Zongyuan Chen

A disposable microfluidic cassette for DNA amplification and detection

A pneumatically driven, disposable, microfluidic cassette comprised of a polymerase chain reaction (PCR) thermal cycler, an incubation chamber to label PCR amplicons with up-converting phosphor (UPT) reporter particles, conduits, temperature-activated, normally closed hydrogel valves, and a lateral flow strip, was constructed and tested. The hydrogel valves, which were opened and closed with the aid of electrically controlled thermoelectric units, provided a simple means to seal the PCR reactor and suppress bubble formation. The hydrogel-based flow control was electronically addressable, leakage-free, and biocompatible. To test the device, a solution laden with genomic DNA isolated from B. cereus was introduced into the microfluidic cassette and a specific 305 bp fragment was amplified. The PCR amplicons were labelled with the phosphor (UPT) reporter particles, applied to the lateral flow strip, bound to pre-immobilized ligands, and detected with an IR laser that scanned the lateral flow strip and excited the phosphor (UPT) particles that, in turn, emitted light in the visible spectrum. The UPT particles do not bleach, they provide a permanent record, and they readily facilitate the filtering of background noise. The cassette described herein will be used for rapid testing at the point of care.

A Microfluidic System for Saliva‐Based Detection of Infectious Diseases

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.

A timer-actuated immunoassay cassette for detecting molecular markers in oral fluids

An inexpensive, hand-held, point-of-care, disposable, self-contained immunoassay cassette comprised of air pouches for pumping, a metering chamber, reagents storage chambers, a mixer, and a lateral flow strip was designed, constructed, and tested. The assay was carried out in a consecutive flow format. The detection was facilitated with up-converting phosphor (UCP) reporter particles. The automated, timely pumping of the various reagents was driven by a spring-loaded timer. The utility of the cassette was demonstrated by detecting antibodies to HIV in saliva samples and further evaluated with a non-contagious, haptenized DNA assay. The cassette has several advantages over dip sticks such as sample preprocessing, integrated storage of reagents, and automated operation that reduces operator errors and training. The cassette and actuator described herein can readily be extended to detect biomarkers of other diseases in body fluids and other fluids at the point of care. The system is particularly suitable for resource-poor countries, where funds and trained personnel are in short supply.

Finger-actuated, self-contained immunoassay cassettes

The building blocks for an inexpensive, disposable, luminescence-based microfluidic immunoassay cassette are described, and their integration in a point-of-care diagnostic system is demonstrated. Fluid motion in the cassette is driven by depressing finger-actuated pouches. All reagents needed for the immunoassay can be stored in the cassette in liquid form. Prior to use, the cassette consists of two separate parts. A top storage component contains pouches, sealed storage chambers, a metering chamber, and needle seats. The bottom processing component contains connection needles, a mixing chamber, and a detection chamber with immobilized proteins. Subsequent to sample introduction, the storage and processing components are mated. The needles form hydraulic connections between the two parts and, in some cases, close valves. The pouches are then actuated sequentially to induce flow of various reagents and facilitate process operations. The cassette is compatible with different detection modalities. Both a cassette with immunochromatographic-based detection and a cassette with microbead-based detection were constructed and evaluated. The immunochromatographic cassette was used to detect antibodies to HIV in saliva samples. The bead-based cassette was used to detect the proinflammatory chemokine IL-8. The experimental data demonstrates good repeatability and reasonable sensitivity.

Thermally-actuated, phase change flow control for microfluidic systems

An easy to implement, thermally-actuated, noninvasive method for flow control in microfluidic devices is described. This technique takes advantage of the phase change of the working liquid itself-the freezing and melting of a portion of a liquid slug-to noninvasively close and open flow passages (referred to as a phase change valve). The valve was designed for use in a miniature diagnostic system for detecting pathogens in oral fluids at the point of care. The paper describes the modeling, construction, and characteristics of the valve. The experimental results favorably agree with theoretical predictions. In addition, the paper demonstrates the use of the phase change valves for flow control, sample metering and distribution into multiple analysis paths, sealing of a polymerase chain reaction (PCR) chamber, and sample introduction into and withdrawal from a closed loop. The phase change valve is electronically addressable, does not require any moving parts, introduces only minimal dead volume, is leakage and contamination free, and is biocompatible.

Lab-On-A-Chip for Oral Cancer Screening and Diagnosis

Oral squamous cell carcinoma (OSCC) is a disfiguring and deadly cancer. Despite advances in therapy, many patients continue to face a poor prognosis. Early detection is an important factor in determining the survival of patients with OSCC. No accurate, cost‐efficient, and reproducible method exists to screen patients for OSCC. As a result, many patients are diagnosed at advanced stages of the disease. Early detection would identify patients, facilitating timely treatment and close monitoring. Mass screening requires a rapid oral cancer diagnostic test that can be used in a clinical setting. Current diagnostic techniques for OSCC require modern laboratory facilities, sophisticated equipment, and elaborate and lengthy processing by skilled personnel. The lab‐on‐chip technology holds the promise of replacing these techniques with miniaturized, integrated, automated, inexpensive diagnostic devices. This article describes lab‐on‐chip devices for biomarker‐based identification of oral cancer. Similar methods can be employed for the screening of other types of cancers.

Lab-on-a-chip technologies for oral-based cancer screening and diagnostics: capabilities, issues, and prospects.

Abstract:  The design of a microfluidic lab‐on‐a‐chip system for point‐of‐care cancer screening and diagnosis of oral squamous cell carcinoma (OSCC) is presented. The chip is based on determining a ∼30‐gene transcription profile in cancer cells isolated from oral fluid samples. Microfluidic cell sorting using magnetic beads functionalized with an antibody against cancer‐specific cell‐surface antigens (e.g., epithelial cell adhesion molecule [EpCAM]) is described. A comprehensive cancer diagnostics chip will integrate microfluidic components for cell lysis, nucleic acid extraction, and amplification and detection of a panel of mRNA isolated from a subpopulation of cancer cells contained in a clinical specimen.

Development of a Microfluidic Device for Detection of Pathogens in Oral Samples Using Upconverting Phosphor Technology (UPT)

Abstract:  Confirmatory detection of diseases, such as HIV and HIV‐associated pathogens in a rapid point‐of‐care (POC) diagnostic remains a goal for disease control, prevention, and therapy. If a sample could be analyzed onsite with a verified result, the individual could be counseled immediately and appropriate therapy initiated. Our group is focused on developing a microfluidic “lab‐on‐a‐chip” that will simultaneously identify antigens, antibodies, RNA, and DNA using a single oral sample. The approach has been to design individual modules for each assay that uses similar components (e.g., valves, heaters, metering chambers, mixers) installed on a polycarbonate base with a common reporter system. Assay miniaturization reduces the overall analysis time, increases accuracy by simultaneously identifying multiple targets, and enhances detector sensitivity by upconverting phosphor technology (UPT). Our microfluidic approach employs four interrelated components: (1) sample acquisition–OraSure UPlink™ collectors that pick‐up and release bacteria, soluble analytes, and viruses from an oral sample; (2) microfluidic processing–movement of microliter volumes of analyte, target analyte extraction and amplification; (3) detection of analytes using UPT particles in a lateral flow system; and (4) software for processing the results. Ultimately, the oral‐based microscale diagnostic system will detect viruses and bacteria, associated pathogen antigens and nucleic acids, and antibodies to these pathogens.

Development of a Generic Microfluidic Device for Simultaneous Detection of Antibodies and Nucleic Acids in Oral Fluids

A prototype dual-path microfluidic device (Rheonix CARD) capable of performing simultaneously screening (antigen or antibody) and confirmatory (nucleic acid) detection of pathogens is described. The device fully integrates sample processing, antigen or antibody detection, and nucleic acid amplification and detection, demonstrating rapid and inexpensive “sample-to-result” diagnosis with performance comparable to benchtop analysis. For the chip design, a modular approach was followed allowing the optimization of individual steps in the sample processing process. This modular design provides great versatility accommodating different disease targets independently of the production method. In the detection module, a lateral flow (LF) protocol utilizing upconverting phosphor (UCP) reporters was employed. The nucleic acid (NA) module incorporates a generic microtube containing dry reagents. Lateral flow strips and PCR primers determine the target or disease that is diagnosed. Diagnosis of HIV infection was used as a model to investigate the simultaneous detection of both human antibodies against the virus and viral RNA. The serological result is available in less than 30 min, and the confirmation by RNA amplification takes another 60 min. This approach combines a core serological portable diagnostic with a nucleic acid-based confirmatory test.

HIV Infection Affects Streptococcus mutans Levels, but Not Genotypes:

We report a clinical study that examines whether HIV infection affects Streptococcus mutans colonization in the oral cavity. Whole stimulated saliva samples were collected from 46 HIV-seropositive individuals and 69 HIV-seronegative control individuals. The level of S. mutans colonization was determined by conventional culture methods. The genotype of S. mutans was compared between 10 HIV-positive individuals before and after highly active antiretroviral therapy (HAART) and 10 non-HIV-infected control individuals. The results were analyzed against viral load, CD4+ and CD8+ T-cell counts, salivary flow rate, and caries status. We observed that S. mutans levels were higher in HIV-infected individuals than in the non-HIV-infected control individuals (p = 0.013). No significant differences in S. mutans genotypes were found between the two groups over the six-month study period, even after HAART. There was a bivariate linear relationship between S. mutans levels and CD8+ counts (r = 0.412; p = 0.007), but not between S. mutans levels and either CD4+ counts or viral load. Furthermore, compared with non-HIV-infected control individuals, HIV-infected individuals experienced lower salivary secretion (p = 0.009) and a positive trend toward more decayed tooth surfaces (p = 0.027). These findings suggest that HIV infection can have a significant effect on the level of S. mutans, but not genotypes.