Use of electrochemical DNA biosensors for rapid molecular identification of uropathogens in clinical urine specimens

Abstract

We describe the first species-specific detection of bacterial pathogens in human clinical fluid samples using a microfabricated electrochemical sensor array. Each of the 16 sensors in the array consisted of three single-layer gold electrodes-working, reference, and auxiliary. Each of the working electrodes contained one representative from a library of capture probes, each specific for a clinically relevant bacterial urinary pathogen. The library included probes for Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, Enterocococcus spp., and the Klebsiella-Enterobacter group. A bacterial 16S rRNA target derived from single-step bacterial lysis was hybridized both to the biotin-modified capture probe on the sensor surface and to a second, fluorescein-modified detector probe. Detection of the target-probe hybrids was achieved through binding of a horseradish peroxidase (HRP)-conjugated anti-fluorescein antibody to the detector probe. Amperometric measurement of the catalyzed HRP reaction was obtained at a fixed potential of -200 mV between the working and reference electrodes. Species-specific detection of as few as 2,600 uropathogenic bacteria in culture, inoculated urine, and clinical urine samples was achieved within 45 min from the beginning of sample processing. In a feasibility study of this amperometric detection system using blinded clinical urine specimens, the sensor array had 100% sensitivity for direct detection of gram-negative bacteria without nucleic acid purification or amplification. Identification was demonstrated for 98% of gram-negative bacteria for which species-specific probes were available. When combined with a microfluidics-based sample preparation module, the integrated system could serve as a point-of-care device for rapid diagnosis of urinary tract infections.

FIG. 1.

Components and performance of the electrochemical sensor. (A) The 16-sensor array (2.5 by 7.5 cm) was microfabricated with a thin, optical-grade layer of gold electrodes deposited on plastic. Each sensor in the array contained three electrodes: a central working electrode, a circumferential reference electrode, and a short auxiliary electrode. (B) The chip mounter with contact pins for simultaneous reading of the current output from each of the sensors in the array. (C) Detection strategy. (1) Bacterial lysis to release 16S rRNA target (black dashed line). (2) Hybridization of the target with the fluorescein (green circle)-labeled detector probe (blue line). (3) Hybridization of the target with the biotin (red circle)-labeled capture probe (orange line). (4) Binding of anti-fluorescein antibody conjugated with HRP to the target-probe sandwich. (5) Generation of current by transfer of electrons to the electron transfer mediator, TMB. (D) Current output in an experiment involving a clinical urine specimen containing K. pneumoniae showing signal stabilization from all 16 sensors in the array within 60 seconds. Probe results were obtained by averaging the log₁₀ current outputs from duplicate sensor readings at 60 seconds.

FIG. 2.

Specificities of Enterobacteriaceae-specific probe pairs. Positive signals were seen for all Enterobacteriaceae species tested but not for gram-positive uropathogens (Eo, Ef, Ss, and Sa) or P. aeruginosa (Pa). (See the footnote to Table 2 for bacterial species abbreviations.) Means and standard deviations of experiments performed in duplicate are shown. NC refers to the negative control experiments performed with capture and detector probes but without bacterial lysate.

FIG. 3.

Direct, species-specific detection of uropathogens in representative clinical urine specimens using the electrochemical sensor array. Current output for each of the probe pairs in the array are shown in nanoamperes. The mean current output of duplicate sensors is shown above each bar; the error bars represent the standard deviations. The probe pair designation is shown below each bar, and the species specificity is given in Table 2. The urinalysis and microbiological characteristics of each specimen are shown to the right of the bar graph. The background signal level was determined by averaging the log₁₀ results of the NC sensors and the sensors with the four lowest species-specific probe pairs (from among EC, PM, KE, PA, and EF). As described in the text, significant signals were 0.30 log unit (5 standard deviations) above background. (A) E. coli in this clinical urine specimen produced significant signals in the UNI, EB, and EC sensors despite high numbers of white blood cells (WBC). RBC, red blood cells. (B) 16S rRNA from as few as 4 × 10⁴ K. pneumoniae cells/ml in urine produced significant signal levels in the UNI, EB, and KE sensors.

FIG. 4.

“UTI Chip” signal interpretation algorithm. The three-step algorithm used to interpret the results of electrochemical-sensor experiments on 78 specimens that met inclusion criteria is shown. Positive signals were those with a mean log of greater than 0.30 log unit (5 standard deviations) over background. “UNI” and “EB” are eubacterial- and Enterobacteriaceae-specific probe signals, respectively. “Bkgnd” is the background signal, as defined in the text. “MaxSpSp” refers to the maximum species-specific signal. Clinical microbiology results are given in shaded boxes. Two-letter species abbreviations are given in the footnote of Table 2. NSG indicates “no significant growth.” NG indicates “no growth.” An asterisk indicates false-positive results, and false-negative results are underlined.