Antibody-Based Array for Tacrolimus Immunosuppressant Monitoring with Planar Plastic Waveguides Activated with an Aminodextran-Lipase Conjugate

Cyclic olefin copolymers (COC; e.g., Zeonor, Topas, Arton, etc.) are materials with outstanding properties for developing point-of-care systems; however, the lack of functional groups in their native form makes their application challenging. This work evaluates different strategies to functionalize commercially available Zeonor substrates, including oxygen plasma treatment, photochemical grafting, and direct surface amination using an amino dextran-lipase conjugate (ADLC). The modified surfaces were characterized by contact angle measurements, Fourier transform infrared-attenuated total reflection analysis, and fluorescence assays based on evanescent wave excitation. The bioaffinity activation through the ADLC approach results in a fast, simple, and reproducible approach that can be used further to conjugate carboxylated small molecules (e.g., haptens). The usefulness of this approach has been demonstrated by the development of a heterogeneous fluorescence immunoassay to detect tacrolimus (FK506) immunosuppressant drug using an array biosensor platform based on evanescence wave laser excitation and Zeonor-ADLC substrates. Surface modification with ADLC-bearing FK506 provides a 3D layer that efficiently leads to a remarkably low limit of detection (0.02 ng/mL) and IC50 (0.9 ng/mL) together with a wide dynamic range (0.07–11.3 ng/mL).

Biotin and FK506 stock solutions were prepared in dimethylsulfoxide (DMSO) (1 mg/mL) and stored at −20 °C.FK506 standard solutions for calibration purposes were prepared daily upon diluting the stock solutions in PBS.Water was purified with a Milli-Q system (Millipore, Bedford, MA).All other chemicals used were of analytical reagent grade.

Instrumentation
The hydrophilicity of the surface was assessed by measuring the water contact angles at room temperature with a goniometer (Dataphysics OCA 15 Plus, Dataphysics Instruments GmbH, Germany).The contact angle between a 2 µL drop of deionized water and the substrate was measured immediately after the contact was made to minimize evaporation.Five drops of water were placed on the substrate, and the contact angle of each drop's left and right sides was optically measured (SCA 20 software, Dataphysics Instruments) and averaged.
Fourier Transform Infrared (FTIR) spectra of the polymers were obtained using a Perkin Elmer Spectrum 100 FTIR spectrometer equipped with a universal ATR sampling accessory (Perkin Elmer, UK).Spectra were recorded with a 4 cm -1 resolution in the 4000 to 400 cm -1 range.
Atomic force microscopy (AFM) was used to evaluate the coating of Zeonor surfaces with the ADLC.Substrates were characterized using a multimode AFM Multimode Nanoscope III A (Bruker) in tapping mode with Si3N4 tips (UCM Central Instrumentation Facilities).The size of the analyzed surfaces was 2 μm x 2 μm.
Fluorescence imaging was carried out using a Leopard Array Biosensor (Hanson Technologies, USA), a commercial version of the NRL Array Biosensor prototype. 3The instrument is equipped with a 635 nm diode laser source (LAS-635-15, Lasermax).The excitation beam is focused on the edge of the microscope slide waveguide.The fluorescence array intensity developed on the slide is filtered using a long-pass (665 nm) and a band-pass filter (700 ± 35 nm) and measured using a CCD camera (Retiga 1300, Q-Imaging).The system is fully automated and uses two six-chamber reservoir modules for the samples and tracers, respectively.The slide is mounted vertically and pressed against a six-channel gasket molded in poly(dimethylsiloxane) (PDMS, NuSil Silicone Technology), forming six assay flow channels.Each channel is connected on one end to a peristaltic pump and on the other to a 2-way valve that switches between the sample and the tracer that flows through the channels. 3The acquired image is analyzed using a proprietary software control interface (HLAB, Hanson Technologies) that controls the microfluidic system.The locations and intensities of the fluorescent spots allow for the identification and quantification of FK506 concentration in the samples. 4A scheme of the array platform is shown in Figure S2b.

COC surface modification
This study tested cyclic olefin copolymer (COC) Zeonor ® (a trademark of Zeon Corp., Tokio, Japan) slides to develop a suitable heterogeneous immunoassay for FK506 analysis.For this purpose, three procedures have been studied for activating the Zeonor slides' surfaces, considering the hydrophobic and unreactive nature of polyolefin materials.

Oxygen plasma activation and functionalization using 3-aminopropyltriethoxysilane
Oxidation of polymer surfaces by oxygen plasma treatment is a popular technique for the creation of functional groups which facilitate the immobilization of a variety of reagents, such as silanes, 5 that will fatherly attach carboxylated FK506 (FK506-CO2H) 6 to the surface.
Oxygen plasma treatment of the Zeonor slides was performed by reactive-ion etching (RIE) using a PlasmaPro NGP80 Etch & Deposition Tool.The surface was oxidized with a 120 W oxygen plasma at 60 mTorr and 10 cm 3 /min for 3−6 min.The activated slides were immersed in a solution containing 3% of APTES in MeOH−water (95:5, v/v) for 1 h at room temperature (RT) 7 (Figure S1a).The aminated slides were rinsed three times with clean MeOH and stored in the same solvent at 4 ºC until use.

Photochemical grafting
COC surfaces were modified using a sequential two-step photografting procedure previously described. 8,9riefly, in 200 µL-size wells placed onto thermoplastic substrates, photo-grafting at 312 nm (Spectroline 312A UV table) was initiated by either benzophenone (BP) or azobisisobutyronitrile (AIBN) in MeOH−water (90:10, v/v) at different concentrations (0.5−5%, w/v) and UV irradiation times (5−20 min).Subsequently, the wells were rinsed with MeOH to remove the excess initiator.Then, a 0.1 mol/L deoxygenated solution of 2-AEM in MeOH−water (90:10, v/v) was added to each activated well and re-illuminated with UV radiation (312 nm) for 30 min.Finally, the wells were extensively washed with water to remove the unreacted 2-AEM.The reaction scheme is depicted in Figure S1b.

Surface amination using an amino dextran-lipase conjugate
Described in the main text.Fluorescence signal (a.u.) Regeneration cycle

Figure S2 ( a )
Figure S2 (a) Scheme of the tacrolimus immunoassay protocol: (1) the sample is incubated with the antibodies on the surface containing the bound hapten; (2) the excess of unbound bioreagents is washed away; (3) the labelled antibodies are injected and, after incubation, the excess is rinsed off and the image is acquired.(b) Scheme of the automated array biosensor platform used for the measurements.Adapted with permission from reference 12.

Figure
Figure S3 (a) Comparison of the hydrophobicity of Zeonor before and after the plasma treatment.(b) Effect of the treatment time on the contact angle of modified Zeonor surface.

Figure S4
Figure S4 Biochip images for the analysis of FK506 using the developed immunoarray.The silanized Zeonor chip was patterned with FK506 (90−900 µg/mL), biotin (30 µg/mL) as positive control, and MES buffer as a negative control.The slide was assayed with different concentrations of FK506 (0.001−10 μg/L) in the presence of 0.4 μg/mL of anti-FK506 and 0.4 μg/mL of anti-biotin (positive control) antibodies.Tracer solution: 2.5 μg/mL AlexaFluor647-labeled anti-mouse IgG and anti-mouse IgM detection antibodies cocktail.(a) Zeonor with 3 min of plasma treatment.(b) Zeonor with 6 min of plasma treatment.

Figure S8 Figure S9
Figure S8 Cross reactivity with the immunosuppressants (a) mycophenolic acid (MPA) and (b) cyclosporin A (CyA) have been analyzed.The assay was performed after functionalizing the ADLC with different concentrations (25-125 µg/mL) of the immunosuppressants.

Table S1 .
Comparison of different methods to activate COC surfaces described in the literature.