技術(shù)原理
石英晶體微天平---QCM技術(shù)
石英晶體微天平(Quartz Crystal Microbalance-QCM)的發(fā)展始于上世紀(jì)60年代初期,它是一種非常靈敏的質(zhì)量檢測儀器,其測量精度可達(dá)納克級,比靈敏度在微克級的電子微天平高100 倍,理論上可以測到的質(zhì)量變化相當(dāng)于單分子層或原子層的幾分之一。石英晶體微天平利用了石英晶體諧振器的壓電特性,將石英晶振電極表面質(zhì)量變化轉(zhuǎn)化為石英晶體振蕩電路輸出電信號的頻率變化,進(jìn)而通過計算機(jī)等其他輔助設(shè)備獲得高精度的數(shù)據(jù)。
ATTANA利用QCM技術(shù),通過石英諧振器頻率的變化感知芯片表面質(zhì)量的變化,生成感應(yīng)曲線,其敏感度可達(dá)到分子水平。實驗時,只需將細(xì)胞,抗原或抗體等包被于芯片表面,將含有與之互作分子的溶液流過芯片表面,石英諧振器即可檢測分子間結(jié)合﹑解離等整個變化過程,生成一張傳感圖,提供動力學(xué)親和力等數(shù)據(jù)分析。
產(chǎn)品應(yīng)用/特點
ATTANA 應(yīng)用
眾所周知,藥物研發(fā)是一個費時費力的過程。為了最大程度的節(jié)省人力物力以及時間,科學(xué)家一直在尋求在藥物研發(fā)的早期驗證并去除不合適的藥物候選物的方法。ATTANA通過高通量驗證藥物候選物,加快了藥物篩選的進(jìn)程,使得在藥物研發(fā)早期提出不適合的候選物,節(jié)省研發(fā)成本與時間成為可能。
ATTANA是世界上第一臺可以在細(xì)胞表面直接檢測無標(biāo)記分子間相互作用的生物傳感器,它的出現(xiàn)填補(bǔ)了傳統(tǒng)研究分子間相互作用儀器及基于細(xì)胞的檢測之間的空白。
細(xì)胞&生化檢測-雙重應(yīng)用
核心應(yīng)用:
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細(xì)胞表面的動力學(xué)反應(yīng)
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抗原抗體反應(yīng)
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競爭力分析
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配體垂釣
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抗原表位定位等
ATTANA特點1 –直接進(jìn)行細(xì)胞層面的檢測
實驗?zāi)康模?/span>
研究為用于特定免疫治療乳腺癌或卵巢癌而設(shè)計異構(gòu)化的曲妥珠單抗的特性。
實驗結(jié)果:
ATTANA的實驗數(shù)據(jù)顯示,異構(gòu)化后的曲妥珠單抗會與細(xì)胞表面抗原進(jìn)行非特異性結(jié)合。
被修飾后曲妥珠單抗的特性:
通過傳統(tǒng)分子間相互作用儀研究方法分別研究曲妥珠單抗(圖A)與修飾后的的曲妥珠單抗(圖B)與包被于芯片上的HER2抗原的結(jié)合力與結(jié)合特性。
該研究隨后轉(zhuǎn)入細(xì)胞層面,將表達(dá)HER2的癌細(xì)胞包被于芯片,發(fā)現(xiàn)盡管曲妥珠單抗(圖C;KD=2.5nM)與HER2的親和力與文獻(xiàn)一致,但修飾后的曲妥珠單抗(圖D)展現(xiàn)了與細(xì)胞表面抗原非特異性結(jié)合的特性。
ATTANA特點2 –針對原樣進(jìn)行高通量檢測
通過Attana,科學(xué)家可以通過對原樣的分析(高致 100%的血清或細(xì)胞裂解液)及實驗中溫度的控制,最大程度地還原體內(nèi)環(huán)境,實驗所得細(xì)胞與分子間的生物化學(xué)反應(yīng)數(shù)據(jù)最為真實可靠。
通過消除樣本的純化、標(biāo)記或預(yù)處理,ATTANA加快了分子及克隆篩選和免疫反應(yīng)的監(jiān)控,最大程度節(jié)約研發(fā)時間。同時,ATTANA的生物傳感器可以被用于研究反應(yīng)特異性﹑親和力﹑動力學(xué)參數(shù)及其他生化結(jié)合特性, 亦可以用于研究不同細(xì)胞﹑抗體﹑蛋白﹑病毒及細(xì)菌的宏觀結(jié)構(gòu)。
應(yīng)用:
實驗?zāi)康模?/span>
使用ATTANA 200系統(tǒng)以及低非特異性結(jié)合表面建立一個快速決定解離常數(shù)(kd)的方法,從而對可以與一個配體發(fā)生反應(yīng)的細(xì)菌裂解液中的支架蛋白進(jìn)行分級。
實驗結(jié)果 &結(jié)論:
建立的方法可以輕易的根據(jù)解離常數(shù)(kd)將反應(yīng)分級。該方法允許在無人為看護(hù)的狀況下,檢測192個樣本(圖A)。在細(xì)胞裂解液與芯片表面間無非特異性結(jié)合被檢測到(圖B)。該方法可以被用作克隆的篩選﹑生物標(biāo)記物的研發(fā)﹑疫苗的篩選制備﹑生物制造研究等生物學(xué)相關(guān)領(lǐng)域。
圖:
含有不同細(xì)胞支架蛋白的E.coli裂解液與配體的結(jié)合曲線。圖A:與包被抗原反應(yīng);圖B:與參照物反應(yīng);圖A-B:與包被抗原反應(yīng)
耗材
芯片
COP-1 sensor chip
COP-1傳感器芯片
LNB-Carboxyl sensor chip
LNB-羧基傳感器芯片
Carboxyl sensor chip
羧基傳感器芯片
Biotin sensor chip
生物素傳感器芯片
Polystyrene sensor chip
聚苯乙烯傳感器芯片
試劑盒
Amine Coupling Kit
氨基偶聯(lián)試劑盒LNB-羧基傳感器芯片或羧基傳感器芯片合用
Mouse IgG Capture Kit
小鼠IgG抗體捕捉試劑盒與LNB-羧基傳感器芯片(或羧基傳感器芯片)以及氨基偶聯(lián)試劑盒合用
Human IgG Capture Kit
人IgG抗體捕捉試劑盒與LNB-羧基傳感器芯片(或羧基傳感器芯片)以及氨基偶聯(lián)試劑盒合用
Streptavidin reagent
與生物素傳感器芯片合用
HBS-T 10x Running Buffer HBS-T 10x緩沖液
產(chǎn)品規(guī)格
參考文獻(xiàn)
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Journal of Molecular Biology (2008) 384, 1143–1156Generation, Affinity Maturation, and Characterization of a Human Anti-Human NKG2D Monoclonal Antibody with Dual Antagonistic and Agonistic ActivityKa Yin Kwong, Sivasubramanian Baskar, Hua Zhang, Crystal L. Mackall and Christoph Rader
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Analytical Chemistry 79 (18): 6897-6902 (Sep. 15, 2007)Photoderivatized Polymer Thin Films at Quartz Crystal Microbalance Surfaces: Sensors for Carbohydrate-Protein InteractionsYuxin Pei, Hui Yu, Zhichao Pei, Matthias Theurer, Carolin Ammer, Sabine André, Hans-Joachim Gabius, Mingdi Yan and Olof Ramstr?m
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Review of Scientific Instruments 78, 083110 (2007)System for in situ studies of atmospheric corrosion of metal films using soft x-ray spectroscopy and quartz crystal microbalanceJ. Forsberg, L.-C. Duda, A. Olsson, T. Schmitt, J. Andersson, J. Nordgren, J. Hedberg, C. Leygraf, T. Aastrup, D. Wallinder, and J.-H. Guo
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2007Flow Injection Assay of the Path ogenic Bacteria Using Lectin-based QCM BiosensorGulnara Safina, Margret van Lier and Bengt Danielsson, Pure and Applied Biochemistry, Chemical Center.
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Sensors and Actuators B 123 (2007) 21–26Quartz crystal microbalance biosensor design II. Simulation of sample transportMats J?nsson, Henrik Anderson, Ulf Lindberg, and Teodor Aastrup
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Sensors and Actuators B 123 (2007) 27–34Quartz crystal microbalance sensor design I. Experimental study of sensor response and performanceHenrik Anderson, Mats J?nsson, Lars Vestling, Ulf Lindberg, and Teodor Aastrup
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Biosensors and Bioelectronics 22 (2006) 42–48Quartz crystal microbalance bioaffinity sensor for rapid identification of glycosyldisulfide lectin inhibitors from a dynamic combinatorial libraryZhichao Pei, Rikard Larsson, Teodor Aastrup, Henrik Anderson, Jean-Marie Lehn and Olof Ramstr?m
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Bioorganic & Medicinal Chemistry Letters 15 (2005) 2707–2710Redox-responsive and calcium-dependent switching of glycosyldisulfide interactions with Concanavalin AZhichao Pei, Teodor Aastrup, Henrik Anderson and Olof Ramstr?m
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Biosensors and Bioelectronics 21 (2005) 60–66Study of real-time lectin–carbohydrate interactions on the surface of a quartz crystal microbalanceZhichao Pei, Henrik Anderson, Teodor Aastrup and Olof Ramstr?m
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Analytical Biochemistry 341 (2005) 89-93Electro-immobilization of proinsulin C-peptide to a quartz crystal microbalance sensor chip for protein affinity purificationErmias Melles, Henrik Anderson, Daniel Wallinder, Jawed Shafqat, Tomas Bergman, Teodor Aastrup and Hans J?rnvall