TW200845055A - Superparamagnetic particles and manufacturing method thereof - Google Patents

Abstract

The present invention provides a method for manufacturing the superparamagnetic particles, which comprises the following steps: blending and stirring divalent-iron powder, urea, and water; executing the recycling reaction to the blending for 1 to 24 hours at 80 to 110 degrees centigrade under the normal pressure, and thereby producing the ferric oxide; cleaning the ferric oxide, blending and stirring it with water, ammonia and organic alcohol, and executing ultrasonic vibration to the mixture; adding tetraethyl silicate to the mixture and having it react for 0.5 to 12 hours at 50 to 70 degrees centigrade; and after lowing the temperature of the mixture to 25 to 30 degrees centigrade, further adding tetraethyl silicate to it to react for 1 to 12 hours, so as to form the ferric oxide particles coated with silicon dioxide of superparamagnetism. Furthermore, this invention also provides the superparamagnetic particles manufactured by the afore-mentioned method.

Description

200845055 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。前述超順磁性粒 子可應用於化學或生化物質的純化與反應，並可應用於 大規模反應與自動化磁珠操作平台。 【先前技術】200845055 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 superparamagnetic particles can be applied to the purification and reaction of chemical or biochemical substances, and can be applied to large-scale reaction and automated magnetic bead operation 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，聚集到磁場的N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。由於氧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< 中，其粒徑等級是以微米為主，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，僅有少數的; =應，，九其原因，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j 來，由於奈米粒子的開發，粒徑大幅縮小，而得以 早期微米粒子無法進人的區域，因此開始大量開發與石 ί 〒已經可達微血管區域等，並廣泛地^ ^生物商夺領域上，例如磁共振造影、溫熱治療等·, 22順磁性之奈米粒子也具有可控制化載體的4 性，故▼動了整個生醫產業自動化的新里程。 式，於Λ成具有超順雜之氧化鐵粒子的^ 在门％•具有二仏鐵離子與三價鐵離子的溶液 液，將酸驗值調整到9 i 14之間，如此即^ ί 5 200845055 化三鐵的沈澱物。但此傳統方式需將三價與二價鐵 的莫耳比例控制在Fe3+: Fe2+= 2 : i ’且需在無氧環 下進行，否則將影響氧化鐵粒子的結晶型態，而且 鐵粒子會在製備過程中持續氧化，進而影響到氧化鐵粒 子的飽和磁化量（saturation magnetization)大小。目々 的解決方式是在氮氣下進行反應、或改在有機相中S 應，以便減緩氧化；但二價鐵離子易氧化成三價 子，需新鮮配置，且不易精準控制反應中的莫 這都會增加大量合成的困難度。此外，四氧化三鎩敖早 中的鐵離子並非全數處於最高的氧化態，所以這些鐵 子會在空氣中慢慢氧化成不具順磁性或磁性較 氧化二鐵。一般來說，會在四氧化三鐵粒子表面包覆^ 分子聚合物或二氧切的無機氧化物，但這類 = 出現無法長時間耐高溫與耐酸性差的缺點，= 到限制。 從，、應用文 、針對先前技術的缺點，本發明利用尿素特 溫熱分解特性，在單純只需二價鐵離子在沾主、、冋 產生具有高飽和磁化量的超順磁性氧粒子夺 抗酸性增強’並可接受高溫高壓滅菌處理礼=2 化矽包覆之氧化鐵粒子可經進一步以：： 與蛋白質或其他化合物 化鐵粒子的應用層面。 《加超順磁性虱 【發明内容】 有鑑於此，本發明之目的係楹 子，製造方法’相較於傳統的磁性粒^ 用單一的二價鐵離子來源，配合尿素特殊的^溫 6 200845055 溫與低溫兩階段的包覆m了=^皮震| ’利用高 =性佳，恤溶液中不4以=;屮； .層：多種表_:以==均勻二氧化 核心氧化的主0性粒子’其 可抗強酸’並可進行高溫高壓滅=乳化夕包復層， 使石夕燒本醇種表面經過酸處理而 環境下具有吸_酸的^力的超順磁性粒子，其於高鹽 美之石^發/月之再一目的係提供一種表面經過具有官能 i等官ii:二Γ二吏其ί:具有胺基、羧基、搭 使之鱼疋白二5粒再利用活化劑或連結劑， 載體，可應用於例純:成為具有專-性的吸附 ⑻ϋ㈣化合物粉末、尿素和水混合攪拌，在 境/進行坦流反應1至 (b)=r:=二氨水和有機 7 200845055 (C)在前述混合物中加入矽酸四乙酯（tetraethyl orthosilicate，TEOS)，在 50°C 至 7(TC 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 石粦酸鹽緣衝液、醋酸鹽緩衝液、MES緩衝液（2-(N-嗎 琳基)乙烧石頁酸（2-(N_morpholino)ethanesulfonic acid)) 或BlS_Tris緩衝液（雙(2-羥基乙基)亞胺基-參(羥基甲基） 甲院 (bis(2-hydroxyethyl)imino-tris(hydroxymethyl) methane)) 〇 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（句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。Superparamagnetic particles with superparamagnetic particles will be magnetically induced by the magnetic field and will accumulate to the N or S pole of the magnetic field. After the magnetic field is removed, the magnetic moment disappears and recovers. Re-dispersed until it is not magnetic. Since iron oxide 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 particle size 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 the fact that the size of the microparticles is still relatively large relative to the fineness of the microparticles is that they cannot effectively enter the fine tissue region. Recently, due to the development of nano-particles, the particle size has been greatly reduced, and early micro-particles have not been able to enter the human area. Therefore, a large number of developments have begun, and the micro-vessel regions have been reached, and extensively In the field, such as magnetic resonance angiography, thermotherapy, etc., 22 paramagnetic nanoparticles also have the property of controllable carriers, so it has moved a new milestone in the automation of the biomedical industry. In the formula, Yu Yucheng has ultra-smooth iron oxide particles. In the door%• solution solution with diterpene iron ions and ferric iron ions, the acid value is adjusted to 9 i 14 , so ^ ί 5 200845055 The precipitate of triiron. However, this traditional method needs to control the molar ratio of trivalent and divalent iron to Fe3+: Fe2+= 2 : i 'and need to be carried out under an anaerobic ring, otherwise it will affect the crystal form of the iron oxide particles, and the iron particles will Oxidation continues during the preparation process, which in turn affects the magnitude of the saturation magnetization of the iron oxide particles. The solution is to carry out the reaction under nitrogen or to change the S in the organic phase to slow the oxidation; but the ferrous ions are easily oxidized to trivalent, which needs to be freshly configured, and it is difficult to precisely control the Mo in the reaction. It will increase the difficulty of a lot of synthesis. In addition, the early iron ions of antimony trioxide are not in the highest oxidation state, so these irons are slowly oxidized in the air to be non-paramagnetic or magnetic rather than iron oxide. In general, the surface of the ferroferric oxide particles is coated with a molecular polymer or a dioxo-cut inorganic oxide, but such a defect has the disadvantage of not being able to withstand high temperatures and acid resistance for a long time, = to a limit. From the application, the application, and the disadvantages of the prior art, the present invention utilizes the characteristic thermal decomposition property of urea, and only requires the formation of the anti-acidity of the superparamagnetic oxygen particles having a high saturation magnetization in the main and the bismuth ions. Enhanced 'and can accept high temperature and high pressure sterilization treatment ceremony = 2 矽 矽 coated iron oxide particles can be further: :: with the application of protein or other compounded iron particles. "Additional superparamagnetic 虱" [Invention] In view of this, the object of the present invention is that the scorpion, the manufacturing method is compared with the conventional magnetic granules, using a single source of divalent iron ions, and the urea is specially used for the temperature 6 200845055 Temperature and low temperature two-stage coating m = ^ Pi Zhen | 'Using high = good, not in the shirt solution = 4; 屮; . Layer: a variety of tables _: with = = uniform oxidation of the core of the main oxidation 0 The granules 'which are resistant to strong acid' and can be subjected to high temperature and high pressure extinction = emulsification eve coating, so that the surface of the smelting alcohol is subjected to acid treatment, and the superparamagnetic particles having the action of absorbing acid in the environment are The high-salt beauty stone ^ hair / month of the other purpose is to provide a surface through the functional i and other officials ii: two Γ two 吏: with an amine group, a carboxyl group, with the fish 疋 white two 5 reuse Agent or binder, carrier, can be applied to purely: to become a specific adsorption (8) ϋ (4) compound powder, urea and water mixing and stirring, in the environment / to carry out the reaction of the reaction 1 to (b) = r: = diamine water and organic 7 200845055 (C) Add tetraethyl orthosilicate (TEOS) to the above mixture, 50 ° C to 7 (reaction at a temperature of TC for 0.5 hour to 12 hours; and (d) after the temperature is lowered to 25 ° C to 30 ° C, additional tetraethyl phthalate is added, and the reaction is carried out for 1 hour to 12 hours to form The cerium oxide coated iron oxide particles have superparamagnet. In a preferred embodiment of the invention, the foregoing manufacturing method further comprises the following steps to produce an activated superparamagnetic particle: (e) The above-mentioned step (d) has the cerium oxide-coated iron oxide particles mixed with an acidic buffer having a pH of less than 6. In a preferred embodiment of the present invention, the aforementioned acidic buffer is sarcophagus. Acid salt buffer, acetate buffer, MES buffer (2-(N-morpholino) ethanesulfonic acid) or BlS_Tris buffer (bis(2-hydroxyethyl) (bis(2-hydroxyethyl)imino-tris(hydroxymethyl) methane)) In a preferred embodiment of the invention, the aforementioned manufacturing method further comprises the following steps, Producing superparamagnetic particles with functional groups on the surface • Sub: (f) The above steps (the sentence has oxidized The coated iron oxide particles into an organic polar solvent; (G) were added with mixing silane-functional group of the compound, so that the surface of the iron oxide particles coated with silicon dioxide having a functional group.

在本發明之較佳實施態樣中，前述極性有機溶劑 ^ 丙酮、DMS0 (二曱亞砜（dimethyl sulfoxide))、DMF N，N 一 甲基甲酿胺（Ν,Ν-dimethylformamide) )、NMP N甲基-2』比嘻咬酮（N methyl 2_pyrr〇lid〇ne))或異丙 醇0 8 200845055 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、十八基和笨基 所組成之組群。In a preferred embodiment of the present invention, the aforementioned polar organic solvent, acetone, DMSO (dimethyl sulfoxide), DMF N, N-methylformamide, NMP In a preferred embodiment of the invention, the aforementioned functional group is selected from the group consisting of an amine group and a sulfhydryl group. N methyl-2′′ is more than N 2 2 py 酮 酮 酮 或 或 或 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 a group consisting of a hydroxyl group, a methyl group, an octyl group, an octadecyl group, and a stupid group.

在本發明之較佳實施態樣中，前述矽烷化物包含丫_ 胺基丙基二乙氧基梦烧（y_amin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 三甲氧基矽烧（glycidoxypropyl trimethoxysilane)、甲基 二乙氧基石夕烧（methyl triethoxy silane)、正辛基三乙氧 基石夕烧（n-octyl triethoxysilane)、十八基三曱氧基石夕烧 (octadecyl trimethoxysilane )或苯基三乙氧基矽烷 (phenyl triethoxysilane )。 丰發明另提供一種超順磁性粒子，其係藉由前述 ^目關^法製得，其中前述超順磁性粒子的主要成分為四 氧化二鐵’二氧化石夕的含量為0.05重量％至40重量％， 且其粒控範圍為100 nm至10 μιη。 在本备明之較佳貫施恶樣中，前述超順 之飽合磁化量大於35emu/g。 『祖于 义本發明並提供一種活化之超順磁性粒子，其係藉 由箣述相關方法製得，在高鹽環境下對核酸分子具有強 吸附力。其中高鹽環境較佳係指0.5M至81V[的NaCl、In a preferred embodiment of the present invention, the decanoate comprises y_aminopropyl propyl ethoxylate (y_amin〇pr〇Pyl triethoxysilane), (3. thiopropyl propyl) trimethoxy decane ( (3-Mercaptopropyl)trimethoxysilane), glycidoxypropyl trimethoxysilane, methyl triethoxy silane, n-octyl triethoxy sulphide (n -octyl triethoxysilane), octadecyl trimethoxysilane or phenyl triethoxysilane. The invention further provides a superparamagnetic particle obtained by the foregoing method, wherein the main component of the superparamagnetic particle is Fe2O2, the content of the dioxide is 0.05% by weight to 40% by weight. %, and its particle control range is from 100 nm to 10 μιη. In the preferred embodiment of the present invention, the supersaturated saturation magnetization is greater than 35 emu/g. The ancestors of the present invention provide an activated superparamagnetic particle which is obtained by a related method and has a strong adsorption force for nucleic acid molecules in a high salt environment. The high salt environment preferably means 0.5M to 81V [NaCl,

Nal、GuHCl 或 GuSCN。 ^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。 早矣= 本發明之較佳實施11樣巾，若前述超順磁性粒 子表面所具有的官能基是胺基，則其 物舌=修飾’使其可與具㈣基官能基之 在本發明之較佳實施態樣中，前述羥基活化劑係 200845055 EDAC、CMC (1_環己基_3_(2_嗎啉基乙基）碳化二亞胺Nal, GuHCl or GuSCN. The invention further provides a superparamagnetic particle having a functional group on its surface which is produced by the aforementioned related method. According to a preferred embodiment of the present invention, if the functional group of the surface of the superparamagnetic particle is an amine group, the tongue is modified to make it compatible with the functional group having In a preferred embodiment, the aforementioned hydroxyl activator is 200845055 EDAC, CMC (1_cyclohexyl_3_(2-morpholinylethyl)carbodiimide

(l-cyclohexyl_3-(2_morpholinoethyl)carbodiimide) )、DCC (二環己基碳化二亞胺（diCyCl〇hexyl 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 馨鏈黴親合素（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鱗 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 )、ID A (亞胺基二乙酸 (iminodiacetic acid))或 EDTA(乙底酸 ethylenediamine- tetraacetic acid) 〇 _ 綜上所述，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 子，並利用改良的一氧化石夕包覆技術，使所製得之有二 氧化石夕包覆的氧化鐵粒子除了極佳的磁反應性外，更具 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 200845055 氧5鐵粒子。將二價鐵化合物粉末、尿素和 ί 8g°c 1贿的常壓環境下進行迴流反 Γ ^，其中二價鐵離子在水中會慢H彳 氣:，素在熱水中會分解成氨水= 氧基會從迴流管排出，而氨水則會提供氫 /夜的I鹼值慢慢升高，產生以下的顏色反應： 黃 褐 色 1^1 f->!-§ l^t_|(l-cyclohexyl_3-(2_morpholinoethyl)carbodiimide), DCC (diCyCl〇hexyl 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 protein or scale with histidine. Acidified protein. In a preferred embodiment of the present invention, the chelating agent is nitrilotriacetic acid, ID A (iminodiacetic acid) or EDTA (ethylenediamine-tetraacetic acid) 〇 In summary, the present invention provides a method for producing superparamagnetic particles. Compared with the conventional coprecipitation method, the present invention can synthesize a supersaturated magnetized superparamagnetic iron oxide particle by using only a single iron ion source. And using the modified nitric oxide coating technology, the iron oxide particles coated with the dioxide dioxide have excellent acid resistance and high temperature resistance. High pressure treatment. 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 a highly saturated magnetization amount of 200845055 oxygen 5 iron particles in the presence of only divalent iron ions. The divalent iron compound powder, urea and ί8g°c 1 are subjected to reflux under normal pressure, wherein the divalent iron ions are slow in the water and H彳 gas: the pigment is decomposed into ammonia in hot water = The oxy group will be discharged from the reflux tube, and the ammonia will provide a hydrogen/night I base value which will gradually increase, resulting in the following color reaction: yellow brown 1^1 f->!-§ l^t_|

Fe2+ Fe3+Fe2+ Fe3+

Fe304 手合遙當驗度達到9以上時，二價鐵離子與三價鐵離 ;· /、沈丨殿反應’開始生成黑色的四氧化三鐵粒子 ，而水中未反應的二價鐵離子繼續反應，在前述晶 二2面开/成四氧化三鐵沈殿，使粒徑逐漸增加。因此， 整尿素與二價鐵離子的莫耳比，來控制晶核生 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 pe2++〇2 + 2 H20-> 4 Fe3++ 4 ΟΗ" 2) C〇_2)2 + H2〇 Κ8〇至 11〇〇C) _> 2腿肩 2ΝΗ3 + 2Η2〇->2ΝΗ4〇Η^>2ΝΗ4+ + 2 0Η' 3) Fe2+ + 2 Fe3+ + g Off Fe304 + 4 H20 全反應式如下：When the Fe304 hand-in-hand test reaches 9 or more, the divalent iron ion and the trivalent iron are separated; · /, Shen Shen Dian reaction 'starts to generate black ferroferric oxide particles, while the unreacted divalent iron ions in the water continue to react. The crystal 2 is opened/formed into a triiron tetroxide, and the particle size is gradually increased. Therefore, the molar ratio of the urea to the divalent iron ions is used to control the number of crystal nuclei; or the reaction time after the formation of the ferroferric oxide is adjusted to control the particle size range of the iron oxide particles to be 100 nm to 10 μm. The reaction mechanism for producing superparamagnetic particles of the present invention is as follows: 1) 4 pe2++〇2 + 2 H20-> 4 Fe3++ 4 ΟΗ" 2) C〇_2)2 + H2〇Κ8〇 to 11〇〇C) _&gt 2 leg shoulder 2ΝΗ3 + 2Η2〇->2ΝΗ4〇Η^>2ΝΗ4+ + 2 0Η' 3) Fe2+ + 2 Fe3+ + g Off Fe304 + 4 H20 The overall reaction formula is as follows:

FeCl2 + 3 CCKNH^ + ^ 〇2 + 6 H20 Fe304 + 6 NH4CI + 3 C02 四氧化三鐵粒子在空氣中會漸漸氧化成不具順磁 或磁性較弱的三氧化二鐵。本發明利用改良的二氧化 200845055 :夕術在四氧化三鐵粒子表面形成包覆層，降低1 ί 先在高溫下反應，生成厚實的二氧化石夕 ^ :後在低溫下反應，將表面空隙填補完整，並在表 = 性的矽烷醇基。由前述方法所製得的有二 並可接之鐵粒子’其核殼結構的抗酸性增強， I了接又间>皿鬲壓滅菌處理。 Θ述有二氧化矽包覆之氧化鐵粒子可經進一步酸 f ’增加其表面的魏醇基，如此可在高鹽環境下作 的载體，但卻不會吸附蛋白質與其他污染 子步經帶有官能基的魏修飾，使氧化鐵粒 鐽2二：有g能基，而與蛋白質或其他化合物進行化學 磁性氧化鐵粒子的應用層面。此類磁性 中不化分子生物檢驗分析之前處理步驟 礤枓一為’粒徑大於5〇 nm的磁性粒子不具超順 於明沾ii遲滞現象（remanentmagnetism)。但在本 法中：首先生成奈米級的氧化鐵晶核，再 表層繼續成長，因此，即便氧化鐵粒子的粒 &已達〇 μιη，仍保有超順磁性，且其飽和磁化量大於 另—方面，可藉由增加氧化鐵粒子的粒捏、 二ί化㈣包覆來增加單難子Μ氧化_ 加;縮短磁反應時間。本發明的超順磁性粒子在外 不引下’磁反應時間小於2秒。如此強的磁力 短磁反應時間，也可以在1公升、甚至1〇〇 a升的大反應體積中進行操作。 载，磁順磁性的粒子在外加磁場移離後，不需加 ΐ。句重新分散在溶液中，繼續反 k項彳寸〖生此夠私加異相反應的均勻性。 -氧化硬的包覆須在氧化鐵粒子製備完成後馬上 12 200845055 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、1-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、氫氧化舒、氫 氧化四甲基銨、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石夕酸四乙 醋’在5〇t：至7〇t：的溫度下反應0·5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 。之後將溫度降至25。〇至FeCl2 + 3 CCKNH^ + ^ 〇2 + 6 H20 Fe304 + 6 NH4CI + 3 C02 The triiron tetroxide particles are gradually oxidized in the air into ferric oxide which is not paramagnetic or weak magnetic. The invention utilizes the improved dioxide oxidation 200845055: the formation of a coating layer on the surface of the ferroferric oxide particles, reducing the temperature of 1 ί firstly reacting at a high temperature to form a thick silica dioxide: after reacting at a low temperature, the surface void Fill in the complete, and in the table = sex stanol groups. The iron particles prepared by the foregoing method have an enhanced acid resistance of the core-shell structure, and the mixture is sterilized by a press. It is stated that the cerium oxide-coated iron oxide particles can further increase the surface of the thiol group via the acid f', so that the carrier can be used in a high salt environment, but does not adsorb proteins and other polluting sub-steps. The Wei modification with a functional group allows the iron oxide particles to be ruthenium 2: has a g-energy group, and is applied to a chemical magnetic iron oxide particle with a protein or other compound. The processing steps before such molecular magnetic bioassay analysis are as follows: magnetic particles having a particle diameter greater than 5 〇 nm do not have super-retentive ii hysteresis (remanentmagnetism). However, in this method, the nano-sized iron oxide crystal nucleus is first formed, and the surface layer continues to grow. Therefore, even if the particles of the iron oxide particles have reached 〇μιη, the superparamagnetism is retained, and the saturation magnetization is greater than the other. On the other hand, it is possible to increase the single-difficult enthalpy oxidation by adding the granule pinch and the bismuth (4) coating of the iron oxide particles; shortening the magnetic reaction time. The superparamagnetic particles of the present invention are not subjected to a magnetic reaction time of less than 2 seconds. Such a strong magnetic short magnetic reaction time can also be operated in a large reaction volume of 1 liter or even 1 〇〇 a liter. The magnetic paramagnetic particles do not need to be twisted after the external magnetic field is removed. The sentence is redispersed in the solution, and the anti-k term is continued. The uniformity of the heterogeneous reaction is sufficient. - The oxidized hard coating shall be carried out immediately after the preparation of the iron oxide particles 12 200845055 to prevent the continued oxidation of the ferroferric oxide. First, the iron oxide particles are dispersed in a mixture of water and an alcohol such as methanol, ethanol, isopropanol, 1-propanol, 1-butanol, 2-butanol, tert-butanol, isobutanol, and the like. Then add ammonia or other test solution (such as sodium hydroxide, hydrazine, tetramethylammonium hydroxide, tetraethylammonium hydroxide), and control the reaction environment to perform ultrasonic vibration. Adding tetrahydroacetic acid of the mixture to the above mixture at a temperature of 5〇t: to 7〇t: for 0.5 hours to 12 hours; by controlling the addition amount of tetraethyl myristic acid, two The thickness of the oxidized oxide coating is limited to from 1 nm to 1 . Then the temperature is reduced to 25. 〇到

3〇°C ’另外加入矽酸四乙酯，反應1小時至I]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L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， 若次泡在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Further, tetraethyl citrate was added at 3 ° C to react for 1 hour to 1 hour, which was completed by a slow reaction rate. The modified core-shell structure L 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 the ceria coated with iron oxide particles has a stanol group. If the sub-bubble is in an acidic buffer solution having a pH of less than 6, the surface of the ceria can be activated, and the number of stanol groups is increased, and such activation is super Shun

磁=粒子在 0.5M 至 8M 之 NaCl、NaI、GuHCl 或 GuSCN 的高鹽環境下，對核酸分子有很強的吸附力，且超順磁 Ϊ子，結ί不會被破壞，至於蛋白質分子與其他污染 ，則可藉由簡單的清洗步驟去除。可對所得之純化核 ^進行聚合酶連鎖反應（polymerase chain reaction， 廿备疋匕放大，其結果如第四圖所示，純化核酸中 並”、、A白貝和其他會抑制pcR反應的物質存在。 节,| f 化矽包覆之氧化鐵粒子移至極性有機溶 jit ，再加入具有官能基的矽烷化物（如厂 氧齡垸），使氧化鐵粒子表面具有“ 基（例如胺基）。之後可利用經基活化劑（如EDAC)做 13 200845055 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1 :超順磁性氧化鐵粒子的小董製備 _ 將0·25克二氯化鐵粉末與6克尿素結晶顆粒溶解 在1〇〇毫升去離子水中，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。 9 實施例2 :超順磁性氧化鐵粒子的大量製備 將60克一氣化鐵粉末與90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，拌3分鐘，並將混合物移入溫度設定為95〇c的油鍋 迴流反應12小時，生成黑色氧化鐵粒子，靜置沈 澱^，將接近澄清的上清液吸出，沉澱物用去離子水清 洗二次，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。 實施例3 :二氧化矽的包復 14 200845055 將30克由實施例！或實施例 分散在225毫升去離子水中，再加』化鐵粒子 水，然後加入900毫升異丙醇，將升28%的氨 應器中持續授拌，並進行超以=在，璃4反 ^的魏四以旨’並將水溫升高至5代’ 小蚪；之後將水溫降至室溫（約25ΐ)，、=反2 的矽酸四乙酯，持續反應丨小時。反應—°毽=升 澱’將透明上清液吸出’细去離子二Magnetic = particles in the high salt environment of 0.5M to 8M NaCl, NaI, GuHCl or GuSCN, have a strong adsorption of nucleic acid molecules, and superparamagnetic scorpion, the knot will not be destroyed, as for protein molecules and Other contamination can be removed by a simple cleaning step. The obtained purified nucleus can be subjected to a polymerase chain reaction (polymerase chain reaction, and the results are as shown in the fourth figure, and the nucleic acid is purified), A white shellfish and other substances which inhibit the pcR reaction. The rhodium-coated iron oxide particles are moved to a polar organic solution jitt, and then a functional group-containing decane compound (such as a plant oxygen age enthalpy) is added to make the surface of the iron oxide particle have a "base (for example, an amine group). It can be further modified with a trans-activator (such as EDAC) 13 200845055 to make superparamagnetic particles and proteins (such as Protein A, Protein G, Antibody, Enzyme or Streptavidin). (Streptavidin)), which is then used to purify the antibody; it can also be further modified with a chelating agent (such as triacetoxy) to sequester the superparamagnetic particles with a divalent or trivalent transition metal ion, and then To purify a protein with histidine or a phosphorylated protein. EXAMPLES Example 1: Preparation of super-paramagnetic iron oxide particles by Xiaodong_ 0. 25 g of iron dichloride powder and 6 g of urea crystal particles Dissolve In 1 ml of deionized water, stir in a glass reactor equipped with a condenser tube for 10 minutes to form a yellow-green clear solution, and transfer to 9 Torr. The crucible oil pan was refluxed for 12 hours to form black iron oxide particles. After standing to settle, the supernatant which was nearly clear 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. The precipitate was kept in an aqueous solution before the coating with cerium oxide. Avoid drying. 9 Example 2: Preparation of superparamagnetic iron oxide particles 60 g of iron-fibride powder and 90 g of urea crystal particles were dissolved in 1 liter of deionized water in a glass reactor equipped with a condenser tube. Quickly, mix for 3 minutes, and transfer the mixture to a pressure cooker set to 95 °c for 12 hours to form black iron oxide particles, leave the precipitate, and aspirate the supernatant. The precipitate is deionized water. After washing twice, the ammonium ions and chloride ions remaining in the reaction are removed. Before the coating with cerium oxide, the precipitate is kept in an aqueous solution to avoid drying. Preparation of yttrium oxide 14 200845055 Disperse 30 grams from the example! or example in 225 ml of deionized water, add iron particle water, then add 900 ml of isopropanol, which will be 28% in ammonia Continue to stir mix, and carry out the super-in =, glass 4 anti-Wei four to the purpose of 'and the water temperature to 5 generations' small 蚪; after the water temperature is reduced to room temperature (about 25 ΐ), = = anti 2 tetraethyl citrate, continuous reaction for a few hours. Reaction - ° 毽 = ascending precipitation 'suck the clear supernatant 'fine deionized two

=醇的味道為止。實施例3製成之有 超順磁粒子之影像圖如第一圖所示。 7匕使之 實施例4 :表面活化處理 將實施例3的有二氧化石夕包覆之氧化鐵粒子靜置 ί ί 加入300毫升i〇 mM的磷 酉欠鹽綾衝液（pH 5·5)，清洗兩次，之後在相 1持續 5小時，再以去離子水清洗，重新分散在酸 鹼值8的缓衝液中’即可製得具有活化之二氧化 的超順磁性粒子。 ^ 實施例5 :表面官能基的修飾 將實施例3的有二氧化;g夕包覆之氧化鐵粒子靜 去除上清液後，從中取30克加入250毫升無水丙_， 洗兩次，之後將前述粒子分散在5〇〇毫升丙酮中， 績攪拌10分鐘，再加入3·5毫升γ-胺基丙基三乙氧基矽 烷，在室溫下反應4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丙酮的味道為 止，即可製得表面具有胺基官能基的超順磁性粒子。 實施例6:核酸的純化 15 200845055= the taste of alcohol. An image of the superparamagnetic particles prepared in Example 3 is shown in the first figure. 7: Example 4: Surface activation treatment The iron oxide particles coated with the oxidized cerium oxide of Example 3 were left to stand. ί ί 300 300 300 300 300 300 300 300 300 300 300 300 300 300 300 300 300 300 300 300 300 300 300 300 300 300 After cleaning twice, then in phase 1 for 5 hours, then washed with deionized water and redispersed in a buffer of pH 8 to obtain superparamagnetic particles with activated dioxide. ^ Example 5: Modification of surface functional groups. After the removal of the supernatant of the iron oxide particles of Example 3, the supernatant was removed, and 30 g of 250 g of anhydrous propylene was added thereto, and washed twice. The above particles were dispersed in 5 ml of acetone, stirred for 10 minutes, and then added with 3.5 ml of γ-aminopropyltriethoxydecane, and reacted at room temperature for 4 hours. After completion of the reaction, the precipitate was allowed to stand, the clear supernatant was aspirated, and washed with deionized water until the taste of acetone was not obtained, and superparamagnetic particles having an amine functional group on the surface were obtained. Example 6: Purification of nucleic acids 15 200845055

取分散在100微升中的lxl05個TSGH-8301細胞加 入400微升的細胞裂解液（i20g GuSCN，100ml 0.1MAdd 1 ll05 of TSGH-8301 cells dispersed in 100 μl to 400 μl of cell lysate (i20g GuSCN, 100ml 0.1M)

Tris-HCl ρΗ6·4，22ml 0.2M EDTA ρΗ8·0，2.4ml Triton X-100)，反應10分鐘。之後加入50微升15〇 mg/ml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100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。將混合物置於磁座上，將上清液吸出，加入8〇〇 微升的清洗液（120g GuSCN，100ml 0.1M Tris-HCl _ ρΗ6·4)，清洗兩次；再加入800微升7〇%乙醇，清洗兩 次。之後將氧化鐵粒子靜置10分鐘，使清洗液中的酒 精揮發；再加入100微升的沖提液（10 mM Tris pH 8)， 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IxlO5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5x105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 :抗體的純化Tris-HCl ρΗ6·4, 22 ml 0.2 M EDTA ρΗ8·0, 2.4 ml Triton X-100), and reacted for 10 minutes. Then, 50 μl of 15 〇mg/ml of superparamagnetic particles having an activated ceria surface prepared in Example 4 was added and 100 μl of the linking solution (isopropyl alcohol) was added and uniformly mixed for 10 minutes. The magnetic particles are linked to the nucleic acid produced after cell lysis. The mixture was placed on a magnetic stand, the supernatant was aspirated, and 8 μL of a washing solution (120 g GuSCN, 100 ml 0.1 M Tris-HCl _ ρΗ6·4) was added and washed twice; then 800 μl 7 加入 was added. % ethanol, washed twice. The iron oxide particles were then allowed to stand for 10 minutes to evaporate the alcohol in the cleaning solution; then 100 μl of the extract (10 mM Tris pH 8) was added and uniformly mixed for 10 minutes. Then the reaction was placed on a magnetic stand. The supernatant was aspirated and placed in a clean container, which was the purified nucleic acid. The results were as shown in Figure 4, where the Μ1 kb nucleic acid standard solution; E1 to E3 were three replicates of IxlO5 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, and C3 is 5x105 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 does not have an egg white matter and other substances which inhibit the PCR reaction. Example 7: Purification of antibodies

取10微升的血清，加入900微升的連結液（10mM 磷酸鹽，150 mMNaCl，pH 7.5)，再加入 20 微升 50 mg/ml 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，150 mM NaQ，pH 7·5)，清洗兩次；再加 16 200845055Take 10 μl of serum, add 900 μl of ligation solution (10 mM phosphate, 150 mM NaCl, pH 7.5), and add 20 μl of 50 mg/ml superparamagnetic particles bound with protein A (using Example 5). The surface-modified amine-based superparamagnetic particles were further modified by a base activator and then 'bonded to protein A' and uniformly mixed for 30 minutes. Place the mixture on the magnetic base, aspirate the supernatant, add 500 μl of washing solution (1 mM sulphate, 150 mM NaQ, pH 7.5), wash twice; add 16 200845055

入100微升的沖提液（0·5 IV[甘胺酸，〇15 MNaC卜pH 2·5)，均勻混合10分鐘，接著將反應物置於磁座上， 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 Tds，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，1 中Μ為蛋白質標準液，S為稀釋2〇倍的企清，為夫赫 磁性粒J吸_蛋白質，W1為第〜欠清洗液的產物' W2為第二次清洗液的產物，E1為第一次沖提液的產 物，其產量大約占總量的60至70%，E2為第二次沖提 液的產物，其產量大約占總量的3〇至4〇%。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7〇%]、IgM [約 6%]、IgA [約 1〇 至 20%]、IgE [約 〇 〇〇1%]和砂[約 〇·2%])^分子量分佈在15〇1^&至9〇〇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 ehain)兩部分，其中各免疫球蛋 白的重鏈大小係在50让1^至751^^範圍内，而數量最 多之IgG的輕鏈大小為25kDa，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，直 之免疫球蛋白（IgM、IgA、IgE^IgD)_t^ =kDa上形成較弱的帶。而電泳片上大於75 ]^^的 ^ ^要是齡解丨段㈣結合而成的半產㈣完整 免疫球I白。 實施例8 :本發明舆市㈣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 200845055 12·5 mg本發明與兩種市售磁性粒子，再分別加入500 的2 M HC1，混合均勻後，將磁性粒子磁吸至底部，量 ，上清液於290nm的吸光值（α29{))。在0至1〇分鐘時， 母2分鐘量測一次；在至6〇分鐘時，每5分鐘量測 一次。由於Fe3+離子的吸收峰在290nm，因此，若前述 上清液的A·值在60分鐘内有上升，則表示該磁性粒 子的二氧化矽層被酸破壞，使氧化鐵裸露並開始氧化， 形成Fe3+。故在前述測定中，a29g值未上升者判定為 「佳」，而值上升者則判定為「不佳」，如下表1和 第三圖所示。 2)耐高溫高壓處理的測定 在2 ml聚丙烯的樣品管中，分別加入ι·25 mg本發 明與兩種市售磁性粒子，再分別加入丨ml去離子水，混 合j勻後，半鎖緊上蓋，放入滅菌鍋中，進行高溫高壓 滅，:溫度設定120°C，持續40分鐘，之後在顯微鏡下 進行觀察磁性粒子表面二氧化矽層脫落的情形，其結果 如了表1和第二圖所示。在下表〗中，「佳」代表表面 二氧化矽層未有脫落的情形，「可」代表表面二氧化矽 >^有$微脫落的情形，而「差」代表表面二氧化石夕層有 嚴重脫落的情形；而在第二圖中，A-1和A_2分別為本 發，之超順磁性粒子在高溫高壓處理前後的外型，表面 氧化咬層未有脫洛的情形，B-1和B-2分別為Qiagen 之磁性粒子在高溫高壓處理前後的外型，表面二氧化矽 層有些微脫落的情形·· C-1和C-2分別為invitrogen之 磁性粒子在高溫高壓處理前後的外型，表面二氧化矽層 有嚴重脫落的情形。 此外，磁性粒子是否耐高溫高壓處理，亦可配合下 文戶ί述L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 200845055 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 磁吸時間的測定係在25 S 3(TC下進行，將磁珠置 於1 ml或40 ml的去離子水中。若去離子水的體積為i πύ，則利用小磁鐵進行測試；若其體積為4〇 -广則利 用大磁鐵進行測試。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，測試結果如下表1所示。與市售產品比較，在反 應體積大小不同的測試中可明顯看出，若反應體積俞 大，則磁性愈強的磁性粒子，其反應操作的時^越短: 本發明 "— -— Qiagen Invitrogen 顏色 黑色 音福氙 粒振（μπι) 2-10 2-10 汽 [i 〜1 儲藏液的pH值 5-6 L 6-7 5 对酸牲 佳 ------- 佳 不佳 耐高溢高愿 佳 可 差 磁吸時間 (操作艟積1 ml) 小於2秒 ——---- 小於4秒 小於1分鐘 磁吸時間 (操作親積40 ml) 小於20秒 小於40秒 5-10分鐘 高潘高壓處理後的磁吸 小於2秒 時間（操作遁積1ml) 註：1) /j、磁鐵的會吾：Q/： 小於6秒 小於1分鐘 2)大磁鐵的重置· 94.2 g (4〇mi的磁吸時間測試用） 【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4,約500 nm )的電子顯微鏡圖（4〇〇,〇〇〇χ放大圖）。 > .本發明之超賴性粒子與料磁性粒子經 南溫商壓處理讀麵魏下的4GGX放大圖。 200845055 磁性粒子的抗酸性比較圖。 的DNA電°泳圖發明之超順磁性粒子進行核酸純化 的蛋二^泳=本發明之超順磁性粒子進行抗體純化 【主要元件符號說明】Add 100 μl of the extract (0·5 IV [glycine, 〇15 MNaC Bu pH 2·5), mix uniformly for 10 minutes, then place the reaction on the magnetic stand, the supernatant is aspirated and placed clean. In a container, add 5 μl of neutralizing solution (1 M Tds, pH 9.0), move the magnetic particles, and repeat the stripping step to obtain the purified antibody. The results are shown in Figure 5, 1 Μ is a protein standard solution, S is 2 稀释 diluted by Qiqing, is a magnetic particle J _ protein, W1 is the product of the first ~ under-cleaning solution 'W2 is the product of the second cleaning solution, E1 is the first The product of the secondary extract has a yield of about 60 to 70% of the total amount, and E2 is the product of the second extract, and its yield is about 3 to 4% of the total. As is apparent from the results of the fifth graph, the superparamagnetic particles prepared by the present invention can be used as an antibody for purification by surface functional group modification. Various immunoglobulins in serum (IgG [about 7〇%], IgM [about 6%], IgA [about 1〇 to 20%], IgE [about 〇〇〇1%] and sand [about 〇·2%] The molecular weight distribution ranges from 15〇1^& to 9〇〇kDa. Before the electrophoresis is confirmed, each sample must be heated to boil, so that the protein can move in the electrophoretic film. The immunoglobulin after heating and boiling is divided into two parts: a heavy chain and a light ehain, wherein the heavy chain size of each immunoglobulin is in the range of 50 to 1^ to 751^^, and the number is the largest. The IgG has a light chain size of 25 kDa. These two proteins form two distinct major bands on E1 and E2. In addition, the immunoglobulins (IgM, IgA, IgE^IgD)_t^ = kDa are formed. Weak band. On the electrophoresis sheet, ^ ^ is greater than 75 ^ ^ ^ ^ ^ is the age of the sputum section (four) combined semi-production (four) complete immune ball I white. Example 8: Comparison of the bismuth (four) particles of the present invention 1) Determination of acid resistance In a plastic colorimetric tube of 1 ml of ultraviolet light, respectively, 17 200845055 12·5 mg of the present invention and two commercially available magnetic particles were added. Then, 500 M 2 of HC 1 was separately added, and after mixing uniformly, the magnetic particles were magnetized to the bottom, and the amount of the supernatant was absorbed at 290 nm (α29{)). At 0 to 1 minute, the mother measures once every 2 minutes; at 6 minutes, it measures every 5 minutes. Since the absorption peak of Fe3+ ions is 290 nm, if the A· value of the supernatant rises within 60 minutes, it means that the ceria layer of the magnetic particles is destroyed by acid, and the iron oxide is exposed and oxidized to form. Fe3+. Therefore, in the above measurement, if the value of a29g is not increased, it is judged as "good", and if the value is increased, it is judged as "poor", as shown in Table 1 and Figure 3 below. 2) Determination of high temperature and high pressure resistance In 2 ml polypropylene sample tube, add ι·25 mg of the present invention and two commercially available magnetic particles, respectively, and then add 丨ml deionized water, mix j and then, semi-lock Close the lid, put it into a sterilizing pot, and perform high temperature and high pressure extinction. The temperature is set at 120 ° C for 40 minutes, and then the surface of the magnetic particle is removed under the microscope. The results are shown in Table 1 and The two figures are shown. In the following table, "good" means that the surface of the cerium oxide layer has not fallen off, "may" represents the surface cerium oxide > ^ has a micro-shedding situation, and "poor" represents the surface of the cerium dioxide layer In the second figure, A-1 and A_2 are the hairs of the hair, the shape of the superparamagnetic particles before and after the high temperature and high pressure treatment, and the surface oxidized bite layer has no detachment, B-1 And B-2 are the appearance of Qiagen magnetic particles before and after high temperature and high pressure treatment, and the surface of the ceria layer is slightly detached. · C-1 and C-2 are the magnetic particles of invitrogen before and after high temperature and high pressure treatment. Appearance, the surface of the ceria layer has severe shedding. In addition, whether the magnetic particles are resistant to high temperature and high pressure treatment can also be observed by the following changes in the magnetic reaction time after the treatment; if the magnetic reaction time becomes slow, it means that the high temperature and high pressure treatment will cause the magnetic particles to oxidize or 18 200845055 Its magnetic structure change ' indicates that the magnetic particles are not resistant to high temperature and high pressure treatment, as shown in Table 1. 3) Determination of magnetic attraction time The measurement of magnetic attraction time is carried out at 25 S 3 (TC), and the magnetic beads are placed in 1 ml or 40 ml of deionized water. If the volume of deionized water is i π ύ, use small The magnet is tested; if it has a volume of 4 〇-wide, it is tested with a large magnet. A large/small magnet is mounted on the magnetic base, and the container containing the magnetic particles and the reaction liquid is placed on the magnetic base to perform the magnetization time. Test, the test results are shown in the following Table 1. Compared with the commercially available products, it is obvious in the test of different reaction volume sizes that if the reaction volume is large, the magnetic particles with stronger magnetic properties, the time of the reaction operation Short: The invention "--- Qiagen Invitrogen color black tone Fu granule vibration (μπι) 2-10 2-10 steam [i ~ 1 storage solution pH 5-6 L 6-7 5 ------ 佳佳佳耐高高高愿佳差差磁时间(Operation hoarding 1 ml) Less than 2 seconds——--- Less than 4 seconds Less than 1 minute Magnetic time (Operational product 40 Ml) less than 20 seconds less than 40 seconds 5-10 minutes high magnetic pressure after the high pressure treatment is less than 2 seconds (operation hoarding 1ml Note: 1) /j, magnet of the meeting: Q /: less than 6 seconds less than 1 minute 2) large magnet reset · 94.2 g (4〇mi magnetic time test) [Simple diagram] Fig. 1 is an electron micrograph of a superparamagnetic particle (particle 4, about 500 nm) coated with cerium oxide (4 〇〇, 〇〇〇χ magnified view). > . The 4GGX magnified image of the super-reactive particles and the magnetic particles of the present invention processed by the south temperature commercial pressure processing surface. 200845055 Anti-acidity comparison chart of magnetic particles. DNA electrophoresis, the superparamagnetic particles of the invention are subjected to nucleic acid purification, and the antibody is purified by the superparamagnetic particles of the present invention.

2020

Claims (1)

200845055 、申請專利範圍： ι· 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8〇。〇 至not:的常壓環境下進行迴流反應！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； (c) 在箣述混合物中加入砍酸四乙酯，在％t至的 溫度下反應〇·5小時至12小時；及 (φ將溫度降至25。〇至30。〇後，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。 2·^請專利範圍第丨項所述之方法，其進 括下列 步驟： (e) 述步驟(d)之有=氧切包覆之氧化鐵粒子與 pH值小於6之酸性緩衝液混合攪拌。 範圍第2項所述之方法’其中前述酸性缓衝 液為％鲅鹽緩衝液、醋酸鹽緩衝液、 Bis_Tds緩衝液。 綾削u 4. ίί請專利範圍第1項所述之方法，其進—步包括下列 (f) 有二氧切包復之氧化鐵粒子移 前述有二 5·如申請專利範圍第4項所述 此基 #1 Λ 1¾ PI TXAyrQp, " / 其中前述極性有機 二由ί: 〇，、ΝΜΡ或異丙醇。 6.如申请專利範圍第4項所 醉 項所述之方法，其中前述官能基係 200845055 甲基、辛基、十八基和 選自由胺基、氳硫基、氫氧基 苯基所組成之組群。 7· 第乙之方法，其中前述魏化物 ^各丫胺基丙基二乙乳基矽烷、（3_氫硫基丙基)三曱 襄一氧丙氧基丙基三甲氧基石夕烧、甲基三乙氧基石夕烧、 "，基三乙氧基矽烷、十八基三曱氧基石夕烷、或笨基三乙 氧基發燒。200845055, the scope of patent application: ι· A method for producing superparamagnetic particles, comprising the following steps: (a) mixing and stirring ferrous compound powder, urea and water at 8 Torr. 〇 到到: The reflux reaction is carried out under normal pressure! After 24 hours, iron oxide particles are formed; (b) after washing the iron oxide particles, water, ammonia water and an organic alcohol solvent are added and stirred for ultrasonic vibration; (c) tetraethyl citrate is added to the mixture. The reaction is carried out at a temperature of %t to 〇·5 hours to 12 hours; and (φ reduces the temperature to 25 〇 to 30. After 〇, additional tetraethyl phthalate is added, and the reaction is carried out for 1 hour to 12 hours, forming two The cerium oxide-coated iron oxide particles have superparamagnetism. 2·^ The method described in the scope of the patent specification includes the following steps: (e) the step (d) has = oxygen-cut coating The iron oxide particles are mixed and stirred with an acidic buffer having a pH of less than 6. The method of the second aspect, wherein the acidic buffer is a % guanidinium buffer, an acetate buffer, or a Bis_Tds buffer. Ίί Please refer to the method described in the first paragraph of the patent, the following steps include the following: (f) the oxidized iron-coated particles having the dioxin-cutting amount are as described above, and the second base is as described in item 4 of the patent application scope. 1 Λ 13⁄4 PI TXAyrQp, " / where the aforementioned polar organic two by ί 6. The method of claim 4, wherein the aforementioned functional group is 200845055 methyl, octyl, octadecyl and selected from the group consisting of an amine group and a sulfonium sulfonate. a group consisting of a hydroxy group and a hydroxyphenyl group. 7. The method of the second method, wherein the carbamide compound is propylaminopropyl decyl decane or (3 hydroxythiopropyl) ruthenium Oxypropoxypropyltrimethoxycarbazide, methyltriethoxylate, ", triethoxy decane, octadecyltrimethoxy oxane, or stupid triethoxylate. •一種超順磁性粒子，其係藉由申請專利範圍第〗至7項 中任項所述之方法所製得，其中前述超順磁性粒子的 主要成分為四氧化三鐵，二氧化矽的含量為〇 〇5重量％ 至40重量〇/❹，且其粒徑範圍為1〇〇11111至1〇μιη。 9·如申凊專利範圍第8項所述之超順磁性粒子，其飽人 化1大於35 emu/g。 10·種活化的超順磁性粒子，其係由如申請專利範圍第2 項所述之方法製得，其在高鹽環境下對核酸分子具有 吸附力。 11· 一種表面具有官能基之超順磁性粒子，其係由如申請專 利範圍第4項所述之方法製得。 12·如申請專利範圍第11項所述之超順磁性粒子，若前述超 順磁性粒子表面所具有的官能基是胺基，則其可進一步 利用羥基活化劑修飾，使其可與具有羧基官能基之蛋白 質或化合物鍵結。 13·如申請專利範圍第12項所述之超順磁性粒子，其中前述 羥基活化劑係EDAC、CMC、DCC或DIC。 14·如申請專利範圍第12項所述之超順磁性粒子，其中前述 經沒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。 15·如申請專利範圍第14項所述之超順磁性粒子，其中前述 22 200845055 %白二=?。A、蛋白質G、抗體、酶或鏈黴親合素 超順磁性粒子，其表面係 •螯人:丨ί利乾圍第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。A superparamagnetic particle obtained by the method according to any one of claims 7-1 to 7, wherein the main component of the superparamagnetic particle is triiron tetroxide and cerium oxide. It is 〇〇5 wt% to 40 wt〇/❹, and its particle size ranges from 1〇〇11111 to 1〇μιη. 9. The superparamagnetic particle according to item 8 of the patent application scope, wherein the saturated one is greater than 35 emu/g. An activated superparamagnetic particle obtained by the method of claim 2, which has an adsorption force to a nucleic acid molecule in a high salt environment. A superparamagnetic particle having a functional group on its surface, which is obtained by the method of claim 4 of the patent application. 12. The superparamagnetic particle according to claim 11, wherein if the functional group on the surface of the superparamagnetic particle is an amine group, it can be further modified with a hydroxyl activator to have a carboxyl function. a protein or compound bond. 13. The superparamagnetic particle of claim 12, wherein the hydroxy activator is EDAC, CMC, DCC or DIC. The superparamagnetic particle according to claim 12, wherein the superparamagnetic particle modified by the virgin activator is further bonded to a protein to purify the antibody. 15. The superparamagnetic particle according to item 14 of the patent application, wherein the aforementioned 22 200845055 % white two =?. A, a protein G, an antibody, an enzyme or a streptavidin superparamagnetic particle, the surface of which is a chelate: a superparamagnetic particle according to item 16, wherein the chelating agent is a triacetate group. Ammonia, IDA or edta. 23 200845055 七、指定代表圖： (一) 本案指定代表圖為：第（一）圖。 (二) 本代表圖之元件符號簡單說明： 無23 200845055 VII. Designated representative map: (1) The representative representative of the case is: (1). (2) A brief description of the component symbols of this representative figure: None 八、本案若有化學式時，請揭示最能顯示發明特徵的化學式： 無8. If there is a chemical formula in this case, please reveal the chemical formula that best shows the characteristics of the invention: