Article pubs.acs.org/ac
Quencher Group Induced High Specificity Detection of Telomerase in Clear and Bloody Urines by AIEgens Yuan Zhuang,† Mengshi Zhang,† Bin Chen,§ Ruixue Duan,† Xuehong Min,† Zhenyu Zhang,† Fuxin Zheng,‡ Huageng Liang,‡ Zujin Zhao,§ Xiaoding Lou,*,† and Fan Xia† †
Key Laboratory for Large-Format Battery Materials and System, Ministry of Education, School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology, Wuhan 430074, China ‡ Department of Urology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China § State Key Laboratory of Luminescent Materials and Devices, South China University of Technology, Guangzhou 510640, China Downloaded by GEORGETOWN UNIV on August 26, 2015 | http://pubs.acs.org Publication Date (Web): August 25, 2015 | doi: 10.1021/acs.analchem.5b02699
S Supporting Information *
ABSTRACT: Telomerase is a widely used tumor biomarker for early cancer diagnosis. On the basis of the combined use of aggregation-induced emission (AIE) fluorogens and quencher, a quencher group induced high specificity strategy for detection of telomerase activity from cell extracts and cancer patients’ urine specimens was creatively developed. In the absence of telomerase, fluorescence background is extremely low due to the short distance between quencher and AIE dye. In the addition of telomerase, fluorescence enhances significantly. The telomerase activity in the E-J, MCF-7, and HeLa extracts equivalent to 5− 10 000 cells can be detected by this method in ∼1 h. Furthermore, the distinguishing of telomerase extracted from 38 cancer and 15 normal urine specimens confirms the reliability and practicality of this protocol. In contrast to our previous results (Anal. Chem. 2015, 87, 6822−6827), these advanced experiments obtain more remarkable specificity.
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pretreatment of samples.42 Among other important telomerase detection methods, fluorescence methods are spotlighted owing to their simplicity, high accuracy, and rapidity. For example, a fluorescence strategy based on a molecular beacon functionalized gold nanoparticle (AuNP) probe has been developed to detect activity of telomerase. 17 However, conventional fluorophores used in these fluorescence strategies are aggregation-caused quenching (ACQ) molecules. They often give bright emission in dilute solution, whereas the emission is weakened or quenched in concentrated solution or in the aggregated state. Thus, the ACQ effect has compelled many fluorescence strategies to operate in a “turn-off” mode, which is insensitive and not suitable for practical use.43−47 In order to address this concern, recently we focus on a group of fluorogens with aggregation-induced emission (AIE)
elomerase, a sensitive and selective biomarker for cancer, can add specific sequence (AATCCG)n to a telomere using its template RNA when combined with template strand primer (TS primer). Due to the high activity in cancer cells and low or no activity in normal cells,1−3 telomerase plays important roles in early detection and diagnosis of cancers.4,5 Over the past decade, a large number of strategies for telomerase activity detection have been reported, such as telomeric repeat amplification protocol (TRAP) 6,7 and strategies based on electrochemistry,8−11 colorimetry,12−14 fluorescence,15−23 chemical luminescence,24 Rayleigh scattering, 25 surface plasmon resonance, 26 and biosense.27−36 However, TRAP is based on PCR, and we need to observe results with the help of polyacrylamide gel electrophoresis (PAGE), leading to the high possibility of false positive or false negative results.37−41 Also, using radioactive 32P-dATP in the PCR process causes serious environmental pollution. On the other hand, although electrochemical methods are sensitive toward targets, they are usually subject to complicated © XXXX American Chemical Society
Received: July 18, 2015 Accepted: August 19, 2015
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DOI: 10.1021/acs.analchem.5b02699 Anal. Chem. XXXX, XXX, XXX−XXX
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Downloaded by GEORGETOWN UNIV on August 26, 2015 | http://pubs.acs.org Publication Date (Web): August 25, 2015 | doi: 10.1021/acs.analchem.5b02699
Analytical Chemistry
Figure 1. Schematic illustration of quencher group induced high specificity fluorescence strategy for detection of telomerase activity. QP combines with Silole-R molecules in part before extension reaction, but complex S-QP maintains relatively low fluorescence background because of the short distance between fluorophore Silole-R and quencher at 5′-end of QP. In the presence of active telomerase extracted from cancer cells or urine specimens of bladder cancer patients, QP extends with repeat unit (TTAGGG)n from the 3′-end due to extensive reaction of telomerase, leading to the aggregation degree of Silole-R apparently increasing. The fluorescence intensity will enhance because of the gradually increasing distance between Silole-R and quencher, making the fluorescence emission of complex S-QP experience an off-to-on process, which could be monitored by fluorescence spectra. Telomerase extracted from 10 000 MCF-7 cancer cells is used in the diagrammatic drawing.
telomerase (extracted from 10 000 MCF-7 cancer cells) is added to this system, Silole-R molecules binding to extension repeat units are relatively far away from the quencher; fluorescence intensity lights up with a signal increase percentage of 1424%. In contrast, we conduct the similar experiments using TP (TS primer without quencher group), and the signal increase percentage is only 586%, lower than that using QP. These results show the importance of quencher group in this telomerase detection system.
property coined by Tang’s group. Fluorogens with AIE property make the molecule nonemissive when dissolved and dispersed, but distinctly emissive when supramolecularly aggregated.48,49 In our previous work, we creatively designed an AIE-based turn-on method to detect telomerase activity using a positive charged quaternized tetraphenylethene salt (TPE-Z).50 The solution containing TPE-Z and TS primer showed weak but not neglectable fluorescent. In the presence of telomerase, the fluorescence enhanced after extension reaction owing to the added negatively charged sites for the dye molecules to bind and aggregate. Although we obtained a high fluorescence signal, the signal-to-background ratio was not satisfactory due to the relatively high detection background, which makes the selectivity of this method seriously restricted. Therefore, reduction of detection background and thus enhancement of selectivity are still of great significance. To solve this problem and obtain admirable selectivity, we molecularly labeled a quencher group Dabcyl (4-(4(dimethylamino)phenylazo)benzoic acid) to produce QP (quencher group-labeled TS primer) for use in this work. The positively charged AIE dye, Silole-R, is a typical AIE molecule with high fluorescence quantum yield. The synthetic route to Silole-R is shown in Scheme S1 (Figure S1). As shown in Figure 1, amphiphilic Silole-R is water-soluble owing to its positive quaternary ammonium group. It shows low background when highly dispersed in aqueous buffer. The positively charged Silole-R can spontaneously bind to the negatively charged QP because of the electrostatic force.51 The emission of Silole-R (λmax = 478 nm) and the absorption of Dabcyl (λmax = 480 nm) have a high degree of overlap (Figure S2). Before telomerase extension reaction, Silole-R aggregate emission is efficiently quenched due to the fluorescence resonance energy transfer (FRET) from Silole-R aggregate to the quencher. After
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EXPERIMENTAL SECTION Materials. Water was purified by a Millipore filtration system. The 1× CHAPS lysis buffer was purchased from Millipore (Bedford, MA). The deoxynucleotide solution mixture (dNTPs), recombinant RNase inhibitor, DEPC-treated water, and S1 nuclease (S1 Nase) were purchased from TaKaRa Bio Inc. (Dalian, China; DEPC = diethylpyrocarbonate). Oligonucleotides were synthesized by TaKaRa Bio Inc. (Dalian, China). 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. Patient samples were donated by Union Hospital, Tongji Medical College, Huazhong University of Science and Technology. 0.05% Trypsin/EDTA was purchased from Multicell Technologies. 3′-Azido-3′-deoxythymidine (AZT) and thrombin (from human plasma) were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. Bst 2.0 WarmStart DNA polymerase (Bst DNA polymerase) was purchased from Biotium. Bull serum albumin (BSA) was purchased from Kayon. Cell Culture. E-J and HeLa cancer cells were cultured in 1640 (GIBCO) medium with 10% fetal calf serum (FBS) and 1% penicillin streptomycin (PS, 10000 IU penicillin and 10 000
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DOI: 10.1021/acs.analchem.5b02699 Anal. Chem. XXXX, XXX, XXX−XXX
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Analytical Chemistry
Figure 2. Working mechanism interpretation of the low background fluorescence assay to detect activity of telomerase. (a) Inhibition of telomerase activity in extracts from 10 000 E-J cancer cells by 1 mM AZT. (b) Fluorescence emission spectra of Silole-R only (black), and Silole-R in the presence of elongated QP by telomerase extracted from E-J cancer cells without (red) and with 18 U S1 Nase (blue) or with twice 10 U S1 Nase (pink). Inset: Plot of the changes in fluorescence intensity at 478 nm with telomerase, S1 Nase, and secondary S1 Nase, successively. (c) Nondenaturating PAGE analysis of low background fluorescence assay: DNA ladder marker (lane M), QP in the absence (lane 1) and presence of telomerase extracted from 20000 E-J cancer cells (lane 2), 20 000 MCF-7 cancer cells (lane 3), and 20 000 HeLa cancer cells (lane 4).
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μg/mL streptomycin, Multicell) in a culture flask at 37 °C in a humidified atmosphere containing 5% CO2. MCF-7 cancer cells were cultured similarly in Dulbecco’s modified Eagle’s medium (DMEM, Multicell). HLF cells were cultured similarly in minimum essential medium (MEM/EBSS, 1×, HyClone). Telomerase Extracted from Cultured Cells. Bladder cancer cells (E-J), breast cancer cells (MCF-7), cervical cancer cells (HeLa), and human lung fibroblast cells (HLF) were first suspended in 1× CHAPS lysis buffer (lysis buffer, Millipore) to make the concentrate 5000 cells/μL and incubated on ice for 30 min. Then, the mixture was centrifuged at 12 000 g for 20 min at 4 °C. The supernatant was transferred, aliquoted, and stored at −80 °C. HeLa cells were first treated with 100 μM AZT (diluted in 1640 medium with 10% FBS and 1% PS) for 48 h. Then, telomerase was extracted in the way described above. Telomerase Extracted from Urines. Fresh urine samples were collected and centrifuged at 850 g for 10 min at 4 °C, washed once using phosphate-buffered saline (PBS, Multicell). The above samples were centrifuged at 2300 g for 5 min at 4 °C. The precipitate was resuspended in 200 μL of ice-cold 1× CHAPS lysis buffer and then incubated on ice for 30 min. Then the mixture was centrifuged at 10 000 g for 20 min at 4 °C. The supernatant was transferred, aliquoted, and stored at −80 °C. Telomerase Extension Reaction and Detection by Silole-R. An appropriate volume of telomerase extracted from a certain number of cells was diluted in 1× CHAPS lysis buffer and then added into the telomerase extension reaction buffer containing dNTPs, QP, and recombinant RNase inhibitor. The solution was incubated at 37 °C for 60 min, and then transferred to 94 °C for 10 min to end the reaction. The whole reaction was conducted under the condition of protection from light. In this process, telomeric repeated sequence was extended to 3′-end of QP in the performance of telomerase, which was not affected by the quencher owing to its existence at the 5′end of QP. Afterward, the Silole-R was added to solution keeping the final concentration at 10 μM with the total volume as 200 μL. The system with more telomerase showed higher fluorescence intensity. Using this method, a high specificity telomerase activity detection strategy was established for early detection of telomerase activity and cancer diagnose.
RESULTS AND DISCUSSION
Quenching of Silole-R by Quencher Group Labeled TS Primer. To confirm that longer ssDNA results in a more significant fluorescence increase, telomerase extracted from E-J cancer cells was used in the presence of QP (TP). Sequences of DNA used in this assay are shown in Table S1. When telomerase extracted from 10 000 E-J cancer cells was added to a solution of QP or TP, a strong emission band with the band maximum at 478 nm was observed. Under the optimized conditions, after addition of telomerase extracts, we observed 1106% signal increase in fluorescence intensity when QP was used. On the contrary, only 577% signal increase was observed when TP was used (Figure S3). The results clearly suggest that the use of quencher group labeled TS primer could cause low detection background and high signal increase ratio. Optimized Condition Selection. A series of control experiments are conducted to ascertain the optimized conditions for telomerase activity detection. As shown in Figure S4, the fluorescence intensity of Silole-R increases rapidly in the first 60 min, and reaches a platform subsequently. Thus, 1 h is chosen as reaction time for this strategy. It is noted that no significant changes of fluorescence intensity are observed when none or only one of the QP, dNTPs, or telomerase is existing in this system (Figure S5). In the absence of Silole-R, fluorescence intensity is far lower than that in the presence of Silole-R (Figure S6). These results above indicate that Silole-R has the potential ability to work as an in vitro bioprobe for activity detection of telomerase by using quencher group labeled TS primer. Working Mechanism Interpretation. A series of confirmatory experiments were conducted to support the mechanism of this strategy. It could be observed that aggregation of Silole-R was induced by interaction between Silole-R and telomerase-triggered QP elongation. 3′-Azido-3′deoxythymidine (AZT, a typical inhibition that could significantly inhibit the activity of telomerase) was used to inhibit the activity of telomerase.52 As shown in Figure 2a, the fluorescence intensity enhancements for active telomerase and AZT-treated telomerase are 1106% and 53%, respectively. The results indicate the potential application of this assay for screening telomerase inhibitors and telomerase-targeted drugs. Moreover, S1 nuclease (S1 Nase, a single-strand-specific C
DOI: 10.1021/acs.analchem.5b02699 Anal. Chem. XXXX, XXX, XXX−XXX
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Analytical Chemistry endonuclease which catalytically hydrolyzes single-strand DNA or RNA to 5′-nucleoside monophosphates)53 was chosen to hydrolyze DNA and verify the influence of DNA length to fluorescence intensity of complex S-QP. After extension reaction, a 441% signal enhancement is observed (Figure 2b). Upon addition of S1 Nase to the elongation product of QP and telomerase extracts, the fluorescence intensity clearly drops by 69% because of cleaved reaction. Subsequently, S1 Nase is added to the system again, making fluorescence intensity decrease nearly as low as the baseline. It suggests that almost all the signal strand DNA was cut into fragmentation, which leads to the lower aggregation degree and thus lower fluorescence intensity of this system. Furthermore, a commercial human telomerase ELISA Kit was used to compare our method with the existing results, consequently showing the calibration curve of standard telomerase with an applicable correlation coefficient (R2 = 0.992) (Figure S7). From Figure S8, we can see the concentrations obtained from commercial telomerase ELISA Kit are in according with the (I/I0) − 1 values of our method. These results above show the credibility of our method. A nondenaturating polyacrylamide gel electrophoresis (PAGE) analysis was also employed to monitor the reaction product (Figure 2c). Strong bands at about 60 bp were observed in the lane of QP in the presence of telomerase extracted from E-J, HeLa, or MCF-7, but no analogous band was observed in the lane of only QP in the absence of telomerase. It could be inferred that the telomerase extension reaction has reliably occurred even if quencher is present at the 5′-end of primer, consistent with the fluorescence experiments above. Specificity Study. Before the specificity of this method was checked, some experiments were conducted to compare the signal-to-background ratio of detection system. Figure S9 shows the fluorescence intensity gradually increase with the number of telomerases (E-J). After addition of telomerase, similar signal increases were observed in the presence of QP or TP. However, the signal-to-background ratios were different. Through comparison of the signal-to-background ratios between the QP-based method and the TP-based method, we confirmed that the quencher group helps increasing signal-to-background ratio, especially in high concentration of telomerase. For the purpose of proving the specificity of this method that the fluorescence enhancements are only related with telomerase activity, control experiments were conducted and involved in inactive telomerase, telomerase extracted from normal cells, or other proteins. With consideration of the thermolability, three sources of telomerase kept at 95 °C for 20 min were used in the control excperiments.54 Distinct difference between heatinactive telomerase and active telomerase confirms that telomerase activity is relevant to this method. Moreover, no notable signal change is observed when using telomerase extracted from human lung fibroblast cells (HLF, a human normal cell line) and HeLa cancer cells which is treated with 100 μM AZT for 48 h before extraction. To test the interference, lysis buffer, Bst DNA polymerase, trypsin, thrombin, and BSA are used in this method, respectively. The results show a notable difference between active telomerase and heat-inactive telomerase, certifying the specificity of our method effectively (Figure 3a). Afterward, for verification of the function of quencher in decreasing the fluorescence background and increasing the signal-to-background ratio, TP without quencher was used in the same experiments. Figure 3b shows that fluorescence intensity increase ratios of TP systems in the presence of active telomerase are apparently lower than
Figure 3. (a) QP and (b) TP were, respectively, challenged by telomerase extracted from 10 000 E-J (A or A′), MCF-7 (C or C′), HeLa (E or E′) cancer cells, 10 000 human lung fibroblast cells (HLF, G or G′), 10 000 HeLa cancer cells which were treated with 100 μM AZT for 48 h before extraction (H or H′), lysis buffer (I or I′), Bst DNA polymerase (J or J′), trypsin (K or K′), thrombin (L or L′), BSA (M or M′), and heat-inactivated telomerase extracted from 10 000 E-J (B or B′), MCF-7 (D or D′), and HeLa (F or F′) cancer cells. Only the telomerase from A, A′, C, C′, E, and E′ showed the high activity. Error bars indicate standard deviation of triplicate tests.
those of QP systems. However, at the same time, ratios of Silole-R in the presence of QP or TP are nearly the same in other control systems including inactive telomerase, telomerase extracted from normal cells, other proteins, and so on. The specific values between fluorescence enhancement ratios are shown in Figure 3a,b, respectively. Obviously, when QP is used, specific values are extra high compared to TP. The specific values between fluorescence enhancement ratios of active E-J telomerase and other interferents are listed in Table S2, in which the same conclusion is obtained. It could be inferred that the signal-to-background ratio clearly increases by using QP. Thus, active telomerase could be distinguished easily from other interferents. The results suggest that using a quencherlabeled primer has an excellent effect in increasing the specificity of the strategy. Detection of Telomerase Activity from Cancer Cells Extracts. In order to find out the detection limit and linearity range of this method, experiments of relationships between the quantity of telomerase and fluorescence intensity are conducted. Different amounts of telomerase extracted from E-J cells (human bladder cancer cells) are serially diluted and D
DOI: 10.1021/acs.analchem.5b02699 Anal. Chem. XXXX, XXX, XXX−XXX
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Analytical Chemistry
Figure 4. Activity detection of telomerase extracted from three types of cancer cells. Emission spectra of complex S-QP in response to telomerase from different numbers of bladder cancer cells (E-J, a), breast cancer cells (MCF-7, c), and cervical cancer cells (HeLa, e). The relationship between fluorescence enhancement and numbers of E-J (b), MCF-7 (d), and HeLa (f) cancer cells. Insets: Linear relationships between fluorescence enhancement and the logarithm of cell numbers. I means fluorescence intensity of complex S-QP at the wavelength of 478 nm in the presence of telomerase from different numbers of cancer cells. The fluorescence intensity is enhanced gradually from 0 to 10 000 cancer cells.
Figure 5. Results of low background telomerase activity detection in real urine specimens. (a, b) Fluorescence intensity enhancement ratios between specimen and blank at 478 nm in response to telomerase extracted from 19 clear urine cancer specimens (a) and 19 bloody urine cancer specimens (b). The horizontal dashed line represents the threshold level according to the definition of I/I0 + 3σ. Error bars indicate standard deviation of triplicate tests. (c) Box chart representation of activity detection of telomerase extracted from urine specimens of 19 clear bladder patients (blue), 19 bloody bladder patients (red), and 15 normal people (gray) by using this low background fluorescence strategy. Inserts: Photographs of clear and bloody urine extracts.
coincubated with QP and dNTPs. Then, the fluorescence spectra are recorded, respectively. Figure 4a shows that fluorescence intensities gradually increase with the addition of telomerase quantities equal to the cells’ numbers 0−10 000 cells. The positive correlation between fluorescence increment at 478 nm and amounts of E-J cells is shown in Figure 4b. Through this method, we can distinctly detect the telomerase activity from as few as 5 cells. Moreover, the strategy was further proved by using telomerase extracted from HeLa and MCF-7 cancer cells, respectively. As Figure 4c−f indicates, all
the results demonstrate that the method is reliable and general for telomerase activity detection. Detecting Bladder Cancer by This Low Background Method. To confirm the application value of our strategy in clinical potentials, telomerases extracted from urine specimens of 38 bladder cancer patients and 15 normal people are used. Bladder cancer is one of the most common genitourinary malignancies with the highest recurrence rate.55−57 Urine specimens from bladder cancer patients and normal people are collected and applied to evaluate the applicability of this low E
DOI: 10.1021/acs.analchem.5b02699 Anal. Chem. XXXX, XXX, XXX−XXX
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Analytical Chemistry background fluorescence method in bladder cancer detection and diagnosis. As shown in Figure 5, Figure S10, Table S3, and Table S4, we use telomerase extracted from 19 clear urine specimens (urine specimens of bladder cancer patients whose urine are clear), 19 bloody urine specimens (gross hematuria, urine specimens of bladder cancer patients whose urines contain more than 4 mL blood in 1000 mL urines and are bloody macroscopically), and 15 normal urine specimens. These specimens are without any treatment before extraction to verify the detection capability of our strategy. The results show that signal increase ratios of 19/19 (100%) for clear urine (Figure 5a) and 17/19 (89.5%) for bloody urine (Figure 5b) are above the threshold level according to the definition of I/I0 + 3σ (the horizontal dashed line; I/I0 means average of fluorescence intensity ratios at 478 nm between 15 normal specimens and their corresponding blank samples, σ means standard deviation of fluorescence intensity ratios at 478 nm between 15 normal specimens and their corresponding blank samples), with the relative deviations (RSD) all less than 8.2%. Compared to positive result rates of our previous work with 100% for clear urine specimens and 72% for bloody specimens, respectively,50 the new low background method using QP obviously maintains excellent detection rate in clear urine specimens and obtains more satisfactory detection rate in bloody urine specimens. Because of the lower background and higher signal increase ratio, the new low background method with QP is more selective and specific to perform well under a complicated condition such as blood. Figure S10 shows the blank and almost invariable fluorescence intensity ratios of normal urine specimens. Moreover, the fluorescence intensity ratio distributions of clear, bloody, and normal urine specimens are shown in Figure 5c, suggesting an apparent difference between urine specimens of bladder cancer patients and normal people. The detection results of cancer and normal specimens are listed in Tables S3 and S4, respectively. Furthermore, to prove the activity of cancer cell extracts and the credibility of our strategy, a bloody urine specimen extract and 10 000 HeLa cancer cell extracts are also detected using a commercial ELISA kit. As shown in Figure S11, the two sources of telomerase possess activity on the same order of activity magnitude compared of our method, showing the credibility of our method. The results above indicate that this low background method could detect activity of telomerase extracted from urines of bladder cancer patients and could be applied as a novel strategy for diagnosis of bladder cancer.
probability of this strategy to detect and diagnosis cancer is also proven. As a result, it is anticipated that the simple, highly sensitive, rapid, excellently specific, and highly applicable strategy could provide an efficient new tool for detection of telomerase activity and clinical diagnosis of cancer.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b02699. Detailed description of the experimental procedures, DNA sequences, and additional figures (PDF)
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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 work is supported by National Basic Research Program of China (973 Program, 2015CB932600, 2013CB933000), the National Natural Science Foundation of China (21375042, 21405054, 51273053), Natural Science Foundation of Hubei Province of China (2014CFB1012), and 1000 Young Talent (to F.X.).
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CONCLUSION In summary, on the basis of the low background fluorescence strategy and the unique optical property of Silole-R, a high specificity telomerase activity detection method is developed. In comparison with complicated pretreatment or expensive fluorophore−quencher double prelabeled probes in other methods, our method is relatively simple (single labeled), highly sensitive (detection limit of 5 cancer cells), rapid (1 h), and especially excellently specific. In addition, the quencherlabeled telomerase primer used in our method can tremendously lower the fluorescence background, and thus increase the signal-to-background ratio. The feasibility of this strategy is demonstrated by using telomerase extracted from different cancer cells including E-J, HeLa, MCF-7, and normal cells HLF. Furthermore, using telomerase extracted from urine of 38 bladder cancer patients (19 patients whose urines are clear; 19 patients whose urines are bloody) and 15 normal people, the F
DOI: 10.1021/acs.analchem.5b02699 Anal. Chem. XXXX, XXX, XXX−XXX
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Downloaded by GEORGETOWN UNIV on August 26, 2015 | http://pubs.acs.org Publication Date (Web): August 25, 2015 | doi: 10.1021/acs.analchem.5b02699
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DOI: 10.1021/acs.analchem.5b02699 Anal. Chem. XXXX, XXX, XXX−XXX
