Synthesis of Steroid–Oligonucleotide Conjugates for a DNA Site-Encoded SPR Immunosensor

Abstract

The excellent self-assembling properties of DNA and the excellent specificity of the antibodies to detect analytes of small molecular weight under competitive conditions have been combined in this study. Three oligonucleotide sequences (N(1)up, N(2)up, and N(3)up) have been covalently attached to three steroidal haptens (8, hG, and 13) of three anabolic-androgenic steroids (AAS), stanozolol (ST), tetrahydrogestrinone (THG), and boldenone (B), respectively. The synthesis of steroid-oligonucleotide conjugates has been performed by the reaction of oligonucleotides carrying amino groups with carboxyl acid derivatives of steroidal haptens. Due to the chemical nature of the steroid derivatives, two methods for coupling the haptens and the ssDNA have been studied: a solid-phase coupling strategy and a solution-phase coupling strategy. Specific antibodies against ST, THG, and B have been used in this study to asses the possibility of using the self-assembling properties of the DNA to prepare biofunctional SPR gold chips based on the immobilization of haptens, by hybridization with the complementary oligonucleotide strands possessing SH groups previously immobilized. The capture of the steroid-oligonucleotide conjugates and subsequent binding of the specific antibodies can be monitored on the sensogram due to variations produced on the refractive index on top of the gold chip. The resulting steroid-oligonucleotide conjugates retain the hybridization and specific binding properties of oligonucleotides and haptens as demonstrated by thermal denaturation experiments and surface plasmon resonance (SPR).

From DNA Nanotechnology to Material Systems Engineering

“…It can also be used for silicon, gold and other materials. This enables plasmonic detection of small molecules, electrochemical screening of enzymes or the study of electron transfer processes in between proteins and metals . For proteome research, DDI on conductive silicon substrates is used .…”

“…The use of nucleic acid hybridization for functionalization of bulk materials is not limited to proteins but can be readily extended to small molecules, such as peptides,[154b] glycans, or hormones as well as to inorganic nanoparticles. The latter is of particular interest for the self‐assembly of DNA‐based materials.…”

DNA Surface Technology: From Gene Sensors to Integrated Systems for Life and Materials Sciences

“…This allows for the convenient removal of IVTT components and makes it possible to investigate the interaction potential of several thousand proteins with other binding partners.W hen the DNAc hips are embedded in microfluidic lab-on-a-chip systems,g lobal reaction parameters can be tightly controlled to improve experimental reliability,t hroughput, and multiplexing capabilities with concomitant reduction of reagent consumption and processing time. [23,27] Them ethod is not limited to glass or polymer surfaces but can also be used for silicon, gold, and other materials.E xemplary applications include the plasmonic detection of small molecules, [28] electrochemical screening of enzymes, [29] or the investigation of electron transfer processes between proteins and metal surfaces. Invented in the early 1990s, [26] DDI takes advantage of surface-bound capture oligonucleotides that selectively hybridize with sets of proteins tagged with complementary oligonucleotides ( Figure 2B).…”

Multimodal plasmonic biosensing nanostructures prepared by DNA-directed immobilization of multifunctional DNA-gold nanoparticles

“…The oligonucleotides with amine and thiol moieties at the 5' end (N1down/up, N2down/up, N3down/up, N4down/up) were ordered to Sigma-Aldrich®. The synthesis of the haptenoligonucleotide conjugates (ST: 8-N1up; THG: hG-N2up, B: 13-N3up and FQ: FQ-N4up) has already been described (Tort et al 2012a). The sequences of these oligonucleotides are shown in table S1 of the Supporting Information document.…”

“…DNA-directed immobilization (DDI), based on the self-organizing capabilities and the highly specificity of the DNA hybridization, allows immobilizing potentially any type of biomolecule onto a DNA-biofunctionalized surface (see (Meyer et al 2014;Seymour et al 2015;Tan et al 2014) for recent review papers), including small organic molecules. Thus, we have reported the possibility of manufacturing multiplexed hapten-microarrays by hybridizing haptenoligonucleotide probes with complementary ssDNA strands immobilized on distinct spots of a DNA microarray (Tort et al 2012a;Tort et al 2009). DDI has also been used to immobilize nanoparticles using DNA biofunctionalized nanoprobes (Loweth et al 1999;Peschel et al 2002;Reichert et al 2000;Wolfgang and Taton 2003).…”