High sensitivity detection of 16s rRNA using peptide nucleic acid probes and a surface plasmon resonance biosensor
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].
Section snippets
Materials
1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and 1.0 M ethanolamine (pH 8.5) were obtained from Biacore AB (Uppsala, Sweden). 11-Mercaptoundecanoic acid (MUA), H2SO4, and H2O2 were purchased from Aldrich (Milwaukee, WI, USA). A gold chip for an SPR sensor, consisting of a 2 nm chromium adhesion layer and 45 nm of gold deposited on a 25 mm-diameter round glass, was purchased from K-MAC (Daejeon, Korea). MW 500 K and 10 K amino dextran were obtained
Results and discussion
The cationic Au nanoparticles were characterized by a DLS method. Particle diameters and zeta potentials were measured three times repeatedly, and the average values and standard deviations were listed in Table 2. The initial Au particles show anionic charge due to citrate coated onto bare Au surface, and the MUA self-assembled also show an anionic charge due to the carboxylic acid residue of MUA. Amino dextran modified particle shows cationic charge by the amine group and larger size than the
Conclusions
By utilizing the neutral backbone property of PNA, a simple signal enhancing method for high sensitivity detection of 16s rRNA on an SPR sensor surface was developed. 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. The lower detection limit of E. coli rRNA was 58.2 ± 1.37 pg mL−1 and decreased about 5500-fold comparing to that without signal amplification. When the detection
Acknowledgements
This research was supported by a grant from the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea (03-PJ1-PG1-CH11-0003) and a grant from the leading foreign research institute recruitment program from the Ministry of Science & Technology (MOST).
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These Authors contributed equally to this work.