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Surface plasmon resonance (SPR) biosensors have become an important label-free optical biomolecular sensing technology and a "gold standard" for retrieving information on the kinetics of biomolecular interactions. Even though biomolecules typically contain an abundance of easily ionizable chemical groups, there is a gap in understanding of whether (and how) the electrostatic charge of a biomolecular system influences the SPR biosensor response. In this work we show that negative static charge present in a biomolecular layer on the surface of an SPR sensor results in significant SPR spectral shifts, and we identify two major mechanisms responsible for such shifts: 1) the formation of an electrical double layer (ionic mechanism), and 2) changes in the electron density at the surface of a metal (electronic mechanism). We show that under low ionic strength conditions, the electronic mechanism is dominant and the SPR wavelength shift is linearly proportional to the surface concentration of biomolecular charges. At high ionic strength conditions, both electric and ionic mechanisms contribute to the SPR wavelength shift. Using the electronic mechanism, we estimated the pKa of surface-bound carboxylic groups and the relative concentration of the carboxyl-terminated alkanethiols in a binary self-assembled monolayer of alkanethiols. The reported sensitivity of SPR to surface charge is especially important in the context of biomolecular sensing. Moreover, it provides an avenue for the application of SPR sensors for fast, label-free determination of the net charge of a biomolecular coating, which is of interest in material science, surface chemistry, electrochemistry, and other fields.

The non-specific binding of non-target species to functionalized surfaces of biosensors continues to be challenge for biosensing in real-world media. Three different low-fouling and functionalizable surface platforms were employed to study the effect of functionalization on fouling resistance from several types of undiluted media including blood plasma and food media. The surface platforms investigated in this work included two polymer brushes: hydroxy-functional poly(2-hydroxyethyl methacrylate) (pHEMA) and carboxy-functional poly(carboxybetaine acrylamide) (pCBAA), and a standard OEG-based carboxy-functional alkanethiolate self-assembled monolayer (AT-SAM). The wet and dry polymer brushes were analyzed by AFM, ellipsometry, FT-IRRAS, and surface plasmon resonance (SPR). The surfaces were functionalized by the covalent attachment of antibodies, streptavidin, and oligonucleotides and the binding and biorecognition characteristics of the coatings were compared. We found that functionalization did not substantially affect the ultra-low fouling properties of pCBAA (plasma fouling of ∼20 ng/cm 2 ), a finding in contrast with pHEMA that completely lost its resistance to fouling after the activation of hydroxyl groups. Blocking a functionalized AT-SAM covalently with BSA decreased fouling down to the level comparable to unblocked pCBAA. However, the biorecognition capability of blocked functionalized AT-SAM was poor in comparison with functionalized pCBAA. Limits of detection of Escherichia coli O157:H7 in undiluted milk were determined to be 6×10 4 , 8×10 5 , and 6×10 5 cells/ml for pCBAA, pHEMA, and AT-SAM-blocked, respectively. Effect of analyte size on biorecognition activity of functionalized coatings was investigated and it was shown that the best performance in terms of overall fouling resistance and biorecognition capability is provided by pCBAA.