Detection of PCR products amplified from DNA of epizootic pathogens using magnetic nanoparticles and SERS

Abstract

Here we present a novel approach using surface-enhanced Raman scattering (SERS) spectroscopy for the sequence-specific detection of DNA utilizing magnetic nanoparticles (MNPs) for the enrichment of the target molecules. To achieve fast and efficient binding of longer DNA strands, e.g. PCR products, the hybridization procedure is performed in solution. To further purify and enrich the DNA strands of interest, MNPs are used for their separation. Following the binding of the target DNA, a dye-modified, short synthetic ssDNA is hybridized, which serves as label for the SERS detection. The SERS spectra are used to identify the bound molecules. The applicability of this approach was first tested with short synthetic oligonucleotides to evaluate its specificity. Afterward, the system was applied to detect PCR products amplified from DNA of specific agents of epizootic diseases. Sequences of the bacterium Mycoplasma mycoides subspecies mycoides small colony type (MmmSC), causing contagious bovine pleuropneumonia (CBPP) were used as PCR targets. To demonstrate the multiplexing capability of SERS, the simultaneous detection of three different PCR products labeled with three dyes was performed.

Surface Enhanced Raman Spectroscopy for DNA Biosensors—How Far Are We?

“…In another label-free experiment, although one that requires a more complicated experimental procedure (similar to the application of labels), three ssDNAs are hybridized. Briefly, thiolated single-stranded DNA with a specific sequence is attached to plasmonic nanoparticles or plasmonic and magnetic nanoparticles [43][44][45][46][47][48][49][50]. Due to hybridization, in the presence of the target DNA, nanoparticles form agglomerates (Figure 3), which can be easily detected from the increase in the intensity of the measured SERS signal in the case of plasmonic agglomerates (the SERS enhancement factor is especially large for species located in the narrow slit between plasmonic structures), or from the formation of the plasmonic-magnetic agglomerates, which can concentrate by the magnetic field [51].…”

Discrimination of clostridium species using a magnetic bead based hybridization assay

“…[13] Compared to chip-based methods [14][15][16][17][18][19][20][21][22] the use of magnetic particles benefits from improved hybridization kinetics. By employing biotin-labeled primers for the amplification of target DNA with polymerase chain reaction (PCR), the PCR products can bind directly onto the magnetic beads.…”

Raman spectroscopy - An essential tool for biophotonics

“…An enzymatic silver reduction results in an arrangement of statistically distributed spiky silver nanoclusters, whose SERS activity can be characterized by recording the electrical conductivity [25]. These enzymatically generated SERS substrates have been successfully applied for the detection of PCR products amplified from DNA of epizootic pathogens [26]. Furthermore, a top-down approach based on electron beam lithography and ion beam etching has been applied for the preparation of tunable plasmonic arrays [27].…”