Abstract
Functional nanomaterials with fluorescent or quenching abilities are important for the development of molecular probes for detection and studies of nucleic acids. Here, we describe a new class of molecular nanoprobes, the NanoCeracQ that uses nanoceria particles as a nanoquencher of fluorescent oligonucleotides for rapid and sensitive detection of DNA sequences and hybridization events. We show that nanoceria forms stable and reversible bionanoconjugates with oligonucleotides and can specifically recognize and detect DNA sequences in a single step. In absence of the target DNA, the nanoprobe produced minimal background fluorescence due to the high quenching efficiency of nanoceria. Competitive binding of the target induced a concentration dependent increase in the fluorescence signal due to hybridization and release of the fluorescent tag from the nanoparticle surface. The nanoprobe enabled sensitive detection of the complementary strand with a detection limit of 0.12 nM, using a single step procedure. The results show that biofunctionalized nanoceria can be used as a universal nanoquencher and nanosensing platform for fluorescent DNA detection and studies of nucleic acid interactions. This approach can find broad applications in molecular diagnostics, sensor development, gene expression profiling, imaging and forensic analysis.
Highlights
The development of rapid and sensitive detection of nucleic acids is of significant scientific and biomedical importance for early diagnosis and screening of disease, to improve patient care, quality and outcomes in the healthcare system[1,2]
Earlier studies have shown that ssDNA has strong affinity for nanoceria surfaces by a mechanism involving the interaction between the phosphate backbone and cerium at the particle surface[24,32,33]
Lack of complete change to a neutral/negative nanoceria surface upon attachment of DNA is due to the highly positive surface of nanoceria which is predominant and strong enough to compensate the negative charge of DNA; the significant decrease in the positive charge at the surface of the DNA-nanoceria complex shows a clear change in surface properties due to DNA attachment
Summary
The development of rapid and sensitive detection of nucleic acids is of significant scientific and biomedical importance for early diagnosis and screening of disease, to improve patient care, quality and outcomes in the healthcare system[1,2]. Some of the simplest fluorescence technologies rely on the fluorescence energy transfer resonance and use a fluorophore and its spectrally matched quencher This “lights on” bind-and-detect fluorescence quenching assays has made them ideal tools for point-of-care measurements in environments with limited resources or access to laboratory facilities. DNA-based assays with one step detection capabilities via fluorescence quenching have found a great deal of interest in recent years for numerous applications[14,15] These methods showed increased convenience in analysis, rapid hybridization kinetics and sensitivity[16,17]. Nanoceria serves as a general biocompatible substrate for reversible attachment of nucleic acids due to the strong interaction between cerium and the phosphate backbone[24] These particles generate strong target-induced fluorescent changes, enhancing sensitivity of analysis. This approach may find applications in diagnostics, forensic analysis, imaging and environmental monitoring
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