Fast Detection of Low-Abundance Proteins Based on Ion Concentration Polarization

Molecular detection and analysis are of fundamental importance in disease prevention, disease diagnosis, medical treatment, drug delivery, food industry, and environmental monitoring. Conventional immunoassays require hours of incubation for low concentration analytes (femtomolar) since the rate-limiting step is the transport of target molecules to the biosensors. Thus, a new technology for fast and sensitive immunoassay is highly desirable for disease monitoring and personalized treatments. The work reported in this thesis is focused on developing silicon microfabrication technologies for sensors that can detect low concentration proteins (femtomolar) in a short time (tens of minutes)./nWe first demonstrate the use of germanium (Ge) films as sacrificial layers that allow the patterning of proteins onto surfaces with commonly used organic solvents. As researchers miniaturize biosensors and microfluidic devices down to submicron scales, high-resolution biomolecule conjugation compatible with these processes is highly desirable. The presented technique is scalable to high volume manufacturing and is compatible with nano- and microfabrication processes, including standard lithography. We achieved nanoscale resolution and misalignment with this technique./nWe then discuss the development of ICP-based preconcentration devices on silica and silicon substrates. We report two nanofabrication strategies in this chapter that enhance the accumulation of protein molecules via 1) an increase in the number of nanochannels per microchannel and 2) an increase in the depth of nanochannels. Increasing the depth of nanochannels leads to the switch from silica substrates to silicon substrates, using deep reactive ion etch (DRIE). We also report the attempts for antibody immobilization in the ICP preconcentrators using previously reported Ge technique. However, due to the instability of antibodies in the drying process, we switched to bead-based immunoassays in our preconcentration devices./nAfter the demonstration of protein preconcentration in our devices, we present a scalable method for fabricating hundreds of vertical nanochannels (6.5 µm deep) employing ion concentration polarization (ICP) enrichment for fast analyte detection. Compared to horizontal nanochannels, massively paralleled vertical nanochannels not only provide comparable electrokinetic functions but also significantly reduce effective fluid resistance in each microfluidic channel, which enables microbead loading for sensing purposes. These nanochannels filter microbeads by size and preconcentrate analytes at the anodic side of the test area via electrokinetic entrapment. The device is capable of enriching protein molecules by >1000 fold in 10 min. We demonstrate fast detection of IL6 down to 7.4 pg/ml with only a 10 min enrichment period followed by a 5 min incubation, which is a 162-fold enhancement in sensitivity compared to that without enrichment. Our results demonstrate the possibility of using silicon/silica-based vertical nanochannels to mimic the function of polymer membranes for protein enrichment.

Advisor: Maharbiz, Michel M