Novel Approaches And Applications Of Bipolar Electrochemiluminescence In Life Analysis

As an important branch of electrochemistry,ECL retains the advantages of the electrochemistry like quick response,high sensitivity,wide detection range as well as good controllability.The introduction of bipolar electrode has made ECL a wireless and portable strategy.Besides,the dividable anode and cathode of a BPE device provides ECL with a greatly expanded detection range,which is particularly important for life analysis.In addition,with the combination of BPE-ECL and microelectrode,the method exhibits huge potential in trace detections.Showing a prospective collection of advantages like high accuracy,high sensitivity,high throughput,high resolution,low background noise and low detection limits,the microelectrode based BPE-ECL is able to cover a number of macro and microscopic detection targets.Based on the principles of BPE and ECL,this doctoral dissertation dedicates to signal amplification,method optimization and apparatus design.The details are discussed as following:1.Electrochemiluminescence aptasensor based on bipolar electrode for detection of adenosine in cancer cellsWe report a novel approach for the detection of adenosine in cancer cells by electrochemiluminescence?ECL?on a wireless indium tin oxide bipolar electrode?BPE?.In this approach,ferrocene?Fc?which is labeled on adenosine aptamer is enriched on one pole of the BPE by hybridization with its complementary DNA?ss-DNA?and oxidized to Fc+under an external voltage of 5.0 V at the two ends of BPE.Then,a reversed external voltage was added on the BPE,making Fc+enriched pole as cathode.The presence of Fc+promotes the oxidation reaction on the anodic pole of the BPE,resulting in a significant increase of ECL intensity using Ru?bpy?32+/tripropylamine?TPrA?system as test solution.The presence of target adenosine was reflected by the ECL signal decrease on the anodic pole caused by the target-induced removal of ferrocene-aptamer on the cathodic pole.The decrease of ECL signal was logarithmically linear with the concentration of ATP in a wide range from 1.0 fM to 0.10 ?M.This ECL biosensing system could accurately detect the level of adenosine released from cancer cells.2.Joint enhancement strategy applied in ECL biosensor based on closed bipolar electrodes for the detection of PSAA highly sensitive electrogenerated chemiluminescence?ECL?biosensor was developed on the basis of a closed bipolar electrode?BPE?apparatus for the analysis of prostate specific antigen?PSA?.Bipolar modifications bring up two different stages of enhancement on the same electrode.Anodic enhancement was conducted by modifying gold nanoparticles?Au NPs?to catalyze the anodic ECL reaction between luminol and hydrogen peroxide.Cathodic introduction of thionine tagged PSA antibody led to a further pertinently enhancement synchronized with the PSA amount variation,because the existence of thionine greatly increased the rate of electron gains on cathode,leading to the corresponding acceleration of anodic ECL reaction.The more thionine modified target molecules were introduced,the faster luminol was oxidized,the higher faraday current approached,and sensitive quantification was realized in correlation with the responsive ECL intensity differences.The quantification resulted in a good determination range between 0.1 pg/mL and 0.1?g/mL.This strategy mainly took advantage of the special structure of closed BPE to realize a simultaneous amplification on both ends of BPE.Moreover,the platform had a potential of providing a multi-functional strategy for the realization of other bio-detections by simply substituting the PSA sandwich structure with other bio-structures.3.Temporal Sensing Platform Based on Bipolar Electrode for the Ultrasensitive Detection of Cancer CellsWe report a bipolar electrode?BPE?sensing platform for the temporal detection of cancer cells.Combining the advantages of anodic dissolution and electrochemiluminescence?ECL?,this strategy shows an ultralow detection limit down to 5 cells/cm2.At the anode working as the reporting pole,Au NPs were assembled through DNA double strand,which served as both catalyzer for the ECL reaction of luminol/H₂O₂ and seeds for the chemical reduction of Ag,the anodic dissolution probe.The duration of Ag layer dissolution was positively correlated with the amount of Ag but negatively related to the controlled potential and the conductivity of the circuit.Therefore,it was possible to amplify a slight conductivity change through tuning the other two factors.As the formation of Au@Ag completely quenched the ECL emission of luminol,the ECL emission recovery reflected the extent of anodic dissolution.Through monitoring the ECL recovery time before and after the incubation of cells on the cathode,a few number of cells could be quantified due to slight difference of the conductivity.This method shows several merits.First,the combination of anodic dissolution and ECL significantly increases the detection sensitivity of BPE device.In addition,this strategy broadens the application of BPE for the ultrasensitive monitoring of cancer cells,which was applied to investigate the capture efficiencies of antibodies and aptamers toward MCF-7 and A549.4.Real Time Detection of Label Free Cytoplasmic Dopamine Based on Nanopore Bipolar Electrode ElectrochemiluminescenceWe fabricated a nanopore electrode?NPE?based closed bipolar electrode?BPE?for intracellular detection of dopamine.A probing tip namely borosilicate glass-sealed platinum wire electrode was fabricated by drawing a glass capillary and a platinum wire?o.d.25?m?laid inside simultaneously using a laser puller procedure.A cavity was formed in the front of the capillary because of the different fusing coefficient between glass and platinum.The point giass tip??150nm?was applied to penetrate single cells,while the capillary body was used as the container of ECL signal molecules.The cytoplasm was siphoned into the cavity to immerse the sensing pole of the Pt wire through which some ECL influence factors could be investigated.The other end of the Pt wire coupled with the sensing pole to function as the reporter of the formed BPE system and herein generated ECL signals could thus be wirelessly detected.With simple modification of dopamine aptamer on the sensing pole of the Pt,an ultrasensitive and selective intracellular detection of dopamine was realized and dopamine expression of nicotine addiction was also verified.With the employment of microelectrode,we for the first time introduced a BPE-ECL strategy in single cell analysis without introduction of non-cytoplasm in the target environment.This technique proposed could be extended to other biological detections with simple modifications or alternative materials,and some extremely minor structures could hopefully be monitored electrically or optically.5.Micro/portable Electrochemiluminescence DetectorWe describes a small ECL detector based on the closed BPE.This detector integrates a dark box which is also used as a container for luminescent agents,a working BPE and a photomultiplier tube?PMT?in a single device which collectively works as a portable but accurate probing system.The BPE is positioned across the bottom of the dark box,intentionally to keep one pole?reporting pole?inside the container and the other?sensing pole?outside.The sensing reaction could thus be separated from the ECL reaction.Additionally,with the introduction of micro BPEs,intracellular analysis could be achieved by simple insertion with a manipulator.The miniaturized detector not only provides simplicity and low cost for ECL instrumentation and detection,but also a feasible way of fine probing with high observability.