Transcript downey_poster - SURF-IT Summer Research

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