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An Ultrasensitive Electrochemiluminescent Sensor for MicroRNA with Multinary Zn-Ag-In-S/ZnS Nanocrystals as Tags Bin Zhang, Fang Zhang, Ping Zhang, Dazhong Shen, Xuwen Gao, and Guizheng Zou Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b00199 • Publication Date (Web): 14 Feb 2019 Downloaded from http://pubs.acs.org on February 16, 2019
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Analytical Chemistry
An Ultrasensitive Electrochemiluminescent Sensor for MicroRNA with Multinary Zn-Ag-In-S/ZnS Nanocrystals as Tags Bin Zhang,† Fang Zhang,† Ping Zhang,‡ Dazhong Shen,‡ Xuwen Gao,† Guizheng Zou†,* †School
of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, China of Chemistry, Chemical Engineering and Materials Science, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Normal University, Jinan 250014, PR China ‡College
ABSTRACT: To screen novel toxic-element-free and biocompatible electrochemiluminophores, electrochemluminescence (ECL) of multinary nanocrystals (NCs) was investigated for the first time with Zn-Ag-In-S (ZAIS) NCs as model. Aqueous soluble ZAIS NCs could bring out efficient reductive-oxidation ECL with tri-n-propylamine as co-reactant, while coating the ZAIS NCs with a ZnS shell could reduce the surface defects of ZAIS NCs, and enable 6.7 folds enhanced ECL of ZAIS/ZnS NCs than ZAIS NCs. ECL of ZAIS/ZnS NCs was about 4.2 folds to that of ternary CuInS2/ZnS NCs, spectrally similar to that of Ru(bpy)32+ with maximum emission around 605 nm, and favorable for less electrochemical interference with a lowered triggering potential (~0.95 V) than that of Ru(bpy)32+. An ultrasensitive ECL microRNA sensor was fabricated with ZAIS/ZnS NCs as tags, which could sensitively and selectively determine microRNA-141 with a wide linearity range from 0.1 fmol/L to 20 pmol/L and a low limit of detection at 50 amol/L (S/N = 3). Multinary NCs might provide a promising alternative to the traditional binary NCs for both electrochemiluminophore screening and NCs ECL modulating.
Electrochemiluminescence (ECL) was a promising analytical technique and has aroused widespread concerns due to its unique superiorities, such as low background, high sensitivity, electrochemical controllability, and wide dynamic range for sensing.1-4 ECL usually generated via charge-transfer between electrochemiluminophore of oxidized states and reduced states.4-6 Since Bard demonstrated that C2O42− could be electrochemically oxidized to produce strong reducing radicals and then react with the electrochemiluminophore of oxidized states to produce ECL,7 ECL between various electrochemiluminophores and co-reactants couples were extensively explored,1,5 as directly adding co-reactant into ECL solution was a simple and effective way for ECL assay.6,8 Investigations on screening novel electrochemiluminophores and co-reactants were crucial to ECL evolution.9-11 For example, the discovery of electrochemiluminophore Ru(bpy)32+ in 1972 and co-reactant n-propylamine (TPrA) in 1987 eventually brought out a serials Ru(bpy)32+/TPrA reagent kits for biomedical and diagnostic ECL assays.5,12,13 Since the breakthrough towards ECL bio-sensing with non-moleculetyped CdSe NCs as electrochemiluminophores,14 ECL of various binary NCs were extensively investigated in the past decades,1,15 such as PbS,16,17 CdSe,18 CdTe,19 and CdSe/ZnSe.20 The environmental and bio-compatible concerns of II-VI NCs made ECL from toxic-element-free Au,21 Ag,22 and C3N423,24 NCs aroused much attention. Recently, our group presented a promising alternative to screening environmental-friendly electrochemiluminophores with ternary I–III–VI CuInS2 (CIS) NCs as the model.25,26 Inspired by these achievements, herein, ECL of water-soluble and toxic-element-free multinary Zn-Ag-In-S (ZAIS) NCs and
its possible sensing application were investigated for the further evolution of ECL, because coating the ZAIS NCs with a ZnS shell could enable dramatically enhanced ECL of ZAIS/ZnS NCs, which was not only much stronger than the ECL of previously reported ternary CIS/ZnS NCs,26 but also in a similar color to the ECL of traditional Ru(bpy)32+,27 and generated at a lowered ECL potential than Ru(bpy)32+ for less electrochemical interference.5,28 From traditional binary II-VI NCs to ternary CIS NCs,26,29 then to multinary ZAIS/ZnS NCs with improved ECL performance step by step, this work might present a promising strategy to screen novel electrochemiluminophores and modulate NCs ECL. EXPERIMENTAL SECTION Chemicals and Materials. All chemicals and reagents were of analytical grade or better, and all aqueous solutions were prepared with double distilled water (DDW) (see Supporting Information). CIS/ZnS was prepared and purified according to previously reported procedures.26 Table 1. The peptide and oligonucleotides sequences Name tDNA miRNA-141 pDNA
Sequence 5′-NH2-(CH2)6-CCA TCT TTA CC-P-3′
5′-SH-(CH2)6-CCA T
5′-UAA CAC UGU CUG GUA AAG AUG G-3′ 5′-AGA CAG TGT TAC GA-(CH2)6-SH-3′
miRNA-155
5′-UUA AUG CUA AUC GUG AUA GGG GU-3′
miRNA-21
5′-UAG CUU AUC AGA CUG AUG UUG A-3′
miRNA-429
5′-UAA UAC UGU CUG GUA AAA CCG U-3′
DNA and microRNA (miRNA) sequences (Table 1) were obtained from Sangon Biological Engineering Technology &
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Services Company Ltd. (Shanghai, China) and purified with high performance liquid chromatography. Probe immobilization buffer (PIB) consisting of 10 mmol/L Tris-HCl, 1.0 mmol/L ethylenediaminetetraacetic acid (EDTA), 100 mmol/L NaCl and 10 mmol/L tris(2-carboxyethyl) phosphine hydrochloride (TCEP) (pH 7.4) was employed to dissolve the probe DNA (pDNA). Tris-acetate-EDTA-Mg2+ buffer (TAE/Mg2+) containing 40 mmol/L Tris, 2.0 mmol/L EDTA, 12.5 mmol/L Mg(CH3COO)2 and 20 mmol/L CH3COOH (pH 8.0) was used for dissolving the miRNA-141 captured trigger DNA (tDNA). Hybridization buffer (HB) prepared with 10 mmol/L Tris-HCl, 1.0 mmol/L EDTA, 200 mmol/L NaCl and 10 mmol/L MgCl2 (pH 8.0) was used to dissolve miRNAs. Preparation of L-Cysteine Capped ZAIS and ZAIS/ZnS NCs. An aqueous synthetic approach was used to prepare ZAIS and ZAIS/ZnS NCs.30 Briefly, 5.5 mL DDW, 2.93 mg of AgNO3, 48.66 mg of In(CH3COO)3, and 22.75 mg of Zn(CH3COO)2·2H2O were loaded into a three-neck flask under continuous stirring. Then, 2 mL 0.6 mol/L L-cysteine was added, and 6 mol/L NaOH was introduced in to adjust pH to 8.5. After adding 6.5 mL of 50 mmol/L thioacetamide (TAA), the mixture was sealed in a Teflon-lined stainless steel autoclave and maintained at 110 °C for 240 min. To prepare ZAIS/ZnS NCs, 10 mL of ZAIS NCs was heated to 100 °C, and refluxed with a condenser. After 200 μL of 50 mmol/L of Zn(CH3COO)2·2H2O solution was injected into the solution under continuous stirring, the mixture was refluxed for 120 min. The resultant ZAIS and ZAIS/ZnS NCs were centrifuged with ethanol for three times, then re-dispersed in DDW and stored at 4 °C. ZAIS and ZAIS/ZnS NCs stock solutions were determined to be 2.5 mg/mL respectively, and directly used for optical characterization.
The freshly-pretreated GCE was firstly immobilized with ABA to form GCE-ABA,33 and then reacted with a drop (20 μL) of 2.0 μmol/L tDNA at 37 °C for 3 h to form GCE-ABA-tDNA with the assistance of 1-ethyl-3-(3-dimethyl-amino-propyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS).29 GCE-ABA-tDNA was then blocked with 1 wt % BSA and incubated with a drop (20 μL) of miRNA-141 at 37 °C for 2 h to form GCE-ABA-tDNA
