CN106501500B - A kind of Liquid Crystal Sensor and its detection method of Visual retrieval protein biomarkers - Google Patents
A kind of Liquid Crystal Sensor and its detection method of Visual retrieval protein biomarkers Download PDFInfo
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- CN106501500B CN106501500B CN201610827578.3A CN201610827578A CN106501500B CN 106501500 B CN106501500 B CN 106501500B CN 201610827578 A CN201610827578 A CN 201610827578A CN 106501500 B CN106501500 B CN 106501500B
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Classifications
-
- 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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- 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
- G01N33/5302—Apparatus specially adapted for immunological test procedures
-
- 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
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
- G01N33/76—Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/575—Hormones
- G01N2333/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g. HCG; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
Abstract
A kind of Liquid Crystal Sensor and its detection method of Visual retrieval protein biomarkers, belong to sensor technical field.Sample loop is constructed in chip surface first.Then primary antibody is fixed in sample ring region.Later, chip is immersed into body fluid sample to be detected, and antibody probe is added in above-mentioned sample liquid, after reaction 30 minutes, the antigen in body fluid sample is specifically bound with primary antibody, antibody probe respectively, forms " sandwich " sandwich immunoassay structure.It is dried after flushing, instills liquid crystal, and chip is put into observation box.Upper and lower two faces of the observation box are the mutually perpendicular polarizing film in polarization direction.It changes since the specific binding of antigen, antibody will lead to liquid crystal aligning on chip, it can be observed that the liquid crystal image in sample loop occurs from " dark " to the variation of " bright ", to judge whether contain HCG in the urine specimen.This method is easy to operate, it is not necessary that antibody is marked, without complex instrument, is suitable for bedside and detects.
Description
Technical field
The present invention relates to a kind of building of Liquid Crystal Sensor and its applications in detection protein biomarkers, belong to
Sensor technical field.
Background technique
Human chorionic gonadotrophin (HCG) is a kind of glycoprotein hormone generated by placenta in human female of becoming pregnant,
Largely exist in the urine of pregnant woman.And HCG is practically free of in unrecognizing factor urine.Normal human urine HCG is feminine gender, <
312U/L.Urinating HCG concentration > 2500U/L is that gestation is positive, and see: (1) pregnant: HCG can be detected for 10 days after becoming pregnant in urine
The positive, there is peak up to 2500U/L or more, 60-70 days in 35-40 days HCG after becoming pregnant, up to 100,000-20 ten thousand U/L.(2) outside palace
Pregnant: urine pregnancy test has 60% positive rate.(3) incomplete abortion: intrauterine still has placenta tissue remaining, and HCG is qualitative for sun
Property.(4) certain tumours such as vesicular mole, suede cancer, male testical teratoma etc..(5) certain endocrine metabolism diseases: such as pituitary
Disease, hyperthyroidism, climacteric syndrome.(6) certain gynecological diseases: such as ovarian cyst, endometrial hyperplasia.(7) take chlorpromazine,
After the drugs such as phenthazine.
Present early pregnancy diagnosis technology has: (1) early pregnancy laminar flow immune test paper, is that one kind that people design is convenient for female
Detect the product oneself whether being pregnant.The detection of early pregnancy test paper is immunized in laminar flow: urine being dripped in the detection hole on test paper, is such as only existed
There is a color band (the aobvious red of the test paper having, the aobvious blue of some test paper) in the check plot of test paper, indicates negative, illustrates not cherish
It is pregnant；Conversely, such as all there is apparent colour band in detection zone and check plot, then it represents that it is positive, illustrate pregnant.Laminar flow is immune early
Pregnant test paper is typically only capable to detect a kind of protein markers molecule, as a kind of primary dcreening operation inspection, it may appear that false positive or false negative.
(2) B-type ultrasonography is most correct reliable method with B ultrasound diagnosis.Most early in pregnant week, be also exactly the menstrual cycle it is expired
One week, it can show that intrauterine has the circular ring of light, also known as gestation ring on B ultrasound screen, the dark space in ring is sheep
Water, wherein being also shown rhythmic fetus heart beating.However the cost of this method is with regard to relatively high.(3) examination of basal body temperature method,
The basal body temperature of normal women of child-bearing age is in cyclically-varying as the menstrual cycle, and this Temperature changing is related with ovulation.If
Continue two weeks or more higher basal body temperatures it is necessary to consider that hospital checks, because you are likely to be pregnancy.If >=20
Day can be identified as early pregnancy.Decline gradually in pregnant early stage BBT curve, indicates that inadequate luteal function or placental function are bad, there is miscarriage
Tendency.This method is apparent, and time-consuming, cumbersome, and full of uncertainty.
Existing early pregnancy diagnosis technology has its advantage and disadvantage, our invention also have can direct visual perception, can be same
The detection of Shi Shixian multiple proteins marker.This method also has many advantages, such as that convenience, environmental protection, reagent consumption are few, and detects
Journey is not needed by complicated instrument, and user oneself can complete by smart phone.
Summary of the invention
The present invention relates to the Liquid Crystal Sensors and its detection method of a kind of Visual retrieval protein biomarkers.
A kind of Liquid Crystal Sensor of Visual retrieval protein biomarkers, which is characterized in that including observation box and core
Piece, there are two the mutually perpendicular polarizing films in polarization direction as watch window, observation box for the upper and lower two opposite face pastes of observation box
Two opposite sides are equipped with notch, for being inserted into chip；Chip is transparent glass-chip, and sample is built on glass-chip
Product annular groove, sample loop trench bottom are fixed with a kind of antibody of protein biomarkers antigen to be detected as first antibody.Make
Used time, using the body fluid to be detected that may contain protein biomarkers as middle layer and by protein bio mark to be detected
Another antibody of object antigen is added in sample annular groove as antibody probe, is reacted, the protein to be detected in body fluid
Biomarker antigen can be specifically bound simultaneously with first antibody and antibody probe, form " sandwich " sandwich immunoassay structure,
It cleans and dries, liquid crystal is added；Finally, chip is put into observation box, the specific binding of antibody and antigen will lead to chip upper liquid
Crystalline substance orientation changes, and then observes that under light background, the liquid crystal image in sample loop occurs from " dark " to the variation of " bright ",
To judge whether contain protein biomarkers to be detected in the urine specimen.
The present invention is for detecting the human chorionic gonadotrophin in urine specimen (HCG), first in chip surface structure
Sample annular groove is built, then HCG monoclonal antibody is fixed in sample annular groove；Later, said chip is immersed into urine to be detected
Sample, and another anti-HCG probe is added, after reaction 30 minutes, HCG antigen in urine specimen can be with HCG anti-on chip the
One antibody and the anti-HCG probe of addition are specifically bound simultaneously.It cleans and dries, liquid crystal is added.Finally, chip is put into observation
Box, upper and lower two faces of the observation box are the mutually perpendicular polarizing film in polarization direction, due to the specific binding meeting of antibody and antigen
Liquid crystal aligning on chip is caused to change, it can be observed that the liquid crystal image in sample loop occurs from " dark " to the change of " bright "
Change, to judge whether contain HCG in the urine specimen.
Chip surface prepares sample annular groove battle array by photo mask method and photo polymerization monomer (such as polyethyleneglycol diacrylate)
Column, are fixed and sample detection for antibody.It is preferred that the internal diameter 2mm of each sample annular groove, outer diameter 2.4mm, height 0.15mm.Pass through
Different antibodies are fixed in above-mentioned sample loop array, which can be applied to while a variety of biomarkers point in test sample
Son.
Contain multiple identical sample annular groove arrays on a single die, first antibody and use pair equipped with same antigen
Identical antibody probe is answered, different body fluid can be measured simultaneously.
Or further contain multiple identical sample annular groove arrays, the first antibody equipped with not synantigen on a single die
With use corresponding antibody probe, can measure same body fluid disparity items or different protein biomarkers to be detected.
Further before the fixed first antibody in chip sample annular groove bottom, oxygen plasma treatment chip sample annular groove bottom table
Inertia DMOAP is modified using the method for UV activation then in the fixed DMOAP of chip sample annular groove bottom surface in face
Chip surface is pre-processed, and the fixation of antibody is subsequently used for.The core that inertia DMOAP is modified using the method for UV activation
Piece surface is pre-processed, the fixation for antibody.The chip of DMOAP modification is by after the ultraviolet light of 254nm, DMOAP alkane
Base chain end can generate a certain amount of aldehyde radical, can use the sample that first antibody molecule is fixed on chip surface by schiff base reaction
In product annular groove.
Dimethyl stearyl [3- (trimethoxy silicon substrate) propyl] ammonium chloride (DMOAP) and air are utilized respectively to liquid crystal
Molecule carries out vertical preorientation.
Observation box material is engineering plastics (such as ABS resin), can be prepared by technologies such as injection molding, 3D printings；Watch window
The mutually perpendicular polarizing film in polarization direction is posted on upper and lower two surfaces, and two lateral layouts are suitble to inserting for chip at groove in box
Enter and fixes.
Liquid crystal material is preferably nematic liquid crystal, such as 4 '-n-pentyls-[1,1 '-phenylbenzene] -4- nitrile (5CB), E7, SLC
7011 etc..
Using the screen of smart phone as light source, the observation box for being inserted into chip is placed in above mobile phone screen, from perpendicular to sight
Examine the image of liquid crystal in the surface observation sample ring region of box.
This method is easy to operate, without complex instrument, is suitable for bedside and detects.Pass through designerantibodies array this method
With the potentiality for detecting multiple proteins marker simultaneously, it can be used for early pregnancy detection and the early diagnosis of a variety of diseases.
Detailed description of the invention
Fig. 1 observation box pictorial diagram.
Fig. 2 chip pictorial diagram (white annulus is sample annular groove).
Fig. 3 positive compares pictorial diagram (background color: yellow and white) with negative findings.
Fig. 4 mobile phone is that background observes pictorial diagram.
Fig. 5 photo mask method prepares sample annular groove schematic diagram.
Fig. 6 is the Liquid Crystal Sensor and its visualizing monitor method schematic diagram of Visual retrieval protein biomarkers.
Specific embodiment
Below with reference to embodiment, the invention will be further described, but the present invention is not limited to following embodiments.
Embodiment 1
The present invention constructs the step of visualization Liquid Crystal Sensor and its detection method is as follows:
(1) clean sheet glass: preferred cleaning process is: 1. sheet glass being immersed in the Decon90 solution of 5%v/v
2-12h；2. ultrasonic cleaning 15 minutes, outwells washing lotion；3. it is rinsed 5 times with deionized water, it is 15 minutes ultrasonic in deionized water；④
It repeats 3., then impregnates slide spare in deionized water.
(2) glass sheet surface prepares sample loop: 1. activating glass sheet surface hydroxyl.Preferred activation process is: by glass
Piece, which is placed in ammonium hydroxide/hydrogen peroxide mixed solution that volume ratio is 3:1, to be impregnated 30 minutes, uses N after being rinsed with deionized water2It blows
It is dry；2. glass sheet surface modifies 3- (silicochloroform base) propyl methacrylate (TPM).Preferred method of modifying is: will
Sheet glass, which is put into heptane/chloroformic solution (volume ratio 4:1) of the TPM containing 5mM, impregnates 2h, cleans 5 with n-hexane followed by
Minute, then cleaned 3-5 times with deionized water, then use N2Drying；3. sample loop mask plate and the TPM sheet glass modified are made into
Sandwich (Fig. 5), distance piece thickness are about 0.15mm；4. preferred sample loop preparation method is to prepare the styrax of 3%w/w
Polyethyleneglycol diacrylate (PEG575) solution of double methyl ethers (DMPA), injects in above-mentioned sandwich, in optical power density
For 13.0mW/cm2Ultraviolet lamp (365nm) under irradiate 6 seconds, with ethyl alcohol clean, then use N2Drying, is shown in Fig. 5.
(3) the fixed DMOAP of chip surface: 1. oxygen plasma treatment surface of glass slide: preferred condition is, and: 90W handles the time
2 minutes.2. chip surface fixes DMOAP: preferred condition is: reacting 5 minutes, spends in the DMOAP aqueous solution of 0.1%v/v
Ionized water uses N after cleaning2Drying, is finally crosslinked 20 minutes in 100 DEG C of vacuum drying ovens.Alkyl chain (C-18) on DMOAP and
Air Interface can induce the vertical orientation of liquid crystal molecule.
(4) chip surface is fixed Anti-HCG antibody (primary antibody): being first 3.5mW/cm in optical power density2Ultraviolet light
It is irradiated chip 100 seconds under (254nm)；Secondly, Anti-HCG antibody (primary antibody) solution is prepared, preferred 8 μ g/mL of antibody concentration, each
The antibody-solutions being added in sample loop are 2.5 μ L.After cleaning, N is used2Drying, 4 DEG C save backup.
(5) size of preferred chip: long 25mm, wide 20mm, thick 1.2mm；The size of preferred observation box: long 30mm,
Wide 20mm, high 6.0mm；Visual field size in polarizing film: long 22mm, wide 13mm；The design of two sides further groove can be protected inside observation box
Card chip can be smoothly inserted into.
(6) chip is immersed in urine specimen, the preferred reaction time is 30 minutes, and reaction temperature is room temperature.
(7) in the urine specimen in step (6), it joined another HCG antibody (secondary antibody), preferred concentration is 10 μ g/
mL。
(8) after chip is cleaned, liquid crystal is added, the amount of liquid crystal being added in preferred each sample loop is 0.3 μ L.
(9) chip is inserted into observation box, right-angle view measurement result.
Observation box material object graph Fig. 1, chip pictorial diagram (white annulus is sample loop) are shown in Fig. 2.Positive and negative findings pair
See that Fig. 3, mobile phone are that background observes graph Fig. 4 in kind according to pictorial diagram (background color: yellow and white), is Visual retrieval albumen
The Liquid Crystal Sensor and its visualizing monitor method of matter biomarker are shown in Fig. 6.
Claims (8)
1. a kind of Liquid Crystal Sensor of Visual retrieval protein biomarkers, which is characterized in that including observation box and chip,
There are two the mutually perpendicular polarizing films in polarization direction as watch window, observation box phase for the upper and lower two opposite face pastes of observation box
Pair two sides be equipped with notch, for being inserted into chip；Chip is transparent glass-chip, and sample is built on glass-chip
Annular groove, sample loop trench bottom are fixed with a kind of antibody of protein biomarkers antigen to be detected as first antibody；In core
Before the fixed first antibody of piece sample loop trench bottom, oxygen plasma treatment chip sample annular groove bottom surface, then in chip sample
Product annular groove bottom surface fixes DMOAP, is located in advance using the method for UV activation to the chip surface that inertia DMOAP is modified
Reason, is subsequently used for the fixation of first antibody.
2. a kind of Liquid Crystal Sensor of Visual retrieval protein biomarkers described in accordance with the claim 1, feature exist
It is in, detection method, in use, using the body fluid to be detected that may contain protein biomarkers as middle layer and will be to be checked
Another antibody for surveying protein biomarkers antigen is added in sample annular groove as antibody probe, is reacted, body fluid
In protein biomarkers antigen to be detected can be specifically bound simultaneously with first antibody and antibody probe, formed " Sanming City
Control " sandwich immunoassay structure, it cleans and dries, liquid crystal is added；Finally, chip is put into observation box, the specificity knot of antibody and antigen
Credit union causes liquid crystal aligning on chip to change, so observe under light background the liquid crystal image in sample loop occur from
" dark " arrives the variation of " bright ", to judge whether contain protein biomarkers to be detected in the body fluid sample.
3. a kind of Liquid Crystal Sensor of Visual retrieval protein biomarkers according to claim 1 or 2, feature
It is, detects the human chorionic gonadotrophin (HCG) in urine specimen, constructs sample annular groove in chip surface first, then
HCG monoclonal antibody is fixed in sample annular groove；Later, said chip is immersed into urine specimen to be detected, and be added another
Kind Anti-HCG antibody probe, after reaction 30 minutes, the HCG antigen in urine specimen can be with HCG first antibody anti-on chip and addition
Anti-HCG antibody probe specifically bind simultaneously；It cleans and dries, liquid crystal is added；Finally, chip is put into observation box, the observation
Upper and lower two faces of box are the mutually perpendicular polarizing film in polarization direction, since the specific binding of antibody and antigen will lead to chip
Upper liquid crystal aligning changes, and observes that the liquid crystal image in sample annular groove occurs from " dark " to the variation of " bright ", to judge
Whether contain HCG in the urine specimen out.
4. a kind of Liquid Crystal Sensor of Visual retrieval protein biomarkers according to claim 1 or 2, feature
It is, chip surface prepares sample annular groove array by photomask and photo polymerization monomer, fixes for antibody and sample detection.
5. a kind of Liquid Crystal Sensor of Visual retrieval protein biomarkers, feature exist according to claim 2
In, on a single die containing multiple identical sample annular groove arrays, the corresponding phase of first antibody and use equipped with same antigen
Same antibody probe, can measure different body fluid simultaneously；
Or containing multiple identical sample annular groove arrays on a single die, first antibody and use equipped with not synantigen correspond to
Antibody probe, the disparity items of same body fluid can be measured.
6. a kind of Liquid Crystal Sensor of Visual retrieval protein biomarkers, feature exist according to claim 5
In disparity items includes different protein biomarkers to be detected.
7. a kind of Liquid Crystal Sensor of Visual retrieval protein biomarkers according to claim 1 or 2, feature
It is, liquid crystal material is nematic liquid crystal.
8. a kind of Liquid Crystal Sensor of Visual retrieval protein biomarkers according to claim 1 or 2, feature
It is, using the screen of smart phone as light source, the observation box for being inserted into chip is placed in above mobile phone screen, from perpendicular to observation box
Surface observation sample annulus region in liquid crystal image.
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