Lab-on-a-Chip for Point-of-Care Diagnostics

Despite global efforts to control the acquired immune deficiency syndrome (AIDS) pandemic, the human immunodeficiency virus (HIV) infection continues to spread relatively unabated in many parts of the world. The diagnosis of HIV infection at the point-of-care and in resource-poor settings poses considerable challenges due to the time delay between sample collection and diagnosis. Here, three microfluidic cassettes are designed and tested for pathogens detection in oral fluids at point-of-care for low-resources setting.

(1) Timer-actuated immunoassay cassette for HIV antibody detection in oral fluids. The cassette, shown in Fig. 1, comprised of air pouches for pumping, a metering chamber, reagents storage chambers, a mixer, and a lateral flow strip. 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 cassette has several advantages over dip sticks such as sample preprocessing, integrated storage of reagents, and automated operation that reduces operator errors and training.

(2) Single-chamber cassette for HIV nucleic acids extraction and isothermal amplification. The single reaction chamber of the cassette, shown in Fig. 2, is equipped with an integrated, flow-through, Flinders Technology Associates (Whatman FTAÒ) membrane for the isolation, concentration, and purification of DNA and/or RNA from saliva. The nucleic acids captured by the membrane are used directly as templates for amplification without elution, thus simplifying the cassette’s flow control. The utility of the integrated, single-chamber cassette was demonstrated by detecting the presence of HIV-1 in oral fluids with a detection limit of less than 10 HIV particles.

(3) Water-activated, self-heating, non-instrumented, cartridge for the detecting the pathogen DNA. In many health care settings, it is uneconomical, impractical, or unaffordable to maintain and access a fully equipped diagnostics laboratory. Examples include home health care, developing-country health care, and emergency situations in which first responders are dealing with pandemics or biowarfare agent release. In those settings, fully disposable diagnostic devices that require no instrument support, reagent, or significant training are well suited. A novel, disposable, water-activated, self-heating, non-instrumented, cartridge in Fig. 3 is designed to amplify and detect the pathogen DNA for low-resources setting. The cartridge is successfully demonstrated to amplify and detect the presence of E. Coli. DNA. The amplification result can directly be observed by naked.

These cassettes are particularly suitable for resource poor regions, where funds and trained personnel are in short supply. The cassette can be readily modified to detect other pathogens in saliva, urine, and other body fluids as well as in water and food.

 

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Fig. 1 Timer-actuated immunoassay cassette for HIV detection in oral fluids (Video)

Fig. 2 Integrated, single-chamber LAMP cassette for HIV detection in oral fluids

Fig. 3 Water-activated, self-heating, non-instrumented, microfluidic-based cartridge for pathogen DNA detection (Video)

 

Reference

    • Changchun Liu, Xianbo Qiu, Serge Ongagna, Dafeng Chen, Zongyuan Chen, William R. Abrams, Daniel Malamud, Paul L. A. M. Corstjens and Haim H. Bau, A timer-actuated immunoassay cassette for detecting molecular markers in oral fluids, Lab on a Chip, 2009, 9(6): 768-776.
    • Changchun Liu, Eran Geva, Michael Mauk, Xianbo Qiu, William R. Abrams, Daniel Malamud and Haim H. Bau, An Isothermal Amplification Reactor with an Integrated Isolation Membrane for Point-of-Care Detection of Infectious Diseases in Oral Fluids, Analyst (Accepted).
    • Biodetection cassette with automated actuator, U. S. patent, 2008, 61/086,573.
    • Devices, and methods for point of care diagnostics utilizing nucleic acid amplification, and especially methods of isothermal nucleic acid amplification. U.S. Provisional Patent Filing, 2010.

     

Biosensors
Biosensors have become an important tool for detection of chemical and biological components for clinical, food and environmental monitoring. Here, three biosensors based on different principles (SPR, electrochemical and fluorescence imaging) are developed and characterized:

(1) Hard-soft microfluidic-based biosensor flow cell for SPR imaging. Surface plasmon resonance (SPR) imaging technique is label free, real-time, and high-throughput analysis method for interaction studies with array format. An ideal microfluidic-based SPR biosensor flow cell should have not only a “soft” interface for high strength sealing with SPR biosensing chips, but also “hard” macro-to-micro interface for tubing connection. Since these properties are exclusive of each other, no one material can provide the advantages of both. Here, a SiO2 thin film, deposited by plasma-enhanced chemical vapor deposition (PECVD) technology, was used as an intermediate layer for irreversibly adhering PDMS to plastic substrate, and develop a hard-soft, compact, robust microfluidic-based, SPR imaging biosensor flow cell in Fig. 4. This novel SPR imaging biosensor flow cell does not only keep the original advantage conventional PDMS-based biosensor flow cell such as the intrinsically soft interface, easy-to-fabrication, and low cost, but also has a rigid, robust, easy-to-use interface to tubing connection and can be operated up to 185 kPa in aqueous environments without failure. Its application was successfully demonstrated with two types of experiments by coupling with SPR imaging biosensor: the real-time monitoring of the immunoglobulin G (IgG) interaction, as well as the detection of sulfamethoxazole (SMOZ) and sulfamethazine (SMZ).

(2) Single agarose bead-based electrochemical biosensor. Fig. 5 is a photograph of a simple, robust, single agarose bead-based electrochemical biosensor. The sensor’s working electrode consists of an electrochemically-etched platinum wire, hermetically heat-fusion sealed in a pulled glass capillary (micropipette). A commercially available, densely functionalized agarose bead was mounted on the tip of the etched platinum wire. The use of a pre-functionalized agarose bead eliminates the tedious and complicated surface functionalization process that is often the bottleneck in the development of electrochemical biosensors. The biotin agarose bead-based, micropipette, electrochemical (Bio-BMP) biosensor can monitor H2O2 concentration in the range from 1×10−6 to 1.2×10−4 M, and the streptavidin bead-based, micropipette, electrochemical (SA-BMP) biosensor was able to detect the amplicons of 1 pg DNA template of B. Cereus bacteria.

(3) Bead array-based microfluidic sensor chips (or electronic taste chips). Bead array-based microfluidic sensor chips have been widely used in many bioanalytical applications due to their high throughput, low consumption of samples and reagents, and high sensitivity. These devices contain, however, wells and features that can easily trap air bubbles. Here, a novel, integrated, bead array-based microfluidic sensor chip with a robust, passive, membrane-based debubbler in Fig. 6 is designed and tested. The debubbler is able to completely filter gas bubbles out of a segmented flow at rates up to 60 µl/s/mm2 of membrane area. As an application demonstration, the integrated microfluidic sensor is successfully used to detect haptenized PCR amplicons of B. Cereus bacteria.

 

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Fig. 4 Hard-soft microfluidic-based biosensor flow cell for SPR imaging

Fig. 5 Single agarose bead-based electrochemical biosensor

Fig. 6 Bead array-based microfluidic sensor chip integrated with debubblers (Video)

 

Reference

    • Changchun Liu, Dafu Cui and Hui Li, A hard-soft microfluidic-based biosensor flow cell for SPR imaging application, Biosensors and Bioelectronics, 2010 26(1): 255-61.
    • Changchun Liu, Michael G. Schrlau and Haim H. Bau, Single bead-based electrochemical biosensor, Biosensors and Bioelectronics, 2009, 25(4): 809-814.
    • Changchun Liu, Jason Thompson and Haim H. Bau, A membrane-based, high-efficiency, microfluidic debubbler, Lab Chip (Accepted).
    • Bead Based Electrochemical Biosensor, U. S. patent, 2009, 61/235,891.

     

BioMEMS Device
BioMEMS devices are the application of MEMS technology in the field of biomedical and health sciences. It is expected to revolutionize the way of biomedical diagnosis, drug delivery, food safety et al. Here, three different BioMEMS microdevices are, respectively, developed for biosensing, nucleic acid analysis and gas chromatography (GC) applications.

(1) High-aspect-ratio, microfluidic-based, surface plasmon resonance (SPR) biosensing device. The deep reactive-ion etched (DRIE) silicon mold is usually used in the field of molding of high-aspect-ratio, PDMS-based microfluidic devices due to its high strength, excellent microfabrication precision and high reliability. One of its major limitations, however, is the difficulty to obtain a uniform etching depth and smooth etching surface due to the lag effect, which makes the subsequent high-aspect-ratio PDMS microfluidic replica sealing being more difficult, sometimes even fail to seal. To address these problems, a PDMS double-casting method is present for rapid fabrication of a high-aspect-ratio, microfluidic-based, surface plasmon resonance (SPR) biosensing device in Fig. 7, which contains micropillar-based microfilter, SPR label-free biosensing channel, and waste channel et al. As an application demonstration, this present SPR biosensing device is successfully applied to detect albumin in urine by a competitive immunoassay method.

(2) Integrated microfluidic chip for blood cell separation and DNA extraction. Sample pretreatment is still a challenge for microfluidic-based nucleic acid analysis. An integrated miniaturized device capable of separating cell, lysing cell and extracting DNA is developed to perform sample pre-treatment of whole blood in a continuous flow mode. A microfabricated, crossflow-based, microfilter in Fig. 8 is proposed to separate blood cells, which could successfully avoid clogging or jamming. After blood cells is lysed, genomic DNA in white blood cells is released and adsorbed on porous matrix fabricated by electrochemical etching technology. The experiment shows that more than 35.7 ng genomic DNA can be purified on the integrated microfluidic device from 1 µL rat whole blood.

(3) Silicon-based Gas Chromatography (GC) chip. Gas Chromatography (GC) is a highly sensitive chemical analysis technique with a broad range of applications. Existing commercial GC systems are generally quite bulky and fragile. A key part of a GC system is the GC column. For environmental testing, in particular atmospheric monitoring, there is a requirement for portable, robust, low power gas chromatography systems. Microfluidics enables miniaturisation of the gas chromatography column and low power methods for column heating. A 6 m length ´ 100 µm width ´100 height µm height, microfabricated GC column in Fig. 9 is fabricated and tested for benzene and toluene separation in 185 s. The microfabricated GC chip offers substantial potential as a field portable GC instrument.

 

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Fig. 7 Integrated, microfluidic-based, SPR biosensor

Fig. 8 SEM image of cross-flow filtration microchannel for blood cell sorting

Fig. 9 SEM image of microfabricated GC column

Reference

    • An integrated microfluidic sensor chip and its detection method, Chinese Patent, 200810118326.9.
    • Xing Chen, Da-fu Cui, Chang-chun Liu, Hui Li and Jian Chen, Continuous flow microfluidic device for cell separation, cell lysis and DNA purification, Analytica Chimica Acta , 2007, 584(2): 237-243.
    • Yutai Li, Dafu Cui and Changchun Liu, MEMS-based microfabricated columns for gas chromatography, 2007, Micronanoelectronic Technology, 44 (7): 407-409, 416.