201223546 六、發明說明： 【發明所屬之技術領域】 \ 本發明係關於一種奈米團簇，特別係關於一種螢光金 奈米團簇聚集體。 【先前技術】 當金屬或半導體粒子的尺寸足夠小時，將產生量子限 制效應，即微粒的電荷和能量是量子化的，這樣的微小粒 子團被稱為量子點。量子點電子排列相當緊密，由於量子 限量化效應可以激發出不同顏色的螢光，量子點吸收能量 較高的光波後產生能階躍升，當電子從高能階的狀態降到 低能階的狀態時，會發射出波長較長（偏紅光系）的光。 不同粒徑的量子點會發射出不同波長的螢光，例如硒化鎘 (CdSe)粒徑在2.1 nm時發出藍色螢光，粒徑5 nm時 發出綠色螢光，當粒徑接近10 nm時，它所激發的螢光就 接近紅色。 相較於傳統的有機染料分子，量子點具有螢光亮度 強、光穩定性佳、以及用單一波長的雷射便可以激發出多 種不同波長的發射波之特性。發射波是一狹窄且對稱的波 形，且可重複激發，因此螢光時效可以持久。這些特性吸 引科學家的重視，奈米量子點的應用也越來越多樣性，深 201223546 具取代傳統染劑的潛力，因此，在生醫工程應用方面，更 有令人期待的發展性。 近年來量子點以其優異的光學特性，已成功地克服過 去生物及醫學光學探針所面臨之瓶頸，儼然成為新一代螢 光探針設計之重要奈米材料。從細胞三維立體影像、長時 間活細胞監控、單分子動態胞内追蹤、長效型光學感測器 研製、癌症診斷與治療皆有突破性進展，加上量子點快速 產業化及其上億商機，已成奈米生物技術極為成功應用之 典範。然而傳統市售以鎘或鉛等有毒重金屬材料為主之水 溶性量子點，其延伸對環境及人體健康可能帶來的衝擊逐 漸受到重視，是目前全面開拓其生醫應用所面臨之窘境。 金屬金係為研究較早之一種奈米材料，在生物學研究 中被稱為膠體金，粒子尺寸在1- 100nm之間。金量子點具 有很高的電子密度，在電子顯微鏡下有很好的襯度，並且 具有相當高之生物相容性，其已被證實經由改變其原子團 簇之大小即可發出不同顏色之螢光，能夠應用在多元之生 醫標定或光學元件的製作上，但由於製程相當不易，合成 時需要利用昂貴的樹狀聚合物（dendrimer)作為金量子點之 包覆材料，耗時且不易大量生產，故限制其廣大生醫應用 之開發。因此，開發出簡易並可大量生產製造之金量子點 形成技術係產業界.亟欲發展之重點。 201223546 【發明内容】 鑒於上述發明背景中，為了符合產業上之要求，本發 明提供螢光金奈米團簇聚集體。 本發明之特徵的在於提供一種螢光金奈米團簇聚集 體(Fluorescent gold nanocluster)，上述螢光金奈米團簇聚集 體表面具有一種二氩硫辛酸(dihydrolipoic acid; DHLA)配 體(ligand)，其中，上述螢光金奈米團簇係藉由上述二氫硫 辛酸配體與上述金奈米團簇間之作用而產生螢光性質，且 上述螢光金奈米團簇聚集體之粒徑範圍為〇.5nm至3nm， 此外，上述螢光金奈米團簇之光激螢光波長範圍為400至 lOOOnm。 本發明之另一特徵在於提供一種螢光金奈米團簇聚 集體（Fluorescent gold nanocluster matrix)，上述營光金奈 米團簇聚集體係由複數個金奈米團簇規則堆疊所形成，上 述金奈米團竊粒徑範圍為0.5nm至3nm，且上述金奈米團 簇表面具有一種烧硫醇(alkanethiol)配體（ligand)，其中，各 個上述金奈米.團簇係透過其表面之烷硫醇配體間之作用 力，相互吸引堆疊以形成上述螢光金奈米團簇聚集體 (Fluorescent gold nanocluster matrix)，並且，上述螢光金奈 .米團簇聚集體係藉由上述金奈米團藤之聚集而產生螢光性 質，此外，上述螢光金奈米團簇聚集體之光激螢光波長範 圍為 400 至 lOOOnm。 201223546 本發明之又一特徵在於提供一種金屬奈米團簇（metal nanocluster)之形成方法，首先提供一混合溶液，上述混合 溶液包含一第一金屬前驅物（metal precursor)、一界面活性 劑（surfactant)、一還原劑（reductant)與一溶劑，於上述混合 溶液中進行一還原反應以形成·一奈来金屬粒子（metal nanoparticle)，再者，於形成上述奈米金屬粒子後加入一第 二金屬前驅物，使得上述第二金屬前驅物之粒子數大於上 述奈米金屬粒子之總數，由於上述奈米金屬粒子與上述第 二金屬前驅物之濃度差異甚大，造成一不平衡之並存系 統，上述奈米金屬粒子因此崩裂為粒徑較小之金屬奈米團 簇（metal nanocluster)以形成一平衡系統，其中，上述金屬 奈米團兹之粒徑範圍係為1 nm至4nm。 根據以上所述之目的，本發明揭示了一種螢光金奈米 團簇聚集體及其形成方法，上述之形成方法能應用於各種 金屬，據以形成各種金屬奈米團簇。其中，上述之螢光金 奈米團鎮聚集體能作為生物探針(bioprobes)，並具有下列 之應用：生物螢光標記（fluorescent biological label)、臨床 醫療影像顯影劑以及臨床醫療檢測、追蹤與治療。 201223546 【實施方式】 本發明在此所探討的方向為一種螢光金$$ 集體。為了能徹底地瞭解本發明’將提出詳盡碟聚 顯然地’本發明的施行並未限定於該領域之技敲者 明： 的特殊細節。另一方面，眾所周知的纽成或步驟 熟習 於細節中’以避免造成本發明不必要之限制。 為迷 十％ a月的較 佳實施例會詳細描述如下’然而除了這些詳細描述之外 本發明還可以廣泛地施行在其他的實施例中，且本發明、 範圍不受限定，其以之後的專利範圍為準。 本發明之一實施例係揭露一種螢光金奈米團鎮 (Fluorescent gold nanocluster)，上述螢光金奈米團簇表面具 有一種二氫硫辛酸（dihydrolipoic acid ; DHLA)配體 (ligand) ’其中，上述螢光金奈米團鎮係藉由上述二氫硫辛 酸配體與上述奈米團簇間之作用而產生螢光性質，且上述 螢光金奈米團藥之粒徑範圍為0.5nm至3nm，此外，上述 螢光金奈米團簇之光激螢光波長範圍為400至lOOOnm。 於本實施例之一較佳範例中，上述螢光金奈米團簇更 包含一間隔物（spacer)，上述間隔物之一端鍵結上述二氫硫 辛酸（dihydrolipoic acid; DHLA)配體，且上述間隔物鍵結 之另一端有一特定基團，其中，上述間隔物包含寡聚物或 高分子，而上述特定基團係包含下列族群中之一者：化學 201223546 官能基、交聯分子、醣類、螢光分子、順磁性分子、生物 分子與藥物。 其中，上述寡聚物或高分子包含下列族群中之一者或 其任意組合··多元醇（polyols )、聚鍵系多元醇（polyether polyols)、聚醋類多元醇（polyester polyols)、聚碳酸酉旨多 元醇（polycarbonate polyols )、聚環己内酯多元醇 (polycaprolactone polyols)、壓克力多元醇（polyacrylate polyols )、聚乙二醇(polyethylene glycol; PEG)、糊精（dextran) 及其共聚物。 於本實施例之另一較佳範例中，上述螢光金奈米團簇 更包含一間隔物，上述間隔物鍵結上述二氫硫辛酸 (dihydrolipoic acid; DHLA)配體，且上述間隔物本身具有 一特定基團，其中，上述間隔物包含下列族群中之一者： 化學官能基、交聯分子、醣類、螢光分子、順磁性分子、 生物分子與藥物。 本發明之再一實施例係揭露一種螢光金奈米團簇聚 集體（Fluorescent gold nanocluster matrix)，上述榮光金奈 米團簇聚集體係由複數個金奈米團簇規則堆疊所形成，上 述金奈米團鎮粒徑範圍為0.5nm至3nm，且上述金奈米團 簇表面具有一種烧硫醇（alkanethiol)配體(ligand)，其中，各 201223546 個上述金奈米團簇係透過其表面之烷硫醇配體間之作用 力，相互吸引堆疊以形成上述螢光金奈米團簇聚集體 (Fluorescent gold nanocluster matrix)，並且，上述螢光金奈 米團簇聚集體係藉由上述金奈米團簇之聚集產生螢光性 質，此外，上述螢光金奈米團簇聚集體之光激螢光波長範 圍為 400 至 lOOOnm。 於本實施例之一較佳範例中，上述螢光金奈米團簇聚 集體表面包覆一間隔物，上述間隔物之一端鍵結上述烷硫 醇配體，且上述間隔物之另一端鍵結有一特定基團，其中， 上述間隔物包含兩性高分子或寡聚物，而特定基團包含下 列族群中之一者：化學官能基、交聯分子、醣類、螢光分 子、順磁性分子、生物分子與藥物等。 上述兩性高分子或寡聚物包含下列族群中之一者或 其任意組合：聚順丁浠二酸酐[poly(maleic anhydride); PMA]、1-十八稀馬來酸酐的聚合物[Poly(maleic anhydride-alt-1 -octadecene) ； PMAO]與聚丙稀酸 (polyacrylic acid ; PAA)及其衍生物。 於本實施例之另一較佳範例中，螢光金奈米團簇聚集 體表面包覆一間隔物，上述間隔物鍵結上述烷硫醇 (alkanethiol)配體，且上述間隔物本身具有一特定基團，其 10 201223546 中，上述間隔物包含下列族群中之一者：化學官能基、交 聯分子、聽類、螢光分子、順磁性分子、生物分子與藥物。 本發明之另一實施例係揭露一種金屬奈米團鎮（metal nanocluster)之形成方法，首先提供一混合溶液，上述混合 溶液包含一第一金屬前驅物（metal precursor)、一界面活性 劑（surfactant)、一還原劑（reductant)與一溶劑，於上述混合 溶液中進行一還原反應以形成一奈米金屬粒子（metal nanopanide)，其中，上述奈米金屬粒子具有表面電漿吸收 之性質。 再者於形成上述奈米金屬粒子後加入一第二金屬前 驅物’使得上述第〔金屬t驅物之粒子數大於上述奈米金 眉粒子之總數，由於上述奈米金屬粒子與上述第二金屬前 驅物之濃度差異甚大，造成平衡之並存系統 ，上述奈 米金属粒子因此崩裂為粒徑較小之金屬奈米團簇（metal nanocluster)以形成一平衡系、统，其中，上述金屬奈米團鎮 之粒徑範圍係為lnm至4nm。 此外’上述第—金屬前驅物與第二金屬前驅物係能為 相Π或不Π 其中，上述第一金屬前驅物(metal precursor) 與第一金屬前驅物係選自下列族群之一者：氣化金 (AuCl3)、四氣金酸（HAuC14)、溴化金（AuBr3)、四溴金酸 201223546 (HAuBq) 0 而上述界面活性劑（surfactant)係選自下列族群之一者 或其任意組合：雙十二烷基二烷基溴化銨鹽 (Didodecyldimethylammonium bromide ; DDAB)、四辛基溪 化録（Tetraoctylammonium bromide ; TOAB)、四丁基漠化 録（Tetrabutylammonium bromide ; TBAB)。還原劑（reductant) 則選自下列族群之一者或其任意組合：四丁基溴化銨 (tetrabutylammonium borohydride ; TBAB)、硼氫化納 (NaBH4)、維生素C (Ascorbic Acid)。 溶劑則係為曱苯 (toluene)或氯仿（chloroform)。 另一方面，於形成上述金屬奈米團簇後進行一配體接 合（Ligand-binding)反應，上述反應係將配體（ligand)接合 於上述金屬奈米團簇之表面，以形成一配體包覆之金屬奈 米團竊（ligand-capped metal nanocluster)。 而上述配體(ligand)係選自下列族群之一者：二氫硫辛 酸(dihydrolipoic acid ; DHLA)、十二烧硫醇（dodecanethiol ; DDT)、 雙（磺酸鈉苯基）苯基磷 [Bis(p-sulfonatophenyl)phenylphosphine ; BSPP]、三苯基 填（triphenylphosphine)。 12 201223546 再者，上述配體接合（Ligand-binding)反應係為一硫 醇配體接合(thiol-related ligand binding)反應，上述反應係 將上述硫醇配體（thiol-related ligand)接合於上述金屬奈米 團簇之表面，以形成一硫醇配體包覆之金屬奈米團簇 (thiol-capped metal nanocluster) ° 上述硫醇配體(thiol-related ligand)係選自下列族群之 一者：二氫硫辛酸(dihydrolipoic acid ; DHLA)、十二烧硫 醇（dodecanethiol ; DDT) 雙硫醇破拍酸 (meso-2,3-dimercaptosuccinic acid ; DMSA )、穀胱甘肽 (glutathione ; GSH)、1,6-己二硫醇（1,6-hexanedithiol)。 其中，上述硫醇包覆之金屬奈米團簇（thiol-capped metal .nanocluster)係為一螢光金屬奈米團竊（Fluorescent metal nanocluster)，上述螢光金屬奈米團簇之粒徑範圍係為 0.5nm 至 3nm 〇 並且，於形成上述螢光金屬奈米團簇（Fluorescent metal nanocluster)後進行一官能基坡覆（functional coating) 反應，以使得上述螢光金屬奈米團簇具有至少一官能基特 性，其中，上述官能基披覆（functionalcoating)反應係為一 生物接枝(Bioconjugation)反應。 13 201223546 上述官能基披覆（functional coating)反應之官能基係 選自下列族群之一者：化學官能基、交聯分子、醣類、螢 光分子、順磁性分子、生物分子與藥物。 根據以上所述之實施例，上述螢光金奈米團簇與螢光 金'不、米團叙聚集體（Fluorescent gold nanocluster matrix)等 聋' 光金屬奈米團鎮係能作為生物探針(bioprobes)，並具有 下歹丨之應用·生物螢光標記（fluorescent biological label)、 ^床醫療影像顯影劑以及臨床醫療檢測、追蹤與治療。 範例一螢光金奈米團簇 ()螢光金奈米圑簇AuNC-DHLA之製備 (1)首先’將雙十二烷基二烷基溴化銨鹽溶於曱苯用以 作為預製傷溶液（100mM)，其次，將氯化金或四溴金酸溶 ;又十二烷基二烷基溴化銨鹽中（25mM)以形成一金屬金 刖驅溶液’接著，混合〇.625mL癸酸與lmL的預製備溶液 並予授拌’隨後於攪拌同時再注入〇.8inL金屬金前驅溶 液’以獲得一暗紅色溶液，其中，加入過量之曱醇直到暗 紅色溶液變成不透明之藍紫色溶液，藉由曱醇誘使金奈米 粒子聚集’此外，使用離心機將溶液中未反應之剩餘反應 試劑以及過小之金奈米粒子移除。 將純化後之金奈米粒子再次溶解於雙十二烷基二烷 基'/臭化知·鹽》谷液中以形成一暗紅色溶液，其次，再加入金 14 201223546 屬金前驅溶液並持續攪拌，直到暗紅色溶液轉為淡黃色透 明液體。此時，上述金奈米粒子已崩裂為粒徑較小之金奈 米團簇。其中.，上述金奈米粒子具有表面電漿性質 (520-530nm)，而金奈米團簇則無此性質，其吸收光譜如第 一圖所示，其中，以氯化金作為第一金屬前驅物，並且， (A)為金奈米粒子；（B)為使用氣化金作為第二金屬前驅物 之金奈米團簇；（C)為使用四溴金酸為第二金屬前驅物之金 奈米團簇。 (II)0.0322g四丁基溴化銨粉末溶於2.5mL的雙十二烷 基二烷基溴化銨鹽溶液，直至粉末完全溶解。再將〇.〇52g 硫辛酸(lipoic acid)加入上述溶液中，直至溶液沒有氣泡產 生，使得硫辛酸還原成二氫硫辛酸，其中，為避免硫辛酸 還原不完全，因此再加入過量之還原劑四丁基溴化銨粉 末，確定已無氣泡產生。再者，將2.5mL金奈米團簇溶液 混入二氫硫辛酸溶液中並持續攪拌，此時，上述混合溶液 會轉為不透明並呈現為黃棕色，據此形成螢光金奈米團簇。 -其中，螢光金奈米團簇AuNC-DHLA之吸收 (absorption)、光激螢光（photoluminescence ; PL)、光激發 螢光.（photoluminescence excitation ; PLE)之光譜圖，如 第二圖所示。 15 201223546 (二）螢光金奈米團簇生物分子接枝 首先，取10μ1螢光金奈米團簇溶液與1〇μ1 X-PEG-amine(3mMin ddH20)均勻混合形成一混合溶液， 接著’加入l-ethyl-3-(3-dimethylaminopropyl) carbodiimide 溶液(EDC，8mM in ddH20)，震盪反應兩小時，據此完成 一生物分子接枝反應，其中，上述之PEG係能為維生素 (biotin)或卵白素(avidin)，上述生物分子接枝反應示意圖如 第三圖所不。此外，將改質之螢光金奈米團簇以膠體電泳 方式（2% agarose，75V)進行純化，以1〇〇kDa分子篩離心 置換於SBB (sodium boi^te buffer，pH=9)中。 範例二螢光金奈米團箱聚集體 (一）螢光金奈米團簇AuNC-DDT之製備 提供-種金奈米團箱溶液，其形成方法如範例一⑴所 述。將上述金奈米團舰液持續攪拌鐘 奸 入帶有硫醇基之碳鏈分子十…化隄滴 ,n , 卞—烷硫醇溶液中（體積比為 M 進行粒子表面配位基修飾置換，此時溶 之關現象，據此，形成螢光金奈_聚集 (二）螢光金奈米團簇生物分子接枝 8〇mM in ddH20 螢光簇溶液與細叫半乳糖溶液 藏合均勻，加入 16 201223546 l-ethyl-3-(3-dimethylaminopropyl) carbodiimide ( EDC ， 30mM in ddH20 )交聯劑溶液，震盪反應兩小時，利用EDC 與帶'胺基半乳糖所產生之醯胺鍵結，將半乳糖分子接枝於 螢光金奈米團簇表面，以lOOkDa分子篩離心，除去過量之 半乳糖。 其中，取20μ1接枝後之螢光半乳糖金奈米團簇，與20μ1凝 集素RCA120 ( lmg/ml)混合均勻，反應20分鐘，觀察凝集 反應是否發生，以判定半乳糖分子是否成功接枝至螢光金 團簇表面。 此外，將改質後之螢光金奈米團簇以膠體電泳方式（2〇/〇 agarose，75V)進行純化，經透析膜回收後，以i〇〇kDa 分子師離心置換於SBB ( sodium borate buffer，pH=9 ) 中。第六圖所示即為以兩性高分子改質之具烷硫醇配體的 螢光金奈米團簇聚集體示意圖。 經由統計分析發現，吸收光譜上升趨勢與螢光光譜相 符，顯示Au-DDT團簇之螢光特性與團簇產生自組裝 (self-assemble)之聚集程度有關，隨Au-S鍵結的產生，團 鎮組裝結構愈趨明顯而導致螢光產生且強度增強，其中， 分別以325nm、345nm及365nm波長激發光激發Au-DDT螢光 團簇，皆在600nm產生紅色放射螢光’且波峰並無產生位 移，表示Au-DDT團簇的紅光為螢光特性，而非一般散射 光；此外，將袜射光分別固定在580ηηι及600nm，測量最適 17 201223546 合之螢光激發光波長，發現兩者皆在325nm位置產生峰值， 顯示Au-DDT螢光金團簇以325nm波長進行激發，能在 600nm得到最大強度之紅色螢光放射，如第四圖所示。 參考第五圖所示，HA11CI4 precursor在370nm具有特性吸收 峰’經TBAB還原劑作用後產生6nm之奈米金粒子，在520nm 出現表面電漿共振吸收峰；繼續加入HAuCU溶液，使奈来 金粒子崩解形成團簇，520nm吸收峰消失，表示團鎮小於 5nm ’除了原本HAuCU在370nm之峰值出現，在3i〇nm產生 新的吸收峰’最後加入DDT分子產生螢光金團簇Au_DDT， 由於碳鏈分子間彼此之疏水性作用，使團簇形成自組装^士 構’吸收光譜在紅外光範圍明顯增加，足見聚集作用之產 生。其中，金奈米粒子與金奈米團簇各階段合成過程產物 之吸收光譜：（A) HAuC14 precursor ( 〇.625mM ) (B)奈米金 粒子（C)金奈米團簇（D)AuNC-DDT螢光奈米金粒子團 簇。 顯然地，依照上面實施例中的描述，本發明可能有許多的 修正與差異。因此需要在其附加的權利要求項之範圍内力口 以理解，除了上述詳細的描述外，本發明還可以廣泛地在 其他的實施例中施行。上述僅為本發明之較佳實施例而 已，並非用以限定本發明之申請專利範圍；凡其它未脫離 本發明所揭示之精神下所完成的等效改變或修飾，均應包 含在下述申請專利範圍内。 13 201223546 【圖式簡單說明】 第一圖為本發明之範例一中，金奈米粒子與金奈米團鎮之 吸收光譜； 第二圖為本發明之範例一中，螢光金奈米團簇AuNC-DHLA 之吸收（absorption)、光激螢光（photoluminescence ; PL)、光 激發營光(photoluminescence excitation ; PLE)之光譜圖； 第三圖為本發明之範例一中，螢光金奈米團簇AuNC-DHLA 生物分子接枝反應示意圖； 第四圖為本發明之範例二中，螢光金團簇AuNC-DDT之光激 發螢光光譜(PLE)與光激發光譜(PL); 第五圖為本發明之範例二中，金奈米粒子與螢光金奈米團 簇AuNC-DDT之吸收光譜；與 第六圖為本發明之範例二中，以兩性高分子改質之具烷硫 醇配體的螢光金奈米團簇聚集體示意圖。 19201223546 VI. Description of the Invention: [Technical Field to Which the Invention Is Applicable] The present invention relates to a nano cluster, and more particularly to a cluster of fluorescent gold nanoclusters. [Prior Art] When the size of the metal or semiconductor particles is small enough, a quantum confinement effect is produced, that is, the charge and energy of the particles are quantized, and such microparticles are called quantum dots. Quantum dot electrons are arranged very tightly. Because quantum quantification effects can excite different colors of fluorescence, quantum dots absorb energy waves with higher energy and produce energy step jumps. When electrons fall from high-energy state to low-energy state, Light with a longer wavelength (reddish light) is emitted. Quantum dots of different particle sizes emit different wavelengths of fluorescence. For example, cadmium selenide (CdSe) emits blue fluorescence at a wavelength of 2.1 nm, and emits green fluorescence at a particle size of 5 nm. When the particle size is close to 10 nm. When it is excited, the fluorescence is close to red. Compared to traditional organic dye molecules, quantum dots have high fluorescence intensity, good light stability, and the ability to excite a variety of different wavelengths of emitted waves with a single wavelength of laser. The transmitted wave is a narrow and symmetrical wave and can be repeatedly excited, so the fluorescence aging can last. These characteristics attract the attention of scientists, and the application of nano-quantum dots is becoming more and more diverse. The deep 201223546 has the potential to replace traditional dyes, so it is more promising for biomedical engineering applications. In recent years, quantum dots have successfully overcome the bottlenecks faced by biological and medical optical probes with their excellent optical properties, and have become an important nanomaterial for the design of a new generation of fluorescent probes. From the three-dimensional image of cells, long-term live cell monitoring, single-molecule dynamic intracellular tracking, development of long-acting optical sensors, cancer diagnosis and treatment, breakthroughs have been made, coupled with the rapid industrialization of quantum dots and its billion business opportunities. It has become a model for the extremely successful application of nanotechnology. However, traditionally, water-soluble quantum dots mainly composed of toxic heavy metals such as cadmium or lead are widely used, and the impact of their extension on the environment and human health is gradually being paid attention to. It is the dilemma facing the full development of its biomedical applications. Metal gold is one of the earliest researched nanomaterials. It is called colloidal gold in biological research and its particle size is between 1 and 100 nm. Gold quantum dots have a high electron density, good contrast under an electron microscope, and a relatively high biocompatibility, which has been shown to emit different colors of fluorescence by changing the size of their clusters. It can be applied to the production of multi-dimensional biomedical calibration or optical components, but because the process is quite difficult, it is necessary to use expensive dendrimer as a coating material for gold quantum dots during synthesis, which is time-consuming and difficult to mass produce. Therefore, it limits the development of its vast number of biomedical applications. Therefore, the development of a simple and mass-produced gold quantum dot formation technology industry is the focus of development. 201223546 SUMMARY OF THE INVENTION In view of the above-described background of the invention, in order to meet industrial requirements, the present invention provides a fluorescent gold nano-clustered aggregate. The present invention is characterized in that a fluorescent gold nanocluster is provided, and the surface of the above-mentioned fluorescent gold nano cluster aggregate has a dihydrolipoic acid (DHLA) ligand (ligand Wherein the fluorescent gold nano-clusters are fluorescently produced by the action between the dihydrolipoic acid ligand and the gold nanoclusters, and the fluorescent gold nano-aggregate aggregates are The particle size ranges from 〇.5 nm to 3 nm, and in addition, the above-mentioned fluorescent gold nanon clusters have a wavelength of from 400 to 100 nm. Another feature of the present invention is to provide a Fluorescent gold nanocluster matrix, wherein the camping gold nano cluster assembly system is formed by stacking a plurality of gold nanoclusters, the gold The nano-cracking particle size ranges from 0.5 nm to 3 nm, and the surface of the above-mentioned gold nano-clusters has an alkanethiol ligand, wherein each of the above-mentioned gold nano-clusters passes through the surface thereof. The interaction between the alkanethiol ligands is attracted to each other to form the above-mentioned Fluorescent gold nanocluster matrix, and the above-mentioned fluorescent Chennai cluster aggregation system is performed by the above-mentioned Chennai The aggregation of the rice vines produces a fluorescent property, and in addition, the wavelength of the fluorescent fluorescence of the above-mentioned fluorescent gold nanon cluster aggregates ranges from 400 to 100 nm. 201223546 Another feature of the present invention is to provide a method for forming a metal nanocluster. First, a mixed solution is provided. The mixed solution comprises a first metal precursor and a surfactant. a reducing agent and a solvent, performing a reduction reaction in the mixed solution to form a metal nanoparticle, and further adding a second metal after forming the nano metal particles a precursor, wherein the number of particles of the second metal precursor is greater than the total number of the nano metal particles, and the concentration of the nano metal particles and the second metal precursor are different, resulting in an unbalanced coexistence system. The metal metal particles thus break up into metal nanoclusters having a smaller particle size to form an equilibrium system, wherein the above-mentioned metal nanospheres have a particle size ranging from 1 nm to 4 nm. In accordance with the above objects, the present invention discloses a fluorescent gold nanoparticle cluster and a method of forming the same, which can be applied to various metals to form various metal nanoclusters. Among them, the above-mentioned fluorescent gold nano-aggregate can be used as bioprobes, and has the following applications: fluorescent biological label, clinical medical imaging developer, and clinical medical detection, tracking and treatment . 201223546 [Embodiment] The present invention is directed to a fluorescent gold $$ collective. In order to be able to fully understand the present invention, it will be apparent that the present invention is not limited to the specific details of the art. On the other hand, well-known steps or steps are familiar with the details to avoid unnecessary limitations of the invention. The preferred embodiment of the present invention will be described in detail below. However, the present invention may be widely practiced in other embodiments in addition to the detailed description, and the scope of the present invention is not limited. The scope shall prevail. One embodiment of the present invention discloses a Fluorescent gold nanocluster having a dihydrolipoic acid (DHLA) ligand on the surface of the fluorescent gold nano-cluster. The above-mentioned fluorescent Jinnaite group system generates fluorescence properties by the action between the above-mentioned dihydrolipoic acid ligand and the above-mentioned nano-clusters, and the particle size range of the above-mentioned fluorescent gold nano-sized drug is 0.5 nm. Up to 3 nm, in addition, the above-mentioned fluorescent gold nano-clusters have a wavelength of light-emitting fluorescence ranging from 400 to 100 nm. In a preferred embodiment of the present embodiment, the fluorescent gold nano-clusters further comprise a spacer, and one of the spacers is bonded to the dihydrolipoic acid (DHLA) ligand, and The other end of the spacer bond has a specific group, wherein the spacer comprises an oligomer or a polymer, and the specific group includes one of the following groups: Chemistry 201223546 Functional group, crosslinking molecule, sugar Classes, fluorescent molecules, paramagnetic molecules, biomolecules and drugs. Wherein the above oligomer or polymer comprises one of the following groups or any combination thereof? Polyols, polyether polyols, polyester polyols, polycarbonate Polycarbonate polyols, polycaprolactone polyols, polyacrylate polyols, polyethylene glycol (PEG), dextran, and copolymerization thereof Things. In another preferred embodiment of the present embodiment, the fluorescent gold nano cluster further comprises a spacer, the spacer is bonded to the dihydrolipoic acid (DHLA) ligand, and the spacer itself There is a specific group, wherein the spacer comprises one of the following groups: a chemical functional group, a crosslinking molecule, a saccharide, a fluorescent molecule, a paramagnetic molecule, a biomolecule, and a drug. According to still another embodiment of the present invention, a fluorescent gold nanocluster matrix is disclosed. The glory gold nano cluster assembly system is formed by stacking a plurality of gold nano clusters. The nanoparticle town has a particle size ranging from 0.5 nm to 3 nm, and the surface of the above-mentioned gold nanoclusters has an alkanethiol ligand, wherein each of the above-mentioned 201223546 gold nanoclusters passes through the surface thereof. The interaction between the alkanethiol ligands, attracting each other to form the above-mentioned Fluorescent gold nanocluster matrix, and the above-mentioned fluorescent gold nano cluster assembly system by the above-mentioned Chennai The aggregation of the rice clusters produces a fluorescent property, and in addition, the above-mentioned fluorescent gold nano cluster aggregates have a wavelength of light-emitting fluorescence ranging from 400 to 100 nm. In a preferred embodiment of the present embodiment, the surface of the fluorescent gold nano-clustered aggregate is coated with a spacer, one end of the spacer is bonded to the alkanethiol ligand, and the other end of the spacer is bonded. There is a specific group, wherein the spacer comprises an amphoteric polymer or oligomer, and the specific group comprises one of the following groups: a chemical functional group, a crosslinking molecule, a saccharide, a fluorescent molecule, a paramagnetic molecule , biomolecules and drugs. The above amphoteric polymer or oligomer comprises one of the following groups or any combination thereof: poly(maleic anhydride); PMA], polymer of 1-octadecene maleic anhydride [Poly ( Maleic anhydride-alt-1 -octadecene) ; PMAO] with polyacrylic acid (PAA) and its derivatives. In another preferred embodiment of the present embodiment, the surface of the fluorescent gold nanoparticle aggregate is coated with a spacer, the spacer is bonded to the alkanethiol ligand, and the spacer itself has a spacer Specific groups, in 10 201223546, the above spacers comprise one of the following groups: chemical functional groups, cross-linking molecules, auditory classes, fluorescent molecules, paramagnetic molecules, biomolecules and drugs. Another embodiment of the present invention discloses a method for forming a metal nanocluster. First, a mixed solution is provided. The mixed solution comprises a first metal precursor and a surfactant. And a reducing agent and a solvent are subjected to a reduction reaction in the mixed solution to form a metal nanopanide, wherein the nano metal particles have the property of surface plasma absorption. Further, after forming the above-mentioned nano metal particles, a second metal precursor is added to make the number of particles of the above-mentioned [metal t-driver larger than the total number of the above-mentioned nano-gold eyebrow particles, due to the above-mentioned nano metal particles and the above-mentioned second metal The concentration of the precursors is very different, resulting in a coexisting system of equilibrium. The above-mentioned nano metal particles are thus broken into metal nanoclusters having a smaller particle size to form a balance system, wherein the above-mentioned metal nano-clusters The particle size range of the town is from 1 nm to 4 nm. In addition, the above-mentioned first metal precursor and second metal precursor system can be opposite or not, wherein the first metal precursor and the first metal precursor are selected from one of the following groups: gas Gold (AuCl3), tetrakisic acid (HAuC14), gold bromide (AuBr3), tetrabromoic acid 201223546 (HAuBq) 0 and the above surfactant is selected from one of the following groups or any combination thereof : Didodecyldimethylammonium bromide (DDAB), Tetraoctylammonium bromide (TOAB), Tetrabutylammonium bromide (TBAB). The reductant is selected from one of the following groups or any combination thereof: tetrabutylammonium borohydride (TBAB), sodium borohydride (NaBH4), and vitamin C (Ascorbic Acid). The solvent is toluene or chloroform. On the other hand, after forming the above-mentioned metal nanoclusters, a Ligand-binding reaction is carried out, and the above-mentioned reaction is to bond a ligand to the surface of the above-mentioned metal nanoclusters to form a ligand. Lagged-capped metal nanocluster. The above ligand is selected from one of the following groups: dihydrolipoic acid (DHLA), dodecanethiol (DDT), bis(sodium sulfonate phenyl)phenylphosphine [ Bis(p-sulfonatophenyl)phenylphosphine; BSPP], triphenylphosphine. 12 201223546 Furthermore, the above Ligand-binding reaction is a thiol-related ligand binding reaction, and the above reaction is to bond the above thiol-related ligand to the above. The surface of the metal nanoclusters forms a thiol-capped metal nanocluster. The above thiol-related ligand is selected from one of the following groups. Dihydrolipoic acid (DHLA), dodecanethiol (DDT) dithiol sulphate (meso-2,3-dimercaptosuccinic acid; DMSA), glutathione (GHSH) 1,6-hexanedithiol (1,6-hexanedithiol). Wherein the thiol-coated metal nanocluster is a Fluorescent metal nanocluster, and the particle size range of the fluorescent metal nanoclusters is a 0.5 nm to 3 nm 〇 and a functional coating reaction after forming the above-mentioned Fluorescent metal nanocluster, so that the above-mentioned fluorescent metal nanoclusters have at least one functional group The base property, wherein the above functional capping reaction is a bioconjugation reaction. 13 201223546 The functional group of the above functional coating reaction is selected from one of the following groups: a chemical functional group, a crosslinking molecule, a saccharide, a fluorescent molecule, a paramagnetic molecule, a biomolecule, and a drug. According to the above embodiments, the above-mentioned fluorescent gold nano-clusters and the fluorescent metal nano-cluster matrix can be used as biological probes. Bioprobes), with squat applications, fluorescent biological labels, bed medical imaging developers, and clinical medical testing, tracking and treatment. Example 1 Preparation of Fluorescent Golden Nano-clusters () Fluorescent Golden Nano-A Clusters of AuNC-DHLA (1) Firstly, 'Double-dodecyldialkylammonium bromide salt is dissolved in toluene for pre-injury Solution (100 mM), followed by gold chloride or tetrabromogold acid; and dodecyldialkylammonium bromide salt (25 mM) to form a metal ruthenium drive solution'. Next, mix 〇.625 mL癸The acid is mixed with 1 mL of the pre-prepared solution and then injected with 〇.8 inL metal gold precursor solution while stirring to obtain a dark red solution, wherein excess sterol is added until the dark red solution becomes an opaque blue-violet solution. The gold nanoparticles are induced to accumulate by sterol. In addition, the unreacted residual reaction reagent and the too small gold nanoparticles in the solution are removed using a centrifuge. The purified gold nanoparticle is redissolved in the dodecyldialkyl '/small salt> solution to form a dark red solution, and secondly, gold 14 201223546 is a gold precursor solution and continues to be added. Stir until the dark red solution turns into a light yellow transparent liquid. At this time, the above-mentioned gold nanoparticles have been broken into clusters of gold nanoparticles having a small particle size. Among them, the above-mentioned gold nanoparticles have surface plasma properties (520-530 nm), while the Jinnai clusters have no such properties, and the absorption spectrum thereof is as shown in the first figure, wherein gold chloride is used as the first metal. Precursor, and (A) is a gold nanoparticle; (B) is a gold nanoparticle using gasified gold as a second metal precursor; (C) is a second metal precursor using tetrabromoic acid The golden nano cluster. (II) 0.0322 g of tetrabutylammonium bromide powder was dissolved in 2.5 mL of the dodecyldialkylammonium bromide salt solution until the powder was completely dissolved. Then add 52g of lipoic acid to the above solution until no bubbles are generated in the solution, so that the lipoic acid is reduced to dihydrolipoic acid. In order to avoid incomplete reduction of lipoic acid, an excess of reducing agent is added. Tetrabutylammonium bromide powder was determined to have no bubble generation. Further, a 2.5 mL of the golden nano cluster solution was mixed into the dihydrolipoic acid solution and stirring was continued, at which time the mixed solution turned opaque and appeared yellowish brown, thereby forming a fluorescent gold nanocluster cluster. - a spectrum of absorption, photoluminescence (PL), photoluminescence excitation (PLE) of the fluorescent gold nano cluster AuNC-DHLA, as shown in the second figure . 15 201223546 (II) Fluorescent gold nano-cluster biomolecular grafting First, a 10 μl fluorescent gold nano-cluster solution is uniformly mixed with 1 μl X-PEG-amine (3mMin ddH20) to form a mixed solution, followed by ' Adding l-ethyl-3-(3-dimethylaminopropyl) carbodiimide solution (EDC, 8 mM in ddH20), shaking the reaction for two hours, thereby completing a biomolecular grafting reaction, wherein the above PEG system can be a vitamin (biotin) or Avidin, the above-mentioned biomolecule grafting reaction diagram is as shown in the third figure. In addition, the modified fluorescent gold nanocapsules were purified by colloidal electrophoresis (2% agarose, 75 V) and centrifuged in SBB (sodium boi^te buffer, pH=9) with 1 〇〇kDa molecular sieve. Example 2 Fluorescent Golden Nano-Box Aggregates (I) Preparation of Fluorescent Golden Nano- Clusters AuNC-DDT A gold-nano cluster solution was provided, which was formed as described in Example 1 (1). The above-mentioned Jinnai group ship fluid is continuously stirred into the carbon chain molecule with thiol group, and the n, 卞-alkanol solution (volume ratio M is used for particle surface ligand modification and replacement). At this time, the melting phenomenon, according to this, the formation of fluorescent Chennai _ aggregation (two) fluorescent gold nano-cluster biomolecule grafting 8 mM in ddH20 fluorescent cluster solution and fine galactose solution uniformity Add 16 201223546 l-ethyl-3-(3-dimethylaminopropyl) carbodiimide ( EDC , 30mM in ddH20 ) crosslinker solution, shake the reaction for two hours, and use EDC to bond with the amine produced by 'aminogalactose. The galactose molecules were grafted onto the surface of the fluorescent gold nano-cluster and centrifuged at 100 kDa molecular sieve to remove excess galactose. Among them, the 20 μl-grafted fluorescent galactose-gold nanoclusters and 20 μl lectin RCA120 were obtained. (lmg/ml) was mixed uniformly, reacted for 20 minutes, and observed whether the agglutination reaction occurred to determine whether the galactose molecule was successfully grafted to the surface of the fluorescent gold cluster. In addition, the modified fluorescent gold nanoclusters were Colloidal electrophoresis (2〇/〇agarose, 75V) Purified, recovered by dialysis membrane, and centrifuged in SBB (sodium borate buffer, pH=9) by i〇〇kDa. The sixth figure shows the modification of amphoteric polymer with alkanethiol ligand. Schematic diagram of the cluster of fluorescent gold nano clusters. It is found through statistical analysis that the upward trend of the absorption spectrum is consistent with the fluorescence spectrum, showing the fluorescence characteristics of the Au-DDT cluster and the self-assemble aggregation of the clusters. Depending on the degree, with the formation of Au-S bond, the assembly structure of the group becomes more and more obvious, resulting in fluorescence generation and intensity enhancement. Among them, the Au-DDT fluorescent clusters are excited by excitation light at 325 nm, 345 nm and 365 nm, respectively. Red fluorescing is generated at 600 nm and there is no displacement of the peak, indicating that the red light of the Au-DDT cluster is a fluorescent characteristic, not a general scattered light; in addition, the stimuli are fixed at 580 ηηι and 600 nm, respectively, and the measurement is optimal 17 201223546 Combined with the wavelength of the fluorescent excitation light, it was found that both peaked at 325 nm, indicating that the Au-DDT fluorescent gold cluster was excited at 325 nm, and the maximum intensity of red fluorescent radiation was obtained at 600 nm, as shown in the fourth figure. Place Referring to the fifth figure, the HA11CI4 precursor has a characteristic absorption peak at 370 nm. After the TBAB reducing agent, 6nm nano gold particles are generated, and a surface plasma resonance absorption peak appears at 520 nm; the HAuCU solution is continuously added to make Neyle gold. The particles disintegrate to form clusters, and the absorption peak at 520 nm disappears, indicating that the group town is less than 5 nm 'except that the original HAuCU appears at the peak of 370 nm, and a new absorption peak is generated at 3i〇nm. Finally, the DDT molecule is added to produce the fluorescent gold cluster Au_DDT, due to The hydrophobic interaction between the carbon chain molecules makes the absorption spectrum of the self-assembled clusters of the clusters increase significantly in the infrared range, which shows the aggregation. Among them, the absorption spectra of the products of the various stages of the synthesis of the gold nanoparticles and the gold nanospheres: (A) HAuC14 precursor (〇.625 mM) (B) nano gold particles (C) gold nano clusters (D) AuNC -DDT fluorescent nano gold clusters. Obviously, many modifications and differences may be made to the invention in light of the above description. It is therefore to be understood that within the scope of the appended claims, the invention may be The above are only the preferred embodiments of the present invention, and are not intended to limit the scope of the claims of the present invention; all other equivalent changes or modifications which are not departing from the spirit of the present invention should be included in the following claims. Within the scope. 13 201223546 [Simple description of the diagram] The first figure is the absorption spectrum of the gold nanoparticles and the Jinnai group in the first example of the invention; the second figure is the fluorescent gold nano group in the first example of the invention. Cluster AuNC-DHLA absorption, photoluminescence (PL), photoluminescence excitation (PLE) spectrum; third diagram is the first example of the invention, fluorescent gold nano Schematic diagram of clustering AuNC-DHLA biomolecule grafting reaction; the fourth graph is the photoexcited fluorescence spectrum (PLE) and photoexcitation spectrum (PL) of the fluorescent gold cluster AuNC-DDT in the second example of the present invention; The figure is the absorption spectrum of the gold nanoparticle and the fluorescent gold nano cluster AuNC-DDT in the second example of the invention; and the sixth figure is the amphoteric sulfur modified by the amphoteric polymer in the second example of the present invention. Schematic diagram of fluorescent gold nano cluster aggregates of alcohol ligands. 19