Ratiometric Fluorescent Bioprobe for Highly Reproducible Detection of

Mar 14, 2016 - Herein, to improve the reproducibility and robustness, a ratiometric fluorescent bioprobe for telomerase activity detection has been de...
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A Ratiometric Fluorescent Bioprobe for Highly Reproducible Detection of Telomerase in Bloody Urines of Bladder Cancer Patients Yuan Zhuang, Qi Xu, Fujian Huang, Pengcheng Gao, Zujin Zhao, Xiaoding Lou, and Fan Xia ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.6b00076 • Publication Date (Web): 14 Mar 2016 Downloaded from http://pubs.acs.org on March 16, 2016

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A Ratiometric Fluorescent Bioprobe for Highly Reproducible Detection of Telomerase in Bloody Urines of Bladder Cancer Patients Yuan Zhuang,‡,a Qi Xu,‡,a Fujian Huang,a Pengcheng Gao,a Zujin Zhao,b Xiaoding Lou,*,a Fan Xiaa a

Hubei Key Laboratory of Bioinorganic Chemistry & Materia Medica, School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology, Wuhan 430074, China. b State Key Laboratory of Luminescent Materials and Devices, South China University of Technology, Guangzhou 510640, China ABSTRACT: Fluorescent bioprobes, as one of the most important tools, hold high promise for real-time analytical sensing of biological molecules and processes in live cells and organisms. Although several excellent bioprobes employ turn-on fluorescence intensity, such a sole responsive signal is readily perturbed by various experimental conditions. Herein, to improve the reproducibility and robustness, a ratiometric fluorescent bioprobe for telomerase activity detection has been developed. The ratiometric fluorescent bioprobe is designed on the use of two fluorescence dyes in parallel. One is red emissive aggregation-caused quenching (ACQ) dye, Cy5, as a control, molecularly labeled on the 5’-end of telomerase substrate oligonucleotides (TS primer). The other is watersoluble aggregation-induced emission (AIE) dye, Silole-R, as reporter of telomerase activity, non-emissive in buffer. In the presence of telomerase, the blue emission is enhanced by the added negatively charged sites for Silole-R to bind and aggregate, while the red emission is almost unchanged as stable internal reference. With the addition of incremental amounts of telomerase, the ratiometric emission intensity ratios (I478/I665) of this bioprobe gradually increase. Furthermore, the distinguishing of telomerase extracts from 20 bladder cancer bloody and 10 normal urine specimens confirms the practicality of this bioprobe. In contrast to previous turn-on bioprobe, these advanced experiments obtain higher reproducibility and positive result rate (100%) towards bladder cancer bloody urine specimens. KEYWORDS: AIEgens, ratiometric, fluorescence, telomerase, urine specimens

Fluorescent bioprobes are essential tools for real-time analytical sensing of biological molecules and processes in live cells and organisms.1-10 Fluorescent bioprobes have a variety of intrinsic advantages, including high sensitivity, simple instrumentation and capacity to high-throughput screening. The bioprobes can provide valuable insights in understanding physiological alterations in pathological settings, thus have been extensively employed as useful tools in developing of targeted therapies and personalized medicine.11-17 Although a wide array of fluorescent bioprobes have been developed, many of them are focused on fluorescence turn-off, in which the fluorescence signal is diminished in the presence of the target analyte.18-19 The sensitivity of fluorescence turn-on models are significantly boosted by the enhancement of a fluorescence signal on an ultra-low background. As a result, turnon bioprobes are especially attractive because of their reduced false-positive responses as compared to their turn-off counterparts.20-25 A novel class of fluorogenic molecules with aggregation-induced emission (AIE) characteristics has sparked intense research interest in biological research field.26-28 The fluorescence turn-on characteristics of the AIE luminogens (AIEgens) upon binding to the targets make them excellent candidates as light-up probes for biosensing applications.29-32 Telomerase is a ribonucleoprotein that can add specific sequence (TTAGGG)n to 3’-end of telomere. As a sensitive bi-

omarker for cancer, telomerase plays important parts in early detection of cancers.33-34 The AIE-based bioprobes for quantification and monitoring of the telomerase activity have been reported in our previous works.35-38 A positively charged AIE dye (TPE-Z) is non-emissive when freely diffused in solution. The fluorescence of TPE-Z is enhanced with the elongation of DNA strand which could light up telomere elongation process. By exploitation of it, we can detect telomerase activity from different cell lines with high sensitivity and specificity.35 Moreover, On the basis of the combined use of quencher and AIEgens, a quencher group induced high specificity strategy for telomerase activity detection from cell extracts and cancer patients’ urine specimens was creatively developed.36 However, these kind of AIE-based bioprobes employ fluorescence turn-on model, such a solely responsive signal is readily perturbed by various experimental conditions, including signal variations due to dye bleaching, fluctuations in source intensity or temperature.39-41 Ratiometric measurements can eliminate these environmental effects and give more precise signal because of their self-referencing capability by calculation of two emission intensity ratio.42-46 Thus, the design and development of ratiometric fluorescence bioprobes are of great concern and significant challenges. Normally, two independent fluorophores are usually preassembled or preconjugated together by sophisticated procedures when designing ratiometric sen-

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sors.47-49 In order to improve the reproducibility and robustness, a simple ratiometric bioprobe showing non-interfering dual emission has been developed. As detailed below, a positive charged AIEgen, Silole-R, is chosen. Owing to its amphiphilic nature, Silole-R is completely soluble in water. Then we molecularly labeled Cy5 at 5’-end of telomerase substrate oligonucleotides (TS primer) to produce Cy5-labeled TS primer (CP) for use in this work. Sequences of oligonucleotides used in this assay are shown in Table S1. The solution containing Silole-R and CP showed nonintrusive dual emission bands at 478 nm of blue emission (Silole-R) and 665 nm of red emission (Cy5) under a single wavelength excitation (Scheme 1). In the presence of telomerase (a cancer marker, over 85% of human cancers express either up-regulation or reactivation of telomerase activity),50-53 the blue emission is enhanced by the added negatively charged sites for Silole-R to bind and aggregate, while the red emission is almost unchanged as stable internal reference. One fluorescence emission spectrum refer to fixed instrument and environment conditions which have the same effect on Cy5 and Silole-R. In order to eliminate the system errors, ratiometric value (I478/I665) is chosen to represent the fluorescence intensity. With the addition of different amounts of telomerase, the ratiometric value (I478/I665) of this bioprobe gradually increase. These fluorescence intensity ratios can be employed as quantitative analysis of telomerase. The ratiometric bioprobe we described here is expected to be facile, reliable and thus readily extrapolated to create a range of similar fluorescent biosensors for early diagnosis. Scheme 1. Schematic illustration of the ratiometric fluorescent bioprobe for telomerase activity detection.

EXPERIMENTAL SECTION Materials. Oligonucleotides were synthesized in TaKaRa Bio Inc. (Dalian, China). The recombinant RNase inhibitor (RRI), deoxynucleotide solution mixture (dNTPs), and RNasefree water were purchased from TaKaRa Bio Inc. Water was purified by a Millipore filtration system. Telomerase extension reaction buffer was consist of 1.5 mM MgCl2 and 20 mM Tris (pH = 7.76). 0.05% Trypsin/EDTA and penicillinstreptomycin (10000 IU penicillin and 10000 µg/mL streptomycin) were purchased from Multicell Technologies. Fetal calf serum (FBS) was purchased from HyClone. 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

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(Bst DNA polymerase) was purchased from Biotium. The 1×CHAPS lysis buffer was purchased from Millipore (Bedford, MA). E-J cells, MCF-7 cells and HeLa cells were obtained from Xiangya Central Experiment Laboratory. Human lung fibroblast (HLF) cells were obtained from China Center for Type Culture Collection. 30% Arc-Bis (29:1) was purchased from Biosharp. Patient samples were donated by Union Hospital, Tongji Medical College, Huazhong University of Science and Technology. Cell Culture. Cells were cultured in corresponding medium contained 10% FBS and 1% penicillin-streptomycin in culture flasks in a humidified constant temperature incubator containing 5% CO2 at 37 ºC. The medium were Dulbecco’s modified Eagle’s medium (DMEM, Multicell) for MCF-7 cancer cells, 1640 medium (Gibco) for HeLa cancer cells, and minimum essential medium (MEM/EBSS, 1X, HyClone) for E-J cancer cells and HLF cells. Telomerase Extracted from Cultured Cells and Urine Samples. Telomerase was extracted according to the previous literature.35,36

RESULTS AND DISCUSSION Characterization of Our Ratiometric Fluorescent Bioprobe. The ratiometric fluorescent bioprobe we proposed in this work is based on the use of two fluorescence dyes in parallel. One is red emissive aggregation-caused quenching (ACQ) dye, Cy5, as a control, molecularly labeled on the 5’end of TS primer. The other is water-soluble aggregationinduced emission (AIE) dye, Silole-R, as a reporter of telomerase activity, non-emissive in buffer. Owing to electrostatic force, the positively charged Silole-R can spontaneously bind to the negatively charged DNA backbone.54 Intensity of blue emission increases along with the increment of the length of DNA strand. The design principle is that, during telomerase-induced primer extension, only the negatively charged sites for Silole-R to bind and aggregate would be changed, while the concentration of Cy5 would remain constant. Therefore, Cy5 is expected to serve as an internal control. Before conducting this protocol, we investigate the optical properties of Cy5 and Silole-R. The as-prepared ratiometric bioprobe has two intrinsic peaks of the fluorescence spectra in the visible range under UV illumination. The symmetric peak at 665 nm of red emission is ascribed to the Cy5 dispersed in buffer solution, and the other broad peak around 478 nm (blue emission) can be attributed to Silole-R aggregates related emission. The emission spectrum of Silole-R (λmax = 478 nm) and the absorption spectrum of Cy5 (λmax = 650 nm) did not overlap (Figure S1). Furthermore, fluorescence emission peak of CP at 665 nm remained unchanged in the absence and presence of TP and Silole-R, guaranteeing stability of internal control (Figure S2). Reproducibility Study. To confirm that the ratiometric fluorescent bioprobe (containing both Cy5 and Silole-R) is highly reproducible relative to the turn-on fluorescence bioprobe (containing only Silole-R), we collected 31 individual measurements of fluorescence spectra before target detection (Figure 1a and 1c, Figure S3a and S3c). Similar to the classic turnon fluorescence bioprobe, the background response in these 31 tests showed wide variation (Figure 1a) with an average of 0.890 and a standard deviation (SD) of 0.160. However, the variation in ratiometric fluorescent bioprobe was significantly reduced (Figure 1c) with an average background ratio response of 0.797 and a SD of 0.064. More importantly, high

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reproducibility can be carried out in the presence of telomerase (Figure 1b and 1d, Figure S3b and S3d). As shown in Figure 1d, when telomerase extracts from 10000 E-J cells (human bladder cancer cells) was introduced and co-incubated with CP and dNTPs, the fluorescence intensity at 478 nm was significantly increased, whereas the intensity of Cy5 still remained nearly the same. The SD in ratiometric fluorescent bioprobe (0.055) was much smaller than that of turn-on fluorescence bioprobe (0.305, Figure 1b). It's worth mentioning that the SD in turn-on fluorescence bioprobe changed from 0.160 (background) to 0.305 (after adding activity telomerase), while the SD values in ratiometric fluorescent bioprobe were 0.064 and 0.055, respectively, indicating the importance of highly reproducibility towards detection background. The detailed data were listed in Table S2 and Table S3. Moreover, the fluorescence spectra and SD values before and after adding target were obtained under various experimental conditions, including different cuvette, slit widths, even operation voltage of fluorescence spectrophotometer (Figure 1e and 1f, Figure S4). Although the spectra were of great difference, the ratio values were nearly invariable. The above data proved that our ratiometric model was far more robust, reliable, and reproducible than the previous bioprobes that relied on sole turn-on responsive signal.35,36

Figure 1. Comparison between turn-on fluorescent bioprobe and ratiometric fluorescent bioprobe. (a, b) Reproducibility of the turn-on fluorescent bioprobe in the absence (a) and presence (b) of telomerase. (c, d) Reproducibility of the ratiometric fluorescent bioprobe in the absence (c) and presence (d) of telomerase. All the gray histograms represent the values of 31 parallel experiments. Red bars and black error bars represent averages and standard deviations (SD) for 31 parallel experiments, respectively. (e, f) I478/I665 values of ratiometric fluorescent bioprobe under different conditions in the absence (e) and presence (f) of telomerase. Error bars indicate standard deviation of triplicate tests. Telomerase were extracted from 10000 E-J cancer cells.

Determination of Telomerase Activity from Cancer Cells Extracts. Through comparison of the reproducibility in different model, the importance of ratiometric bioprobe designing comes out. It can provide reference for high reproducibility application. For the purpose of finding out the detection limit and linearity range of our ratiometric bioprobe, experiments of relationships between the quantity of telomerase and fluorescence intensity were conducted. As shown in Figure 2a, the emission intensity ratio, I478/I665, gradually increased with increasing amounts of telomerase (extracts from E-J cells, human bladder cancer cells). During this process, aggregation of Silole-R was induced by interaction between Silole-R and telomerase-triggered CP elongation, and subsequently modulated the emission through an AIE process. As a result, the red fluorescence at 665 nm stem from Cy5 kept constant while the blue fluorescence at 478 nm stem from Silole-R increased, depending on the activity of telomerase, allowing ratiometric detection of telomerase. The positive correlation between I478/I665 and amounts of E-J cells was shown in Figure 2a and 2b. By using our ratiometric bioprobe, we could distinctly

Figure 2. Activity detection of telomerase extracts from three types of cancer cells by using the ratiometric fluorescent bioprobe. (a, c, e) Emission spectra of this bioprobe in response to telomerase extracts from 0, 5, 50, 500, 5000, and 10000 bladder cancer cells (E-J, a), cervical cancer cells (HeLa, c), and breast cancer cells (MCF-7, e). (b, d, f) The relationship between emission intensity ratio I478/I665 and number of E-J (b), HeLa (d), and MCF-7 (f). Insets: Linear relationships between the emission intensity ratio I478/I665 and the logarithm of cells number.

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detect the telomerase activity down to 5 cells. In order to check the reliability and generality of this bioprobe, telomerase extracts from HeLa and MCF-7 cancer cells were introduced (Figure 2c-2f). Positive correlations between I478/I665 and amounts of HeLa and MCF-7 cancer cells were also shown in insets of Figure 2. Specificity Study. To ensure that the observed signals were due to the specific elongation of telomerase to CP, a series of control experiments were carried out with heat-treated inactivated telomerase, telomerase extracts from normal cells and other proteins. As shown in Figure 3, high emission intensity ratio changes ((I478/I665)-(I478/I665)0) were observed in E-J, HeLa, and MCF-7 cancer cells, in consistent with the overexpressed telomerase in human tumors. In contrast, low emission intensity ratio changes were observed when using heat-treated inactivated telomerase and telomerase extracts from human lung fibroblast cells (HLF, a human normal cell line). Furthermore, to test the interference, lysis buffer, trypsin, thrombin, bull serum albumin (BSA) and Bst DNA polymerase were tested by using this bioprobe. The results displayed notable difference between active telomerase and other potential interferences, certifying the specificity of our bioprobe effectively. Additionally, to verify that the fluorescence intensity of Cy5 in CP was unaffected by extension reaction, fluorescence spectra of CP in the absence and presence of telomerase were recorded. The spectra did not change, indicating that telomerase has negligible influence on Cy5 (Figure S5). Moreover, we utilized non-denaturating polyacrylamide gel electrophoresis (PAGE) analysis to investigate the resultant DNA in active telomerase treated system. We discovered a strong band at ~60 bp indicative of telomerase products but cannot see any telomerase products in control samples (Figure S6), matched with the obtained fluorescence results.

Figure 3. Emission intensity ratio change ((I478/I665)-(I478/I665)0) in responses to telomerase extracts from 10000 E-J (A), HeLa (C), and MCF-7 cells (E), heat-inactivated telomerase extracts from 10000 E-J (B), HeLa (D), and MCF-7 cells (F), human lung fibroblast (HLF) cells (G), lysis buffer (H), trypsin (I), thrombin (J), BSA (K), and Bst DNA polymerase (L), respectively. Error bars indicate standard deviation of triplicate tests.

Diagnosis of Bladder Cancer by Ratiometric Method. Bodily fluids including blood, urine and sweat could be used for biomarker detection. Among them, urine is noninvasive, which can provide very valuable information about related diseases. Bladder cancer is one of the most common genitourinary malignancy with the highest recurrence rate. To confirm the application value of our strategy in bladder cancer detec-

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tion and diagnosis, telomerases extracts from urine specimens of bladder cancer patients and normal subjects are collected and applied. In our previous turn-on models, both of two positive result rates towards clear specimen were 100%, but the positive result rates towards bloody specimen (gross hematuria, more than 4 mL blood in 1000 mL urines) were still not satisfied (72% and 89.5%).35,36 Due to the high reproducibility of this bioprobe, we wondered if it could perform better in bloody specimen detection. As shown in Figure S7, although the bloody specimen without the presence of fluorescence bioprobes shows several ultraviolet absorption peak, the absorbance at 360 nm, 478 nm, and 665 nm are all relatively low. What is more, urine sample shows no apparent emission peak. It means that urine sample has little influence on our detection. Firstly, an initial baseline was established by injecting telomerase extracts from 10 normal urine specimens into our dual emissive model (Figure S8). Subsequently, upon introduction of telomerase extracts from 20 bladder cancer bloody urine specimens, it was observed that the emission intensity ratio (I478/I665) obviously increased (Figure 4a). The emission intensity ratio distributions of bladder cancer bloody and normal urine specimens were shown in Figure 4b, suggesting an apparent difference between them. These results showed that the result of 20/20 (100%) bladder cancer urine specimens are above the threshold level. Table S4 and Table S5 exhibited the analysis results of these samples, revealing that this bioprobe obtains excellent detection rate in bloody urine specimens and possess the potential in practical applications.

Figure 4. Results of ratiometric telomerase activity detection in urine specimens. (a) Emission intensity ratio (I478/I665) in response to telomerase extracts from 20 bladder cancer bloody urine specimens. The horizontal dashed line represents the threshold level (I0+3σ; I0: average of (I478/I665) from 10 normal specimens; σ: standard deviation of (I478/I665) from 10 normal specimens). Error bars indicate standard deviation of triplicate tests. Insert: Photograph of a bloody urine sample. (b) Box chart representation of telomerase activity detection from urine specimens of 20 bladder cancer patients (red) and 10 normal subjects (gray) by using this ratiometric detection strategy.

Reproducibility of Urine Sample Diagnosis. We have also carried out additional experiments to show the utility of our ratiometric bioprobe in bladder cancer diagnosis. Sample No. 2 was chosen to test the reproducibility of this bioprobe. The sample was measured for 4 times using ratiometric bioprobe and turn-on bioprobe, respectively. We can see from Figure 5 and Figure S9 that the standard deviation of ratiometric bioprobe towards sample No. 2 was 0.031, lower than that 0.283 when using turn-on bioprobe. The novel ratiometric fluorescent bioprobe obviously maintained excellent reproducibility

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in bladder cancer bloody urine specimen detection. Furthermore, to prove the credibility of our bioprobe, extracts from 10000 E-J cancer cells, 10000 HeLa cancer cells, and a bladder cancer urine specimen were also detected using a commercial ELISA Kit. As shown in Figure S10, these extracts possess the same order of activity magnitude, in good agreement with our method. The results above demonstrated that this ratiometric bioprobe could be applied as a reliable method with high reproducibility for diagnosis of bladder cancer.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work is supported by National Basic Research Program of China (973 program, 2015CB932600, 2013CB933000), the National Natural Science Foundation of China (21525523, 21375042, 21574048, 21405054) and 1000 Young Talent (to Fan Xia).

REFERENCES

Figure 5. Reproducibility comparison between ratiometric and turn-on fluorescent bioprobe. Results of No. 2 urine specimen under different conditions by using ratiometric (a) and turn-on (b) bioprobe.

CONCLUSION In summary, we have demonstrated a ratiometric bioprobe for telomerase activity detection. The solution containing Silole-R and CP shows nonintrusive dual emission bands at 478 nm of blue emission (Silole-R) and 665 nm of red emission (Cy5) under a single wavelength excitation. With the addition of different amounts of telomerase, the dual emission intensity ratios (I478/I665) of this bioprobe gradually increases. These fluorescence intensity ratios can be employed for quantitative analysis of telomerase. By importing an internal control fluorescence dye, we have achieved high reproducibility with satisfactory sensitivity, selectivity, and rapidity (~ 1 h). The feasibility of this ratiometric bioprobe is demonstrated by using telomerase extracts from different cancer cells including E-J, HeLa, MCF-7, and normal cells HLF. Moreover, by checking telomerase extracts from 20 bloody bladder cancer and 10 normal urine specimens, 100% positive result rate towards bloody urine specimens is also achieved. We thus deem the present contribution will bring new material for the construction of bioprobe and promote the application in clinical diagnostics.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at http://pubs.acs.org. Detailed description of the experimental procedures, DNA sequences, and additional figures.

AUTHOR INFORMATION Corresponding Author *Phone: +86-27-87559484. E-mail: [email protected].

Author Contributions ‡ These authors contributed equally.

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