A Disposable DNA Sample Preparation Microfluidic Chip
The Nucleic Acid (NA) probe assays typically include PCR to amplify the number of copies of DNA to a detectable level. The PCR technique requires a relatively pure DNA sample in aqueous solution, free of inhibitors during the PCR process. Therefore, the extraction and purification of nucleic acids from biological samples are the critical steps that should be carefully handled in the NA assays. In this work we have proposed a new microfluidic chip to address this sample preparation process for NA probe assays.
The proposed microfluidic system is composed of three parts: microfilter, micromixer and DNA purification chip. In the microfilter, red blood cells are separated from whole blood sample. In the mixer, the plasma blood sample is mixed with lysis reagents to release the DNA into the solution and the lysed solution is mixed with chaotropic salt. In the DNA purification chip, the DNA's in the solution will bind to the exposed SiO2 surface at a high concentration of chaotropic salt. After DNA has been adequately extracted in the binding process, wash solutions flow through the microchannel of the chip to wash away the remaining sample fluid. Finally, the DNA can be eluted from the chip by flowing through appropriate chemical buffer solutions.
Figure 1. Schematic diagram of the proposed microfluidic system for sample preparation of Nucelic acid probe assay.
Microfilter using Metallic Microseives
MEMS microfilter improvement is driven by the need to perform absolute separation of micro-sized particles from micro-scale fluid volumes. Filtration is an essential step in many biological and medical applications. For example, routine blood tests require separating blood plasma from whole blood. By using microfabrication techniques it has recently become possible to create integrated miniaturized fluid handling devices. Promising applications include miniaturized systems for the filtration, fractionation, and manipulation of biological cells and nucleic acids.
In this work, we have designed the microfilter to separate red blood cells from whole blood. When designing filters to effect blood cell separation, it is known that the deformability of the cells plays a major role in the separation efficiency. The reported sizes of blood cells are often based on morphological studies of the stained cells. However, filtration of the spherical and discoid white and red blood cells is influenced by the cell concentration, applied pressure, viscosity of the medium, and the size of the filter port. Red blood cells with relatively stable discoid architecture cannot pass through a gap smaller than 3mm. The proposed microfilter has a metallic microsieve.
Passive Micromixer in Three Dimensional Microchannels
Rapid mixing is essential in many of the microfluidic systems targeted for use in biochemistry analysis, drug delivery, and sequencing or synthesis of nucleic acids. Biological processes such as cell activation, enzyme reactions, and protein folding often involves reactions that require mixing of reactants for initiation. Mixing is also necessary in many microfabricated chemical systems that carry out complex chemical synthesis.
The proposed mixer consists of two-layer channels and the mixing occurs in a two-step process. The first step is segmentation where a heterogeneous mixture of two fluids is formed by convection and the second step is the inter-diffusion of molecules between domains. The proposed mixer has a separated serpentine flow path in order to increase the chaotic advection as well as has the repeated segments to increase the interfacial area.
The micro mixer has been fabricated using polydimethylsiloxane (PDMS) and SU-8. We have tested the mixer using phenolphthalein, a pH indicator that changes its color from transparent to red for higher pH values than 8. We have monitored the mixing at various flow rates at each segment of the fabricated microchannel. In the micro scale, we have demonstrated that the fabricated mixer can achieve the mixing successfully for low Reynolds numbers below 50.
Figure 2. The proposed micromixer of two-layer channels.
Figure 3. (a) Photographs at each consecutive segment stage of the fabricated mixer at a flow rate of 2mL/min. (b) Normalized average optical intensity in each stage of the mixer.
DNA Purification Chip on Photosensistive Glass
A DNA purification chip has been designed to maximize the DNA binding surface area on a photosensitive glass substrate. The fabricated glass surface consists of a number of pillars with a height of 200mm. The chip has a total internal surface area of about 2cm2 which will be effectively used as DNA binding sites.
We have tested two experiments: high concentration inputs (600ng/200mL) and low concentration inputs (100copies/200mL). For the high concentration sample, the presence of the target DNA can be detected without PCR. For the low concentration input test, PCR must be performed to first amplify a target sequence in order to detect the presence of the target DNA. The binding capacity of the chip is about 15ng/cm2. The fabricated purification chip can extract the DNA from low concentration inputs at 100copies/200mL.
Figure 4. (a) Schematic diagram of DNA purification chip, (b) SEM pictures of photosensitive glass microstructures
Figure 5. Photograph of electrophoretic gel analysis results showing the eluted DNA from the DNA purification chip where the lane M denotes the marker: (a) high concentration sample(600ng/200ml) without PCR and (b) low concentraion sample (5pg/200ml) with PCR
- Joon-Ho Kim, Byoung-Gyun Kim, Hyukjun Nam, Dae-Eun Park, Kwang-Seok Yun, Jun-Bo Yoon, Jichang You and Euisik Yoon, "A Disposable DNA Sample Preparation Microfluidic Chip for Nucleic Acid Probe Assay", IEEE International MEMS Conference 2002, Las Vegas, USA, Jan., 2002.