Stuck on Silicon: Using pH to Combat Molecular Adhesion

The Problem:•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.

26nm

Single molecule detection apparatus and close up showing molecules clumping together and also adhering to the walls of the channel.

Q-β Bacteria Phage injecting its RNA into a cell. Q-β Bacteria Phage diameter with negative charges around

the capsid.

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.

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).

a. b. c.

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.

Samantha Downey, Mikhail Rudenko, Holger Schmidt, David DeamerJack Baskin School of Engineering, University of California, Santa Cruz

Surf-IT, Summer 2008

The Solution: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.

1. BSA has a high affinity for the silicon at pH 9, which is also true for the phage.

3. Phage with BSA which shows that Phage does not adhere with BSA. This corresponds with image (c.) on

the left.

2. Phage alone at three pH levels showing how adhesion increases as the pH nears 9. Corresponds to image (b.) to

the left.