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Wearable All-Solid-State Potentiometric Microneedle Patch for Intradermal Potassium Detection Marc Parrilla, Maria Cuartero, Sara Padrell Sánchez, Mina Rajabi, Niclas Roxhed, Frank Niklaus, and Gaston A. Crespo Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04877 • Publication Date (Web): 13 Dec 2018 Downloaded from http://pubs.acs.org on December 17, 2018
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Analytical Chemistry
Wearable All-Solid-State Potentiometric Microneedle Patch for Intradermal Potassium Detection Marc Parrillaa, María Cuarteroa, Sara Padrell Sánchezb,c, Mina Rajabid, Niclas Roxhedd, Frank Niklausd and Gastón A. Crespoa,* aApplied
Physical Chemistry Division, Department of Chemistry, School of Engineering Science in Chemistry, Biochemistry and Health, KTH Royal Institute of Technology, Teknikringen 30, SE-100 44, Stockholm, Sweden. bDepartment of Clinical Science, Intervention and Technology, Karolinska Institutet, K 57, SE-141 86, Stockholm, Sweden. cDivision of Obstetrics and Gynecology, Karolinska Universitetssjukhuset, 14186 Stockholm, Sweden. dDepartment of Micro and Nanosystems, School of Electrical Engineering and Computer Science, KTH Royal Institute of Technology, Malvinas väg 10, SE-100 44, Stockholm, Sweden. *Corresponding author: Gastón A. Crespo (
[email protected]) ABSTRACT: A new analytical all-solid-state platform for intradermal potentiometric detection of potassium in interstitial fluid is presented here. Solid microneedles are modified with different coatings and polymeric membranes to prepare both the potassiumselective electrode and reference electrode needed for the potentiometric readout. These microneedle-based electrodes are fixed in an epidermal patch suitable for insertion into the skin. The analytical performances observed for the potentiometric cell (Nernstian slope, limit of detection of 10–4.9 potassium activity, linear range of 10–4.2 to 10–1.1, drift of 0.35 ± 0.28 mV h–1), together with a fast response time, adequate selectivity and excellent reproducibility and repeatability, are appropriate for potassium analysis in interstitial fluid within both clinical and harmful levels. The potentiometric response is maintained after several insertions into animal skin, confirming the resiliency of the microneedle-based sensor. Ex vivo tests based on the intradermal detection of potassium in chicken and porcine skin demonstrate that the microneedle patch is suitable for monitoring potassium changes inside the skin. In addition, the dimensions of the microneedles modified with the corresponding layers necessary to enhance robustness and provide sensing capabilities (1000 µm length, 45° tip angle, 15 µm thickness in the tip and 435 µm in the base) agree with the required ranges for a painless insertion into the skin. In vitro cytotoxicity experiments showed that the patch can be used for at least 24 h without any side effect for the skin cells. Overall, the developed concept constitutes important progress in the intradermal analysis of ions related to an electrolyte imbalance in humans, which is relevant for the control of certain types of diseases.
INTRODUCTION Wearable analytical devices are extremely attractive for diagnostics owing to the uniqueness of on-body observations accomplished by the integration of different kinds of sensors into conventional objects such as garments, sweat-bands, epidermal patches, contact lenses, dental implants, glasses and microneedle patches.1,2 In addition, wearable analytical devices are a critical component in decentralizing the chemical information, thus providing new advances in personalized and preventive medicine, which eventually contributes to the improvement of human well-being in a way that does not have any precedent to this day. Consequently, the analysis of human samples using traditional instrumentation, mostly by centralized clinical laboratories, is being gradually replaced by innovative point-of-care devices that allow for real-time monitoring of significant clinical parameters in patients, resulting in rapid diagnosis.3 This paradigm shift is promoting the advancement of digital healthcare (eHealth) through three of the established requirements: more efficient clinical analyses, enhanced quality of care and evidenced-based decision-making.4 Among all the sensing concepts available to be integrated into wearable analytical devices, electrochemical sensing enables the detection of clinically relevant molecules in a robust, reliable, simple and cost-effective manner.5 In addition, the potential for miniaturizing electrochemical sensors, even to the nanoscale, makes their integration in many types of wearable
substrates possible, including transdermal patches.6 For example, electrochemical sensors integrated into microneedlebased patches are able to reach the interstitial fluid in the dermis layer of human and animal skin to carry out on-body transdermal detection of different analytes (Table S1).7,8 More specifically, the chemical composition of the interstitial fluid contains valuable real-time information about the physiological status of the individual.9 In this context, the majority of the microneedle-based electrochemical devices were conceived for glucose detection using amperometric readout (Table S1). These sensors were successfully applied to the in vivo detection of glucose both in animals and humans.10 In addition, alcohol,11 lactate12 and benzoquinone13 were detected with microneedlebased sensors operating in amperometry mode, although in vivo applications of such devices have not been demonstrated yet. Regarding the ion detection using potentiometric sensors, to our knowledge, only two devices based on microneedles have been published in the literature until today. Miller et al. reported on a microfluidic chip capable of extracting interstitial fluid by means of hollow microneedles and driving it up to the final potentiometric detection of potassium in a traditional flow cell.14 Hence, this work does not involve a direct intradermal detection. Potential drawbacks such as the involvement of complex extraction mechanisms (e.g., vacuum suction, microfluidic valves or liquid pre-filling), and a delay in the measured values due to the time required for the molecules to diffuse from the interstitial fluid to the electrode are involved in this approach. In contrast, Mani et al. have recently
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demonstrated the in vivo measurement of pH in rats based on tungsten microneedles modified with a Ag/AgCl thick coating or a ZnO thin film.15 We have identified two main difficulties responsible for this lack of wearable devices for intradermal ion detection. First, the difficulty of preparing potentiometric electrodes using microneedles as a platform assuring that: (i) the potentiometric response of the working and reference electrodes remain unaltered inside the skin and (ii) there is no toxic effect to the individual caused by either the contact with the electrode materials or their detachment. Second, the dilemma about the appropriate protocol for the validation of intradermal measurements carried out ex vivo and in vivo,8 which is essential to evaluate that the accuracy of the analytical device lies within the established limits of tolerance for a trustable clinical decision-making process based on real-time observations. We propose herein a wearable microneedle patch for intradermal potentiometric detection of potassium in interstitial fluid. Ex vivo experiments measuring potassium concentration inside chicken and porcine skin demonstrate the potential of the microneedles for in vivo application. Moreover, the observed dimensions of the tailor-made microneedles as well as in vitro cytotoxicity tests support the feasibility of the final application. Thus, the new microneedle patch is a promising concept for the continuous and real-time intradermal monitoring of potassium in individuals. Furthermore, we have identified the critical aspects to provide a final prototype to be used in vivo at hospitals, and even at home to control the treatment of several diseases in which potassium ion is involved (i.e. electrolyte imbalance-related disorders). EXPERIMENTAL SECTION Preparation of the all-solid-state microneedle-based potassium-selective electrode. Figure 1a illustrates the microneedle patch fabricated as described elsewhere (see Supporting Information for more details).16 Figure 1b presents all the coatings used for the preparation of the solid microneedle-based potassium-selective electrode. Before fixing the microneedle in the patch, a thick film of carbon was deposited on top of the bare microneedle tip by dip coating them in a commercial carbon ink (Henkel, Germany). Then, the film was cured in the oven (120 °C, 10 min, according to the manufacturer instructions). Subsequently, the carbon-coated microneedle was embedded into the polydimethylsiloxane (PDMS) substrate, and the external part of the microneedle was glued by Loctite Super Glue (Henkel Norden AB) to the substrate to avoid detachment and finally left to dry at room temperature for 4 h. Then, a thin film of functionalized multiwalled carbon nanotubes (f-MWCNTs) was deposited on top of the carbon-modified microneedle by drop casting 10 4 µL of a f-MWCNTs solution in THF (1 mg mL–1).17 The excess of f-MWCNTs after each drop deposition was carefully cleaned using the micropipette. Each layer was allowed to dry for 2 min before depositing the next layer. Finally, the potassium membrane cocktail was drop cast on top of the f-MWCNTs film (4 µL) and left to dry at room temperature for 4 h (see Supporting Information for the membrane compositions, Tables S2 and S3). The microneedle-based potassium-selective electrode was conditioned overnight.
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Preparation of the all-solid-state microneedle-based reference electrode. Figure 1b schematizes all the coatings used for the preparation of the microneedle-based reference electrode. The first layer of Ag/AgCl was deposited on top of the bare microneedle by dip coating of the commercial Ag/AgCl ink (Henkel, Germany). Then, the layer was cured in the oven (120 °C, 10 min, according to the manufacturer). This step allows having a pseudo-reference electrode.18 Then, the Ag/AgCl-coated microneedle was embedded into the substrate following the same procedure as for the potassium-selective electrode. Finally, the poly(vinyl butyral) (PVB) reference membrane cocktail19 was drop cast on the top of the modified microneedle (3 4 µL), removing the excess of cocktail from the bottom of the microneedle, and allowing each layer to dry for 10 min. The reference membrane was left to dry overnight. The reference electrode was conditioned for 12 h in 3 M KCl and left to dry in air for 1 h. Finally, 4 µL of polyurethane (PU) was drop cast onto the microneedle, the excess removed and left to dry at room temperature for 4 h. This last step avoids the leaching of the salt (KCl) and improves the potential stability of the reference electrode.20
Figure 1. a) Illustration of the microneedle patch. b) Modification of the bare microneedles with different coatings. For the working electrode (WE), 1: stainless steel, 2: carbon coating, 3: f-MWCNTs layer, 4: potassium-selective membrane. For the reference electrode (RE), 5: stainless steel, 6: Ag/AgCl layer, 7: PVB membrane and 8: hydrophilic polyurethane. c) Image of the microneedle patch inserted into the skin. The dimensions of the microneedles allow for potassium detection in the interstitial fluid at the dermis level of the skin. d) Dynamic responses observed for potassium cation before and after several insertions into animal skin. The inset shows the corresponding calibration graphs. Before the first insertion: slope of 52.6 mV, LOD of 10–4.9 and LRR of 10–4.2– 10–1.2. After the 10th insertion: slope of 54.5 mV, LOD of 10–4.9 and LRR of 10–4.2–10–1.2. e) Correspondence of the potassium activity measured during ex vivo experiments in chicken skin with a previous calibration graph. To modify the potassium concentration inside the skin, the holder with the dermis part was in contact with artificial interstitial fluid with increasing potassium concentrations (1: from 10–7.15 to 10–4.15, 2: 10–3.65, 3: 10–3.15, 4: 10–2.65, 5: 10–2.15, 6: 10–1.66, 7: 10–1.17 and 8: 10–0.71).
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Analytical Chemistry
RESULTS AND DISCUSSION To demonstrate the possibility of monitoring potassium levels once the microneedle-based potassium-selective sensor and the reference electrode (termed as potentiometric cell) are inserted into the dermis of the skin (Figure 1c), we have conducted ex vivo experiments in which a piece of either chicken or porcine skin is exposed to increasing changes in potassium concentration in an external solution (artificial interstitial fluid background), in which the dermis is immersed in turn (see Figure S1). Previous to this experiment, the electrode was externally calibrated for potassium in a background of artificial interstitial fluid placed in a beaker (slope of 52.2 mV, limit of detection (LOD) of 10–4.9 and linear range of response (LRR) of 10–4.2–10–1.2, Figure 1d). This calibration was repeated after several insertions (up to 10) into animal skin (Video S1) to confirm that the integrity of the sensor was not affected by any physical effect (i.e. membrane detachment from the substrate and/or physical wrinkling or other deformation). Similar analytical parameters were obtained after the first and tenth insertions showing that the membrane remained attached in the microneedle after the entire test. In further studies, different type of skins as well as a higher number of insertions may be used to confirm that none of the membranes (in the WE and RE) is detached from the microneedle. Moreover, scanning electron microscopy (SEM) images could confirm the state of the membrane after the insertions. In addition, it would be important to quantify that the force needed to detach the microneedles from the substrate is less than the force needed for the insertion. This was already reported for the bare microneedles16 and analogous experiments would be necessary for the modified patch. Figure 1e shows the potentiometric response once the electrodes were inserted into the chicken skin while it was exposed to increasing potassium concentrations, following the described experimental set-up (Figure S1). As observed, it took almost 30 min to reach a steady-state potential after each addition of potassium. However, when these potentials are correlated with the previous calibration performed in the beaker, the corresponding potassium activities are lower than the known bulk activities in the external solution (Figure 1e). Differences close to –1.5 orders of magnitude were calculated for the logarithmic activity of potassium in the external solution ranging from –2.15 to –0.7 (steps 5–8 in Figure 1e). Moreover, this difference found in the potassium activity with respect to the external solution varies for different types of skin (e.g. for the porcine skin, differences of –0.8 and –1.3 orders of magnitude were found for logarithmic potassium activity in the external solution of –2 and –1.2, respectively, see Figure S2). Indeed, other factors such as the skin physiological stage, degree of lipids, thickness, as well as storage and conditioning processes have been found to affect the ion and molecules diffusion into the skin, and therefore, it will affect the final concentration of the target ion in the skin at a given time.21,22 Consequently, it is expectable that the potassium concentration inside the skin differs from the concentration fixed in the external solution. Notably, the time needed to reach the steady-state potential after each potassium addition in the external solution, is related to the
time needed for the potassium diffusion across the skin and, in principle, the response time of the sensor once inserted into the skin should be similar as that presented in traditional experiments in the beaker (t95=
