Loop-mediated isothermal amplification (LAMP) is a sensitive method for nucleic acid analysis, and has been demonstrated as a ideal technique for use in miniaturized microfluidic systems. While LAMP assays are often detected using absorbance or fluorescence, we demonstrate an integrated system for LAMP assays through the detection of precipitate formation using a modified commercial DVD drive. This integrated DVD drive system is able to automate the sedimentation process and scattering-based detection of accumulated precipitate, as well as maintain the constant temperature needed for LAMP analysis.
HIV is a pandemic that currently threatens over 33 million lives worldwide and HIV/AIDS remains one of the major causes of death globally. The continued monitoring of the CD4+ T-lymphocytes count in HIV patients is necessary for proper treatment, although this testing is too expensive and complex for limited resource settings. We report on a novel integrated centrifugal (CD) microfluidic system for rapid and low-cost HIV diagnosis through automated counting of CD4+ T-cells for point-of-care applications. We demonstrate the integrated T-cell immunocapture and detection mechanism using a novel system comprised of a modified commercial DVD drive and polymer disc.
We present a novel "Lab-on-DVD" system and demonstrate its capability for rapid and low-cost HIV diagnostics by counting CD4+ cells isolated from whole blood. We show that a commercial DVD drive can, with certain modifications, be turned into an improved DVD-based laser scanning microscope (DVD-LSM). The system consists of a multi-layered disposable polymer disc and a modified commercial DVD reader with rotational control for sample handling, temperature control for optimized bioassay, a photodiode array for detection, and software for signal processing and user interface - all the necessary components required for a truly integrated lab-on-a-chip system, with the capability to deliver high-resolution images down to 1 mm in size. Using discs modified with antibodies, we specifically captured CD4+ cells from whole blood, demonstrating single cell resolution imaging. The novel integrated DVD platform with sub-micron image resolution brings, for the first time, affordable cellular diagnostic testing to the point-of-care and should be readily applicable at resource-limited settings.
Isothermal rolling circle amplification (RCA) is a simple and versatile isothermal enzymatic nucleic acid amplification techniques, and has been demonstrated as a ideal tool for biomedical research. We demonstrate for the first time the detection of padlock probes mediated and rolling circle amplification of DNA on centrifugal microfluidic system. DNA spotted on DVD substrate is amplified in-situ and novel bead-based detection of the amplified product is demonstrated using a modified commercial DVD reader for "image based" molecular diagnostics.
We demonstrate the incorporation of low-cost and easy-to-fabricate microfluidic chambers on digital versatile discs (DVDs) to perform DNA assays using standard, commercially available and unmodified DVD drives. This simple microarray-based method is applied for the screening of seven prevalent strains of genetically modified organisms (GMOs) in maize and soybean food samples. The detection limit of our system was found to be 7 μg/g, well exceeding the EU regulation limits for GMOs in food products.
A new advancement in massive DNA-based screening in limited-resource settings is demonstrated through the incorporation of easy-to-fabricate microfluidic chambers on digital versatile discs (DVDs) to perform isothermal recombinase polymerase amplification (RPA) in a microarray format. Standard un-modified DVD discs and commercial drives are used for the low-cost detection method. DNA primers were printed in a microarray format on the polycarbonate surfaces of DVDs with integrated control spots to guarantee the absence of false-negatives and false-positives. The solid-phase amplification assay, including the washing protocols and development reaction, was performed by the dispensation of solutions through the inlet and by controlling the flow-movement by DVD drive centrifugation. The final disc with reaction products was inserted into a DVD player and microarray images were captured and automatically processed. This simple approach was applied for the screening of genetically modified organisms (GMOs) in food samples. The limit of detection was 7 mu g g(-1), which is well below the EU regulation limit for GMOs in food products. Therefore, the only required materials for food safety monitoring were standard store-bought DVDs, plastic chambers, tips, pipettes, an oven, and a standard DVD drive. The proposed strategy allows an integrated microarray system with low manipulation, reduced sample volume, and portability, which are beneficial for low-resource settings.