Phospholipid/cholesterol/decanethiol mixtures for direct assembly of immunosensing interfaces

Abstract

a b s t r a c tIn this work, a simple yet robust method to prepare lipid-based biosensing interfaces on gold using common lipids (a phospholipid and cholesterol) and an alkanethiol is reported. The lipids were carefully chosen to tailor the biophysical properties of the bilayer. The simplicity of the method relies on the incorporation of a small percentage of decanethiol in the lipid vesicles for a direct formation of a thiol-linked supported lipid bilayer, which is advantageous in several respects. It prevents the use of specially synthesized thiolipids and preserves the natural fluidity and dynamics of the lipids. As a consequence the whole arrangement is extremely stable regarding ionic strength changes and solution flow during surface plasmon resonance experiments. Moreover, we show that this interface is very effective on suppressing the nonspecific adsorption of proteins on the surface, and enables the covalent attachment of the recognition antibody. The subsequent detection of specific interaction toward antigen was monitored in real-time by SPR and confirmed by ellipsometric measurements. This lipid-based biosensing platform is versatile and can be adapted to the biorecognition reaction of interest.

Nanostructured interfaces with site-specific bioreceptors for immunosensing

“…Finally, the anti-IgG detection was followed, and a variation of 8480 is detected in the grayscale value. This significant grayscale increase represents an anti-IgG/IgG ratio of 3.2, which corresponds to more than 2 anti-IgG molecules linked per each IgG, which is in agreement with the fact that the anti-IgG (secondary antibody) can react with several sites on the IgG (primary antibody) [18]. Furthermore, this evidence is corroborated by previous results obtained for covalently adsorbed IgG on SAM [8], proving that this is a proper platform to monitor the antiIgG detection by properly oriented IgG and, therefore, to be used as an immunosensor.…”

“…S1c, Supplementary Material), which can be attributed to the presence of the nanostructured gold on the substrate. In the absence of AuNPs, k values remain low (from 0.047 to 0.145), revealing that the organic films formed are nearly transparent, as expected for thin protein layers [18]. The addition of the AuNPs to the system make difficult the use of conventional ellipsometry at a fixed wavelength, since their optical properties are profoundly distinct from those of bulk gold surfaces [40].…”

“…To obtain the optical parameters of the bare gold, a two-phase model was used and to estimate the thickness of the protein layer, a three-phase model was employed, as reported in the literature [12,39], fixing the real part of the refractive index at 1.5 [40]. The low extinction coefficient values (k) obtained in the measurements, except when AuNPs were attached to the surface, indicate the presence of nearly transparent films, as expected for thin organic layers [18]. The modified gold surfaces were incubated with 0.1 mg/mL IgG and anti-IgG in PBST for 1 h at room temperature.…”

“…DTC have a resonant bidentate structure along N-C-S 2 and establish strong linkages to gold substrates [16]. This methodology has been used to immobilize a wide range of molecules and biocompounds [13,[17][18][19][20][21] and, importantly, to prepare immunosensor interfaces for the detection of biological molecules relying on the specific antibody-antigen reaction. Furthermore, the ability to prevent protein nonspecific adsorption to the biorecognition surfaces has been also demonstrated [12].…”

A novel in‐capillary assay for dynamically monitoring fast binding between antibody and peptides using CE

“…Although several elegant techniques have been developed for the study of antigen-antibody/peptide-protein interactions, such as surface plasmon resonance [1][2][3][4][5], ELISA [6][7][8][9], isothermal calorimetry [10,11], and microarrays [12][13][14][15][16], high-performance size-exclusion chromatography [17][18][19][20] etc., they require complex and expensive instruments, hindering ease of operation and desired separation. Therefore, a simple and rapid method is urgently needed to effectively Article Related Abbreviations: FL, fluorescence detection; FAM-DYKD, FAM-DYKDHDGDYKDHDIDYKDDDDK; M2, monoclonal anti-FLAG M2 antibody detect and monitor the binding process between antibodies and peptides.…”