Paper-based/Ratiometric Electrochemiluminescence Analysis

As the combination product of electrochemical and chemical luminescence,electrogenerated chemilumiescence(ECL)mainly refers to the electron transfer between the electrode products or one electrode product and one component in system after the application of voltage or current,which would bring the formation of excited states.These excited states will return to the ground state and emit light.Without the requirement of any additional light source in the process of detection,ECL effectively avoids the problems of scatter light and optical purity,bringing low optical background signal,which has been widely used in biological analysis.In recent years,the ratiometry analysis has attracted wide interests.Similar to the dual-wavelength fluorescence ratiometry,ECL also improves the sensitivity of detection through ratiometry analysis and reduces the impact of environmental changes.ECL ratiometry mainly includes double-potential and dual-wavelength signal ratio analysis.The double-potential ECL ratiometry has been widely developed in biological and chemieal analysis.However,the development of dual-wavelength ECL ratiometry is limited to a certain extent.In addition,the construction of paper-based ECL sensor keeps the advantages of paper-based device,such as low price,easy modification,portability,small volume consumption,convenient operation and so on.In this paper,based on the detection proeess and construction materials of ECL method,a novel paper-based/ratiometry ECL analysis method was designed for the detection of tumor markers and tumor cells.Based on the advantages of paper-based devices,paper-based ECL biosensors were designed for the detection of prostate specific antigen(PSA),HL-60 cancer cells and viral DNAs.In addition,relied on the resonance energy transfer between g?C3N4 nanosheets and Ru(bpy)32+,dual-wavelength ECL ratiometry method was successfully constructed for miRNA-21 detection.1.Paper-based electrochemluminescence biosensor for cancer cells detectionA novel paper-based electrochemiluminescence(ECL)sensor was developed for the detection of HL-60 cancer cells.In this approach,a section of plastic transparent tape was punched with a hole(6mm diameter)and then attached on the conductive surface of ITO glass to provide an effective working area for electrochemical measurements.A piece of filter paper(13mm×8mm)connected to a sheet of ITO working electrode was applied as a thin-layer electrochemical cell.Pt wire and Ag/AgCl wire were fixed on the surface of filter paper with a clasp and used as the counter electrode and reference electrode,respectively.This designed device successfully reduced the detection volume to 20 ?L.Then,Au nanoparticles(Au NPs)and graphene were coated on the surface of ITO to immobilize capture HS-DNA.The aptaFer of HL-60 cancer cells,which was labeled with Ru(bpy)32+-conjugated silica nanoparticles(RuSi@Ru(bpy)32?)was hybridized with the capture DNA to form bioconjugates on the ITO electrode surface.In the presence of HL-60 cells,the aptamer would conjugate with glycoprotein at cell surface and RuSi@Ru(bpy)32?NPs-aptamer released.After the dissociation of RuSi@Ru(bpy)32+NPs-aptamer from the surface of ITO electrode,ECL signal declined.This simple,portable and disposable testing platform could bring the detection limit of HL-60 cancer cells down to 56 cells/mL.2.Paper-based bipolar electrode for electrochemiluminescence detection of cancer biomarkerIn order to further reduce the price,closed bipolar electrode(BPE)was constructed on the paper?based analytical device to sensitive electrochemiluminescence detect PSA.Compared with the traditional three electrode system,BPE has many advantages,such as simple structure,without the need of external wire,small size of the battery and easy miniaturization of the sensor device.This paper-based BPE device consisted of two hydrophilic cells(sensing cell and reporting cell)patterned by hydrophobic wax.These two hydrophilic cells were connected by a carbon ink BPE as electronic conductors,which could effectively separate the reactants and the detecting solution,avoiding their mutual interferences.At the same time,multi-walled carbon nanotubes(MWCNTs)were modified on the cathode of BPE to improve ECL responses.After the assembly of antibody and antigen,silica nanospheres carrying antibody and glucose oxidase were assembled onto the surface of electrode through the sandwich structure.In the presence of glucose,oxidase could catalyze glucose to produce H2O2 in situ,which accelerated the electron transport on the cathode surface.Therefore,ECL signal from the anode surface was obviously enhanced and sensitively detected PSA.The detection limit was down to 1 pg/mL.This paper-based BPE device provides a new opportunitie and direction for the BPE analytical application.3.Paper-based bipolar electrodes array for electrochemiluminescence detection of multiplex pathogenic DNAsA novel paper-based bipolar electrodes(BPEs)array device was fabricated for multiplex electrochemiluminescence(ECL)detection of pathogenic DNAs.This paper-based BPE array device consisted of 15 unitsglucose and each unit consisted of six sensing cells and two reporting cells patterned using hydrophobic wax.In the presence of complementary target DNA,eapture DNA assembled on the eathodes of BPEs would hybridize with Pt NPs labeled probe DNA.The introduction of Pt NPs could catalyze the reduction of dissolved O2 at cathodes,bringing an enhanced ECL signal from Ru(bpy)32+/tripropyIamine(TPrA)at the anodes of BPEs.Due to the relative large contacting area of the paper-based cells exposing to air,the dissolving O2 lost in reduction reaction could be promptly replenished,which ensured the stability of ECL signal.This obtained paper-based BPE array sensor showed excellent performances for the multiplex analysis of the syphilis(Treponema pallidum)gene,the immunodeficiency virus gene(HIV)and hepatitis B virus gene(HBV).4.Dual-wavelength electrochemilurmnescence ratiometry for microRNA detectionThe double-signal ratiomet:ry mainly depends on the ratio of two signals,not the single signal,which could effectively reduce the changes of external environment,for examples,path length,background light and scattering.Therefore,this double-signal ratiometry has better accuracy than the single-signal detection in the analytical application.Based on the resonance energy transfer(RET)from graphite-like carbon nitride nanosheet(g-C3N4 NS)to Ru(bpy)32+,a dual-wavelength electrochemiluminescence(ECL)ratiometric approach was reported for sensitive detection of miRNA-21.In this approach,Au nanoparticles(Au NPs)funetionalized g-C3N4 NS nanohybrid(An-g-C3N4 NH)coated on glassy carbon electrode(GCE)could exhibit strong and stable ECL emissions with emission peak eentered at 460 nm.The ECL emission at such wavelength matched well with the absorption peak of Ru(bpy)32+as well as impeccably stimulate the emission of Ru(bpy)32+at the wavelength of 620 nm,producing ECL-RET with high efficiency.Thus,based on the ECL signals quenching at 460 nm and increasing at 620 nm,a dual-wavelength ratiometric ECL-RET system was achieved.This system was then utilized for determination of target miRNA-21.with the attachment of thiol-modified molecular beacon on Au-g-C3N4 NH,target miRNA-21 hybridized with the molecular beacon to form DNA-RNA duplex.The obtained DNA-RNA duplex was further cleaved by duplex-specific nuclease to release target miRNA-21 which would take part in the next cycle for further hybridization.Finally,Ru(bpy)32+was introduced to the electrode surface through the hybr:idization between probe DNA-Ru(bpy)32+and the rest single-strand DNA.By measuring the ratio of ECL460nm/ECL62Onm,we could accurately quantify the concentration of miRNA-21 in a wide range from 1.0 fM to 1.0 nM.This work provides an important reference for the study of dual-wavelength ECL ratiometry and also exhibits potential capability in the detection of nucleic acids.