Potential-Resolved Electrochemiluminescence Analysis

Electrochemiluminescence(ECL)has become a research hotspot as a new,efficient and sensitive analytical technique.Conventional ECL analysis is usually limited to a single-signal mode,which may introduce interference from substrate and detection system,including microenvironmental contamination,complex operational steps,and electron transfer kinetic hysteresis.In order to overcome the interference from the background signal and improve the accuracy and sensitivity of the ECL analysis,scientists have gradually focused on the dual-signal ratiometic sensing mode in recent years.If the excitation potentials of the two ECL reagents are wide enough during the ECL process,it is possible to realize the dual-potential ECL ratiometic sensing.In addition,since the optical signal is measured in the ECL analysis,when the optical signal is strong enough,the visualization can be realized,and the naked eye can be read directly without additional signal reading devices,which made the analysis easier.At present,many visual signals are quantitatively or semi-quantitatively analyzed based on the monochrome intensity.However,since the naked eye is not sensitive to the monochrome intensity,the detection sensitivity is not high,and it is easy to cause a false positive.Multi-color ECL analysis can solve this problem.In general,multi-color ECL analysis requires at least two ECL reagents with different emission wavelengths.If the excitation potentials of the two ECL reagents are different,the color of the mixed ECL could be modulated by the different potentials.Based on this,we can use some special design to make the color of the mixed ECL change with the substrate concentration,so as to achieve the visual ECL analysis.In this paper,based on the potential-resolved ECL ratio and multi-color sensing modes,we combined the ECL system such as luminol,Ru(bpy)32+,iridium complex,and graphene-like carbon nitride with the bipolar electrode(BPE)technology to construct the dual-potential ratiometic biosensor,spatial-resolved ECL ratiometry,multi-color biosensor and array.The details are summarized as follows:1.A ratiometric electrochemiluminescence detection for cancer cells using g-C3N4 nanosheets and Ag-PAMAM-luminol nanocompositesA dual-signaling electrochemiluminescence(ECL)ratiometric sensing approach for the detection of HL-60 cancer cells was reported for the first time.G-C3N4 nanosheets and Ag-PAMAM-luminol nanocomposits(Ag-PAMAM-luminol NCs)were prepared and served as reductive-oxidative and oxidative-reductive ECL emitters respectively.DNA probe functionalized Ag-PAMAM-luminol NCs would hybridize with aptamers modified onto magnetic beads.In the presence of HL-60 cells,the aptamer would conjugate with the target cell and release Ag-PAMAM-luminol NCs.After magnetic separation,released Ag-PAMAM-luminol NCs would hybridize with capture DNA on g-C3N4 nanosheets.ECL from g-C3N4 nanosheets coated on ITO electrode at-1.25 V(vs SCE)could be quenched by Ag-PAMAM-luminol NCs due to the spectra overlap between the absorption spectrum of Ag NPs and the ECL spectrum of g-C3N4 nanosheets.Meanwhile,Ag-PAMAM-luminol brought the ECL signal of luminol at +0.45 V(vs SCE).Thus,the concentration of HL-60 cancer cells could be quantified by both the quenching of ECL from g-C3N4 nanosheets and the enhancement of ECL from luminol.By measuring the ratio of ECL intensities at two excitation potentials,this approach could achieve sensitive and reliable detection for cancer cells in a wide range from 200 cells/mL to 9000 cells/mL with the detection limit of 150 cells(S/N=3).2.Spatial-resolved electrochemiluminescence ratiometry based on bipolarelectrode for bioanalysisA spatial-resolved electrochemiluminescene ratiometry based on a closed biopolar electrode is reported for the highly sensitive detection of prostate specific antigen(PSA).Au@g-C3N4 NCs as one ECL emitter were firstly coated on the cathode of BPE,while the anode of the BPE served for calibration via another ECL substance,Ru(bpy)32+ adding in the reservoir.The electroneutrality across the BPE makes the reactions on each pole of BPE electrically coupled.Thus one electrochemical sensing reaction at one pole of BPE could be quantified at both ends.A composite,Pt-PAMAM-DNAzyme was assembled on the surface of cathode via DNA hybridization between probe DNA and PSA aptamer.It acted as an ECL quencher of g-C3N4 via resonance energy transfer and catalyzing the reduction of O2,the co-reactant of g-C3N4.Meanwhile,it could promote the ECL of Ru(bpy)32+ at anode,since the catalytic reduction of O2 at the cathode increased the faradiac current flowing through the BPE.Based on this signal composite,an ECL "off-on"phenomenon was observed at the cathode,after Pt-PAMAM-DNAzyme was "peeled off" by PSA.Conversely,at the anode,an "on-off" ECL changing was obtained.Therefore,a sensitive ratiometry for PSA detection was achieved with a linear range from 0.1 to 200.0 ng/mL.Since the two ECL emitters were physically separated,the ratiometric system was relatively simple and neither optical filters nor spectrometer were required.The strategy combining the ECL ratiometry and BPE broadens the applications of BPE-ECL and shows good perspective in clinical application.3.Bipolar electrode based multi-color electrochemiluminescence biosensorWe report a multi-color ECL device based on closed bipolar electrode(BPE)for the visualized sensing of prostate-specific antigen in human blood serum.As the emission color of concomitant electrochemiluminophores is potential resolved,similar to a three-electrode system,selective excitation of ECL could be achieved by tuning the interfacial potential at the poles of BPE.Via modulating the resistance of BPE,multi-color ECL emission of Ru(bpy)32+ and Ir(ppy)3 mixture as the co-reactant was observed at the anode and the principle was elaborated.The concept was utilized to the quantification of clinical biomarkers with the color variation.A PSA concentration dependent silver bridge was constructed in the gap of the BPEs as an electric conductivity modulator.On the basis of multi-color BPE-ECL device,the cut-off values(4.0 ng/mL and 10.0 ng/mL)of human PSA could be recognized by the green-yellow-red ECL emission changing with naked eyes.As the first multi-color ECL device in biological analysis,BPE may raise the application of potential-resolved ECL to a new level.4.Bidirectional electrochemiluminescence color switch:An application in detecting multi-markers of prostate cancerA selective excitation of Firpic and Ru(bpy)32+ through tuning electrode potential is reported in this work.Bidirectional color change from blue-green to red could be observed along with increase and decrease of the potential,which was ascribed to the dual-potential excitation property of Firpic.Similar to three-electrode system,selective excitation of ECL could be achieved at the anode of bipolar electrode.Both increase and decrease of the faradic reactions at the cathode of the BPE could induce ECL reporting color at the other pole switched from blue-green to red.We applied a closed BPE device for the bioanalysis of multicolour ECL since the organic solvent containing electrochemiluminophores could be separated from the bioanalytes.On the basis of BPE arrays coupled with the ECL switch,the detection of three biomarkers of prostate cancer,PSA,miRNA-141 and sarcosine were integrated in a same device.The cut-off values of the biomarkers could be recognized directly by naked eye.Such device holds great potential in early diagnosis of prostate cancer.