Simultaneous detection of dopamine and ascorbic acid by a thread-based microfluidic device and multiple pulse amperometry

doi:10.21203/rs.3.rs-2114017/v1

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Simultaneous detection of dopamine and ascorbic acid by a thread-based microfluidic device and multiple pulse amperometry

https://doi.org/10.21203/rs.3.rs-2114017/v1

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In this work, the simultaneous determination of ascorbic acid (AA) and dopamine (DA) was proposed using a simple and inexpensive microfluidic device. Microfluidic devices are totally suitable to couple with electrochemical detectors such as screen-printed electrodes (SPE) and offer many advantages: portability, high sample throughput and low reagent consumption. Here, a 3D-printed microfluidic device (µTED) was developed using textile threads as microfluidic channels and an unmodified SPE as amperometric detector. By using the multiple pulse amperometry (MPA), two potential values (0.65 V and − 0.10 V) were chosen. The current signals generated in the reduction of dopamine o-quinone were used to calculate a correction factor for the ascorbic acid oxidation signals, allowing the simultaneous quantification. The proposed microfluidic device provided a stable flowrate of carrier solution and low consumption of samples and reagents (volume of injection of 2.0 µL). Using the best set of experimental conditions, a linear dynamic range (LDR) between 50 and 900 µmol L^− 1 for DA and AA were achieved. Sensitivities of 2.24 µA L µmol^− 1 and 5.09 µA L µmol^− 1 as well as limits of detection (LOD) of 2.60 µmol L^− 1 and 1.54 µmol L^− 1 were calculated for AA and DA, respectively. The proposed method was successfully applied for simultaneous determination of AA and DA in commercial blood serum sample spiked at three concentration levels. In addition, MPA showed to be a very simple way to perform simultaneous determination of AA and DA without the need of previous separations or the use of chemically modified electrodes.

Dopamine

Ascorbic acid

Simultaneous determination

Microfluidic device

Multiple pulse amperometry

Diagnostic techniques research mainly focus on accurate and faster responses, as new diseases and pathological agents emerges, such characteristics are essential to provide crucial answers and define the best treatment for the patient. Developed countries performs clinical trials in well-equipped laboratories, which is not the case in most of developing countries (Choi 2016). Thus, more practical procedures and the possibility of performing clinical trials in remote places is a growing demand in recent years. The laboratory feasibility to reach the sample provides faster results, as well as lower risk of contamination, greater practicality, lower cost and ease of constant monitoring of patients (Choi 2016; Hu et al. 2016).

In this regard, microfluidic analysis devices fit all these characteristics and shows an increasingly use on analytical applications due to its portability, low consumption of reagents and samples, and reliability of results (Ochiai et al. 2017). Traditionally, microfluidic platforms are buildup based on silicon and glass (Sugioka et al. 2014; Wang et al. 2016) or polymers elastomers and thermoplastics (Wu et al. 2009; Ogończyk et al. 2010; Young et al. 2011; Kunstmann-Olsen et al. 2016). Some alternatives are proposed in order to reduce costs and instrumentation such as the paper-based microfluidic devices (µPADs). However, these devices have some limitations, such as the need to establish hydrophobic barriers to delimit the microfluidic channel, and the low mechanical strength that hinders the assembly of these devices (Agustini et al. 2016a). To overcome these limitations, cotton threads are a good alternative, as they have good mechanical resistance, even wet, and does not need any kind of barrier to delimit the microfluidic channel. Furthermore, in the threads the transport of liquids occurs spontaneously by capillarity, meaning that a pump system is not necessary (Agustini et al. 2017).

Microfluidics allows the integration of many analytical stages, as well as a sensing system, resulting in smaller devices, which implies on downsizing the detection system in order to be integrated. However, traditional analytical techniques (i.e. fluorescence or mass spectroscopy) usually sacrifices analytical performance in order to achieve miniaturization. In the other hand, coupling microfluidics to electrochemical detection systems grants a synergistic interaction that enhances their inherent characteristics (Nyholm 2005; Fernández-la-Villa et al. 2019). In view of such synergism, Agustini et al. (2016b) proposed a microfluidic device based on textile threads (µTED). This device consists of the use of cotton threads as microfluidic channels, the device was applied with an electrochemical sensing system (amperometric) to simultaneously detect acetaminophen and diclofenac.

The importance of quantifying dopamine and ascorbic acid in biological samples is undeniable (Viry et al. 2010; Liu et al. 2012). Dopamine plays an essential role as a neurotransmitter acting in the nervous, hormonal, cardiovascular and endocrine systems, thus, it participates in the control of cognitive functions such as stress, behavior, and attention. Ascorbic acid, in turn, is an antioxidant used to prevent infertility, mental illness, scurvy, liver disease and cancer. The fact that the two substances coexist in several biological samples, such as extracellular fluid, blood serum, tears, and urine, makes it essential to develop simple, fast, low-cost and portability analytical methods, capable of performing simultaneous determination of these species for purposes diagnosis and control of diseases (Wang et al. 2014).

However, electrochemically quantify these two analytes simultaneously is quite difficult, as their oxidation takes place in the same potential region, around 600 mV (vs. Ag/AgCl). Some works report modified electrodes in order to overcome this problem and differentiate its oxidation peaks, such as carbon nanotubes (Thirumalai et al. 2018), metallic nanoparticles (He et al. 2017), conductive polymers (H.Narouei 2017), molecular imprint polymers (Zaidi 2018), and graphene (Zhang et al. 2017).

There are few reported works that describe electrochemical methodologies for quantifying dopamine and ascorbic acid, simultaneously, without any previous step of sample preparation (separation) or electrode modification. For simultaneous determinations, multiple pulse amperometry (MPA) combined with a hydrodynamic system is a good alternative. This technique allows the application of more than one potential by an alternating sequence, making it possible to monitor the current for different potentials (Agustini et al. 2016b; Bavol et al. 2018). MPA has recently been used in the simultaneous determination of different drugs (Gimenes et al. 2015; Chaves et al. 2015). In addition, this technique is also used to quantify antioxidants (Corrêa Ribeiro et al. 2018), biomarker metabolites such as cotinine (Alecrim et al. 2016), and synthetic dyes such as indigo carmine and allura red (Deroco et al. 2018).

In light of it, the present work exploits the synergistic relationship between microfluidics and electrochemical techniques through the application of the microfluidic device - µTED for simultaneous determination of DA and AA by MPA, in order to contribute to the development of point-of-care and point-of-need systems and boost the next generation of lab-on-a-chip platforms.

2.1 Reagents and solutions

All chemical reagents used were analytical grade and used as received from the manufacturers. All solutions were prepared with deionized water (Mill-Q, Direct-Q3, Millipore) with resistivity higher than 18.2 MΩ cm. Used chemicals include L-Ascorbic acid (Sigma Aldrich), Dopamine Hydrochloride (Sigma Aldrich), Sulfuric acid P.A. (Alphatec), Uric acid (Sigma Aldrich), Anhydrous glucose (Reagen), Sodium chloride P.A. (F. Maia) and Serum from human male AB plasma (Sigma Aldrich).

2.2 Construction of the µTED device

The µTED device was built based on ABS polymer (Acrylonitrile butadiene styrene) using a 3D printer GTMax3D Graber i3. The device was first designed by Agustini et al. 2016b with the dimensions detailed in Fig. 1A. A screen-printed electrode (SPE) consisted of graphite working and counter electrodes, and an Ag/AgCl reference electrode was used for electrochemical measurements. The electrode was developed on a polyester substrate, using graphite and Ag/AgCl inks. Nine hydrophilic threads were placed on the device forming the microfluidic path. The device design is detailed in Fig. 1B.

To assemble the measurement platform, first the SPE (B) was connected to the potentiostat (A) and put on the detection region (C). The µTED microfluidic path was assembled by fixing the hydrophilic threads on the input (E) and output (F) reservoirs with a double-sided tape. The carrier solution was inserted on the input reservoir, which travels to the to the output reservoir through the microfluidic path due to hydrophilic and gravitational forces. The solution drags any species injected in the proper region through the detection region, resulting in a transient signal if any species are electrochemically converted by the Multiple pulse amperometry (MPA) technique.

2.3 Electrochemical procedures

Electrochemical measurements were performed using a Metrohm µAutolab Type III potentiostat/galvanostat using NOVA 1.10 and GPES 4.9 softwares. Cyclic voltammetry (CV) measurements were performed from − 0.40 to 0.70 V, and scan rate of 50 mV s^− 1. MPA was performed at different oxidation and reduction applied potentials in pulses of 200 ms. The measurements were performed using 0.20 mol L^− 1 H₂SO₄ solution as supporting electrolyte and 2.0 µL of sample solution.

2.4 Analytical curve and interference study

In order to perform the simultaneous quantification of DA and AA, three different analytical curves were built: Curve 1: DA; Curve 2: DA + AA; Curve 3: AA, with concentration of both species ranging from 50 µmol L-1 to 900 µmol L^− 1 and n = 3.

The effect of uric acid, glucose and sodium chloride on DA and AA signals were evaluated. Each possible interferent was studied on three levels of concentration: 0.01 mmol L^− 1; 0.1 mmol L^− 1; 1.0 mmol L^− 1, with 0.1 mmol L^− 1 of DA and AA. Every solution was prepared on H₂SO₄(0.2 mol L^− 1).

2.5 Blood serum sample study

Blood serum samples were diluted 2x on H₂SO₄ 0.2 mol L^− 1 and fortified with DA and AA on three levels: 0.02 and 0.05; 0.1 and 0.2; 0.3 and 0,6 mol L^− 1 of DA and AA respectively

2.6 Injection volume study

A solution containing 0.10 mmol L^− 1 of DA and AA was analyzed varying the injected solution volume, in order to verify the trend of current intensities as a function of the solution volume, which was varied from 0.5 to 4.0 µL with 0.5 µL increments.

3.1 Electrochemical Behavior of DA and AA

The electrochemical behavior of DA and AA was investigated using non-modified screen-printed electrode (SPE) through cyclic voltammetry in 0.20 mol L^− 1 H₂SO₄ (Gimenes et al. 2010a). Cyclic voltammograms recorded for each analyte separately are presented in Fig. 2A, and electrochemical reactions of both analytes in Fig. 2B.

Figure 2A shows the ascorbic acid oxidation at 0.39 V with no reversible reaction occurring, while the dopamine oxidation at 0.52 V leads to the reduction of the oxidation product (o-quinone dopamine) at 0.19 V. To discover the optimized potential to be applied in the multiple pulse amperometry to yield a higher current signal, a range of potential was studied through standard amperometry with both DA and AA in solution. The oxidation potential was varied between 0.40 V and 0.70 V (vs. Ag/AgCl), while the reduction potential was studied between 0.15 V and − 0.15 V (Fig. S1). Based on the transient current obtained in the resulted amperometry for each potential, 0.65 V and − 0.10 V were chosen as optimized potentials for the simultaneous determination of DA and AA. These values were employed in the MPA tests and optimization using the µTED device.

For the simultaneous determination of DA and AA by MPA, each analyte and the mixture of both at the concentration of 100 µmol L^− 1 in 0.20 mol L^− 1 H₂SO₄ were injected in triplicates on the microfluidic system and the resulted transient signals are shown in Fig. 3A. The resulted signals follow the expected: The DA provided an oxidation response when the potential of 0.65 V is applied, as well as a reduction response when − 0.10 V is applied; the AA showed the oxidation response to 0.65 V and a small reduction peak on -0.10 V and the mixture oxidation response was the sum of both separate responses. This confirms the possibility to simultaneously determine the concentration of both analytes based only on mixture signals, as the anodic contribution of the DA can be isolated from the total response through a ratio of the cathodic signal to be determined after the method optimization.

It is important to note that a chemical reaction can occur between the product of dopamine oxidation and ascorbic acid, leading to the reduction of dopamine o-quinone back to dopamine and the formation of dehydroascorbic acid. When occurring chemically and spontaneously, the current resulting from the electrochemical conversion suffers an impact that can lead to a divergence in the calculated concentrations (Gimenes et al. 2010b). To investigate the impact of this reaction in the electrochemical response, a study was performed fixing the DA (0.1 mmol L^− 1) concentration while increasing the AA (A: 0.1; B: 0.2; C: 0.4; D: 0.6; E: 0.8; F: 1.0; mmol L^− 1) concentration in the mixture. The resulted amperograms are shown in Fig. 3B. After processing the resulted signals, it is possible to observe a deviation in the DA reduction signal when the AA concentration is 10 times higher than the DA. Thus, on the proposed method conditions, ascorbic acid concentrations higher than 8 times the dopamine should be avoided and can be considered a limitation in sample analysis.

3.2 System optimization

The volume of solution containing the analytes or sample injected close to the detection region of the µTED was studied. The volume was varied between 0.50 µL and 4.0 µL, and the oxidation and reduction signal of the triplicates displayed in Fig. S1. The current peaks observed for the addition of the mixture increase with the volume until 2.5 µL, caused by the higher quantity of molecules able to be electrochemically converted. Additionally, solution volumes higher than

2.0 µL led to an overflow of the microfluidic path, decreasing the reproducibility of the signal, and increasing the time needed to recover the baseline. The pulse time was also studied, as this parameter defines the data sampling during the analysis and directly impacts in the shape of the transient signals, as in the MPA technique the pulse alternates between the two set potentials to measure both amperograms simultaneously. The variation of the oxidation and reduction currents in the range of 100 to 250 ms is not expressive. Therefore, the determining factor for choosing the pulse time for further analysis was the data collection capacity of the software used. Shorter pulse time results on greater amount of data to be collected and this limits the total amperometry time. In order to be able to perform amperometry for up to 3000 seconds, without having a significant decrease in the signals, it was decided to establish 200 ms as the pulse time for the next analyses. (Fig. S2).

3.3 Analytical curve and interference study

The simultaneous determination of DA and AA requires the separation of the anodic signal contribution of both analytes. To discover the relation between the cathodic and the anodic signals provided by dopamine on optimized conditions, different concentrations of the analyte were evaluated in triplicate. There was verified that the reduction current was approximately 40% of the oxidation current signal of DA, which was used as correction factor. This value allows the estimation of the anodic signal contribution of AA and DA in a mixture since only DA can provides a cathodic signal. Thus, the anodic current can be calculated through the Equations (1) and (2).

$$Anodic signal contribution \left(DA\right)= \frac{- Cathodic signal (AA+DA)}{0.4}$$

$$Anodic signal \left(AA\right)=Anodic signal \left(AA+DA\right)-Anodic signal contribution \left(DA\right)$$

Concentrations between 50 and 900 µmol L^− 1 dopamine and ascorbic acid were evaluated in triplicates. The transient signals recorded and the analytical curve obtained after correction step are shown in Fig. 4. The analytical curve for DA is based on the dopamine o-quinone reduction signal, resulting in a sensitivity of 2.24 µA L µmol^− 1 with R² of 0.997, which leads to limits of detection (LOD, S/N = 3) and quantification (LOQ, S/N = 10) of 1.54 µmol L^− 1 and 5.13 µmol L^− 1, respectively. The AA analytical curve resulted in a sensitivity of 5.09 µA L µmol^− 1 and R² of 0.983, with LOD and LOQ of 2.60 µmol L^− 1 and 8.65 µmol L^− 1.

To assure that the signal processing to retrieve the AA contribution to the oxidation signal has no deviation from a curve with AA only, the same concentrations of AA were evaluated and compared to the curve obtained from the mixture. The analytical curve obtained for AA through the proposed method deviated less than 2.0% from the separated curve, confirming that the simultaneous quantification of both analytes is possible without generating any major disadvantages to the method.

To apply the proposed method in biological samples is necessary to explore the possible interference from species present in the sample. The possible interference by uric acid, glucose and sodium chloride were evaluated at three concentration levels (0.01, 0.10 and 1.0 mmol L^− 1), and the variation in the oxidation and reduction signals of 0.10 mmol L^− 1 DA and AA were recorded (Figure S3). While the uric acid presented no interference in all concentration levels, 1.0 mmol L^− 1 sodium chloride caused an average increase of 11% in the oxidation and reduction signals, and 1.0 mmol L^− 1 glucose led to a 10% increase in the oxidation signal and 20% in the reduction signal. At lower levels of concentration (0.01 mmol L^− 1), none of the tested species showed a major deviation in the analysis, which confirms the viability of the proposed method in the determination of DA and AA in biological samples. This result demonstrates that MPA coupled with the microfluidic system can provide an easy solution to the simultaneous determination of these analytes, without deep modification of the electrode, separation techniques or complicated sample preparation. This method can be used in several other combination of analytes and simplify its quantification.

3.4 Blood serum sample analysis

The blood serum sample was evaluated through the proposed method. The commercial sample was 2x diluted in 0.20 mol L^− 1 H₂SO₄ supporting electrolyte to reduce the matrix interference and the viscosity. DA and AA were added in three samples at different levels of concentration: I (20 µmol L^− 1 DA and 50 µmol L^− 1 AA), II (100 µmol L^− 1 DA and 200 µmol L^− 1 AA) and III (300 µmol L^− 1 DA and 600 µmol L^− 1 AA). The triplicates of the samples were added between a crescent and decrescent analytical curve (50 − 900 µmol L^− 1) to evaluate the presence of memory effects. The result of the study is shown in Fig. 6. The sensibility of the crescent and decrescent analytical curves suggests no memory effects including after the addition of the sample. The first addition of the sample without the enrichment of any of the analytes suggest the presence of DA and AA in the sample. The enriched samples at three concentration levels led to a recovery around 60%, which means the sample matrix influenced in the obtained response, which can be a result of the complexity of the serum sample. However, this does not disqualify the application of the proposed method, which was able to detect the presence of both AA and DA in human blood serum at the same order of concentrations normally described for this biological fluid. It is important to highlight that the distinction of electroactive species in biological media usually makes use of modified electrodes. On the other hand, we have used a low-cost and reproducible screen-printed electrode (SPE) without the need for modification, which allow its easy surface regeneration and reuse. Thus, the proposed method allowed the distinction and simultaneous determination of two similar electroactive species using the multiple pulse amperometry (MPA) alternative technique.

The proposed method describes the determination of dopamine and ascorbic acid using multiple-pulse amperometry technique in a lab-made microfluidic system (µTED). The µTED allows a high analytical frequency while the MPA enables the simultaneous quantification of targets with similar electrochemical behavior. The simultaneous determination of dopamine and ascorbic acid was possible without the use of chemically modified electrodes, separation techniques or sample preparation, simplifying the procedure for the quantification of these analytes. This method can be employed in the separation of several targets of interest, mainly drugs and components that share similar electrochemical reactions, and the high analytical frequency makes it a solid alternative for quality control methods.

Acknowledgments

This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES-Brazil, Finance Code 001), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq - 402943/2016-3; 309803/2020-9, 408309/2018-0, 311290/2020-5), and São Paulo Research Foundation (FAPESP, Process 2019/00473-2).

Conflicts of interest

The authors declare that they have no conflicts of interest.

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Simultaneous detection of dopamine and ascorbic acid by a thread-based microfluidic device and multiple pulse amperometry

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Abstract

Figures

1 Introduction

2 Material And Methods

2.1 Reagents and solutions

2.2 Construction of the µTED device

2.3 Electrochemical procedures

2.4 Analytical curve and interference study

2.5 Blood serum sample study

2.6 Injection volume study

3 Results And Discussion

3.1 Electrochemical Behavior of DA and AA

3.2 System optimization

3.3 Analytical curve and interference study

3.4 Blood serum sample analysis

Conclusions And Outlook

Declarations

References

Supplementary Figures

Additional Declarations

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