Article pubs.acs.org/ac
Turning Nonspecific Interference into Signal Amplification: Covalent Biosensing Nanoassembly Enabled by Metal-Catalyzed CrossCoupling Hao Li,† Fang Wang,‡ Shenguang Ge,† Haiyun Liu,† Mei Yan,*,† and Jinghua Yu† †
Institute for Advanced Interdisciplinary Research, University of Jinan, Jinan 250022, China Shandong Academy of Pharmaceutical Sciences, Jinan 250101, China
‡
S Supporting Information *
ABSTRACT: In this work, a new method of protein detection in complicated samples is proposed. This method employs probe-target recognition to induce cross-linking among the probe, the target, and the nonspecific proteins in the complicated sample as a means to convert interference into effective signal amplification. This also eliminates the necessity of multistep signal amplification in a separate solution system. On the basis of this strategy, a simple and robust assay for the activity of serum cathepsin B is established. Peptide probes immobilized on a sensing slide can recognize cathepsin B, and this can induce thiol-alkyne covalent coupling between the probe and cathepsin B. Meanwhile, applying electrochemical potential scanning to this sensing surface, Cu binding fragments of the probe peptide can be released into the solution phase to act as an electrochemical catalyst for oxidative dityrosine cross-linking among all proteins including the captured cathepsin B and the nonspecific proteins. A continuous nanoassembly covalently anchored on the sensing surface can gradually form, allowing violent detergent rinsing to remove residual interference. Using this method, not only sensitivity in the picomolar range can be achieved for serum analysis, the results of the analysis can also reliably discriminate benign and cancerous ovarian conditions. These results may suggest prospective application of this method in early screening of cancer in the future.
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Attempting to deal with this problem, we have designed a biosensing method (Scheme 1) using target-specific noncovalent recognition to induce covalent cross-linking of the original “interfering” proteins in the sample, as a means of signal amplification. First, since the excessive existence of the “interfering” proteins is inevitable, we reason whether or not, they can be recruited for signal amplification, rather than merely interfering with the performance. Previously we have demonstrated using peptide complexed copper ion as the electrochemical catalyst for dityrosine cross-linking between protein and peptide,16 here this reaction could be utilized for constructing fluorescent nanoassembly from the “interfering” proteins, mainly proteins, for signal amplification. This may enable one-pot and reagentless biosensing, since it is no longer necessary to separate target-recognition and signal amplification into two phases to avoid the “interfering” proteins, which now act as signal amplifier, thereby, at the same time also eliminating the need of adding more signal amplifying reagents. This covalent signal amplification should be coupled with covalent target capturing, to form an undisrupted continuous covalent nanoassembly on the sensing interface, and this would allow violent detergent rinsing to exclude residual inferring of
he ultimate goal of developing novel biosensing method is biomedical application. In detecting complicated samples, one noncircumventable issue that must be tackled at first is the interference from nonspecific species.1,2 Although these are weak and noncovalent, the sheer number of interfering species, existing in far excess to the analyte, usually low-abundance biomarkers, can compromise many sophisticated designs aimed at improving the analytical performance.3−5 In fact, the adoption of a complicated nanoassembly or cycling of biomacromolecules for signal amplification may really invite more interference,6−10 since these may provide more potential binding sites for multiple noncovalent interactions with the interfering species. Therefore, to restrict the interference within certain limits, interface biosensing scheme is combined with thorough or even violent rinsing, as in classic designs such as ELISA which remains to be the most feasible for application, up to date. This is because only the strong noncovalent antibody− antigen interactions can withstand the rinsing. While most of the artificial ligands recently developed11−15 may have relatively weak binding with their targets, the conformation required for molecular recognition may even be disrupted by the denaturant rinsing which is necessary for clinical detection. Therefore, in detecting complicated or clinical samples, the presence of excessive interfering species is inevitable, but the comparatively weak binding strength of novel targeting ligands has left us with limited choice to overcome this interference. © XXXX American Chemical Society
Received: April 6, 2017 Accepted: May 18, 2017
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DOI: 10.1021/acs.analchem.7b01269 Anal. Chem. XXXX, XXX, XXX−XXX
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Scheme 1. Proposed Method Using Target Recognition to Induce Covalent Assembly for Sensitive Detection of Cathepsin B: (a) Peptide-Modified Sensing Surface, (b) Recognition and Covalent Capture, (c) Electrochemical Cleavage and Cross-Linking, and (d) Fluorescent Readouta
a
Not drawn to scale.
serum samples of benign conditions and 15 of cancerous condition) after elected consent by the local ethical committee. A total of 15 serum samples of healthy individuals were also collected as the control. Electrode Treatment. Transparent Au deposited ITO slides (layer thickness 95%, by Congbeibio Co, Ltd. Cathepsin B was from R&D System. 11-Mercaptoundecanoic acid (MUA) and 9-mercapto-1-nonanol (MN) were from Sigma-Aldrich. All the other chemicals were of analytical-grade. The solutions of the peptide probe were prepared by dissolving the powder to 10 μM with 10 mM phosphate buffer solution (PBS) (pH 7.4). The lyophilized powder of cathepsin was added to the “blank” blood serum sample to prepare the standard samples for establishing the working curve; the specifics are as described in the Results and Discussion. All solutions were prepared with double-distilled water, which was purified with a Milli-Q purification system (Branstead) to a specific resistance of 18 MΩ cm. For the detection of clinical samples, serum samples were collected from patients at Shandong Tumor Hospital (15 B
DOI: 10.1021/acs.analchem.7b01269 Anal. Chem. XXXX, XXX, XXX−XXX
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Figure 1. Validation of the proposed design. (a) Isothermal titration calorimetry (ITC) data obtained by titrating 0.5 mM of the Cu(II) into 0.05 mM probe. The solution adopted is 10 mM PBS, pH 7.4. The top row displays the raw data of power versus time. The bottom row is the corresponding data by integrating enthalpy values versus the molar ratio of titrant/titrand. These data are fit using Origin 7.0 software, and the resulting fitting curve is also shown in the lower row. (b) Surface plasma resonance (SPR) sensorgram obtained by flowing the target protein (10 nM) across a sensing surface modified by the designed probe (red curve) or by a probe containing the original propeptide sequence of the target protein. The injection starts from point 1, and points 2−4 indicate the starting of the dissociation step, the addition of detergent, and the beginning of acidic regeneration, respectively. (c) Fluorescent response of dityrosine tagged probe after gradually longer duration of electrochemically controlled Cu self-cleavage of the probe. Inset shows the relationship between time of reaction and the residual signal, error bars indicate standard deviation (n = 3). (d) Electrochemical impedance spectra (EIS) recorded during the detection procedure.
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RESULTS AND DISCUSSION The detailed design of the method (Scheme 1) can be described as follows. The peptide probe, its sequence shown as in Scheme 1a and in Figure S1, is immobilized with MUA (the green stock of the probe in Scheme 1a). The probes contain a cathepsin B targeting sequence (the violet portion in Scheme 1a) and two successive sequences bound with Cu ion (the gray portion with green balls in Scheme 1a). Upon incubation with complex samples containing cathepsin B and nonspecific proteins, cathepsin B can be captured by the probe (Scheme 1b). The cathepsin B targeting sequence is derived from the propeptide of cathepsin B precursor.21 This sequence can fit into the substrate crevice, so in our modified sequence, the position directly opposite to the catalytic cysteine of the active center is occupied by azidoalanine, which can covalently conjugate with the active cysteine, as a special property of the cysteine protease family.22 Thus, molecular recognition of cathepsin B by the probe can induce thiol-alkyne cross-linking (Scheme 1b, framed inset), covalently binding cathepsin B together with the probe. Then, electrochemical scanning can be applied to the biosensing surface to release the terminal Cu-
solutions were degassed prior to titration. The data were analyzed using Origin 7.0 software. The above electrochemical scanning was carried out on a CHI660D potentiostat (CH Instruments) with a conventional three-electrode system: the slides as the working electrode, a saturated calomel electrode (SCE) as the reference electrode, and a platinum wire as the counter electrode. The electrolyte cell was a homemade type of roughly 100 μV. Fluorescence emission spectra of surfaces were measured using a QM-4/2005 fluorescence spectrometer (Photon Technology International, Inc., Birmingham, NJ) equipped with a xenon lamp. This light source and the detector were in the same plane at right angle to each other. The slides were kept in a water-filled cuvette at 60 °C from the base of the cuvette for fluorescence measurements. The surface with gold film was kept away from the light source and toward the detector. Activating peak wavelength for dityrosine was 325 nm, while that for 3,4-dihydroxyphenylalanine (DOPA) was around 360 nm. SPR measurements were performed with an Autolab ESPRIT system (Echo Chemie B.V., The Netherlands) equipped with a 670 nm monochromatic p-polarized light resource. C
DOI: 10.1021/acs.analchem.7b01269 Anal. Chem. XXXX, XXX, XXX−XXX
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Figure 2. (a) Morphology of the cross-linked nanoassembly characterized with scanning electrochemical microscopy; inset shows, under UV illumination, the fluorescent dots formed from the droplets of serum sample containing different amount of target protein. (b) Quantitative fluorescent response of dityrosine obtained in detecting serum samples containing different amounts of target protein. Inset shows the peak response as a function of target concentration, and the result of linear regression between peak response and the logarithm of target concentration has also been displayed. Error bars indicate standard deviation (n = 3). (c) Fluorescent response of dityrosine showing the specificity of the proposed method, all species are at 10 nM, and the blank is 10 000× diluted serum. (d) The quantitative results obtained without the detergent rinsing, and the meaning of curves and data points is the same as in part b.
(Figure 1). The interaction between cupric ion and the peptide probe, studied by isothermal titration calorimetry (ITC), shows 2:1 binding of cupric ion to the peptide (Figure 1a), consistent with the fact that the DAHK and GHK motifs have comparable binding strength toward cupric ion,23 and these sites may have no much cooperation between them. The robustness of the noncovalently induced covalent capturing is verified using surface plasma resonance (SPR). The covalently immobilized target protein, after molecular recognition and cross-coupling with the probe, can resist detergent rinsing (Figure 1b, red curve), while the captured proteins can be removed from the surface by the rinsing, if the original propeptide is used as a control (Figure 1b, black curve). The copper self-cleavage is studied using dityrosine as a fluorescent reporter to label the GHK terminal of the peptide probe (Figure 1c), and the gradual loss of signal readout can be observed after electrochemical scanning applied to the chip for gradually longer duration, indicating controlled cleavage catalyzed by electrochemically activated copper. The biosensing interface upon each step of the detection procedure, as well as under several control conditions, has been investigated with electrochemical impedance spectra (EIS) (Figure 1d). The slide before peptide modification appear as a straight line (curve 1), indicating nearly no resistance to electron transfer. Peptide modification gives rise to a semicircle of moderate diameter on the spectra (curve 2), while electrochemical scanning inducing copper self-
binding GHK sequence by copper self-cleavage of the subterminal Cu-binding DAHK sequence (Scheme 1c). DAHK23 is linked to the N-terminal of the cathepsin B targeting sequence by the side chain carboxyl group of the Nterminal aspartic acid moiety of DAHK. DAHK, with Cu ion, can electrochemically generate reactive oxygen species as we have demonstrated previously.16 This can induce decarboxylation of the aspartic acid moiety, radical-mediated N-terminal cyclization of AHK, and tandem beta-scission between D and A.24 As DAHK is destroyed, the terminal GHK, with Cu ion, can be released into the solution phase and disperse among the nonspecific proteins. As an artificial catalyst, also as demonstrated previously,16 these can induce dityrosine crosslinking among the tyrosine-moieties of cathepsin B and the numerous nonspecific proteins (Scheme 1c, framed inset). Using covalently captured cathepsin B as the surface anchoring points, a cross-linked nanoassembly can gradually form. Violent detergent and chelator rinsing can then be applied to the biosensing surface and the numerous cross-linkages, the fluorescently active dityrosine, may generate amplified fluorescent signal readout (Scheme 1d). In the absence of the target enzyme, the nanoassembly also forms but can be removed via the rinsing step since robust covalent anchoring points are not formed. The proper functioning of the designed peptide probe has first been characterized with various experimental techniques D
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healthy female volunteer, also contains considerable amount of cathepsin B, calibrated with a standard ELISA kit, at around 3.5 M. So the blank serum is diluted serially, and in all samples the target enzymes are supplemented with recombinant targets to the uniform 10 nM. The resulted signal responses are compared (Figure S4), which is very similar down to 1000fold dilution. This indicates satisfactory recovery rates of the added recombinant proteins. And it can also be concluded that down to 1000-fold dilution the proposed cross-linking of serum interfering proteins is effective as the means of signal amplification. Therefore, in establishing the calibration working curve, standard samples over 3.5 nM could be prepared by adding recombinant proteins while samples of concentrations lower than that could be prepared by dilution. Under these optimized conditions, the analytical performance of the proposed method is examined. Quantitative results can also be obtained in detecting different amounts of the target protein (Figure 2b). The signal readout varies with the exponential of the target concentration, and a linear detection range can be established between 3.2 pM and 32 000 pM, with a limit of detection (LOD) as low as around 1 pM (Figure 2b, inset). Various control species (Figure 2c) can only induce signal readout leveling with that of the background of blank serum control (10 000× diluted), confirming the essential role of the specifically recognized and covalently immobilized target proteins as the anchoring points of the surface assembly. As a comparison, without the detergent rinsing, the quantitative performance such as the detection range and LOD can be greatly handicapped (Figure 2d). Accumulating data has suggested a link between serum cathepsin activity of the invasion/metastasis of ovarian cancer.25,26 In fact, in malignant melanoma and breast, colon, lung, stomach, hepatocellular, and other cancers, elevation of extracellular cathepsin B activity is reported.27 Under normal conditions, cathepsin B is restricted inside the lysosome, but it is now suggested that these enzymes can be released into the extracellular matrix for remodeling it toward invasion and metastasis.27 Upon dissemination, these cathepsin enzymes can also enter into the circulating blood. Therefore, as a potential circular tumor marker, cathepsin B could be evaluated less invasively, as compared with biopsy, and the serum assay may also facilitate the early detection and timely diagnosis of invasion. Employing the above developed method, one-pot and reagentless electrochemical and fluorescent assays can be conducted for serum samples of ovarian conditions. The results are summarized in Figure 3. Statistically significant differences can be observed between groups of healthy volunteers, patients with benign conditions and ovarian cancer, but the attempt to find significant difference between the different stages of the cancer development is not successful (not shown).
cleavage can decrease the diameter of the semicircle (curve 3), indicating lost of the terminal sequence of the peptide, similar to the above results (Figure 1c). Complicated sample prepared by spiking recombinant cathepsin B with the serum of a healthy volunteer is employed for target recognition. The covalent capturing results in an evident increase in the impedance (curve 4), while violent rinsing only slightly reduces the impedance (curve 5), which may be due to the decreased resistance of the misfolded protein in a more open conformation. If the original propeptide is used as a control, violent rinsing can reduce the impedance back to that of the peptide modified electrode (curve 6), coherent with the above SPR results (Figure 1b). Following the standard procedure as proposed (Scheme 1), the peptide modified slide is incubated with the sample followed by electrochemical scanning, and a dramatic increase of the impedance is observed (curve 7), after violent rinsing the impedance only slightly decreases (curve 8), suggesting formation and immobilization of the cross-linked surface structure. Again using the original propeptide as the control, before rinsing some evident increase of impedance can be observed (curve 9) but can be completed abrogated by the rinsing (curve 10). These results may validate the designed method as proposed in Scheme 1. Also, it is noted that SPR results in Figure 1b may show interesting kinetic features of the noncovalently induced covalent cross-linking (red curve), compared with the noncovalent peptide-target recognition (blue curve). In the association phase (from point 1 to 2), the cross-linking appears as a slow process superimposed on the relatively fast molecular recognition, which seems not to be able to reach saturation in a short time. In the dissociation phase (from 2 to 3), the thiolalkyne cross-linking shows complete irreversibility due to its high stability except strong acidic conditions. So as mentioned above, detergent rinsing fails to remove the target protein (point 3), but acidic regeneration of the surface may be able to destroy the linkage (point 4). The surface nanoassembly exists in a gel-like status and fluoresces on illumination with UV light (Figure 2a). The emission strength may show parallel with the abundance of the target enzyme detected. On the basis of the fluorescent signal readout, essential experimental conditions of this method can be optimized. First, using peptide probes of different concentration to modify the slide, the influence of the varied surface density of the probe is investigated (Figure S1). The final readout approaches saturation with higher surface density achieved by probes of 10 μM or higher concentration, since higher surface density can provide more in-solution GHK-Cu catalytic fragments and also more captured targets as the anchoring points. The assembly process is also optimized through the electrochemical control, and excessive formation of the surface structure should be avoided as this may impair the fluorescent signal readout. So the duration and range of potential scanning of the electrochemical treatment have been optimized (Figure S2a,b), and the moderate duration, with a relatively large range of potential scanning are selected. Although potential scanning of large over potential may denature the proteins in the sample, this only facilitates the cross-linking by exposing more tyrosine moieties originally folded inside the core of the proteins. After fixing the parameters for the assembly process, the incubation time for the recognition and cross-coupling with the target protein has also been optimized (Figure S3). Another important issue is that the “blank” serum, all from one batch of blood of one
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CONCLUSION The presence of interfering proteins in complicated samples such as the clinical sample, with often excessive abundance as compared with the target biomarker, necessitates relatively thorough rinsing to eliminate such interference. However, this is not quite compatible with the novel targeting ligands recently developed, such as the aptamers, which often do not have an antibody-like binding strength to resist the violent rinsing. Therefore, here we propose to use the noncovalent target recognition to induce covalent cross-linking which only is truly immune to denaturant rinsing. Specifically, electrochemically E
DOI: 10.1021/acs.analchem.7b01269 Anal. Chem. XXXX, XXX, XXX−XXX
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(4) Ray, S.; Reddy, P. J.; Jain, R.; Gollapalli, K.; Moiyadi, A.; Srivastava, S. Proteomics 2011, 11, 2139−2161. (5) Zhai, G.; Wu, X.; Luo, Q.; Wu, K.; Zhao, Y.; Liu, J.; Xiong, S.; Feng, Y.-Q.; Yang, L.; Wang, F. Talanta 2014, 125, 411−417. (6) Cao, X.; Ye, Y.; Liu, S. Anal. Biochem. 2011, 417, 1−16. (7) Ding, L.; Bond, A. M.; Zhai, J.; Zhang, J. Anal. Chim. Acta 2013, 797, 1−12. (8) Lei, J.; Ju, H. Chem. Soc. Rev. 2012, 41, 2122−2134. (9) Zhao, Y.; Chen, F.; Li, Q.; Wang, L.; Fan, C. Chem. Rev. 2015, 115, 12491−12545. (10) Zhou, S.; Yuan, L.; Hua, X.; Xu, L.; Liu, S. Anal. Chim. Acta 2015, 877, 19−32. (11) Chen, A.; Yang, S. Biosens. Bioelectron. 2015, 71, 230−242. (12) Citartan, M.; Gopinath, S. C. B.; Tominaga, J.; Tan, S.-C.; Tang, T.-H. Biosens. Bioelectron. 2012, 34, 1−11. (13) Ruigrok, V. J. B.; Levisson, M.; Hekelaar, J.; Smidt, H.; Dijkstra, B. W.; van der Oost, J. Int. J. Mol. Sci. 2012, 13, 10537−10552. (14) Sun, H.; Zu, Y. Molecules 2015, 20, 11959−11980. (15) Wang, Y.-X.; Ye, Z.-Z.; Si, C.-Y.; Ying, Y.-B. Chin. J. Anal. Chem. 2012, 40, 634−642. (16) Li, H.; Huang, Y.; Yu, Y.; Wang, Y.; Li, G. Biosens. Bioelectron. 2016, 80, 560−565. (17) Ambeba, E.; Linkov, F. Future Oncol. 2011, 7, 1399−1414. (18) Huang, Y.-k.; Yu, J.-c.; Kang, W.-m.; Ma, Z.-q.; Ye, X.; Tian, S.b.; Yan, C. PLoS One 2015, 10, e0142080. (19) Nolen, B. M.; Lokshin, A. E. Int. J. Biol. Markers 2011, 26, 141− 152. (20) Summers, T.; Langan, R. C.; Nissan, A.; Bruecher, B. L. D. M.; Bilchik, A. J.; Protic, M.; Daumer, M.; Avital, I.; Stojadinovic, A. J. Cancer 2013, 4, 210−216. (21) Redecke, L.; et al. Science 2013, 339, 227−230. (22) Ekkebus, R.; van Kasteren, S. I.; Kulathu, Y.; Scholten, A.; Berlin, I.; Geurink, P. P.; de Jong, A.; Goerdayal, S.; Neefjes, J.; Heck, A. J. R.; Komander, D.; Ovaa, H. J. Am. Chem. Soc. 2013, 135, 2867− 2870. (23) Trapaidze, A.; Hureau, C.; Bal, W.; Winterhalter, M.; Faller, P. JBIC, J. Biol. Inorg. Chem. 2012, 17, 37−47. (24) Lee, S. H.; Oe, T. Drug Metab. Pharmacokinet. 2016, 31, 27−34. (25) Gashenko, E. A.; Lebedeva, V. A.; Brak, I. V.; Tsykalenko, E. A.; Vinokurova, G. V.; Korolenko, T. A. Int. J. Circumpolar Health 2013, 72, 21215. (26) Warwas, M.; Haczynska, H.; Gerber, J.; Nowak, M. Clin. Chem. Lab. Med. 1997, 35, 301−304. (27) Nishikawa, H.; Ozaki, Y.; Nakanishi, T.; Blomgren, K.; Tada, T.; Arakawa, A.; Suzumori, K. Gynecol. Oncol. 2004, 92, 881−886.
Figure 3. Box charts showing the distribution of the detected level of cathepsin b in serum samples of benign and malignant ovarian conditions, with those obtained from normal cases without ovarian conditions as the control. The raw data is included as a column scatter plot to the left of each box. A curve corresponding to normal distribution is also displayed on top of the scatter plot.
controlled and Cu ion catalyzed oxidative dityrosine crosscoupling is employed to generate a robust covalent nanoassembly from both the target protein and the original “interfering” proteins in serum samples, and this allows violent rinsing, eliminating the necessity of the change of buffer system between target capturing and signal generation. Using this method, not only sensitivity in the picomolar range can be achieved for serum analysis, the results of the analysis can also reliably discriminate benign and cancerous ovarian conditions. These results may suggest prospective application of this method in early screening of cancer in the future.
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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.7b01269. Probe sequence and conditions optimization of major steps of the detection procedure (PDF)
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AUTHOR INFORMATION
Corresponding Author
*Fax: +86-531-82765969. E-mail:
[email protected]. ORCID
Shenguang Ge: 0000-0002-0537-6491 Haiyun Liu: 0000-0002-0637-771X Mei Yan: 0000-0002-7509-4262 Jinghua Yu: 0000-0001-5043-0322 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work is supported by the National Natural Science Foundation of China (Grant Nos. 81602737, 51502112, and 21475052).
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REFERENCES
(1) Dodig, S. Biochem. Med. 2009, 19, 50−62. (2) Vanderstichele, H.; Stoops, E.; Vanmechelen, E.; Jeromin, A. Alzheimer's Res. Ther. 2012, 4, 39. (3) Narimatsu, H.; Sawaki, H.; Kuno, A.; Kaji, H.; Ito, H.; Ikehara, Y. FEBS J. 2010, 277, 95−105. F
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