Surface Plasmon Resonance (SPR) and its Application

DR. BARKHA GUPTA
ASSISTANT PROFESSOR (VETERINARY BIOCHEMISTRY)
DEPARTMENT OF VETERINARY PHYSIOLOGY AND BIOCHEMISTRY
POST GRADUATE INSTITUTE OF VETERINARY EDUCATION AND RESEARCH (PGIVER), JAIPUR RAJASTHAN UNIVERSITY OF VETERINARY AND ANIMAL SCIENCES (RAJUVAS), BIKANER
YouTube Channel: Barkha’s Vet Sphere

1. DR. BARKHA GUPTA
ASSISTANT PROFESSOR (VETERINARY BIOCHEMISTRY)
DEPARTMENT OF VETERINARY PHYSIOLOGY AND BIOCHEMISTRY
POST GRADUATE INSTITUTE OF VETERINARY EDUCATION AND RESEARCH (PGIVER), JAIPUR
RAJASTHAN UNIVERSITY OF VETERINARY AND ANIMAL SCIENCES (RAJUVAS), BIKANER
YouTube Channel: Barkha’s Vet Sphere

Surface Plasmon
Resonance (SPR) and
its Application

2. Surface Plasmon Resonance (SPR)
Label-free method

Surface sensitive spectroscopic technique

It is a non-destructive means of sensing and Surface Plasmons
have been used for gas sensing, biosensing, immuno-sensing
and electrochemical studies

Used to detect the binding of biological molecules onto arrays
of probe biomolecules covalently attached to chemically-
modified gold surfaces

Rapidly monitors dynamic processes to a wide range of
biomedically relevant interfaces

2

3. Development of reliable, sensitive and high-throughput
label-free detection techniques has become imperative for
proteomic studies due to drawbacks (Cost, Stability i.e.
Shorter Half-life and Complexity) associated with label-based
technologies.
Label-free detection methods, which monitor inherent
properties of the query molecule such as mass, optical and
dielectric properties, promise to simplify bioassays.
Need?
3

4. Label-free Detection Techniques

4
LABEL-FREE
DETECTION
TECHNIQUES
Surface plasmon
resonance
(SPR)-based
techniques
Ellipsometry
techniques
Interference
-based
techniques
Scanning Kelvin
Nanoprobe
(SKN)
Atomic Force
Microscopy (AFM)
Enthalpy array
Microcantilever
Electrochemical impedance
spectroscopy (EIS) aptamer
array
Ellipsometry
OI-RD
SRIB
AIR
BioCD
SPR
SPRi
Nanohole
array

5. Overview of Label-free Techniques
5
1. 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.
2. 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.
3. 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.
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.

6. Overview of Label-free Techniques
6

4.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.
5.Atomic force microscopy (AFM): Vertical or horizontal deflections of cantilever measured by high-resolution
scanning probe microscope, thereby providing significant information about surface features.
6.Enthalpy array: Thermodynamics and kinetics of molecular interactions measured in small sample volumes
without any need for immobilization or labelling of reactants.
7.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.
8.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.

7. SPR
Surface plasmon resonance (SPR), an optical biosensor technique is based on the phenomenon of
evanescent wave. This utilises a property of gold and other materials; specifically that a thin layer
of gold on a high refractive index glass surface can absorb laser light, producing electron waves
(surface plasmons) on the gold surface.
SPR causes a reduction in the intensity of reflected light at a specific angle of reflection. This
angle varies with the refractive index close to the surface on the side opposite from the reflected
light (sample side). Change in angle is converted to resonance signal, which is directly
proportional to mass bound at surface.
SPR response values are expressed in resonance units (RU). One RU = 0.0001
o
. For most
proteins this is about a change in conc. of 1pg/mm
2
on the sensor surface.
A surface-plasmon-resonance is excited at a metal-dielectric interface by a monochromatic, p-
polarized light beam, such as He-Ne laser beam.
 The surface plasmon is sensitive to changes in the environment near the interface and therefore
has potential as a sensing probe. Sensitive detection method that monitors variations in thickness
and refractive index in ultra-thin films.



7

8. How does SPR work?


This Kretschmann Experimental System uses a metal film thin enough to
monitor the plasmon
A plasmon can be thought of as a ray of light bound onto a surface -
propagating among the surface and presenting itself as an electromagnetic
field
http://www.biochem.mpg.de/oesterhelt/xlab/spfs.html

9

9. What is SPR?
Surface Plasmon: Longitudinal charge density wave along the
interface of two media, where one is metal and other is dielectric


10

10. Definitions of the components

11
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 inter-band transitions which fall in the visible range. In most
other metals, these transitions lie in the ultraviolet region, thereby making them
unsuitable for SPR.

11. Definitions of the components

12
7.Prism: The prism placed in contact with the glass slide surface helps in reflecting the incident
light from the surface
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.
Configurations of SPR devices that are capable of generating and measuring SPR:
 Prism coupled total reflection systems
 Optical fibres
 Grating coupled systems
 Optical waveguide systems

12. Surface Plasmon Resonance (SPR)
Flow cell system
Gold film
Free antigen
Glass slide
Bound antibodies
Incident light
Reflected light
Prism
.
Flow cell system
Gold film Glass
slide
Antigen-
Antibody
complex
Incident light
Reflected light
Change in angle of reflection
% Reflectivity

Reflection angle
Prism
13

13. Surface Plasmon Resonance (SPR)
14
% Reflectivity

Reflection angle
Bound antibodies
Flow cell system
Gold film
Incident light
Reflected light
Change in angle of reflection
Free antigen
Glass slide
Antigen-Antibody
complex
Prism

14. Advantage of using gold film
Gold: Non-magnetic, surface plasmon wave is p-polarized, and due to its electromagnetic and
surface propagating nature, creates enhanced evanescent wave
15

Gold-thiol Chemistry
Evanescent wave
In total internal reflection, the reflected
photons create an electric field on the
opposite site of the interface. The plasmons
create a comparable field that extends into
the medium on either side of the film. This
field is called the evanescent wave because
the amplitude of the wave decreases
exponentially with increasing distance from
the interface surface, decaying over a
distance of about one light wavelength.
Gold thiol adsorption
A thiol compound and a gold surface is one of the
well-established combinations used when making a
self-assembled monolayer (SAM)

15. Typical SPR Signal
16

16. Sensogram
17
Good
This simple principle forms the basis of the Sensogram – a continuous,
real-time monitoring of the association and dissociation of the
interacting molecules.
The sensorgram provides quantitative information in real-time on
specificity of binding, active concentration of molecule in a sample,
kinetics and affinity. Molecules as small as 100 Da can be studied in a
typical SPR reaction.
Bad

17. SPR: Kinetics of Association phase
C= Concentration of analyte
Rmax = maximum analyte binding capacity of the surface in RU
R = SPR signal at time t in RU
18 A
surface
+B AB
Ka
Kd
Km
A
bulk dR/dt = K
aC (R
max - R) - K
dR
or dR/dt = K
aC R
max - (K
d+ K
aC)R

18. SPR: Kinetics of Association phase
19
K
d is not very reliable as K
aC >>K
d

19. SPR: Kinetics of Dissociation phase
R
t is response at time t in RU
R
0 is response at an arbitrary starting point
K
D = k
d /k
a
20 ln (R
0/R
t) = K
d (t-t
0) dR/dt = - K
dR
After integration and logarithm

20. K
D is the equilbrium binding constant, which can either be determined by steady-
state affinity fitting (if your binding curves reach equilibrium, i.d. are characterized
by rapid association and dissociation binding kinetics) or from kinetic binding data
k
d is the dissociation rate constant.
From kinetic binding data, K
D (unit M) is calculated by dividing the dissociation rate
constant k
d (unit s
-1
) by the association rate constant k
a (unit M
-1
s
-1
)

21
(i)Preparation of the surface via immobilization of a probe molecule (Ligand)
(ii)Verification of the activity of the prepared sensor
(iii) Incubation of the sensor with a target-containing sample (A) to form a complex
(iv) Dissociation of the complex to reuse the sensor, or to further analyze the target by
employing stringent washing of several different pH solutions or different ionic
strengths
(v)Elution of captured proteins for further analysis
Sequential steps of SPR

21. Applications of SPR
Physical applications: measure dielectric properties, adsorption processes, surface
degradation or hydration of
Thin organic monolayers or bilayers
Polymer films

Biological applications: as biosensors for specific biological interactions including
adsorption and desorption kinetics, antigen-antibody binding and epitope mapping for
determination of
Biomolecular structure and interactions of proteins, DNA & Viruses
Lipid Bilayers
Non-specific biomolecular interactions- bio-compatibility
Tissue engineering

22

22. SPR: Physical Applications
23
Thin organic monolayers or bilayers
Polymer films

23. SPR: Biological Applications
24
Epitope mapping
Tissue engineering

24. 25

25. The livestock sector which contributes the largest economy of India, harbors many bacterial,
viral, and fungal diseases impacting a great loss to the production and productive potential
which is a major concern in both small and large ruminants. Hence, SPR based biosensor
assay is an accurate, sensitive, and rapid diagnostic aid which may help in prevention of
Livestock diseases.
26

26. Applications of SPR-Based Biosensors
1.Biomedical Applications
1.1. Interaction Analyses
1.2. Conformational Change Studies
1.3. Mutation Detection
2. High-Throughput Screening (HTS); Drug Screening/discovery
3. Proteomics Researches
3.1. Tumor-Associated DNA Markers
3.2. Disease-Related Protein Biomarkers
4. Cellular Analysis and Cell-Based Detection

27

27. 28

28. Peptide nucleic acids (PNAs) are excellent probes able to detect the W1282X point mutation of the cystic fibrosis (CF) gene when
biospecific interaction analysis (BIA) by surface plasmon resonance (SPR) and biosensor technologies is performed. BIA is an
easy, fast, and automatable approach for detecting mutations of CF, allowing real-time monitoring of hybridization between 9-mer CF
PNA probes and target biotinylated PCR products generated from healthy, heterozygous subjects and homozygous W1282X
samples and immobilized on streptavidin-coated sensor chips. This method is the first application of PNAs, BIA, and SPR to a
human hereditary mutation, and demonstrates the feasibility of these approaches for discriminating between normal and mutated target
DNA. This could be of great interest for molecular pre-implantation diagnosis to discriminate homozygous CF embryos from
heterozygous and healthy embryos. Other advantages of the methodology are (a) that it is a nonradioactive methodology and (b)
here gel electrophoresis and/or dot-spot analysis are not required. More importantly, the demonstration that SPR-based BIA could be
associated with microarray technology allows us to hypothesize that the method could be used for the development of a protocol
employing multispotting on SPR biosensors of many CF-PCR products and a real-time simultaneous analysis of hybridization to PNA
probes. SPR could be an integral part of a fully automated diagnostic system based on the use of laboratory workstations,
biosensors, and arrayed biosensors for DNA isolation, preparation of PCR reactions, and identification of point mutation.
29

29. The partitioning of apolipoprotein A-I (apoA-I) molecules in plasma between HDL-bound and -unbound states is an
integral part of HDL metabolism. Surface plasmon resonance (SPR) technique to monitor in real time the reversible
binding of apoA-I to HDL. Biotinylated human HDL 2 and HDL 3 were immobilized on a streptavidin-coated SPR
sensor chip, and apoA-I solutions at different concentrations were flowed across the surface. The wild-type (WT) human
and mouse apoA-I/HDL interaction involves a two-step process; apoA-I initially binds to HDL with fast association and
dissociation rates, followed by a step exhibiting slower kinetics. The isolated N-terminal helix bundle domains of human
and mouse apoA-I also exhibit a two-step binding process, consistent with the second slower step involving opening of the
helix bundle domain. The results of fluorescence experiments with pyrene-labeled apoA-I are consistent with the N-
terminal helix bundle domain interacting with proteins resident on the HDL particle surface. Dissociation constants ( Kd )
measured for WT human apoA-I interactions with HDL 2 and HDL 3 are about 10 µM, indicating that the binding
is low affinity. This Kd value does not apply to all of the apoA-I molecules on the HDL particle but only to a relatively
small, labile pool.
30

30. Dissociation of biotin from streptavidin is very difficult due to their high binding affinity. The re-use of streptavidin-
modified surfaces is therefore almost impossible, making devices containing them (e.g. surface plasmon resonance (SPR)
sensor chips) expensive. A new protocol is described for reversible and site-directed immobilization of proteins
with streptavidin affinity tags on the streptavidin-coated SPR biosensor chip (SA chip). Two streptavidin affinity
tags, nano-tag and streptavidin-binding peptide (SBP tag), were applied. They both can specifically interact with
streptavidin but have weaker binding force compared to the biotin–streptavidin system, thus allowing association and
dissociation under controlled conditions. The SA chip surface could be regenerated repeatedly without loss of activity by
injection of 50 mM NaOH solution. The fusion construct of a SBP tag and a single-chain antibody to mature bovine
prion protein (scFv-Z186-SBP) interacts with the SA chip, resulting in a single-chain-antibody-modified surface. The
chip showed kinetic response to the prion antigen with equilibrium dissociation constant KD≈4.01×10−7. All results
indicated that the capture activity of the SA chip has no irreversible loss after repeated immobilization and
regeneration cycles. The method should be of great benefit to various biosensors, biochips and immunoassay
applications based on the streptavidin capture surface.
31

31. 32

32. 33
1. CMD 200 d sensor surface 2. EDC/NHS surface activation
3. Laccase immobilization
5. Interaction between laccase and BC 4. Surface blocking

33. A modular platform is developed for the fast, reagent-free and site-specific immobilization of azide-containing ligands
by strain-promoted cycloaddition onto a cyclooctyne-modified SPR sensor surface. The usefulness of the concept was
shown with a papain model system without loss of surface quality. Furthermore, azide-containing green fluorescent protein
(GFP) was also effectively immobilized. The application of cycloaddition chemistry for the modification of SPR chips
based on initial functionalization of the chip surface with bicyclo[6.1.0]nonyne (BCN), followed by ligand immobilization
via strain-promoted azide–alkyne cycloaddition (SPAAC). The BCN functionalized SPR chips are modular in nature and can
be smoothly functionalized in reagent-free fashion with azide containing compounds. In particular, immobilization of small
molecule inhibitors of papain allow rapid and quantitative protein detection. The power of the approach is further
demonstrated by attachment of a whole protein, i.e. azide functionalized green fluorescent protein (Anl-GFP). Cyclooctyne-
modified SPR chips enable smooth and site-selective immobilization of ligands and prove to be more robust than
traditionally functionalized systems.


34

34. RNA aptamers that bind the opium alkaloid codeine were generated using an iterative in vitro selection process. The
binding properties of these aptamers, including equilibrium and kinetic rate constants, were determined through a rapid, high-
throughput approach using surface plasmon resonance (SPR) analysis to measure real-time binding. The approach
involves direct coupling of the target small molecule onto a sensor chip without utilization of a carrier protein. Two highest
binding aptamer sequences, FC45 and FC5 with Kd values of 2.50 and 4.00 mM, respectively, were extensively studied.
Corresponding mini-aptamers for FC5 and FC45 were subsequently identified through the described direct coupling Biacore
assays. These assays were also employed to confirm the proposed secondary structures of the mini-aptamers. Both aptamers
exhibit high specificity to codeine over morphine, which differs from codeine by a methyl group. Finally, the direct
coupling method was demonstrated to eliminate potential non-specific interactions that may be associated with indirect
coupling methods in which protein linkers are commonly employed. Therefore, in addition to presenting the first RNA
aptamers to a subclass of benzylisoquinoline alkaloid molecules, this work highlights a method for characterizing small
molecule aptamers that is more robust, precise, rapid and high through put than other commonly employed
techniques.
35

35. Protein-protein interactions are pivotal to most, if not all, physiological processes, and understanding the nature of such
interactions is a central step in biological research. Protein-protein interactions (PPI); the formation and dissociation of
protein complexes, are key events in many biological processes (e.g., replication, transcription, translation,
signaling, cell-cell communication. SPR is particularly suitable for studying membrane proteins since it consumes small
amounts of purified material, and is compatible with lipids and detergents. This protocol describes an SPR experiment
characterizing the kinetic properties of the interaction between a membrane protein (an ABC transporter) and a
soluble protein (the transporter's cognate substrate binding protein).
Illustration of the steps of an SPR experiment using a Ni-NTA sensor
chip. (A) Injection of the nickel over the NTA matrix on flow cells Fc=1
and Fc=2 (shown are unsubtracted curves). The increase in mass is low
(~30-50 RUs). (B-D) Illustration of the Fc=2-1 sensogram recorded
during an SPR experiment. In cycle 2 as the his-tagged ligand is injected
over the Ni-charged surface, mass gradually accumulates. Injection is
terminated when reaching a final RU value ranging between 1,000-5,000
RU (depending on the ligand's MW). A baseline is formed, reflecting the
stable binding of the ligand to the chip. In cycle 3, a blank injection is
preformed, injecting the components of analyte sample excluding only
the analyte. In this injection often a low response is recorded (1-5 RUs).
In cycle 4 the analyte is injected using the same regime as the blank
injection. The increasing RUs reflect the mass change on the surface, i.e.,
the binding of the analyte to the ligand. The signal rapidly increases and
then plateaus as the system reaches equilibrium. When the injection
ends, the analyte dissociates from the ligand, resulting in a decrease in
the signal and return to baseline levels. 36

36. 37

37. Molecularly imprinted polymer (MIP) technique is a powerful mean to produce tailor made synthetic recognition
sites. Here precipitation polymerization was exploited to produce a library of MIP nanoparticles (NPs) targeting the N
terminus of the hormone Hepcidin-25, whose serum levels correlate with iron dis-metabolisms and doping. Biotinylated
MIP NPs were immobilized to NeutrAvidin™ SPR sensor chip. The response of the MIP NP sensor to Hepcidin-25 was
studied. Morphological analysis showed MIP NPs of 20–50 nm; MIP NP exhibited high affinity and selectivity for the
target analyte: low nanomolar Kds for the interaction NP/Hepcidin-25, but none for the NP/non regulative Hepcidin-20.
The MIP NP were integrated as recognition element in SPR allowing the detection of Hepcidin-25 in 3 min. Linearity was
observed with the logarithm of Hepcidin-25 concentration in the range 7.2–720 pM. LOD was 5 pM. The response for
Hepcidin-20 was limited. Hepcidin-25 determination in real serum samples spiked with known analyte concentrations was
also attempted. The integration of MIP NP to SPR allowed the determination of Hepcidin-25 at picomolar
concentrations in short times out performing the actual state of art.
38

38. 39

39. 40

40. •Elucidate the function of novel proteins in proteomics research
•Identify binding partners to any target molecule – ligand fishing
•Determine the nature of protein complexes and their function
•Unravel the intricate molecular interactions involved in cell signaling pathways
•Study membrane associated molecules in near native environments
•Determine expression levels of selected proteins under different conditions, for
example, health, disease, drug treatment
•Find antagonists to critical binding events
•Investigate the binding properties of protein isoforms
•Identify disease markers in clinical samples
•Optimize therapeutic antibodies
•Evaluate ion selectivity in signaling pathways
•Identify DNA damage – mutations, DNA adducts etc
Life Science Research Applications
41

41. •Proteomics – target identification and ligand fishing
•Target and assay validation for High Throughput Screening (HTS)
•‘Hit’ to lead characterization – rapid affinity ranking and detailed kinetics of
interaction for small molecules binding to target proteins
•Lead optimization through interaction kinetic based QSAR (Quantitative
Structure Activity Relationship)
• Early ADME (Absorption, Distribution, Metabolism, Excretion) – rapid in
vitro assays for defined ADME parameters, such as protein binding studies
•Clinical trials – stratification of patient response to therapeutics
•Production and manufacturing – process and batch QC of therapeutic
antibodies and proteins
Drug Discovery and Development Applications
42

42. •Quality control - Rapid and reliable vitamin analysis in health and nutrition
food manufacturing
•Food Safety - Reliable screening for veterinary drug residues such as
antibiotics and β-Agonists in dairy and meat surveillance schemes and industry
and Optimization of screening assays for monitoring hazardous natural toxins
in food and feed
• R&D - Assay construction for the identification of hormone disrupters and to
investigate their mechanism of action
• Process control - Monitoring of specific analytes in food and beverage
production
Food Analysis Applications
43

43.  The combined use of surface plasmon resonance (SPR) and modified oligonucleotides have expanded diagnostic
capabilities of SPR-based biosensors and have allowed detailed studies of molecular recognition processes.
Functional and conformationally restricted DNA analogs (e.g., aptamers and PNAs) when used as
components of SPR biosensors contribute to enhance the biosensor sensitivity and selectivity. At the same
time, the SPR technology brings advantages that allows for better exploration of underlying properties of non-
natural nucleic acid structures such us DNAzymes, LNA and HNA.
 Aptamers and PNAs (DNA analogs) exhibit many advantages as recognition elements in biosensing when
compared with traditional antibodies and DNA probe, respectively. The SPR investigation of DNA analogs and
mimics, such us DNAzyme. Peptide Nucleic Acid (PNA), Locked Nucleic Acid (LNA), Hexitol Nucleic Acid
(HNA) and Phosphoramidates morpholino (MORFs) oligomers has significantly improved our
understanding of biomolecular interactions.
 The combined use of SPR biosensing with DNA analogs is expected to provide significant improvements in those
fields that benefit from the enhanced target detection sensitivity and selectivity combined with the label-free
detection protocol and capabilities for high-throughput analyses. The above mentioned benefits have been
already shown to be useful to limit biochemical sample manipulation needs and PCR amplification of nucleic acid
targets thus reducing analysis time and costs. More specifically the combined use of SPR biosensors and DNA
analogs can provide breakthroughs in important areas such as non-invasive early disease detection and
biomarker discovery.
44

44. Nucleic acid aptamers find widespread use as targeting and sensing agents in nature and biotechnology.
Their ability to bind an extensive range of molecular targets, including small molecules, proteins, and
ions, with high affinity and specificity enables their use in diverse diagnostic, therapeutic, imaging, and
gene-regulatory applications. Methods are descibed for characterizing aptamer kinetic and equilibrium
binding properties using a surface plasmon resonance-based platform. This aptamer characterization
platform is broadly useful for studying aptamer–ligand interactions, comparing aptamer properties,
screening functional aptamers during in vitro selection processes, and prototyping aptamers for
integration into nucleic acid devices. SPR enables facile control over multiple binding conditions and
aptamer and ligand properties, allowing detailed study of the effects on aptamer binding of pH, temperature,
or ion dependence; ligand specificity; ligand structure activity relationships using ligand analogues; and
aptamer sequence-activity relationships using introduced point mutations. In addition, this method is directly
extendable to protein ligands and aptamers containing either modified nucleotides or expanded genetic
alphabets. This robust and rapid aptamer characterization platform provides a versatile tool that can be
readily combined with other methods for advancing the study, design, and engineering of functional
nucleic acids. 45

45. 46

46. Advantages of SPR
47
Label-free Detection
Non-invasive detection
Real-time monitoring of binding events and Direct measurements of
affinity and kinetic constants of biomolecular interactions such as antibody–
antigen, protein-protein, enzyme-substrate or inhibitor, protein-DNA, receptor-
drug, protein-polysaccharide , protein-virus , and living cell-exogenous stimuli
Rapid, sensitive (Small quantities of purified reagents are required) and specific
detection of chemical and biological analytes.
Lab-on-a-chip (LOC) and point-of-care testing (POCT) - optical Biosensing
technologies

47. SPR detection is not yet able to reduce the time-consuming Sequential steps of the
analysis
Can’t easily discriminate between specific and non-specific interactions with the
sensor surface. Elaborate washing does not completely remove the non-specifically
bound material; thus, reference material or control samples are needed to correct for the
non-specific binding
Lack of controlled immobilization of ligands on the sensor surface
Not good for studying small analyte because SPR is mass sensitive, the sensitivity for
high molecular weight molecules is good, but binding of low molecular weight
compounds is more difficult to detect
Limited sensor area leading to a diminished capacity
Hardly amenable for miniaturized, portable platforms required in point-of-care
(POC) testing
SPR instruments are still bulky and costly, and this remains as an obstacle to the
commercialization of SPR technology
Limitations of SPR
48

48. Recent Advances of SPR Technology
SPR imaging (SPRi) as a powerful optical, label-free monitoring tool for highly
sensitive, multiplexed detection (different types of ligands can be immobilized on a
single SPRi-Biochip. It also enables the study of many parameters at the same time
(concentration, immobilization pH, etc.), making it possible to compare, rank and select
molecules easily) and monitoring of biomolecular events than conventional SPR
The microarrays design of the SPRi chips incorporating various metallic nanostructures
make these optofluidic devices more suitable for diagnosis and lab-on-a-chip (LOC)
technologies and near-patient testing (POCT) than the traditional SPR sensors.
 High resolution SPR imaging
 SPR- Phase Imaging (SPR-PI)
 Localized Surface Plasmon Resonance (LSPR) - Nanodevices

49

49. The Nanodiagnostics (decorated gold nanospheres and nanoshells, nanobarcodes
and nanobiosensors) have a promising future to shift the paradigm from organized
laboratories and skilled personnel to point-of-care testing and lab-on-chip technologies,
which are user friendly and can provide instant diagnosis right at the bedside of human
patients and at the doorstep of livestock owners (Tumor detection, tissue imaging,
intracellular imaging, immuno-histochemistry, infectious agent detection, multiplexed
diagnostics and fluoro-immunoassays)
The application of biosensor technology can rightly be utilized for developing assays for
pregnancy diagnosis in bovines specifically targeting some pregnancy associated
biomarkers which are available in body fluids like milk, serum, plasma or urine.
Biosensors are immensely useful in many different applications and future research aims at
improving the sensitivity and throughput of these devices for greater reproducibility and
applicability to larger sets of data acquisition.
Future Prospects
50

50.  LSPR AG
 Bruker Daltonics
 Sofchip manufactures
 Plasmetrix
 Carterra
 Nicoya Lifesciences
 BioNavis Ltd
 Creoptix
 Pall ForteBio

 Biacore
 Biosensing Instrument Inc.,
 Plexera LLC
 Reichert's Technologies
 BiOptix
 HORIBA Scientific
 Bio-Rad Laboratories, Inc.
 PhotonicSys
 IBIS Technologies BV
SPR Suppliers
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51. Facility Available
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Outsource from:
 IVRI, Barely
 NDRI, Karnal
 JNU, Delhi
 ATPC, RCB, Faridabaad
 IIT, Dehli, IIT, Kanpur & IIT Bombay

52. THANKS
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