CN103289929B - The fast separating process of bacillus cereus - Google Patents
The fast separating process of bacillus cereus Download PDFInfo
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- CN103289929B CN103289929B CN201310219397.9A CN201310219397A CN103289929B CN 103289929 B CN103289929 B CN 103289929B CN 201310219397 A CN201310219397 A CN 201310219397A CN 103289929 B CN103289929 B CN 103289929B
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Abstract
The invention discloses the sharp separation novel method of bacillus cereus (Bacillus cereus), better, bacterium carries out follow-up study provides basis, relates to biological technical field.Method comprises multi-arm well star polymkeric substance and the multi-arm well star polymkeric substance of antibody covalent coupling, antibody modification wrap the object bacterium caught by the multi-arm well star polymkeric substance that long-chain biological element molecule, antibody are co-modified with long-chain biological element in sample liquid again, Streptavidin is modified nanometer magnetic bead identification and the long-chain biological elementization multi-arm well star polymkeric substance in coupling sample liquid, being separated and the step such as the resuspended of bacterium that is captured; Re-suspension liquid directly can carry out subsequent analysis, and compared with traditional bacterial magnetic separation method, the method is more suitable for carries out Magneto separate to bacterium in complex matrices, improves object bacterium separation efficiency in sample.
Description
Technical field
The present invention relates to biological technical field, specifically relate to the food-borne pathogens separation method based on nanometer magnetic bead.
Background technology
Food-borne pathogenic fungi pollution is one of significant problem of China's food safety.According to WHO statistics, developed country about has the people of 1/3rd to infect food origin disease every year, and the whole world has 2,200,000 people to die because suffering from food origin disease every year.In China, annual food poisoning number of cases is 20 ~ 400,000 people, and except mishap, major part causes by food-borne pathogens.Bacillus cereus (Bacillus cereus) is as one of common food-borne pathogens, main pollution meat, milk, vegetables, fish, rice and wheaten food etc., regarded as by Norway and Holland the sex pheromone the most easily detected in food at present, the poisoning caused by it happens occasionally, the health of harm people.Its main toxicity symptom caused shows as vomiting, diarrhoea, similar with some other microbial symptom of causing a disease, thus is usually left in the basket.Therefore, the technology pole of bacillus cereus is necessary fast, in efficiently concentrating sample separation in development.
Immunity magnetic separation technique is one of important component part of food-borne pathogens rapid screening technology, and this technology can efficient capture, object bacteria in concentrated enrichment liquid, improves pathogenic microbes detect sensitivity.In recent years, object bacteria antibody is connected on magnetic bead by the immunomagnetic separation (IMS) based on magnetic micro-beads, then the magnetic bead being connected with antibody is dropped in sample liquid object bacteria is caught, enrichment, Magneto separate (concrete principle is shown in Fig. 2 A).But, many limitation should be there is based on the isolation technique of micron order immunomagnetic beads at present: 1) specific surface area of micron magnetic bead is relatively little, reduces magnetic capture efficiency; 2) due to the particle properties of micron magnetic bead self, combined by heterogeneous reaction (multiphase reaction) between itself and bacterial cell, usually need the time more grown to go specificity to catch bacterial cell in food substrate; 3) micron magnetic bead monodispersity is poor, self assemble easily occurs in food substrate liquid or forms precipitation; 4) traditional immune magnetic separation technique, often antibody molecule is directly coupled on immunomagnetic beads, this process usually can cause the activity of antibody to reduce widely and cause the direction in space of antibody to change the space steric effect increased between antibody, thus reduce the capture rate 5 of antibody) food substrate character is complicated and wherein the miscellaneous bacteria concentration of non-object pathogenic bacterium is large, micron magnetic bead easily produces non-specific adsorption, is difficult to realize the specific isolation to object bacterium in food sample liquid; 6) excessive concentration of micron magnetic bead can cause the breakage of bacterial cell (magnetic field causes cell surface magnetic bead to be attracted each other, and cell is squeezed and even breaks), causes the failure be separated; 7), during magnetic bead coupled antibody, activated for tool antibody is connected in magnetic bead surfaces by general hydrophobic adsorbent or the chemical coupling mode of adopting.Too closely, the hydrophobic or strong hydrophilicity group of magnetic bead nature and remained on surface thereof easily causes antibody space conformation to change, and causes antibody bioactive to decline for antibody and magnetic bead surfaces distance.
Summary of the invention
For the defect of prior art, the object of this invention is to provide a kind of easy, capture rate is high, disengaging time is short, (be less than 30T/m) under low gradient magnetic can from the food substrate of complexity fast, the method for specific isolation bacillus cereus.
The sharp separation novel method of bacillus cereus, comprise the following steps: (1) gets 2.8mg multi-arm well star polymer dissolution in 2mL 0.02M, pH 6.5 phosphoric acid buffer PBS, add 0.6mg N-hydroxysuccinimide NHSS, 0.4mg ethyl 3-(3-dimethylamino) carbodiimide hydrochloride EDC, room temperature is placed on blending instrument and stirs, activation 15min; Getting 6mg bacillus cereus specific antibody adds in above-mentioned reaction solution, and room temperature is placed on blending instrument and stirs 30min; Decompression is spin-dried for solvent, deionized water dissolving, and dialyse 1d in PBS and deionized water; Dialysis terminates the solution lyophilize obtained to obtain multi-arm well star polymer-antibody complex; (2) get 15mg long-chain biological element, 3.6mg NHSS, 2.4mg EDC is dissolved in 2mL PBS (0.02M, pH 6.5) damping fluid; Add 0.55mg multi-arm well star polymer-antibody complex, room temperature is placed on blending instrument and stirs 30min; Decompression is spin-dried for solvent, deionized water dissolving, and dialyse 1d in PBS and deionized water; Dialysis terminates the solution lyophilize obtained to be obtained long-chain biological element-multi-arm well star polymer-antibody complex; (3) 1mL testing sample solution is got, add 0.1mg bacillus cereus antibody and long-chain biological element co-modified multi-arm well star polymkeric substance and step (2) long-chain biological element-multi-arm well star polymer-antibody complex, be placed on blending instrument, with the rotating speed incubated at room 15min of 30rpm; Add the nanometer magnetic bead that 0.1mg is modified with Streptavidin, be placed on blending instrument, with the rotating speed of 30rpm incubated at room 15min again, conventional magnetic frame is separated 3min; (4), after magneticseparation, after 0.1%PBST washing, the nanometer magnetic bead-Streptavidin-long-chain biological element-multi-arm well star polymer-antibody-bacillus cereus antigenic compound of catching bacillus cereus is namely obtained with PBS damping fluid is resuspended.
Described multi-arm well star polymkeric substance is multi-arm well star polyamide-amide, and its molecular weight is 70000Da.Structure is as Fig. 1.
The described nanometer magnetic bead particle diameter having modified Streptavidin is 20-50nm, is preferably 30nm.
Multi-arm well star polymkeric substance realizes the covalent coupling of multi-arm well star polymkeric substance and antibody by carboxyl that is amino and bacillus cereus specific antibody.
Multi-arm well star polymkeric substance, by carboxyl that is amino and long-chain biological element molecule, realizes the covalent coupling of multi-arm well star polymkeric substance and long-chain biological element; Add excessive long-chain biological element to ensure amino sites exposed on closed multi-arm well star polymkeric substance.
Concrete principle is shown in Fig. 2 B.
Present method is specially adapted to the separation of complex sample, as food samples, whole blood sample etc.Food samples comprises the food material after all kinds of fresh or freeze cutting, as products such as fresh vegetables, meat, seafood and milks.Sample preparation is treatment process conveniently.
Technical solution of the present invention is adopted to have following beneficial effect:
1, the present invention by means of the Cascaded amplification effect of multi-arm well star polymkeric substance, magnetic bacterium signal exponentially level is expanded, the separation of magnetic bacterium just can be realized under lower magneticstrength, and within the identical time, compared with routine immunization Beads enrichment method, be separated to object bacterium ability stronger, be specially adapted to the separation of complex sample, as food samples, whole blood sample etc.For the defect that object bacterium speed is slow, magnetic field requirements is high in the simple 20-50nm immuno magnetic cell separation complex matrices sample adopted after antibody modification, multi-arm well star polymkeric substance is adopted to realize the amplification of nanometer magnetic bead magnetic signal, thus improve object bacterium separation efficiency in complex matrices sample, achieve object bacterium specificity sharp separation in the food substrate that (to be less than 30T/m) under low gradient magnetic complicated.
2, this programme is for be coupled on multi-arm well star polymkeric substance by antibody molecule, avoid in ordinary method antibody molecule is coupled to magnetic bead surfaces cause antibody activity reduce and sterically hindered large shortcoming.
3, the present invention adopts multi-arm well star polymkeric substance, reaction soln can be made more stable, not easily precipitate, add the chance that antibody molecule contacts with object bacteria, is conducive to improving capture rate; Simultaneously, multi-arm well star polymkeric substance is connected with a large amount of long-chain biological element molecules, the nanometer magnetic bead can modified in conjunction with Streptavidin, thus makes on multi-arm well star polymkeric substance in conjunction with a large amount of nanometer magnetic beads, achieve the Cascaded amplification of magnetic bacterium signal, be conducive to the disengaging time shortening magnetic bacterium.
4, after replacing micron order magnetic particle with nanometer magnetic bead (20-50nm), because nanometer magnetic bead particle diameter is little, specific surface area is large, the steric hindrance be combined with bacterial surface antigen is little, the covering efficiency of bacterium surface magnetic bead significantly improves, and the bacterium of magnetic nano particle subcovering can keep normal shape, nanometer magnetic bead also has dispersed and stability preferably in complex matrices, and therefore the use of nanometer magnetic bead can overcome above-mentioned all defects owing to using micron magnetic bead to cause.
5, the present invention is in sepn process, introduce multi-arm well star high-polymer molecular, multi-arm well star polymkeric substance is connected with a large amount of long-chain biological element molecules, can special and high-affinity ground be dispersed in coupling in matrix solution and have the identification of Streptavidin nanometer magnetic bead, thus make on multi-arm well star polymkeric substance in conjunction with a large amount of nanometer magnetic beads, considerably increase the magnetic bead quantity of target bacteria surface bonding, achieve the target bacteria that sharp separation is caught under magnetic field.Compared with traditional bacterial magnetic separation method, be nanometer magnetic bead more stable in matrix because of what add, the method is more suitable for carries out Magneto separate to bacterium in complex matrices, improves object bacterium separation efficiency in complex matrices sample.
6, during magnetic bead coupled antibody, activated for tool antibody is connected in magnetic bead surfaces by general hydrophobic adsorbent or the chemical coupling mode of adopting.Too closely, the hydrophobic or strong hydrophilicity group of magnetic bead nature and remained on surface thereof easily causes antibody space conformation to change, and causes antibody bioactive to decline for antibody and magnetic bead surfaces distance.But this experimental program introduces multi-arm well star polymkeric substance in coupling process, it has certain space size, thus makes antibody molecule away from magnetic bead and magnetic bead surfaces, avoids the disadvantageous effect of magnetic bead nature and surperficial antagonist molecule.Meanwhile, the multi-arm well star polymkeric substance of introducing but can not affect antibody space conformation, thus serves the bioactive effect of protection antibody molecule.
Accompanying drawing explanation
The structural representation of Fig. 1 amidized multi-arm well star polymkeric substance;
The operational flowchart of the conventional magnetic separation technique (A) of Fig. 2 and magnetic separation technique involved in the present invention (B);
Embodiment
In order to make the present invention clearly understand, below in conjunction with embodiment, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
Long-chain biological element is for buying in the carboxylated long-chain biological element of Thermo Fisher Scientific company of the U.S. (EZ-Link Sulfo-NHS-LC-Biotin, molecular weight 556.59).
The nanometer magnetic bead (30nm) being modified with Streptavidin is bought in Ocean NanoTech company of the U.S..
Amidized multi-arm well star polymkeric substance is multi-arm well star polyamide-amide, and its molecular weight is 70000Da, purchased from Weihai Chen Yuan new chemical materials company limited.
Conventional magnetic frame is separated magneticstrength and is less than 30T/m.
N-hydroxysuccinimide NHSS, ethyl 3-(3-dimethylamino) carbodiimide hydrochloride EDC etc. is conventional reagent, repeats no more.
0.1%PBST compound method: 8.0g NaCl, 0.2g KCl, 0.24g KH
2pO
4, 1.44g Na
2hPO
4be dissolved in 800mL distilled water, adjust pH to 7.4 with 5M NaOH, then be settled to 1000mL and namely obtain 0.01M PBS.Add Tween 20 with the volume ratio of 1/1000 (V/V) again, namely obtain 0.1%PBST.
Embodiment 1
1. multi-arm well star polymer-antibody complex, prepare in accordance with the following steps:
(1) get 2.8mg multi-arm well star polymkeric substance multi-arm well star polyamide-amide and be dissolved in 2mL PBS (0.02M, pH 6.5), add 0.6mg NHSS, 0.4mg EDC, room temperature is placed on blending instrument and stirs, activation 15min;
(2) getting 6mg bacillus cereus specific antibody adds in above-mentioned reaction solution, and room temperature is placed on blending instrument and stirs 30min;
(3) above-mentioned solution decompression is spin-dried for solvent, deionized water dissolving, dialyse 1d in PBS and deionized water; Dialysis terminates the solution lyophilize that will obtain.
2. long-chain biological element-multi-arm well star polymer-antibody complex is prepared in accordance with the following steps:
(1) get 15mg long-chain biological element, 3.6mg NHSS, 2.4mg EDC is dissolved in 2mL PBS (0.02M, pH 6.5) damping fluid;
(2) join in above-mentioned solution by 0.55mg multi-arm well star polymer-antibody complex, room temperature is placed on blending instrument and stirs 30min;
(3) above-mentioned solution decompression is spin-dried for solvent, deionized water dissolving, dialyse 1d in PBS and deionized water; Dialysis terminates the solution lyophilize that will obtain.
3. enrichment is caught: get testing sample solution 1mL, add 0.1mg long-chain biological element-multi-arm well star polymer-antibody complex, be placed on blending instrument, form long-chain biological element-multi-arm well star polymer-antibody-bacillus cereus antigenic compound with the rotating speed incubated at room 15min of 30rpm; Add the nanometer magnetic bead that 0.1mg is modified with Streptavidin, be placed on blending instrument, with the rotating speed of 30rpm incubated at room 15min again, centrifuge tube is inserted conventional magnetic frame and be separated 3min;
4., after deionized water cleans gently, mix with PBS damping fluid and resuspendedly namely obtain the mixture nanometer magnetic bead-Streptavidin-vitamin H-multi-arm well star polymer-antibody-bacillus cereus antigen being enriched with bacillus cereus.
Embodiment 2 concentration effect is tested
(1) getting 1mL concentration is 10
4the bacillus cereus of cfu/mL is in 1.5mL sterile centrifugation tube, and the centrifugal 5min of 12000rpm, abandons supernatant, resuspended by the aseptic PBS solution of equal-volume.
(2) enrichment is caught: arrange technical solution of the present invention group (bacillus cereus antibody and the plain co-modified multi-arm well star polymkeric substance group of long-chain biological), the nanometer magnetic bead group of bacillus cereus specific antibody modification, the micron magnetic bead group enrichment object bacterium of bacillus cereus specific antibody modification respectively.
(3), after Magneto separate, supernatant liquor is poured in sterile centrifugation tube, separate catch bacillus cereus immunomagnetic beads then with PBST cleaning twice, to mix, and with the resuspended immunomagnetic beads mixture of the aseptic PBS solution of 1mL.
(4) capture rate calculates: after the object bacterium re-suspension liquid of each group of enrichment is carried out gradient dilution, count each gradient with flat board, and by the capture rate of capture rate formulae discovery object bacteria, each experiment in triplicate.The calculation formula of each group of capture rate is as follows: (total number of bacterial colony of being adsorbed by enrichment/all total plate count) × 100%.
The scheme that object bacterium is caught in described each group of enrichment is as follows:
A. technical solution of the present invention group (bacillus cereus antibody and the plain co-modified multi-arm well star polymkeric substance group of long-chain biological) enrichment catches object bacterium scheme as embodiment 1, specific as follows:
0.1mg bacillus cereus antibody and the co-modified multi-arm well star polymkeric substance of vitamin H and vitamin H-multi-arm well star polymer-antibody complex are joined containing in object bacteria centrifuge tube, is placed on blending instrument, with the rotating speed incubated at room 15min of 30rpm.Then add the nanometer magnetic bead that 0.1mg is modified with Streptavidin, be placed on blending instrument, with the rotating speed of 30rpm incubated at room 15min again.Finally, centrifuge tube is inserted conventional magnetic frame and be separated 3min.
B. it is specific as follows that object bacterium scheme is caught in the nanometer magnetic bead group enrichment that bacillus cereus specific antibody is modified:
The nanometer magnetic bead that the bacillus cereus specific antibody prepared by 0.1mg is modified joins containing in object bacteria centrifuge tube, is placed on blending instrument, with the rotating speed incubated at room 15min of 30rpm.Finally, centrifuge tube is inserted conventional magnetic frame and be separated 3min.
The nanometer magnetic bead preparation that described bacillus cereus specific antibody is modified: (1) gets 10mg nanometer magnetic bead (30nm, there is no coupling Streptavidin) use dehydrated alcohol successively, 1M NaOH, 1M HCl respectively washs once, PBS (0.02M, pH 4.0) wash three times, aseptic PBS is resuspended.Add NHSS 0.4mg, EDC 0.35mg, be placed on blending instrument and keep magnetic bead to suspend, 37 DEG C of activation 2h.(2) magnetic frame reclaims magnetic bead, and after PBS (0.02M, pH 4.0) washs three times, magnetic bead is resuspended in aseptic PBS, adds 80 μ g bacillus cereus specific antibodies, be placed in 37 DEG C of coupling 2h on blending instrument by every mg magnetic bead.(3) add thanomin room temperature and close 2h.Magnet stand reclaims magnetic bead, and PBS washs three times, and 10ml PBS is (containing 0.05%NaN
3, 0.5%BSA, pH 7.4) and resuspended immunomagnetic beads for subsequent use in 4 DEG C of Refrigerator stores.
C. it is specific as follows that object bacterium scheme is caught in the micron magnetic bead group enrichment that bacillus cereus specific antibody is modified:
The micron magnetic bead that the bacillus cereus specific antibody prepared by 0.1mg is modified joins containing in object bacteria centrifuge tube, is placed on blending instrument, with the rotating speed incubated at room 15min of 30rpm.Finally, centrifuge tube is inserted conventional magnetic frame and be separated 3min.
The micron magnetic bead preparation that described bacillus cereus specific antibody is modified: (1) gets 10mg micron magnetic bead (1150nm, there is no coupling Streptavidin) use dehydrated alcohol successively, 1M NaOH, 1M HCl respectively washs once, PBS (0.02M, pH 4.0) wash three times, aseptic PBS is resuspended.Add NHSS 0.4mg, EDC 0.35mg, be placed on blending instrument and keep magnetic bead to suspend, 37 DEG C of activation 2h.(2) magnetic frame reclaims magnetic bead, and after PBS (0.02M, pH 4.0) washs three times, magnetic bead is resuspended in aseptic PBS, adds 80 μ g bacillus cereus specific antibodies, be placed in 37 DEG C of coupling 2h on blending instrument by every mg magnetic bead.(3) add thanomin room temperature and close 2h.Magnet stand reclaims magnetic bead, and PBS washs three times, and 10ml PBS is (containing 0.05%NaN
3, 0.5%BSA, pH 7.4) and resuspended immunomagnetic beads for subsequent use in 4 DEG C of Refrigerator stores.
Each group of capture rate is as follows:
Experimental result shows, the capture rate of the micron magnetic bead group that bacillus cereus specific antibody is modified is apparently higher than the capture rate of nanometer magnetic bead group, this illustrates contrast nanometer magnetic bead group, because micron magnetic bead volume is large, magnetic strong, and the object bacteria that just energy separation and concentration is more at short notice.But, the capture rate of technical solution of the present invention group is far longer than again the micron magnetic bead group of bacillus cereus specific antibody modification, this shows that technical solution of the present invention can increase object bacteria nano surface magnetic bead fraction of coverage by multi-arm well star polymkeric substance, thus magnetic is improved greatly, and then achieve (3min) high efficiency separation enrichment bacillus cereus at short notice.
Experiment is caught in embodiment 3 enrichment
Conventional magnetic frame disengaging time is 30min, and all the other are with embodiment 2.
Each group of capture rate is as follows:
Experimental result shows, 3min is separated in comparative example 2, when reaching 30min when disengaged, the capture rate of three groups is obtained for raising, particularly the capture rate of the nanometer magnetic bead group of bacillus cereus specific antibody modification improves the most obvious, this shows to improve the capture rate of nanometer magnetic bead group widely by time expand, but the capture rate of bacillus cereus antibody and long-chain biological element co-modified multi-arm well star polymkeric substance group when it is still separated (3min) lower than the short period of time.This shows that technical solution of the present invention can (3min) high efficiency separation enrichment bacillus cereus at short notice.
Embodiment 4
Aseptic meat is pulverized, makes testing sample solution in the usual way, add bacillus cereus and regulate bacterium colony concentration to 10
4cfu/mL is for subsequent use.
The bacillus cereus antibody prepared and the co-modified dendrimer (0.1mg) of long-chain biological element are joined in sample solution respectively, is placed on blending instrument, with the rotating speed incubated at room 15min of 30rpm.Then add the nanometer magnetic bead (0.1mg) being modified with Streptavidin, be placed on blending instrument, with the rotating speed of 30rpm incubated at room 15min again.Finally, conventional magnetic frame is separated 3min.After Magneto separate, supernatant liquor is poured in sterile centrifugation tube, separate catch bacillus cereus immunomagnetic beads then with PBST cleaning twice, to mix, and with the resuspended immunomagnetic beads of the aseptic PBS solution of 1mL.Capture rate such as embodiment 2 method obtains, and all the other are with embodiment 2.The results are shown in Table 1, show the bacillus cereus in this programme energy efficiently concentrating sample separation.
Embodiment 5
Germ-free milk is sample testing sample solution, adds bacillus cereus and regulates bacterium colony concentration to 10
4cfu/mL.All the other are with embodiment 4.
Embodiment 6
Aseptic grates vegetables, makes testing sample solution in the usual way, adds bacillus cereus and regulates bacterium colony concentration to 10
4cfu/mL.All the other are with embodiment 4.
Embodiment 7
Testing sample is aseptic whole blood, adds bacillus cereus and regulates bacterium colony concentration to 10
4cfu/mL.All the other are with implementing 4.
The comparison of bacillus cereus separating effect in the different actual sample of table 1
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (2)
1. the fast separating process of bacillus cereus, it is characterized in that comprising the following steps: (1) gets 2.8mg multi-arm well star polymer dissolution in 2mL 0.02M, pH 6.5 phosphoric acid buffer PBS, add 0.6mg N-hydroxysuccinimide, 0.4mg EDC, room temperature is placed on blending instrument and stirs, activation 15min; Getting 6mg bacillus cereus specific antibody adds in above-mentioned reaction solution, and room temperature is placed on blending instrument and stirs 30min; Decompression is spin-dried for solvent, deionized water dissolving, and dialyse 1d in PBS and deionized water; Dialysis terminates the solution lyophilize obtained to obtain multi-arm well star polymer-antibody complex; (2) get 15mg long-chain biological element, 3.6mg N-hydroxysuccinimide, 2.4mg EDC is dissolved in 2mL 0.02mol/L, in pH 6.5PBS damping fluid; Add 0.55mg multi-arm well star polymer-antibody complex, room temperature is placed on blending instrument and stirs 30min; Decompression is spin-dried for solvent, deionized water dissolving, and dialyse 1d in PBS and deionized water; Dialysis terminates the solution lyophilize obtained to be obtained long-chain biological element-multi-arm well star polymer-antibody complex; (3) get 1mL testing sample solution, add the long-chain biological element-multi-arm well star polymer-antibody complex obtained in 0.1mg step (2), be placed on blending instrument, with the rotating speed incubated at room 15min of 30rpm; Add the nanometer magnetic bead that 0.1mg is modified with Streptavidin, be placed on blending instrument, with the rotating speed of 30rpm incubated at room 15min again, conventional magnetic frame is separated 3min; The described nanometer magnetic bead particle diameter being modified with Streptavidin is 20-50nm; (4), after magneticseparation, after 0.1%PBST washing, the nanometer magnetic bead-Streptavidin-long-chain biological element-multi-arm well star polymer-antibody-bacillus cereus antigenic compound of catching bacillus cereus is namely obtained with PBS damping fluid is resuspended;
Described multi-arm well star polymkeric substance is multi-arm well star polyamide-amide, and its molecular weight is 70000Da.
2. method according to claim 1, is characterized in that the described nanometer magnetic bead particle diameter having modified Streptavidin is 30nm.
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