Scalene waveform for co-detection of guanosine and adenosine using

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Scalene waveform for co-detection of guanosine and adenosine using fast-scan cyclic voltammetry Michael T. Cryan, and Ashley Elizabeth Ross Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b00450 • Publication Date (Web): 02 Apr 2019 Downloaded from http://pubs.acs.org on April 5, 2019

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Analytical Chemistry

Scalene waveform for co-detection of guanosine and adenosine using fast-scan cyclic voltammetry Michael T. Cryan1 and Ashley E. Ross1,2 1University

of Cincinnati Department of Chemistry 312 College Dr. 404 Crosley Tower Cincinnati, OH 45221-0172 Office #: 513-556-9314 Email: [email protected] 2Corresponding

author

Keywords: electrochemistry, purines, neuromodulators, nucleosides, carbon-fiber microelectrodes

1 ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract: Guanosine and adenosine are important neuromodulators in the brain and work in cooperation to mitigate the effects of stroke, traumatic injury, and other neurological events. Both purines can act on a slow (minutes to hours) and rapid (milliseconds to seconds) timescale. A guanosine-adenosine interaction has been proposed in which guanosine modulates adenosine levels, and the two work together to control glutamate neurotransmission. Traditional methods to co-detect purines, such as HPLC with microdialysis, are robust but lack the temporal resolution necessary to quantify release in real-time. Fast-scan cyclic voltammetry (FSCV) has been used to detect guanosine and adenosine independently, but co-detection has not been possible. Here, we developed a novel “scalene waveform” to co-detect guanosine and adenosine with nanomolar limits of detection in real-time with FSCV. The scalene waveform uses a slow rate (100 V/s) on the forward scan and the conventional rate (400 V/s) on the back scan; potentials go from -0.4 V to 1.45 V and back to -0.4 V. The scan rates were optimized to increase the separation of the oxidative peaks for guanosine and adenosine. The temporal separation of the primary peaks was increased 4.6 ± 0.1-fold at the scalene waveform compared to the traditional waveform. Both exogenously-applied guanosine and adenosine and endogenous transient release were detected at the scalene waveform in rat brain slices. We show the first method for co-detecting guanosine and adenosine using FSCV, which can be used to study the guanosine-adenosine interaction and better understand their cooperative therapeutic effects.


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Introduction: Guanosine and adenosine are important purinergic nucleosides in the brain, involved in neuroprotection and neuromodulation.1–6 Adenosine controls cerebral blood flow, neuronal excitability, and protects the brain from oxidative stress and ischemia.7 The modulatory and protective effects of guanosine are not as well-understood, but it is believed to play a role in nociception and can ameliorate the consequences of seizures, spinal injury, mood disorders, and neurodegenerative disease.3 Guanosine has also been shown to assuage the deleterious effects of Parkinson’s and Alzheimer’s by modulating glutamate transport.8 Over the last decade or more, extracellular signaling on the milliseconds to seconds timescale has been suggested for both nucleosides.4,9–12 Fast-scan cyclic voltammetry (FSCV) at carbon-fiber microelectrodes offers subsecond temporal resolution and has been the technique of choice for studying rapid purinergic signaling in the brain.9,10,13,14 FSCV has been used to show that adenosine can modulate dopamine release on a millisecond timescale, providing evidence that purinergic neuromodulation can be rapid.12 A “guanosine-adenosine interaction” has been suggested in which guanosine modulates the level of adenosine in the extracellular space by slowing down its reuptake into the cell,15 and the two act together to control glutamate neurotransmission.11,16 Despite these known interactions, guanosine and adenosine cannot be rapidly co-detected with FSCV due to their similar oxidation peak locations.10 A method that could monitor rapid concentration fluctuations of guanosine and adenosine simultaneously and in real-time would be advantageous for investigating their dual role in brain function. Here, we show a novel “scalene”-shaped waveform for FSCV which results in an increase in oxidative peak separation between guanosine and adenosine. High-performance liquid chromatography (HPLC) and microdialysis sampling have remained popular methods for multi-purine detection. Use of HPLC for determination of nucleosides is a powerful technique that requires no derivatization and offers low limits of detection.18,19 Microdialysis sampling is often coupled to separation techniques to obtain regional 3 ACS Paragon Plus Environment


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information on extracellular purine levels in vivo;20,21 however, microdialysis probes have been shown to cause local tissue damage upon implantation and result in acutely-elevated levels of guanosine and adenosine.21,22 Although robust, chromatography with microdialysis sampling in vivo does not have the temporal resolution necessary to measure signaling dynamics in real-time. Fast-scan cyclic voltammetry (FSCV) is commonly used to study neurotransmission with subsecond time resolution;13,23–26 however, detection of multiple purine signaling molecules simultaneously has not been explored. Independent waveforms exist for guanosine and adenosine detection, but they are not currently amenable to co-detection.10,13 In FSCV, waveform parameters can be tailored in order to maximize the selectivity for a specific analyte.27,28 This is an attractive method for increasing analyte selectivity because it does not require expensive or time-consuming electrode modifications. Traditionally, a triangle waveform is used with FSCV; however, several modified waveforms have been developed that use unconventional scan rates and waveform shapes in order to render the waveform selective for its target analyte.27–30 Multiplescan rate waveforms have been used for the detection of neuropeptides.27 In addition, plateau times at the switching potential have been shown to aid in distinguishing potential interferents from each other by inducing unique changes in their respective cyclic voltammograms.28 By exploiting the differences in adenosine and guanosine kinetics at the electrode surface, we have developed a novel “scalene”-shaped waveform for co-detection of these purines. In this paper, we present a scalene waveform for simultaneous detection of guanosine and adenosine using FSCV at unmodified carbon-fiber microelectrodes. The waveform sweeps from -0.4 V to 1.45 V at a 100 V/s rate on the forward scan, with the conventional 400 V/s rate on the back scan.

The oxidative peak separation in time for guanosine and adenosine was

optimized, showing significantly improved resolution as compared to the conventional triangle waveform. Oxidative peaks for guanosine, adenosine, and dopamine can be simultaneously detected with this waveform, which allows triple analyte detection with FSCV. Signal stability over the course of 30 mins was assessed, and the viability of the co-detection signal in rat caudate 4 ACS Paragon Plus Environment

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putamen slices was examined. Spontaneous transient events were detected with this waveform in the brain, proving its use for sensing endogenous release. Co-detection of these purines using FSCV will allow the guanosine-adenosine interaction to be explored with subsecond resolution for the first time.

Methods: Reagents. For details on the reagents used, see Supporting Information. Fast-scan

cyclic

voltammetry

at

carbon-fiber

microelectrodes.

Fast-scan

voltammograms were obtained using a WaveNeuro potentiostat equipped with a 1 MΩ headstage (Pine Instruments, Durham, NC, USA). High-Definition Cyclic Voltammetry (HDCV) software (University of North Carolina-Chapel Hill, courtesy of R. Mark Wightman) with a PC1e-6363 multifunction I/O device (National Instruments, Austin, TX, USA) was used for data acquisition and analysis. Data was collected at carbon-fiber microelectrodes (see Supporting Information for electrode fabrication details). The traditional waveform scanned from -0.4 V to 1.45 V (against Ag/AgCl) and back at 400 V/s and 10 Hz. The scalene waveform scanned from -0.4 V to 1.45 V and back, using 100 V/s on the forward scan and 400 V/s on the back scan. A frequency of 10 Hz was used for the scalene waveform. Scan rates at and below 400 V/s were filtered with a 2 kHz lowpass filter; 600 and 800 V/s scan rates were filtered using a 5 kHz lowpass filter. All data was background-subtracted to remove any non-faradaic current. Electrodes were calibrated using flow injection analysis as previously reported31 via a Fusion 100 Two-Channel Chemyx syringe pump (Stafford, TX, USA) and a flow rate of 1 ml/min. Details on statistical analysis are provided in the Supporting Information. Ex vivo experiment. All animal procedures described here were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Cincinnati and performed in accordance with The Guide for the Care and Use of Laboratory Animals (the Guide) by the National Research Council. Male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA, USA) weighing 250-350 g were housed in a vivarium and provided food and 5 ACS Paragon Plus Environment


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water ad libitum. Brain slices of the caudate putamen were collected as previously described.12 Rats were anesthetized using isoflurane (Henry Shrein, Melville, NY, USA) and euthanized by decapitation immediately before the experiment. The brain was removed and placed in ice-cold oxygenated (95% O2 and 5% CO2) aCSF for 2 mins. The brain was then mounted onto the stage for slicing using super glue. Coronal slices of the caudate putamen at a 400 µm thickness were prepared with a Leica VT1000 S vibratome (Chicago, IL, USA) set to a speed of 90 and frequency of 3. After slicing, tissue was allowed to recover in oxygenated aCSF for approximately 1 hr prior to experimentation. For testing, slices were placed in a perfusion chamber (Warner Instruments, Hamden, CT, USA) maintained at 37° C and perfused with oxygenated aCSF at 2 ml/min using a peristaltic pump (Watson-Marlow, Wilmington, MA, USA). The microelectrode was implanted into the tissue approximately 75 µm beneath the surface using a micromanipulator. The electrode was allowed to equilibrate for 20 mins. For exogenous delivery, a Parker Hannifin Picospritzer III (Hollis, NH, USA) was used to deliver 100 µM adenosine and 50 µM guanosine approximately 100 µm away from the working electrode via a fabricated glass picopipette with a 15-20 µm opening at the tip, similar to previous reports.10,26 Picospritzer pressure was set at 30 psi with ejections lasting 800 ms. Exogenous guanosine and adenosine were successfully co-detected in a total of 8 slices.

Results and Discussion: Traditional waveforms for adenosine and guanosine A triangular waveform is normally used with FSCV to oxidize and reduce electroactive analytes of interest. Traditional waveforms that are specific for guanosine and adenosine have been previously developed, which include scanning to 1.3 V for guanosine or 1.45 V for adenosine.7,13,28 Adenosine is not detectable at the guanosine waveform due to the lower switching potential (Fig. S1);10 therefore, to detect both analytes you must use a 1.45 V switching potential (“adenosine waveform”, Fig. 1). The adenosine waveform scans from -0.4 V to 1.45 V and back at 400 V/s and frequency of 10 Hz. Fig. 1 shows example data for 5 µM guanosine, 5 6 ACS Paragon Plus Environment

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µM adenosine, and a mixture of equal concentration of both analytes at the traditional adenosine waveform. Cyclic voltammograms (CVs) are opened up and plotted as current vs. waveform time for better visualization of peak position. Guanosine undergoes a primary oxidation on the forward scan at 1.36 V (4.6 ms, Fig. 1A), and the secondary peak occurs at 0.88 V (3.22 ms). Adenosine’s primary peak occurs on the back scan at 1.23 V (5.16 ms, Fig. 1B) and the secondary peak is at 1.1 V (3.86 ms) on the forward scan. There is minimal separation between the primary peaks of guanosine and adenosine when analyzed in a mixture (Fig. 1C). The difference in time (∆t) between the primary peaks was used to assess the degree of peak separation and the ∆t on average was 0.69 ± 0.02 ms (n = 6) with the traditional waveform. The CVs and color plots show that the peaks are not well-resolved and some coalescence exists. In addition, the primary peak for guanosine overlaps the smaller secondary peak for adenosine (1.1 V), which in turn merges with guanosine’s secondary oxidation peak (0.88 V). In this work, we have developed a novel, multi-scan rate waveform that increases the temporal separation of the primary peaks for guanosine and adenosine, allowing for co-detection of these species in real-time.



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Figure 1: Minimal separation is observed between the primary peaks for guanosine and adenosine using the traditional “adenosine waveform”. The triangle waveform goes from -0.4 V to 1.45 V and back at a rate of 400 V/s. (A) False color plot and unfolded cyclic voltammogram for 5 µM guanosine using the traditional waveform. Guanosine oxidizes at 1.36 V on the forward scan (1° peak on CV), with secondary oxidation at 0.88 V (2° peak on CV). (B) False color plot and unfolded CV for 5 µM adenosine at the traditional waveform. Adenosine oxidation is seen on the back scan at 1.23 V (1° peak on CV), with a secondary peak at 1.1 V (2° peak on CV). (C) False color plot and respective unfolded CV for the 5 µM guanosine (GN) and adenosine (AD) mixture. On average, the ∆t between the two peaks using the triangle waveform was 0.69 ± 0.02 ms. The waveform shape is shown (red dotted trace) to aid in visual analysis of peak position. Scalene waveform for simultaneous adenosine and guanosine detection A scalene triangle waveform was developed to further separate the primary oxidative peaks of guanosine and adenosine. Oxidative peak positions shifted by varying the forward scan rate. Shifting in peak position as a result of varying scan rates is common, especially for 8 ACS Paragon Plus Environment

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irreversible oxidation steps like the primary oxidations for both guanosine and adenosine. The primary peaks for guanosine and adenosine shifted and were more resolved when a forward scan rate of 100 V/s was used instead of 400 V/s, while maintaining the same potential limits (Fig. 2). At the slower forward scan rate, the primary oxidation peak for guanosine appears on the forward scan at 1.2 V (15.9 ms, Fig. 2A) compared to 1.36 V (4.6 ms) with the traditional waveform. The secondary oxidation peak was observed at 0.66 V (10.6 ms). Adenosine’s primary peak was observed on the back scan at 1.29 V (18.9 ms, Fig. 2B) compared to 1.23 V (5.16 ms) with the traditional waveform, and the secondary peak was at 0.83 V (12.3 ms) on the forward scan using the new waveform.



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Figure 2: The scalene waveform significantly separates the primary peaks for guanosine and adenosine. (A) False color plot and respective opened CV for 5 µM guanosine. Primary oxidation is observed at 1.2 V (15.9 ms), with secondary oxidation happening at 0.66 V (10.6 ms). A small switching error is observed. (B) False color plot and opened CV for 5 µM adenosine at the scalene waveform. Primary oxidation is observed at 1.29 V on the back scan (18.9 ms), with secondary oxidation at 0.83 V (12.3 ms). (C) False color plot and opened CV for the 5 µM guanosine (GN) and adenosine (AD) mix using the scalene waveform. Average ∆t between the two primary peaks was 3.15 ± 0.09 ms, representing a 4.6 ± 0.1-fold increase in separation as compared to the triangle waveform. The waveform shape is shown (blue dotted trace) to aid in visual analysis of peak position. The scalene waveform substantially enhances the temporal separation between both the primary and secondary peaks of adenosine and guanosine (Fig. 2C). The ∆t between the two primary oxidative peaks was on average 3.15 ± 0.09 ms (n = 8) using this new waveform, representing a 4.6 ± 0.1-fold increase in separation relative to the traditional triangle waveform. The ∆t for the secondary oxidation peaks of guanosine and adenosine with the scalene waveform was on average 2.96 ± 0.20 ms (n = 8), which is an improvement compared to the traditional waveform where the secondary peaks overlap. Overall, the scalene waveform provides better peak resolution, with an improved return to baseline between the two primary oxidative peaks. Adenosine and guanosine electrode interaction The improved separation of guanosine and adenosine’s oxidative peaks at the scalene waveform can be attributed to a difference in their respective kinetics at the electrode. Guanosine’s primary oxidative peak occurs on the forward scan, whereas adenosine’s primary oxidative peak occurs on the back scan for the traditional adenosine waveform (Fig. 1). The difference in peak location indicates that adenosine electron transfer is much slower than guanosine. We have previously shown that functional groups on the purine ring can modulate the interaction at carbon-fiber and that guanine and adenine interact differently.32 In addition, both guanosine and adenosine’s primary oxidation reactions are irreversible.10,13 Irreversible reactions result in shifts in peak potential as a function of scan rate. By manipulating the scan rate on the forward scan where guanosine oxidizes, we hypothesized that we could preferentially shift guanosine’s peak with limited effect on adenosine. 10 ACS Paragon Plus Environment

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Not only are electron transfer rates different for adenosine and guanosine, but the dominating interaction at the electrode is different. A scan rate experiment using the traditional waveform provided insight into how guanosine and adenosine each interact with the electrode surface (Fig. 3). Guanosine’s oxidative current increases linearly with the square root of scan rate (Fig. 3A); this plot has a higher r2 value than that of current vs. scan rate (Fig. S2A), suggesting that guanosine is diffusion-limited. However, we have previously reported that a combination of adsorption and diffusion is likely, but diffusion may be the dominant interaction.10 Adenosine was shown to be primarily adsorption-controlled, which agrees with earlier characterization studies (Fig. 3B and Fig. S2B).13,28Adsorption-limited processes are typically slower interactions than diffusion-limited which could contribute to adenosine’s oxidation peak showing up on the back scan. It is important to note that a plateau in current for guanosine at scan rates faster than 400 V/s was observed, suggesting that guanosine migration to the electrode becomes kinetically limited. This was not observed for adenosine. Overall, using the slow forward scan rate exploits the analytes’ differences in kinetics at the electrode and gives rise to further separation of their oxidation potentials. Specific optimization of scan rate is shown in the next section.

Figure 3: Guanosine is primarily diffusion-limited and adenosine is adsorption-limited. Scan rate experiments were done using the traditional adenosine waveform and both analytes were tested at 5 µM. (A) Current increases with respect to square root of scan rate for guanosine (n = 4). A higher r2 value was observed compared to the current vs. scan rate (Fig. S2A), suggesting diffusion. (B) Current as a function of scan rate is linear for adenosine, suggesting adsorptioncontrolled interactions (n = 7).



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Scalene waveform optimization The traditional waveform for FSCV was modified to improve guanosine and adenosine oxidative peak separation. To exploit their differences in kinetics and interaction at the electrode, we chose to test the extent to which varying the forward scan rate affected peak position and current, while maintaining the back scan at 400 V/s (Fig. 4A and C). Experimental scan rates for the forward scan were 50, 75, 100, 200, 400, and 600 V/s. A forward scan rate of 800 V/s was also tested but the oxidation peaks were almost completely overlapped (data not shown). Decreasing the forward scan rate from 600 V/s to 50 V/s resulted in a significant improvement in temporal peak separation (Δt, Fig. 4A, n = 8). Maximum separation was observed at 50 V/s; however, the total waveform time increases with slower scan rates, which causes a decrease in the holding time in between waveform applications (using 10 Hz waveform application frequency). A decrease in holding time between waveform application results in less time for preconcentration, causing a significant decrease in current detected forguanosine at 100 V/s compared to 400 V/s (Fig. 4C, two-way ANOVA, p < 0.0001 with Bonferroni post-test). This provides additional evidence that while guanosine is primarily diffusion-limited, some adsorption at the electrode exists.10 By setting the forward rate to 100 V/s, the ∆t was significantly different than that at the conventional 400 V/s scan rate (one-way ANOVA, p < 0.0001 with Bonferroni post-test), and a decrease in current was also observed (Fig. 4C, n = 8). Average peak separation in time using 100 V/s on the forward scan was 3.15 ± 0.09 ms (n = 8), compared to 0.69 ± 0.02 ms (n = 5) with 400 V/s. Because adenosine still oxidizes on the back scan with the scalene waveform, its current was not as impacted by the forward scan rate (Fig. 4C, two-way ANOVA, p > 0.05 with Bonferroni post-test). Scanning at a rate of 200 V/s on the forward scan does recover some of the sensitivity; however, the peak separation is not adequate enough for confident distinction between the two peaks (Fig. 4A). A forward scan rate of 100 V/s was chosen as optimal for peak separation, ease of color plot interpretation, and sensitivity loss.


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Figure 4: The scalene waveform uses multiple scan rates to enhance peak separation while maintaining detection ability for guanosine and adenosine. (A) The forward scan rate was varied from 50 to 600 V/s while holding the back scan constant at 400 V/s (n = 8). Analytes were tested at 5 µM. Increasing forward scan rate gave rise to a decrease in peak separation (Δt). 100 V/s was chosen as optimal (Δt = 3.15 ± 0.09 ms), which is significantly different than the peak separation at 400 V/s (one-way ANOVA, p < 0.0001 with Bonferroni post-test). (B) Increasing the back scan rate had no significant effect on peak separation (one-way ANOVA, p > 0.05 with Bonferroni post-test, n = 8). (C) Increasing the forward scan rate increases the peak current for both guanosine (GN) and adenosine (AD). Current at 100 V/s is significantly less than that at 400 V/s for guanosine (two-way ANOVA, p < 0.001 with Bonferroni post-test) but not for adenosine (p > 0.05), showing a loss in sensitivity for guanosine, but is compensated for by better separation at the slower scan rate (n = 8). (D) Increasing the back scan rate had no significant effect on current for guanosine (two-way ANOVA, p > 0.05 with Bonferroni post-test, n = 8) but did impact adenosine current (p < 0.01) The extent to which the back scan affected adenosine peak position was tested due to its position on the back scan (Fig. 4B). The back scan rates 400, 600, and 800 V/s were tested while maintaining the forward scan rate at 100 V/s. Increasing the back scan rate had no significant effect on the ∆t between guanosine and adenosine peaks (one-way ANOVA, p > 0.05, n = 8); therefore 400 V/s was chosen as optimal for the scalene waveform. In addition, increasing the cathodic scan rate did not reclaim any of the sensitivity lost on the forward scan (Fig. 4D, two-way ANOVA, p > 0.05, n = 8) for guanosine. A small increase in adenosine current was observed by



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increasing the back scan rate (two-way ANOVA, p < 0.01 with Bonferroni post-tests). Despite the small increase in current for adenosine, going faster than 400 V/s on the back scan resulted in a decrease in data interpretability because the peaks were shifted to the top of the color plot (data not shown). Decreasing the back scan rate below 400 V/s would likely result in a further decrease in sensitivity. Sensitivity at the scalene waveform Nanomolar limits of detection for guanosine and adenosine are maintained at the scalene waveform (Fig. 5). Two calibration curves were generated for each analyte: one for the analyte alone and one with both purines in a mixture of equal concentrations. Experimental concentrations ranged from 100 nM to 100 µM and the linear ranges were 100 nM to 10 µM. The plots for adenosine (Fig. 5B) do not appear to pass near the origin at the lower concentrations. This is attributed to the presence of a non-faradaic “switching error” located at the switching potential. Switching errors have been previously reported with the triangle waveform, and can interfere with accurately quantitating the true faradaic current arising from analytes which oxidize at the switching potential like adenosine, histamine, and hydrogen peroxide.33 Switching errors are particularly more troublesome at low concentrations of analyte, when the faradaic current is smaller than that of the non-faradic switching error peak; however they can be minimized through longer electrical pretreatment times. It is important to note that switching errors are not observed on every electrode. The limit of detection (LOD) was defined as three times the average noise. In a mixture, the LOD for guanosine is 66 ± 13 nM (Fig. 5A, n = 5), and the LOD for adenosine is 137 ± 27 nM (Fig. 5B, n = 5). These LODs were compared to those achieved when the waveform was used to separately detect both analytes. The LOD for guanosine alone is 60 ± 20 nM (Fig. S3A, n = 5), and the LOD for adenosine alone is 129 ± 28 nM (Fig. S3B, n = 5). The LODs obtained for guanosine and adenosine in a mixture were not significantly different from those observed when they were in separate solutions (paired t-test, p > 0.05). 14 ACS Paragon Plus Environment

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Figure 5: When mixed together, both guanosine and adenosine show detection limits comparable to those observed when they are separated. (A) Concentration curve for guanosine in a mixture with an equal concentration of adenosine. Linear region shown on right (n = 5). (B) Concentration plot for adenosine when mixed with an equal concentration of guanosine, with linear region on right (n = 5).

The scalene waveform maintained high sensitivity for both adenosine and guanosine. Sensitivity is defined as the slope of the linear region and is expressed in terms of nA/µM. Sensitivity for guanosine alone in solution was 4.8 nA/µM and was 4.1 nA/µM in a mixture with adenosine. Adenosine’s sensitivity alone in solution was 2.4 nA/µM and decreased to 2.0 nA/µM in a mixture with guanosine. Sensitivities for both analytes did decrease slightly when mixed with the other purine—this is likely due to the analytes competing for a limited number of sites for oxidation on the electrode. However, the decreases in sensitivity were not significantly different (paired t-test, p > 0.05). It is important to note that the LODs for the scalene waveform are higher than when using the traditional triangle waveform.10,13 Small (lower than 100 nM) transients would be difficult to detect using the scalene waveform; however, adenosine transient concentrations have been shown to be region-dependent and range from low nanomolar to micromolar.9,16 In addition, the adenosine-guanosine interaction is particularly important during insults to the brain where purine levels are elevated.11,15,19 Multi-analyte detection at the scalene waveform 15 ACS Paragon Plus Environment


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Triple detection of guanosine, adenosine, and dopamine is possible at the scalene waveform. Dopamine oxidizes at 0.6 V using the traditional waveform, and is historically wellcharacterized with FSCV.25,34–36 With the scalene waveform, the oxidative peak shifted to 0.46 V (Fig. 6A, n = 6). Dopamine is observed at 8.66 ms when plotting the CV as current vs. waveform time (Fig. 6B, n = 6). In future studies, this waveform could be used to study purinergic and dopaminergic interactions in the brain.

Figure 6: Triple detection of guanosine, adenosine, and dopamine was observed using the scalene waveform. (A) Example false color plot showing 5 µM each of dopamine, guanosine, and adenosine. Dopamine oxidation occurs at 0.46 V when the modified waveform is used. (B) Opened cyclic voltammogram for the purine-dopamine mix. ∆t between dopamine and guanosine, its nearest neighbor, is 6.96 ms. The biologically complex environment of the brain necessitates confident and selective voltammetric measurements. Additional analytes that are likely to be encountered within the brain were tested using this waveform: GTP, ATP, histamine, and hydrogen peroxide (Fig. S4). GTP undergoes oxidation at 1.2 V on the forward scan, resulting in peak overlap with guanosine (Fig. S4D). However, due to the presence of negatively-charged phosphate groups, current for GTP is lower by 2.7 ± 0.1-fold (n = 9). ATP oxidizes on the back scan at 1.31 V using the scalene waveform (Fig. S4E), showing coalescence with the oxidative peak for adenosine. Analogous to 16 ACS Paragon Plus Environment

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the relationship between guanosine and GTP, current for ATP was on average 1.8 ± 0.1-fold less than adenosine (n = 9). Histamine is electroactive and oxidizes at 1.26 V on the back scan similar to adenosine (Fig. S4C). Current for histamine is low at this waveform, and was on average 1.9 ± 0.2-fold less than adenosine (n = 9). Hydrogen peroxide also oxidizes on the back scan at 1.30 V (Fig. S4F) but similar to histamine, its current was on average 2.7 ± 0.6-fold less than adenosine (n = 9). Although current for histamine and hydrogen peroxide are lower, their oxidative peaks do overlap with adenosine’s. Thus, this waveform is potentially capable of separating guanosine from one of these three analytes in a mixture. This could be advantageous depending on the brain region of interest. Likewise, pharmacological manipulation could be used in vivo to parse out which analyte is giving rise to the signal on the back scan. In vitro stability Guanosine and adenosine co-detection is stable over longer sampling times using the scalene waveform. Previously, both adenosine and guanosine were shown to be stable after rapidly repeated injections,10,13 and this stability was not expected to change at the scalene waveform. A bolus of a mixture of adenosine and guanosine (5 µM each) was introduced at the electrode every 5 minutes for 30 minutes to better assess longer-term stability (Fig. S5). Current for both analytes was normalized to the first injection to visualize any changes in signal over time. Current did not significantly decrease over the course of 30 minutes for either analyte (two-way ANOVA, p > 0.05 with Bonferroni post-test) which permits use of this waveform to study fluctuations in the brain over time. Ex vivo detection in the brain Co-detection of adenosine and guanosine was validated in live slices of rat caudate putamen. Both electrically stimulated and transient adenosine release has been wellcharacterized in the caudate putamen, and may be a region of interest for purine co-



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detection.9,37,38 To validate co-detection in tissue, an electrode was placed within the medial region of the caudate, 100 µm away from a picospritzing pipette. The picospritzing pipette was backfilled with 100 µM adenosine and 50 µM guanosine. Large concentrations are common for exogenous application due to diffusional loss in tissue, with only a few micromolar actually reaching the electrode.7 A short bolus was administered in the tissue to mimic a transient event. Co-detection was successful in live brain slices (Fig. 7A, n = 8). The CV for adenosine and guanosine in tissue was comparable to in vitro detection (Fig. 2). Current rapidly returned to baseline, indicating that both purines can be transported back into the cell in this region.

Figure 7: Co-detection was achieved in live rat brain slices. (A) Guanosine and adenosine were both detected in tissue following exogenous application. Current vs. time (upper), false color plot (mid), and the cyclic voltammogram immediately following exogenous application (lower) are shown. The purple arrow marks exogenous application. (B) Transient, unstimulated release was observed a few seconds after exogenous application. Current vs. time (upper), false color plot (mid), and a normalized, superimposed cyclic voltammogram for the transients (lower) are depicted. Unstimulated transients on the color plot are circled in white. The purple arrow denotes time of picospritzing, and the asterisks represent transient release. The CV for exogenous application is shown in black and the transient CV in green. CVs are normalized to aid in peak comparison. The large decrease in current observed is likely due to an ionic shift. Exogenous is labelled as “Exo” and endogenous is labelled as “Transient”.


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Spontaneous release events were detected at the scalene waveform. Spontaneous transient adenosine and hydrogen peroxide release has been well characterized9,16,39,40 in the striatum. Transient release at the switching potential was observed using the scalene waveform in 5 of 8 slices (Fig. 7B). A secondary peak was not observed, making it difficult to confidently identify the analyte. Hydrogen peroxide does not exhibit a secondary peak; likewise, at low concentrations of adenosine, the secondary peak is often below the limits of detection.38In addition to the transient events at the switching potential, transient events at 1.2 V on the forward scan were observed in 3 out of 8 slices. Interestingly, this transient event occurs at the same potential as guanosine, and the CV was similar to that of exogenous application (Fig. 7B). Other analytes that oxidize at the switching potential such as hydrogen peroxide, histamine, and ATP oxidize on the back scan similar to adenosine (Fig. S4) and are therefore not likely the culprit. To date, guanosine transients have not been explored nor characterized. Further pharmacological studies are needed to verify that spontaneous guanosine transients occur in this region, and to confidently identify the analyte at the switching potential as adenosine or hydrogen peroxide. These results show that the scalene waveform is able to detect both exogenous and endogenous release in the brain and is a powerful new analytical tool for co-detection with FSCV.

Conclusion: Here, we have characterized a novel, multi-scan rate waveform for FSCV that is capable of co-detecting guanosine and adenosine in real time with improved peak resolution. This was accomplished by using a slow, 100 V/s scan rate on the forward scan. This scan rate exploited the differences in guanosine and adenosine’s kinetics to improve the temporal and spatial separation of the oxidative peaks. Nanomolar LODs were observed for both analyte separately and were maintained within a mixture. We also show that this waveform could be expanded to co-detect guanosine with other analytes like histamine or hydrogen peroxide. The applicability of this waveform was confirmed in live brain slices. Both guanosine and adenosine were detected 19 ACS Paragon Plus Environment


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in rat caudate putamen slices after exogenous application, and endogenous, transient release ofwas also observed albeit further identification is needed. The scalene waveform will allow detection of the close interaction between adenosine and guanosine and their therapeutic effect within the central nervous system during neurochemical injury to be studied in real-time.

Supporting Information: Supplemental methods on the reagents used, electrode fabrication, and statistics. CVs at the traditional guanosine and adenosine waveforms. Adsorption and diffusion plots for guanosine and adenosine, respectively. Concentration curves for guanosine and adenosine when the analytes are separately detected using the scalene waveform. Example CVs for common biological interferents GTP, ATP, histamine, and hydrogen peroxide. Stability of waveform over time. References (1) (2) (3) (4) (5) (6)

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Dynamics in the Rat Dorsal Striatum Using Fast-Scan Cyclic Voltammetry https://doi.org/10.1021/cn4000499.

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Scalene waveform for co-detection of guanosine and adenosine using

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