TWI493042B - Early detection method for detecting HIV-1 from saliva samples and glycan-immobilized metal nanoparticles - Google Patents
Early detection method for detecting HIV-1 from saliva samples and glycan-immobilized metal nanoparticles Download PDFInfo
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本發明係關於一種聚糖固定化金屬奈米粒子於HIV-1檢測之應用，特別是關於一種自唾液檢體檢測HIV-1之早期檢測方法。The present invention relates to the use of a glycan-immobilized metal nanoparticle for the detection of HIV-1, and more particularly to an early detection method for detecting HIV-1 from a saliva sample.
人類免疫缺陷病毒(Human Immunodeficiency Virus,HIV)是一種感染人類免疫系統細胞的慢病毒(Lentivirus)，屬反轉錄病毒的一種。普遍認為，人類免疫缺陷病毒的感染導致愛滋病(AIDS,Acquired Immune Deficiency Syndrome，後天免疫缺乏症候群，或譯作「愛滋病」)，愛滋病是後天性細胞免疫功能出現缺陷而導致嚴重隨機感染及/或繼發腫瘤並致命的一種疾病。愛滋病自1981年在美國被識別並發展為全球大流行至2003年底，已累計導致兩千餘萬人死亡。人類免疫缺陷病毒通常也俗稱為「愛滋病病毒」或「愛滋病毒」。人類免疫缺陷病毒(HIV)分為兩型：HIV-1與HIV-2。多數國家的HIV感染是由HIV-1造成的，並且感染HIV-1後超過90%的患者會在10-12年內發病成為愛滋病；HIV-2主要分布在西部非洲，其感染往往沒有相關的病症。Human Immunodeficiency Virus (HIV) is a lentivirus that infects cells of the human immune system and is a type of retrovirus. It is widely believed that human immunodeficiency virus infection causes AIDS (Acquired Immune Deficiency Syndrome, or acquired AIDS), and AIDS is a defect in acquired cellular immune function that leads to severe random infection and/or secondary A disease in which a tumor is deadly. AIDS has been identified and developed into a global pandemic in the United States in 1981. By the end of 2003, it had caused more than two million deaths. Human immunodeficiency virus is also commonly known as "AIDS" or "HIV." Human immunodeficiency virus (HIV) is divided into two types: HIV-1 and HIV-2. HIV infection is caused by HIV-1 in most countries, and more than 90% of patients infected with HIV-1 become AIDS within 10-12 years; HIV-2 is mainly distributed in West Africa, and its infection is often not related. Illness.
生物細胞之表面被聚糖所覆蓋。聚糖在生體內具多樣之機能。病毒可以辨識存在於細胞表層之聚糖，並當成低親和性之受容體，利用此特性成為對細胞感染之第一步驟。病毒對高等動植物進行感染，動植物會產生感染症狀。此感染症會對同動植物種進行傳染。例如人類之流感的流行將危害健康，家畜、農作物或觀賞用動植物的場合也因為傳染造成生產損失。因此對感染動植 物之病毒，可以簡便且高感度來測定，對於感染症之早期診斷、醫療或預防是非常重要的。The surface of the biological cell is covered by glycans. Glycans have diverse functions in the living body. The virus recognizes the glycans present in the cell surface and acts as a low-affinity receptor. Using this property becomes the first step in cell infection. The virus infects higher plants and animals, and the animals and plants produce symptoms of infection. This infection can affect the same plant species. For example, the spread of human flu will endanger health, and the production of livestock, crops or ornamental animals and plants will also cause production losses due to infection. Therefore, the infection The virus can be measured easily and with high sensitivity, and is very important for early diagnosis, medical treatment or prevention of infectious diseases.
人類免疫缺陷病毒第一型(Human immunodeficiency virus type 1,HIV-1)不只存在於血液，也會存在於母乳、***、***分泌液、唾液、汗等體液。唾液中之HIV-1因量微，現在被開發的HIV-1感染唾液篩檢法，主要是以西方墨點法(Western blotting)來檢測唾液中抗HIV-1抗體。Human immunodeficiency virus type 1 (HIV-1) is not only found in the blood, but also in body fluids such as breast milk, semen, vaginal secretions, saliva, and sweat. The amount of HIV-1 in saliva is small, and the currently developed HIV-1 infection saliva screening method mainly uses Western blotting to detect anti-HIV-1 antibodies in saliva.
然而，因為唾液中之抗HIV-1抗體也非常微量，故檢測的精度並不高。唾液中之抗HIV-1抗體檢查與血液檢査同樣，在感染後大約3個月才會出現抗HIV-1抗體，此潛伏期間無法藉由前述方法發現HIV-1感染。在此我們著眼於即使是以西方墨點法也可以診斷且非侵入性、感度高的HIV-1診斷法之開發，亦即利用聚糖固定化金屬奈米粒子來捕捉病毒之濃縮法，以及PCR基因増幅法來檢測唾液中HIV-1。However, since the anti-HIV-1 antibody in saliva is also very small, the accuracy of detection is not high. Anti-HIV-1 antibody test in saliva As with blood tests, anti-HIV-1 antibodies appear only about 3 months after infection, and HIV-1 infection cannot be detected by the aforementioned method during this incubation period. Here we focus on the development of HIV-1 diagnostics that can be diagnosed and non-invasive and highly sensitive, even with Western blotting, that is, the use of glycan-immobilized metal nanoparticles to capture the concentration of the virus, and PCR gene swath method to detect HIV-1 in saliva.
緣此，本發明之一目的即是提供一種本發明者為由特定構造之聚糖與配體化合物相結合之配體複合體，並將其固定於金屬，形成所謂的聚糖固定化金屬奈米粒子。並利用病毒與聚糖之結合性，利用將病毒濃縮之方法，簡單且非侵入的由唾液檢體中將HIV-1病毒粒子捕捉濃縮、以達成HIV-1感染症之早期診斷法。Accordingly, it is an object of the present invention to provide a ligand complex which is a combination of a specific structure of a glycan and a ligand compound, and which is immobilized on a metal to form a so-called glycan-immobilized metal naphthalene. Rice particles. By using the combination of the virus and the glycan, the HIV-1 virion is easily and non-invasively captured by the method of concentrating the virus, and the HIV-1 virion is captured and concentrated to achieve an early diagnosis of HIV-1 infection.
本發明之實施例中，提供一種自唾液檢體檢測HIV-1之早期檢測方法，其步驟包括：提供一可辨識HIV-1病毒之聚糖固定化金屬奈米粒子；將該聚糖 固定化金屬奈米粒子與一唾液檢體接觸形成一混合液；將該混合液進行濃縮；以及經由一檢測手段判定該唾液檢體中是否含有HIV-1病毒；其中，該聚糖固定化金屬奈米粒子係由一連結子化合物之氨基與一聚糖之還原末端相結合所形成之配體複合體，該連結子化合物係為具有分子內氨基、硫原子、主鏈上有碳-氮鍵結之碳氫鏈之化合物，該配體複合體係藉由硫原子與一金屬奈米粒子結合，該聚糖係為可與HIV-1病毒相結合性之聚糖。In an embodiment of the present invention, an early detection method for detecting HIV-1 from a saliva sample is provided, the method comprising: providing a glycan-immobilized metal nanoparticle capable of recognizing HIV-1 virus; The immobilized metal nanoparticles are contacted with a saliva sample to form a mixed solution; the mixture is concentrated; and a detection means is used to determine whether the saliva sample contains HIV-1 virus; wherein the glycan immobilized metal The nanoparticle is a ligand complex formed by combining an amino group of a linker compound with a reducing end of a glycan having an intramolecular amino group, a sulfur atom, and a carbon-nitrogen bond on the main chain. A compound of a hydrocarbon chain which is bonded to a metal nanoparticle by a sulfur atom, which is a glycan which can bind to the HIV-1 virus.
本發明所揭露之聚糖固定化金屬奈米粒子，可適用於生命科學、醫療診斷、生物科技等總總分野。具有粒徑均一、高水分散性、且容易固定化於聚糖等優點之安定的聚糖固定化金屬奈米粒子。依上述構成、聚糖固定化金屬奈米粒子可將HIV-1濃縮、迅速、簡便且且高感度檢出或測定病毒。The polysaccharide-immobilized metal nanoparticle disclosed in the invention can be applied to the total field of life science, medical diagnosis, biotechnology and the like. The stabilized glycan-immobilized metal nanoparticles having uniform particle size, high water dispersibility, and easy immobilization on polysaccharides. According to the above configuration, the glycan-immobilized metal nanoparticles can concentrate HIV-1, quickly, easily, and with high sensitivity to detect or measure the virus.
較佳地，其中該連結子化合物具有如式(1)之結構：
較佳地，其中該聚糖係為肝素、硫酸化肝素、擁有硫酸化肝素之雙糖構造、軟骨素E或硫酸葡聚糖。Preferably, the glycan is heparin, sulfated heparin, a disaccharide structure possessing sulfated heparin, chondroitin E or dextran sulfate.
較佳地，其中該聚糖固定化金屬奈米粒子之平均粒徑介於1nm~100nm。Preferably, the average particle diameter of the polysaccharide-immobilized metal nanoparticles is between 1 nm and 100 nm.
較佳地，其中該金屬奈米粒子為金、銀、銅、鋁、白金、氧化鋁、SrTiO3 、LaAlO3 、NdGaO3 、ZrO3 。Preferably, the metal nanoparticle is gold, silver, copper, aluminum, platinum, alumina, SrTiO 3 , LaAlO 3 , NdGaO 3 , ZrO 3 .
較佳地，其中該檢測手段係為聚合酶連鎖反應(PCR)、即時聚 合酶連鎖反應(Real time PCR)、北方墨點法(northern blotting)、免疫層析法(Immunochromato)、酵素連結免疫吸附分析法(ELISA)。Preferably, wherein the detection means is a polymerase chain reaction (PCR), real-time polymerization Real time PCR, northern blotting, immunochromatography, enzyme-linked immunosorbent assay (ELISA).
依上述之構成、上述聚糖固定化金屬奈米粒子之平均粒徑，相對於HIV-1之大小，為最適當粒徑。因此HIV-1所結合之上述混和物，可利用離心來容易地捕捉收集病毒。也因此可以迅速、簡便且且高感度檢出或測定病毒。According to the above configuration, the average particle diameter of the above-described glycan-immobilized metal nanoparticles is the most suitable particle size with respect to the size of HIV-1. Therefore, the above-mentioned mixture in which HIV-1 is combined can be easily collected by centrifugation to collect the virus. Therefore, it is possible to detect or measure the virus quickly, simply, and with high sensitivity.
經由本發明所採用之技術手段，可由簡單且非侵入性的採取之唾液檢體來捕獲濃縮HIV-1、使得HIV-1檢查診斷達到迅速、簡便且高感度之效果。Through the technical means adopted by the present invention, the concentrated and non-invasive saliva sample can be used to capture the effect of concentrating HIV-1, making the HIV-1 test diagnosis rapid, simple and highly sensitive.
本說明書之所謂「聚糖固定化金屬奈米粒子」、配體複合體藉由硫原子與金屬奈米粒子結合之構造的粒子。In the present specification, the "glycan-immobilized metal nanoparticle" and the ligand complex are particles having a structure in which a sulfur atom and a metal nanoparticle are combined.
於本說明書中所謂「配體複合體」是由可與任何金屬結合之上述連結子化合物、以及可與病毒蛋白質等形成特異的相互作用之聚糖所構成之化合物、而上述連結子化合物為其氨官能基與具還原末端之聚糖相結合之化合物。因此，上述配體複合體必須對蛋白質等物質，不會發生非特異性之疏水性相互作用。In the present specification, the "ligand complex" is a compound composed of the above-described linker compound which can bind to any metal, and a glycan which can form a specific interaction with a viral protein or the like, and the above-described linker compound A compound in which an amino functional group is combined with a glycan having a reduced terminal. Therefore, the ligand complex described above must not interfere with a non-specific hydrophobic interaction with a substance such as a protein.
配體複合體所含之上述連結子化合物因分子內具有硫原子，此硫原子可與金屬形成金屬-硫鍵結(例如、Au-S鍵結)、可牢固的結合於金屬。因此、配體複合體所含聚糖可以藉由連結子化合物，間接的固定於金屬上。The above-described linker compound contained in the ligand complex has a sulfur atom in the molecule, and the sulfur atom can form a metal-sulfur bond (for example, Au-S bond) with the metal, and can be firmly bonded to the metal. Therefore, the glycan contained in the ligand complex can be indirectly immobilized on the metal by the linker compound.
上述連結子化合物如一般式(1)所示。具有分子內氨(氨)基、主鏈上具有碳-氮鍵結。連結子化合物之氨基與具還原末端之聚糖相結合、硫原子與金屬奈米粒子結合，因此可將聚糖分子配 列集合於金屬奈米粒子上。又，上述氨基可與具還原末端之聚糖進行還原氨基化反應、可將上述聚糖簡便的導入。The above-mentioned linker compound is represented by the general formula (1). It has an intramolecular ammonia (ammonia) group and a carbon-nitrogen bond on the main chain. The amino group of the linker compound is combined with the glycan having a reducing terminal, and the sulfur atom is bonded to the metal nanoparticle, thereby allowing the glycan molecule to be coordinated The columns are assembled on the metal nanoparticles. Further, the amino group can be subjected to a reductive amination reaction with a glycan having a reducing terminal, and the above glycan can be easily introduced.
在式(1)中，a、b、c、d各自獨立地為0以上6以下之整數。上述X為末端具有芳香族氨基，且主鏈上有碳-氮鍵結合之碳氫誘導鏈、含3鏈以上之支鏈結構。又其直鏈構造部位含有1鏈之構造。In the formula (1), a, b, c, and d are each independently an integer of 0 or more and 6 or less. The above X is a hydrocarbon-inducing chain having a carbon-nitrogen bond in the main chain and a branched chain structure containing 3 or more chains. Further, the linear structure portion has a structure of one chain.
上述金屬奈米粒子之平均粒徑以1nm以上100nm以下之膠體狀(colloid)金屬粒子為較適合。平均粒徑1nm以下之奈米粒子製作困難、而平均粒徑100nm以上之膠體狀物容易沈降、有不易與病毒反應之疑慮。又，病毒之大小大約在100nm左右，若使用比病毒大的粒子，則將降低與病毒之結合效率。平均粒徑之算出方法等將說明如後。The colloidal metal particles having an average particle diameter of the above-mentioned metal nanoparticles of 1 nm or more and 100 nm or less are suitable. It is difficult to produce nano particles having an average particle diameter of 1 nm or less, and a colloidal material having an average particle diameter of 100 nm or more is likely to settle, and there is a fear that it is difficult to react with a virus. Further, the size of the virus is about 100 nm, and if a particle larger than the virus is used, the efficiency of binding to the virus is lowered. The method of calculating the average particle diameter and the like will be described later.
上述金屬可以為金、銀、銅、鋁、白金、氧化鋁、SrTiO3 、LaAlO3 、NdGaO3 、ZrO3 等。其中以金較適合。以金之取得之容易性來看、HAuCl4 及其塩類較適合、HAuCl4 則特別合適。上述金屬奈米粒子之獲得方法並無特別限定，例如使用既有且公開之方法，將HAuCl4 及其塩類溶解於甲醇、水或這類之混合溶媒等，以檸檬酸酸等還原金屬離子(例如金離子)成為金屬(例如金)即可得到。上述HAuCl4 及其塩類之具體例，例如氯化鈉金鈉鹽NaAuCl4 。The above metal may be gold, silver, copper, aluminum, platinum, alumina, SrTiO 3 , LaAlO 3 , NdGaO 3 , ZrO 3 or the like. Among them, gold is more suitable. In view of the ease of obtaining gold, HAuCl 4 and its anthraquinones are more suitable, and HAuCl 4 is particularly suitable. The method for obtaining the metal nanoparticle is not particularly limited. For example, HAuCl 4 and its anthraquinone are dissolved in methanol, water or a mixed solvent of the like by a method disclosed and disclosed, and metal ions are reduced by citric acid or the like ( For example, gold ions can be obtained as a metal (for example, gold). Specific examples of the above-mentioned HAuCl 4 and its anthracene are, for example, sodium chloride sodium sodium salt NaAuCl 4 .
上述配體複合體之構造為在上述連結子化合物之氨基上，導 入具還原末端之聚糖。換言之，上述配體複合體為具有如上述連結子化合物藉由氨基與還具還原末端之聚糖相結合之構造。此聚糖之導入，例如上述連結子化合物之氨基(-NH2 基)與糖進行還原氨基化反應即可得。亦即，因聚糖中之平衡所產生之醛基(-CHO基)或酮基(-CRO基、R碳氫基)可與上述連結子化合物之氨基反應。因此，藉此反應形成Schiff base塩基，將其繼續還原，則可容易地將聚糖導入於氨基位置。The ligand complex has a structure in which a glycan having a reducing end is introduced onto an amino group of the above-described linker compound. In other words, the above ligand complex has a structure in which the above-described linker compound is bonded to a glycan having a reducing terminal by an amino group. The introduction of the glycan, for example, the amino group of the above-mentioned linker compound (-NH 2 group) can be obtained by reductive amination reaction with a sugar. That is, an aldehyde group (-CHO group) or a ketone group (-CRO group, R hydrocarbon group) which is produced by the balance in the glycan may react with the amino group of the above-mentioned linker compound. Therefore, by this reaction, a Schiff base thiol group is formed, and if it is continuously reduced, the glycan can be easily introduced into the amino group position.
上述配體複合體之調製、可將上述連結子化合物與上述聚糖以莫耳比1：1~50：1之比例混合為合適。The preparation of the above ligand complex may be carried out by mixing the above-mentioned linker compound with the above-mentioned glycan at a molar ratio of 1:1 to 50:1.
又，上述「具還原末端之聚糖」為氨基位置碳原子未被置換之單糖鏈、寡糖鏈、或者是多糖鏈。亦即，上述「具還原末端之聚糖」為還原聚糖。上述具還原末端之聚糖，無論是市售商品或天然物、合成調製品、或是由市售或天然取得多聚糖之分解物均可使用。Further, the "glycan having a reducing terminal" is a monosaccharide chain, an oligosaccharide chain or a polysaccharide chain in which an amino group carbon atom is not substituted. That is, the above "polysaccharide having a reduced terminal" is a reduced glycan. The above-mentioned glycan having a reducing end can be used, whether it is a commercially available product or a natural product, a synthetic preparation, or a commercially available or natural decomposed product of a polysaccharide.
上述具還原末端聚糖其具體例為葡萄糖、半乳糖、甘露醣、麥芽糖、異麥芽糖、乳糖、潘糖、纖維二醣、密二糖(melibiose)、甘露寡糖、殼低聚糖(chitooligosaccharide)、Laminan-oligosaccharide、葡萄糖胺、乙醯葡萄糖胺(N-acetyl-glucosamine)、葡萄糖醛酸(glucuronic acid)、肝素、硫酸化肝素、硫酸軟骨素、硫酸皮膚素(Dermatan sulfate)、硫酸葡聚糖(Dextran sulfate)等等。又，雖舉例含此類之聚糖，但不限定於此，以選擇濃縮對象病毒可以辨識之聚糖為佳，將其導入上述連結子化合物均適宜。Specific examples of the above-mentioned reduced terminal glycans are glucose, galactose, mannose, maltose, isomaltose, lactose, panose, cellobiose, melibiose, mannooligosaccharide, chitooligosaccharide. , Laminan-oligosaccharide, glucosamine, N-acetyl-glucosamine, glucuronic acid, heparin, sulfated heparin, chondroitin sulfate, Dermatan sulfate, dextran sulfate (Dextran sulfate) and so on. Further, although such a glycan is exemplified, it is not limited thereto, and it is preferred to select a glycan which can be recognized by the virus to be concentrated, and it is suitable to introduce the glycoside into the above-mentioned linker compound.
上述連結子化合物為在分子內具有氨基、硫原子、主鏈上有碳-氮鍵結之碳氫鏈構造之化合物即可，並無特別限定，公知之連 結子化合物，或者例如由本案發明人到目前為止所開發之連結子化合物即非常適用。The linker compound is not particularly limited as long as it has an amino group, a sulfur atom or a hydrocarbon chain structure having a carbon-nitrogen bond in the main chain, and is not particularly limited. A knot compound, or a linker compound developed, for example, by the inventors of the present invention, is very suitable.
上述碳氫鏈可以是主鏈至少含有1個碳-氮鍵結，甚者，亦可以是一部分之碳或是氫被其他原子或取代基所置換者。例如，碳-碳鍵結(C-C鍵結)至少1個被碳-氮鍵結(C-N鍵結)置換之碳氫鏈，其他碳-素鍵結之一部分再被碳-氮鍵結、碳-氧鍵結(C-O鍵結)、醯胺鍵結(CO-NH鍵結)等置換之碳氫鏈亦可。上述氨基用於與聚糖結合，上述硫原子則用於與金屬結合。The above hydrocarbon chain may have at least one carbon-nitrogen bond in the main chain, or may be a part of carbon or hydrogen replaced by another atom or substituent. For example, a carbon-carbon bond (CC bond) has at least one hydrocarbon chain replaced by a carbon-nitrogen bond (CN bond), and one of the other carbon-nuclear bonds is partially carbon-nitrogen bonded, carbon- A hydrocarbon chain such as an oxygen bond (CO bond) or a guanamine bond (CO-NH bond) may be substituted. The above amino group is used for binding to a glycan, and the above sulfur atom is used for bonding with a metal.
上述氨基為易於與聚糖結合，位於上述碳氫鏈之末端位置較合適。上述氨基可以是被修飾之氨基。例如被Acetyl、methyl、formyl等修飾之氨基或、芳香族氨基。當然，未修飾之氨基亦可。其中以芳香族氨基較合適。還原氨化反應之最適條件為pH3~4之條件下，氨基必須避免質子化。因此，芳香族之共軛，在pH3~4條件下，非共有電子對存在於氮原子上之芳香族氨基較合適。The above amino group is easily bonded to the glycan, and is preferably located at the end of the above hydrocarbon chain. The above amino group may be a modified amino group. For example, an amino group or an aromatic amino group modified by Acetyl, methyl, foryl or the like. Of course, unmodified amino groups can also be used. Among them, an aromatic amino group is suitable. The optimum conditions for the reductive amination reaction are pH 3 to 4, and the amino group must be prevented from protonation. Therefore, in the aromatic conjugate, under the condition of pH 3-4, non-common electrons are suitable for the aromatic amino group present on the nitrogen atom.
關於上述硫原子，上述碳氫鏈存在一部分的碳被硫置換的狀態、以及為了金屬-硫容易形成結合，上述碳碳氫鏈最好含有disulfide鍵結(S-S鍵結)或Thio基(SH基)。In the above sulfur atom, a state in which a part of carbon of the hydrocarbon chain is replaced by sulfur and a metal-sulfur easily form a bond, and the carbon hydrocarbon chain preferably contains a disulfide bond (SS bond) or a Thio group (SH group). ).
上述聚糖固定化金屬奈米粒子是由上述配體複合體與含上述金屬溶液所混和製造，因此非常容易將聚糖固定化。Since the above-described glycan-immobilized metal nanoparticles are produced by mixing the above ligand complex with the above metal solution, it is very easy to immobilize the glycan.
上述聚糖固定化金屬奈米粒子之平均粒徑微奈米等級(大於1nm，小於1μm)、平均粒徑為1nm以上100nm以下較合適。The average particle diameter of the above-described glycan-immobilized metal nanoparticles is preferably a nano-nano grade (greater than 1 nm, less than 1 μm) and an average particle diameter of 1 nm or more and 100 nm or less.
本說明書中所述之「粒徑」，粒子以透過型電子顕微鏡觀察之場合、是測量粒子之二次元形狀之最大內接圓之直徑。例如、粒子之二次元形狀為實質的圓形狀時，則測量圓之直徑。實質上為楕圓形狀之場合則測量楕圓之短徑，實質上為正方形狀之場合則 測量正方形之邊長，實質上為長方形狀之場合則測量長方形之短邊長。「平均粒徑」為複數個粒子之上述粒徑之平均值。於本說明書中，平均粒徑是否為所定之範值，是將20個粒子以透過型電子顕微鏡觀察，測定各粒子之上述粒徑，再求得20個粒子之上述粒徑平均值。The "particle diameter" described in the present specification is the diameter of the largest inscribed circle in which the particle is measured in the shape of the secondary element of the particle when it is observed by a transmission electron microscope. For example, when the shape of the secondary element of the particle is a substantially circular shape, the diameter of the circle is measured. When the shape is substantially round, the short diameter of the circle is measured, and when it is substantially square, When the side length of the square is measured and the shape is substantially rectangular, the short side length of the rectangle is measured. The "average particle diameter" is an average value of the above particle diameters of a plurality of particles. In the present specification, whether or not the average particle diameter is a predetermined value is obtained by observing 20 particles with a transmission electron microscope, measuring the particle diameter of each particle, and obtaining the average particle diameter of 20 particles.
與本發明相關之方法為將含有聚糖固定化金屬奈米粒子與檢體之混和物加以離心。上述檢體中若含有HIV-1之場合，HIV-1會與聚糖固定化金屬奈米粒子之聚糖相結合。檢體只要是可與聚糖固定化金屬奈米粒子接觸者均可，並無特別限定。例如、唾液、鼻粘膜、血液之體液等。這些體液可直接做為檢體使用，例如添加MEM培養基或生理食塩水等所調製之液體也可使用。也可以將聚糖固定化金屬奈米粒子與添加水、生理食塩水或磷酸緩衝液等調製之溶液相混和，使檢體與聚糖固定化磁性金屬奈米粒子接觸。A method related to the present invention is to centrifuge a mixture containing the glycan-immobilized metal nanoparticles and the sample. When HIV-1 is contained in the above sample, HIV-1 is bound to the glycan of the immobilized metal nanoparticle of the glycan. The sample is not particularly limited as long as it can be in contact with the manganese-immobilized metal nanoparticles. For example, saliva, nasal mucosa, body fluids of blood, and the like. These body fluids can be directly used as a sample, and for example, a liquid prepared by adding MEM medium or physiological chyme water can also be used. The glycan-immobilized metal nanoparticles may be mixed with a solution prepared by adding water, physiological chylohydrate or a phosphate buffer, and the sample may be brought into contact with the glycan-immobilized magnetic metal nanoparticles.
病毒具有辨識聚糖之性質，使用濃縮對象之病毒可以辨識之聚糖，調製聚糖固定化金屬奈米粒子，使其與檢體接觸若檢體中含病毒，則病毒將會與上述聚糖形成特異的(特定的、專一性)結合。The virus has the property of identifying the glycan, and the glycan which can be recognized by the virus of the concentrated object is used to prepare the glycan-immobilized metal nanoparticle to be in contact with the sample. If the sample contains a virus, the virus will be combined with the above-mentioned glycan. Form a specific (specific, specific) combination.
本發明之濃縮對象病毒為HIV-1。The concentrated target virus of the present invention is HIV-1.
所謂HIV-1所特異辨識之聚糖有肝素、硫酸化肝素、擁有硫酸化肝素之雙糖構造、軟骨素E、硫酸葡聚糖。選擇這些聚糖中之任一者，調製聚糖固定化金屬奈米粒子，與檢體相接觸，檢體中若含有病毒，病毒將會與上述聚糖行特異的結合。The glycans specifically identified by HIV-1 include heparin, sulfated heparin, a disaccharide structure possessing sulfated heparin, chondroitin E, and dextran sulfate. The glycan-immobilized metal nanoparticle is prepared by selecting any one of these glycans, and is in contact with the sample. If the virus is contained in the sample, the virus will specifically bind to the above-mentioned glycan.
上述HIV-在檢體中含有0.1~10copies/ml左右、利用本發明之方法，加上離心分離，可以短時間且簡便地進行病毒濃縮，並可提供RT-PCR測定檢測出病毒之存在。The above-mentioned HIV-containing 0.1 to 10 copies/ml in the sample, and the method of the present invention, together with centrifugation, can carry out virus concentration in a short time and easily, and can provide an RT-PCR assay to detect the presence of a virus.
上述所謂「加上離心分離」是利用10000×g之離心。The above-mentioned "plus centrifugal separation" is performed by centrifugation at 10,000 x g.
藉離心將與病毒結合之聚糖固定化金屬奈米粒子沉積。病毒DNA之回收方法並不限定於此沉積法，依照公知的方法也可以。例如，上述沉澱物以滅菌水或蒸留水水洗後，適宜進行懸浮，將含上述沉澱物之滅菌水或蒸留水加熱至90℃以上、最好是加熱至100℃、可將上清中之病毒DNA回收。The nanoparticle deposition of the immobilized metal nanoparticles is combined with the virus by centrifugation. The method for recovering viral DNA is not limited to this deposition method, and may be carried out according to a known method. For example, after the precipitate is washed with sterilized water or distilled water, it is suitably suspended, and the sterilized water or distilled water containing the precipitate is heated to 90 ° C or higher, preferably to 100 ° C, and the virus in the supernatant can be used. DNA recovery.
上述混和物為上述聚糖固定化金屬奈米粒子之聚糖與檢體相接觸所得。藉此若檢體中存在HIV-1時，HIV-1會辨識上述聚糖並結合。所謂上述聚糖固定化金屬奈米粒子與檢體相接觸，可以將聚糖固定化金屬奈米粒子添加水、生理食塩水、或磷酸緩衝液所調製之溶液，與檢體混和而得。混和後、可以吸注管的方式攪拌，使病毒與聚糖確實結合，而且也可縮短病毒與聚糖結合所需時間。The above mixture is obtained by contacting the glycan of the above-described glycan-immobilized metal nanoparticles with a sample. Thus, if HIV-1 is present in the sample, HIV-1 recognizes and binds the above glycans. When the glycan-immobilized metal nanoparticles are brought into contact with the sample, a solution prepared by adding the water, physiological chylohydrate, or a phosphate buffer solution to the glycan-immobilized metal nanoparticles can be mixed with the sample. After mixing, the tube can be agitated to allow the virus to bind to the glycan, and the time required for the virus to bind to the glycan can also be shortened.
又，所謂上述「辨識」是指病毒表面可以結合分子內糖部位之蛋白質(聚糖辨識部位)可與聚糖結合。上述結合可以是氫鍵結、離子鍵結、靜電的相互作用鍵結、凡德瓦力鍵結等。Further, the above-mentioned "identification" means that a protein (glycan recognition site) which can bind to a sugar moiety in a molecule on the surface of a virus can bind to a glycan. The above bonding may be hydrogen bonding, ionic bonding, electrostatic interaction bonding, van der Waals bonding, and the like.
藉本發明相關方法確認病毒濃縮結果之方法並無特別限定，也可用公知之方法。例如，將回收之病毒以聚合酶連鎖反應(PCR)、即時聚合酶連鎖反應(Real time PCR)、北方墨點法(northern blotting)、免疫層析法(Immunochromato)、酵素連結免疫吸附分析法(ELISA)等方法確認。其中為了迅速地將DNA定量，可使用RT-PCR。使用RT-PCR之場合，可將濃縮前之Ct(Threshold Cycle)值與濃縮後之Ct值相減，依本發明之方法濃縮之相差部分，若與離心分離濃縮之相差部分相同，則可解決本發明欲解决之課題。特別是Ct為35以上時、大多是非由對象病毒之核酸而是由夾雑物而來的核酸所產生之増幅。因此，檢視病毒由來之DNA被増 幅，可以RT-PCR反應40cycle後之溫度解離曲綫(Tm曲綫)、如此可判定是否是病毒而來之核酸所產生之増幅。The method for confirming the virus concentration result by the method of the present invention is not particularly limited, and a known method can also be used. For example, the recovered virus is polymerase chain reaction (PCR), real time polymerase chain reaction (Real time PCR), northern blotting, immunochromatography, enzyme-linked immunosorbent assay ( Confirmed by methods such as ELISA). Among them, in order to rapidly quantify DNA, RT-PCR can be used. When RT-PCR is used, the Ct (Threshold Cycle) value before concentration can be subtracted from the concentrated Ct value, and the phase difference portion concentrated by the method of the present invention can be solved if the difference is the same as that of the centrifugal separation and concentration. The problem to be solved by the present invention. In particular, when Ct is 35 or more, it is often caused by a nucleic acid which is not a nucleic acid of a target virus but a nucleic acid which is caused by a sputum. Therefore, the DNA of the virus is detected. The amplitude can be determined by RT-PCR reaction 40 cycles after the temperature dissociation curve (Tm curve), so that it can be determined whether it is a virus caused by the virus.
又，本發明並不只限定以上說明之各構成、專利範圍所示，其範圍內可做種種之變更，不同之實施形態只要利用到本技術說明之技術手段，或將其適當組合所得之實施形態均包含於本發明之技術的範圍。Further, the present invention is not limited to the above-described respective configurations and patent ranges, and various modifications can be made within the scope of the invention. The embodiments of the present invention may be used in accordance with the technical means of the present specification or an appropriate combination thereof. All are included in the scope of the technology of the present invention.
就本發明、以實施例為基礎與以具體的說明、但本發明不限定於此實施例The present invention is based on the embodiments and specific descriptions, but the invention is not limited to the embodiments.
實施例1：利用本發明相關之方法，由唾液檢體中將HIV-1捕捉濃縮、HIV-1之高感度測定。Example 1: Using the method of the present invention, HIV-1 capture concentration and high sensitivity of HIV-1 were measured from saliva samples.
首先，調製聚糖固定化金奈米粒子調整用試劑20nM NaAuCl4‧2H2O(7.956mg/ml)、50nM NaBH4(7.6mg/4ml)、5nM DS25配體複合體(12.5mg/ml)。First, a reagent for adjusting the glycan-immobilized gold nanoparticles was prepared, 20 nM NaAuCl4‧2H2O (7.956 mg/ml), 50 nM NaBH4 (7.6 mg/4 ml), and 5 nM DS25 ligand complex (12.5 mg/ml).
將20nM NaAuCl4‧2H2O 2.08ml加入超純水33.45mL、然後將4.08ml之50nM NaBH4加入並攪拌5秒。接下來再加入5nM DS25配體複合體1.52mL並攪拌30秒。室溫下遮光攪拌30分。然後以分子量1000cut之透析膜進行透析(超純水、PBS、超純水之順序)，最後以分子量3500cut之透析膜進行透析(超純水)。然後分成15mL X 3予以冷凍乾燥。然後將其溶解於超純水使其濃度為10mg/ml。再以4200rpm離心30分鐘，將沉澱濃度調整到UV530nm之吸光度為0.5、則完成聚糖DS25固定化金奈米粒子(以下也稱為「DS25固定化金奈米粒子」。平均粒徑5nm)溶液1之調製。2.08 ml of 20 nM NaAuCl4‧2H2O was added to 33.45 mL of ultrapure water, then 4.08 ml of 50 nM NaBH4 was added and stirred for 5 seconds. Next, 1.55 mL of 5 nM DS25 ligand complex was added and stirred for 30 seconds. Shake for 30 minutes at room temperature. Then, dialysis was carried out with a dialysis membrane having a molecular weight of 1000 cut (sequence of ultrapure water, PBS, ultrapure water), and finally dialyzed against a dialysis membrane having a molecular weight of 3,500 cut (ultra-pure water). It was then lyophilized into 15 mL of X3. It was then dissolved in ultrapure water to a concentration of 10 mg/ml. Further, the mixture was centrifuged at 4,200 rpm for 30 minutes, and the concentration of the precipitate was adjusted to a UV 530 nm absorbance of 0.5 to complete the solution of the glycan DS25-immobilized gold nanoparticles (hereinafter also referred to as "DS25 immobilized gold nanoparticles" (average particle diameter: 5 nm)). 1 modulation.
圖1為將平均分子量2500之硫酸化葡聚糖(DS25)之聚糖固定化金奈米粒子，以穿透型電子顕微鏡所觀察之圖像。Fig. 1 is an image observed by a penetrating electron micromirror with a glycan-immobilized gold nanoparticle having an average molecular weight of 2,500 sulfated dextran (DS25).
又，上述「含DS25之配體複合體」為平均分子量2500之硫酸化葡聚糖水溶液，及上述一般式(1)所示之連結子化合物(n1 =0、q=0)之N，N-dimethylacetoamide(DMAC)溶液(以下簡稱為「DMAC溶液」)，添加醋酸後之DS25、上述連結子化合物、NaBH3CN之莫耳比為1：1：10，將添加醋酸之上述DS25水溶液、添加醋酸之DMAC溶液、NaBH3CN混合並於37℃下攪拌3日。Further, the "DS25-containing ligand complex" is a sulfated dextran aqueous solution having an average molecular weight of 2,500, and N of the linker compound (n 1 =0, q = 0) represented by the above general formula (1). N-dimethylacetoamide (DMAC) solution (hereinafter referred to as "DMAC solution"), the molar ratio of DS25, the above-mentioned linker compound and NaBH3CN after adding acetic acid is 1:1:10, and the above-mentioned DS25 aqueous solution of acetic acid is added, and acetic acid is added. The DMAC solution, NaBH3CN were mixed and stirred at 37 ° C for 3 days.
HIV-1患者唾液檢體以PBS稀釋為1/2，調整其體積成500μl，取10μl做為濃縮前之對照(control)。上述溶液1添加10μl得到溶液2、並於室溫(25.5℃)下持續攪拌並靜置30分鐘。The saliva samples of HIV-1 patients were diluted to 1/2 with PBS, and the volume was adjusted to 500 μl, and 10 μl was taken as a control before concentration. 10 μl of the above solution 1 was added to obtain a solution 2, and stirring was continued at room temperature (25.5 ° C) and allowed to stand for 30 minutes.
上述溶液2以10000G離心10分鐘進行分離，可得到上清及沉澱。沉澱部分添加超純水10μl於100℃下加熱5分鐘後，以10000G離心10分鐘進行分離得到上清液。The solution 2 was separated by centrifugation at 10,000 G for 10 minutes to obtain a supernatant and a precipitate. 10 μl of ultrapure water was added to the precipitated portion and heated at 100 ° C for 5 minutes, and then centrifuged at 10,000 G for 10 minutes to obtain a supernatant.
圖2為由調製上述溶液2到調製提供RT-PCR之上清為止之步驟。Figure 2 is a step of providing RT-PCR supernatant from the preparation of the above solution 2 to modulation.
將這些上清液2μl添加於表1所示各試劑，並添加表1所示量混合之PCR用試劑20μl以供RT-PCR測定用。所使用RT-PCR裝置為Thermal cycler Dice Real Time System TP800(登録商標型番為TP800、TAKARABIO株式會社製)。所使用試劑為Takara One Step SYBR ®(Prime Script ® RT-PCR Kit Ⅱ)，使用試螢光色素為SYBR Green。所使用引子(Primer)為581F(5’-tggtaactagagtccctcagacc-3’)以及620T(5’-agctcctctggtttccctttc-3’)。2個拷貝數(copy)之HIV-1基因利用將宿主細胞之DNA INTEGRATE之U1細胞之DNA製作檢量線，以測 定樣品中HIV-1之DNA絶對定量。RT-PCR的條件為：反轉譯反應45℃ 2分鐘、初期熱變性處理設定95℃ 1分鐘、PCR cycle數為95℃ 1秒、60℃ 1秒、72℃ 5秒為1cycle、進行40循環(cycle)。2 μl of these supernatants were added to each reagent shown in Table 1, and 20 μl of the PCR reagent mixed in the amounts shown in Table 1 was added for RT-PCR measurement. The RT-PCR device used was a Thermal cycler Dice Real Time System TP800 (registered trademark model: TP800, manufactured by TAKARABIO Co., Ltd.). The reagent used was Takara One Step SYBR ® (Prime Script ® RT-PCR Kit II), and the test fluorescent pigment was SYBR Green. The primers used were 581F (5'-tggtaactagagtccctcagacc-3') and 620T (5'-agctcctctggtttccctttc-3'). Two copies of the HIV-1 gene are prepared by using a DNA of the host cell DNA INTEGRATE U1 cells to make a calibration curve. Absolute quantification of DNA of HIV-1 in the sample. The conditions of the RT-PCR were: reverse translation at 45 ° C for 2 minutes, initial heat denaturation treatment at 95 ° C for 1 minute, PCR cycle number at 95 ° C for 1 second, 60 ° C for 1 second, and 72 ° C for 5 seconds for 1 cycle, for 40 cycles ( Cycle).
關於RT-PCR，與未添加DS25固定化金奈米粒子之唾液檢體液(圖2A)相比較，DS25固定化金奈米粒子與唾液檢體中之HIV-1形成複合體後之沉澱(圖2B)，由其Tm圖(圖4)可得知RNA較明確地被檢出。Regarding RT-PCR, the precipitation of DS25-immobilized gold nanoparticles and HIV-1 in saliva samples was compared with saliva liquid (Fig. 2A) without addition of DS25 immobilized gold nanoparticles (Fig. 2A) 2B), from its Tm map (Fig. 4), it can be known that RNA is more clearly detected.
利用以U1細胞之DNA所製作之檢量線(圖3)，對稀釋唾液檢體HIV-1溶液之HIV-1量進行測定。10、100 CCID50 /ml分別顯示為10、100copies/ml。根據此次進行之RT-PCR法顯示可以正確地檢出HIV-1 DNA。此方法所檢出各唾液檢體中之HIV-1 DNA之結果，在0.1-10copies/ml範圍時，藉由DS25固定化金奈米粒子濃縮後之HIV-1 DNA之檢出效率顕著提高，亦即可由濃縮後之檢體來檢測HIV-1 DNA(圖4)。The amount of HIV-1 in the diluted saliva sample HIV-1 solution was measured using a calibration curve (Fig. 3) prepared from the DNA of U1 cells. 10, 100 CCID 50 /ml is displayed as 10, 100 copies / ml. According to the RT-PCR method performed this time, HIV-1 DNA can be detected correctly. The result of detecting HIV-1 DNA in each saliva sample by this method is that the detection efficiency of HIV-1 DNA concentrated by DS25 immobilized gold nanoparticles is increased in the range of 0.1-10 copies/ml. It is also possible to detect HIV-1 DNA from the concentrated sample (Fig. 4).
進一步地，使用平均分子量17500肝素之固定化金屬奈米粒子，以上述同樣之方法進行唾液中之HIV-1測定時，即使是0.1copies/ml之超低濃度，也可以測定出HIV-1(圖4)。Further, when the HIV-1 assay in saliva is carried out in the same manner as described above using the immobilized metal nanoparticles having an average molecular weight of 17,500 heparin, HIV-1 can be measured even at an ultra-low concentration of 0.1 copies/ml. Figure 4).
使用肝素固定化金奈米粒子之病毒濃縮，即使濃度只有0.1copies/ml之HIV-1唾液檢體也可以RT-PCR法檢出。因此藉由聚糖固定化金奈米粒子將病毒濃縮與RT-PCR法組合，是一種可以由唾液中檢出微量HIV-1之優異方法。唾液為簡單且非侵入性的即可採取之檢體，在今日常被運用於各種感染症、腫瘍疾患、心疾患等之診斷。因為HIV-1感染者唾液中之HIV-1量為血液中之HIV-1量的約20%，0.1copies/ml濃度HIV-1也可由唾液檢體來檢出，此表示藉聚糖固定化金奈米粒子濃縮病毒與RT-PCR法組合 之方法，其感度不輸血液檢體之檢測。做為使用唾液檢體篩檢法，雖然測定唾液中之抗HIV-1抗體之診斷套組(Kit)已經有市售商品，但是此方法為被稱為西方墨點法(Western blotting)，由感染到抗體檢出之可能所需期間(約3個月)，無法診斷HIV-1感染之有無。然而利用聚糖固定化金奈米粒子濃縮病毒與RT-PCR組合之方法，即使以西方墨點法也可以由唾液檢體判定HIV-1感染之有無。HIV-1在分泌體液中、血液、母乳、***、***分泌液感染HIV-1並傳播，但是多數之口腔內，只要無出血症狀，藉由唾液感染HIV-1是非常困難。因此本方法可做為簡便、有效且安全的HIV-1感染之早期診斷法。The virus was concentrated using heparin-immobilized gold nanoparticles, and even HIV-1 saliva samples having a concentration of only 0.1 copies/ml were detected by RT-PCR. Therefore, the combination of virus concentration and RT-PCR by immobilization of gold nanoparticles by glycans is an excellent method for detecting trace amounts of HIV-1 from saliva. Saliva is a simple and non-invasive specimen that can be used today for diagnosis of various infectious diseases, swollen diseases, heart diseases, and the like. Since the amount of HIV-1 in the saliva of HIV-1 infected persons is about 20% of the amount of HIV-1 in the blood, the 0.1copies/ml concentration of HIV-1 can also be detected by the saliva sample, which means that the glycan is immobilized. Combination of Golden Nanoparticle Concentrated Virus and RT-PCR The method is that the sensitivity does not change the detection of the blood sample. As a saliva sample screening method, although a diagnostic kit for measuring anti-HIV-1 antibodies in saliva has been commercially available, this method is called Western blotting. It is not possible to diagnose the presence or absence of HIV-1 infection during the period of time (about 3 months) during which the antibody is detected. However, by using a method in which a glycan-immobilized gold nanoparticle-concentrated virus is combined with RT-PCR, the presence or absence of HIV-1 infection can be determined from a saliva sample even by the Western blot method. HIV-1 infects and spreads HIV-1 in secretory body fluids, blood, breast milk, semen, and vaginal secretions, but in most oral cavity, it is very difficult to infect HIV-1 by saliva as long as there is no bleeding symptoms. Therefore, the method can be used as an early diagnosis method for HIV-1 infection which is simple, effective and safe.
由以上實施例可知，本發明所提供之自唾液檢體檢測HIV-1之早期檢測方法確具產業上之利用價值，惟以上之敘述僅為本發明之較佳實施例說明，凡精於此項技藝者當可依據上述之說明而作其它種種之改良，惟這些改變仍屬於本發明之精神及以下所界定之專利範圍中。It can be seen from the above embodiments that the early detection method for detecting HIV-1 from saliva samples provided by the present invention has industrial use value, but the above description is only for the description of the preferred embodiments of the present invention. The skilled artisan can make various other modifications in light of the above description, but such changes are still within the spirit of the invention and the scope of the invention as defined below.
第1圖顯示將平均分子量2500之硫酸葡聚糖(DS25)固定化形成聚糖固定化金奈米粒子，經透過型電子顕微鏡(TEM)之顯示結果；第2圖顯示實施例所提供RT-PCR測定用之流程圖；第3圖顯示使用U1細胞之DNA所製作之檢量線；第4圖顯示實施例之RT-PCR測定結果。Fig. 1 shows the results of immobilization of dextran sulfate (DS25) having an average molecular weight of 2,500 to form glycan-immobilized gold nanoparticles, which are displayed by a transmission electron micromirror (TEM); and Fig. 2 shows the RT-provided by the examples. A flow chart for PCR measurement; Figure 3 shows a calibration curve prepared using DNA of U1 cells; and Figure 4 shows the results of RT-PCR measurement of the examples.
<110> 世錡生命科學有限公司<110> Shiyi Life Science Co., Ltd.
<120> 自唾液檢體檢測HIV-1之早期檢測方法及其聚糖固定化金屬奈米粒子<120> Early detection method for detecting HIV-1 from saliva samples and glycan-immobilized metal nanoparticles
<160> 2<160> 2
<170> PatentIn version 3.5<170> PatentIn version 3.5
<210> 1<210> 1
<211> 23<211> 23
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<221> misc_feature<221> misc_feature
<223> RT-PCR之引子對581F<223> RT-PCR primer pair 581F
<400> 1 <400> 1
<210> 2<210> 2
<211> 21<211> 21
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<221> misc_feature<221> misc_feature
<223> RT-PCR之引子對620T<223> RT-PCR primer pair 620T
<400> 2 <400> 2
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| CN1451042A (en) * | 1999-01-25 | 2003-10-22 | Musc研究发展基金会 | Method of monitoring HIV drug resistance |
| TW200533632A (en) * | 2004-02-18 | 2005-10-16 | Japan Science & Tech Agency | Carbohydrate-ligand conjugate and method for analyzing protein with the ligand conjugate |
| CN101943699A (en) * | 2010-08-31 | 2011-01-12 | 广州万孚生物技术有限公司 | Test strip for detecting HIV antibodies in spittle and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1451042A (en) * | 1999-01-25 | 2003-10-22 | Musc研究发展基金会 | Method of monitoring HIV drug resistance |
| TW200533632A (en) * | 2004-02-18 | 2005-10-16 | Japan Science & Tech Agency | Carbohydrate-ligand conjugate and method for analyzing protein with the ligand conjugate |
| CN101943699A (en) * | 2010-08-31 | 2011-01-12 | 广州万孚生物技术有限公司 | Test strip for detecting HIV antibodies in spittle and preparation method thereof |
Non-Patent Citations (1)
| Title |
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| H. A. Harrop and C. C. Rider, "Heparin and its derivatives bind to HIV-1 recombinant envelope glycoproteins, rather than to recombinant HIV-1 receptor, CD4", Glycobiology, 1998, Vol. 8, No. 2, p. 131~137. * |
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