The Quartz Crystal Microbalance (QCM) as a Biosensor
Thesis Proposal
Mentors:
Dr. Ruth Baltus
Dr. Linda Luck
David Bogdan
March 4, 2005
Introduction
Biosensors are an increasingly important technology in the detection of
compounds ranging from pesticides to biological weapons. Typically, biosensors consist
of a biological macromolecule that is immobilized on the surface of a signal transducer,
as shown in Figure 1. As the macromolecule binds specifically to the ligand being
Figure 1. Schematic for generic biosensor[1]
detected, the signal transducer can measure a physical change due to the binding event.
The transducer usually detects a change in resistance, pH, heat, light, or mass and then
converts that data to an electrical signal to be collected and processed.
One promising type of detector is the Quartz
Crystal Microbalance (QCM). The QCM is a
piezoelectric mass-sensing device. A QCM device works
by sending an electrical signal through a gold-plated
quartz crystal (shown in Figure 2), which causes a
vibration at some resonant frequency. The QCM then
Figure 2. Crystals used in a QCM [2]
measures the frequency of oscillation in the crystal. When used as a biosensor, the QCM
can detect changes in frequency of the crystal due to changes in mass on the surface of
the crystal.
While there has been significant research on the use of the QCM as a biosensor,
there are many possibilities that have not been explored and many problems that have not
been solved. This study will look at the use of the QCM as a biosensor with the leucineisoleucine-valine (LIV), leucine-specific (LS) and glucose-galactose receptor (GGR)
periplasmic binding proteins of gram-negative E. coli. The study will also look at the
feasibility of using the QCM with a flow-cell, and the effects of temperature on detection
using a QCM.
Background
Gram-negative bacteria have both inner and outer membranes, and it is between
these two membranes that periplasmic binding proteins exist. Over 100 of these proteins
have already been discovered and examined by X-ray crystallography [2]. The proteins
bind to ligands as either part of active transport systems or as signals for chemotaxis
systems [3]. Periplasmic binding proteins are particularly well suited for use in
biosensors because they have a high specificity for ligand binding, along with being both
soluble and stable [4]. The proteins have a monomeric structure, but have two large
lobes that bend on a hinge-like peptide strand [3,4]. Binding occurs by the two lobes
closing around the substrate, which is held in by low-energy hydrogen bonds. By closing
around the substrate, the protein makes a conformational change, which is the signal for
the active transport or chemotaxis system [3].
This conformational change is the likely explanation for how the quartz crystal
microbalance can detect the binding of very small molecules, such as glucose
(MW=180.16) or leucine (MW=131.17), when being bound by much larger proteins such
as GGR (MW=33,370 [4]) and LIV (MW=36,744 [1]). It is known that there are a
number of interfacial properties, such as mass, the viscosity of the layer, the stiffness of
the layer, and the change in conductivity of the layer, that influence the frequency of the
crystal vibration, but how this occurs on the molecular level is not yet understood [4].
Determining how the temperature at which the binding event is detected affects the
overall shift in frequency of the crystal could help explain whether or not the
conformational change of the protein, and therefore the stiffness of the layer, is the keydetermining factor in the detection of such small ligands. If the stiffness of the layer is
the key factor, higher temperatures should loosen the proteins, and a smaller frequency
shift should be observed.
For the QCM to measure a binding event, the receptor protein must be attached to
the surface of the crystal. Previous studies have accomplished this by using thiolcontaining compounds [4]. The thiocompounds create a self-assembled monolayer on the
surface of the crystal, bound by gold-sulfur bonds. The receptor protein is then
covalently bound to the thiocompounds, and an orderly monolayer of the protein is
obtained. Another method that has been used in previous studies here at Clarkson
University is genetically engineering the receptor protein to contain the amino acid
cysteine, the only amino acid that contains sulfur. The sulfur in the cysteine can then
bind to the surface of the gold itself, creating a self-assembled monolayer without the use
of any intermediate compound. By changing one of the amino acids in the protein to a
cysteine at a carefully chosen point on the molecule, the protein can still function and
bind to the ligand despite being immobilized on the surface of the crystal.
Three genetically altered proteins from gram-negative E. coli will be used in this
study. Both the LS and LIV proteins are structurally similar to eukaryotic
neuroreceptors, including the metabotropic glutamate receptor and the N-methyl-Daspartate receptor, making them important models for future study [1]. While very
similar in structure, the LS protein binds specifically to L-leucine, D-leucine, and Lphenylalanine, while the LIV protein binds to L-leucine, L-isoleucine, L-valine, Lthreonine, L-serine, and L-alanine [1]. GGR, the third protein used, binds to glucose and
galactose. Glucose biosensors are particularly important in monitoring diabetes. All
three of these proteins will be modified to contain a cysteine in the 1st amino acid
position. Structurally, this position as at the very tip of one of the two lobes, minimizing
the effect on the binding site located in the middle of the two lobes.
Previous studies using these proteins have used a “batch” type experimental setup.
For this setup, the resonant frequency of the blank crystal is found in buffer, and then the
crystal is removed for protein immobilization. After immobilization, the crystal is placed
back in the buffer for a frequency measurement, and then taken back out for ligand
binding, before placing back in buffer for the final frequency measurement [1]. While
this technique was successful, for applications in biosensing a flow-cell would be ideal
for detection in fluids, such as blood. In addition to being more practical for use outside
of a laboratory, a flow-cell would provide the opportunity for real-time monitoring of the
binding event, as opposed to pre- and post measurement.
Methodology
A. Flow-cell
To test the use of a flow-cell, two different experimental setups were used. In the
first setup, a syringe pump was used. The syringe pump was simply connected to the
Maxtek, Inc., flow cell with 1/8” rubber tubing, and the exit stream of the flow cell
simply poured into a beaker through another section of tubing. This setup was only used
to test the stability of the crystal at different flow rates, using water as a medium.
The second setup used a High Performance Liquid Chromatography (HPLC)
pump. In this setup, the pump had four feed lines and each could draw from a separate
reservoir. The pump was controlled digitally and could switch between different feeds
and flow rates quickly. Air damages the pump heads, so if any air is present in the feed
lines, the air must be drawn out by syringe. The pump outlet was again attached to the
flow cell through 1/8” rubber tubing. The flow cell was housed in an aluminum box to
block any electronic interference and to dampen physical vibrations around the cell. The
flow cell outlet was attached to rubber tubing that emptied into a beaker.
When experiments involving the 3,3’-Ditiobis(sulfosuccinimidylpropionate)
(DTSSP), a thiocompound, or cysteine were run, the crystal was cleaned before use. To
clean, the crystal was first soaked in 1.2M NaOH solution for 10 minutes, and then 1.2M
HCl solution for 10 minutes. To completely remove the cysteine, the crystal was soaked
in 12M HCl for an additional 5 minutes.
Before any experiment could be run, the Maxtek, Inc., QCM had to be warmed up
for at least two and a half hours. For the QCM to warm up, the cell had to be plugged
into the machine, and the crystal had to be in the cell. With the flow cell, this also meant
that the pump had to be running during this initial warm-up period. The frequency was
monitored during this period to determine when the frequency stabilized.
Before testing using actual proteins, both DTSSP and cysteine were used because
they readily form gold-sulfur bonds. For the DTSSP attachment experiments, the DTSSP
was dissolved in distilled water. Before the DTSSP solution was pumped through the
cell, water was used to establish the stable frequency for ten minutes. After the cell was
stable for 10 minutes, the feed was switched to the DTSSP solution. The DTSSP was
tested at .25, .5, and 1 ml/min, all at the micro-molar range. After the frequency
stabilized again, the feed was switched back to water for at least 30 minutes.
For the Cysteine attachment experiments, a GGR buffer was used. This buffer
was 100mM KCl, 10mM Tris (pH 7.1), and 0.5mM Ca++. For these experiments, the
buffer was used to stabilize the crystal and establish the stable frequency. After ten
minutes of being stabilized, the feed was switched to a 100mM or 1M solution of
Cysteine in the buffer.
The Cysteine was tested at .25 and .5 ml/min.
B. LIV, LS, and GGR Batch Experiments
LIVE1C with Leucine
Before any experiment could be run, the QCM was warmed up for at least two to
two and a half hours. For the QCM to warm up, the holder had to be plugged into the
machine, and the crystal had to be in the holder. During this time the QCM software
recorded frequency measurements, and at the end of the warm-up period a stable baseline
frequency was found.
The experiments were setup so that the crystal holder and crystal were submerged
in 200mL of buffer in a 250mL beaker. For the LIVE1C and LSD1C experiments,
10mM K2HPO4 with a pH of 6.9 was used. For the GGRA1C and GGRQ26C
experiments, 100mM KCl, 10mM Tris, 0.5 mM CaCl2 with a pH of 7.1 was used.
During the experiment the beaker was covered with Parafilm.
Once a stable reading was found, the holder was taken out of the buffer and
unplugged from the QCM machine. For the first LIVE1C with leucine experiments,
400μL of 1g/L LIVE1C solution was placed on top of the crystal and was left to incubate
for 1.5 hours. The solution was then rinsed off thoroughly, and the holder was returned
to the beaker of buffer, plugged back in, and the QCM was turned back on to collect data.
It took approximately 15 minutes to obtain a stable frequency again.
Once the frequency was obtained, the holder was taken out and unplugged again.
400μL of 500μM leucine (in K2HPO4) solution was then placed on top of the crystal
surface, and left to incubate for an hour. The solution was then rinsed off thoroughly,
and the holder was returned to the beaker of buffer, plugged back in, and the QCM was
turned back on to collect data. It took approximately 15 minutes to obtain a stable
frequency again.
Between each experiment, the crystal used was cleaned. To clean, the crystal was
first soaked in 1.2M NaOH solution for 30 minutes, and then 1.2M HCl solution for 5
minutes. For the last step, the top of the crystal was soaked in 12M HCl for an additional
2 minutes.
LIVE1C with L-phenylalanine and leucine
This experiment followed the same procedure as the LIVE1C with leucine
experiments, except that before incubating the crystal with leucine solution, 400μL of
500μM L-phenylalanine was placed on the crystal for an hour, and then the frequency
was measured.
LIVE1C with leucine Time Optimization
This experiment was to determine if less protein could be used in the detection
process, and if the results could be obtained in less time. This experiment followed the
same procedure as the LIVE1C with leucine experiments, except that the concentration
and incubation times were changed. The concentration of LIVE1C was cut in half to
.5g/L. The incubation time for LIVE1C was broken into three 15-minute periods, were
the frequency was measured in-between each period. The incubation time for the leucine
solution was broken down into two 15-minute periods, with frequency measurement
taken in-between the two.
LSD1C with leucine
This experiment followed the same procedure as the LIVE1C with leucine
experiments, except that in place of LIVE1C, LSD1C was used. The concentration of
LSD1C used was .407g/L. The incubation times were also cut in half, to 45 minutes for
the protein and 30 minutes for the ligand.
GGRA1C with glucose
This experiment followed the same procedure as the LIVE1C with leucine
experiments, except that a .5g/L solution of GGRA1C incubated for 45 minutes was
substituted for LIVE1C, and 1mM glucose solution incubated for 30 minutes was
substituted for the Leucine solution. Also, the holder was left plugged into the QCM the
entire time. On the second of two GGRA1C experiments, 20μM DTT was added to the
protein solution.
C. Temperature Testing
This experiment will follow the LIVE1C with leucine experiments, but the beaker
used for the measurement cell will be a jacketed beaker. A temperature regulating pump
system will be used to pump water through the jacketed beaker, keeping it at either an
elevated temperature (40C) or at a decreased temperature (10C). Higher temperatures are
known to denature the proteins, while a cooling fluid would need to be used at
temperatures near or below 0C.
The effects of temperature will be studied in two ways. First, a single crystal will
be used, and baseline frequencies will be measured at two or three different temperatures,
for example 0C in an ice-water bath, 24C at room temperature, and 40C in the jacketed
beaker. Then the crystal will be prepared with LIVE1C, and then measured in at all three
temperatures. If successful, the experiment could continue with the binding and
measuring of the ligand at all three temperatures.
Another method that will be used will involve two crystals being run
simultaneously at different temperatures. In this experiment, the exact same protein and
ligand samples could be used, and the results from the two temperatures could be
compared.
If either of these methods prove successful with LIVE1C and leucine, other
protein-ligand combinations could also be tested at various temperatures.
Timeline
-Spring Semester 2004 – Flow Cell Experiments
-Fall Semester 2004 – LIV, LS, GGR Experiments
-Spring Semester 2005 – Temperature Experiments
 Week 10 – LIV at 40C and 24C
 Week 11 – LIV at 10C and 24C
 Weeks 12-15 – LIV, LS, or GGR temperature testing depending on initial success
of LIV experiments
-Fall Semester 2005 – Write Thesis
Expectations
Initial testing of the flow-cell has shown little success, and further testing will
need to be done with actual proteins to determine the feasibility of the concept. The
pump systems used were not ideal for this application, but with a peristaltic-type pump a
flow-cell could be developed in the future. An ideal pump would be able to circulate
very small amounts of fluid from multiple sources.
While many of the protein-ligand binding experiments have already been
completed, there is still much left to be done. Previous work has shown that GGR in
particular can successfully and reproducibly be used in the detection of glucose while
immobilized on a QCM crystal [4]. This study is expected to build on those results by
showing that the proteins LS and LIV can also successfully and reproducibly be used in
the detection of specific ligands while immobilized on a QCM crystal.
The temperature testing of LIV is the current focus of the study. It is expected
that once a suitable method for comparing different temperatures is found, higher
temperatures will decrease the actual frequency shift measured due to the decreased
stiffness of the protein layer. Following this logic, lower temperatures should increase
the frequency shift, as the protein layer should become stiffer.
References
[1] Carmon, Kendra. (2004) Development of a QCM Biosensor Using Receptor Proteins
to Detect the Binding of Small Ligands. Thesis.
[2] Dwyer, Mark A. and Hellinga, Homme W. (2004) Periplasmic Binding Proteins: A
Versatile Superfamily for Protein Engineering. Current Opinion in Structural
Biology. 14, 495-504.
[3] Felder, C.B., Graul, R.C., Lee, A.Y., Merkle, H.P., and Sadee, W. (1999) The Venus
Flytrap of Periplasmic Binding Proteins: An Ancient Protein Module Present in
Multiple Drug Receptors. AAPS PharmSci. 1 (2), Article 2.
[4] Carmon, K.S., Baltus, R.E. Luck, L.A. (2004) A Piezoelectric Quartz Crystal
Biosensor: The Use of Two Single Cysteine Mutants of the Periplasmic E.coli
Glucose/Galactose Receptor as Target Proteins for the Detection of Glucose.
Biochemistry 43, 14249-14256.