Plasma Treatment of PDMS for Microcontact Printing (μCP) of Lectins Decreases Silicone Transfer and Increases the Adhesion of Bladder Cancer Cells

The present study investigates silicone transfer occurring during microcontact printing (μCP) of lectins with polydimethylsiloxane (PDMS) stamps and its impact on the adhesion of cells. Static adhesion assays and single-cell force spectroscopy (SCFS) are used to compare adhesion of nonmalignant (HCV29) and cancer (HT1376) bladder cells, respectively, to high-affinity lectin layers (PHA-L and WGA, respectively) prepared by physical adsorption and μCP. The chemical composition of the μCP lectin patterns was monitored by time-of-flight secondary ion mass spectrometry (ToF-SIMS). We show that the amount of transferred silicone in the μCP process depends on the preprocessing of the PDMS stamps. It is revealed that silicone contamination within the patterned lectin layers inhibits the adhesion of bladder cells, and the work of adhesion is lower for μCP lectins than for drop-cast lectins. The binding capacity of microcontact printed lectins was larger when the PDMS stamps were treated with UV ozone plasma as compared to sonication in ethanol and deionized water. ToF-SIMS data show that ozone-based treatment of PDMS stamps used for μCP of lectin reduces the silicone contamination in the imprinting protocol regardless of stamp geometry (flat vs microstructured). The role of other possible contributors, such as the lectin conformation and organization of lectin layers, is also discussed.


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Table S1 Determination of the sol fraction.Changes in mass of three PDMS pieces after swelling in EtOH and drying.Data presented as mean ± maximum error (0.0001 g + SD).

µCP of lectins with flat PDMS stamps sonicated in cyclohexane
A set of substrates covered with PHA-L and WGA imprinted with cyclohexane (CHX) treated PDMS cuboids were prepared.The flat PDMS stamps were sonicated in CHX for (15 min) and dH2O (15 min), and dried with N2.Subsequently, these stamps were used to deposit lectins on APTES-functionalized glass slides following the protocol described in Section 2.8.

Analysis of fluorescence micrographs reflecting lectin distribution
Fluorescence microscopy (see Section 2.11 for details) was used to visualize and compare lectin layers prepared with different protocols (DC, µCP with PDMSOz, µCP with PDMSEtOH, and µCP with PDMSCHX).The camera acquisition time was 6 ms for all lectin types.
For each case, 3 substrates were prepared and a minimum of 6 images were taken.The Supporting Information

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distribution of fluorescence intensity and its mean ± SD of each image were obtained using CellSens software (Olympus).Mean fluorescence intensities were then calculated for all data sets and normalized to the background signal (glass + APTES).S2.In the case of WGA, high intensities of ion peaks corresponding to the amino acids: Alanine (Ala), Histidine (His), Glutamic Acid (Glu), and Glutamine (Gln) were found.PHA-L showed a higher concentration of Arginine (Arg), Serine (Ser), Valine (Val), Threonine (Thr), Isoleucine (Ile), Leucine (Leu), Phenylalanine (Phe), and Tryptophan (Trp).The highest intensity contrast was given by C4H6NO+ and C5H12N+ ion peaks characteristic of Glu/Gln and Ile/Leu, respectively.In the case of WGA, the intensity of the C4H6NO+ ion was higher than in the PHA-L spectrum.The signal ratio (C4H6NO+WGA/ C4H6NO+PHA-L) was of 1.6.The intensity of the characteristic signal of the Ile/Leu was ~3folds higher for PHA-L than WGA .Both lectins showed a high and comparable (4.75(10) 10 -2 and 4.01(6) 10 -2 ) intensity of the Lysine (Lys) signal (C5H10N+, m/z = 84).
Supporting Information S9 The surface distribution of WGA lectin within a single lectin pattern is shown in Figure S9.
ToF-SIMS data confirmed selective protein deposition independent of stamp preparation protocols.

Figure
Figure S1 100 µL drop of ultra-pure water deposited on PDMS cuboids before and after UV ozone Figures S2 and S3 show representative fluorescence images of WGA-TRITC and PHA-L-FITC layers, and normalized fluorescence intensities obtained for drop-cast and imprinted lectin layers.

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Figure S2 Representative fluorescence images of the substrate (a) and WGA-TRITC lectins

Figure
Figure4Sshows mean number of HCV29 and HT1376 cells per 1 mm 2 on lectin layers

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Figure S4 Adhesion of HCV29 and HT1376 cells to PHA-L and WGA-modified substrates prepared using DC or µCP.Data presented as mean ± SD.

Figure
Figure S7 Schematic illustration of printing of lectin patterns with a PDMS stamp.

Table S2 ToF
-SIMS examination of the amino acids composition of the WGA and PHA-L lectins.Comparison of the intensity of secondary ions derived from different amino acids acquired for WGA and PHA-L layers drop cast on APTES-modified glass substrates.The intensity of selected peaks from each spectrum was normalized to the sum of amino acids derived ions.