Printed Combinatorial Sensors for Simultaneous Detection of Ascorbic Acid, Uric Acid, Dopamine, and Nitrite.

In this study, an effective and simple direct printing method was developed to create sensing devices on screen-printed carbon electrodes (SPCEs) to detect multiple species simultaneously. Two sensing materials, graphene oxide nanoribbons (GONRs) and poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS), were printed on one SPCE for detection of multiple biochemical substances. Printed layers of the GONRs and PEDOT:PSS mixture (GONRs & PEDOT:PSS) on SPCE showed embedment of GONRs in the PEDOT:PSS layer and diminished the electrochemical activity of GONRs. In contrast, by printing the GONRs and PEDOT:PSS at separate locations (GONRs + PEDOT:PSS) on the same SPCE, the electrochemical activities of both GONRs and PEDOT:PSS can be preserved. Thus, without synthesizing new materials, the modified electrode is able to simultaneously detect ascorbic acid (AA), uric acid (UA), dopamine (DA), and nitrite (NO2-), with high anodic oxidation currents and well-separated voltammetric peaks, in differential pulse voltammetry measurements. The detection limits for the four analytes are 41 nM (AA), 30 nM (DA), 11 nM (UA), and 18 nM (NO2-), respectively. The electrode can either detect single species separately or simultaneously determine specific concentrations of the four species in aqueous mixtures, and this can be further extended for many other electrochemical sensing applications.

Introduction

The recent demands on health care require fast and efficient diagnosis or screening of biochemical substances to manage chronic illnesses. In the human body, there are plenty of biochemical substances and thus simultaneous detection or monitoring of these substances is crucial to disease screening. For example, ascorbic acid (AA), dopamine (DA), uric acid (UA), and nitrite (NO2–) regularly coexist in many biological matrices and thus were considered as important molecules in human physiological processes. AA is a vital component in human diet and plays an important role in tissue repair.[1] DA is a common neurotransmitter in many mammalian central nervous systems and is responsible for the functions of central nervous, renal, and hormonal systems.[2−4] UA is another important biomolecule in body fluids and is associated with several diseases, such as gout, hyperuricaemia, and Lesch–Nyhan syndrome.[5,6] NO2– in physiological systems is associated with NO, a neurotransmitter or a neuromodulator in the central nervous system. Because NO can be oxidized easily to NO2– in biological circumstances, as fast as in a few seconds,[7−11] NO2– is also a commonly found substance in the human body. The balance of coexisting UA, AA, DA, and NO2– in the human body is an important index for physiological functions, and thus a sensitive biosensor to simultaneously detect these four molecules with accurate concentration determination is highly desirable for analytical or diagnostic applications.

Electrochemical techniques are the most commonly used approach to quantitatively detect DA, AA, UA, or NO2– with great accuracy. However, simultaneous detection to these four molecules in the same mixture solution has been a challenge in this research field. Because the oxidation potentials of DA, AA, UA, and NO2– are very close, selective determinations of these four molecules are technically difficult for bare electrodes. The recent development in sensing material synthesis has shown the possibility to detect these four molecules at the same time with chemically modified electrodes. The most commonly used sensing material is functionalized carbon nanotube (CNT). For example, Yang et al. functionalize multiwalled CNTs (MWCNTs) with hexadecyl trimethyl ammonium bromide and show that the functionalized MWCNT can yield simultaneous electrochemical determination for AA, UA, DA, and NO2–.[12] Similarly, after being adsorbed on MWCNT, metal ions, such as lanthanum(III)[13,14] or iron(III)[15] have also been reported to simultaneously detect these four molecules electrochemically with great accuracy. Besides functionalized CNT, gold nanoparticles can also show similar electrochemical capability on a surface-modified glassy carbon electrode.[16]

Although the above materials are capable of simultaneously determining DA, AA, UA, and NO2– mixtures, the synthetic steps to formulate new sensing materials are highly challenging. It is of great interest for scientists or engineers to prepare electrodes by combining several known sensing materials on one electrode for simultaneous determination of DA, AA, UA, and NO2– mixtures. In previous studies,[17,18] graphene oxide nanoribbons (GONRs) can selectively detect AA, DA, and UA in aqueous mixtures. For NO2–, recent research shows that poly(3,4-ethylenedioxythiophene) (PEDOT) has a superior electrocatalytic property toward NO2– and can be used to determine NO2– concentration quantitatively.[19,20] Among various types of PEDOT derivatives, PEDOT:polystyrene sulfonate (PSS) has not only great conductivity but also good electrochemical characteristics for sensing. Intuitively, the mixture of PEDOT:PSS and GONRs might show a combined or synergetic capability to selectively detect DA, AA, UA, and NO2–. To codeposit two materials on an electrode, electrochemical methods are regularly used. However, due to the physical limitations, these methods can only yield layered electrodes without patterns. Recently, printing sensing materials over chemically inert electrodes have been reported to provide an alternative sensor preparation.[21] These printing methods not only can deposit materials with accurate amounts but can also deliver inks to specific locations with patterns. It would be of scientific interest to utilize these printing methods to deposit GONRs and PEDOT derivatives and investigate the combinatorial effects of these two sensing materials for simultaneous detection of DA, AA, UA, and NO2–.

In this study, an effective and simple direct printing method will be developed to deposit GONRs and PEDOT derivatives on the same screen-printed carbon electrodes (SPCEs) to create a combinatorial sensor for UA, AA, DA, and NO2–. The electrochemical behaviors of electrode surfaces modified with either mixed or separate sensing materials will be carefully examined to understand the mutual influence between these two materials. The ability of the prepared combinatorial electrodes for simultaneous detection of multiple biofactors will also be inspected to investigate the feasibility of combining multiple sensing materials together as one versatile sensor.

Results and Discussion

Material Characterization and Surface Morphology of Printed Sensors

After the microwave process, MWCNTs are unzipped into GONR. Transmission electron microscopy images of MWCNT and GONR before and after the unzipping process are illustrated in Figure a,b. As shown in Figure b, graphene sheet structures are found on both sides of the nanotube, whereas the central core of the nanotube remains slightly dark and tubelike. This type of core–shell heterostructure is termed as a MWCNT/GONR nanomaterial, which is consistent with the morphology of the GONRs reported previously.[17] The unzipped GONRs can be stably dispersed in water (Figure S2) and are used in the following dispensing step.

Figure 1

Transmission electron microscope images of (a) as-received MWCNTs and (b) individual GONRs; scanning electron microscopy images of (c) pristine SPCE, (d) PEDOT:PSS-modified SPCE, (e) GONRs-modified SPCE, and (f) (GONRs & PEDOT:PSS) mixture-modified SPCE; (g) picture of the SPCE; (h) optical microscope image of SPCE modified with (GONRs + PEDOT:PSS).

The SPCEs can be modified with specific materials and/or designed patterns. All optical microscope images are shown in Figure S1. The original SPCE has a rough surface (Figure c), with large carbon grains of several microns in size. After being modified with PEDOT:PSS (Figure d), the surface is covered with plastic polymers and becomes smooth. Similarly, after printing over the circular electrode, GONRs also uniformly spread over the SPCE surface (Figure e). The GONR dispersion is compatible with PEDOT:PSS solution (Figure S2), and their aqueous mixture is also used to modify the SPCE. In Figure f, one can observe that GONRs are embedded in the PEDOT:PSS polymer thin film, and thus the electrochemical reactivity of GONRs might be reduced due to the less exposed area to analyte solutions.

The optical image of an electrode with (GONRs + PEDOT:PSS) printed in separate (concentric) areas is shown in Figure h: the circle is modified with GONRs and the outer annulus is modified with PEDOT:PSS. With the digital printing procedure, one can control the concentric radius (0.106 cm in this case) so that the annulus area (0.0352 cm2) is nearly the same as the circular area (0.0355 cm2). Through this printing method, one can simply control the material deposit areas so that the two sensing materials can be separated on the SPCE to avoid direct physical or chemical interferences. However, the electrochemical interferences, which involve electron transfer near the modified SPCE surfaces, might still exist because the two substances are still on the same SPCE. Fortunately, GONRs and PEDOT:PSS are both anodic sensing material so that there are insignificant electrochemical reactions around the overlapping concentric boundary, as will be shown below.

Electrochemical Performances of the Prepared Electrodes

The electrochemical responses of the aforementioned surface-modified electrodes to various biochemical substances are examined via cyclic voltammetry (CV). Figure shows the cyclic voltammograms of these electrodes for 1 mM AA, UA, DA, and NO2–, respectively, in phosphate-buffered saline (PBS) solution. All electrodes show no electrochemical responses in PBS solution. The GONRs have a good ability to detect AA, UA, and DA (Figure a), which agrees with the detection capability of GONRs reported previously,[17,18] but exhibit little response to NO2–. On the other hand, PEDOT:PSS shows preferentially high peak currents for DA and NO2– (Figure b) but shows relatively low peak currents for AA and UA. Besides, PEDOT:PSS shows electrochemical properties for NO2– sensing similar to those in the literature.[19,20]

Figure 2

Cyclic voltammograms of (a) GONRs, (b) PEDOT:PSS, (c) (GONRs & PEDOT:PSS) mixture, and (d) (GONRs + PEDOT:PSS) electrodes produced when sensing 1 mM AA, UA, DA, and NO2–. The scan rate is 50 mV s–1.

The coexistence of these two sensing materials on SPCE can simultaneously help to detect all acids at the same time with specific electrochemical signals. First, the GONRs and PEDOT:PSS are mixed together and printed on the SPCE. Figure c shows the cyclic voltammograms of the (GONRs & PEDOT:PSS) mixture on SPCE. The results confirm that the (GONRs & PEDOT:PSS) mixture can detect all bioacids. However, the peak current for AA is reduced, compared to that of bare GONRs. Because the amounts of GONRs on both electrodes are the same, the poor electrochemical response to AA might be a result of embedded GONRs in the PEDOT:PSS polymer (cf. Figure f) after drying. In a previous study,[18] it was concluded that the dangling bonds on the edges of the GONRs are responsible for the enhanced electrochemical sensing after the unzipping process. But in the mixture, GONRs are embedded in PEDOT:PSS with little exposed areas to the test fluids and thus lead to low responses to AA, which can only be detected by GONRs. To avoid this problem, same amounts of GONRs and PEDOT:PSS are printed in separate areas on the same electrode (Figure h). The cyclic voltammograms of this (GONRs + PEDOT:PSS) electrode exhibit high oxidation peak currents for all four testing substances (Figure d). Moreover, the peak currents of this (GONRs + PEDOT:PSS) electrode are nearly the same as the superposition of peak currents from individual GONRs and PEDOT:PSS electrodes in Figure a,b, indicating that PEDOT:PSS and GONRs in different areas can express their individual detecting abilities without interfering with each other.

Differential Pulse Voltammetry (DPV) for Simultaneous Detection

DPV is performed to test the ability for simultaneous detection of the (GONRs + PEDOT:PSS) electrode for AA, UA, DA, and NO2–. First, the DPV curves (Figure a) show that the (GONRs + PEDOT:PSS) electrode can clearly distinguish these four substances separately. All peaks are separate by at least 100 mV. When all test substances are mixed in the same PBS solution, the (GONRs + PEDOT:PSS) electrode can still detect these four substances clearly (Figure b). The voltammetric peaks have nearly the same voltage positions as of those in CV, with separations for AA–DA, DA–UA, and UA–NO2– of 220.5, 115.1, and 405.9 mV, respectively. These separations between the voltammetric peaks are clear enough to distinguish these four substances.

Figure 3

(a) DPV curves of the (GONRs + PEDOT:PSS) electrode in 0.1 M PBS (pH 7.0) solutions containing AA, DA, UA, and NO2–, respectively. All analytes have the same concentration of 1 mM. (b) The DPV curve of the (GONRs + PEDOT:PSS) electrode in 0.1 M PBS (pH 7.0) solution containing 1 mM AA, 0.033 mM DA, 0.33 mM UA, and 0.33 mM NO2–.

The (GONRs + PEDOT:PSS) electrode also shows great sensitivity for each individual substance in the mixture. Figure shows the DPV curves of the (GONRs + PEDOT:PSS) electrode in a mixture solution containing AA, DA, UA, and NO2–. When one increases the concentration of DA (Figure b), the peak current at 220 mV increases. There are two regions for the oxidation current (IDA) versus the DA concentration (CDA) (Figure f). In the low concentration region of DA, the linear regression equation is IDA = 5.02 + 0.82CDA, with a correlation coefficient of R2 = 0.9906. In the high concentration region, the linear regression yields a relationship of IDA = 67.25 + 0.16CDA, with a correlation coefficient of R2 = 0.9901 (Table S2). The existence of two slopes may be explained by monolayer adsorption followed by multilayer adsorption, as reported previously in the literature,[22] and the lower sensitivity in the high concentration region could be attributed to limited active sites of GONRs and PEDOT:PSS. Similar behavior of the two sensitive regions are also observed for UA (Figure g) and NO2– (Figure h). In particular, AA has only one region from 250 to 1500 μM (Figure e), possibly because the second region is located in higher concentrations. Besides, the oxidation currents also show linear responses for those substances when the concentrations of AA, DA, UA, and NO2– increase at the same time (Figure S4). The electrochemical responses of the (GONRs + PEDOT:PSS) electrodes are quite stable. As shown in Figure S3, the same sensor was tested five times for repeatability (Figure S3a), and five sensors were tested to ensure the reproducibility (Figure S3b). Table S1 summarizes the normalized standard deviations of peak currents in Figure S3 and indicates that when the electrode is submerged in an aqueous mixture containing all four substances, good repeatability and reproducibility (<5%) can be obtained in DPV tests to steadily provide distinguishable peaks for AA, DA, UA, and NO2–. We also check the application in urine samples. The urine sample was diluted to 100 times with 0.1 M PBS (pH 7.0) before measurements. The recoveries ranged between 96 and 109.2% (Table S3), showing that the proposed method could be effectively used in a real sample for determination of AA, DA, UA, and NO2–.

Figure 4

DPV curves of the (GONRs + PEDOT:PSS) electrode in 0.1 M PBS (pH 7.0) with the AA/DA/UA/NO2– mixture: (a) 0.25–1.5 mM AA + 0.005 mM DA + 0.25 mM UA + 0.25 mM NO2, (b) 0.5 mM AA + 0.0005–0.8 mM DA + 0.5 mM UA + 0.5 mM NO2–, (c) 0.5 mM AA + 0.0025 mM DA + 0.0005–0.2 mM UA + 0.5 mM NO2–, and (d) 0.5 mM AA + 0.005 mM DA + 0.1 mM UA + 0.001–2.5 mM NO2–. (e) The oxidation current of AA (IAA) vs AA concentration (CAA) for (a). (f) The oxidation current of DA (IDA) vs DA concentration (CDA) for (b). (g) The oxidation current of UA (IUA) vs the UA concentration (CUA) for (c). (h) The oxidation current of NO2– (INO) vs the NO2– concentration (CNO) for (d).

Amperometric Responses

The (GONRs + PETDOT:PSS) electrode can also yield in fairly good sensitivity and detection limits for AA, DA, UA, and NO2–. The amperometric responses of the (GONRs + PEDOT:PSS) electrode to AA, DA, UA, and NO2– are depicted in Figure , and the results are summarized in Table . All oxidation currents show great linearity to specific analyte concentrations at fixed potentials. For AA (Figure a,e), the linear regression equation is given by IAA = 0.0016 + 0.1002CAA, with a correlation coefficient of R2 = 0.9991. With a signal-to-noise ratio of 3, the detection limit of AA using the (GONRs + PEDOT:PSS) electrode is found to be 41 nM (Table ). Similar results are also observed for UA, DA, and NO2–. All detection limits of AA, UA, and DA using the (GONRs + PEDOT:PSS) electrode are similar to that of the previous data for GONR.[17] The performances of the printed (GONRs + PEDTO:PSS) sensor have a similar or even better detection limit for AA, DA, UA, and NO2– compared to the literature (Table S4). These results indicate that the GONR and PEDOT:PSS in separate areas on the (GONRs + PEDOT:PSS) electrode retain their own electrochemical activities, and the overlapping boundaries have little effects on the electrochemical detection.

Figure 5

Amperometric responses of the (GONRs + PEDOT:PSS) electrode after the subsequent addition of (a) AA (0 V), (b) DA (0.3 V) (c) UA (0.4 V), and (d) NO2– (0.75 V) in a 0.1 M PBS solution. The oxidation currents vs the concentration of various substances: (e) AA, (f) DA, (g) UA, and (h) NO2–.

Table 1

Analytical Parameters for the Amperometric Determination of AA, DA, UA, and NO2– Using a (GONRs + PEDOT:PSS) Electrode

				detection limit (μmol L–1)
analyte	linear range (μmol L–1)	linear regression equation, Ipa (μA), C (μmol L–1)	correlation coefficient (R2)	exp.	cal.
AA	0.05–16.55	IAA = 0.0016 + 0.1002CAA	R2 = 0.9991	0.05	0.041
DA	0.05–16.55	IDA = −8.809 + 0.129CDA	R2 = 0.9997	0.05	0.030
UA	0.05–16.55	IUA = 0.0009 + 0.1251CUA	R2 = 0.9991	0.05	0.011
NO2–	0.05–16.55	INO2– = −5.164 + 0.1117CNO2–	R2 = 0.9956	0.05	0.018

Conclusions

An effective and simple printing method is developed to prepare electrodes with two sensing materials for simultaneous detection of multiple species. GONRs and PEDOT:PSS are deposited on the same SPCE to simultaneously detect multiple biochemical substances. Direct deposition of the GONRs/PEDOT:PSS mixture on SPCEs leads to embedment of GONRs in the PEDOT:PSS layer and diminishes the electrochemical activity of GONRs. By printing the two materials with accurate amounts on separate areas of the same electrode, the electrochemical activities of both GONRs and PEDOT:PSS are preserved. Thus, without synthesizing new materials, the modified electrodes are able to simultaneously detect AA, DA, UA, and NO2–, with high anodic oxidation currents and well-separated voltammetric peaks in DPV measurements. The corresponding peak separations were 220.5 mV (AA–DA), 115.1 mV (DA–UA), and 405.9 mV (UA–NO2–). The detection limits for the four analytes are 41, 30, 11, and 18 nM, respectively, in amperometric current–time measurements. The electrode can either detect single species separately or determine the specific concentrations of aqueous mixtures of the four species. This new combinatorial approach for surface modification can act as a novel platform for simultaneous detection of multiple substances and can be further extended for many other electrochemical sensing applications.

Experiments

Chemicals

Phosphoric acid (H3PO4), sulfuric acid (H2SO4), and potassium permanganate (KMnO4) were obtained from J.T. Baker. MWCNTs (product name, NCT tube) were purchased from Mitsui & Co., Ltd. AA, UA, DA, nitrite acid, and Nafion (5 wt % aqueous solution) were purchased from Sigma-Aldrich. PEDOT:PSS (Clevies, PH1000, the solid content is 1.0 wt % (in water) and the PEDOT:PSS ratio is 1:2.5) was purchased from Heraeus Co., Ltd.

Microwave-Assisted Synthesis of GONRs

The experimental procedure for the synthesis of GONRs has been reported in detail in our previous study.[17] Briefly, 0.05 g of MWCNTs was suspended in a 9:1 (v/v) H2SO4/H3PO4 mixture and treated in a microwave reactor for 2 min, then addition of KMnO4 (0.25 g) was made to the solutions. Finally, the reaction mixture was treated in a microwave reactor for 8 min and filtered through a membrane (pore size 0.1 μm; Millipore). The resulting solid products were washed with water and then dried in an oven at 100 °C for an hour.

Preparation of GONR Ink and Modified Electrodes by Printing Technology

GONR ink was prepared by mixing 6 mg of GONR powders, 2 mL of ethanol, 3 mL of deionized water, and 60 μL of Nafion. The mixture was then sonicated to form a stable suspension. The (GONRs + PEDOT:PSS) ink was a mixture of the GONRs ink and PEDOT ink, with a 1:1 volume ratio.

The printing system mainly consists of a desktop robot (DT-200F; Dispenser Tech Co., Ltd.) and a pneumatic controller (9000E; Dispenser Tech Co., Ltd.). A nozzle with an inner diameter of 0.06 mm (34G), with a 5 mL syringe, was used to print the inks over the SPCE.

Instrumentation

The morphologies of modified electrodes were investigated by using a scanning electron microscope (Nova NanoSEM 230). Transmission electron microscope (Hitachi H-7100) was used to characterize sample morphologies.

Electrochemical Measurements

Electrochemical experiments were carried out using a CHI 6271E electrochemical workstation (CHI). A three-electrode system was used with a standard silver/silver chloride (Ag/AgCl) electrode as the reference electrode. The modified electrode served as the working electrode with a platinum wire as the counter electrode in the electrochemical experiments. In this study, all potentials were reported with respect to the Ag/AgCl electrode. AA, UA, DA, and NO2– were added to an electrochemical cell containing PBS (pH = 7.0), with 0.1 M KCl at room temperature.