Sensitive and Bidirectional Detection of Urine Telomerase Based on

Aug 28, 2014 - Telomerase, a valuable biomarker, is highly correlated with the development of most of human cancers. Here, we ..... Greider , C. W. Pr...
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Sensitive and Bidirectional Detection of Urine Telomerase Based on the Four Detection-Color States of Difunctional Gold Nanoparticle Probe Ruixue Duan, Boya Wang, Tianchi Zhang, Zhenyu Zhang, Shaofang Xu, Zhifei Chen, Xiaoding Lou, and Fan Xia* Key Laboratory for Large-Format Battery Materials and Systems, Ministry of Education, School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology (HUST), Wuhan, China S Supporting Information *

ABSTRACT: Telomerase, a valuable biomarker, is highly correlated with the development of most of human cancers. Here, we develop a bidirectional strategy for telomerase activity detection and bladder cancer diagnosis based on four detection-color states of difunctional gold nanoparticle (GNP) probes such as blue, purple, red, and precipitate. Specifically, we define the red GNP probe as origin, which represents urine extracts with inactive telomerase and implies normal individuals. The forward direction is corresponding to the detection of a relatively high concentration of active telomerase, in which system GNP probes assemble obviously and precipitate, predicting bladder cancer samples. The negative direction is corresponding to extracts with a relatively low concentration (purple) and without any telomerase (blue), which can be differentiated by naked eyes or UV−vis spectrum, indicating bladder cancer and normal individuals, respectively. More importantly, this noninvasive strategy shows great sensitivity and selectivity when tested by 18 urine specimens from bladder cancer patients, inflammation, and normal individuals.

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great potential to be applied in resource-constrained countries.25 For example, Qu et al. developed a fast and simple method for telomerase activity using primer-modified GNPs.26,27 Nevertheless, it should be noted that this colorimetric method employed the G-quadruplex folded by telomerase products to protect and stabilize GNPs, which is an indirectly detection method against sensitivity. In addition, most of these colorimetric biosensors are associated with a dramatic color change from red-to-blue or blue-to-red, which, herein, we call a unidirectional detection system. The unidirectional character based on simple color change is a limit to the design of versatile sensing schemes and is also disadvantageous to distinguishing and reading out the visual results easily and quickly, especially when multiple detection states of GNPs are required. Herein, we design a difunctional GNP probe with four detection-color states, develop a bidirectional and direct method with accuracy and simplicity for detection of urine telomerase activity, and successfully apply it in painless diagnosis of bladder cancer (Figure S1). We demonstrate that this strategy is bidirectional, like a number axis, according to the four detection-color states of the GNP probe and values of

uman telomerase, a reverse transcriptase, contains an endogenous RNA template and can catalyze repeated units TTAGGG to the ends of telomers.1 Telomerase activity is correlated with tumor aggressiveness because it can be detectable in most immortal cell lines and primary human tumors.2 Telomeric Repeat Amplification Protocol (TRAP) has been developed as a golden method for telomerase activity detection. Although TRAP and other modified TRAP assays are very sensitive, they suffer from numerous artifacts from PCR amplification.3−6 For example, they are susceptible to inhibition by highly concentrated cell extracts, are time-consuming, and require sophisticated optimization. Moreover, it is also limited by the risk of a false positive from the exponential amplification. In addition, it is worth mentioning that the sensitivity can be limited by G-quadruplex.7 In this context, other alternative approaches for telomerase activity detection based on fluorescence,8,9 colorimetry,10,11 electrochemistry,12,13 electrochemiluminescence,14 chemical luminescence,15 and surface plasmon resonance16 have been developed. Since the use of a gold nanoparticle (GNP) for biological uses was first demonstrated by Mirkin et al. in 1996,17 GNP has played an important role in constructing promising platforms for colorimetric detection of DNA,18,19 protein,20−22 small molecules,23 and metal ions24 due to its high extinction coefficients and strong distance dependent optical properties. The GNP-based colorimetric assays have shown inherent superiorities, such as being simple, easy to read out, and having © 2014 American Chemical Society

Received: June 26, 2014 Accepted: August 28, 2014 Published: August 28, 2014 9781

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CHAPS lysis buffer, and then incubated on ice for 30 min. The mixture was centrifuged at 10 000g for 20 min at 4 °C. The supernatant was transferred, aliquoted, and stored at −80 °C. Telomerase Extracted from Urine Samples. Fresh urine samples were collected and centrifuged at 850g for 10 min at 4 °C and washed once using phosphate-buffered saline (PBS). Centrifuged the above samples at 2300 g for 5 min at 4 °C. The precipitate was resuspended in 200 mL of ice-cold 1X CHAPS lysis buffer and then incubated on ice for 30 min. Centrifuged the mixture at 10 000g for 20 min at 4 °C. The supernatant was transferred, aliquoted, and stored at −80 °C. Telomerase Extracted from Tissues. The tissue sample was placed in a sterile mortar and frozen by adding liquid nitrogen. The sample was pulverized by grinding with a matching pestle. The thawed sample was transferred to a sterile 1.5 mL microcentrifuge tube, and it was resuspended in an appropriate amount of 1X CHAPS Lysis Buffer and then incubated on ice for 30 min. The mixture was centrifuged at 10 000g for 20 min at 4 °C. The supernatant was transferred, aliquoted, and stored at −80 °C. Preparation of GNP Probe. Briefly, 0.33 μL of 500 mM acetate buffer (pH 5.2) and 0.5 μL of 10 mM TCEP were added to the mixture of 0.4 nmol TS primers and 3.6 nmol reporter probes to activate the thiolated DNA. The mixture was incubated for 1 h at room temperature. Then 1 mL of GNPs (the concentration of the GNPs was increased by centrifugation and resuspension to obtain a final concentration of 1 nM) was added to the above solution. The mixture was incubated overnight at room temperature. 500 mM tris acetate (pH 8.2) buffer was added dropwise to the sample until a concentration of 5 mM tris-acetate was reached. 1 M NaCl dropwise was added to reach a final salt concentration of 0.1 M NaCl. The tube was wrapped in foil and placed on an orbital shaker for at least another day. The solution was centrifuged at 4 °C for 20 min and 12 000 rpm. To remove excess thiol-DNA, the solution was centrifuged (14 000 rpm, 80 min), and the supernatant was carefully removed four times before use. The deposited GNP probes were rinsed with 1 mL of buffer containing 25 mM Tris, 100 mM NaCl, and 0.005% Tween-20. Tween-20 was used to reduce the sticking of GNP probes to the Eppendorf tube. The GNP probes were stored at 4 °C. Telomerase Extension Reaction. Telomerase extracts were diluted in lysis buffer with the respective number of cells; the extracts (5 μL) were added to 45 μL extension solution containing 1× NEB buffer 2, 1 mM dNTP, 1 mM MgCl, 0.2 mg/mL BSA, 0.4 U/μL RNase inhibitor, and TS primered labeled GNP probes. The solution was incubated at 37 °C for 120 min. After standing for 2 h, the resulting mixtures were heated at 95 °C for 5 min to deactivate the telomerase; finally, it was incubated at 37 °C for 20 min to realize reassembly between the reporter probe and telomerase products. For control experiments, telomerase extracts were heat treated (95 °C for 15 min).

A650/A520 of detection samples (Figure 1a). Origin is the red GNP probes, and the value of A650/A520 here is less than

Figure 1. (a) The number axis theory for clinical estimate of bladder cancer without pain based on proposed method. N, normal individuals; C, bladder cancer. (b) Telomerase extraction from urine samples. (c) The scheme for telomerase detection.

0.23. The forward direction of the number axis represents precipitated GNP probes, and the range of values of A650/ A520 is about 0.23−0.60. The negative direction of the number axis represents purple and blue GNP probes. The value of A650/A520 of blue GNP probes is greater than 0.60, and the range value of A650/A520 of purple GNP probes is also about 0.23−0.60. Then, we advance a theory for bladder cancer diagnosis on the basis of the relationship between the GNP probe states and telomerase activity and successfully test it by 18 urine samples. The colors of GNP probes keeping red (A650/A520 < 0.23) or changing to blue (A650/A520 >0.60) will indicate that the samples may be from normal persons; on the other hand, purple color or precipitate will imply that the samples may be from bladder cancer patients.

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EXPERIMENTAL SECTION Materials. The deoxynucleotide solution mixture (dNTPs), BSA, RNase inhibitor, and DEPC-treated water were purchased from TaKaRa Bio Inc. (Dalian, China; DEPC = diethylpyrocarbonate). Oligonucleotides were synthesized by TaKaRa Bio Inc. (Dalian, China). Patient samples were donated by union hospital, Tongji Medical College, Huazhong University of Science and Technology. Primer SH-TTTTTTTTTT AAT CCG TCG AGC AGA GTT Reporter probe CCCTAA CCCTAA CCC-SH Telomerase Extracted from Cultured Cells. MCF-7 cells were cultured in DMEM medium supplemented with 15% fetal calf serum. After the cells were harvested with trypsin, 1 × 106 cells were collected, pelleted in a 1.5 mL EP tube at 1000g for 5 min. Cells were washed once with phosphate buffered saline (pH 7.4), pelleted again, dispensed in 200 μL of ice-cold 1X

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RESULTS AND DISCUSSION The Scheme for Telomerase Detection. Figure 1 illustrates a schematic of the strategy for telomerase detection performed here. Telomerase primer (TS primer) and a reporter probe that can hybridize with the telomerase products are copreconjugated to the surface of a GNP, forming a GNP probe (red). In the presence of the telomerase, the primer is elongated. The elongated primer can bind to the reporter probes from another one or two or even more GNP probes.

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Meanwhile, the elongated primer from hybridized GNP probes can also hybridize with others in the same manner, and then the complicated network structure formed. In this proposed method (Figures 1c and 2), if the extracted sample consists

Figure 3. (a) Colorimetric response of the proposed detection system to various concentrations of telomerase. Samples from left to right represent the telomerase equivalent to 0, 6, 23, 94, and 375 MCF-7 cells μL−1 in lysis buffer, respectively, and water samples. (b) Corresponding UV/vis spectra of our detection system in the presence of various concentrations of telomerase.

aggregation of GNP probes. It is mainly because ions from the lysis buffer screen the repulsive interactions of the citrate ions around GNPs intensively,28 leaving it unable to stabilize the nanoparticles against aggregation and thus leading to a characteristic blue color. Likewise, for the purple GNPs, the red shift and broadened surface plasmon band can also be observed from UV−vis spectra of telomerase samples equivalent to 6 and 23 MCF-7 cells μL−1 in lysis buffer. Although the value of A650/A520 of purple GNPs is enhanced with the decrease of telomerase concentration, they are still obviously smaller than that of blue GNPs. This phenomenon is because telomerase and ions from telomerase lysis buffer have a combined effect on GNP probes. Telomerase elongates the TS primer and initiates the orderly assembly of GNP probes, whereas ions from telomerase lysis buffer induce unorderly aggregation of GNP probes. Therefore, the aggregation of purple GNPs is less intensive compared to that of blue GNPs. However, for the precipitated GNP probes induced by extracted samples containing high concentration of telomerase, the value of maximal absorption increases remarkably, and a weak red shift can be observed compared to purple GNP probes. The different maximal absorptions and red shifts between purple and precipitated GNP probes are caused by assembly layers and interparticle distance, respectively, and the large distances provided by extended telomerase products significantly reduce the red shift effect.29 Moreover, the value of A650/A520 is enhanced with the increment of telomerase concentration, which we have further demonstrated by introducing DMSO (Figures S2 and S3). More importantly, we document that our detection system is bidirectional based on the four detection-color states of the GNP probe and values of A650/A520 of detection samples, like a number axis. The original point is the red GNP probes, which is interference-free for inactive telomerase, and the value of A650/A520 is less than 0.23 (the threshold line is calculated by the evaluation of the average response of the red GNP probes plus 3 times the standard deviation). In the forward direction where the concentration of active telomerase is relative high, the GNP probes will undergo solution-to-precipitate change with the increment of the concentration of active telomerase,

Figure 2. TEM images from four states of GNP probes. The scale bar is 50 nm. The blue GNPs are induced by the sample containing only lysis buffer. The red GNPs are induced by the heat-treated telomerase extracted from 375 cells μL−1 in lysis buffer. The purple GNPs are induced by the telomerase equivalent to about 50 cells μL−1 in lysis buffer, and the precipitate GNPs are induced by the telomerase equivalent to 375 cells μL−1 in a lysis buffer.

of inactive telomerase, the GNP probes will be protected, and its color will still be red; for the sample containing a high concentration of active telomerase, the GNP probes will assemble and precipitate; however, for the extracted sample with a low concentration of active telomerase or without any telomerase, the color of GNP probes will experience red-topurple or red-to-blue change, respectively, which can be monitored by naked eyes or UV−vis spectra. Detection of Telomerase from MCF-7 Cells and Demonstration of the Bidirectional Character. For the proof-of-concept experiment reported herein, telomerase activity equivalent to 0, 6, 23, 94, and 375 MCF-7 cells μL−1 in lysis buffer, respectively, and water samples are investigated first using the present strategy. The detection systems are incubated at 37 °C for 2 h, then heated at 95 °C for 5 min to deactivate the telomerase, finally incubated at 37 °C for 20 min to realize reassembly between the reporter probe and telomerase products. Figure 3a shows pure lysis buffer changes the color of GNP from red to blue, and telomerase samples equivalent to 6, and 23 MCF-7 cells μL−1 in lysis buffer change the color from red to purple. The color of GNP probe will still be red when tested by water samples. Interestingly, for higher telomerase concentration, the bottom of the tube appears precipitate. The above phenomenon is confirmed by UV−vis spectroscopy (Figure 3b). The water samples (Figure 3b) or inactive telomerase samples (Figure 2) keep the GNPs stable and dispersed, producing the red color due to the intense surface plasmon resonance absorption of the nanoparticles at 520 nm. For the blue GNPs, the UV−vis spectrum of pure lysis buffer samples displays the obviously characteristic red shift and broadening of the surface plasmon band, indicating the 9783

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and the range values of A650/A520 are about 0.23−0.60. Meanwhile, in the negative direction where the concentration of the active telomerase is relatively low, the color of GNP probes suffers red-to-purple change with the decrease of the concentration of telomerase. When the quantity of telomerase is zero, the color of GNP probes will change to blue. The values of the purple and blue GNP probes can also be distinguished by the value of A650/A520. The value of A650/A520 of blue GNP probes is greater than 0.60 (the threshold line is calculated by the evaluation of the average response of the blue GNP probes plus three times the standard deviation). Thus, the range value of A650/A520 from purple GNP probes is also about 0.23− 0.60. In comparison with traditional colorimetric methods based on GNPs, our approach is more convenient and accurate because it can directly and sensitively estimate the analysis samples that contain high quantity telomerase, or low quantity telomerase, and samples without any telomerase or with inactive telomerase via the obvious four detection states and bidirectional characters of GNPs by naked eyes. Specificity and Stability Investigation. To validate the specificity of the detection system, we prepare several different protein targets, such as 100 μg BSA, 70 μg thrombin, and 5 μg telomerase from bladder cancer patients’ tissue. Figure 4a

Figure 5. (a) Photograph showing colorimetric assay to analyze telomerase activities extracted from 18 urine samples. (b) Corresponding patient IDs; clinical outcomes provided by union hospital, Tongji Medical College, Huazhong University of Science and Technology; the detection states of GNP probes, and the values of A650/A520. (N, normal; C, bladder cancer; I, inflammation).

A650/A520 are less than 0.60, which demonstrates that both samples contain low quantity of telomerase and signifies bladder cancer patients. A4 triggers a red-to-blue change, which can be further proved by its UV−vis spectrum, predicting that the target of it does not contain any telomerase and implying a normal or inflammation specimen. Other samples from Figure 5 meet the requirement of a forward direction system; the more telomerase products there are, the more GNP probes precipitate and the greater the values of A650/A520 are, implying bladder cancer specimens. These results are consistent with clinical diagnosis. Therefore, we have demonstrated this painless method is promising for bladder cancer diagnosis and proved it can distinguish between cancer and noncancer samples selectively according to our number axis theory.

Figure 4. (a) Photographs showing colorimetric detection of inactive telomerase, BSA, thrombin, telomerase extraction from tissue. (b) Photographs showing colorimetric detection of the above samples after one month. (c) Corresponding UV/vis spectra of various samples after one month. Control: inactive tissue telomerase.

shows that high concentration of BSA and thrombin will not aggregate or assemble the GNPs probe; whereas, in the presence of tissue sample, precipitate is observed. If deactivate the tissue sample (Control sample), the precipitate will not appear. To investigate the stability of this system, we store these samples for 1 month at room temperature. Figure 4b,c shows high concentrations of BSA and thrombin will not affect the stability even after 1 month. Clinical Applicability. Bladder cancer is the second most common genitourinary malignancy, and the incidence increases with age.30 To verify our proposed theory in this study and evaluate the applicability in bladder cancer diagnosis, 18 urine specimens from bladder cancer patients, inflammation patients, and normal individuals are investigated (Figure 5). A1, B1, C1, and A4 are used as controls. A1 represents a water sample. B1 is tested by urine sample froma normal person, C1 and A4 are tested by inflammation samples. Others of Figure 5 are from bladder cancer samples. According to our number axis theory for cancer diagnosis, the color of GNP probes from B1 and C1 are still red and the values of A650/A520 are less than 0.23, so we can conclude that the targets of B1 and C1 do not have telomerase activity and may be from normal or inflammation persons. In the negative direction system, A5 and C3 induce the GNP probes to undergo red-to-purple change and the values of

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CONCLUSION In conclusion, we have proposed a direct and bidirectional approach with high accuracy to detect telomerase activity by designing a difunctional GNP probe. By taking advantages of four-color-state and bidirectional performances of GNP probes here, our bidirectional method has excellent discrimination ability to evaluate the concentration and activity of telomerase quickly compared to the traditional colorimetric method. In comparison with the golden method, the TRAP-based method, our strategy is accuracy and simplicity by avoiding numerous artifacts and sophisticated optimization. Besides, this system is stable, and samples can also be distinguished even after one month. More importantly, we have proven its capabilities from 18 urine specimens from bladder cancer patients, normal, and inflammation individuals, establishing a noninvasive method for bladder cancer diagnosis. Therefore, this naked eye system with good accuracy, cost, selectivity, stability, and applicability will make it a promising tool for the detection of telomerase activity 9784

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and diagnosis of bladder cancer and even other cancers in resource-constrained countries.

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ASSOCIATED CONTENT

S Supporting Information *

Experimental procedures and analytical data are provided. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by initiatory financial support from HUST, the National Basic Research Program of China (2013CB933000), the 1,000 Young Talents program (to F. X.), Youth Scientist of National 973 Basic Research Program (SQ2015CC070115), and the National Natural Science Foundation (21375042).

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