In the past few years, a large number of microfluidic prototype devices and systems have been developed, specifically for biochemical warfare detection systems and portable diagnostic applications. The BioMEMS team at the University of Cincinnati has been working on the development of a remotely accessible generic microfluidic system for biochemical detection and biomedical analysis, based on the concepts of surface-mountable microfluidic motherboards, sandwich immunoassays, and electrochemical detection techniques [9.74,75]. The limited goal of this work is to develop a generic MEMS-based microfluidic system and to apply the fluidic system to detect bio-molecules, such as specific proteins and/or antigens, in liquid samples. Figure 9.22 illustrates the schematic diagram of a generic microfluidic system for biochemical detection using a magnetic bead approach for both sampling and manipulating the target bio-molecules [9.76,77].
The analytical concept is based on sandwich im-munoassay and electrochemical detection [9.78], as illustrated in Fig. 9.23. Magnetic beads are used as both substrates of antibodies and carriers of target antigens.
A simple concept of magnetic bead-based bio-sampling with electromagnet for the case of sandwich immunoas-say is shown in Fig. 9.24.
Antibody-coated beads are introduced on the electromagnet and separated by applying magnetic fields. While holding the antibody-coated beads, antigens are injected into the channel. Only target antigens are immobilized and, thus, separated on the magnetic bead surface due to antibody/antigen reaction. Other antigens get washed out with the flow. Next, enzyme-labeled secondary antibodies are introduced and incubated, along with the immobilized antigens. The chamber is then rinsed to remove all unbound secondary antibodies. Substrate solution, which will react with enzyme, is injected
into the channel, and the electrochemical detection is performed. Finally, the magnetic beads are released to the waste chamber, and the bio-separator is ready for another immunoassay. Alkaline phosphatase (AP) andp-aminophenyl phosphate (PAPP) were chosen as enzyme and electrochemical substrate, respectively. Alkaline phosphatase makes PAPP turn into its electrochemical product, p-aminophenol (PAP). By applying potential, PAP gives electrons and turns into 4-quinoneimine (4QI), which is the oxidant form of PAP.
For the successful immunoassay, the biofilter [9.76] and the immunosensor were fabricated separately and integrated together. The integrated biofilter and immunosensor were surface-mounted using a fluo-ropolymer bonding technique [9.79] on a microfluidic motherboard, which contains microchannels fabricated by glass etching and glass-to-glass direct bonding technique. Each inlet and outlet were connected to sample reservoirs through custom-designed microvalves. Figure 9.25 shows the integrated microfluidic biochemical detection system for magnetic bead-based immunoassay.
After a fluidic sequencing test, full immunoassays were performed in the integrated microfluidic system to prove magnetic bead-based biochemical detection and sampling function. Magnetic beads (Dynabeads® M-280, Dynal Biotech Inc.) coated with biotinylated sheep anti-mouse immunoglobulin G (IgG) were injected into the reaction chamber and separated on the surface of the biofilter by applying magnetic fields. While holding the magnetic beads, antigen (mouse IgG) was injected into the chamber and incubated. Then
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