Introduction

Traditional bulk experiments represent measurements of ensemble averages by which a vast multitude of duplicate systems are probed and average responses are recorded [6, 7]. Tools comparable in size to the basic compartment of life allow precise understanding of biological functions [1-3]. Single-molecule studies have revealed the molecular behaviors hidden in traditional bulk experiments [8, 9]. These studies rely on sophisticated optical strategies that are necessary to limit the detection volume for single-molecule studies [10, 11] and surface immobilization strategies for direct observation. Rondelez et al. proposed a microchamber array to trap individual molecules for enzymatic studies [4]. The activity of single enzymes was measured with conventional microscope setup by confining products of single enzymes into the ultra-small volume (femtoliter) of the microchambers. This simple and powerful platform can be extended for further biological reactions at single-molecule level. Meanwhile, cell-based biological assays traditionally probe cell ensembles thereby completely averaging over relevant individual cell characteristics and even sometimes misleading the interpretation of cell properties [12-15]. Several methods are proposed to array single cells for interpretation of individual behaviors [14, 16-18]. However, all these approaches focus on how cell behavior arisen from the environmental stimuli [13]. No methodology was proposed for direct analysis of individual intracellular materials to study heterogeneity of single cells.

The ultimate goal of the thesis is to realize further biological analyses for precise understanding of biological functions, which is impossible to achieve with pre-existing methodologies, by using the concepts of microchamber array, confined ultrasmall volumes, statistical manipulation and parallelized analysis of individual differences. A highly efficient protein synthesis system is developed in order to achieve for the first time protein synthesis with the smallest amount of DNA possible: a single molecule which is statistically confined. This required also to specially treating the surface of the device for biocompatibility with cell-free protein synthesis. This ultrasensitive system allows to generate a pure protein sample in each microchambers and will be further developed for new generation high-density protein chips. On the other hand, a novel concept of microchamber array platform, parallelized analysis of individual differences, is proposed for direct analysis of individual intracellular materials. By integrating electrostatic functions into the array, single cells are stably trapped at the floor of microwells. All trapped cells are simultaneously lysed inside the sealed microwells that can lead to parallel manipulation and analysis of populations of cells and their lysate. Quantification of intracellular ATP concentrations among a population of single cells demonstrates the feasibility of this new concept. The approach should be of interest for many biological studies about heterogeneity of single cells.

Microchamber array for biochemical reaction at single-molecule level

Protein synthesis with single-DNA molecules has significant advantages in that we can get the pure proteins from individual DNA molecules. Kinpara et al. performed protein synthesis with micro-size chamber array (minimum volume of the chamber was 1 pL) [20]. With this method, a minimum of 10 molecules of DNA per chamber was necessary in order to observe the signal from protein synthesis. The purpose of this section is to develop a novel cell-free protein synthesis platform with sufficient high-performance to achieve protein synthesis from statistically confined single-DNA molecules. The dense compartmentalization of the array would allow the development of high-density protein chips.

PDMS chip having 190 fL microchamber array is fabricated with conventional molding replica technique and coated with a 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer to prevent protein adsorption [21, 22]. DNA plasmids containing Emerald GFP gene was added into commercially available cell-free synthesis system. MPC coating increases protein synthesis efficiency about 3 times. In order to verifying a number of DNA molecules confined within each microchamber, rate of protein synthesis is investigated statistically. Occupancy distribution is fitted with Poisson distribution. The fitted expected value λ=1.38 is comparable with the introduced concentration of the DNA molecules, about 1.25 DNA molecules per chamber. About 40 percent of chambers contain only single DNA molecules.

This is the first report that performs cell-free protein synthesis inside the microchamber array with the minimal quantity of DNA molecules possible as a starting material: a single molecule. A single DNA molecules are trapped into microchamber array statistically. Fine tuning cell-free protein synthesis system and finding the appropriate coating surface reagent for biocompatibility of the system were critical to achieve this result. With this method, pure proteins can be synthesized from individual DNA molecules in an array format, which will allow extreme compaction of the pure protein spots (about 10000 spots in 1mm2 area) for high-density protein chips.

Novel microchamber array platform for study of heterogeneity of single cell

The goal of this section is to realize the concept of parallelized analysis of individual differences with new microchamber array platform. The concept is demonstrated by quantifying individual ATP concentration ([ATP]). To understand fully how ATP levels influence cellular functions and faith, a direct measurement of its concentration at the single-cell level is needed. This requires single-cell trapping, cell lysis and readout of the ATP amount contained in each cell. This is a difficult task and so far no methodology exists for this purpose. We have successfully reach this goal described in the section below.

The device for the parallelized analysis of individual cells contains a large number of arrayed microwells that can lead to parallel manipulation and analysis of populations of cells (50 by 70 microwells inside 0.9mm2 area). The microwell array was fabricated on inter-digitated ITO electrodes with negative-type photoresist. Electric fields are highly localized at the edge of the electrodes at the bottom of the microwell. This set up efficiently induces dielectrophoresis (DEP) force to trap cells into the microwell with stability and electroporation (EP) for lysis. The microwells physically restrict not only the available space for cells during trapping but also prevent the diffusion of intracellular materials after cell lysis.

Microwells were gradually occupied with single cells by DEP trapping and after 3 minutes almost all of them contained single cells. In order to demonstrate confinement of intracellular materials, the microwell array is closed by pressing the PDMS membrane with rounded plastic tips and electric pulses are applied to lyse trapped cells with EP. 100 percent of cells inside the microchambers are lysed at the same time since we can apply homogeneous and high electric fields to each cell. The intracellular materials are tightly confined inside the closed microchamber array. By lysing trapped cells with luciferin-luciferase (LL) reaction reagents inside the microchabmer array, intracellular [ATP] can be quantified after calibration of light emitted (luminescence) from the microchamber array. Individual intracellular [ATP] is obtained from randomly selected 100 microchambers. The mean value of intracellular [ATP] obtained in device, 1.6mM, is comparable with the values obtained in bulk measurements with the luminometer, 1.4mM. Measured individual [ATP] is distributed from 0.8 to 2.2mM. It is impossible to see this individual difference with traditional bulk measurements which probes cell ensembles thereby completely averaging over relevant individual cell characteristics.

In this section, a novel concept of microchamber array is developed to realize parallelized quantification of absolute intracellular [ATP] individually. To do so, cells are trapped into the microchamber array and lysed simultaneously. Tightly enclosed microchambers confine individual intracellular materials for certain periods of time (30 min). Individual intracellular [ATP] is quantified by lysing single cells with LL reaction reagents inside the microchambers, which is impossible to realize with the existing methodologies.

Conclusions

Microchamber array platform is desirable for biology analyses because of its ultrasmall confined volumes, statistical manipulation and parallelized analysis of individual differences. In the thesis, further biological analyses, which are impossible to perform with conventional methodologies, are realized by using concepts of microchamber array platform. Microchamber array platform is improved with anti-absorption coating for cell-free protein synthesis with statistically confined single DNA molecules. This platform will allow the fabrication of high-density RNA and protein chips. Moreover, a novel concept of microchamber array platform is realized by integrating additional electrostatic functions, which provides the essential first steps in parallel analysis of individual intracellular materials. Such integrated microchamber array platform promise to answer biological questions about heterogeneity of single cell, difficult to realize with the exiting methodologies.

本論文は、pL(ピコリットル)やfL(フェムトリットル)といった極めて微小な体積で生物学的な分析を行うことができるマイクロチャンバアレイについて、その用途を広げると同時に新しい概念に基づく分析法を実現するための技術を構築しようとするものである。微小体積を扱う方法には、マイクロチャンバに加えて、脂質二重膜や液滴を使う方法など、いくつかの手法が提案されているが、本論文では、デッドボリュームが小さいこと、微小体積が正確に定義できること、機能を付加できること等の優位性に着目してマイクロチャンバをとりあげ、その新たな機能や用途について検討している。

マイクロチャンバの概念については従来から提案されてきたものであり、たとえばfL(フェムトリットル)オーダーのチャンバについては、これまでに酵素の機能解析を一分子単位で行うことを目的とした研究がなされている。これに対し、本研究では、チャンバ内で直接タンパク質を合成することによって、極めて高密度かつ純度の高いタンパク質アレイを製作するための技術基盤の構築を試みている。また、ある細胞集団について、個々の細胞それぞれについて同時並行的に生物学的解析を行う新しい概念の解析方法を提案し、マイクロチャンバアレイを用いて、これを実現する方法を示すに至っている。

従来の生物学的な実験手法においては、一般に、ある体積に含まれる分子や細胞を集団として取り扱い、その平均値を計測することが行われてきた。これに対して近年では実験技術の進展に伴い、一分子や一細胞単位で解析を行うことが可能になってきている。その一方で、特定の集団に含まれる分子や細胞には、個々にばらつきがあることが指摘されているにもかかわらず、一分子あるいは一細胞解析を多数同時並行して行う方法は確立していない。本論文は、このような問題に対し、一細胞ずつ個別的に、かつ多数の細胞を同時に解析する新しい概念の解析法を提案するものである。

具体的には、マイクロチャンバアレイの各チャンバ内部において生体外タンパク質合成反応を行う方法について検討を加え、特にリン脂質ポリマーの一種であるMPCポリマーによるコーティングを行うことによって、確率的にDNA一分子からEmGFPやYFPなどの蛍光タンパク質を合成することに成功している。これにより、合成されるタンパク質の純度の高さが保証されるとともに、チャンバ間距離が数マイクロメートルほどの高密度アレイの実現が期待できる。

一方、マイクロチャンバアレイに電極構造を組み込むことによって、捕捉した細胞内に含まれる物質の解析を多数同時並行的に行うことができるデバイスを新たに提案している。電極構造を付加することによって、誘電泳動を用いて細胞を能動的に捕捉した上でチャンバを密閉し、電気的に細胞を破砕する機能が実現できる。このデバイスを用いて、個別細胞の内部に含まれるATPの濃度を計測し、同一の細胞集団であっても一定の分布があることを見出している。

本論文の第1章では、研究の目的と背景、ならびに論文の構成について述べており、微小体積の液体を対象とする実験手法を概観したのちに、マイクロチャンバアレイに関わる新しいコンセプトの提示を行っている。

第2章では、マイクロチャンバアレイの機能や特徴を概説するとともに、電気的機能を付加する意義について述べている。

第3章では、すでに提案されているマイクロチャンバアレイの内部で生体外タンパク質合成反応を試み、DNA一分子から蛍光タンパク質が合成可能であることを示し、理論上2500倍以上高密度なタンパク質アレイが実現可能であると考察している。

第4章では、電極集積型マイクロチャンバアレイを提案し、デバイスの全体構造や、チャンバ及び電極のサイズ等、設計パラメータに関する検討結果について述べた後に、実際に細胞を捕捉し、破砕する機能の検証を行っている。

第5章では、新しい解析法のコンセプトに基づき、細胞内のATP濃度計測を一細胞単位で、かつ多数同時並行的に行うことを試みている。特定の細胞集団における細胞毎のATP濃度の分布が計測可能であること、またATP合成を阻害する物質で処理することにより、濃度の平均値のみならず、分布の変化も捉えられることを実証している。

第6章においては、本論文で提案したマイクロチャンバアレイの新たな用途ならびに新しい概念の計測手法の位置づけについて考察を加えた後、第7章において結論と今後の見通しについて述べている。

以上のように、本論文は、マイクロチャンバアレイの応用を、一分子や一細胞のみに焦点を絞る用途だけでなく、それらを多数アレイ化して分析する新しい分析法の概念を提案し、その具体的な実現方法を提示したものである。本論文で創出された技術は、新しい実験手法として広く基礎的な生命科学分野における貢献が期待されるだけでなく、近い将来の一分子あるいは一細胞レベルでの網羅的診断及び解析手法を実現する技術的基盤を与えるものであり、工学に資するところがきわめて大きい。よって本論文は博士(工学)の学位請求論文として合格と認められる。