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Construction of AIEgens-Based Bioprobe with Two Fluorescent Signals for Enhanced Monitor of Extracellular and Intracellular Telomerase Activity Yuan Zhuang, Chunli Shang, Xiaoding Lou, and Fan Xia Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 16 Jan 2017 Downloaded from http://pubs.acs.org on January 16, 2017

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Analytical Chemistry

Construction of AIEgens-Based Bioprobe with Two Fluorescent Signals for Enhanced Monitor of Extracellular and Intracellular Telomerase Activity *

Yuan Zhuang,a,‡ Chunli Shang,a,‡ Xiaoding Lou,a, Fan Xiaab,

*

a

Hubei Key Laboratory of Bioinorganic Chemistry & Materia Medica, Key Laboratory of Material Chemistry for Energy Conversion and Storage, Ministry of Education, School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology, Wuhan 430074, P. R. China. b Shenzhen Institute of Huazhong University of Science and Technology, Shenzhen 518000, P. R. China *E-mail: [email protected]; [email protected]. Fax: 86-27-8755 9485.

ABSTRACT: Detections of telomerase activity in vitro and in living cells are of great importance for clinical diagnosis of cancer. In this work, an AIEgens-based bioprobe with two fluorescent signals for enhanced monitor of extracellular and intracellular telomerase activity is designed. After addition of telomerase, two positively charged AIEgens (Silole-R and TPE-H) bind to quencher group labeled primer (QP) and the extension repeated units, leading enhancement of two telomerase-triggered fluorescent signals. Furthermore, by combination the wider linear range in vitro and lower background in living cells imaging, the bioprobe is used to detect telomerase extracted from various cell lines (MCF-7, HeLa, E-J, and HLF), 50 bladder cancer patients’ urine samples, 10 normal people’s urine samples, and also applied in mapping telomerase activity inside living cells (MCF-7, HeLa, MDA-MB-231, and HT1080). The results showed that this well designed strategy can successfully detect telomerase activity in vitro and in living cells with high sensitivity, indicating the potential application of this method in cancer cells bioimaging and clinical cancer diagnosis.

rescence enhanced along with the elongation of template substrate oligonucleotides (TS primer). Telomerase from different cultured cell lines and cancer patients’ urine samples were detected successfully by using this AIE turn-on method. The detection limit was down to 10 cells.41 Moreover, by combination of AIEgens Silole-R and a quencher group labeled TS primer, a lower fluorescence background were obtained, leading to the maintained high sensitivity and higher specificity.42 However, the emission colors of TPE-Z and Silole-R are both blue, which are indistinguishable in bioimaging of living cells and easily confused with biological autofluorescence due to the high background.45,46 This problem limits their further application. To avoid this restriction and broaden the application of AIEgens with different color emission in bioimaging, another positively charged AIEgen named TPE-H is introduced to combine with Silole-R in telomerase activity detection. As described above, Silole-R is highly sensitive for telomerase detection, but due to its blue emission (at 478 nm), it could be disturbed by autofluorescence of organism. Thus the sole Silole-R is not suitable for mapping telomerase activity in living cells. On the other hand, TPE-H shows relatively low sensitivity, which causes the narrow linear range and high detection limit, but its red emission (at 605 nm) escape from blue autofluorescence interference. To study the relationship between fluorescence of the two AIEgens and the length of quencher group labeled oligonucleotide (QP), experiments were carried

INTRODUCTION As one of the most common cancer biomarkers, telomerase has an intimate association with the cellular immortality and carcinogenesis. Telomerase can add specific sequence (TTAGGG)n to a telomere using its template RNA.1-4 In a majority of human tumors (~85-90%), telomerase has a high activity expression; but in most normal somatic cells, it shows activity depression.5 Thus, detection of telomerase activity is of great importance in early detection and diagnosis of cancer.6-22 Fluorescent biosensors are essential tools for real-time analytical sensing of biological molecules and processes in living cells,23-34 and have been widely applied in telomerase activity detection.35-40 However, most fluorescence biosensors are “turn-off” mode, in which fluorescence intensity decreases along with the addition of target analyte increases. On the other hand, turn-on biosensors reduce the false-positive responses, which makes them more attractive. To address this concern, a group of fluorogens with aggregation-induced emission (AIE) property attract our attention. The AIE luminogens (AIEgens) are non-emissive when dissolved and dispersed, but distinctly emissive when supramolecularly aggregated. In our previous work, a series of telomerase activity detection protocols using AIEgens have been established.41-44 An AIEgen with positive charge called TPE-Z showed weakly fluorescent when highly dissolving in solution. After adding different amounts of active telomerase, the fluo1

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out. As shown in the Figures S1 and S2, both the fluorescence intensity values I605 and I478+I605 enhanced along with the length of QP. These results indicate that the intensity of TPEH is directly affected by the length of oligonucleotide no matter whether Silole-R exists. Thus, TPE-H could be used as telomerase detection bioprobe in favor of biological imaging and observation. Combination of the blue fluorescent Silole-R and red fluorescent TPE-H can make up their defects and establish a novel two fluorescent signals based telomerase detection strategy with high sensitivity and specificity (Scheme 1). Both Silole-R and TPE-H show weak fluorescence in 1% ethanol aqueous solution. Primer used in this work called QP is molecularly labeled with a quencher group Dabcyl at its 5’end. Table S1 shows sequences of oligonucleotide used in this assay. Mixed solution of Silole-R, TPE-H and QP exhibits unique and well-resolved dual emission bands at 478 nm (Silole-R) and 605 nm (TPE-H) under the same excitation wavelength of 405 nm. To study the relationship between fluorescence intensity and FRET distance, the fluorescence spectra of Silole-R in the presence of quencher labeled oligonucleotide with different lengths were measured. As shown in the Figure S3, the fluorescence was efficiently quenched in the presence of quencher labeled oligonucleotide whose length is lower than 30 nt. Then, in the presence of 42-nt quencher labeled oligonucleotide, the fluorescence substantially enhanced, indicating the disappearing of FRET effect because of the long distance (larger than 10 nm). Thus, the Silole-R and TPE-H can be efficiently quenched and will not be affected after elongation reaction by the quencher due to the short distance (≤ 6.12 nm) between AIEgens and the quencher. After addition of telomerase extracts from cultured cancer cells or cancer patients’ urine samples, more positively charged AIEgens bind to the negatively charged DNA strand. Thus, the aggregation degrees of both Silole-R and TPE-H gradually increase along with the elongated QP, showing up the simultaneous enhancements of dual emission bands at 478 Scheme 1. Schematic illustration of two fluorescent signals bioprobe for detection of extracellular and intracellular telomerase activity.

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and 605 nm. At the same time, the short distance between Silole-R and TPE-H leads to the energy transferring and fluorescence enhancement from Silole-R to TPE-H. Moreover, the AIEgens bound to the extended repeated units keep away from the quencher, leading to the decay of quenching effect and the increase of signal/noise ratio. Through this process, the low sensitivity of TPE-H can be offset by Silole-R. Thus, with the support of Silole-R, TPE-H, which is originally not suitable for quantificational telomerase detection but bioimaging, can be applied to monitor to detect telomerase activity in living cells.

EXPERIMENTAL SECTION Materials. Water was purified by a Millipore filtration system. Oligonucleotides were synthesized by TaKaRa Bio Inc. (Dalian, China). The deoxynucleotide solution mixture (dNTPs), RNase-free water, and recombinant RNase inhibitor (RRI) were purchased from TaKaRa Bio Inc. (Dalian, China). 30% Arc-Bis (29:1) was purchased from Biosharp. 1×CHAPS lysis buffer was purchased from Millipore (Bedford, MA). MCF-7, HeLa, and E-J cell lines were obtained from Xiangya Central Experiment Laboratory. MDA-MB-231 and HT1080 cell lines were obtained from Typical Culture Preservation Commission Cell Bank, Chinese Academy of Sciences (Shanghai, China). Human lung fibroblast (HLF) cell lines was obtained from China Center for Type Culture Collection. 0.05% Trypsin/EDTA and phosphate buffered saline (PBS) were purchased from Multicell Technologies. Thrombin (from human plasma) was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. Bull serum albumin (BSA) was purchased from Kayon. Bst 2.0 warmsmart DNA polymerase (Bst DNA polymerase) was purchased from Biotium. Patient samples were donated by Union Hospital, Tongji Medical College, Huazhong University of Science and Technology. Telomerase Extracted from Cultured Cells. Telomerase of bladder cancer cells (E-J), breast cancer cells (MCF-7), cervical cancer cells (HeLa), and human lung fibroblast cells (HLF) were extracted in the way described below. The AZTtreated HeLa cells were treated with 100 µM of AZT diluted in medium for 48 h before extraction. After pretreating (if necessary), cells were suspended in lysis buffer with the concentration of 5000 cells/µL and incubated on ice for 30 min. Then the mixture was centrifuged at 12000 g and 4 ºC for 20 min to remove the useless precipitates. The transferred supernatant was stored at -80 ºC. Telomerase Extracted from Urines. Fresh urine samples were first centrifuged at 850 g and 4 ºC for 10 min and washed once using PBS. Then they were centrifuged at 2300 g and 4 ºC for 5 min. The precipitate was resuspended in 200 µL of ice-cold lysis buffer, and then incubated on ice for 30 min. The mixture was centrifuged at 10000 g and 4 ºC for 20 min to remove the useless precipitates. The transferred supernatant was stored at -80 ºC. Imaging of Telomerase Activity in Living Cells. 0.5 mL of HeLa (MCF-7, MDA-MB-231, HT1080) cells (4×104 mL-1) was seeded in a 20-mm confocal dish for 24 h. Then, 1 mL of appropriate medium without (or with) addition of 20 µM of 3’Azido-3’-deoxythymidine (AZT) was added into experimental (or control) group for 48 h, respectively. 3.6 µM of QP was transfected according to the instruction using 10 µL of lipofec2

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Analytical Chemistry lomerase-triggered QP elongated. Subsequently, the intensities of Silole-R and TPE-H increased separately, and at the same time partial energy transferred from Silole-R to TPE-H. As a result, the value of IB+R gradually increased along with the cell number. By using this two fluorescent signals bioprobe, the detection limit was as few as 250 cells. Afterwards, telomerase extraction of E-J cells and HeLa cells were distinctly detected similarly, and parallel results were obtained (Figures S5-7), indicating the universality and reasonability of this two fluorescent signals based detection strategy.

tamine 2000 in 250 µL of Opti-MEM at 37 ºC for 1 h. Subsequently, Opti-MEM transfection mixtures were removed from the cells dish. Then, 1.0 mL of PBS containing 5.0 µM of Silole-R (or 5.0 µM of Silole-R and 10.0 µM of TPE-H) was added to the cell dish for 10 min, then removed from the dish. Cells were washed for three times using PBS before imaging observation. Laser Scanning Confocal Microscopy (LSCM) Imaging. An Olympus biological confocal laser scanning microscope (model: FV1200) was used to collect cellular images. Excitation laser was 405 nm; fluorescence emission ranges were collected at 410-510 nm for Silole-R and 555-655 nm for TPE-H. A 60× oil immersion objective lens was used to get images. The fluorescence intensity values in the LSCM imaging assay were analyzed by quantifying the average fluorescence intensity of LSCM images with the image processing software (Olympus FV10-ASW V4.0).

RESULTS AND DISCUSSION Characterization of the Two Fluorescent Signals Biosensor. The two fluorescent signals bioprobe proposed in this work was based on use of two positively charged fluorescence dyes with AIE properties in parallel. One was the watersoluble AIEgen Silole-R, having a blue emission spectrum with the maximum peak intensity at 478 nm. The other was the water-insoluble AIEgen TPE-H having a red emission spectrum with the maximum peak intensity at 605 nm, whose fluorescence is weak in the ethanol solution. The aggregation degree of TPE-H changed with different ratios of water/ethanol solution has been reported.47 The emission reached a plateau when water fraction was higher than 90%, indicating TPE-H molecules aggregated in high water contents solution. To avoid the interference to Silole-R, water/ethanol mixture with water fraction of 99% was chosen as detection medium. Then, the UV-vis absorption and fluorescence emission spectra of Silole-R and TPE-H were measured. As we can see from Figure 1a, high degree of overlap existed between emission spectrum of Silole-R (λmax = 478 nm) and absorption spectrum of TPE-H (λmax = 428 nm), verifying the energy transferring from Silole-R to TPE-H. Detection of Telomerase Activity from Cancer Cells Extracts. To find out the linearity range of this method, experiments between quantity of telomerase and fluorescence intensity were conducted. This AIEgens-based bioprobe had two intrinsic emission peaks in the visible range under the same UV illumination. The peak at 478 nm of blue emission was ascribed to Silole-R, and the other at 605 nm of red emission belonged to TPE-H. It was clear that the sole fluorescent dye TPE-H could not get satisfactory sensitivity, but the sole Silole-R was just the opposite when adding telomerase extracted from MCF-7 cancer cells (Figure 1b, Figure 1c and Figure S4). To cover the shortage of each other, dual fluorescent dyes Silole-R and TPE-H were used under the same excitation wavelength of 360 nm (maximum excitation of Silole-R), and the value of IB+R [IB+R = (I478)/(I478)0 + (I605)/(I605)0] was chosen as the specific value of this bioprobe. As shown in the Figures 1d and 1e, when different concentrations of telomerase, which equal to 0-20000 MCF-7 cells, were added, the value of IB+R gradually increased. Presumably, in this process, Silole-R and TPE-H aggregated and closed to each other because more AIE dyes were adsorbed by static electricity interaction as the te-

Figure 1. Comparison between single fluorescent signal and two fluorescent signals methods in telomerase activity detection. (a) Energy transferring certification between Silole-R and TPE-H. The emission of Silole-R (λmax = 478 nm, blue dashed line) and the absorption of TPE-H (λmax = 428 nm, red solid line) have an apparent overlap (marked in purple hachure). (b) Fluorescence spectra of TPE-H based detection for telomerase extracted from different amounts of MCF-7 cancer cells with the number of 0, 2000, 4000, 6000, 8000, 10000, and 20000, respectively. (c) Plot of the changes in (I605)/(I605)0 value with different MCF-7 cell numbers. (d) Fluorescence spectra of two fluorescent signals (Silole-R and TPE-H) based detection for telomerase extracted from different amounts of MCF-7 cancer cells (0, 250, 2000, 4000, 10000, and 20000). (e) Plot of the changes value IB+R with different MCF-7 cell numbers. IB+R refers to [(I478)/(I478)0 + (I605)/(I605)0]. The subscript 0 refers to corresponding background intensity value prior to the extension reaction. Linear ranges are marked in gray squares in parts c and e. 3

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real urine specimens of 10 normal people and 50 bladder cancer patients were tested.

Working Mechanism Interpretation. To retrieve the suitable optimal conditions for this method, a number of control experiments were conducted. As shown in Figure S8, IB+R increased rapidly in the first 60 min, subsequently remained a plateau for at least 50 min. Thus, 60 min was chosen as the reaction time. Afterwards, 3'-Azido-3'-deoxythymidine (AZT), a common telomerase activity inhibitor, was used to significantly inhibit the telomerase activity (Figures 2a and S9). Compared to the telomerase-triggered QP without AZT, the IB+R value successively decreased with the increasing addition of AZT. After adding 1 mM of AZT, the IB+R value was almost as low as the baseline value (in the absence of telomerase or AZT). Then, S1 nuclease (S1 Nase), a single-strand-specific endonuclease which catalytically hydrolyzes single-strand nucleic acid to 5'-nucleoside monophosphates, was chosen to hydrolyze DNA and verify the relationship between elongation of QP and enhancement of IB+R. As shown in the Figures 2b and S10, after adding telomerase extracted from 10000 E-J cancer cells, the value of IB+R increased well above the baseline value. Then 18 U of S1 Nase was added into this elongation-reacted system and reacted for 10 min, showing up apparent reduction of IB+R. Subsequently, another 18 U was added into the same system and reacted for another 10 min. Succedent fluorescence measurement showed a further decrease of IB+R value, nearly as low as the baseline. These results above indicated that elongation of QP can directly lead to the enhancement of fluorescence intensity IB+R value. In addition, a non-denaturating polyacrylamide gel electrophoresis (PAGE) analysis was conducted to prove the prospective product and estimate whether quencher groups disturb the elongation reaction. Figure 2c showed that strong bands at around 60 bp appeared only in the presence of active telomerase, indicating quencher group had no interference to elongation reaction. This result proved the prospective telomerasetriggered elongation reaction. Specificity Study. In order to test the specificity of this two fluorescent signals biosensor, heat-inactivated E-J cells, HeLa cells and MCF-7 cells telomerase (kept at 95 ºC for 20 min before used), telomerase extracted from human lung fibroblast cells (HLF, a normal human lung cell line) and AZT-treated HeLa cells telomerase (treated with 100 µM of AZT for 48 h before telomerase extraction) were tested by this method. As shown in Figure 2d, comparison with corresponding active telomerase, these inactive system showed lower intensity value, which demonstrated the necessity of telomerase activity in this detection process. Moreover, several interferents including lysis buffer, trypsin, thrombin, bull serum albumin (BSA), and Bst DNA polymerase were tested to verify the antiinterference performance. Apparent differences between these interferences and active telomerase were observed. The apparent differences between positive groups (active telomerases) and negative groups (inactivated telomerases and interferents) credibly proved the satisfactory specificity of this method. Determination of Telomerase Extracted from Real Urine Samples of Bladder Cancer Patients. Bladder cancer is a common genitourinary malignancy, and it has the highest recurrence rate.48-50 Fresh urines from bladder cancer patients contain live cancer cells, which can be used to extract telomerase. To evaluate the clinical diagnosis applicability of this two fluorescent signals strategy, telomerase extracts of

Figure 2. Working mechanism interpretation of the two fluorescent signals method to detect activity of telomerase. (a) Histogram of changes in normalized IB+R with inhibition of 10000 E-J cells telomerase activity by 0, 0.5 and 1 mM of AZT. (b) Histogram of normalized IB+R changing with gradual hydrolyzation of elongated QP by S1 Nase. (c) Non-denaturating PAGE analysis of this two fluorescent signals method: DNA ladder marker (lane M), QP in the absence (lane 1) and presence of telomerase extracts of 50000 E-J cancer cells (lane 2), 50000 MCF-7 cancer cells (lane 3) and 50000 HeLa cancer cells (lane 4). (d) Fluorescence intensity change IB+R responses of this sensing system to active telomerase extracts from 10000 E-J (A), HeLa (C) and MCF-7 (E) cancer cells, heat-inactivated telomerase extracts from 10000 E-J (B), HeLa (D) and MCF-7 (F) cancer cells, telomerase extracts from 10000 HLF cells (G) and AZT-treated HeLa cancer cells (H), lysis buffer (I), trypsin (J), thrombin (K), BSA (L) and Bst DNA polymerase (M), respectively. Error bars indicate SD of triplicate tests.

To reliably prove the activity of telomerase used in this work, a commercial human telomerase Elisa Kit was used to detect telomerase extracts from cancer cell MCF-7, a clear cancerous urine specimen and normal HLF cell (Figures 3a and S11). The comparative results between the standardized concentration from Elisa Kit and IB+R value from our two fluorescent signals method exhibited the same tendencies. Without any other pretreatment before telomerase extraction, telomerase from 20 clear urine specimens (urine specimens of bladder cancer patients whose urines are clear) and 30 bloody urine specimens (gross hematuria, urine specimens of bladder cancer patients whose urines are bloody macroscopically and contain more than 4 mL of blood in 1000 mL of urines) were tested using our method (Figures S12a and S12b). Then, a threshold level baseline was calculated from measurement results of 10 normal people (shown in Figure S12c) according to (IB+R)N + 3σ [(IB+R)N means average of fluorescence intensity IB+R values of the 10 normal specimens, σ means standard deviation of fluorescence intensity IB+R values of the 10 normal specimens], which was expressed as the dashed line in Figures S12a and S12b. Apparently, both the clear and bloody cancerous specimens were well above the normal specimens, with all 4

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Analytical Chemistry

of the relative standard deviations (RSDs) less than 7.1% for clear specimens and 8.3% for bloody specimens. By using this two fluorescent signals method, the proportions of positive results are 90.0% (18/20) for clear urine specimens and 80.0% (24/30) for bloody urine specimens, respectively. Analytical results of urine specimens are listed in Figure 3b, Table S2 and Table S3. In evidence, the results above indicated that this two fluorescent signals strategy could detect activity of telomerase extracted from urines of bladder cancer patients, and could be used as a promising strategy for clinical bladder cancer diagnosis.

Figure 3. Detection of telomerase activity from patients’ urine specimens. (a) Histogram for telomerase concentrations obtained from commercial telomerase Elisa Kit and our two fluorescent signals method in detecting the activity of telomerase extracted from 10000 MCF-7 cells (A), clear specimen No.1 (B) and 10000 normal HLF cells (C). (b) Box chart representation of activity detection of telomerase from urine specimens of 20 clear bladder cancer patients (blue), 30 bloody bladder cancer patients (red) and 10 normal people (gray) by using this two fluorescent signals detection strategy.

Figure 4. Telomerase detection in living cells by this two fluorescent signals based detection strategy. (a-d) Confocal images of HeLa cells (4×104 mL-1, 0.5 mL) after transfection with 3.6 µM of QP for 1 h, then incubation with only 5.0 µM of Silole-R (a, b) or with 5.0 µM of Silole-R and 10.0 µM of TPE-H (c, d). Cells were pretreated without (a, c) or with (b, d) 20.0 µM AZT for 48 h before transfection. Scale bar: 30 µm. (e, f) Corresponding fluorescence intensity of Silole-R and TPE-H in parts a, b (e) and c, d (f). (g) 3D surface projections of Z-stack images for part c. Scale bar: 30 µm.

Imaging of Telomerase in Living Cells. Due to the sensitivity of Silole-R and bioimaging feasibility of TPE-H, this two fluorescent signals biosensor was used to detect telomerase activity of living cancer cells. The improved stability of the probe was demonstrated in our previous work37 because the modified quencher group can protect probe from enzymatic degradation. A series of experiments for cellular telomerase activity detection were conducted. Telomerase-positive and AZT-treated HeLa (4×104 mL-1, 0.5 mL) cells were respectively transfected by QP (3.6 µM, 250 µL) for 60 min, then subsequently dyed by Silole-R (5.0 µM, 500 µL) for 10 min before confocal observation. The fluorescence images showed that a weak fluorescence change was observed (Figures 4a, 4b and 4e). Then the mixture of Silole-R (5.0 µM) and TPE-H (10.0 µM) was added similarly after transfection, and the fluorescence images with two channels of blue and red emission were taken. As shown in the Figures 4c and 4d, compared with fluorescence intensity in AZT-treated inactivated HeLa cells, the blue emission in telomerase-positive HeLa cells was still almost indistinguishable and undiversified (1.965 times enhancement, Figure 4f), but the red emission intensity apparently increased (8.081 times enhancement). This phenomenon was obviously caused by the difference of telomerase activity in the cells, proving the bioimaging feasibility of TPE-H in telomerase activity detection. 3D surface projections of Zstack images for Figure 4c also proved this result (Figure 4g). The red emission mainly existed around nuclear, certifying the distribution of telomerase in cytoplasm.51 To testify the

Figure 5. Telomerase detection in MCF-7 and MDA-MB-231 cancer cells. (a-d) Confocal images of MCF-7 cells (a and b, 4×104 mL-1, 0.5 mL) and MDA-MB-231 cells (c and d, 4×104 mL1 , 0.5 mL) after transfection with 3.6 µM of QP for 1 h, then incubation with 5.0 µM of Silole-R and 10.0 µM of TPE-H. Cells were pretreated without (a, c) and with (b, d) 20.0 µM of AZT for 48 h before transfection. Scale bar: 30 µm. (e, f) Corresponding fluorescence intensity of Silole-R and TPE-H in parts a, b (e) and c, d (f). 5

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Notes

universality, MCF-7, MDA-MB-231, and HT1080 cancer cells were measured in the same way (Figures 5 and S13-S15). After transfected and dyed by Silole-R and TPE-H, red emission showed apparent enhancement from cells without pretreatment to cells treated with AZT, while blue emission had no significant changes (Figures 5, S13 and S14). However, when only dyed with Silole-R, it could not be distinguished between untreated and AZT-treated MCF-7 cells (Figure S15).The results further indicated the feasibility and universality in different living cell lines of our method. As a result, the two fluorescent signals biosensor strategy could be used to distinguishing the cancer cells with high telomerase activity from telomeraseinactive cells in various cell lines, even screening of telomerase-targeting drug.

The authors declare no competing financial interest.

ACKNOWLEDGMENT This research is supported by National Natural Science Foundation of China (21525523, 21574048, 21375042, 21405054), National Basic Research Program of China (973 program, 2015CB932600, 2013CB933000), the Special Fund for Strategic New Industry Development of Shenzhen, China (Grant No. JCYJ20150616144425376) and 1000 Young Talent (to Fan Xia).

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CONCLUSION In summary, we have developed a two fluorescent signals based biosensor with wider linear range and lower background for telomerase detection in both solution and living cells. This method is established under the energy transferring between AIEgens Silole-R and TPE-H, making up the defects of two dyes when used alone. Compared with the sole AIEgen SiloleR, this method is suitable for telomerase activity detection in living cells by bioimaging, and potential application in telomerase-targeting drug screening. This two fluorescent signals method also has advantages of high sensitivity and specificity towards sole AIEgen TPE-H based strategy. The feasibility of this method is demonstrated by using telomerases extracted from different cancer cells (HeLa, MCF-7, E-J, HLF and AZT-treated HeLa cells) and other interferents. Moreover, telomerase from urines of 10 normal people and 50 bladder cancer patients are tested to verify the probability to cancer diagnosis. More importantly, through detecting of telomerase activity by bioimaging in living cancer cells (HeLa, MCF-7, MDA-MB-231 and HT1080), the ability of detection in living cells is also proven. As a result, it is anticipated that the two fluorescent signals based biosensor could provide an efficient tool for telomerase activity detection in both solution and living cells, and clinical diagnosis of cancer.

ASSOCIATED CONTENT Supporting Information Experimental procedures of AIEgens synthesis, telomerase extension reaction, fluorescence measurements, PAGE analysis, telomerase activity detection using commercial ELISA Kit; DNA sequences; fluorescence spectra of the sole AIEgen; reaction time study; AZT inhibition study; S1 Nase degradation study; values of patients’ samples; 3D surface projections of Z-stack images; images of HT1080 cells; images of MCF-7 cells incubated with Silole-R. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. *E-mail: [email protected]. Fax: 86-27-8755 9485.

Author Contributions ‡

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Y. Z. and C. S. contributed equally.

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