Purine functional group type and placement modulate the interaction

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Purine functional group type and placement modulate the interaction with carbon-fiber microelectrodes Gary N Lim, and Ashley Elizabeth Ross ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.8b01504 • Publication Date (Web): 18 Jan 2019 Downloaded from http://pubs.acs.org on January 22, 2019

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Purine functional group type and placement modulate the interaction with carbon-fiber microelectrodes Gary N. Lim, Ashley E. Ross* *Corresponding author 312 College Dr. 404 Crosley Tower Cincinnati, OH 45221-0172 Office #: 513-556-9314 Email: [email protected] Keywords: adenine, guanine, carbon-fiber microelectrode, fast-scan cyclic voltammetry, adenosine, guanosine

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ABSTRACT Purine detection in the brain with fast-scan cyclic voltammetry (FSCV) at carbon-fiber microelectrodes (CFME) has become increasingly popular over the last decade; despite the growing interest, an in-depth analysis of how purines interact with the CFME at fast-scan rates has not been investigated. Here, we show how the functional group type and placement in the purine ring modulate sensitivity, electron transfer kinetics and adsorption on the carbon-fiber surface. Similar investigations of catecholamine interaction at CFME with FSCV have informed the development of novel catecholamine-based sensors and is needed for purine-based sensors. We tested purine bases with either amino, carbonyl or both functional groups substituted at different positions on the ring and an unsubstituted purine. Unsubstituted purine showed very little to no interaction with the electrode surface, indicating that functional groups are important for interaction at the CFME. Purine nucleosides and nucleotides, like adenosine, guanosine and ATP, are most often probed using FSCV due to their rich extracellular signaling modalities in the brain. Because of this, the extent to which the ribose and triphosphate groups affect the purine-CFME interaction was also evaluated. Amino functional groups facilitated the interaction of purine analogs with CFME more than carbonyl groups, permitting strong adsorption and high surface coverage. Ribose and triphosphate groups decreased the oxidative current and slowed the interaction at the electrode which is likely due to steric effects and electrostatic repulsion. This work provides insight into the factors that affect purine-CFME interaction and conditions to consider when developing purine-targeted sensors for FSCV.


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Fast-scan cyclic voltammetry (FSCV) at carbon-fiber microelectrodes (CFMEs) is an electroanalytical tool used to detect rapid neurochemical fluctuations in the brain with exquisite spatial and temporal resolution.1–3 Catecholamines, like dopamine (DA),4–6 epinephrine,7,8 and norepinephrine,7,8 have been well-characterized with FSCV over the last several decades. In recent years, FSCV has expanded to include indolamines, like serotonin9,10 and melatonin,11 neuropeptides,12,13 and to purines.14–17 Purine-targeted detection has become increasingly interesting

due

to

their

rich

involvement

in

neuromodulation,

neuroprotection,

and

neuroinflammation.18–21 Adenosine (AD) and adenosine triphosphate (ATP) oxidation at CFME’s have been carefully characterized14–16 and most recently, guanosine (GN).17 Oxidation of both purine nucleosides occur at the purine moiety (Scheme 1), adenine (6-aminopurine) and guanine (2-amino-6-oxopurine), respectively. However, experimental results have indicated that adenosine and guanosine interact differently with the carbon-fiber surface.22 A systematic investigation into the extent to which purines interact with the electrode surface, similar to previous investigations into catecholamine interaction,5 has not been done so far. This study aims to investigate how the functional group type and its substitution position affect the degree of purine adsorption on the electrode surface, relative electron transfer (ET) kinetics and sensitivity. These results will provide a better understanding of purine interaction with the CFME at fast-scan rates and will help inform the development of new highly selective and sensitive purine sensors. Previous investigations into analyte interaction at the electrode surface with FSCV have primarily focused on DA.5,6 DA is oxidized in a 2-electron, 2-proton quasi-reversible process yielding dopamine-o-quinone (DOQ).5 Bath et. al showed that the amino group in DA interacts with the oxides on the CMFE.5 This result was later supported by observing an increase in sensitivity to DA detection after electrochemical overoxidation of the CFME to increase surface oxides.6,23 Langmuir adsorption isotherms have been used to model the rate of adsorption and desorption of DA on the electrode at the traditional 1.0 V waveform,5 and later at the extended 1.4 V waveform,6 and to evaluate the strength of DA adsorption on the electrode. The saturation


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surface coverage (s) was estimated to be 41 pmol/cm2 at the traditional 1.0 V waveform. When an extended waveform (scanning from -0.4 to +1.4 V vs Ag/AgCl and back) was applied, s more than doubled (92 pmol/cm2).6 The high s at the extended waveform is attributed to the oxidation of the carbon-fiber rendering more oxide groups on the surface, thus, resulting in more sites for interactions with DA. These results revealed that DA adsorbs on the electrode surface and the amino group on DA facilitates this interaction. DA is a catecholamine while purine is a heterocyclic amine consisting of a fused pyrimidine and imidazole ring. Although both are amine functionalized compounds, their structures are very different which can yield different interactions with the carbon-fiber surface. To the best of our knowledge, no in-depth analysis into the extent to which functional groups modulate purine interaction and adsorption at a CFME with FSCV has been investigated. Here, we tested the extent to which functional group type and placement on the purine ring changes the kinetics and interaction at the CFME surface. Purines substituted with either an amino, carbonyl or both groups at varying substitution positions were tested. The rate-limiting step at the electrode (adsorption- vs. diffusion-controlled) was evaluated by analyzing how current varies as a function of scan rate. Furthermore, adsorption isotherms were constructed to estimate the degree of interaction of adsorption-controlled analogs. Purine-targeted sensors with FSCV are often used to detect purine nucleosides or nucleotides. Because of this, the extent to which the ribose and triphosphate groups affect the electrode interaction was also examined. This work answers fundamental questions of how amino and carbonyl groups modulate the interaction of purines with the carbon surface and how this interaction changes when a ribose and triphosphate groups are added. A deeper understanding of how substituted purines interact with the electrode surface could aid in developing better purine-based sensors for FSCV detection.


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EXPERIMENTAL SECTION Reagents. Purine, hypoxanthine, and xanthine were purchased from Sigma-Aldrich (St. Louis, MO, USA) while the rest of the reagents were from Fisher Scientific (USA). All stock solutions were 10 mM. Stock solutions of guanine, guanosine, 2,6-diaminopurine, hypoxanthine and isoguanine were dissolved in 1.0 M HCl, xanthine was dissolved in 1.0 M NaOH, and adenine, adenosine, ATP, GTP, 2-aminopurine were dissolved in 0.1 M HCl. All stock solutions were stored at 4 C and diluted daily in Tris buffer for testing. The Tris buffer consisted of 15 mM Tris (hydroxymethyl) aminomethane, 1.25 mM NaH2PO4, 2.0 mM Na2SO4, 3.25 mM KCl, 140 mM NaCl, 1.2 mM CaCl2 dehydrate, and 1.2 mM MgCl2 hexahydrate at pH 7.4. All aqueous solutions were made with deionized water (Milli-Q, Millipore, Billerica, MA, USA). Fast-scan cyclic voltammetry. All FSCV measurements were performed using a WaveNeuro with a 5M headstage (Pine Instruments, Durham, NC, USA). See supplemental methods for instructions on carbon-fiber microelectrode fabrication. Data were collected and analyzed using HDCV software (UNC-Chapel Hill, Mark Wightman) with a computer interface board (National Instruments PC1e-6363, Austin, TX, USA). A triangular waveform was applied scanning the CFME from -0.40 to +1.45 V (vs Ag/AgCl) and back at 400 V/s with a repetition rate of 10 Hz. For scan rates 400 V/s and below, a 2 kHz low pass filter was used. For scan rates exceeding 400 V/s, a 5 kHz low pass filter was used. Non-Faradaic currents in all data were removed through background subtraction. Experiments were conducted in a flow cell using an HPLC six-port valve (VICI, Houston, TX, USA). A bolus of the analyte was injected by pneumatically actuating the valve. A 1 mL/min flow rate was controlled using a Fusion 200 TwoChannel Chemyx Syringe pump (Stafford, TX, USA). Langmuir isotherm modelling. A Langmuir adsorption isotherm was used to model the interaction of purine analogs with the surface of the carbon-fiber microelectrodes at equilibrium following:6,24


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Γsβ[purine]

Γpurine = 1 + β[purine] (1) purine and s are the surface concentration and saturation surface coverage of adsorbed purine analogs on the electrode, respectively, and  is the thermodynamic equilibrium constant for adsorption. purine was determined using equation 2 since oxidation of purine analogs involved both reversible and irreversible steps:25

ip =

n′2θF2 (2) 2.718RTνAΓ

ip is peak current obtained from the oxidation of the purine analogs, n’ is the number of electrons transferred prior to the chemically irreversible steps,  is the total number of electrons transferred in the redox process divided by n’, F is the Faraday constant,  is the scan rate, A is the area of the electrode surface (1.68  10-5 cm3, for 75-µm length electrode), R is the ideal gas constant and T is the temperature (25 °C). For purine analogs following the adenine oxidation scheme: n’=2 and =3, while for guanine oxidation scheme: n’=2 and =2 (Scheme 1). For dopamine which undergoes a one-step reversible oxidation process, equation 3 was used to determine its surface coverage:25

ip =

n2F2 (3) 4RT νAΓ

n is the number of electrons in the reversible process (n=2 for dopamine). At low concentrations, purine is much smaller than s, thus, a linearized form of equation 1 can be used:26

Γpurine = b[purine] (4) where b = s (equation 5). The slope of equation 4, b, is an equilibrium coefficient that dictates adsorption strength.26 Statistics. All stats were conducted with GraphPad Prism v. 7.0 (GraphPad Software Inc., La Jolla, CA, USA). Statistical p-values were significant at 95% confidence level (p < 0.05). Values


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are reported as the mean ± standard error of the mean (SEM), and n represents the number of electrodes. RESULT AND DISCUSSION Adenine and guanine oxidation scheme. Purines, like adenosine and guanosine, are popular target analytes for FSCV detection. Adenosine detection using FSCV is the most welldocumented, and its oxidation scheme is well-characterized at CFMEs.14,15,18,27 Guanosine, a recently explored analyte with FSCV, has also been well-characterized with FSCV detection.17 Evidence suggests that these purines interact with carbon-fiber electrode surfaces differently despite their structural similarity. Specifically, their degree of adsorption, relative electron transfer (ET) rate, and sensitivity differ. A better understanding of which structural components on the purine ring interact with the electrode surface is necessary in order to develop better sensors for FSCV in the future which target these purines. Therefore, a detailed investigation into how functional groups on the purine ring modulate electrode interaction was explored. Oxidation of adenosine and guanosine occurs at the purine moiety, adenine and guanine, respectively. Although both are purine nucleobases, each undergoes a different electrochemical oxidation mechanism (Scheme 1). Adenine involves a 3-step oxidation process with a total loss of 6 electrons and 6 protons (Scheme 1A).28 With traditional electrochemical techniques, the first oxidation peak (oxidation of the pyrimidine moiety of adenine) appears at around 1.0 V vs Ag/AgCl.29 In FSCV, it is observed on the back scan (1.40 V vs Ag/AgCl)14,15 as denoted by the red trace in its CV (Figure 1B). This shift is due to the faster scan rates used in FSCV and indicative of slow ET. The use of fast scan rates in FSCV also results in further oxidation of product I. The subsequent products appear after the formation of product I and at lower potentials (Figure S-1A). Products II and III appear at 1.15 and 0.63 V vs Ag/AgCl, respectively, on the forward scan. The first and second steps are irreversible due to the lack of reduction peaks in the cathodic scan.30 Furthermore, products I and II readily oxidize compared to adenine, thus, their respective products are expected to appear at less positive potentials.14


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Scheme 1. Oxidation scheme of adenine (A) and guanine (B).

In a similar manner as adenine, the first oxidation of guanine is an irreversible 2-electron, 2-proton step (Scheme 1B). However, guanine only involves a 2-step process compared to the 3-step oxidation of adenine.31 Oxidation of guanine initially forms the 8-oxoguanine (product I, resulting from the oxidation of the imidazole moiety) which is then further oxidized to product II. The primary oxidation peak of guanine is around 0.75 V vs Ag/AgCl with slow-scan cyclic voltammetry. In FSCV, the primary oxidation peak of guanine is observed at 1.17 V vs Ag/AgCl on the forward scan (Figure 1E, blue trace). This indicates that guanine is easier to oxidize than adenine. The secondary oxidation product is observed immediately following 8-oxoguanine production at 0.80 V on the forward scan, similar to adenine (Figure S-1B). The extent to which functional groups on the purine ring modulate sensitivity, ET rate, and adsorption to CFME’s were investigated. Adenine (6-aminopurine) and guanine (2-amino, 6oxopurine) readily oxidizes at the CFME; however, they undergo slightly different oxidation mechanisms (Scheme 1) and interact uniquely with the carbon-fiber surface as depicted by the position of the primary oxidation peak and kinetics observed at the electrode. Furthermore, guanine generates more current than adenine at the same concentration. Because of these observations, the effect of amino and carbonyl functional groups, and their placement on the purine ring were compared. To do this, purine analogs containing an amino (adenine (A), 2aminopurine (2AP) and 2,6-diaminopurine (DAP)), carbonyl (hypoxanthine (HX) and xanthine (X))


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or both functional groups (guanine (G) and isoguanine (IG)) at varying substitution positions (Figure 1) were investigated using FSCV. Unsubstituted purine (P) was also tested to determine if the purine ring alone interacts with the CFME. Changes in electrode material could affect the interaction of purines with the surface, so all experiments were done at T-650 CFME’s. T-650 CFME’s have been shown to be highly disordered with surface oxide functionalities as evidenced by Raman Spectroscopy and XPS.23,32 Positive interactions at the electrode indicates favorable interactions with surface oxide modalities. The adenosine waveform (-0.4 to 1.45 V and back at a scan rate of 400 V/s) was used so that all analytes could be readily oxidized and directly compared.22 Previous literature has suggested that applying potentials greater than or equal to 1.3 V significantly change the chemical composition of the electrode by increasing the surface oxide functionalities6,23,33; therefore, it is expected that this waveform would facilitate a greater degree of disorder at the surface. Qualitative assessment of purine analog detection at CFME. Qualitative evaluation of purine analog detection at CFMEs revealed that amino groups on the purine are more favorable for interaction than carbonyl groups. The unsubstituted purine generated less than 10 nA of current indicating very little to no interaction with the carbon-fiber (Figure 1A). For substituted purines, amine substituted purines generated more current compared to the carbonyl functionalized analogs substituted at the same position as observed between A vs HX and DAP vs X. Adenine (6-aminopurine) produced almost 3-fold more current than HX (6-oxopurine) (Figure 1B and G, respectively) while DAP was about 5-fold higher than X (2,6-dioxopurine) (Figure 1D and H, respectively). This suggests that amino groups are more favorable than carbonyl groups for interactions with the CFME. This has also been observed for catecholamine interaction at CFMEs with FSCV.5,6 Furthermore, the number of amino substituents also affects its CFME interaction as illustrated by DAP having about 1.2- and 1.5-fold more current than A and 2AP, respectively. Although amino groups favor interaction with CFME, it is worth noting that carbonyl groups weakly interact with the oxides of the carbon-fiber. This is validated by HX and


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X generating about 3-fold more current than unsubstituted purine. A previous study also showed that both uric acid and ascorbic acid current increases at an overoxidized CFME surface, supporting that carbonyl groups can interact with surface oxides.6 Quantitative determination of actual sensitivity and surface coverage of each purine analog at CFME is discussed in a later section.

Figure 1. The type of functional group and its placement in the purine ring influence the shape of the CV and position of the oxidation peak. The adenosine waveform was applied for all purines (-0.4 V to 1.45 V and back at a rate of 400 V/s, 10 Hz). Backgroundsubtracted cyclic voltammograms of 5 M solutions of purine analogs revealed that unsubstituted purine (A) showed very little to no interaction with the electrode surface generating less than 10 nA of current. For substituted analogs, the primary oxidation peaks of 6-substituted purines (adenine (B) and hypoxanthine (G)) are on the back scan (red trace). For 2-substituted (2-aminopurine (C)) and disubstituted purines (2,6diaminopurine (D), guanine (E), isoguanine (F) and xanthine (H)), is the primary peaks are on the forward scan (blue trace). Structures are shown in the inset. Refer to Figure S-2 for the corresponding false color plots.

Oxidative peak position was analyzed as a function of substitution position. For 6substituted purines, it was observed that the primary oxidation peak is on the back scan as seen for A and HX (Figure 1B and G, respectively, red trace). However, for 2-substituted and 2,6-


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disubstituted purines, the primary oxidation peak is on the forward scan (Figure 1C-F and H, blue trace). The contrast in the position of the primary oxidation peak is likely attributed to their ET kinetics at the carbon-fiber surface. Slow and fast ET rates result in primary oxidation peaks on the back and forward scan, respectively. The rate of ET is dictated by the oxidation mechanism that these purine analogs undergo. The 6-substituted analogs follow a similar scheme as adenine (Scheme 1A), hence, results in a slow ET rate. Disubstituted analogs oxidize via guanine oxidation (Scheme 1B) and show faster ET kinetics. The 2-substituted analogs oxidize at the pyrimidine moiety similar to adenine,34 however, they have a lower oxidation potential in slowscan cyclic voltammetry35 which suggests a faster ET rate. Thus, the 2-substituted analog’s primary oxidation peak is on the forward scan in FSCV. Adsorption- vs diffusion-controlled oxidation of purine analogs at CFME. To assess whether electrochemical oxidation of purine analogs at CFME is facilitated by adsorption or diffusion, scan rates were varied from 50 to 1000 V/s while keeping the other parameters of the waveform constant. Current vs scan rate and current vs square root of scan rate plots were constructed (Figure S-3 and S-4, n=4, respectively). The r2 values of both plots for the purine analogs were very similar (Table S-1), making it difficult to ascertain adsorption from diffusion. In addition, current vs scan rate and square root of scan rate plots of P, HX and X showed a plateaued response at 500, 800 and 1000 V/s scan rates (Figure S-3A and D). This suggests that at 500 V/s the migration of these purine analogs between sites in the carbon-fiber surface has started to become kinetically limited. A similar result was observed with DA oxidation at conical carbon microelectrodes above 1000 V/s.36 Dopamine and ascorbic acid were used as controls to evaluate these results since they are known to undergo adsorption- and diffusion-controlled oxidation reactions, respectively.4,37 Calculated r2 values of 0.98 (DA) and 0.98 (AA) were obtained for the current vs scan rate plot while 0.99 (DA) and 1.00 (AA) for the square root plot (Table S-1, n=4). This illustrates that the traditional scan rate experiment can be difficult to interpret with FSCV since the r2 values are very close and do not differentiate adsorption- from


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diffusion-controlled oxidation. Log-log plots have previously been used to better analyze scan rate experiments with FSCV, so this approach was used to better analyze purine interactions at the electrode surface.11,36 A slope of 0.5 estimates a diffusion-controlled process while a slope of 1.0 predicts adsorption.38 A slope between 0.5 and 1.0 indicates a contribution of both processes.11 Evaluation of this method was also conducted using dopamine and ascorbic acid as a control. Slopes of 0.91 and 0.59 for dopamine and ascorbic acid, respectively (Figure S-5), indicates adsorption-limited for dopamine and diffusion-limited for ascorbic acid, as supported by literature.9,37 Therefore, the log-log plot is a better method for discerning adsorption- vs diffusioncontrolled reactions with FSCV.

Figure 2. The interaction of unsubstituted (A) and carbonyl substituted purines (D) with the CFME is purely diffusion-controlled as revealed by the slope of the log-log plots (m=0.5). For substituted purines with at least one amino group (B and C) the slope ranges from 0.89 to 0.93 indicating both adsorption- and diffusion-controlled interaction. The slopes were very close to 1.0 which suggests that adsorption is the dominant interaction. (n=4). See Table 1 for slope values. The slopes of the log-log plot showed that amine substituted purine analogs are a combination of adsorption and diffusion interactions while unsubstituted purine and carbonyl


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functionalized purine analogs are purely diffusion-controlled (Table 1). Purine, hypoxanthine and xanthine (Figure 2A and D) have slopes at approximately 0.5 indicating diffusion-controlled oxidation processes. This agrees with the previous discussion that purine has very little to no interaction with the CFME. For HX and X, the dipole-dipole repulsion between the carbonyl groups of the purine analogs and oxide groups of the carbon-fiber only allows diffusion interaction.22 However, possible hydrogen-bonding between functional groups can still occur. For substituted purines with at least one amine substituent, the slopes range from 0.84 to 0.93 (Figure 2C and D, Table 1, n=4). This demonstrates that both adsorption and diffusion interactions exist at the CFME; however, the slopes are closer to 1.0, indicating that adsorption is likely the dominating interaction at the electrode. Table 1. Analyte interaction with the CFME and its sensitivity with FSCV detection. sensitivityc slopea decay rateb DA --0.92  0.02 0.061  0.010 AA ----0.59  0.004 P ----0.41  0.06 A 0.84  0.03 0.072  0.012 14.63  0.09 2AP 0.85  0.02 0.062  0.011 10.99  0.58 DAP 0.92  0.03 0.070  0.010 24.90  0.27 G 0.92  0.04 0.069  0.011 16.77  0.14 IG 0.93  0.03 0.087  0.012 19.88  0.17 HX 0.44  0.06 0.036  0.020 6.78  0.56 X 0.55  0.08 0.033  0.015 6.43  0.11 aslopes from log-log plots (Figure 2, n=4) bnA/Hz, decay rates from frequency experiment (Figure 3, n=4) cnA/M (r2=1.0 for all analytes), slopes from concentration curve plots (Figure 4, n=6-10) Time for analyte adsorption is directly affected by the waveform application frequency. Increasing the frequency of waveform application results in a decrease in the preconcentration time for adsorption on the electrode surface resulting in a decrease in current. Analytes which adsorb on the electrode surface are more impacted by frequency than diffusion-controlled analytes.5,15 Current as a function of waveform application frequency were fit with an exponential decay and the decay rates were compared to assess the extent to which current depended on


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frequency (Figure 3). As a control, the adsorption-controlled analyte DA and the diffusioncontrolled analyte AA were used as standards for comparing decay rates (Figure S-6 and Table 1, n=4). DA current decreases as the applied frequency increases at a decay rate of 0.061 nA/Hz while increasing frequency had very little effect on AA current. The amine functionalized purines (A, 2AP, DAP, G and IG) showed a more rapid decrease in current as a function of waveform application frequency compared to the diffusion-controlled purines (HX and X) (Figure 3 and Table 1, n=4). Average decay rates of adsorption-controlled purines are approximately twice as fast as diffusion-controlled analogs. This supports the results from the scan rate experiment, suggesting that amine substituted purine analogs (A, 2AP, DAP, G and IG) are more governed by adsorption than carbonyl substituted analogs (HX and X).

Figure 3. Adsorption-controlled purine analogs (A and B) are more affected by waveform application frequency than diffusion-controlled purines (C). Decay rates for adsorption-controlled purines (A and B) are twice as fast as diffusion-controlled purines (C). See Table 1 for decay rates. (n=4). Sensitivity of purine analogs with FSCV. The kinetic interactions at the electrode surface can impact the apparent sensitivity of the analyte. Sensitivity as a function of the substituent and placement on the ring was investigated. Unsubstituted purine generated less than 10 nA of current, hence, its sensitivity with FSCV was not examined. To determine sensitivity for


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each purine, current as a function of analyte concentration was tested (Figure 4). All purine analogs except hypoxanthine (linear range to 100 µM) were linear in the range of 50 or 100 nM to 10 µM. The slope of the linear region of the curve indicates the sensitivity and is expressed in nA/µM (Table 1). The highest slope and therefore, the analyte which has the greatest sensitivity with FSCV was DAP (Table 1). DAP has two amine substituents allowing for a stronger interaction with the carbon-fiber. The slopes also indicate that FSCV is about 1.4 times more sensitive to G and IG than A and 2AP. This is likely because there are two sites for interaction for G and IG. This further supports that the carbonyl groups do interact with the oxides of the carbon-fiber albeit weak.6 The technique is least sensitive to HX and X which both only have carbonyl groups, hence, less favorable interaction with CFME. These results show that FSCV is more sensitive to amine substituted purine analogs especially in the presence of more than one amino group. FSCV is least sensitive to carbonyl functionalized purines regardless of the number of carbonyl substituents. Langmuir adsorption isotherms of the purine analogs were constructed in order to further understand the interaction of purine analogs with the carbon-fiber surface.

Figure 4. FSCV is more sensitive to amine substituted purines (A and B) than carbonyl substituted purines (C and D). Within amine substituted analogs, FSCV is more sensitive to disubstituted purines than monosubstituted purines (G vs 2AP and IG vs A) which is likely because disubstituted purines have two sites for interaction with the CFME. FSCV is most sensitive to DAP


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which has two amino functional groups, hence, two sites for electrostatic interaction. (n=6-10). See Table 1 for slope values. Langmuir adsorption isotherm. A Langmuir adsorption isotherm was used to further investigate the interaction of adsorption-controlled purine analogs (A, 2AP, DAP, G and IG) with the electrode surface at equilibrium (Figure 5). HX and X are diffusion-controlled; hence, both are not expected to cover the electrode surface with uniform monolayers, and were not considered in the adsorption isotherm. The catecholamine DA was also investigated at the traditional DA waveform (-0.4 to 1.3 V and back at 400 V/s, Figure S-7) to serve as a control for the analysis.39–42 The diffusion component is sometimes subtracted prior to analyzing the Langmuir isotherm; however, the diffusion component is minor and estimation of adsorption interaction can still be made.6,24,26 Our control, DA, had a saturation surface coverage (s) of 39.7 pmol/cm2 which is in the same magnitude as prior reports for DA using the same waveform.5,6 This shows that our current model is a good approximation of the adsorption isotherm.

Figure 5. Adsorption-controlled purine analogs form a uniform monolayer on the carbon-fiber surface. Disubstituted purines showed stronger adsorption on the electrode surface than monosubstituted purines, especially for DAP. DAP is about 1.35 and 2.40 fold stronger than G and IG, and A and 2AP, respectively. (n=6-10). See Table 2 for calculated adsorption strength (b), equilibrium constant () and saturation coverage (s). Adsorption isotherms revealed that disubstituted purine analogs (DAP, G and IG) have about 3-fold more surface coverage than monosubstituted purines (A and 2AP) (Table 2). This is likely due to the two sites of interaction in disubstituted purines and further supports that hydrogenbonding interaction between the carbonyl functional groups of purines and oxide groups of the


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carbon-fiber exists. Among disubstituted purines, the adsorption of DAP (3.35  10-4 cm) is about 1.35-fold stronger than G and IG (2.28  10-4 and 2.68  10-4 cm, respectively). The relatively stronger interaction of DAP with CFME is due to the presence of two amine substituents that allow two sites for electrostatic interaction.22,26 This is also why DAP adsorbs on the electrode about 2.40 times stronger than monoamine substituted purines (A and 2AP, 1.31  10-4 and 1.48  10-4 cm, respectively). However, dopamine covers the electrode surface 5.64-fold more with an estimated adsorption strength 2.12-fold higher than purines (Table 2). This is likely due to its small molecular size, packing more dopamine on the electrode surface. The thermodynamic equilibrium constant () for dopamine adsorption is 3.62-fold less than purine analogs. This suggests a relatively faster desorption rate of dopamine than purines. The slow desorption rates of purine analogs are likely attributed to the cascading oxidation reactions (Scheme 1) following its primary oxidation. Table 2. Langmuir adsorption isotherm parametersa (n=4-10). b* s+ ‡ A 1.31 4.99 3.92 2AP 1.48 4.53 2.93 DAP 3.35 15.1 4.84 G 2.28 14.6 3.76 IG 2.68 14.6 3.96 DAb 4.70 39.7 1.07 * -4 + 2 units:  10 cm, pmol/cm , and ‡ 10-2 cm3/pmol aadsorption isotherm is determined using adenosine waveform unless otherwise stated badsorption isotherm is determined using dopamine waveform The adsorption isotherm revealed that amino functional groups promote the interaction of purine analogs with CFME. Disubstituted purines functionalized with both amino and carbonyl groups showed stronger interaction with the electrode surface as indicated by the higher b values than the monoamine substituted analogs (G vs 2AP and IG vs A). In addition, diamine substituted purine (DAP) has a stronger adsorption on the carbon-fiber surface since there are two sites of electrostatic interaction and thus, has the highest s among purine analogs. These results


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indicated that the functional group type and placement indeed modulate the interaction of purine analogs with the carbon-fiber surface. Purine nucleobases, nucleosides and nucleotides detection with FSCV. The purine bases and analogs are not usually investigated with FSCV since they do not exhibit a rapid mode of signaling in the brain. The nucleosides and nucleotides are typically the target of investigation due to their rich signaling modalities in the brain. Therefore, it is important to identify how the ribose and triphosphate groups affect purine analog-CFME interaction. Unwrapped CVs of the purine nucleobases, nucleosides and nucleotides are plotted as current (nA) vs time (msec) for better visualization of the peak position (Figure 6). The primary oxidation peak of adenine (A) appears at 4.77 msec (denoted by the dotted line on Figure 6A). The primary peak is delayed on average by 272  20.6 sec when a ribose (ribofuranose) is attached to adenine via -N9glycosidic bond yielding adenosine (AD) and resulted in a 70.3  1.3% decrease in current (Figure 6A and C, n=5). The 272 sec-shift is likely due to the steric effect that the ribose ring imposes towards the adsorption of AD onto the CFME, hence, slower response time and a decrease in oxidative current.25 When an additional triphosphate group is bonded at the 5’-position of the ribose forming adenosine triphosphate (ATP), the oxidation peak remained in the same position with an additional 36.2  1.8% decrease in current on average (Figure E, n=5). The decrease in current could be attributed to the higher molecular weight of ATP, thus, slower diffusion to the CFME and because ATP is negatively charged which decreases the electrostatic interaction at the electrode.15,22


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Figure 6. Ribose and triphosphate groups decrease the oxidative current and delay the response time at the electrode. Example background-subtracted unwrapped CVs of 5 M solutions of purine nucleobases (adenine (A) and guanine (B)), nucleosides (adenosine (C) and guanosine (D)) and nucleotides (adenosine triphosphate (E) and guanosine triphosphate (F)) are shown for better visualization of peak position. Dotted lines denote the position of the primary oxidation peak. Ribose and triphosphate groups also cause a decrease in oxidative current and a delay in response time for guanosine (GN) and guanosine triphosphate (GTP) compared to guanine (G). The presence of a ribose group in GN lowered the primary oxidative current of G by about 15.5  1.8% with a delay of 462  4.9 sec in response time (Figure 6B and D, n=5). This is likely due to the steric effect from the ribose group. The larger delay in response time of GN compared to AD is likely due to the intramolecular hydrogen-bonding between the NH2 group of G moiety and the OH group of the ribose at the 5’-position. As a result, this hinders the adsorption of GN on the electrode and slows down the interaction.43 The presence of triphosphate groups in GTP had little effect on the response time with respect to GN (Figure 6E), similar to AD vs ATP. However, an


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additional 57.9  1.8% decrease in oxidative current was observed (n=5). G oxidizes at the imidazole moiety (Scheme 1B) and the triphosphate groups (highly electronegative) along with the ribose group inductively withdraws the electron density of the imidazole moiety.44 This makes the oxidation of G more difficult resulting in the decrease of oxidative current for GTP. Furthermore, GTP is negatively charged which prompts electrostatic repulsion with the oxides of the CFME, similar to ATP. The oxidation mechanism of the nucleosides and nucleotides are dictated by its purine moiety. However, it appears that the ET kinetics and interaction with the CFME is greatly affected by the ribose and triphosphate groups. Hence, it is important to consider the extent of how ribose and triphosphate groups affect the interaction of purine nucleosides and nucleotides with the CFME when designing new sensors.

CONCLUSIONS We show that functional group type and position on the purine ring modulate sensitivity, ET kinetics and adsorption on CFME with FSCV. Unsubstituted purine has very little to no oxidative current at the CFME which provides additional evidence that functional groups are needed for interaction at the electrode surface. Interactions with the carbon-fiber are stronger with amine functionalized purines than carbonyl functionalized purines substituted at the same position as demonstrated by A vs HX and DAP vs X. Disubstituted purines functionalized with both an amino and carbonyl group (G and IG) interacted more strongly with the electrode surface than monosubstituted purines with a single amino (A and 2AP) or carbonyl (HX).

Additionally,

disubstituted purines adsorbed more strongly on the carbon-fiber surface than monoamine substituted purines. The ribose groups, as in the nucleosides, decrease the observed oxidative current and delay the response time. A further decrease in oxidative current is observed in the presence of additional triphosphate groups (nucleotides). These results demonstrate the effect that functional group type and placement has on the interaction of purine analogs (bases, nucleosides and nucleotides) with the CFME. This paper provides a conceptual advancement in


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our understanding of purine interactions at bare carbon-fiber and could be specifically informative for designing modified carbon-fiber electrodes or selecting new sensor materials for purine detection with FSCV. Treatments to either increase surface oxide groups or introduce new functionalities onto the electrode could be beneficial depending on the purine of interest.

Supporting Information: The Supporting Information is available free of charge on the ACS Publications website. Color plots of adenine and guanine with corresponding CVs at specific times to show order oxidation peaks, color plots of purine analogs corresponding to CVs in Figure 1, current as a function of scan rate plots for purine analogs, current as a function of the square root of scan rate plots for purine analogs, log-log plots of current vs. scan rate for dopamine and ascorbic acid, current as a function of increasing frequency of waveform application for dopamine and ascorbic acid, Langmuir adsorption isotherm for dopamine, table showing r2 values from adsorption and diffusion plots, and supplemental methods on carbon-fiber microelectrode fabrication.

ACKNOWLEDGEMENTS The authors would like to acknowledge the chemistry department at the University of Cincinnati for funding this work.

CONFLICTS OF INTEREST There are no conflicts of interest to declare for this work.


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Purine functional group type and placement modulate the interaction

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