Multiplexed Covalent Patterns on Double‐Reactive Porous Coating

Abstract We have conceptualized and demonstrated an approach based on the combination of hydrophobicity, a substrate‐independent dip coating as porous material with double residual chemical reactivities for implementing multiplexed, miniaturized and unclonable bulk‐infused patterns of different fluorophores following distinct reaction pathways. The embedded hydrophobicity (∼102°) restricted the unwanted spreading of beaded aqueous ink on the coating. The constructions of micropatterns on porous dip‐coating via ink‐jet printing or microchannel cantilever spotting offered orthogonal read‐out and remained readable even after removal of the exterior of the coating.


Atomic Force Microscopy (AFM)
. AFM images was acquired on a Oxford Instrument, MFP-3D Origin system in tapping mode with silicon tip (AC160TS-R3, nominal frequency 325 kHz, nominal force constant 40 N/m). Three different regions of the prepared dip-coating were analyzed.

Optical Imaging.
The digital images were captured by using Nikon Coolpix b700 digital camera. Thickness of the material was measured using stylus surface profilometer . Bright field, fluorescence microscopic images and z-stacking images were captured by using a ZEISS Axio Vert.A1 inverted microscope and Zeiss LSM 880 Laser Scanning Confocal Microscope. Additional fluorescence microscopy for the micropatterns was performed on a Nikon Eclipse 80i upright fluorescence microscope (Nikon, Germany) equipped with an Intensilight illumination (Nikon, Germany), a Nikon DS Qi2 camera, and Cy5 and FITC filters (Nikon Y-2E/C). The spot diameters were measured by the built-in NIS-element software (Nikon, Germany) on the microscope for the images presented in ESI Fig. S6. The S.D. from these diameters was reported as error.
3.6 X-ray photoelectron spectroscopy (XPS) measurements. The X-ray photoelectron spectroscopy (XPS) measurements were carried out under an ultra-high vacuum conditions with a base pressure of 1×10 -9 mbar. Core-level spectra were recorded under normal emission with a Scienta R4000 hemispherical electron analyzer using Al-Kα radiation (1486.6 eV). Firstly, for every sample the survey XPS spectrum was measured and no unexpected contaminations were observed in these spectra. For a precise determination of the N 1s lines position and necessary correction the XPS Peak 41 software was used.

Fabrication of Spatially Selective Pattern Interfaces.
Chemically reactive porous and moderately rough interfaces were separately and manually exposed to aqueous droplet of TMRC as well as FITC respectively to develop luminescent circular spot on the dipcoating. The substrate was thoroughly washed with ethanol and DI-water to remove loose bound and unreacted TMRC and FITC. The larger QR-Codes (Fig. 3) were obtained by printing an aqueous solution of FITC (concentration of 0.1 mg/ml) with a commercial consumer grade inkjet printer (Canon PIXMA G2021) with printing resolution of 4800 × 1200 dpi onto dip-coated paper. The initial micropatterns (Fig. 3) were spotted via µCS on a Nano eNabler System (Bioforce Nanosciences). Firstly, the microchannel-cantilever was cleaned by ozone treatment for 5 minutes. Then, the selected ink, i.e., aqueous solution of tetramethylrhodamine cadaverine (TMRC, 0.1mg/ml) was loaded to microchannel cantilever (SPT-S-30, Bioforce Nanosciences), and the cantilever was adjusted systematically to touch only the upper periphery of the substrate (to avoid spillages of the ink) for delivering the ink solution to the dipcoating. The written pattern using TMRC was prepared on the dip-coated hydrophobic polymeric substrate which was previously manually marked with a marker to maintain the design of the pattern along a fixed line and position and used as a reference for the next superimposed pattern prepared by fluorescein isothiocyanate (FITC, 0.1 mg/ml) on that substrate after the air drying of the previously prepared micro-patterns. Then the whole patterned substrate was placed under laser scanning confocal microscope to capture the fluorescence images. The miniaturized DataMatrix Codes (Fig. 3) and spot size trials (ESI Fig. S6) were spotted by μCS on a NLP 2000 instrument (Nanoink, Inc.). The microchannel-cantilever (SPT-S-C10S) was purchased from Bioforce Nanosciences. Prior to use, the microchannel-cantilever was plasma-cleaned by oxygen plasma (0.2 mbar, 100 W, 20 sscm O2, 2 min) on a Diener plasma system Atto. Streptavidin_alexa 647 (ThermoFisher Scientific, Germany, 1 µg µL -1 in DMSO, magenta channel) and FITC (Sigma-Aldrich, Germany, 1 µg µL -1 in DMSO, green channel) were used as fluorescent dyes. The microchannel-cantilever reservoir was then filled with 0.5 μL of ink, and the ink was pushed into the pen by blowing with a nitrogen stream. All patterning was done at room temperature, with control humidity and dwell time. All DataMatrix patterns were printed at 30% RH and 0.5 S of dwell time. The dot arrays were printed at different humidity (20%, 45%, 70% RH) and dwell time (0.1, 0.5, 1, 2, 5 sec). The DataMatrix and dot arrays were designed by in-built software in NLP 2000 instrument. After printing, the samples were washed immediately with DI water (18.2 MΩ cm, Arium water system, Sartorius, Germany) to make sure to remove unbound and excess ink. The samples were then dried with nitrogen before further analysis by optical microscopy.

Abrasion Test.
For abrasion tests, a double-sided adhesive tape (1 × 1 cm) was first attached onto a microscopic glass slide, and then, the patterned dip coated substrate (3 × 1 cm) was brought in contact to the adhesive tape with an applied load of 500 g. The external load is applied to facilitate a uniform and homogeneous contact between the substrate and the tape. After 20 min, the adhesive tape was peeled off of the substrate. After removal of the adhesive surface, it was found that top portion of the coating was transferred to that adhesive surface partially, and the interiors of the coating was arbitrarily exposed. Fluorescent patterns were observed to be readable even after 25 cycles of adhesive tape peeling test. To perform sand paper abrasion test, an abrasive sand paper with 4 cm length and 2.5 cm width was exposed to freshly prepared coating with external load of 100 g and applied a back and forth motion for 25 times.

Before Sand-paper abrasion
A After Sand-paper Abrasion B C Fig. S2 A-B) Digital images of the chemically dip-coating before (A) and after (B) sand paper abrasion for 25 times with back and forth motion under 100 g load. C) ATR spectra of the chemically reactive coating before (black) and after (red) sand paper abrasion. ATR-FTIR signatures at 1408 cm -1 and 1733 cm -1 for carbonyl and vinylic C-H deformation respectively, revealed the presence of residual acrylate group.   Figure S4.   Test-patterns for influence of dwell time and relative humidity on the µCS process. The humidity and dwell time were systematically varied during the spotting process as denoted in the florescence images.

Fig. S14
A fluorescence image of a DataMatrix code exposed for optimal visibility of the feature borders. 6 selected features of the DataMatrix are enlarged on the right, to exemplify the unclonable dot shapes.

Fig. S15
Fluorescence images of a 4×4 dot array in air (left) and in water (right). While the intensity and sharpness of the optical images is slightly reduced due to observing through the water film, no substantial change besides this optical effect is noticable, implying the stability of the printed pattern as well the coating itself under humidity and liquid water.