Development of the Automatically Celluar Metabolic Analyzer with Ablility of Simultaneous Measurement of Three Metabolic Products

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Project title

Code/計畫編號

NSC102-2221-E019-015-MY2

Translated Name/計畫中文名

可同步偵測三種細胞代謝指標之自動化細胞代謝感測裝置雛形機開發

Project Coordinator/計畫主持人

Shih-Hao Huang

Funding Organization/主管機關

National Science and Technology Council

Department/Unit

Department of Mechanical and Mechatronic Engineering

Website

https://www.grb.gov.tw/search/planDetail?id=3095519

Year

2013

Start date/計畫起

01-08-2013

Expected Completion/計畫迄

01-07-2014

Bugetid/研究經費

815千元

ResearchField/研究領域

醫學工程

本計畫以二年時間開發具有多重藥物注入與可同步偵測三種細胞代謝指標(OCR, OCAR, CDPR)之自動化細胞代謝感測裝置雛形機，可即時同步偵測有氧呼吸O2 (Oxygen Comsumption Rate, OCR)、以及糖酵解作用 H+( Extracelluar Acidification Rate, ECAR)、 CO2 產率 (Carbon Dioxide Production Rate, CDPR)，相較於商用機台，具有低售價、低細胞消耗量、可控制細胞培養微環境與多細胞共培養、與分析多種待測藥物…等優點。本計畫第1年度已完成：(a)細胞培養微陣列晶片、(b)氣動密閉作動裝置、(c)磷光相位差光學檢測系統、(d)完成細胞有氧呼吸 (OCR)細胞代謝指標的量測與多重藥物注入的功能的測試。第2年度已完成：(a)可同步偵測有氧呼吸O2 (OCR)、以及糖酵解作用H+ (ECAR)定量測試，(b)完成透過二種細胞代謝指標(OCR, ECAR)與代謝抑制藥物，快速解析待測細胞或胚胎的基礎代謝率、ATP轉換、氫離子滲漏（膜的完整性）、極限呼吸率，(c)結合光纖微陣列感測達到具有多重藥物注入與可同步偵測二種細胞代謝指標(OCR, OCAR)之自動化細胞代謝感測裝置雛形機開發:含量測程式、人機介面、與自動化裝置。此外，第二年計畫亦著重藉由濃度梯度產生微流體裝置整合至細胞或胚胎培養微陣列晶片中，可依據需求注入多種藥物，形成具有不同濃度梯度之測試藥物，藉由氣動密閉作動裝置具有可氣壓上下致動的閥座密封細胞培養微陣列晶片中的微陣列孔洞，使得每一個微陣列孔洞具有不同濃度之測試藥物，可大幅縮短實驗所需之時程與人為的操作。在應用方面，利用此開發的細胞代謝OCR感測裝置雛形機，應用於評估鹿角菜膠(carrageenan)是否可以有效對抗第二型登革熱病毒(Dengue virus Type II, DV2)的藥物開發與篩選的應用研究上。該裝置能夠即時監控細胞感染後不同階段的耗氧率(OCR)，而耗氧率可反映細胞代謝的活性以評估藥物或抗體抑制病毒的能力，目前本系統已成功偵測定量細胞下的耗氧率並可重複實驗在不同病毒multiplicity of infection (m.o.i)下細胞的氧氣代謝情況，更進一步完成以不同藥物處理病毒方式分析抗病毒之效能。近年來斑馬魚已成為研究脊椎動物胚胎發育的重要模式動物，用斑馬魚來探討藥物作用的機制，如一些心血管及抗癌症等藥物，都已經發現在斑馬魚胚胎及哺乳類系統皆能產生相似的生理及形態的反應。因此，利用此開發的細胞代謝OCR感測裝置雛形機應用於斑馬魚模式動物對致癌毒物與抗癌藥物之快速篩選，已完成利用代謝抑制藥物作用下對胚胎發育耗氧量的定量量測，並針對各時期(3、7、12、24、36、48 hpf, hours post-fertilization)胚胎粒線體與非粒線體呼吸量進行分析。研究結果發現在胚胎發育期過程其胚胎呼吸量隨胚胎成長時間呈現線性變化。在代謝抑制藥物 (oligomycin、FCCP、 sodium azidee)作用下可得知:在胚胎發育初期，主要是進行非粒線體呼吸代謝來產生所需能量，非粒線體呼吸代謝在胚胎早期具有保護細胞分裂不受氧分子傷害使初期胚胎順利發展，提供胚胎初期約70%的能量。ATP合成酶在胚胎後期進行粒線體有氧呼吸作用下為胚胎主要能量來源。總結，應用測試結果驗證本計畫提出的自動化細胞代謝感測裝置雛形機的能夠大量對不同藥物或專一性抗體進行效能評估，進而達到藥物快速篩選、疾病檢測，希望對生物醫學與人類疾病治療有所貢獻。In this project, we have spent two-year efforts to successfully achieve the automatically celluar metabolic analyzer with ability of simultaneous measurement of three metabolic products, i.e., oxygen consumption rate (OCR), extracelluar acidification rate, OCAR), carbon dioxide production Rate (CDPR). The device consists of three main components including: (1) A microwell array for single cell culture. Cells were seeded onto the sensitive dyes inside a microwell. (2) Pneumatically-driven lid to hermetically seal the microwell. This component also contains microfluidic channels for automatic drug injection. (3) A optical measuring system for measurements of cellular OCR, OCAR, and CDPR based on phase-based lifetime detection. This system includes a digital micromirror device (DMD) to project an illumination patterns, which excites the sensitive dyes for measurement of OCR, OCAR, and CDPR. We have achieved that a microfluidic device combined with a light modulation system was developed to assess the inhibitory effect of carrageenan on Dengue virus (DENV) infection via real-time monitoring of cellular oxygen consumption rates (OCRs). Measuring cellular OCRs, which can reflect cellular metabolic activity, enabled us to monitor the process of viral infection in real time and to rapidly determine the antiviral activity of potential drugs/chemical compounds. The time variation of the cellular OCR of single cells that were infected in situ by DENV at different multiplicity of infection (m.o.i.) values was first successfully measured within a microfluidic device. By monitoring cellular OCRs, we could rapidly evaluate the inhibitory effect of carrageenan on DENV infection, obtaining a result within 7 h and showing that carrageenan had strong and effective anti-DENV activity. Our proposed platform enables to perform time-course or dose-response measurements of changes in cellular metabolic activity caused by diseases, chemical compounds, and drugs via monitoring of the cellular OCR, with rapid and real-time detection. Besides, a microfluidic device combined with a light modulation system was developed to detect the oxygen consumption rate (OCR) of a single developing zebrafish embryo via phase-based phosphorescence lifetime detection. The total basal respiration (OCR, in pmol O2/min/embryo) of a single developing zebrafish embryo within a sealed microwell has been successfully measured from the blastula stage (3 hours post-fertilization, 3 hpf) through to hatching (48 hpf). Total basal respiration increased in a linear and reproducible fashion with embryonic ages. By sequentially adding pharmacological inhibitors of bioenergetic pathways, the respiratory measurements of a single zebrafish embryo at key developmental stages enable us to monitor changes in mitochondrial function in vivo that are coordinated with embryonic development. We have successfully measured the metabolic profiles of a single developing zebrafish embryo from 3 hpf to 48 hpf within a microfluidic device. The total basal respiration is partitioned into the non-mitochondrial respiration, mitochondrial respiration, respiration due to ATP turnover, and respiration due to proton leak. The changes of these respirations are correlated with zebrafish embryonic development stages. Overall, our proposed platform provides the potential to study bioenergetic metabolism in a developing organism and for a wide range of biomedical applications that relate mitochondrial physiology and disease. Through our proposed device, scientists can have new insights into obesity, diabetes, cancer, cardiovascular and neurodegenerative diseases.

Keyword(s)

耗氧量
微流體
磷光相位差
oxygen consumption rates
microfluidic device
drug discovery

DSpace CRIS

Development of the Automatically Celluar Metabolic Analyzer with Ablility of Simultaneous Measurement of Three Metabolic Products

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