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| Home > Publications database > Label-free detection of biomolecules by a field-effect transistor microarray biosensor with bio-functionalized gate surfaces |
| Dissertation / PhD Thesis/Book | PreJuSER-52661 |
2006
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag
Jülich
Please use a persistent id in citations: http://hdl.handle.net/2128/2597
Report No.: Juel-4227
Abstract: The aim of this work was to bio-functionalize the SiO$_{2}$ gate of ion-sensitive field-effect transistor (ISFET) to covalent bind DNA sequences via a series of chemical reactions. On the modified surface, the detection of the DNA hybridization and in particular single nucleotide polymorphism (SNP) detection was achieved. Furthermore, to explore the working principle of the field-effect detection, polyelectrolyte multilayers (PEMs) buildup and recognition reaction of biotin and streptavidin were also analyzed with the ISFET biosensors. Up to now, labeling detection systems dominated the DNA detection bioassays. Here either the probe or the catcher DNA are labeled with fluorescence-, radioactive- or enzymatic-labels. In recent years, many new approaches for signal generation that avoid labeling have been reported. Based on the demand of a fast, cheap, highly sensitive, label-free and direct electronic readout, ISFET biosensor are ideally suited. In my work, I started with bio-functionalization of SiO$_{2}$ control substrates, leading to a covalent binding of the probe biomolecules, i.e. single stranded DNA and biotin. In this surface modification process, a step-by-step protocol was setup, firstly cleaning/activation with MeOH/HCl for 30 mins generated the highest -OH bond density. Secondly, silanization with 3-aminopropyltriethoxysilane (APTES) in gas phase left a thinner, more homogeneous silane layer. Crosslinking with succinic anhydride solution for 2 hours controlled the following DNA immobilization. The DNA position-specific microarrays for hybridization detection were fabricated by a custom-made aligned microspotter system. The DNA-DNA hybridization has higher efficiency in higher ionic strength solution (SSC buffer solution) and higher selectivity for SNP detection in lower ionic strength TE buffer solutions. All bioassay protocols were successfully transferred to the fully encapsulated ISFET devices and were “mild” enough for the encapsulation material of the chips. In the direct current (DC) readout, ISFETs as a potentiometric biosensors monitor the change of the solid/liquid interface potential caused by the attachment of biomolecules. To investigate the detection principle of the ISFETs in general, polyelectrolyte multilayers (PEMs) were used as a model system. During the layer-by-layer buildup, the thickness of the PEMs and the outer charge of the layer system are changing, which can be recorded by the ISFETs. The recorded results confirmed the surface charge sensitivity of the ISFETs. With increasing distance away from the surface, the charge detection of the ISFETs decreased exponentially. To reduce the long-term drift of the FET readout and exclude possible side effects from temperature, pH changes, and buffer solutions etc., a reference chip or a reference channel were used to perform differential readout detections. The DNA immobilization between covalent binding and electrostatic adsorption caused a gate voltage change of 4mV. It confirmed that the covalent binding of DNA immobilization introduced a higher surface coverage compared to the electrostatic adsorption. The differential DNA hybridization between a perfect matched (PM) and a fully mismatched (FMM) probe sequence also gave a clear signal. However, the SNP is barely distinguishable in DC readout as potential changes. Therefore, ISFETs as impedimetric biosensors were developed to record the impedance change at the gate input in an alternating current (AC) mode. In the optimized detection solution, a reliable recording of ex-situ SNP was achieved and in-situ detection showed DNA hybridization kinetically. In the first proof of principle experiment, the adding of gold-nanoparticle (AuNPs) to the target DNA did not enhance the selectivity of the SNPs detection, which confirmed the valid charge sensitive of ISFET in electrical double layer. Furthermore, in-situ and ex-situ measurements of biotin/streptavidin binding revealed distinct effects on the transfer function curves.
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