As sensor technology is developing rapidly to handle multiple analytes in complex media and a chip-scale sensor system is becoming smaller, there is an urgent need to develop a corresponding protein immobilization technique suitable for long-term chip storage without losing its bioactivity and for the convenient generation of multiple functional spots in one flow channel. Currently, protein arrays are often generated using spotting methods. The biggest drawback limiting widespread application of protein arrays is perhaps the poor stability (lifetime) of protein chips once "spotting". Recent studies show that while some antibodies are more suited (or stable) on antibody arrays than others, many protein arrays will start to lose their bioactivity gradually after two weeks. Furthermore, surface chemistries for protein immobilization currently available, such as physical adsorption, covalent immobilization, protein A or G, and biotin/streptavidin, are not selective and will provide only one functional spot within one flow channel.
Intellectual Merit: Recently, it is demonstrated in proof-of the-concept experiments by the PI's group that the DNA-direct protein immobilization technique is well suited to achieving the goals for chip stability and functionality. The DNA-directed antibody immobilization is achieved via ssDNA-antibody conjugates, each of which consists of an antibody chemically linked to an ssDNA and is designed such that the ssDNA has a sequence complementary to one of the ssDNA sequences attached to the surface. Before detection, a cocktail of different ssDNA-antibody conjugates is applied to the chip pre-functionalized with ssDNA probe molecules. Each antibody conjugate will be self-immobilized to a designated spot via DNA hybridization. In this way, the chip will be stored as a DNA chip and used as a protein chip. This will not only resolve the long-term storage issue of a protein chip, but also provide a simple and convenient way to create multiple functional spots in one flow channel. Furthermore, it has been shown that this new platform is 50 times more sensitive than the commonly used biotin/streptavidin platform. This work will focus on engineering aspects to realize this technology for various lab-on-a-chip biosensors. It consists of five tasks - (a) developing a new surface platform convenient for the fast generation of DNA arrays and synthesizing ssDNA-antibody conjugates, (b) designing multiple ssDNA sequences with high specificity, (c) testing the specificity and cross-activity of multiple ssDNA-antibody conjugates using a state-of-the-art eight-channel SPR sensor, (d) patterning a chip with different ssDNA sequences using a modified inkjet printer, and (e) realizing this technology on a glass substrate.
Broader Impact: This technology will not only remove the key obstacle of long-term chip stability faced in conventional protein arrays, but also provide a selective protein immobilization technique to create multiple functional spots in one flow channel. The integration of this proposed protein immobilization technique with biosensors (all types) will make biosensors very powerful, including their robustness for multiple channels, stability for long-term chip storage, and high sensitivity and specificity in addition to one universal chip for all applications and convenient chip regeneration. The PI's group is collaborating with a variety of researchers to solve real-world problems using sensors. Support of this work will allow these people to access sensor technology to solve their problems. Many of undergraduate students in the PI's laboratory have been involved in sensor-related work. Support of this proposal will provide more multiple-disciplinary research and educational opportunities to these students and will provide valuable information to a new course on biological interfaces in biosensors and biomaterials under development by the PI.
