了解單分子層面復(fù)雜的生物過程是預(yù)防和**癌癥及其他**的關(guān)鍵，單分子聲力譜儀作為即時(shí)可用的單分子檢測(cè)儀器，實(shí)現(xiàn)了活體測(cè)量技術(shù)的突破以及分子水平上相互作用的成像。
AFS技術(shù)
聲學(xué)力譜（AFS），是一種用于高度并行操縱單分子的方法。AFS單分子聲力譜儀是**款商業(yè)化的獨(dú)立儀器，包括一個(gè)易于操作和測(cè)量的智能化專業(yè)顯微鏡。AFS技術(shù)是采用功能強(qiáng)大而成本低廉的芯片裝置，能夠高精度施加力同時(shí)對(duì)數(shù)千個(gè)的生物分子進(jìn)行檢測(cè)。
原理
AFS技術(shù)由玻璃微流控芯片和透明的壓電變換器構(gòu)成來產(chǎn)生共振聲波。AFS芯片上的聲波（超聲波）可同時(shí)并行地在數(shù)千個(gè)生物分子（如DNA，RNA或蛋白質(zhì)）施加亞皮牛頓（pn）到數(shù)百皮牛頓（pn）的力，同時(shí)具有亞毫秒的響應(yīng)時(shí)間及固有穩(wěn)定性。
AFS作為一個(gè)理想的工具在核酸蛋白、**蛋白和抗原抗體層面上破譯分子間相互作用的詳情。AFS技術(shù)能夠使科學(xué)家研究蛋白質(zhì)的結(jié)構(gòu)與功能相互關(guān)系，新的生物機(jī)制和細(xì)胞力學(xué)，也可用于研究和識(shí)別自由能圖，動(dòng)力學(xué)速率和在反應(yīng)過程中的中間態(tài)。
AFS作為一個(gè)高度集成化的系統(tǒng)，包括倒置光學(xué)顯微鏡與軟件、工作站及相關(guān)電子器件，用戶界面友好，操控方便。
主要技術(shù)特點(diǎn)
AFS的多功能設(shè)計(jì)
AFS作為一個(gè)高度靈活的單分子檢測(cè)儀器，其顯微鏡平臺(tái)給用戶定制和個(gè)性化需求提供了多重選擇。
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通過在200毫米、150毫米、75毫米和50毫米不同鏡筒透鏡安裝距離來改變光學(xué)放大倍數(shù)
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可拆卸側(cè)板便于對(duì)光學(xué)路徑的簡(jiǎn)單調(diào)整
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標(biāo)準(zhǔn)25毫米網(wǎng)格便于外部安裝射流功能的組件（如注射泵）
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特殊設(shè)計(jì)能夠快速簡(jiǎn)單連接外部放大器
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專用芯片底座能夠使射流和電子連接**地接到AFS芯片、簡(jiǎn)單快速地固定或重新固定
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滑動(dòng)裝置用于準(zhǔn)確定位和鎖定物鏡上的樣品
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配裝夾具以確保工作臺(tái)上流體組件的固定，以便實(shí)現(xiàn)多種流動(dòng)形式，如重力或注射器驅(qū)動(dòng)的流動(dòng)
AFS的應(yīng)用
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動(dòng)態(tài)力譜
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恒力測(cè)試
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力-距離曲線
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生物聚合物力學(xué)
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鍵斷裂
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微流變學(xué)
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細(xì)胞力學(xué)
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水凝膠力學(xué)性能
AFS規(guī)格參數(shù)
AFS是一個(gè)真正的測(cè)量單分子的工具，包括一個(gè)專用的倒置光學(xué)顯微鏡，射流模塊和電子組件，集成在一個(gè)小箱體內(nèi)（300mm×375mm×200mm）。
AFS專用顯微鏡
單色LED照明
CFI消色差物鏡（可升級(jí)）
電動(dòng)Z軸物鏡臺(tái)（可升級(jí)）
USB 3.0 CMOS攝像頭（可升級(jí)）
手動(dòng)XY樣品臺(tái)
信號(hào)發(fā)生器（可升級(jí)）
AFS芯片
AFS系統(tǒng)包含三種AFS芯片，每個(gè)AFS芯片都會(huì)提前校準(zhǔn)并且給出芯片的共振頻率
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加載速率范圍：10-4 PN / s到103 PN / s
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AFS芯片尺寸：45 mm x 15 mm x 1.275 mm
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*大加載力：大于200 pN（4.5μm的聚苯乙烯微球，采用電壓放大器）
**的AFS芯片設(shè)計(jì)
我們將不斷地改進(jìn)AFS芯片。與AFS用戶密切合作，提高芯片的性能、功能和可用性。
AFS工作站及軟件（可升級(jí)）
AFS包括labview軟件包和一個(gè)強(qiáng)大的工作站。AFS軟件能夠?qū)崟r(shí)以納米級(jí)分辨率并行測(cè)試數(shù)以千計(jì)的微球。
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在25赫茲實(shí)時(shí)并行三維跟蹤60-300個(gè)微球
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在25赫茲實(shí)時(shí)并行二維跟蹤300-1500個(gè)微球
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在25赫茲實(shí)時(shí)跟蹤（X,Y）位置精度為2納米
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在25赫茲實(shí)時(shí)跟蹤（Z）位置精度為5納米
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通過參照微球的差值跟蹤進(jìn)行（Z）方向漂移較正
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自動(dòng)對(duì)照表校準(zhǔn)
AFS放大器（可選）
AFS的功能可通過一個(gè)電壓放大器擴(kuò)展。電壓增加，應(yīng)用與生物分子的*大加載力相應(yīng)增加。
放大器可以很容易通過后面板連接到AFS。AFS計(jì)算機(jī)已經(jīng)預(yù)裝所有相關(guān)的軟件和驅(qū)動(dòng)程序以適用于電壓放大器。
The essence of the AFS? technology lies in a glass microfluidic chip with two piezo elements that generate resonant acoustic waves (ultrasound). These resonant acoustic waves are used to exert forces on micrometer sized particles with a different density than the surrounding medium (e.g. a polystyrene microsphere or a living cell).
In the compact AFS? chip the acoustic waves are employed to exert forces from sub-pN to hundreds of pNs on thousands of biomolecules (such as DNA, RNA or proteins) or cells in parallel, with sub-millisecond response time and inherent stability.
Further reading:
Sitters et al. ‘Acoustic Force Spectroscopy’, Nature Methods (2015)
AFS? enables scientists to investigate the mechanical properties of biological systems by probing them via synthetic microspheres or by directly probing the system itself (e.g. a cell). Due to the planar nature of the acoustic waves, experiments can be performed in a highly parallel fashion. This makes AFS? an ideal tool to study a wide variety of biological systems, including, but not limited to: Antibody – antigen affinity, protein unfolding, cell manipulation, microrheology, lipid membrane interactions and DNA-protein interactions.
Highly multiplexed antibody-antigen affinity characterization
Statistical analysis and high data throughput are of crucial importance to characterize the strength of antibody-antigen interactions. Due to the massive multiplexing capabilities of AFS? a large number of bonds can be probed in parallel, greatly reducing measurement times.
Measuring DNA extension curves in parallel
AFS? has the powerful and unique capability of performing single-molecule measurements in a highly parallel fashion. The importance of parallelity lies in the fact that many independent measurements are often needed to distinguish heterogeneous behavior and rare events from intrinsic stochasticity caused by thermal fluctuations.
In this example we perform a single-molecule experiment with 20 DNA molecules (8.4 kbps in length) in parallel. The ability of multiplexing mechanical measurements at the single-molecule level can be readily applied to different assays (e.g. RNA or proteins).
Measuring RNA polymerase activity
With its inherent multiplexing capabilities and force stability AFS? is the ideal tool to study rare DNA processing events on sub-second to hour timescales. RNA polymerase (RNAP) activity is typically of complex nature: Stochastically occurring elongation is frequently interrupted by pausing events of different nature.
Courtesy of Anatoly Arseniev, Georgii Pobegalov & Mikhail Khodorkovskii at Peter the Great St. Petersburg Polytechnic University, Russia
Acoustic Forces
Range from 0 to 40 pN (4.5 μm diameter polystyrene microspheres)*
Static and dynamic force measurements with 14 bit resolution
Loading rates ranging from 10-4 pN/s to 103 pN/s
* Upgradable to 500 pN using external amplifier, includes bypass feature
Imaging
Monochromatic LED Illumination
CFI Achromat Air Objective (interchangeable)
CMOS Camera (upgradable)
Motorized Z Objective Stage
Manual XY Sample Stage
Workstation & Software
Powerful workstation including LabView-based open-source software capable of:
? Constant forces
? Dynamic force ramps
? User-defined time varying force profiles
? Real-time parallel 2D tracking of 1500 microspheres at 25 Hz
? Real-time parallel 3D tracking of 300 microspheres at 25 Hz
? Real-time position accuracy of 2 nm (XY) and 5 nm (Z) at 25 Hz