The purpose of the study is to develop a high-resolution biosensor to detect biomolecules in a microfluidic system. The microfluidic system is also called 'Lab-on-a-chip' because functions of chemistry laboratory such as reaction, separation and detection can be integrated on a small chip. It can handle an extremely tiny amount of biomolecules in a micro-scale chamber or a channel in much faster reaction time. Therefore it has successfully been applied to biotechnology. However, its detection method depends mainly on optical means; this results in the need for a rather bulky optical system and expensive fluorescent-regents or the difficulty in continuous monitoring for long-time. Electrical detection using biosensors integrated in the microfluidics system can provide a good solution to the problem. I have compared electrochemical sensors and mechanical sensors and chose a mechanical biosensor based on a resonating microcantilever because of its high sensitivity, easy functionalization and simple structure. The microcantilever has high mass sensitive in vacuum and air but not in water due to excess damping. The thesis proposes the biosensor using the microcantilever (80μm x 20μm x 5μm) resonating at air/ liquid interface to realize high sensitivity and easy integration to the microfluidics. The cantilever is fabricated on a SOI wafer and placed on the bottom of a microchannel, which is actuated efficiently by a photothermal laser. Its surface facing to liquid layer is functionalized for label-free detection, while opposite side is exposed to air to improve the resonance characteristics. However, the resonance frequency of the new cantilever is susceptible to a surface tension on the meniscus surface at a micro-slit and liquid pressure changes in the microchannel, besides loaded mass on the surface. The thesis explores methods to eliminate such undesired effects and confirms the performance of the biosensor.

The thesis consists of 8 chapters. Chapter 1 introduces background, purpose and significance of the thesis, and chapter 2 explains a principle and characteristics of the biosensor. Chapter 3, 4 and 5 shows resonance frequency shift by loaded mass (ch. 3), surface tension (ch. 4) and liquid pressure in a microchannel (ch. 5). Chapter 6 is related on a construction of the novel biosensor system summarizing results in ch. 3, 4, and 5. Chapter 7 shows its possibility as a biosensor and the thesis is concluded in chapter 8.

Chapter 2 shows the concept of design and its characteristics. The novel cantilever improves 62% of resonating amplitude, 50% of quality factor (15) and eight times higher signal-to-noise ratio, because liquid damping acts on only one-side and there is no obstacle between laser detector and the cantilever. According to improved results, the novel cantilever is characterized by about 25 times higher frequency resolution with mass sensitivity down to 1.54fg (femto-gram).

Chapter 3 verifies the resonance frequency shift by mass loading on the cantilever. To apply the silicon resonator to a biosensor, the surface should be functionalized using antibodies that are cross-linked to amino-group of APTES (self-assembled monolayer) on SiO2 layer sputtered on the cantilever. The functionalizing process is optimized using three different concentrated fluorescent antibodies, 20, 40, and 80μg/ml, under a fluorescent microscopic observation and verified their each reaction kinetics by the resonance frequency measurement.

Chapter 4 describes that a surface tension changes is susceptible to the resonance frequency theoretically and experimentally. Though the micro-slit keeps liquid with its meniscus surface, it affects on additional elastic property causing changes of the resonance frequency. Different width of micro-slit down to 10μm and several concentration of surfactant (Tween20) are tested to understand the effect on surface tension to the resonance frequency. The narrower width of a micro-slit and lower concentration of surfactant has the higher resonance frequency, which is correlated to theoretical calculation.

In chapter 5, the resonance frequency is stabilized during injecting flow into the microchannel. A peristaltic pump and push/ pull type of syringe pump are tested to balance liquid pressure between an inlet and outlet of the microchannel using microscopic method and resonance frequency measurement. Finally, we optimize fluidic resistance adjusting length and diameter of PTFE tube linked to both side of a microchannel, to achieve an imaginary ground level of liquid pressure at the cantilever.

Chapter 6 suggests method to measure the loaded mass using the novel biosensor and to reduce side effect introduced in chapter 4 and 5. In this regard, chapter 7 shows the cantilever as a biosensor to detect low concentration of insulin (6.3ng/ml, highest concentration level in vivo) and other applications.

In chapter 8, significance and prospect of the novel sensor is explained to conclude the thesis. Moreover, I want to introduce new type of sensor to measure the surface tension in a microfluidic system, which was a drawback for a mass sensitive biosensor. In addition, I prospect that the cantilever resonating at air/ liquid can apply to various purposes such as AFM to work in liquid.

本論文は「A high resolution biosensor using microcantilever resonating at air/ liquid interface (気液界面で共振するカンチレバーを用いた高感度バイオセンサー)」と題し英文で書かれており8章と付録からなる。マイクロ流体システムに集積化して細胞などから分泌される生体分子を実時間で測定するセンサを目指して、気液界面で振動するカンチレバーの液体側の表面で対象分子を捕獲し、共振周波数の変化で検出するデバイスを扱っている。

第1章は序論であり、本研究の研究背景を述べている。マイクロ流体システム中での生体分子濃度の変化を、高感度実時間で検出するセンサが必要であることを述べ、これまでの研究を総括してその問題点を明らかにするとともに、気液界面で共振振動するカンチレバーに基づくデバイスを解決策として提案し、本論文の目的と研究の意義を提示している。

第2章では、提案したカンチレバーの共振特性を変化させる要因について、表面への標的分子捕獲に伴う質量変化以外に、副次的に作用する要因を検討し、その影響を低減する設計指針を述べている。作製プロセスを考案し、実際に気液界面で共振振動するカンチレバーの特性を測って、液中で動作させた場合に比べて性能が向上することを確認した。

第3章では、センサの動作原理である質量変化に伴うカンチレバーの共振周波数変化について、理論と実験の両面から詳細に検討している。さらに、標的分子の選択的捕獲に用いる抗体の表面修飾について、共振周波数変化とカンチレバー表面の蛍光強度変化とを対応させて確認を行い、両者がよく一致することを示した。

第4章では、カンチレバーの周囲で液体の漏れを防ぐ、メニスカスの影響を論じている。メニスカスのサイズや液体の表面張力が共振周波数に及ぼす効果を、理論と実験の両面から検討し、スリットの最適寸法として6μmを得た。また、今回の測定対象とする液体では表面張力の影響も無視できることを確認した。

第5章では、マイクロ流路内の圧力が共振周波数に与える影響を検討し、チップ外の流路とポンプの最適化でそれを無視できる範囲に低減できることを示した。

第6章では、振動検出系と信号処理系を含めたセンサシステムの構成を示している。

第7章は提案したデバイスのバイオセンサー応用であり、インシュリンを対象に検出を試みた結果、濃度340 ppm (2 ng/ml)まで検出可能であることが示された。

第8章は結論であり、本論文で得た成果をまとめ、マイクロ流体システム内の低濃度の生体分子を検出可能なセンサにするために、今後の研究で明らかにすべき課題を述べている。

以上これを要するに、本論文は、マイクロ流体システムに集積化可能で、生体分子の濃度変化を実時間かつ高感度に検出可能なセンサを得るために、気液界面で振動するカンチレバーへの標的物質の結合に伴い共振周波数が変化するデバイスを提案し、その設計製作法と副次的作用の共振周波数への影響の低減法を示すとともに、実際に低濃度の生体物質を検出可能なことを示したもので、電気工学に貢献するところが少なくない。

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。