Plasmonic Hepatitis B Biosensor for the Analysis of Clinical Saliva

A biosensor for the detection of hepatitis B antibodies in clinical saliva was developed. Compared to conventional analysis of blood serum, it offers the advantage of noninvasive collection of samples. Detection of biomarkers in saliva imposes two major challenges associated with the low analyte concentration and increased surface fouling. The detection of minute amounts of hepatitis B antibodies was performed by plasmonically amplified fluorescence sandwich immunoassay. To have access to specific detection, we prevented the nonspecific adsorption of biomolecules present in saliva by brushes of poly[(N-(2-hydroxypropyl) methacrylamide)-co-(carboxybetaine methacrylamide)] grafted from the gold sensor surface and post modified with hepatitis B surface antigen. Obtained results were validated against the response measured with ELISA at a certified laboratory using serum from the same patients.


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

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 COOstemming 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.

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 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. 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.