CN106959235A - A kind of blood preseration device - Google Patents
A kind of blood preseration device Download PDFInfo
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- CN106959235A CN106959235A CN201710383751.XA CN201710383751A CN106959235A CN 106959235 A CN106959235 A CN 106959235A CN 201710383751 A CN201710383751 A CN 201710383751A CN 106959235 A CN106959235 A CN 106959235A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
- G01N1/4005—Concentrating samples by transferring a selected component through a membrane
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
- G01N1/4005—Concentrating samples by transferring a selected component through a membrane
- G01N2001/4016—Concentrating samples by transferring a selected component through a membrane being a selective membrane, e.g. dialysis or osmosis
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- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a kind of blood preseration device.Blood preseration device provided by the present invention, including hemofiltration film (1), molecular weight mwco membrane (2) and water accepting layer (3)；The lower section of the hemofiltration film (1) sets the molecular weight mwco membrane (2), and the lower section of the molecular weight mwco membrane (2) sets the water accepting layer (3).Blood preseration device provided by the present invention, can effectively improve the dry blood formation time, improve the stability of albumen.The present invention can isolate the mixture comprising target protein or nucleic acid by the second layer molecular weight mwco membrane according to the suitable interception of the molecular weight of target protein and nucleic acid selection.In addition, the blood preseration device of the present invention includes water accepting layer, scraps of paper drying time can be greatly shortened, the stability of protein is improved.
Description
Technical field
The invention belongs to clinical disease diagnosis detection field, and in particular to a kind of blood preseration device.
Background technology
Dry blood paper disk method (dried blood spot, DBS), i.e., collect whole blood sample in paperboard, in blood specimen collection
There is certain advantage in method than conventional method.The less blood volume of its demand, can reduce animal and use, facilitate blood specimen collection, deposit
Accumulating is defeated, is an effective tool during orphan disease examination.Dry blood paper disk method early in the sixties in last century just by
For in the neonatal screening of PKU.With the development of tandem mass spectrum technology etc., dry blood paper disk method is metabolized class in heredity
Application value in the diagnosis and examination of disease is increasingly highlighted.
For from the angle of Sample preservation, dried blood spot is more more stable than whole blood, so its storage and cost of transportation are far low
In whole blood.In addition, the dry blood scraps of paper need the sample size gathered less, therefore the dry blood scraps of paper are diagnosed needing multiple repairing weld
Detection in have specific advantage.Due to the simplification and higher stability of dry blood paper disk method, in many extensive diseases
(such as neonatal genetic screening) is it has emerged that extremely wide application prospect in examination.It is external to do extensively
The blood scraps of paper are applied in disorder in screening sample transport, and the means of transportation of traditional whole blood is instead of to a certain extent.It is domestic
The dry blood scraps of paper are gradually begun to use to carry out the transport of blood product, multiple hospitals are ended using dry blood scraps of paper transport blood sample
Grow sick (HIV), hepatitis C, the glycogen storage disease such as disorder in screening such as Pompeii's disease.
But for the detection field of protein in blood, common blood test method and dry blood paper disk method also have not
Foot.Common blood test needs centrifugal separation plasma and haemocyte, operates comparatively laborious；And dry blood paper disk method, it is existing dry
Blood piece preparation method be by droplet of blood on the certain area of filter paper, then dry, set when using using special punching naturally
The standby dried blood spot for being acquired certain area is tested.The dried blood spot of this single layer structure is inconvenient in subsequent use
(glairy measure is vulnerable to interference of haemocyte intracellular composition etc.), and drying process is longer (about 4 are more than hour), egg
White matter is easily degraded, and can be influenceed by air humidity.
The content of the invention
In order to make up the deficiency in above field, the invention provides a kind of blood preseration device, solve in separation blood
Protein the problem of need cumbersome step, while greatly reducing the time of blood drying, improve the stability of protein.
The present invention is achieved through the following technical solutions：
A kind of blood preseration device, including hemofiltration film (1), molecular weight mwco membrane (2) and water accepting layer (3)；The hemofiltration film
(1) lower section sets the molecular weight mwco membrane (2), and the lower section of the molecular weight mwco membrane (2) sets the water accepting layer (3)；
The hemofiltration film (1) is used for washed corpuscles and blood plasma；The material of the hemofiltration film (1) be selected from whatman903 or
Fusion5 or cellulose acetate film；
The molecular weight mwco membrane (2) is used to retain protein or nucleic acid；The material of the molecular weight mwco membrane (2) is selected from
Cellulose ester membrane or ceramic membrane；
The material of the water accepting layer 3 is selected from silica gel, filter paper, CaCl2Or water-absorbing resin；
The upper surface of the molecular weight mwco membrane (2) scribbles 0.5-2.6mg/cm2Casein.
Preferably, the upper surface of the molecular weight mwco membrane (2) scribbles 1.3mg/cm2Casein.
The molecular weight for the compound that the material selection of the molecular weight mwco membrane is retained as needed is chosen.
Also include getting stuck (5) and sealing ring (4), the hemofiltration film (1), molecular weight mwco membrane (2) and water accepting layer (3) pass through
Sealing ring (4), which is sealed in, to get stuck in (5).
Get stuck (5) are made up of fluid-tight hard material, and described get stuck (5) are used to support the hemofiltration film (1), divided
Son amount mwco membrane (2) and water accepting layer (3).
The sealing ring (4) is made up by ring-type and of fluid-tight hard material, and the sealing ring (4) is used for the filter
Blood film (1), molecular weight mwco membrane (2), water accepting layer (3), which are fixed on, to get stuck in (5).
The aperture of the cellulose acetate film is selected from 0.22 μm, 0.45 μm, 0.8 μm or 3 μm.
The molecular cut off of the cellulose ester membrane is 20000.
The material of the water accepting layer is that mass ratio is 1:1 water-absorbing resin and CaCl2Mixture.
The water-absorbing resin particle diameter is 150-250 mesh.
Blood preseration device provided by the present invention, can effectively improve the dry blood formation time, improve the stability of albumen.
Blood test field, the inspection to protein and nucleic acid is particularly important, and very many diseases can be by whetheing there is target in blood
Protein and nucleic acid and made a definite diagnosis.The present invention can be by according to the suitable retention of the molecular weight of target protein and nucleic acid selection
The second layer molecular weight mwco membrane of amount, isolates the mixture comprising target protein or nucleic acid.The present invention need not be separated first
Haemocyte, while the protein smaller than target protein or nucleic acid molecular weight and nucleic acid can be screened out in advance, molecular weight retention
The mixture that film is truncated to can directly redissolve chemical examination, the step of enormously simplify blood test.Meanwhile, in molecular weight mwco membrane
Surface, can be coated with protein stabilizing agent or nucleic acid stability agent, to improve according to the characteristic of target protein or nucleic acid on its surface
The stability of object.0.5-2.6mg/cm is coated with using surface2The cellulose ester membrane BNP albumen of casein is relatively stablized, and returns
High income；Wherein it is coated with 1.3mg/cm2The cellulose ester membrane BNP albumen of casein is most stable, rate of recovery highest.In addition, this
The blood preseration device of invention includes water accepting layer, can greatly shorten scraps of paper drying time, improves the stability of protein.
Brief description of the drawings
Fig. 1 is the structural representation of the blood preseration device of embodiment 1；
Fig. 2 is to include the diagrammatic cross-section got stuck with the blood preseration device of sealing ring；
Fig. 3 is to include the diagrammatic cross-section got stuck with the blood preseration device of sealing ring；
Fig. 4 is the blood preseration device of embodiment 2；
Fig. 5 is BNP measurement result figures after dry blood device before processing；
The molecule mwco membrane of 1- hemofiltration films, 2- retention albumen or nucleic acid, 3- water accepting layers, 4- sealing rings, 5- gets stuck.
Embodiment
Following examples material therefor：Whatman903, Fusion5, cellulose acetate film, cellulose ester membrane, ceramic membrane,
Silica gel, filter paper, CaCl2, water-absorbing resin, casein be commercially commercially available.
Embodiment 1, prepare blood preseration device
The present invention provides a kind of blood preseration device, in all of the embodiments illustrated, all has following structure special as shown in Figure 1
Levy：Including hemofiltration film 1, molecular weight mwco membrane 2 and water accepting layer 3；The lower section of the hemofiltration film 1 sets the molecular weight mwco membrane 2,
The lower section of the molecular weight mwco membrane 2 sets the water accepting layer 3；The hemofiltration film 1 is used for washed corpuscles and blood plasma；The filter
The material of blood film 1 is selected from whatman903 or Fusion5 or cellulose acetate film；The molecular weight mwco membrane 2 is used to retain egg
White matter or nucleic acid；The material of the molecular weight mwco membrane 2 is selected from cellulose ester membrane or ceramic membrane；The material choosing of the water accepting layer 3
From silica gel, filter paper, CaCl2Or water-absorbing resin.The compound that the material selection of the molecular weight mwco membrane 2 is retained as needed
Molecular weight is chosen.Meanwhile, on molecular weight mwco membrane surface, egg can be coated with its surface according to the characteristic of target protein or nucleic acid
White matter stabilizer or nucleic acid stability agent, to improve the stability of object.
In a further embodiment, as shown in Figures 2 and 3, also including 5 and the sealing ring 4 of getting stuck, the hemofiltration film 1, point
Son amount mwco membrane 2 and water accepting layer 3 are sealed in by sealing ring 4 to get stuck in 5.Described get stuck 5 is made up of fluid-tight hard material,
It is described to get stuck 5 for supporting the hemofiltration film 1, molecular weight mwco membrane 2 and water accepting layer 3.The sealing ring 4 is for ring-type and by impermeable
The hard material of water is made, and the sealing ring 4 is used to the hemofiltration film 1, molecular weight mwco membrane 2, water accepting layer 3 are fixed on and got stuck
In 5.
Embodiment 2, each layer are preferred
First, hemofiltration film is preferred
For the performance of relatively more several hemofiltration UF membrane blood plasma and haemocyte, the first layers of blood separating film selects Whatman respectively
903#, Fusion5 (GE), cellulose acetate film (aperture is respectively 0.22 μm, 0.45 μm, 0.8 μm, 3 μm), glass fibre membrane,
Polyester fiber film；The second layer is from cellulose ester membrane (molecular cut off 20000 leads to kind biology)；Third layer selects anhydrous CaCl2
(traditional Chinese medicines).Surface of the present invention is dropped in 0.5ml Mouse whole bloods, to the material 0.5ml of the cellulose ester membrane retention of the second layer
PH7.510mM PBS determines CD44 after redissolving with mouse CD44 molecule (CD44) ELISA Kit, and filter is shown if CD44 is positive
Blood film can effective blood cell filtration, as a result show that above-mentioned several filter membranes can blood cell filtration well, it is contemplated that price factor,
It is preferred that Whatman903#.
2nd, molecular weight retention membrane material is preferred
The preparation of reagent：
PH7.5 10mM PBS preparation：Weigh 0.27g potassium dihydrogen phosphates (KH2PO4) (traditional Chinese medicines, 10017608), 1.42g
Disodium hydrogen phosphate (Na2HPO4) (traditional Chinese medicines, 100203008), 8g sodium chloride (NaCl) (traditional Chinese medicines, 10019308), 0.2g potassium chloride
(KCl) (traditional Chinese medicines, 10016308) adjust pH to 7.5, then with volumetric flask constant volume extremely in 900mL ultra-pure waters after being completely dissolved
1L。
Experiment is divided into following steps：
Set up BSA (60KD, amresco) Goat IgG (150KD, open safe biology) BNP (Hytest) assay method
Be coated with microwell plate the anti-BSA of 1.0 μ g/ml (60KD) Goat IgG (150KD) BNP antibody PBS (10mM,
PH7.5), 100 μ L/ holes, 2-8 DEG C, 24 hours；
Above-mentioned solution is abandoned, PBS (10mM, pH7.5) solution containing 3%BSA, 200 μ L/ holes, 2-8 DEG C, 24 hours is added；
Abandon above-mentioned solution, add determinand solution or calibration object solution (0ng/ml, 0.5ng/ml, 3ng/ml, 10ng/ml,
30ng/ml, 100ng/ml), 100 μ L/ holes, 37 DEG C, 30 minutes；
Above-mentioned solution is abandoned, with the PBS (10mM, pH7.5) containing 0.05%Tween-20,5 times are rinsed, every time 200 μ L/ holes；
Add HRP mark anti-BSA (60KD) Goat IgG (150KD) BNP antibody (1/10000), 100 μ L/ holes, 37
DEG C, 15 minutes；
Above-mentioned solution is abandoned, with the PBS (10mM, pH7.5) containing 0.05%Tween-20,5 times are rinsed, every time 200 μ L/ holes；
Add under 100 μ L/ holes TMB nitrite ions (biopanda), room temperature condition and react after 10min, add 50 μ L/ hole 2M
H2SO4。
With ELIASA (thermo) mensuration absorbance.
With calibration object concentration and its absorbance, using LogX-LogY fitting a straight lines, standard curve is set up, by sample to be tested
Absorbance bring this equation into, you can calculate the concentration value of sample to be tested.
BSA (60KD) Goat IgG (150KD) the dry blood device determination of recovery rates of BNP：
Take the μ g/mL of 10 μ L 1 BSA (60KD) Goat IgG (150KD) BNP solution be added to 10mL hyclones (four
Ji Qing), above-mentioned solution 0.5mL is taken to drip in following dry blood devices after mixing, it is stand-by after drying.By the second layer in dry blood device point
Sub- mwco membrane is removed, and is put into 2ml centrifuge tubes, and the albumen ELISA kit adsorbed thereon is dissolved with 0.5ml PBS solutions
(laboratory self-control) determines the rate of recovery (rate of recovery=addition protein concentration/dry blood determines concentration * 100% after redissolving) of albumen,
Test result such as following table：
By the analysis of the protein retained on the cellulose ester membrane to the second layer, suitable cellulose ester membrane is chosen
During with ceramic capillary filter membrane, the interception efficiency to target protein is very high, it is contemplated that production cost preferred cellulose ester film.
3rd, water accepting layer absorbent material is preferred
0.5mL hyclones are taken, are dripped in following dry blood devices, the change of weight is recorded with weight method, when weight is constant,
That is drying time.(note：Device third layer from top to down with it is secondary be filter paper, calcium chloride (or silica gel or water-absorbing resin, or calcium chloride,
Any two kinds of mixture in silica gel, water-absorbing resin, mixed proportion is 1：1) result of the test shows, water-absorbing resin and CaCl2 1:
1 mixture drying time is most short, and effect is optimal.
4th, water-absorbing resin is selected
By above-mentioned preferred whatman903# filter membranes, cellulose ester membrane (1KD), water-absorbing resin conduct described in following table is chosen
Water accepting layer.0.5mL hyclones are taken, are dripped in above-mentioned dry blood device, with reference to the above method, drying time, result of the test are recorded
Show, water-absorbing resin particle diameter is optimal in 150-250 mesh effects.
5th, cellulose membrane is surface-treated
By following table institute example solution coating on cellulose ester membrane (1KD) surface, dry rear stand-by.Will be above-mentioned preferred
Whatman903# filter membranes, cellulose ester membrane (1KD), water-absorbing resin (150-250 mesh) and CaCl21：1 mixture, according to Fig. 1 institutes
Show and prepare save set.Take the μ g/mLBNP solution of 10 μ L 1 to be added to 10mL hyclones (Chinese holly), taken after mixing above-mentioned molten
Liquid 0.5mL is dripped in following dry blood devices, it is to be dried after, by dry blood device be placed on 37 DEG C carry out within 0 day, 1 day, 3 days, 6 days it is old
Change experiment, with reference to experiment two, the test b NP rate of recovery.As a result show to be coated with 0.5-2.6mg/cm using surface2Casein
The cellulose ester membrane BNP albumen of (being purchased from sigma, cat#C8654) is relatively stablized, and the rate of recovery is high；Wherein it is coated with 1.3mg/cm2Junket
The cellulose ester membrane BNP albumen of albumen is most stable, rate of recovery highest.
6th, prepared by dried blood spot
By preferred hemofiltration film whatman903#, (surface is coated with 1.3mg/cm to cellulose ester membrane (1KD)2Casein),
Water-absorbing resin (150-250 mesh) and CaCl21：1 mixture prepares dry blood device (see Fig. 4).
0.5mL (diameter d=40mm) or 0.1ml (diameter d=12mm) are dripped in following dry blood devices, treated after drying
With.
Second layer molecule mwco membrane in dry blood device is removed, is put into 2ml centrifuge tubes, is dissolved with isometric PBS solution
The albumen adsorbed thereon.
7th, dried blood spot application effect
BNP assay methods are as follows,
It is coated with the PBS (10mM, pH7.5) of the anti-BNP antibody of 1.0 μ g/ml in microwell plate, 100 μ L/ holes, 2-8 DEG C, 24 is small
When；
Above-mentioned solution is abandoned, PBS (10mM, pH7.5) solution containing 3%BSA, 200 μ L/ holes, 2-8 DEG C, 24 hours is added；
Abandon above-mentioned solution, add determinand solution or calibration object solution (0ng/ml, 0.5ng/ml, 3ng/ml, 10ng/ml,
30ng/ml, 100ng/ml), 100 μ L/ holes, 37 DEG C, 30 minutes；
Above-mentioned solution is abandoned, with the PBS (10mM, pH7.5) containing 0.05%Tween-20,5 times are rinsed, every time 200 μ L/ holes；
Add the anti-BNP antibody (1/10000) of HRP marks, 100 μ L/ holes, 37 DEG C, 15 minutes；
Above-mentioned solution is abandoned, with the PBS (10mM, pH7.5) containing 0.05%Tween-20,5 times are rinsed, every time 200 μ L/ holes；
Add under 100 μ L/ holes TMB nitrite ions (biopanda), room temperature condition and react after 10min, add 50 μ L/ hole 2M
H2SO4。
With ELIASA (thermo) mensuration absorbance.
With calibration object concentration and its absorbance, using LogX-LogY fitting a straight lines, standard curve is set up, by sample to be tested
Absorbance bring this equation into, you can calculate the concentration value of sample to be tested.
Take and dripped to containing finite concentration BNP solution 0.5mL in above-mentioned dry blood device, it is stand-by after drying.Then dry blood is filled
Put middle second layer molecule mwco membrane to remove, be put into 2ml centrifuge tubes, the albumen adsorbed thereon is dissolved with 0.5ml PBS solutions, is used
The above method determines BNP.Replication 0.5ng/ml samples 10 times, calculate repeated CV% (the CV%=marks of its measurement result
Quasi- difference/average value × 100%).
Sample to be tested is divided into two parts, a copy of it prepares dry blood device, it is to be dried after, then redissolved, with another
Sample is determined with above-mentioned kit method together, calculates both correlations, and 40 result of the tests show (Fig. 5), and sample is dry
There is the uniformity of height after blood device before processing.
Claims (9)
1. a kind of blood preseration device, it is characterised in that：Including hemofiltration film (1), molecular weight mwco membrane (2) and water accepting layer (3)；Institute
The lower section for stating hemofiltration film (1) sets the molecular weight mwco membrane (2), and the lower section of the molecular weight mwco membrane (2) sets described inhale
Water layer (3)；
The hemofiltration film (1) is used for washed corpuscles and blood plasma；The material of the hemofiltration film (1) be selected from whatman903 or
Fusion5 or cellulose acetate film；
The molecular weight mwco membrane (2) is used to retain protein or nucleic acid；The material of the molecular weight mwco membrane (2) is selected from fiber
Cellulose ester film or ceramic membrane；
The material of the water accepting layer 3 is selected from silica gel, filter paper, CaCl2Or water-absorbing resin；
The upper surface of the molecular weight mwco membrane (2) scribbles 0.5-2.6mg/cm2Casein.
2. blood preseration device according to claim 1, it is characterised in that：The upper surface of the molecular weight mwco membrane (2)
Scribble 1.3mg/cm2Casein.
3. blood preseration device according to claim 1, it is characterised in that：Also include getting stuck (5) and sealing ring (4), institute
State hemofiltration film (1), molecular weight mwco membrane (2) and water accepting layer (3) and be sealed in by sealing ring (4) and got stuck in (5).
4. blood preseration device according to claim 3, it is characterised in that：Get stuck (5) are by fluid-tight hard material
Material is made, and described get stuck (5) are used to support the hemofiltration film (1), molecular weight mwco membrane (2) and water accepting layer (3).
5. blood preseration device according to claim 3, it is characterised in that：The sealing ring (4) is for ring-type and by impermeable
The hard material of water is made, and the sealing ring (4) is used for the hemofiltration film (1), molecular weight mwco membrane (2), water accepting layer (3) is solid
It is scheduled on and gets stuck in (5).
6. blood preseration device according to claim 1, it is characterised in that：The aperture of the cellulose acetate film is selected from
0.22 μm, 0.45 μm, 0.8 μm or 3 μm.
7. blood preseration device according to claim 1, it is characterised in that：The molecular cut off of the cellulose ester membrane is
20000。
8. the blood preseration device according to claim 1, it is characterised in that：The material of the water accepting layer is mass ratio
For 1:1 water-absorbing resin and CaCl2Mixture.
9. the blood preseration device according to claim 1 or 8, it is characterised in that：The water-absorbing resin particle diameter is
150-250 mesh.
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| CN106959235B (en) | 2019-08-09 |
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