A Point-of-Care Micro-Laboratory for Direct Pathogen Identification in Body Fluids

Joseph C. Liao1*, Yanbao Ma2, Vincent Gau3, Mitra Mastali4,5, Chien-Pin Sun2, Yang Li1, Edward R.B. McCabe6, Elliot M. Landaw7, David Bruckner8, Bernard M. Churchill1, David A. Haake4,5, Chih-Ming Ho2

Departments of 1Urology, 4Medicine, 6Pediatrics, 7Biomathematics, 8Pathology and Laboratory Medicine, UCLA School of Medicine, Los Angeles, USA

2Institute for Cell Mimetic Space Exploration and Department of Mechanical and Aerospace Engineering, UCLA School of Engineering and Applied Science, Los Angeles, USA

3GeneFluidics, Inc., USA 5Division of Infectious Diseases, 111F, Veterans Affairs Greater Los Angeles Healthcare, USA

Abstract—We describe a prototype micro-laboratory for rapid genetic identification of bacterial pathogens from infected human body fluids. The core module of the detection platform is a microfabricated electrochemical sensor array. Picomolar amperometric detection is achieved through formation of a DNA sandwich between capture and detector probe pairs and bacterial 16S rRNA, coupled with an oxidoreductase cnzymatic transducer. Using the sensor array functionalized with a panel of species-specific DNA oligonucleotide probes, detection of bacterial urinary pathogens have been demonstrated in a pilot clinical validation study demonstrating 98% sensitivity for Gram-negative pathogen detection, using conventional urine culture as the standard. Genotypic species identification is achieved within 45 minutes, compared to 1-2 days needed for standard bacterial culture technique. A biofilter has also been fabricated and validated with clinical urine specimens. Efficient concentration of the pathogens is demonstrated and replaces the need for conventional centrifugation. A microfluidic mixer to facilitate reagent mixing is also under development as part of sample preparation module. Preliminary experimental data indicate good correlation with computational simulation. These are critical milestones towards our development of an integrated point-of-care platform for pathogen detection in body fluids.

Keywords- electrochemical DNA sensor; lab-on-a-chip; microfluidics

I. INTRODUCTION Electrochemical DNA biosensors offer high sensitivity and

possibility for genetic identification of pathogens from complex, turbid body fluids (e.g. urine, blood, saliva) without PCR amplification [1-4]. The advantages of portability, low power consumption and ease of integration with sample preparation modules make electrochemical DNA biosensors an attractive core of an integrated micro-laboratory for pathogen identification. Currently, standard approach for the diagnosis of bacterial infections requires time-consuming process of cultivation in a centralized laboratory, which takes minimum of 1 to 2 days. Such delay in diagnosis may lead to unnecessary treatment, inappropriate use of antimicrobial agents, and promotion of multi-drug resistant pathogens.

Urine is the most common body fluids submitted to clinical laboratories for analysis. Majority of urine specimens are sent to rule out possible urinary tract infection (UTI), the most

common bacterial infection in humans [5, 6]. UTI is a major cause of patient morbidity and health care expenditure for all age groups, accounting annually in United States over 7 million office visits and over 1 million hospital admissions [7], at the annual cost of $3.5 billion [8, 9]. Processing of urine specimens by clinical microbiological laboratories is an important component of these health care costs. A significant proportion of the urine specimens submitted to a clinical microbiology laboratory, however, are negative. A point-of-care diagnostic system to determine the causative urinary pathogen (uropathogen) may potentially revolutionize management of UTI by expediting diagnosis and treatment of the infected patients, rapid triage of the uninfected, and chronic surveillance in the high-risk patients.

In this report, we summarize our progress to date on the development of key components for a point-of-care micro-laboratory for uropathogen detection. Milestones accomplished are: characterization of a microfabricated electrochemical sensor array, design of a panel of DNA probes against the most common uropathogens, a pilot clinical validation study, development of a biofilter for sample concentration, and a micro-mixer to enable rapid reagent mixing.

II. ELECTROCHEMICAL SENSOR

A. Sensor charcterization Figure 1 shows the GeneFluidics electrochemical sensor

array. Each array measures 2.5 x 7.5 cm and consists of 16 gold sensors. The microfabrication process is as described previously with modifications [3]. Each sensor contains 3 electrodes: working, reference and auxiliary. The sensor surface is coated with an alkanethiolate self-assembled monolayer to enable functionalization with DNA oligonucleotide probes. Cyclic voltammetry of all 16 sensors within an array using a multi-channel potentiostat is shown in Figure 2.

This project was funded by the US National Institute of Biomedical Imaging and Bioengineering (NIBIB) Grant RO1 EB00127 and the American Foundation for Urologic Disease.

All authors are members of the UCLA Urosensor Bioengineering Research Partnership: http://www.urology.medsch.ucla.edu/uropathogen-partners.html

*Contact author: jliao1@gmail.com. Present address: Department of Urology (S287), Stanford University Medical Center, 300 Pasteur Avenue, Stanford, CA 94305-5118.

Figure 1. Electrochemical sensor array. Adapted from [1]

Amperometric detection strategy is based on sandwich hybridization of target nucleic acids by capture and detector DNA oligonucleotide probes (Figure 3). After hybridization and immobilization onto the sensor surface, an oxidoreductase enzyme, horseradish peroxidase (HRP) is coupled to the detector probe for signal amplification. An electroreduction current is generated at fixed potential (-100 mV), which is correlated with number of hybridization events. Picomolar detection sensitivity is achieved using a synthetic DNA oligonucleotide target (Figure 4).

Pathogen-specific probes A panel of oligonucleotide DNA probes specific against

16S rRNA of the most common uropathogens was developed [10, 11]. 16S rRNA is an essential component of bacterial ribosome and is present in all eubacteria. Sequence analysis of 16S rRNA reveals interposing regions of sequence conservation and diversity. Areas of sequence diversity across different bacterial species enable design of species-specific probes. Probes designed target Escherichia coli (EC), Proteus mirabilis (PM), Pseudomonas aeruginosa (PA), Klebsiella-

Enterobacter species (KE), and Enterococcus species (EF). In addition, a universal probe (UNI) and a probe that recognizes enteric Gram-negative pathogens (ENTBC). The specificity of the probes was verified using a library of uropathogen isolates collected from UCLA Clinical Microbiology Laboratory.

B. Pilot clinical validation study We have tested 78 clinical urine specimens in a pilot

clinical study [11]. Blinded urine specimens were obtained from the UCLA Clinical Microbiology Laboratory and 1 ml pellet obtained through centrifugation. Manual processing of the urine pellet were performed based experimental protocol outlined in Figure 3 and assayed using the sensor. Results of the 45-minute assay were compared with the standard culture method (1-2 days). Overall, 98% sensitivity against the Gram-negative bacteria for which species-specific probes were available. A representative experiment is shown in Figure 5 demonstrating detection of E. coli. This is the first report to our knowledge of bacterial pathogen detection in human body fluids using a microfabricated sensor, as well as the clinical validity of this approach.

III. BIOFILTER

A. Fabrication A major challenge to integrate the biosensor into a point-of-

care, lab-on-a-chip platform is the ability to concentrate body

Figure 5. Representative experiment showing a patient-derived urine specimen tested against the electrochemical sensor array containing library of uropathogen probes. Positive signals seen for E. coli probe (EC), and the positive controls: universal probe (UNI) and enteric Gram-negative probe (ENTBC). Other probes yielded signals in the range of background (NC). Culture confirmed presence of E. coli in the urine specimen > 105 cells/cc. Each probe was tested in duplicates.

Figure 3. Schematic of the amperometric detection mechanism based on DNA sandwich. The target is hybridized with the capture and detector probes and immobilized on the sensor surface. The electrochemical signal transducer horseradish peroxidase (HRP) binds to the detector probe moiety (fluorescein). The electroreduction current is generated by electron transfer to the mediator tetramethylbenzidine (TMB) at fixed potential (-100 mV). Not drawn to scale.

Figure 2. Cyclic voltammetry of the 16 sensors coated with alkanethioloate self-assembled monolayer.

Figure 4. Detection sensitivity of the electrochemical sensor array using a synthetic oligonucleotide target in molar concentration (nA, nano-ampere). NC = negative control containing no target.

fluids without centrifugation. To address this, a prototype membrane biofilter driven by a miniature peristaltic pump was developed that concentrates uropathogens directly from urine specimens. The acrylic filter chamber (Figure 6) was fabricated with a computer numerically controlled machine (outer diameter 25 mm, inner diameter 10 mm, height 40 mm). Grooves 250 µm wide and 0.75 mm deep were cut on bottom to form a screen to support the filter membrane. Polyethersulfone membrane with 0.45 µm pore size was used for micro-filtration. A miniature peristaltic pump drives the biofilter. A small magnetic stirring bar is used to reduce clogging.

B. Concentration of clinical urine specimens Eight urine specimen derived from patients with UTI were

tested (filtration volume 2-10 cc). Total filtration time was 2-5 minutes. Figure 7 shows the result of a representative experiment comparing biofilter (5 min filtration) and centrifugation (10000 rpm, 2 min) of a clinical urine specimen. Urine specimen without concentration was used as control. All three samples were assayed with the electrochemical sensor. The biofilter also served as a hybridization chamber for the target bacterial 16S rRNA and the detector probe. The hybridization products were deposited directly on the sensor array for genotypic detection. Pseudomonas aeruginosa was identified and confirmed by bacterial urine culture (data not shown). The entire assay took approximately 60 min. These results suggest the feasibility of using the biofilter to efficiently concentrate clinical urine specimens without need for centrifugation.

IV. MICROMIXER The rate-limiting factor of the assay time is dependent on

passive diffusion between the reagents and the sample. These steps include bacterial cell wall lysis, DNA/RNA hybridization, and the attachment of the signal transducing enzyme. These biochemical reactions can be expedited significantly by active mixing in the detection system. We have designed an active micromixer based on pressure disturbance to be integrated with the biofilter and the sensor [12]. The geometry of the micromixer has been optimized by numerical simulations.

The mixing is investigated by using computational fluid dynamic (CFD) analysis. Different geometries and orientation of inlets of the micromixers working at different working conditions were simulated. Based on the simulation result, the micromixer was designed and fabricated by using PDMS (Dow Corning) with SU8-50 as the mold. The PDMS channel was sandwiched by two microscope slides. The performance of the micromixer was tested by mixing aqueous solutions and the flow field was visualized by using yellow and blue dyes. Figure 8 shows a comparison of concentration distribution between numerical results based on CFD (Reynolds number = 0.119 and Strouhal number = 1.26) and experimental results. Preliminary testing with dye demonstrated efficient mixing in less than 1.5 mm. Testing using lysed bacterial samples with DNA probes are planned.

V. SYSTEM INTEGRATION A schematic of the integrated pathogen micro-laboratory

including the sensor, biofilter, and micromixer is shown in Figure 9. The steps include: 1) concentration of urine specimen with the biofilter; 2) release of lysis buffer into the urine pellet; 3) release of detector probes into the bacterial lysate containing the 16S rRNA; 4) elution of bacterial lysate and the detector probe into the micromixer module; 5) deposition of the 16S rRNA:detector probe hybrid onto the electrochemical sensor array functionalized with the species-

Figure 8. Active micromixer based on pressure disturbance. Comparison between computational fluid dynamic simulation (top) and experimental results (bottom).

Figure 6. Schematic of the biofilter for pathogen concentraiton (left) and filtration of a cloudy clinical urine specimen (right).

Figure 7. Net concentration effect of filtration and centrifugation relative to unprocessed infected urine sample. Negative control (“No cell”) contained no target.

specific capture probes; 6) washing; 7) addition of the HRP signal transducer; 8) addition of the HRP substrate and mediator; 9) amperometric reading. Active efforts are currently underway to integrate these modular components.

In this report, we have demonstrated the important modular components of an integrated micro-laboratory for rapid genetic detection of bacterial pathogens in urine. We anticipate this will be a useful platform for point-of-care diagnosis of UTI’s and relative ease in adaptation for detection of other pathogens in other body fluids.

ACKNOWLEDGMENT We thank the other members of the UCLA Urosensor

Bioengineering Research Partnership for their insights and helpful suggestions: Marc Suchard, Jane Babbit, Annette Møller, Jefferey Gorbein, Linda McCabe, Yao Hua Zhang, and Linda Gibson. We also thank James Matsunaga of UCLA/VAGLAHS; Susan K. Haake of the UCLA School of

Dentistry; and John Kibler, Ashish Pradhan of GeneFluidics for expert assistance.

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Figure 9. Schematic of the integrated prototype micro-laboratory for rapid urinary pathogen detection including the sensor, biofilter, and micro-mixer.