Development of an advanced electrochemical DNA biosensor for bacterial pathogen detection - PubMed (original) (raw)

Development of an advanced electrochemical DNA biosensor for bacterial pathogen detection

Joseph C Liao et al. J Mol Diagn. 2007 Apr.

Abstract

Electrochemical sensors have the capacity for rapid and accurate detection of a wide variety of target molecules in biological fluids. We have developed an electrochemical sensor assay involving hybridization of bacterial 16S rRNA to fluorescein-modified detector probes and to biotin-modified capture probes anchored to the sensor surface. Signal is generated by an oxidation-reduction current produced by the action of horseradish peroxidase conjugated to an anti-fluorescein monoclonal Fab. A previous study found that this electrochemical sensor strategy could identify uropathogens in clinical urine specimens. To improve assay sensitivity, we examined the key steps that affect the current amplitude of the electrochemical signal. Efficient lysis and release of 16S rRNA from both gram-negative and -positive bacteria was achieved with an initial treatment with Triton X-100 and lysozyme followed by alkaline lysis, resulting in a 12-fold increase in electrochemical signal compared with alkaline lysis alone. The distance in nucleotides between the target hybridization sites of the detector and capture probes and the location of fluorescein modification on the detector probe contributed to a 23-fold change in signal intensity. These results demonstrate the importance of target-probe and probe-probe interactions in the detection of bacterial 16S rRNA using an electrochemical DNA sensor approach.

PubMed Disclaimer

Figures

Figure 1

Figure 1

Flow chart showing the steps involved in the process of bacterial detection using the electrochemical sensor. Step 1: Bacterial lysis to release the 16S rRNA target. Step 2: Primary hybridization of the 16S rRNA target with the fluorescein-modified detector probe. Step 3: Secondary hybridization of the target-detector probe hybrid to the capture probe on the sensor surface. Step 4: Redox reaction generated by addition of anti-fluorescein horseradish peroxidase (αF-HRP), TMB substrate, and H2O2. The amount of time required for each step is shown. Washing occurs only before and after the addition of αF-HRP. The entire assay was performed within 50 minutes.

Figure 2

Figure 2

Effect of lysis conditions on electrochemical signal intensity. Enterococcus cells (105) were exposed to NaOH, Triton X-100 (Tx), and/or lysozyme (Lzm) at room temperature followed by direct electrochemical detection of released 16S rRNA using _Enterococcus_-specific capture (EF207C) and detector (EF165D) probes. The lysis conditions were as follows: 1) NaOH for 10 minutes; 2) Triton X-100 for 5 minutes, followed by NaOH for 5 minutes; 3) Triton X-100 and lysozyme for 10 minutes; 4) NaOH for 5 minutes, followed by Triton X-100 and lysozyme for 5 minutes; and 5) Triton X-100 and lysozyme for 5 minutes, followed by NaOH for 5 minutes. Mean and SD of experiments performed in duplicate are shown. Background signal was determined in negative control experiments performed with capture and detector probes but without bacterial lysate. Conditions 3, 4, and 5 produced results that were significantly greater than negative control (P < 0.01).

Figure 3

Figure 3

Electrochemical signal intensity as a function of the distance (in nucleotides) between the capture and detector probe hybridization sites on the 16S rRNA target. Current output (in nanoamperes) was measured using capture probe EC434C and various 3′-fluorescein-modified detector probes hybridized to 16S rRNA released from 4.2 × 107 E. coli. Mean and SD of experiments performed in duplicate are shown. Background signal was determined in negative control (NC) experiments performed with capture and detector probes but without bacterial lysate. There was a negative correlation (r = −0.84) between signal intensity and the number of nucleotides between the capture and detector probe hybridization sites. The signal intensity obtained using capture and detector probes hybridizing to adjacent (0-nucleotide gap) sites produced an electrochemical signal significantly greater than that obtained using detector probes hybridizing ≥3 nucleotides away from the capture probe hybridization site (P < 0.01).

Figure 4

Figure 4

Electrochemical signal intensity as a function of location of probe hybridization and fluorescein modification. A: Signal intensity was measured using the _Enterococcus_-specific capture probe EF C207 paired with 3′- or 5′-fluorescein-modified detector probes hybridizing to the enterococcal 16S target at a site adjacent to (EF D171) or six nucleotides removed from (EF D165) the capture probe. B: Signal intensity was measured using 3′- or 5′-fluorescein-modified detector probe EC393D paired with the _E. coli_-specific capture probes hybridizing to the E. coli 16S target at a site adjacent to (EC430C) or six nucleotides removed from (EC434C) the detector probe. The configurations of the capture and detector probes are shown schematically. Mean and SD of experiments performed in duplicate are shown.

Figure 5

Figure 5

Sensitivity of the electrochemical sensor assay as a function of locations of probe hybridization and fluorescein modification. A: Electrochemical sensor results from fivefold serial dilutions of enterococcal cells using capture probe EF207C paired with detector probes EF165D or EF171D modified by fluorescein at the 5′ and 3′ positions, respectively. B: Electrochemical sensor results from fivefold serial dilutions of E. coli cells using capture probe EC434C paired with detector probes EC393C or EC399C modified by fluorescein at the 5′ and 3′ positions, respectively. The dashed horizontal lines indicate current output thresholds for duplicate results significantly greater than negative control (P < 0.01). 3′-Fluorescein modification of the detector probe combined with continuity between the detector and capture probe hybridization sites resulted in a 23-fold improvement in sensitivity of electrochemical sensor assay for detection of enterococci (from 232,000 down to 9900 cells) and E. coli (from 6500 down to 280 cells).

Figure 6

Figure 6

Effects of a mixture of detector probes on electrochemical signal intensity. Lysates containing 16S rRNA from either Enterococcus or E. coli were hybridized with detector probes specific for Enterococcus, E. coli, or a mixture of both detector probes. In each experiment, the detector probe-16S rRNA hybrids were applied to electrochemical sensors functionalized with an _Enterococcus_-specific capture probe. Mean and SD of experiments performed in duplicate are shown. Background signal was determined in negative control (NC) experiments performed with capture and detector probes but without bacterial lysate. Experiments with the Enterococcus lysate show that there was no significant loss of signal intensity for detection of 16S rRNA target when hybridization was performed with a mixture of detector probes. Experiments with the E. coli lysate show that there was also no loss of capture probe specificity using a mixture of detector probes. Similar results were obtained with other two-, three-, and five-detector probe mixtures (data not shown).

Figure 7

Figure 7

Results testing the specificity of the electrochemical sensor array. Lysates of nine ATCC strains were combined with a mixture of seven detector probes and applied to the surface of the 16-sensor array (top left) functionalized with seven different capture probes (see Materials and Methods for details and Table 1 for abbreviations). Negative control (NC) sensors to which no cell lysates were applied were used to measure background signal intensity. Mean and SD of log signal intensity from duplicate sensors are plotted on the vertical axes in antilog (nanoamperes) scale.

Figure 8

Figure 8

Relationship of variability to signal intensity. The mean signal intensity and SD for each of the 156 duplicate experiments reported in this study are included in this log-log plot, after excluding seven points with 0 SD. The pattern of the vertical scatter of the duplicates, the strong relationship between SD and mean, and analysis of the logarithm of the signal (see text) support a log normal distribution.

Similar articles

Cited by

References

    1. Drummond TG, Hill MG, Barton JK. Electrochemical DNA sensors. Nat Biotechnol. 2003;21:1192–1199. - PubMed
    1. Gau JJ, Lan EH, Dunn B, Ho CM, Woo JC. A MEMS based amperometric detector for E. coli bacteria using self-assembled monolayers. Biosens Bioelectron. 2001;16:745–755. - PubMed
    1. Gooding JJ. Electrochemical DNA hybridization biosensor. Electroanalysis. 2002;14:1149–1156.
    1. Palecek E, Jelen F. Electrochemistry of nucleic acids and development of DNA sensors. Crit Rev Anal Chem. 2002;3:261–270.
    1. Wang J. Electrochemical nucleic acid biosensors. Anal Chim Acta. 2002;469:63–71.

Publication types

MeSH terms

Substances

LinkOut - more resources