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The Combination of Biological and Physical Chemistry - Silicon Nanowire Field Effect Transistor Detects Protein-Protein Interactions

The combination of nanotechnology and biology can stimulate many new ideas and breakthroughs. After many years of cooperation and efforts, the research teams led by Department of Chemistry Professor Yi-Tsong Chen (co-hired by the Institute of Atomic and Molecular Sciences of the Academia Sinica) and by Institute of Zoology Associate Professor Chien-Yuan Pan successfully applied the silicon nanowire field effect transistors to the detection of protein-protein interactions. In this interdisciplinary cooperation project, the two research teams invented various experiment models and published a number of important papers to achieve their research goal. The most recent one being: "Label-free detection of protein-protein interactions using a calmodulin-modified nanowire transistor', which was published in the Proceedings of the National Academy of Sciences USA (PNAS) on January 19th, issue 107, pp. 1047-1052.

Many activities of life are carried out through the interaction of proteins. Therefore, to ascertain whether there is interaction between proteins is very important to the understanding the physiological activities of the cells. Many researches in life science are based on how the proteins interact among one another; and various techniques are developed to verify whether there is indeed interaction. The most common method used is called "immunoprecipitation", whose basic principle is that - if there is interaction between two proteins, then if an antibody is applied to cause one protein to precipitate, then the other protein shall precipitate as well because of interaction. However, many similar experiments require a good specificity of antibodies, as well as a large number of proteins, and it usually takes a few days to complete the experiment. For research purposes, this leaves a lot to be desired.

Over the past few years, Professor Chen's research team explored deeply the basic principles of SiNW-FET and its possible applications. Possessing the properties of semi-conductors, the silicon nanowire field effect transistors are very sensitive to the changes in the environment because of its high surface area/volume ratio. If molecules come into contact with the surface of SiNW-FET, they will affect the surface potentials of silicon nanowires, causing the SiNW-FET to change its conductivity. If SiNW-FET is used as a biological sensor, because biochemical molecules or biological cells generally are in a liquid state, in the experiment extra attention must be paid to the complex electrochemical interface of the solution and SiNW-FET. But through careful design, SiNW-FET now can be used as a sensor for detecting chemical molecules or biological systems, and the advantages include: high sensitivity, dedicated options, immediate response, no marker detection, very small sample demand, and rapid screening.

Professor Pan's laboratory primarily uses electrophysiological methods to explore the message transmission between nerve cells, while at the same time studies a type of calcium-binding protein, and how it affects the activity of different types of calcium ion channels. Although some calcium-binding proteins are found to control the currents of calcium ion channels, but so far there is no clear evidence to prove that there is direct interaction between protein and calcium ion channels. Therefore, after discussion with Professor Chen, although we understand that the interaction among proteins is much smaller than that between antibodies and antigens, but using SiNW-FET to detect the interaction between proteins would constitute a great challenge in experiment. After detailed assessment, Professor thinks that SiNW-FET can be used for the exploration.

Previously, the research teams of Professor Chen and Professor Pan had collaborated to use carbon nanotube field effect transistors (referred to as CNT-FET) or SiNW-FETs to detect the combination of antigens and antibodies. In these experiments, chemical modifications are made on the surface of CNT-FET or SiNW-FETs, to which the antibody is connected, and when the antigen combines with the antibody, because of the changes in the electric field and electric position, electrical signals can be sensitively produced on CNT-FETs or SiNW-FETS. Consequently, we can measure the conductivity of CNT-FETs and SiNW-FETS to determine whether antigen has been combined with antibody. In past experiments, the research team had successfully used the CNT-FET or SiNW-FET systems to detect the presence of 100 pM antigens in the solution, and was able to detect antigens released by single cell. Because this method of detection has a very high degree of sensitivity, and requires a small amount of antibodies and antigens, it has great potentials and can be developed into a widely used technique for chip inspection.

However, in the development of this system, because the combination of antibody and antigen is too strong, preventing the re-use of the same chip, so waste and inconvenience are often incurred. Also, because the same chip can not be reused, quantitative measurements becomes very difficult. To solve this problem, after a series of brainstorming, the research team decided to use glutathione (glutathione, referred to as GSH) and glutathione S-transferase (referred to as GST) special features of reversible combination, first apply GSH to SiNW-FET, then the specific GST fusion proteins can combine with the GSH on SiNW-FETs through GST. We use this to modify the specific protein on SiNW-FET, and we will be able to rapidly screen the proteins that can react with it. This kind of device arrangement not only maintains a high degree of sensitivity, but also has the advantage in that after every experiment, we can wash off the specific proteins on SiNW-FET with high concentration GSH cleaning lotion, so the entire chip can be used again. It can be combined with the GST fusion protein to carry out the next experiment. Under this design, the number of GSH on the surface of SiNW-FET remains unchanged, and the measurements of SiNW-FET can be calibrated, and quantitative measurements can be taken.

In the paper published in PNAS, the two professors' research teams combined an important calcsium binding protein - the calmodulin (CaM) with GST to form a fusion protein (referred to as CaMN-GST). Experiments results show that, when CaM combines with cardiac troponin I, at least 1uM of calcium ions is needed for the activation of its interaction. To understand whether CaM has direct interactions with calcium ion channels? We must design the experiment, first let the calcium ion channels be expressed on the cell membranes, then purify the cell membranes, and let the extracts containing calcium ion channels flow into the SiNW-FET decorated with CaM, and by observing the changes in electrical conductivity CaM's direct interaction with calcium ion channels is proved. These positive experiment results made the whole research team very excited, because the experiment model uses very small amount of protein to prove that there is interaction between proteins and the amount of time required for this experiment is much shorter than the traditional munoprecipitation method.

In the future, Professor Chen and Professor Pan's research teams, will further make use of these nano techniques and devices to conduct more biochemical activity measurements and apply them to high throughput screening. They welcome any interested research teams to join them, exploring the research, development, and applications of this type of nano technology together.

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