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Second-Derivative-Based Background Drift Removal for a Tonic Dopamine Measurement in Fast-Scan Cyclic Voltammetry
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  • Seongtak Kang
    Seongtak Kang
    Department of Electrical Engineering and Computer Science, Daegu Gyeongbuk Institute of Science and Technology (DGIST), 333, Techno jungang-daero, Hyeonpung-myeon, Dalseong-gun, Daegu 42988, Republic of Korea
  • Jeongrak Park
    Jeongrak Park
    Department of Brain and Cognitive Science, DGIST, 333, Techno jungang-daero, Hyeonpung-myeon, Dalseong-gun, Daegu 42988, Republic of Korea
  • Yunho Jeong
    Yunho Jeong
    College of Transdisciplinary studies, DGIST, 333, Techno jungang-daero, Hyeonpung-myeon, Dalseong-gun, Daegu 42988, Republic of Korea
    More by Yunho Jeong
  • Yong-Seok Oh
    Yong-Seok Oh
    Department of Brain and Cognitive Science, DGIST, 333, Techno jungang-daero, Hyeonpung-myeon, Dalseong-gun, Daegu 42988, Republic of Korea
    More by Yong-Seok Oh
  • Ji-Woong Choi*
    Ji-Woong Choi
    Department of Electrical Engineering and Computer Science, Daegu Gyeongbuk Institute of Science and Technology (DGIST), 333, Techno jungang-daero, Hyeonpung-myeon, Dalseong-gun, Daegu 42988, Republic of Korea
    Brain Engineering Convergence Research Center, DGIST, 333, Techno jungang-daero, Hyeonpung-myeon, Dalseong-gun, Daegu 42988, Republic of Korea
    *Email: [email protected]
Open PDFSupporting Information (1)

Analytical Chemistry

Cite this: Anal. Chem. 2022, 94, 33, 11459–11463
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https://doi.org/10.1021/acs.analchem.2c01047
Published August 8, 2022

Copyright © 2022 American Chemical Society. This publication is available under these Terms of Use.

Abstract

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The dysregulation of dopamine, a neuromodulator, is associated with a broad spectrum of brain disorders, including Parkinson’s disease, addiction, and schizophrenia. Quantitative measurements of dopamine are essential for understanding dopamine functional dynamics. Fast-scan cyclic voltammetry (FSCV) is the most popular electrochemical technique for measuring real-time in vivo dopamine level changes. Standard FSCV has only analyzed “phasic dopamine” (changes in seconds) because the gradual generation of background charging current is inevitable and is the primary noise source in the low-frequency band. Although “tonic dopamine” (changes in minutes to hours) is critical for understanding the dopamine system, an electrochemical technique capable of simultaneously measuring phasic and tonic dopamine in an in vivo environment has not been established. Several modified voltammetric techniques have been developed for measuring tonic dopamine; however, the sampling rates (0.1–0.05 Hz) are too low to be useful. Further investigation of the in vivo applicability of previously developed background drift removal methods for measuring tonic dopamine levels is required. We developed a second-derivative-based background removal (SDBR) method for simultaneously measuring phasic and tonic neurotransmitter levels in real-time. The performance of this technique was tested via in silico and in vitro tonic dopamine experiments. Furthermore, its applicability was tested in vivo. SDBR is a simple, robust, postprocessing technique that can extract tonic neurotransmitter levels from all FSCV data. As SDBR is calculated in individual-scan voltammogram units, it can be applied to any real-time closed-loop system that uses a neurotransmitter as a biomarker.

This publication is licensed for personal use by The American Chemical Society.

Copyright © 2022 American Chemical Society
Dopamine is a neuromodulator that conveys important information such as cognition, reward and pleasure, and voluntary motor movements. (1−4) Dysregulation of the dopamine system is associated with a broad spectrum of brain disorders such as Parkinson’s disease, addiction, and schizophrenia. (3,5,6) Dopamine levels in the target areas of the brain display highly dynamic changes, with fluctuations on different time scales. (7,8) These changes include rapid transients, which are ramps that may last for several seconds (phasic) and oscillations on the time scale of minutes to hours (tonic). (4,9) Quantitative analysis of dopamine levels is crucial for learning about the functional role of dopamine dynamics in a normal brain as well as studying brain-disorder pathology in preclinical and clinical studies. (10,11)
Fast-scan cyclic voltammetry (FSCV) involving a carbon fiber microelectrode (CFM) is a well-established electrochemical technique that can effectively measure dopamine-level changes in the brain. (12−17) FSCV measures faradaic current changes based on the dopamine oxidation peak voltage exhibited in a voltammogram, after subtracting the background current. (18,19) The high scan rate of FSCV is sensitive enough to measure rapid changes in dopamine levels (phasic dopamine); however, it also generates a progressively large background charging current (capacitive current), making it difficult to analyze voltammetry beyond 2 min. (15,20) The steady rise in the amplitude of the dopamine peak in FSCV due to this background charging current is called background drift. FSCV background drift makes it difficult to measure slow changes in dopamine levels (tonic dopamine). (21−23)
Attempts to measure tonic dopamine levels in the brain in real-time are still challenging. Modified voltammetric techniques have been proposed for measuring tonic dopamine in vivo. (24−27) These modified voltammetric techniques measure tonic dopamine levels; however, the low temporal resolution (10–20 s) makes it difficult to analyze detailed dopamine signaling for understanding neuropsychiatric disorders. The high-pass filtering technique can measure phasic dopamine with background drift removed, but also removes tonic dopamine levels, which have a similar frequency band to that of the background drift. (23) Recently, tonic dopamine measurement using background drift removal was attempted using modified FSCV. (28,29) These methods attempted to estimate the background with an additional voltage waveform; however, estimating the background of the in vivo system with an electrode-specific training set still needs further investigation. An integration-based method estimates tonic dopamine by integrating around the dopamine peak of the background-subtracted voltammogram. (30) In this integration-based method, the user must set the integration potential range, causing potential bias in the estimated dopamine level, which can lead to inadequate estimates of tonic dopamine levels.
In this study, we developed a second-derivative-based background drift removal (SDBR) method for measuring phasic and tonic dopamine levels using standard FSCV. SDBR extracts tonic dopamine information by applying a second derivative from the dopamine oxidation peaks in the background-subtracted voltammogram. The efficacy of SDBR has been tested from in silico to in vitro and in vivo.

Materials and Methods

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Data Acquisition and Analysis

CFM and Ag/AgCl reference electrodes (Pinnacle Technology Inc., Lawrence, KS) were used for FSCV data acquisition. Voltametric scans were electrochemically performed using a triangular waveform that ranged from −0.4 to +1.3 V, with a scan rate of 400 V/s and a waveform frequency of 10 Hz. The data acquisition was performed using High Definition Cyclic Voltammetry software (HDCV, University of North Carolina at Chapel Hill) in conjunction with a WaveNeuro FSCV system (Pine Research Instrumentation, Durham, NC). (31) All data were analyzed in MATLAB R2020b (Mathworks, Natick, MA).

In Vitro Experiment

CFM and Ag/AgCl reference electrodes were placed in a beaker filled with 0.05 M phosphate-buffered saline (PBS) with a pH of 7.4. All experimental procedures were performed in Faraday cages to ensure environmental stability. The performance verification, including sensitivity, and selectivity tests were conducted through in vitro experiments. Figure S1 describes the selectivity test. After each drop of dopamine (dopamine hydrochloride, Sigma-Aldrich) solution, it was mixed with a stirrer for 2 min. After mixing, the power supply of the stirrer was turned off to eliminate noise.

Surgery and In Vivo Dopamine Measurements

Adult C57BL/6J 35 g male mice were purchased from Charles River Laboratories (Yokohama, Japan) and used for the in vivo experiments. All animal care and experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at Daegu Gyeongbuk Institute of Science and Technology (DGIST-IACUC-21091701–0003). To evaluate the effectiveness of SDBR in vivo, tonic dopamine levels in the striatum of healthy and Parkinsonian mice were measured for 60–120 min using standard FSCV. In Parkinsonian/healthy mice, 10–20 min after baseline recording, levodopa/reserpine (Sigma-Aldrich) was intraperitoneally injected, respectively. The surgical details are provided in the Supporting Information.

Second-Derivative-Based Background Drift Removal (SDBR) Method

We modeled the background-subtracted voltammogram around the dopamine oxidation peak generated for each scan based on the following characteristics. First, the voltammogram near the dopamine oxidation peak after background subtraction is symmetrical and has a Gaussian shape. (15) Second, the amplitude current of the dopamine oxidation peak of the background-subtracted voltammogram has a linear correlation with the dopamine concentration. (15,32) Third, we assumed that the background charging current generated in a narrow range around the dopamine oxidation peak (dopamine oxidation peak voltage ±40 mV) varies with time, but is independent of voltage. Furthermore, we modeled a background-subtracted voltammogram at specific scan times (t) and voltages (V) around the dopamine oxidation peak voltage (eq 1).
(1)
where VoltgramBS denotes a background-subtracted voltammogram, peakv is the dopamine oxidation peak, ConcDA is the dopamine concentration, and Chargc is the background charging current. If V is set to peakv to observe the current of the dopamine oxidation peak of the voltammogram:
(2)
In eq 2, which represents the current of peakv in general background-subtracted voltammogram, the Chargc remains constant. We eliminated the Chargc in the proposed model and quantified the intrinsic curvature of the dopamine oxidation peak by applying the second derivative to each background-subtracted voltammogram (eq 3).
(3)
where VoltgramSDBR denotes the SDBR-applied voltammogram. If we observe the dopamine peak current after the second derivative of the modeled voltammogram by setting V to peakv:
(4)
Note that this second derivative, the tonic dopamine level, can be obtained irrespective of the time-varying charging current level. Additional details are provided in the Supporting Information (Figure S2). The dopamine oxidation peak voltage of each sensor was defined as the voltage with a maximum SDBR value in the range of 0.4 to 0.7 V in the in vitro test. Because the reduction peak voltage has lower sensitivity than the oxidation peak voltage, it was only used to identify the presence of dopamine during phasic dopamine signaling in vivo experiments (Figure S3). To improve the signal-to-noise ratio of the SDBR result, the SDBR values of the five voltage channels adjacent to the dopamine peak voltage were averaged.

Results and Discussion

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SDBR In Vitro Experiments

It was confirmed in vitro that SDBR can remove the capacitive current, which is the main noise source in the conventional background subtraction method. Figure 1 shows the in vitro test results of the background subtraction method and the SDBR. Dopamine (200 nM) was dropped into the PBS solution every 20 min and stirred for 2 min. Standard FSCV measures the faradaic current caused by dopamine around the CFM, and the capacitive current change is gradually generated owing to the high scan rate (Figure 1A). In previous studies, a background subtraction technique was applied to observe phasic dopamine levels (Figure 1B).

Figure 1

Figure 1. In vitro test of SDBR to record the tonic and phasic dopamine with standard FSCV. (A) Raw FSCV color plot in vitro test. The black dotted line denotes the timing of the 200 nM dopamine drop. Each circled number and the corresponding colored-line are the voltammogram at a specific time, detailed in (E)–(H). (B) Background-subtracted color plot and current change of dopamine peak over time. (C) SDBR applied color plot and current change of dopamine peak over time. (D) A calibration plot obtained by SDBR (R2 = 0.996), slope = 0.0319 ± 0.0003 pA/V2 nM–1, limit of detection = 8.16 ± 0.08 nM (n = 5 electrodes). (E, F) Comparison of background subtraction and SDBR for three voltammograms measuring the same concentration at 2 min intervals. (G, H) Three voltammograms were measured 10 min after each drop of dopamine solution for comparison of background subtraction and SDBR. (E, G) Left: the background-subtracted voltammogram, right: the enlarged voltammogram of the red dotted box in the left image. (F, H) Left: the result of applying SDBR to the raw voltammogram, right: the enlarged SDBR result of the blue dotted box in the left image.

However, capacitive currents that cause a continuous current rise, even at the same dopamine concentration, make it difficult to analyze changes in tonic dopamine levels. This continuous increase in the capacitive current with time is described by eq 2. In contrast, SDBR results were flat with similar values at the same concentration during the 1 h experiment without any background drift (Figure 1C, eq 4). Figure 1D shows the SDBR calibration plot. The SDBR signal correlated with tonic dopamine concentrations (62.5–1000 nM; n = 5 electrodes; R2 = 0.996).
The limit of detection was 8.16 ± 0.08 nM, which is sufficient for dopamine measurements in vivo. Figure 1E shows the voltammogram changes at 2 min intervals (①, ②, and ③ in Figure 1A) for the same dopamine concentration (200 nM). The voltammograms at 2 min intervals had a similar shape near the dopamine oxidation peak, but the amplitude steadily increased owing to the capacitive current (Figure 1E). Despite time passing, the SDBR values of the dopamine oxidation peak were similar when they had the same concentration (Figure 1F). Figure 1G shows the voltammograms 10 min after each drop of 200 nM dopamine (different concentrations of dopamine) (①, ④, and ⑤, shown in Figure 1A). The peak currents expressed in the three background-subtracted voltammograms were not linearly correlated to the dopamine concentration because the capacitive current contaminated them. SDBR linearly expressed three different dopamine level changes in dopamine oxidation peak (Figure 1H).

SDBR In Vivo Experiments

It was confirmed through in vitro experiments that SDBR can measure changes in tonic dopamine levels without any background drift. To confirm the practicality of SDBR in the in vivo environment, the FSCV results were measured in the striatum of normal mice and the Parkinson’s disease (PD) model (6-OHDA) mice following levodopa and reserpine intraperitoneal injection (Figure 2). When saline solution was intraperitoneal (IP) injected into healthy mice (n = 3), the estimated change in dopamine concentration was maintained within 10 nM for 120 min (Figure 2A). Figure 2B illustrates the measured dopamine concentration changes in the striatum after injecting levodopa into PD model mice (n = 3). Tonic dopamine levels maintained below 5 nM for 20 min and increased to almost 60 nM after levodopa IP injection.

Figure 2

Figure 2. Measurement of tonic dopamine level change using SDBR during in vivo pharmacological stimulation. (A) SDBR results were measured in the striatum of healthy mice (n = 3) following saline intraperitoneal (IP) injection. (B) SDBR measured in the striatum of PD model mice (n = 3) following levodopa (10 mg kg–1) IP injection. (C) SDBR measured in the striatum of healthy mice (n = 3) following reserpine (5 mg kg–1) IP injection. Bold blue line represents mean concentrations over time, and the light blue line around the bold line represents the standard error of the mean (SEM). The upper black bar indicates significant differences compared to saline injection (2-way ANOVA, p < 0.0001, Dunnett’s multiple comparison test). Data from Figures 1 and 2 are freely available online. (36)

Additionally, we injected reserpine, which is a vesicular monoamine transporter (VMAT) inhibitor into healthy mice (n = 3) to observe tonic dopamine levels decreasing (Figure 2C). Before reserpine injection, tonic dopamine level changes remained near 0 nM, and then dropped to close to 60 nM after injection. All experiments showed statistically significant changes in tonic dopamine levels after IP injection (levodopa, reserpine). Three types of in vivo experiments showed that SDBR can reliably extract changes in tonic dopamine concentration in vivo.
As a summary, a comparison between SDBR and the tonic dopamine measurement methods described in this paper is summarized in Table 1. Using SDBR, we can simultaneously acquire phasic and tonic dopamine because SDBR extracts tonic information from the FSCV data primarily for measuring phasic dopamine. SDBR is applicable to real-time systems because it has a short latency (approximately 4 μs for a single voltammogram with 850 data points). Also, since SDBR uses standard FSCV as it is, it has the versatility to extract tonic dopamine information from all FSCV data measured with standard FSCV. Although the sensitivity (limit of detection) of SDBR to measure tonic dopamine level is not superior to other techniques in Table 1, it is enough to measure changes in tonic dopamine levels in vivo. Also, since SDBR uses standard FSCV, it still has a high sensitivity to phasic dopamine signals.
Table 1. Comparison between SDBR and the Tonic Dopamine Measurement Methods Using FSCVa
methodtemporal resolutionsimultaneous availability of phasic dopamineneed to modify the waveform of standard FSCV?limit of detection (nM)ref
FSCAV20 spartiallyyes3.7 ± 0.5 (24)
CBM-FSCV10 snoyes5.8 ± 0.9 (26)
convolution-based current removal1 syesyes<40 (28)
M-CSWV10 snoyes0.17 ± 0.03 (25)
SWV15 snoyes2.03 ± 0.09 (27)
SDBR0.1 syesno8.16 ± 0.08proposed
a

Some of the contents of Table 1 referred to the previously reported review article. (3)

Conclusions

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SDBR is a novel technology that extracts tonic dopamine level changes while maintaining the high temporal resolution of FSCV. This is achieved by applying the second derivative to the voltammogram measured with standard FSCV. Simultaneous measurement of phasic dopamine by FSCV and extracted tonic dopamine through SDBR will contribute to a better understanding of all dopamine systems and brain diseases related to dopamine signaling. SDBR effectively extracts tonic dopamine information by applying a simple second-derivative operation to individual voltammograms. SDBR has the potential to be applied to real-time closed-loop therapy systems that use dopamine levels as a biomarker. (33−35) In addition, because SBDR is a postprocessing technology that does not require any modification to the standard FSCV system, it can be universally applied to existing FSCV measurement data. SBDR will provide additional tonic-level changes that can be used to accelerate advances in FSCV-related research.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.analchem.2c01047.

  • Author contributions, selectivity test, surgery, and in vivo dopamine measurement experiment details and in silico test (PDF)

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Author Information

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  • Corresponding Author
    • Ji-Woong Choi - Department of Electrical Engineering and Computer Science, Daegu Gyeongbuk Institute of Science and Technology (DGIST), 333, Techno jungang-daero, Hyeonpung-myeon, Dalseong-gun, Daegu 42988, Republic of KoreaBrain Engineering Convergence Research Center, DGIST, 333, Techno jungang-daero, Hyeonpung-myeon, Dalseong-gun, Daegu 42988, Republic of KoreaOrcidhttps://orcid.org/0000-0001-9109-3860 Email: [email protected]
  • Authors
    • Seongtak Kang - Department of Electrical Engineering and Computer Science, Daegu Gyeongbuk Institute of Science and Technology (DGIST), 333, Techno jungang-daero, Hyeonpung-myeon, Dalseong-gun, Daegu 42988, Republic of KoreaOrcidhttps://orcid.org/0000-0002-6635-4819
    • Jeongrak Park - Department of Brain and Cognitive Science, DGIST, 333, Techno jungang-daero, Hyeonpung-myeon, Dalseong-gun, Daegu 42988, Republic of Korea
    • Yunho Jeong - College of Transdisciplinary studies, DGIST, 333, Techno jungang-daero, Hyeonpung-myeon, Dalseong-gun, Daegu 42988, Republic of Korea
    • Yong-Seok Oh - Department of Brain and Cognitive Science, DGIST, 333, Techno jungang-daero, Hyeonpung-myeon, Dalseong-gun, Daegu 42988, Republic of Korea
  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF), funded by the Korean government (MSIT; No. 2017M3A9G8084463).

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  1. Mason L. Perillo, Bhavna Gupta, Akash Saxena, Alexandra P. Veltri, Wen Li, James R. Siegenthaler, Erin K. Purcell. Biological and Mechanical Limitations for Chronic Fast‐Scan Cyclic Voltammetry Sensor Design. Advanced Materials Technologies 2025, 51 https://doi.org/10.1002/admt.202401808
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  3. Jeongrak Park, Seongtak Kang, Yaebin Lee, Ji-Woong Choi, Yong-Seok Oh. Continuous long-range measurement of tonic dopamine with advanced FSCV for pharmacodynamic analysis of levodopa-induced dyskinesia in Parkinson’s disease. Frontiers in Bioengineering and Biotechnology 2024, 12 https://doi.org/10.3389/fbioe.2024.1335474
  4. Han Hee Jung, Jeongdae Ha, Jeongrak Park, Seongtak Kang, Jinmo Kim, Han Na Jung, Samhwan Kim, Junwoo Yea, Hyeokjun Lee, Saehyuck Oh, Janghwan Jekal, Soojeong Song, Jieun Son, Tae Sang Yu, Youngjeon Lee, Jinyoung Won, Kyung Seob Lim, Yoon Kyeung Lee, Hohyun Keum, Taeyoon Lee, Young Min Song, Jae‐Woong Jeong, Jong‐Cheol Rah, Ji‐Woong Choi, Sheng Xu, Yong‐Seok Oh, Kyung‐In Jang. Highly Deformable Double‐Sided Neural Probe with All‐in‐One Electrode System for Real‐Time In Vivo Detection of Dopamine for Parkinson's Disease. Advanced Functional Materials 2024, 34 (4) https://doi.org/10.1002/adfm.202311436
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Analytical Chemistry

Cite this: Anal. Chem. 2022, 94, 33, 11459–11463
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https://doi.org/10.1021/acs.analchem.2c01047
Published August 8, 2022

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  • Abstract

    Figure 1

    Figure 1. In vitro test of SDBR to record the tonic and phasic dopamine with standard FSCV. (A) Raw FSCV color plot in vitro test. The black dotted line denotes the timing of the 200 nM dopamine drop. Each circled number and the corresponding colored-line are the voltammogram at a specific time, detailed in (E)–(H). (B) Background-subtracted color plot and current change of dopamine peak over time. (C) SDBR applied color plot and current change of dopamine peak over time. (D) A calibration plot obtained by SDBR (R2 = 0.996), slope = 0.0319 ± 0.0003 pA/V2 nM–1, limit of detection = 8.16 ± 0.08 nM (n = 5 electrodes). (E, F) Comparison of background subtraction and SDBR for three voltammograms measuring the same concentration at 2 min intervals. (G, H) Three voltammograms were measured 10 min after each drop of dopamine solution for comparison of background subtraction and SDBR. (E, G) Left: the background-subtracted voltammogram, right: the enlarged voltammogram of the red dotted box in the left image. (F, H) Left: the result of applying SDBR to the raw voltammogram, right: the enlarged SDBR result of the blue dotted box in the left image.

    Figure 2

    Figure 2. Measurement of tonic dopamine level change using SDBR during in vivo pharmacological stimulation. (A) SDBR results were measured in the striatum of healthy mice (n = 3) following saline intraperitoneal (IP) injection. (B) SDBR measured in the striatum of PD model mice (n = 3) following levodopa (10 mg kg–1) IP injection. (C) SDBR measured in the striatum of healthy mice (n = 3) following reserpine (5 mg kg–1) IP injection. Bold blue line represents mean concentrations over time, and the light blue line around the bold line represents the standard error of the mean (SEM). The upper black bar indicates significant differences compared to saline injection (2-way ANOVA, p < 0.0001, Dunnett’s multiple comparison test). Data from Figures 1 and 2 are freely available online. (36)

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    • Author contributions, selectivity test, surgery, and in vivo dopamine measurement experiment details and in silico test (PDF)


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