Development of Aptasensors for Steroidal Hormones
Authors:   , Alsager Omar Ahmed 
Published: December 2021

<p>Aptamers are synthetic nucleic acid single stranded (ss)DNAs or RNAs that can bind with high affinity and specificity to a broad range of targets, including proteins and low molecular weight molecules. This work presents the design, development and implementation of novel aptamer based sensors (aptasensors) for the detection of a target of environmental and medical significance - 17-β estradiol (E2). By combining a previously isolated E2 binding 75-mer ssDNA aptamer with a variety of different signal transducers, E2 was successfully detected and quantified below the environmental and biological relevant concentrations. By applying the same aptamer to different sensor formats, the advantages and disadvantages of each signal transduction mechanism were compared.  Target-induced conformational switch within an aptamer molecule can be transduced via labelling different sections of the aptamer with pairs of fluorescent dyes or with a redox probe, however those strategies require detailed knowledge of specific aptamer conformations and target interaction sites. Herein, a label free method is developed - size based aptasensor described in Chapter 2. The new method only depends on the general property that small molecule binding aptamers adopt a more compact folded structure when they bind to their target. Dynamic light scattering (DLS) and tunable resistive pulse sensing (TRPS) were used to probe recognition events between E2 and aptamers conjugated to carboxylated polystyrene nanoparticles (NPs). Upon E2 recognition, a distinct reduction in size and a less negative surface potential of the conjugated particles were observed, which can be correlated to the concentration of E2 in the lower nanomolar range (as low as 5 nM).  On-site monitoring of E2 requires rapid and sensitive screening methods with minimal instrumentation. Previously, gold nanoparticles (AuNPs) were exploited in the construction of colorimetric aptasensors for different targets. Aggregation assays produce colorimetric signals observed by naked-eye when target-bound aptamers dissociate from AuNP surfaces, triggering aggregation. However, it is unknown how the length of aptamer sequences affects their dissociation from AuNP surfaces and subsequent aggregation. Chapter 3 demonstrates the benefit of editing aptamer sequences with specific regard to the way signals are transduced in AuNP based colorimetric assays. The 20 flanking nucleotides to the 35-mer inner core of the parent 75-mer aptamer were eliminated. The 35-mer aptamer has a lower dissociation constant KD (14 nM vs. 25 nM), improved discrimination against other steroidal molecules and greatly improve the sensitivity for E2 detection from 5 nM to 200 pM. In fact, this simple strategy enabled facile detection of E2 in urine at 5 nM, approaching levels of biological relevance.  There is a pressing demand for methods with accurate and rapid performance to detect and quantify E2, at levels comparable or even below the biological concentrations to eliminate pre-concentration and sample purification process. Existing electrochemical aptasensors feature DNA probes covalently tethered to various surfaces including gold and conducting polymer electrode. An electrochemical impedance spectroscopy (EIS) based sensor was created using nanoporous conducting polymer electrodes functionalized with the 75-mer aptamer. The one fM detection limit found is one order of magnitude lower than the recorded biological level. As a novel alternative approach, sensing electrodes were also created via the non-specific adsorption of the 35-mer onto Au and Au nanoparticle electrodes. This approach, described in Chapter 4, led to the same level of detection as the conducting polymer aptasensor, but via a mechanism with similarities to the colorimetric sensor. Non-specific adsorption of aptamers to Au was found to play additional favourable roles including self-passivation and stabilization of Au nanoparticle based electrodes. Sensing with this format might remove the need for laborious surface passivation with alkylthiol molecules encountered with the conventional covalent attachment of the DNAs through thiol-linkers.  In general, the reported aptasensors provide efficient means to detect the steroidal molecule E2 as well as advance the understanding of aptasensors by comparing the performance of the same aptamer in various sensing platforms. Long aptamers sequences appeared to be more efficient in signal transduction when specific surface tethering is involved, as in the size-based assay, and the electrochemical assay with aptamers covalently tethered to the electrode. Here, the non-binding flanking nucleotides, i.e. nucleotides adjacent to the target binding pocket, appeared to amplify the sensing signals. However, shorter truncated sequences showed better performance when signal generation depends on surface dissociation of non-specifically adsorbed aptamer sequences, as in the colorimetric assay, and the electrochemical sensor constructed from adsorbed aptamers. These insights can be readily applied to aptasensors for the growing range of targets.</p>