An Electrolyte-Gated Graphene Field-Effect Transistor for Detection of Gadolinium(III) in Aqueous Media

Abstract

In this work, an electrolyte-gated graphene field-effect transistor is developed for Gd³⁺ ion detection in water. The source and drain electrodes of the transistor are fabricated by photolithography on polyimide, while the graphene channel is obtained by inkjet-printing a graphene oxide ink subsequently electro-reduced to give reduced graphene oxide. The Gd³⁺-selective ligand DOTA is functionalized by an alkyne linker to be grafted by click chemistry on a gold electrode without losing its affinity for Gd³⁺. The synthesis route is fully described, and the ligand, the linker and the functionalized surface are characterized by electrochemical analysis and spectroscopy. The as functionalized electrode is used as gate in the graphene transistor so to modulate the source-drain current as a function of its potential, which is itself modulated by the concentration of Gd³⁺captured on the gate surface. The obtained sensor is able to quantify Gd³⁺ even in a sample containing several other potentially interfering ions such as Ni²⁺, Ca²⁺, Na⁺ and In³⁺. The quantification range is from 1 pM to 10 mM, with a sensitivity of 20 mV dec⁻¹ expected for a trivalent ion. This paves the way for Gd³⁺ quantification in hospital or industrial wastewater.

1. Introduction

Chelated with hydrophilic ligands, Gadolinium(III) is widely used in hospitals as a contrast agent for brain MRI (magnetic resonance imaging). It is typically injected intravenously in the form of a complex before imaging, for a total of 15 tons each year worldwide. If chelated Gd³⁺ is considered safe, free Gd³⁺ is highly toxic (lethal dose of 0.34 mmol.kg⁻¹ of body mass) because of its chemical similarities to Ca²⁺ (in particular, a similar ion radius), which has important roles in muscle or arteries contraction as well as in nerve transmission and, as other heavy metals, accumulates in the body. Additionally, chelated Gd³⁺ is metabolized and released later by patients in urine in the form of free Gd³⁺ ions, which then recover their toxicity. Several warnings have been given by the US Food and Drug Administration (FDA), the World Health Organization (WHO) and the European Medicines Agency (EMA) [1] in the last 15 years. It is, therefore, necessary to develop routine and costless on-site analytical devices able to perform continuous or frequent Gd³⁺ detection directly in hospital waste waters, in central water treatment plants, and in plants that synthesize the MRI Gd chelates that would like to control their releases in the environment.

Many Gadolinium MRI chelating agents are reported in the literature, as cyclic ionic ones such as Gadoterate (Gd³⁺ complexed with 1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-tetraacetic acid, DOTA) or Gadobutrol (Gd³⁺ complex with 2,2′,2″-(10-((2R,3S)-1,3,4-trihydroxybutan-2-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate, DO3A-butrol) or linear ones such as Gadopentetate, and many others (it is known that cyclic ionic chelating agents are the most efficient). We may not consider these complexing agents only for Gd delivery in MRI, but, conversely, they can serve as selective capture probe for Gd³⁺ sensors. However, few Gadolinium sensors have been reported to date. Among optical sensors, Edogun et al. described a DNA-based fluorescence assay for lanthanide (among which Gd³⁺) ions, relying on the unquenching of a fluorophore upon complexation [2]. More recently, Pallares et al. described a luminescent assay based on the competitive decomplexation of linear chelate of Europium by Gadolinium(III) [3]. Among potentiometric sensors, Ganjali et al. [4] described a Gd³⁺ sensor using a Schiff base and displaying a sensitivity of ca. 20 mV.dec⁻¹ and a detection limit of 3 × 10⁻⁶ M. Zamani et al. [5,6] also reported similar potentiometric electrodes showing a sensitivity of 20 mV.dec⁻¹ and a LOD of 5 × 10⁻⁷ M. Very recently, Gadhari et al. [7] reported a DOTA-based potentiometric sensor for Gd³⁺ presenting the same sensitivity of 20 mV.dec⁻¹ and a LOD of 7 × 10⁻⁹ M.

There are other ways than conventional potentiometry to detect ions, however. Among them, field-effect transistors (FETs) and more particularly electrolyte-gated FETs have emerged during the last decade. The functioning of these transistors is particular: the conventional dielectric material that usually sits between the gate electrode and the semiconducting layer is replaced by an electrolyte, solid [8] or liquid [9,10], in direct contact with the gate and the semiconductor. For a p-type (n-type) semiconductor, a negative (positive) gate bias causes an accumulation of anions (cations) at the electrolyte/semiconductor interface and the accumulation of the same number of positive (negative) charges within the semiconductor, forming a channel of positive (negative) charge carriers. These opposite charges in close vicinity generate very high interface capacitances (in the range 1–100 µF cm⁻²) that allow EG-FETs to operate at very low voltages, typically over a 0–2 V range. In these devices, conservation of charges applies so that the same amount is trapped at the gate and at the semiconductor interface. It means that the transistor can be controlled by functionalization of the electrolyte/semiconductor interface and by that of the gate/electrolyte interface, the latter being generally easier to achieve. Therefore, with a view to making a sensor, functionalization of the gate by a capture probe is a possibility [11,12], which is emphasized in this work.

Several EG-FETs based on organic semiconductors have been described for ion sensing [13,14]. One of their drawbacks is the relatively poor stability of the organic semiconductor immersed in aqueous media. For this reason, electrolyte-gated graphene FETs (EGGFETs) deserve investigation [15]. It is important to keep in mind that graphene is not a semiconductor, however; it is a semimetal for which valence and conduction bands meet at the so-called Dirac point of the Brillouin zone, which corresponds to the Charge Neutrality Point (CNP, corresponding to the minimum charge carriers’ density into the rGO) at which positive (holes) and negative (electrons) charge carriers are found in equal quantity. For this reason, when graphene is employed in field-effect transistors, there is no threshold voltage (V_th) identifiable on the transfer curves, as for conventional FETs, but the transfer curves show an ambipolar behavior, with a p-branch (source-drain current due to holes transport) for applied gate bias leading to move the Fermi level E_F below the CNP, and a n-branch (source-drain current due to electrons transport) for applied gate bias leading to move E_F higher than the CNP. The energetic position of the Fermi level at equilibrium depends on the chemical state of graphene. Pristine graphene is supposed to show a minimum source/drain current $I_D^{min}$ at zero gate voltage (V_G = 0 V). However, defects induced by the synthesis protocol, for instance, induce some structural defects or intercalation of doping species able to significantly shift the value of V_G at which $I_D^{min}$ is observed [16].

EGGFETS are particularly relevant for sensing applications because of the robustness of graphene in aqueous environments compared to organic semiconductors, but mostly because of its sensitivity to electrostatic interactions, due to the ideally 2D structure (i.e., a very high surface-to-volume ratio). Thus, ion sensors based on EGGFETs have been reported. For example, Maehashi et al. [17] described in 2013 an ionophore-modified EGGFET for selective K⁺ detection, demonstrating a left-shift (toward lesser positive potentials) of the CNP with a sensitivity of ca. −8 mV.dec⁻¹ between 10 nM and 1.0 mM. Very recently, Wang et al. [18] reported an aptamer-based EGGFET for Cu²⁺ detection presenting a detection range from 10 nM to 3 μM and a sensitivity of the CNP position estimated at ca. −30 mV.dec⁻¹.

These latter two EGGFETs were fabricated by conventional methods, i.e., subtractive photolithography in a clean room. For the sake of cost saving and simplicity, in view of applications, it is today more than ever pertinent to develop energy-saving additive manufacturing processes for these devices, such as inkjet printing. However, pristine graphene is extremely tricky to print directly as it is, because it cannot be processed in solution (or even stable suspension) without the use of surfactants or other additives that adsorb on the graphene flakes and modify their electrical properties. For this reason, the use of graphene oxide (GO) is preferred, which can form stable suspensions in water without any additive [19]. Due to GO being poorly conductive, it is necessary to retrieve part of the electrical conductivity of graphene by reduction from GO to rGO (reduced graphene oxide) after printing. We showed in a previous work that the electrochemical reduction of GO leads to notable change in the position of the CNP, which can be shifted in a controlled manner over a range of 1 V from its initial position. This makes it possible to finely tune the doping level of rGO as a function of the Coulomb charge passed, and also makes it possible to modulate the mobility of charge carriers over more than one order of magnitude [20].

In this work, an EGGFET fabricated on polyimide (Kapton^®_, DuPont, Wilmington, DE, USA) wafer was used instead of a conventional silicon wafer, onto which GO was inkjet-printed on top and in between the source-drain interdigitated contacts, then electroreduced into rGO to form the conductive channel (Scheme 1). This device was applied for the detection and quantification of free Gd³⁺. For the sake of simplicity, we used a gold wire as gate, functionalized by a DOTA derivative and dipped into the aqueous electrolyte that covers the channel of the transistor. After estimation of the association constant of free Gd³⁺, the resulting transistor was electrically characterized in 0.1 M KCl, then changes in its transfer curves were recorded for various concentrations of added Gd³⁺.

2. Materials and Methods

2.1. Instruments and Procedures

Chemicals, reagents, synthesis and routine characterization procedures are detailed in Appendix A, Appendix B and Appendix C.

Isothermal Titration Calorimetry (ITC) was performed using a high-sensitivity MicroCal PEAQ-ITC Malvern-Panalytical apparatus. Several solutions were prepared before-hand: 50 µM DOTA, 50 µM DO3A-alkyne (Product 7, see Appendix A, Appendix B and Appendix C), 500 µM GdCl₃, 6H₂O, 50 µM CaCl₂. The DOTA or DO3A-alkyne solutions were inserted into the calorimeter and the system was let to equilibrate for 5 min. The measurements were started by one injection of 0.4 µL of Gd³⁺ (or Ca²⁺) followed by 18 injections of 2 µL of the same ion, every 3 min. The electrochemical characterizations of the adsorption in the gold threads were made by Cyclic Voltammetry (CV) on an Autolab PGSTAT30 potentiostat using an equimolar solution of K₄FeCN₆/K₃FeCN₆ (10⁻² M) in 0.1 M KCl. Experiments were performed using Au wires as working electrode (∅ = 1 mm), a lab-made Ag/AgCl reference electrode and a stainless-steel grid as counter electrode.

The transistor electrodes were patterned from a one-step photolithographic process followed by e-beam-assisted evaporation of 100 nm gold layer directly on the polyimide substrate, to define an interdigitated source-drain layout with a channel width/length (W/L) ratio of 3000 (W = 30,000 μm, L = 10 μm). The ink was formulated from a commercial GO powder (from Nanografi Co. Inc, Ankara, Turkey) suspended under sonication into a mix of 50% (v/v) DI-water, 30%(v/v) 1-propanol and 20% (v/v) ethylene glycol. It was printed on the interdigitated source and drain electrodes using a Dimatix inkjet printer 2850 (DMP 2850). Three layers were subsequently printed to form a homogeneous GO channel.

The reduction of printed GO to reduced (rGO) was performed on a Biologic SP-240 potentiostat by chronocoulometry using a Ag/AgCl reference electrode, a stainless-steel grid as counter electrode and the short-circuited source and drain contacts as working electrode. A controlled constant potential of −1.3 V was applied up to the appropriate reduction charge (0.5 mC). Transistor characterizations were performed with a Keithley 4200 A semiconductor characterization system, and the transfer curves with 0.1 M KCl or PBS as electrolyte were acquired for various concentrations of Gd³⁺ and/or other supposed interfering ions.

2.2. Protocol for DO3A-Alkyne Immobilization

Au electrodes were cleaned with acetone and isopropanol then shortly passed under an H₂ flame to remove all organic impurities. Some electrodes were coated with an insulating varnish then polished on diamond polishing cloths at one end to leave only an Au disk of the diameter of the wire. 1-azido-6-thiohexane (Product 3) was dissolved in 1 mL MilliQ water + a minimum (50 µL) of acetonitrile and the Au wires were dipped into this solution for 24 h, then thoroughly washed with ultrapure water under sonication and dried under argon before being stored in a 2 mL Eppendorf under argon atmosphere. For coupling of the modified DO3A-alkyne (Product 7, see Appendix A, Appendix B and Appendix C), an Eppendorf was filled with sodium ascorbate (20 mg, 0.101 mmol) in 1 mL degassed MilliQ water. In another Eppendorf, copper(II) sulphate pentahydrate (0.005 g, 0.02 mmol) was dissolved in 1 mL degassed MilliQ water. DO3A-alkyne was then dissolved in 250 µL of the sodium ascorbate solution +25 µL of the copper(II) sulphate pentahydrate solution and the Au wire was put in the Eppendorf and left for 24 h. After this step, Au electrodes were washed with ultrapure water and dried under argon before being stored back in an Argon-flushed Eppendorf.

3. Results and Discussion

3.1. Determination of the Affinity Constants between Gd³⁺ and DOTA or DO3A-Alkyne

Au electrodes were cleaned with acetone and isopropanol and then shortly passed under an H₂ flame to remove all organic impurities. Some electrodes were coated with an insulating varnish. Before using the DO3A-alkyne chelator in the EGGFET sensor, we estimated by microcalorimetry the formation constants of DOTA and DO3A-alkyne complexes with Gd³⁺ and Ca²⁺, the latter being, as explained in the introduction, a known competitor of Gd³⁺. The heat variations were plotted as a function of the [DOTA]/[Gd³⁺], [DOTA]/[Ca²⁺] and [DO3A-alkyne]/[Gd³⁺] molar ratios, from which the formation constants K_f were estimated by adjusting the isotherms. For complexation of Ca²⁺ by DOTA, the heat changes were extremely low, indicating a very low affinity; therefore, the formation constant K_f(DOTA/Ca2+) was not quantified.

Conversely, for complexation of Gd³⁺ by DOTA and DO3A-alkyne, heat changes were both very significant and made it possible to estimate the respective formation constants K_f(DOTA/Gd3+) and K_{f(DO3A-alkyne/Gd3+)}. In our experimental conditions, T = 25 °C, MilliQ water (pH 5.5) and [DOTA] = 50 × 10⁻⁶ M, K_f(DOTA/Gd3+) was estimated at (2.5 ± 0.6) × 10⁶ and K_f(DO3A/Gd3+) was estimated at (1.4 ± 0.3) × 10⁶. These values are consistent with the literature and demonstrate the high affinity of DOTA and DO3A-alkyne for Gd³⁺; these constants are expected significantly greater in higher ionic strength media [21,22,23,24]. Therefore, DO3A-alkyne is suitable for surface modification and for Gd³⁺ detection. The microcalorimetry results and the corresponding adjustments are given in Figure 1.

3.2. Functionalization of the Gate Electrode

Gate functionalization was performed as described in Section 2 and illustrated in Figure 2. XPS, FTIR and cyclic voltammetry techniques were used to characterize the grafting and coupling steps. XPS and FTIR experiments were carried out on commercial planar Au electrodes (sputtered gold on glass plates). Data analyses are detailed in Appendix C. In summary, complete chemisorption of the spacer (1-azido-6-thiohexane) and the ligand (DO3A-alkyne) on Au are confirmed by XPS analysis (Figure A1). Secondly, the atomic composition of the electrode surface after the click coupling was consistent with the addition of DO3A-alkyne to the electrode. This indicates that the surface coupling conditions were sufficiently mild to proceed at the solid-liquid interface [25]. Additionally, the N_1s XPS spectrum unambiguously confirmed the attachment of DO3A-alkyne to the spacer. However, FTIR spectra of the DO3A-alkyne-functionalized surface (Figure A6) showed that most but not all azido groups reacted upon coupling with the DO3A-alkyne. Cyclic voltammetry (Appendix C, Figure A7) showed that simple adsorption of DO3A in these conditions does not lead to any electrochemical blocking of the interface. Conversely, grafting of the 1-azido-6-thiohexane leads to a partial blocking; when followed by coupling with DO3A-alkyne, the blocking is even more pronounced. These results show the efficiency of the 1-azido-6-thiohexane grafting and that of the DO3A-alkyne coupling, under the form of a dense monolayer, so that the surface density of DO3A could be estimated in the range 5 × 10⁻¹¹–1 × 10⁻¹⁰ mol.cm⁻² (values based on spatial calculations).

3.3. Gd³⁺ Sensing Using the EGGFET

We previously described the electrical behavior of EGGFETs [20]. The transfer characteristics of such transistors is expected to consist of a U-shape curve with a p-branch on the left-hand side corresponding to the contribution of holes to conductivity, and a n-branch on the right-hand side corresponding to the contribution of electrons to conductivity. The minimum of the source/drain current $I_D^{min}$ is obtained for a specific value of the gate voltage V_G* which relies on the doping state of rGO at equilibrium. For a p-doped material (i.e., E_F below CNP), the value of V_G* is expected to be found at positive gate voltage, as shown on Figure 3). For a 1 mm gate wire (area of 7.85 × 10⁻³ cm², while the rGO area is 5 × 10⁻³ cm²), we found V_G* = 0.84 V. For smaller gates (0.5 and 0.25 mm in diameter, i.e., 1.96 × 10⁻³ cm² and 0.49 × 10⁻³ cm², respectively), V_G*was shifted to lower voltages but, as expected from the conservation of charges between the gate/electrolyte and the electrolyte/rGO interface, the gating effect was lower as well as the drain current (results not shown). For this reason, all experiments were performed with 1 mm gates. Under these conditions, the amplitude of the drain current changes was of about 10 μA upon a gate voltage amplitude of 1 V. It must be noted that the output curves are very stable with time: after two runs of gate voltage sweep, the transfer characteristic is stable over several hours of experiment.

As for organic electrolyte-gated transistors [27], the potential difference between the source and the gate electrode is the sum of the potential drop at the gate/electrolyte and at the electrolyte/graphene interfaces. Therefore, if we assume that nothing is changed at the graphene interface, any change at the gate results in a change in the gate voltage, i.e., in a translation of the transfer characteristic along the gate voltage axis [20]. It has been shown [13] that, for ion detection on electrolyte-gated or electrochemical FETs, the gate shift is proportional to the logarithm of the ion concentration as expected with the Nernst law. This behavior can be directly translated into a V_G* change in our case:

V_G* = a log [Gd³⁺] + V_G*⁰,

(1)

Δ V_G*= V_G* − V_G*⁰ = a log [Gd³⁺],

(2)

with a, the proportionality constant (assimilated to a sensitivity) and V_G*⁰ the position of the minimum of the transfer curve on the V_G axis in the absence of Gd³⁺ in the electrolyte. Figure 4a shows the series of transfer curves obtained for increasing Gd³⁺ concentrations while Figure 4b shows the evolution of V_G* as a function of these concentrations. We found a value of a = 29 mv dec⁻¹ for the shift of V_G* in these conditions. The Figure 4c shows a blank experiment obtained under the same conditions as for Figure 4b but using a bare gate electrode instead of a DO3A-alkyne-functionalized one. In these conditions, the shift of V_G* was found poorly dependent on the Ga³⁺ concentration, with a slope of ca. −8 mV.dec⁻¹. Note that, in Figure 4a, the drain current $I_D^{min}$ was subtracted from the measured drain current I_D for all V_D to make easier the comparison between each minimum. Note that, to some extent, the position of minimum of the transfer curve moves with the value of the drain voltage. For the sake of comparison, we took care to set a constant value of V_D for all our measurements.

Another experiment was performed on a DO3A-alkyne-functionalized EGGFET for which the concentration of interfering ion Ca²⁺ was changed, and on a DO3A-functionalized EGGFET for which the concentration of Gd³⁺ was changed in a medium containing all the potential interfering ions Ni²⁺, Ca²⁺, Na⁺ et In³⁺ at 10⁻⁶ M, plus 0.1 M KCl (Figure 5). These experiments show a V_G* that is insensitive to the concentration of Ca²⁺ ions, whereas it shifts of ca. 20 mV.dec⁻¹ for Gd³⁺ in a mixture of other ions.

As shown, even if precisely determined, and considering the standard deviation, the limit of detection of our DO3A-functionalized EGGFET is below 10⁻¹² M and the limit of quantification sits between 10⁻¹² and 10⁻¹¹ M. The dynamic range is very large, from 10⁻¹² to 10⁻² M, and the sensor is selective to Gd³⁺. The average concentration of Gd(III) in effluents of a hospital offering MRI and radiotherapies sits between 10 and 20 μg·L⁻¹, corresponding to 60–120 nM.

We did not have the opportunity to work on real samples such as hospital wastewater, for regulatory reasons. However, the results given here show that the approach consisting of functionalizing an EGGFET by DOTA or DOTA derivatives, which was yet not reported, is promising.

4. Conclusions

We developed here a polyimide-supported EGGFET in which the channel is made of reduced graphene oxide, obtained by electro-reduction of an inkjet-printed lab-made graphene oxide ink. The gold gate electrode was functionalized by a DOTA-like ligand, known to be specific to Gd³⁺ ion. The synthesis of the DOTA-alkyne ligand and of the azido-functionalized alkylthiol spacer are described. The ligand is grafted on the Au gate of the transistor through a spacer so to guarantee a sufficient accessibility of the DOTA to Gd³⁺ while keeping it sufficiently close to the electrode in order to efficiently influence its potential. The constants of formation of the DOTA-Gd³⁺ complex and of its modified form DO3A-alkyne-Gd³⁺ were evaluated using isothermal titration calorimetry (ITC), showing K_f values in the order of 10⁶ in our operating conditions, values that make them pertinent for being used as efficient ligands in the developed EGGFETs. Their electrical characteristics were measured with or without Gd³⁺, with or without DO3A-alkyne, and with or without potential interfering ions. The results show that the device is poorly sensitive to Gd³⁺ if a non-functionalized bare Au gate is used, whereas it is sensitive to Gd³⁺ using a DO3A-alkyne-modified gate, even in a mixture of potentially interfering monovalent, bivalent or trivalent ions of comparable size, with a shift of the transfer curve along the gate voltage axis of 20 mV.dec⁻¹ in these conditions. These results pave the way for using DOTA- or DO3A-alkyne-modified EGGFETs for quality control of hospital or industry wastewaters. The next step in the development will be the amplification of the gate voltage shift into a drain current change, at constant V_D and V_G, and application on real hospitals wastewaters. The selectivity against Pb²⁺, an ion that can also be found in hospital wastewaters and for which DOTA-like ligands were shown to present good affinity [24], will be also investigated.