CN108169495A - A kind of micro-fluidic chip and its application - Google Patents
A kind of micro-fluidic chip and its application Download PDFInfo
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- CN108169495A CN108169495A CN201810149379.0A CN201810149379A CN108169495A CN 108169495 A CN108169495 A CN 108169495A CN 201810149379 A CN201810149379 A CN 201810149379A CN 108169495 A CN108169495 A CN 108169495A
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Abstract
The present invention provides a kind of micro-fluidic chip and its application, the micro-fluidic chip includes the top plate layer (1), the first cavity layer (2), flow channel layer (3), the second cavity layer (4) and the backplane level (5) that are cascading from top to bottom；The top plate layer (1) is provided at least one well and at least one air hole (16)；First cavity layer (2) is provided at least one liquid storage cylinder and at least one reaction chamber (25)；Second cavity layer (4) is provided at least one waste liquid chamber (41)；The flow channel layer (3) is provided with runner, for top plate layer (1), the first cavity layer (2) to be connected with the second cavity layer (4).The micro-fluidic chip detection speed of the present invention is fast, high degree of automation, process control, cost are relatively low, available for Molecular Detection.
Description
Technical field
The invention belongs to immunoassay fields, are related to a kind of micro-fluidic chip and its application more particularly to a kind of cadmium ion
Detect micro-fluidic chip and its application.
Background technology
Heavy metal cadmium (Cd) is a kind of common environmental contaminants, is mainly derived from the industrial activities such as mining and smelting.It releases
The cadmium being put into environment is enriched in the cadmium of soil through in the aquatic animals and plants in crops and water, entering people eventually by diet
Body.Gastrointestinal tract is about 5-10% to the absorptivity of cadmium, and lung is then up to 10-50% to the absorptivity of cadmium.Life of the cadmium in human body
Object long half time reaches 10-30, and damage is respectively provided with to liver, kidney, lung, bone, cardiovascular system, immune system and nervous system and is made
With.The main harm of cadmium includes renal toxicity and bone toxicity.Wherein, kidney is the first target organs of cadmium exposure, and cadmium is by inducing oxygen
Change stress make renal tubular cell that cloudy swelling, apoptosis, necrosis, hyperplasia and tube chamber atrophy occur, and cause reabsorption dysfunction；
The cadmium of high dose with calcium competition by directly inhibiting transhipment of the cell to calcium, while lead to 1,25- dihydroxies by influencing renal function
The synthesis of base vitamine D3 declines and influences bone calcium metabolism indirectly, eventually leads to bone calciprivia, causes osteoporosis.Recent research knot
Fruit proves that cadmium is also one of risk factors of angiocardiopathies such as atherosclerosis, hypertension.In addition, it is exposed to height for a long time
The cadmium of concentration may induce cancer, and cadmium is included in " human carcinogen " by international cancer research institution (IARC).
Traditional detection of heavy metal ion method includes atomic absorption spectrography (AAS), inductively coupled plasma mass spectrometry, volt-ampere
Method, the chromatography of ions and electric atomizing atomic absorption spectrography (AAS) etc., however these detection methods must use large-scale analyzer
Device, is not used to Site Detection, and costly, treating capacity is limited, detection time is long, accuracy is limited by instrument by examining, unfavorable
In promoting and applying in daily life.Micro-fluidic chip has the advantages that high specificity, high sensitivity, speed are fast, at low cost, inspection
It is easy to carry to survey instrument, is increasingly becoming the common technology of detection field.
105424784 A of CN disclose detection of heavy metal ion micro-fluidic chip and detection method in a kind of water, described micro-
Fluidic chip includes microchannel and micro-valve；The microchannel includes micro- logical with reference to microchannel, ion blotting microchannel, electrophoresis sample introduction
Road, electrophoretic separation microchannel, connection microchannel, waste liquid discharge microchannel and buffer solution discharge microchannel；The micro-valve includes ginseng
End micro-valve according to a mouthful micro-valve, ion blotting outlet micro-valve, waste liquid outlet micro-valve, buffer solution cut-off micro-valve and sample introduction is brought out.However,
The accuracy of the micro-fluidic chip is relatively low, it is impossible to the automation for carrying out heavy metal ion quantitatively detects, and process is cumbersome, take compared with
It is long.
Therefore it provides a kind of high sensitivity, high specificity, accuracy are good, the micro-fluidic chip of detection process automation,
Molecular Detection field is of great significance.
Invention content
In view of the deficiencies of the prior art, the present invention provides a kind of micro-fluidic chip and its application, the micro-fluidic chip inspection
Degree of testing the speed is fast, high degree of automation, process control, cost are relatively low, available for Molecular Detection.
For this purpose, the present invention provides following technical scheme：
In a first aspect, the present invention provides a kind of micro-fluidic chip, the micro-fluidic chip includes from top to bottom layer successively
Top plate layer 1, the first cavity layer 2, flow channel layer 3, the second cavity layer 4 and the backplane level 5 of folded setting；
The top plate layer 1 is provided at least one well and at least one air hole 16；
First cavity layer 2 is provided at least one liquid storage cylinder and at least one reaction chamber 25；
Second cavity layer 4 is provided at least one waste liquid chamber 41；
The flow channel layer 3 is provided with runner, for top plate layer 1, the first cavity layer 2 to be connected with the second cavity layer 4.
The micro-fluidic chip of the present invention is connected liquid storage cylinder, reaction chamber with waste liquid chamber by runner, realizes molecule oneself
Dynamicization controllably detects, while sets well in top plate layer, and detection reagent can be added into liquid storage cylinder, is a kind of safety, nothing
Pollution, efficient automatic detection device.
Preferably, the liquid storage cylinder is mounted with detection reagent.
Preferably, the detection reagent include in detection antibody, cleaning solution or luminescence-producing reaction liquid any one or at least
Two kinds of combination.
The detection reagent of the present invention is loaded according to the difference of type in different liquid storage cylinders, the exit setting of liquid storage cylinder
There is pump valve system, ensure that detection reagent does not flow back, avoid pollution, extend the shelf-life of detection reagent.
In the present invention, the shape and number of liquid storage cylinder can arbitrarily be set, and those skilled in the art can be according to practical need
The shape and number of the type adjustment liquid storage cylinder for detection reagent of summing, micro-fluidic chip of the invention include 4 tubular liquid storage cylinders
For loading detection reagent.
Preferably, the reaction chamber 25 is coated with capture antibody.
In the present invention, test substance is fixed in reaction chamber by the coated capture antibody of reaction inner cavity surface, passes through measure
The signal value of reaction chamber carries out the detection of test substance.
Preferably, the detection antibody is cadmium ion monoclonal antibody.
In the present invention, cadmium ion monoclonal antibody is as detection antibody, and high specificity, high sensitivity, micro-fluidic chip can
For the content of heavy metal cadmium ion in measure food, soil, water source, animal and human body fluid.
Preferably, the capture antibody is anti-mouse IgG antibody.
In the present invention, anti-mouse IgG antibody is the secondary antibody of cadmium ion monoclonal antibody, can be with cadmium ion monoclonal antibody
Antibody-antigene reaction occurs.
In the present invention, cleaning solution is the 25mmol/L of the pH=7.2 containing 0.2mol/L NaCl and 0.1%Tween-20
Tris-HCL buffer solutions, chemiluminescence exciting liquid include chemiluminescence preexciting liquid A and chemiluminescence exciting liquid B, wherein, chemistry
The preexciting liquid A that shines is H2O2And HNO3Mixed liquor, H2O2Mass fraction be 0.1-5%, HNO3A concentration of 0.1-5mol/
The mixed liquor of L, chemiluminescence exciting liquid B for Triton X-100 and NaOH, the wherein a concentration of 0.1- of Triton X-100
A concentration of 0.1-1.0mol/L of 2.0mol/L, NaOH.
Preferably, the flow channel layer 3 includes first flow 31, second flow channel 32 and third flow channel 33.
Preferably, the well 11 is connected with the liquid storage cylinder by first flow 31.
Preferably, the second flow channel 32 is in clip shape.
In the present invention, second flow channel is set as clip shape, the flow path of sample to be tested is increased, extends and treat
Time of the test sample sheet in runner ensure that sample to be tested fully reacts before reaction chamber is reached with detection antibody, improve
The detection accuracy of micro-fluidic chip.
Preferably, it is provided with reaction chamber 25 between the second flow channel 32 and the third flow channel 33.
Preferably, the reaction chamber 25 is connected with the waste liquid chamber 41 by third flow channel 33.
Preferably, the waste liquid chamber 41 is connected with the air hole 16.
Preferably, the micro-fluidic chip is prepared by high molecular material.
Preferably, the high molecular material include crystalline silicon, glass, dimethyl silicone polymer, polymethyl methacrylate,
In polyethylene terephthalate or polyester any one or at least two combination, preferably dimethyl silicone polymer.
Preferably, the liquid storage cylinder and flow channel layer 3 are prepared by light tight high molecular material, and preferably light tight poly- two
Methylsiloxane.
Preferably, the top plate layer 1 and backplane level 5 are prepared by light transmission high molecular material, preferably the poly- diformazan of light transmission
Radical siloxane.
In the present invention, in order to ensure the reaction product in the liquid storage cylinder and runner inside micro-fluidic chip not by extraneous visible
The interference of light and detection light source builds liquid storage cylinder and runner using light tight dimethyl silicone polymer, and reaction chamber is needed to detection
Light is penetrating, and therefore, top plate layer and backplane level are made of light transmission dimethyl silicone polymer.
Preferably, the cadmium ion monoclonal antibody is modified with luminous marker.
Preferably, it is different to include acridinium ester, fluorescein isothiocynate, RB 200, tetramethyl for the luminous marker
In thiocyanic acid rhodamine, lanthanide chelate or phycoerythrin any one or at least two combination, preferably acridinium ester.
In the present invention, luminous marker is directly modified in cadmium ion monoclonal antibody, not to cadmium ion monoclonal antibody
Conformation impact, as signal amplifying system, enhance the detection sensitivity of micro-fluidic chip.
Cadmium ion monoclonal antibody preferably is marked using chemiluminescent labels in the present invention, chemical luminous system is simple,
Reaction can be carried out rapidly in alkaline environment, do not need to catalyst, acridinium ester is easily combined with protein, photon yield not by
Conformation influences, and the photon energy levels of generation are high, and signal is strong, and signal to noise ratio is high, and stability is good, and exciting liquid is at low cost.
Preferably, the cadmium ion monoclonal antibody is immunized animal by cadmium ion comlete antigen and is prepared.
Preferably, the cadmium ion comlete antigen is the compound of cadmium ion, chelating agent and carrier protein.
Preferably, the chelating agent includes ethylenediamine tetra-acetic acid, diethylenetriamine pentaacetic acid, diethylenetriamine pentaacetic acid derivative
Object, Cyclen-N, N, N, N- tetraacethyl or Cyclen -1,4,7,10-
In tetraacethyl-N-hydroxy-succinamide ester any one or at least two combination, preferably Isosorbide-5-Nitrae, 7,10- tetraazacyclododecanes
Dodecane -1,4,7,10- tetraacethyls-N-hydroxy-succinamide ester.
Preferably, it is pure to include poly-D-lysine, human serum albumins, keyhole limpet hemocyanin or ox blood for the carrier protein
In albumen any one or at least two combination, preferably poly-D-lysine.
In the present invention, cadmium ion haptens is formed by chelating agent and carrier protein has immunogenicity and immunoreactivity
Cadmium ion comlete antigen.
Preferably, the cadmium ion comlete antigen be cadmium ion-Isosorbide-5-Nitrae, 7,10- tetraazacyclododecanands-Isosorbide-5-Nitrae, 7,10- tetra-
Acetic acid-N-hydroxy-succinamide ester-poly-l-lysine complex.
In the present invention, preferred 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-n-hydroxysuccinimide
Ester (DOTA-NHS) is as chelating agent, the NHS reactive groups of DOTA-NHS and the amino orienting response of carrier protein, the nitrogen of heterocycle
The atom complex stable with the generation of heavy metal cadmium ion coordination, coupling stability is stronger, and poly-D-lysine (PLL) is as carrier
Albumen not only increases the immunogenicity of cadmium ion, and the antibody generated has higher potency to cadmium ion.
In the present invention, the course of work of the micro-fluidic chip is as follows：
(1) sample to be tested enters first flow 31 by well 11, meanwhile, the acridinium ester label cadmium ion in liquid storage cylinder 21
Monoclonal antibody enters first flow 31, and incubation is mixed with sample to be tested, forms antigen antibody complex, flows through second flow channel 32
With third flow channel 33, into reaction chamber 25；
(2) it is coated on the anti-mouse IgG antibody capture antigen antibody complex of 25 inner surface of reaction chamber in advance, resists antigen
Nanocrystal composition is fixed in reaction chamber 25；
(3) reaction chamber is cleaned using the cleaning solution in liquid storage cylinder 22, after removing the substance being not associated in reaction chamber, liquid storage cylinder
Chemiluminescence exciting liquid A and chemiluminescence exciting liquid B in 23 and 24 enter reaction chamber 25, excite in cadmium ion monoclonal antibody
Acridinium ester shine；
(4) chemiluminescence intensity of reaction chamber 25 is measured, measures the cadmium ion in sample to be tested.
Second aspect formulates standard curve the present invention provides a kind of using micro-fluidic chip as described in relation to the first aspect
Method includes the following steps：
(1) the cadmium ion titer of configuration various concentration at least known to 6 parts；
(2) the cadmium ion titer described in step (1) by well 11 enter first flow 31, with acridinium ester label cadmium from
Sub- monoclonal antibody mixing is incubated, and antigen antibody complex is obtained, into reaction chamber 25；
(3) it is coated on the antigen antibody complex described in the anti-mouse IgG antibody capture step (2) of reaction chamber 25 in advance；
(4) after cleaning, chemiluminescence reaction liquid enters reaction chamber 25, measures the chemiluminescence intensity of reaction chamber 25；
(5) using chemiluminescence intensity as ordinate, a concentration of abscissa of cadmium ion titer draws chemiluminescence intensity
With four parametric plot of relationship of concentration of cadmium ions, the standard curve is obtained.
In the present invention, the cadmium ion storing solution of a concentration of 1mg/mL is diluted to concentration using 3% nitric acid (v/v) is respectively
The cadmium ion titer of 0.2ng/mL, 0.5ng/mL, 1ng/mL, 2ng/mL, 5ng/mL and 10ng/mL.
The third aspect, the present invention provides a kind of methods for detecting cadmium ion, and the method is using as described in relation to the first aspect
Micro-fluidic chip.
Preferably, it the described method comprises the following steps：
(1 ') sample to be tested enters first flow 31 by well 11, mixed with acridinium ester label cadmium ion monoclonal antibody
It closes and is incubated, antigen antibody complex is obtained, into reaction chamber 25；
The antigen-antibody that (2 ') are coated in advance described in the anti-mouse IgG antibody capture step (1 ') of reaction chamber 25 is compound
Object；
(3 ') after cleaning, chemiluminescence reaction liquid enters reaction chamber 25, measures the chemiluminescence intensity of reaction chamber 25；
The concentration of cadmium ion in the sample to be tested is calculated according to standard curve in (4 ').
Fourth aspect, the present invention provides a kind of micro-fluidic chips as described in relation to the first aspect to be used for Molecular Detection, preferably
To be used for detection of heavy metal ion.
Preferably, the heavy metal ion includes cadmium ion, iron ion, manganese ion, copper ion, zinc ion, chromium ion, mercury
In ion, nickel ion, lead ion or arsenic ion any one or at least two combination, preferably cadmium ion.
Compared with prior art, the present invention has the advantages that：
(1) micro-fluidic chip of the invention is realized by the way that fluid channel is set to connect liquid storage cylinder, reaction chamber with waste liquid chamber
The automation of molecule controllably detects, while sets well, establishes a kind of safe and pollution-free, efficient automatic detection dress
It puts；
(2) the present invention micro-fluidic chip in, by the use of acridinium ester label cadmium ion monoclonal antibody as detect antibody,
Anti-mouse IgG antibody is as capture antibody, by the high degree of specificity of antibody-antigene reaction and the High sensitivity of chemical luminous system
Degree is combined, and realizes the quick and precisely detection of cadmium ion；
(3) cadmium ion of the invention detection micro-fluidic chip high specificity, pair heavy metal ion similar to cadmium ion is not
Generate cross reaction, high sensitivity, lowest detection 0.01ng/mL.
Description of the drawings
Fig. 1 is micro-fluidic chip vertical view, wherein, 11- prepare liquid wells, 12- detection antibody wells, 13- cleanings
Liquid well, 14- chemiluminescence exciting liquid A wells, 15- chemiluminescence exciting liquid B wells, 16- air holes, 21- detections
Antibody stock chamber, 22- cleaning solution liquid storage cylinders, 23- chemiluminescence exciting liquid A liquid storage cylinders, 24- chemiluminescence exciting liquid B liquid storage cylinders,
25- reaction chambers, 31- first flows, 32- second flow channels, 33- third flow channels, 41- waste liquid chambers；
Fig. 2 is top plate layer vertical view, wherein, 1- top plate layers, 11- prepare liquid wells, 12- detection antibody wells, 13-
Cleaning solution well, 14- chemiluminescence exciting liquid A wells, 15- chemiluminescence exciting liquid B wells, 16- air holes；
Fig. 3 is the first cavity layer vertical view, wherein, the first cavity layers of 2-, 21- detection antibody stock chambers, the storage of 22- cleaning solutions
Sap cavity, 23- chemiluminescence exciting liquid A liquid storage cylinders, 24- chemiluminescence exciting liquid B liquid storage cylinders, 25- reaction chambers；
Fig. 4 is flow channel layer vertical view, wherein, 3- flow channel layers, 16- air holes, 25- reaction chambers, 31- first flows, 32- the
Two runners, 33- third flow channels；
Fig. 5 is the second cavity layer vertical view, wherein, the second cavity layers of 4-, 41- waste liquid chambers；
Fig. 6 is backplane level vertical view, wherein, 5- backplane levels.
Specific embodiment
For the technological means and its effect that the present invention is further explained is taken, with reference to embodiments with attached drawing to this hair
It is bright to be further described.It is understood that the specific embodiments described herein be used only for explain the present invention rather than
Limitation of the invention.
In the examples where no specific technique or condition is specified, according to the described technology of document in the art or condition,
Or it is carried out according to product description.Reagents or instruments used without specified manufacturer, be can be by regular channel commercially available from
The conventional products of acquisition.
The preparation of 1 cadmium ion comlete antigen of embodiment
(1) by 10mg bifunctional chelating agent Cyclen -1,4,7,10- tetraacethyl-N- hydroxyl ambers
Amber imide ester (DOTA-NHS), which is dissolved in 1mL dimethyl sulfoxides (DMSO), forms metal-chelating agent solution, 30mg poly-D-lysines
(PLL) it is dissolved in 1mL 4- hydroxyethyl piperazineethanesulfonic acids (HEPES) buffer solution；
(2) DOTA-NHS solution is slowly dropped into carrier protein solution at room temperature, is stirred in drop, adjust pH to 9.0,
It is stirred overnight at room temperature, reaction solution uses 15mL centrifugal ultrafiltration pipe ultrafiltration, HEPES buffer solution in colourless or micro- yellow transparence every other day
Washing 3 times is concentrated into the coupling compound of 2mL, as bifunctional chelating agent and albumen；
(3) after the coupling compound of bifunctional chelating agent and albumen being diluted 5 times with HEPES buffer solution, it is slowly dropped into 1mL
In the heavy metal cadmium solion of a concentration of 1mg/mL, reaction 5 hours is stirred at room temperature, with 15mL centrifugal ultrafiltration pipe ultrafiltration, HEPES
Buffer solution washs 3 times, obtains the complete immunogene Cd-DOTA-PLL of cadmium ion.
The preparation of 2 cadmium ion monoclonal antibody of embodiment
(1) the 60 complete immunogene Cd-DOTA-PLL of μ g cadmium ions are dissolved in 200 μ L sterile salines, it is complete with 200 μ L
Full Freund's adjuvant mixing, fully emulsified backward Balb/c female mices subcutaneous abdomen carry out multi-point injection；After two weeks using immune
The mixed liquor of former Cd-DOTA-PLL and incomplete Freund's adjuvant carry out booster immunization, and it is subcutaneous for nape part to be immunized position, from the
It is immunized and starts three times, latter week is immunized from mouse orbit blood sampling detection serum titer；4th time immune not to add adjuvant, directly adopts
Peritoneal immunity is carried out with immunogene Cd-DOTA-PLL；
(2) it is immunized after terminating 3 days, the splenocyte of immune mouse is merged with SP2/0 myeloma, by 3 times or more Asias gram
Grand, screening obtains being capable of the hybridoma of stably excreting preventing from heavy metal cadmium ion monoclonal antibody, collects hybridoma training
Supernatant is supported, centrifugation removal cell fragment is saved backup in -20 DEG C；
(3) it selects through producing Balb/c mouse, intraperitoneal injection 0.5mL atoleines are dense by the cell in endless full nutrient solution
Degree is adjusted to 106/ mL, seven days pneumoretroperitoneum injection 1mL positive colony hybridomas of mouse of every pretreatment, after 7-10 days,
Mouse web portion significantly increases, and pierces through abdominal cavity, acquires ascites；
(4) ascites is centrifuged, abandons fat deposit and cellular layer, collect intermediate clear layer, it is thick with octanoic acid-ammonium sulfate salting-out process
Ascites is put forward, is further purified with protein G pillar affinity chromatographies, obtains the cadmium ion monoclonal antibody of purifying.
The preparation of the cadmium ion monoclonal antibody of 3 a word used for translation ingot ester of embodiment label
(1) cadmium ion monoclonal antibody is placed in bag filter, in the label buffer solution (0.1mol/L not less than 1L
Na2CO3-NaHCO3Buffer solution, pH=10) in dialyse, during which buffer fluid exchange at least 3 times；
(2) 0.52mg a word used for translation ingot esters (NSP-DMAE-NHS) are weighed, is dissolved in 894 μ L dimethylformamides (DMF), is made into
The NSP-DMAE-NHS DMF solutions of 1mmol/L；
(3) cadmium ion monoclonal antibodies of the 500 μ g by dialysis is placed in centrifuge tube, it is a concentration of adds in 100 μ L
The NSP-DMAE-NHS DMF solutions of 1mmol/L add in label buffer solution polishing volume to 1000 μ L, are protected from light and react at room temperature
30min adds in 100 μ L, 10% lysines, and the reaction was continued 40min terminates reaction, obtains being marked with the cadmium ion list of acridinium ester
Clonal antibody (NSP-DMAE-NHS-Ab)；
(4) NSP-DMAE-NHS-Ab and free NSP-DMAE-NHS is detached using Sephadex G-50 columns (1 × 25cm),
Elution chromatography column is balanced with purification buffer (0.1mol/L PBS, pH=6.3)；
(5) protein peak is detected with chromatograph in separation process, the chemiluminescence intensity and 280nm for measuring efflux respectively are inhaled
Shading value；
(6) eluent that shading value is high and absorbance is big is collected, is dispensed after adding in 1%BSA, the label purified has
The cadmium ion monoclonal antibody of pyridine ester.
The construction of 4 micro-fluidic chip of embodiment
The vertical view of the micro-fluidic chip of the present embodiment as shown in Figure 1, the vertical view of top plate layer 1 as shown in Fig. 2, the first chamber
The vertical view of body layer 2 as shown in figure 3, flow channel layer 3 vertical view as shown in figure 4, the second cavity layer 4 vertical view as shown in figure 5,
The vertical view of backplane level 5 is as shown in Figure 6.
The micro-fluidic chip include be cascading from top to bottom top plate layer 1, the first cavity layer 2, flow channel layer 3,
Second cavity layer 4 and backplane level 5；
The top plate layer 1 is provided with 5 wells and 1 air hole 16, wherein, well is respectively sample to be tested sample-adding
Hole 11, acridinium ester label cadmium ion monoclonal antibody well 12, cleaning solution well 13, chemiluminescence exciting liquid A wells
14 and chemiluminescence exciting liquid B wells 15；
First cavity layer 2 is provided with 4 liquid storage cylinders and 1 reaction chamber 25, wherein, liquid storage cylinder is respectively acridinium ester mark
Note cadmium ion monoclonal antibody liquid storage cylinder 21, cleaning solution liquid storage cylinder 22, chemiluminescence exciting liquid A liquid storage cylinders 23 and chemiluminescence swash
Lotion B liquid storage cylinders 24,25 inner surface of reaction chamber are coated with anti-mouse IgG antibody；
Second cavity layer 4 is provided with 1 waste liquid chamber 41；
The flow channel layer 3 is provided with runner, for top plate layer 1, the first cavity layer 2 to be connected with the second cavity layer 4；
The flow channel layer 3 includes first flow 31, second flow channel 32 and third flow channel 33；
The well 11 is connected with the liquid storage cylinder by first flow 31；
The second flow channel 32 is in clip shape；
Reaction chamber 25 is provided between the second flow channel 32 and the third flow channel 33；
The reaction chamber 25 is connected with the waste liquid chamber 41 by third flow channel 33；
The waste liquid chamber 41 is connected with the air hole 16.
The course of work of the micro-fluidic chip of the present invention is as follows：
(1) sample to be tested enters first flow 31 by well 11, meanwhile, the acridinium ester label cadmium ion in liquid storage cylinder 21
Monoclonal antibody enters first flow 31, and incubation is mixed with sample to be tested, forms antigen antibody complex, flows through second flow channel 32
With third flow channel 33, into reaction chamber 25；
(2) it is coated on the anti-mouse IgG antibody capture antigen antibody complex of 25 inner surface of reaction chamber in advance, resists antigen
Nanocrystal composition is fixed in reaction chamber 25；
(3) reaction chamber is cleaned using the cleaning solution in liquid storage cylinder 22, after removing the substance being not associated in reaction chamber, liquid storage cylinder
Chemiluminescence exciting liquid A and chemiluminescence exciting liquid B in 23 and 24 enter reaction chamber 25, excite in cadmium ion monoclonal antibody
Acridinium ester shine；
(4) chemiluminescence intensity of reaction chamber 25 is measured, measures the cadmium ion in sample to be tested.
The measure of 5 sample to be tested concentration of cadmium ions of embodiment
The present embodiment carries out the detection of concentration of cadmium ions using micro-fluidic chip as described in Example 4, the specific steps are：
(1) standard curve is formulated using micro-fluidic chip：It is respectively 0.2ng/mL, 0.5ng/mL, 1ng/ that 6 parts of concentration, which are configured,
The cadmium ion titer of mL, 2ng/mL, 5ng/mL and 10ng/mL enter first flow 31, with acridinium ester label by well 11
The mixing of cadmium ion monoclonal antibody is incubated, and is obtained antigen antibody complex, into reaction chamber 25, is consolidated by anti-mouse IgG antibody
It is scheduled in reaction chamber 25, after cleaning solution cleaning reaction chamber, chemiluminescence exciting liquid A and chemiluminescence exciting liquid B enter reaction chamber
25, measure the chemiluminescence intensity of reaction chamber 25, using chemiluminescence intensity as ordinate, a concentration of horizontal seat of cadmium ion titer
Mark draws four parametric plot of relationship of chemiluminescence intensity and concentration of cadmium ions, obtains standard curve；
(2) sample to be tested is added in by well 11, measures the chemiluminescence signal of reaction chamber 25 in the same way, according to
The concentration of cadmium ion in sample to be tested is calculated in standard curve.
In the present embodiment, the equation of standard curve is：
Y=(A-D)/[1+ (x/C) ^B]+D (R2=0.998)
Wherein, A=885443.1666, B=-1.25923, C=1.98661, D=18963.13843.
The concentration of cadmium ion is as shown in table 1 in 10 samples to be tested.
Table 1
The Evaluation on specificity of 6 micro-fluidic chip of embodiment
Using the micro-fluidic chip of the embodiment 4 heavy metal ion iron ion similar to cadmium ion, manganese ion, copper ion,
Zinc ion, chromium ion, mercury ion, nickel ion, lead ion and arsenic ion are detected, and make cross reaction curve, evaluate miniflow
Control the specificity of chip.
The results are shown in Table 2, and the micro-fluidic chip of embodiment 4 has higher specificity to cadmium ion, to other huge sum of moneys
Belong to the equal no cross reaction of ion.
Table 2
The sensitivity evaluation of 7 micro-fluidic chip of embodiment
20 retests are carried out to cadmium ion zero standard solution using the micro-fluidic chip of embodiment 4, measurement result takes
Average value adds 2 times of standard deviations, the as sensitivity of micro-fluidic chip.
As a result it shows：The micro-fluidic chip of embodiment 4 is 0.01ng/mL to the sensitivity of cadmium ion.
In conclusion the present invention micro-fluidic chip by the way that fluid channel is set to connect liquid storage cylinder, reaction chamber with waste liquid chamber,
The automation for realizing molecule controllably detects, while sets well, establishes a kind of safe and pollution-free, efficient automation
Detection device；In the micro-fluidic chip of the present invention, by the use of acridinium ester label cadmium ion monoclonal antibody as detection antibody, resist
Mouse IgG antibody is as capture antibody, by the height sensitivity of the high degree of specificity of antibody-antigene reaction and chemical luminous system
It is combined, the cadmium ion detection micro-fluidic chip high specificity of preparation, high sensitivity, process is simple, and detection time is short, cost
It is low, it is of great significance in detection of heavy metal ion field.
Applicant states that the present invention illustrates the method detailed of the present invention, but not office of the invention by above-described embodiment
It is limited to above-mentioned method detailed, that is, does not mean that the present invention has to rely on above-mentioned method detailed and could implement.Technical field
Technical staff it will be clearly understood that any improvement in the present invention, equivalence replacement and auxiliary element to each raw material of product of the present invention
Addition, selection of concrete mode etc., all fall within protection scope of the present invention and the open scope.
Claims (10)
1. a kind of micro-fluidic chip, which is characterized in that the micro-fluidic chip includes the top plate being cascading from top to bottom
Layer (1), the first cavity layer (2), flow channel layer (3), the second cavity layer (4) and backplane level (5)；
The top plate layer (1) is provided at least one well and at least one air hole (16)；
First cavity layer (2) is provided at least one liquid storage cylinder and at least one reaction chamber (25)；
Second cavity layer (4) is provided at least one waste liquid chamber (41)；
The flow channel layer (3) is provided with runner, for top plate layer (1), the first cavity layer (2) to be connected with the second cavity layer (4).
2. micro-fluidic chip according to claim 1, which is characterized in that the liquid storage cylinder is mounted with detection reagent；
Preferably, the detection reagent includes any one in detection antibody, cleaning solution or luminescence-producing reaction liquid or at least two
Combination；
Preferably, the reaction chamber (25) is coated with capture antibody；
Preferably, the detection antibody is cadmium ion monoclonal antibody；
Preferably, the capture antibody is anti-mouse IgG antibody.
3. micro-fluidic chip according to claim 1 or 2, which is characterized in that the flow channel layer (3) is including first flow
(31), second flow channel (32) and third flow channel (33)；
Preferably, the well (11) is connected with the liquid storage cylinder by first flow (31)；
Preferably, the second flow channel (32) is in clip shape；
Preferably, it is provided with reaction chamber (25) between the second flow channel (32) and the third flow channel (33)；
Preferably, the reaction chamber (25) is connected with the waste liquid chamber (41) by third flow channel (33).
4. according to claim 1-3 any one of them micro-fluidic chips, which is characterized in that the waste liquid chamber (41) with it is described
Stomata (16) is connected.
5. according to claim 1-4 any one of them micro-fluidic chips, which is characterized in that the micro-fluidic chip is by macromolecule
Material preparation obtains；
Preferably, the high molecular material include crystalline silicon, glass, dimethyl silicone polymer, polymethyl methacrylate, poly- pair
In ethylene terephthalate or polyester any one or at least two combination, preferably dimethyl silicone polymer；
Preferably, the liquid storage cylinder and flow channel layer (3) are prepared by light tight high molecular material, preferably light tight poly- diformazan
Radical siloxane；
Preferably, the top plate layer (1) and backplane level (5) are prepared by light transmission high molecular material, preferably the poly- diformazan of light transmission
Radical siloxane.
6. according to claim 1-5 any one of them micro-fluidic chips, which is characterized in that the cadmium ion monoclonal antibody is repaiied
It is decorated with luminous marker；
Preferably, the luminous marker includes acridinium ester, fluorescein isothiocynate, RB 200, the different sulphur cyanogen of tetramethyl
In sour rhodamine, lanthanide chelate or phycoerythrin any one or at least two combination, preferably acridinium ester.
7. according to claim 1-6 any one of them micro-fluidic chips, which is characterized in that the cadmium ion monoclonal antibody by
Cadmium ion comlete antigen is immunized animal and is prepared；
Preferably, the cadmium ion comlete antigen is the compound of cadmium ion, chelating agent and carrier protein；
Preferably, the chelating agent includes ethylenediamine tetra-acetic acid, diethylenetriamine pentaacetic acid, diethylenetriamine pentaacetic acid derivative, 1,
4,7,10- tetraazacyclododecanands-N, N, N, N- tetraacethyl or Cyclen -1,4,7,10- tetrems
In acid-N-hydroxy-succinamide ester any one or at least two combination, preferably Isosorbide-5-Nitrae, 7,10- tetraazacyclododecanes 12
Alkane -1,4,7,10- tetraacethyls-N-hydroxy-succinamide ester；
Preferably, the carrier protein includes poly-D-lysine, human serum albumins, keyhole limpet hemocyanin or bovine serum albumin(BSA)
In any one or at least two combination, preferably poly-D-lysine；
Preferably, the cadmium ion comlete antigen be cadmium ion-Isosorbide-5-Nitrae, 7,10- tetraazacyclododecanands-Isosorbide-5-Nitrae, 7,10- tetrems
Acid-N-hydroxy-succinamide ester-poly-l-lysine complex.
8. a kind of using the method for formulating standard curve such as claim 1-6 any one of them micro-fluidic chip, feature exists
In including the following steps：
(1) the cadmium ion titer of configuration various concentration at least known to 6 parts；
(2) the cadmium ion titer described in step (1) by well (11) into first flow (31), with acridinium ester label cadmium from
Sub- monoclonal antibody mixing is incubated, and antigen antibody complex is obtained, into reaction chamber (25)；
(3) it is coated on the antigen antibody complex described in the anti-mouse IgG antibody capture step (2) of reaction chamber (25) in advance；
(4) after cleaning, chemiluminescence reaction liquid enters reaction chamber (25), measures the chemiluminescence intensity of reaction chamber (25)；
(5) using chemiluminescence intensity as ordinate, a concentration of abscissa of cadmium ion titer draws chemiluminescence intensity and cadmium
Four parametric plot of relationship of ion concentration, obtains the standard curve.
A kind of 9. method for detecting cadmium ion, which is characterized in that the method is using the micro-fluidic core as described in claim 1-7
Piece；
Preferably, it the described method comprises the following steps：
(1 ') sample to be tested by well (11) into first flow (31), it is mixed with acridinium ester label cadmium ion monoclonal antibody
It closes and is incubated, antigen antibody complex is obtained, into reaction chamber (25)；
(2 ') are coated on the antigen antibody complex described in the anti-mouse IgG antibody capture step (1 ') of reaction chamber (25) in advance；
(3 ') after cleaning, chemiluminescence reaction liquid enters reaction chamber (25), measures the chemiluminescence intensity of reaction chamber (25)；
The concentration of cadmium ion in the sample to be tested is calculated according to standard curve in (4 ').
10. a kind of if claim 1-7 any one of them micro-fluidic chip is for Molecular Detection, be preferably used for heavy metal from
Son detection；
Preferably, the heavy metal ion include cadmium ion, iron ion, manganese ion, copper ion, zinc ion, chromium ion, mercury from
In son, nickel ion, lead ion or arsenic ion any one or at least two combination, preferably cadmium ion.
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| CN201810149379.0A CN108169495A (en) | 2018-02-13 | 2018-02-13 | A kind of micro-fluidic chip and its application |
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| CN113976192A (en) * | 2021-08-23 | 2022-01-28 | 上海荧辉医疗器械有限公司 | Microsphere-labeled microfluidic chip and microsphere protein labeling method |
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