Supporting Information: Plasmonic hepatitis B biosensor for the analysis of clinical saliva

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1 Supporting Information: Plasmonic hepatitis B biosensor for the analysis of clinical saliva Tomáš Riedel, *, Simone Hageneder, František Surman, Ognen Pop-Georgievski, Christa Noehammer, Manuela Hofner, Eduard Brynda, Cesar Rodriguez- Emmenegger, *,, Jakub Dostálek *, Institute of Macromolecular Chemistry AS CR v.v.i., Heyrovsky Sq. 2, Prague 6, Czech Republic Biosensor Technologies, AIT-Austrian Institute of Technology GmbH, Muthgasse 11, 1190 Vienna, Austria Molecular Diagnostics, Health and Environment Dpt., AIT-Austrian Institute of Technology GmbH, Muthgasse 11, 1190 Vienna, Austria DWI Leibniz Institute for Interactive Materials and Institute of Technical and Macromolecular Chemistry, RWTH Aachen University, Forckenbeckstraße 50, Aachen, Germany Corresponding Author * Tomáš Riedel, riedel@imc.cas.cz, Phone: , Fax: Cesar Rodriguez-Emmenegger, rodriguez@dwi.rwth-aachen.de, Phone: , Fax: Jakub Dostálek, jakub.dostalek@ait.ac.at, Phone: +43 (0) , Fax: +43 (0) Characterization of the brush architecture X-ray photoelectron spectroscopy (XPS) XPS measurements were carried out with a K-Alpha + spectrometer (ThermoFisher Scientific, East Grinstead, UK). The samples were analyzed using a micro-focused, monochromated Al Kα X-ray source (400 µm spot size) at an angle of incidence of 30 (measured from the surface) and an emission angle normal to the surface. The kinetic energy of the electrons was measured using a 180 hemispherical energy analyzer operated in the constant analyzer energy mode (CAE) at 200 ev and 50 ev pass energy for the survey and high resolution spectra respectively. Data acquisition and processing were performed using Thermo Advantage software. The XPS spectra were fitted with Voigt profiles obtained by convolving Lorentzian and Gaussian functions. The analyzer transmission function, Scofield sensitivity factors, and effective attenuation lengths (EALs) for photoelectrons were applied for quantification. EALs were calculated using the standard TPP-2M formalism. All spectra were referenced to the C1s peak of hydrocarbons at ev. The BE scale was controlled by the well-known position of the photoelectron C-C and C-H, C-O and C(=O)-O peaks of

2 polyethylene terephthalate and Cu 2p, Ag 3d, and Au 4f peaks of metallic Cu, Ag and Au, respectively. The BE uncertainty of the reported measurements and analysis is in the range of ±0.1 ev. The XPS analysis verified the covalent structure of the poly(hpma-co-cbmaa) brushes grown from the initiator SAM on Au via SI-ATRP. The core level C 1s spectrum (Figure 2 a) of the brushes is characterized by C-C, C-H (285.0 ev), C*-C(=O)- from the secondary chemical shifts induced to the carbon atoms in the vicinity to amide and charged carboxylic groups (285.5 ev), C-N (286.1 ev), C- OH (286.9 ev), C(=O)-NH (288.0 ev) and C(=O)-Ocarboxylate (288.7 ev). The high resolution N 1s spectrum of the brushes is characterized by a prevailing amide contribution (400.1 ev) and a contribution characteristic for the quaternary ammonium cation of the CBMAA comonomer (403.0 ev). The corresponding O 1s spectrum was fitted with two contributions arising from the C=O (531.4 ev) and C-O (532.6 ev) moieties of HPMA and CBMAA. Table S1 reports the surface atomic concentration of the identified moieties. Based on the obtained XPS results, the composition of the poly(hpma-co-cbmaa) brushes was estimated to be 87% HPMA and 13% CBMAA. Figure S1. High resolution C 1s, O 1s and N 1s XPS spectra of poly(hpma-co-cbmaa) brushes grown from the sensor surface by SI-ATRP. Measured spectra are presented with black lines, while their corresponding fitted envelopes are presented in red. The individual contributions of different functional groups are represented with dotted blue lines.

3 Table S1. Surface concentration of chemical moieties present on the surfaces of poly(hpmaco-cbmaa) brushes as determined by XPS. C1s N1s O1s Moiety Binding Surface energy concentration [ev] [atomic %] C-C, C-H ± 0.7 C*-C=O ± 0.6 C-N ± 0.2 C-O ± 1.2 C(=O)-NH ± 0.4 C(=O)-O ± 0.8 NH-C(=O) ± 0.1 N + (CH 3 ) ± 0.1 O=C ± 0.4 O-C ± 0.6

4 Fourier transform infrared grazing angle specular reflectance spectroscopy (FTIR- GASR) FTIR-GASR was carried out using a Nicolet Nexus 870 with a SAGA attachment. In total 256 scans at a resolution of 2 cm 1 were recorded for each sample and processed with OMNIC software. The spectrometer was purged continuously with dry air. The FTIR GASR spectrum of poly(hpma-co-cbmaa) brushes (Fig. S2) exhibits both a band at 1376 cm 1 and a shoulder band at 1610 cm 1 corresponding to the symmetric and asymmetric stretching modes of COO stemming from CBMAA as well as it shows the amide I and amide II bands at 1527 and 1653 cm 1 originating from the HPMA. Figure S2. FTIR GASR spectrum of the poly(hpma-co-cbmaa) brush.

5 Atomic force microscopy (AFM) Multimode AFM Nanoscope IIIa (Digital Instruments) was used to investigate the topography of the polymer brushes. The scans were performed in tapping mode in water. Area of 5x5 µm2 was scanned at a rate 0.5 Hz and analyzed using Gwyddion software. The AFM images showed smooth and homogenous coverage with the polymer brush without any uncovered areas (Fig. S3). The roughness (root mean square) of the surface was Rq = 1.4±0.1 nm. Figure S3. A topographical image of the poly(hpma-co-cbmaa) brush. Scale bar is 1 µm. Post-modification of brushes with protein ligand The selection of the buffer was optimized based on the following premises: (1) Favor activation of carboxylic groups (formation of N-hydrosuccynimide active ester) over the hydrolysis of the active ester, and (2) the amidation of proteins to the carboxylic groups of the brushes over the hydrolysis of the N-hydrosuccynimide. The active ester is more prone to

6 hydrolysis at basic ph compared to slightly acidic conditions, that is why we performed the activation in SA buffer and water. Moreover, the SA buffer better solubilizes any residual EDC or NHS which could remain on the surface. The immobilization of the antigen (pi 4.6) [Lee, Y. S.; Kim, B. K.; Choi, E. C., Physicochemical properties of recombinant hepatitis B surface antigen expressed in mammalian cell (C127). Archives of Pharmacal Research 1998, 21 (5), ] is conducted at 7.4 in HEPES buffer. The copolymer brushes are composed of HPMA (neutral) a betaine (quaternary ammonium always positively charge and carboxylate: negatively charged above ph 4) and some betaines activated (only the quaternary ammonium). Thus the surface in the immobilization procedure can only be positively charged. At ph 7.4 (HEPES) the antigen is slightly negatively charged (pi 4.6). Therefore the antigen will be attracted to the surface increasing the efficiency of the covalently coupling. The sample is then incubated in PBS so that any unreacted succinimide group is hydrolyzed.