and “Signal-on” Electrochemical Cisplatin Sensor - ACS Publications

of cisplatin via the intrastrand GNG motif (N = A), generating a bend in the probe which results in an increase in the MB current. Although both high-...
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A Tunable “Signal-off” and “Signal-on” Electrochemical Cisplatin Sensor Yao Wu, and Rebecca Y Lai Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02353 • Publication Date (Web): 11 Aug 2017 Downloaded from http://pubs.acs.org on August 12, 2017

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A Tunable “Signal-off” and “Signal-on” Electrochemical Cisplatin Sensor Yao Wu and Rebecca Y. Lai* Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE 68588-0304, USA. *Fax: +1 402 472 9402. Tel.: +1 402 472 5340. E-mail: [email protected] Abstract: We report the first electrochemical cisplatin sensor fabricated with a thiolated and methylene blue (MB)-modified oligoadenine(A)-guanine(G) DNA probe. Depending on the probe coverage, the sensor can behave as a “signal-off” or “signal-on” sensor. For the high-coverage sensor, formation of intrastrand Pt(II)-AG adducts rigidifies the oligo-AG probe, resulting in a concentration-dependent decrease in the MB signal. For the low-coverage sensor, the increase in probe-to-probe spacing enables binding of cisplatin via the intrastrand GNG motif (N = A), generating a bend in the probe which results in an increase in the MB current. Although both high-coverage “signal-off” and low-coverage “signal-on” sensors are capable of detecting cisplatin, the “signal-on” sensing mechanism is better suited for real time analysis of cisplatin. The low-coverage sensor has a lower limit of detection, wider optimal AC frequency range, and faster response time. It has high specificity for cisplatin and potentially other Pt(II) drugs, and does not cross-react with satraplatin, a Pt(IV) prodrug. It is also selective enough to be employed directly in 50% saliva and 50% urine. This detection strategy may offer a new approach for sensitive and real time analysis of cisplatin in clinical samples.

Since its serendipitous discovery in 1965 by Rosenberg in the course of examining the effect of electric fields on the growth of Escherichia coli cells, cisdiamminedichloroplatinum(II) (cisplatin) has been widely used in clinical settings as a front-line treatment against various neoplasias, in particular, testicular, ovarian, bladder, and head and neck cancers.1-3 The therapeutic properties of cisplatin are attributed to the chloride ligand displacement reactions that generate various aquated derivatives, owing to the lower chloride concentration in the cell cytoplasma when compared to the extracellular fluid.4 These aquated derivatives form Pt(II) adducts with nucleic acids predominantly at purine N7 positions. Among the DNA adducts, 60-65% are intrastrand GG adducts, 25-30% are AG adducts, 5-10% are GNG (N = any nucleotide) adducts, and 1-3% are antiparallel interstrand GG adducts.4-6 Formation of these bulky DNA adducts prevents cell replication and activation of other cellular repair mechanisms, which leads to cell death.4-6 Cisplatin is one of the most widely used chemotherapeutic drugs, but its effectiveness could be compromised because of inherent or acquired cellular resistance to the drug after long term exposure.7,8 Its benefits are also hampered by toxic side effects, including nephrotoxicity, neurotoxicity, and the induction of nausea and vomiting.9-11 Hence, it is necessary to monitor extracellular and/or intracellular cisplatin concentrations to ensure therapeutic effectiveness. To date, a variety of analytical methods have been used to detect cisplatin in biological samples. The most popular technique is high performance liquid chromatography coupled with different detectors such as UV-vis and mass spectrometry.12,13 Atomic absorption spectrocopy14 and inductively coupled plasma-mass spectrometry15 have also been used to analyze the platinum content from cisplatin. Despite the availabil-

ity of the aforementioned techniques, there is still a need for more convenient and cost-effective methods for clinical analysis of cisplatin. In recent years, the use of biomolecules such as glutathione-s-transferases,16 N-acetyl-L-cysteine-capped quantum dots,17 and G-quadruplex DNA18,19 to detect cisplatin has been reported. While these sensors have demonstrated high sensitivity and selectivity, some are operational only at high temperature and incapable of real time analysis due to the need of an exogenous reagent. To fulfill the unmet need for real time analysis of cisplatin, we have designed and fabricated a reagentless electrochemical sensor capable of real time analysis of cisplatin in complex biological samples. It can be employed as either a “signal-off” or “signal-on” sensor by simply altering the DNA probe coverage. For the first time, the biochemical mechanism of action of cisplatin involving the formation of specific DNA adducts is exploited in the design of a folding- and dynamics-based electrochemical sensor. With further optimization, this sensor could find applications in clinical analysis of cisplatin. EXPERIMENTAL SECTION Materials and Reagents. 6-mercapto-1-hexanol (C6-OH), tris-(2-carboxyethyl) phosphine hydrochloride (TCEP), nitric acid, sulfuric acid (H2SO4), sodium perchlorate (NaClO4), 4(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), cisplatin, carboplatin, tetracycline, ampicillin, trimethoprim, nitrofurantoin, sulfamethoxazole, amoxicillin, and levofloxacin, guanidine hydrochloride (GHCl) were used as received (Sigma-Aldrich, St. Louis, MO). Satraplatin was purchased from Watson International Ltd. (Suzhou, China). The synthetic urine solution was obtained from Ricca Chemical Company (Arlington, TX). Synthetic stimulated human parotid saliva

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was purchased from US Biocontract Inc. (San Diego, CA). All other chemicals were of analytical grade. All of the solutions were made with deionized (DI) water purified through a Synergy Ultrapure Water System (18.2 MΩ•cm, Millipore, Billerica, MA). The DNA probe immobilization buffer consisted of 20 mM Tris, 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, and 1 mM CaCl2 (pH 7.4) (Phys2). The target interrogation buffer consisted of 50 mM HEPES with 0.1 M NaClO4 (pH 5.0) (HEPES). The 50% saliva and 50% urine samples were made by mixing a buffer containing 0.2 M HEPES and 0.2 M NaClO4 with the saliva or urine in a 1:1 ratio. The pH of the mixed samples was adjusted to pH 5. A stock solution containing 4 mM cisplatin was dissolved in DI water with 1% (v/v) 1 M NaOH and stored overnight prior to being used in the experiments. The DNA probe purchased from Biosearch Technologies, Inc. (Novato, CA) was used as received. MB-P was modified at the 5’ terminus with a C6-disulfide linker and at the 3’ terminus with a methylene blue (MB) redox label (Supporting Information Figure S1). MB-P: 5’ HS-(CH2)6-AGAGAG-MB 3’ Sensor Preparation. Prior to sensor fabrication, gold disk electrodes with a geometric area of 0.0314 cm2 (CH Instruments, Austin, TX) were polished with 0.1 µm diamond slurry (Buehler, Lake Bluff, IL), rinsed with DI water, and sonicated in a low-power sonicator for ~5 min to remove bound particulates. They were then electrochemically cleaned by a series of oxidation and reduction cycles in 0.5 M H2SO4. The real surface area of each electrode was estimated on the basis of the amount of charge consumed during the reduction of the gold surface oxide in 0.05 M H2SO4 using a reported value of 400 µC cm-2. The roughness factor (real area/geometric area) of the electrodes used in this study ranged from 1.0 - 1.5. Two slightly different methods were used in the fabrication of the high-coverage and low-coverage sensors. The first step in the fabrication process involved the mixing of 1 µL of 200 µM MB-P with 1 µL of 10 mM TCEP. This mixture was left at room temperature (RT) (~23 oC) for 1 hr to ensure complete reduction of the disulfide bond on the probes. This solution was then diluted with Phys2 to the specific concentration needed for probe immobilization. For the high-coverage sensor, a probe solution containing 5 µM MB-P in Phys2 and 50 mM MgCl2 was drop casted onto freshly cleaned electrodes for 24 hr at RT. The partially-modified electrodes were subsequently passivated with 2 mM C6-OH in Phys2 for 1 hr at RT to displace nonspecifically MB-P. The addition of extra MgCl2 was required to achieve a high probe density. A slightly different process was used to fabricate the low-coverage sensor. The probe solution contained 5 µM MB-P without the excess MgCl2. This solution was drop casted onto the gold electrodes for 16 hr at ~4 oC (in the fridge), the electrodes were rinsed with Phys2 and subsequently passivated with 2 mM C6-OH in Phys2 for 3 hr at ~4 oC. The as-fabricated sensors, independent of the probe coverage, were placed in 4 M GHCl for 4 min. The sensors were then rinsed with DI water, dried with a stream of nitrogen gas, and placed in an aliquot of pure buffer or mixed buffer solution (HEPES, 50% saliva, or 50% urine). GHCl, a chaotropic agent, was used to disrupt the interactions between the neighboring DNA probes, this pretreatment step was necessary to

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improve probe uniformity on the sensor surface. The sensors fabricated without this pretreatment step showed less reproducible response to the target. The density of electroactive MB-P on the electrode surface, Γ, was determined by integrating the charge under the MB reduction peak in cyclic voltammograms (CVs) collected at slow scan rates (Equation 1). Γ = Q/nFA (1) where Q is the integrated charge of the reduction peak in the CVs, n is the number of electrons transferred per redox event (n = 2 for MB), F is the Faraday’s constant, and A is the real electrode area. Γ is presented as an average value from CVs recorded at three different scan rates (10, 20 and 50 mV s-1). CVs were collected at different scan rates (0.01 - 100 V s-1) to determine the electron transfer rate constant (ks) of MB. The increase in CV peak separation (∆Ep = Ep,a-Ep,c) as a function of increasing scan rate (v) reflects control of the voltammetry by the rate of heterogeneous electron transfer reactions of the MB label on the surface-immobilized probes. When ∆Ep is larger than 200/n mV, a graph of ∆Ep versus log v yields a straight line which is in accordance with the Laviron equation (Equation 2).20 log ks = α log(1-α) + (1-α)logα - log(RT/nFv) - α(1-α)nF∆Ep/2.3RT (2) -1 where ks is the electron transfer rate constant (s ), α is the electron transfer coefficient, v is the CV scan rate (Vs-1), n is the number of electrons transferred in the reaction, and ∆Ep is the difference between the anodic potential and cathodic potential (V). α can be determined from the slope of the straight line, and ks can be calculated with the help of the intercept. Electrochemical Measurements. Electrochemical measurements were performed at RT using a CHI 1040A Electrochemical Workstation (CH Instruments, Austin, TX). Alternating current voltammetry (ACV) and CV were used in sensor characterization and target interrogation. AC voltammograms were collected over a wide range of frequencies (1–1000 Hz) with an amplitude of 25 mV. CVs were recorded using scan rates between 0.01 and 20 Vs−1. MB-P-modified gold disk electrodes were used as working electrodes. A platinum wire was used as the counter electrode and an Ag/AgCl (3 M KCl) electrode served as the reference electrode (CH Instruments, Austin, TX). Prior to the addition of cisplatin, AC voltammograms of the sensors were recorded in HEPES, 50% saliva, or 50% urine. In most experiments, 50 µM cisplatin was added to the solution after sensor equilibration. The concentration of cisplatin used in the selectivity experiment in 50% urine was 200 µM. AC voltammograms were collected every 10 min until no further change in the MB peak current was observed (i.e., signal saturation). A similar sensor interrogation procedure was used to obtain the dose-response curves. The concentrations used in the calibration experiments were 200 nM, 500 nM, 1 µM, 2 µM, 5 µM, 10 µM, 30 µM, 50 µM, and 100 µM. The equilibration time at each target concentration was 1 hr. The limit of detection (LOD) is defined as the lowest target concentration capable of generating a signal change that is significantly different (i.e., s/n = 3) from the signal fluctuation in the absence of the target. The signal fluctuation or “noise” was determined

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from ACVs recorded in the absence of the target over a period of 1 hr. The ratio between the MB peak current in the presence and absence of the target was used to calculate the % signal enhancement (SE) and % signal suppression (SS) (Equations 3 and 4). %SE = [(I-I0)/I0] * 100 (3) %SS = [(I0-I)/I0] * 100 (4) where I is the baseline-subtracted peak current in the presence of the target and I0 is the baseline-subtracted peak current in the target-free solution. All experiments were performed at RT and without mechanical stirring. Unless mentioned otherwise, all experimental results presented here are averaged from three different sensors (n =3). RESULTS AND DISCUSSION Sensor Design. The designs and proposed sensing mechanisms of the cisplatin sensors are shown in Scheme 1. The biorecognition probe is a DNA sequence with three adenines (A) and three guanines (G) (5’ AGAGAG 3’). The probe has no secondary structures according to Mfold.21 Although an oligo-G probe (e.g., 5’ GGGGGG 3’) is also suitable for this application, it was not used because of the challenges in forming a high probe coverage yet stable monolayer. Scheme 1. Schematic illustrations of the high probe coverage “signal-off” (A) and low probe coverage “signal-on” (B) electrochemical cisplatin sensors.

For the high probe coverage sensor, in the absence of cisplatin (aquated cisplatin), electron transfer between the MB label and the electrode is relatively efficient. In the presence of cisplatin, preferential formation of intrastrand Pt(II)-AG adducts rigidifies the probes, resulting in a concentration-dependent reduction in the MB current (i.e., “signal-off” sensor behav-

ior). However, if the probe coverage is adequately low, the sensor can function as a “signal-on” sensor. The increase in probe-to-probe spacing enables the formation of intrastrand Pt(II)-GNG adducts (N = A in this study) which can create a kink or bend in the probe. This change in the probe structure alters the electron transfer kinetics between the MB label and the electrode, resulting in an increase in the MB current. It is worth noting that while the binding motifs presented here are likely to be the more prominent ones, the presence of other adducts capable of producing the same “signal-off” or “signalon” effect cannot be precluded. These aspects are currently being investigated in our laboratory. Sensor Characterization. We first characterized the sensors using ACV, one of the most commonly used sensor interrogation techniques for this class of folding- and dynamicsbased electrochemical biosensors.22-27 In the absence of cisplatin, a well-defined MB peak was recorded for both sensors at a potential consistent with the redox potential of MB in a pH 5 buffer (HEPES) (Figure 1A,C).28 Addition of 50 µM cisplatin resulted in ~68% SS and ~164% SE for the high-coverage and low-coverage sensors, respectively, verifying the proposed probe coverage-dependent sensing mechanisms. Signal saturation was achieved in ~25 min for the low-coverage sensor with a DNA probe density of 1.85±0.06 × 1013 molecules cm-2 (Supporting Information Figure S2A). A much longer response time was needed for the high-coverage sensor with a probe density of 3.06±0.06 × 1013 molecules cm-2, complete signal saturation could not be reached in 70 min (Supporting Information Figure S2B). The difference in response time is expected, due to steric hindrance, target binding could take a longer time to reach saturation for the high-coverage sensor. This behavior has been reported in previously developed folding- and dynamics-based electrochemical biosensors. 22-27,29,30 To better understand the sensing mechanisms, we analyzed the AC frequency-dependent current responses of the sensors before and after the addition of 50 µM cisplatin (Supporting Information Figure S3). For this class of sensors, the MB peak current should be proportional to the AC frequency when the frequency is significantly lower than the electron transfer rate. But as the applied frequency reaches a value above which electron transfer cannot keep up with the rapidly changing potential, the MB peak current diminishes relative to the background current.31 For the high-coverage sensor, in the absence of cisplatin, the MB peak current increased between 1 and 200 Hz, followed by a steady decrease at frequencies beyond 200 Hz. In the presence of cisplatin, the increase in the MB current was more drastic between 1 and 200 Hz, but the decline in the current between 200 and 750 Hz was more gradual. Since the %SS is determined using both pre- and post-binding MB currents, it should also be dependent on the applied frequency. For the high-coverage sensor, the optimal interrogation frequency was 1 Hz (Figure 1B), the %SS was significantly lower at frequencies beyond 1 Hz. For the low-coverage sensor, the MB current increased rapidly between 1 and 5 Hz, followed by a sharp decrease between 10 and 200 Hz. The current remained steady at frequencies beyond 200 Hz. In the presence of cisplatin, the MB current increased drastically between 1 and 100 Hz and remained relatively high between 100 and 750 Hz. These results are quite similar to those obtained for the “signal-on” E-DNA

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Figure 1. AC voltammograms of the high-coverage (A) and low-coverage (C) cisplatin sensors in the absence and presence of 50 µM cisplatin in HEPES. AC frequency-dependent sensor responses of the high-coverage (B) and low-coverage (D) sensors in the presence of 50 µM cisplatin.

sensors, suggesting similarities in the binding motif.22,23 The AC frequency-dependent %SE plot is shown in Figure 1D. Although the sensor displayed high %SE over a wide frequency range (e.g., 200 - 750 Hz), 200 Hz was chosen as the optimal frequency and was used in all the experiments in this study. For both sensors, the MB peak shape was less welldefined at high AC frequencies, thereby preventing accurate quantification of the peak current. In addition to ACV, CV was also used to characterize the sensors. The heterogeneous electron-transfer rate constants (ks) for MB were determined using the Laviron equation.20 For the high-coverage sensor, the calculated ks values in the absence and presence of cisplatin were 0.9 ± 0.0 s-1 and 0.7 ± 0.1 s-1, respectively. The change in electron transfer kinetics was more noticeable for the low-coverage sensor, the ks values before and after target binding were 2.7 ± 0.1 s-1 and 6.9 ± 0.1 s-1, respectively. Overall, these results are consistent with the ACV data and the sensing mechanisms. Sensor Sensitivity. ACV was used to obtain dose-response curves for the two sensors. Both sensors responded to the target in a concentration-dependent manner (Figure 2). For the high-coverage sensor, the LOD was 500 nM and the linear dynamic range was between 0.5 and 5 µM. The LOD of the low-coverage sensor was 200 nM, but the linear dynamic range was more limited (0.2 - 2 µM). The calibration data were also fitted to a one site binding model, the dissociation constants (Kd) were 5.0±1.4 µM and 8.9±2.9 µM for the highcoverage and low-coverage sensors, respectively. These values are consistent with the shape of the dose-response curves. This model is deemed suitable because it is unlikely that more than one cisplatin can bind to a short, surface-confined DNA probe. Formation of Pt(II)-AG and Pt(II)-GAG adducts most likely occurs at the distal end of the probes (i.e., 3’-end) where steric crowding is less severe.

Figure 2. Dose-response curves for the high-coverage (A) and low-coverage (B) cisplatin sensors in HEPES. The concentrations of cisplatin were 200 nM, 500 nM, 1 µM, 2 µM, 5 µM, 10 µM, 30 µM, 50 µM, and 100 µM. The inset figures show the doseresponse curves at lower target concentrations. The data were collected at 1 Hz for the high-coverage sensor and 200 Hz for the low-coverage sensor.

It is worth noting that both LODs are low enough for detection of cisplatin in urine samples from chemotherapy patients.32-34 According to one study, depending on the method and schedule of cisplatin administration, the concentration of

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free Pt in urine samples of gastrointestinal cancer patients can be as high as 1.13±0.1 µg/mL (5.79±0.51 µM as cisplatin).34 Shown in Supporting Information Table S1 are the LODs and linear dynamic ranges of recently developed cisplatin sensors.12-19 As can be seen, the LODs of the current sensors are comparable to other cisplatin sensors. Although a lower LOD can be achieved using several other techniques, these techniques require multiple steps (e.g., chromatographic separations) and are incapable of real-time detection of cisplatin. Sensor Specificity and Selectivity. Based on the above results, the low-coverage “signal-on” sensor has several advantages over the high-coverage “signal-off” sensor, these attributes include a lower LOD, a wider optimal AC frequency range, and most importantly, a shorter response time. Furthermore, “signal-on” sensors are, in general, more desirable for real-world biosensing applications. Thus, here we focused on analyzing the low-coverage sensor’s response to other Ptbased drugs and several commonly prescribed antibiotics. The sensor was tested against 50 µM levofloxacin, amoxicillin, sulfamethoxazole, nitrofurantoin, trimethoprim, amoxicillin, tetracycline, satraplatin, and carboplatin (Figure 3). The sensor’s responses to the seven antibiotics were minimal, the only interfering drug was carboplatin (~84% SE). It is not unexpected since carboplatin is a Pt(II) drug that is structurally similar to cisplatin. The difference in the sensor’s response to cisplatin and carboplatin is likely due to the difference in the leaving group upon binding to the DNA bases. The leaving group of carboplatin is 1,1-cyclobutanedicarboxylate, it is larger in size and is a poorer leaving group when compared to the chloride ligands of cisplatin.35 It is worth mentioning that the sensor did not respond to satraplatin, a new Pt(IV) prodrug that is under preclinical and clinical evaluation, the SE was ~3%.36,37 These results clearly show that the sensor does not cross-react with Pt(IV) species as well as various antibiotics. It is well-suited for analyzing cisplatin, but it could also be used to detect other Pt(II) anticancer drugs such as carboplatin. It can potentially be used to trace the conversion of Pt(IV) drugs such as satraplatin to its reactive Pt(II) species (e.g., JM 118) in clinical research.38

Another attribute of folding- and dynamics-based electrochemical biosensors is their selectivity, most of them have been employed in realistically complex samples.22-27,29 Two complex biological samples of clinical relevance, saliva and urine, were used in this study. ACVs of the low-coverage sensor obtained at 200 Hz in the absence and presence of cisplatin in 50% saliva and 50% urine are shown in Figure 4. The sensor responded well to cisplatin in 50% saliva, the SE was ~109% (Figure 4A). The %SE was higher at higher frequencies such as 500 Hz (Supporting Information Figure S4B). The AC frequency-dependent current and %SE profiles resemble those obtained in HEPES, suggesting similarity in sensor behavior in the two media (Figure 1D, Supporting Information Figure S3B, S4A, S4B). The sensor’s response to cisplatin in 50% urine was less optimal, the SE was ~70%. Higher %SE can be obtained at higher frequencies (e.g., 750 Hz), however, the response is likely to be less reproducible because of the challenges in peak current quantification (Supporting Information Figure S4D). The AC frequency-dependent current profiles are dissimilar to those obtained in HEPES or 50% saliva, this change could be attributed to the differences in the ionic strength (Supporting Information Figure S4C). Overall, despite the differences in the %SE, this sensor has proven to be functional in both complex biological samples.

Figure 4. AC voltammograms of the low-coverage sensor in the absence and presence of 50 µM cisplatin in 50% saliva (A) and 200 µM cisplatin in 50% urine (B). The data were collected at 200 Hz.

Figure 3. The low-coverage “signal-on” sensor’s responses to 50 µM cisplatin, carboplatin, satraplatin, tetracycline, ampicillin, trimethoprim, nitrofurantoin, sulfamethoxazole, amoxicillin, and levofloxacin in HEPES using ACV at 200 Hz.

CONCLUSION In summary, we have demonstrated, for the first time, the use of an oligo-AG DNA probe for real time detection of cisplatin. The sensing mechanism can be tuned by simply changing the probe coverage, binding of cisplatin via the formation of intrastrand Pt(II)-AG adducts is more likely for a sensor with a high probe coverage, whereas binding via the formation

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of intrastrand Pt(II)-GAG adducts is possible if the coverage is adequately low. Although both high-coverage “signal-off” and low-coverage “signal-on” sensors are capable of detecting cisplatin, the “signal-on” sensing mechanism is more desirable. The low-coverage sensor has a lower LOD, faster response time, and a wider optimal AC frequency range. It has high specificity for cisplatin and potentially other Pt(II) drugs, and does not cross-react with satraplatin. It can be employed directly in diluted saliva and urine samples. With further optimization, this sensor could find applications in real time analysis of cisplatin in clinical samples.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Structure of the DNA probe. Binding curves for the sensors. AC frequency-dependent current responses in HEPES. Table showing analytical performance of different cisplatin sensors. AC frequency-dependent current responses and %SE plots for the lowcoverage sensor in 50% saliva and 50% urine (Figures S1−S4, and Table S1) (PDF).

AUTHOR INFORMATION Corresponding Author *Fax: +1 402 472 9402. Tel.: +1 402 472 5340. E-mail: [email protected]

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT The authors acknowledge the National Science Foundation (CHE-0955439) for financial support.

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