The Application Of Ratiometric Sensing Mode In Electrochemical Biomolecule Detection

Currently,the electroanalysis technique and the electrochemical sensors have been successfully applied in nuclear acids detection,immunosensing,organic matters sensing and metal ions detection etc.However,the classic electrochemical sensors only based on single detection signals which suffered from low repeatability and poor anti-interference ability.This shortage seriously limited its actual application and further development.As a frequently applied technique in florescence analysis and electrochemiluminescence,the ratiometric detection technique is also introduced in the area of electroanalysis recently.The ratiometric detection possess dual signals-the detection signal and reference signal and the output signal are based on the ratios of the dual signals.The application of reference signal largely enhanced the repeatability and anti-interference ability,which could make up the disadvantages of classic electroanalysis which only process single signal.In this thesis,three kinds of novel dual-signal ratiometric electrochemical sensing strategy were constructed.Based on the three kinds of sensing strategy,the electrochemical detection for DNA,the bio-macromolecule and dopamine and ascorbic acid,the small biomolecules,were realized.The thesis included the three parts as below:?1?The graphene oxide was reduced by chemical reduction method to obtain the reduced graphene oxide and the sodium flavin mononucleotide?FMNS?was applied as the dispersing agent for dispersing the rGO.Compared with the sole rGO,the dispersion ability of FMNS/rGO was obviously enhanced.In addition,due to the excellent electrochemical redox performance of FMNS,the obtained FMNS/rGO possesses stable and obvious redox signal.Based on its redox signal and the assistance of electrochemical indicator-the Meldola's blue,a novel ratiometric electrochemical sensor was developed.In this work,the FMNS was not only applied as the provider of signal,but also worked as the immobilization sites for DNA probes.When the probe was hybridized with the target DNA,the increased DNA loading amount would result in the decrease of the redox signal of FMNS.Meanwhile,the formation of DNA double helix could provide the possibility for MDB to cutting in the DNA duplex and result in the increase of the signal of MDB.Thus,the successful hybridization of target DNA could cause the opposite change of the signal from MDB and FMNS at the same time.Based on the relationships between the change of the ratios of the dual signal and the concentration of target DNA,a novel ratiometric electrochemical DNA sensor was constructed.A linear range from 10^-6 mol/L to10^-11 mol/L,a linear relationship of y=0.2785x+3.5421,R²=0.9913 and a detection limit of 2.68 pM were obtained.Besides,in this section,the detection performances of the two classic detection mode:based on the single signals of the redox signal of the interface and the electrochemical indicator and the dual-signal ratiometric mode were compared.?2?The ascrobic acid was considered as a kind of important small bio-molecule.The classic detection method for ascorbic acid was directly monitoring the oxide peak on electrode.Due to the influence of interference such as the dopamine,uric acid etc.In usual,it usually thought that the direct determination of dopamine need to use the chemical modified electrode to separate the redox potentials of ascorbic acid and interferences.In this chapter,a novel method based on bare electrodes for electrochemical ascorbic acid detection was reported.Different from the classic strategies,in this chapter,the reduction peak of Cu?II?ion was employed as the indirect report signal for ascorbic acid detection.The ascorbic acid could reduce the Cu?II?ions in relatively short times which could cause the concentration decrease of it.As a result,the reduction peak of it would go down.In addition,the common interference such as dopamine and uric acid could not induce obvious interference in given times.Thus,the electrochemical detection of ascorbic acid could be realized by adopting unmodified electrodes instead of adopting the chemical modified electrodes,which avoided complex and time-wasting procedures such as materials preparation and electrode modification.By further adding same concentrations of reference substance in every sample,a novel ratiometric electrochemical ascorbic acid detection method was established.A linear range of 2-100?M,a linear relationship of y=0.134 x+5.091 and a detection limit of 0.2?M were obtained.In addition,the performance of the ratiometric signal mode and classic single signal mode for this work was also compared.?3?The 2D-metal organic frameworks?2D MOFs?Copper-porphyrin?Cu-TCPP?was synthesized and applied as the precursor on electrode surface.The nanogold was electrodeposited on it and the conductive polymer poly-Xanthurenic acid?PXA?was further electrochemical polymerized to obtain the Cu/TCPP/Au/PXA nanocomposite.Compared with the sole materials Cu-TCPP,nanogold and PXA,the electrochemical performance of the nanocomposite-the Cu/TCPP/Au/PXA was obviously enhanced.In addition,the Cu/TCPP/Au/PXA was further applied in the electrochemical detection of dopamine.In order to construct the ratiometric sensing strategy for dopamine detection,the method of adding fixed concentrations of redox substance in detection solution was applied.A kind of common medicine-paracetamol was founded to possess well-separated peak potential with the dopamine on the Cu/TCPP/Au/PXA modified electrode and was applied as the reference substance in this experiment.By applying it as the reference,the electrochemical detection for dopamine-a kind of important neurotransmitter was realized.A linear range of 5-125?M,a linear relationship of y=0.0214x+0.5271 and a detection limit of 1?M were realized.In addition,the differences between the classic single signal electrochemical dopamine detection method and the ratiometric electrochemical method was compared.