CN105259356B - The full-automatic ELISA detection means and its detection method of microspironema pallidum on a kind of micro-fluidic chip - Google Patents
The full-automatic ELISA detection means and its detection method of microspironema pallidum on a kind of micro-fluidic chip Download PDFInfo
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Abstract
The invention discloses a kind of full-automatic ELISA detection means of microspironema pallidum on micro-fluidic chip, including micro-fluidic chip, dc source, fluorescence detecting system and control system, the micro-fluidic chip includes liquid storage tank I, liquid storage tank II, liquid storage tank III, liquid storage tank IV, liquid storage tank V, liquid storage tank VI, liquid storage tank VII and main channel, the liquid storage tank I, the liquid storage tank II, the liquid storage tank III, the liquid storage tank IV and the liquid storage tank V are connected by sample inlet channel with the entrance of the main channel respectively, the outlet of the main channel is connected with sample export passage, the other end of the sample export passage is connected with the liquid storage tank VII.The invention also discloses a kind of detection method using the full-automatic ELISA detection means of microspironema pallidum on a kind of above-mentioned micro-fluidic chip.The present invention has mechanization degree height, operation sequence simple, small volume, precise structure, and reliability height, cost is low, the features such as being easy to implement Automated condtrol.
Description
Technical field
The present invention relates to a kind of microspironema pallidum (Treponemapallidum, TP) fully-automated synthesis and the device separated
And its detection method, the present invention mainly realized on micro-fluidic chip ELISA modes in blood microspironema pallidum it is complete from
The full-automatic ELISA detection means and its detection method of microspironema pallidum on dynamic detection, specifically a kind of micro-fluidic chip.
Background technology
Microspironema pallidum (Treponemapallidum, TP) is similar to fine and closely woven spring, and helical buckling rule, two ends point is straight,
It is transparent, not easy coloring, thus also known as spirochaeta pallida, it, which infects human body, can cause chronic sexually transmitted disease --- syphilis.Syphilis is passed
Metachromia is strong, and harmfulness is big, whole body can be caused respectively to organize after infection, the infringement of organ, and its incidence of disease has rise trend in recent years, because
This selects a kind of microspironema pallidum detection simplicity, and accurate method assists clinical diagnosis and treatment to be very important.
Conventional treponemal body detecting method mainly has at present：Microspironema pallidum pathogen detection, such as dark-field microscope
Inspection technique, silver-plated development process and direct immunofluorescence；Non-syphilitic leptospira antigen serum test, such as Venereal Disease Research Laboratory are tried
Test, Rapid plasma reagintest, toluidine red are tested；TP antigen serum test, such as fluorescence syphilis
Helicoid antibody absorption test, microspironema pallidum hemagglutination test (HA test) and EUSA；Protocols in Molecular Biology
Deng.
Microspironema pallidum pathogen detection method only needs to direct smear microscopy, and operation is easier, but the detection method
Implementation but limit by materials and corresponding special microscope, and sample requirement is higher, and especially testing result sensitiveness is relatively low,
Therefore clinical practice is less.
Non-syphilitic leptospira antigen serum test is poor due to Sensitivity and Specificity, is typically limited to only Primary Screening Test and treatment
Effect observation；Wherein Venereal Disease Research Laboratory Test, because required reagent of its experiment needs now with the current, preparation is more numerous and diverse,
Therefore domestic laboratory is seldom used；Although and the judgement that Rapid plasma reagintest infects to curative effect and again has
Certain values, but because its specificity is poor, it is only used as primary dcreening operation means, it is impossible to individually make a definite diagnosis syphilis；Although toluidine red experiment has
Help understand the state of an illness active level of patient, but a Syphilis Detection In Screening means can only be used as.
The specificity and sensitiveness of TP antigen serum test are higher, can be used as syphilis confirmatory test.Wherein
Fluorescent treponemal antibody absorbed test, be most sensitive detection method in the experiment of all conveyor screws, its specificity is also very
Height, is considered to detect " goldstandard " of syphilis by medical field；Microspironema pallidum hemagglutination test (HA test) is frequently as human body syphilis spiral shell
The specificity verification experiment of body-sensing dye is revolved, but it is worth to syphilis therapeutic effect without judgement, and the detection method is to I phase syphilis
Positive rate is not so good as fluorescent treponemal antibody absorbed test；EUSA is in microspironema pallidum gene work
On the basis of journey is succeeded in developing, a kind of new Serology test gradually grown up, it was applied to detection or once
Through infecting the patient of syphilis, therefore available for the examination and diagnosis of infection by Treponema pallidum person.
Protocols in Molecular Biology, mainly round pcr, can directly detect the specific DNA piece of the microspironema pallidum in sample
It is disconnected, difficult diagnosis or atypical syphilitic are carried out making a definite diagnosis very big reference value.But there is the non-of primer in this method
Specificity, it is low to serum/whole blood sensitiveness, easily suppressed by materials such as the tissues and cell fragment in sample, false negative easily occur
As a result the shortcomings of.
To sum up analyze, comprehensive, quick, accurate, sensitive and cheap microspironema pallidum detection means and detection method are right
The examination of syphilis, the diagnosis of syphilis and treatment, and control spreading for syphilis significant.
The content of the invention
According to the shortcoming and defect of existing microspironema pallidum detection means set forth above, and provide a kind of micro-fluidic
The full-automatic ELISA detection means of microspironema pallidum on chip.
The technological means that the present invention is used is as follows：
The full-automatic ELISA detection means of microspironema pallidum on a kind of micro-fluidic chip, including micro-fluidic chip, direct current
Source, fluorescence detecting system and control system,
The micro-fluidic chip includes liquid storage tank I, liquid storage tank II, liquid storage tank III, liquid storage tank IV, liquid storage tank V, liquid storage tank
VIth, liquid storage tank VII and main channel, the liquid storage tank I, the liquid storage tank II, the liquid storage tank III, the liquid storage tank IV and described
Liquid storage tank V is connected by sample inlet channel with the entrance of the main channel respectively, the outlet of the main channel and sample export
Passage is connected, and the other end of the sample export passage is connected with the liquid storage tank VII, and the main channel includes side wall I, side wall
II and the Mixed Zone, detection zone, the separated region that are sequentially connected with,
The side wall I between the separated region and the sample export passage is provided with TP collection channels,
The other end of the TP collection channels is connected with the liquid storage tank VI,
The full-automatic ELISA detection means of microspironema pallidum also includes electromagnet I and electricity on a kind of micro-fluidic chip
Magnet II,
One end of the electromagnet I is attached on the side wall II in the Mixed Zone,
One end of the electromagnet II is attached on the side wall I in the separated region,
It is provided with the liquid storage tank I in platinum electrode I, the liquid storage tank II and is provided with platinum electrode II, is set in the liquid storage tank III
Have to be provided with platinum electrode IV, the liquid storage tank V in platinum electrode III, the liquid storage tank IV and be provided with platinum electrode V, the liquid storage tank VI
Interior be provided with platinum electrode VI, the liquid storage tank VII is provided with platinum electrode VII,
The platinum electrode I is connected by relay I with the positive pole of the dc source, and the platinum electrode II passes through relay
II is connected with the positive pole of the dc source, and the platinum electrode III is connected by relay III with the positive pole of the dc source,
The platinum electrode IV is connected by relay IV with the positive pole of the dc source, and the platinum electrode V passes through relay V and institute
The positive pole connection of dc source is stated, the platinum electrode VI is connected by relay VI with the negative pole of the dc source, the platinum
Electrode VII is connected with the negative pole of the dc source,
Described one end of electromagnet I is connected by relay VII with the positive pole of the dc source, the electromagnet I it is another
End is connected with the negative pole of the dc source, and described one end of electromagnet II is connected by the positive pole of relay VIII and the dc source
Logical, the other end of the electromagnet II is connected with the negative pole of the dc source；
The fluorescence detecting system includes light activating system and optical detection system, and the smooth activating system includes Laser emission
Head and light source driving circuit, the optical detection system include the photoelectricity testing part of detection fluorescence signal, filtering device and signal
Process circuit, the laser beam emitting head is located at the underface of the detection zone, and the photoelectricity testing part is located at the detection
The surface in region, the light source driving circuit and the signal processing circuit are connected by relay Ⅸ with positive source；
The control system includes NI data collecting cards and PC processing terminals, the input of the NI data collecting cards and institute
State the connection of fluorescence detecting system output end, the output ends of the NI data collecting cards respectively with the relay I, the relay
II, the relay III, the relay IV, the relay V, the relay VI, the relay VII, the relay
Device VIII is connected with the relay Ⅸ, and the PC processing terminals can respectively be controlled according to preset time with the fluorescence signal detected
NI data collecting cards processed export corresponding voltage signal, control relay closure or disconnection, so that corresponding circuits on control chip
And the break-make of electromagnet.
The main channel is straight channel.
The electromagnet I and the electromagnet II are rectangular, and the electromagnet I is attached in the Mixed Zone
The side wall II on one end length it is corresponding with the length of the Mixed Zone,
The electromagnet II is attached at the length of one end on the side wall I in the separated region and described point
Length from region is corresponding.
The width of the main channel is 200 μm -500 μm, and the depth of the main channel is 20 μm -30 μm, the mixed zone
The length in domain is 1cm, sufficient mixing can be realized under this length, the length of the separated region is 0.5cm, because if
The length of the separated region is long or too short, the efficiency of separation can be all influenceed, if too short, it is impossible to which providing enough magnetic force makes
Magnetic bead is deflected, it is impossible to realize separation, and if long, the probability that the multiple magnetic beads of synchronization flow through separated region rises, right
During one Beads enrichment, other magnetic beads can be influenceed to move, cause some to be not bound with TP magnetic bead and together deflect inflow target
In passage.
50 μm -100 μm of the width of the sample inlet channel, the depth of the sample inlet channel is 20 μm -30 μm；
50 μm -100 μm of the width of the TP collection channels, the depth of the TP collection channels is 20 μm -30 μm；
50 μm -100 μm of the width of the sample export passage, the depth of the sample export passage is 20 μm -30 μm, is led to
Cross above-mentioned size and limit and cause Electronic control efficiency preferably, it is possible to prevente effectively from the influence of other factors.
The relay I, the relay II, the relay III, the relay IV, the relay V is described
Relay VI, the relay VII, the relay VIII and the relay Ⅸ are electromagnetic relay.
The electromagnet I and the electromagnet II are integrated in the micro-fluidic chip.
The photoelectricity testing part is photomultiplier or single photon counting module.
The invention also discloses a kind of full-automatic ELISA detections using microspironema pallidum on a kind of above-mentioned micro-fluidic chip
The detection method of device, with following steps：
S1, TP antibody is coated on magnetic bead surfaces, obtains the coated magnetic bead of TP antibody；
S2, the coated magnetic bead of TP antibody, blood sample, enzyme labelled antibody, enzyme fluorogenic substrate, cleaning fluid be separately added into
Into the liquid storage tank I, the liquid storage tank II, the liquid storage tank III, the liquid storage tank IV and the liquid storage tank V, and to described
Buffer solution is added dropwise in liquid storage tank VI and the liquid storage tank VII；
The enzyme labelled antibody refers to the enzyme labelled antibody that can be reacted with TP, and the enzyme fluorogenic substrate has fluorescent characteristic, can
To be combined with the enzyme labelled antibody.
S3, the startup control system, the control system control the relay I and the relay VII to close respectively
Close, connect the dc source, the coated magnetic bead of TP antibody carries out electric osmose and transported, when the coated magnetic bead fortune of the TP antibody
Move to being fixed on during the Mixed Zone by the effect of electromagnet I on the side wall II；
S4, the control system control the relay I to disconnect and control the closure of relay II, the blood sample
Carry out electric osmose to transport, fully reacted with the coated magnetic bead of TP antibody when the blood sample moves to the Mixed Zone
Generation antigen-antibody is combined, and obtains the coated magnetic bead of antigen antibody complex, and the control system controls the relay afterwards
II disconnects and controls the closure of relay V, and the cleaning fluid cleans the coated magnetic bead of antigen antibody complex, purpose
It is to remove other uncombined materials, obtained waste liquid is transported in the liquid storage tank VII；
S5, the control system control the relay V to disconnect and control the closure of relay III, and the enzyme mark resists
Body carries out electric osmose and transported, when the enzyme labelled antibody moves to the Mixed Zone with the coated magnetic of the antigen antibody complex
Pearl is sufficiently mixed, and obtains the magnetic bead with enzyme, afterwards the control system control the relay III disconnect and described in control after
Electrical equipment V is closed, the cleaning fluid cleaning magnetic bead for carrying enzyme, it is therefore an objective to remove other uncombined enzyme labelled antibodies, now
The enzyme amount carried on the magnetic bead with enzyme is related to the amount of TP in the blood sample, and obtained waste liquid is transported to described
In liquid storage tank VII；
S6, the control system control the relay V to disconnect and control the closure of relay IV, the enzyme fluorescence
Substrate carries out electric osmose and transported, and is fully mixed with the magnetic bead with enzyme when the enzyme fluorogenic substrate moves to the Mixed Zone
Close, obtain the magnetic bead for fluoroscopic examination, the control system controls the relay IV to disconnect and control the relay afterwards
Device V is closed, and the cleaning fluid cleaning is described to be used for the magnetic bead of fluoroscopic examination, it is therefore an objective to be used for the magnetic of fluoroscopic examination described in removing
Undesired impurities on pearl, obtained waste liquid is transported in the liquid storage tank VII；
S7, the control system control the relay VII to disconnect and control the closure of relay Ⅸ, described for glimmering
The magnetic bead of light detection is released, and opens the fluorescence detecting system, the laser beam emitting head and the photoelectricity testing part enter
Enter working condition, the magnetic bead for fluoroscopic examination carries out electric osmose and transported, and the magnetic bead for fluoroscopic examination passes through described
Detection zone carries out fluoroscopic examination,
When being not detected by fluorescence signal, the magnetic bead for fluoroscopic examination enters institute by the sample export passage
State in liquid storage tank VII,
When detecting fluorescence signal, signal is sent into the NI data collecting cards and the PC by the photoelectricity testing part
Processing terminal, the PC processing terminals are stored to signal, shown, and NI data collecting cards are exported according to programme-control
Corresponding voltage signal, the control system controls the relay VIII and the closure of the relay VI respectively, described for glimmering
The magnetic bead of light detection carries out electric osmose and transported, and is deflected in the presence of electromagnetic force, is entered by the TP collection channels
In the liquid storage tank VI, i.e., when the magnetic bead for fluoroscopic examination moves to the separated region by the work of electromagnet II
Deflected, and entered by the TP collection channels in the liquid storage tank VI with to the side wall I；
S8, the PC processing terminals are analyzed and processed to detected fluorescence signal, according to fluorescent pulse number with
Intensity calculates TP content.
The magnetic bead in the step S1 is prepared from by SPIO, and the diameter of the magnetic bead is less than
5μm。
Compared with prior art, the present invention has following advantage and effect：
(1) present invention realizes the full-automatic detection for being rapidly completed microspironema pallidum mainly on the basis of micro-fluidic chip
With separating, mechanization degree is high, operation sequence is simple, overcomes in conventional test in laboratory method, and instrument is heavy, operating process
Complicated, costly, detection cycle is long, and human factor influence；
(2) microfluidic platform detected using micro-fluidic chip as microspironema pallidum, while required auxiliary equipment is also used
Small volume, the controlling equipment and detection means of precise structure drive for example with electrode drive instead of pump, using photomultiplier
Or single photon counting module and then replacement CCD or ICCD etc., small with taking up space, equipment is light and handy, is easy to carry, available for existing
The advantages of field detection.
(3) magnetic bead is fixed in Mixed Zone using electromagnet, and in separated region by controlling corresponding electromagnetism
Tie Tong breaks, and with electromagnetic force to realize magnetic bead sorting, this method has reliability height, cost is low, simple to operate, be easy to implement certainly
The features such as dynamicization is controlled.
The present invention can be widely popularized in fully-automated synthesis with the field such as separating for the foregoing reasons.
Brief description of the drawings
The present invention is further detailed explanation with reference to the accompanying drawings and detailed description.
Fig. 1 is the structural representation of micro-fluidic chip in embodiment of the invention.
Fig. 2 be the present invention embodiment on a kind of micro-fluidic chip microspironema pallidum full-automatic ELISA inspections
Survey the structural representation of device.
Embodiment
As depicted in figs. 1 and 2, on a kind of micro-fluidic chip microspironema pallidum full-automatic ELISA detection means, it is including micro-
Fluidic chip 1, dc source 2, fluorescence detecting system 3 and control system 4,
The material of the cover plate of the micro-fluidic chip 1 is PDMS, and the substrate of the micro-fluidic chip 1 is slide,
The micro-fluidic chip 1 includes the 1-1 of liquid storage tank I, the 1-2 of liquid storage tank II, the 1-3 of liquid storage tank III, the 1-4 of liquid storage tank IV, storage
The 1-5 of liquid pool V, the 1-6 of liquid storage tank VI, the 1-7 of liquid storage tank VII and main channel 1-8, the 1-1 of liquid storage tank I, the 1-2 of the liquid storage tank II,
The 1-3 of liquid storage tank III, the 1-4 of the liquid storage tank IV and the 1-5 of the liquid storage tank V respectively by sample inlet channel 1-9 with it is described
Main channel 1-8 entrance connection, the outlet of the main channel 1-8 is connected with sample export passage 1-10, and the sample export leads to
The road 1-10 other end is connected with the 1-7 of liquid storage tank VII, the main channel 1-8 include side wall I 1-8-1, the 1-8-2 of side wall II and
Mixed Zone 1-8-3, detection zone 1-8-4, the separated region 1-8-5 being sequentially connected with,
The 1-8-1 of side wall I between the separated region 1-8-5 and the sample export passage 1-10 is provided with
TP collection channels 1-11, the other end of the TP collection channels 1-11 is connected with the 1-6 of liquid storage tank VI,
The full-automatic ELISA detection means of microspironema pallidum also includes the 1-12 of electromagnet I on a kind of micro-fluidic chip
With the 1-13 of electromagnet II,
One end of the 1-12 of electromagnet I is attached on the side wall II in the Mixed Zone 1-8-3,
One end of the 1-13 of electromagnet II is attached on the side wall I in the separated region 1-8-5,
It is interior provided with the 1-2-1 of platinum electrode II provided with platinum electrode I 1-1-1, the 1-2 of liquid storage tank II in the 1-1 of liquid storage tank I,
It is interior provided with the 1-4-1 of platinum electrode IV, the storage provided with platinum electrode III 1-3-1, the 1-4 of liquid storage tank IV in the 1-3 of liquid storage tank III
It is interior provided with the 1-6-1 of platinum electrode VI, the liquid storage tank VII provided with platinum electrode V 1-5-1, the 1-6 of liquid storage tank VI in the 1-5 of liquid pool V
The 1-7-1 of platinum electrode VII is provided with 1-7,
The 1-1-1 of platinum electrode I is connected by the 4-1 of relay I with the positive pole of the dc source 2, the platinum electrode II
1-2-1 is connected by the 4-2 of relay II with the positive pole of the dc source 2, and the 1-3-1 of platinum electrode III passes through the 4- of relay III
3 connect with the positive pole of the dc source 2, and the 1-4-1 of platinum electrode IV passes through the 4-4 of relay IV and the dc source 2
Positive pole is connected, and the 1-5-1 of platinum electrode V is connected by the 4-5 of relay V with the positive pole of the dc source 2, the platinum electrode
VI 1-6-1 is connected by the 4-6 of relay VI with the negative pole of the dc source 2, the 1-7-1 of platinum electrode VII and the direct current
The negative pole connection in source 2,
Described 1-12 one end of electromagnet I is connected by the 4-7 of relay VII with the positive pole of the dc source 2, the electromagnetism
The 1-12 of iron I other end is connected with the negative pole of the dc source 2, and described 1-13 one end of electromagnet II passes through the 4-8 of relay VIII
Connected with the positive pole of the dc source 2, the other end of the 1-13 of electromagnet II is connected with the negative pole of the dc source 2；
The fluorescence detecting system 3 includes light activating system 3-1 and optical detection system 3-2, the smooth activating system 3-1 bags
Laser beam emitting head and light source driving circuit are included, the optical detection system 3-2 includes the photoelectricity testing part of detection fluorescence signal, filter
Optical device and signal processing circuit, the laser beam emitting head are located at the underface of the detection zone 1-8-4, the Photoelectric Detection
Device is located at the surface of the detection zone 1-8-4, the light source driving circuit and the signal processing circuit by after
The 4-9 of electrical equipment Ⅸ is connected with positive source；
The control system 4 includes NI data collecting card 4-10 and PC processing terminals 4-11, the NI data collecting cards 4-
10 input is connected with the output end of fluorescence detecting system 3, the output end of the NI data collecting cards 4-10 respectively with institute
Relay I 4-1, the 4-2 of relay II, the 4-3 of relay III, the 4-4 of relay IV, the 4-5 of relay V are stated,
The 4-6 of relay VI, the 4-7 of relay VII, the 4-8 of relay VIII is connected with the 4-9 of relay Ⅸ.
The main channel 1-8 is straight channel.
The 1-12 of electromagnet I and the 1-13 of the electromagnet II are rectangular, and the 1-12 of electromagnet I is attached at positioned at institute
The length for stating one end on the side wall II in the 1-8-3 of Mixed Zone is corresponding with the length of the Mixed Zone 1-8-3,
The 1-13 of electromagnet II is attached at the length of one end on the side wall I in the separated region 1-8-5
Degree is corresponding with the length of the separated region 1-8-5.
The width of the main channel 1-8 is 200 μm, and the depth of the main channel 1-8 is 20 μm, the Mixed Zone 1-8-
3 length is 1cm, and the length of the separated region 1-8-5 is 0.5cm.
50 μm of the width of the sample inlet channel 1-9, the depth of the sample inlet channel 1-9 is 20 μm；
50 μm of the width of the TP collection channels 1-11, the depth of the TP collection channels 1-11 is 20 μm；
50 μm of the width of the sample export passage 1-10, the depth of the sample export passage 1-10 is 20 μm.
The sample inlet channel 1-9, the TP collection channels 1-11 and the sample export passage 1-10 are straight-through
Road,
The axis of the axis of the main channel 1-8 and the sample export passage 1-10 is located along the same line,
The 4-1 of relay I, the 4-2 of relay II, the 4-3 of relay III, the 4-4 of relay IV, it is described after
Electrical equipment V 4-5, the 4-6 of relay VI, the 4-7 of relay VII, the 4-8 of relay VIII and the 4-9 of the relay Ⅸ is equal
For electromagnetic relay.
The 1-12 of electromagnet I and the electromagnet II 1-13 is integrated in the micro-fluidic chip.
The photoelectricity testing part is photomultiplier or single photon counting module.
A kind of detection side using the full-automatic ELISA detection means of microspironema pallidum on a kind of above-mentioned micro-fluidic chip
Method, with following steps：
S1, TP antibody is coated on magnetic bead surfaces, obtains the coated magnetic bead of TP antibody；
S2, the coated magnetic bead of TP antibody, blood sample, enzyme labelled antibody, enzyme fluorogenic substrate, cleaning fluid be separately added into
To the 1-1 of liquid storage tank I, the 1-2 of the liquid storage tank II, the 1-3 of the liquid storage tank III, the 1-4 of the liquid storage tank IV and the liquid storage tank
In V 1-5, and buffer solution is added dropwise to the 1-6 of the liquid storage tank VI and 1-7 of the liquid storage tank VII；
S3, the startup control system 4, the control system 4 control the 4-1 of relay I and the relay respectively
VII 4-7 is closed, and connects the dc source 2, the coated magnetic bead of TP antibody carries out electric osmose and transported, when TP antibody coating
Magnetic bead be fixed on the 1-8-2 of side wall II by the 1-12 of electromagnet I effects when moving to the Mixed Zone 1-8-3
On；
S4, the control system 4 control the 4-1 of relay I to disconnect and control the 4-2 of relay II to close, described
Blood sample carries out electric osmose and transported, and is coated with when the blood sample moves to the Mixed Zone 1-8-3 with the TP antibody
Magnetic bead fully react generation antigen-antibody combine, obtain the coated magnetic bead of antigen antibody complex, afterwards the control system 4
Control the 4-2 of relay II to disconnect and control the 4-5 of relay V to close, the cleaning fluid cleans the antigen-antibody and answered
The coated magnetic bead of compound, obtained waste liquid is transported in the 1-7 of liquid storage tank VII；
S5, the control system 4 control the 4-5 of relay V to disconnect and control the 4-3 of relay III to close, described
Enzyme labelled antibody carries out electric osmose and transported, and is answered when the enzyme labelled antibody moves to the Mixed Zone 1-8-3 with the antigen-antibody
The coated magnetic bead of compound is sufficiently mixed, and obtains the magnetic bead with enzyme, and the control system 4 controls the 4-3 of relay III afterwards
Disconnect and control the 4-5 of relay V to close, the cleaning fluid cleaning magnetic bead for carrying enzyme, obtained waste liquid is conveyed
Into the 1-7 of liquid storage tank VII；
S6, the control system 4 control the 4-5 of relay V to disconnect and control the 4-4 of relay IV to close, described
Enzyme fluorogenic substrate carries out electric osmose and transported, and enzyme is carried with described when the enzyme fluorogenic substrate moves to the Mixed Zone 1-8-3
Magnetic bead be sufficiently mixed, obtain the magnetic bead for fluoroscopic examination, afterwards the control system 4 control the 4-4 of relay IV break
Open and control the 4-5 of relay V to close, the cleaning fluid cleaning is described to be used for the magnetic bead of fluoroscopic examination, obtained waste liquid quilt
It is transported in the 1-7 of liquid storage tank VII；
S7, the control system 4 control the 4-7 of relay VII to disconnect and control the 4-9 of relay Ⅸ to close, and open
The fluorescence detecting system 3, the magnetic bead for fluoroscopic examination carries out electric osmose and transported, and the magnetic bead for fluoroscopic examination leads to
Cross the detection zone 1-8-4 and carry out fluoroscopic examination,
When being not detected by fluorescence signal, the magnetic bead for fluoroscopic examination enters by the sample export passage 1-10
Enter in the 1-7 of liquid storage tank VII,
When detecting fluorescence signal, the control system 4 controls the 4-8 of relay VIII and the relay VI respectively
4-6 is closed, and the magnetic bead for fluoroscopic examination carries out electric osmose and transported, and is deflected in the presence of electromagnetic force, passes through institute
TP collection channels 1-11 is stated to enter in the 1-6 of liquid storage tank VI；
S8, the PC processing terminals 4-11 are analyzed and processed to detected fluorescence signal, according to fluorescent pulse
Number calculates TP content with intensity.
The magnetic bead in the step S1 is prepared from by SPIO, and the diameter of the magnetic bead is less than
5μm。
The foregoing is only a preferred embodiment of the present invention, but protection scope of the present invention be not limited thereto,
Any one skilled in the art the invention discloses technical scope in, technique according to the invention scheme and its
Inventive concept is subject to equivalent substitution or change, should all be included within the scope of the present invention.
Claims (1)
1. the detection method of the full-automatic ELISA detection means of microspironema pallidum, the miniflow on a kind of use micro-fluidic chip
Control the full-automatic ELISA detection means of microspironema pallidum on chip, including micro-fluidic chip, dc source, fluorescence detecting system
And control system, it is characterised in that：
The micro-fluidic chip includes liquid storage tank I, liquid storage tank II, liquid storage tank III, liquid storage tank IV, liquid storage tank V, liquid storage tank VI, storage
Liquid pool VII and main channel, the liquid storage tank I, the liquid storage tank II, the liquid storage tank III, the liquid storage tank IV and the liquid storage tank
V is connected by sample inlet channel with the entrance of the main channel respectively, and outlet and the sample export passage of the main channel connect
Logical, the other end of the sample export passage is connected with the liquid storage tank VII, and the main channel includes side wall I, side wall II and suitable
Mixed Zone, detection zone, the separated region of secondary connection,
The side wall I between the separated region and the sample export passage is provided with TP collection channels,
The other end of the TP collection channels is connected with the liquid storage tank VI,
The full-automatic ELISA detection means of microspironema pallidum also includes electromagnet I and electromagnet II on the micro-fluidic chip,
One end of the electromagnet I is attached on the side wall II in the Mixed Zone,
One end of the electromagnet II is attached on the side wall I in the separated region,
It is provided with the liquid storage tank I in platinum electrode I, the liquid storage tank II and is provided with platinum electrode II, platinum is provided with the liquid storage tank III
It is provided with electrode III, the liquid storage tank IV in platinum electrode IV, the liquid storage tank V and is provided with platinum electrode V, is set in the liquid storage tank VI
Have and platinum electrode VII be provided with platinum electrode VI, the liquid storage tank VII,
The platinum electrode I is connected by relay I with the positive pole of the dc source, the platinum electrode II by relay II with
The positive pole connection of the dc source, the platinum electrode III is connected by relay III with the positive pole of the dc source, described
Platinum electrode IV is connected by relay IV with the positive pole of the dc source, the platinum electrode V by relay V with it is described straight
The positive pole connection of power supply is flowed, the platinum electrode VI is connected by relay VI with the negative pole of the dc source, the platinum electrode
VII connects with the negative pole of the dc source,
Described one end of electromagnet I is connected by relay VII with the positive pole of the dc source, the other end of the electromagnet I with
The negative pole connection of the dc source, described one end of electromagnet II is connected by relay VIII with the positive pole of the dc source,
The other end of the electromagnet II is connected with the negative pole of the dc source；
The fluorescence detecting system include light activating system and optical detection system, the smooth activating system include laser beam emitting head and
Light source driving circuit, the optical detection system includes the photoelectricity testing part of detection fluorescence signal, filtering device and signal transacting
Circuit, the laser beam emitting head is located at the underface of the detection zone, and the photoelectricity testing part is located at the detection zone
Surface, the light source driving circuit and the signal processing circuit are connected by relay Ⅸ with positive source；
The control system include NI data collecting cards and PC processing terminals, the input of the NI data collecting cards with it is described glimmering
Optical detection system output end is connected, the output ends of the NI data collecting cards respectively with the relay I, the relay II,
The relay III, the relay IV, the relay V, the relay VI, the relay VII, the relay VIII
Connected with the relay Ⅸ；
The main channel is straight channel；
The electromagnet I and the electromagnet II are rectangular, and the electromagnet I is attached at the institute in the Mixed Zone
The length for stating one end on side wall II is corresponding with the length of the Mixed Zone,
The electromagnet II is attached at the length of one end on the side wall I in the separated region and the Disengagement zone
The length in domain is corresponding；
The width of the main channel is 200 μm -500 μm, and the depth of the main channel is 20 μm -30 μm, the Mixed Zone
Length is 1cm, and the length of the separated region is 0.5cm；
The width of the sample inlet channel is 50 μm -100 μm, and the depth of the sample inlet channel is 20 μm -30 μm；
The width of the TP collection channels is 50 μm -100 μm, and the depth of the TP collection channels is 20 μm -30 μm；
The width of the sample export passage is 50 μm -100 μm, and the depth of the sample export passage is 20 μm -30 μm；
The relay I, the relay II, the relay III, the relay IV, the relay V, the relay
Device VI, the relay VII, the relay VIII and the relay Ⅸ are electromagnetic relay；
The electromagnet I and the electromagnet II are integrated in the micro-fluidic chip；
The photoelectricity testing part is photomultiplier or single photon counting module；
Methods described, with following steps：
S1, TP antibody is coated on magnetic bead surfaces, obtains the coated magnetic bead of TP antibody；
S2, the coated magnetic bead of TP antibody, blood sample, enzyme labelled antibody, enzyme fluorogenic substrate, cleaning fluid be added separately to institute
State in liquid storage tank I, the liquid storage tank II, the liquid storage tank III, the liquid storage tank IV and the liquid storage tank V, and to the liquid storage
Buffer solution is added dropwise in pond VI and the liquid storage tank VII；
S3, the startup control system, the control system control the relay I and the closure of the relay VII, connect respectively
Lead to the dc source, the coated magnetic bead of TP antibody carries out electric osmose and transported, when the coated magnetic bead of TP antibody is moved to
It is fixed on during the Mixed Zone by the electromagnet I effect on the side wall II；
S4, the control system control the relay I to disconnect and control the closure of relay II, and the blood sample is carried out
Electric osmose is transported, and generation is fully reacted with the coated magnetic bead of TP antibody when the blood sample moves to the Mixed Zone
Antigen-antibody is combined, and obtains the coated magnetic bead of antigen antibody complex, and the control system controls the relay II to break afterwards
The closure of relay V is opened and controls, the cleaning fluid cleans the coated magnetic bead of antigen antibody complex, what is obtained is useless
Liquid is transported in the liquid storage tank VII；
S5, the control system control the relay V to disconnect and control the closure of relay III, and the enzyme labelled antibody enters
Row electric osmose is transported, and is filled when the enzyme labelled antibody moves to the Mixed Zone with the coated magnetic bead of the antigen antibody complex
Divide mixing, obtain the magnetic bead with enzyme, the control system controls the relay III to disconnect and control the relay afterwards
V closure, the cleaning fluid cleaning magnetic bead for carrying enzyme, obtained waste liquid is transported in the liquid storage tank VII；
S6, the control system control the relay V to disconnect and control the closure of relay IV, the enzyme fluorogenic substrate
Carry out electric osmose to transport, be sufficiently mixed when the enzyme fluorogenic substrate moves to the Mixed Zone with the magnetic bead with enzyme,
The magnetic bead for fluoroscopic examination is obtained, the control system controls the relay IV to disconnect and control the relay V afterwards
Closure, the cleaning fluid cleaning is described to be used for the magnetic bead of fluoroscopic examination, and obtained waste liquid is transported in the liquid storage tank VII；
S7, the control system control the relay VII to disconnect and control the closure of relay Ⅸ, open the fluorescence inspection
Examining system, the magnetic bead for fluoroscopic examination carries out electric osmose and transported, and the magnetic bead for fluoroscopic examination passes through the detection
Region carries out fluoroscopic examination,
When being not detected by fluorescence signal, the magnetic bead for fluoroscopic examination enters the storage by the sample export passage
In liquid pool VII,
When detecting fluorescence signal, the control system controls the relay VIII and the closure of the relay VI, institute respectively
State and transported for the magnetic bead progress electric osmose of fluoroscopic examination, and deflected in the presence of electromagnetic force, collect logical by the TP
Road is entered in the liquid storage tank VI；
S8, the PC processing terminals are analyzed and processed to detected fluorescence signal, according to fluorescent pulse number and intensity
Calculate TP content；
The magnetic bead in the step S1 is prepared from by SPIO, and the diameter of the magnetic bead is less than 5 μm.
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| CN107655879B (en) * | 2017-09-01 | 2018-12-18 | 北京华科泰生物技术有限公司 | For detecting the micro-fluidic chemiluminescence detection system of the magnetic particle of sexual gland series |
| CN109655611A (en) * | 2018-12-20 | 2019-04-19 | 天津瑞普生物技术股份有限公司 | Micro-fluidic chip immunodiagnosis kit and preparation method thereof |
| CN110887822A (en) * | 2019-11-13 | 2020-03-17 | 深圳中山泌尿外科医院 | Method for detecting embryo secretory protein |
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| CN103278628A (en) * | 2013-06-12 | 2013-09-04 | 齐齐哈尔医学院 | Method and device for detecting and separating HP (helicobacter pylori) ELISA (enzyme-linked immuno sorbent assay) |
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| CN103105352A (en) * | 2013-01-28 | 2013-05-15 | 大连海事大学 | Device and method for rapidly detecting surviving unicellular organisms in ship ballast water |
| CN103278628A (en) * | 2013-06-12 | 2013-09-04 | 齐齐哈尔医学院 | Method and device for detecting and separating HP (helicobacter pylori) ELISA (enzyme-linked immuno sorbent assay) |
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Effective date of registration: 20171109 Address after: Bu Kui 161006 North Main Street in Qigihar City, Heilongjiang province Jianhua District No. 333 Patentee after: Qiqihar Medical University Address before: Bu Kui 161000 North Main Street in Qigihar City, Heilongjiang province Jianhua District No. 333 Patentee before: Zhang Xiaojie |