Detecção condutométrica sem contato: uma nova ferramenta para monitoramento de interações biomoleculares em microssistemas analíticos / Contactless conductivity detection: a new tool for monitoring biomolecular interactions on analytical microsystems

AUTOR(ES)
DATA DE PUBLICAÇÃO

2008

RESUMO

The study reported in this thesis shows the application of a capacitively coupled contactless conductivity detection (C4D) for monitoring biomolecular interactions on analytical microsystems. Initially, the analytical performance of the microsystems fabricated in glass, poly(dimethylsiloxane) (PDMS) and polyester-toner (PT) was investigated in order to choose the best material (in terms of fabrication facilities, costs and repeatability) for the biomolecular assays. Among all substrate materials studied, devices fabricated in PT showed suitability for quick experiments, in which the analytical repeatability is not the most important parameter. The devices fabricated in PDMS and sealed against a glass plate presented the best results in terms of repeatability and the analytical performance was similar to that one of glass devices. For this reason, PDMS/glass devices were chosen for showing the goals of this thesis. On the other hand, PT devices were employed to study the geometrical design of the C4D system. The instrumentation for monitoring binding assays was basically composed of two C4D systems, a software written in LabVIEW and a solution pumping system. In order to find the suitable detection cell configuration for this dual-C4D system, designs containing three, four and five electrodes were evaluated on PT devices. The optimal design was composed of three electrodes symmetrically spaced. In this configuration, one electrode is used for applying an excitation sinusoidal wave and the other two for picking up the resulting signal. The dimensions of the electrodes (width and gap) were optimized by chemometric tools. The avidin-biotin complex was used as a binding model for showing the feasibility of the proposed system. For the biomolecular microsystems, electrodes were fabricated on glass surface using photolithographic, sputtering and lift-off processes. Detection electrodes were insulated with a 50-nm silicon oxide layer deposited by plasmaenhanced chemical vapor deposition. The SiO2 layer was functionalized by immersing the cleaned surface in a 3-aminopropyltriethoxy-silane solution in ethanol for 3 h. For biotinylation of the amino-silane layer, 10 ?L of photobiotin dissolved in deionized water (0.1 mg/mL) was dropped on the modified glass surface and exposed to a 365 nm UV radiation at intensity of 10 mW/cm2 for 15 min. Detection was carried out by passing a sinusoidal excitation signal from the function signal generator to the first electrode and picking up the resulting signal at the two receiver electrodes. To reduce electrical noise pickup, all measurements were carried out in a Faraday cage. The data acquisition was obtained in a software written in LabVIEW and the conductivity sensorgrams were recorded in real-time. The microfluidic network was fabricated in PDMS by soft lithography and irreversibly sealed against the electrodes plate. Solutions were handled into microfluidic channels using a peristaltic pump or two syringe pumps. Buffer and avidin-containing solution was injected into the microchannels and conductivity changes were monitored over time. Avidin solutions were allowed to remain in contact with the surface until a stable conductivity had reached equilibrium. Avidin-free buffer solutions were then injected to rinse off non-specifically bound analytes. Two solenoid valves were used to allow an automatic dispensing of the sample/buffer solution into microchannels. The limit of detection found for avidin-biotin system was 75 nmol L-1.

ASSUNTO(S)

biomolecular interactions detecção condutométrica sem contato biossensores microssistemas analíticos analytical microsystems biosensors interações biomoleculares contactless conductivity detection

ACESSO AO ARTIGO

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