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Stuck on Silicon: Using pH to Combat Molecular Adhesion Samantha Downey, Mikhail Rudenko, Holger Schmidt, David Deamer Jack Baskin School of Engineering, University of California, Santa Cruz Surf-IT, Summer 2008 The Solution: The Problem: Ideas: •Find where the surface and molecule have the same charge. •The molecule has a negative charge at certain pH levels. •Especially at pH 9, which is the level used to dye the molecule. •We will find a pH where the silicon surface will have the same charge, preventing adhesion. •Find a molecule that will bind to the surface and block the phage. •Bovine Serum Albumin (BSA) can create a hydrophilic bond to the surface and block interactions between the phage and surface. Q-β Bacteria Phage injecting its RNA into a cell. Q-β Bacteria Phage diameter with negative charges around the capsid. BSA/Fluorescamine 120 100 Method: Using zwitterionic buffers ranging from pH 5 to pH 10, we tested the affinity of the following molecules on the SiN and SiO wafer surfaces. •BSA dyed with Fluorescamine. •Q-β Bacteria Phage dyed with Alexa Fluoro. •We also tested the dyes Rhodamine GG , Fluorescamine, and Alexa Fluoro to ensure they did not adhere to the surface, giving false positives. A spectro-fluorometer was used for detection and set to the proper excitation and emission wavelengths for each sample, at a set time interval. Fluorescent Counts/Sec 80 counts(avg.) 60 40 20 0 5 6 7 8 9 10 1. BSA has a high affinity for the silicon at pH 9, which is also true for the phage. Phage Adhesion 3000 2500 Fluorescent Counts/Sec Single molecule detection apparatus and close up showing molecules clumping together and also adhering to the walls of the channel. 26nm 2000 pH 8.1 1500 pH 8.5 pH 8.7 1000 500 0 T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 Trial 2. Phage alone at three pH levels showing how adhesion increases as the pH nears 9. Corresponds to image (b.) to the left. Phage/BSA 160 140 120 a. b. c. a. Phage bound to the wall of the channel, b. phage bouncing off the surface at optimal pH (see graph 2) , c. phage bouncing off BSA molecule on SiN surface(see graph 3). Fluorescent Counts/Sec •Work is being done to create small, portable single molecule detectors. •Bio-molecules used in the development of these detectors become bound to the channel before reaching the detector. •This is most likely caused by charges on the surfaces of the phage capsid and in the silicon surfaces inside the channel. •Molecules can also clump together and block the channel. •The molecule of most interest in this experiment is the Q-β Bacteria Phage. The following is the data received from the various trials listed under Method. One can see that adding BSA at pH 9 reduces adhesion of phage to SiN, but phage alone shows little adhesion at pH 8. Therefore, using a buffer of ~pH 8 to transport the molecule through the channel is ideal. 100 80 pH 9 60 40 20 0 T1 T2 T3 T4 T5 T6 T7 T8 T9 Trial 3. Phage with BSA which shows that Phage does not adhere with BSA. This corresponds with image (c.) on the left. T10