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.Urinary tract infection (UTI) is the most common urological disease in the United States and the second most common bacterial infection of any organ system (12, 32). UTI is a major cause of patient morbidity and health care expenditure for all age groups, accounting for over 7 million office visits and more than 1 million hospital admissions per year (39). Catheterassociated UTI accounts for 40% of all nosocomial infections and more than 1 million cases per year (22,41,49). The total cost of UTI to the United States health care system in the year 2000 was approximately $3.5 billion (13,19,20). An important component of these health care costs involves the processing of urine specimens by clinical microbiology laboratories. Urine is the type of body fluid most frequently submitted to clinical microbiology laboratories for culture. A major drawback of microbiological culture systems is the time lag of approximately 2 days between specimen collection and pathogen identification. The primary cause of the delay between specimen collection and pathogen identification is the time to colony formation after the specimen is plated on solid culture media.

Motion-based DNA detection using catalytic nanomotors

“…Th e practical utility of this new motion-driven DNA assay was illustrated using the same capture and detector probe for the detection of 16S rRNA which is released from E. coli pathogenic bacteria and obtained from a previously reported sample preparation 20,21 . Figure 3b and the corresponding Supplementary Movie 4 show catalytic nanomotors ' racing ' following hybridization assays using diff erent bacterial lysate solutions corresponding to diff erent E. coli cell concentrations: 0, 7 × 10 3 and 7 × 10 5 CFU μ l − 1 , leading to average distance signals of 32, 56 and 96 μ m, respectively ( Fig.…”

“…Similarly, magnetic Au -Ni -Au -Pt nanomotors were prepared by introducing a ferromagnetic Ni segment. Following an initial deposition of gold segment at 0.75 C, Ni was electrodeposited at − 1.0 V for 2 C (versus Ag / AgCl) from a plating solution (20 4)). Subsequently, the second gold segment (0.75 C) and a platinum segment were electrodeposited as above.…”

Bacterial Detection & Identification Using Electrochemical Sensors

“…We typically perform the hybridization steps at ambient temperature (~20 °C) 5,6 . However, the hybridization steps (3.2 and 3.3) can also be performed at higher temperatures in a hybridization oven if the chip is placed in a covered chamber containing moistened filter paper to prevent evaporation 7,8 . Optimal signal is obtained with capture-detector probe pairs that hybridize to adjacent sites on the nucleic acid target without a gap between the hybridization sites 8 .…”

“…However, little attention has been given to the performance of DNA biosensors in raw biological samples. The electrochemical sensor assay described here can be performed directly on serum and urine samples without a significant loss of sensitivity 7 . The use of thiolated capture probes and the mixed monolayer described here renders the sensor surface resistant to non-specific protein absorption, retaining sensitivity in raw biological specimens including serum or urine 10 .…”

Optimization of an amperometric biosensor for the detection of hepatitis C virus using fractional factorial designs

“…2 Recent engineering advances have enabled the development of electrochemical DNA biosensors with molecular diagnostic capabilities. 4 Electrochemical DNA biosensors offer several advantages compared to alternative molecular detection approaches, including the ability to analyze complex body fluids, high sensitivity, compatibility with microfabrication technology, a low power requirement, and compact instrumentation compatible with portable devices. [5][6][7] The development of electrochemical transduction schemes for DNA bionsensor (so-called genosensors) has recently received increasing attention using photoelectrochemistry, potentiometry, and DNA-modified electrodes.…”