The development of electrochemical aptamer biosensors based on polyamidoamine dendrimer-streptavidin supramolecular architecture

M.Tech. (Chemistry)Abstract: This dissertation reports on the development of an electrochemical aptasensor for hydrogen peroxide and thrombin based on a novel polyamidoamine dendrimer-streptavidin supramolecular architecture. Polyamidoamine dendrimer (PAMAM) was electrodeposited on a glassy carbon electrode using cyclic voltammetry at a potential window of -200 mV to +1100 mV at scan rate 50 mV s-1. The electrode was labelled GCE/PAMAM. A 50 μL volume of 100 μg/mL streptavidin (strept) was drop coated on the GCE/PAMAM electrode for 2 h. The electrode was labelled GCE/PAMAM/Strept and also referred to as the supramolecular platform. The platform was characterised using various techniques such as voltammetry, electrochemical impedance spectroscopy, electron microscopy etc. The supramolecular architecture (GCE, PAMAM)/ strept) was conjugated to an enzyme called horseradish peroxidase (HRP) to make the biosensor GCE/PAMAM/Strept-HRP. The biosensor i.e. GCE/PAMAM/Strept-HRP was used for the detection of hydrogen peroxide. The GCE/PAMAM/Strept-HRP biosensor was studied using electrochemical techniques (CV, SWV, EIS and chronoamperometry) and microscopic techniques (HR-SEM). The platform was conducting and electroactive exhibiting reversible electrochemistry (E°′ = 450 mV and Ipa/Ipc ≈ 1 in Fe(CN)63-/4-). The second part of the work involves the detection of thrombin using a GCE/PAMAM/Strept/TBA modified electrode. Biotinylated aptamer probe was immobilised on the platform through biotin-streptavidin interaction. The aptasensor was monitored using CV, SWV and EIS. The selectivity of the GCE/PAMAM/Strept/TBA based aptasensor was evaluated using interfering proteins of BSA, lysozyme, and haemoglobin that also co-exist with thrombin in the blood. The relative response obtained shows insignificant effect for the interfering proteins with BSA and haemoglobin having a relative response of 9.2% and 7.6% respectively. This confirms the excellent specificity of the proposed aptasensor

An Interfacial Affinity Interaction-Based Method for Detecting HOTAIR lncRNA in Cancer Plasma Samples

Long non-coding RNA Homeobox transcript antisense intergenic RNA (HOTAIR) is recognized as a participant in different processes of normal cell development. Aberrant overexpression of HOTAIR contributes to the initiation, growth, and invasiveness of ovarian cancer. Using the affinity interaction of target HOTAIR lncRNA sequences towards a screen-printed gold electrode (SPE-Au), herein we report on a novel, rapid and simple method to detect HOTAIR sequences. HOTAIR lncRNA sequences were first extracted from ovarian cancer cell lines and patient plasma samples and were magnetically captured and purified by complimentary capture probe-functionalized magnetic beads. Isolated target HOTAIR lncRNAs were directly adsorbed onto unmodified screen-printed gold electrodes (SPE-Au) for direct quantification with [Fe(CN)6]3−/4− redox couple. Our assay achieved a linear dynamic range of 100 nM and 1 pM for detecting pre-clinical model HOTAIR lncRNA samples (%RSD ≤ 5%, for n = 3) and was highly specific, showing clear distinction between HOTAIR lncRNA targets and non-specific miR-891 and miR-486 (100 nM) (%RSD ≤ 5%, for n = 3). The method was tested using ovarian cancer-specific cell lines (SKOV3 and OVCAR3) and mesothelial cell line (MeT-5A)-derived lncRNAs. The analytical performance of our method was validated using RT-qPCR. Finally, the method was tested using clinical samples from ovarian cancer patients and the resulting electrochemical responses show a clear distinction between the ovarian carcinoma and benign samples

An amplification-free method for the detection of HOTAIR long non-coding RNA

The discovery of large transcripts of long RNAs that have limited protein coding capacity, known as long non-coding RNAs (lncRNAs) present new concepts on RNA-mediated gene regulation. Increasing evidence suggests that large intervening ncRNAs regulate key pathways in cancer genesis and metastasis. Among the most characterized lncRNAs, homeobox (HOX) transcript antisense intergenic RNA (HOTAIR) acts as an oncogenic molecule in different cancer cells, and thus its expression level serves as a potential biomarker for diagnostic and therapeutic purposes in several human cancers, such as breast, prostate, liver and ovarian cancer. This paper reports a simple and sensitive sensor platform for the detection of HOTAIR. Extracted HOTAIR sequences from ovarian cancer cells and plasma samples derived from ovarian cancer patients were magnetically isolated and purified, followed by a sandwich hybridization event at a screen-printed gold electrode. This event was monitored by amperometry using the hydrogen peroxide/horseradish peroxidase/hydroquinone (HO/HRP/HQ) system. The catalytic enhancement of the amperometric signal enabled our assay to achieve a detection limit of 1.0 fM with a good inter-assay reproducibility (relative standard deviation (%RSD) = < 5.0%, n = 3). The method was used for the analysis of specific HOTAIR in cell line and a small cohort of plasma samples derived from patients with ovarian cancer. The analytical performance of the method was also demonstrated using a standard RT-qPCR. We believe that the proof of the concept assay demonstrated here could be a cost-effective alternative platform for screening cancer-related lncRNAs in routine clinical settings