present embodiment is pre-flock/nanofilter, promptly is mounted with pre-filter membrane (for example the normal pore size is the degerming filter membrane of 0.2 μ m) above above-mentioned nanofilter.The suspending liquid of getting pre-filter membrane below, nanofiltration membrane top is the separate targets thing, usefulness for further analysis.
The nanometer piece-rate system of present embodiment preparation can be separated or/and concentrate the nano particle in the sample (for example virion is or/and object/affine nano-particle compound).
The nanometer piece-rate system of present embodiment preparation, brief note is Z112 in following examples.
Embodiment 1.1.3. contains the nanofilter of nanometer ultra filtration membrane
In the present embodiment, used nanometer separation vessel contains ultra filtration membrane, and the molecular interception value that for example is selected from Biomax company is 500,000 ultra filtration membrane (for example production code member is the high flow rate poly (ether-sulfone) ultrafiltration membrane of PBVK1500).Ultra filtration membrane can be connected with other parts in a different manner, thereby makes different nanometer piece-rate systems.
A scheme in the present embodiment is the nanometer piece-rate system, contains: 1). ultra filtration membrane, 2). the suction concentrator.For example, leave above one in the transparent plastic anchor clamps of application of sample mouth, ultra filtration membrane is positioned at the anchor clamps center.Plastic wall is arranged before it, have on the wall be communicated with the application of sample mouth flow through structure (for example total volume 1ml).The thick water-absorption fiber layer of 3mm is arranged thereafter.Behind the water-absorption fiber layer plastic wall is arranged.
The nanometer separation vessel of present embodiment preparation, its principle of work is: make sample (for example contain target antibody/be the human immunoglobulin dilution of the affine nano-particle compound of aglucon with the antigen of target antibody) enter through the application of sample mouth and flow through structure, to be retained in the ultra filtration membrane top by its big molecule of holding back (for example above-mentioned compound), and under the effect of water-absorption fiber layer, the molecule of reduced size is flowed out by ultra filtration membrane.
The nanometer separation vessel of present embodiment preparation can separate or/and concentrate the big molecule in the sample (for example virion is or/and object/affine nano-particle compound).
The nanometer piece-rate system of present embodiment preparation, brief note is Z113 in following examples.
Embodiment 1.2. contains the preparation of the filtering system that can cancel filter medium
In the present embodiment, used filter medium is respectively: macropore filter membrane and nanofiltration magnetic nanometer.Wherein: the average pore size score of macropore filter membrane is from intended particle particle diameter 50nm (for example, PULL company aperture is 0.2 μ m degerming filter membrane) greatly at least; The nanofiltration magnetic nanometer, for particle diameter greater than the average pore size of macropore filter membrane but can form magnetic nanometer (for example, the Fe of size 250nm of diameter that on average dam less than the filtering medium layer of separate targets particle diameter
3O
4Particle).Nanometer piece-rate system in the present embodiment, nanofiltration medium wherein (magnetic nanometer filtering medium layer) can be cancelled at filter process.
More detailed description is stated embodiment as follows.
Embodiment 1.2.1 contains the filtering system that can cancel filter medium
Nanofiltration system in the scheme of present embodiment contains: 1). macropore filter membrane, 2). nanofiltration magnetic nanometer, 3). filter cylinder.Wherein, filter cylinder contains the top cover of feed tube and the bottom of filter membrane support portion and drain pipe is arranged, and it is used to hold macropore filter membrane and nanofiltration magnetic nanometer when nanofiltration.In the present embodiment, filter cylinder is the stainless steel cylinder of homemade internal diameter 5mm, and spiral marking connection between its top cover and the bottom can be dismantled, with civilian dress, remove filter membrane.
The nanometer separation vessel of present embodiment preparation, its principle of work during as simple filter is: make the nanofiltration magnetic nanometer in the filter cylinder, enough (filtering medium layer for example (if necessary to form thickness on the macropore filter membrane, but externally-applied magnetic field keeps the stable of this filtering medium layer), again sample (30 times of dilute serums that for example contain object/affine nano-particle compound) is entered filter cylinder through the application of sample mouth, to be retained in this filtering medium layer top by its separate targets particle of holding back (for example above-mentioned compound), and the molecule of reduced size will be flowed out through drain pipe by this filtering medium layer and macropore filter membrane.Be concentrated or/and behind the purifying at the separate targets particle, thereby utilize externally-applied magnetic field that above-mentioned filter medium layer scattering is cancelled, make the separate targets particle to contact and through the macropore filter membrane, flow out through drain pipe again.This nanometer separation vessel, the folk prescription that can carry out the separate targets particle helps the robotization of entire separation system to continuous separation.
The nanometer piece-rate system of present embodiment preparation, brief note is Z121 in following examples.
Embodiment 1.2.2 contains the affinity chromatography system that can cancel filter medium
A scheme of the affinity chromatography system that adds, subtracts filter medium in the present embodiment, it contains: 1). the macropore filter membrane; 2). the nanofiltration magnetic nanometer; 3). affine nano particle; 4). filter cylinder.Wherein, filter cylinder contains the top cover of feed tube and the bottom of filter membrane support portion and drain pipe is arranged, and it is used to hold macropore filter membrane, nanofiltration magnetic nanometer and affine nano particle when affine absorption.In the present embodiment, filter cylinder is the stainless steel cylinder of homemade internal diameter mm, and spiral marking connection between its top cover and the bottom can be dismantled, with civilian dress, remove filter membrane.In the present embodiment, affine nano particle is selected from the affine nano particle of embodiment 2 preparations.
The nanometer separation vessel of present embodiment preparation, its principle of work is: make in the filter cylinder nanofiltration with magnetic nanometer on the macropore filter membrane, forms thickness enough (for example 1mm) filtering medium layer (if necessary, but externally-applied magnetic field keeps the stable of this filtering medium layer), make non magnetic affine nano particle on this filtering medium layer, form affinity chromatography layer (for example 0.5mm), again sample (for example 20 times of dilute serums) is entered filter cylinder through the application of sample mouth, make affinity chromatography layer adsorption analysis object, and the molecule of the reduced size that will not adsorb is by the affinity chromatography layer, filtering medium layer and macropore filter membrane flow out through drain pipe.After affine absorption is finished, can add cleansing solution if necessary, the reduced size molecule that flush away does not adsorb.Utilize externally-applied magnetic field make above-mentioned filtering medium layer loose from, thereby object/affine nano-particle compound can be contacted and through the macropore filter membrane, flow out through drain pipe again.This nanometer separation vessel need not carry out the wash-out of object, can realize that again folk prescription to continuous separation, helps the robotization of entire separation system.
The nanometer piece-rate system of present embodiment preparation, brief note is Z122 in following examples.
Embodiment 1.3. contains the preparation of the nanometer piece-rate system of centrifugal separation system
The nanometer piece-rate system of present embodiment preparation contains centrifugal separation system.More detailed description is stated embodiment as follows.
Embodiment 1.3.1. contains the preparation of the nanometer piece-rate system of simple centrifugal separation system
In the present embodiment, centrifugal separation system is a centrifuge tube, preferably can be used for centrifugal force 20000g, maximum volume is less than or equal to the centrifuge tube of 1.5ml.
The nanometer piece-rate system of present embodiment preparation, its principle of work is: sample (30 times of dilute serums that for example contain object/affine nano-particle compound) is added in the centrifuge tube, centrifugal under high centrifugal force (for example greater than 15000g) condition.The supernatant that inclines, required target nanoparticle is retained in the sediment.If necessary, add damping fluid in the sediment after, the ultrasonic suspending liquid that forms the separate targets nano particle.
The nanometer piece-rate system of present embodiment preparation, brief note is Z131 in following examples.
Embodiment 1.3.2. contains the preparation of the nanometer piece-rate system of molecular exclusion gel
In the present embodiment, the nanometer piece-rate system contains molecular exclusion gel separation system, and Syphancryl S-200 HR separator tube for example is housed, and the gel bottom surface is the filter membrane of 0.2 μ m.
This system works principle is: sample (30 times of dilute serums that contain object/affine nano-particle compound) is added in the separator tube of emptying, through after centrifugal, particle and most of unbinding protein material greater than 0.2 μ m are trapped, collect the suspending liquid of the evaluating objects thing/affine nano-particle compound of the purifying in the centrifuge tube, as the sample that carries out next step analysis.
The nanometer piece-rate system of present embodiment preparation, brief note is Z132 in following examples.
Embodiment 2. is used for the preparation of the magnetic levitation adsorption system of analytical equipment
The magnetic levitation adsorption system for preparing in the present embodiment comprises magnetic levitation adsorption system (for example prefilter+magnetic levitation adsorption column) that contains pre-flock and the magnetic levitation adsorption system (magnetic levitation adsorption column) that does not contain pre-flock.The purpose of pre-flock (for example being the prefilter of 0.2 μ m-0.4 μ m by the aperture) is to remove score from the big component of object.The system for preparing in the present embodiment except that said system, can also comprise parts such as connecting pipe.
The scheme of a magnetic levitation adsorption column in the present embodiment contains: 1). the affine particle of magnetic, 2). pillar.Wherein, pillar contains the top cover of feed tube (also can also have pre-filter membrane), and it is used to limit the motion of the affine particle of magnetic to pillar wall vertical direction when affine absorption.In the present embodiment, filter cylinder is the glass tube of homemade internal diameter 2mm.In the present embodiment, the affine particle of magnetic contains the 50nm gold-magnetic particles and is separately fixed at HBs Ab1 and HCV Ag1 (seeing Table 2) on the gold-magnetic particles.Wherein, gold-magnetic particles is GoldMag
TM-@ gold-magnetic particles (Xida Beimei Gene Co., Ltd., Shanxi), its nuclear is nanometer Fe
3O
4, the nuclear surface is the shell of gold, product has magnetic Fe concurrently
3O
4The superparamagnetism of nano particle and the immobilized performance of collaurum surface biological molecule.Thereon fixing of antibody carries out with reference to product description.
The magnetic levitation adsorption column of present embodiment preparation, its principle of work is: under the outside magnetic field effect, the affine particle of magnetic is fixed in the pillar endpiece has the post of (for example 1mm), forms the affine adsorbed layer of the affine particle of enrichment magnetic.Again sample (for example 20 times of dilute serums) is entered pillar through the application of sample mouth, flow through affine adsorbed layer, and carry out affine absorption there, and the molecule that will not adsorb directly flows out.After affine absorption is finished, can add not adsorbate of cleansing solution flush away if necessary.Withdraw externally-applied magnetic field then, separate targets thing/affine magnetic nanometer compound is flowed out from pillar.This magnetic levitation adsorption column can realize that folk prescription to continuous separation, helps the robotization of entire separation system.
Obviously, the magnetic levitation adsorption column of present embodiment preparation is different from magnetic stabilization fluid bed.Magnetic stabilization fluid bed pillar endpiece has filtration, and the separate targets thing is behind the affine particle wash-out from magnetic, and structure flows out after filtration.And the magnetic levitation adsorption column of present embodiment preparation, separate targets thing/affine magnetic nanometer compound flows out from the pillar lower end.
The piece-rate system of present embodiment preparation, brief note is Z200 in following examples.
Embodiment 3: the preparation of affine nano particle
In the present embodiment, affine nano particle preparation comprises: provide activation nano particle and preparation to contain the aglucon/activation nanoparticle mixture of affine nano particle.
1. preparation activates nano particle
In the present embodiment, the activation nano particle is respectively the activation nano particle of inorganic nano-particle subbase and the activation nano particle of organic nano particle base.The latter is commercial activation nano particle (K2-005, Merck company).Among the former, the affine particle (GoldMag that is magnetic
TM-@ gold-magnetic particles, 50nm, Xida Beimei Gene Co., Ltd., Shanxi) be commercial activation nano particle.Other is self-control, and preparation method and institute obtain the kind that activates nano particle another patent (Shen disannul a call CN200610020637.2) with reference to us.
In the present embodiment, obtain different active nano particles by the conditioned reaction parameter.Active nano particle among the present invention, reactive group fixing on the 1g nano particle greater than 70 μ mol, be preferably greater than 140 μ mol, or 1m
2Activated group fixing on the nanoparticle surface greater than 0.5 μ mol, be preferably greater than 1 μ mol.Table 1 has been listed the composition of the part activation nano particle for preparing in the present embodiment.
Table 1
| The activation nano particle | Nano particle | The coupling agent of coupling group is provided | Activated group | ? | ? |
| Kind | Quantity * | Kind | Quantity ** | ? | ? |
| A1 | Monox (LUDOX AS-40) | The 3-aminopropyl trimethoxysilane | 0.62N％ | The glycocoll base | 0.34N％ |
| A2 | Monox (LUDOX AS-40) | The 3-aminopropyl trimethoxysilane | 0.62N％ | The N base | 0.68N％ |
| A3 | Monox (LUDOX AS-40) | The 3-aminopropyl trimethoxysilane | 0.62N％ | The arginine base | 0.83N％ |
| A4 | Monox (LUDOX AS-40) | Aminopropyl triethoxysilane | 0.56N％ | The glycocoll base | 0.28N％ |
| A5 | Monox (LUDOX AS-40) | 3-isocyanates propyl-triethoxysilicane | 0.43N％ | Amino diazanyl | 0.59N％ |
| A6 | Silicon oxide nanoparticle (SS1) | The 3-aminopropyl trimethoxysilane | 0.51N％ | The glycocoll base | 0.28N％ |
| A7 | The aluminium oxide nano particle | The 3-aminopropyl trimethoxysilane | 0.53N％ | The glycocoll base | 0.24N％ |
*: the N number percent after the coupling in the ultimate analysis
*: contain the N number percent in the ultimate analysis of nano particle of coupling group before the activation of the N number percent in the ultimate analysis nitrogen content (N%) relevant=activation nano particle ultimate analysis-with activated group
2. preparation contains the aglucon/activation nanoparticle mixture of affine nano particle
In the present embodiment, the preparation method of aglucon/activation nanoparticle mixture generally comprises:
1). aglucon and activation nano particle are provided
In the present embodiment, the above-mentioned activation nano particle that provides is provided used activation nano particle; Used aglucon is as shown in table 2.
Table 2
| Affine nano particle aglucon | The reactor probe | The source |
| Hepatitis C virus antigen 1 (HCV Ag 1) | Hepatitis C virus antigen 2 (HCV Ag 2) | Hepatopathy research institute of The People's Hospital of Peking University |
| Anti-hepatitis virus surface antibody 1 (HBS Ab1) | Anti-hepatitis virus surface antibody 2 (HBS Ab2) | Hepatopathy research institute of The People's Hospital of Peking University |
| HIV1+2Ag1 | ?HIV1+2?Ag2 | Henan Li Li biotech company |
In the table 2, affine nano particle aglucon has not exclusively the same site with the reactor probe, can match with sandwich assays target antibody or target antigen.
2). preparation contains the aglucon/activation nanoparticle mixture of affine nano particle
Among the present invention, term " affine nano particle " is meant that aglucon is combined in the compound that forms on the nano particle.One of preferred combination is the combination of not dissociating, for example covalent bonding in high salt (for example 0.5M NaCl) solution.
The preparation method is: obtain activation nano particle sediment by centrifuging, it is evenly distributed on makes the equally distributed suspending liquid of activation nano particle in the damping fluid again, and mix, react with aglucon solution.By regulate known reaction controlled condition (reactant concentration for example, reaction medium, temperature of reaction, the reaction time, or the like) can control reaction.In the present embodiment: the concentration of nano particle (w/v) is regulated between 5% to 5 ‰ during reaction, and aglucon concentration is regulated between 0.1mg/ml to 1mg/ml; Reaction medium is damping fluid (the 0.1M carbonate buffer solution of PBS or pH9.3); Temperature of reaction is regulated between room temperature to 40 ℃; Reaction time was regulated between 0.5 to 24 hour.The professional and technical personnel can obtain required optimal conditions by regulating these parameters.
3). separate affine nano particle from aglucon/activation nanoparticle mixture
After reaction is finished, a small amount of suspending liquid is carried out centrifugal (2-8 ℃ 20000g), is got supernatant and measure the dense change of aglucon, to measure the fixedly quantity of aglucon.Among the present invention, term " fixedly aglucon " is meant the aglucon that is not fixed in aglucon/activation nanoparticle mixture on the nano particle.Wherein, the mensuration of supernatant protein concentration is used conventional determining method.
If necessary, also whole suspending liquid can be carried out centrifugal (2-8 ℃ 20000g), is got precipitation, to reduce in aglucon/activation nanoparticle mixture fixedly aglucon.
In aglucon/activation nanoparticle mixture, also may contain non-affine nano particle.Among the present invention, term " non-affine nano particle " is meant the nano particle that is not fixed with aglucon, though or be fixed with its content of aglucon very little, so that there is no the nano particle of the significant reaction of no affine nano particle.In the present embodiment, the ratio of non-affine nano particle is measured by known affinity chromatography.For example, under the situation of HBs Ab, HBs Ag is fixed on the activation chromatography glue, more a small amount of suspending liquid is carried out affinity chromatography, and measure the amount (comparing precipitation capacity) of nano particle in the chromatography supernatant, can get this ratio by centrifuging as aglucon.If necessary, also whole suspending liquid can be carried out above-mentioned affinity chromatography, to obtain high affine nano particle with affinity chromatography purity (100%).The method of carrying out of the preparation method of affinity chromatography glue and affinity chromatography is a conventional method.
If necessary, the conventional deactivating process for the treatment of of also available biological chip base carries out Passivation Treatment to activation nanoparticle surface in the potpourri.
In the present embodiment, obtain different aglucon/activation nanoparticle mixture by the conditioned reaction parameter.Utilize self-control activation nano particle, in the aglucon for preparing under optimal conditions/activation nanoparticle mixture: the content of free aglucon is less than 50%, some in addition less than 20%; The content of non-affine nano particle is less than 50%, some in addition less than 20%.Yet, utilizing in weak activation nano particle or the preparation under the same conditions of disactivation nano particle, the content of free aglucon can be greater than 50%, and the content of non-affine nano particle can be greater than 50%.Below we will see that this is of practical significance.
Part aglucon/activation the nanoparticle mixture of present embodiment preparation is listed in the table 3.
Table 3
| Aglucon/activation nanoparticle mixture | The activation nano particle * | Aglucon ** | Free probe molecule content (%) *** | Hang down affine/no affine particle ratio (%) **** |
| ?B1 | ?A1 | ?HBs?Ab1 | ＜20 | ＜20 |
| ?B2 | ?A2 | ?HBs?Ab1 | ＜20 | ＜20 |
| ?B3 | ?A3 | ?HBs?Ab1 | ＜20 | ＜20 |
| ?B4 | ?A4 | ?HBs?Ab1 | ＜20 | - |
| ?B5 | ?A4 | ?HCV?AG1 | ＜20 | - |
| ?B6 | ?A4 | ?HIV1+2?Ag1 | ＜20 | - |
| ?B7 | ?A5 | ?HBs?Ab1 | ＜20 | - |
| ?B8 | ?A6 | ?HBs?Ab1 | ＜20 | ＜20 |
| ?B9 | ?A7 | ?HBs?Ab1 | ＜20 | - |
| ?B10 | ?K2-005 | ?HBs?Ab1 | - | - |
| ?B11 | GoldMag TM-@ gold-magnetic particles | ?HBs?Ab1 | - | - |
| ?B12 | GoldMag TM-@ gold-magnetic particles | ?HCV?Ag1 | - | - |
*: reference table 1;
*: reference table 2;
* *: (the total addition of centrifuged supernatant middle probe molecule content/probe molecule) * 100%;
* *: (affinity chromatography is through the centrifugation amount of the equal-volume liquid of the centrifugation amount/probe molecule/activation nanoparticle mixture of liquid) * 100%
The preparation method of embodiment 4. analytical equipments
In the present embodiment, the preparation of analytical equipment comprises:
1. the preparation of biochip reaction device
In the present embodiment, used base is amino hydrazine slide, its method for making is with reference to Melnyk O etc., Peptide arrays for highly sensitive and special antibody-binding fluorescencearrays, Bioconjug Chem.13:713-20.2002.The preparation method of present embodiment is suitable for the chip slapper base made by following material or derivatives thereof equally: silicon chip, silica gel, pottery, metal oxide, metal, other polymeric material and their compound.In like manner, chip used base of present embodiment sheet base that can also be nanostructure-containing.
The preparation method comprises:
1). point sample
The reactor probe of listing in the table 2 is made solution, and concentration is selected between 0.5-2.0mg/ml.Point sample method routinely adds to the chip slapper base with the reactor probe solution respectively again, forms probe points.2 kinds of non-current reactors have been prepared: only be fixed with a kind of non-current reactor of reactor probe and be fixed with the non-current reactor of multiple reactor probe.In the reactor, 2 points of every kind of reactor probe points.All probe points form M * N probe array on the chip slapper base.Wherein M is greater than 1, and N is greater than 1.Used point sample instrument is DY-2003 biochip point sample instrument (CAS Electrical Engineering Research Institute's preparation).By regulate known reaction controlled condition (reactant concentration for example, reaction medium, temperature of reaction, the reaction time, or the like) can control reaction.The professional and technical personnel can obtain required optimal conditions by regulating these parameters.
2). hatching
Behind the point sample, down and carry out immobilized reactant on the sheet base in the certain hour (1-24 hour) in uniform temperature (in 4-37 ℃ select).
3). passivation
After the hatching, make chip uniform temperature (in 4-37 ℃ select) down and in the certain hour (1-15 hour) and the passivator (calf serum or milk or other known passivator) of finite concentration (1-30mg/ml) react, to reduce the non-special adsorption activity on the chip.
The method of present embodiment is suitable for various chips certainly, for example single reaction chamber chip, many reaction tanks chip, the chip that flows, non-current chip, or the like.
The non-current biochip of present embodiment preparation, be the non-current chip of many reaction tanks, the preparation method is with reference to the embodiment 1 in our another patented claim " high integration analysis chip of minimized height for reactor and application thereof " (PCT application number CN20040169).In brief: be coated on the chip slapper base on the reactor boundary position with high hydrophobic organosilicon coating (Chengdu morning twilight chemical research institute), after drying at room temperature, solidify, form the high hydrophobic convex body of height 25-115 μ m, width 2.0-2.5mm by supplier's operation instruction.3mm * 3mm rectangle can be got in the surface that high hydrophobic convex body surrounds.On this surface of substrate, laterally have 12 sheet Ji Chi, 4 sheet Ji Chi are vertically arranged, have 48 sheet Ji Chi.Then, sheet Ji Chi is carried out above-mentioned " point sample " operation and other operation, make non-current chip.
The mobile biochip of present embodiment preparation, the preparation method is with reference to the embodiment 9 or 10 in our another patented claim " high integration analysis chip of minimized height for reactor and application thereof " (PCT application number CN20040169).Say it: above-mentioned " point sample ", " hatching " and " passivation " operation are carried out in the zone (wide 4mm, long 15mm) of reserving stationary probe on the sheet base.End face element in the present embodiment be reusable, corrosion resistant plate liquid in-out mouth, liquid in-out pipe and that above-mentioned zone adapts arranged.Its with face that the sheet base contacts on hermetically-sealed construction is arranged.Its hermetically-sealed construction be with sheet base above-mentioned zone outside regional corresponding elastomeric layer (dried certainly silicone rubber solution, Chengdu morning twilight chemical research institute) (bed thickness is less than 0.5mm).Its liquid inlet and outlet is corresponding into and out of the liquid zone with the reaction tank on the element of bottom surface.Use mechanical jig pressure to link, thereby make non-current chip forming sealing between end face element and the sheet base.
The partial reaction device of present embodiment preparation is listed in the table below 4.
Table 4
| The reactor numbering | Probe (reference table 2) | Type of reactor | ? |
| Antigen of probe | Probe antibody | ? | ? |
| R1 | HCVAg1 | - | Flow-type |
| R2 | HIV 1+2?Ag1 | - | Flow-type |
| R3 | - | HBs?Ab1 | Flow-type |
| R4 | HCVAg1、HIV 1+2Ag1 | HBs?Ab1 | Flow-type |
| R5 | HCVAg1 | - | Non-current |
| R6 | HIV 1+2?Ag1 | - | Non-current |
| R7 | - | HBsAb1 | Non-current |
| R8 | HCVAg1、HIV 1+2Ag1 | HBsAb1 | Non-current |
| R9 | HCVAg1 | HBs?Ab1 | Flow-type |
2. prepare the biochip Mk system
In the present embodiment, Mk system is a label, and it comprises the label aglucon and the mark substance of combination with it.In the present embodiment, used label aglucon is the affine nano particle aglucon in the table 2, and used mark substance is rhodamine (Molecular probes company) or collaurum.
In the present embodiment, rhodamine label preparation method is the preparation method of known rhodamine label.Purifying splashes into the swivelling pipe that gel is housed with mix products if necessary, and is centrifugal under the 4000r/min condition, gets collector liquid.
In the present embodiment, golden label preparation method is known golden label preparation method.Wherein, used gold particle is 50nm GoldMag
TM-@ gold-magnetic particles (Xida Beimei Gene Co., Ltd., Shanxi).
3. the piece-rate system of evaluating objects thing is provided
In the present embodiment, the piece-rate system of evaluating objects thing comprises respectively:
1). the nanometer piece-rate system, it is selected from the nanometer piece-rate system (Z111, Z112, Z113, Z121, Z122, Z131, Z132) of embodiment 1 preparation;
2). the magnetic levitation adsorption system, it is selected from the magnetic levitation adsorption system (Z200) of embodiment 2 preparations; With
3). affine nano particle and nanometer piece-rate system, wherein: affine nano particle is selected from embodiment 3; The nanometer piece-rate system is selected from above-mentioned nanometer piece-rate system.
In the analytical equipment, except stationary installation (for example infusion set, signal extracting device, reactor fixed sturcture, or the like), all or part of consumptive material constitutes kit.The part kit of present embodiment preparation is listed in the table 5.
Table 5
| Kit | Reactor | Piece-rate system | Mk system | ? | ? |
| The separate targets thing | System | Affine particle | ? | ? | ? |
| K1 | ?R1 | Target antibody/aglucon antigen nano-particle compound | Z111 | ?B5 | The rhodamine mark |
| K2 | ?R2 | Target antibody/aglucon antigen nano-particle compound | Z112 | ?B6 | The rhodamine mark |
| K3 | ?R3 | Virion | One of Z111-Z113 | ?- | The rhodamine mark |
| K4 | ?R3 | Target antigen | Z200 | ?B11 | The rhodamine mark |
| K5 | ?R4 | Virion, target antibody/aglucon antigen nano-particle compound | Z112 | ?B5、?B6 | The rhodamine mark |
| K6 | ?R4 | Virion, target antibody/aglucon antigen nano-particle compound, target antigen/aglucon antibody nano-particle compound | Z112 | ?B1、?B5、?B6、 | The rhodamine mark |
| K7 | ?R5 | Target antibody/aglucon antigen nano-particle compound | One of Z111-Z113, or Z121, or Z122 | ?B5 | The rhodamine mark |
| K8 | ?R6 | Target antibody/aglucon antigen nano-particle compound | One of Z111-Z113 | ?B6 | The rhodamine mark |
| K9 | ?R7 | Virion | One of Z111-Z113 | ?- | The rhodamine mark |
| K10 | ?R7 | Virion, target antigen/aglucon antibody nano-particle compound | Z131 | ?B10 | The rhodamine mark |
| K11 | ?R7 | Virion | Z132 | ?- | The rhodamine mark |
| K12 | ?R7 | Virion | One of Z111-Z113 | ?B11 | Silver is strengthened golden mark |
| K13 | ?R7 | Virion, target antigen/aglucon antibody nano-particle compound | Z131 | ?B11 | Silver is strengthened golden mark |
| K14 | ?R8 | Virion, target antibody/aglucon antigen nano-particle compound, target antigen/aglucon antibody nano-particle compound | Z112 | ?B1、?B5、?B6、 | The rhodamine mark |
| K15 | ?R9 | Target antigen, target antibody | Z200 | ?B11 | The rhodamine mark |
Embodiment 5: the application of analytical equipment of the present invention and comparative studies (1)
In the present embodiment, the biochip kit of the present invention that is used for studying is selected from the biochip kit that embodiment 4 prepares.The conventional method of comparative studies is:
1). sampling
In the present embodiment, sample is respectively: HCV antibody positive serum, HIV
1+2The antibody positive human serum, HBs Ag positive serum, HCV antibody positive gamma globulin, and negative serum (HCV antibody, HIV
1+2Antibody and all negative serum of HBs Ag).All samples are all through using classical ELISA method to detect in advance.
2). the contrast chip agent box is provided
With the biochip kit that compares, wherein biochip reaction device and label are the same with the biochip reaction device and the label of biochip kit of the present invention.
3). object separates
To biochip kit of the present invention, during experiment, own dilute sample is separated earlier.The separate targets thing is the evaluating objects thing, or/and evaluating objects thing/affine nano-particle compound (separating the formation reaction that also comprises described compound this moment).Separation comprises: 1). the separation of not washing, and for example, 100 times of sample (containing or do not contain affine nano particle) dilutions concentrate 100 times with volume through nanofilter again, and object concentration increases greatly with the ratio of other small size biomolecule concentration; 2). the separation of washing, for example, 20 times of sample (containing or do not contain affine nano particle) dilutions, filter through nanofilter, and add cleansing solution flush away small size biomolecule, and concentrate again or do not concentrate, the object concentration and the ratio of other small size biomolecule concentration are increased greatly.
In the present embodiment, separation method is difference according to the different piece-rate systems in the kit, the principle of work of each piece-rate system in the reference example 1,2.In the present embodiment, in the formation of above-mentioned object/affine nano-particle compound reaction, the concentration of affine nano particle (w/v) is 10,000/between 1%.In the present embodiment, when above-mentioned object/affine nano-particle compound separated, compound maximum concentration (w/v) was less than 10%, preferably less than 5%.
In the present embodiment, separated product is the suspending liquid that contains the separate targets thing.
3). object is fixed and is detected
In the present embodiment,, object can be fixed on the reactor probe by above-mentioned product is added reactor. in the present embodiment, above-mentioned object/affine nano-particle compound in reactor fixedly the time concentration be 10,000/to 1%.Above-mentioned product is added reactor, both can be continuous adding, also can be discontinuous adding.More detailed description is as follows:
(1). non-current chip
The suspending liquid of 3-5 μ l separate targets thing is added in the reaction tank of described chip.With the biochip kit that compares, do not contain piece-rate system, do not separate, directly 3-5 μ l sample diluting liquid is added in the reaction tank of described chip.Wash with cleansing solution after 30 minutes 37 ℃ of reactions.Label (conventional concentration) addition is 5 μ l, and 37 ℃ of reactions were washed with cleansing solution after 30 minutes.The reaction of rhodamine mark, the dry laggard line scanning of reactor.Scanner is confocal laser scanner (GMS of Afymetrix company 418 chip scanners), scanning excitation wavelength 532nm, wavelength of transmitted light 570nm, laser intensity 35/50-55/70, the treated software of the signal that reads (JAGUAR II) is handled, and obtains the result after averaging then.The golden mark that silver amplifies is that catalyzer forms silver precipitation spot with the gold, and naked eyes can be surveyed.
(2) mobile chip
Suspending liquid with 10-15 μ l separate targets thing adds in the flowing reactive pond of described chip.With the biochip kit that compares, do not contain piece-rate system, do not separate, directly 10-15 μ l sample diluting liquid is added in the reaction tank of described chip.Flow velocity is between 10-15 μ l/ hour during application of sample.Behind the application of sample, add washing lotion with same flow velocity or high flow velocities.Taking off substrate then washs again.Add label (conventional concentration) solution at each reactor again, addition is about 10 μ l, 37 ℃ of temperature of reaction, 5 minutes reaction time.The reaction of rhodamine mark, the dry laggard line scanning of reactor.Scanner is confocal laser scanner (GMS of Afymetrix company 418 chip scanners), scanning excitation wavelength 532nm, wavelength of transmitted light 570nm, laser intensity 35/50-55/70, the treated software of the signal that reads (JAGUARII) is handled, and obtains the result after averaging then.The golden mark that silver amplifies is that catalyzer forms silver precipitation spot with the gold, and naked eyes can be surveyed.
Compare with the contrast chip agent box, chip agent box of the present invention all obtains higher sensitivity.For example, when positive is diluted to N times, utilize the contrast chip agent box can not survey the positive.And utilize chip agent box of the present invention, and even when being diluted to mN times (m＞1), still can measure the positive, i.e. sensitivity (or measuring lower limit) can improve m doubly.The numeral of m and N is decided by used positive and chip agent box.Generally, utilize chip agent box of the present invention, m is greater than 4, and is indivedual even greater than 10 or more.
More detailed description is stated embodiment as follows.
Embodiment 5.1. comprises the analysis that the object nano particle separates
In the present embodiment, analyze and comprise the steps: with described nanometer piece-rate system the evaluating objects thing particle that may exist in the separation acquisition sample; Isolated described evaluating objects thing particle is fixed in the reactor.
In the present embodiment, used biochip kit is selected from the prepared product of embodiment 4, for example the K3 in the table 5, K9, K11 and K12.
Embodiment 5.2. comprises the analysis that object/affine nano-particle compound separates
In the present embodiment, analyze and comprise the steps: that with described affine nano particle the evaluating objects thing reaction with existing in the sample forms object/affine nano-particle compound; With described nanometer piece-rate system, separate described object/affine nano-particle compound; Isolated described object/affine nano-particle compound is fixed in the reactor.
In the present embodiment, used biochip kit is selected from the prepared product of embodiment 4, for example the K1 in the table 5, K2, K7 and K8.
Embodiment 5.3. comprises object nano particle and analysis that object/affine nano-particle compound separates
In the present embodiment, analyze and comprise the steps: that with described affine nano particle the evaluating objects thing reaction with existing in the sample forms object/affine nano-particle compound; With described nanometer piece-rate system, compartment analysis object particle and described object/affine nano-particle compound; Isolated described evaluating objects thing particle and object/affine nano-particle compound are fixed in the reactor.
In the present embodiment, used biochip kit is selected from the prepared product of embodiment 4, for example the K5 in the table 5, K6, K13 and K14.
Embodiment 5.4. comprises the analysis that the magnetic levitation adsorption system is separated
In the present embodiment, analysis comprises the steps: with described magnetic levitation adsorption system compartment analysis object; Isolated described evaluating objects thing is fixed in the reactor.
In the present embodiment, used biochip kit is selected from the prepared product of embodiment 4, for example K15 in the table 5 and K4.
Embodiment 6: the comparative studies of analytical equipment of the present invention and application (2)
In the present embodiment, the biochip kit of the present invention that is used for studying is selected from the biochip kit that embodiment 4 prepares.The conventional method of comparative studies is identical with embodiment 5, the contrast chip agent box difference that only provides.
In the present embodiment, wherein all contain piece-rate system and affine nano particle, just the composition difference of aglucon/nanoparticle mixture.The preferred biochip kit of the present invention, in aglucon/nanoparticle mixture, the content of free aglucon is less than 50%, some in addition less than 20%; The content of non-affine nano particle is less than 50%, some in addition less than 20%.Yet, utilizing in weak activation nano particle or the preparation under the same conditions of disactivation nano particle, the content of free aglucon can be greater than 50%, and the content of non-affine nano particle can be greater than 50%.The latter is used to contrast in the chip agent box.
In the present embodiment, under equal conditions, the sensitivity (or measuring lower limit) that utilizes the preferred biochip kit of the present invention to be obtained is more than 200% of sensitivity (or measuring lower limit) that utilizes above-mentioned contrast chip agent box to be obtained.
Claims (20)
1. analytical equipment, its composition comprises analysis reactor and piece-rate system, and described piece-rate system comprises nanometer piece-rate system and one or more affine nano particles, wherein:
Described nanometer piece-rate system is selected from and contains nanofiltration system and the nanometer centrifugation systems of stablizing filter medium, and described stable filter medium is meant the filter medium that does not have metamorphosis in filter process;
Described affine nano particle is selected from the aglucon/activation nanoparticle mixture that contains affine nano particle, and wherein: described affine nano particle is meant that aglucon is combined in the compound that forms on the nano particle;
Described activation nano particle is meant the compound that activated group and nano particle form by covalent bond, and described nano particle is selected from inorganic nano-particle and organic nano particle; It is characterized in that: in described aglucon/activation nanoparticle mixture: the content of free aglucon less than 50% and the content of non-affine nano particle less than 50%, wherein: described non-affine nano particle is not for being fixed with the nano particle of aglucon basically; Described free aglucon is not for being fixed on the aglucon on the nano particle.
2. according to the described analytical equipment of claim 1, it is characterized in that: the average pore size of described stable filter medium is 15-100nm.
3. according to claim 1 or 2 described analytical equipments, it is characterized in that: described stable filter medium comprises hollow fiber nanometer chimney filter or nanofiltration membrane or giant molecule ultra filtration membrane.
4. according to the described analytical equipment of claim 1, it is characterized in that: described nanometer centrifugation systems contains centrifuge tube.
5. according to the described analytical equipment of claim 1, it is characterized in that: described nanometer centrifugation systems contains molecular exclusion gel separation system.
6. according to the described analytical equipment of claim 1, it is characterized in that: the content of described non-affine nano particle is less than 20% of described nano particle total amount.
7. according to the described analytical equipment of claim 1, it is characterized in that: in the described activation nano particle, activated group fixing on the 1g nano particle is greater than 70 μ mol or 1m
2Activated group fixing on the nanoparticle surface is greater than 0.5 μ mol.
8. according to the described analytical equipment of claim 7, it is characterized in that: described activated group is selected from: amino, general formula is-RNH
2Group.
9. according to the described analytical equipment of claim 8, it is characterized in that: when described activated group is selected from amino, described activated group be evenly distributed density greater than 1.85 μ mol/m
2Nanoparticle surface.
10. according to the described analytical equipment of claim 7, it is characterized in that: described activated group comprises amino diazanyl.
11. according to claim 1 or 7 described analytical equipments, it is characterized in that: described aglucon comprises the polypeptide aglucon.
12., it is characterized in that: in its at least one analysis reactor, both be fixed with probe antibody, and be fixed with antigen of probe again according to the described analytical equipment of claim 1.
13. according to claim 1 or 12 described analytical equipments, it is characterized in that: existing aglucon is the affine nano particle of antigen in its at least one analysis reactor, it is the affine nano particle of antibody that aglucon is arranged again.
14., it is characterized in that: for having the biochip kit of above-mentioned piece-rate system and reactor according to the described analytical equipment of claim 1.
15. according to the described analytical equipment of claim 14, it is characterized in that: described analysis reactor comprises non-current reactor.
16. according to the described analytical equipment of claim 14, it is characterized in that: described analysis reactor comprises flow reactor.
17. an analytical approach comprises the steps:
One of-sampling and claim 1-16 described analytical equipment;
-with described affine nano particle, with the evaluating objects thing reaction that may exist in the sample, formation object/affine nano-particle compound; Use described nanometer piece-rate system then, separate targets thing/affine nano-particle compound; Again isolated object/affine nano-particle compound is fixed in the reactor.
18., it is characterized in that according to the described analytical approach of claim 17: in the formation of above-mentioned object/affine nano-particle compound reaction, the concentration of described affine nano particle (w/v) is 10,000/between 1%.
19. according to the described analytical approach of claim 18, it is characterized in that: when above-mentioned object/affine nano-particle compound separated, described compound maximum concentration (w/v) was less than 10%.
20. according to the described analytical approach of claim 18, it is characterized in that: when above-mentioned object/affine nano-particle compound separated, described compound maximum concentration (w/v) was less than 5%.
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| Title |
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| 吕晓龙.纳米级分离技术研究－中空纤维分离膜的制备盒应用.《天津大学学报》.2000,第33卷(第3期),407-409. * |
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