Transdermal Microneedle Sensor Arrays based on

Palladium:Polymer Composites

Aaron McConville and James Davis[*]

School of Engineering, Ulster University, Jordanstown, Northern Ireland, BT37 0QB

Abstract

The solvent based casting of metal particle-polymer mixtures has been investigated as a rapid means through which to producea 10x10 array of pyramidal(200x200x350 micron) microneedlesfor electroanalytical sensing applications. The incorporation of nano particulate palladium powder within either a polycarbonate or polystyrene binder is shown to result in mechanically robust microneedles. The electrochemical properties of the resulting structures have been investigated and their application for transdermal sensing applications has been demonstrated through the use of epidermal / skin mimic.

Keywords: palladium, composite, microneedle, transdermal, sensing array

Text Words: 2954

References: -447

Figures 3x200 = 600

Total Words: 3107

1.0 Introduction

The application of microneedles for drug delivery applications is well established[1-4] but there has been an increasing interest in the transfer of the technology for sensing purposes[5-10]. The near painless transdermal insertion of the microneedle array offers considerable advantages over conventional sampling routes[4] but, in most cases, the devices are produced using relatively sophisticated microfabrication and micromachining processes that can severely compromise the accessibility of the technology[5-10]. The availability of silicone moulds for the production of polymer composite microneedles however,has revolutionised research into transdermal drug delivery[3,11-12] and, it could be envisaged that such systems would be an invaluable tool through which to develop electrochemical sensors. It can be postulated that the incorporation of metallic micro and nano particles within the composite formulation could speed the production of conductive microneedle arrays suitable for a range of biomedical applications. In this communication, the production of palladium microneedles has been investigated and their potential applicability for transdermal electroanalysis is demonstrated.

Palladium modified electrodes have been extensively exploited in a wide variety of electrochemical sensing applicationsin environmental[13,14], industrial[15,16] and biomedical contexts[17,18]and are typically employed in the form ofmicro-nano particulate catalytic clusters within composite constructions[13-16]. The latter typically involvescarbon [13], carbon nanotubes[14] or graphene/graphene oxide[15-17] but,as yet, there are few reports of the use of Pd in microneedle architectures. Chandrasekaren et al. produced intricate Pd and Pd-Co alloy microneedles through electrodeposition onto micromachined substrates but their electroanalytical performance was not investigated[19]. It was therefore of interest to determine the viability of constructing microneedle structures based on a more accessiblemicroparticulatePd composite.Theproposed strategy centred on the encapsulation of Pd powder (<1 micron diameter) within either polycarbonate or polystyrene binders. While the exploitation of these polymeric systems in microneedle fabrication is well established[3,20,21], the challenge here was to determine whether or not the inclusion of the metallic particles, at the ratio necessary to ensure adequate conductivity for electroanalytical purposes,would compromise the mechanical integrity and cohesiveness of the needle structure. The proposed approach is based on solvent casting mixtures of powder and polymeric binder into a silicone mould which, upon release, should produce a 10 by 10 array of needles with the dimensions indicated in Figure 1A.Ideally, the needles should be of sufficient length and mechanical rigidity to penetrate the epidermis of the skin(typically 100 m) without triggering the sensory cells[22]. In this communication, the formulation of a skin mimic (Figure 1B) based on a calcium alginate hydrogel loaded with ferrocyanide as a redox probe was used to determine the electroanalytical properties of the composite Pd microarray. A parafilm™ barrier (~130 m thick) was employed to act as the epidermal layer such that the ferrocyanide probe would only be accessible upon the needles successfully puncturing the top film.

Figure 1. A) Microneedle dimensions and B) Skin mimic employing a non porousparafilm™ barrier and alginate hydrogel loaded with a ferrocyanide redox probe.

2.0 Experimental Details

Materials: Palladium powder (<1 μm), polystyrene (avg. MW 192,000), L-cysteine (97%), potassium ferrocyanide (≥99%), acetone (≥99.8%), dichloromethane (DCM, ≥99.8%), and Parafilm M® were obtained from Sigma-Aldrich Company Ltd (Dorset, England). Potassium chloride (99%) was obtained from Alfa Aesar (Lancashire, England) and silicone MPatch™ microneedle templates were purchased from Micropoint Technologies Pte Ltd (CleanTech Loop, Singapore). All chemicals purchased were of analytical grade and used without any further purification.

Microneedle Fabrication: Microneedle templates were cleaned prior to each use by way of sonication in acetone for 600s and allowed to dry at room temperature. Needle casting involved a three stage process in which an initial 50 μL aliquot of DCM was introduced into a separate beaker, along with 50 mg of Pd powder. The Pd suspension was pipetted into the template and allowed to dry at room temperature - the intention being to precoat the needle surface with a greater quantity of interfacial Pd and enhance the electroanalytical performance. The second stage casting solution was prepared by dispersing 50 mg of Pd within 50 µL of a 50% w/v solution of polystyrene in DCM to form the bulk of the needles and initial baseplate. The final stage involved the introduction of a further 50 µL of 50% PS/DCM containing 50mg Pdto form the top of the baseplate backing. The templates were subjected to centrifugal force at 3000 rpm for 300s to facilitate the removal of air bubbles and enable efficient packing of the polymer/particle mixture within the needle structure. Following this, the remaining DCM was allowed to evaporate at room temperature and pressure for 4 hours prior to demoulding of the microneedle array using adhesive tape.Electrical connection to the MN array was achieved through using silver conductive paint (RS Components Ltd, Northants England) to bond a length of silver wire (0.5mm diameter, Goodfellow UK) to the front surface of one corner of the microneedle array to maximise the usable electrochemical surface area.

In order to improve the electron transfer kinetics of the microneedle array, surface modification by way of a self-assembling monolayer was employed. Due to the presence of thiol functional groups which can be readily adsorbed onto metallic surfaces, the array was submerged in a solution of 10mM L-cysteine for 60 minutes. The array was then rinsed with distilled water prior to any electrochemical analysis.

Alginate Preparation:Alginic acid sodium salt slowly was added to 0.1M potassium chloride to producea 1.5% w/v viscous solution. The mixture(typically 10mL) was stirred for 4 hours at 45°C after which a 5 mL solution of 0.2M calcium chloride in deionised water was added drop-wise against the edge of the beaker until the alginate solution was completely covered.The solutions were covered with parafilm and left overnight to induce complete cross-linking. In the case of gels containing the redox probe, solid potassium ferrocyanide was added to prior to adding the calcium cross linker to provide the required concentration (0.5 mM, 1 mM or 2 mM) based on a 15 mL total volume)and stirred untildissolution was complete.

Characterisation: The fabricated microneedle arrays were characterised by way of digital optical microscopy (Nexus Aigo GE-5, Brunel Microscopes Ltd, England), focused ion beam scanning electron microscopy (Quanta 200 3D FIB/SEM, FEI Company, USA), and electrochemically (PG581 Portable Potentiostat, Bio-Logic SAS, France). Electrochemical measurements were carried out at room temperature 20oC (+/- 2oC). Microneedles were sputtered under vacuum prior to scanning electronic microscopy using a 80:20 Pd/Au target at 30mA for 3 minutes (Emitech K500X Sputter Coater, Quorum Technologies Ltd, England). An accelerating voltage of 5kV was used to obtain the micrographs.

3.0 Results and Discussion

Scanning electron micrographs of the resulting palladium-polymermicroneedles are shown in Figure 2A-C. The needle array is lifted directly from the mould and, as such, mechanical flexing of the latter is minimised and, it can be seen, the integrity of the needle structure is preserved upon removal. While the needles exhibitwell defined geometry, there is, however, a degree of distortion where the sides of the pyramid can be seen to curve inwards. It is likely that this artefact is caused by shrinkage of the polymer resulting from the gradual removal of the solvent. No difference in morphology was observed when comparing Pd:Polycarbonate or Pd:polystyrenestructures with both displaying a granular surface highlighted in Figures 2A-C and is attributed, primarily, to the particulate nature of the Pd component.The effect of the latter is emphasized when compared with the relatively smooth morphology of MNs composed solely of polymer. One such example is highlighted in Figure 2D where the homogeneity of a polystyrenecasting solution ensures more efficient filling of the needle void and results in greater definition.

It could be anticipated that the surface roughness observed in Figures 2A-C could be addressed through increasing the proportion of polymer but, it must be recognised that a compromise is often required whereby structural improvements come at the cost of conductivity and poor electroanalytical performance. In this case, the 50:50 ratio proved to be the optimal arrangement although there are differences in the tip structure of the metal composite and homopolymer needles with the former being notably blunt in comparison. The mean tip diameter of the composite was found 7.2 micron compared to 4.4 micron for the polystyrene needles. Nevertheless, the tip width of the metal composite possesses sufficient needle definition andthough it could be anticipated that the use of smaller metal particles would further improve the capacity for skin puncture. The Pd powder in this case was obtained from commercial sources and is classified as submicron but will consist of a spectrum of particle sizes and it can be expected that - were greater control over the particle size available – the granularity exhibited in Figure 2A-C would diminish. It should also be noted, however, that this same texture provides an enhanced surface area which could be analytically useful for both sensing purposes and for the electrochemically controlled release of reagents.

Figure 2. Scanning electron micrographs of the palladium:polycarbonate microneedle (A-C) and comparison with a polystyrene homopolymer needle produced from the same mould (D).

Cyclic voltammograms detailing the response of the Pd composite MN towards ferrocyanide (2mM, 0.1M KCl, 50 mV/s) are detailed in Figure 3A. It should be noted that the voltammograms shown in Figure 3A represent the response of the whole assembly (base plate and needle structures)submerged in aqueous solution and not in the alginate. It can be seen that the unmodified MN structureinitially exhibits poor electron transfer kinetics with the response to ferrocyanide being noticeably irreversible with no appreciable reduction process observed.Vagaries in the surface oxide layer are known to influence the electrochemical performance[23,24] but it has been previously shown that the electrode response can be markedly improved through modification with thiol monolayers– in an analogous manner to that employed in gold functionalisation[25,26]. Feliciano-Ramos et al. in particular, have shown that Palladium readily forms the latter upon immersion in cysteine solutions[25] and, given the potential biocompatibility of the amino acid modifier, their approach was adoptedin this study. The voltammetric response of the cysteine modified MN array towards ferrocyanide is compared with the untreated assembly in Figure 3A and it can be seen that there is a significant improvement. The oxidation process (+0.4 V) is sharper and has shifted markedly to less positive potentials by 220 mV. The reduction process (+0.11 V) is more defined though there remains a degree of irreversibility with a peak separation of some 300 mV.

The voltammograms highlighted in Figure 3A represent the response from the entire composite but it would be expected that in a real world application – only the needle tip would be within reach of any subcutaneous target analyte. The ability of the needle to puncture the epidermal barrier and the subsequent response of the MN tips were therefore investigated using a skin mimic based on the schematic detailed in Figure 1B. In this scenario, the electrochemistry of the ferrocyanide redox probe would only be accessible once the MN had successfully pierced the impermeable top layer. A preliminary assessment to ensure that this was the case was conducted using a smooth Pd film with no MN protrusions. The counter and reference electrodes were placed directly in the hydrogel layer and the Pd electrode pressed firmly onto the top layer of the Parafilm™. As expected, the latter isolated the working electrode and no response was observed as indicated by the flat line in Figure 3B. Replacing the smooth Pd film with the cysteine modified MN array however, resulted in the puncture of the top layer with the effect that the protruding needle tips could interrogate the hydrogel. Cyclic voltammograms detailing the response of the needle array towards various concentrations ferrocyanide (0.5mM, 1mM, 2mM) encapsulated within the alginate hydrogel described in Section 2.0 are shown in Figure 3B. It can be seen that there is a marked increase in the peak separation in comparison to the response detailed in Figure 3A. This can be attributed to a degree of uncompensated resistance within the needle component of the composite assembly. The current response is also markedly smaller which would be expected given that it is only the tip of the needle structures which are active rather than the base plate.

Figure 3 A) Cyclic voltammograms detailing the response of the Pd:polycarbonate microneedles to ferrocyanide (2mM, 0.1M KCl, 50mV/s) in aqueous solution before and after modification with cysteine. B) Cyclic voltammogram detailing the response of the microneedle tips after puncturing the parafilm layer (130m) and accessing the ferrocyanide redox probe encapsulated within a calcium alginate gel. Voltammograms recorded for 0.5mM, 1mM and 2mM ferrocyanide loaded alginate gel containing 0.1M KCl. No response was observed with a smooth Pd electrode placed directly on the skin mimic confirming the puncture capability of the microneedle array.

4.0 Conclusion

There are a multitude of advantages to the adoption of microneedle arrays within clinical diagnostics but it is clear that the development of such technologies requires the provision of a toolset that is accessible and which can enable rapid prototyping. The ability to form electroanalyticallyviable microneedle arrays through the casting of a metallic powder-polymer mixture has been demonstratedin principle and it is easy to envisage the further adaptation of the approach through the substitution of the palladium component with other nano particulate species. The increasing availability of the latter, the generic nature of the manufacturing step outlined and the ability to rapidly customise the cast formulations provides a foundation tuning the sensing system to a range of transdermal sensing applications. There is an inevitable compromise between the ratio of metallic conductor and polymeric binder but, with more rigorous control over particle size, further improvements in performance could be achieved. At present, only the working electrode is composed of microneedles but it could be expected that the same fabrication approach could be used to produce a separate counter/reference system which could be positioned alongside the former and provide a complete MN sensing system.

Acknowledgements

The authors thank the Department of Employment and Learning (DEL) Northern Ireland for supporting this work.

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