Several Electrochemical Biosensors Based On Different Signal Amplification Strategies

Electrochemical biosensors combine the sensitivity of the electrochemicaltransducers with the high specificity of the biological recognition processes. Becauseof their simplicity, cheap cost, fast response and high stability, electrochemicalbiosensors have been widely used in analytical chemistry. Among them, theenzyme-based and DNA-based taypes are the two of common electrochemicalbiosensors.Taking advantage of the superior biocatalytic property of enzymes, theenzyme-based electrochemical biosensors have experienced three developmentprocesses. Among them, the third-generation, which is based on the direct electrontransfer between enzyme and electrode surface, has been considered as one of themost promising construction methods of the enzyme electrodes. Therefore, rationalelectrode materials become very important. They need large surface area to load moreenzymes, excellent biocompatibility to keep their bioactivity, and goodelectroconductivity to promote the direct electron transfer between enzymes andelectrode surface.Due to the introduction of aptamers, DNA-based electrochemical biosensors cannot only be used to detect the DNA hybridization and damage, but also can be used forthe detection of small molecules, proteins, drugs and so on. With the development inanalytical field, needs for the ultrasensitive sensors grow rapidly. Recently, variousnanomaterials have been used in the DNA-based electrochemical sensors. Theirunique electrochemical properties may improve the analytical performances of thesensors.In this dissertation, we have developed several electrochemical biosensors. Goldnanomaterials and graphene have been used as the signal amplifiers for theimprovement of the sensor performance. Moreover, we developed a newdual-signaling amplification strategy for the electrochemical aptasensor. The mainworks are summarized as follows:(1) A facile electrodeposition method has been successfully adopted to fabricatedendritic gold nanostructure (DenAu) on glassy carbon (GC) electrode and theprepared DenAu/GC electrode has large surface area. A sensitive third-generationsuperoxide radical (O2-) biosensor was then constructed by assembled L-cysteine (cys) onto the DenAu/GC electrode to immobilize large amounts of superoxide dismutase(SOD). The direct electron transfer of SOD was successfully realized and a pair ofquasi-reversible redox peaks of SOD was observed at the SOD/cys/DenAu/GCelectrode in the phosphate buffer solution (25mM, pH7.2). Due to the high loadingof SOD on the electrode, the resulted biosensor exhibited good analytical performancefor O2-detection, such as a low detection limit of2.1nM, a good stability andreproducibility, especially a wide linear range up to540M.(2) By using biobarcode nanoparticles, we have successfully constructed aDNA-based biosensor for amplified electrochemical detection of hydroxyl radical(·OH). Thiolated DNA1(SH-DNA1) was firstly immobilized on the planar gold (Au)electrode.·OH generated from Fenton reaction could induce oxidative damage of theDNA layer adsorbed on the electrode surface, which was monitored by anintercalating probe, methylene blue (MB). In order to enhance the sensitivity of thebiosensor, DNA2-functionalized Au nanoparticles (DNA2-AuNPs) were used toamplify the response signal. The developed DNA-based biosensor could detect·OHquantitatively with wide linear range up to10mM and low detection limit of3μM,and exhibited satisfactory selectivity. On the other hand, this electrochemicalbiosensor could have potential application in the evaluation of antioxidant capacity.(3) Taking the advantage of the ultrahigh electron transfer ability of AuNPs andits unique interaction with the single-strand aptamer, a novel electrochemicalaptasensor for the sensitive detection of adenosine triphosphate (ATP) has beendeveloped. Specifically, because of the interaction between gold andnitrogen-containing bases, ATP aptamer (ABA) which is immobilized onto goldelectrode can directly adsorb AuNPs. The negative electrochemical species[Fe(CN)6]3-/4-may transfer electrons to electrode through adsorbed AuNPs. In thepresence of the target ATP, the formed ABA-ATP complex cannot capture AuNPs andresults in a large electron-transfer resistance owing to the negatively charged ATP andthe phosphate backbones of ABA. So, a simple and sensitive method for the detectionof ATP can be developed by using AuNPs as the signal amplifier. Experimental resultsdemonstrate that the developed electrochemical method can detect as low as0.54nMATP without any requirement of the modification to AuNPs. This method may also begeneralized for detecting a series of targets by using other functional DNA in thefuture.(4) Based on the superior electrocatalytic property of graphene (GN) to ascorbicacid (AA) oxidation, a novel label-free electrochemical aptasensor has been developed and used for the sensitive detection of ATP. Briefly, GN is attached to the thiolatedATP binding aptamer (ABA) modified gold electrode through π-π stacking interaction,resulting in a significant oxidation signal of AA. In the presence of ATP, the formationof ATP-ABA complex leads to the release of GN from sensing interface, resulting in asharp decrease of the oxidation peak current of AA and an obviously positive shift ofthe related peak potential. Taking both the change values of the peak current and peakpotential of AA oxidation as the response signals, ATP can be detected sensitively. Itcan be expected that GN, as nanocatalyst, should become the very promisingamplifying-elements in DNA-based electrochemical biosensors.(5) A simple electrochemical aptasensor for sensitive and selective determinationof ATP has been developed based on a new dual-signalling amplification strategy.This aptasensor features both ‘‘signal-on’’ and ‘‘signal-off’’ elements. The ferrocene(Fc)-labeled aptamer probe (Fc-P) is designed to hybridize with the thiolatedmethylene blue (MB)-modified DNA probe (MB-P) on gold electrode to form rigidduplex DNA. In the presence of ATP, the interaction between ATP and the aptamerleads to the dissociation of the duplex DNA structure and thereby the Fc-P releasesfrom the sensing interface. The single-stranded MB-P could thus tend to form ahairpin structure through the hybridization of the complementary sequences at both itsends. As a result, the oxidation peak current of Fc decreases, that of MB increases,and the changes of dual signals are linear with the concentration of ATP. When “ΔI=ΔIMB+|ΔIFc|”(ΔIMBand ΔIFcare the change values of the oxidation peak currents ofMB and Fc, respectively.) is used as the response signal for quantitativelydetermination of ATP, the detection limit (1.9nM) is much lower than that by usingeither MB-P or Fc-P alone. This new dual-signalling aptasensor is readily regeneratedand shows good response toward the target. It will have important applications in thesensitive and selective electrochemical determination of other small molecules andproteins.