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

Abstract

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.

Introduction

The 16s rRNAs are commonly used genetic markers for identification of organisms that can be analyzed directly without PCR amplification due to a relatively high number of copies [1]. There have been several reports on the direct detection of 16s rRNA using oligonucleotide probes. Guschin et al. and Bavykin et al., for example, used gel element microarrays to directly and specifically detect fragmented RNA from simple model microbial communities [2], [3]. Small et al. recently described an oligonucleotide array for the direct detection of 16s rRNA [4]. However, significant sensitivity limitation compared to PCR-based assays was observed. The 16s rRNA does not readily hybridize to oligonucleotide probes because its secondary structure prevents probes from interacting with complementary rRNA. To overcome these constraints, Small et al. used biotinylated oligonucleotides near the position of the capture probe in the hybridization buffer, which appeared to relax structural interference and increase hybridization efficiency [4]. However, the detection sensitivity was 14 μg mL⁻¹ of total RNA representing 7.5 × 10⁶ cells, which is still unsatisfactory. Recently 16s rRNA has been analyzed directly using an surface plasmon resonance (SPR) sensor system without labeling, but the lower detection limit was 2 μg mL⁻¹ of 16s rRNA [5], [6].

In this report, a signal amplification method for high sensitivity detection of 16s rRNA was developed using peptide nucleic acid (PNA) probes with an SPR biosensor system. Since first designed in 1991 by Nielsen et al. [7], PNA has been used for molecular diagnostics [8], [9], antisense [10], and the other sensing technologies [11] due to its superior chemical and biological stability as well as its hybridization efficiency. PNA has a non-ionic backbone structure composed of N-(2-aminoethyl)-glycine units without pentose sugar moieties or phosphate groups [7], [8], [9], [10], [11]. Changes in surface property upon hybridization of DNA or RNA, from neutral to negative, allow selective electrostatic adsorption of positively charged materials such as fluorescent polymers [12], conjugated polymers [13], photoactive polymers [14], conductive polymers [15] and surface enhanced Raman scattering (SERS)-active Ag nanoparticle clusters [16]. Kerman et al. designed ferrocene-conjugated chitosan nanoparticles (Chi-Fc) that can be used as an electroactive indicator of PNA and DNA hybridization [17]. Nanoparticles modified with positively charged chitosan attached to the negatively charged phosphate backbone of DNA and gave rise to a high electrochemical oxidation signal from ferrocene. In this study, we used a surface plasmon resonance biosensor to detect the amount of hybridization with similar signal amplification strategy. The SPR biosensor is sensitive to changes in the thickness or refractive index of biomaterials at the interface between a thin Au film and an ambient medium and thus able to characterize biomolecular interactions in real time without labeling [18]. Signal enhancement methods for protein interactions [19] and small molecule detection [20] in an SPR biosensor by surface characterized Au nanoparticles have been reported. If Au nanoparticles are adhered onto the SPR sensor surface, the SPR angle is shifted remarkably through strong optical coupling between the gold film and nanoparticles [18], [21], [22]. Herein, hydrophilic cationic Au nanoparticles were prepared and designed to specifically interact with 16s rRNA to amplify the hybridization reaction between PNAs and E. coli 16s rRNA. The analysis using the SPR sensor enables to measure the amounts of binding at each analytical step, while other reports adopting the same analytical strategy such as fluorescence [12] and SERS [13] is possible only to detect end points. It is expected that the analytical method described in this report is convenient and sensitive in the detection of nucleic acids without requiring a second hybridization and additional labeling of samples [22].