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Supporting Information

Electrochemiluminescence Enhanced by Restriction of Intramolecular Motions (RIM): Tetraphenylethylene Microcrystals as a Novel Emitter for Mucin 1 Detection

Ming-Hui Jiang, Sheng-Kai Li, Xia Zhong, Wen-Bin Liang, Ya-Qin Chai, Ying Zhuo* and 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 400715, PR China

Table of Contents for Supporting Information

1.1 Reagents and Material. S-3

1.2 Apparatus. S-4

1.3 Zeta Potential Measurement of TPE MCs and Pd NPs. S-5

1.4 The ECL relative quantum efficiency of TPE. S-5

1.5 Raman spectra of the free TPE monomers and aggregate TPE MCs. S-6

1.6 Optimization of the Experimental Conditions. S-7

1.7 CV and ECL Characterizations of the Proposed Biosensor. S-8

1.8 Polyacrylamide Gel Electrophoresis (PAGE) Analysis. S-10

1.1 Reagents and Material.

Tetraphenylethylene (TPE) was brought from Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan). Potassium tetrachloropalladate(II) hydrate (K2PdCl4) was obtained from Adamas Reagent Co. Ltd. (Beijing, China). Tetrabutylammonium hexafluorophosphate (TBAPF6), hexanethiol (HT), sodium borohydride (NaBH4), Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) were provided by Sigma-Aldrich Co. (St. Louis, MO, USA). L-ascorbic acid (AA) was purchased from Chemical Reagent Co. Ltd. (Chongqing, China). Triethylamine (TEA), cetyltrimethylammonium bromide (CTAB), cetyltrimethyl ammonium chloride (CTAC), tetrahydrofuran (THF) and ethylenediaminetetraacetic acid (EDTA) were received from Kelong Chemical Inc. (Chengdu, China). Human Mucin 1 (MUC1) was provided by North Connaught Biotechnology (Shanghai, China). Exonuclease I (Exo I) was obtained from New England Biolabs (Ipswich, MA). The HPLC-purified oligonucleotides in this work were synthesized at Sangon Biotech Co. Ltd. (Shanghai, China) and its sequences were listed in Table 1. In addition, the buffers involved were presented as follows. The 1× TE buffer (pH 8.0) containing 10 mM Tris-HCl and 1.0 mM EDTA was employed for dissolving and storaging S3. Tris-HCl buffer (20 mM Tris-HCl, 200 mM NaCl, pH 7.4) was prepared for the dissolution, storage and annealing hybridization of S0, S1 and S2. 10× Exo I digestion reaction buffer (pH 7.9) was prepared with 200 mM Tris-HCl, 500 mM NaCl, 30 mM MgCl2. The phosphate buffered saline (PBS, 0.1 M, pH 7.4) for ECL test was composed of 0.1 M Na2HPO4, 0.1 M KH2PO4 and 0.1 M KCl. In this work, all the solutions were prepared using ultrapure water, which derived from Milli-Q water purification system with an electric resistance of 18.2 MΩ.

Table S1. The Sequences of Oligonucleotides Used in This Work

|name |Sequences(5′→3′) |

|S0 |5'-AGTAGCAGTTGATCCTTTGGATACCCTGGATCG-3' |

|S1 |5'-CGATCCAGGGTCCGAGCCGGTCGAACTGCTACTAT15CGCACAGAGGAGGGCCGTAAGTTAGTTGGAG-3' |

|S2 |5'-CGATCCAGGGTCCGAGCCGGTCGAACTGCTACTA |

| |T15CTCCAACTAACTTACGGCCCTCCTCTGTGCG-3' |

|S3 |5'-Fc-TAGTAGCAGrAGCCCTGGATCGTTTT(CH2)6-SH-3' |

1.2 Apparatus.

The ECL emission and cyclic voltammetric (CV) measurements were monitored, respectively, with a model MPI-E multifunctional analyzer (Xi'An Remax Electronic Science & Technology Co. Ltd., Xi'An, China) and a CHI660C electrochemical work station (Shanghai CH Instruments, China). The traditional three-electrodes system used with a modified glassy carbon electrode (GCE, Φ = 4 mm) as working electrode, aplatinum wire as auxiliary electrode and Ag/AgCl (saturated KCl) or saturated calomel electrode (SCE) as reference electrode in the test process. For photophysical characterization of tetraphenylethylene (TPE), a RF-5301PC spectrofluorophotometer (Shimadzu , Tokyo, Japan) with an excitation source provided by the 150 W xenon lamp (Ushio Inc., Japan) was utilized. The ECL emission spectrum of TPE MCs was obtained on a CHI 760E combined with a Newton EMCCD spectroscopy detector (Andor Co., Tokyo, Japan). A scanning electron microscope (SEM, S-4800, Hitachi, Tokyo, Japan) was used to characterize the surface appearance of different nanomaterials. The zeta potentials of particles were tested with a dynamic laser light scattering (ZEN3600, Malvern). The powder X-ray diffraction patterns of TPE MCs was collected via a XD-3 X-ray diffractometer (XRD, Purkinje, China).

1.3 Zeta Potential Measurement of TPE MCs and Pd NPs.

In order to study the interaction of layer-by-layer assembly in the fabrication process for biosensor, the zeta potentials of TPE MCs and Pd NPs were determined by a dynamic laser light scattering. As shown in Figure S1, the zeta potential value of TPE MCs (curve a) was positive with an average of 25.83 mV, which was opposite to that of Pd NPs (curve b) with an average of -12.27 mV. Thus, this result certified that the Pd NPs could be adsorbed on the surface of TPE MCs layer via electrostatic interaction.

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Figure S1. Zeta potential of (a) TPE MCs and (b) Pd NPs under three parallel experiments.

1.4 The ECL relative quantum efficiency of TPE.

We used the bare GCE as a standard to measured the ECL relative quantum efficiency (Φ’)1. Firstly, the cyclic voltammetric response of bare GCE in 0.1 M TBAPF6 THF solution (containing 1 mg/mL TPE monomer and 20 mM TEA) and in 0.1 M PBS (containing 1 mg/mL TPE MCs and 20 mM TEA) were obtained, respectively. Then, during the scanning process, the charge for the oxidation of TPE (Q) was acquired by the integral difference between the solution containing TPE and the blank solution. According to equation 1, the number of electrochemically oxidized TPE molecules (nTPE) could be calculated roughly. Among them, z is the number of transferred electrons for per molecule, and F is Faraday’s constant. Subsequently, the ECL quantum efficiency (Φ) for two cases were calculated according to equation 2.

nTPE = Q/z F (equation 1)

Φ = k IECL/nTPE (equation 2)

And, the results of ECL relative quantum efficiency for TPE were presented in Table S2, where the ECL quantum efficiency obtained at 0.1 M PBS containing 1 mg/mL TPE MCs and 20 mM TEA was about 12.7 times higher than that of at 0.1 M TBAPF6 THF solution containing 1 mg/mL TPE monomer and 20 mM TEA. Therefore, compared with the TPE monomer dissolved in organic phase, the TPE MCs increased the ECL efficiency to a certain degree.

Table S2. The ECL relative quantum efficiency of TPE

|State |IECL |Q |Φ’(Φ①/Φ②) |

|① TPE MCs in PBS |1122 |0.22 |12.7 |

|② TPE monomer in THF solution |68 |0.28 | |

1.5 Raman spectra of the free TPE monomers and aggregate TPE MCs.

To support the speculation that the formation of microcrystals could decrease the rate of non-radiative decay, the Raman spectra of the free TPE monomers and aggregate TPE MCs were performed, respectively (Figure S2). According to the reported work, the bands in the region 1200-1300 can be assigned to C-H in-plane bending vibrational modes2. Notably, it could be found that the TPE monomers (curve a in Figure S2) exhibited a obvious C-H vibrational band in 1200-1300, while the TPE MCs (curve b in Figure S2) showed a very weak band in the same region. Therefore, the result just support our hypothesis that the formation of microcrystals could decrease the rate of non-radiative decay.

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Figure S2. Raman characterization of the free TPE monomers (curve a) and aggregate TPE MCs (curve b).

1.6 Optimization of the Experimental Conditions.

To achieve the best assay performance of the developed biosensor, two crucial experimental conditions were optimized. Figure S3A first showed the effect of different reaction time of TICEA. It could be seen that the ECL intensity consecutively increased with the extension of reaction time and trended to a plateau at 90 minutes. Thus, the reaction time of TICEA was determined to be 90 minutes. On the other hand, in the presence of Pb2+, the incubation time between the S1/S2 and S3-Fc played an important role, which would directly influence the recovery of ECL signal. As a result, with the increase of incubation time, a gradual increased ECL response was presented in Figure S3B. And then, the ECL signal reached a platform at 90 minutes, which was served as the optimal incubation time in the work.

[pic]

Figure S3. Experimental optimizations of the proposed biosensor for MUC1 detection. (A) The reaction time of TICEA and (B) The incubation time between the S1/S2 and S3-Fc in the presence of Pb2+.

1.7 CV and ECL Characterizations of the Proposed Biosensor.

To verify the successful fabrication of the proposed ECL biosensor, the cyclic voltammetric (CV) studies were implemented in the presence of [Fe(CN)6]3-/4- solution (5 mM, acting as the redox probe), as displayed in Figure S4A. A pair of obvious redox peaks were observed on the bare GCE (curve a), while them were decreased after the modification of TPE MCs (curve b) due to the electrostatic hindrance of TPE MCs. After the PdNPs were decorated on the surface of the above modified electrode, the redox peak current slightly increased (curve c), indicating that the large surface area and superior conductivity of PdNPs were beneficial to electron transport. However, the consecutive decline of redox peak currents were presented when the Fc-S3 (curve d) and HT (curve e) were successively incubated on the modified electrode for the reason that the negatively charged DNA and HT hindered the electron transfer. Subsequently, with the S1/S2 and Pb2+ solution incubated on the above modified electrode, an increased redox peak current was acquired (curve f).

In addition, the ECL intensity-time responses were used to further characterize the biosensor. As the curve a showed in Figure S4B, a weak ECL signal was observed on the TPE MCs modified GCE in 0.1 M PBS (pH 7.4). And, the ECL intensity was increased significantly (curve b) with the introduction of TEA solution (20 mM) in the test buffer. When the PdNPs were coated on the surface of above modified electrode, a slight augment of the ECL signal was presented in curve c. As expected, the ECL intensity dramatically decreased (curve d) after successful modification with the Fc-S3, which was ascribed to the efficient quenching effect of Fc molecule on TPE MCs luminescence. After blocking with HT, the ECL signal further decreased (curve e) owing to the insulation of HT. However, when the above modified electrode was incubated with the S1/S2 resulted from TICEA process and the Pb2+ solution, the ECL response was enhanced again (curve f). It could be speculated that the quenching probe of Fc-S3 was irreversibly self-cleaved for dissociating the Fc molecule from the electrode surface and achieving the ECL signal recovery. The results of the CV measurement and ECL characterization consistently proved the successful fabrication of the proposed biosensor.

[pic]

Figure S4. (A) CV characterizations of (a) bare GCE, (b) TPE MCs/GCE, (c) PdNPs/TPE MCs/GCE, (d) Fc-S3/PdNPs/TPE MCs/GCE, (e) HT/Fc-S3/PdNPs/TPE MCs/GCE, (f) S1/S2 (Pb2+)/HT/Fc-S3/PdNPs/TPE MCs/GCE. (B) ECL response profiles of (a) TPE MCs/GCE (tested in 0.1 M PBS), and (b) TPE MCs/GCE, (c) PdNPs/TPE MCs/GCE, (d) Fc-S3/PdNPs/TPE MCs/GCE, (e) HT/Fc-S3/PdNPs/TPE MCs/GCE and (f) S1/S2 (Pb2+)/HT/Fc-S3/PdNPs/TPE MCs/GCE (tested in 0.1 M PBS containing 20 mM TEA).

1.8 Polyacrylamide Gel Electrophoresis (PAGE) Analysis.

The successful operation of the target-activated bipedal DNA walker was confirm by PAGE. The final concentration of DNA strands and target MUC1 were 1 μM and 100 ng/mL, respectively. Other auxiliary reagents were added according to the preceding testing process. As shown in Figure S5, the bright strand in lane 2 (S0-S1/S2-S0) posed a higher position compared with that in lane 1 (S1/S2), because S0-S1/S2-S0 as a three-stranded DNA duplex structure had a larger molecular weight. However, both lane 1 and lane 2 exhibited a dim stripe at the bottom, which could be attributed to a spot of single strands that didn’t hybridize. Subsequently, the stator in lane 3 containing S0-S1/S2-S0 + Exo I (Exo I, a sequence-independent nuclease that catalyze the stepwise hydrolysis of single-stranded DNAs along the 3’-terminus to 5’-terminus direction, and it also successfully digested the aptamer DNA in the protein-aptamer complex3.) showed no difference compared to the bright strand of lane 2, indicating the nicking process was not occurred on hybridized S0 due to the absence of target MUC1. After introducing the target MUC1 into the above mixture, a bright strand of lane 4 (S0-S1/S2-S0+ MUC1 + Exo I) appeared in a lower position comparing with that of lane 3. This result demonstrated the aptamer (S0)-target complex could be digested by Exo I to output the S1/S2. Comparing the PAGE images of lane 5 (lane 4 + S3 + cofactor Pb2+) to lane 4, two dim new bands (in the red dotted line box) in last place were observed, which proved some short DNA sequences were produced and the DNAzyme cleavage process was occurred as expected.

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Figure S5. Native PAGE analysis of different samples. lane 1: S1/S2; lane 2: S0-S1/S2-S0; lane 3: S0-S1/S2-S0+ Exo I; lane 4: S0-S1/S2-S0+ MUC1 + Exo I; lane 5: lane 4 + S3 + cofactor Pb2+.

Reference

Qiao, Y. L.; Li, Y.; Fu, W.; Guo, Z. H.; Zheng, X. W. Enhancing the electrochemiluminescence of luminol by chemically modifying the reaction microenvironment. Anal. Chem. 2018, 90, 9629-9636.

Krishnakumar, V.; Balachandran, V. FTIR, FT-Raman spectral analysis and normal coordinate calculations of 2-hydroxy-3-methoxybenzaldehyde thiosemicarbozone. Indian J. Pure Ap. Phy. 2004, 42, 313-318.

Wu, Z.; Zhen, Z.; Jiang, J. H.; Shen, G. L.; Yu, R. Q. Terminal protection of small-molecule-linked DNA for sensitive electrochemical detection of protein binding via selective carbon nanotube assembly. J. Am. Chem. Soc. 2009, 131, 12325-12332.

*Corresponding author. Tel: +86-23-68253172; Fax: +86-23-68253172.

E-mail address: yingzhuo@swu.. (Y. Zhuo).

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