Surface Modification and Characterization of Polycarbonate Microdevices for Capture of Circulating Biomarkers, Both in Vitro and in Vivo

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. First, 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 ultraviolet–visible (UV–vis) spectroscopy 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 X-ray photoelectron spectroscopy (XPS) and attenuated total reflectance fourier transform infrared (ATR-FT-IR) spectroscopy showed nitrogen species present at the surface for all acid concentrations used, but subsurface 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 antifouling 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 nonspecific 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

Capture of the Circulating <i>Plasmodium falciparum</i> Biomarker HRP2 in a Multiplexed Format, via a Wearable Skin Patch

Herein we demonstrate the use of a wearable device that can selectively capture two distinct circulating protein biomarkers (recombinant P. falciparum r<i>Pf</i>HRP2 and total IgG) from the intradermal fluid of live mice <i>in situ</i>, for subsequent detection <i>in vitro</i>. The device comprises a microprojection array that, when applied to the skin, penetrates the outer skin layers to interface directly with intradermal fluid. Because of the complexity of the biological fluid being sampled, we investigated the effects of solution conditions on the attachment of capture antibodies, to optimize the assay detection limit both <i>in vitro</i> and on live mice. For detection of the target antigen diluted in 20% serum, immobilization conditions favoring high antibody surface density (low pH, low ionic strength) resulted in 100-fold greater sensitivity in comparison to standard conditions, yielding a detection limit equivalent to the plate enzyme-linked immunosorbent assay (ELISA). We also show that blocking the device surface to reduce nonspecific adsorption of target analyte and host proteins does not significantly change sensitivity. After injecting mice with r<i>Pf</i>HRP2 via the tail vein, we compared analyte levels in both plasma and skin biopsies (cross-sectional area same as the microprojection array), observing that skin samples contained the equivalent of ∼8 μL of analyte-containing plasma. We then applied the arrays to mice, showing that surfaces coated with a high density of antibodies captured a significant amount of the r<i>Pf</i>HRP2 target while the standard surface showed no capture in comparison to the negative control. Next, we applied a triplex device to both control and r<i>Pf</i>HRP2-treated mice, simultaneously capturing r<i>Pf</i>HRP2 and total IgG (as a positive control for skin penetration) in comparison to a negative control device. We conclude that such devices can be used to capture clinically relevant, circulating protein biomarkers of infectious disease via the skin, with potential applications as a minimally invasive and lab-free biomarker detection platform

CD8⁺ T cell immunogenicity of Nanopatch-delivered viral vector vaccines in prime boost schedules.

<p>Mice (n = 5/6) were primed with 5×10⁷ PFU MVA.PbCSP (no TH or SC) (A) or 5×10⁹ VP ChAd63.ME-TRAP +10% ^w/_v TH+SC (B), either by coated Nanopatch or ID injection. Two weeks post-MVA.PbCSP priming, a boost immunisation of MVA.PbCSP was given and 8 weeks after ChAd63.METRAP a boost immunisation of MVA.ME-TRAP (no TH or SC) was given (dose 5×10⁷ PFU, given either ID or by Nanopatch). One week post-MVA (A) or 3 weeks post-ChAd63 (B) or 2 weeks post-boost (C+D), blood was taken for analysis of Pb9-specific IFN-γ secreting cells. SFC = spot forming cells. PBMC = peripheral blood mononuclear cells.</p

Imaging of skin penetration by Nanopatch microprojections.

<p>(A) The size of a single Nanopatch relative to a forefinger. (B+C) SEM images of microprojection morphology after dry-etch fabrication. (D) Representative cryo-SEM images of the mouse ear skin surface following application of a single Nanopatch. D(i) shows a far field view of the corner of the patched area, with micro-channel openings characteristic of microprojection penetration, adjacent to unbroken skin. D(ii) shows perforated area in higher magnification, with a single micro-channel next to a hair follicle inset. Scale bar inset = 10 µm. (E) Representative micrographs of ear tissue sections following delivery of Nanopatch coated with a fluorescent dye. BF: brightfield image, F: fluorescence image, BF+F: both brightfield and fluorescent images overlaid. SC = <i>stratum corneum</i>; VE = viable epidermis; D = dermis.</p

Viral viability throughout formulation and during Nanopatch dry-coating.

<p>(A+B) ChAd63.ME-TRAP (1×10⁹ VP) and MVA.GFP (1×10⁷ PFU) were mixed with combinations of MC and PS20, with or or without the disaccharides TH+SC (10% ^w/_v each sugar). Formulations were added to DF-1 cell monolayers (A; MVA, n = 4) or HEK-293A cells (B; ChAd63, n = 5) to evaluate viral titre, which was compared to unformulated virus. (C+D) Formulations containing ChAd63.ME-TRAP (C; 1×10⁹ VP) and MVA.GFP (D; 1×10⁷ PFU) were coated onto Nanopatch and immediately eluted into D-MEM. Eluates (n = 4/5) were added to cell monolayers in infectivity assays as before. Eluted viral titres were compared against unformulated, liquid virus. Negative control wells contained D-MEM only. NS = not significant. nd = no data. IFU = infectious units, PFU = plaque forming units.</p