Label‐free detection of 16S ribosomal RNA hybridization on reusable DNA arrays using surface plasmon resonance imaging

Abstract

In this paper, we describe the detection of bacterial cell-extracted 16S ribosomal RNA (rRNA) using an emerging technology, surface plasmon resonance (SPR) imaging of DNA arrays. Surface plasmon resonance enables detection of molecular interactions on surfaces in response to changes in the index of refraction, therefore eliminating the need for a fluorescent or radioactive label. A variation of the more common SPR techniques, SPR imaging enables detection from multiple probes in a reusable array format. The arrays developed here contain DNA probes (15-21 bases) designed to be complementary to 16S rRNA gene sequences of Escherichia coli and Bacillus subtilis as well as to a highly conserved sequence found in rRNAs from most members of the domain Bacteria. We report species-specific hybridization of cell-extracted total RNA and in vitro transcribed 16S rRNA to oligonucleotide probes on SPR arrays. We tested multiple probe sequences for each species, and found that success or failure of hybridization was dependent upon probe position in the 16S rRNA molecule. It was also determined that one of the probes intended to bind 16S rRNA also bound an unknown protein. The amount of binding to these probes was quantified with SPR imaging. A detection limit of 2 micro g ml-1 was determined for fragmented E. coli total cellular RNA under the experimental conditions used. These results indicate the feasibility of using SPR imaging for 16S rRNA identification and encourage further development of this method for direct detection of other RNA molecules.

High-throughput SPR sensor for food safety

“…The selected sequences are known to specifically identify these pathogens. 5 -(CGC TCC AGC CTA ACT GAA)-3 (Herman and Deridder, 1992) and 5 -(TCC AGC CTA ACT GAA CCA TA)-3 (Romero et al, 1995) are specific for B. abortus, 5 -(GTC CCC CTC TTT GGT CTT GC)-3 (Nelson et al, 2002) and 5 -(TAT TAA CTT TAC TCC CTT CC)-3 (Huijsdens et al, 2002) are specific for E. coli, and 5 -(CCT AAA AGG TTA CTC CAC CGG CT)-3 (Greisen et al, 1994) is specific for S. aureus. The DNA targets were used instead of ribosomal RNA fragments mainly because of better stability of the DNA.…”

“…SPR biosensor-based detection of bacterial pathogens has been demonstrated employing immunoreactions between bacteria and specific antibodies immobilized on the SPR sensor surface (Oh et al, 2002;Taylor et al, 2006). Alternatively, bacterial pathogens can be measured by detecting their specific nucleic acids, such as rRNA sequences which can be extracted from bacterial pathogens (Nelson et al, 2002). This approach provides high specificity, stability and speed compared to antibody-based detection of whole bacteria (Wang et al, 2007).…”

“…This approach provides high specificity, stability and speed compared to antibody-based detection of whole bacteria (Wang et al, 2007). Furthermore, it takes advantage of the well-developed DNA array technology that is compatible with high-throughput operation format desired in many important applications (Boozer et al, 2006;Nelson et al, 2002). The most common approach to parallelized SPR sensing is SPR imaging (Rich and Myszka, 2006;Schaferling and Nagl, 2006;Wassaf et al, 2006).…”

Genetic and OtherDNA‐Based Biosensor Applications

“…Often, species-specific genes, or more generic type sequences such as rRNA sequences, are used as targets. [13][14][15] The latter sequences have the advantage that they are relatively stable and are present in high amounts in bacterial cells. On the other hand, they are largely conserved, even among different taxonomic units, and the specificity must be well assessed before using them as a marker.…”

Surface Plasmon Resonance Imaging-Based Protein Array Chip System for Monitoring a Hexahistidine-Tagged Protein during Expression and Purification

“…SPRI detects the specific binding of unlabeled biomolecules to arrayed capture molecules attached to a modified gold thin film by measuring changes in the local index of refraction upon adsorption (11). It has been proven to be a valuable tool for investigating molecular interactions without the use of any fluorescent or radiochemical labels (4,(6)(7)(8). Use of the SPR principle can be further expanded to the detection of microbial cells with immobilized antibodies or protein ligands on an SPR sensor chip (9,12).…”