TWI541505B - 以游離輻射製備檢驗試劑載體之方法 - Google Patents
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Description
本發明係有關於一種以游離輻射製備奈米檢驗試劑之方法，尤指涉及一種利用高分子聚乙二醇(Polyethylene Glycols,PEG)以化學鍵結修飾具有磁性分子之奈米碳管表面所構成之磁性奈米複合材，特別係指作為可專一性地標定多種癌症之奈米檢驗試劑，可提高偵測靈敏度並降低偽訊號產生，以達到特定疾病診斷之目的。
通常要將一般材料接上疾病(尤指癌症)之專一性抗原/抗體(Antigen/Antibody)並不容易且吸附力不強，抗原/抗體容易在處理過程中脫落或破壞其蛋白質結構，失去其活性。因此，有許多用來改良吸附生物材料之方法被提出，例如載體之開發一直為分離純化技術主要之研究方向，近年來奈米碳載體備受重視，其中奈米碳官能基化後之磁性載體之技術，在奈米碳表面會產生官能基，影響對於生物材料之吸附效應。即當抗原或係抗體與官能基化之磁性奈米碳結合後，另外再加入磁性分子以加熱方式使其貼附於奈米微粒上，於一外加磁場之存在下，則可輕易地操控它移至某處或者另一狀態。自1991年代時，奈米碳在製備巴克球(Bucky Ball，碳60)之實驗產物中被發現，由於奈米碳管在物性、化性或材料特性上具有相當優越之性能，包含質量輕、高韌性、可撓曲性高、表面積大、高熱傳導性等特性，因此衍生出許多廣泛之應用，受到相當矚目。另外
，為了研究分離生物性材料之技術，開始有一些學者將磁性載體技術應用於分離技術中，作為一種利用磁場將目標物分離之分離技術，目前已廣泛應用於醫療診斷、DNA、RNA純化、蛋白質、酵素之固定化、免疫分析與環境分析、及磁性流體等領域中，例如將其與特定之抗原/抗體結合後，這些磁性載體就具有高生物選擇性，並可以吸附特定疾病及病毒所具有之DNA、RNA或抗原/抗體。
有鑑於此，在尚未導致癌症之非誘發細胞病變效應(Noncytopathic,NCP)時期，對疾病之偵測及診斷將有助於早期診斷病灶，進而提高治癒癌症之機率，一般臨床上對疾病之偵測及診斷主要依賴基於抗體-抗原交互作用之免疫分析，且目前已包括有酵素連接免疫吸收分析(Enzyme-Linked Immunosorbent Assay,ELISA)、化學冷光呈色(Chemiluminesce)、聚合酶連鎖反應(Polymerase Chain Reaction，PCR)、表面電漿共振(Surface Plasmon Resonance,SPR)、電化學石英晶體微天平(Electrochemical QCM,EQCM)或免疫聚合酶連鎖反應(Immuno-PCR)等檢驗方式提出。例如比較商業化之磁性載體，其係以生物玻璃基材作為磁性載體，惟其於水裡面會水解化掉，且當PH值改變時，其基材也會變化，故其穩定度不好；再者，該磁性載體上之OH基易與水分子結合，因此亦衍生非專一性結合(Non-Specific Binding)之問題，進而降低其靈敏度。故，一般習用者係無法符合使用者於實際使用時之所需。
本發明之主要目的係在於，克服習知技藝所遭遇之上述問題並提供一種利用高分子聚乙二醇以化學鍵結修飾具有磁性分子之奈米碳管表面所構成之磁性奈米複合材，藉此作為可專一性地標定多種癌症之奈米檢驗試劑，可提高偵測靈敏度並降低偽訊號產生，以達到特定疾病組織之磁振造影及
診斷之目的，係可應用於臨床大量例行活體外定量量測癌症診斷與治療之評估方法者。
為達以上之目的，本發明係一種以游離輻射製備奈米檢驗試劑之方法，係為一奈米碳管，經酸處理及鈷-60游離輻射照射，造成該奈米碳管表面改質，形成不同之官能基接枝後，加入磁性分子以鈷-60游離輻射照射方式使該磁性分子吸附於該奈米碳管上，再利用高分子聚乙二醇以化學鍵結修飾此具有磁性分子之奈米碳管表面，可使抗體或抗原得以輕易被吸附至此奈米碳管上之磁性奈米複合材，其包括：一磁性奈米碳管；以及一接枝官能性分子，係分佈於該磁性奈米碳管上，並接有一高分子聚乙二醇，用以將一活性分子結合於該磁性奈米碳管表面。
1‧‧‧磁性奈米複合材
11‧‧‧奈米碳管
111‧‧‧磁性分子
12‧‧‧接枝官能性分子
121‧‧‧聚乙二醇
2‧‧‧活性分子
3‧‧‧樣品
31‧‧‧待測物
4‧‧‧二級抗體
41‧‧‧放射性物質
第1圖，係本發明磁性奈米複合材之製作流程示意圖。
第2圖，係本發明磁性奈米複合材之結構示意圖。
第3圖，係本發明之使用態樣流程示意圖
請參閱『第1圖及第2圖』所示，係分別為本發明磁性奈米複合材之製作流程示意圖、及本發明磁性奈米複合材之結構示意圖。如圖所示：本發明係一種以游離輻射製備奈米檢驗試劑之方法，係為一奈米碳管11(Carbon Nano Tube,CNT)，經酸溶液處理及鈷-60游離輻射照射，造成該奈米碳管11表面改質，形成不同之官能基接枝後，加入磁性分子111以鈷-60游離輻射照射方式使該磁性分子111吸附於該奈米碳管11上，再利用高分子聚乙二醇(Polyethylene Glycols,PEG)121以化學鍵結修飾此具有磁性分子111之奈米碳管11表面，可使抗體或抗原得
以輕易被吸附至此奈米碳管11上之磁性奈米複合材1，其包括：一奈米碳管11，係分佈有一磁性分子111，其中，該磁性分子111係為可合成四氧化三鐵(Fe3O4)之二價鐵(Fe2+)；以及一接枝官能性分子12，係分佈於該磁性奈米碳管11上，並接有一高分子聚乙二醇121，用以將一活性分子結合於該磁性奈米碳管11表面，其中，該接枝官能性分子12係含有一官能基，並可為羧基(-COOH)、胺基(-NH2)、硫醇基(-SH)、羥基(-OH)、醛基(-COH)或酯基(-COO-)，俾利由酸接枝官能化。
當本發明於製備時，於一較佳實施例中，係將一群奈米碳管11進行硝酸、硫酸或醋酸之酸溶液處理，及利用鈷-60游離輻射照射，使該奈米碳管11上形成一接枝官能性分子12，可於末端長出COOH之官能基，之後再將一磁性分子111加入該奈米碳管11中，以鈷-60游離輻射照射方式使該磁性分子111附載於該奈米碳管11上，最後以高分子聚乙二醇121修飾表面以提高水溶性、生物相容性並可避免被血清中之蛋白吸附達到專一性標的之目的，使每一奈米碳管11在皆具有磁性同時，亦可藉由外圍接有之聚乙二醇121提高專一性結合率(Specific Binding)，使本發明以此游離輻射製備而成之磁性奈米複合材1，作為可專一性地標定多種癌症之奈米檢驗試劑，可提高偵測靈敏度並降低偽訊號產生，以達到特定疾病診斷之目的。
請參閱『第3圖』所示，係本發明之使用態樣流程示意圖。如圖所示：本發明係可以上述磁性奈米複合材1應用於臨床大量例行活體外定量量測癌症診斷與治療之評估，其至少包含下列步驟：(A)提供一磁性奈米複合材1，該磁性奈米複合材1外圍係接有高
分子聚乙二醇121，其表面並結合有一活性分子2可捕捉樣品3中之待測物31，其中，該活性分子2係可為抗原(Antigen)、核酸、寡聚核苷酸(Oligonucleotide)、蛋白質、醣類或抗體(Antibody)；(B)將一樣品3加入該磁性奈米複合材1中，使該樣品3中之待測物31與該磁性奈米複合材1表面之活性分子2產生特異性反應而進行捕捉；以及(C)再加入一標識有放射性物質41之二級抗體(Secondary Antibody)4進行反應，使具有該放射性物質41之二級抗體4標示該待測物31，俾以此標示作為指標進行訊號偵測。
當本發明於運用時，於一較佳實施例中，係以上述磁性奈米複合材1作為載體之結構，利用其接枝官能性分子12及其上高分子聚乙二醇121將一抗原2結合於該奈米微粒11表面；繼之，藉由在一容器底部放置之磁鐵，使該磁性奈米複合材1中之磁性分子111對磁場起反應而往該磁鐵方向移動聚集，待將剩下沒反應之抗原2清洗去除後，即形成純粹帶有抗原2之磁性奈米複合材1；接著，加入一樣品3(即癌症病患之血清)於其中，利用對疾病具專一性吸附之磁性奈米複合材1，能僅將該血清中可與抗原2相對應之待測物31，即一級抗體(Primary Antibody)接枝於該磁性奈米複合材1上，於其中，同樣施以一磁場將該磁性奈米複合材1聚集吸附住，未被吸附住之血清中之其他物質即被分離去除；最後，將此接上一級抗體之磁性奈米複合材1再接枝上二次抗體4，並再施以一磁場將其聚集吸附住，未被吸附住之二次抗體4即被分離去除，隨後即可利用該二次抗體4上標識之放射性物質41，例如碘-125(I-125)檢測其訊號。
其中，若上述癌症病患血清中並無相對應之一級抗體時，該磁性奈米複合
材1上之抗原2即不會進行捕捉，而隨後加入之二級體抗4亦同樣不會捕捉，自然在後續檢測放射性物質41之訊號時即可避免訊號之偽陽性結果。
綜上所述，本發明係一種以游離輻射製備奈米檢驗試劑之方法，可有效改善習用之種種缺點，係為一奈米碳管(Carbon Nano Tube,CNT)，經酸處理及鈷-60游離輻射照射，造成該奈米碳管表面改質，形成不同之官能基接枝後，加入磁性分子以鈷-60游離輻射照射方式使該磁性分子吸附於該奈米碳管上，再利用高分子聚乙二醇(Polyethylene Glycols,PEG)以化學鍵結修飾此具有磁性分子之奈米碳管表面，可使抗體或抗原得以輕易被吸附至此奈米碳管上之磁性奈米複合材，進而使本發明之產生能更進步、更實用、更符合使用者之所須，確已符合發明專利申請之要件，爰依法提出專利申請。
惟以上所述者，僅為本發明之較佳實施例而已，當不能以此限定本發明實施之範圍；故，凡依本發明申請專利範圍及發明說明書內容所作之簡單的等效變化與修飾，皆應仍屬本發明專利涵蓋之範圍內。
11‧‧‧奈米碳管
111‧‧‧磁性分子
12‧‧‧接枝官能性分子
121‧‧‧聚乙二醇
Claims (9)
- 一種以游離輻射製備奈米檢驗試劑之方法，係為一奈米碳管(Carbon Nano Tube,CNT)，經酸溶液處理及鈷-60游離輻射照射，造成該奈米碳管表面改質，形成不同之官能基接枝後，加入可合成四氧化三鐵(Fe3O4)之二價鐵(Fe2+)之磁性分子以鈷-60游離輻射照射方式使該磁性分子吸附於該奈米碳管上，再利用高分子聚乙二醇(Polyethylene Glycols,PEG)以化學鍵結修飾此具有磁性分子之奈米碳管表面，可使抗體或抗原得以輕易被吸附至此奈米碳管上之磁性奈米複合材，其包括：一磁性奈米碳管；以及一接枝官能性分子，係分佈於該磁性奈米碳管上，並接有一高分子聚乙二醇，用以將一活性分子結合於該磁性奈米碳管表面。
- 依據申請專利範圍第1項所述之以游離輻射製備奈米檢驗試劑之方法，其中，該接枝官能性分子係經酸溶液處理及鈷-60游離輻射照射所形成。
- 依據申請專利範圍第1項所述之以游離輻射製備奈米檢驗試劑之方法，其中，該接枝官能性分子係含有一官能基，並可為羧基(-COOH)、胺基(-NH2)、硫醇基(-SH)、羥基(-OH)、醛基(-COH)或酯基(-COO-)。
- 依據申請專利範圍第1項所述之以游離輻射製備奈米檢驗試劑之方法，其中，該酸溶液處理係為硝酸、醋酸或硫酸。
- 依據申請專利範圍第1項所述之以游離輻射製備奈米檢驗試劑之方法，其中，該活性分子係為抗原(Antigen)、核酸、寡聚核苷酸(Oligonucleotide)、蛋白質、醣類或抗體(Antibody)。
- 依據申請專利範圍第1項所述之以游離輻射製備奈米檢驗試劑之方法，係可應用於臨床大量例行活體外定量量測癌症診斷與治療之評估，其至少包含下列步驟：(A)提供一磁性奈米複合材，該磁性奈米複合材外圍係接有高分子聚乙二醇，其表面並結合有一活性分子可捕捉樣品中之待測物；(B)將一樣品加入該磁性奈米複合材中，使該樣品中之待測物與該磁性奈米複合材表面之活性分子產生特異性反應而進行捕捉；以及(C)再加入一標識有放射性物質之二級抗體(Secondary Antibody)進行反應，使具有該放射性物質之二級抗體標示該待測物，俾以此標示作為指標進行訊號偵測。
- 依據申請專利範圍第6項所述之以游離輻射製備奈米檢驗試劑之方法，其中，該樣品係為癌症病患之血清。
- 依據申請專利範圍第6項所述之以游離輻射製備奈米檢驗試劑之方法，其中，該待測物係為一級抗體(Primary Antibody)。
- 依據申請專利範圍第6項所述之以游離輻射製備奈米檢驗試劑之方法，其中，該放射性物質係可為碘-125(I-125)。
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| JP2013143158A JP5674870B2 (ja) | 2013-04-02 | 2013-07-09 | 磁性ナノ複合材の作製方法及び磁性ナノ複合材を用いた測定方法 |
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