Abstract
Surface Plasmon Resonance assays are being developed as alternative biodetection methods for a great number of pesticides and toxins. These substances typically have low molecular weight, making it necessary to perform competitive inhibition immunoassays. In most of the cases, the strategy is to immobilize a protein derivative of the analyte, which usually involves the appearance of nonspecific protein binding which limits the detection range of the assay. In this work we present results of a poly-L-lysine (Au-MUA-PLL) based sensor platform for quantitative determination of 2,4-dinitrophenol as model system for small molecular weight substances detection. The prepared sensor chip was characterized by means of Atomic Force Microscopy, Surface Plasmon Resonance, and Surface Enhanced Raman Spectroscopy. Experiments verified the absence of nonspecific protein adsorption to Au-MUA-PLL surfaces and the improvement of the competitive inhibition assays performance in comparison with single and mixed thiol self-assembled monolayers. The possibility of directly immobilizing 2,4-dinitrophenol to the poly-L-lysine containing platforms leads to an improvement in the detection of the soluble analyte by the competitive inhibition assay avoiding undesirable nonspecific protein adsorption. Therefore, Au-MUA-PLL surfaces constitute a suitable alternative for quantitative detection of small molecules when nonspecific adsorption cannot be avoided.
Highlights
Biosensors based in Surface Plasmon Resonance (SPR) have become increasingly employed in analytical determinations of a broad range of biological molecules, pesticides, environmental pollutants, explosives, pharmacological compounds, and substances employed in medical diagnosis [1,2,3,4,5,6,7]
Similar competitive inhibition assays in Bovine Serum Albumin (BSA)-DNP covered single thiol self-assembled monolayer (SAM) resulted in poor degree of signal inhibition (36% for anti-DNP 38 nM incubated during 20 minutes with DNP 2 mM) or no inhibition at all in BSADNP immobilized over Au-mercaptoundecanoic acid (MUA)/DTT
PLL films yielded a thickness of 30 ± 1 Aand, according to Atomic Force Microscopy (AFM) imaging, they cover the MUA self-assembled monolayer following the nanocrystalline gold surface
Summary
Biosensors based in Surface Plasmon Resonance (SPR) have become increasingly employed in analytical determinations of a broad range of biological molecules, pesticides, environmental pollutants, explosives, pharmacological compounds, and substances employed in medical diagnosis [1,2,3,4,5,6,7]. In order to expand the analyte concentration working range, it is of utmost importance to minimize nonspecific antibody binding, either to the sensor platform or to the analyte derivative. With the aim of minimizing the nonspecific protein binding, the coating of the sensor chips should reduce both the electrostatic and the steric interactions with proteins In this sense, poly-L-lysine (PLL) appears as interesting coating to investigate as it is a polycation at usual working pHs. In this sense, poly-L-lysine (PLL) appears as interesting coating to investigate as it is a polycation at usual working pHs This homopolypeptide has been covalently attached via amide bonds to an alkanethiol self-assembled monolayer (SAM) on a gold surface [21], resulting in layers with typical thickness that are in accordance with the formation of a PLL monolayer [21]. The improvement of competitive inhibition immunoassays performed of SPR sensor chips coated with MUA-PLL is demonstrated
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