Nanoparticle-Assisted Nuclear Relaxation-Based Detection of Oligonucleotides.

Nuclear relaxation-based techniques rely on the capability of paramagnetic center to affect the transverse relaxation time (T2) of the water protons. These changes are sensitive to the microenvironment of the paramagnetic center, which can be used to detect a variety of targets. In this work, we present an "on-off" oligonucleotide detection scheme in aqueous solutions, which uses gadolinium phthalocyanine (GdTcPc)-grafted silica nanoparticles as the paramagnetic centers. A probe oligonucleotide strand was conjugated to the GdTcPc to act as a recognition element. In the presence of the target oligonucleotide, which was complementary to the probe, an increase in the ΔT2 value, which was measured by subtracting the characteristic T2 value of the background solution from that of the sample using a benchtop relaxometer, was observed. The magnitude of this increase was proportional to the target oligonucleotide concentration. A linear range was obtained from 30 to 140 nM, with a detection limit of 15 nM. The developed nuclear relaxation-based detection scheme is shown to be a simple, fast, and selective method to detect oligonucleotide and could be useful in point-of-care diagnostic applications.

Detection of Sortase A and Identification of Its Inhibitors by Paramagnetic Nanoparticle-Assisted Nuclear Relaxation.

Sortase A is a virulence factor responsible for the attachment of surface proteins to Staphylococcus aureus and other Gram-positive bacteria. Inhibitors of this enzyme are potential anti-infective agents. Herein, a new highly selective magnetic relaxation-based method for screening potential sortase A inhibitors is described. A 13-amino acid-long peptide substrate of sortase A is conjugated to SiO2-EDTA-Gd NPs. In the presence of sortase A, the LPXTG motif on the peptide strand is cleaved resulting in a shortened peptide as well as a reduced water T2 value whose magnitude is dependent on the concentration of sortase A. The detection limit is determined to be 76 pM. In contrast, the presence of sortase A inhibitors causes the T2 to remain at a higher value. The proposed method is used to characterize inhibition of sortase A by curcumin and 4-(hydroxymercuri)benzoic acid with an IC50 value of 12.9 ± 1.6 µM and 130 ± 1.76 µM, respectively. Furthermore, this method was successfully applied to detect sortase A activity in bacterial suspensions. The feasibility to screen different inhibitors in Escherichia coli and S. aureus suspensions was demonstrated. This method is fast and potentially useful to rapidly screen possible inhibitors of sortase A in bacterial suspensions, thereby aiding in the development of antibacterial agents targeting Gram-positive bacteria.

Nanoparticle assisted nuclear relaxation-based oligonucleotide detection.

We present a proof-of-concept "on-off" detection scheme, which uses gadolinium phthalocyanine (GdTcPc)-grafted silica nanoparticles as paramagnetic centers, capable of modifying the transverse relaxation time (T2) of water protons in solution. A DNA strand (as probe) was conjugated to the GdTcPc to act as a recognition element. In the presence of the target DNA, which was complementary to the probe, an increase in the T2 value was detected, with magnitude proportional to the target DNA concentration. The linear range was observed from 30 to 140 nM, with limit of detection of 15 nM. The developed nuclear relaxation-based detection scheme is shown to be a simple, fast and selective method to detect DNA and could be useful in point-of-care diagnostic applications.