A Microfluidic System for Saliva-Based Detection of Infectious Diseases

A Microfluidic System for Saliva-Based Detection of Infectious Diseases ZONGYUAN CHEN, a MICHAEL G. MAUK, a JING WANG, a WILLIAM R. ABRAMS, b PAUL L. A. M. CORSTJENS, c R. SAM NIEDBALA, d DANIEL MALAMUD, b AND HAIM H. BAU a a University of Pennsylvania School of Engineering and Applied Science, Philadelphia, Pennsylvania, USA b New York University College of Dentistry, New York, New York, USA c Leiden University Medical Center, Leiden, the Netherlands d Department of Chemistry, Lehigh University, Bethlehem, Pennsylvania, USA ABSTRACT: A lab-on-a-chip system for detecting bacterial pathogens in oral fluid samples is described. The system comprises : (1) an oral fluid sample collector; (2) a disposable, plastic microfluidic cassette ( chip ) for sample processing including immunochromatographic assay with a nitrocellulose lateral flow strip; (3) a platform that controls the cassette operation by providing metered quantities of reagents, temperature regulation, valve actuation; and (4) a laser scanner to interrogate the lateral flow strip. The microfluidic chip hosts a fluidic network for cell lysis, nucleic acid extraction and isolation, PCR, and labeling of the PCR product with bioconjugated, upconverting phosphor particles for detection on the lateral flow strip. KEYWORDS: microfluidics; lab-on-a-chip; infectious diseases; diagnostics; immunochromatography INTRODUCTION AND BACKGROUND Currently, point-of-care (POC) testing for infectious diseases is routinely performed using immunochromatographic assays of serum, urine, or saliva. These assays are typically implemented as easy-to-use nitrocellulose lateral flow test strips. Similar test strips are widely employed for a variety of applications, such as home pregnancy testing and detecting drugs of abuse. Serologic lateral flow strips that detect HIV antibodies in oral fluid are completely selfcontained, requiring no addition of reagents or accessory instrumentation. 1 Address for correspondence: Haim H. Bau, 216 Towne Building, Mechanical Engineering and Applied Mechanics, University of Pennsylvania, Philadelphia, PA 19104-6315. Voice: 215-898-8363; fax: 215-573-6114. bau@seas.upenn.edu Ann. N.Y. Acad. Sci. 1098: 429 436 (2007). C 2007 New York Academy of Sciences. doi: 10.1196/annals.1384.024 429

430 ANNALS OF THE NEW YORK ACADEMY OF SCIENCES The strips include a control test function for quality assurance, and can provide test results in less than 10 min. The lateral flow test strip device provides convenient, fast, and low-cost POC and home testing. However, there is considerable incentive for improving sensitivity and increasing functionality. For example, a more quantitative assay could be used to determine viral loads. Additionally, parallel testing of nucleic acid (DNA and RNA) and antigen targets, from both viral and bacterial pathogens, as well as detection of antibodies, would enable simultaneous robust, multipurpose, and confirmatory testing from the same sample with the same test device. However, multiplexed nucleic acid testing and immunoassays require elaborate sample processing, which complicates implementation as a simple-to-use POC device. Microfluidic lab-on-a-chip devices in the form of miniaturized networks of conduits, chambers, and valves formed in a plastic substrate chip facilitate sample metering, cell and virus lysis, isolation of nucleic acids and proteins, amplification of nucleic acids using PCR or analogous techniques, and labeling of analytes for detection. Thus, a lateral flow strip combined with a microfluidic cassette for sample processing would significantly expand the range of applications and tasks that could be performed by immunochromatographic methods. Most lateral flow test strips use colloidal gold, dye, or latex beads as reporters to allow visual inspection of test results. 1 Lateral flow strip tests that incorporate upconverting phosphors as reporters have also been developed. 2 5 Upconverting phosphor technology (UPT) is based on sub-micron-sized ceramic particles coated with lanthanides that absorb infrared light (excitation) and emit visible light (response signal). The particles can be functionalized with antigens and antibodies for use as labels on lateral flow strips. UPT particles avoid interference from background fluorescence and do not exhibit photobleaching effects. UPT reporters have been shown to improve the limit of detection in various bioassays by 10-fold or more. 3 6 In one particular application, a POC diagnostics system (OraSure Uplink TM ) includes an FDAapproved oral fluid sample collector, a molded plastic cartridge that houses the lateral flow strip and features a loading port for the sample collector, and a tabletop reader instrument with laser scanner and photomultiplier tube detector to interrogate the lateral flow strip. 7 Also, UPT-based immunochromatography flow strips have been adapted for detecting nucleic acid targets, 3 5 such as UPT-labeled PCR products, thus opening the way for applying this technology for nucleic acid testing. This UPT lateral flow system thus provides an excellent platform for developing a microfluidics system for multiplexing nucleic acid based diagnostics and immunoassays. The approach is to develop a disposable credit card sized plastic cassette that hosts a microfluidic circuit integrating components for (1) sample metering; (2) enzymatic and chemical lysis using lysozyme, proteinase K, chaotropic salts, and detergents; (3) solidphase extraction using a porous silica membrane embedded in the cassette; (4) PCR amplification; (5) labeling of the PCR product with UPT phosphor; and

CHEN et al. 431 (6) immunochromatography assay of the PCR amplicon with a nitrocellulose lateral flow strip mounted in the cassette. Efforts to add microfluidic capabilities to lateral flow strip assays have already met with success. Chen et al. 8 developed a consecutive lateral flow assay microfluidic chip with UPT reporters for use with the UPlink TM laser scanner. The chip includes functions for sample introduction, buffer distribution, metering, mixing, and thermopneumatic pumping. After a serum sample is loaded on the chip, the chip is connected to an instrumentation platform that provides fluidic power, temperature control, and valve actuation. Automated processing on the chip comprises a sequence of steps including sample aliquoting and metering, dilution with buffer, and blotting on a nitrocellulose strip followed by a separate wash step of the strip with buffer, followed by addition of buffer with UPT label. The UPT labels are dry-stored and preloaded on the chip in a lyosphere. The chip is then inserted into the UPlink TM reader for interrogation of the strip by IR (980 nm) and signal readout. Along similar lines, Wang et al. 9 reported a disposable microfluidic cassette for DNA amplification and detection combining a microfluidic PCR chamber and an incubation chamber to label the PCR product with UPT particles, integrated with a lateral flow strip to capture UPT-labeled PCR amplicon for detection in the laser scanner. Here, we report a microfluidic cassette for PCR-based detection of Gram-positive bacteria using a lateral flow strip with UPT reporters. The cassette provides complete processing including sample introduction and metering, enzymatic cell lysis, nucleic acid isolation, PCR amplification with pathogen-specific primers conjugated for labeling and capture on a lateral flow strip, and blotting of labeled PCR product on the lateral flow strip affixed to the cassette. The microfluidic system is tested with diluted B. cereus, a safe surrogate for anthrax, but the processing steps are sufficiently generic and the chip design is sufficiently flexible so that it can be adapted for detection of different viral and bacterial pathogens. CHIP DESIGN, FABRICATION, AND OPERATION FIGURE 1A is a photograph of a microfluidic chip for PCR-based detection of bacteria targets. The chip is made as a thermally bonded three-layer laminate of polycarbonate sheets with channels and chambers defined in two of the layers by CNC machining. Cross-section dimensions of the conduits range from 250 to 500 m. The microfluidic cassette is mounted in a molded plastic cartridge compatible with insertion into the UPlink TM laser scanner/reader unit (FIG. 1B). Phase-change ice valves actuated by small, electrically controlled thermoelectric elements on the processing platform are used for flow control and sealing the 10- L PCR chamber. 10 Flow is halted when the fluid in a channel freezes at the section of conduit in contact with a cooled thermoelectric element, and flow resumes when the thermoelectric element is heated to thaw

432 ANNALS OF THE NEW YORK ACADEMY OF SCIENCES FIGURE 1. (A) Polycarbonate microfluidic cassette for bacteria detection. The cassette features conduits, chambers, and phase-change valve sites for sample metering, lysis, solidphase extraction, and isolation of nucleic acids with a porous silica membrane-binding phase, PCR, labeling, and nitrocellulose lateral flow test strip. The cassette measures 85 35 mm and is 3 mm thick. (B) Microfluidic cassettes with lateral flow strips are mounted in a molded plastic cartridge for the UPlink TM laser scanner/reader unit. the fluid. The PCR chamber is also heated and cooled by a thermoelectric element. The chip is interfaced with programmable syringe pumps, reservoirs for buffers and reagents, and electronic controllers for the thermoelectric elements, controlled by a PC with LabVIEWTM software (FIG. 2).

CHEN et al. 433 FIGURE 2. Schematic of supporting instrumentation for microfluidic cassette including buffer and reagent reservoirs, programmable syringe pumps, thermoelectric elements for actuating phase-change ice valves and heating/cooling PCR chamber, controlled power supplies, and PC. The sample is introduced through a loading port and 50 L of sample aliquoted in a metering chamber. The sample is then subjected to a twostep lysis process. The sample is first enzymatically digested for 30 min at 37 C with lysozyme (20 mg/ml lysozyme, 1.2% Triton X-100, 2mM EDTA, 20 mm Tris-Cl), and then incubated with SDS detergent (0.1%), proteinase K (10 mg/ml) and 6M guanidinium chloride at 60 C for 15 min. The lysate is forced through a porous silica membrane embedded in the cassette. The chaotropic salts induce nucleic acids binding to the silica. The silica-bound nucleic acids are washed with ethanol-based solutions to remove debris. Next, the nucleic acids are desorbed from the silica by elution with low-salt, phneutral buffer, and a 5- L elution fraction is mixed with PCR reagents (0.3 U Taq polymerase, 200 M dntp, 0.1 g/ul BSA, 50 mm Tris-Cl, 1.5 3.5 mm MgCl 2 ) and primers (forward: 5 -TCT CGT TTC ACT ATT CCC AAG T-3 conjugated to dioxygenin; reverse: 5 AAG GTT CAA AAG ATG GTA TTC AGG-3 conjugated to biotin) and sealed in a 10- L PCR chamber, where it

434 ANNALS OF THE NEW YORK ACADEMY OF SCIENCES is thermally cycled 25 times (95 C, 15 sec; 55 C, 25 sec; 72 C, 20 sec). The 305-bp amplicon is labeled by incubation at room temperature for 15 min with 150 ng of avidin-conjugated UPT particles. The UPT-labeled PCR product is applied to the sample pad of a lateral flow strip and captured by immobilized anti-dig antibodies at the test line stripe. The strip is then scanned with the UPlink TM reader. RESULTS To assess microfluidic lysis and nucleic acid isolation for producing a DNA template for PCR from cell culture samples, nucleic acid eluted from the chip silica membrane was PCR-amplified and compared to PCR-amplified nucleic acid isolated by a standard benchtop protocol (QIAGEN DNeasy TM Kit). In the benchtop protocol, a 50- L sample of B. cereus ( 10 6 cells/ml) was subjected to a two-step enzymatic lysis process with lysozyme, chaotropic salts, and detergents. The nucleic acid was then isolated from the lysate in successive FIGURE 3. Comparison of on-chip and benchtop lysis and nucleic acid isolation. To determine the efficiency of on-chip lysis and nucleic acid isolation relative to benchtop lysis and nucleic acid isolation, elutions from the silica membrane on the chip and from a spin column were amplified by PCR and analyzed by gel electrophoresis. Control: PCR of pure B. cereus genomic DNA. Benchtop: B. cereus (50 L, 10 6 cells/ml) was subjected to two-step lysis (lysozyme digestion followed by treatment with chaotropic salt and proteinase K according to QIAGEN DNeasy TM protocol). Nucleic acid was isolated from lysate using QIAGEN DNeasy TM spin column. Aliquots of 5 L from two successive 50- L elutions (E1 and E2) from the spin column were mixed with 5 L of PCR mix (Taq, dntp, buffer, primers, BSA), amplified for 25 cycles, and run on an agarose gel (1.5%, ethidium bromide staining). Chip runs (two replicates, I and II): Similar B. cereus samples (50 L, 10 6 cells/ml) were loaded into the chip and subjected to two-step lysis and nucleic acid isolation using the silica-membrane component of the chip. Aliquots (5 L) from successive 50- L elutions (E1, E2, E3...) were mixed with 5 L of PCR mix (Taq, dntp, buffer, primers, BSA), amplified for 25 cycles on a benchtop thermal cycler, and run on an agarose gel (1.5%, ethidium bromide staining). Weak or absent PCR band for first elution (E1) for chip runs suggests a PCR inhibitor is contained in the initial elution. Subsequent elutions from the chip membrane, however, showed strong PCR amplification.

CHEN et al. 435 FIGURE 4. A representative UPlink TM laser scan of the lateral flow strip for measurement of UPT-labeled PCR amplicon from bacteria (50 Lof 10 6 cells/ml) processed on a microfluidic cassette. 50- L elutions from a QIAGEN DNeasy TM spin column (chaotrope silica method), and amplified by benchtop PCR. Similarly, a 50- L sample of B. cereus was loaded into the microfluidic cassette for on-chip lysis and nucleic acid isolation. Successive 50- L elutions from the silica membrane embedded in the chip were collected and amplified by benchtop PCR. In each case, 5- L aliquots from successive 50- L elution of the spin column and from the chip silica membrane were mixed with 5 L of PCR mix (Taq, dntp, buffer, primers, BSA), amplified for 25 cycles, loaded and run on a 1.5% agarose gel with ethidium bromide stain (FIG. 3). The weak or absent PCR product from the initial elution of the chip indicates the presence of an inhibitor, probably residual ethanol from the silica membrane wash step. This initial elution fraction should be avoided for providing a PCR template, but otherwise the subsequent elutions from the chip yielded easily detected PCR product. There appears to be some benefit to removing inhibitors from the silica membrane by a vacuum drying step prior to elution (data not shown). To test the chip for complete sample processing (i.e., on-chip lysis, nucleic acid isolation, PCR amplification, labeling, and lateral flow assay), the chip was loaded with a 50- L aliquot of B. cereus ( 10 6 cells/ml), and operated according to the sequence detailed above. A typical UPlink TM scan of the chip flow strip is shown in FIGURE 4, indicating a detectable B. cereus specific PCR product produced from the sample. DISCUSSION AND CONCLUSIONS We have demonstrated a microfluidic cassette that can prepare UPT-labeled microfluidic PCR product for detection on a lateral flow strip assay interfaced with the cassette, thus showing the feasibility of microfluidic PCR-based nucleic acid testing of raw samples. The current development effort is focused on

436 ANNALS OF THE NEW YORK ACADEMY OF SCIENCES Gram-positive bacterial targets (B. cereus, a safe surrogate for anthrax), but the system can be readily adapted for detection of viral and bacterial pathogens, multiplexed analysis of several pathogens, and serologic testing for antibodies. The device will serve as a module in a comprehensive, POC saliva-based diagnostic system that will also include modules for antigen and antibody assays. ACKNOWLEDGMENTS The work was supported by NIH Grants UO1DE0114964 and UO1DE017855, and in collaboration with OraSure Technologies, Inc. (Bethlehem, PA, USA). REFERENCES 1. KETEMA, F., H.L. ZINK, K.M. KREISEL, et al. 2005. A 10-minute, US Food and Drug Administration-approved HIV test. Expert Rev. Mol. Diagn. 5: 135 143. 2. NIEDBALA, R.S., H. FEINDT, K. KARDOS, et al. 2001. Detection of analytes by immunoassay using up-converting phosphor technology. Anal. Biochem. 293: 22 30. 3. CORSTJENS, P.L.A.M., M. ZUIDERWIJK, M. NILSSON, et al. 2003. Lateral-flow and up-converting phosphor reporters to detect single-stranded nucleic acids in a sandwich-hybridization assay. Anal. Biochem. 312: 191 200. 4. CORSTJENS, P.L.A.M., M. ZUIDERWIJK, A. BRINK, et al. 2001. Use of up-converting phosphor reporters in lateral flow assays to detect specific nucleic acid sequences: a rapid, sensitive DNA test to identify human papilloma type 16 infection. Clin. Chem. 47: 1885 1893. 5. HAMPL, J., M. HALL, N.A. MUFTI, et al. 2001. Upconverting phosphor reporters in immunochromatographic assays. Anal. Biochem. 288: 176 187. 6. CORSTJENS, P.L.A.M., S. LI, M. ZUIDERWIJK, et al. 2005. Infrared up-converting phosphors for bioassays. IEEE Proc. Nanobiotechnol. 152: 64 72. 7. MALAMUD, D., H. BAU, S. NIEDBALA & P. CORSTJENS. 2005. Point detection of pathogens in oral samples. Adv. Dent. Res. 18: 12 16. 8. CHEN, Z., P.L.A.M. CORSTEJENS, M. ZUIDERWIJK, et al. 2005. A disposable microfluidic point-of-care device for detection of HIV: a new upconverting phosphor technology application. Proceedings of the 9th Int. Conf. Miniaturized Sys. Chem. Life Sci. 791 793. 9. WANG, J., Z. CHEN, P.L. CORSTJENS, et al. 2006. A disposable microfluidic cassette for DNA amplification and detection. Lab. Chip 6: 46 53. 10. WANG, J., Z. CHEN, M. MAUK, et al. 2005. Self-actuated, thermo-responsive hydrogel valves for lab on a chip. Biomed. Microdevices 7: 313 322.