High sensitivity detection of 16s rRNA using peptide nucleic acid probes and a surface plasmon resonance biosensor.

A signal enhancing method allowing highly sensitive detection of E. coli 16s rRNA was developed using peptide nucleic acid (PNA) as a capture probe and a surface plasmon resonance (SPR) sensor as a detector. 16s rRNA has been used as a genetic marker for identification of organisms, and can be analyzed directly without PCR amplification due to the relatively high number of copies. PNA has a neutral backbone structure, therefore hybridization with 16s rRNA results in the ionic condition being changed from neutral to negative. A cationic Au nanoparticle was synthesized and used for signal amplification by ionic interaction with 16s rRNA hybridized on the PNA probe-immobilized SPR sensor chip. This method resulted in a detection limit of E. coli rRNA of 58.2+/-1.37 pg mL(-1). Using this analytical method, Staphylococcus aureus was detected without purification of rRNA.

[1]  M. Egholm,et al.  Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide. , 1991, Science.

[2]  F. Brockman,et al.  Direct Detection of 16S rRNA in Soil Extracts by Using Oligonucleotide Microarrays , 2001, Applied and Environmental Microbiology.

[3]  Douglas R Call,et al.  Detection of bacterial pathogens in environmental samples using DNA microarrays. , 2003, Journal of microbiological methods.

[4]  D A Stahl,et al.  Oligonucleotide microchips as genosensors for determinative and environmental studies in microbiology , 1997, Applied and environmental microbiology.

[5]  Noriaki Hara,et al.  SPR sensor chip for detection of small molecules using molecularly imprinted polymer with embedded gold nanoparticles. , 2005, Analytical chemistry.

[6]  G. Bazan,et al.  Application of cationic conjugated polymers in microarrays using label-free DNA targets , 2007, Nature Protocols.

[7]  Ole Brandt,et al.  Peptide nucleic acids on microarrays and other biosensors. , 2004, Trends in biotechnology.

[8]  A. Warsinke,et al.  Gold nanoparticle-enhanced surface plasmon resonance measurement with a highly sensitive quantification for human tissue inhibitor of metalloproteinases-2 , 2005 .

[9]  Mark R Liles,et al.  Label-free detection of 16S ribosomal RNA hybridization on reusable DNA arrays using surface plasmon resonance imaging. , 2002, Environmental microbiology.

[10]  Martin Moskovits,et al.  A heterogeneous PNA-based SERS method for DNA detection. , 2007, Journal of the American Chemical Society.

[11]  Min-Gon Kim,et al.  Surface plasmon resonance analysis of Alzheimer's beta-amyloid aggregation on a solid surface: from monomers to fully-grown fibrils. , 2008, Analytical chemistry.

[12]  M. Saito,et al.  Electroactive chitosan nanoparticles for the detection of single-nucleotide polymorphisms using peptide nucleic acids , 2008, Analytical and bioanalytical chemistry.

[13]  Zhiqiang Gao,et al.  Detection of Nucleic Acids Using Enzyme‐Catalyzed Template‐Guided Deposition of Polyaniline , 2007 .

[14]  J. Gershoni,et al.  Biotin binding to avidin. Oligosaccharide side chain not required for ligand association. , 1987, The Biochemical journal.

[15]  Alan J. Heeger,et al.  DNA detection using water-soluble conjugated polymers and peptide nucleic acid probes , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[16]  L. Tondelli,et al.  Comparison of novel delivery systems for antisense peptide nucleic acids. , 2005, Journal of controlled release : official journal of the Controlled Release Society.

[17]  M. Fischer,et al.  Exploring computational lead optimisation with affinity constants obtained by surface plasmon resonance for the interaction of PorA epitope peptides with antibody against Neisseria meningitidis. , 2001, Biochimica et biophysica acta.

[18]  Lin He,et al.  Colloidal Au-Enhanced Surface Plasmon Resonance for Ultrasensitive Detection of DNA Hybridization , 2000 .

[19]  F. Pellestor,et al.  The peptide nucleic acids, efficient tools for molecular diagnosis (Review). , 2004, International journal of molecular medicine.

[20]  R. Corn,et al.  Surface plasmon resonance imaging measurements of DNA and RNA hybridization adsorption onto DNA microarrays. , 2001, Analytical chemistry.

[21]  M. Natan,et al.  Colloidal Au-enhanced surface plasmon resonance immunosensing. , 1998, Analytical chemistry.

[22]  E C Nice,et al.  Instrumental biosensors: new perspectives for the analysis of biomolecular interactions. , 1999, BioEssays : news and reviews in molecular, cellular and developmental biology.

[23]  A. Mirzabekov,et al.  Portable System for Microbial Sample Preparation and Oligonucleotide Microarray Analysis , 2001, Applied and Environmental Microbiology.

[24]  Luc Bissonnette,et al.  Detection of target DNA using fluorescent cationic polymer and peptide nucleic acid probes on solid support , 2005, BMC biotechnology.