Supporting Information. Hollow Porous Polymeric Nanospheres of Self-Enhanced. Ruthenium Complex with Improved Electrochemiluminescent
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1 Supporting Information Hollow Porous Polymeric Nanospheres of Self-Enhanced Ruthenium Complex with Improved Electrochemiluminescent Efficiency for Ultrasensitive Aptasensor Construction Anyi Chen, Min Zhao, Ying Zhuo, Yaqin Chai, Ruo Yuan Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing , China Corresponding authors at: Tel.: , fax: addresses: (Y. Zhuo), Yuan). S1
2 Table of Contents for Supporting Information Table S1 Figure S1 Figure S2 Figure S3 Figure S4 Sequence information for the nucleic acids. TEM image of the Ru-HPNSs. UV-vis absorption spectra of the reagents. SEM image of Ru(mcbpy)(bpy) 2 Cl 2 particles. Fluorescent intensities and emission spectra of Ru(mcbpy)(bpy) 2 Cl 2 in different concentrations. Figure S5 Figure S6 Elemental analysis of the Ru-HPNSs. CV and ECL curves of Ru(mcbpy)(bpy) 2 Cl 2 in different concentrations. Figure S7 Optimization of the layers of {PEI-Ru/PAA} n. Figure S8 Table S2 Table S3 Calculation of the limit of detection. Analysis of MUC1 in clinical samples. Comparison of different methods for MUC1 detection. S2
3 Table S1 Sequence Information for the Nucleic Acids Used in This Study. Name Sequences* (5 3 ) H1 GCA GTT GAT CCT TTG GAT ACC CTG GTT CTC TCT CTG TAT CCA AAG H2 HS-(CH 2 ) 6 -TTT CTT TGG ATA CAG AGA GAG ATG TGT AGC TCT CTC TG H3 HOOC-TTT CAG AGA GAG CTA CAC ATC TCT CTC TGT ATC CAA AG Materials and Reagents. HPLC-purified DNA oligonucleotides were synthesized by Sangon Inc. (Shanghai, China) and the sequences were showed in Table S1. MUC1 (16 ng/ml) from human breast cancer cell surface were bought from Shanghai North Connaught Biotechnology Co. Ltd. (Shanghai, China). Bis(2,2-bipyridyl)(4-methyl-4-carboxypropyl-2,2-bipyridyl) ruthenium(ii) hydrochloride (Ru(mcbpy)(bpy) 2 Cl 2 ) was acquired from Suna Tech Inc. (Suzhou, China). Polyethyleneimine (PEI, branched, 70 kda) was purchased from Macklin Biochemical Co., Ltd (Shanghai, China). N-hydroxy succinimide (NHS) and 1-ethyl-3-(3-di-methylaminopropyl) carbodiimide (EDC) hydrochloride, polyacrylic acid (PAA), tetraethyl orthosilicate (TEOS) and chloroplatinic acid (H 2 PtCl 6 ) was purchased from J&K Scientific Ltd. (Beijing, China). The buffers involved in this work were prepared as follows: The 1 TE buffer (10 mm Tris-HCl, 1.0 mm ethylenediaminetetraacetic acid (EDTA), ph 8.0) was used for the dissolution and storage of H1 and H2. HEPES buffer (10 mm HEPES, 150 S3
4 mm NaCl, 10 mm MgCl 2, ph 7.0) was used for the dissolution and storage of H3 and the annealing of hairpin H1, H2 and H3. The 0.1 M phosphate buffered saline (PBS, ph 7.4) was prepared with 0.08 M Na 2 HPO 4, 0.02 M KH 2 PO 4 and 0.1 M KCl. Deionized water (specific resistance of 18.2 MΩ cm) was used to prepare all the aqueous solutions. Apparatus. The ECL measurements were performed on a model MPI-A ECL analyzer (Xi An Remex Electronic Science & Technology Co. Ltd., Xi An, China) with a conventional three-electrode system used with Ag/AgCl (saturated KCl) as the reference electrode, a platinum wire as auxiliary electrode, and a modified glassy carbon electrode (GCE, Φ = 4 mm) as the working electrode in the experiment. Electrochemical measurement was carried out with a CHI660C electrochemistry workstation (Shanghai Chenhua Instruments, China). The transmission electron microscope (TEM) images were achieved by a JEM 1200EX TEM (JEOL Ltd, Tokyo, Japan). The surface appearance of the materials was characterized by S4800 scanning electron microscope (Hitachi, Tokyo, Japan) at an acceleration voltage of 25 kv. X-ray photoelectron spectroscopy (XPS, Thermoelectricity Instruments, U.S.A.) were used for elemental analysis. UV vis absorption spectra were carried out on a UV-2450 UV vis spectrophotometer (Shimadzu, Tokyo, Japan). Fluorescence spectra were carried out on FL-5700 spectrophotometer (Hitachi, Tokyo, Japan) at room temperature. Native polyacrylamide gel electrophoresis was performed with BG-verMIDI standard S4
5 vertical electrophoresis apparatus (Baygene, Beijing, China), surface plasmon resonance (SPR) spectra were measured with a time-resolved SPR system (DyneChem. Preparation of SiO 2 Nanoparticles. The SiO 2 nanoparticles were prepared referring to the reported works 1,2 with slight variation. Ammonium hydroxide (3 ml, 30%) was mixed with 50 ml absolute ethanol, followed by rapid addition of the precursor TEOS (1.5 ml) with vigorously stirring. After the colorless solution turned into ivory emulsion, the solution was sequentially stirred for 12 h. Then the ivory emulsion was collected by centrifugation (12000 rpm, 5 min) and washed with absolute ethanol, deionized water, and absolute ethanol in succession for 3 times. Finally, the products were dried with vacuum dryer for further use. Native Polyacrylamide Gel Electrophoresis (PAGE). The samples were loaded into the notches of the freshly prepared native polyacrylamid gel (16%), and electrophoresis was performed at 120 V for 180 min in 1 TBE buffer. After dying with ethidium bromide (EB), the gel was transferred to dark box for gel imaging. S5
6 Figure S1 TEM image of the Ru-HPNSs with relative low magnification. Figure S2 UV-vis absorption spectra of PAA, PEI, Ru(mcbpy)(bpy)2Cl2, and PEI-Ru, where Ru(II) stands for Ru(mcbpy)(bpy)2Cl2. S6
7 Figure S3 SEM image of Ru(mcbpy)(bpy) 2 Cl 2 particles. Figure S4 (A) Fluorescent intensities and (B) fluorescent emission spectra of Ru(mcbpy)(bpy) 2 Cl 2 in different concentrations. S7
8 Elemental analysis of the Ru-HPNSs Elemental analysis of the Ru-HPNSs was based on the deconvolution of XPS peaks associated with nitrogen, ruthenium and carbon. As shown in Figure S5A, the C1s-Ru3d core levels spectrum of XPS for the Ru-HPNSs displayed three remarkable peaks at ev, ev, and ev. The peak at ev was well corresponding to the binding energy associated with Ru3d 5/2 photoelectrons, indicating the successfully immobilization of Ru complex. The peaks at ev, and ev could be deconvoluted into several components associated with the carbon contribution (C1s) and Ru3d 3/2 photoelectrons. According to the structure of PAA, the C1s-Ru3d core level should present a defined peak centred at associated with the carboxyl group from PAA. 3 However, the deconvolution of the experimental spectrum showed an XPS component at ev associated with amide group, 4 indicating the crosslinking of PAA and PEI. The other components centred at ev, ev ev and ev were ascribed to the carbon atoms involved in the groups of CH 2 (PAA), CH (PAA), CH 2 -amide (PEI) and CH 2 -amine (PEI), respectively. 3, 5, 6 Figure S5B displayed the N1s core levels spectrum of XPS for the S8
9 Ru-HPNSs, exhibiting a single peak at ev. The analysis of spectrum showed that this single peak involved two XPS components at ev and ev. Differing from the main XPS component at ev associated with PEI, the contribution at ev was assigned to the nitrogen atoms from bipyridine involved in ruthenium complexation 5, 7. While another XPS component of at ev associated with PEI wasn t observed, which reconfirmed the crosslinking of PEI and PAA. Figure S5C demonstrated the full regeion of XPS for the Ru-HPNSs, which surveyed the atomic ratios of C, N, O, Ru. According to the atomic ratios, the molar ratio between Ru and PEI was calculated to be about 75:1, exhibiting good immobilization of Ru luminophores. Figure S5 XPS analysis for (A) the C1s-Ru3d core levels spectrum, (B) the N1s core levels spectrum, and (C) the full region of XPS for the Ru-HPNSs. S9
10 Figure S6 (A) CV and (B) ECL curves of Ru(mcbpy)(bpy) 2 Cl 2 in different concentrations range from 1.0 mm to 20 mm. S10
11 Optimization of the layers of PEI-Ru In order to prepare Ru-HPNSs with efficient ECL for improving the sensitivity of MUC1 detection, the layers of PEI-Ru were the key factors in the preparation process. Specifically, a batch of PAA n-1 /PEI-Ru n (n=1, n=2, n=3, n=4, n=5) encapsulated SiO 2 composites were prepared and further etched by HF to obtain the Ru-HPNSs as the detection probes. Then, the developed aptasensors were incubated with 100 pg/ml MUC1 and above-prepared detection probes. As shown in Figure S7, it was seen that the ECL intensity increased with the increase of n from 1 to 3, and then tended to a stable state gradually with the increase of n from 3 to 5. Thus, 3 layers of PEI-Ru were selected for the assays ECL intensity / a.u N Figure S7 Optimizations of the layers of Ru-PEI in {PEI-Ru/PAA} n (n=1, n=2, n=3, n=4, n=5) on ECL intensity of the MUC1 aptasensor. S11
12 Limit of detection calculation. The calibration plot at low concentrations of MUC1 was shown in Figure S8B. To calculate the limit of detection, an ECL measurement for blank samples was executed with three parallel tests first, which exhibited average ECL intensity (I B ) of 71 with standard deviation (s B ) of With signal-to-noise ratio value (k) of 3, the smallest detectable signal (I L ) could be calculated as I L = I B + k s B = 137 (1) Then the point with ECL intensity of 137 was picked out on the calibration plot at low concentration of MUC1. Thus the limit of detection was calculated to be 0.31 fg/ml. Figure S8 Calibration plot to (A) whole linear range and (B) low concentrations of MUC1. S12
13 Analysis of MUC1 in clinical samples To discuss the feasibility of the developed aptasensor in real samples, standard addition method-based recovery experiments were carried out using the human serum as real samples. Firstly, the healthy human whole blood samples from Xinqiao Hospital of Chongqing were centrifuged at 3500 rpm for 25 min to get purified human serum, and then the human serum was diluted for 40-fold with PBS buffer for MUC1 dissolution. As displayed in Table S2, the recovery was in the range of 91% ~ 105%, which confirmed that the proposed sensor could determine MUC1 in human serum. Table S2 Recovery of MUC1 in human serum samples by the proposed sensor. Sample number Added (M) Found (M) a Recovery (%) ± ± ± ± ± a Mean value ± SD of three measurements. S13
14 Table S3 Different aptasensors with different methods for MUC1 detection. Method Linear range LOD Ref. Fluorescence 0.04 ~ 10 µm 28 nm 8 Fluorescence 0.01 nm ~ 5.0 nm 3.33 pm 9 Electrochemical 1.0 ~100 nm 1.0 pm 10 Electrochemical 1.0 nm ~ 1.0 µm nm 11 ECL 10 fg/ml ~ 30 ng/ml 2.8 fg/ml 12 ECL 1.0 fg/ml ~ 100 pg/ml 0.31 fg/ml This work S14
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