A bifunctional derivative of the thrombin-binding aptamer with a redox-active Fc moiety and a thiol group at the termini of the aptamer strand was synthesized. The ferrocene-labeled aptamer thiol was self-assembled through S-Au bonding on a polycrystalline gold electrode surface and the surface was blocked with 2-mercaptoethanol to form a mixed monolayer. By use of a fluorescent molecular beacon, the effect of counterions on quadruplex formation was established. The aptamer-modified electrode was characterized electrochemically by cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS). The modified electrode showed a voltammetric signal due to a one-step redox reaction of the surface-confined ferrocenyl moiety of the aptamer immobilized on the electrode surface in 10 mM N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES) buffer of pH 8.0. An increase in the DPV current signal was evident after blocking with 2-mercaptoethanol, effectively removing aptamer nonspecifically absorbed rather than bound to electrode surface or due to the formation of the aptamer-thrombin affinity interaction. The impedance measurement, in agreement with the differential pulse voltammetry (DPV), showed decreased Faradaic resistances in the same sequence. The "signal-on" upon thrombin association could be attributed to a change in conformation from random coil-like configuration on the probe-modified film to the quadruplex structure. The DPV of the modified electrode showed a linear response of the Fc oxidation signal to the increase in the thrombin concentration in the range between 5.0 and 35.0 nM with a linear correlation of r = 0.9988 and a detection limit of 0.5 nM. The molecular beacon aptasensor was amenable to full regeneration by simply unfolding the aptamer in 1.0 M HCl, and could be regenerated 25 times with no loss in electrochemical signal upon subsequent thrombin binding.

A novel impedimetric aptasensor using a mixed self-assembled monolayer composed of thiol-modified thrombin binding aptamer and 2-mercaptoethanol on a gold electrode is reported. The changes of interfacial features of the electrode were probed in the presence of the reversible redox couple, Fe(CN)6(3-/4-), using impedance measurements. The electrode surface was partially blocked due to the self-assembly of aptamer or the formation of the aptamer-thrombin complex, resulting in an increase of the interfacial electron-transfer resistance detected by electrochemical impedance spectroscopy or cyclic voltammetry. The aptasensor was regenerated by breaking the complex formed between the aptamer and thrombin using 2.0 M NaCl solution, and the immobilized aptamer subsequently was used for repeated detection of thrombin. The aptamer-functionalized electrode showed a linear response of the charge-transfer resistance to the increase of thrombin concentration in the range of 5.0-35.0 nM and the thrombin was easily detectable to a concentration of 2.0 nM.

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We present the rapid prototyping of electrochemical sensor arrays integrated to microfluidics towards the fabrication of integrated microsystems prototypes for point-of-care diagnostics. Rapid prototyping of microfluidics was realised by high-precision milling of polycarbonate sheets, which offers flexibility and rapid turnover of the desired designs. On the other hand, the electrochemical sensor arrays were fabricated using standard photolithographic and metal (gold and silver) deposition technology in order to realise three-electrode cells comprising gold counter and working electrodes as well as silver reference electrode. The integration of fluidic chips and electrode arrays was realised via a laser-machined double-sided adhesive gasket that allowed creating the microchannels necessary for sample and reagent delivery. We focused our attention on the reproducibility of the electrode array preparation for the multiplexed detection of tumour markers such as carcinoembryonic antigen and prostate-specific antigen as well as genetic breast cancer markers such as estrogen receptor-alpha, plasminogen activator urokinase receptor, epidermal growth factor receptor and erythroblastic leukemia viral oncogene homolog 2. We showed that by carefully controlling the electrode surface pre-treatment and derivatisation via thiolated antibodies or short DNA probes that the detection of several key health parameters on a single chip was achievable with excellent reproducibility and high sensitivity.

An electrochemical molecular beacon aptasensor for fast and sensitive assay of thrombin is developed and characterized. A bifunctional derivative of the thrombin-binding aptamer, 15-base long with a redox-active ferrocene moiety and a pendant hexanethiol linker group at the termini of the aptamer strand was immobilized on Au electrode surface. The electrochemical behavior of the aptasensor was studied using cyclic voltammetry (CV), chronoamperometry (CA), chronopotentiometry (CP), square-wave voltammetry (SWV) and chronopotentiometric stripping analysis (PSA) techniques. Taken together, these experiments support the switch "on" mechanism of the system when the thrombin binds to the immobilized aptamer. The variation of response to the concentration of thrombin, the target protein, was evaluated by square-wave voltammetry and chronopotentiometric stripping analysis in HEPES buffer solution (pH 8.0, 0.01 M). The aptasensor showed a specific response to thrombin in the range 1.0 to 35 nM with a detection limit of subnanomolar level concentrations. The reaction time, reproducibility, specificity and stability of the aptasensor were also studied.

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