Selective Amplification of SPR Biosensor Signal for Recognition of rpoB Gene Fragments by Use of Gold Nanoparticles Modified by Thiolated DNA

Abstract

An experimental approach for improving the sensitivity of the surface plasmon resonance (SPR) DNA hybridization sensor using gold nanoparticles (GNPs), modified by specific oligonucleotides, was elaborated. An influence of the ionic strength on the aggregation stability of unmodified GNPs and GNPs modified by the thiolated oligonucleotides was investigated by monitoring a value of light extinction at 520 nm that can be considered as a measure of a quantity of the non-aggregated GNPs. While the unmodified GNPs started to aggregate in 0.2 × saline-sodium citrate (SSC), GNPs modified by the negatively charged oligonucleotides were more stable at increasing ionic strength up to 0.5 × SSC. A bioselective element of the SPR DNA hybridization sensor was formed by immobilization on the gold sensor surface of the thiolated oligonucleotides P2, the sequence of which is a fragment of the rpoB gene of Mycobacterium tuberculosis. The injections into the measuring flow cell of the SPR spectrometer of various concentrations of GNPs modified by the complementary oligonucleotides T2-18m caused the pronounced concentration-dependent sequence-specific sensor responses. The magnitude of the sensor responses was much higher than in the case of the free standing complementary oligonucleotides. According to the obtained experimental data, the usage of GNPs modified by specific oligonucleotides can amplify the sensor response of the SPR DNA hybridization sensor in ~1200 times.

Surface plasmon resonance investigation of DNA hybridization on a sensor surface using gold nanoparticles modified by specific oligonucleotides

“…Additionally, our prediction that at use of AuNPs modified by specific oligonucleotides a response of the DNA hybridization sensor based on the SPR spectrometer "Plasmon 6" can be amplified by over 1000 times [9] was confirmed also. The efficient hybridization of the oligonucleotides of various lengths immobilized on AuNPs with the oligonucleotides immobilized on the sensor surface as well as the possibility of thermal and chemical regene rations allow multi-use of the sensor along with a strong amplification of the sensor signal.…”

“…Indeed, AuNPs with a higher surface density of the immobilized oligonucleotides demonstrated a higher stability. Their level of light extinction at 520 nm remained unchanged up to 0.5×SSC [9]. These results allow using the modified AuNPs for DNA hybridization because the concentration of the buffer solution can provide rather stringent and highly selective conditions for the hybridization process [18,21].…”

“…Taking into account that in the field of SPR sensors, the limit of detection (LOD) is usually defined as the concentration of analyte that produces sensor response corresponding to 3 standard deviations of sensor response measured for a blank sample [1,24], LOD for AuNPs modified by T2-11m or T2-18m was calculated to be ~ 10 pM. Previously, the detection of 20 nM T2 (free standing) by the same P2-based DNA-sensor was shown [9]. Therefore, the calculated LOD is in a good agreement with our earlier prediction that at using AuNPs modified by specific oligonucleotides, a response of the DNA hybridization sensor based on the SPR spectrometer "Plasmon 6" can be amplified by over 1000 times [9].…”

“…Previously, the detection of 20 nM T2 (free standing) by the same P2-based DNA-sensor was shown [9]. Therefore, the calculated LOD is in a good agreement with our earlier prediction that at using AuNPs modified by specific oligonucleotides, a response of the DNA hybridization sensor based on the SPR spectrometer "Plasmon 6" can be amplified by over 1000 times [9]. Additionally, this LOD value is 2 orders of magnitude smaller, than the level of detection of T2, which was achieved using the impedimetric P2-based DNA-sensor [25].…”

“…It allows us to expand the range of ionic strength of solutions in which the use of nanoparticles becomes possible. If the ionic strength of the solution is high enough to ensure the efficient DNA hybridization, and the AuNPs do not undergo aggregation, it becomes possible to use AuNPs as molecular labels to enhance the sensor signal [3][4][5]9].…”