TWI356428B

TWI356428B -

Info

Publication number: TWI356428B
Application number: TW96116233A
Authority: TW
Priority date: 2007-05-08
Filing date: 2007-05-08
Publication date: 2012-01-11
Also published as: TW200845055A

Description

1356428 九、發明說明：【發明所屬之技術領域】本發明係提供一種超順磁性粒子之製造方法，以及利用該方法所製造的超順磁性粒子。前述超順磁子可應用於化學或生化物質的純化與反應，並可應大規模反應與自動化磁珠操作平台。【先前技術】1356428 IX. Description of the Invention: [Technical Field of the Invention] The present invention provides a method for producing superparamagnetic particles, and superparamagnetic particles produced by the method. The aforementioned superparamagnetics can be applied to the purification and reaction of chemical or biochemical substances, and can be applied to large-scale reactions and automated magnetic bead operating platforms. [Prior Art]

具有超順磁性之磁性粒子（superparamagnetic particles)’在外加磁場的條件下，會受到磁場的誘導而產生磁性，t集到磁％的]Sf極或s極；將磁場移離後，其磁性瞬間消失，回復到不具有磁性的狀態，重新分散。由於氡化鐵粒子具有生物相容性、且無細胞毒性，所以是所有磁性粒子中最被廣為使用的材料。在具有超順磁性之磁性粒子的早期應用與研丨中，其粒役等級是以微米為主，且其應用是以體外應) 為主，在分離純化領域的應用尤其如此，僅有少數内應，，究其原因，在於微米粒子的大小相對於細微| 織仍是較大的，故無法有效地進入細微組織區域。近] 來，/由於奈米粒子的開發，粒徑大幅縮小，而得以進, 早期微米粒子無法進人的區域，因此開始大量開發與; J+ii!範圍已經可達微血管區域等’並廣泛二在生物醤予領域上，例如磁共振造影、溫熱治療等.丨 ^ 之奈米粒子也具有可控制化載體咖性，故▼動了整個生醫產業自動化的新里程。、常用於合成具有超順磁性之氧化鐵粒子的: 式’疋簡單的共沈澱法（ co_precipitation )。共外資、本 _ 在同時具有二價鐵離子與三價鐵離子的溶液’將酸鹼值調整到9至14之間，如此即可得到四」 5 1356428Superparamagnetic particles with superparamagnetic particles will be magnetically induced under the condition of external magnetic field, and will be magnetized to the Sf pole or s pole of magnetic %. After the magnetic field is removed, its magnetic moment Disappeared, returned to a state that is not magnetic, and redispersed. Since iron telluride particles are biocompatible and non-cytotoxic, they are the most widely used materials among all magnetic particles. In the early application and research of magnetic particles with superparamagnetism, the granulation grade is mainly micron, and its application is mainly in vitro, especially in the field of separation and purification, only a few The reason for this is that the size of the microparticles is still large relative to the fineness, so that it cannot be efficiently entered into the fine tissue region. Recently, due to the development of nano-particles, the particle size has been greatly reduced, and it has been able to enter, and early micro-particles can not enter the human area, so a large number of developments have begun; J+ii! has reached the microvascular region, etc. Second, in the field of biomedication, such as magnetic resonance imaging, thermotherapy, etc. 丨 ^ Nano particles also have controllable carrier coffee, so the movement of the entire biomedical industry automation miles. It is commonly used to synthesize iron oxide particles with superparamagnetism: a simple coprecipitation method (co_precipitation). A total of foreign capital, this _ in the solution of both ferric ions and ferric ions at the same time ' adjust the pH value between 9 and 14, so that you can get four" 5 1356428

化三鐵的沈殿物。但此傳統方式需將三價與二價鐵離子的莫耳比例控制在Fe3+ : Fe2+ = 2 : 1，且需在無氧環境下進行，否則將影響氧化鐵粒子的結晶型態’而且氧化鐵粒子會在製備過程中持續氧化，進而影響到氧化鐵粒子的飽和磁化1 ( saturation magnetization)大小。目前的解決方式是在氮氣下進行反應、或改在有機相中反應，以便減緩氧化；但二價鐵離子易氧化成三價鐵離子，需新鮮配置，且不易精準控制反應中的莫耳比例，這都會增加大量合成的困難度。此外，四氧化三鐵粒子中的鐵離子並非全數處於最高的氧化態，所以這些鐵離 ^會在空氣中慢慢氧化成不具順磁性或磁性較弱的三 ^化二鐵。一般來說，會在四氧化三鐵粒子表面包覆高分子聚合物或二氧化矽的無機氧化物，但這類粒子常會出現無法長時間耐高溫與耐酸性差的缺點，使其應用受到限岳ΐί。 ~1^±先前技術的缺點，本發明利用尿素特殊的高性’在單純只需二價鐵離子存在的情況下， S有ί飽和磁化量的超順磁性氧化鐵粒子，並在表The three temples of the three irons. However, this traditional method needs to control the molar ratio of trivalent and divalent iron ions to Fe3+ : Fe2+ = 2: 1, and it needs to be carried out in an oxygen-free environment, otherwise it will affect the crystalline form of iron oxide particles and iron oxide. The particles will continue to oxidize during the preparation process, which in turn affects the saturation magnetization of the iron oxide particles. The current solution is to carry out the reaction under nitrogen or change the reaction in the organic phase to slow the oxidation; but the ferrous ions are easily oxidized to ferric ions, which need to be freshly configured, and it is difficult to accurately control the molar ratio in the reaction. This will increase the difficulty of a lot of synthesis. In addition, the iron ions in the triiron tetroxide particles are not all in the highest oxidation state, so these iron ions are slowly oxidized in the air to become non-paramagnetic or weakly magnetized. Generally, the surface of the ferroferric oxide particles is coated with an inorganic oxide of a high molecular polymer or cerium oxide. However, such particles often have the disadvantage of not being able to withstand high temperature and acid resistance for a long time, and their application is limited. Ϊ́ί. ~1^± Disadvantages of the prior art, the present invention utilizes the special highity of urea' in the presence of purely divalent iron ions, S has a superparamagnetic iron oxide particle of saturation magnetization, and

/ 二私化矽來形成核-殼結構，使磁性氧化鐵粒子的強，並可接受高溫高壓滅菌處理。前述有二氧氧ϊ鐵粒子可經進一步酸處理，來作為吸附 ^蛋白亦可進—步經帶有官能基的雜修飾，而 ^鐵他化合物進行化學鍵結，增加超順磁性氧化鐵粒子的應用層面。【發明内容】子之此’本發明之目的係提供-種超順磁性粒用單彳較於傳統的磁性粒子，本發明只需使、-貝鐵離子來源’ g己合尿素特殊的高溫熱分解 6 1356428 特性’即可在常壓高溫的開放系統下化量及超順磁性的氧化鐵粒子，二j-、有向飽和磁並利用改進的二氧化矽包覆技術’將發明溶、水和驗液的混合溶液中進行在j·機醇勻的二氧化梦，此-核-殼結構耐高溫一層均抗酸性佳，在酸性溶液中不I有=錢理，且其本發明的方法中，只需屬離子釋出。在不同粒徑的粒子，而/ Two private 矽 to form a core-shell structure, so that the magnetic iron oxide particles are strong, and can be subjected to high temperature and high pressure sterilization. The foregoing bismuth oxyhydroxide iron particles may be further subjected to acid treatment to act as an adsorption protein, and may be subjected to a hetero-modification with a functional group, and the ferrous compound is chemically bonded to increase the superparamagnetic iron oxide particles. Application level. SUMMARY OF THE INVENTION The object of the present invention is to provide a superparamagnetic particle with a single ruthenium compared to a conventional magnetic particle, and the present invention only needs to make a high temperature of the -be-iron ion source Thermal decomposition 6 1356428 characteristics 'can be used in an open system at normal pressure and high temperature and superparamagnetic iron oxide particles, two j-, directional saturation magnetization and improved cerium oxide coating technology' will dissolve the invention, In the mixed solution of water and the test solution, the dream of oxidizing in the alcohol of the machine is carried out, and the high-temperature layer of the core-shell structure is excellent in acid resistance, and is not in the acidic solution, and the invention thereof In the method, only the ions are released. In particles of different particle sizes, and

石夕層可進行多種表面料，來進行純=㈡一乳化枋r本^^之另一目的係提供一種超順磁性粒子，盆核心乳化，粒子的主要成分是具有超順磁性的四氧^ 二鐵’士軋化鐵粒子的表面有均勻的二氧化石夕包覆層，可抗強酸，並可進行高溫高壓滅菌處理。 u之又—目的係提供—種表面經過酸處理而使矽烷醇基（Si-OH)增加的超順磁性粒子，環境下具有吸附核酸的能力。The Shixia layer can be used for a variety of surface materials to carry out pure = (di) emulsification 枋r. The other purpose is to provide a superparamagnetic particle, the core of the basin is emulsified, and the main component of the particle is tetraoxygen with superparamagnetism. The surface of the iron-iron particles of the two irons has a uniform coating of sulphur dioxide, which is resistant to strong acid and can be autoclaved. The purpose of u is to provide a superparamagnetic particle whose surface is acid-treated to increase the stanol group (Si-OH), and has the ability to adsorb nucleic acid under the environment.

本發明之再一目的係提供一種表面經過具有官能基之矽烷（silane)修飾而使其表面具有胺基、羧基、醛基等官能基的超順磁性粒子，再利用活化劑或連結劑，使之與蛋白質或化合物鍵結，而成為具有專一性的吸附載體’可應用於例如抗體之純化。為達上述目的’本發明提供一種超順磁性粒子之製造方法，其包含下列步驟： (a) 將二價鐵化合物粉末、尿素和水混合攪拌，在 80°C至110°C的常壓環境下進行迴流反應1至 24小時’生成氧化鐵粒子； (b) 清洗前述氧化鐵粒子後再加入水、氨水和有機醇溶劑混合攪拌，進行超音波震盪； 7 1356428 (c) 在前述混合物中加入石夕酸四乙醋（tetraethyl orthosilicate，TEOS)，在 50°C 至 70°C 的溫度下反應0.5小時至12小時；及 (d) 將溫度降至25°C至30°C後，另外加入矽酸四乙酯，反應1小時至12小時，形成有二氧化矽包覆之氧化鐵粒子，其具有超順磁性。在本發明之較佳實施態樣中，前述製造方法進一步包括下列步驟以製得一活化之超順磁粒子： (e) 將前述步驟(d)之有二氧化矽包覆之氧化鐵粒 • 子與pH值小於6之酸性緩衝液混合攪拌。在本發明之較佳實施態樣中，前述酸性緩衝液為磷酸鹽緩衝液、醋酸鹽緩衝液、MES緩衝液（2-(N-嗎琳基）乙烧續酸（2-(N-morpholino)ethanesulfonic acid)) 或Bis-Tris緩衝液（雙(2-羥基乙基)亞胺基-參(羥基曱基) 曱烧 (bis(2-hydroxyethyl)imino-tris(hydroxymethyl) methane)) ° 在本發明之較佳實施態樣中，前述製造方法進一步包括下列步驟’以製得表面具有官能基之超順磁粒 • 子： (f) 將前述步驟（d)之有二氧化矽包覆之氧化鐵粒子移入極性有機溶劑中； (g) 加入具有官能基之矽烷化物混合攪拌，使前述有二氧化矽包覆之氧化鐵粒子表面具有官能基。在本發明之較佳實施態樣中，前述極性有機溶劑為丙酮、DMS0 (二甲亞颯（dimethyl sulfoxide) )、DMF (N，N-—甲基曱醯胺（N，N-dimethylformamide) )、NMP (N-曱基-2-0比略咬酮（N_methyl-2-pyrrolidone))或異丙醇。 8 1356428 在本發明之較佳實施態樣中，前述官能基係選自由胺基、氫硫基、氫氧基、甲基、辛基、十八基和苯基所組成之經群。在本發明之較佳實施態樣中，前述矽烷化物包含γ-胺基丙基二乙氧基發烧（<y_amjn〇pr〇pylA further object of the present invention is to provide a superparamagnetic particle having a surface modified by a silane having a functional group and having a functional group such as an amine group, a carboxyl group or an aldehyde group on the surface thereof, and then using an activator or a linking agent. The adsorption carrier which is bonded to a protein or a compound to become a specific one can be applied to, for example, purification of an antibody. In order to achieve the above object, the present invention provides a method for producing superparamagnetic particles, which comprises the following steps: (a) mixing and stirring a ferrous compound powder, urea and water in an atmospheric environment at 80 ° C to 110 ° C The reflux reaction is carried out for 1 to 24 hours to form iron oxide particles; (b) the iron oxide particles are washed, and then water, ammonia water and an organic alcohol solvent are added and stirred for ultrasonic vibration; 7 1356428 (c) added to the aforementioned mixture Tetraethyl orthosilicate (TEOS), reacted at a temperature of 50 ° C to 70 ° C for 0.5 to 12 hours; and (d) after the temperature is lowered to 25 ° C to 30 ° C, additional Tetraethyl phthalate is reacted for 1 hour to 12 hours to form cerium oxide-coated iron oxide particles having superparamagnetism. In a preferred embodiment of the present invention, the foregoing manufacturing method further comprises the steps of: preparing an activated superparamagnetic particle: (e) oxidizing iron oxide coated with cerium oxide according to the aforementioned step (d). The mixture is mixed with an acidic buffer having a pH of less than 6. In a preferred embodiment of the present invention, the acidic buffer is phosphate buffer, acetate buffer, MES buffer (2-(N-Merlinyl) ethyl benzoate (2-(N-morpholino) Ethylsulfonic acid)) or Bis-Tris buffer (bis(2-hydroxyethyl)imino-tris(hydroxymethyl) methane) ° In a preferred embodiment of the present invention, the foregoing manufacturing method further comprises the following steps of: preparing a superparamagnetic particle having a functional group on the surface; (f) coating the above step (d) with cerium oxide The iron oxide particles are transferred into a polar organic solvent; (g) the decano compound having a functional group is added and stirred to have a functional group on the surface of the cerium oxide-coated iron oxide particles. In a preferred embodiment of the present invention, the polar organic solvent is acetone, DMSO (dimethyl sulfoxide), DMF (N, N-methylformamide) , NMP (N-methyl-2-pyrrolidone) or isopropanol. 8 1356428 In a preferred embodiment of the invention, the aforementioned functional group is selected from the group consisting of an amine group, a thiol group, a hydroxyl group, a methyl group, an octyl group, an octadecyl group, and a phenyl group. In a preferred embodiment of the invention, the decanoate comprises a gamma-aminopropyldiethoxy fever (<y_amjn〇pr〇pyl)

triethoxysilane )、（3-氫硫基丙基）三甲氧基矽烷 ((3-Mercaptopropyl)trimethoxysilane)、環氧丙氧基丙基二甲氧基石夕炫（glycidoxypropyl trimethoxysilane)、曱基二乙氧基石夕烧（methyl triethoxysilane)、正辛基三乙氧基石夕烧（n-octyl triethoxysilane)、十八基三曱氧基石夕院 (octadecyl trimethoxysilane )或苯基三乙氧基矽烧 (phenyl triethoxysilane ) ° 本發明另提供一種超順磁性粒子，其係藉由前述相關方法製得，其中前述超順磁性粒子的主要成分為四氧化三鐵，二氧化矽的含量為0,05重量％至4〇重量％，且其粒控範圍為l〇〇nmi l〇pm。在本發明之較佳實施態樣中，前述超順磁性粒子之飽合磁化量大於35 emu/g。Triethoxysilane ), (3-Mercaptopropyl) trimethoxysilane, glycidoxypropyl trimethoxysilane, fluorenyl diethoxysilane Methyl triethoxysilane, n-octyl triethoxysilane, octadecyl trimethoxysilane or phenyl triethoxysilane ° The invention further provides a superparamagnetic particle obtained by the aforementioned related method, wherein the main component of the superparamagnetic particle is triiron tetroxide, and the content of ceria is from 0,05 wt% to 4 wt%. , and its particle control range is l〇〇nmi l〇pm. In a preferred embodiment of the invention, the superparamagnetic particles have a saturation magnetization of greater than 35 emu/g.

本發明並提供一種活化之超順磁性粒子，其係藉由前述相關方法製得，在高鹽環境下對核酸分子具有^ 吸附力。其中高鹽環境較佳係指〇.5厘至8M的N^C1、 Nal、GuHCl 或 GuSCN。本發明又提供一種表面具有官能基之超順磁性粒子，其係由前述相關方法製得。在本發明之較佳實施態樣中，若前述超川貝磁性粒子表面所具有的官能基是胺基，則其可進一步活化劑修飾，使其可與具有絲官能基 = 物鍵結。負次化口在本發明之較佳實施態樣中，前述經基活化劑係 9 1356428 EDAC、CMC ( 1-環己基-3-(2-嗎啉基乙基）碳化二亞胺 (l-cyclohexyl-3-(2-morpholinoethyl)carbodiimide) )' DCC (二環己基碳化二亞胺（dicyclohexyl carbodiimde))或 DIC(二異丙基碳化二亞胺（diisopropyl carbodiimide))。在本發明之較佳實施態樣中，前述經羥基活化劑修飾後之超順磁性粒子進一步與蛋白質鍵結後，係可用以純化抗體。在本發明之較佳實施態樣中，前述蛋白質係蛋白質 A (Protein A)、蛋白質 G (Protein G)、抗體、酶或鲁鍵徽親合素（Streptavidin)。在本發明之較佳實施態樣中，前述表面具有官能基之超順磁性粒子的表面係進一步利用螯合劑修飾，使其可與過渡金屬離子螯合，純化帶有組胺酸之蛋白或磷酸化蛋白。在本發明之較佳實施態樣中，前述螯合劑為三乙酸基氨（nitrilotriacetic acid )、IDA (亞胺基二乙酸 (iminodiacetic acid))或 EDTA(乙底酸 ethylenediamine-tetraacetic acid) 〇 • 综上所述，本發明提供一種超順磁性粒子之製造方法，相較於傳統的共沈澱法，本發明只需使用單一鐵離子源’即可合成高飽和磁化量的超順磁性氧化鐵粒，，並利用改良的二氧化矽包覆技術，使所製得之有二氧化矽包覆的氧化鐵粒子除了極佳的磁反應性外，更異有絕佳的抗酸性’且可耐高溫高壓處理。而利用本方法所製得之超順磁性粒子用途廣泛，可應用於如核酸與蛋白質的純化。【實施方式】本發明利用尿素特殊的高溫熱分解特性，在單純只需二價鐵離子存在的情況下，產生具有高飽和磁化量 10 磁性氧化鐵粒子。將二價鐵化合物粉末、尿素和 ^混合授掉’ 18〇°C至110°c的常壓環境下進行迴流反二至24】、4 ’其中二價鐵離子在水中會慢慢氧化成 ^貝，離子三同時尿素在熱水中會分解成氨水與二氧化石！1 ^/、中^氧化碳會從迴流管排出，而氨水則會提供氫乳基’使溶液的酸鹼值慢慢升高，產生以下的顏色反應：里 4 色 ! _黃槁紅頁深色'^濁邕-管〇赞->褐 vJ匕巴色色色The present invention also provides an activated superparamagnetic particle which is obtained by the aforementioned related method and which has an adsorption force to a nucleic acid molecule in a high salt environment. Preferably, the high salt environment refers to N^C1, Nal, GuHCl or GuSCN of 厘5 PCT to 8 M. The present invention further provides a superparamagnetic particle having a functional group on its surface, which is obtained by the aforementioned related method. In a preferred embodiment of the present invention, if the functional group on the surface of the above-mentioned super-chuanbei magnetic particle is an amine group, it may be further modified with an activator so that it can bond with a silk functional group. Negative secondary port In a preferred embodiment of the invention, the aforementioned radical activator is 9 1356428 EDAC, CMC (1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide (l- Cyclohexyl-3-(2-morpholinoethyl)carbodiimide) ) DCC (dicyclohexyl carbodiimide) or DIC (diisopropyl carbodiimide). In a preferred embodiment of the invention, the superparamagnetic particles modified by the hydroxy activating agent are further bonded to the protein to be used to purify the antibody. In a preferred embodiment of the invention, the protein is Protein A, Protein G, Antibody, Enzyme or Streptavidin. In a preferred embodiment of the present invention, the surface of the superparamagnetic particle having a functional group on the surface is further modified by a chelating agent to chelate with a transition metal ion to purify a histidine-containing protein or phosphoric acid. Protein. In a preferred embodiment of the present invention, the chelating agent is nitrilotriacetic acid, IDA (iminodiacetic acid) or EDTA (ethylenediamine-tetraacetic acid) 〇• As described above, the present invention provides a method for producing superparamagnetic particles, which can synthesize a supersaturated magnetized superparamagnetic iron oxide particle by using a single iron ion source as compared with the conventional coprecipitation method. And using the improved cerium oxide coating technology, in addition to excellent magnetic reactivity, the iron oxide particles coated with cerium oxide are more excellent in acid resistance and can withstand high temperature and high pressure. deal with. The superparamagnetic particles obtained by the method are widely used, such as purification of nucleic acids and proteins. [Embodiment] The present invention utilizes a special high-temperature thermal decomposition property of urea to produce magnetic iron oxide particles having a high saturation magnetization amount in the presence of only divalent iron ions. The ferrous compound powder, urea and mixture are mixed and subjected to a normal pressure environment of '18 ° C to 110 ° C to carry out reflux to two to 24], 4 'where the divalent iron ions are slowly oxidized in water to ^ Shell, ion three simultaneous urea will decompose into ammonia and dioxide in hot water! 1 ^ /, medium ^ carbon oxide will be discharged from the return pipe, and ammonia will provide hydrogen lactate 'to make the pH value of the solution slowly increase, resulting in the following color reaction: 4 colors! _ yellow 槁 red page deep Color '^邕邕-管〇赞->Brown vJ匕色色色

Fe2+ Fe 3+Fe2+ Fe 3+

Fe304 當酸鹼度達到9以上時，二價鐵離子與三價鐵離 f·會產生共沈殿反應，開始生成黑色的四氧化三鐵粒子晶核；而水中未反應的二價鐵離子繼續反應，在前述晶核，面形成四氧化三鐵沈澱，使粒徑逐漸增加。因此，可藉由，整尿素與二價鐵離子的莫耳比，來控制晶核生成的數量·’或調整四氧化三鐵生成後的反應時間，將氧化鐵粒子的粒徑範圍控制在100 nm至10 μιη。本發明製成超順磁粒子之反應機制如下： 1) 4 Fe2++〇2 + 2 h204 Fe3++ 4 ΟΗ' 2) CO(NH2)2 + h2〇 —加熱(80至110oC) > 2 NH3 + C〇2 2 NH3 + 2 H2〇 2 NH4OH ^ 2 NH4+ + 2 OH* 3) Fe2++2Fe3+ + 8 0H、Fe304 + 4H20 全反應式如下： 3 FeCl2 + 3 0〇(ΝΗ2)2 + ^ (¾ + 6 H20 ^ Fe304 + 6 NH4CI + 3 0¾ 四氧化三鐵粒子在空氣中會漸漸氧化成不具順磁性或磁性較弱的三氧化二鐵。本發明利用改良的二氧化 U56428 $包覆技術在四氧化三鐵粒子表面形成包覆層，降低其氧化程度。首先在高溫下反應，生成厚實的二氧化矽層’然後在低溫下反應，將表面空隙填補完整’並在表面生成具有活性的矽烷醇基。由前述方法所製得的有二氧化矽包覆之氧化鐵粒子’其核-殼結構的抗酸性增強，並可接受高溫高壓滅菌處理。When the acidity and alkalinity reaches 9 or more, the ferrous iron ion and the ferric iron leave f· will produce a common sedimentation reaction, and begin to form a black ferrocene tetraoxide particle nucleus; while the unreacted ferrous ion in the water continues to react. In the foregoing crystal nucleus, a surface of the ferroferric oxide precipitates to gradually increase the particle size. Therefore, the number of crystal nuclei generated can be controlled by the molar ratio of the whole urea to the divalent iron ions, or the reaction time after the formation of the ferroferric oxide can be adjusted, and the particle size range of the iron oxide particles can be controlled to 100. Nm to 10 μιη. The reaction mechanism for producing superparamagnetic particles of the present invention is as follows: 1) 4 Fe2++〇2 + 2 h204 Fe3++ 4 ΟΗ' 2) CO(NH2)2 + h2〇-heating (80 to 110oC) > 2 NH3 + C〇 2 2 NH3 + 2 H2〇2 NH4OH ^ 2 NH4+ + 2 OH* 3) Fe2++2Fe3+ + 8 0H, Fe304 + 4H20 The overall reaction formula is as follows: 3 FeCl2 + 3 0〇(ΝΗ2)2 + ^ (3⁄4 + 6 H20 ^ Fe304 + 6 NH4CI + 3 03⁄4 The triiron tetroxide particles will gradually oxidize into non-paramagnetic or weak magnetic ferric oxide in air. The present invention utilizes a modified U56428 $ coating technique in tetraoxide The surface of the iron particles forms a coating layer to reduce the degree of oxidation. First, the reaction is carried out at a high temperature to form a thick ceria layer, which then reacts at a low temperature to fill the surface voids and form an active stanol group on the surface. The cerium oxide-coated iron oxide particles prepared by the foregoing method have an enhanced acid resistance of the core-shell structure and can be subjected to high temperature and high pressure sterilization treatment.

前述有二氧化矽包覆之氧化鐵粒子可經進一步酸處理’增加其表面的矽烷醇基，如此可在高鹽環境下作為吸附核酸的載體，但卻不會吸附蛋白質與其他污染物；亦可進一步經帶有官能基的矽烷修飾，使氧化鐵粒子表面具有官能基，而與蛋白質或其他化合物進行化學鍵結，增加超順磁性氧化鐵粒子的應用層面。此類磁性材料漸漸成為自動化分子生物檢驗分析之前處理步驟中不可或缺的載體。The above-mentioned cerium oxide-coated iron oxide particles can be further acid-treated to increase the stanol group on the surface, so that it can serve as a carrier for adsorbing nucleic acid in a high salt environment, but does not adsorb proteins and other pollutants; Further, it may be modified by a functional group-containing decane to have a functional group on the surface of the iron oxide particle, and chemically bond with a protein or other compound to increase the application level of the superparamagnetic iron oxide particle. Such magnetic materials are becoming an indispensable carrier in the processing steps prior to automated molecular bioassay analysis.

一般認為，粒徑大於50 nm的磁性粒子不具超順磁性’或有磁遲滞現象（remanent magnetism )。但在本發=的製造方法中，首先生成奈米級的氧化鐵晶核，再 I"曼t艾在晶核表層繼續成長，因此，即便氧化鐵粒子的粒徑已達10 μιη，仍保有超順磁性，且其飽和磁化量大於 35 emu/g。另一方面’可藉由增加氧化鐵粒子的粒徑、或藉由二氧化矽的包覆來增加單顆粒子中的氧化鐵粒子數目，縮短磁反應時間。本發明的超順磁性粒子在外加磁場的吸引下，磁反應時間小於2秒。如此強的磁力不僅可以縮短磁反應時間，也可以在丨公升的大反應_中進行操作。 d _ 具有超順磁性的粒子在外加磁場移離後，不需加 ^ 性便即消失，而㈣重新分散在溶液中，繼續反應。运項特性能夠增加異相反應的均句性。二氧化石夕的包覆須在氧化鐵粒子製備完成後馬上 12 1356428It is generally believed that magnetic particles having a particle diameter of more than 50 nm are not superparamagnetic or have a magnetic magnetism. However, in the manufacturing method of the present invention, first, a nano-sized iron oxide crystal nucleus is formed, and then I"Man tA continues to grow on the surface of the crystal nucleus. Therefore, even if the particle size of the iron oxide particle has reached 10 μm, it remains. Superparamagnetic and its saturation magnetization is greater than 35 emu/g. On the other hand, the magnetic reaction time can be shortened by increasing the particle diameter of the iron oxide particles or by coating with cerium oxide to increase the number of iron oxide particles in the single particles. The superparamagnetic particles of the present invention have a magnetic reaction time of less than 2 seconds under the attraction of an external magnetic field. Such a strong magnetic force not only shortens the magnetic reaction time, but also operates in a large reaction of 丨 liters. d _ Superparamagnetic particles disappear after the external magnetic field is removed, and (4) redisperse in the solution and continue to react. The transport feature can increase the homography of the heterogeneous reaction. The coating of the oxidized stone shall be immediately after the preparation of the iron oxide particles. 12 1356428

進行’以防止四氧化三鐵繼續氧化。首先將氧化鐵粒子分散在水與醇類（如甲醇、乙醇、異丙醇、1-丙醇、L 丁醇、2-丁醇、第三丁醇、異丁醇等）的混合液中，再加入氨水或其他驗性溶液（如氫氧化鈉、氫氧化卸、氫氧化四曱基銨、氫氧化四乙基銨）’將反應環境控制在驗性’進行超音波振盪。在前述混合物中加入發酸四乙酯’在50°C至70°C的溫度下反應〇.5小時至12小時；藉由控制石夕酸四乙酯的添加量，可將二氧化石夕包覆層的厚度限制在從1 nm到1 μπι。之後將溫度降至25°C至 3〇°C ’另外加入矽酸四乙酯，反應1小時至12小時，其係藉由較慢的反應速率，將表面空隙填補完整。本發明之氧化鐵粒子-二氧化矽的改良式核-殼結構，在2M的濃鹽酸下可耐酸超過60分鐘；且高溫高壓滅菌不會影響氧化鐵的超順磁性與其表面的二氧化結構。 ^ 包覆氧化鐵粒子的二氧化矽表面具有矽烷醇基，若浸泡在pH值小於6之酸性緩衝溶液中，可使二氧化矽表面活化，矽烷醇基的數量增加，而這類活化的超順Perform ' to prevent the continued oxidation of ferroferric oxide. First, the iron oxide particles are dispersed in a mixture of water and an alcohol such as methanol, ethanol, isopropanol, 1-propanol, L-butanol, 2-butanol, tert-butanol, isobutanol, or the like. Add ammonia water or other test solution (such as sodium hydroxide, hydroxide, tetramethylammonium hydroxide, tetraethylammonium hydroxide) to control the reaction environment to perform ultrasonic vibration. Adding tetraethyl methacrylate to the above mixture and reacting at a temperature of 50 ° C to 70 ° C for 5 hours to 12 hours; by controlling the amount of tetraethyl sulphate added, the sulphur dioxide can be packaged The thickness of the coating is limited from 1 nm to 1 μm. Thereafter, the temperature is lowered to 25 ° C to 3 ° C. The addition of tetraethyl citrate is carried out for 1 hour to 12 hours, which completes the surface voids by a slower reaction rate. The modified core-shell structure of the iron oxide particle-cerium oxide of the present invention is resistant to acid for more than 60 minutes under 2M concentrated hydrochloric acid; and high temperature and high pressure sterilization does not affect the superparamagnetism of the iron oxide and the surface oxidation structure thereof. ^ The surface of cerium oxide coated with iron oxide particles has a stanol group. If immersed in an acidic buffer solution with a pH of less than 6, the surface of cerium oxide can be activated and the number of stanol groups can be increased. Shun

磁=粒子在 0.5M 至 8M 之 NaCl、NaI、GuHCl 或 GuSCN 的同鹽環境下，對核酸分子有很強的吸附力，且超順磁粒子之結果不會被破壞，至於蛋白質分子與其他污染丨可藉由簡單的清洗步驟去除。可對所得之純化核 ^ 進f 聚合酶連鎖反應（polymerase chain reaction，來將它放大’其結果如第四圖所示，純化核酸中、蛋白質和其他會抑制pCR反應的物質存在。劑c如將^包覆之氧化鐵粒子移至極性有機溶胺美丙美-I ^萁再加入具有官能基的矽烷化物（如γ-基使氧化鐵粒子表面具有官能 ’又後可利用羥基活化劑（如EDAC)做 13 1356428 進一步修飾’使超順磁性粒子與蛋白質（如蛋白質A (Protein A)、蛋白質G (Protein G)、抗體、酶或鏈黴親合素（Streptavidin))鍵結，之後用以純化抗體；其亦可利用螯合劑（如三乙酸基氨）做進一步修飾，使超順磁性粒子與二價或三價的過渡金屬離子螯合，之後用以純化帶有組胺酸（histidine)之蛋白或磷酸化蛋白。實施例實施例ί :超順磁往氧化鐵粒子的小量製備 • 將0.25克二氣化鐵粉末與6克尿素結晶顆粒溶解在100宅升去離子水中，在裝有冷凝管的玻璃反應器中快速攪拌10分鐘，形成黃綠色澄清溶液，移入9〇。〇的油鍋進行迴流反應12小時，生成黑色氧化鐵粒子，靜置沈澱後，將接近澄清的上清液倒出’沉澱物用去離子水清洗三次，去除反應剩下來的銨離子與氯離子。進行二氧化矽包覆前，將前述沉澱物保持在水溶液中，避免乾燥。 φ 實施例2 :超順磁性氧化鐵粒子的大量製備將60克二氯化鐵粉末與9〇克尿素結晶顆粒溶解在1公升去離子水中，在裝有冷凝管的玻璃反應器中快速攪拌3分鐘，並將混合物移入溫度設定為95它的油鍋進行迴流反應12小時，生成黑色氧化鐵粒子，靜置沈澱後’將接近澄清的上清液吸出，沉澱物用去離子水清洗三次，去除反應剩下來的銨離子與氯離子。進行二氧化石夕包覆前’將前述沉殿物保持在水溶液中，避免乾燥。實施例3:二氧化矽的包覆 14Magnetic = particles in the same salt environment of 0.5M to 8M NaCl, NaI, GuHCl or GuSCN, have strong adsorption to nucleic acid molecules, and the results of superparamagnetic particles will not be destroyed, as for protein molecules and other pollution丨 can be removed by a simple cleaning step. The resulting purified nuclear polymerase chain reaction can be amplified. The results are as shown in the fourth figure, and the purification of nucleic acids, proteins and other substances that inhibit the pCR reaction are present. Transferring the coated iron oxide particles to a polar organic amine amide-I^萁 and then adding a decyl compound having a functional group (such as a γ-group to make the surface of the iron oxide particles have a function) and then using a hydroxyl activator ( For example, EDAC) 13 13356428 further modified 'to bind superparamagnetic particles to proteins (such as Protein A, Protein G, antibodies, enzymes or Streptavidin), and then use Purification of the antibody; it may also be further modified with a chelating agent (such as triacetoxy) to sequester the superparamagnetic particles with a divalent or trivalent transition metal ion, followed by purification with histidine (histidine) Protein or phosphorylated protein. EXAMPLES Example ί: Miniparamagnetic preparation of superparamagnetic iron oxide particles • Dissolve 0.25 g of iron dihydrate powder and 6 g of urea crystal particles in 100 liters of deionized water. The glass reactor equipped with a condenser tube was rapidly stirred for 10 minutes to form a yellow-green clear solution, which was transferred to 9 Torr. The oil pan of the crucible was refluxed for 12 hours to form black iron oxide particles, and after standing still, it was close to the clarification. The supernatant was poured out and the precipitate was washed three times with deionized water to remove the ammonium ions and chloride ions remaining in the reaction. Before the coating with cerium oxide, the precipitate was kept in an aqueous solution to avoid drying. φ Example 2: Large-scale preparation of superparamagnetic iron oxide particles 60 g of iron dichloride powder and 9 g of urea crystal particles are dissolved in 1 liter of deionized water, rapidly stirred in a glass reactor equipped with a condenser tube for 3 minutes, and the mixture is The temperature was set to 95. The oil pan was refluxed for 12 hours to form black iron oxide particles. After standing to precipitate, the near-clear supernatant was aspirated, and the precipitate was washed three times with deionized water to remove the remaining ammonium. Ions and chloride ions. Before the coating of the dioxide is carried out, the above-mentioned sediments are kept in an aqueous solution to avoid drying. Example 3: Coating of cerium oxide 14

八畔*將30 ί由實施例1或實施例2製得的氧化鐵粒子 1扯,225毫升去離子水中，再加入22.5毫升μ%的氨二二後;^入900毫升異丙醇，將此溶液封閉在玻璃反窃中持縯攪拌，並進行超音波震盪1〇分鐘。加入4 $ 的矽酸四乙酯，並將水溫升高至5(TC，持續反廄2 小時，之後將水溫降至室溫（約25。〇，再加入45毫^升 =矽酸四乙醋，持續反應丨小時。反應完成後，靜置沈澱，將透明上清液吸出，並用去離子水清洗至無氨異丙醇的味道為止。實施例3製成之有二氧化超順磁粒子之影像圖如第一圖所示。實施例4 :表面活化處理Eight sides* to 30 ί of iron oxide particles 1 prepared in Example 1 or Example 2, 225 ml of deionized water, and then 22.5 ml of μ% ammonia dihydrate; then into 900 ml of isopropanol, This solution was closed in a glass anti-theft and stirred for 1 minute. Add 4 $ of tetraethyl citrate and raise the water temperature to 5 (TC, continue to simmer for 2 hours, then lower the water temperature to room temperature (about 25. 〇, then add 45 mM = citric acid) After the reaction was completed, the precipitate was allowed to stand, the clear supernatant was aspirated, and washed with deionized water until the taste of ammonia-free isopropanol was obtained. Example 3 was prepared with superoxide superoxide. The image of the magnetic particles is shown in the first figure. Example 4: Surface activation treatment

將實施例3的有二氧化矽包覆之氧化鐵粒子去除上清液後’從中取30克加入3〇()毫升1〇mM ，鹽緩衝液（ΡΗ5·5)，清洗兩次，之後在相同的緩持續搜拌5小時，再以去離子水清洗，重新分散驗值8的缓衝液中，即可製得具有活化之二氧化二的超順磁性粒子。面After removing the supernatant of the cerium oxide-coated iron oxide particles of Example 3, 30 grams of 3 mM (1 ml of mM, salt buffer (ΡΗ5·5) was added thereto, and washed twice, after The same slow continuous search for 5 hours, and then washed with deionized water, redistribution of the buffer value of 8 to obtain a superparamagnetic particle with activated dioxide. surface

實施例5:表面官能基的修飾將實施例3的有二氧化矽包覆之氧化鐵粒子去除上清液後’從中取30克加入250毫升無水内胸置清洗兩次’之後將前述粒子分散在500毫升丙_中，技續攪拌10分鐘，再加入3.5毫升γ-胺基丙基三乙氧基，烷，在室溫下反應4小時。反應完成後，靜置沈澱，矽透明上清液吸出，並用去離子水清洗至無丙酮的$、曾$ 止，即可製得表面具有胺基官能基的超順磁性粒子"7為實施例6 :核酸的纯化 15 1356428Example 5: Modification of surface functional group After removing the supernatant of the cerium oxide-coated iron oxide particles of Example 3, 'take 30 g from the solution and add 250 ml of anhydrous inner tube to wash twice', then disperse the aforementioned particles. In 500 ml of propylene, the mixture was stirred for 10 minutes, and then 3.5 ml of γ-aminopropyltriethoxy, alkane was added, and the mixture was reacted at room temperature for 4 hours. After the reaction is completed, the precipitate is allowed to stand, and the clear supernatant is aspirated, and washed with deionized water until the amount of acetone-free, and the superparamagnetic particles having an amine functional group on the surface can be obtained. Example 6: Purification of nucleic acids 15 1356428

取分散在100微升中的lxl〇5個TSGH-8301細胞加入400微升的細胞裂解液㈠2〇g guscN，100ml 0.1MAdd 1 x 10 ml of TSGH-8301 cells dispersed in 100 μl to 400 μl of cell lysate (1) 2 μg guscN, 100 ml 0.1 M

Tris-HCl PH6.4，22ml 0.2M EDTA pH8.〇，2.4ml Triton X-100)，反應10分鐘。之後加入50微升15〇 mg/mi由實施例4製備而得之具有活化之二氧化矽表面的超順磁性粒子’並加入1〇〇微升的連結液（異丙醇），均勻混合10分鐘，使磁性粒子與細胞裂解後產生的核酸產生連結。將昆合物置於磁座上’將上清液吸出，加入8〇〇微升的清洗液（120g GuSCN，100ml 0.1M Tris-HCl pH6·4)，清洗兩次；再加入800微升70%乙醇，清洗兩次。之後將氧化鐵粒子靜置丨〇分鐘，使清洗液中的酒精揮發；再加入1〇〇微升的沖提液（10niMTrispH8)，均勻混合10分鐘’接著將反應物置於磁座上，將上清液吸出’置入乾淨的容器中’此即純化後的核酸，結果如第四圖所示，其中Μ為1 kb的核酸標準液；E1至E3 為lxlO5個細胞的三次重複實驗；C1至C3為不同數目之細胞的比較，其中C1為lxl〇5個細胞，C2為2xl〇5 個細胞’ C3為5xl〇5個細胞。由第四圖之結果可知，利用本發明之活化後之超順磁粒子來純化之核酸並無蛋白質和其他會抑制PCR反應的物質存在。實施例7:抗想的純化取10微升的血清，加入900微升的連結液（10 mM 磷酸鹽，15〇1^价(：1，？117.5)，再加人20微升5〇11^/1111 鍵結有蛋白質A的超順磁性粒子（利用實施例5之表面修飾胺基之超順磁粒子進一步利用經基活化劑修飾後’再與蛋白質A鍵結），均勻混合30分鐘。將混合物置於磁座上，將上清液吸出，加入500微升的清洗液（1〇 mM鱗酸鹽，i5〇mMNaa，pH7.5)，清洗兩次；再加 1356428 入100微升的沖提液（0.5 Μ甘胺酸，0.15 M NaC卜pH 2.5)，均勻混合1〇分鐘，接著將反應物置於磁座上，將上清液吸出，置入乾淨的容器中，再加入5微升的中和液（1 M Tris ’ pH 9.0) ’移動磁性粒子，重複進行一次沖提步驟，即得純化後的抗體，結果如第五圖所示，其中Μ為蛋白質標準液，S為稀釋20倍的血清，F為未被磁性粒子吸附的蛋白質’ W1為第一次清洗液的產物， W2為第二次清洗液的產物，Ε1為第一次沖提液的產物，其產量大約占總量的60至70%，Ε2為第二次沖提液的產物，其產量大約占總量的30至40%。由第五圖之結果顯示，利用本發明製備之超順磁粒子，經表面官能基修飾後可作為抗體純化之用。血清中各種免疫球蛋白（IgG [約70%]、IgM [約 6%]、IgA [約 10 至 20°/〇]、IgE [約 0.001%]和 IgD [約 0.2%])的分子量分佈在15〇 kDa至900 kDa，在進行電泳確認前，須將各樣本加熱沸騰，使蛋白能在電泳膠片中移動。加熱沸騰後的免疫球蛋白會分成重鏈（heavy chain)和輕鏈（light chain)兩部分，其中各免疫球蛋白的重鏈大小係在50 kDa至75 kDa範圍内，而數量最多之IgG的輕鏈大小為25 kDa ’這兩個大小的蛋白會在 E1與E2上形成兩條明顯的主帶，此外，其他數量較少之免疫球蛋白（IgM、IgA、IgE和IgD)的輕鏈，會在 25 kDa上下形成較弱的帶。而電泳片上大於75 kDa的帶’主要是熱分解片段重新結合而成的半產物或完整的免疫球蛋白。實施例8:本發明舆市售磁性粒子的比較 1)耐酸性的測定在1 ml的紫外光等級的塑膠比色管中，分別加入 17 1356428 12.5 mg本發明與兩種市售磁性粒子，再分別加入5〇〇的2 M HC1，混合均勻後，將磁性粒子磁吸至底旦，上清液於290 nm的吸光值（α29(^在0至1〇分°鐘時里每2分鐘量測^次；在10至6〇分鐘時，每5分鐘量測久。由於Fe離子的吸收蜂在290 nm，因此，若前述Tris-HCl pH 6.4, 22 ml 0.2 M EDTA pH 8. 〇, 2.4 ml Triton X-100), reaction for 10 minutes. Then, 50 μl of 15 〇mg/mi of the superparamagnetic particle having the activated ceria surface prepared in Example 4 was added and 1 〇〇 microliter of the linking solution (isopropyl alcohol) was added, and uniformly mixed 10 In minutes, the magnetic particles are linked to the nucleic acids produced by cell lysis. Place the compound on the magnetic base'. The supernatant was aspirated, and 8 μL of the washing solution (120 g GuSCN, 100 ml 0.1 M Tris-HCl pH 6.4) was added and washed twice; then 800 μl 70% was added. Ethanol, washed twice. The iron oxide particles are then allowed to stand for a few minutes to evaporate the alcohol in the cleaning solution; then 1 liter of microliters of the extract (10 niMTris pH 8) is added and uniformly mixed for 10 minutes. Then the reaction is placed on the magnetic base and placed thereon. The clear liquid is sucked out into the cleaned container, and the purified nucleic acid is shown in the fourth figure, wherein the Μ is a 1 kb nucleic acid standard solution; E1 to E3 are three repeated experiments of lxlO5 cells; C1 to C3 is a comparison of different numbers of cells, wherein C1 is lxl〇5 cells, C2 is 2xl〇5 cells' C3 is 5xl〇5 cells. As is apparent from the results of the fourth graph, the nucleic acid purified by using the activated superparamagnetic particles of the present invention has no protein and other substances which inhibit the PCR reaction. Example 7: Resistant purification Take 10 microliters of serum and add 900 microliters of ligation solution (10 mM phosphate, 15 〇 1 valence (: 1, 117.5), plus 20 microliters of 5 〇 11 ^/1111 Superparamagnetic particles bonded with protein A (super-paramagnetic particles of the surface-modified amine group of Example 5 were further modified by a radical activator and then bonded to protein A), and uniformly mixed for 30 minutes. The mixture was placed on a magnetic stand, the supernatant was aspirated, 500 μl of washing solution (1 mM mM sulphate, i5 mM mM Naa, pH 7.5) was added and washed twice; then 1356428 was added to 100 μl. Eluate (0.5 Μ glycine, 0.15 M NaC Bu pH 2.5), mix evenly for 1 minute, then place the reaction on the magnetic base, aspirate the supernatant, place in a clean container, add 5 micro The neutralized solution (1 M Tris 'pH 9.0) 'moves the magnetic particles and repeats the stripping step to obtain the purified antibody. The result is shown in the fifth figure, where Μ is the protein standard solution and S is the dilution. 20 times serum, F is a protein not adsorbed by magnetic particles' W1 is the product of the first cleaning solution, W2 is the second The product of the cleaning solution, Ε1 is the product of the first extract, the yield is about 60 to 70% of the total, and Ε2 is the product of the second extract, which accounts for about 30-40% of the total. The results of the fifth graph show that the superparamagnetic particles prepared by the present invention can be used as an antibody for purification by surface functional groups. Various immunoglobulins in serum (IgG [about 70%], IgM [about 6 The molecular weight distribution of %], IgA [about 10 to 20°/〇], IgE [about 0.001%] and IgD [about 0.2%] is between 15〇kDa and 900 kDa, and each sample must be heated before confirmation by electrophoresis. Boiling, allowing the protein to move in the electrophoretic film. The heated immunoglobulin after boiling is divided into two parts, a heavy chain and a light chain, wherein the immunoglobulin has a heavy chain size of 50 kDa to In the 75 kDa range, the highest number of IgGs has a light chain size of 25 kDa. These two sizes of proteins form two distinct major bands on E1 and E2. In addition, other small amounts of immunoglobulin (IgM) Light chains of IgA, IgE, and IgD) form weaker bands above and below 25 kDa. The band greater than 75 kDa is mainly a semi-product or a complete immunoglobulin recombined with thermal decomposition fragments. Example 8: Comparison of commercially available magnetic particles of the present invention 1) Determination of acid resistance in 1 ml of UV In the light grade plastic colorimetric tube, 17 1356428 12.5 mg of the present invention and two commercially available magnetic particles are respectively added, and then 5 〇〇 of 2 M HCl is added separately, and after mixing uniformly, the magnetic particles are magnetically attracted to the base. The absorbance of the supernatant at 290 nm (α29 (^ is measured every 2 minutes at 0 to 1 〇 °°; at 10 to 6 minutes, every 5 minutes). Since the Fe ion is absorbed at 290 nm, therefore, if

土J液，A29。值*鐘内有上升，則表示該磁性粒子的二氧化矽層被酸破壞，使氧化鐵裸露並，成Fe'故在前述測定中，a29。值未上d匕「佳」’❿A29。值上升者則判定為「不佳」，第二圖所示。 2)对高溢高屋處理的測定Soil J liquid, A29. If the value * rises in the clock, it means that the ceria layer of the magnetic particles is destroyed by the acid, and the iron oxide is exposed to Fe', so in the above measurement, a29. The value is not on d匕 "good" ❿A29. The value riser is judged as "poor", as shown in the second figure. 2) Determination of high overflow high house treatment

在2 ml聚丙稀的樣品管中’分別加入1^5 本發明與兩種市售磁性粒子，再分別加入1 mi去離子水，混合均勻後，半鎖緊上蓋，放入滅菌鍋中，進行言二言^ 度設定12(TC，持續4〇分鐘，之後在&=下進仃觀察磁性粒子表面二氧化石夕層脫落的情形，其结果如下表1和第二圖所示。在下表1中，「佳/代^面二氧化矽層未有脫落的情形，「可」代表表面二^化矽 ^有些微脫落的情形，而「差」代表表面二氧化矽層有嚴重脫洛的情形；而在第二圖中，A-1和八_2分別為本發明之超順磁性粒子在高溫高壓處理前後的外型，表面二氧化矽層未有脫落的情形；Β-1和Β-2分別為〇ia2en 之磁性粒子在高溫高壓處理錢的外型，表層有些微脫落的情形：C-1和02分別為Invitr〇gen之磁性粒子在高溫高壓處理前後的外型，表面二氧化石夕層有嚴重脫落的情形。曰此外’磁性粒子是否而ί高溫高壓處理，亦可配合下文所述’而由處理後的磁反應時間變化來觀察；若磁反應時間變慢，表示高溫高壓處理會造成磁性粒子氧化或 18 1356428 使其磁性結構改變’表示該磁性粒子不耐高溫高壓處理’如表1所示。 3)磁吸時間的測定Add 2^5 of the present invention and two commercially available magnetic particles in a 2 ml polypropylene sample tube, and add 1 mi of deionized water separately. After mixing evenly, the upper cover is half-locked and placed in a sterilizing pot. In the second sentence, the degree is set to 12 (TC, lasts for 4 minutes, and then the surface of the magnetic particle is peeled off under the &= observation. The results are shown in Table 1 and Figure 2 below. In the case of "1", "the good/generation surface of the cerium oxide layer does not fall off, "可可" represents the surface of the surface of the ^ 矽 ^ some slightly detached, and "poor" represents the surface of the cerium oxide layer has severe detachment In the second figure, A-1 and 八_2 are the appearances of the superparamagnetic particles of the present invention before and after high temperature and high pressure treatment, and the surface cerium oxide layer is not detached; Β-1 and Β -2 is the shape of the magnetic particles of 〇ia2en treated at high temperature and high pressure, and the surface layer is slightly detached: C-1 and 02 are the appearances of magnetic particles of Invitr〇gen before and after high temperature and high pressure treatment, surface oxidation The stone layer has a serious shedding. 曰In addition, 'magnetic particles are ί High temperature and high pressure treatment can also be observed with the change of magnetic reaction time after treatment; if the magnetic reaction time is slow, it means that the high temperature and high pressure treatment will cause the magnetic particles to oxidize or 18 1356428 to change the magnetic structure' The magnetic particles are not resistant to high temperature and high pressure treatment as shown in Table 1. 3) Determination of magnetic attraction time

磁吸時間的測定係在25至30°C下進行，將磁珠置於1 ml或40 ml的去離子水卜若去離子水的體積為i ml，則利用小磁鐵進行測試；若其體積為4〇 mi，則利 ^大磁鐵進行顺。在磁座上安裝大/小磁鐵再把裝有磁性粒子和反應液體的容器放到磁座上，進行磁吸時間 =，測試結果如下表i所示。與市售產品比較，在反應體積大小額的測試中可明顯看出，若反應體積愈大，則磁性愈強的磁性粒子，其反應操作的時間越短。表1，本發明與市售;^性粒子的特性比較The magnetic induction time is measured at 25 to 30 ° C. The magnetic beads are placed in 1 ml or 40 ml of deionized water. If the volume of deionized water is i ml, the test is carried out using a small magnet; For 4〇mi, the big magnet is smooth. Install a large/small magnet on the magnetic base and place the container containing the magnetic particles and the reaction liquid on the magnetic base for the magnetization time =. The test results are shown in Table i below. Compared with commercially available products, it is apparent in the test of the size of the reaction volume that, if the reaction volume is larger, the magnetic particles having stronger magnetic properties have a shorter reaction operation time. Table 1. Comparison of characteristics of the present invention and commercially available particles

2) *磁: ) 【圖式簡單說明】第一圖：本發明有二氧化矽包覆之超順磁性粒子 (粒徑約500 nm)的電子顯微鏡圖（4〇〇,〇〇〇χ放大圖）。第二圖：本發明之超順磁性粒子與市售磁性粒子經高溫高壓處理前後在顯微鏡下的_Χ放大圖。 19 1356428 第三圖：本發明與市售磁性粒子的抗酸性比較圖。第四圖：使用本發明之超順磁性粒子進行核酸純化的DNA電泳圖。第五圖：使用本發明之超順磁性粒子進行抗體純化的蛋白質電泳圖。【主要元件符號說明】2) *Magnetism: ) [Simple description of the diagram] First: The electron micrograph of the superparamagnetic particle (particle size about 500 nm) coated with cerium oxide (4 〇〇, 〇〇〇χ magnification) Figure). Fig. 2 is a magnified view of the superparamagnetic particles of the present invention and commercially available magnetic particles under a microscope before and after high temperature and high pressure treatment. 19 1356428 Third Figure: Comparison of the acid resistance of the present invention with commercially available magnetic particles. Figure 4: DNA electropherogram of nucleic acid purification using the superparamagnetic particles of the present invention. Figure 5: Electropherogram of a protein purified using the superparamagnetic particles of the present invention. [Main component symbol description]

2020

Claims

υ^ϋ428 十、申請專利範圍： 1. -種超順雜粒子之製造方法，其包含下列步驟： ⑷物粉末、尿素和水混合㈣，在8代 (b)=====水、氨水和有機醇溶 ⑷ΐΐϊΐί物中加入石夕酸四乙醋’在5〇°CS7〇°C的 ^度下反應0.5小時至12小時；及 (d) ϊ;ΐΤ;25。至和吏，另外加入石夕酸四乙醋， Lfnii2㈣，形成有二氧化矽包覆之氧化鐵粒子’其具有超順磁性。 2. 凊專利範圍第1項所述之方法’其進-步包括下列 (e) ^述步驟⑷之有二氧切包覆之氧化鐵粒子食 3. pH值小於6之酸性緩衝液混合攪拌。 /、 Ϊ 圍衝第液2、項gfgf ί =;其中前述酸性緩衝 Bis-Tris緩^衝液醋酸鹽緩衝液、贿緩衝液或 4. ^請專利範圍第1項所述之方法，其進—步包括下列 ⑺步驟⑷之有二氧化矽包覆之氧化鐵粒子移入極性有機溶劑中；秒 (g)加入具有官能基之矽烷化物混合攪拌，氧化矽包覆之氧化鐵粒子表面具有官能基。一圍第4項所述之方法，其中前述極性有機冷月彳為丙酿I、DMSO、DMF、ΝΜΡ或異而醇。 6.如申請專利範圍第4項所述之方法，其中前述官能基係 21 1356428 選自由胺基、氫硫基、氫氧基、甲基、辛基、十八基和苯基所組成之組群。 a 7·如申請專利範圍第4項所述之方法，其中前述矽烷化物包s y-胺基丙基二乙氧基％烧、（3-氫硫基丙基)三甲氧基矽烷、環氧丙氧基丙基三甲氧基石夕烷、甲基三乙氧^矽烷: 正辛基二乙氧基矽烷、十八基三甲氧基石夕烷、或二氧基矽烷。 +土一Q 8. —種超順磁性粒子，其係藉由申請專利範圍第丨至7項令任:項所述之方法所製得，纟中前述超順磁性粒子的主要成分為四氧化三鐵，二氧化矽的含量為〇〇5重量％至40重量％，且其粒徑範圍為1〇〇11111至 9. 如申請專利範圍第8項所述之超順磁性粒子，豆飽合磁化量大於35emu/g。 '、 1〇馆^化的超順磁性粒子，其係由如申請專利範圍第2 吸附f方法製得，其在高鹽環境下雜酸分子具有強 u.巧表面具有官能基之超順磁性粒子，其利範圍第4項所述之方法製得。 ^ ° 第11項所述之超順磁性粒子，若前述超利用面所具有的官能基是胺基，則其可進一步質物=料’使其可與具錢基官能基之蛋白 i3tm範圍第12項所述之超順磁性粒子，其中前述 i432，edac、cmc、dc(^dic。經經^、、舌n，第12項所述之超順磁性粒子，其中前述 15結後三係可順磁性粒子進一步與蛋白質鍵申°月專利範圍第14項所述之超順磁性粒子，其令前述 22 1356428 蛋白質係蛋白質A、蛋白質G、抗體、酶或鏈黴親合素 (Streptavidin ) ° 16. 如申請專利範圍第11項所述之超順磁性粒子，其表面係進一步利用螯合劑修飾，使其可與過渡金屬離子螯合，純化帶有組胺酸之蛋白或磷酸化蛋白。 17. 如申請專利範圍第16項所述之超順磁性粒子，其中前述螯合劑為三乙酸基氨、IDA或EDTA。υ^ϋ428 X. Patent application scope: 1. A method for manufacturing super-compact particles, which comprises the following steps: (4) mixing of powder, urea and water (4), in 8 generations (b) ===== water, ammonia And the organic alcohol soluble (4) ΐΐϊΐ 加入石石石石石石石石石石石石石石石在在在在在在在在在在在在在在在在在在在在在 0.5 0.5 0.5 0.5 0.5 0.5 To the oxime, an additional tetrahydroacetic acid, Lfnii 2 (four), is formed with cerium oxide-coated iron oxide particles, which have superparamagnetism. 2. The method described in the first paragraph of the patent range includes the following steps: (e) the step (4) of the oxidized iron oxide coated with the dioxygenated coating. 3. The acidic buffer mixed with a pH of less than 6 . /, Ϊ Circulating liquid 2, item gfgf ί =; wherein the above-mentioned acidic buffered Bis-Tris buffer buffer, brittle buffer or 4. ^ Please refer to the method described in the first item of the patent scope, The step includes the following step (7): (2) the cerium oxide-coated iron oxide particles are transferred into the polar organic solvent; and the second (g) is added to the decanoate having a functional group to be stirred, and the surface of the cerium oxide-coated iron oxide particles has a functional group. The method of item 4, wherein the polar organic cold glutinous rice is propylene I, DMSO, DMF, hydrazine or iso-alcohol. 6. The method of claim 4, wherein the aforementioned functional group 21 1356428 is selected from the group consisting of an amine group, a thiol group, a hydroxyl group, a methyl group, an octyl group, an octadecyl group, and a phenyl group. group. The method of claim 4, wherein the decane compound comprises s y-aminopropyl diethoxy%, (3-hydrothiopropyl)trimethoxynonane, epoxy Propoxypropyltrimethoxy aspartame, methyltriethoxyoxane: n-octyldiethoxydecane, octadecyltrimethoxyindol, or dioxydecane. +土一Q 8. A kind of superparamagnetic particles, which is obtained by the method described in the scope of the patent application No. 7 to 7 in which the main component of the above superparamagnetic particles is tetraoxide. The content of the triiron and the cerium oxide is from 5% by weight to 40% by weight, and the particle size ranges from 1 to 11111 to 9. The superparamagnetic particles according to claim 8 of the patent application, the bean is saturated. The amount of magnetization is greater than 35 emu/g. ', 1 〇 ^ 的的的超超超超超超超超超超超超超超超超超超超超超超超超超超超超超超超超超超超超超超超超超超超超The particles are produced by the method described in the fourth item. ^ ° The superparamagnetic particles according to Item 11, if the functional group of the above-mentioned super-utilizing surface is an amine group, it can further be a substance = material to make it compatible with the nitrile-containing protein i3tm range 12 The superparamagnetic particle according to the above item, wherein the above i432, edac, cmc, dc (^ dic., via ^,, n, the superparamagnetic particle according to item 12, wherein the above-mentioned 15 after the three lines are compliant The magnetic particles are further combined with the superparamagnetic particles described in claim 14 of the patent application, wherein the 22 1356428 protein is protein A, protein G, antibody, enzyme or streptavidin. The superparamagnetic particle according to claim 11 is further modified by a chelating agent to chelate with a transition metal ion to purify a histidine-containing protein or a phosphorylated protein. The superparamagnetic particle of claim 16, wherein the chelating agent is triacetinamide, IDA or EDTA.

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