Label-free detection techniques

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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

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Master Layout (Part 1)
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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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Surface plasmon resonance (SPR)-based techniques
Ellipsometry techniques
Microcantilever
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Scanning Kelvin Nanoprobe (SKN)
Interference-based techniques
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Enthalpy array
Electrochemical impedance spectroscopy (EIS)
aptamer array
Atomic Force Microscopy (AFM)
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Definitions of the componentsPart 1 Overview
of label-free techniques
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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. 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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Definitions of the componentsPart 1 Overview
of label-free techniques
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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 label-free detection
technique. 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. 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
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SPRi
Nanohole array
SPR
Surface plasmon resonance (SPR)-based techniques
Ellipsometry
Ellipsometry techniques
Microcantilever
OI-RD
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SRIB
Scanning Kelvin Nanoprobe (SKN)
Interference-based techniques
AIR
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BioCD
Enthalpy array
Electrochemical impedance spectroscopy (EIS)
aptamer array
Atomic Force Microscopy (AFM)
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Action
Audio Narration
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.
Each heading must appear one at a time and the
user must be allowed to click on them to
understand the details.
As given in the previous two slides.
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Master Layout (Part 2)
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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Bound antibodies
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Definitions of the componentsPart 2 Surface
plasmon resonance (SPR)
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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. 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. 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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Definitions of the componentsPart 2 Surface
plasmon resonance (SPR)
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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.
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Part 2, Step 1
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Bound antibodies
Gold film
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Glass slide
Prism
Incident light
Reflected light
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Action
Audio Narration
Description of the action
As shown in animation.
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 gold-coated
glass array surface. Incident light striking the
surface is constantly reflected at a particular
angle in this state.
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.
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Part 2, Step 2
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Free antigen
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Flow cell system
Bound antibodies
Gold film
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Glass slide
Prism
Incident light
Reflected light
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Action
Audio Narration
Description of the action
The green shapes must enter the grey box slowly
from the side.
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.
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.
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Part 2, Step 3
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Antigen-Antibody complex
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Flow cell system
Glass slide
Gold film
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Prism
Reflected light
Incident light
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Change in angle of reflection
Action
Audio Narration
Description of the action
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.
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.
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Master Layout (Part 3)
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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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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, 1401314018.
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Definitions of the componentsPart 3 Surface
plasmon resonance imaging (SPRi)
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1. Light source A broad beam, monochromatic,
polarized light is used to illuminate the entire
array surface at the same time. 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. 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. 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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Part 3, Step 1
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Immobilized antibodies
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Gold coated array surface
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Action
Audio Narration
Description of the action
The brown colored Y shaped objects must bind to
the yellow surface as shown.
A gold coated glass array surface is used for
immobilization of antibodies complimentary to the
target protein of interest.
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.
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Part 3, Step 2
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Immobilized antibodies
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Gold coated array surface
CCD camera
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Scanner mirror
Light source
SPRi image
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Action
Audio Narration
Description of the action
As shown in animation.
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.
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.
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Part 3, Step 3
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Antigens
Immobilized antibodies
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Gold coated array surface
CCD camera
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Scanner mirror
Light source
SPRi image
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Action
Audio Narration
Description of the action
The green dots must bind to the brown Y shaped
objects and spots must appear on the grey surface
below.
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.
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.
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Master Layout (Part 4)
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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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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, 79887992.
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Definitions of the componentsPart 4 Spectral
reflectance imaging biosensor (SRIB)
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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.
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Part 4, Step 1
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Surface
Height relative to surface (nm)
CCD camera
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Position on sample (mm)
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Illumination
Change in OPD
SiO2 coating
Silicon surface
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Action
Audio Narration
Description of the action
As shown in animation.
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.
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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Interactivity option 1Step No 1
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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 a-fetoprotein.
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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.
Antibodies against liver-specific proteins
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Gold coated array surface
Results
Boundary/limits
Interacativity Type Options
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.
User must drag and drop the shapes shown in the
next slide in their correct positions indicated
by their dotted outlines.
Drag and drop.
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Lausted, C., Hu, Z., Hood, L. Quantitative Serum
Proteomics from Surface Plasmon Resonance
Imaging. Mol. Cell Proteomics 2008, 7, 2464-2474.
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Interactivity option 1Step No 2
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Antigen-antibody binding interaction
Optics system for SPRi
Antibodies against liver-specific proteins
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Scanner mirror
Gold coated array surface
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CCD camera
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Light source
SPRi image
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Questionnaire
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• 1. Which of the following label-free techniques
  relies on thermodynamic changes occurring due to
  molecular interactions?
• 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
• 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
• 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?
• Answers a) Electrical conductivity b) Phase
  difference c) Temperature d)? Refractive index

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Links for further reading

• Research papers
• 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, 54125425.
• Yu, X., Xu, D., Cheng, Q., Label-free detection
  methods for protein microarrays. Proteomics 2006,
  6, 54935503.
• 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, 65046510.
• 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, 15211528.
• 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, 7584.