The Applications Of Multiple Amplifying Biotechniques In Electrochemical Aptasensor

Electrochemical aptasensor is one kind of novel biosensor, which combines the recognition of aptamer and the conduction of electrochemical signal.It plays a vital role in detection of target protein and judgment of early diagnosis. The sort of electrochemical aptasensor not only possesses high specificity and selectivity of aptamer, but also has advantages of analysis, such as high-sensitivity, fast, simple, living detection and low cost. Recently, owing to improve the sensitivity of aptasensor in the detection of target protein, multiple signal amplifying biotechniques are widely employed to construct aptasensor. Hence, it is meaningful to work on effective, reliable and high sensitive electrochemical aptasensor by using amplifying biotechniquc and electrochemical biosensors.1. A novel electrochemical aptasensor for thrombin detection based on the hybridization chain reaction with hemin/G-quadruplex DNAzyme-signal amplificationA novel signal amplification electrochemical aptasensor for the sensitive and selective detection of thrombin was successfully fabricated. The amplification method was based on the hybridization chain reaction (HCR) and a pseudobienzyme electrocatalytic system. HCR-based double stranded DNA (dsDNA) polymers not only constructed an effective carrier for anchoring larger amounts of electron mediator methylene blue (MB) into the DNA duplexes, but also resulted in the formation of hemin/G-quadruplex DNAzymes nanowires by intercalating hemin into two induced single-stranded DNA (ssDNA). With the addition of NADH into the electrolytic cell, the hemin/G-quadruplex acting as an NADH oxidase and HRP-mimicking DNAzyme for the pseudobienzyme amplifying system could in situ biocatalyze the formation of H2O2 with local concentrations and low transfer loss resulting in dramatic signal enhancements. The binding event can be detected by a decrease in the integrated charge of MB which electrostatically absorbed onto dsDNA polymers. In the presence of thrombin, the dsDNA polymers associated with MB and hemin/G-quadruplex structures were removed from the electrode surface, leading to a significant decrease of redox current. DPV signals of MB provided quantitative measures of the concentrations of thrombin, with a linear calibration range of 0.01-50 nmol·L-1 and a detection limit of 2 pmol·L-1.Moreover, the resulting aptasensor also exhibited good specificity, acceptable reproducibility and stability, indicating that the present strategy was promising for simple and sensitive protein analysis.2. A highly sensitive electrochemical aptasensor for thrombin detection using functionalized mesoporous silica@ multiwalled carbon nanotubes as signal tags and DNAzyme signal amplificationWe demonstrated a novel and sensitive sandwich-type pseudobienzyme aptasensor for thrombin detection. Greatly amplified sensitivity was based on mesoporous silica-multiwalled carbon nanotube (mSiO2@MWCNT) nanocomposites as enhanced materials and a pseudobienzyme electrocatalytic system. Firstly, the mSiO2@MWCNT nanocomposites not only have good biocompatibility and a suitable microenvironment for stabilizing the aptamer assembly, but also can load large amounts of electron mediator thionine (Thi), platinum nanoparticles (PtNPs) and hemin/G-quadruplex bioelectrocatalytic complex. Moreover, in the presence of H2O2 in an electrolytic cell, the synergistic reaction of PtNPs and hemin/G-quadruplex bioelectrocatalyzed the reduction of H2O2, dramatically amplifying the response signals of electron mediator Thi and improving the sensitivity. Secondly, dendrimer functionalized reduced graphene oxide (PAMAM-rGO) as the biosensor platform enhanced the surface area for the immobilization of abundant primary aptamers as well as facilitated electron transfer from Thi to the electrode, thus amplifying the detection response in the range between 0.1 pmol·L-1 and 80 nmol·L-1 with a detection limit of 0.05 pmol·L-1.. This new design avoided the fussy labeling process and the spatial distribution of each sequentially acting enzyme, which provided an ideal candidate for the development of a sensitive and simple bioanalytical platform.3. Amplified amperometric aptasensor for selective detection of protein using catalase-functional DNA-PtNPs dendrimer as a synergetic signal amplification labelBased on the synergetic amplification of a catalase (CAT) enzyme-functional DNA-platinum nanoparticles (PtNPs) dendrimer through autonomous layer-by-layer assembly, a new strategy was constructed an electrochemical aptasensor for sensitive detection of platelet-derived growth factor BB (PDGF-BB) Firstly, polyamidoaminedendrimer (PAMAM) with a hyper-branched and three-dimensional structure was served as nanocarriers to coimmobilize a large number of PDGF-BB binding aptamer (PBA Ⅱ) and ssDNA 1 (S,) to form PBA Ⅱ-PAMAM-S1 bioconjugate. In the presence of PDGF-BB, the bioconjugate was self-assembled on the electrode by sandwich assay. Following that, the carried S1 propagated a chain reaction of hybridization events between CAT-PtNPs-S1 and CAT-PtNPs-ssDNA 2 (S2) to form a CAT-functional PtNPs-DNA dendrimer, which successfully immobilized substantial CAT enzyme and PtNPs with superior catalysis activity. In this process, the formed negatively charged double-helix DNA could cause the intercalation of hexaammineruthenium(Ⅲ) chloride (RuHex) into the groove viaelectrostatic interactions. Thus, numerous RuHex redox probes and CAT were decorated inside/outside of the dendrimer. In the presence of H2O2 in electrolytic cell, the synergistic reaction of CAT and PtNPs towards electrocatalysis could further amplify electrochemical signal. By monitoring the change in the current, we can indirectly determine the concentration of target PDGF-BB in the sample with the range of 0.05 pmol·L-1-35 nmol·L-1 with a relatively low detection limit of 0.02 pmol·L-1..