This dissertation outlines the design, fabrication, and optimization of microfluidic systems for the ultra-sensitive detection of proteins. In particular, it focuses on measuring the concentration of cancer markers in complex media. The Biobarcode Assay (BCA) was adapted and implemented in a microfluidic device in order to achieve this goal. The assay initially demonstrated by the Mirkin Group at Northwestern University, achieves extraordinary sensitivity through the use of dual functionalized gold nanoparticle probes. These particles are encoded with "barcode" DNA sequences that are unique to the target of interest and antibodies for attaching themselves to the target.
The assay protocol first binds the target protein to either to a magnetic microparticle or to the wall of a microfluidic channel using monoclonal antibodies. The gold probes are then introduced into the device and allowed to attach to the target protein. Once the sandwich is formed, the sample is purified and the barcode DNA is released from the captured nanoparticle probes. The presence of the target protein is determined by identifying the DNA sequence that is released. Because each nanoparticle probe carries several hundred DNA per protein binding event, there is substantial signal amplification.
Using this approach, a chip-based system was developed that achieved a 500 attomolar detection limit for prostate specific antigen (PSA). Further modification and optimization has led to multiplexed detection of PSA and HCG with 10 fM sensitivity. Improvements in the protocol have also reduced the chip-based assay time to below 90 minutes.
Efforts to reduce the complexity and cost of the system led to the investigation of an electrical detection scheme for identifying the barcode DNA. The detector consists of a gap between two electrodes that is patterned with single stranded DNA, complimentary to half of the target sequence. The functionalized gap is then exposed to the target sequence and gold nanoparticles functionalized with the other half of the complimentary sequence. The target DNA immobilizes the gold nanoparticles in the gap. Silver deposition, which is facilitated by these nanoparticles, bridges the gap and leads to conductivity changes. While detection of DNA using simple current measurement was possible in a microfluidic format, this work revealed that the BCA, in its current state, is incompatible with this technique. The reducing agents employed to release the barcode DNA from the nanoparticle probes destroy the gold electrodes, preventing the gap from being bridged.
A similar approach was then employed to directly detect protein targets. Instead of DNA, monoclonal antibodies were patterned in the gap. The detector was then exposed to the protein target and gold nanoparticles functionalized with polyclonal antibodies. The target protein was used to immobilize the nanoparticle probes and silver staining was once again employed to bridge the gap It was determined that this technique can be used to detect protein targets both in buffer and serum samples with a 100 pM sensitivity limit.
|School:||University of Illinois at Urbana-Champaign|
|School Location:||United States -- Illinois|
|Source:||DAI-B 69/02, Dissertation Abstracts International|
|Subjects:||Molecular biology, Biomedical research, Chemical engineering|
|Keywords:||Cancer markers, Chip-based detection, Lab-on-a-chip, Protein detection|
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