###### Abstract
Electrochemical assays have the advantage of making use of low-cost instrumentation, ease of implementation, and offer good prospects for high sensitivity and selectivity. Electrochemical assays can be developed for a very wide range of biomarkers by using the appropriate recognition molecules and designing an assay resulting in a current or charge response that is related to biomarker concentration. As an alternative to antibodies, aptamers possess several appealing features for application in biosensors, such as their stability, high specificity and sensitivity, ease of re-use and small size compared to other agents such as antibodies [8]. Whereas antibody probes can be subject to irreversible denaturation, aptamers can be reliably regenerated. The desired aptamer is developed using the systematic evolution of ligands by the exponential enrichment (SELEX) process. SELEX starts with an initial library of oligonucleotides and through a number of cycles identifies an oligonucleotide that has a strong affinity for the target. The oligonucleotidestides in the initial pool consist of a random internal region of base pairs and constant flanking regions of typically 15 to 20 base pairs long needed for the polymerase chain reaction (PCR) amplification step [9]. A random region of 20 base pairs would present the possibility for 420 = 1.1 \(\times\) 1012 possible sequences. The number of nucleotides in the random region can be as high as 60. The initial pool does not contain all of the possibilities, especially for longer lengths of the random region [10]. A SELEX cycle consists of allowing the target to bind to oligonucleotides in the pool, followed by a selection of those that have bound to the target, and their amplification in advance of the next cycle. A typical process when the target is a protein involves allowing the protein to mix with the oligonucleotide pool in solution so that the sequences with higher affinities can bind to the protein. Nitrocellulose membranes are frequently used to capture the oligonucleotide-protein complexes and pass through the free oligonucleotides [11]. The proteins can also be captured on activated sepharose and the unbound oligonucleotides removed [12]. The oligonucleotides sequences that are bound to the captured proteins are released so that they can be subjected to amplification [13]. While aptamers are identified from pools of ssDNA oligonucleotides, aptamers based on RNA are also known [14]. There have been many variations in the SELEX process reported [15]. The affinities achieved for protein targets using aptamers are as high as those achieved by most antibodies, and are in the 1-100 nM range [10]. In their use as capture agents on gold electrode surfaces, or for example, as reporter agents on gold nanoparticles, aptamers with thiolated ends can readily be prepared. Attemers designed for immobilization on gold typically have a linker such as a short alkyl chain attached to one end of the oligonucleotide and a disulfide with mercaptohexanol on the other end [16]. Reduction with dithiothreitol or with tris(2-carboxyethyl)phosphine (TCEP) is used to produce the free thiol. The disulfide form can directly modify a gold surface but will result in lower aptamer surface density. Derivatives of the 15-mer and 29-mer aptamers against thrombin have been reported in which the 5'-end was modified with two dithianes with a phosphodiester bridge and a short polyethylene glycol (PEG) linker resulting in especially strong and flexible binding to the gold surface [17]. When carbon-based electrodes are used, a strategy for aptamer immobilization is to generate carboxyl groups on the surface and conjugate amine-terminated aptamers using EDC/NHS chemistry [18]. Immobilization of aptamers on glassy carbon electrodes has also been achieved using \(\pi\)-stacking on a spread graphene-porphyrin composite [19]. The adsorption of a graphene/aptamer complex onto glassy carbon electrodes modified by poly(o-phenylenediamine) has been reported [20].