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 Figure.
Left: A typical SPR setup. An incident light is directed onto a
SPR sensor chip via a prism, and the reflected beam is detected
via a photodetector or imager. At an appropriate angle (resonance
angle), the incident light excites the surface plasmons in the sensor
chip (metal film) and the intensity of the reflected light drops
to a minimum. The electromagnetic field created by the SPR penetrates
into the fluidic medium and probes molecular binding processes taking
place on the surface and index of refraction changes in the fluidic
medium. Right: Reflectivity vs. incident angle plot shows a sharp
drop in the reflection intensity due to SPR, also referred to as
the SPR "dip". The angular position of the dip is often measured
and used to define SPR sensitivity. |
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Dependence
upon Prism Material:
It is worth noting that the resonance angle depends not
only on molecular binding and the index of refraction of the fluid
medium, but also on the index of refraction of the prism, the dielectric
constant of the metal film as well as on the wavelength of light
used to excite the surface plasmons. So when comparing the sensitivities
of different instruments in terms of degree angles, one should be
aware of the prism material, the metal film as well as the wavelength
of light.
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Example 1:
If the prism is made of BK7 (n=1.515)
glass and the wavelength of incident light is 635 nm, then the angular
shift due to a protein binding layer (n= 1.5) of 3 nm on a gold
sensor chip is 0.75 deg. If keeping everything the same, except
replacing the BK7 glass prism with a SF10 glass (n=1.723) prism,
then the same protein binding layer leads to an angular shift of
0.35 deg (a weaker response). So if two instruments report the same
angular shift, the one using BK7 prism is actually more sensitive
in terms of measuring molecular binding.
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Dependence
upon Wavelength:
The penetration length of the evanesce field created by SPR into
the fluid medium increases with the wavelength. Longer wavelengths
(e.g., near infrared) have the “seeming” advantage of
being able to probe further beyond the sensor surface, however this
results in a significant loss of surface sensitivity.
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Example 2:
Two SPR instruments both use BK7 glass prisms,
gold sensor chips, and have similar angular sensitivity, but one
uses 635 nm light and the other uses 890 nm light. For a protein
binding layer of 3 nm, the first instrument produces 0.75 deg angular
shift, but the second instrument leads to only 0.2 deg angular shift
(a much weaker response). So for two instruments having similar
angular shifts, the one using 635 nm light is actually more sensitive
in terms of measuring molecular binding. Although longer wavelengths
allow for slightly deeper detection into the solution bulk, this
results in a significantly lower sensitivity for measuring molecular
binding on the sensor surface.
Example 3: Two SPR instruments
claim to have similar sensitivities since they have matching values
for angular sensitivity. However, one instrument uses a BK7 glass
prism and 635 nm light, while the other instrument uses a SF10 glass
prism and 890 nm light. For a protein binding layer of 3 nm, the
first instrument produces 0.75 deg angular shift. Not surprisingly
though, the second instrument results in a much weaker response
of 0.15 deg angular shift. So for two instruments having similar
angular sensitivity, the one using 635 nm light and BK7 glass is
actually 5 times more sensitive in terms of measuring molecular
binding.
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Relative
Index of Refraction Unit:
Another quantity often used to describe SPR sensitivity
is the relative change in the index of refraction of the fluid medium,
known as RIU. Unlike angular shift, the unit of RIU is more relevant
to applications that demand an accurate measurement of the index
of refraction of a bulk fluid. As a result, RIU may not be the most
convenient unit for applications that aim to study molecular binding
events. A relationship between RIU and angular shift in degrees
is possible if one knows the exact instrumental conditions (e.g.,
wavelength of incident light and material of prism glass). And just
like with angular shift, a SPR instrument that has the best sensitivity
in terms of RIU does not always mean that it has the best sensitivity
in terms of detecting molecular binding.
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Example 4:
If an SPR instrument uses a BK7 glass prism,
gold sensor chip, and 635 nm incident light, then a 0.010 RIU change
in aqueous buffer solution results in ~1.55 deg angular shift. However,
this conversion between RIU and angular shift is not universal,
as it depends upon the instrumental conditions (e.g., wavelength
of incident light and prism material). By increasing the wavelength
to 890 nm but keeping all the other experimental parameters the
same, then a new relation is observed in which a 0.010 RIU change
in aqueous buffer solution results in a smaller ~0.99 deg angular
shift. Additionally, if both the wavelength is increased to 890
nm and the prism glass changed to SF10 then a 0.010 RIU change in
aqueous buffer solution results in ~0.61 deg angular shift. Thus
the comparison of sensitivity between units of degree angular shift
and RIU requires careful consideration of instrumental conditions.
Example 5: How do the following
SPR instruments compare? The first instrument has a BK7 glass prism
with 635 nm light and sensitivity of 0.1 mDeg. A second instrument
has a SF10 glass prism with 890 nm light and sensitivity of 1 µRIU.
From Example 4, we learned that 0.010 RIU corresponds to 0.61 deg
for this configuration. Using this relationship 1 µRIU corresponds
to a 0.06 mDeg sensitivity. Does this mean that the second instrument
is more sensitive than the first? No, remember that angular sensitivities
alone do not tell the complete story. We must determine the surface
binding sensitivity to make the fairest comparison. From Example
3, we learned that the configuration of the second instrument is
5 times less sensitive than the configuration of the first instrument.
Thus, a 0.06 mDeg sensitivity, actually corresponds to an equivalent
surface binding sensitivity of 0.3 mDeg. As a result, the first
instrument is experimentally more sensitive than the second.
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Surface
coverage:
If one is interested in using SPR to detect molecular binding
taking place on a sensor surface, then the surface coverage in terms
of mass, e.g., pg/mm², is an appropriate way to
define sensitivity. The unit, RU (termed Resonance
Unit or Response Unit) is defined as 1 RU = 1 pg/mm², and is also
often used to determine surface coverage.
However, this description cannot be ubiquitously used. For instance,
SPR really measures the optical polarizability and size and density
of molecules bound to the surface, which are related to but different
from an SPR measurement in terms of mass per unit surface area.
The polarizability depends on the wavelength of light, especially
if the wavelength is close to the optical absorption band of the
molecule (e.g. chromophores, UV-vis labels, etc.). Since most proteins
have similar polarizabilities, the SPR signal may be considered
approximately proportional to the coverage of molecules bound to
the sensor surface, and pg/mm² is a useful way to quantify SPR sensitivity.
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Example 6:
A monolayer of cytochrom c leads to an angular
shift of ~0.5 Deg. The corresponding mass coverage is ~3000 pg/mm².
For an angular sensitivity of 0.1 mDeg, the corresponding mass sensitivity
is 0.6 pg/mm² or 0.6 RU.
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Molar
concentration:
Some vendors provide sensitivity in terms of lowest detectable molar
concentration. This is a convenient and attractive measure
of SPR instrument sensitivity. However, the reality is that a highly
sensitive instrument cannot faithfully guarantee the detection of
an extremely low analyte concentration. This is because sensitivity
and detection level are two different (although related) analytical
“figures of merit” [1] which are often mistakenly mixed.
The lowest detectable molar concentration depends upon several significant
experimental factors such as the molecular weight, optical property,
and binding affinity of the analyte as well as the surface coverage
of the capture molecules. Background noise also plays a key role
in determining the lowest detection level (more about this later).
Molecules with large molecular weight and polarizability are easier
to detect than those with small molecular weight and polarizability.
A high affinity and surface coverage of the capture molecules also
facilitate the detection of analyte molecules per given concentration.
Additional factors influencing the lowest detectable molar concentration
include sensor chip preparation (e.g., the thickness of the modifier
layer and its refractive index), temperature, and buffer solution
performance. Moreover, numerous experimental strategies exist which
can amplify SPR binding responses (e.g., labels, competitive binding
assays, enzymatic reactions, etc.). As a result, SPR sensitivity
in terms of lowest detectable molar concentration
can be misleading, and very unforgiving to beginning SPR users.
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Example 7:
A sensor chip is functionalized
with 5 x 10-16 mol/mm² anti-PNA (peanut agglutinin), PNA
molecular weight is about 100 kDa, and PNA-anit PNA equilibrium
dissociation constant, K, is about 20 nM. For a SPR instrument with
sensitivity of 0.1 mDeg, or corresponding mass sensitivity of ~0.6
pg/mm², the minimum detectable concentration will be: ~0.5
nM at equilibrium. Clearly, if factors such as surface coverage
and equilibrium dissociation constant were different, then the minimum
detectable concentration would also change. Thus, the evaluation
of sensitivity in terms of analyte concentration should be carefully
considered, keeping in mind that chip and experimental conditions
play critical roles.
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Determination
of detection levels:
The definition of “lowest detectable level”
is often not clearly spelled out. The lowest detection level is
largely governed by the background noise. Some choose the peak-to-peak
value of the noise in the SPR signal, while others use root-mean-square
or standard deviation. In analytical chemistry, an often used definition
of detection limit is three times the standard deviation of the
background (blank) noise. Second, the noise of a measured physical
quantity usually occurs at various time scales, so SPR sensitivity
should be given together with the time scale of the measurement.
Filters, such as time averaging and smoothening of data, can remove
certain noises and improve the sensitivity and detection level.
This practice tends to slow down the response time.
So one must also make sure the response time is fast enough for
an application when choosing an instrument. Third, the noise level
may be influenced by electronic amplification (or gain control).
A higher gain may improve signal to noise ratio, but this usually
affects the dynamic range (detection range) of the instrument. Finally,
when comparing with imaging SPR or other pixel-based detectors,
the sensitivity depends on how many pixels the SPR signal is averaged
over and for how long. More pixels and more time lead to better
sensitivity, but it may sacrifice spatial resolution and response
time.
Reference.
[1] Skoog, D. A.; Holler, F. J.; Crouch, S. R. Principles of Instrumental
Analysis, 6th ed.; Thomson Higher Education, Belmont, CA, 2007.
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