CN1102881A

CN1102881A - Molecular sieve concentration article

Info

Publication number: CN1102881A
Application number: CN 94111357
Authority: CN
Inventors: 景桂英; 陈明朗
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 1994-06-18
Filing date: 1994-06-18
Publication date: 1995-05-24

Abstract

The production process of molecular sieve for concentration use includes gel liquid compounding based on the conventional proportion of polyacrylamine gel, gel deairing and filtering, mixing with TEMED liquid, polymerization by dropping gel liquid into mould, dewatering of gel grains together with the mould in 50-70% dewatering agent for 4-8 hr, dewatering of gel grains in anhydrous dewatering agent for 3-5 hr and tripe repetitions of water absorption and dewatering of gel grains. The dry gel grains thus produced is suitable for concentration of small amount of sample.

Description

Molecular sieve concentration article

The present invention relates to molecular sieve concentration technique field, be exclusively used in sample concentration, remove the ion in the sample, moisture content and small-molecule substance, keep needed protein, nucleic acid and other macromolecular substances, improve sample concentration, be particularly suitable for concentrating of small amount of sample.

Sample Preconcentration Technique is a purposes experimental technique very widely, adopts the method for physics or chemistry to carry out sample concentration usually, freeze-drying is arranged, the precipitation method, dialysis, polyglycol absorption process and silica gel absorption process etc. in the prior art.Though these experimental techniques all have certain concentrated effect, they in use are subjected to some condition restriction again, the use inconvenience that has, and what have can have a negative impact or cause sample loss etc. sample.As freeze-drying is that they need vacuum condition with the protein solution freeze drying, though organic solvent or inorganic salts can make protein precipitation, but it can also cause some sex change of protein; In addition, utilize the principle of osmotic pressure, sample is packed in the bag filter, absorbing moisture with polyglycol dry powder or silica gel, also is the sample concentration method of using always, but it needs the long time, and because the non-specific adsorption effect, sample loss is very big, especially is not suitable for concentrating of small amount of sample; Also have the extensive stock concentrator to come out at present, it is to utilize a tough semi-permeable diaphragm filter of physical property to replace common dialysis membrane, though the use of this commodity concentrator is more convenient, it costs an arm and a leg, and its film is stronger to the non-specific adsorption of albumen.

The objective of the invention is to, at the defective of above-mentioned existing Sample Preconcentration Technique, development provides a kind of easy to use, does not need any specific condition, does not cause protein denaturation, and the time is short, and price is low, effective molecular sieve concentration article.

A kind of molecular sieve concentration article provided by the present invention, its production method comprises:

1, take by weighing acrylamide (Acr) in the conventional ratio of polyacrylamide gel, methylene diacrylamide (Bis), Ammonium Persulfate 98.5 (Ap) adds water to scale, and mixing is mixed with glue, and other measures tetramethylethylenediamine (TEMED) liquid;

2, glue is bled add TEMED liquid again after filtering, mixing is made glue 1;

3, glue 1 is poured in the encapsulating bottle 2, the liquid outlet 3 of encapsulating bottle bottom connects one section emulsion tube 4, and the other end of emulsion tube links to each other with dropper 6 with valve 5;

4, glue is poured in the mould 7, mould must possess the characteristics of strong alkali-acid resistance and alcohol ketone simultaneously;

5, treat the abundant polymerization of glue after, gel is put into the alcohols of 50-70% or ketone dehydrant 4-8 hour together with mould, take out mould, micelle is poured in anhydrous alcohols or the ketone dehydrant soaked 3-5 hour, the taking-up micelle is put into and was dried into dried micelle in 30 ℃ of incubator 3-5 hours;

6, dried micelle is soaked in the water, becomes behind the transparent adhesive tape 5 dewatered dryings set by step again, so triplicate takes out dried micelle, and pack is sealed.

Used alcohols or ketone dehydrant in the production method, for well, the material of mould is the best with plastics or glass with methyl alcohol, ethanol, propyl alcohol or acetone, butanone.

Molecular sieve concentration article provided by the present invention is compared with existing Sample Preconcentration Technique has remarkable advantage and good effect, it has utilized polyacrylamide gel to have the characteristic of regulatable three dimensional network pore structure and the principle of molecular sieving effect, during use dried micelle is dropped in the sample solution, just moisture content and the little molecule of Bi Qi mesh diameter in can absorbent solution, keep bigger protein, nucleic acid and other macromolecular substances of molecular weight, thereby reach the purpose of concentrating sample.Because dried micelle mesh size can be controlled by the concentration and the degree of crosslinking that change glue, can be during use according to the requirement of solute molecule size, select the dried micelle of suitable model to get final product, therefore, the concentrated dried glue of molecular sieve has easy to use, and suction is strong, and concentrated effect is remarkable, advantages such as production technology is simple, and is with low cost.Because dried micelle can be made Any shape (Fig. 3, Fig. 4), as a post particle shape, the tubular c taper of b etc., especially taper and sample contact area are little, can not lose sample, are fit to small amount of sample and concentrate.In addition, can customize mould, produce the molecular sieve concentration article of different size, model, shape in batches.

Fig. 1, molecular sieve concentration article technological process of production figure

Fig. 2, encapsulating bottle 2 structural representations

Fig. 3, dried micelle photo in kind

Fig. 4, mould 7 structures, shape synoptic diagram

Introduce embodiments of the invention in detail below in conjunction with accompanying drawing.

1, glue liquid: take by weighing 20 gram Acr, 0.5 gram Bis, 0.17 gram AP adds water to 100ml, mixing prepare the standard glue (formula rate see reference document P.109 show 3-9) of 100m120%, other measures TEMED liquid 10 μ l; With the standard glue with suction filtration device (laboratory is general) bleed filter after, add TEMED liquid, mixing is made into glue 1.

2, encapsulating: pour glue 1 into encapsulating bottle 2, the liquid outlet 3 of encapsulating bottle bottom links to each other with one section emulsion tube 4, and emulsion tube links to each other with dropper 6 with valve 5; Glue in the encapsulating bottle is splashed in the mould 7, and mould 7 is made by the material of plastics or glass or other strong alkali-acid resistances and alcohol ketone, and the micelle model can be made particle shape in the mould, cylindricality or taper (see figure 3) and other Any shape;

3, rough dried micelle: after the abundant polymerization of the glue in the mould to be formed (about 0.5-1 hour), put into 50-70% methyl alcohol, ethanol, propyl alcohol or acetone, butanone or other alcohols ketones dewatering agent 4-8 hour together with mould after, take out mould, micelle wherein is syneresis, micelle poured in anhydrous alcohols or the ketone dehydrant soaked 3-5 hour, at this moment micelle all becomes milky, again micelle is taken out and place oven dry in 3-5 hour in 30 ℃ of incubators, make the thick finished product of dried micelle.

4, refined dry micelle; Rough dried micelle is soaked in the water, allows it inhale permeable part, become transparence, dewater by rough dried micelle program 3 again, three times so repeatedly, can make the refined dry micelle, pack is sealed, and is used for pilot production.

5, concentration test: making concentration by above-mentioned prescription is 20% dried micelle, can be used for molecular weight for greater than 10 ⁴Protein or the concentrating of nucleic acid samples, concentrating of different molecular weight sample can be used concentration and the degree of crosslinking that changes glue, repeats the prepared dried glue of above-mentioned production run.The inventor is used for the sample concentration of bovine serum albumin(BSA) with the dried glue of 20% concentration, and the result is as follows:

1) dried glue water absorbing force is measured: water absorbing force=(dry weight before suction back weight in wet base-suction)/(dry weight before the suction) * 100%

Weight change before and after the dried glue suction of table 1 molecular sieve

Project	Dry weight before the suction	Suction back weight in wet base	Water absorbing capacity	Water absorbing force %
					X±S n	106.15± 5.709 40	499.25± 26.98 40	393.1± 23.85 40	370 －

Experimental result table 1 shows that the water-intake capacity that molecular sieve concentrates dried glue is very strong, and milky dried micelle immerses very fast imbibition in the water, becomes transparent adhesive tape.As calculated, 1 gram dry weight can absorb 3.7 gram moisture content, and water absorbing force is 370%.Through correlation analysis, dry weight and the related coefficient between the water absorbing capacity that molecular sieve concentrates glue are r=0.46, and statistical test, related coefficient be (P＜0.01) extremely significantly.Regression equation between dry weight and the water absorbing capacity is: =186.2+1.94x, promptly every 100g dry weight can absorb about 380g water.

2) concentrated effect: get 5 test tubes, add respectively equivalent protein sample solution and etc. the molecular sieve of weight concentrate dried glue, after concentrating beginning 15 ', 30 ', 60 ', 80 ' and 240 ', take out dried glue successively, with the charged arteries and veins in district, dye through Coomassie brilliant blue R-250, thin layer scanning draws the thin layer scanning figure that charged arteries and veins is distinguished in bovine serum albumin(BSA) (BSA) sample concentration front and back, and the pairing area in different concentration time samples scanning peak sees Table 2 among the figure.

The area of table 2 BSA scanning peak correspondence

Concentration time

Project stoste contrast 15min 30min 60min 80min 240min

The BSA peak

Area 2603.4 2940.6 3450.8 4462.9 5538.7 6532.1

Ratio % 77.8 84.7 85.0 86.8 83.4 80.7

The result finds out that along with the increase of concentration time, the pairing area in BSA sample scanning peak also strengthens gradually, and this interpret sample concentration has improved, and it is to improve gradually along with the prolongation of concentration time.Therefore, suitable concentration time or suitable dried glue weight are selected in the purpose and the requirement that can concentrate per sample in use.

3) mensuration of Acr level of residual monomers

The Acr monomer is a kind of neural poison, and it can absorb and operator's health is had a negative impact through skin.Yet molecular sieve provided by the present invention concentrates dried glue and dewaters through repeated multiple times, and the Acr level of residual monomers has been reduced to very low in the three-dimensional mesh of its inside.Adopt ultraviolet light absorption method to survey the Acr content of monomer at 262nm place, before the dehydration in the bright glue Acr content of monomer be 3.1mg/ml, and after the dehydration in the dried glue Acr level of residual monomers only be about 0.2mg/ml, be 1/15 of bright glue.Low like this content of monomer can not have adverse effect to the operator, can not produce sample yet and pollute, and also can increase the further content of monomer that reduces in the dried glue of dehydration number of times as requested.

Claims

1, molecular sieve concentration article, its production method comprises:

1.1 take by weighing acrylamide (Acr) in the conventional ratio of polyacrylamide gel, methylene diacrylamide (Bis), Ammonium Persulfate 98.5 (Ap) adds water to scale, and mixing is mixed with glue night, and other measures tetramethylethylenediamine (TEMED) liquid;

Add TEMED liquid 1.2 glue bled again after filtering, mixing is made glue 1;

1.3 glue 1 is poured in the encapsulating bottle 2, and the liquid outlet 3 of encapsulating bottle bottom connects one section emulsion tube 4, the other end of emulsion tube links to each other with dropper 6 with valve 5;

1.4 glue is poured in the mould 7, and mould must possess the characteristics of strong alkali-acid resistance and alcohol ketone simultaneously;

1.5 after treating the abundant polymerization of glue, gel is put into the alcohols of 50-70% or ketone dehydrant 4-8 hour together with mould, take out mould, micelle poured in anhydrous alcohols or the ketone dehydrant soaked 3-5 hour, take out micelle and put into and be dried into dried micelle in 30 ℃ of incubator 3-5 hours;

1.6 dried micelle is soaked in the water, become behind the transparent adhesive tape again 1.5 dewatered dryings set by step, so triplicate takes out dried micelle, and pack is sealed.

2, molecular sieve concentration article according to claim 1 is characterized in that, production stage 1.5 used dewatering agents are methyl alcohol, ethanol, propyl alcohol or acetone, butanone.

3, molecular sieve concentration article according to claim 1 and 2 is characterized in that, production stage 1.4 used mould materials are plastics or glass.