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Surface modification and characterization of polycarbonate microdevices for capture of circulating biomarkers, both in vitro and in vivo Bernard W-L. Yeow, Jacob W. Coffey, David A. Muller, Lisbeth Grondahl, Mark A. F. Kendall, and Simon Robert Corrie Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac402942x • Publication Date (Web): 01 Oct 2013 Downloaded from http://pubs.acs.org on October 8, 2013

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Analytical Chemistry

Surface modification and characterization of polycarbonate microdevices for capture of circulating biomarkers, both in vitro and in vivo Bernard Yeow1, Jacob W. Coffey1, David A. Muller1,3, Lisbeth Grøndahl4, Mark A. F. Kendall1,2,3 and Simon R. Corrie1,3 1

The University of Queensland, Australian Institute for Bioengineering and Nanotechnology, Delivery of Drugs and 2 2 Genes Group (D G ), St Lucia, QLD, Australia, 4072. 2

University of Queensland Diamantina Institute (UQDI), Wooloongabba, QLD, Australia, 4012

3

Australian Infectious Diseases Research Centre, St Lucia, QLD, Australia, 4072.

4

The University of Queensland, School of Chemistry and Molecular Biosciences, St Lucia, QLD, Australia, 4072.

ABSTRACT: Herein we report the fabrication, characterization and testing of a polymer microprojection array, for the direct and selective capture of circulating biomarkers from the skin of live mice. Firstly we modified polycarbonate wafers using an electrophilic aromatic substitution reaction with nitric acid to insert aromatic nitro-groups into the benzene rings, followed by treatment with sodium borohydride to reduce the nitro-groups to primary amines. Initial characterization by UV-VIS suggested that increasing acid concentration led to increased depth of material modification, and that this was associated with decreased surface hardness and slight changes in surface roughness. Chemical analysis with XPS and ATR-FTIR showed nitrogen species present at the surface for all acid concentrations used, but sub-surface nitrogen species were only observed at acid concentrations >35 %. The nitrogen species were identified as a mixture of nitro, imine and amine groups, and following reduction there was sufficient amounts of primary amine groups for covalent attachment of a polyethylene glycol anti-fouling layer and protein capture probes, as determined by colorimetric and radiometric assays. Finally, the modification scheme was applied to polycarbonate microprojection arrays, and we show that these devices achieve flank skin penetration depths and biomarker yields comparable with our previously reported gold-coated silicon arrays, with very low non-specific binding even in 10% mouse serum (in vitro) or directly in mouse skin (in vivo). This study is the first demonstration showing the potential utility of polymer microprojections in immunodiagnostics applications.

1.

Introduction

Microprojection arrays (MPAs) are emerging as innovative medical devices with a myriad of applications spanning delivery, diagnostic and surgical applications. The most popular application to date has been in delivery of vaccines to the skin (reviewed by Kim et. al. (2012)1). Vaccines targeted directly to the thousands of antigenpresenting cells (APCs) have potent and long-lasting immune responses; delivering either antigens alone (e.g. for influenza2 and HPV3) or when adjuvants are codelivered by the patch to the skin4. A number of novel applications have recently emerged, including as replacements for surgical staples and sutures,5 pest control,6 and UV-mediated drug delivery.7 Silicon and other metals have been the traditional material of choice for MPA fabrication, however polymers have unique properties for tailored application (for a

detailed recent review of microneedle technology, see Kim et. al. (2012)1). Both silicon and metals have considerable stiffnesses (Young’s Moduli) and breaking stresses, however silicon is brittle while metals are more ductile. Most polymers, on the other hand, are generally less mechanically strong, with bending as the main failure mode.8 However, if the polymer properties can be chosen (or tailored) to penetrate into the skin and target the desired tissue strata, then subsequent bending failure may be more desirable.1 One example of where tailoring polymer properties results in a distinct advantage is in the fabrication of microneedles that dissolve in vivo, releasing drug/vaccine payloads along with biocompatible sugars.1 Indeed, the ability to blend, mix and process polymers in a variety of ways has led to several recent improvements in MPA technology – including the ability to trigger drug release with UV excitation,7 and to tailor mechanical properties to mimic biological structures.6

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Our group introduced the concept of using MPAs as immunodiagnostic devices - selectively capturing circulating proteins and antibodies from the dermal vasculature for applications in rapid diagnostics.9-11 Others have introduced innovative MPA-like devices with integrated electrochemical detection capabilities, however to our knowledge the devices have only been developed for in vitro testing.12 To date we have used silicon to fabricate our MPAs, however to our knowledge there have not been any reports of using solid polymer MPAs for analytical applications. Polymers hold significant advantages over silicon, mainly related to the ease and scale of manufacturing (compression/injection molding of polymers vs. semiconductor processing of silicon). However, when silicon devices are coated with thin gold films, gold-thiol interactions provide a simple route to fabricate selective biomarker capture surfaces, incorporating high density anti-fouling layers (e.g. Biacore technology). Therefore, the development of polymer surface modifications and anti-fouling coatings, with comparable density and biomolecular specificity to that of the gold layers, is a key challenge in realising the potential of polymer-based devices in this field. Polycarbonate (PC) has been used as a biocompatible polymer substrate for microneedle fabrication by several groups since 2007. Han et. al. (2007) used UV-lithography to produce a master structure, and then a hot embossing process using a PDMS mold to produce polycarbonate microneedles 500-1500 µm long, with spacing between needles of 500-2000 µm.13 The authors showed microscopy images of needles piercing skin, but it was not until later that Oh et. al. (2008) showed that similar PC microneedles were capable of skin penetration to deliver drugs 14 and immunization against delivered ovalbumin antigen15 PC has a combination of useful properties for microfabrication and skin application – a moderate glass transition temperature (~420K), good melt viscosity (~1100 Pa. s at 540K), relatively high modulus (~2.4 MPa), high impact strength (~850 MPa), and good light transmission.16 Plasma treatment is commonly used to make PC more hydrophilic, however there are surprisingly few strategies for modifying the surface of PC materials for attachment of biomolecules. Several studies report plasma approaches for developing biologically patterned surfaces17, whiles others have used solutionphase chemical treatments18, all to varying effects. However, none yet have reported the detailed characterization of PC materials following surface modification, or the interplay between chemical and physical/mechanical properties in the context of biomedical devices that operate in high force/stress applications.

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PC via electrophilic substitution using nitric acid treatment and reduction of nitro groups to primary amines as depicted in Scheme 1. After confirming the presence and functionality of primary amines at the surface, we show that ELISA assays can be performed on flat surfaces or on PC-based MPA devices on the skin of live mice, with skin penetration depth and biomarker capture yield comparable to our standard gold-coated silicon devices, even in highly complex sample environments which contain high background sera concentrations (10% mouse sera or direct insertion into mouse skin). 2.

Materials and Methods

Mili-Q water quality water (18.2MΩ.cm) was generated with Millipore Gradient A 10 purification system. Ethanol (EtOH, >99 % purity), nitric acid (HNO3, 70 % concentration), phosphoric acid (>99 % purity) and propan-2-ol (>99.5 %, purity) was purchased from SigmaAldrich. 1 mm-thick polycarbonate wafers (Safeguard Hard Polycarbonate), were purchased from Dotmar Engineering Plastic Products. Sodium borohydride (NaBH4, >99 % purity) was purchased from Merck Millipore. 2,2′-Azino-bis(3-ethylbenzothiazoline-6sulfonic acid) diammonium salt (ABTS), 3,3',5,5'Tetramethylbenzidine (TMB; ELISA Systems), Goat antimouse-IgG-HRP, Sodium dodecyl sulfate (SDS, >99 % purity), Sulfo-NHS-LC-biotin (NHS-Biotin), HRPConjugated Streptavidin (HRP-STP), phosphate buffered saline (PBS), Tween® 20 (T), bovine serum albumin (BSA) and 2-(N-morpholino)ethanesulfonic acid monohydrate (MES) were obtained from Sigma-Aldrich and Thermo Scientific. FluVax2009® was obtained from CSL Ltd, (Parkville, Australia).

In this study, we report for the first time, the fabrication, modification and testing of polycarbonate MPA devices for capturing circulating biomarkers from the skin of live mice. We based our surface modification strategy on the method used by Jain et al.19, extending their work to include a detailed surface modification of


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Analytical Chemistry Surface modification of PC. PC samples were prepared by thorough washing with propanol and Milli-Q water. Samples were reacted in 40 mL nitric acid (concentration 4 % - 70 % v/v in Milli-Q water) for 1 hour at 70 °C. The modified samples were washed thorough with Milli-Q water and refluxed in 40 mL of sodium borohydride (30 %w/v in aqueous solution) for 2 hours to reduce aromatic nitro groups (-NO2) to amine groups (-NH2). At the end of the treatment the samples were washed thoroughly with Milli-Q water. Samples are named PC-X for samples treated with X % nitric acid and PC-X-R for samples at same concentration that had undergone a subsequent reduction. Treated PC MPA samples that have undergone both the nitration and reduction step are named PC-XMPA. Assay to detect accessible primary amines. PC samples were placed in the wells of a 24-well plate (Thermo Scientific) and incubated with 1 mL Sulfo-NHS-LC-biotin reagent (0.12mg/mL in PBS) for 60 minutes. The samples were then thoroughly washed with a mixture of PBS - 0.05 % Tween (PBS-T) 5 times before addition of 0.5 mL HRP Streptavidin (1:10000) solution for 60 minutes. The modified samples were washed with PBST 5 times and transferred to a new 24-well plate. 50 µL TMB substrate was added to each sample and left for 5 minutes, followed by addition of 50 µL of 1 M phosphoric acid to stop the color development.

Scheme 1: Electrophilic aromatic substitution of PC using nitric acid, followed by reduction of aromatic nitro groups to primary amines.

Fabrication of Microprojection Arrays. Gold-coated silicon microprojection arrays were fabricated using a deep reactive ion etching process as described in Jenkins et. al. (2012)10 (Au/Si-MPAs or simply “Au/Si samples” to describe flat surfaces.). Polycarbonate (PC) foils (Dotmar Engineering Plastic Products, Acacia Ridge, QLD, Australia) were either diced (ADT 7100 Precision Dicing Saw) and used as provided (PC samples), or used as feed material for a compression molding process. To fabricate polycarbonate MPAs (PC-MPA), a PDMS mold was fabricated by pouring a 1:14 Sylgard-184 mixture over a silicon MPA master, allowing the mold to cure for 2 days at room temperature and then for 2 hours at 60°C. Compression molding was performed in a EVG-520is semi-automated hot embossing tool (bottom platen; polycarbonate foil; PDMS mold; 2 mm-thick glass plate; 2 mm-thick graphite pad; top platen) using a recipe optimized based on molding time, force and temperature (Recipe: 1) evacuate chamber; 2) apply 200 N touch force; 3) heat plates to 200 °C; 5) Piston force 2500 N for 10 minutes; 6) cool plates to 150 °C; 7) remove force; 8) vent chamber and de-mold).

Dissolution of PC surface with acetone swabs. A swab test was conducted using acetone to remove layers of the samples and sample thickness was measured before and after the acetone swab with an electronic digital caliper (RS Components, accuracy +/- 0.03mm). The swab was conducted using a cotton bud soaked with acetone and swabbed across the sample. The swab process was stopped when the sample became transparent. The sample was then cleaned with propan-2-ol to remove any excess acetone and debris. Atomic Force Microscopy (AFM). An Asylum Research MFP 3D Atomic Force Microscopy system was used to measure surface roughness and perform scratch tests on PC samples. Both measurements were performed using Tap 300 cantilevers (Budget Sensors (Bulgaria)). The applied force was 3 µN and the velocity 500 nm/s. The scratching was done in Litho mode of the MFP 3D AFM and the images were obtained in Tapping Mode. All the cantilevers used had been calibrated against a glass slide prior to the measurements. Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) Spectroscopy. A Nicolet 5700 FTIR spectrometer was used to analyze the IR spectra of PC samples using a diamond internal reflection element (IRE) in ATR mode. Spectra were recorded in the 4000 –



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500 cm-1 wavelength region with 64 scans at 2 cm-1 resolution. No ATR correction was applied. Ultraviolet-Visible (UV-VIS) Spectroscopy. A Cary 60 UV-Vis spectrophotometer (Agilent Technologies) was used to measure absorbance of visible light (800 to 400 cm-1) by placing the sample in between the beam source and detector. Untreated samples were washed with propan-2-ol and MilliQ water while treated samples were washed with MilliQ water before drying in a vacuum desiccator. For comparative purposes, photographs of PC samples analyzed by UV-Vis spectroscopy were taken using a 5-megapixel iSight camera (Iphone 4S, Apple inc.) X-Ray Photoelectron Spectroscopy (XPS). XPS survey and high-resolution spectra were collected from an Axis Ultra XPS spectrometer (Kratos Analytical) at analyzer pass energies of 160 and 40 eV, respectively. The spectrometer has a monochromatic Al Kα X-ray source operating at 15 kV, 10 mA (150 W) for all data acquisitions. Binding energies were charge-corrected to 285.0 eV for aliphatic carbon. High-resolution spectra were resolved using CASAXPS program. Peak fitting was performed in CASAXPS, using fitting parameters (peak binding energy, full width half maximum, etc.) from reference spectra.20 Attachment of PEG and proteins to PC or Au/Sisurfaces. Au/Si and PC samples were coated with polyethylene glycol (PEG) layers to reduce non-specific protein adsorption. 5 kDa hetero-bifunctional x-PEGCOOH groups (Creative PEGworks; x = HS- for Au-coated surfaces; x = NHS- for aminated PC surfaces) were dissolved in 0.6 M potassium sulphate and incubated with the samples for 48 hours in a 60 °C oven. The samples were then thoroughly rinsed in water and 0.1 M MES buffer in preparation for protein attachment. To convert carboxylic acid groups on the sample surfaces to activated esters, samples were incubated in an equimolar mixture of EDC/NHS (5 mM) in 0.1 M MES at room temperature for 90 minutes. After two washing cycles in 0.1 M MES buffer, the samples were then incubated overnight at 4 oC in 20 µg/mL of a protein solution of PBS (either ovalbumin or FluVax®). Plate and MPA ELISA. Traditional plate ELISA was performed on Nunc Maxisorp plates as previously described.21 Briefly, plates were coated with 5 µg/mL FluVax® diluted in 0.1 M sodium carbonate overnight at 4 °C. After 2 hours blocking in 1 %BSA/PBS (pH 9.6), mouse

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sera samples diluted in PBST were added to the plates and incubated on an orbital rotator for 90 minutes at room temperature. Goat anti-mouse-IgG-HRP at 1:1000 in PBST (90 minutes at room temperature) was used as the detection antibody, with TMB as the enzyme substrate (5 min incubation). 1 M phosphoric acid was used to stop the color change reaction and the plates were read immediately in a plate reader (FLUOstar Omega, BMG LABTECH) at 450 nm. 6 plate washes in PBST were performed in between each step. MPA ELISAs were performed as described above, except that MPAs were added to 1 %BSA/PBS-blocked plates for incubation with diluted mouse sera. Also, MPAs were transferred into fresh wells prior to substrate addition. Radiometric analysis. MPAs coated with 14C-labelled ovalbumin (ARC Radiochemicals, St Louis, MO, USA) were placed face-up in scintillation vials filled with 10 mL Ultima GoldTM (Perkin Elmer) scintillation liquid. Samples were analyzed for 10 minutes in a TriCarb 2810 TR liquid scintillation analyzer (Perkin Elmer) with automated quench correction and color compensation. Application of Microprojection Arrays to mice. All experiments involving mice were performed in accordance with animal ethics regulations of The University of Queensland. 14 days prior to MPA application, mice were vaccinated with FluVax® 2009 (CSL Ltd, Australia) via intramuscular injection (n = 5). At day 14, vaccinated mice and control mice (n = 5) were anesthetized and either Au- or PC-MPAs dynamically applied to the flank skin using a custom-designed applicator.22 For depth penetration measurements, MPAs were coated with fluorescent reporter probes that transfer to the skin upon application, leaving fluorescent dye tracks which can subsequently be measured histologically as described in previous work.11 After removal from the skin, MPAs were washed 6 times in PBST, then analyzed via MPA-ELISA. Data Analysis. All experiments were performed in triplicate redundancy unless otherwise noted. Error bars in figures were calculated as standard error of the mean, except for depth penetration measurements, in which standard deviation is presented (due to relatively larger sample size). Statistical tests were performed as described throughout the text.


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Figure 1: Characterization of PC samples following nitric acid treatment. (a) UV-VIS spectra and associated digital images; (b) measurements of sample thickness before and after acetone swabbing (see text for details); (c) AFM roughness measurements; (d) AFM scratch test results (insert Control sample after (horizontal) scratch).

3.

Results

Bulk material characterization following HNO3 treatment. We first investigated bulk material properties following nitric acid treatment, hypothesizing that at high acid concentration, sample degradation would occur in preference to surface modification. The most striking observation was the “yellowing” of the material in a dosedependent fashion. UV-VIS spectra suggested that at least 35 % nitric acid concentration was required to produce a measurable change in absorbance in comparison to the untreated samples, and that the absorbance increased across the visible and near-infrared wavelength range. The UV-VIS spectra were consistent with visual observation as shown in Figure 1a. A key question arising from the dose-dependent absorbance changes was if increasing nitric acid concentration resulted in a surface-specific modification or a bulk material change. Bulk changes could affect both the chemical and physical properties, both of which could be relevant to MPA applications. A “swab test” was designed using acetone (which dissolves polycarbonate)

to remove surface layers from the 35-70% treated samples. Swabbing was performed only to the point at which the samples became transparent (on both sides), then the thickness of the material was measured and the results presented in Figure 2b. The results suggest that the color change of all samples was depth dependent, with more intense color (higher UV-VIS absorbance) related to a deeper level of material modification. Interestingly, treating with higher nitric acid concentrations also resulted in increased sample thickness prior to swabbing, indicating a swelling effect. No significant changes in the wafer morphology were observed by SEM (data not shown) and AFM was performed to determine if nitric acid treatment resulted in increases in surface roughness (Figure 2c). The untreated samples revealed a surface roughness of ~20 nm. Using a one-way ANOVA, there was no significant difference in surface roughness between untreated and HNO3-treated PC wafers (p-value = 0.563), however there was a marginally increased variance in surface roughness (p-value = 0.055; Bartlett’s test).



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Figure 2: Chemical analysis of treated PC samples via (a) XPS (insert showing N 1s atomic % versus nitric acid concentration for samples that had undergone both the nitric acid reaction step as well as the reduction step, and (b) ATR-FTIR of samples that had undergone the nitric acid reaction step as well as the reduction step.

Table 1. XPS elemental analysis of PC samples Survey Scans

C 1s Region b

O 1s Region

C 1s

O 1s

N 1s

C-C

(%)

(%)

(%)

(%)

(%)

Peak

284.5

(FWHM)

(0.9-1.2)

PC reference

a

C-C

c

C-O

d

O-C

O=C

(%)

O2C=O (%)

(%)

(%)

285

286.2

290.4

533.9

532.3

(0.9-1.2)

(0.9-1.2)

(0.9-1.2)

(1.3-1.5)

(1.3-1.5)

88.9

11.1

0

52.4

19.6

9.8

7.1

7.3

3.8

PC untreated

90.7±0.9

9.3±0.9

0

58.6±4.7

18.7±3.3

7.6±0.8

5.8±1.3

5.8±1.1

3.5±0.3

PC-35

83.8±1.9

15.3±0.8

0.9±0.7

42.1±7.1

23.8±6.8

10.3±1.9

6.6±2.6

9.8±1.9

6.1±1.9

PC-35-R

85.1±1.5

13.5±1.4

1.4±0.6

44.5±6.7

21.5±3.3

9.9±0.7

8.5±3.4

8.1±0.3

5.9±0.7

a

Ref = standard PC reference spectra

b

20

Aromatic C-C

c

Aliphatic C-C

d

Data includes both O2C=O and pi-pi* as presented in the reference spectra

This increase in the variance of roughness measurement as a function of HNO3-treatment may suggest small changes, even if there is no difference in the means. Scratch tests were also performed using AFM, on representative samples, to measure the depth of the scratch created by the cantilever at 3 µN of force. The result showed that the samples treated with 50 % and 70 % nitric acid concentration had scratch depths of ~15 nm more than the control sample. Samples treated with 17.5 % and 35 % nitric acid did not suggest any changes compared to the untreated sample (Figure 1d). Thus, at high acid concentrations (50-70 %), this result suggests that some degradation of the polymer surface occurred, leading to decreased surface hardness of the material. Surface and bulk material analysis before and after HNO3 treatment and reduction XPS analysis of untreated PC samples was consistent with reference material,20 composed of 90 % carbon and

10 % oxygen as shown in Table 1. Following nitric acid treatment a small peak appeared in the N 1s region, at an atomic percentage of ~0.5-1.5 %. In these samples, ~3-5 % decrease in overall carbon was observed along with a corresponding increase in oxygen as well as an increase in O-C/O=C ratio, consistent with some degradation at the surface (Figure 2a; Table 1). The N/O2C=O ratio increased approximately linearly with acid concentration in solution (R2 = 0.86; Figure 2a inset). High resolution analysis of the N 1s region of acid-treated PC samples consistently revealed a peak at 399 eV, representative of the formations of amines and/or imines. We also occasionally observed a second peak at 401 eV, assigned to protonated primary amines, and also a third peak at 407 eV which was assigned to aromatic nitro groups (both peaks < 0.1 % relative atomic percentages). Treatment of these samples with sodium borohydride (PC-R samples) did not reveal any systematic changes in the XPS spectra, however this was expected due to the very small and inconsistent aromatic nitro- group peaks (< 0.1 %), and the fact that


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primary amines, secondary amines and imines are indistinguishable via XPS without chemical derivitization. ATR-FTIR analysis of PC samples was used to further investigate the elemental composition, with a specific focus on classifying the nitrogen species. Several characteristic peaks were observed in the untreated PC samples, including stretch vibration at 1767 cm-1 (C=O), 1157 cm-1 (O-C-O) and 2960 cm-1 (C-H). Furthermore, at acid concentrations of 35% or higher, ATR-FTIR revealed extra peaks assigned to aromatic nitro groups (~1535 cm-1 and ~1350 cm-1) and imines (~1670 cm-1)23. Again, no systematic differences were observed following sodium borohydride reduction. Confirmation of amine functionality Importantly, neither XPS nor ATR-FTIR spectroscopy definitively identified the presence of functional amines at the surface of the treated PC samples. We therefore devised a surface-specific test to confirm the presence of primary amine groups accessible in aqueous solutions (conceptually similar to the required application), incubating samples with NHS-reactive biotin (NHS-Bt) for subsequent colorimetric detection via streptavidinHRP (SAHRP). Figure 3 shows that SAHRP was only bound to biotinylated surfaces that had undergone nitric acid treatment and reduction. Interestingly it appears that some surfaces (in this case biotin-modified PC surfaces without SAHRP) can in fact react with the ELISA substrate to produce the colored product even in the absence of the peroxidase enzyme. We have observed similar reactions on some silicon and other metal surfaces, and such phenomena are noted in product literature. However, this confirms that primary amines are present on the surface of the modified wafers.

chemistry to covalently attach a radiolabelled ovalbumin protein. PEG attachment was confirmed via XPS with the C-O peak (286.2 eV) increasing from 9 ± 1.5 % (different sample to that reported in Table 1, but the levels are consistent) to 15.5 ± 1 % (p-value 0.047; t-test with equal variances). Table 2 shows that full monolayer surface coverage of subsequent attached ovalbumin was achieved with respect to theory. Importantly, protein incubated with samples in the absence of EDC/NHS activation did not yield detectable radiometric signal (as a result of significant washing), confirming that covalent amide bonds were formed. Since there did not appear to be systematic differences in protein surface density as a function of surface chemistry (e.g. N 1s atomic %), we infer that FluVax® probes attached in subsequent experiments also result in relatively equivalent surface densities.

Table 2. Surface density measurements from radiometric analysis of surface-bound 14C-ovalbumin Surface

Surface Density 2 (ovalbumin/nm )

Au-MPA

0.038 ± 0.001

PC-17-MPA

0.033 ± 0.002

PC-35-MPA

0.028 ± 0.003

a

Theory

0.029 – 0.044

a

Based on the assumption that ovalbumin protein is a 3 rod structure dimensions are 7 × 5 × 4.5 nm and hence can assume an upright or flat orientation

ELISA tests performed on Nunc Maxisorp, Gold and modified PC surfaces

Figure 3: Column chart showing NHS-biotin (NHS-Bt) attachment to acid-treated and reduced PC-17-R samples via colorimetric detection using streptavidin-HRP (SAHRP). Building upon our amine-functionalized surfaces, we next attached anti-fouling polyethylene glycol chains (x-PEGCOOH) to both PC- and Au-samples and used EDC/NHS

In order to test our modified PC surfaces (PC-17-R and PC-35-R) in a relevant in vitro system, we attached FluVax® to the PEG layers and incubated them in seracollected from FluVax-immunised mice in comparison to naïve controls to see if anti-FluVax-IgG could be detected specifically. Supplementary Figure 1 shows that these “indirect” ELISA approaches are highly specific for the anti-FluVax-IgG (in contrast to “direct” ELISAs where a protein is the target), and that there is negligible difference in total antibody levels between naïve and immunized mice. These data were compared to PEG-coated gold surfaces and Nunc Maxisorp surfaces as a standard. Figure 4a shows the results of ELISA assays performed on the three surfaces following incubation in diluted mouse serum. To investigate non-specific binding phenomena, we included two controls: (a) PEG-coated surfaces with or without FluVax® probes attached; and (b) serum from FluVax®-vaccinated mice, or from naïve animals (both of which contain similar amounts of total IgG). For all three surfaces, the combination of 10% naïve serum and no FluVax® probes presented resulted in very



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low background signals. For all three materials, there was evidence of non-specific binding of FluVax®-IgG to PEGsurfaces, with the most significant signal observed on the PC-17-R sample. While for probe-immobilized surfaces incubated in FluVax®-serum, both PC surfaces yielded approximately double the absorbance signal in comparison to the gold surface, likely because the gold film was deposited on only one side of the silicon wafer. We compared ELISA titration data on the PEG-coated gold and PC surfaces to the standard Nunc Maxisorp ELISA plate. Figure 4b shows the results of the titrations, including the maximum non-specific binding levels observed when surfaces were incubated in 10 % naive serum. Titration experiments on the Nunc Maxisorb surface showed very high signals, saturating at ~1.7 % positive serum, with a high affinity curve. The same titration assays performed on the gold and PC surfaces yielded significantly lower signals and slopes, indicating poor affinity related to the surface or surface modification chemistry. Interestingly, it was observed that the gold surface showed the lowest detectable titer (defined as when signal is higher than 3 standard deviations above the 10 % naïve serum signal for each surface), even in comparison to the Maxisorp surface. Figure 4: ELISA assay results on Maxisorp, Au/Si or PC surfaces; (a) direct comparison between Au/Si and PC samples at 10 % serum dilutions; (b) comparison between Au/Si, PC and Maxisorp surfaces in ELISA format with titrations of mouse sera.


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Figure 5: Results from in vivo application of PC- and Au/Si-MPAs on the flank skin of live mice. SEM images show the geometry and appearance of the (a) Au/Si-MPAs before application to mice skin; (b) PC-MPAs before nitric acid treatment or application to mouse skin; (c) PC-35-MPAs before application to mouse skin; (d) PC-35-MPAs after application to mouse skin. (e) Depth penetration measurements show equivalent penetration depths for Au/Si- and untreated PC-MPAs; (f) MPA-ELISA results showing direct capture of anti-FluVax®-IgG from the flank skin of vaccinated mice in comparison to naïve controls.

Fabrication and in vivo testing of PC microprojection arrays To test the surface modification strategy in a real biological system, we took the same PC wafer material and molded it into microprojection arrays using a standard micromolding approach. This consisted of taking a PDMS negative mold of a silicon MPA and then hot embossing the PC in the presence of the PDMS mold. Optimization of the molding time, pressure and temperature resulted in PC microprojections with dimensions consistent with the respective silicon array. Figure 5 a-b shows the comparison between the silicon and PC-MPAs respectively. The projection lengths are approximately 80 µm in both cases, base diameters ~40 µm, however the newly-made PC projections are considerably less sharp. Interestingly, acid treatment of the PC samples resulted in some bending of the projection tips (Figure 5c), a phenomena we have previously observed only after PC-MPA application to mouse skin (Figure 5d). However, the penetration depths

of the PC-MPAs and Si-MPAs into mouse flank skin were not significantly different from each other (Figure 5e; pvalue = 0.95; Mann-Whitney test, not assuming Gaussian distribution), suggesting that the energy/strain rate applied to cause MPA penetration was sufficient to overcome the effect of the tip sharpness or lack thereof. In order to determine if these PC-MPAs were able to capture circulating biomarkers from the dermal tissue of mice, we coated PC-17-MPA, PC-35-MPA and Au-MPA with PEG and FluVax® probes and applied them to the flank skin of mice. After 10 minute application time, we observed significant absorbance signals for MPAs applied to FluVax®-vaccinated mice in comparison to naïve animals (Figure 5f). Directly comparing Au- and PC-MPA samples, we kept the substrate incubation time to 5 minutes for all samples, to be consistent with all data in Figure 4 and Supp Fig 1. However, this resulted in the PCMPA samples producing the maximum absorbance reading of 3.5 A. U. in 4/5 of animals for both PC-17-MPA and PC-35-MPA samples. Therefore, the error bars in



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Figure 5f for PC-MPA samples under-estimate the true variability in the mice, which is better represented by the lower signals from the Au-MPAs. However, this data confirms that amine-functionalized PC-MPA devices are mechanically and chemically suited to detect biomarkers in the skin of live mice. 4.

Discussion

The combination of surface-specific XPS spectroscopy with ‘bulk’ material analysis with ATR-FTIR and UV-VIS spectroscopy reveal distinct differences between the surface and the bulk polymer following nitric acid treatment. UV-VIS and FTIR spectroscopy both revealed concentration-specific increases in absorbance both (a) generally across the UV-VIS spectrum and (b) specifically in the case of aromatic nitro groups and imine groups. However, XPS and functional tests involving biotin/SAHRP reveal high nitrogen content at the surface, for all nitric acid concentrations, a significant portion of which appears to be present as primary amines. Combining this data, we suggest that increasing the concentration of nitric acid in the electrophilic aromatic substitution reaction results in chemical modification of the polymer at increased depths, but subsequent reduction only of surface-accessible groups, hence the lack of amines observed in the ATR-FTIR analysis (Figure 6). Increasing the depth of chemical modification may result in increased physical/mechanical property changes which are undesirable in our application – hence we suggest a relatively low acid concentration (e.g. 17 %) for surface-specific treatment.

Figure 6: Schematic showing the proposed functional group distribution in modified PC materials as a function of acid concentration and temperature.

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groups to primary amine groups is clearly limited to the accessible surface. While our results confirm that surface-modified PC microprojection devices provide similar analytical performance in comparison to gold/silicon and Maxisorp ELISA surfaces, there is a lack of studies investigating the effects of different surface modification techniques on the resultant mechanical properties of these materials. For in vitro diagnostics applications, changes in mechanical properties may not be particularly relevant because the materials are not put under specific mechanical stresses. However, in the case of MPA devices for diagnostics applications in vivo, bending (elastic modulus) and failure properties of the projection materials may be crucial. Szyndler et al. (2013)6 identified in the difficulty in designing mechanically responsive nano/micro-sized devices in a recent study. Specifically, due to the small size of these devices, the geometry must be controlled to ensure that their mechanical property limits are not overcome/exceeded. In our study, the polymer microprojections penetrated skin to the dermal layer and specifically captured a circulating marker with low nonspecific binding. The performance was consistent with our previously developed Au/Si-MPA devices. 5.

Conclusions

In this study, we have fabricated the first surfacemodified polymer MPAs designed for analytical applications, and applied these devices to capture specific biomarkers from live mice either in vitro (using serum) or in vivo (in flank skin). Introducing amine functional groups into the polycarbonate surface via electrophilic aromatic substitution allowed us to attach anti-fouling PEG coatings and capture probes for biomarker capture. We found that these devices exhibit similar detection limits to Maxisorp ELISA surfaces, penetrate skin to depths comparative with solid silicon projections, and capture biomarkers from mouse skin at comparative levels to previously developed gold-coated silicon MPA devices.

AUTHOR INFORMATION Corresponding Author

A previous report used essentially the same reaction scheme to modify a porous PC membrane, but reported detection of primary amines via ATR-FTIR analysis. Jain et. al. (2012)19 modified the membranes for antibody attachment via gluteraldehyde chemistry. Detection of primary amines following the reduction step would be expected for a membrane material, as the porous network facilitates penetration of reacting species throughout the material. In the current study, while there is some evidence for swelling of the largely non-porous PC wafers at high acid concentrations (Figure 1b), reduction of nitro

* Simon R. Corrie, Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St Lucia, QLD, 4072, Australia. Email: [email protected]; Telephone: +617 3346 4209.

Notes Authors M.A.K. and S.R.C. are inventors on a patent that relates to this work that has recently been licensed to Vaxxas Pty Ltd. M.A.K. is a co-founder of Vaxxas and Chief Technical Officer.

Author Contributions


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The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript

ACKNOWLEDGMENT The authors acknowledge funding from the Australian Research Council (ARC DECRA Fellowship (S.R. C.); Future Fellowship (M.A.K.)). We also acknowledge the facilities, and the scientific and technical assistance, of the Australian National Fabrication Facility (ANFF Queensland Node – particularly Dr Elena Teran for AFM) and the Australian Microscopy & Microanalysis Research Facility (AMMRF) at the Centre for Microscopy and Microanalysis (CMM – particularly Dr Barry Wood for XPS), The University of Queensland and HistoTechnology at the Queensland Institute for Medical Research (particularly G. Rees for cryosectioning). Finally we would like to acknowledge technical assistance from Mr Matthew Heidecker, Ms Anna Kervison and manuscript review by Dr Michael Crichton

REFERENCES (1) Kim, Y.-C.; Park, J.-H.; Prausnitz, M. R. Advanced Drug Delivery Reviews 2012, 64. 1547-1568. (2) Fernando, G. J. P.; Chen, X. F.; Prow, T. W.; Crichton, M. L.; Fairmaid, E. J.; Roberts, M. S.; Frazer, I. H.; Brown, L. E.; Kendall, M. A. F. Plos One 2010, 5. 11; Chen, X.; Fernando, G. J. P.; Crichton, M. L.; Flaim, C.; Yukiko, S. R.; Farmaid, E. J.; Kendall, M. Journal of Controlled Release 2011, 152, 349-355.. (3) Corbett, H. J.; Fernando, G. J. P.; Chen, X.; Frazer, I. H.; Kendall, M. A. F. PLOS One 2010, 5. e13460. (4) Fernando, G. J. P.; Chen, X.; Primiero, C. A.; Yukiko, S. R.; Fairmaid, E. J.; Corbett, H. J.; Frazer, I. H.; Brown, L. E.; Kendall, M. A. F. Journal of Controlled Release 2012, 159; Ng, H.-I.; Fernando, G. J. P.; Kendall, M. A. F. Journal of Controlled Release 2012, in press. (5) Yang, S. Y.; O'Cearbhail, E. D.; Cisk, G. C.; Park, M. K.; Cho, W. K.; Villiger, M.; Pomahac, B.; Karp, J. M. Nature Communications 2013, 4. 1702. (6) Szyndler, M. W.; Haynes, K. F.; Potter, M. F.; Corn, R. M.; Loudon, C. Journal of the Royal Society Interface 2013, 10. (7) DeMuth, P. C.; Min, Y.; Huang, B.; Kramer, J. A.; Miller, A. D.; Barouch, D. H.; Hammond, P. T.; Irvine, D. J. Nature Materials 2013, 12. 367-376. (8) Park, J. H.; Prausnitz, M. R. Journal of the Korean Physical Society 2010, 56. 1223-1227. (9) Bhargav, A.; Muller, D. A.; Kendall, M. A. F.; Corrie, S. R. ACS Applied Materials and Interfaces 2012, 4. 2483-2489; Corrie, S. R.; Fernando, G. J. P.; Crichton, M. L.; Brunck, M. E. G.; Anderson, C. D.; Kendall, M. A. F. Lab on a Chip 2010, 10. 26552658. (10) Jenkins, D.; Corrie, S.; Flaim, C.; Kendall, M. A. F. RSC Advances 2012, 2. 3490-3495. (11) Muller, D. A.; Corrie, S. R.; Coffey, J.; Young, P. R.; Kendall, M. A. F. Analytical Chemistry 2012, 84. 3262-3268. (12) Miller, P. R.; Skoog, S. A.; Edwards, T. L.; Lopez, D. M.; Wheeler, D. R.; Arango, D. C.; Xiao, X.; Brozik, S. M.; Wang, J.; Polsky, R.; Narayan, R. J. Talanta 2012, 88. 739-742; Miller, P. R.;

Gittard, S. D.; Edwards, T. L.; Lopez, D. M.; Xiao, X.; Wheeler, D. R.; Monteiro-Riviere, N. A.; Brozik, S. M.; Polsky, R.; Narayan, R. J. Biomicrofluidics 2011, 5; Windmiller, J. R.; Valdes-Ramirez, G.; Zhou, N.; Zhou, M.; Miller, P. R.; Jin, C.; Brozik, S. M.; Polsky, R.; Katz, E.; Narayan, R.; Wang, J. Electroanalysis 2011, 23. 23022309; Harper, J. C.; Brozik, S. M.; Flemming, J. H.; McClain, J. L.; Polsky, R.; Raj, D.; Ten Eyck, G. A.; Wheeler, D. R.; Achyuthan, K. E. Acs Applied Materials & Interfaces 2009, 1. 1591-1598. (13) Han, M.; Hyun, D. H.; Park, H. H.; Lee, S. S.; Kim, C. H.; Kim, C. Journal of Micromechanics and Microengineering 2007, 17. 1184-1191. (14) Oh, J.-H.; Park, H.-H.; Do, K.-Y.; Han, M.; Hyun, D.-H.; Kim, C.-G.; Kim, C.-H.; Lee, S. S.; Hwang, S.-J.; Shin, S.-C.; Cho, C.-W. European Journal of Pharmaceutics and Biopharmaceutics 2008, 69. 1040-1045. (15) Jin, C. Y.; Han, M. H.; Lee, S. S.; Choi, Y. H. Biomedical Microdevices 2009, 11. 1195-1203. (16) Madkour, T. M. In Polymer Data Handbook, Mark, J. E., Ed.; Oxford Unviversity Press, Inc.: New York, 1999. (17) Bergemann, C.; Quade, A.; Kunz, F.; Ofe, S.; Klinkenberg, E.-D.; Laue, M.; Schroeder, K.; Weissmann, V.; Hansmann, H.; Weltmann, K.-D.; Nebe, B. Plasma Processes and Polymers 2012, 9. 261-272; Thomson, D.; Hayes, J. P.; Thissen, H. In Biomems and Nanotechnology, Nicolau, D. V.; Muller, U. R.; Dell, J. M., Eds., 2003, pp 161-167. (18) Banuls, M.-J.; Garcia-Pinon, F.; Puchades, R.; Maquieira, A. Bioconjugate Chemistry 2008, 19. 665-672; Jackeray, R.; Abid, Z. C. K. V.; Singh, G.; Jain, S.; Chattopadhyaya, S.; Sapra, S.; Shrivastav, T. G.; Singh, H. Talanta 2011, 84. 952-962. (19) Jain, S.; Chattopadhyay, S.; Jackeray, R.; Abid, C. K. V. Z.; Kohli, G. S.; Singh, H. Biosensors & Bioelectronics 2012, 31. 37-43. (20) Beamson, G.; Briggs, D. High Resolution XPS of Organic Polymers – The Scienta ESCA300 Database; John Wiley & Sons: Chichester, 1992. (21) Fernando, G. J.; Stenze, l. D. J.; Tindle, R. W.; Merza, M. S.; Morein, B.; Frazer, I. H. Vaccine 1995, 13. 1460-1467. (22) Crichton, M. L.; Ansaldo, A.; Chen, X.; Prow, T. W.; Fernando, G. J.; Kendall, M. A. Biomaterials 2010. (23) Simons, W. W. The Sadtler handbook of infrared spectra; Sadtler Research Laboratories: Philadelphia, 1978.



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