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Technical Notes
103 - Surface Plasmon Resonance
vs. Quartz Crystal Microbalance
Surface Plasmon Resonance (SPR) and Quartz Crystal
Microbalance (QCM) are two powerful label-free detection
techniques that are receiving increasing attention
for many applications, ranging from biomedical, electrochemical
and surface studies to chemical and biosensors. An
often asked question is: Which one is better or more
suitable for my applications? This note will discuss
the differences between the two techniques and compare
some of the key performance parameters, such as sensitivity,
response time, detection limit, accuracy and multiplexity.
The goal is to provide researchers with necessary
information to make a rational choice based on their
applications.
Basic principles
QCM is basically a mass loading
sensor. It is sometimes also referred to as Quartz
Crystal Nanobalance because nanograms of material
can be “weighed”(Fig.
1). The most critical component is a quartz crystal
cut in the so-called AT orientation forming a keyhole
shaped disk or chip. The surfaces of the chip are
patterned with electrodes, which serve two purposes:
1) drive the crystal into oscillation or resonance
electrically and 2) provide a sensor surface on
which molecular binding takes place. The most pronounced
oscillation or resonance mode is the shear mode
(displacement along the chip surface). The resonance
frequency decreases upon adsorption of molecules
onto the sensor surface. The frequency change (Δf)
is proportional to the mass (Δm) of the adsorbed
molecules per unit area, expressed
in the Sauerbrey equation:
where CQCM is the mass sensitivity
and equal to Vρ/2f2 (f is the resonance frequency,
V and ρ are the shear modulus and density of
quartz, respectively).
BI-SPR Technology
. Flow
Injection SPR . Electrochemical
SPR . Gas
Phase SPR
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Figure
1: Schematic of QCM chip.
SPR is based
on a different principle but can also provide the mass of molecules
adsorbed on a sensor chip – a glass slide covered with a thin
layer of metal (e.g., Au). In a typical SPR setup, a light beam
is directed onto the metal film through an optical prism and surface
plasmons in the metal film are excited if the incident angle (resonance
angle) is appropriate. The resonance angle is extremely sensitive
to the polarizability and number density of the adsorbed molecules.
For most biological molecules (e.g., proteins), the polarizabilities
are similar, so the shift in the resonance angle is proportional
to mass of the adsorbed molecules and given by
(2)
where CSPR is mass sensitivity for SPR.
The above descriptions for QCM and SPR are both simplified. However,
additional considerations should be noted to better understand the
differences between QCM and SPR. For QCM, molecular adsorption onto
the sensor chip introduces not only a mass change but also dissipation
of mechanical energy due to the internal friction of the adsorbed
layer. This dissipation of energy is particularly acute when the
measurement is conducted in the liquid phase (more discussion below).
In the case of SPR, the conformational change of molecules bound
to the surface may cause additional angular shift. These spurious
contributions to the mass loading information for both QCM and SPR
may be utilized to obtain additional information. |
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Mass sensitivity
Sensitivity and limit of detection of a sensor are related but different
parameters, although they are often mistakenly interchanged. A detailed
discussion of SPR sensitivity and detection limit is given in Technical
Note #102. The sensitivity of mass detection-based sensors is
the mass of detected target molecules per unit area of the sensor
surface for a given change of the sensor signal (frequency in QCM
or angular shift in SPR). For QCM, the sensitivity is CQCM
which depends on the resonance frequency, mass density, and shear
modulus of the quartz crystal. For a typical 5 MHz-QCM, CQCM
can be ~ 20 ng/cm2 per Hz. A higher resonance frequency will
lead to greater sensitivity of QCM. Unfortunately, a higher resonance
frequency requires the already thin crystal to be thinner, which
consequently makes the crystal become extremely fragile. For SPR,
the sensitivity is CSPR, which also depends upon
several parameters, such as polarizability of the target molecules,
wavelength of light, and prism index of refraction (see Technical
Note #102). A typical value for CSPR is ~1
ng/cm2 per mdeg.
Limit of Detection
Limit of detection (LOD) describes the smallest amount of target
molecules adsorbed onto the sensor that can be detected. Clearly
LOD is determined by the mass sensitivity but also by how accurately
one can measure the sensor output signal (frequency in QCM and angular
shift in SPR). If the resolutions of the frequency and angular shift
in QCM and SPR are δf and δθ, then the LODs of
the two sensors are given by
LODQCM= CQCM δf (3)
and LODSPR= CQCMδθ (4)
respectively. For a frequency resolution of 0.1 Hz, LOD for QCM
is about 2 ng/cm2 for a response time of 1 sec.
This may be improved by averaging over a longer time period, say
5 sec., but this reduces the response time. It is also worth pointing
out that stability of 0.1 Hz over an entire injection peak is extremely
challenging to obtain for most QCM devices (vide infra). A SPR instrument
can detect 0.1 mdeg angular shift for a response time of 1 sec.,
which corresponds to a LOD of ~0.1 ng/cm2 is considerably
better than that of QCM.
LOD discussed above is given in terms of detectable mass per unit
area. Another useful way to define LOD is total detectable mass,
denoted by LODM. The two are related by LODM=LOD × Sensor
Area. A typical QCM sensor has surface area of ~1 cm2, so the total
mass LOD is 2 ng. In contrast, the sensing area
for SPR is determined by the size of the illumination spot*, which
is often as small as 10-5 cm2. A corresponding SPR total mass LOD
is 1 fg, making SPR a million times more sensitive
than QCM. This means that in order for QCM to maintain a competitive
sensitivity level, the sensing area must be kept considerably larger
than that of SPR sensors.
It is also important to note that any external pressure (e.g., an
O-ring or a gasket) applied to the QCM’s sensor surface suppresses
the oscillation of the crystal, degrading its sensing performance.
Thus, the entire metal-coated sensor surface (cf. Fig 1) of QCM
sensors must be exposed to solution, drastically increasing its
cell volume and sample consumption.
Kinetics and Binding Affinity
One of the most important tasks in studying molecular binding/interaction
processes is to determine the binding kinetics, which not only allows
one to extract information about the how strong a binding/interaction
is but also shed light on the binding/interaction mechanism. To
correctly extract binding kinetic information, the sample solution
must be able to enter and leave the sensing area sharply, with little
dispersion. This task can be readily accomplished for SPR sensors
using microfluidic flow channels and proper valving techniques.
A similar fluid handling strategy cannot be easily implemented for
QCM. This is because of the relatively large internal cell volume
of QCM sensors (cf. Fig 1) which have to be fully exposed to sample
solution.
Accuracy
The Sauerbrey equation is very accurate for gas-phase measurements.
However, in the liquid environment, any nonrigidity of the molecules
attached to the crystal surface will make the frequency-mass relationship
deviate from the Sauerbrey equation. Any surface imhogeneity, solvation
of the film, and solution composition or viscosity change can also
affect the response in QCM. These terms are collectively referred
to as the “viscoelastic effect”. Although some of the
effects also affect the SPR response (e.g., solution composition
change and film solvation), they can be largely corrected by using
a reference channel (see description below).
Multiple Channels for Background Subtraction
and Multiplexity
For most biosensor applications or affinity studies, it is highly
desirable to have two detection channels. In this way, one channel
can be used for sample analysis and the second channel can be used
as a reference or control. Referencing is best achieved by keeping
everything in both channels as similar as possible, except for the
testing variable. For SPR, two channels on the same sensor chip
can be easily defined by pressing a patterned gasket to the sensor
surface. This has the advantage of utilizing the same sensor chip
and driving source (light in this case) as well having greater uniformity
in sensor surface due to their close proximity. For QCM, this method
cannot be implemented as the QCM sensor would be damped, drastically
degrading its performance. Referencing with QCM may be achieved
through the used of two QCM chips. This method of referencing has
several drawbacks: the two QCM crystals must be perfectly identical
to properly reference each other, the delivery of sample solution
to both sensors must be identical, and instrumentation is considerably
more complex. As a consequence, the use of referencing to correct
for spurious effects such as temperature variation, electronic noise,
mechanical drift, and the abovementioned viscoelastic effect is
difficult to achieve. This makes maintaining the LOD, accuracy,
and reproducibility of QCM measurements challenging.
Sensor Chip Functionalization and
Sample Volume
Sensor chips should to be pre-functionalized with an appropriate
layer of ligand/probe or a layer of matrix to reduce non-specific
adsorption. The procedures for both QCM and SPR are similar as both
utilize gold coated surfaces. However, for in-situ or real-time
pre-functionalization, QCM requires more reagents due to its larger
surface area and cell volume. Moreover, uniformity of the surface
functionalization becomes more difficult to achieve as the surface
area increases.
Summary
Both QCM and SPR are sensitive label-free detection methods that
have found many applications in surface adsorption and bioaffinity
studies. However, SPR is more sensitive, better for kinetic studies
and more easily extended to multiple channels. SPR also requires
far less sample volume.
Table 1. Comparison between SPR and QCM
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