Rapid detection of bacterial pathogens is critical toward judicious management of infectious diseases. Herein, we demonstrate an in situ electrokinetic stringency control approach for a self-assembled monolayer-based electrochemical biosensor toward urinary tract infection diagnosis. The in situ electrokinetic stringency control technique generates Joule heating induced temperature rise and electrothermal fluid motion directly on the sensor to improve its performance for detecting bacterial 16S rRNA, a phylogenetic biomarker. The dependence of the hybridization efficiency reveals that in situ electrokinetic stringency control is capable of discriminating single-base mismatches. With electrokinetic stringency control, the background noise due to the matrix effects of clinical urine samples can be reduced by 60%. The applicability of the system is demonstrated by multiplex detection of three uropathogenic clinical isolates with similar 16S rRNA sequences. The results demonstrate that electrokinetic stringency control can significantly improve the signal-to-noise ratio of the biosensor for multiplex urinary tract infection diagnosis.
Urinary tract infection; Stringency control; Multiplex detection; Matrix effects; Self-assembled monolayers
Transforming microfluidics-based biosensing systems from laboratory research into clinical reality remains an elusive goal despite decades of intensive research. A fundamental obstacle for the development of fully automated microfluidic diagnostic systems is the lack of an effective strategy for combining pumping, sample preparation, and detection modules into an integrated biosensing platform. Herein, we report a universal electrode approach, which incorporates DC electrolytic pumping, AC electrokinetic sample preparation, and self-assembled monolayer based electrochemical sensing on a single microfluidic platform, to automate complicated molecular analysis procedures that will enable biosensing applications in non-traditional healthcare settings. Using the universal electrode approach, major microfluidic operations required in molecular analyses, such as pumping, mixing, washing, and sensing can be performed in a single platform. We demonstrate the universal electrode platform for detecting bacterial 16S rRNA, a phylogenetic marker, toward rapid diagnostics of urinary tract infection. Since only electronic interfaces are required to operate the platform, the universal electrode approach represents an effective system integration strategy to realize the potential of microfluidics in molecular diagnostics at the point of care.
System Integration; Electrochemical detection; Electrokinetics; 16S rRNA; Urinary Tract Infection
Multidrug-resistant pathogens are an emerging global health problem. In addition to the need of developing new antibiotics in the pipeline, the ability to rapidly determine the antibiotic resistance profiles of bacteria represents one of the most crucial steps toward the management of infectious diseases and the prevention of multidrug-resistant pathogens. Here, we report a single cell antimicrobial susceptibility testing (AST) approach for rapid determination of the antibiotic resistance of bacterial pathogens. By confining individual bacteria in gas permeable microchannels with dimensions comparable to a single bacterium, the antibiotic resistance of the bacteria can be monitored in real-time at the single cell level. To facilitate the dynamic loading of the bacteria into the confined microchannels for observation, AC electrokinetics is demonstrated for capturing bacteria to defined locations in high-conductivity AST buffer. The electrokinetic technique achieves a loading efficiency of about 75% with a negligible effect on the bacterial growth rate. To optimize the protocol for single cell AST, the bacterial growth rate of individual bacteria under different antibiotic conditions has been determined systematically. The applicability of single cell AST is demonstrated by the rapid determination of the antimicrobial resistant profiles of uropathogenic clinical isolates in Mueller-Hinton media and in urine. The antibiotic resistance profiles of bacteria can be determined in less than one hour compared to days in standard culture-based AST techniques.
To develop a portable point-of-care system based on biosensors for common infectious diseases such as urinary tract infection, the sensing process needs to be implemented within an enclosed fluidic system. On chip sample preparation of clinical samples remains a significant obstacle to achieve robust sensor performance. Herein AC electrokinetics is applied in an electrochemical biosensor cassette to enhance molecular convection and hybridization efficiency though electrokinetic induced fluid motion and Joule heating induced temperature elevation. Using E. coli as an exemplary pathogen, we determined the optimal electrokinetic parameters for detecting bacterial 16S rRNA in the biosensor cassette based on the current output, signal-to-noise ratio, and limit of detection. In addition, a panel of six probe sets targeting common uropathogenic bacteria was demonstrated. The optimized parameters were also validated using patient-derived clinical urine samples. The effectiveness of electrokinetic for on chip sample preparation will facilitate the implementation of point-of-care diagnosis of urinary tract infection in the future.
This study reports a hybrid electrokinetic technique for label-free manipulation of pathogenic bacteria in biological samples toward medical diagnostic applications. While most electrokinetic techniques only function in low-conductivity buffers, hybrid electrokinetics enables effective operation in high-conductivity samples, such as physiological fluids (~1 S m−1). The hybrid electrokinetic technique combines short-range electrophoresis and dielectrophoresis, and long-range AC electrothermal flow to improve its effectiveness. The major technical hurdle of electrode instability for manipulating high conductivity samples is tackled by using a Ti–Au–Ti sandwich electrode and a 3-parallel-electrode configuration is designed for continuous isolation of bacteria. The device operates directly with biological samples including urine and buffy coats. We show that pathogenic bacteria and biowarfare agents can be concentrated for over 3 orders of magnitude using hybrid electrokinetics.
This study reports a multifunctional electrode approach which directly implements electrokinetic enhancement on a self-assembled-monolayer-based electro-chemical sensor for point-of-care diagnostics. Using urinary tract infections as a model system, we demonstrate that electrokinetic enhancement, which involves in situ stirring and heating, can enhance the sensitivity of the strain specific 16S rRNA hybridization assay for 1 order of magnitude and accelerate the time-limiting incubation step with a 6-fold reduction in the incubation time. Since the same electrode platform is used for both electrochemical signal enhancement and electrochemical sensing, the multifunctional electrode approach provides a highly effective strategy toward fully integrated lab-on-a-chip systems for various biomedical applications.
A significant barrier to efficient antibiotic management of infection is that the standard diagnostic methodologies do not provide results at the point of care. The delays between sample collection and bacterial culture and antibiotic susceptibility reporting have led to empirical use of antibiotics, contributing to the emergence of drug resistant pathogens. As a key step toward the development of a point of care device for determining the antibiotic susceptibility of urinary tract pathogens, we report on a biosensor based antimicrobial susceptibility test.
Materials and Methods
For assay development bacteria were cultured with or without antibiotics, and growth was quantitated by determining viable counts and electrochemical biosensor measurement of bacterial 16S rRNA. To determine antibiotic susceptibility directly from patient samples, urine was cultured on antibiotic plates for 2.5 hours and growth was determined by electrochemical measurement of bacterial 16S rRNA. For assay validation 252 urine samples were collected from patients at the Spinal Cord Injury Service at Veterans Affairs Palo Alto Health Care System. The biosensor based antimicrobial susceptibility test was completed for samples containing gram-negative organisms. Pathogen identification and antibiotic susceptibility results were compared between our assay and standard microbiological analysis.
A direct biosensor quantitation of bacterial 16S rRNA can be used to monitor bacterial growth for a biosensor based antimicrobial susceptibility test. Clinical validation of a biosensor based antimicrobial susceptibility test with patient urine samples demonstrated that this test was 94% accurate in 368 pathogen-antibiotic tests compared to standard microbiological analysis.
This biosensor based antimicrobial susceptibility test, in concert with our previously described pathogen identification assay, can provide culture and susceptibility information directly from a urine sample within 3.5 hours.
urinary tract infections; biosensing techniques; microbial sensitivity tests; point-of-care systems
Electrothermal flow is a promising technique in microfluidic manipulation toward laboratory automation applications, such as clinical diagnostics and high throughput drug screening. Despite the potential of electrothermal flow in biomedical applications, relative little is known about electrothermal manipulation of highly conductive samples, such as physiological fluids and buffer solutions. In this study, the characteristics and challenges of electrothermal manipulation of fluid samples with different conductivities were investigated systematically. Electrothermal flow was shown to create fluid motion for samples with a wide range of conductivity when the driving frequency was above 100 kHz. For samples with low conductivities (below 1 S/m), the characteristics of the electrothermal fluid motions were in quantitative agreement with the theory. For samples with high conductivities (above 1 S/m), the fluid motion appeared to deviate from the model as a result of potential electrochemical reactions and other electrothermal effects. These effects should be taken into consideration for electrothermal manipulation of biological samples with high conductivities. This study will provide insights in designing microfluidic devices for electrokinetic manipulation of biological samples toward laboratory automation applications in the future.
Urine is the most abundant and easily accessible of all body fluids and provides an ideal route for non-invasive diagnosis of human diseases, particularly of the urinary tract. Electrochemical biosensors are well suited for urinary diagnostics due to their excellent sensitivity, low cost, and ability to detect a wide variety of target molecules including nucleic acids and protein biomarkers. We report the development of an electrochemical immunosensor for direct detection of the urinary tract infection (UTI) biomarker lactoferrin from infected clinical samples. An electrochemical biosensor array with alkanethiolate self-assembled monolayer (SAM) was used. Electrochemical impedance spectroscopy was used to characterize the mixed SAM, consisted of 11-mercaptoundecanoic acid and 6-mercapto-1-hexanol. A sandwich amperometric immunoassay was developed for detection of lactoferrin from urine, with a detection limit of 145 pg/ml. We validated lactoferrin as a biomarker of pyuria (presence of white blood cells in urine), an important hallmark of UTI, in 111 patient-derived urine samples. Finally, we demonstrated multiplex detection of urinary pathogens and lactoferrin through simultaneous detection of bacterial nucleic acid (16S rRNA) and host immune-response protein (lactoferrin) on a single sensor array. Our results represent first integrated sensor platform capable of quantitative pathogen identification and measurement of host immune response, potentially providing clinical diagnosis that is not only more expeditious but more informative than the current standard.
Electrochemical biosensor; Amperometry; Urinary diagnostics; Urinary tract infections; Biomarkers
This study reports the use of microfluidics, which intrinsically has a large surface-to-volume ratio, toward rapid antimicrobial susceptibility testing at the point of care. By observing the growth of uropathogenic E. coli in gas permeable polymeric microchannels with different dimensions, we demonstrate that the large surface-to-volume ratio of microfluidic systems facilitates rapid growth of bacteria. For microchannels with 250 micrometer or less in depth, the effective oxygenation can sustain the growth of E. coli to over 109 cfu/ml without external agitation or oxygenation, which eliminates the requirement of bulky instrumentation and facilitates rapid bacterial growth for antimicrobial susceptibility testing at the point of care. The applicability of microfluidic rapid antimicrobial susceptibility testing is demonstrated in culture media and in urine with clinical bacterial isolates that have different antimicrobial resistance profiles. The antimicrobial resistance pattern can be determined as rapidly as 2 hours compared to days in standard clinical procedures facilitating diagnostics at the point of care.
Cartridge-based microfluidics is a promising technology for clinical diagnostics. By miniaturizing the fluid-handling processes required for genomic and proteomic analyses, reagent and specimen volume is minimized along with the size of the system. We demonstrate an automated microfluidic system capable of performing six multiplexed genomic and proteomic analyses simultaneously, by means of an integrated electrochemical sensor and embedded controls.
microfluidics; electrochemical sensor; multiplexed assay; quantitative; molecular analysis; point of care; clinical diagnostics
Many bacterial pathogens are becoming drug resistant faster than we can develop new antimicrobials. To address this threat in public health, a metamodel antimicrobial cocktail optimization (MACO) scheme is demonstrated for rapid screening of potent antibiotic cocktails using uropathogenic clinical isolates as model systems. With the MACO scheme, only 18 parallel trials were required to determine a potent antimicrobial cocktail out of hundreds of possible combinations. In particular, trimethoprim and gentamicin were identified to work synergistically for inhibiting the bacterial growth. Sensitivity analysis indicated gentamicin functions as a synergist for trimethoprim, and reduces its minimum inhibitory concentration for 40-fold. Validation study also confirmed that the trimethoprim-gentamicin synergistic cocktail effectively inhibited the growths of multiple strains of uropathogenic clinical isolates. With its effectiveness and simplicity, the MACO scheme possesses the potential to serve as a generic platform for identifying synergistic antimicrobial cocktails toward management of bacterial infection in the future.
We have previously demonstrated the clinical validity of the rapid detection of uropathogens by use of a DNA biosensor. This assay involves the hybridization of capture and detector probe pairs with bacterial 16S rRNA target molecules to form a DNA-RNA sandwich on the sensor surface. Horseradish peroxidase-conjugated antibody binds to the detector probe to enzymatically amplify the hybridization signal. These previous studies involved the hybridization of bacterial 16S rRNA target sequences with 35-mer oligonucleotide probe pairs at 65°C. Achievement of point-of-care technology will be greatly facilitated by ambient-temperature detection. The purpose of this study was to examine the effects of probe length and target location on signal intensity using hybridization temperatures of 20 to 25°C. Signal intensity was found to vary dramatically with hybridization location in the species-specific bulge region of 16S rRNA helix 18. Probe pairs of as short as 10 nucleotides in length were able to produce a significant electrochemical signal, and signal intensity was correlated with probe length for probes of 10 to 20 nucleotides in length. The sensitivity of the Escherichia coli-specific 15-mer probe pairs was approximately 330 cells. These shorter probes allowed differentiation of Klebsiella pneumoniae from Proteus mirabilis 16S rRNA target sequences differing by a single nucleotide. A panel of oligonucleotide probe pairs ranging from 11 to 23 nucleotides in length was able to distinguish among seven groups of urinary tract pathogens. In conclusion, we have developed short oligonucleotide probe pairs for the species-specific identification of uropathogens at ambient temperature by use of an electrochemical sensor.
Electrochemical biosensors have revolutionized glucose monitoring but have not yet fulfilled their promise of a low cost, direct detection replacement for genetic amplification tests such as PCR [K. Kerman, M. Kobayashi, E. Tamiya, Recent trends in electro-chemical DNA biosensor technology, Meas. Sci. Technol. 15 (2004) R1-R11; A. Chaubey, B.D. Malhotra, Mediated biosensors. Biosens. Bioelectron. 17 (6-7) (2002) 441-456]. It has been anticipated that the integration of nanoscale chemical structures such as self-assembled monolayers with electrochemical biosensors would increase sensitivity by decreasing inherent system noise. We have designed a novel biosensing approach incorporating such integration and achieved rapid, ultra-low concentration sensitivities without target amplification. Raw samples are mixed with lysis buffer to allow hybridization of nucleic acid targets with anchor and signal probes before immobilizing a signaling enzyme proximate to the biosensor surface. A bias potential is subsequently applied and the secondary byproduct of a cyclic peroxidase reaction measured. Further studies have demonstrated the application of our approach in protein, clinical chemistry, and ionic assays.
Electrochemical detection; Cyclic enzymatic reaction; Bionanotechnology; Genetic assay; Immunoassay; Simultaneous multi-channel detection
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.