CN108254549A - A kind of detection method and system, chip of the molecular amounts of marker to be measured - Google Patents
A kind of detection method and system, chip of the molecular amounts of marker to be measured Download PDFInfo
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- CN108254549A CN108254549A CN201810121368.1A CN201810121368A CN108254549A CN 108254549 A CN108254549 A CN 108254549A CN 201810121368 A CN201810121368 A CN 201810121368A CN 108254549 A CN108254549 A CN 108254549A
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- magnetic bead
- heaven
- ascending
- chip
- hole
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
Abstract
The present invention provides a kind of detection method and system, chip of the molecular amounts of marker to be measured, including：By the magnetic bead of pan coating primary antibody, the sample comprising marker to be measured, biotin labeling secondary antibody, be marked with enzyme Streptavidin add in reaction tank in, obtain being attached in the second compound on magnetic bead；The magnetic bead of attached second compound is added in into chip surface, and the magnetic bead is made to fall into the ascending to heaven in hole of the chip；Detection substrate is added in into the chip surface, and the detection substrate is made to flow into the ascending to heaven in hole of the chip, third preset time is incubated, the detection substrate is made to be reacted with the second compound on the magnetic bead, sends out light；Obtain luminous hole count of ascending to heaven；According to acquired luminous hole count of ascending to heaven, the molecular amounts of marker to be measured in the sample are calculated.The present invention can realize the detection of every milliliter of winged gram quantity grade, relatively traditional elisa technique, and detection sensitivity improves 100 times or more.
Description
Technical field
The present invention relates to field of biological detection, the detection method and system of the molecular amounts of espespecially a kind of marker to be measured,
Chip.
Background technology
The digitized detection method of current marker to be measured, application it is wider be enzyme-linked immunosorbent assay (enzyme-
Linked immuno sorbent assay, abbreviation ELISA).
Traditional ELISA method is detected using elisa plate, since the reacting hole volume of elisa plate is bigger,
In several hectolambdas, reaction needs many substrates that can just be detected, can realize the marker to be measured of every milliliter of pik magnitude
Detection, but can not achieve the detection of lower concentration.
Invention content
The object of the present invention is to provide a kind of detection methods and system, chip of the molecular amounts of marker to be measured, utilize
The monomolecular reaction volume that the miniature aperture of chip is provided can be detected accurately and treat mark as low as hundreds of protein moleculars
Will object, realizes the detection of every milliliter of winged gram quantity grade, relatively traditional elisa technique, and detection sensitivity improves 100 times or more.
Technical solution provided by the invention is as follows：
A kind of detection method of the molecular amounts of marker to be measured, including：Step S100 by the magnetic bead of pan coating primary antibody,
The secondary antibody of sample, biotin labeling comprising marker to be measured is marked in the Streptavidin addition reaction tank of enzyme, is obtained attached
The second compound being connected on magnetic bead；The magnetic bead of attached second compound is added in chip surface, and make the magnetic by step S200
Pearl falls into the ascending to heaven in hole of the chip；Detection substrate is added in the chip surface by step S300, and makes the detection substrate
Ascending to heaven in hole for the chip is flowed into, third preset time is incubated, makes the detection substrate and second on the magnetic bead compound
Object reacts, and sends out light；Step S400 obtains luminous hole count of ascending to heaven；Step S500 according to acquired luminous hole count of ascending to heaven,
Calculate the molecular amounts of marker to be measured in the sample.
In the above-mentioned technical solutions, it obtains combining the second compound of marker to be measured in reaction tank, and is attached in
On each magnetic bead；By the hole of ascending to heaven of magnetic bead and chip, the second compound for combining marker to be measured is made to be dispersed in each fly
It rises in hole；By the statistics to luminous hole count of ascending to heaven, the molecular amounts of more accurately marker to be measured can be obtained, it is opposite to pass
The elisa technique of system, detection sensitivity improve 100 times or more.
Further, the step S100 is specifically included：Step S110 by the magnetic bead of pan coating primary antibody, include mark to be measured
The sample of object, the secondary antibody of biotin labeling are added in reaction tank, are incubated the first preset time, are made the mark to be measured in the sample
Object, the secondary antibody and the anti-binding on the magnetic bead form the first compound；Step S120 cleans the reaction tank
Operation；Step S130 adds in the Streptavidin for being marked with enzyme in the reaction tank, is incubated the second preset time, makes the chain
Mould Avidin is combined to form the second compound with the first compound on the magnetic bead；Step S140 carries out the reaction tank clear
Wash operation.
In the above-mentioned technical solutions, by obtaining combining the second compound of marker to be measured in reaction tank, and it is attached
It is connected on each magnetic bead, by the follow-up experiment to each magnetic bead, is conducive to subsequently more accurately count point of marker to be measured
Subnumber amount.
Further, following any one step is further included before the step S100：Step S010 is made described by coupling procedure
Primary antibody is coated with magnetic bead surfaces, obtains the magnetic bead of coating primary antibody；Or, step S020 makes the primary antibody using the hydrophobicity of magnetic bead surfaces
Magnetic bead surfaces are adsorbed onto, obtain the magnetic bead of coating primary antibody.
In the above-mentioned technical solutions, the primary antibody can combine marker to be measured, by the way that each magnetic bead is allowed to be coated with primary antibody, so as to
Marker to be measured, secondary antibody etc. is made to be attached on magnetic bead and forms compound, convenient for subsequently through the follow-up experiment to each magnetic bead, more
Accurately count the molecular amounts of marker to be measured.
Further, the step S120 and/or the step S140 include：Step S10 adds in the bottom of the reaction tank
Enter magnetism, adsorb magnetic bead, sop up the liquid in the reaction tank；Phosphate buffer solution is added in the reaction tank by step S20
It is interior；Step S30 removes the magnetism of the bottom of the reaction tank, and magnetic bead is resuspended；Step S40 adds in magnetic in the bottom of the reaction tank
Property, magnetic bead is adsorbed, sops up the liquid in the reaction tank；Step S50 repeating said steps S20- step S40, until cleaning
After number of operations reaches preset times, terminate cleaning operation.
In the above-mentioned technical solutions, the detection by fully washing away the substance of non-specific binding and being not bound with
Secondary antibody, Streptavidin etc., so as to avoid the introducings such as the substance of non-specific binding and extra reagent to experimental result
Interference.
Further, the step S300 includes：The detection substrate of beta galactosidase is added in the chip by step S310
Surface, and the detection substrate is made to flow into the ascending to heaven in hole of the chip；Step S320 be incubated third preset time, make the β-
The detection substrate of galactosidase is reacted with the second compound on the magnetic bead, sends out light.
In the above-mentioned technical solutions, by adding in substrate, the hole of ascending to heaven of the magnetic bead containing attached second compound is made to shine,
Convenient for more obviously showing and statistical test result.
Further, the step S400 includes：Step S410 obtains the photo of the chip containing all holes of ascending to heaven；Step
Rapid S420 detects the luminous point on the photo, obtains the luminous hole count of ascending to heaven.
In the above-mentioned technical solutions, a kind of statistical method according to luminous point is provided, than traditional depth according to color
It is shallow more accurate to judge.
The present invention also provides a kind of chip, including：Chip body；The one side of the chip body flies comprising at least 40,000
Rise hole, each blind hole that the first surface of chip described in Kong Weicong is recessed to second surface of ascending to heaven；First table
Face is opposite with the second surface；For cylinder, the ranging from 2um-10um of base diameter is high ranging from the hole of ascending to heaven
2um-10um。
In the above-mentioned technical solutions, monomolecular reaction volume is provided by the hole of ascending to heaven of chip, is advantageously implemented every milliliter
The detection of the marker to be measured of pik magnitude, improves the sensitivity of detection.
Further, the chip body is evenly arranged with hole of ascending to heaven on one side.
In the above-mentioned technical solutions, by being uniformly arranged hole of ascending to heaven, reaction is made uniformly to carry out, the shadow between the hole that avoids ascending to heaven
It rings, convenient for statistics, estimation result.
The present invention also provides a kind of detecting system of the molecular amounts of marker to be measured, including：Said chip；It further includes：
Magnetic bead, reaction tank；The reaction tank, for when in compound synthesis phase, the magnetic bead of pan coating primary antibody to include
It the sample of marker to be measured, the secondary antibody of biotin labeling and is marked with the Streptavidin of enzyme and is carried out in the reaction tank instead
It should；The chip, for when in detection-phase, the magnetic bead falls within the ascending to heaven in hole of the chip.
In the above-mentioned technical solutions, a kind of detecting system of the molecular amounts of marker to be measured is provided.The system utilizes
The monomolecular reaction volume that the hole of ascending to heaven of chip is provided can obtain the molecular amounts of more accurately marker to be measured, relatively
Traditional elisa technique, detection sensitivity improve 100 times or more.
Further, when cylinder is in the hole of ascending to heaven：The diameter of the bottom in the hole of ascending to heaven and the diameter of the magnetic bead
Ratio, between 1.2~1.6；The ratio of the height and the diameter of the magnetic bead in the hole of ascending to heaven, between 0.5~1.5 it
Between.
In the above-mentioned technical solutions, by limiting the proportionate relationship in the aperture in hole of ascending to heaven and the diameter of magnetic bead and ascending to heaven
The proportionate relationship of the height and the diameter of magnetic bead in hole, makes a hole of ascending to heaven only accommodate a magnetic bead, so as to make statistical result more accurate.
It, being capable of band by a kind of detection method and system, chip of the molecular amounts of marker to be measured provided by the invention
Carry out following at least one advantageous effect：
1st, the present invention can promote detection sensitivity, relatively traditional elisa technique, detection sensitivity improve 100 times with
On.
2nd, the present invention makes the hole of ascending to heaven containing the magnetic bead for attaching the second compound shine by adding in substrate, can be brighter
Aobvious display and statistical test result.
Description of the drawings
Below by a manner of clearly understandable, preferred embodiment is described with reference to the drawings, a kind of marker to be measured is divided
The detection method and system of subnumber amount, above-mentioned characteristic, technical characteristic, advantage and its realization method of chip give furtherly
It is bright.
Fig. 1 is a kind of flow chart of one embodiment of the detection method of the molecular amounts of marker to be measured of the present invention；
Fig. 2 is a kind of flow of another embodiment of the detection method of the molecular amounts of marker to be measured of the present invention
Figure；
Fig. 2 a are a kind of another embodiments of the detection method of the molecular amounts of marker to be measured of the present invention to anti-
Pond is answered to carry out the particular flow sheet of cleaning operation；
Fig. 3 is a kind of structure diagram of one embodiment of chip of the present invention；
Fig. 4 is a kind of structural representation of one embodiment of the detecting system of the molecular amounts of marker to be measured of the present invention
Figure；
Fig. 5 is the vertical view of one embodiment of the chip of the present invention；
Fig. 6 is the vertical view of another embodiment of the chip of the present invention.
Drawing reference numeral explanation：
1000. detecting systems, 1100. chips, 1200. magnetic beads, 1300. reaction tanks, 1110. chip bodies, 1111. ascend to heaven
Hole.
Specific embodiment
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, control is illustrated below
The specific embodiment of the present invention.It should be evident that the accompanying drawings in the following description is only some embodiments of the present invention, for
For those of ordinary skill in the art, without creative efforts, other are can also be obtained according to these attached drawings
Attached drawing, and obtain other embodiments.
To make simplified form, part related to the present invention is only schematically shown in each figure, they are not represented
Its practical structures as product.In addition, so that simplified form is easy to understand, there is identical structure or function in some figures
Component only symbolically depicts one of those or has only marked one of those.Herein, "one" is not only represented
" only this " can also represent the situation of " more than one ".
In one embodiment of the invention, as shown in Figure 1, a kind of detection method of the molecular amounts of marker to be measured,
Including：
Step S100 by the magnetic bead of pan coating primary antibody, the sample comprising marker to be measured, biotin labeling secondary antibody, mark
The Streptavidin that note has enzyme is added in reaction tank, obtains being attached in the second compound on magnetic bead；
The magnetic bead of attached second compound is added in chip surface, and the magnetic bead is made to fall into the chip by step S200
It ascends to heaven in hole；
Detection substrate is added in the chip surface by step S300, and the detection substrate is made to flow into ascending to heaven for the chip
In hole, third preset time is incubated, the detection substrate is made to be reacted with the second compound on the magnetic bead, sends out light；
Step S400 obtains luminous hole count of ascending to heaven；
Step S500 calculates the molecular number of marker to be measured in the sample according to acquired luminous hole count of ascending to heaven
Amount.
Specifically, need to be ready to reagent and sample before experiment, primary antibody be it is a kind of can be with marker specificity knot to be measured
The albumen of conjunction；Secondary antibody is also a kind of albumen that can be specifically bound with marker to be measured, secondary antibody biotin labeling, and biotin is
A kind of vitamin, such as vitamin B7；Streptavidin is marked with enzyme, and the purpose of enzyme label is to subsequently add in detection substrate
When by developing the color or shining detect the presence of compound, enzyme can be beta galactosidase or other with catalytic activity
Enzyme；Sample includes marker to be measured, such as serum specimen.
Magnetic bead surfaces is allowed to be coated with primary antibody first, then by the magnetic bead, sample comprising marker to be measured, biotin labeling
Secondary antibody is marked in the Streptavidin addition reaction tank of enzyme, generates the second compound.Marker to be measured and an anti-binding, two
It is anti-to be combined again with marker to be measured, obtain the first compound；It is marked with the Streptavidin of enzyme and the combination energy that biotin is very high
Power, and reacted with the first compound, the second compound is obtained, since primary antibody is coated on magnetic bead, so in reaction process
The compound gradually formed is also coated on magnetic bead therewith.
The magnetic bead of attached second compound is added in into chip surface, by high speed centrifugation, magnetic bead is made to fall into ascending to heaven for chip
In hole.Since the hole size of ascending to heaven of chip can only accommodate a magnetic bead, so each hole of ascending to heaven at most falls into a magnetic bead.
The detection substrate of beta galactosidase is added in chip surface, and makes the hole of ascending to heaven of the detection substrate inflow chip
It is interior, it is incubated third preset time, such as 15~30 minutes, in hole of ascending to heaven, if having attached the second compound on magnetic bead, this
Two compounds can send out faint light, i.e. fluorescence after being reacted with the detection substrate of beta galactosidase, then comprising the magnetic bead
Hole of ascending to heaven can shine；If fluorescence, i.e. ascending to heaven comprising corresponding magnetic bead cannot be sent out without forming the second compound on magnetic bead
Hole cannot shine.
Only in hole of ascending to heaven, monomolecular beta galactosidase, which can react, generates enough substrates, the substrate of generation
The fluorescence sent out can be detected by instrument.And traditional elisa plate is since reaction pore volume is in several hectolambdas, reaction needs very much
Substrate can just be detected, so can not achieve monomolecular detection.
The hole count of ascending to heaven to shine on chip is counted, for example is counted using third party's instrument, each luminous point represents one
A marker, so as to calculate the total amount of marker to be measured.If sample is added in reaction tank after diluting, according to diluted
Ratio calculates the molecular amounts of marker to be measured that raw sample contains.
By the statistics to luminous hole count of ascending to heaven, the molecular amounts of more accurately marker to be measured can be obtained, relatively
Traditional elisa technique, detection sensitivity improve 100 times or more.
In another embodiment of the present invention, as shown in Fig. 2, Fig. 2 a, a kind of inspection of the molecular amounts of marker to be measured
Survey method, including：
Step S010 makes the primary antibody be coated with magnetic bead surfaces by coupling procedure, obtains the magnetic bead of coating primary antibody；Or, step
S020 makes the primary antibody be adsorbed onto magnetic bead surfaces using the hydrophobicity of magnetic bead surfaces, obtains the magnetic bead of coating primary antibody；
Step S110 adds the secondary antibody of the magnetic bead of pan coating primary antibody, the sample comprising marker to be measured, biotin labeling
Enter in reaction tank, be incubated the first preset time, make marker to be measured, the secondary antibody and one on the magnetic bead in the sample
Anti-binding forms the first compound；
Step S120 carries out cleaning operation to the reaction tank；
Step S130 adds in the Streptavidin for being marked with enzyme in the reaction tank, is incubated the second preset time, makes institute
Streptavidin is stated to be combined to form the second compound with the first compound on the magnetic bead；
Step S140 carries out cleaning operation to the reaction tank；
The magnetic bead of attached second compound is added in chip surface, and the magnetic bead is made to fall into the chip by step S200
It ascends to heaven in hole；
The detection substrate of beta galactosidase is added in the chip surface by step S310, and flows into the detection substrate
The chip is ascended to heaven in hole；
Step S320 is incubated third preset time, makes the detection substrate of the beta galactosidase and the on the magnetic bead
Two compounds react, and send out light；
Step S410 obtains the photo of the chip containing all holes of ascending to heaven；
Step S420 detects the luminous point on the photo, obtains the luminous hole count of ascending to heaven；
Step S500 calculates the molecular number of marker to be measured in the sample according to acquired luminous hole count of ascending to heaven
Amount.
The step S120 and/or the step S140 include：
Step S10 adds in magnetism in the bottom of the reaction tank, adsorbs magnetic bead, sops up the liquid in the reaction tank；
Step S20 adds in phosphate buffer solution in the reaction tank；
Step S30 removes the magnetism of the bottom of the reaction tank, and magnetic bead is resuspended；
Step S40 adds in magnetism in the bottom of the reaction tank, adsorbs magnetic bead, sops up the liquid in the reaction tank；
Step S50 repeating said steps S20- step S40 until after the number of operations of cleaning reaches preset times, terminate
Cleaning operation.
Specifically, relatively previous embodiment, the present embodiment, instead of step S100, uses step with step S110-S140
S310-S320, with step S410-S420 instead of step S400, increases step S000, step S10- instead of step S300
S50。
There are many methods for magnetic bead coating primary antibody, and one kind is by coupling procedure, utilizes the carboxyl and antibody Fc of magnetic bead surfaces
Amino chemical reaction in segment, forms covalently coupled；Antibody divides Fab segments and Fc segments, wherein Fab segments and antigen binding
It is related, so the coupled combination for not interfering with antibody and detecting marker using Fc segments.Another kind utilizes magnetic bead surfaces
Hydrophobicity actively adsorbs primary antibody to material surface.
The secondary antibody of the magnetic bead of pan coating primary antibody, the sample comprising marker to be measured, biotin labeling is added in into reaction tank
It is interior, it is incubated the first preset time, such as 15 minutes, makes the anti-binding shape on marker to be measured in sample, secondary antibody and magnetic bead
Into the first compound.In order to ensure only adsorbing a marker to be measured on each magnetic bead, need to comprising marker to be measured
Sample carries out appropriate dilution, it is proposed that marker number to be measured is less than the ratio of hole count of ascending to heaven, control marker to be measured and magnetic bead
Less than 0.5.
Later, magnetism is added in reaction tank bottom, for example magnet is placed in bottom, adsorbs magnetic bead, then sops up in reaction tank
Liquid, the liquid got rid of in reaction tank can also be centrifuged.Cleaning solution, such as phosphate buffer solution are added in reaction tank
(PBST adds in 0.05%Twen20 in the solution) removes reaction tank bottom magnet, stirs magnetic bead, magnetic bead is made to hang again,
Magnet is placed in reaction tank bottom again, adsorbs magnetic bead, then the liquid or centrifugation sopped up in reaction tank gets rid of the liquid in reaction tank,
This calculates a cleaning action.If preset times are 3 times, above-mentioned cleaning action is repeated, until the number of operations of cleaning reaches
Preset times.By the operation cleaned above, the secondary antibody that is not bound in abundant washing reaction pond.
The Streptavidin for being marked with enzyme is added in the reaction tank, the second preset time, such as 15 minutes is incubated, makes
The Streptavidin is combined to form the second compound with the first compound on the magnetic bead.
Later, added in reaction tank bottom magnetic, for example bottom places magnet, adsorbs magnetic bead, then sops up or centrifugal drying
Fall the liquid in reaction tank.Cleaning solution is added in reaction tank, for example (PBST is added in phosphate buffer solution in the solution
0.05%Twen20), reaction tank bottom magnet is removed, magnetic bead is stirred, magnetic bead is made to hang again, then is placed in reaction tank bottom
Magnet adsorbs magnetic bead, sops up or centrifuge the liquid got rid of in reaction tank, this calculates a cleaning action.If preset times are 3
It is secondary, then above-mentioned cleaning action is repeated, until the number of operations of cleaning reaches preset times.By the operation cleaned above, fully
The Streptavidin being not bound in washing reaction pond.
The magnetic bead of attached second compound is added in into chip surface, by high speed centrifugation, magnetic bead is made to fall into ascending to heaven for chip
In hole.The detection substrate of beta galactosidase is added in, is allowed to flow into ascending to heaven in hole for chip, is incubated third preset time, such as
15 minutes, the detection substrate of beta galactosidase is made to be reacted with the second compound on magnetic bead, sends out light.
Chip is positioned under digital microscope, is taken pictures around porose place, then containing porose all
The luminous point for being spliced into a photo, detecting on photo of the seamless no coincidence of photo in place, each point represent a marker,
So as to calculate the total amount of marker to be measured.If sample is added in reaction tank after diluting, according to diluted ratio, calculate
The molecular amounts of marker to be measured that raw sample contains.
For the above method, it is exemplified below：Early detection and long-term tracking to serum p-tau-181.In serum
The occurrence and development of p-tau-181 concentration and senile dementia have critically important correlation.
First, it will specifically bind in the antibody coating to magnetic bead of Tau albumen.
The magnetic bead after coating, the blood plasma of patient 10ul, two antibody of biotin labeling, this antibody are added in reaction tank
The phosphorylation in 1 site of Tau protein 18s can be specifically bound.After being incubated 15min in reaction tank, magnetic is added in reaction tank bottom
Property, magnetic bead is adsorbed, sops up the liquid in reaction tank.PBST solution is added in, removes reaction tank bottom magnetic, magnetic bead is resuspended, then add
Enter magnetism, sop up the liquid in reaction tank.It repeats this process three times, fully washs the secondary antibody being not bound with.
The Streptavidin for being marked with beta galactosidase is added in, after being incubated 15min in reaction tank, in reaction tank bottom
The compound magnetic, absorption magnetic bead is formed is added in, sops up the liquid in reaction tank.Add in PBST solution, removal reaction tank bottom
Magnetism is resuspended magnetic bead, adds magnetism, sop up the liquid in reaction tank.Repeat this process three times, fully washing is not bound with
Streptavidin.
The magnetic bead for forming compound is added in into chip surface, magnetic bead can fall into the aperture of chip surface under high speed centrifugation
It is interior.The detection substrate of beta galactosidase is added in chip surface, detection substrate inflow chip is ascended to heaven in hole, in hole and multiple
Fluorescence can be sent out after closing the beta galactosidase reaction of object, the magnetic bead without forming compound with p-tau-181 cannot be sent out
Go out fluorescence.Fluorescence signal in detection chip calculates amounts of the p-tau-181 in serum.
In another embodiment of the present invention, as shown in Fig. 3, Fig. 5, a kind of chip 1100, including：
Chip body 1110；
The one side of the chip body 1110 includes at least 40,000 holes 1111 of ascending to heaven, the hole 1111 of ascending to heaven be from
The blind hole that the first surface of the chip 1100 is recessed to second surface；The first surface and the second surface phase
It is right；
Cylinder is in the hole 1111 of ascending to heaven, the ranging from 2um-10um of base diameter, high ranging from 2um-10um.
Specifically, Fig. 3 be chip structure diagram, Fig. 5 be chip vertical view (be intended merely to facilitate understanding in figure,
Schematically several holes of ascending to heaven are drawn, and be not drawn into the hole of ascending to heaven of actual quantity).
Chip includes chip body and at least 40,000 holes of ascending to heaven.If the hole number of ascending to heaven on fruit chip is very little, then need
Macrodilution will be carried out containing the sample of marker to be measured, because marker to be measured is very little, will also result in larger test error；If
Hole number of ascending to heaven is too many, and the cost of chip can be higher, can also reduce the sensitivity of detection, when most of magnetic beads do not shine,
During the quantity of a small amount of luminous magnetic bead of detection, error can improve, and reduce detection sensitivity.About 40,000 holes of ascending to heaven, had both met survey
The needs of examination, and make chip cost moderate.
Using micro-processing technology, the aperture of size of ascending to heaven is gone out on chip.Ascend to heaven hole be mainly used for a protein molecular or
The diameter of the detection of viruses molecule, usual viruses molecule or protein molecular is bigger than general molecule, which also needs and primary antibody, two
Anti- that reactions is waited to form compound, about in 0.1um or so, which is attached on magnetic bead the size of compound, and hole of ascending to heaven needs to hold
The magnetic bead for having attached compound is received, in addition it is also contemplated that the Kong Tai little that ascends to heaven can cause magnetic bead to be not easily accessible in hole and too small
It ascends to heaven the difficulty of processing in hole etc., so the size in hole of ascending to heaven need to do appropriate amplification in more than size.But hole is not ascended to heaven also not
Can be too big, since the detection substrate that single enzyme is catalyzed within certain reaction time is as many, so the volume in hole of ascending to heaven
Influence the power of the detection signal of light.When hole shape of ascending to heaven is cylinder, it is proposed that the ranging from 2um-10um of base diameter is high
The ranging from 2um-10um of degree.Hole shape of ascending to heaven may be square or cuboid etc., consider that magnetic bead is spherical, hole of ascending to heaven
The volume that shape is consumed by cylinder is minimum, and in this way convenient for going out more holes on the chip of equal volume, it is advantageous to fly
It is cylinder to rise hole shape.
Each hole of ascending to heaven is recessed to obtain from the first surface of chip for blind hole to second surface.It ascends to heaven because magnetic bead need to be fallen into
Hole is detected the reaction in stage, so hole of ascending to heaven is necessary for blind hole, ensure magnetic bead carrying and magnetic bead and detection substrate it is anti-
It should.
With base diameter 3.2um, for high 4.0um, the volume in hole of ascending to heaven is：Floor space * cylinders height=π * r2* h=
3.14*(3.2/2)2* 4=32.1536 cu μ ms=32.1536fL, i.e., 32.1536 ascend to heaven.
In another embodiment of the present invention, as shown in figure 3, a kind of chip 1100, including：
Chip body 1110；
The one side of the chip body 1110 includes at least 40,000 holes 1111 of ascending to heaven, the hole 1111 of ascending to heaven be from
The blind hole that the first surface of the chip 1100 is recessed to second surface；The first surface and the second surface phase
It is right；
Cylinder is in the hole 1111 of ascending to heaven, the ranging from 2um-10um of base diameter, high ranging from 2um-10um；
The chip body 1110 is evenly arranged with hole 1111 of ascending to heaven on one side.
Specifically, relatively previous embodiment, embodiment adds constraints：The one side of the hole in chip body of ascending to heaven
It is uniformly arranged.
By being uniformly arranged hole of ascending to heaven, make test marker to be measured molecular amounts reaction in each hole uniformly into
Row, the influence between the hole that avoids ascending to heaven, convenient for statistics, estimation result.
When the shape of chip body is cuboid, hole of ascending to heaven arranged on the chip by array way (as shown in figure 5,
Only several holes of ascending to heaven schematically have been drawn in figure), this structure can make to ascend to heaven hole count most with the shape of chip body
Bigization makes magnetic bead equably fall into hole of ascending to heaven, and makes testing result more effective.
When the shape of chip body is cylinder, hole of ascending to heaven is outside as the center of circle using the center of chip body on the chip
Radial arrangement (as shown in fig. 6, only schematically having drawn several holes of ascending to heaven in figure)；This structure can be with chip sheet
The shape of body makes hole count maximization of ascending to heaven, magnetic bead is made equably to fall into hole of ascending to heaven, makes testing result more effective.
In another embodiment of the present invention, as shown in figure 4, a kind of detecting system of the molecular amounts of marker to be measured
1000, including：Chip 1100 described in any of the above-described embodiment；It further includes：Magnetic bead 1200, reaction tank 1300；
The reaction tank 1300, for when in compound synthesis phase, the magnetic bead of pan coating primary antibody to include
It the sample of marker to be measured, the secondary antibody of biotin labeling and is marked with the Streptavidin of enzyme and is carried out in the reaction tank 1300
Reaction；
The chip 1100, for when in detection-phase, the magnetic bead 1200 to fall within ascending to heaven for the chip 1100
In hole.
Specifically, provide a kind of detecting system of the molecular amounts of marker to be measured, the system include chip, magnetic bead and
Reaction tank.
Compound synthesis phase refers to the generation phase of the first compound and the second compound.In compound synthesis phase,
Magnetic bead is in reaction tank.It is carried out in reaction tank by the secondary antibody of the magnetic bead and marker to be measured, biotin labeling of coating primary antibody
Reaction forms the first compound；It is carried out in reaction tank instead by the first compound and Streptavidin that are attached on magnetic bead again
Should, form the second compound.
Detection-phase refers to addition detection substrate, makes the stage of marker luminescence display to be measured.In detection-phase, magnetic bead is
In ascending to heaven in hole for chip.It is carried out in the hole of ascending to heaven of chip instead by the second compound and detection substrate that are attached on magnetic bead
Should, fluorescence is sent out, so as to which corresponding hole of ascending to heaven be made to shine.
The monomolecular reaction volume that the detecting system is provided using the hole of ascending to heaven of chip can obtain more accurately to be measured
The molecular amounts of marker, relatively traditional elisa technique, detection sensitivity improve 100 times or more.
In another embodiment of the present invention, as shown in figure 4, a kind of detecting system of the molecular amounts of marker to be measured
1000, including：Chip 1100 described in any of the above-described embodiment；It further includes：Magnetic bead 1200, reaction tank 1300；
The reaction tank 1300, for when in compound synthesis phase, the magnetic bead of pan coating primary antibody to include
It the sample of marker to be measured, the secondary antibody of biotin labeling and is marked with the Streptavidin of enzyme and is carried out in the reaction tank 1300
Reaction；
The chip 1100, for when in detection-phase, the magnetic bead 1200 to fall within ascending to heaven for the chip 1100
In hole 1111；
When cylinder is in the hole 1111 of ascending to heaven：
The ratio of the diameter of the bottom in the hole 1111 of ascending to heaven and the diameter of the magnetic bead 1200, between 1.2~1.6 it
Between；
The ratio of the height and the diameter of the magnetic bead 1200 in the hole 1111 of ascending to heaven, between 0.5~1.5.
Specifically, relatively previous embodiment, embodiment adds the proportionate relationships of ascend to heaven hole size and magnetic bead size
Constraint.
By limiting the proportionate relationship in the aperture in hole of ascending to heaven and the diameter of magnetic bead, in the horizontal direction it is only capable of a hole of ascending to heaven
A magnetic bead is accommodated, so as to make statistical result more accurate.
Proportionate relationship by the height and the diameter of magnetic bead that limit hole of ascending to heaven, makes a hole of ascending to heaven be only capable of holding in vertical direction
A magnetic bead is received, so as to make statistical result more accurate.
This system further includes：Magnet；The magnet, for when performing cleaning to reaction tank, being added in reaction tank bottom
Magnetism adsorbs magnetic bead.It further includes：Video camera；The video camera, for obtaining the photograph of the chip containing all holes of ascending to heaven
Piece.
It should be noted that above-described embodiment can be freely combined as needed.The above is only the preferred of the present invention
Embodiment, it is noted that for those skilled in the art, in the premise for not departing from the principle of the invention
Under, several improvements and modifications can also be made, these improvements and modifications also should be regarded as protection scope of the present invention.
Claims (10)
1. a kind of detection method of the molecular amounts of marker to be measured, which is characterized in that including：
Step S100 by the magnetic bead of pan coating primary antibody, the sample comprising marker to be measured, biotin labeling secondary antibody, be marked with
The Streptavidin of enzyme is added in reaction tank, obtains being attached in the second compound on magnetic bead；
The magnetic bead of attached second compound is added in chip surface, and the magnetic bead is made to fall into ascending to heaven for the chip by step S200
In hole；
Detection substrate is added in the chip surface by step S300, and makes the hole of ascending to heaven of the detection substrate inflow chip
It is interior, third preset time is incubated, the detection substrate is made to be reacted with the second compound on the magnetic bead, sends out light；
Step S400 obtains luminous hole count of ascending to heaven；
Step S500 calculates the molecular amounts of marker to be measured in the sample according to acquired luminous hole count of ascending to heaven.
2. the detection method of the molecular amounts of marker to be measured according to claim 1, which is characterized in that the step
S100 is specifically included：
Step S110 adds in the secondary antibody of the magnetic bead of pan coating primary antibody, the sample comprising marker to be measured, biotin labeling anti-
In Ying Chi, the first preset time is incubated, makes marker to be measured, the secondary antibody and the resistive connection on the magnetic bead in the sample
It closes and forms the first compound；
Step S120 carries out cleaning operation to the reaction tank；
Step S130 adds in the Streptavidin for being marked with enzyme in the reaction tank, is incubated the second preset time, makes the chain
Mould Avidin is combined to form the second compound with the first compound on the magnetic bead；
Step S140 carries out cleaning operation to the reaction tank.
3. the detection method of the molecular amounts of marker to be measured according to claim 1, which is characterized in that the step
Following any one step is further included before S100：
Step S010 makes the primary antibody be coated with magnetic bead surfaces by coupling procedure, obtains the magnetic bead of coating primary antibody；Or,
Step S020 makes the primary antibody be adsorbed onto magnetic bead surfaces using the hydrophobicity of magnetic bead surfaces, obtains the magnetic bead of coating primary antibody.
4. the detection method of the molecular amounts of marker to be measured according to claim 2, which is characterized in that the step
S120 and/or the step S140 include：
Step S10 adds in magnetism in the bottom of the reaction tank, adsorbs magnetic bead, sops up the liquid in the reaction tank；
Step S20 adds in phosphate buffer solution in the reaction tank；
Step S30 removes the magnetism of the bottom of the reaction tank, and magnetic bead is resuspended；
Step S40 adds in magnetism in the bottom of the reaction tank, adsorbs magnetic bead, sops up the liquid in the reaction tank；
Step S50 repeating said steps S20- step S40 until after the number of operations of cleaning reaches preset times, terminate cleaning
Operation.
5. the detection method of the molecular amounts of marker to be measured according to claim 1, which is characterized in that the step
S300 includes：
The detection substrate of beta galactosidase is added in the chip surface by step S310, and is made described in the detection substrate inflow
Chip is ascended to heaven in hole；
Step S320 is incubated third preset time, makes the detection substrate of the beta galactosidase and second on the magnetic bead multiple
Object reaction is closed, sends out light.
6. the detection method of the molecular amounts of marker to be measured according to claim 1, which is characterized in that the step
S400 includes：
Step S410 obtains the photo of the chip containing all holes of ascending to heaven；
Step S420 detects the luminous point on the photo, obtains the luminous hole count of ascending to heaven.
7. a kind of chip, which is characterized in that including：
Chip body；
The one side of the chip body includes at least 40,000 holes of ascending to heaven, the first table of each chip described in Kong Weicong of ascending to heaven
The blind hole being recessed towards second surface；The first surface is opposite with the second surface；
Cylinder is in the hole of ascending to heaven, the ranging from 2um-10um of base diameter, high ranging from 2um-10um.
8. chip according to claim 7, it is characterised in that：
The chip body is evenly arranged with hole of ascending to heaven on one side.
9. a kind of detection system of the detection method of molecular amounts using any markers to be measured of the claims 1-6
System, which is characterized in that including any chips of right 7-8；It further includes：Magnetic bead, reaction tank；
The reaction tank, for when in compound synthesis phase, the magnetic bead of pan coating primary antibody to include mark to be measured
It the sample of object, the secondary antibody of biotin labeling and is marked with the Streptavidin of enzyme and is reacted in the reaction tank；
The chip, for when in detection-phase, the magnetic bead falls within the ascending to heaven in hole of the chip.
10. detecting system according to claim 9, it is characterised in that：
When cylinder is in the hole of ascending to heaven：
The ratio of the diameter of the bottom in the hole of ascending to heaven and the diameter of the magnetic bead, between 1.2~1.6；
The ratio of the height and the diameter of the magnetic bead in the hole of ascending to heaven, between 0.5~1.5.
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| CN201810121368.1A CN108254549A (en) | 2018-02-07 | 2018-02-07 | A kind of detection method and system, chip of the molecular amounts of marker to be measured |
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| CN201810121368.1A CN108254549A (en) | 2018-02-07 | 2018-02-07 | A kind of detection method and system, chip of the molecular amounts of marker to be measured |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110361370A (en) * | 2019-07-31 | 2019-10-22 | 深圳中山泌尿外科医院 | A kind of single embryo's secretory protein quantitative detecting method based on Microfluidic droplet |
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| CN102884431A (en) * | 2010-03-01 | 2013-01-16 | 匡特里克斯公司 | Ultra-sensitive detection of molecules or particles using beads or other capture objects |
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