MicroRNAs (miRNA) are a class of biomarkers whose relevance in oncology is increasing enormously, especially for cancers with high degree of invasiveness such as glioblastoma and lung cancer. Detection of miRNA circulating in blood appears a potential strategy for early diagnosis and patient monitoring, besides providing insights into the cellular path of disease. In the present PhD thesis, a biosensor based on Electrolyte-Gated Organic Field-Effect Transistor (EGOFET) is presented. A dual Gate/common Channel architecture was designed and prototyped for quantifying the concentration of one of the strands of miRNA-21 in saline buffer. The organic electronic device allows one to measure the differential response of the gate functionalized with oligonucleotide probe, with respect to a reference electrode functionalized with thiol based self-assembled monolayer. Both electrodes were immersed in the electrolyte above the transistor Channel. Hybridization with oligonucleotide in the picomolar regime induces a sizable reduction of the current flowing through the transistor Channel. The device signal is reported at various Gate voltages, showing maximum sensitivity at low Gate voltages, with a limit of detection as low as 35 pM. I modeled the dose curves with an analytical function derived from a thermodynamic model of the reaction equilibria relevant in the experiment and for the device configuration. I have also shown that the experimental dependence on analyte concentration emerges from the interplay of the different equilibria of reactions occurring on the surface of the Gate electrode and in solution. The binding free energy characteristic of the hybridization on the device surface is found to be approximately 20% lower with respect to the reaction in solution, hinting to partially inhibiting effect of the surface and presence of competing reactions. Impedance spectroscopy and surface plasmon resonance (SPR) performed on the same oligonucleotide pair were correlated to the electronic current transduced by the EGOFET and confirmed the selectivity of the biorecognition probe covalently bound on the gold surface. The aggregation of α-synuclein is a critical event in the pathogenesis of neurological diseases, such as Parkinson or Alzheimer. An EGOFET device was integrated with a microfluidics fabricated with 3D printing and replica molding allowing to detect amounts of α-synuclein in the range from 2.5 pM to 250 nM. The lower limit of detection (LOD) measures the attainable performance of the integrated device as a tool for prognostics and diagnostics. In the prototype device, the Gate electrode is the effective sensing element as it is functionalized with anti-(α-synuclein) antibodies using two alternative strategies: i) an amino-terminated self-assembled monolayer activated by glutaraldehyde, and ii) the His-tagged recombinant protein G. In both approaches, comparable sensitivity values were achieved, featuring very low LOD values at the pM level. The microfluidics engineering is central to achieve a controlled functionalization of the gate electrode and avoid contamination or physisorption in the transistor Channel where the organic semiconductor is patterned. The demonstrated sensing architecture, being a disposable stand-alone chip, can be operated as a label-free tool to explore in-vitro protein aggregation that takes place during the progression of neurodegenerative illnesses.
ORGANIC ELECTRONIC TRANSISTORS AND MICROFLUIDICS FOR CANCER BIOMARKER SENSING / Selvaraj, Meenu. - (2022 Jan 13).
ORGANIC ELECTRONIC TRANSISTORS AND MICROFLUIDICS FOR CANCER BIOMARKER SENSING
SELVARAJ, MEENU
2022-01-13
Abstract
MicroRNAs (miRNA) are a class of biomarkers whose relevance in oncology is increasing enormously, especially for cancers with high degree of invasiveness such as glioblastoma and lung cancer. Detection of miRNA circulating in blood appears a potential strategy for early diagnosis and patient monitoring, besides providing insights into the cellular path of disease. In the present PhD thesis, a biosensor based on Electrolyte-Gated Organic Field-Effect Transistor (EGOFET) is presented. A dual Gate/common Channel architecture was designed and prototyped for quantifying the concentration of one of the strands of miRNA-21 in saline buffer. The organic electronic device allows one to measure the differential response of the gate functionalized with oligonucleotide probe, with respect to a reference electrode functionalized with thiol based self-assembled monolayer. Both electrodes were immersed in the electrolyte above the transistor Channel. Hybridization with oligonucleotide in the picomolar regime induces a sizable reduction of the current flowing through the transistor Channel. The device signal is reported at various Gate voltages, showing maximum sensitivity at low Gate voltages, with a limit of detection as low as 35 pM. I modeled the dose curves with an analytical function derived from a thermodynamic model of the reaction equilibria relevant in the experiment and for the device configuration. I have also shown that the experimental dependence on analyte concentration emerges from the interplay of the different equilibria of reactions occurring on the surface of the Gate electrode and in solution. The binding free energy characteristic of the hybridization on the device surface is found to be approximately 20% lower with respect to the reaction in solution, hinting to partially inhibiting effect of the surface and presence of competing reactions. Impedance spectroscopy and surface plasmon resonance (SPR) performed on the same oligonucleotide pair were correlated to the electronic current transduced by the EGOFET and confirmed the selectivity of the biorecognition probe covalently bound on the gold surface. The aggregation of α-synuclein is a critical event in the pathogenesis of neurological diseases, such as Parkinson or Alzheimer. An EGOFET device was integrated with a microfluidics fabricated with 3D printing and replica molding allowing to detect amounts of α-synuclein in the range from 2.5 pM to 250 nM. The lower limit of detection (LOD) measures the attainable performance of the integrated device as a tool for prognostics and diagnostics. In the prototype device, the Gate electrode is the effective sensing element as it is functionalized with anti-(α-synuclein) antibodies using two alternative strategies: i) an amino-terminated self-assembled monolayer activated by glutaraldehyde, and ii) the His-tagged recombinant protein G. In both approaches, comparable sensitivity values were achieved, featuring very low LOD values at the pM level. The microfluidics engineering is central to achieve a controlled functionalization of the gate electrode and avoid contamination or physisorption in the transistor Channel where the organic semiconductor is patterned. The demonstrated sensing architecture, being a disposable stand-alone chip, can be operated as a label-free tool to explore in-vitro protein aggregation that takes place during the progression of neurodegenerative illnesses.File | Dimensione | Formato | |
---|---|---|---|
Tesi di dottorato - SELVARAJ MEENU 20211129165458.pdf
accesso aperto
Tipologia:
Tesi di dottorato
Licenza:
PUBBLICO - Pubblico con Copyright
Dimensione
7.99 MB
Formato
Adobe PDF
|
7.99 MB | Adobe PDF | Visualizza/Apri |
I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.