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Label-free detection techniques
Development of reliable, sensitive and high-throughput
label-free detection techniques has become imperative
for proteomic studies due to drawbacks associated
with label-based technologies. Label-free detection
methods, which monitor inherent properties of the
query molecule, promise to simplify bioassays.
Harini Chandra
Affiliations
Master Layout (Part 1)
1
This animation consists of 4 parts:
Part 1 – Overview of label-free techniques
Part 2 – Surface plasmon resonance (SPR)
Part 3 – Surface plasmon resonance imagining (SPRi)
Part 4 – Spectral reflectance imaging biosensor (SRIB)
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Microcantilever
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4
5
Surface plasmon
resonance (SPR)based techniques
LABEL-FREE
DETECTION
TECHNIQUES
Scanning
Kelvin
Nanoprobe
(SKN)
Enthalpy
array
Atomic
Force
Microscopy
(AFM)
Ellipsometry
techniques
Interferencebased techniques
Electrochemical
impedance spectroscopy
(EIS) aptamer array
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Definitions of the components:
Part 1 – Overview of label-free techniques
1. Label-free detection: Label-free detection techniques monitor inherent properties of
the query molecules such as mass, optical and dielectric properties. Unlike label-based
detection methods, these techniques avoid any tagging of the query molecules thereby
preventing changes in structure and function. They do not involve laborious procedures
but have their own pitfalls such as sensitivity and specificity issues.
2. Surface plasmon resonance-based techniques
i) Surface plasmon resonance (SPR): Detects any change in refractive index of
material at the interface between metal surface and the ambient medium.
ii) Surface plasmon resonance imaging (SPRi): Image reflected by polarized light
at fixed angle detected.
iii) Nanohole array: Light transmission of specific wavelength enhanced by coupling
of surface plasmons on both sides of metal surface with periodic nanoholes.
3. Ellipsometry-based techniques
i) Ellipsometry: Change in polarization state of reflected light arising due to
changes in dielectric property or refractive index of surface material measured.
ii) Oblique incidence reflectivity difference (OI-RD): Variation of ellipsometry that
monitors harmonics of modulated photocurrents under nulling conditions.
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4. Interference-based techniques: Interferometry is based on the principle of
transformation of phase differences of wave fronts into readily recordable intensity
fluctuations known as interference fringes. The various detection strategies that make
use of this principle include:
i) Spectral reflectance imaging biosensor (SRIB): Changes in optical index due
to capture of molecules on the array surface detected using optical wave interference.
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2
Definitions of the components:
Part 1 – Overview of label-free techniques
ii) Biological compact disc (BioCD): Local interferometry i.e. transformation of
phase differences of wave fronts into observable interference fringes, used for detection
of protein capture.
iii) Arrayed imaging reflectometry (AIR): Destructive interference of polarized light
reflected from silicon substrate captured and used for detection.
5. Electrochemical impedance spectroscopy (EIS) -aptamer array: Aptamers are
short single-stranded oligonucleotides that are capable of binding to a wide range of
target biomolecules. EIS combined with aptamer arrays can offer a highly sensitive labelfree detection technique.
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6. Atomic force microscopy (AFM): Vertical or horizontal deflections of cantilever
measured by high-resolution scanning probe microscope, thereby providing significant
information about surface features.
7. Enthalpy array: Thermodynamics and kinetics of molecular interactions measured in
small sample volumes without any need for immobilization or labelling of reactants.
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8. Scanning Kelvin nanoprobe (SKN): A non-contact technique that does not require
specialized vacuum or fluid cell, SKN detects regional variations in surface potential
across the substrate of interest caused due to molecular interactions.
9. Microcantilever: These are thin, silicon-based, gold-coated surfaces that hang from a
solid support. Bending of cantilever due to surface adsorption is detected either
electrically by metal oxide semiconductor field effect transistors or optically by changes in
angle of reflection.
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Part 1, Step 1:
SPRi
Nanohole array
SPR
Microcantilever
Surface plasmon
resonance (SPR)based techniques
Ellipsometry
techniques
Ellipsometry
OI-RD
2
SRIB
3
Interferencebased techniques
AIR
BioCD
Enthalpy
array
4
Action
5
LABEL-FREE
DETECTION
TECHNIQUES
Scanning
Kelvin
Nanoprobe
(SKN)
Each heading
must appear one
at a time and the
user must be
allowed to click
on them to
understand the
details.
Atomic
Force
Microscopy
(AFM)
Electrochemical
impedance spectroscopy
(EIS) aptamer array
Description of the action
First show the central heading in the circle. Then show
each of the arrows appearing and their respective
colored boxes as shown. User must be allowed to click
on any of these headings to read the definitions as
given in the previous two slides. Once the user is
done, he must be provided with a ‘NEXT’ option, which
when clicked, must highlight the boxes indicated
‘SPR’, ‘SPRi’, ‘nanohole array’ and ‘SRIB’.
Audio Narration
As given in the previous two
slides.
Master Layout (Part 2)
2
Free antigen
3
% Reflectivity
1
This animation consists of 4 parts:
Part 1 – Overview of label-free techniques
Part 2 – Surface plasmon resonance (SPR)
Part 3 – Surface plasmon resonance imagining (SPRi)
Part 4 – Spectral reflectance imaging biosensor (SRIB)
Antigen-Antibody
complex
Reflection angle
Flow cell system
Bound antibodies
Gold film
Glass slide
Prism
Incident light
4
Reflected light
Change in angle of reflection
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2
Definitions of the components:
Part 2 – Surface plasmon resonance (SPR)
1. Flow cell system: A fluidic device that allows entry of antigens and
continuously removes unbound antigens from the system.
2. Free antigen: Antigens that have not bound to their complimentary antibody
are in their free state.
3. Bound antibodies: Test proteins such as antibodies that are capable of
specifically capturing the desired target protein with high affinity are
immobilized on to the gold-coated glass microarray slide.
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4. Antigen-Antibody complex: The complex formed due to binding interaction
between the free antigen and its corresponding bound antibody.
5. Glass slide: The array surface most commonly used for SPR applications. It
is suitably coated with a metal film like gold or silver.
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6. Gold film: A thin film of gold is used to coat the glass array surface due to its
favourable electronic interband transitions which fall in the visible range. In
most other metals, these transitions lie in the ultraviolet region, thereby making
them unsuitable for SPR.
7. Prism: The prism placed in contact with the glass slide surface helps in
reflecting the incident light from the surface.
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2
3
4
5
Definitions of the components:
Part 2 – Surface plasmon resonance (SPR)
8. Incident light: Light falling on the gold-coated array surface with its
immobilized antibodies has a particular wavelength and is known as the incident
light.
9. Reflected light: Some of the energy of the light incident on the array surface
gets absorbed for molecular transitions while the remaining light of lower energy
(and higher wavelength) gets reflected from the array surface at a specific angle.
10. Change in angle of reflection: Any changes in the angle of reflected light
are indicative of biomolecular binding interactions on the array surface. The
angle at which minimum intensity of reflected light is obtained is known as the
SPR angle and serves as a quantitative measure of biomolecules binding to the
array surface.
% Reflectivity
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Part 2, Step 1:
2
Reflection angle
Bound antibodies
Glass slide
Gold film
3
Prism
Incident light
4
Action
As shown in
animation.
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Description of the action
First show appearance of grey rectangle surface
followed by yellow coating with their respective
labels. Then show the Y shaped object binding
to the surface. Next, light beam must strike the
surface and a different color beam must be
reflected from it as shown followed by
appearance of the graph on the right.
Reflected light
Audio Narration
SPR is a highly sensitive spectroscopic tool
that is increasingly being used for label-free
detection studies. Test proteins such as
antibodies are immobilized onto the goldcoated glass array surface. Incident light
striking the surface is constantly reflected at a
particular angle in this state.
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Part 2, Step 2:
Free antigen
2
Flow cell system
Bound antibodies
Gold film
Glass slide
3
Prism
Incident light
Reflected light
4
Action
5
The green
shapes must
enter the grey
box slowly from
the side.
Description of the action
Show the grey rectangle appearing followed
by the arrows and the label ‘flow cell system’.
Then, show the green freeform shapes
appearing and entering the grey rectangle
area slowly from the side. Their movement
must not be sharp, point-to-point but more of
a meandering and slow entry.
Audio Narration
Unlabelled free antigens or other query
proteins enter via the flow cell and move
towards the immobilized antibodies or
other test proteins. There is no change in
reflected light upon entering into the
system.
% Reflectivity
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Part 2, Step 3:
Antigen-Antibody
complex
2
Reflection angle
Flow cell system
Gold film
Glass
slide
Prism
3
Incident light
Change in angle of reflection
4
Action Description of the action
5
Reflected light
The green
shapes must
bind to the
green Y
shaped objects
and must
continuously
enter and leave
the system.
Show the green shapes binding to the Y
shaped objects. Once binding occurs, there
must be a change in the reflected light beam
as shown and change in reflection angle must
be shown followed by appearance of new pink
curve in the graph.
In the background, the green shapes must
continue to enter and leave the system if not
bound by the Y shaped objects.
Audio Narration
Binding of antigen to antibody immediately brings about a
change in the angle of reflection of light due to changes in the
refractive index of the medium. These changes can be
continuously monitored to characterize biomolecular interactions
in real-time. The SPR angle i.e. the angle at which minimum
intensity of reflected light is obtained is indicative of the amount
of biomolecule binding to the surface. The graph represents
change in reflection intensity before and after antigen binding.
1
This animation consists of 4 parts:
Part 1 – Overview of label-free techniques
Part 2 – Surface plasmon resonance (SPR)
Part 3 – Surface plasmon resonance imagining (SPRi)
Part 4 – Spectral reflectance imaging biosensor (SRIB)
Antigen-antibody
complex
2
Immobilized
antibodies
3
% Reflectivity
Master Layout (Part 3)
Reflection angle
Gold coated
array surface
CCD camera
Scanner
mirror
4
Light source
5
SPRi image
Lokate, A. M., Beusink, J. B., Besselink, G. A., Pruijn, G. J., Schasfoort, R. B., Biomolecular interaction monitoring of
autoantibodies by scanning surface plasmon resonance microarray imaging. J. Am. Chem. Soc. 2007, 129, 14013–14018.
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Definitions of the components:
Part 3 – Surface plasmon resonance imaging (SPRi)
1. Light source: A broad beam, monochromatic, polarized light is used to illuminate the
entire array surface at the same time.
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2. Scanner mirror: A mirror which can reflect the light from the light source on to the
biochip surface.
3. Gold-coated array surface: Similar to SPR, a gold-coated glass array surface or
sometimes a gold-coated hydrogel array are used as the biochip for immobilization of the
capture molecule of interest.
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4. Immobilized antibodies: Test proteins such as antibodies that are capable of
specifically capturing the desired target protein from a mixture are immobilized on to the
gold-coated microarray slide.
5. Antigen-antibody complex: The complex formed due to specific binding interactions
between the antigens and their corresponding immobilized anitbodies.
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6. CCD camera: A charge-coupled device (CCD) camera continuously monitors any
changes that occur on the array surface and is capable of providing real-time kinetic data.
This digital imaging technology is widely used for scientific and medical applications
where high quality image data is required.
7. SPRi image: Reflected light from a spot will reach a minimal value when the spot
meets the optimal SPR conditions, thereby resulting in a dark spot. In this way, the SPRi
image is formed for the multiple spots across the array surface.
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1
Part 3, Step 1:
2
Immobilized
antibodies
Gold coated
array
surface
3
4
Action
5
The brown
colored Y shaped
objects must bind
to the yellow
surface as
shown.
Description of the action
First show appearance of only the yellow
surface with its label. Then show the brown
colored Y shaped objects binding to this surface
as depicted in the animation.
Audio Narration
A gold coated glass array surface is used for
immobilization of antibodies complimentary to
the target protein of interest.
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Part 3, Step 2:
Immobilized
antibodies
2
3
Gold coated
array surface
CCD camera
Scanner
mirror
Light source
4
Action
As shown in
animation.
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Description of the action
First show the purple can, the grey
‘mirror’, the two orange ovals and the
green ‘camera’. Then show the yellow
light rays moving as shown in the
animation until the grey surface below is
reached.
SPRi image
Audio Narration
A broad beam, monochromatic, polarized light
originating from a suitable light source is used to
illuminate the entire biochip surface with the help of
mirrors placed at suitable angles that will reflect the
light onto the surface. Reflected light from each spot
on the array surface is captured by means of a CCD
camera and used to generate the SPRi image.
Antigens
Immobilized
antibodies
2
3
Gold coated
array surface
Light source
4
Action Description of the action
5
Reflection angle
CCD camera
Scanner
mirror
The green dots
must bind to
the brown Y
shaped objects
and spots must
appear on the
grey surface
below.
% Reflectivity
1
Part 3, Step 3:
Show the green dots binding to the brown Y
shaped objects. Once binding occurs, there
must be a change in the color of reflected
light beam as shown and spots must appear
on the grey surface shown below as
depicted in the animation.
SPRi image
Audio Narration
Binding of target antigen to the antibody is detected in
real-time due to changes in intensity of reflected light
from every spot on the array surface. Multiple
biomolecular interactions can be studied
simultaneously in a HT manner and changes occurring
on the array surface can provide kinetic data about the
interactions.
Master Layout (Part 4)
Surface
Height relative to
surface (nm)
1
This animation consists of 4 parts:
Part 1 – Overview of label-free techniques
Part 2 – Surface plasmon resonance (SPR)
Part 3 – Surface plasmon resonance imagining (SPRi)
Part 4 – Spectral reflectance imaging biosensor (SRIB)
2
CCD
camera
Position on
sample (mm)
3
Illumination
4
Change in OPD
SiO2 coating
Silicon surface
5
Ozkumur, E., Needham, J. W., Bergstein, D. A., Gonzalez, R. et al., label-free and dynamic detection of biomolecular
interactions for high-throughput microarray applications. Proc. Natl. Acad. Sci. USA 2008, 105, 7988–7992.
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3
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Definitions of the components:
Part 4 – Spectral reflectance imaging biosensor (SRIB)
1. Illumination: A tunable laser beam of a specific wavelength that has been made
spatially incoherent by passing the beam through spinning ground-glass disks is used to
illuminate the array surface immobilized with biomolecules.
2. Silicon surface: A silicon wafer having a thermally grown surface coating of silicon
oxide (SiO2) is used as the solid support for immobilization of biomolecules.
3. SiO2 coating: The thermally grown and polished SiO2 layer can be used as the
reflecting surface instead of conventional glass microscopic slides due to its superior
uniformity and smoothness. Reproducible functionalization of these surfaces is also easily
achievable due to the known chemical composition and surface chemistry.
4. Change in OPD: Light incident on the SiO2 surface gets reflected at a specific
wavelength, the magnitude of which depends on the optical path length difference (OPD)
between the top of the surface and the buried SiO2 -Si interface. Any binding of
biomolecules to the top surface results in a further increase in OPD which exhibits itself as
a characteristic shift in spectral reflectivity and as an intensity difference at a particular
wavelength.
5. CCD camera: : A charge-coupled device (CCD) camera continuously monitors any
changes that occur on the SiO2 array surface and is capable of providing high throughput,
real-time kinetic data. This digital imaging technology is widely used for scientific and
medical applications where high quality image data is required.
CCD
camera
2
Surface
Height relative to
surface (nm)
1
Part 4, Step 1:
Position on
sample (mm)
3
Illumination
Change in OPD
SiO2 coating
Silicon surface
4
Action
5
As
show
n in
anima
tion.
Description of the action
First show appearance of brown surface, blue coating layer and
then the orange light beam. Show the first set of curved green
arrows and then the pink camera on top capturing it with
appearance of 1st two grey blocks. Then show the first part of graph
on the right. Next show a blue layer appearing and the second
green arrows on the surface, the next two cameras and grey
blocks. Then show the second part of graph. Finally show the last
two blue blocks and the third set of green arrows and last 2
cameras and 3 grey blocks. Finally show the last part of the graph.
Audio Narration
A SiO2 coated Si surface is functionalized with the
biomolecule of interest. The magnitude of total
reflected light at a particular wavelength depends
entirely on the OPD between the top surface and the
SiO2-Si interface. Binding of the target to the
immobilized biomolecule further increases the OPD
and is seen as a shift in the spectral reflectivity. SRIB
therefore serves as a useful tool for HT, real-time
detection of biomolecular interactions.
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2
3
Interactivity option 1:Step No: 1
SPR imaging has been used for serum proteomics studies to characterize the
antigens present in patients with hepatocellular carcinoma (HCC) (Lausted et al.,
2008). Antibodies specific to liver protein targets were arrayed on a gold-coated
surface and ten probed with human serum samples from HCC as well as non-HCC
patients. The authors detected 39 significant protein changes in this study, 10 of
which were already known including the commonly used liver cancer marker afetoprotein.
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Antibodies against
liver-specific
proteins
Gold coated array surface
Interacativity Type
Drag and drop.
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Antigens from
human serum
samples
Arrange the optics
system for the
experiment in their
correct positions.
Then click on the
light source to view
the SPRi image.
Options
User must drag and
drop the shapes
shown in the next
slide in their correct
positions indicated
by their dotted
outlines.
Boundary/limits
Results
Once the user places the shapes in
their correct positions, he must be
allowed to click on the purple cylinder
(light source) which must result in the
emission of the light rays as shown
followed by appearance of the final
grey surface at the bottom.
Lausted, C., Hu, Z., Hood, L. Quantitative Serum Proteomics from Surface Plasmon Resonance Imaging. Mol. Cell Proteomics
2008, 7, 2464-2474.
1 Interactivity option 1:Step No: 2
2
Antigen-antibody
binding interaction
Optics system for SPRi
Antibodies against
liver-specific
proteins
3
4
5
Scanner
mirror
Gold coated
array surface
Light source
CCD camera
SPRi image
1
Questionnaire
1. Which of the following label-free techniques relies on thermodynamic changes occurring
due to molecular interactions?
2
Answers: a) SPR b) SRIB c) Enthalpy array d) SKN
2. Which of the following is not an interference-based detection technique?
Answers: a) SRIB b) SKN c) AIR d) BioCD
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3. Surface features of an object can be studied in detail using which of the techniques below?
Answers: a) AIR b) SPRi c) Nanohole array d) AFM
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4. A change in the optical path length difference upon binding of target to the immobilized
biomolecule occurs in which technique?
Answers: a) SRIB b) SPR c) AFM d) Ellipsometry
5. Surface plasmon resonance detects changes in which of the following properties?
5
Answers: a) Electrical conductivity b) Phase difference c) Temperature d) Refractive index
Links for further reading
Research papers:
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Ray, S., Mehta, G., Srivastava, S. Label-free detection techniques for protein microarrays:
Prospects, merits and challenges. Proteomics 2010, 10, 731-748.
Ramachandran, N., Larson, D. N., Stark, P. R., Hainsworth, E., LaBaer, J., Emerging tools for
real-time label-free detection of interactions on functional protein microarrays. FEBS J. 2005,
272, 5412–5425.
Yu, X., Xu, D., Cheng, Q., Label-free detection methods for protein microarrays. Proteomics
2006, 6, 5493–5503.
Lee, H. J., Nedelkov, D., Corn, R. M., Surface plasmon resonance imaging measurements of
antibody arrays for the multiplexed detection of low molecular weight protein biomarkers. Anal.
Chem. 2006, 78, 6504–6510.
Yuk, J. S., Kim, H. S., Jung, J. W., Jung, S. H. et al., Analysis of protein interactions on protein
arrays by a novel spectral surface plasmon resonance imaging. Biosens. Bioelectron. 2006,
21, 1521–1528.
de Boer, R. A., Hokke, C. H., Deelder, A. M., Wuhrer, M., Serum antibody screening by
surface plasmon resonance using a natural glycan microarray. Glycoconj. J. 2008, 25, 75–84.