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Ultra-Thin Plasma-Polymerized Functional Coatings for Biosensing: Polyacrylic Acid, Polystyrene and Their CoPolymer

Recently, many efforts have been done to chemically functionalize sensors surface to achieve selectivity towards diagnostics targets, such as DNA, RNA fragments and protein tumou‐ ral biomarkers, through the surface immobilization of the related specific receptor. Especially, some kind of sensors such as microcantilevers (gravimetric sensors) and one-dimensional photonics crystals (optical sensors) able to couple Bloch surface waves are very sensitive. Thus, any kind of surface modifications devoted to functionalize them has to be finely controlled in terms of mass and optical characteristics, such as refractive index, to mini‐ mize the perturbation, on the transduced signal, that can affect the response sensitivity towards the detected target species. In this work, the study and optimization of ultra-thin plasma polymers and copolymers, compatible with these constrains and obtained from the vapours of acrylic acid containing a carboxylic (−COOH) group and styrene (an aromatic molecule with a vinyl as substituent at the ring), are reported. The obtained plasma polyacrylic acid (PPAA), plasma polystyrene (PPST) and their copolymer (PPAA–ST), characterized through optical contact angle analysis (OCA), Fourier transform infrared (FTIR) spectroscopy in attenuated total reflection (ATR-FTIR), X-ray photoelectrons spectroscopy (XPS), and atomic force microscopy (AFM), are shown to match specific and critical requirements, such as low thickness (∼40 nm) and refractive index (∼1.5), high surface density of reactive groups (10 15 –10 16 COOH/cm 2 ), bio-antifouling properties where required, reproducibility, chemical resistance and stability.


Introduction
In the last decades, one of the most critical issues in biosensing devices was the proper surface chemical modification aimed to covalently immobilize a bioreceptor (e.g. a capture antibody) or probe (a single-stranded DNA, ss-DNA), able to selectively bind a target, by matching an antigen or an oligonucleotide sequence (DNA or RNA fragment) of interest for biodiagnostics, respectively [1][2][3]. Polymeric thin coatings, such as polyacrylic acid, polydi(ethylene glycol) monovinyl ether, maleic anhydride, obtained by a plasma-enhanced chemical vapour deposition (PECVD) [4][5][6][7][8], at low-pressure conditions, are quite suitable for the chemical functionalization of a sensing surface [9,10], owing to the following reasons: (i) they do not change the chemical properties of the bulky material of a sensing device so leaving unaffected its working principle; (ii) they are conformal and (iii) do not need particular chemistry exposed to the sensor surface for adhesion; and (iv) they can show different features in terms of density of functional groups, wettability (hydrophilicity/hydrophobicity) and chemical stability by simply varying the process parameters (modulation of the plasma discharge, monomer or mixture of monomers/process gas, reactant vapour partial pressure, etc.), even though the same reagent is used as precursor (acrylic acid, vinyl acetic acid, allyl alcohol, allylamine) [11][12][13].
The chemical stability, in aqueous solution or other solvents (according to the biological protocol/technological procedure), is one of the mandatory goal to achieve, dealing with the plasma polymers exposing monotype functional groups suitable for covalent binding, as reported for polyacrylic acid and polyallylamine [14][15][16][17][18][19]. To enhance this last aspect, plasma copolymerization can be adopted to better tune the distribution and density of functional groups by mixing the monomer containing the functional groups (e.g. acrylic acid, allylamine, allyl alcohol) of interest with a precursor which does not contain specific functionalities (e.g. ethylene, styrene) and which has the function to dilute the reactive groups at the surface [20]. This last kind of monomers can be used alone to get a plasma polymer with bio-antifouling properties so allowing, for reference purpose, the detection of a blank response entirely unaffected by unspecific adhesion of biological materials, as shown by Sardella et al. for surface micro-domains of plasma-polymerized polyacrylic acid and polyethylene oxide [21]. The described polymers are also compatible with microscale lateral patterning based on photolithography combined with multiple subsequent plasma deposition steps. [22] Concerning high-performing sensors, such as gravimetric (microcantilevers) [23] or optical (Bloch surface waves (BSW)-based one-dimensional photonic crystals) [24][25][26][27] microdevices, the limited thickness of the functionalizing coating represents a crucial task to reach in order to avoid losses of sensitivity in the biodetection, as, for the first type, additional masses have to be minimized and, for the second type, the perturbation of refractive index at the surface of the sensing device has to be finely tuned.
In this work, the study, optimization and characterization will be presented of ultra-thin plasma polymers and copolymers obtained from the vapours of acrylic acid containing a carboxylic (−COOH) group and styrene (an aromatic molecule with a vinyl as substituent at the ring). A particular attention will be devoted to match specific and critical requirements, such as low thickness and refractive index, high surface density of reactive groups, bioantifouling properties where required, reproducibility, and chemical resistance and stability.
Several plasma process conditions, amount ratio of the two kinds of precursors and thicknesses, ranging from 10 to 50 nm, were considered and the related coatings were characterized by means of OCA, ATR-FTIR, XPS and AFM. Through these techniques, it was possible to assess the chemical composition of the obtained films, the hydrophilicity and hydrophobicity, the effect of solvents on the chemical structure and thickness of coatings due to eventual dissolution.
The −COOH surface density of plasma-polymerized acrylic acid (PPAA) and copolymer (PPAA-ST) obtained by mixing styrene moieties to acrylic acid vapours during the plasma process was quantified by means of colorimetric titration through toluidine blue O (TBO) [28,29].
Finally, the surface activity, towards test fluorophore-labelled proteins, versus binding or antifouling properties of the three selected polymers (PPAA, PPST and their copolymer) was studied by fluorescence microscopy.

Plasma polymerization system
Plasma treatments were carried out at room temperature in a low-pressure plasma-enhanced CVD reactor (chamber base pressure = 28 mTorr; RF = 13.56 MHz) purchased from IONVAC s.r.l., Rome, Italy [30]. The system is equipped with a delivery frame suitable to inject organic vapours coming from liquid reactants (monomeric precursors) stored in reservoirs intercepting the carrier gas lines.
The parameters of the plasma processes will be reported and discussed along the text in the Findings paragraph.

Characterization techniques
OCA measurements were used to investigate the surface properties of the differently functionalized samples in terms of hydrophilicity and hydrophobicity The OCAH 200 instrument (DataPhysics Instruments GmbH), used for the characterization, is equipped with a chargecoupled device (CCD) camera and an automatic dosing system for the liquids. The selected ones for the sessile droplet method in static mode analysis (droplet volume = 1.5 μl) were deionized water MilliQ grade (H 2 O) and diiodomethane (CH 2 I 2 , Sigma Aldrich). The Young-Laplace method was applied to fit the drop profiles and the SCA20 software was used to calculate contact angles between fitted function and base line. A data set of at least three droplets for each liquid was used to evaluate the standard deviation for each kind of sample.
ATR-FTIR spectra were collected in the range 4000-600 cm −1 , at 4 cm −1 resolution. Sixty-four scans were accumulated for each spectrum on a Nicolet 5700 FTIR spectrometer (ThermoFisher) equipped with a room temperature working deuterated triglycine sulphate (DTGS) detector and a diamond crystal ATR accessory.
A PHI 5000 Versaprobe scanning X-ray photoelectron spectrometer (monochromatic Al Kalpha X-ray source with 1486.6-eV energy) was employed to check the material surface chemistry. High resolution (pass energy: 23.5 eV) and survey spectra (pass energy: 187.85 eV) have been collected using a beam size of 100 μm. A combination of an electron gun and an Ar ion gun has been used as a neutralizer system to compensate the positive charging effect during the analysis, due to not perfectly conductive surfaces. Fitting procedure and deconvolution analysis have been done using the Multipak 9.6 dedicated software. All core-level peak energies were referenced to C1s peak at 284.5 eV (C-C/C-H sp 2 bonds). AFM measurements were performed by the WiTec AlphaSNOM system that implements a combined AFM and scanning near-field optical microscopy (SNOM) in collection/illumination and scattering mode by employing microfabricated, hollow pyramidal probes. The profile step measurements by AFM module of the deposited plasma polymers were collected by tapping mode.
Wide-field fluorescence microscopy was performed by a LEICA DM-LM microscope (objective 20×) equipped with a Hg vapour arc lamp (50 W) and L5-type combination filters suitable for fluorescein-like fluorophore. The coated samples (Si and SiO 2 ) were incubated with a 0.1 mg/ ml proteinA, Alexa Fluor 546 aqueous solution (dispensed volume = 10 μl) and rinsed in PBS, pH 7.3 + H 2 O to remove unbounded protein.
Colorimetric titration of -COOH functionalities of PPAA and PPAA-ST films was performed by means of TBO. The amino group contained in TBO molecule reacts specifically with a surface carboxylic group according to a 1:1 ratio [28]. Functionalized samples (c-Si substrates were used) were contacted with 3 ml of 0.5 mM TBO aqueous solution (pH = 10) at 37°C, for 5 h, in the dark. Then samples were copiously rinsed with 0.1 mM NaOH solution to remove the unreacted dye. Afterwards, samples were transferred in 1.5 ml of 50% v/v acetic acid solution and shaken for 10 min, in order to completely release the TBO linked to the carboxylic functional groups. Colorimetric dosing of TBO (and consequently of carboxylic groups) was performed by using a 2100-C microplate reader (Ivymen Optic System), by recording the optical densities (OD) of the solutions recovered after the reaction with the samples at 630 nm. A solution of 50% v/v acetic acid was used as reference background. By applying the Lambert-Beer law to the OD recorded for the different samples, on the basis of the comparison with an internal calibration obtained with TBO solutions (at known concentrations and volumes), and through the normalization for the geometric surface of the test samples, the evaluation of carboxylic functionalities density (in terms of groups/cm 2 ) can be performed.
In order to get information on the chemical characteristics of PPAA, PPST and PPAA-ST films, flat substrates, that is silicon (CZ/1-0-0, boron/P-type), Corning (Danville) glass and polyethylene (low-density PE; thickness 2.3 mm; Good Fellow) were used for the most of characterizations. For OCA, the different substrates were used in order to verify that the coating has the same properties without any dependence on substrate chemical composition, even for low thicknesses. For FTIR-ATR spectroscopy, silicon was not used owing to its refractive index higher than the ATR module diamond crystal. PE and Corning glass substrates have absorptions in different spectral range hiding thin film vibrational absorption features in different complementary parts of the selected range of spectra collection. The use of both substrates allows the investigation of the plasma polymer vibrational modes in the whole 400-4000 cm −t . For XPS and AFM, only silicon was used.

Plasma polymerized acrylic acid
The advantages of using acrylic acid as a precursor for plasma polymerization are the quite low vapour pressure that makes controllable the flux of vapours by manual metering valve (calculated AA vapour flow was approximately of 3 sccm), the nearly absent toxicity of reactants, the presence of an unsaturation (vinylic double bond) allowing the radical polymerization mechanism and a carboxylic functionality (−COOH). This chemical group is particularly suitable to biosensing applications because it forms easily amidic bridges by reacting with amino groups of biomolecules (e.g. receptor antibodies) with (binding efficiency is enhanced) or without further chemical activation.
In previous works of the authors [4,26,30,31], films with thicknesses included between 80 and 120 nm (processes performed by T Dep between 10 and 20 min) and prepared by means of both continuous wave (CW) and modulated wave (MW) plasma discharge were widely studied. The coating obtained by CW discharge were shown to not retain enough carboxylic functionalities while, among MW coatings, only the film obtained with an RF starting power of 200 W, a duty cycle of 0.1 with a t on of 10 ms and a t off of 90 ms with Ar as carrier gas, were optimal in terms of stability and reproducibility. A step of immersion in d-H 2 O is required after plasma synthesis in order to remove surface oligomers (left at the end of plasma process) so making the film chemically stable for the following interaction steps (biosensing) in aqueous media. Moreover, to enhance the adhesion among the substrate and the deposited film an Ar plasma treatment was required before the film deposition. Thick films allowed a complete and reliable surface characterization that consisted of OCA, FTIR-ATR spectroscopy, XPS analysis, fluorescence microscopy, quartz microbalance system and colorimetric titration. By means of the last, an exceptional value of 1.15 ± 0.35 × 10 16 groups/cm 2 was found. Moreover, from ellipsometric characterization, it also aroused that the optimal film has a refractive index of 1.51 measured on 40-nm thick layers, after d-H 2 O rinsing and from electrokinetic characterization based on the zeta (ζ) potential measurements, that allows the evaluation of acid-base properties, came that PPAA isoelectric point is at pH = 2.1. This indicates that the surface charge originates from the dissociation of the carboxylic acid groups of the PPAA chains and so from the deprotonation of -COOH species [4].
In the following, the characterization of PPAA ultra-thin films is reported in order to define the minimum thickness that can be reached without affecting the chemical resistance mainly in aqueous media and the functional group number and reactivity.
For these processes, Argon (Ar) is the AA vapour-carrier gas (flow =20 sccm). It is made bubble in liquid AA in order to enhance vapours formation and transport them into the chamber. The in-chamber total pressure in presence of the Ar-AA mixture is about at 220-240 mTorr, with a partial pressure of AA vapours of about 25 mTorr. The plasma parameters were an RF starting power of 200 W, a duty cycle of 0.1 with a t on of 10 ms and a t off of 90 ms.
After the film deposition, characterizations were performed before and after rinsing the substrates in de-ionized water (d-H 2 O) for 20 min.
OCA angles performed on bare silicon (Si) and Corning glass with d-H 2 O and CH 2 I 2 (diiodomethane) and related calculation of surface energy (SE, W sl ) divided in its disperse (W d sl )and polar (W h sl ) components, are reported in Table 1   The FTIR-ATR spectra of Figure 2, collected on both PPAA-deposited PE and Corning glass, confirm that the species exposed at surface are carboxylic groups and that they are also visible for the thinner film.  In the following, the correlation between T Dep and film thickness in the range 0-50 nm was performed by means of AFM for profilometric purposes, in order to get a calibration of selected process and information about deposition rate.
From Table 4 arises that the deposition rate has not a linear behaviour. For low times of deposition, the rate is not reproducible (several processes at the same deposition time were performed) due the starting step of pulsed discharge stabilization that is not controllable.

Plasma-polymerized styrene
Styrene is an aromatic molecule (C 6 H 6 -CH=CH 4 ) which, by decomposing, during plasma process, and reassembling in form of cross-linked chains, allows to get a polymer with mechanical toughness, thermal stability, dielectric properties and chemical resistance/ inertness. These properties make it a good bio-antifouling surface. Moreover, it can be used to dilute other functional monomers, thus allowing to introduce aliphatic fragments/aromatic rings among carboxylic groups arising from a chemically functional monomer, such as acrylic acid. A plasma copolymer can be so obtained, as reported in the next paragraph.
The explored processes, by changing different parameters such as the discharge mode (CW and MW), were performed at 2 and 10 min of deposition time. With respect to AA, the monomer flow, measured when the manual valve intercepting the reservoir is open, is not easily controllable and it is highly sensitive to external temperature variations. This could create problems for the reproducibility of the processes and does not allow the measure of the vapour flow of styrene as in the case of acrylic acid. XPS analysis was carried out on selected spectra, obtained with time of deposition of 10 min, in order to confirm the presence of intact benzene ring and to evaluate the content of oxygen that, even if unwanted, seems to be embedded in the coatings (due to quite low vacuum produced by the chamber pumping system). This side effect is supported by data reported in literature.
The analysis has been performed on the MW-obtained coatings only. Figure 5 reports the C1s HR spectrum (a) and the O1s HR spectrum (b) of process PPST_2 of Table 5, obtained at P = 200W; DC = 0.1; t on /t off = 20 ms/180 ms. Peaks have been deconvoluted using Gaussian-Lorentz functions, while backgrounds have been removed with Tougaard functions. Fitting peaks are: • the lower energy peak (284.3 eV) has been assigned to ring carbons;, • the peak at 284.5 eV has been attributed to the presence of C atoms linked as C-C/C-H, • the peak at 285.5 eV is commonly attributed to C-O-C, • the higher energy peak (~291 eV) is due to π-π * transitions arising from the presence of the aromatic rings. It is called "shake-up" satellite because related to "shake-up" excitations taking place in the π orbitals on the benzene rings.
Concerning the O1s HR spectrum, only one fitting peak was found, related to C-O-C chemical bonds. The processes of the PPST_2 kind are the most reproducible, without a dependence on the deposition time, for this reason it is the most representative to single out the features of the HR C1s and O1s spectra of all the PPST processes.
For all processes, XPS peaks, in the energy range corresponding to valence band photoelectrons, are similar to that of a standard polystyrene. In fact, an extensive amount of information is also available from the valence band region. This is because the shifts in this region arise from changes in chemical bonding, rather than the chemical shifts caused by the environment found in the core region.  Table 6. O/C atomic ratio referred to elements percentage obtained from survey spectra of PPST films.
From Table 6 comes that for all processes the content of oxygen is quite negligible. Figure 6a shows that, for the most stable process in terms of water resistance, the energy band affected by the rinsing in water is the one relate to ring carbons, so suggesting that the chemical weakness of PPST is the cross-linking among the benzene rings. However, this effect does not affect the hydrophobic/antifouling properties of the PPST coating.

Figure 6b
reports the intensity distribution of components of C1s spectrum of three selected processes ( Table 6). The comparison puts in evidence that the process at t on /t off = 20/180 produces a higher amount of ring carbons with respect to the others.  The process PPST_2 10 min; P = 200W; DC = 0.1; 20 ms/180 ms is the most performing in terms of low grade of oxidation, good retention of aromatic chemistry that ensures inertness and discourage electrostatic interaction.
To investigate on the bio-antifouling properties of it, it was deposited on the silica (SiO 2 ) surface of a glass substrate (a sector of the sample was masked) and incubated for 30 min with a solution of PtA labelled with a fluorescent marker (Alexa Fluor 546). After rinsing and drying under N 2 flux, according to the "Experimental" Section, the sample was observed by fluorescence microscopy. It is evident from Figure 7a that selected PPST coating strongly limits unspecific adsorption of biomolecules, as fluorescent emission from PPST is lower or nearly absent with respect to the one coming from SiO 2 . Moreover, concerning the lateral patterning involving PPST and PPAA is evident from Figure 7b that PPAA is highly PtA chemisorbing and PPST is bio-antifouling.
Also for PPST such as for PPAA is mandatory to verify that the compositional characteristics are retained also for thin layer. For this reason, the OCA measurements were performed on coatings obtained by different deposition time on the standard test substrates: Si and Corning glass.
The measured angles and related surface energies for coated Si and Corning substrates are reported in Tables 7 and 8, respectively. OCA H₂O are quite high and confirm the hydrophobic properties of all the films. But a slight decrease of it is observed by incrementing the deposition time (here expressed in seconds). This trend suggests that longer is the deposition time, larger is the side oxidation effect produced by the process, and that is confirmed by FTIR-ATR spectra. Moreover, it is confirmed by the increase of the polar component of surface energy W h sl . OCA measurements were performed also on films after acetone rinsing, in view of photolithographic approaches for lateral patterning which involve the production of alternated chemical domains PPAA/PPST. OCA H₂O slightly increases (see Figure 8) but the films chemically resists to the treatment. The FTIR-ATR spectra of Figure 9, collected on the OCA characterized samples, on both PPST deposited PE and Corning, show the band associated to the C=C stretching in the aromatic ring at 1600 cm −1 and below 1200 cm −1 , the features due to the aliphatic backbone chain absorption modes. Most of spectra are present between 1730 and 1645 cm −1 absorption due to slight oxidation occurring during the process, and only for the thicker films, at 2980-2870 cm −1 , the signals related to aliphatic C-H stretching mode are visible. An effect of thickness growth is observed as a slight increase of all described fingerprints. Finally, an effect of enhancement of aliphatic C-H stretching absorption, due to acetone rinsing is observed on the PPST_2-600s (10 min), coating (pink dashed curve), in accordance with OCA results (increase of hydrophobicity). Water rinsing seems to leave unaffected the PSST coating (related spectra are the dotted curves). In order to get a calibration of thicknesses of PPST in the lower range possible, according to deposition time, AFM was used with a profilometric approach and measured thicknesses are reported in Table 9.  For T Dep lower than 2 min, the rate is not reproducible due to the starting step of pulsed discharge stabilization that is not controllable. For this reason, the minimum deposition time allowing reproducible thicknesses is 120 s (2 min).

Copolymer
Plasma copolymerization is based on the activation of a plasma process in a gaseous mixture where the vapours of a monomer containing chemical active functionalities (acrylic acid) are mixed, in the reaction chamber, with the vapours of a chemically inert aliphatic chain/aromatic rings monomer, styrene, to achieve a sort of controlled "dilution" of the first in the second.
Thus, this mechanism allows to increase the film stability by lowering the number of carboxylic groups at surface and incrementing the cross-linking degree of the polymer in terms of backbone. Due to the higher reticulation, the film becomes more suitable for biological applications where the sample has to be in contact with aqueous biological fluids for a long time. Some preliminary results were obtained [4] from ellipsometric and electrokinetics measurements. For the former, the high T Dep copolymerized coating shows a thickness that is the double of that evaluated for the PPAA coating, obtained with the same discharge parameters and duty cycle. For the latter, the isoelectric point of the thick film (T Dep = 7.5 min) obtained in the same operating conditions of PPAA-ST-1 ( Table 10) was close to pH = 4, typical of inert surfaces.
Four kinds of processes (three in MW and one in CW mode), performed at the same T Dep (5 min), are here below reported and characterized by means of OCA, ATR-FTIR and XPS analysis, in accordance to the parameters optimized for PPAA and PPST and by taking advantage from the previous preliminary copolymerization attempts.   Figure 10 are shown the images of main OCA H₂O contact angle related to PPAA-ST_1, PPAA-ST_2 and PPAA-ST_3 processes of Table 10. Figure 10. OCA H₂O images for the first three processes listed in Table 12, on Si.
FTIR-ATR spectra related to processes listed in Table 10 are reported in Figure 11.
As observed in Figure 11, spectra related to plasma copolymers reveal a sum of the features of both the reference polymers (PPAA and PPST). In particular, the spectrum of the film obtained with a t on /t off = 50/50 show characteristics similar to pure PPST. The others, except for the lower intensity of the signals related to the O-H and C=O stretching vibrations, show all the spectral features of carboxylic functionalities of pure PPAA. Under water rinsing, the most show good chemical resistance (except PPAA-ST_4). Under acetone rinsing, only the t on /t off = 50/50 process (PPAA-ST_2) is not dissolved (not shown).
Finally, XPS analysis was carried out in order to evaluate the chemical nature of the resulting copolymers (PPAA-ST): to get the evidence of both benzene rings/aliphatic chain presence and carboxylic functionalities at different grade of ratio. The analysis has been performed on the MW-obtained coatings only, listed in Table 10. Figure 12 reports the C1s HR spectrum (a) and the O1s HR spectrum (b) of process PPAA-ST_2. Peaks have been deconvoluted using Gaussian-Lorentz functions, while backgrounds have been removed with Tougaard functions. Fitting peaks are: • The lower energy peak (284.3 eV) has been assigned to ring carbons, • The peak at 284.5 eV has been attributed to the presence of C atoms linked as C-C/C-H, • The peak at 285.9 eV is commonly attributed to C-O-C/C-O-H, • The peak at 288.6 is commonly attributed to COOH, O-C=O, • The higher energy peak (~291 eV) is due to π-π * transitions arising from the presence of the aromatic rings, • The peak at 532.2 eV is attributed to C-O-C, • The peak 533.5 eV is commonly attributed to COOH/C=O. For process PPAA-ST_2, the C1s component related to π-π * transition is higher than in spectrum related to process PPAA-ST_3 performed by a different DC and t on /t off ratio (0.1 and 20/180). In this last (Figure 13), all the components, of both HR C1s and HR O1s, are related to the higher embedding of acrylic acid species during chain propagation. The comparison of all HR-C1s spectra (Figure 14) shows that the content of carbon and related components are different each other, as function of processes parameters. In particular, after rinsing of PPAA-ST_2 sample in acetone (spectrum PPAA-ST_2I), a little part of π-π * transition component is lost and the carboxylic component increases, but this is the only kind of tested coating that quite resists to acetone treatment (in view of the photolithographic process for lateral patterning).

Figure 14.
Comparison of HR C1s spectra for the first three processes listed in Table 10. Figure 15. Comparison between all PPAA-ST processes valence band (left) HR spectra and reference valence bands spectra of polystyrene and a plasma-polymerized acrylic acid, reported in literature [32,33] (right-image re-plotted by authors).
Notwithstanding evident differences among the spectra, all of them are quite consistent, in the low energy range of valence band, with the literature reference for plasma-polymerized acrylic acid, as shown in Figure 15 (red spectrum in the graph on the left).
The trend of O/C ratio ( A colorimetric titration with TBO has been performed on PPAA-ST coatings deposited on Si according to different process parameters, after H 2 O rinsing. Through this approach, it is possible to quantitatively estimate the surface density of -COOH groups exposed at the film surface and available for biomolecules grafting. for DC = 0.5, the content of carboxylic reacting groups is under technique limit of detection, while for DC = 0.1 and CW process, the content is comparable with PPAA carboxylic groups density and so promising for biomolecules grafting.  In order to get a calibration of thicknesses of PPAA-ST in the lower range possible, already reported for PPAA (10-50 nm) according to deposition time, AFM was used with a profilometric approach.   From Table 15 arises that the deposition rate has not a linear behaviour and is dramatically huge with respect the other plasma polymers. The same behaviour is observed for the other processes of Table 10.
For PPAA-ST, the minimum deposition time possible, providing about 40-nm thick coatings, is 30 s, but this is quite uncontrollable due to the starting step of stabilization occurring when pulsed plasma discharge is activated.

Conclusions
In this work, the reliability to obtain, through a methodic approach, ultra-thin plasma polymerized coatings, about 40-nm thick, able to retain the chemical properties of the pristine precursor, for PPAA and PPST, in terms of binding and bio-antifouling, respectively, notwithstanding the quite low thickness and thus the short extension of macromolecular crosslinking in Z direction. PPAA coatings expose accessible carboxylic groups at a different grade of surface density (up to about 10 16 -COOH groups/cm 2 ) and are stable in several aqueous media. The most performing PPST coating, with hydrophobic and so antifouling properties (that discourage proteins adsorption), resists also to solvent immersion, such as acetone.
Finally, an ultra-thin plasma copolymer, PPAA-ST, that merges the properties of PPAA and PPST was developed in order to enhance chemical stability of the chemically functional coating by diluting hydrophilic carboxylic functionalities. Moreover, the introduction of a higher content of aliphatic/aromatic components provided the reinforcement of the coating carbon backbone.
It is worth of note that, for a particular AA/ST ratio (PPAA-ST_1 process with DC = 10%; t on = 10 ms, t off = 90 ms), notwithstanding the increase in aliphatic fragments/aromatic rings content and thus in chemical resistance, the number of carboxylic groups exposed at the surface was shown to be only one order of magnitude lower than the one obtained for PPAA.