201229490 六、發明說明： 【發明所屬之技術領域】 [0001] 本發明涉及一種用於單分子檢測的分子載體。 [先前技術3 [0002] 單分子檢測（Sing 1 e Mo 1 ecu 1 e Detect ion，SMD )技 術有別於一般之常規檢測技術，觀測到的為單個分子之 個體行為，單分子檢測技術在環境安全、生物技術、感 測器、食品安全等領域應用廣泛。單分子檢測達到分子 探測之極限，為人們長期以來追求之目標。與傳統的分 〇 析方法相比’單分子檢測法研究體系處於非平衡狀態下 的個體行為或平衡狀態下之波動行為因此特別適合研究 化學及生化反應動力學、生物分子之相互作用、結構與 功能資訊、重大疾病早期診斷、病理研究及高通量藥物 篩選等。 [〇〇〇3]目刖，已知有許多方法用於單分子檢須《j，而分子載體之 結構對單分子檢測技術發展成檢測結果起著十分重要之 〇 影響作用。先前技術中之複數種單分子檢測方法中，分 子載體結構係將膠體銀塗覆在玻璃表面 ，銀顆粒通過膠 體黏附於破縣面’然後將所述黏附有銀顆粒的玻璃經 過超聲波洗梅’在玻璃表面形D散祕難，形成分 子載體° _將㈣物分子設置於分子載體表面，通過 拉曼檢測系統向其分子載體上的待測物分子提供鐳射光 輻射it射光巾的光子與待測物分子發生碰撞，從而改 變光子的方向，產生拉曼散射。另外，光子與待測物分 子發生此量父換，改變了光子的能量和頻率，使該光子 100100547 表單編號Α〇1〇ι 第3頁/共45頁 1002000957-0 201229490 具有待測物分子的結構資訊。通過感測器接收來自待測 物分子的輻射訊號，形成拉曼圖譜，利用電腦對所述待 測物分子進行分析。 [0004] 然而，先前技術中，由於所述玻璃的表面為一平整的平 面結構，產生的拉曼散射訊號不夠強，從而使所述單分 子檢測的解析度低，不適用於低濃度及微量樣品的檢測 ，從而應用範圍受到限制。 【發明内容】 [0005] 有鑒於此，提供一種能提高單分子檢測解析度的分子載 體實為必要。 [0006] 一種用於單分子檢測的分子載體，其包括一基底，其中 ，所述基底一表面設置有複數三維奈米结構及一金屬層 包覆於三維奈米結構表面及相鄰三維奈米結構之間基底 的表面。 [0007] 相較於先前技術，本發明通過設置金屬層，在外界入射 光電磁場的激發下，金屬表面電漿發生共振，而由於金 屬層設置在三維奈米結構表面，可起到表面增強拉曼散 射（SERS)的作用，使得輻射訊號增強，從而可以提高 單分子檢測的解析度及準確度。 【實施方式】 [0008] 下面將結合附圖及具體實施例對本發明作進一步的詳細 說明。 [0009] 請參閱圖1、圖2及圖3，本發明第一實施例提供一種用於 單分子檢測的分子載體10，所述分子載體10包括一基底 100100547 表單編號A0101 第4頁/共45頁 1002000957-0 201229490 100、形成於基底100表面的複數三維奈米結構102及設 置於所述三維奈米結構102表面及相鄰三維奈米結構102 之間的基底100表面的金屬層101。 [0010] θ [0011] 所述基底100可為絕緣基底或半導體基底。具體地，所述 基底100的材料可為矽、二氧化矽、氮化矽、石英、玻璃 、氮化鎵、砷化鎵、藍寶石、氧化鋁或氧化鎂等。所述 基底100的形狀不限，只需具有兩個相對設置的平面即可 ，本實施例中，所述基底100的形狀為一平板狀。所述基 底100的大小、厚度不限，可根據實際單分子檢測的需要 選擇。本實施例中，所述基底100的材料為二氧化矽。 ❹ 所述三維奈米結構102設置於所述基底100的一表面。該 三維奈米結構102與基底100為一體成型結構。所述三維 奈米結構102的結構類型不限，可為凸起結構或凹陷結構 。所述凸起結構為從所述基底100的表面向外延伸出的突 起的實體，所述凹陷結構為從所述基底100的表面向内凹 入形成凹進的空間。所述三維奈米結構102的結構類型可 根據實際需求及實驗條件控制。如圖3所示，本實施例中 ，所述三維奈米結構102為一半球狀的凸起結構，所述半 球狀三維奈米結構102的直徑為30奈米〜1 000奈米，高度 為50奈米〜1000奈米。優選地，所述半球狀凸起結構的底 面直徑為50奈米〜200奈米，高度為100奈米~500奈米。 所述相鄰的每兩個半球狀凸起結構之間的距離相等，可 為0奈米〜50奈米。所述兩個半球狀凸起結構之間的距離 係指所述半球狀凸起結構的底面之間的距離，所述半球 狀凸起結構之間的距離為零奈米係指所述兩個半球狀凸 100100547 表單編號Α0101 第5頁/共45頁 1002000957-0 201229490 起結構相切，其底面緊密相連，中間沒有間隔。本實施 例中，所述半球狀三維奈米結構1〇2之間的距離為1〇奈米 〇 [0012] [0013] 所述複數三維奈米結構1〇2在基底1〇〇一表面以陣列形式 設置。所述陣列形式設置減述複數三維奈米結構1〇2可 以按照等間距行列式排布'同心圓環排布或六角形密堆 排布等方式排歹卜而i，所述複數三維奈米結構m以陣 列形式排布形成一或複數相互間隔的單一圖案。所述單 圖案可為二角形、平行四邊形、體形、菱形、方形、 矩形或圓形等。如圖4所示，所述三維奈米結構1〇2以陣 列形式形成四個不同的圖案〇 ........ 所述金屬層101包覆於所述主雄奈米結構】_的表面及相 鄰的三維奈米結構102之間基底1〇〇的表面。具體的，所 述金屬層101為金屬材料形成的一連續的層狀結構，可為 單層層狀結構或複數層層狀結構。所述金屬層1〇1基本均 勻沈積於所述複數三維奈米結構102表面及相鄰的三維奈 米結構102之間的基底1〇〇的表面。所述相鄰的三維奈米 結構1 02之間形成一間隙（Gap )，此處金屬層1 〇 1的表 面存在表面電漿共振，從而產生拉曼散射增強。所述金 屬層101可通過電子束蒸發、離子束濺鍍等方法沈積於所 述三維奈米結構102的表面及相鄰的三維奈米結構102之 間的基底100的表面。所述金屬層101的厚度為2奈米 〜200奈米’優選的，所述金屬層1〇1的厚度均一。所述金 屬層101的材料不限’可為金、銀、銅、鐵或鋁等金屬。 可以理解，本實施例中所述金屬層101的材料並不限於以 100100547 表單編號A0101 第6頁/共45頁 1002000957-0 201229490 上幾種，任何常溫下為固_金屬材料都可以。本實施 例中所述金屬層m優選為厚度⑽奈米的銀。 [0014] Ο ❹ 由於所述基底m具有複數三維奈米結構咖，主要有以 :幾個優點：第―，由於分子載劃中金制1Q2直接形 基底_的表面，無須額外的_層«㈣n =，金屬層可魏容易通過腐料方式去除，然後根據 早分子檢測的需要而沈财同材料的金屬層，所述基底 ⑽可以重複《，而所述金屬層1Q1可以根據實際檢測 早分子的需要而自由進行更換，μ對基底m表面的三 維奈米結構U)2產生影響，即為—“自由平臺” .^了 所述金屬層m直接包覆於所述三維奈米結構⑽的表人面 ，所述三維奈米結構102具有較大的表面積，使所述金屬 層ιοί中的奈米金屬顆粒可以不需要黏接層，就能牢固的 附著在所述三維奈储表面及相㈣三維奈米結 構102之間基底刚的表面，當所述分子載體_於檢測 單分子時，可以減少黏接層等其他化素在檢測過程 中產生的干擾’避免黏接層導電性介質對表面電浆共振 分佈產生影響；再次，由於金屬層1G1設置在三雉奈米結 構102的表面’在外界入射光電磁場的激發下金屬表面 電漿發生共振吸收，而三維奈米結構起到表面增強拉曼 散射的作用，可提高SERS增強因數，增強拉曼散射。所 述SERS增強因數與三維奈米結構1〇2之間的間距相關，所 述二維奈米結構102之間的距離越小，SERS增強因數越大 。所述SERS增強因數理論值可為1〇5至1〇15，從而可以得 到更好的單分子檢測結果。本實施例中所述分子載體1 〇 100100547 表單編號A0101 第7頁/共45頁 1002000957-0 201229490 的SERS增強因數大於101()。 [0015] 請一併參閱圖5及圖6，本發明進一步提供一種應用所述 分子載體10的單分子檢測方法，所述檢測方法主要包括 以下步驟： [0016] 步驟（S11 )，提供一分子載體，所述分子載體包括一基 底，所述基底一表面設置有複數三維奈米結構，在所述 三維奈米結構表面及相鄰三維奈米結構之間的基底的表 面形成有金屬層，所述金屬層附著於所述基底的表面； [0017] 步驟（S12)，在所述金屬層遠離基底的表面組裝待測物 分子； [0018] 步驟（S13)，利用檢測器對組裝在基底上的所述待測物 分子進行檢測。 [0019] 具體的，步驟（S11 )，提供一分子載體10。 [0020] 所述分子載體10的製備方法主要包括：步驟（S111 )， 提供一母板1001;步驟（S112)，在所述母板1001表面 形成三維奈米結構102，形成所述基底100 ;步驟（S113 )，在所述基底100的表面形成一金屬層101，形成所述 分子載體10。 [0021] 在步驟（S111 )中，該母板1001可為絕緣材料或半導體 材料。本實施例中所述母板1001的材料為二氧化矽。所 述母板1001的厚度為200微米〜300微米。所述母板1001 的大小、厚度和形狀不限，可根據實際需要選擇。 [0022] 進一步，可以對所述母板1001的一表面進行親水處理。 100100547 表單編號A0101 第8頁/共45頁 1002000957-0 201229490 [0023] 首先’清洗所述母板1001的表面，清洗時採用超淨間標 準工藝清洗。然後，在溫度為30°C~100°C，體積比為NHq • H20 : H2〇2 : H2〇 = x : y : z的溶浪中溫浴30分鐘~60分 鐘，鮮所述母板1001的表面進行親水處理，之後用去離 子水沖洗2次~3次。其中，X的取值為0.2〜2，y的取值為 0. 2〜2，z的取值為卜20。最後，用氮氣對所述母板1001 表面進行吹幹。 [0024] 進一步，還可以對所述母板1〇〇1的表面進行二次親水處 理’其具體包括以下步驟··將親水處理過後的所述母板 1001在2wt%〜5wt%的十二烷基硫酸鈉溶液（SDS)中浸泡2 小時〜24小時》可以理解’在SDS中浸泡過後的所述母板 1001的表面有利於後續奈米微球的鋪展並形成有序排列 的大面積奈米微球。 [0025] [0026]201229490 VI. Description of the Invention: [Technical Field of the Invention] [0001] The present invention relates to a molecular carrier for single molecule detection. [Prior Art 3 [0002] Single-molecule detection (Sing 1 e Mo 1 ecu 1 e Detect ion, SMD) technology is different from the general conventional detection technology, and the observed individual behavior of a single molecule, single molecule detection technology in the environment It is widely used in safety, biotechnology, sensors, food safety and other fields. Single molecule detection has reached the limit of molecular detection and has long been the goal pursued by people. Compared with the traditional decanting method, the single-molecule detection method is not suitable for studying chemical and biochemical reaction kinetics, biomolecule interaction, structure and volatility in the non-equilibrium state. Functional information, early diagnosis of major diseases, pathological research and high-throughput drug screening. [〇〇〇3] It is known that there are many methods for single molecule detection "j, and the structure of molecular carriers plays an important role in the development of single molecule detection technology into detection results. In the plurality of single molecule detection methods in the prior art, the molecular carrier structure coats the colloidal silver on the surface of the glass, and the silver particles adhere to the broken surface by the colloid, and then the glass adhered with the silver particles is subjected to ultrasonic washing. It is difficult to form a molecular carrier on the surface of the glass. The molecular carrier is placed on the surface of the molecular carrier, and the photon of the laser beam is irradiated to the analyte molecule on the molecular carrier through the Raman detection system. The analyte molecules collide, changing the direction of the photons and producing Raman scattering. In addition, the photon and the analyte molecule undergo this amount of parental change, changing the energy and frequency of the photon, so that the photon 100100547 form number Α〇1〇ι page 3 / total 45 pages 1002000957-0 201229490 with the molecule of the analyte Structural information. The radiation signal from the molecule to be tested is received by the sensor to form a Raman spectrum, and the molecule of the object to be detected is analyzed by a computer. [0004] However, in the prior art, since the surface of the glass is a flat planar structure, the generated Raman scattering signal is not strong enough, so that the resolution of the single molecule detection is low, and is not suitable for low concentration and trace amount. The detection of the sample, so that the scope of application is limited. SUMMARY OF THE INVENTION [0005] In view of the above, it is necessary to provide a molecular carrier capable of improving the resolution of single molecule detection. [0006] A molecular carrier for single molecule detection, comprising a substrate, wherein a surface of the substrate is provided with a plurality of three-dimensional nanostructures and a metal layer is coated on the surface of the three-dimensional nanostructure and adjacent three-dimensional nanometers The surface of the substrate between the structures. [0007] Compared with the prior art, the present invention resonates the metal surface plasma by the metal layer under the excitation of the external incident photoelectric field, and the surface layer is enhanced because the metal layer is disposed on the surface of the three-dimensional nanostructure. The effect of the sniffer (SERS) is to enhance the radiation signal, which can improve the resolution and accuracy of single molecule detection. [Embodiment] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. Referring to FIG. 1, FIG. 2 and FIG. 3, a first embodiment of the present invention provides a molecular carrier 10 for single molecule detection, the molecular carrier 10 comprising a substrate 100100547 Form No. A0101 Page 4 of 45 Page 1002000957-0 201229490 100, a plurality of three-dimensional nanostructures 102 formed on the surface of the substrate 100, and a metal layer 101 disposed on the surface of the substrate 100 between the surface of the three-dimensional nanostructures 102 and the adjacent three-dimensional nanostructures 102. [0010] The substrate 100 may be an insulating substrate or a semiconductor substrate. Specifically, the material of the substrate 100 may be tantalum, cerium oxide, tantalum nitride, quartz, glass, gallium nitride, gallium arsenide, sapphire, aluminum oxide or magnesium oxide. The shape of the substrate 100 is not limited, and only needs to have two oppositely disposed planes. In the embodiment, the shape of the substrate 100 is a flat shape. The size and thickness of the substrate 100 are not limited and can be selected according to the needs of actual single molecule detection. In this embodiment, the material of the substrate 100 is cerium oxide. The three-dimensional nanostructures 102 are disposed on a surface of the substrate 100. The three-dimensional nanostructure 102 and the substrate 100 are integrally formed. The structure of the three-dimensional nanostructure 102 is not limited and may be a convex structure or a concave structure. The raised structure is a raised body that extends outwardly from the surface of the substrate 100, the recessed structure being recessed inwardly from the surface of the substrate 100 to form a recessed space. The structure type of the three-dimensional nanostructure 102 can be controlled according to actual needs and experimental conditions. As shown in FIG. 3, in the embodiment, the three-dimensional nanostructure 102 is a semi-spherical convex structure, and the hemispherical three-dimensional nanostructure 102 has a diameter of 30 nm to 1 000 nm, and the height is 50 nm ~ 1000 nm. Preferably, the hemispherical convex structure has a bottom surface diameter of 50 nm to 200 nm and a height of 100 nm to 500 nm. The distance between each adjacent two hemispherical convex structures is equal and may range from 0 nm to 50 nm. The distance between the two hemispherical convex structures refers to the distance between the bottom surfaces of the hemispherical convex structures, and the distance between the hemispherical convex structures is zero nanometer refers to the two Hemispherical convex 100100547 Form number Α0101 Page 5 / Total 45 pages 1002000957-0 201229490 The structure is tangent, the bottom surfaces are closely connected, and there is no space in between. In this embodiment, the distance between the hemispherical three-dimensional nanostructures 1〇2 is 1〇 nanometer [0012] [0013] The plurality of three-dimensional nanostructures 1〇2 are on the surface of the substrate 1 Array form settings. The array form is provided with a description of the complex three-dimensional nanostructures 1〇2, which may be arranged in an equidistant determinant arrangement, such as a concentric annular arrangement or a hexagonal dense arrangement, and the plurality of three-dimensional nanometers. The structures m are arranged in an array to form a single pattern or a plurality of spaced apart patterns. The single pattern may be a quadrangle, a parallelogram, a body, a diamond, a square, a rectangle or a circle. As shown in FIG. 4, the three-dimensional nanostructures 1〇2 form four different patterns in an array. The metal layer 101 is coated on the main male nanostructure. The surface and the surface of the substrate 1 between the adjacent three-dimensional nanostructures 102. Specifically, the metal layer 101 is a continuous layered structure formed of a metal material, and may be a single layer structure or a plurality of layer structures. The metal layer 1〇1 is deposited substantially uniformly on the surface of the substrate 1〇〇 between the surface of the plurality of three-dimensional nanostructures 102 and the adjacent three-dimensional nanostructures 102. A gap (Gap) is formed between the adjacent three-dimensional nanostructures 102, where the surface of the metal layer 1 〇 1 has surface plasma resonance, thereby producing Raman scattering enhancement. The metal layer 101 may be deposited on the surface of the three-dimensional nanostructure 102 and the surface of the substrate 100 between adjacent three-dimensional nanostructures 102 by electron beam evaporation, ion beam sputtering or the like. The thickness of the metal layer 101 is preferably from 2 nm to 200 nm, and the thickness of the metal layer 1〇1 is uniform. The material of the metal layer 101 is not limited to a metal such as gold, silver, copper, iron or aluminum. It can be understood that the material of the metal layer 101 in the embodiment is not limited to 100100547 Form No. A0101 Page 6 / Total 45 pages 1002000957-0 201229490, any solid material can be used at room temperature. The metal layer m in the present embodiment is preferably silver having a thickness of (10) nm. [0014] ❹ ❹ Since the substrate m has a plurality of three-dimensional nanostructure coffee, there are mainly several advantages: the first, because the surface of the gold 1Q2 direct-shaped substrate _ in the molecular mapping, no additional layer _ (4) n =, the metal layer can be easily removed by the smear method, and then the metal layer of the same material can be deposited according to the needs of early molecular detection, the substrate (10) can be repeated, and the metal layer 1Q1 can be detected according to the actual needs of the early molecule. And freely changing, μ has an influence on the three-dimensional nanostructure U)2 on the surface of the substrate m, that is, "free platform". The metal layer m is directly coated on the surface of the three-dimensional nanostructure (10) The three-dimensional nanostructures 102 have a large surface area, so that the nano metal particles in the metal layer can be firmly adhered to the three-dimensional nano-reservoir surface and phase (four) three-dimensional without an adhesive layer. The surface of the base between the nanostructures 102, when the molecular carrier is used to detect a single molecule, can reduce the interference generated by other chemical elements such as the adhesive layer during the detection process, and avoid the conductive layer on the surface of the adhesive layer. The slurry resonance distribution has an effect; again, since the metal layer 1G1 is disposed on the surface of the three-dimensional nanostructure 102, the metal surface plasma is resonantly absorbed by the external incident photoelectric magnetic field, and the three-dimensional nanostructure acts as a surface-enhanced Raman The effect of scattering increases the SERS enhancement factor and enhances Raman scattering. The SERS enhancement factor is related to the spacing between the three-dimensional nanostructures 1〇2, and the smaller the distance between the two-dimensional nanostructures 102, the larger the SERS enhancement factor. The theoretical value of the SERS enhancement factor can be from 1 〇 5 to 1 〇 15, so that a better single molecule detection result can be obtained. In the present embodiment, the molecular carrier 1 〇 100100547 Form No. A0101 Page 7 of 45 1002000957-0 201229490 has a SERS enhancement factor greater than 101(). [0015] Please refer to FIG. 5 and FIG. 6 together, the present invention further provides a single molecule detection method using the molecular carrier 10, the detection method mainly includes the following steps: [0016] Step (S11), providing a molecule a carrier comprising a substrate, a surface of which is provided with a plurality of three-dimensional nanostructures, and a metal layer is formed on a surface of the substrate between the surface of the three-dimensional nanostructure and the adjacent three-dimensional nanostructure. Attaching a metal layer to the surface of the substrate; [0017] step (S12), assembling the analyte molecule on the surface of the metal layer away from the substrate; [0018] Step (S13), assembling the substrate on the substrate by using a detector The analyte molecule is detected. [0019] Specifically, in step (S11), a molecular carrier 10 is provided. [0020] The preparation method of the molecular carrier 10 mainly includes: step (S111), providing a mother board 1001; step (S112), forming a three-dimensional nanostructure 102 on the surface of the mother board 1001 to form the substrate 100; In step (S113), a metal layer 101 is formed on the surface of the substrate 100 to form the molecular carrier 10. [0021] In the step (S111), the mother board 1001 may be an insulating material or a semiconductor material. The material of the mother board 1001 in this embodiment is cerium oxide. The mother board 1001 has a thickness of 200 μm to 300 μm. The size, thickness and shape of the motherboard 1001 are not limited and can be selected according to actual needs. [0022] Further, a surface of the mother board 1001 may be subjected to a hydrophilic treatment. 100100547 Form No. A0101 Page 8 of 45 1002000957-0 201229490 [0023] First, the surface of the mother board 1001 is cleaned and cleaned using a clean room standard process. Then, at a temperature of 30 ° C ~ 100 ° C, the volume ratio is NHq • H20 : H2 〇 2 : H2 〇 = x : y : z of the lava bath for 30 minutes to 60 minutes, fresh the mother board 1001 The surface was treated with hydrophilicity and then rinsed twice with deionized water for 3 to 3 times. Wherein, the value of X is 0.2 to 2, and the value of y is 0. 2 to 2, and the value of z is Bu 20. Finally, the surface of the mother board 1001 was blown dry with nitrogen. [0024] Further, the surface of the mother substrate 1〇〇1 may be subjected to a secondary hydrophilic treatment, which specifically includes the following steps: the hydrophilic treatment of the mother substrate 1001 at 2 wt% to 5 wt% of twelve Soaking in Sodium Alkyl Sulfate Solution (SDS) for 2 hours to 24 hours" It can be understood that the surface of the mother board 1001 after soaking in SDS is beneficial to the spreading of subsequent nanospheres and forming an ordered array of large areas. Rice microspheres. [0026] [0026]
在步驟（S112)中，在所述母板1〇〇1表面形成三維奈米 結構102爪成所述基底1〇〇的方法具體包括以下步驟： 步驟（S1121)，在所述母板—表面形成掩膜 層 108。 _母板1001所述細層⑽為一單層奈米微球形成的層狀結 構。可以理解，採用單層奈米微球作為掩膜層108，可以 在奈米微球對應的位置製備得到&起結構。 _所述在母板1001的表面形成一單層奈米微球作為掩膜層 108具體包括以下步驟： [0029]首先，製備一含有奈米微球的混合液。 100100547In the step (S112), the method of forming the three-dimensional nanostructure 102 on the surface of the mother substrate 1〇〇1 into the substrate 1〇〇 specifically includes the following steps: Step (S1121), at the mother board-surface A mask layer 108 is formed. The fine layer (10) of the mother substrate 1001 is a layered structure formed by a single layer of nanospheres. It can be understood that by using a single layer of nanospheres as the mask layer 108, a structure can be prepared at a position corresponding to the nanospheres. The formation of a single layer of nanospheres as a mask layer 108 on the surface of the mother substrate 1001 specifically includes the following steps: [0029] First, a mixed liquid containing nanospheres is prepared. 100100547
表單編號A010I 第9頁/共45頁 1002000957-0 201229490 [0030] [0031] [0032] [0033] 本實施例中’在直徑為15厘米的表面皿中依:欠加入i5〇毫 升的純水、3微升〜5微升的的奈米微球 、及當量的〇. lwt%〜3wt%_S後形成混合物，將上述混 合物靜置分賴〜6〇分鐘。待奈米微球充分分散於混合物 中後，再加入1微升〜3微升的4W，SDS，以調節奈米微 球的表面張力，有利於形成單層奈米微球陣列。其中， 不米微球的直徑可為60奈米〜5〇〇奈米，具體地，奈米微 球的直徑可為100奈米、200奈米、3〇〇奈米或4〇〇奈米’ 上述直徑偏差為3奈米〜5奈米。優選的奈米微球的直徑為 奈米或400奈米。所述奈米微球可為聚合物奈米微球 或矽奈米微球等。所述聚合翁奈米微球的材料可為聚笨 乙烯（PS)或聚曱基丙烯酸甲瘤（PMMA)。可以理解，所述 表面皿中的混合物可依實際需求而按比例調製。 其次’在所述母板1001的一表面形成一單層奈米微球混 合液，並使所述單層奈米微球以陣列形式設置於所述母 板1001的表面。 本實施例中採用提拉法或旋塗法在所述母板1001的表面 形成一單層奈米微球溶液。通過控制提拉法的提速或旋 塗法的轉速，所述單層奈米微球可以呈六角密堆排布、 簡單立方排布或同心圓環排布等。 所述採用提拉法在母板1001的表面形成單層奈米微球溶 液的方法包括以下步驟：首先’將經親水處理後的所述 母板1 0 01緩慢的傾斜的沿著表面皿·的側壁滑入表面皿的 混合物中，所述母板1 0 01的傾斜角度為9。至1 5 °。然後 ，將所述母板1001由表面孤的混合物中緩慢的提起。其 100100547 表單編號A0101 第1〇真/共45頁 1002000957-0 201229490 [0034] Ο ο [0035] 100100547 中，上述滑下和提起速度相當，均為5毫米/小時〜1〇毫米 /小時。該過程t，所述奈米微球的溶液令的奈米微球通 過自組裝形成呈六角密堆排布的單層奈米微球。 本實施例中，採用旋塗法在母板1001的表面形成單層奈 米微球溶液，其包括以下步驟：首先，將親水處理過後 的母板1001在2wa的十二烷基硫酸鈉溶液中浸泡2小時 ~24小時，取出後在所述母板1〇〇1的表面上塗覆3微升~5 微升的聚苯乙烯。其次，以旋塗轉速為4〇〇轉/分鐘〜5〇〇 轉/分鐘的速度旋塗5秒〜30秒。然後，以旋塗轉速為8〇〇 轉/分鐘〜1 000轉/分鐘的速度旋塗3〇秒〜2分鐘後。再次 ’將旋塗轉速提高至14〇〇轉/分鐘~15〇〇轉/分鐘，旋塗 10秒〜20秒，除去邊緣多餘的微球。最後，將分佈有奈米 微球的母板1001的表面進行乾燥後即可在所述母板iOOj 的表面上形成呈六角密堆排布的單層奈米微球，進而形 成所述掩膜層108。此外，在形成所述掩膜層之後還 可以進一步對母板1001的表面進行烘烤。所述烘烤的溫 度為50U00X：，烘烤的時間ll分鐘〜5分鐘。 本實施例中，所述奈米微球的直徑可為4〇〇奈米。請參閱 圖7 ’所述單層奈米微球中的奈米微球以能量最低的排布 方式排布，即六角密堆排布。所述單層奈米微球排布最 密集，佔空比最大。所述單層奈米微球中任意三個相鄰 的奈米微球呈一等邊三角形。可以理解，通過控制奈米 微球溶液的表面張力，可以使單層奈米微球中的奈米微 球呈簡單立方排布。 步驟（S1122)，採用反應性蝕刻氣體11〇對所述母板 表單編號A0101 第11頁/共45頁 1002000957-0 [0036] 201229490 1001的表面進行姓刻，在所述母板1〇〇1的表面形成複數 三維奈米結構102。 [0037] [0038] [0039] 100100547 所述採用反應性蚀刻氣體UG對母板_丨的表面進行姓刻 的步驟在-微波錢系統中進行。所述微波電衆系統為 反應離子蝕刻（Reacticm-I0n-Etching，RIE)模式。 所述反應性姓刻氣體11〇基本不與所述奈米微球發生反應 ’但所述反應性#刻氣體UQ對母板表面進行姓刻 ’形成複數二維奈米結構m，得到所述基底1〇〇。 本實施例中’將形成有單層奈米微球的母板1〇〇1的表面 放置於微波電漿系統中，且該微波電漿系统的一感應功 率源產生反應性触刻氣體丨丨〇。該反奉桂钱g氣體丨丨〇以 較低的離子能量從產生區域擴散1漂移至所述母板丨0 0 i 的表面。所述反應性姓刻氣體對所述單層奈米微球之間 的母板1001的表面進行蝕刻，而不與所述奈米微球進行 反應’從而形成所述三維奈米結構1 0 2。可以理解，通過 控制反應性蝕刻氣體11 〇的蝕刻時間可以控制三維奈米結 構102間的間距及三維奈米結構102的高度。 本實施例中’所述微波電漿系統的工作氣體包括六氟化 硫（SF6)和氬氣（Ar)或六氟化硫（SF6)和氧氣（〇2 )。其中，六氟化硫的通入速率為10標況毫升每分〜60標 況毫升每分，氬氣或氧氣的通入速率為4標況毫升每分 〜20標況毫升每分。所述工作氣體形成的氣壓為2帕〜1〇帕 。所述電漿系統的功率為40瓦~70瓦。所述採用反應性蝕 刻氣體110蝕刻時間為1分鐘〜2. 5分鐘。優選地，所述微 波電漿系統的功率與微波電漿系統的工作氣體的氣壓的 表單編號A0101 第12頁/共45頁 1002000957-0 201229490 數值比小於20 : 1。 [0040] 進一步，所述反應性蝕刻氣 中還可以加入三氟甲烷 (CHF )、四氟曱烷（cf )哎龙、 3 4 X其浥合氣體等其他氣體以 調節姓刻速率。所述三氟曱燒、四氟甲烧（％ )或其混合氣體的流量可為20襟况毫升每分〜4〇標況毫升 每分的。 [0041] Ο [0042] [0043] Ο [0044] [0045] [0046] 100100547 可以理解，通過控制所述蚀刻的條件及蝕刻氣氛，可以 得到不同的凸起的三雉条米結構丨〇2，如半橢球狀凸起結 構等。如果所述掩膜層108為—具有複數開孔的連續膜， 則可以得到凹陷的三維条米結構10 2，如半球狀凹陷結構 、半橢球狀凹陷結構、例金字塔狀凹陷結構等。 步驟（S1123)，去除所述掩膜層1 ，得到所述基底 100。 採用四氫呋喃（THF )、今嗣、丁鋼、環己烷、正己烷、 甲醇或無水乙醇等無番成低"毒環保容劑作為剝離劑，溶 解奈米微球，可以去陳条米微畔今释’保留形成在母板 ..... ： . . ：!…… 1001表面的三維奈米結構102 ° 本實施例中，通過在丫網中超聲清洗去除聚笨乙烯奈米 微球。 步驟S113，在所述三雉奈米結構102表面及相鄰三維奈米 結構102之間的基底1〇〇的表面形成一金屬層丨〇1，形成 所述分子載體10。 所述金屬層101可採用電手束蒸發、離子束濺射等方式， 表單編號A0101 第13貢/我45頁 1002000957-0 201229490 在所述基底100表面垂直蒸鍍金屬薄膜。由於所述基底 100表面形成有三維奈米結構102，從而，在三維奈米結 構102及相鄰三維奈米結構1〇2之間的間隙中的基底1〇〇 表面形成金屬薄膜，進而形成所述分子載體1〇。所述金 屬層101的厚度為2奈米〜200奈米，所述金屬層101的材 料不限，可為金、銀、銅' 鐵或鋁等金屬。本實施例中 所述金屬層101厚度優選為2〇奈米。 [0047] 步驟S12，在所述金屬層1〇1遠離基底的表面組裝待測物 分子。 [0048] 所述組裝待測物分子主要包括一下步驟： .:::，： …： [0049] 首先，提供一待測物分子的溶液，所逑待测蠢溶液的分 、· 、 _γ 子/辰度可為10 mmol/L〜10 12mm〇i/L可根據實際需要製 備，本實施例中所述分子濃度為⑺-丨^卽丨/匕； [0050] 其次，將所述形成有金屬層101的分子載體1〇浸入待測物 溶液中’浸泡時間可為2min〜6〇min，優選的為lOmin， 使所述待測物分子均勻的分散於所述金屬層1〇1的表面； [0051] 最後，將所述分子载體10取出，用水或乙醇對所述分子 載體進行沖洗5~ 15次，然後利用乾燥裝置如吹風機等將 所述分子載體10吹幹’使殘留的水或乙醇蒸發，將所述 待測物分子組裝在金屬層1〇丨的表面。 [0052] 步驟S1 3，利用檢測器對所述待測物分子進行檢測。 [0053] 將所述組裝有待測物分子的分子載體1 〇置於檢測裝置中 ’利用檢測器如拉曼光譜儀對所述待測物分子進行檢測 100100547 表單編號A0101 第14頁/共45頁 1002000957-0 201229490 。本實施例中，所述拉曼光譜儀的檢測參數為He Ne :激 發波長633奈米，激發時間l〇sec，設備功率為9 ， 工作功率為9. OmWxO. 05x1。 [0054] ❹Form No. A010I Page 9/45 pages 1002000957-0 201229490 [0033] [0033] In the present embodiment, 'in a 15 cm diameter watch glass: owing to add 5 ml of pure water 3 μl to 5 μl of nanospheres, and an equivalent amount of 〇. lwt% to 3 wt% _S, a mixture is formed, and the mixture is allowed to stand for ~6 〇 minutes. After the nanospheres are fully dispersed in the mixture, 1 microliter to 3 microliters of 4W, SDS is added to adjust the surface tension of the nanospheres, which is advantageous for forming a single layer of nanosphere arrays. Wherein, the diameter of the non-meter microspheres may be from 60 nanometers to 5 nanometers. Specifically, the diameter of the nanospheres may be 100 nanometers, 200 nanometers, 3 nanometers or 4 nanometers. 'The above diameter deviation is 3 nm ~ 5 nm. Preferred nanospheres have a diameter of nanometers or 400 nanometers. The nanospheres may be polymer nanospheres or nanobelt microspheres or the like. The material of the polymerized inonazole microspheres may be polystyrene (PS) or polymethyl methacrylate (PMMA). It will be appreciated that the mixture in the watch glass can be scaled as needed. Next, a single layer of nanosphere mixture is formed on one surface of the mother substrate 1001, and the single layer of nanospheres is disposed in an array on the surface of the mother substrate 1001. In the present embodiment, a single layer of nanosphere solution is formed on the surface of the mother substrate 1001 by a pulling or spin coating method. The single-layer nanospheres may be arranged in a hexagonal close-packed arrangement, a simple cubic arrangement or a concentric annular arrangement by controlling the speed of the pulling method or the rotational speed of the spin coating method. The method for forming a single-layer nano microsphere solution on the surface of the mother substrate 1001 by using the pulling method comprises the following steps: firstly, the hydrophilically treated mother substrate 101 is slowly tilted along the surface plate. The side wall slides into the mixture of the watch glass, and the mother board 101 has an inclination angle of 9. To 1 5 °. The mother board 1001 is then slowly lifted from the surface of the isolated mixture. Its 100100547 Form No. A0101 No. 1 true/Total 45 pages 1002000957-0 201229490 [0034] ο ο [0035] In 100100547, the above-mentioned sliding down and lifting speeds are equivalent, both 5 mm / h ~ 1 mm / h. In the process t, the solution of the nanospheres causes the nanospheres to form a single layer of nanospheres arranged in a hexagonal close-pack by self-assembly. In this embodiment, a single layer of nanosphere solution is formed on the surface of the mother board 1001 by spin coating, which comprises the following steps: First, the hydrophilically treated mother board 1001 is placed in a 2 wa sodium dodecyl sulfate solution. After soaking for 2 hours to 24 hours, after taking out, apply 3 μl to 5 μl of polystyrene on the surface of the mother board 1〇〇1. Next, spin-coat at a speed of 4 rpm/min to 5 rpm/min for 5 seconds to 30 seconds. Then, spin-coat at a speed of 8 rpm/min to 1 000 rpm for 3 sec seconds to 2 minutes. Again, the spin speed was increased to 14 rpm to 15 rpm, and spin coating was applied for 10 seconds to 20 seconds to remove excess microspheres. Finally, after drying the surface of the mother substrate 1001 on which the nanospheres are distributed, a single layer of nanospheres arranged in a hexagonal close-packed layer can be formed on the surface of the mother substrate iOOj to form the mask. Layer 108. Further, the surface of the mother board 1001 may be further baked after the mask layer is formed. The baking temperature is 50 U00X: and the baking time is ll minutes to 5 minutes. In this embodiment, the diameter of the nanospheres may be 4 nanometers. Referring to Figure 7, the nanospheres in the single-layer nanospheres are arranged in the lowest energy arrangement, that is, the hexagonal close-packed arrangement. The single-layer nanospheres are densely packed and have the largest duty cycle. Any three adjacent nanospheres in the single layer of nanospheres have an equilateral triangle. It can be understood that by controlling the surface tension of the nanosphere solution, the nanospheres in the single layer of nanospheres can be arranged in a simple cubic shape. Step (S1122), using the reactive etching gas 11〇, the surface of the mother board form No. A0101, page 11/45 pages, 1002000957-0 [0036] 201229490 1001 is engraved on the mother board 1〇〇1 The surface forms a plurality of three-dimensional nanostructures 102. [0039] [0039] 100100547 The step of performing a surname on the surface of the mother board using the reactive etching gas UG is performed in a microwave money system. The microwave electric system is a Reacticm-I0n-Etching (RIE) mode. The reactive surrogate gas 11 〇 does not substantially react with the nano microspheres 'but the reactive gas engraved gas UQ on the surface of the mother plate is engraved to form a complex two-dimensional nanostructure m, The substrate is 1 〇〇. In the present embodiment, the surface of the mother substrate 1〇〇1 on which the single-layer nanospheres are formed is placed in a microwave plasma system, and an inductive power source of the microwave plasma system generates a reactive etch gas. Hey. The anti-Bangang money gas 漂移 drifts from the generation region diffusion 1 to the surface of the mother substrate 丨0 0 i with a lower ion energy. The reactive surging gas etches the surface of the mother substrate 1001 between the single-layer nano microspheres without reacting with the nano microspheres to form the three-dimensional nanostructure 1 0 2 . It will be appreciated that the spacing between the three-dimensional nanostructures 102 and the height of the three-dimensional nanostructures 102 can be controlled by controlling the etching time of the reactive etching gas 11 。. The working gas of the microwave plasma system in this embodiment includes sulfur hexafluoride (SF6) and argon (Ar) or sulfur hexafluoride (SF6) and oxygen (?2). Among them, the rate of sulphur hexafluoride is 10 standard liters per minute ~ 60 standard milliliters per minute, the access rate of argon or oxygen is 4 standard milliliters per minute ~ 20 standard conditions per minute. The working gas forms a gas pressure of 2 Pa to 1 Pascal. The plasma system has a power of 40 watts to 70 watts. 5分钟。 The reactive etching gas 110 etching time is 1 minute ~ 2. 5 minutes. Preferably, the power of the microwave plasma system is the same as the gas pressure of the working gas of the microwave plasma system. Form No. A0101 Page 12 of 45 1002000957-0 201229490 The numerical ratio is less than 20:1. [0040] Further, other gases such as trifluoromethane (CHF), tetrafluorodecane (cf), or sulphur gas may be added to the reactive etching gas to adjust the surname rate. The flow rate of the trifluorosulfonium, tetrafluoromethane (%) or a mixed gas thereof may be 20 liters per minute to 4 liters per minute. [0044] [0044] [0046] 100100547 It can be understood that by controlling the etching conditions and etching atmosphere, different raised three-dimensional structure 丨〇 2 can be obtained. , such as a semi-ellipsoidal convex structure. If the mask layer 108 is a continuous film having a plurality of openings, a recessed three-dimensional strip structure 10 2 such as a hemispherical recessed structure, a semi-ellipsoidal recessed structure, a pyramidal recessed structure, or the like can be obtained. In step (S1123), the mask layer 1 is removed to obtain the substrate 100. Use tetrahydrofuran (THF), yttrium, butadiene steel, cyclohexane, n-hexane, methanol or absolute ethanol, etc. as a stripping agent to dissolve nanospheres, you can go to Chen Mi Mi The current release 'remains formed on the mother board..... : . . :!...... 1001 surface three-dimensional nanostructure 102 ° In this example, the polystyrene nanospheres are removed by ultrasonic cleaning in a mesh . In step S113, a metal layer 丨〇1 is formed on the surface of the substrate 1 between the surface of the three-dimensional nanostructure 102 and the adjacent three-dimensional nanostructure 102 to form the molecular carrier 10. The metal layer 101 may be subjected to electric hand beam evaporation, ion beam sputtering or the like, and the metal film is vertically vapor-deposited on the surface of the substrate 100 in the form No. A0101, No. 13 Gong/Me 45, 1002000957-0 201229490. Since the three-dimensional nanostructure 102 is formed on the surface of the substrate 100, a metal thin film is formed on the surface of the substrate 1 in the gap between the three-dimensional nanostructure 102 and the adjacent three-dimensional nanostructure 1〇2, thereby forming a The molecular carrier is 1 〇. The thickness of the metal layer 101 is from 2 nm to 200 nm, and the material of the metal layer 101 is not limited, and may be metal such as gold, silver or copper 'iron or aluminum. The thickness of the metal layer 101 in this embodiment is preferably 2 nanometers. [0047] Step S12, assembling the analyte molecules on the surface of the metal layer 1〇 away from the substrate. [0048] The assembly of the analyte molecules mainly includes the following steps: .:::,:: [0049] First, a solution of the analyte molecules is provided, and the fractions, ..., and _γ of the solution to be tested are determined. The ratio of 10 mmol/L to 10 12 mm〇i/L can be prepared according to actual needs, and the molecular concentration in the present embodiment is (7)-丨^卽丨/匕; [0050] Next, the formation is The molecular carrier 1 of the metal layer 101 is immersed in the solution to be tested, and the soaking time may be 2 min to 6 min, preferably 10 min, so that the molecules of the analyte are uniformly dispersed on the surface of the metal layer 1〇1. [0051] Finally, the molecular carrier 10 is taken out, and the molecular carrier is washed 5 to 15 times with water or ethanol, and then the molecular carrier 10 is dried by a drying device such as a hair dryer or the like to make residual water. Or the ethanol is evaporated, and the molecules of the analyte are assembled on the surface of the metal layer. [0052] Step S13, detecting the molecule of the analyte by using a detector. [0053] placing the molecular carrier 1 组装 assembled with the analyte molecule in a detection device 'Detecting the molecule of the analyte using a detector such as a Raman spectrometer 100100547 Form No. A0101 Page 14 of 45 1002000957-0 201229490. In this embodiment, the detection parameter of the Raman spectrometer is He Ne : excitation wavelength 633 nm, excitation time l 〇 sec, device power is 9, working power is 9. OmWxO. 05x1. [0054] ❹
[0055] 本發明提供的單分子檢測方法，具有以下優點：首先， 先前技術的單分子檢測方法為在基底上沈積點接層然 後在黏接層上形成金屬奈米結構作為分子載體，因此所 述黏接層對單分子檢測產生一定的影響，而本發明所述 三維奈米結構通過反應離子蝕刻的方法直接形成於基底 之上’金屬層直接沈積於基底的表面，因此可以防止黏 接層等化學因素對檢測結果產生影響;其次，本發明所 述單分子檢測方法中的分子載體具有三維奈米結構，分 子載體中的金屬層直接沈積於基底表面，而先前技術中 金屬奈米結構必須通過黏接層固定於基底表面從而使 得單分子檢測的結果受到影響；再次，所述三維奈米結 構的形狀、大小、間距等可以通過控制製備條件等方便 的進行控制，即可操作性高；第四’通適表三維奈米釺 構表面設置金屬層，可以提高單分子檢測的解析度尤 其為對於染料、生物分子、螢光材料及六代聯笨等不能 用常規檢測方法檢測的物質，也均可以利用本方去進/ 檢測。 請一併參閱圖8至圖9，本發明第二實施例提供一種分子 载體20，所述分子載體20包括一基底2〇n 、 刃 <υυ、形成於基底 200表面的複數三維奈米結構2〇2及設置於所述—維齐乂 結構202表面及相鄰三維奈米結職之二表卡 面的金屬層20卜所述分子載體2G的結構與第—實施例中 100100547 表單編號Α0101 第15頁/共45頁 1002000957-0 201229490 所述分子載體20的結構基本相同，其不同在於，所述分 子載體20中的三維奈米結構202為凸起的半橢球狀結構。 [0056] 所述半橢球狀三維奈米結構202的底面為圓形，其直徑為 50奈米〜1 000奈米，高度為50奈米〜1 000奈米。優選地， 所述半橢球狀凸起結構的底面直徑為50奈米〜200奈米， 高度為100奈米~500奈米。所述相鄰的每兩個半橢球狀凸 起結構之間的距離相等，所述兩個半橢球狀凸起結構之 間的距離係指所述半橢球狀凸起結構的底面之間的距離 ，可為0奈米〜50奈米。本實施例中，所述半球狀三維奈 米結構202之間的距離為40奈米。 [0057] 所述金屬層201沈積於所述三維奈米結構202的表面及相 鄰的三維奈米結構202之間基底200的表面。具體的，所 述金屬層201為單層層狀結構或複數層層狀結構。所述金 屬層201基本均勻沈積於所述複數三維奈米結構202表面 及相鄰的三維奈米結構202之間的基底200的表面。所述 分子載體20的SERS增強因數理論值可為105~1015，本實 施例中所述分子載體20的SERS增強因數約為106。 [0058] 請一併參閱圖10至圖11，本發明第三實施例提供一種分 子載體30，所述分子載體30包括一基底300、形成於基底 30 0表面的複數三維奈米結構302及設置於所述三維奈米 結構302表面及相鄰三維奈米結構302之間的基底300表 面的金屬層301。所述分子載體30的結構與第一實施例中 所述分子載體30的結構基本相同，其不同在於，所述分 子载體30中的三維奈米結構202為凹陷的倒金字塔結構。 100100547 表單編號A0101 第16頁/共45頁 1002000957-0 201229490 [0059] Ο 所述凹陷的倒金字塔結構係指所述基底3〇〇的表面向内凹 入形成凹進的空間呈倒金字塔形。所述倒金字塔形三維 奈米結構302的底面的形狀不限，可為三角形 '矩形及正 方形等其他幾何形狀。所述三維奈米結構3〇2凹入基底 300表面的尚度為50奈米〜ι〇〇〇奈米，所述倒金字塔三維 奈米結構302的頂端形成的夾角α可為15度〜7〇度。本實 施例中，所述三維奈米結構3〇2的底面為一正三角形，所 述正三角形的邊長為50奈米~1()〇〇奈米。優選的，所述倒 金字塔形三維奈米結構3〇2的底面邊長為50奈米〜200奈 米’凹入基底表面的高度為1〇〇奈米~5⑽奈米，所述頂端 形成的夾角α為30度。所述相鄰的每兩個倒金字塔三維 奈米結構302之間的距離相等，所述每兩個倒金字塔三維 奈米結構302之間的距離係指所述倒金字塔三維奈米結構 的底面之間的距離’可為〇奈米~50奈米。 [0060] Ο 所述金屬層301沈積於所述倒金字塔形三維奈米結構302 的表面及相鄰的三維奈米結構302之間基底300的表面。 具體的，所述金屬層301為單層層科結構或複數層層狀結 構。所述金屬層301基本均勻沈積於所述複數三維奈米結 構302表面及相鄰的三維奈米結構302之間的基底300的 表面。所述分子載體30的SERS增強因數理論值可為 105〜1015，本實施例中所述分子載體30的SERS增強因數 約為108。 請參閱圖12，圖12為本實施例中所述分子載體的三維奈 米結構分別為半球狀、倒金字塔狀及半橢球狀結構時， 用於檢測若丹明分子的拉曼光譜。 100100547 表單編號Α0101 第Π頁/共45頁 1002000957-0 [0061] 201229490 [0062] 請參閱圖13、圖14及圖15，本發明第四實施例提供一種 用於單分子檢測的分子載體40，所述分子載體40包括一 基底400、設置於基底400上的複數三維奈米結構402， 及設置於所述三維奈米結構402表面及相鄰三維奈米結構 402之間的基底400的金屬層401。所述金屬層401附著於 所述三維奈米結構402及三維奈米結構402之間基底400 的表面。本發明第二實施例所述的分子載體40與第一實 施例中所述分子載體10的結構基本相同，其不同在於， 所述分子載體40中的三維奈米結構402為一階梯狀結構。 [0063] 所述階梯狀結構設置在所述基底400表面。所述階梯狀結 構為階梯狀凸起結構。所述階梯狀凸起結構為從所述基 底40 0表面向外延伸出的階梯狀突起的實體。所述階梯狀 凸起結構可為一複數層台狀結構，如複數層三棱臺、複 數層四棱臺、複數層六棱臺或複數層圓柱等。優選地， 所述階梯狀凸起結構為複數層圓柱結構。所述階梯狀凸 起結構的最大尺度為小於等於1000奈米，即其長度、寬 度和高度均小於等於1 000奈米。優選地，所述階梯狀凸 起結構結構長度、寬度和高度範圍為10奈米〜500奈米。 [0064] 本實施例中，所述三維奈米結構402為一階梯狀凸起的雙 層圓柱結構。具體地，所述三維奈米結構402包括一第一 圓柱404及一設置於該第一圓柱4 04表面的第二圓柱4 06 。所述第一圓柱404靠近基底400設置。所述第一圓柱 404的側面垂直於基底400的表面。所述第二圓柱406的 側面垂直於第一圓柱404的上表面，所述上表面係指所述 第二圓柱406遠離基底400的表面。所述第一圓柱404與 100100547 表單編號A0101 第18頁/共45頁 1002000957-0 201229490 [0065] Ο[0055] The single molecule detection method provided by the present invention has the following advantages: First, the prior art single molecule detection method is to deposit a dot layer on a substrate and then form a metal nanostructure on the adhesion layer as a molecular carrier, thereby The adhesive layer has a certain influence on the single molecule detection, and the three-dimensional nanostructure of the present invention is directly formed on the substrate by reactive ion etching. The metal layer is directly deposited on the surface of the substrate, thereby preventing the adhesion layer. The chemical factors affect the detection results. Secondly, the molecular carrier in the single molecule detection method of the present invention has a three-dimensional nanostructure, and the metal layer in the molecular carrier is directly deposited on the surface of the substrate, whereas the prior art metal nanostructure must The result of the single molecule detection is affected by the adhesion layer being fixed on the surface of the substrate; again, the shape, size, spacing, and the like of the three-dimensional nanostructure can be conveniently controlled by controlling the preparation conditions, and the operability is high; The fourth 'common table three-dimensional nano-structured surface is provided with a metal layer, which can improve the single molecule test Especially for the resolution of the substance can not be detected by conventional detection methods dyes, biomolecules, and fluorescent materials with stupid six generations, but also can be to utilize the feed side / detection. Referring to FIG. 8 to FIG. 9 together, a second embodiment of the present invention provides a molecular carrier 20 comprising a substrate 2〇n, a blade, and a plurality of three-dimensional nanoparticles formed on the surface of the substrate 200. Structure 2〇2 and a metal layer 20 disposed on the surface of the-dimensional 乂 乂 structure 202 and the adjacent surface of the two-dimensional nanometer, the structure of the molecular carrier 2G and the 100100547 form number in the first embodiment Α0101 Page 15 of 45 1002000957-0 201229490 The molecular carrier 20 has substantially the same structure, except that the three-dimensional nanostructure 202 in the molecular carrier 20 is a convex semi-ellipsoidal structure. [0056] The bottom surface of the semi-ellipsoidal three-dimensional nanostructure 202 is circular, having a diameter of 50 nm to 1 000 nm and a height of 50 nm to 1 000 nm. Preferably, the semi-ellipsoidal convex structure has a bottom surface diameter of 50 nm to 200 nm and a height of 100 nm to 500 nm. The distance between each adjacent two semi-ellipsoidal convex structures is equal, and the distance between the two semi-ellipsoidal convex structures refers to the bottom surface of the semi-ellipsoidal convex structure The distance between 0 nanometers ~ 50 nanometers. In this embodiment, the distance between the hemispherical three-dimensional nanostructures 202 is 40 nm. [0057] The metal layer 201 is deposited on the surface of the substrate 200 between the surface of the three-dimensional nanostructure 202 and the adjacent three-dimensional nanostructures 202. Specifically, the metal layer 201 is a single layered layer structure or a plurality of layered structures. The metal layer 201 is deposited substantially uniformly on the surface of the substrate 200 between the surface of the plurality of three-dimensional nanostructures 202 and the adjacent three-dimensional nanostructures 202. The theoretical value of the SERS enhancement factor of the molecular carrier 20 may be 105 to 1015, and the molecular carrier 20 of the present embodiment has a SERS enhancement factor of about 106. [0058] Referring to FIG. 10 to FIG. 11 together, a third embodiment of the present invention provides a molecular carrier 30. The molecular carrier 30 includes a substrate 300, a plurality of three-dimensional nanostructures 302 formed on the surface of the substrate 30, and a setting. A metal layer 301 on the surface of the substrate 300 between the surface of the three-dimensional nanostructure 302 and the adjacent three-dimensional nanostructure 302. The structure of the molecular carrier 30 is substantially the same as that of the molecular carrier 30 of the first embodiment, except that the three-dimensional nanostructure 202 in the molecular carrier 30 is a concave inverted pyramid structure. 100100547 Form No. A0101 Page 16 of 45 1002000957-0 201229490 [0059] The depressed inverted pyramid structure means that the surface of the substrate 3 is recessed inwardly to form a recessed space in an inverted pyramid shape. The shape of the bottom surface of the inverted pyramidal three-dimensional nanostructure 302 is not limited, and may be other geometric shapes such as a triangle 'rectangular shape and a square shape. The dimension of the surface of the three-dimensional nanostructure 3〇2 recessed into the substrate 300 is 50 nm~ι〇〇〇N, and the angle α formed by the top end of the inverted pyramid three-dimensional nanostructure 302 may be 15 degrees~7 〇度. In this embodiment, the bottom surface of the three-dimensional nanostructure 3〇2 is an equilateral triangle, and the side length of the equilateral triangle is 50 nm to 1 () 〇〇 nanometer. Preferably, the inverted pyramidal three-dimensional nanostructure 3〇2 has a bottom side length of 50 nm to 200 nm. The height of the concave substrate surface is 1 〇〇 nanometer to 5 (10) nm, and the top end is formed. The angle α is 30 degrees. The distance between each adjacent two inverted pyramid three-dimensional nanostructures 302 is equal, and the distance between each two inverted pyramid three-dimensional nanostructures 302 refers to the bottom surface of the inverted pyramid three-dimensional nanostructure The distance between 'can be 〇 nano ~ 50 nm. [0060] The metal layer 301 is deposited on the surface of the inverted pyramidal three-dimensional nanostructure 302 and the surface of the substrate 300 between adjacent three-dimensional nanostructures 302. Specifically, the metal layer 301 is a single layer layer structure or a plurality of layer structure. The metal layer 301 is deposited substantially uniformly on the surface of the substrate 300 between the surface of the plurality of three-dimensional nanostructures 302 and the adjacent three-dimensional nanostructures 302. The theoretical value of the SERS enhancement factor of the molecular carrier 30 may be 105 to 1015, and the molecular carrier 30 of the present embodiment has a SERS enhancement factor of about 108. Please refer to FIG. 12. FIG. 12 is a diagram showing the Raman spectrum of the rhodamine molecule when the three-dimensional nanostructures of the molecular carrier are hemispherical, inverted pyramidal, and semi-ellipsoidal structures, respectively. 100100547 Form No. 101 0101 Page / Total 45 Page 1002000957-0 [00021] Referring to FIG. 13, FIG. 14, and FIG. 15, a fourth embodiment of the present invention provides a molecular carrier 40 for single molecule detection, The molecular carrier 40 includes a substrate 400, a plurality of three-dimensional nanostructures 402 disposed on the substrate 400, and a metal layer of the substrate 400 disposed between the surface of the three-dimensional nanostructure 402 and the adjacent three-dimensional nanostructure 402. 401. The metal layer 401 is attached to the surface of the substrate 400 between the three-dimensional nanostructure 402 and the three-dimensional nanostructure 402. The molecular carrier 40 according to the second embodiment of the present invention has substantially the same structure as the molecular carrier 10 of the first embodiment, except that the three-dimensional nanostructure 402 in the molecular carrier 40 has a stepped structure. [0063] The stepped structure is disposed on a surface of the substrate 400. The stepped structure is a stepped convex structure. The stepped raised structure is an entity of stepped protrusions extending outwardly from the surface of the base 40 0 . The stepped convex structure may be a plurality of layered structures, such as a plurality of triangular prisms, a plurality of quadrangular prisms, a plurality of layers of hexagonal prisms or a plurality of layers of cylinders. Preferably, the stepped convex structure is a plurality of layered cylindrical structures. The stepped convex structure has a maximum dimension of 1000 nm or less, that is, its length, width and height are less than or equal to 1,000 nm. Preferably, the stepped raised structure has a length, a width and a height ranging from 10 nm to 500 nm. [0064] In the embodiment, the three-dimensional nanostructure 402 is a double-layered cylindrical structure with a stepped protrusion. Specifically, the three-dimensional nanostructure 402 includes a first cylinder 404 and a second cylinder 406 disposed on the surface of the first cylinder 04. The first cylinder 404 is disposed adjacent to the substrate 400. The side of the first cylinder 404 is perpendicular to the surface of the substrate 400. The side of the second cylinder 406 is perpendicular to the upper surface of the first cylinder 404, and the upper surface refers to the surface of the second cylinder 406 away from the substrate 400. The first cylinder 404 and 100100547 Form No. A0101 Page 18 of 45 1002000957-0 201229490 [0065] Ο
[0066] 第二圓柱406形成一階梯狀凸起結構，所述第二圓柱406 設置在所述第一圓柱404的範圍内。優選地，所述第一圓 柱404與第二圓柱406同軸設置。所述第一圓柱404與第 二圓柱406為一體結構，即所述第二圓柱406為第一圓柱 4 0 4的頂面延伸出的圓柱狀結構。 所述第一圓柱404的底面直徑大於第二圓柱406的底面直 徑。所述第一圓柱404的底面直徑為30奈米〜1 000奈米， 高度為50奈米~1000奈米。優選地，所述第一圓柱404的 底面直徑為50奈米~200奈米，高度為100奈米〜500奈米 。所述第二圓柱406的底面直徑為10奈米~500奈米，高 度為20奈米~500奈米。優選地，所述第二圓柱406的底 面直徑為20奈米〜200奈米，高度為100奈米~300奈来。 所述第一圓柱404及第二圓柱406的尺寸可以根據實際需 要製備。本實施例中，所述第一圓柱404與第二圓柱406 同軸設置。所述第一圓柱404的底面直徑為380奈米，高 度為105奈米。所述第二圓柱406的底面直徑為280奈米 ，高度為55奈米。所述相鄰的第一圓柱404之間的距離為 可為0奈米〜50奈米；所述相鄰兩第二圓柱406之間的距離 為10奈米~100奈米。 所述雙層圓柱的三維奈米結構402的製備方法與第一實施 例中所述三維奈米結構102的製備方法基本相同，其不同 在於，採用反應性蝕刻氣體對母板的表面進行蝕刻的同 時，對所述掩膜層進行腐蝕。通過控制蝕刻時間與蝕刻 方向，一方面，所述反應性蝕刻氣體對所述單層奈米微 球之間的所述母板的表面進行蝕刻，從而形成第一圓柱 100100547 表單編號Α0101 第19頁/共45頁 1002000957-0 201229490 404 ;另一方面，所述反應性蝕刻氣體同時對所述母板的 表面上的單層奈米微球進行腐蝕，形成更小直徑的奈米 微球，即單層奈米微球中的每一奈米微球被蝕刻削減為 比所述第一圓柱404直徑更小的奈米微球，使所述反應性 蝕刻氣體可以對所述第一圓柱404進行進一步蝕刻，從而 形成所述第二圓柱406，進而形成所述複數階梯狀的三維 奈米結構402。 [0067] 所述金屬層401沈積於所述三維奈米結構402的表面及相 鄰的三維奈米結構402之間基底400的表面。具體的，所 述金屬層401為由複數分散的奈米金屬顆粒鋪展形成的單 層層狀結構或複數層層狀結構。所述奈米金屬顆粒分散 於所述複數三維奈米結構402表面及相鄰的三維奈米結構 402之間的基底400的表面。 [0068] 相對於第一實施例，本發明第二實施例提供的分子載體 40，由於所述三維奈米結構402為一凸起的雙層圓柱結構 ，相鄰的雙層圓柱結構之間形成兩個距離不同的間隙（ Gap)，即相鄰的第一圓柱404之間形成一間隙，相鄰的 第二圓柱406之間形成另一間隙。因此，當所述分子載體 用於單分子檢測時，在檢測器發出的鐳射的激發下，相 鄰的第一圓柱404之間間隙處的金屬層401產生表面電磁 耦子共振，同時第二圓柱406之間間隙處的金屬層401產 生電磁耦子共振，增強了金屬層表面的拉曼散射，因此 可以進一步的提高SERS增強因數，增強拉曼光譜，提高 所述單分子檢測的解析度，使得單分子檢測結果更加的 準確。 100100547 表單編號A0101 第20頁/共45頁 1002000957-0 201229490 [0069] 請參閱圖16及圖17 ’本發明第ί實施例提供一種用於單 分子檢測的分子載體50 ’所述分子載體5〇包括一基底5〇〇 、設置於基底500上的複數二維奈米結構5〇2及設置於所 述三維奈米結構502表面及相鄰三維奈米結構5〇2之間的 基底500的金屬層501。本發明第五實施例所述的分子載 體50與第四實施例中所述分子載體50的結構基本相同， 其不同在於，所述分子載體50中的三維奈米結構502為一 階梯狀凹陷結構。 [0070] 所述階梯狀凹@ 錄構為從基底500表面向基底500内凹陷 〇 形成的階梯狀一的空間。所述階梯狀凹陷結構可為一 ^邊，如複數層三棱臺、複數層四棱臺、複 複數層台狀、结攝 數層六棱臺 或旅數層圓柱等。 译選地，所述階梯狀凹陷 結構為複數層圓 圓柱結構係指# 。所述階梯狀00 ，即其長度、露 ，所述階梯狀四 社結構。所謂階梯狀凹陷結構為複數層 述階梯狀凹陷的空間為複數層圓柱形狀 陪結構的最大尺度為小於等於奈米 度和高度均'i、於拿於1000奈米。優選地 陪結構結樽焉虞、冕度和高度範圍為10[0066] The second cylinder 406 forms a stepped raised structure, and the second cylinder 406 is disposed within the range of the first cylinder 404. Preferably, the first cylinder 404 is disposed coaxially with the second cylinder 406. The first cylinder 404 and the second cylinder 406 are of a unitary structure, that is, the second cylinder 406 is a cylindrical structure extending from the top surface of the first cylinder 410. The diameter of the bottom surface of the first cylinder 404 is larger than the diameter of the bottom surface of the second cylinder 406. The bottom surface of the first cylinder 404 has a diameter of 30 nm to 1 000 nm and a height of 50 nm to 1000 nm. Preferably, the first cylinder 404 has a bottom surface diameter of 50 nm to 200 nm and a height of 100 nm to 500 nm. The bottom surface of the second cylinder 406 has a diameter of 10 nm to 500 nm and a height of 20 nm to 500 nm. Preferably, the second cylinder 406 has a bottom surface diameter of 20 nm to 200 nm and a height of 100 nm to 300 nm. The dimensions of the first cylinder 404 and the second cylinder 406 can be prepared according to actual needs. In this embodiment, the first cylinder 404 and the second cylinder 406 are coaxially disposed. The bottom surface of the first cylinder 404 has a diameter of 380 nm and a height of 105 nm. The bottom surface of the second cylinder 406 has a diameter of 280 nm and a height of 55 nm. The distance between the adjacent first cylinders 404 may be 0 nm to 50 nm; the distance between the adjacent two second cylinders 406 is 10 nm to 100 nm. The preparation method of the double-layered cylindrical three-dimensional nanostructure 402 is basically the same as the preparation method of the three-dimensional nanostructure 102 in the first embodiment, except that the surface of the mother board is etched by using a reactive etching gas. At the same time, the mask layer is etched. By controlling the etching time and the etching direction, on the one hand, the reactive etching gas etches the surface of the mother board between the single-layer nano microspheres to form a first cylinder 100100547 Form No. 1010101 Page 19 / Total 45 pages 1002000957-0 201229490 404; on the other hand, the reactive etching gas simultaneously etches a single layer of nanospheres on the surface of the mother board to form smaller diameter nanospheres, ie Each nanosphere of the single layer of nanospheres is etched into nanospheres having a smaller diameter than the first cylinder 404, such that the reactive etching gas can be applied to the first cylinder 404 Further etching, thereby forming the second cylinder 406, thereby forming the plurality of stepped three-dimensional nanostructures 402. [0067] The metal layer 401 is deposited on the surface of the substrate 400 between the surface of the three-dimensional nanostructure 402 and the adjacent three-dimensional nanostructure 402. Specifically, the metal layer 401 is a single layered structure or a plurality of layered structures formed by spreading a plurality of dispersed nano metal particles. The nano metal particles are dispersed on the surface of the substrate 400 between the surface of the plurality of three-dimensional nanostructures 402 and the adjacent three-dimensional nanostructures 402. [0068] With respect to the first embodiment, the molecular carrier 40 provided by the second embodiment of the present invention is formed by forming a three-dimensional nanostructure 402 with a convex double-layered cylindrical structure. Two gaps (Gap) are formed at different distances, that is, a gap is formed between the adjacent first cylinders 404, and another gap is formed between the adjacent second cylinders 406. Therefore, when the molecular carrier is used for single molecule detection, under the excitation of the laser emitted by the detector, the metal layer 401 at the gap between the adjacent first cylinders 404 generates surface electromagnetic coupling resonance while the second cylinder The metal layer 401 at the gap between 406 generates electromagnetic coupling resonance, which enhances the Raman scattering of the surface of the metal layer, thereby further improving the SERS enhancement factor, enhancing the Raman spectrum, and improving the resolution of the single molecule detection, Single molecule detection results are more accurate. 100100547 Form No. A0101 Page 20/45 Table 1002000957-0 201229490 [0069] Please refer to FIG. 16 and FIG. 17 'The third embodiment of the present invention provides a molecular carrier 50' for the single molecule detection. The invention comprises a substrate 5〇〇, a plurality of two-dimensional nanostructures 5〇2 disposed on the substrate 500, and a substrate 500 disposed between the surface of the three-dimensional nanostructure 502 and the adjacent three-dimensional nanostructures 5〇2. Metal layer 501. The molecular carrier 50 according to the fifth embodiment of the present invention has substantially the same structure as the molecular carrier 50 of the fourth embodiment, except that the three-dimensional nanostructure 502 in the molecular carrier 50 is a stepped recessed structure. . [0070] The stepped recess is configured as a stepped space formed by recessing the surface of the substrate 500 into the substrate 500. The stepped recessed structure may be an edge, such as a plurality of layers of triangular prisms, a plurality of layers of quadrangular prisms, a plurality of layers of a platform, a knotted layer of hexagonal prisms, or a plurality of layers of columns. In the selected place, the stepped recessed structure is a plurality of layers of circular cylindrical structure means #. The stepped shape 00, that is, its length, dew, and the stepped four-compartment structure. The so-called stepped depression structure is a plurality of layers of the stepped depression. The space of the complex layer is a cylindrical shape. The maximum dimension of the accompanying structure is less than or equal to the degree of nanometer and height, and is taken at 1000 nm. Preferably, the structure has a knot, a twist and a height range of 10
[0071][0071]
^•米~500奈米 &述三維奈米結構502的形狀為一雙層圓柱 ^實施例中’所 结構為一圓柱狀結構空間，具體包括一 吉構，所述圓柊π ，及一與所述第一圓柱空間504連通的 赛一圓柱空間5ϋ 。所述第一圓枉空間504與第二圓柱空 事二圓枉空間5〇6 必。所述第一圓柱空間504靠近基底500的 治506同轴設I 表面設置。所 間506的直径 述第一圓柱空間504的直徑大於第二圓柱空 户斤述第一圓柱空間504的直徑為30奈米 100100547 表單編號Α0101 第21頁/共45頁 1002000957-0 201229490 〜1 000奈米，高度為50奈米〜1 000奈米。所述第二圓柱空 間506的直徑為10奈米〜500奈米，高度為20奈米〜500奈 米。所述第二圓柱空間506及第二圓柱空間506的尺寸可 以根據實際需要製備。 [0072] 所述複數三維奈米結構502在所述基底500上的表面以陣 列形式設置。所述以陣列形式設置指所述複數三維奈米 結構502可以按照簡單立方排布、同心圓環排布或六角形 密堆排布等方式排列，而且所述以陣列形式設置的複數 三維奈米結構502可以形成一單一圖案或複數圖案。所述 相鄰的兩個三維奈米結構502之間的距離相等。具體的， 所述相鄰的第一圓柱空間504之間的距離為可為1奈米 ~ 1 000奈米，優選為10奈米〜50奈米；所述相鄰兩第二圓 柱空間506之間的距離為15奈米〜900奈米，優選的為20 奈米〜100奈米。所述複數三維奈米結構502在所述基底 500上的表面設置的形式及相鄰的兩個三維奈米結構502 之間的距離可以根據實際需要製備。本實施例中，所述 複數三維奈米結構502呈六角形密堆排布形成一單一正方 形圖案。 [0073] 所述雙層圓柱狀空間的三維奈米結構502的製備方法與第 四實施例中所述三維奈米結構402的製備方法基本相同， 其不同在於，所述掩膜層為一具有複數開孔的連續膜。 所述反應性蝕刻氣體對開孔中的基板的表面進行蝕刻的 同時，對所述掩膜層進行腐钱。一方面，所述反應性姓 刻氣體對所述開孔的所述基板的表面進行蝕刻，從而形 成第一圓柱空間504 ;另一方面，所述反應性蝕刻氣體同 100100547 表單編號A0101 第22頁/共45頁 1002000957-0 201229490 時對所述基板的表面上的掩膜層進行腐蝕，使所述開孔 變大，使所述反應性姓刻氣體對所述基板钱刻範圍更大 ，從而形成所述第一圓柱空間5 0 4，最後在開孔對應的位 置製備得到階梯狀凹陷結構。可以理解，通過控制反應 性姓刻氣體的姓刻時間可以控制三維奈米結構5〇2間的間 距，也可以控制三維奈米結構502中所述第一圓柱空間 504及第二圓柱空間506的尺寸。所述具有複數開孔的連 續膜可以通過奈米壓印、模板沈積等方式製備。 [0074] ❹ Ο [0075] 本發明第五實施例提供的分子載體50與第四實施例所提 供的分子載體40所起的作用基本相同。由於所述三維奈 米結構502為一雙層圓柱狀空間，因此所述雙層圓柱狀空 間具有兩個不同的間隙，即第一圓柱空間504形成一間隙 ，第二圓柱空間506形成另一間隙。因此，當所述分子載 體用於單分子檢測時，在外界入射光電磁場的激發下， 第一圓柱空間504中的余屬層產生表面電磁耦子共振，同 時第二圓柱空間506的金屬層產生電磁刼子共振，增強拉 曼散射，因此可以進一步的提高SERS增強因數，提高所 述單分子檢測的解析度，使得單分子檢測結果更加的準 確。 综上所述，本發明確已符合發明專利之要件，遂依法提 出專利申請。惟，以上所述者僅為本發明之較佳實施例 ，自不能以此限制本案之申請專利範圍。舉凡習知本案 技藝之人士援依本發明之精神所作之等效修飾或變化， 皆應涵蓋於以下申請專利範圍内。 【圖式簡單說明】 100100547 表單編號Α0101 第23頁/共45頁 1002000957-0 201229490 [0076] 圖1為本發明第一實施例提供的分子載體的結構示意圖。 [0077] 圖2為本發明第一實施例提供的分子載體沿Π - Π方向的 剖視圖。 [0078] 圖3為本發明第一實施例提供的半球狀三維奈米結構陣列 的掃描電鏡照片。 [0079] 圖4為本發明第一實施例提供的分子載體中包括複數圖案 的三維奈米結構陣列的結構示意圖。 [0080] 圖5為本發明應用分子載體的單分子檢測方法的流程圖。 [0081] 圖6為本發明第一實施例提供的分子載體中三維奈米結構 的製備流程示意圖。 [0082] 圖7為在基底表面六角形密堆排布之單層奈米微球的掃描 電鏡照片。 [0083] 圖8為本發明第二實施例提供的半橢球狀三維奈米結構陣 列的掃描電鏡照片。 [0084] 圖9為本發明第二實施例提供的半橢球狀三維奈米結構陣 列的剖面示意圖。 [0085] 圖1 0為本發明第三實施例提供的倒金字塔狀三維奈米結 構陣列的掃描電鏡照片。 [0086] 圖1 1為本發明第三實施例提供的倒金字塔狀三維奈米結 構陣列的剖面示意圖。 [0087] 圖12為本發明分子載體中不同三維奈米結構用於檢測若 丹明分子時得到的拉曼光譜。 100100547 表單編號A0101 第24頁/共45頁 1002000957-0 201229490 [0088] 圖1 3為本發明第四實施例提供的雙層圓柱狀三維奈米結 [0089] 構陣列的掃描電鏡照片。 圖14為本發明第四實施例提供的分子載體的結構示意圖 〇 [0090] 圖1 5為本發明第四實施例提供的分子載體沿X V - X V方 向的剖視圖。 [0091] 圖16為本發明第五實施例提供的分子載體的結構示意圖 〇 f) i [0092] 圖17為本發明第五實施例提供的分子載體沿X YU-XW的 剖視圖。 [0093] 【主要元件符號說明】 分子載體：10，20，30，40，50 [0094] 基底：100，200，300，400，500 [0095] 金屬層：101，201，301，401，501 [0096] ❹ 母板：1001 [0097] 三維奈米結構：102，202，302，402，502 [0098] 掩膜層：108 [0099] 反應性蝕刻氣體：110 [0100] 第一圓柱：404 [0101] 第二圓柱：406 [0102] 第一圓柱空間：504 100100547 表單編號A0101 第25頁/共45頁 1002000957-0 201229490 [0103] 第二圓柱空間：50 6 100100547 表單編號AOiOl 第26頁/共45頁 1002000957-0^•米~500nm& The shape of the three-dimensional nanostructure 502 is a double-layered cylinder. In the embodiment, the structure is a cylindrical structure space, specifically including a structure, the circle 柊π, and a A race-cylindrical space 5ϋ communicating with the first cylindrical space 504. The first circular space 504 and the second cylindrical empty two circular space 5〇6 are necessary. The first cylindrical space 504 is disposed adjacent to the surface 506 of the substrate 500. The diameter of the first cylindrical space 504 is larger than the diameter of the first cylindrical space 504. The diameter of the first cylindrical space 504 is 30 nm 100100547. Form number Α 0101 Page 21 / Total 45 pages 1002000957-0 201229490 ~ 1 000 Nano, height is 50 nm ~ 1 000 nm. The second cylindrical space 506 has a diameter of 10 nm to 500 nm and a height of 20 nm to 500 nm. The dimensions of the second cylindrical space 506 and the second cylindrical space 506 can be prepared according to actual needs. [0072] The surface of the plurality of three-dimensional nanostructures 502 on the substrate 500 is arranged in an array. The arrangement in the form of an array means that the plurality of three-dimensional nanostructures 502 can be arranged in a simple cubic arrangement, a concentric annular arrangement or a hexagonal dense arrangement, and the plurality of three-dimensional nanometers arranged in an array form. Structure 502 can form a single pattern or a plurality of patterns. The distance between the adjacent two three-dimensional nanostructures 502 is equal. Specifically, the distance between the adjacent first cylindrical spaces 504 may be 1 nm to 1 000 nm, preferably 10 nm to 50 nm; and the adjacent two second cylindrical spaces 506 The distance between 15 nm and 900 nm is preferably 20 nm to 100 nm. The form of the surface of the plurality of three-dimensional nanostructures 502 on the substrate 500 and the distance between the adjacent two three-dimensional nanostructures 502 can be prepared according to actual needs. In this embodiment, the plurality of three-dimensional nanostructures 502 are arranged in a hexagonal close-packed form to form a single square pattern. [0073] The method for preparing the three-dimensional nanostructure 502 of the double-layered cylindrical space is substantially the same as the method for preparing the three-dimensional nanostructure 402 of the fourth embodiment, except that the mask layer has A continuous film of multiple openings. The reactive etching gas etches the surface of the substrate in the opening, and the mask layer is rotted. In one aspect, the reactive surrogate gas etches the surface of the apertured substrate to form a first cylindrical space 504; on the other hand, the reactive etching gas is the same as 100100547 Form No. A0101 Page 22 / a total of 45 pages 1002000957-0 201229490 when the mask layer on the surface of the substrate is etched to make the opening larger, so that the reactive surname gas is larger in the range of the substrate, thereby The first cylindrical space 504 is formed, and finally a stepped recessed structure is prepared at a position corresponding to the opening. It can be understood that the spacing between the three-dimensional nanostructures 5〇2 can be controlled by controlling the surname time of the reactive surname gas, and the first cylindrical space 504 and the second cylindrical space 506 in the three-dimensional nanostructure 502 can also be controlled. size. The continuous film having a plurality of openings can be prepared by nanoimprinting, template deposition, or the like. [0075] The molecular carrier 50 provided by the fifth embodiment of the present invention functions substantially the same as the molecular carrier 40 provided by the fourth embodiment. Since the three-dimensional nanostructure 502 is a double-layered cylindrical space, the double-layered cylindrical space has two different gaps, that is, the first cylindrical space 504 forms a gap, and the second cylindrical space 506 forms another gap. . Therefore, when the molecular carrier is used for single molecule detection, the surface layer of the first cylindrical space 504 generates surface electromagnetic coupling resonance under the excitation of the external incident photoelectric field, and the metal layer of the second cylindrical space 506 is generated. Electromagnetic forceps resonance, enhanced Raman scattering, can further improve the SERS enhancement factor, improve the resolution of the single molecule detection, and make the single molecule detection result more accurate. In summary, the present invention has indeed met the requirements of the invention patent, and the patent application is filed according to law. However, the above description is only a preferred embodiment of the present invention, and it is not possible to limit the scope of the patent application of the present invention. Equivalent modifications or variations made by those skilled in the art in light of the spirit of the invention are intended to be included within the scope of the following claims. [Simple Description of the Drawings] 100100547 Form No. Α0101 Page 23 of 45 1002000957-0 201229490 [0076] FIG. 1 is a schematic structural view of a molecular carrier according to a first embodiment of the present invention. 2 is a cross-sectional view of the molecular carrier according to the first embodiment of the present invention taken along the Π-Π direction. 3 is a scanning electron micrograph of a hemispherical three-dimensional nanostructure array according to a first embodiment of the present invention. 4 is a schematic structural view of a three-dimensional nanostructure array including a plurality of patterns in a molecular carrier according to a first embodiment of the present invention. 5 is a flow chart of a single molecule detection method using a molecular carrier according to the present invention. 6 is a schematic diagram showing a preparation process of a three-dimensional nanostructure in a molecular carrier according to a first embodiment of the present invention. [0082] FIG. 7 is a scanning electron micrograph of a single layer of nanospheres arranged in a hexagonal close-packed surface on the surface of the substrate. 8 is a scanning electron micrograph of a semi-ellipsoidal three-dimensional nanostructure array according to a second embodiment of the present invention. 9 is a schematic cross-sectional view showing a semi-ellipsoidal three-dimensional nanostructure array according to a second embodiment of the present invention. 10 is a scanning electron micrograph of an inverted pyramidal three-dimensional nanostructure array according to a third embodiment of the present invention. 11 is a schematic cross-sectional view showing an inverted pyramidal three-dimensional nanostructure array according to a third embodiment of the present invention. 12 is a Raman spectrum obtained by using different three-dimensional nanostructures in the molecular carrier of the present invention for detecting rhodamine molecules. 100100547 Form No. A0101 Page 24 of 45 1002000957-0 201229490 [0088] FIG. 13 is a scanning electron micrograph of a double-layered cylindrical three-dimensional nano-junction according to a fourth embodiment of the present invention. Figure 14 is a schematic view showing the structure of a molecular carrier according to a fourth embodiment of the present invention. [0090] Figure 15 is a cross-sectional view of the molecular carrier according to a fourth embodiment of the present invention taken along the X V - X V direction. 16 is a schematic structural view of a molecular carrier according to a fifth embodiment of the present invention. 〇 f) i FIG. 17 is a cross-sectional view of a molecular carrier according to a fifth embodiment of the present invention taken along X YU-XW. [Description of main component symbols] Molecular carrier: 10, 20, 30, 40, 50 [0094] Substrate: 100, 200, 300, 400, 500 [0095] Metal layer: 101, 201, 301, 401, 501母 Motherboard: 1001 [0097] Three-dimensional nanostructure: 102, 202, 302, 402, 502 [0098] Mask layer: 108 [0099] Reactive etching gas: 110 [0100] First cylinder: 404 [0101] Second cylinder: 406 [0102] First cylindrical space: 504 100100547 Form number A0101 Page 25 / Total 45 pages 1002000957-0 201229490 [0103] Second cylindrical space: 50 6 100100547 Form number AOiOl Page 26 / Total 45 pages 1002000957-0