Octet國(guó)內(nèi)文獻(xiàn)盤點(diǎn)：小分子應(yīng)用

Octet® 非標(biāo)記分子互作分析系統(tǒng)采用實(shí)時(shí)的非標(biāo)記分析技術(shù)，用于測(cè)定親和力、動(dòng)力學(xué)和抗體/蛋白質(zhì)定量。這種無(wú)流路的儀器平臺(tái)基于生物層干涉（BLI）技術(shù)而設(shè)計(jì)，與傳統(tǒng)的非標(biāo)記技術(shù)相比，具有眾多優(yōu)勢(shì)。

2024年上半年（截至6月30日在線發(fā)表）的國(guó)內(nèi)論文中，使用賽多利斯Octet® 數(shù)據(jù)的國(guó)內(nèi)論文呈現(xiàn)井噴狀態(tài)。陳老師統(tǒng)計(jì)了2024年上半年約200篇國(guó)內(nèi)論文（預(yù)計(jì)總數(shù)超過(guò)200篇），這些文章分布于130個(gè)單位， 詳細(xì)列表在文末哦~老規(guī)矩，讓我們先按照單位和應(yīng)用進(jìn)行統(tǒng)計(jì)。
 

圖1. 2024上半年Octet® 文章第一作者單位分布
 

圖2. 2024H1 Octet® 文章應(yīng)用分布
 
中國(guó)藥科大學(xué)、北京協(xié)和醫(yī)學(xué)院、南京大學(xué)、復(fù)旦大學(xué)、浙江大學(xué)和中山大學(xué)等單位發(fā)表的文章均在4篇以上。其中，中國(guó)藥科大學(xué)論文數(shù)達(dá)到了10篇。這表明，Octet® 因其操作簡(jiǎn)單、成本低、數(shù)據(jù)準(zhǔn)確和儀器穩(wěn)定性好等優(yōu)點(diǎn)，可以幫助科學(xué)家快速取得科研成果。

從應(yīng)用方向上看，依舊涉及到多個(gè)方向。其中小分子的應(yīng)用超過(guò)蛋白和抗體應(yīng)用的文章數(shù)量，牢牢占據(jù)應(yīng)用方向榜首！BLI技術(shù)對(duì)溶劑（DMSO）不敏感，假陽(yáng)性低，高靈敏度等優(yōu)點(diǎn)可以幫助研究人更快，更高質(zhì)量的獲得了小分子檢測(cè)的結(jié)果。


今天，我們就來(lái)挑選幾篇代表性文章來(lái)一一解讀：


浙江大學(xué)——細(xì)胞與分子互作^[1]
 
全球氣候變化伴隨著二氧化碳（CO₂）富集和高溫（HT）脅迫。研究發(fā)現(xiàn)，番茄外源質(zhì)外糖 （Glc）處理增強(qiáng)了番茄在環(huán)境CO₂條件下對(duì)HT脅迫的恢復(fù)力�；�于細(xì)胞的生物層干涉法（Octet® ）、亞細(xì)胞定位和分裂熒光素酶測(cè)定表明，Glc 與番茄 G 蛋白信號(hào)轉(zhuǎn)導(dǎo) 1 （RGS1） 調(diào)節(jié)因子結(jié)合并誘導(dǎo) RGS1 內(nèi)吞作用，從而以濃度依賴性方式誘導(dǎo) RGS1-G 蛋白α亞基 （GPA1） 解離，這是 CO₂-Glc 誘導(dǎo)的耐熱性升高所必需的。這些信息增強(qiáng)了對(duì)Glc-G蛋白信號(hào)傳導(dǎo)功能在應(yīng)對(duì)全球氣候變化的脅迫恢復(fù)力中的理解，并將有助于開發(fā)植物恢復(fù)力的基因工程方法。
 

圖3. 將原生質(zhì)體固化在Octet® 傳感器上，結(jié)合各種單糖，發(fā)現(xiàn)表達(dá)RGS1的細(xì)胞主要結(jié)合葡萄糖，但是將RGS1突變后，與葡萄糖結(jié)合變?nèi)?br />  
Octet® 因?yàn)榻�入即讀的方法，更適合檢測(cè)比較大的分析物，比較細(xì)胞。


西湖大學(xué)——血清抗體濃度測(cè)定^[2]
 
第三劑滅活新型冠狀病毒疫苗的抗體反應(yīng)對(duì)疫苗的保護(hù)作用至關(guān)重要。西湖大學(xué)工學(xué)院對(duì)接種疫苗的15人的九個(gè)月里七個(gè)時(shí)間點(diǎn)進(jìn)行了微量取樣和靜脈穿刺取樣，并建立了基于Octet® 的光纖生物層干涉法測(cè)量(FO-BLI)來(lái)測(cè)試血清中的中和抗體，可以在幾分鐘內(nèi)完成測(cè)試。研究結(jié)果揭示發(fā)現(xiàn)在第三次給藥后，野生型變體的血清NAb水平在7天后增加了2.9倍，在一個(gè)月內(nèi)增加了3.3倍，隨后下降，并在三個(gè)月后變得不可檢測(cè)。針對(duì)原始株與變異株的中和能力檢測(cè), FO-BLI與假病毒中和試驗(yàn)高度相關(guān)(Pearson 相關(guān)系數(shù)為0.983)�；�于FO-BLI的濃度測(cè)試因?yàn)槠淇焖�，并且與功能性匹配，有潛力在疾病預(yù)防和疫苗開發(fā)中有著更大的應(yīng)用。
 

圖4. A圖：Octet® 建立血清中和抗體的檢測(cè)方法，將受體ACE2結(jié)合在傳感器上，與HRP偶聯(lián)的不同病毒株的RBD和血清的混合物孵育，然后用底物放大信號(hào)；B圖：這種方法測(cè)試的標(biāo)準(zhǔn)抗體的靈敏度可以達(dá)到20ng/mL；
 

D圖：FO-BLI的檢測(cè)結(jié)果與病毒中和實(shí)驗(yàn)PVNT（IC50）有良好的相關(guān)性

使用生物層干涉技術(shù)，可以快速建立濃度檢測(cè)方法。
 
 
浙江工業(yè)大學(xué)/北大醫(yī)學(xué)院——分子垂釣^[3]

研究者建立了一種基于Octet® 的垂釣技術(shù)，研究人員將GNAS蛋白固化至SA傳感器表面，再與中藥參芪降糖顆粒（SJG）提取物結(jié)合，隨后提取物被解離至洗脫液中，將洗脫液使用UHPLC-Q/TOF-MS/MS分析GNAS垂釣出的小分子。通過(guò)垂釣組與對(duì)照組的相對(duì)含量變化篩選26個(gè)小分子可能與GNAS有相互作用。其中7個(gè)化合物用Octet® 進(jìn)一步驗(yàn)證，并顯示出強(qiáng)的結(jié)合活性，均是SJG的主要成分。其中，黃芪中的Formononetin、高麗參的三七皂苷Ft1和人參皂苷中的甲素人參F2為“君“、地黃中的Catapol為“臣”，五味子中的五味子素A和茜草中的沒(méi)食子酸為“佐使”，符合中醫(yī)配伍“君臣佐使“原則。
 

圖5. Octet® 驗(yàn)證小分子和GNAS的結(jié)合
 
非流路設(shè)計(jì)可以多次垂釣富集豐度低結(jié)合弱的小分子，提高垂釣的成功率。


中國(guó)醫(yī)學(xué)科學(xué)院基礎(chǔ)醫(yī)學(xué)研究所——脂肪肝新靶點(diǎn)^[4]
 
肝細(xì)胞對(duì)預(yù)防非酒精性脂肪性肝�。∟AFLD）和其他慢性代謝性疾病具有重要意義。中國(guó)醫(yī)學(xué)科學(xué)院基礎(chǔ)醫(yī)學(xué)研究所黃波團(tuán)隊(duì)研究發(fā)現(xiàn)，糖原合成使用其中間代謝物尿苷二磷酸葡萄糖（UDPG）來(lái)拮抗脂肪生成，從而引導(dǎo)小鼠和人肝細(xì)胞將葡萄糖碳儲(chǔ)存為糖原。在這項(xiàng)機(jī)制研究中，發(fā)現(xiàn)UDPG通過(guò)SLC35F5轉(zhuǎn)運(yùn)進(jìn)入高爾基體后，誘導(dǎo)site-1蛋白酶S1P的泛素化降解，從而阻斷活性形式SREBP1c的生成，最終抑制脂肪酸的合成。通過(guò)Octet® 非標(biāo)記分子互作系統(tǒng)對(duì)小鼠和人的脂肪酸合成過(guò)程中關(guān)鍵酶（S1P）與UDPG結(jié)合這一重要步驟進(jìn)行了驗(yàn)證，并提供了結(jié)合的直觀證據(jù)。
 

圖6. 通過(guò)SA傳感器固化小鼠（a）和人（b）的S1P蛋白分別與不同濃度UDPG結(jié)合解離曲線

原文大量pulldown以及CO-IP數(shù)據(jù)，但作者仍選擇用Octet® 非標(biāo)記互作系統(tǒng)來(lái)證明S1P與UDPG直接相互作用，可見Direct Binding是趨勢(shì)。
 
 
Octet® 分子互作分析系統(tǒng)的優(yōu)勢(shì)在于

• 非標(biāo)記Direct Binding是趨勢(shì)，結(jié)果更準(zhǔn)確
• 快速測(cè)定親和力，更加定量化地表征分子互作
• 無(wú)洗滌步驟，可測(cè)弱親和力（解離快）
• 寫入了美國(guó)藥典，文章多，認(rèn)可度廣
• 萬(wàn)金油技術(shù)，可以用與檢測(cè)DNA，小分子，蛋白質(zhì)等各種生物分子
• 多種實(shí)驗(yàn)方案，除了直接測(cè)試，還有競(jìng)爭(zhēng)法，垂釣等
• 操作簡(jiǎn)便，耗材及維護(hù)成本低

Octet® 讓分子互作不再?gòu)?fù)雜！祝愿大家可以用賽多利斯神器發(fā)好文章！
    
《生物層干涉技術(shù)小分子應(yīng)用文集》詳細(xì)介紹了生物層干涉（BLI）技術(shù)的原理，及其在小分子領(lǐng)域的多個(gè)應(yīng)用場(chǎng)景，并對(duì)BLI技術(shù)應(yīng)用于小分子研究的20余篇代表性文獻(xiàn)進(jìn)行了整理匯總：內(nèi)容涵蓋從防御機(jī)制到垂釣驗(yàn)證，再到小分子篩選、表征、優(yōu)化等多個(gè)方面，全展示了BLI技術(shù)在小分子研究中的強(qiáng)大潛力和實(shí)踐成果。
  
 
-參考文獻(xiàn)-
[1] Glucose-G protein signaling plays a crucial role in tomato resilience to high temperature and elevated CO2 | Plant Physiology | Oxford Academic (oup.com)
[2] Vaccines | Free Full-Text | Dynamic Profiling and Prediction of Antibody Response to SARS-CoV-2 Booster-Inactivated Vaccines by Microsample-Driven Biosensor and Machine Learning (mdpi.com)
[3] Zhang H, et al. Discovery of drug targets based on traditional Chinese medicine microspheres (TCM-MPs) fishing strategy combined with bio-layer interferometry (BLI) technology. Anal Chim Acta. 2024 May 29;1305:342542.
[4] Hepatic glycogenesis antagonizes lipogenesis by blocking S1P via UDPG.Science,2024

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