Real-time, Quantitative Lighting-up Detection of Telomerase in Urines of Bladder Cancer Patients by AIEgens

Transcription

1 Supporting Information Real-time, Quantitative Lighting-up Detection of Telomerase in Urines of Bladder Cancer Patients by AIEgens Xiaoding Lou,, Yuan Zhuang,, Xiaolei Zuo,, Yongmei Jia, Yuning Hong, Xuehong Min, Zhenyu Zhang, Xuemei Xu, Nannan Liu, Fan Xia, *, and Ben Zhong Tang Key Laboratory for Large-Format Battery Materials and System, Ministry of Education, School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology (HUST), Wuhan , China. Division of Physical Biology & Bioimaging Center, Shanghai Synchrotron Radiation Facility, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai , China School of Chemistry, The University of Melbourne, Parkville, Victoria 3010, Australia. Department of Chemistry, HKUST Jockey Club Institute for Advanced Study Division of Life Science, Institute of Molecular Functional Materials and Division of Biomedical Engineering, The Hong Kong University of Science and Technology (HKUST), Clear Water Bay, Kowloon, Hong Kong, China. These authors contribute equally. * Correspondence and requests for materials should be addressed to F. X. ( xiafan@hust.edu.cn). ABSTRACT: As a biomarker for early cancer diagnosis, telomerase are one of the promising targets for cancer therapeutics. Inspired by the fluorescent emission principle of aggregation-induced emission fluorogens, we creatively designed an AIE-based turn-on method to detect telomerase activity from cell extracts. A positively charged fluorogen (TPE-Z) is not fluorescent when freely diffused in solution. The fluorescence of TPE-Z is enhanced with the elongation of the DNA strand which could light up telomere elongation process. By exploitation of it, we can detect telomerase activity from different cell lines (E-J, HeLa, MCF-7, and HLF) with high sensitivity and specificity. Moreover, our method is successfully employed to demonstrate the applications in bladder cancer diagnosis (41 urine specimens from bladder cancer patients and 15 urine specimens from normal people are detected). The AIE-based method provides a simple one-pot technique for quantification and monitoring of the telomerase activity and shows great potential for future use in clinical tests. S1

2 Table of the contents: 1. Experimental section 1.1 Fluorescence measurement 1.2 Investigation of telomerase inhibition 1.3 Non-denaturating polyacrylamide gel electrophoresis (PAGE) analysis 1.4 Telomerase activity detection using commercial kit 2. Figures&Tables Figure S1. Schematic illustration of AIE-based turn on technique for different length of single-stranded DNA Figure S2. Time-dependent emission spectra of TPE-Z in the presence of telomerase Figure S3. FL spectra of TPE-Z in the presence of different concentration of TS primer Figure S4. FL spectra of TPE-Z in the presence of different concentration of dntps Figure S5. FL spectra of TPE-Z in the absence and presence of TS primer with telomerase extracted from 25000MCF-7 cancer cells Figure S6. FL spectra of TPE-Z in the absence and presence of TS primer with telomerase extracted from E-J cancer cells Figure S7. FL spectra of TPE-Z in the absence and presence of TS primer with telomerase extracted from HeLa cancer cells Figure S8. FL spectra of TS primer and telomerase in the absence and presence of TPE-Z Table S1. Comparison between the current telomerase activity detection assay and other previously reported optical methods Table S2. Oligonucleotides used in this study Figure S9. FL spectra of TPE-Z in the presence of different oligonucleotides Figure S10. Non-denaturating PAGE analysis of telomerase extension assay Figure S11. ELISA Kit assay of extracts from E-J cancer cells and urine samples Table S3. Activity of telomerase extracted from patients urine specimens using human telomerase (TE) ELISA Kit. Figure S12. Fluorescence responses to telomerase extracts from HeLa cells treated with AZT S2

3 Figure S13. Results of AIE-based telomerase activity detection in real normal urine samples Figure S14. Photograph of patients clear and bloody urine specimen Table S4. Corresponding disease information to the bladder cancer urine samples Table S5. Corresponding information of the urine samples from normal people Figure S15. Classification of patients urine samples 3. References S3

4 1 Experimental section 1.1 Fluorescence measurement The fluorescence detection was carried out on a Cary Eclipse Fluorescence Spectrophotometer (Agilent Technologies). The excitation wavelength was 350 nm and the emission wavelengths were in the range from 400 to 600 nm with the slit widths of both excitation and emission of 10 nm. 1.2 Investigation of telomerase inhibition In the telomerase inhibition experiments, 1 mm AZT was added into telomerase extracts from MCF-7 cells to incubated at 37 ºC for 30 min before use. 1.3 Non-denaturating polyacrylamide gel (PAGE) analysis Telomerase extracts from E-J cells, HeLa cells and MCF-7 cells were incubated with 4.8 mm dntps, 20 μm TS primer and 40 U RNase inhibitor in telomerase extension reaction buffer with the total volume of 50 μl at 37 ºC for 60 min. For the control experiment, 20 μm TS primer in telomerase extension reaction buffer with the total volume of 50 μl was incubated at 37 ºC for 60 min. A 10% non-denaturing PAGE analysis of 5 μl mixture was carried out in 1X TEB buffer at 90 V constant voltage for about 1 h. Then the gels were stained with 3X Gel Red in 100 ml 1X TBE buffer for 30 min and imaged with Gel-Pro TRANSILLUMINATOR 2020D (Carestream Health). 1.4 Telomerase activity detection using commercial kit We purchased the Human telomerase (TE) ELISA Kit from Shanghai Qiaodu Biotechnology CO.,LTD. First, we performed a relative standard curve using standard telomerase activity samples (40IU/L, 20IU/L, 10IU/L, 5IU/L, 2.5IU/L, 0IU/L) from Human telomerase (TE) ELISA Kit. The telomerase activity of different samples are evaluated by comparison with the standard curve of ELISA Kit that can generate the same level of signal as the samples. S4

5 2 Figures & Tables Figure S1. The scheme and fluorescence emission spectra of TPE-Z in the presence of (a) Ex-0 (18-nt), (b) Ex-4 (42-nt), (c) Ex-6 (54-nt). Inset: fluorescence response of TPE-Z at 478 nm in the presence of different length of single-stranded DNA. The excitation wavelength is 350 nm. The schematic and fluorescence emission spectra of 0.5X commercial SYBR Green I in the presence of (d) Ex-0 (18-nt), (e) Ex-4 (42-nt), (f) Ex-6 (54-nt). Inset: fluorescence response of SYBR Green I at 520 nm in the presence of different length of single-stranded DNA. The excitation wavelength is 497 nm. S5

6 Figure S2. (a) Fluorescence emission spectra of TPE-Z in the presence of telomerase extracted from E-J cancer cells with different telomerase extension reaction time in the range from 0 to 180 min. (b) Relationship between fluorescence intensity (478 nm) and telomerase extension reaction time. The concentration of TPE-Z is 7.8 μm. S6

7 Figure S3. (a) Fluorescence emission spectra of TPE-Z in the presence of telomerase extracted from HeLa cancer cells with different concentrations of TS primer in the range from 0 to 1.00 μm. (b) Relationship between (I/I 0 )-1 at 478 nm and concentration of TS primer. The concentration of TPE-Z is 7.8 μm. S7

8 Figure S4. (a) Fluorescence emission spectra of TPE-Z in the presence of telomerase extracted from E-J cancer cells with different concentrations of dntps in the range from 0.05 to 0.35 mm. (b) Relationship between fluorescence intensity (at 478 nm) and concentrate of dntps. The concentration of TPE-Z is 7.8 μm. S8

9 Figure S5. Fluorescence emission spectra of TPE-Z in the absence (spuare) or presence (circle) of TS primer with telomerase extracted from E-J cancer cells. The concentration of TPE-Z is 7.8 μm. S9

10 Figure S6. Fluorescence emission spectra of TPE-Z in the absence (spuare) and presence (circle) of TS primer with telomerase extracted from HeLa cancer cells. The concentration of TPE-Z is 7.8 μm. S10

11 Figure S7. Fluorescence emission spectra of TPE-Z in the absence (spuare) and presence (circle) of TS primer with telomerase extracted from MCF-7 cancer cells. The concentration of TPE-Z is 7.8 μm. S11

12 Figure S8. Fluorescence emission spectra of TS primer in the presence of telomerase extracted from E-J cancer cells without (square) and with (circle) TPE-Z. The concentration of TPE-Z is 7.8 μm. S12

13 Table S1. Comparison between the Current Telomerase Activity Detection Assay and Other Previously Reported Optical Methods. Method System Detection limit Time Ref. Colorimetry assay TS-primer modified AuNPs 1 HeLa cell/µl 1.5 h [1] Catalytic beacons / hemin 500 HeLa cells 1.5 h [2] telomere complementary 500 HeLa cells 1.5 h [3] oligonucleotide modified AuNP probe Colorimetry and SERS 10 cells/ml 5 h [4] dual-mode Chemiluminescence MB-AuNPs / hemin 100 HeLa cells 2.5 h [5] assay DNAzyme-functionalized AuNPs 1000 HeLa cells 2 day [6] / TS-primer modified Au-plate / hemin Fluorescence assay T7 Exonuclease / taqman probe 5 HeLa cells 3 h [7] Exonuclease III / graphene oxide 250 HeLa cells 2 h [8] / linear DNA probe Circular strand-displacement 4 HeLa cells 3 h [9] Polymerization ZnPPIX / G-quadruplexes 380 HeLa cells/µl 3.5 h [10] Exonuclease III / LNA probe 30 MCF-7 cells 3 h [11] Cascade signal amplification 3 cells 3 h [12] CdSe / ZnS QDs / telomerase 18±3 cells/ μl 3.5 h [13] primer / hemin FAM-modified ODN / EB 400 cells/ μl 2 h [14] CdSe / ZnS QDs / telomerase 1000 cells 3.5 h [15] primer/texas red-dutp ZnP / telomerase / G-quadruplex 380 cells/μl 3.5 h [16] AIE-based turn on technique Our work 10 cells 1 h S13

14 Table S2. Oligonucleotides Used in This Study. Name Sequence (5' to 3') Length (nt) TS primer Ex-0 Ex-1 Ex-2 Ex-4 Ex-6 AATCCG TCGAGC AGAGTT CCGAAT AGCTCG GTTAGA CCGAAT AGCTCG GTTAGA TTAGGG CCGAAT AGCTCG GTTAGA TTAGGG TTAGGG CCGAAT AGCTCG GTTAGA TTAGGG TTAGGG TTAGGG TTAGGG CCGAAT AGCTCG GTTAGA TTAGGG TTAGGG TTAGGG TTAGGG TTAGGG TTAGGG S14

15 Figure S9. (a) Fluorescence emission spectra of TPE-Z in the absence and presence of different oligonucleotides (TS primer, Ex-0, Ex-1, Ex-2, Ex-4 and Ex-6). (b) Linear relationship between (I/I 0 )-1 at 478 nm and the lengths of oligonucleotides. The 18-nt DNA in this figure is Ex-0. The concentration of TPE-Z is 7.8 μm. S15

16 Figure S10. Non-denaturating PAGE analysis of telomerase extension assay: lane M, 20 bp DNA markers; lane 1, 20 μm TS primer in the absence of cancer cell extract; lane 2, 20 μm TS primer in the presence of cell extract from HeLa cells; lane 3, 20 μm TS primer in the presence of cell extract from E-J cells. S16

17 Figure S11. Linear relationship between optical density at wavelength of 450 nm and standard telomerase activity using Human Telomerase (TE) ELISA Kit. We can see that 8000 E-J cancer cells are active (11.50 IU/L), and the telomerase extracted from 3 clear and 2 bloody clear urine specimens are also possess activity in the same order of activity magnitude compared of our method. Error bars indicate standard deviation of triplicate tests. S17

18 Table S3. Activity of telomerase extracted from patients urine specimens using human telomerase (TE) ELISA Kit. Telomerase Clear sample Bloody sample Sample No. (I/I 0 )-1 Activity (IU/L) (our method) (commercial kit) S18

19 Figure S12. Fluorescence responses of this sensing system to telomerase extracts from HeLa cancer cells which are treated without (A) and with (B) 100 μm AZT. S19

20 Figure S13. Results of AIE-based telomerase activity detection in real normal urine samples. Fluorescence intensity at 478 nm of detection system in response to telomerase extracted from urine specimens of 15 normal people. The horizontal dashed line represents the threshold level according to the definition of I 0 +3σ. Error bars indicate standard deviation of triplicate tests. S20

21 Figure S14. The photograph of telomerase extracts from urine specimens of bladder cancer patients whose urine are bloody (left) and clear (right) under the daylight lamp. S21

22 Table S4. Corresponding disease information of the bladder cancer urine samples Sample Region Urine (I/I 0 )-1 Relative standard Detection No. of patients appearance at 478 nm deviations (RSD) 1 Nanchang Clear 1.24 Yes Nanchang Clear 1.01 Yes Nanchang Clear 2.02 Yes Nanchang Clear 0.41 Yes Nanchang Clear 0.73 Yes Nanchang Clear 2.21 Yes Nanchang Clear 2.34 Yes Nanchang Clear 0.36 Yes Nanchang Clear 0.75 Yes Nanchang Clear 0.69 Yes Nanchang Clear 1.09 Yes Nanchang Clear 2.05 Yes Nanchang Clear 1.57 Yes Wuhan Clear 0.33 Yes Wuhan Clear 2.19 Yes Nanchang Clear 1.02 Yes Wuhan Clear 2.17 Yes Wuhan Clear 0.47 Yes Wuhan Clear 1.80 Yes Nanchang Clear 0.68 Yes Nanchang Clear 1.41 Yes Nanchang Clear 1.00 Yes Nanchang Clear 0.75 Yes Nanchang Bloody 0.40 Yes Nanchang Bloody 0.48 Yes Nanchang Bloody 0.13 Yes Nanchang Bloody 0.21 Yes Nanchang Bloody No Wuhan Bloody 0.13 Yes Wuhan Bloody No Nanchang Bloody 0.05 Yes Nanchang Bloody 0.08 Yes Nanchang Bloody 0.82 Yes Nanchang Bloody 1.98 Yes Wuhan Bloody 0.21 Yes Wuhan Bloody 0.04 Yes Wuhan Bloody No Wuhan Bloody No Wuhan Bloody 0.08 Yes Wuhan Bloody No Nanchang Bloody 0.51 Yes S22

23 Table S5. Corresponding information of the urine samples from normal people Sample No. Region of sample (I/I 0 )-1 Relative standard at 478 nm deviations (RSD) 1 Wuhan Wuhan Wuhan Wuhan Wuhan Wuhan Wuhan Wuhan Wuhan Wuhan Wuhan Wuhan Wuhan Wuhan Wuhan S23

24 Figure S15 Classification of patients urine samples. S24

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