Supporting Information. Ultrasensitive Bioanalysis of Lipopolysaccharide

Transcription

1 Supporting Information MoS 2 Quantum Dots as New Electrochemiluminescence Emitters for Ultrasensitive Bioanalysis of Lipopolysaccharide Min Zhao, An-Yi Chen, Dan Huang, Ya-Qin Chai, Ying Zhuo, 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: yingzhuo@swu.edu.cn (Y. Zhuo), yuanruo@swu.edu.cn (R. Yuan). S-1

2 Table of Contents Table S1.. S-3 Preparation of Circular Recognition Probe (CRP)... S-3 Native Polyacrylamide Gel Electrophoresis... S-3 Figure S S-4 Figure S2.... S-4 Electrochemical and ECL Characterization of the ECL Aptasensor...S-4 Optimal Conditions for the ECL Aptasensor..... S-7 Table S2.. S-8 Preliminary Analysis of Real Samples S-8 References.. S-9 S-2

3 Table S1. Sequence information for the nucleic acids used in this study. Name primer padlock probe Sequences (5 3 ) NH 2 -(CH 2 ) 6 -TTT TTT TTT TCA GAT GAT ATA AAG GG PO 4 -TCA TCT GCT TCT GCC CGC CTC CTT CCT AGC CGG ATC GCG CTG GCC AGA TGA TAT AAA GGG TCA GCC CCC CAG GAG ACG AGA TAG GCG GAC ACT CCC AAC CCG CCC TAC CCC TTT ATA (The blue underlined bases, orange underlined bases and pink underlined bases stand for LPS aptamer (I), complementary sequence of primer (c-primer) domain (II) and C-rich domain (III), respectively) Preparation of Circular Recognition Probe (CRP). Firstly, padlock probe (1.0 µm), dissolved with the Tris buffer (20 mm Tris-HCl and 5 mm MgCl 2 ), was heated to 95 C for 10 min and then naturally cooled down to room temperature to form the dumbbell-like secondary structure. Next, with the aid of the T4 DNA ligase (1 U/µL) and 1 T4 DNA ligase reaction buffer, the dumbbell-like padlock probe was ligated in the intramolecular reaction. Ultimately, in order to make the T4 DNA ligase inactivate, the above-mentioned system was terminated by a thermal treatment at 65 C for 15 min. The resultant circular recognition probe (CRP) was stored at 4 C when not in use. Native Polyacrylamide Gel Electrophoresis. The electrophoresis experiment of the different samples in the lanes of the fresh polyacrylamide gel (16 %) was executed at 120 V for 2.5 h in 1 TBE buffer. Next, the gel was further stained with ethidium bromide (EB) for 25 min, and then transferred to a dark box for photographing with a digital camera under UV light. S-3

4 EDX and FESEM of MoS Figure S1. EDX of MoS The inset: FESEM and composition of MoS DPVs of TEA Figure S2. DPVs of bare GCE detected in ph 8.0 PBS containing 20 mm TEA. Electrochemical and ECL Characterization of the ECL Aptasensor. In order to affirm the successful stepwise fabrication of the ECL aptasensor, the cyclic voltammograms (CVs) was performed in 0.1 M PBS (ph 8.0) containing 5 mm [Fe(CN) 6 ] 3-/4-. As the curve a displayed in Figure S3A, a pair of well-defined oxidation and reduction peaks of [Fe(CN) 6 ] 3-/4- was observed on the bare GCE. S-4

5 However, the currents decreased after the modification of the MoS layer own to the electronic hindrance of CS (curve b). When the primer was cross-linked onto the surface of the MoS with the aid of the glutaraldehyde, the peak currents were remarkably reduced (curve c), because of the repellence of [Fe(CN) 6 ] 3 /4 from approaching the modified electrode by the abundant negative charges of the DNA backbone. Next, a successive decrease of the peak currents was observed after blocking with the nonconductive BSA (curve d). When the mixture containing the target LPS (0.1 ng/ml), CRP, phi 29 DNA polymerase and dntps was dropped onto the surface of the modified electrode to initiate RCA, the peak currents were obviously declined (curve e). Because the large DNA molecule was generated to repel [Fe(CN) 6 ] 3 /4 closing to the sensing surface, indicating the successful formation of the aptamer recognition-driven target-cycling synchronized RCA. Sequentially, the prepared sensing surface was incubated with hemin and detected in 0.1 M PBS (ph 8.0). As the inset exhibited in Figure S3A, a notable reduction peak of hemin at V was observed (curve f), which illustrated the product of RCA with a G-rich sequence was obtained and further reacted with hemin to form the hemin/g-quadruplex. Moreover, ECL was also used to characterize the aptasensor. As demonstrated in Figure S3B (curve a), a weak ECL signal was observed from the MoS layer modified GCE. With the addition of TEA into the testing buffer, the ECL response dramatically raised (curve b). When the primer was cross-linked onto the modified electrode, an obvious decrease of the ECL was observed due to the DNA molecule S-5

6 hindering the electron transfer (curve c) 1. After blocking with MCH, the ECL sequentially decreased (curve d). Followed by the target-cycling synchronized RCA process, the ECL response sequentially decreased owing to the electronic inhibition of the large DNA molecule from RCA (curve e). When the modified electrode was incubated with hemin, a sharply reduced ECL response (curve f) was noticed as hemin quenched the ECL signal of MoS 2 QDs/TEA system via competing coreactant and electron transfer mechanism. Figure S3. CVs profiles (A) of the fabrication of the ECL aptasensor: (a) bare GCE, (b) MoS (c) primer/mos (d) MCH/primer/MoS (e) RCA/MCH/primer/MoS (tested in 5 mm [Fe(CN) 6 ] 3-/4- ) and the inset: (f) hemin/rca/mch/primer/mos (tested in 0.1 M PBS). The ECL intensity-time profiles (B) of (a) MoS (tested in 0.1 M PBS), and (b) MoS (c) primer/mos (d) MCH/primer/MoS (e) RCA/MCH/primer/MoS and (f) hemin/rca/mch/primer/mos (tested in 0.1 M PBS containing 20 mm TEA). S-6

7 Optimal Conditions for the ECL Aptasensor. The reaction time of RCA was a key factor to affect the sensing performance to complete the aptamer recognition-driven target-cycling synchronized RCA. As displayed in Figure S4A, it showed that ECL was increased with the reaction time from 0.5 ~ 2 h and then remained a relatively stable trend. Thus, 2 h was selected for the subsequent research. Additionally, considering the quenching of hemin toward the MoS 2 QDs/TEA ECL system, the effect of hemin concentration upon ECL was also investigated. Figure S4B depicted ECL was increased with the increasing hemin concentration from 0.01 mm ~ 0.1 mm and then reached a platform, indicating that 0.1 mm hemin attained saturation for the formation of hemin/g-quadruplex complex with the RCA product. Thus, the results showed that the optimum concentration of hemin was 0.1 mm. Figure S4. The effects of the reaction time of RCA (A) and the hemin concentration (B) toward the ECL responses of the developed aptasensor. S-7

8 Table S2. Comparison of the proposed method with some reported methods for LPS detection. Detection method Linear range LOD Ref Fluorescence 45.6 pg/ml ~ ng/ml 0.1 pg/ml 2 EIS 1.0 pg/ml ~ 1.0 ng/ml 1.0 pg/ml 3 CV 1.0 ng/ml ~ 100 ng/ml 4 DPV 1.0 ng/ml ~ 100 µg/ml 1.73 ng/ml 5 ECL 1.0 fg/ml ~ 100 ng/ml 0.18 fg/ml 6 ECL 0.1 fg/ml ~ 50 ng/ml 0.07 fg/ml This work Preliminary Analysis of Real Samples. To investigate the feasibility of the developed aptasensor in clinical application, the recovery experiments of different LPS concentrations in human serum were performed. The recovery was the ratio of the detected concentration calculated from the linear equation (defined as found) and the actual concentration (defined as added). As exhibited in Table S3, it could be seen that the recoveries were in the range from 99.30% to 103.7%, suggesting that the aptasensor has a promising potential application in real samples. Table S3. Recovery results of the developed aptasensor in human serum. Sample number Added / (ng/ml) Found / (ng/ml) Recovery / % S-8

9 References (1) Zhang, P.; Zhuo, Y.; Chang, Y. Y.; Yuan, R.; Chai, Y. Q.; Anal. Chem. 2015, 87, (2) Lan, M.; Wu, J.; Liu, W.; Zhang, W.; Ge, J.; Zhang, H.; Sun, J.; Zhao, W.; Wang, P.; J. Am. Chem. Soc. 2012, 134, (3) Su, W. Q.; Lin, M.; Lee, H.; Cho, M.; Choe, W. S.; Lee, Y.; Biosens. Bioelectron. 2012, 32, (4) Priano, G.; Battaglini, F.; Anal. Chem. 2005, 77, (5) Xie, P. Y.; Zhu, L. J.; Shao, X. L.; Huang, K. L.; Tian, J. J.; Xu, W. T.; Sci. Rep. 2016, 6, (6) Xie, S. B.; Dong, Y. W.; Yuan, Y. L.; Chai, Y. Q.; Yuan, R.; Anal. Chem. 88, 2016, S-9