| Summary: | 博士 === 國立清華大學 === 工程與系統科學系 === 106 === The growing number of challenges in a wide variety of areas, including biomedicine (medical diagnosis, proteomics, drug screening, and toxicity), food production, chemical analysis, and environmental monitoring, has pushed the challenging demands for novel biosensors. Among different kinds of developed biosensing technologies, silicon nanowires field-effect transistor (SiNW-FET)-based biosensors stand out due to their attractive features, such as label-free and real-time detection, ultrahigh sensitivity, exquisite selectivity, multiplexing, and high integration density. The focus of this thesis is the fabrication of multiple-parallel-connected (MPC) SiNW-FETs and their potential applications for sensing biologically relevant ions. Compared with a traditional SiNW-FET, whose conducting channel is composed of only a single or a few SiNWs, an MPC SiNW-FET system possesses remarkably higher detection sensitivity (i.e., larger transconductance) and a better signal-to-noise ratio (SNR) in electrical measurements.
The homeostasis of potassium ion (K+) is essential for the proper function of various cellular activities, such as fluid-electrolyte balance, acid-base balance, apoptosis, neurotransmission, and muscle contraction. To detect K+, an MPC SiNW-FET was modified with K+-specific DNA-aptamers (aptamer/SiNW-FET) for the real-time detection of the K+ efflux from cultured cortical neurons. The aptamer/SiNW-FET showed a wide linear working range of detecting K+ from 10-9 M to 10-6 M and high sensitivity against K+ with association constant of 2.18 ± 0.44 × 106 M-1. Moreover, the aptamer/SiNW-FET showed either less or negligible response to other alkali metal ions. When neurons were placed atop the aptamer/SiNW-FET in a Na+/K+-free buffer, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionicacid (AMPA) stimulated neurons exhibited the escalated K+ efflux in a dose-dependent manner, which is greatly suppressed by 6,7-dinitroquinoxaline-2,3-dione, an AMPA receptor antagonist. In addition, when neurons were stimulated under a normal physiological buffer, the intracellular K+ concentration in the isolated cytosolic fraction was decreased by 75 %. These findings demonstrated that the aptamer/SiNW-FET is sensitive and selective for detecting K+ and the K+ concentration inside and outside of the neurons could be greatly altered to modulate the neuron excitability.
Zinc ion (Zn2+) is vital for various biological activities, such as apoptosis, DNA synthesis, enzyme activities, gene expression, immune system function, and neurotransmission. The objective for designing the Zn2+-specific sensor is to study the link between extracellular Zn2+ concentration ( ) and the amyloid-β (Aβ) fibrilization. Moreover, the exact surrounding neurons under (patho)physiological conditions is not settled. To address these concerns, a SiNW-FET was modified with the Zn2+-sensitive fluorophore, FluoZin-3 (FZ-3/SiNW-FET), to quantify the in real time. The FZ-3/SiNW-FET device has a dissociation constant of ~12 nM against Zn2+ and has a linear working range spanned from 10-11 M to 10-6 M with no appreciable conductance change for other biologially relevant divalent ions (Fe2+, Ca2+, Mn2+, or Mg2+). By placing a coverslip seeded with cultured embryonic cortical neurons atop an FZ-3/SiNW-FET, the AMPA stimulation elevated the to ~110 nM. Blockers against the AMPA receptor or exocytosis have greatly suppressed this elevation, demonstrating that the Zn2+ stored in the synaptic vesicles was the major source responsible for the elevation of . In addition, a SiNW-FET modified with Aβ could bind Zn2+ with a dissociation constant of ~633 nM and respond to the Zn2+ released from AMPA-stimulated neurons. These results showed that can reach a level high enough to bind Aβ and thus raises the possibility that Zn2+-induced Aβ fibrilization could be one of the factors for the onset of Alzheimer’s disease. These extraordinary results demonstrate the almost endless capabilities of SiNW-FET biosensors to explore the biological underpinnings of human diseases.
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