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Spectrum-Resolved Triplex-Color Electrochemiluminescence Multiplexing Immunoassay with Highly-Passivated Nanocrystals as Tags Jie Zhou, Lei Nie, Bin Zhang, and Guizheng Zou Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04424 • Publication Date (Web): 11 Oct 2018 Downloaded from http://pubs.acs.org on October 11, 2018
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Analytical Chemistry
Spectrum-Resolved Triplex-Color Electrochemiluminescence Multiplexing Immunoassay with Highly-Passivated Nanocrystals as Tags Jie Zhou,†,§ Lei Nie,‡,§, Bin Zhang†, and Guizheng Zou†,* School of Chemistry and Chemical Engineering, ‡ School of Pharmaceutical Sciences, Shandong University, Jinan, 250100, China †
* Corresponding author Email:
[email protected] ABSTRACT: Nanocrystals (NCs) were extensively employed in optical-multiplexing with their size-dependent and narrow waveband photoluminescence (PL). Herein, to achieve NCs-based novel optical-multiplexing strategy with improved sensitivity, a spectrum-resolved triplex-color electrochemiluminescence (ECL) multiplexing immunoassay (MIA) was proposed for the first time by extensively exploring the color-different monochromatic ECL from dual-stabilizers-capped CdTe and CdSe NCs and the inherent lower background of ECL than PL. As a proof of concept, carcinoembryonic antigen (CEA), prostate specific antigen (PSA), and alpha fetoprotein (AFP) were adopted as model analytes, and formed three antigen-NCs pairs at glassy carbon electrode surface via one-pot immune-reaction with CdSe(550), CdTe(650), and CdTe(776) NCs as tags respectively. The spectral ECL responses of three antigen-NCs pairs were simultaneously measured via onepot ECL assay, and the maximum ECL intensity of each tag was directly utilized to determine corresponding antigen without any assistance from ether optical filters or signal deconvolution. This ECL-MIA displayed negligible cross-reactivity, high colorselectivity, and excellent sensitivity with limits of detection down to 1, 10, and 0.01 pg/mL for CEA, PSA, and AFP respectively, which might provide a promising alternative to NCs PL multiplexing.
acquired by Roche Diagnostics Corp.) for a number of assays with a particular focus on immunoassays.24-26 Because only one ECL tag is employed, this system is not readily multiplexed. Although great efforts have been made for ECL multiplexing in potentialresolved27-31 and spatial-based32-37 way, only very limited ECL multiplexing was conducted in the spectrally resolved way,38 similar to that of PL multiplexing. The reason might lie in two aspects: one is poor monochromaticity of ECL from both traditional ECL chemicals24,39 and the promising NCs type electrochemiluminophores,40 the spectral overlap of ECL among different electrochemiluminophores would limit the degree of multiplexing, and interfere the interpretation of results; the other is that there is no dispersing component in traditional ECL setups to resolve ECL of different wavebands.24,41 Our group has been engaged in spectrum-resolved ECL for more than ten years.42 To overcome the limits on ECL multiplexing, we not only developed a dual-stabilizers-capped strategy to synthesize monochromatic electrochemiluminophores of different wavebands, i.e. highly-passivated CdTe and CdSe NCs,43-46 but also assembled a CCD based ECL spectrum analyzer,47,48 which was qualified for spectrum-based quantitative ECL assay,49-51 and enabled spectrumbased color-selective ECL assay in both one color and dual-colors resolved models.49,50,52
Semiconductor nanocrystals (NCs) received tremendous attention for their luminescent sensing application,1,2 since they were successfully employed to bind biomolecules in bioconjugate style.3,4 The size-tunable photoluminescence (PL) wavelength and narrow PL band made NCs ideal candidates for optical multiplexing,5,6 fluorescent multiplexing with NCs as labels have been extensively carried out in the past decades.7-12 Several extended multiplexing strategies with some special methods to light up NCs were also developed, such as fluorescence,9 bioluminescence,13,14 chemiluminescence,15 and electrochemiluminescence (ECL)16-18 resonance energy transfer. To avoid the inherent interference from the unselective photoexcitation for PL based multiplexing, electrochemical multiple immunoassay with NCs as tags aroused much attention.19-21 As controllable light emission generated via electrochemical redox,2224 ECL is superior to fluorescence in terms of signal-to-noise ratio due to the absence of excitation-light-source. A microbead-based ECL system along with Ru(bpy)32+/tri-n-propylamine based ECL reagent kits has been commercialized by IGEN9 (technology later
Figure 1. Absorbance (a) and PL spectra (b) of (A) CdSe550, (B) CdTe650, and (C) CdTe776 NCs; ECL spectra of (D) GCE|ABAAb1(CEA, PSA, AFP)Ab2(CEA)|CdSe550, (E) GCE|ABAAb1(CEA, PSA, AFP)Ab2(PSA)|CdTe650, and (F) GCE|ABA-
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Ab1(CEA, PSA, AFP)Ab2(AFP)|CdTe776 formed with 20 L samples merely containing one target such as 10 ng/mL CEA for D, 100 ng/mL PSA for E, and 100 pg/mL AFP for F, respectively. ECL spectra are acquired by collecting all the ECL photons over 64 s in traditional accumulated way by scanning potential in 0.10 M pH7.4 PBS containing 0.10 M (NH4)2S2O8 from 0 to -1.60 V at 50 mV/s for one cycle. Herein, a spectrum-based triplex-color resolved ECL multiplexing immunoassay (MIA) was proposed for the first time to achieve NCs-based optical-multiplexing strategy with improved sensitivity by extensively exploring color-different monochromatic ECL between dual-stabilizers-capped CdTe and CdSe NCs, in which three existing monochromatic electrochemiluminophores, i.e. dual-stabilizers-capped CdSe550, CdTe650, and CdTe776 NCs were utilizied as tags (Figure 1A-C). The CdSe550, CdTe650, and CdTe776 NCs not only demonstrated well-defined first excitonic absorption peak around 535, 621 and 735 nm respectively, but also displayed strong PL around 550, 650, and 776 nm respectively, which indicated they were monodispersed and promising ECL tagging.46,48-50
PSA, AFP)>Ab2(CEA, PSA, AFP)|NCs(550,
650, 776) under the same condition. As dual-stabilizers-capped CdTe and CdSe NCs could preserve their highly-passivated surface-states in immunecomplexes, and give off monochromatic ECL spectrally similar to their PL, 49,50,52 it was clear that CEA, PSA and AFP were simultaneously immobilized onto GCE|ABA-Ab1(CEA, PSA, AFP) surface, and formed three surface-confined Ag-NCs pairs for ECLMIA.46,48 Actually, step by step electrochemical impedance spectroscopy (EIS) characterization on the fabricating procedure (Figure S1) also provided obvious evidence that Ab2|NCs conjugates were loaded onto GCE surface via the proposed immunoreaction strategy.52 Single component immunoassay (SIA) was firstly carried out as control, in which all the immune-complexes were formed with samples merely containing one target and displayed strong ECL in PBS containing (NH4)2S2O8 respectively (Figure S2-S4). ECL spectra of CdSe550, CdTe650, and CdTe776 in the immunecomplexes for ECL-SIA were much closer to their PL spectra respectively with almost identical maximum emission wavelengths and emission waveband respectively (Figure 1 and Figure 2A-C), which indicated all the three electrochemiluminophores preserved their ECL nature in complicated immuno-complexes, and were qualified for spectrum-based ECL assay.52
Scheme 1. Schematic Illustration of Spectral ECL-MIA: (A) fabricating procedure of ECL-MIA; (B) fabricating Ab2|NCs conjugates; (C) the traditional accumulated ECL spectra of (a) GCE, (b) GCE|ABA, (c) GCE|ABA-Ab1(CEA, PSA, AFP)Ab2(CEA, PSA, AFP), and (d) GCE|ABA-Ab1(CEA, PSA, AFP)Ab2(CEA, PSA, AFP)|NCs(550, 650, 776) in 0.10 M pH7.4 PBS containing 0.10 M (NH4)2S2O8. ECL spectra were acquired by collecting all the ECL photons over 64 s by scanning potential from 0 to -1.6 V at 50 mV/s for one cycle. The immune-complexes confined on GCE surface were formed with 20 L sample containing 10 ng/mL CEA, 100 ng/mL PSA, and 100 pg/mL AFP. Scheme 1 displayed the procedure of spectrum-based triplexcolor ECL-MIA: carcinoembryonic antigen (CEA), prostate specific antigen (PSA), and alpha fetoprotein (AFP) were adopted as model analytes; three Ab2|NCs conjugates, i.e. Ab2(CEA)|CdSe550, Ab2(PSA)|CdTe650, and Ab2(AFP)|CdTe776 were simultaneously immobilized onto glassy carbon electrode (GCE) via three parallel sandwich-typed immune-reaction in one-pot, while the immune-complexes were bound onto GCE via linker molecule p-aminobenzoic acid (ABA);52 ECL of three electrochemiluminophores were simultaneously conducted in presence of (NH4)2S2O8, ECL multiplexing signal was acquired on our home-made ECL spectrum acquiring system to perform ECLMIA.49-52 As displayed in Scheme 1C, no ECL was detected from GCE, GCE|ABA, and GCE|ABA-Ab1(CEA, PSA, AFP)Ab2(CEA, PSA, AFP) in co-reactant containing PBS, while strong ECL with three maximum emission wavelengths around 550, 650, and 776 nm was obtained with GCE|ABA-Ab1(CEA, PSA, AFP)
