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Nanoparticle-Aided Amplification of Fluorescence Polarization for Ultrasensitively Monitoring Activity of Telomerase Yanfang Gao, Jing Xu, Baoxin Li, and Yan Jin* Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an 710062, China S Supporting Information *
ABSTRACT: To realize facile and reliable analyzing telomerase activity in homogeneous, herein, for the first time, a fluorescent polarization (FP) strategy was developed for polymerase chain reaction (PCR) free monitoring activity of human telomerase at single-cell level ground on gold nanoparticle (GNP) enhancement of FP. First, thiolated telomerase substrate (TS) primer is modified to the surface of GNP via Au−S bond. In the presence of telomerase, TS primer was extended via adding hexamer repeats (GGGTTA), leading to the formation of a long elongation DNA. Several short carboxyfluorescein (FAM)-modified complementary DNA (FcDNA) can hybridize with the hexamer repeats, resulting in a sharp increase in FP value. Because of the GNP enhancement and self-amplification of telomerase, telomerase activity accounting to one HeLa cell can be rapidly detected in homogeneous solution. Telomerase activities of various cell lines were also favorably estimated. Meanwhile, the inhibition efficiency of telomerase inhibitor was studied, which holds great potential in screening telomerase-targeted anticancer drugs as well. So, a facile method was put forward to reliably and ultrasensitively detect telomerase activity. KEYWORDS: fluorescence polarization, telomerase activity, GNP enhancement, PCR-free, homogeneous solution, single-cell level methods have been developed, such as immunoassay,14 fluorescence,15−19 electrochemical method,20−22 colorimetric method,23−25 chemiluminescence,26,27 surface enhanced Raman scattering (SERS)s,28 and so on. These methods provide available platform for telomerase activity assay, but these techniques still have their own limitations. For instance, our group19 has reported a dual amplification fluorescence strategy for sensitive detecting telomerase activity. Zhang’s group26 has reported an isothermal circular strand-displacement polymerization for telomerase activity assay. Those PCR-free methods can sensitively detect telomerase activity of tumor cells. However, they use other protein enzymes for signal amplification. An alternative protocol based on nanotechnology is developed. Zhang’s group16 develops a telomerase activity detection biosensor based on single quantum dot, and the detection limit is 185 cells, which is still insufficient in sensitivity without amplification. Wang’s group28 develops an “elongate and capture” procedure using both color and SERS as the signal output, which could susceptibly evaluate telomerase activity low to 1 tumor cell per milliliter. But it needs magnetic separation, which makes it prolix and complicated. Thus, the challenge is to exploit a convenient, simple, and rapid method to directly detect telomerase activity in homogeneous solution.
1. INTRODUCTION Human telomeric DNA consists of the G-rich repeated sequence (TTAGGG)n, which is subject to successive reduction in length during normal DNA replication process. Most somatic cells experience progressively shortens of telomeric repeats during consecutive cell divisions, which result in cell consenescence and apoptosis. Meanwhile, telomerase activity is undetectable in adult somatic cell. While, in above 85% of tumors cells, telomerase has up-expression levels.1−4 Telomerase, a ribonucleoprotein enzyme, can synthesize singlestranded telomeric repeats (TTAGGG)n to keep the stability of telomere length by utilizing its RNA subunit as template and its protein subunit as polymerase. Thus, active telomerase can promote cancer cells immortality.5,6 So, the strong relationship between the telomerase activity and tumors makes it as diagnostic and prognostic cancer biomarker.7 Hence, the reliable and ultrasensitive detection of telomerase activity is of great theoretical and practical importance for cancer diagnosis and therapy. Telomerase was discovered in 1985.8 Since then, various methods emerged to analyze telomerase activity. Among them, classical telomere repeat amplification protocol (TRAP) is considered as a momentous approach for evaluating telomerase activity. Nevertheless, it involves the precise control of temperature cycling, the limits of polymerase, and numerous artifacts for polymerase chain reaction (PCR) processing, resulting in this technique being trying and time-consuming.9−13 To overcome the above disadvantages, alternative © 2016 American Chemical Society
Received: February 23, 2016 Accepted: May 17, 2016 Published: May 17, 2016 13707
DOI: 10.1021/acsami.6b02271 ACS Appl. Mater. Interfaces 2016, 8, 13707−13713
Research Article
ACS Applied Materials & Interfaces
Scheme 1. Fluorescence Polarization Strategy for Detection of Human Telomerase Activity Based on Gold Nanoparticle (A) and without Gold Nanoparticle Enhancement (B)
The fluorescence polarization (FP) technique can quickly and homogeneously analyze molecular interactions in biological/chemical systems.29,30 FP, a self-calibration method, is insensitive to factors such as light path, geometry, fluorescence fluctuation, and photobleaching, compared with intensity signal. Meanwhile FP is a one-pot method; there is no need of complex separation, which makes it simpler for measurement.29−31 Herein, we present a novel FP assay for highly sensitive and specific monitoring activity of telomerase. As is well-known, FP value inversely proportioned to the rate of molecular rotation, which is greatly affected by molecular volume and weight. The molecular rotation of short carboxyfluorescein (FAM)-labeled complementary DNA (FcDNA) will be significantly limited when it hybridized with the long elongation product of telomerase substrate (TS) primer. Meanwhile, by using gold nanoparticle (GNP) as carrier of TS primer, the change in FP is greatly amplified. So, activity of telomerase could be ultrasensitively monitored via GNP enhancement and self-amplification of telomerase.
the expectant ultima concentrations. All telomerase extension reaction was performed just like the procedures described in previous work.34 2.4. Procedures for Fluorescence Polarization Assay. The telomerase elongation products were put into 10 mM Tris-HCl buffer containing 1 mM MgCl2 (pH 7.4). Then, 1 μL of 1 μM F-cDNA was put into the mixture and reacted for 5 min at ambient temperature. After that, the FP measurement was immediately performed using fluorometer F-7000. The FP value (mP, 1 P = 1000 mP) can be obtained according to the following Equation,35 in which IP is the fluorescence intensity of parallel light, and IS is the fluorescence intensity of vertical light.
mP = 1000
IP − IS IP + IS
2.5. Conventional Telomere Repeat Amplification Protocol Assays. Telomerase extracts accounting to 500 cells were used to accomplish telomerase extension reaction. Then the elongated products (5 μL) mixed with 400 nM TS primer, 200 μM dNTPs, 400 nM ACX primer, and 2.5 U of Taq DNA polymerase in 1 × PCR buffer. PCR process and the postgel electrophoresis were performed according to previous report.34
2. EXPERIMENTAL SECTION
3. RESULTS AND DISCUSSION 3.1. Principle. Scheme 1 illustrates the sensing mechanism of this FP strategy. The FP value P is sensitive to the change in rotary motion of fluorescent molecules, which depends on its molecular weight (molecular volume) at permanent solution viscosity and temperature.31,35 The degree of variation relies on the size of the complex and the force of the binding interaction. Herein, a dye-tagged short complementary DNA of telomeric DNA (F-cDNA) is designed as fluorescent reporter. Thiolated telomerase substrate (SH-TS) is self-assembled onto the surface of GNP by Au−S bond. In the absence of telomerase, the molecular volume of F-cDNA is small, and it rotates fast. So, the FP value is comparatively tiny. Nevertheless, when telomerase was added, TS primer on the GNP is enzymatically elongated by adding G-rich repeats, (TTAGGG)n, to form a long telomeric DNA. Numerous F-cDNA can hybridize with the telomeric repeats, which obviously increased the molecular volume, resulting in a high FP value (Scheme 1A). Therefore, change in FP value can specifically and sensitively reflect the activity of telomerase. Scheme 1B illustrates the FP enhancement of F-cDNA caused by telomerase. However, the change in
2.1. DNA-Functionalized Gold Nanoparticle Preparation. GNP was synthesized via the reduction of HAuCl4 by citrate according to a recorded literature method.32 The DNA-functionalized GNPs were prepared based on previous method.33 Briefly, 10 nM Au colloid incubated with 2 μM SH-TS at room temperature for 16 h. Then, a high concentration of NaCl (1 M) was stepwise put into the above mixture to make the final concentration of NaCl reach 0.2 M. After it stood by for 24 h, the mixture was centrifuged for 30 min at 12 000 rpm and washed three times to purify DNA-functionalized GNP. Then the nanoparticle was suspended in 10 mM phosphate-buffered saline buffer and kept at 4 °C for further use. 2.2. Preparation of Telomerase Extracts. CCRF-CEM cell line was cultivated according to the previous reported work.34 The other cell lines, including HeLa cell line, AGS cell line, and HL-7702 cell line, were cultivated in Dulbecco's Modified Eagle Medium medium. A Petroff−Hausser cell counter was used for counting cell number. Telomerase extracts were performed according to our previous work.34 2.3. Telomerase Extension Reactions. The telomerase substrate was elongated based on our previous work.34 CHAPS lysis buffer was used to perform blank control experiment. Heat-deactivated control experiment was implemented by heat-treated telomerase extracts (95 °C for 10 min). For the inhibition test, a series volume of aloe− emodin (AE) solutions were first added to the TRAP buffer to obtain 13708
DOI: 10.1021/acsami.6b02271 ACS Appl. Mater. Interfaces 2016, 8, 13707−13713
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ACS Applied Materials & Interfaces
Figure 1. Prepared GNP was investigated by TEM image (A) and DLS characterization (B). UV−vis spectra of GNP and GNP-TS (C). Zetapotentials of GNP and GNP-TS in H2O (D).
FP is lower compared with Scheme 1A due to the lack of GNP. So, the introduction of GNP led to a significant amplification of change in FP, demonstrating that the GNP-aided FP strategy makes it possible to ultrasensitively detect telomerase activity in homogeneous solution. 3.2. Characterization of Gold Nanoparticle and Probe. As an enhancer of FP strategy, the property of GNP needs to be studied. As shown in Figure 1A, the transmission electron microscopy (TEM) image of the spherical GNP revealed distribution with uniform size, and the average diameter of GNP is ∼16 nm. Dynamic light scattering (DLS) experiment further confirmed that the average hydration diameter of GNP is 17.0 nm (Figure 1B). UV−vis spectra (Figure 1C) and zetapotential assay (Figure 1D) were utilized to test the selfassembly of SH-TS on the surface of GNP. As depicted in Figure 1C, UV−vis spectroscopy of GNP-TS displayed two characteristic absorption peaks, including an absorption peak of DNA at 260 nm and a absorption peak of GNP at 522 nm. Compared with unmodified GNP, the absorption peak of TSmodified GNP slightly red-shifted. Figure 1D showed GNP-TS presented a more negative potential in comparison with GNP, revealing the coupling of negatively charged DNA with GNP. 3.3. Fluorescence Polarization Assay for Telomerase Activity Detection. To verify the principle, Figure 2 depicts the change in FP value of F-cDNA. The FP value of F-cDNA/ GNP-TS mixture in lysis buffer was selected as blank (column a). FP value of F-cDNA obviously increased when it incubated with TS primer and telomerase, which was extracted from 500 HeLa cells (column b). More importantly, the change in FP value further increased with the help of GNP (column c). The increase in FP value is mainly ascribed to the telomeraseinduced increase. The enlargement of the molecular volume V
Figure 2. Fluorescence polarization changes of F-cDNA under different conditions. GNP-TS with lysis buffer (a), TS with HeLa cell extracts (b), GNP-TS with HeLa cell extracts (c), GNP-TS with heat-inactive HeLa cell extracts (d), GNP-TS with HL-7702 cell extracts (e). Error bars show standard deviation of three replicate tests at least.
is attributed to the hybridization between F-cDNA and telomerase elongation product, which is attached to the surface of GNP. So, the preliminary result reveals it is possible to use GNP-enhanced FP strategy for detecting telomerase activity. Furthermore, more control experiments were conducted to ensure reliability. When F-cDNA is incubated with GNP-TS and heat-inactive telomerase, the change in FP value is negligible (column d). Meanwhile, normal cell extracts generated ignorable change in FP value (column e), indicating that telomerase activity in normal cells is very low. These results demonstrated that change in FP value is attributed to the telomerase-induced elongation of TS primer. F-RDNA, a random sequence DNA, was used to repeat the above 13709
DOI: 10.1021/acsami.6b02271 ACS Appl. Mater. Interfaces 2016, 8, 13707−13713
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Figure 3. FP response of F-cDNA corresponding to different telomerase activity for the pure TS (A) and GNP-TS system (C). Linear relationship between ΔFP value and number of HeLa cells for the pure TS (B) and GNP-TS (D) system. Error bars show standard deviation of three replicate tests at least.
increasing of GNP-TS concentration to 2 nM, and then the ΔFP value decreased. Hence, 2 nM of GNP-TS was selected for the succeeding assay. Then, an important parameter of telomerase extension time, which might affect the length of telomerase extension products, was evaluated. As displayed in Figure S3B, as the telomerase extension time increases, the ΔFP value increased and then reached equalization after 60 min. Thus, 60 min was selected as the extension time. As shown in Figure S3C, the ΔFP value reached maximum in the presence of 10 nM F-cDNA. As the increase of F-cDNA concentration, the proportion of free F-cDNA was larger, leading to FP value change that was inconspicuous, which decreased the sensitivity of the FP assay. So, the optimal concentration of F-cDNA is 10 nM. At last, the detection time was investigated. Figure S3D showed that the ΔFP value varied quickly within 1 min and reached maximum after 5 min, when 10 nM F-cDNA incubated with telomerase elongation product. Therefore, the detection time was 5 min throughout the experiment. 3.5. Sensitivity of Telomerase Activity Assay. The change of FP signal of F-cDNA was used to estimate telomerase activity of HeLa cells. HeLa cell extracts were successively attenuated by using lysis buffer as a source for telomerase. Figure 3A depicted the change in FP value of FcDNA, when pure TS primer was used as telomerase substrate. The ΔFP value gradually enhanced as the HeLa cell increased from 30 to 1000. Figure 3B shows the ΔFP value increased linearly as the number of HeLa cells ranged from 50 to 1000
experiment. As revealed in Figure S1, in the presence of telomerase, neither GNP-TS nor TS can cause the change in FP value of F-RDNA. These results demonstrated that there is no nonspecific binding. Therefore, it is a reliable approach to monitor telomerase activity in homogeneous solution. Since GNP can be used as fluorescence quencher, there may be the possibility that GNP could quench the fluorescence of FcDNA, which could lead the heterogeneity of the experiment. We investigate the influences of GNP on fluorescence intensity and FP values. As shown in Figure S2A, the samples S2−S8 had the alike fluorescence intensity of S1 (free F-cDNA). Similarly, as shown in Figure S2B, there were almost the same FP values among the samples S1−S4, S7, and S8. Only when 500 HeLa cells incubated with TS does FP value of F-cDNA change (S5). A more obvious increase in FP value was obtained when GNPTS incubated with 500 HeLa cells extract due to the enhancement of GNP (S6). Once again, the above results demonstrated that changes in FP value of F-cDNA are ascribed to the specific hybridization between F-cDNA and telomerase elongation product. And GNP will not affect the fluorescence intensity of F-cDNA. 3.4. Optimization of Experimental Condition. To make the detection platform more sensitive, as shown in Figure S3, some main factors were investigated. As is well-known, the RNA template of telomerase functioned only by binding onto telomere DNA or telomerase substrate primer. So, the concentration of GNP-TS primer was first investigated. As indicated in Figure S3A, the ΔFP value enhanced with the 13710
DOI: 10.1021/acsami.6b02271 ACS Appl. Mater. Interfaces 2016, 8, 13707−13713
Research Article
ACS Applied Materials & Interfaces
Figure 4. (A) Comparison of telomerase activity of different cell lines. Error bars show standard deviation of three replicate tests at least. (B) Classical TRAP assay for telomerase activity. Lines right to left show CCRF-CEM, AGS, HeLa, HL-7702, and heat-inactive HeLa, respectively.
telomerase activity. AE is a natural Chinese medicine monomer and an effective G-quadruplex-binding ligand as reported by our previous work.38 Here, AE was used as an inhibitor to study the inhibition effect of G-quadruplex ligand on the telomerase activity. So, AE was mixed with extracts of HeLa cell from 500 cells. As shown in Figure 5, the half-maximal prohibitive
with a correlation coefficient of 0.994. However, as depicted in Figure 3C, the ΔFP value of F-cDNA varied more remarkably as HeLa cells increased from 0 to 1000 when telomerase incubated with GNP-TS. As illustrated in Figure 3D, these results demonstrated the ΔFP value has a positive correlation with the number of HeLa cells from 10 to 1000 and the correlation coefficient is 0.986. Owing to the GNP enhancement, telomerase activity can be remarkably measured to one HeLa cell. The sensitivity should be comparative or superior to some established approaches (shown in Table S1). On the contrary, Figure S4A depicts the ΔFP value of F-cDNA has little fluctuation as the amount of heat-inactive telomerase increased. Furthermore, as shown in Figure S4B, the ΔFP value also has little change when F-cDNA incubated with GNP-TS and the extracts from different number of normal cell. These confirmed other nonspecific factors had no influence on the ΔFP value. Thus, these results forcefully guarantee the high sensitivity and assurance of the telomerase activity assay. 3.6. Evaluation of the Generality of the Telomerase Assay. To further evaluate the generality of the strategy for telomerase activity assay, the difference in telomerase activity of disparate cell lines was studied. Herein, HeLa cancer cell, CCRF-CEM cell, AGS cancer cell, and HL-7702 normal cell were selected as model target to evaluate telomerase activity. As shown in Figure 4A, all the cancer cell lines can induce high ΔFP value. Conversely, normal cell HL-7702 generated a tiny change in FP value. The heat-inactivate extracts of HeLa cell rendered ignorable change in FP value. Above results were in accordance with previous reports.6,34 Furthermore, to evaluate the accuracy of this method, classical TRAP-silver staining assay was used to analyze the corresponding cell extracts. As shown in Figure 4B, CCRF-CEM cell, AGS cell, and HeLa cell showed obvious telomerase amplification products band. On the contrary, normal cell HL-7702 and heat-inactive cell extracts showed no band of telomerase amplification products, which were consistent with the FP assay results (Figure 4A). What is more, compared with TRAP, FP assay can discriminate expression levels of telomerase activity among various cell lines. 3.7. Evaluation of Inhibition Effect of Telomerase Inhibitors. As a universal cancer biomarker, telomerase inhibitors may become potential anticancer agents.36 Telomerase needs to bind its RNA template onto the unfolded stranded chromosome or the end of the primer. Formation of Gquadruplex at the end of telomeric DNA overhang can effectively block the binding of telomerase.37 Hence, Gquadruplex selective ligands have a potential to inhibit
Figure 5. Inhibition effects of AE on telomerase activity of HeLa cells. Error bars show standard deviation of three replicate tests at least.
concentration (IC50) value of AE on HeLa cells was evaluated to be 5.89 μM. Different G-quadruplex binding abilities can affect telomerase inhibitory activities. Above results conformably suggested that the proposed strategy is applicable for evaluating potential inhibitors of telomerase.
4. CONCLUSION In summary, we develop an FP strategy to reliably detect activity of human telomerase at single-cell level in homogeneous solution ground on GNP enhancement and selfamplification of telomerase. It demonstrates several analytical advantages. First, FP is a powerful technique for studying biological interaction in homogeneous solution because it can be slightly affected by dye fluctuation and surrounding disturbance, which renders FP assay a dramatic selective sensing technology to study telomerase activity in complex matrices. Second, only a single labeled DNA probe was used to report telomerase activity, leading to simple and fast detecting telomerase activity within 5 min. Third, because of the GNP enhancement of FP value change, the telomerase activity could be PCR-free and reliably monitored at single-cell level. 13711
DOI: 10.1021/acsami.6b02271 ACS Appl. Mater. Interfaces 2016, 8, 13707−13713
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Additionally, the good practicability of GNP-enhanced FP assay has been verified by discriminating difference in telomerase activity of different cell lines and evaluating the inhibition efficiency of telomere-binding ligand. So, it has important theoretical and practical significance in the development of cancer diagnosis and telomerase-related research.
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b02271. Experimental reagents, apparatus, FP changes of FRDNA under different conditions, effects of GNP on fluorescence intensity, factors effect on FP changes, comparison of different assays for telomerase activity detection, additional references. (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Fax: 86-29-81530727. Phone: 86-29-81530726. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was fiscally supported by the following grants: the National Natural Science Foundation of China (No. 21075079 and 21375086), the Program for Changjiang Scholars and Innovative Research Team in University (IRT_14R33), Program for Innovative Research Team in Shaanxi Province (No. 2014KCT-28), and the Fundamental Research Funds for the Central Universities (GK201505046).
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