1296045 九、發明說明： 【發明所屬之技術領域】 /本毛明是關於一種以生物分子，例如去氧核糖 稱DNA)或蛋白質製作奈米圖案的方法，特別是_」種利^ 粒子束照射技術使生物分子性質改變，再利用與 互補或受質分子反應以製作奈米尺度_的方法服慨物的 【先前技術】1296045 IX. Description of the invention: [Technical field to which the invention pertains] / Ben Maoming is a method for producing a nanopattern by using a biomolecule such as deoxyribose called DNA or a protein, in particular, _" Technology that changes the properties of biomolecules, and then uses the method of reacting with complementary or accepting molecules to make nanoscales. [Prior Art]
=DNA具紐基配對的特性，以及日益成熟的dna操 作技術及卫具’麟DNA在齡的奈米技術上，扮演重要 角色，並可廣泛的應用在光學、電子、生物電子、物a 以及生醫等高科技產業領域。 ^曰曰乃 傳統的奈米顯微技術’係利用粒子束照射技術，如以電子束 微影術（Electron Beam Lithography)製作奈米圖案，需傕用 阻劑（Resist)以接受電子束（Electr〇nBeam，e_Beam)的照射 製圖。但阻劑的處理過程中往往需要經過加熱或化學侵蝕等步 t =31加生物分子的不穩定性，進而影響後續成品或 製品的製造良率或品質穩定性。 【發明内容】 為了簡化操作步驟與成本，並且避免額外阻劑對生物分子的 致毒性、致突變性、或致變性，以及避免阻劑殘留或可能產生 的副產品對樣本的汙染，本發明提供了一種無需額外阻劑，而 可以直接處理生物分子的奈米圖案製作方法。 本發明係為一種以生物分子薄膜（尤其是DNA)為材料， 再施以粒子束照射使DNA產生性質改變，之後再以帶有桿幟 物的互補DNA參與雜合(hybridizati〇n)反應，經粒子束照射後 的DNA喪失與其互補DNA雜合之能力。因此可在原DNA薄 1296045 膜上區分出接有標幟物的標示區與未接有標幟物的护 照射的圖案。其中照射處理用的粒子束 子束、紫外線光子束、x-ray或輻射線粒子束等；而聚焦 束照射係依設計好的座標與劑量做照射，以表現出直g的= 此外，藉由控制粒子束照射的劑量可控制變性的DNA的 量，在雜合反應後，DNA薄膜上將產生密度不同之標織物 示區，並呈現出具漸層的標幟物圖案。 不=DNA has the characteristics of New Zealand pairing, as well as the increasingly mature DNA manipulation technology and the Guardian 'Lin DNA plays an important role in the nanotechnology of age, and can be widely used in optics, electronics, bioelectronics, and a Biomedical and other high-tech industries. ^曰曰 is a traditional nano-microscopy technology that uses particle beam irradiation techniques, such as electron beam lithography (Electron Beam Lithography) to make nano-patterns, using a resist (Resist) to accept electron beams (Electr照射nBeam, e_Beam) illuminating the map. However, in the process of resisting, it is often necessary to pass the heating or chemical etching step t = 31 to increase the instability of the biomolecule, thereby affecting the manufacturing yield or quality stability of the subsequent finished product or product. SUMMARY OF THE INVENTION In order to simplify the operation steps and costs, and to avoid the toxicity, mutagenicity, or denaturation of the additional resist to the biomolecule, and to avoid contamination of the sample by residual agent residues or possible by-products, the present invention provides A nanopatterning method that can directly process biomolecules without the need for an additional resist. The invention relates to a biomolecular film (especially DNA) as a material, and then subjected to particle beam irradiation to change the properties of the DNA, and then participates in the hybridizati〇n reaction with the complementary DNA with the rod object. The DNA after irradiation with the particle beam loses the ability to hybridize with its complementary DNA. Therefore, the labeling area with the label and the pattern of the illuminating without the label can be distinguished on the original DNA thin 1296045 film. The particle beam, the ultraviolet photon beam, the x-ray or the radiation particle beam for the irradiation treatment, and the focused beam irradiation is irradiated according to the designed coordinates and the dose to express the straight g = in addition, by controlling The dose of the particle beam can control the amount of denatured DNA. After the hybrid reaction, the DNA film will produce a different density fabric area and exhibit a gradient pattern. Do not
【實施方式】 敬請參考第一圖，就由實施例對本發明做進一步的說明。 在本發明的一較佳實施方式，係選用一塊7x7mm2的玻璃板 (Pyrex 7740)為基材，分別以丙酮及異丙醇清洗，再以去離 子水（DeionizedWater)沖洗該玻璃板表面，接著置於烘箱烘 乾。該玻璃板先後分別熱蒸鑛(Thermal Evaporation)厚度50nm 的鉻（Cr)附著層，以及厚度350nm的金（Au)表面層。該 金表面層於活性離子钱刻機（Reactive i〇n Etcher)中以氧電漿 (Oxygen_Plasma)清理表面2分鐘，以增加其表面親水性。 含硫基的單股DNA係購自MDBio公司，其序列為 5’-HS-(CH2)6-(；T)20_3’（以下簡稱 HS-20T)。將此 DNA 配置於 1·〇Μ的KH2P〇4溶液中成為濃度為ΐ〇μΜ的HS-20T溶液。再 將此溶液塗佈於該玻璃板的該金表面層上，置於室溫下反應數 小時，因硫-金的化學反應使HS-20T固著於金表面層上。 HS-20T固著後，形成一 DNA薄膜。以去離子水沖洗薄膜，並 以氮氣吹乾，隨後以電子束照射該DNA薄膜上指定的區域使 之變性。電子束係由場發射掃描式電子顯微鏡（Field_Emissi〇n Scanning Electron Microscope ; FEI Sirion 200)所產生，加速 電壓為30keV，電子束流（Beam Current)約為20pA。電子束 在該DNA膜上，依曝光劑量不同，照射出15塊8χ8μπι2不同 1296045 劑量大小的方形區域。 本發明之第一實施例，係選用接有Hex螢光染劑之互補單 股DNA序列S’-HexD^，（以下簡稱Hex-20A)作為標幟 物。Hex-20A與玻璃板上HS_2〇T之接合反應係於TE-1MNaa 溶液（10mM Tris_HCl、lmM EDTA、1M NaCl)中作用數小 時，其中Hex-20A的濃度為ι〇μΜ。反應過後，再以去離子水 沖洗玻璃板洗去未接合的Hex_20A，再以蓋玻片覆蓋於玻璃板 上。以100W的水銀燈照射玻璃板，其中Hex染劑會因為水銀 燈的照射成為激發態而發出螢光，因此影像可藉由倒立螢光顯 你支鏡（Inverted Fluorescence Microscope ; Olympus 1X71)觀察， 並可藉由攝影機（CCD Camera ; Sony D70)記錄下來，如第 二圖所示。 藉由電子束的照射使HS-20T的DNA失去與互補的 Hex-20A雜合的能力。第二圖中玻璃板上由深到淺的暗色方形 區域即是DNA薄膜接受不同劑量電子束照射曝光的區域。不 同的電子束劑量可產生不同數量的變性DNA，在雜合反應 後，DNA薄膜上就產生密度不同之Hex螢光標示區，在螢光 影像中呈現出具漸層的圖案。第二圖中的背景明亮區域即為未 受電子束照射，因此未產生變性的HS-20T。未變性的HS-20丁 可有效地與Hex-20A雜合形成標示區，因此在Hex螢光染劑 被激發後呈現明亮的反應。 本發明之第二實施例（請參考第三圖），係選用金奈米顆粒 做為標幟物。在電子束照射HS-20T薄膜上特定區域後，以濃 度為ΙΟμΜ並接有生物素(Biotin)的互補單股DNA序列 5’-Biotin_(A)2(r3’（ Biotin-20A ;購自 MDBio，Inc·)進行雜合 反應。之後再加入 0.1 mg/ml Streptavidin ( Sigma-Aldrich Co.) 與玻璃板上的biotin接合並以去離子水清洗玻璃板。再與含有 金奈米顆粒（直徑約13nm)的水溶液反應1〇分鐘，使金奈米 顆粒透過Streptavidin與Biotin_20A產生接合反應。待金奈米 1296045 ^粒^應後清洗吹乾，以掃描式電子顯平 示。第三圖中不同深淺的暗色區^ 顆* f照射後變性或部分變性的HS-20T:因二無二二f Z 】妾合形成弱標示區或未標示區，而呈現暗色’?…不只 圖。mDNAs^_對亮賴奸束騎缝的曲線 ίϋ，度則是取最亮方形區域的平均亮度為A1^i# 為二亮度為A15、以及第n個方形區域的平均亮i 度值為為.（A1 — An)/(A1 _ A15) ’並令最亮的相對Ϊ X值為1’攻暗的相對亮度值為〇。電子束的面積昭射 lu = Μ。其中實心圓是Hex _的螢光影像亮度從 翻之方法’無論是顧版_，或是Ϊί^ 幟物’都可以得到相近似的亮度曲線，並可說明雜ί 巧效果可赠賴射所㈣，而達職階圖像效果，口 火階圖像效果有別於傳統微影技術所使用的阻劑， 非線性的性質，它通常只表現黑白兩種狀態的圖像。第四 下角則疋其對照射劑量的半對數（Semfl〇g)圖，其中在替: 衫像效果上，劑量常數為l.OSxio^c/cm2;在掃描式電子料 鏡影像效果上，劑量常數為3·01χ103μσ^。 Τ々减 透過本發明的應用’利用現今技術層次已相當成熟的聚焦 子束照射技術，已經可製作出線寬僅數奈米的任意圖形。 技術加上生物上成熟的DNA合成、雜合以及銜接各式奈米 粒或生物分子的技術，便可以將奈米金屬或半導體顆 如··硒化鎘CdSe、或硫化鎘CdS)、碳60、DNA、或一此功 能性的蛋白質等生物分子排成設計好的圖案，第五圖即為二^ 際例子，用以證明以DNA分子製作奈米圖案的能力。如此， 未來將可以充份結合奈米材料的特殊性質或生物分子的特有 功能’以製作微透鏡(micro-lens)、zone plate、生物晶片、齐米 6 1296045 導線、生化制元件或分子電子元件等高科技製品。 之發賴—詳魄明’細上所财，僅為本創作 ί已’當不能限定本創作實施之綱，即凡依本 之直以圍所作之均等變化與修飾等，皆應仍屬本創作 之專利涵盍範圍意圖保護之範疇。 【圖式簡單說明】 圖係本發明較佳實施方式的流程圖。 係本發明第—實施例的奈糊_像圖。 ^二，本發明第二實施_奈米_影像圖。 ίϋ=ί發明實施例的照射劑量相對於影像亮度曲線圖。 弟五圖係本發明製作奈米圖像之較佳實施例。 【主要元件符號說明】[Embodiment] The present invention will be further described by way of examples with reference to the first drawings. In a preferred embodiment of the present invention, a 7x7mm2 glass plate (Pyrex 7740) is used as a substrate, which is respectively washed with acetone and isopropyl alcohol, and then washed with deionized water (Deionized Water), and then placed. Dry in an oven. The glass plate was successively a Thermal Evaporation chromium (Cr) adhesion layer having a thickness of 50 nm and a gold (Au) surface layer having a thickness of 350 nm. The gold surface layer was cleaned with oxygen plasma (Oxygen_Plasma) for 2 minutes in a reactive ionizer to increase its surface hydrophilicity. The sulfur-containing single-stranded DNA was purchased from MDBio, and its sequence was 5'-HS-(CH2)6-(;T)20_3' (hereinafter referred to as HS-20T). This DNA was placed in a KH2P〇4 solution of 1·〇Μ to form a HS-20T solution having a concentration of ΐ〇μΜ. Further, this solution was applied onto the gold surface layer of the glass plate, and allowed to react at room temperature for several hours, and HS-20T was fixed on the gold surface layer by a sulfur-gold chemical reaction. After HS-20T is fixed, a DNA film is formed. The film was rinsed with deionized water and dried with nitrogen, followed by denaturation of the designated area on the DNA film by electron beam irradiation. The electron beam system was generated by a field emission scanning electron microscope (Field_Emissi〇n Scanning Electron Microscope; FEI Sirion 200) with an acceleration voltage of 30 keV and a beam current of about 20 pA. Electron beam On the DNA film, 15 square regions of different sizes of 12,960,45 doses were irradiated depending on the exposure dose. In the first embodiment of the present invention, a complementary single-stranded DNA sequence S'-HexD^ (hereinafter referred to as Hex-20A) to which Hex fluorescent dye is attached is used as a label. The binding reaction of Hex-20A to HS_2〇T on the glass plate was carried out in TE-1MNaa solution (10 mM Tris_HCl, lmM EDTA, 1 M NaCl) for several hours, wherein the concentration of Hex-20A was ι〇μΜ. After the reaction, the unbonded Hex_20A was washed with a rinse-dried glass plate and covered with a cover glass on the glass plate. The glass plate is illuminated by a 100W mercury lamp, in which the Hex dye will be excited by the illumination of the mercury lamp, so the image can be observed by Inverted Fluorescence Microscope (Olympus 1X71) and can be borrowed. Recorded by the camera (CCD Camera; Sony D70), as shown in the second figure. The DNA of HS-20T loses its ability to hybridize to complementary Hex-20A by irradiation with an electron beam. The dark square area from deep to shallow on the glass plate in the second figure is the area where the DNA film is exposed to different doses of electron beam. Different electron beam doses can produce different amounts of denatured DNA. After the hybrid reaction, Hex fluorescent label regions with different densities are produced on the DNA film, and a gradation pattern is exhibited in the fluorescent image. The bright background of the background in the second figure is that it is not irradiated with electron beams, so no denatured HS-20T is produced. Undenatured HS-20 butyl can effectively hybridize with Hex-20A to form a labeling region, thus exhibiting a bright response after the Hex fluorescent dye is activated. In the second embodiment of the present invention (please refer to the third figure), the gold nanoparticles are used as the target. After electron beam irradiation on a specific region of the HS-20T membrane, the complementary single-stranded DNA sequence 5'-Biotin_(A)2 (r3' (Biotin-20A; purchased from MDBio) was ΙΟμΜ and conjugated with biotin (Biotin). , Inc.) was subjected to a heterozygous reaction. Then 0.1 mg/ml Streptavidin (Sigma-Aldrich Co.) was added to the biotin on the glass plate and the glass plate was washed with deionized water. The aqueous solution of 13 nm) was reacted for 1 〇 minutes, and the gold nanoparticles were conjugated with Biotin_20A through Streptavidin. After the Genna 1296045 ^ granules were cleaned and dried, the scanning electrons were flattened. The dark color area ^ f * After denaturation or partial denaturation of HS-20T: due to the two or two f Z 】 形成 形成 形成 形成 形成 形成 形成 形成 形成 形成 形成 弱 弱 弱 弱 弱 弱 弱 弱 弱 弱 弱 弱 弱 弱 弱 弱 弱 弱 弱 弱 弱 弱 弱 弱 弱 弱 弱 弱 弱 弱The curve of the slap-up stitch is ϋ, the degree is the average brightness of the brightest square area is A1^i#, the second brightness is A15, and the average brightness value of the nth square area is (A1 - An ) / (A1 _ A15) 'and let the brightest relative Ϊ X value be 1 'the relative brightness value of the dimming 〇. The area of the electron beam is radiant lu = Μ. The solid circle is the brightness of the Hex _ fluorescent image. The method of turning the 'either _ _ _ or Ϊ ^ ^ ^ ^ can get a similar brightness curve. It can also be explained that the effect of the miscellaneous effect can be given to the film (4), and the effect of the image of the grade is different from that of the traditional lithography technology. The nonlinear nature, it usually only shows black and white. The image of the state. The fourth lower corner is the semi-logarithmic (Semfl〇g) map of the irradiation dose, wherein in the effect of the shirt image, the dose constant is l.OSxio^c/cm2; in the scanning electronic material On the mirror image effect, the dose constant is 3·01χ103μσ^. Through the application of the present invention, it is possible to produce an arbitrary pattern with a line width of only a few nanometers by using the focused beamlet irradiation technique which is quite mature at the current technical level. Technology plus biologically mature DNA synthesis, heterozygous, and the technology of binding various nano-particles or biomolecules, you can use nano metal or semiconductor particles such as cadmium selenide CdSe, or cadmium sulfide CdS), carbon 60, DNA, or a functional protein Biomolecules arranged in a pattern designed, namely two fifth ^ inter FIG example, to demonstrate the ability of the DNA molecule to produce nano patterns. In this way, the future will be able to fully combine the special properties of nanomaterials or the unique functions of biomolecules to make micro-lens, zone plates, biochips, zimi 6 1296045 wires, biochemical components or molecular electronic components. And other high-tech products. The reliance on the details - the detailed description of the 'final wealth, only for this creation ί has 'when it can not limit the implementation of this creation, that is, all the changes and modifications made according to this straight line should still belong to this The scope of the patents created is intended to protect the scope. BRIEF DESCRIPTION OF THE DRAWINGS The drawings are a flow chart of a preferred embodiment of the present invention. It is a map of the first embodiment of the present invention. ^ Second, the second embodiment of the invention _ nano_image map. ϋ ϋ = ί ί ί ί ί ί ί ί ί ί ί ί ί ί The fifth figure is a preferred embodiment of the present invention for making a nano image. [Main component symbol description]