Vincent Gau

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.

Antimicrobial susceptibility testing using high surface-to-volume ratio microchannels.

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 Escherichia 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 microm or less in depth, the effective oxygenation can sustain the growth of E. coli to over 10(9) 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 h compared to days in standard clinical procedures facilitating diagnostics at the point of care.

Electrochemical molecular analysis without nucleic acid amplification.

Abstract 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 electrochemical 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.

Matrix Effects—A Challenge toward Automation of Molecular Analysis

Many components in biological matrices influence the result of an analysis, affecting assay sensitivity and reproducibility. Improved matrix management becomes critical as requirements for higher assay sensitivity and increased process throughput become more demanding. There are several robotic laboratory automation systems that are commercially available, which serve to minimize matrix interference by performing purification and extraction protocols. However, there is an unmet need of inline matrix effect reduction solutions to reduce the processing time and cost for automated sample preparation. In microfluidics, effective matrix management is essential for developing fully integrated systems capable of meeting these requirements. This review surveys current biological matrix management techniques for liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods and binding assays with a view toward building automatable processes. For some systems, simple sample-preparation methods, such as dilution and protein precipitation (PPT), are sufficient, whereas other systems require labor-intensive methods, such as liquid-liquid extraction (LLE) and solid-phase extraction (SPE). To achieve high throughput, PPT, LLE, and SPE have been adopted to 96-well-plate format. Online SPE has also been coupled with LC-MS/MS to automate sample preparation and analysis of urine, plasma, and serum matrices. However, offline processing of whole blood is still required to obtain plasma and serum. The ultimate goal of implementing sample preparation to reduce matrix effects within untreated sample is to achieve reproducibility and sensitivity required by the application; therefore, inline sample preparation integrated with molecular analysis will be highly significant for laboratory automation. Electrokinetic methods have the potential of handling whole-blood, urine, and saliva samples and can be incorporated into microfluidic systems for full automation. Optimization of analysis conditions and the use of appropriate standards have likewise assisted in reducing or correcting matrix effects and will also be discussed.

A Biosensor Platform for Rapid Antimicrobial Susceptibility Testing Directly From Clinical Samples

PURPOSE 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. RESULTS 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. CONCLUSIONS 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.

Hybrid electrokinetic manipulation in high-conductivity media

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.

Electrochemical immunosensor detection of urinary lactoferrin in clinical samples for urinary tract infection diagnosis

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 also more informative than the current standard.

Oral fluid nanosensor test (OFNASET) with advanced electrochemical-based molecular analysis platform.

Abstract:  High‐impact diseases, including cancer, cardiovascular disease, and neurological disease, are challenging to diagnose without supplementing clinical evaluation with laboratory testing. Even with laboratory tools, definitive diagnosis often remains elusive. The lack of three crucial elements presents a road block to achieving the potential of clinical diagnostic tests: (1) definitive disease‐associated protein and genetic markers, (2) easy and inexpensive sampling methods with minimal discomfort for the subject, and (3) an accurate and quantitative diagnostic platform. Our aim is to develop and validate a solution for requirement (3) and also to develop a portable system. Requirements (1) and (2) will be addressed through the utilization of novel and highly specific oral cancer saliva proteomic and genomic biomarkers and the use of saliva as the biofluid of choice, respectively. The Oral Fluid NanoSensor Test (OFNASET) technology platform combines cutting‐edge technologies, such as self‐assembled monolayers (SAM), bionanotechnology, cyclic enzymatic amplification, and microfluidics, with several well‐established techniques including microinjection molding, hybridization‐based detection, and molecular purification. The intended use of the OFNASET is for the point of care multiplex detection of salivary biomarkers for oral cancer. We have demonstrated that the combination of two salivary proteomic biomarkers (thioredoxin and IL‐8) and four salivary mRNA biomarkers (SAT, ODZ, IL‐8, and IL‐1b) can detect oral cancer with high specificity and sensitivity. Our preliminary studies have shown compelling results. We sequentially delivered a serial dilution of IL‐8 antigen, probe solution, wash, enzyme solution, wash, and mediator solution to sensor reaction chambers housed in a prototype cartridge and demonstrated strong signal separation at 50 pg/mL above a negative control.

Single Cell Antimicrobial Susceptibility Testing by Confined Microchannels and Electrokinetic Loading

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 1 h compared to days in standard culture-based AST techniques.

Electrothermal Fluid Manipulation of High-Conductivity Samples for Laboratory Automation Applications.

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, relatively 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 greater than 100 kHz. For samples with low conductivities (below I S/m), the characteristics of the electrothermal fluid motions were in quantitative agreement with the theory. For samples with high conductivities (greater than l 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.