Transparent, Hydrophobic Fluorinated Ethylene Propylene Offers Rapid, Robust, and Irreversible Passive Adsorption of Diagnostic Antibodies for Sensitive Optical Biosensing

Abstract

Current literature data is scarce and somehow contradictory in respect to the suitability of “nonstick” fluoropolymer surfaces for immobilization of biomolecules. We have previously shown empirically that transparent Teflon fluorinated ethylene propylene (FEP) offers rapid and sensitive optical biosensing of clinically relevant biomarkers. This study shows for the first time a comprehensive experimental analysis of passive adsorption of diagnostic IgG antibodies on actual Teflon FEP microfluidic strips. Full equilibrium isotherms and kinetics for passive adsorption were studied and modeled employing a protein titration method using hundreds of multibore microfluidic strips for a range of temperatures, pH, ionic strengths, and inner diameters, using both polyclonal and monoclonal antibody systems. Results were benchmarked against other plastic hydrophobic and glass hydrophilic capillary surfaces. For the first time, it was shown quantitatively that the hydrophobicity of fluoropolymer surfaces encourages the passive adsorption of diagnostic antibodies for biosensing and is insensitive to the temperature of incubation and to ionic buffer strength. The mass of captured antigen increased with increasing antibody surface coverage up to ∼400 ng/cm2, with an optimal adsorbed antibody activity for 45–69% of full monolayer coverage, matching results of other biosensing surfaces. The equilibrium was reached fast, within 5–10 min, and surprisingly both the kinetics and equilibrium of antibody adsorption were dependent on the inner diameter of microcapillaries. This is a novel and relevant result that will generally impact on the design of miniaturized microfluidic biosensing devices. The antibody surface densities obtained with hydrophobic plastic surfaces were 2- to 4-fold lower than for a hydrophilic, glass surface, however the former presented a monolayered adsorption with a higher level of irreversibility, as shown by the adsorption and desorption rates around 1 order of magnitude smaller than for glass, which is highly desirable for biosensing with surface-coated biomolecules.

Gravity-Driven Microfluidic Siphons: Fluidic Characterization and Application to Quantitative Immunoassays

“…We adapted a serotyping microplate ELISA that uses serotype-specific anti-NS1 antibody clones 46 to siphon MCF devices. Multiplex capture strips with duplicate pairs of capillaries were coated internally through passive adsorption 40 with serotype-selective capture antibodies ( Table S1 ) against DENV NS1 ( Figure 5 A). Capture of viral antigen was detected using a pan-NS1 detection antibody.…”

“…MCF strips (47 mm long) were prepared as follows: a 1.4 m length of MCF was coated with a panel of serotype-specific capture antibodies against NS1 produced in-house ( Table S1 ) at 60 μg/mL for DENV1 and 40 μg/mL for DENV2–4 in duplicate capillaries; these concentrations were selected as sufficient to yield a stable half-monolayer of antibody on the surface of Teflon FEP microcapillaries as characterized previously. 40 A positive control capillary was coated with 1 μg/mL recombinant DENV2 NS1, and a negative capillary was filled with PBS alone. These multiplexed MCF strips were incubated at RT for 1 h, and the antibody solution replaced with 0.1 mg/mL PVOH (MW 146,000–186,000 g/mol, Sigma Aldrich, UK); the strips were coated for a further 2 h and finally blocked by filling up with 4% w/v BSA (GE Healthcare Life Sciences, UK) and incubated overnight at 4 °C before cutting into 47 mm test strips that were fitted into siphon holders.…”

Antibody Surface Coverage Drives Matrix Interference in Microfluidic Capillary Immunoassays

“…It has been previously shown that antibody surface coverage is related to immobilized antibodies’ functionality since it interferes with their orientation and steric hindrance. 31 Therefore, we explored the impact of an antibody monolayer on the serum matrix effect, since it would favor the binding of high-affinity components—antigens. As matrix interference is usually dependent on diagnostic antibodies, 8 we have tested three different immunoassays: a direct mouse IgG/anti-mouse IgG, a sandwich human PSA, and a sandwich human IL-1β, covering a range of high-performance immunoassays.…”

“…Similar results were observed for the IL-1β sandwich assay, where matrix interference was fully eliminated with antibody surface coverages in the range of 40–200 μg/mL ( Figure 2 B). Based on a previous FEP adsorption study in the same MCF material, 31 it is known that these CapAb concentrations promote the formation of half of the antibody monolayer with antibodies oriented “end-on” with Fab regions in line, enhancing antigen capture in microcapillary bioassays. This agrees with findings in the literature for a thyroxin assay, where the replacement of an antibody coverage with low affinity by high affinity eliminated the matrix effect of serum samples, 36 explained by the low-affinity binding of the interfering factor(s) to the immobilized antibody.…”

“…Also, the whole inner section of the cylindrical/elliptical capillaries is homogeneously coated with the capture antibody in contrast to immobilization on a single surface as it happens for many other microfluidic devices, which offers advantages in studying surface coverage and specific/nonspecific surface binding. 31 …”