Surface plasmon resonance (SPR)-based
• Surface plasmon resonance (SPR)-based biosensors are very powerful
tools for the study of Label-free biomolecular interaction, chemical
detection and immunoassays.
• A biosensor is an analytical device for the analysis of biomaterial
samples to gain an understanding of their bio-composition, structure
and function by converting a biological response into an electrical
signal It should basically consist of a transducer (electrochemical ,
piezoelectric, or optical) and a recognition element which captures a
specific analyte. (check handout on electrochemical methods).
• Various optical sensing methods have been exploited in biosensors
including chemiluminescence, fluorescence, light absorption and
scattering, reflectance, and Surface plasmon resonance , which can be
classified into two categories: label-based (the two former) and label-
free (the others).
SPR biosensing principle: the analyte of interest is detected
by measuring the change of the refractive index
in the target solution.
• Avidin is a highly stable, tetrameric glycoprotein (molecular weight 66–69
kDa) that contains terminal N-acetyl glucosamine and mannose moieties.
Each of the four subunits contains 128 amino acids and binds to biotin with
high specificity and affinity
• Avidin is originally derived from the eggs of aves, reptiles and amphibians.
• Avidin-biotin interaction is considered one of the most specific and stable
non-covalent interactions, which is about 103 to 106 times higher than an
• the strong interaction between avidin and biotin may pose a limitation in
releasing the tagged biomolecules from the biotin or avidin
• Several genetically and chemically engineered avidin and its analogues
have been studied to enhance knowledge about the functional and
structural characteristics of avidins, which may lead to more successful
• The biggest advantage of this system is its high affinity interaction, which is
robust and stable against manipulation, proteolytic enzymes, temperature,
pH, harsh organic reagents, and other denaturing reagents.
• Therefore, the avidin-biotin interaction serves as a great tool in the
biomedical and nanotechnological applications.
• On the other hand, biotin-based conjugates are easy to synthesize and
have less impact on the activity of the biomolecules.
• Compared to other covalent and non-covalent interactions, the
avidin-biotin system provides enormous advantages such as
amplification of weak signals, efficient operation, highly stability and
enables the use of highly diluted primary antibodies.
• Therefore, avidin has been a very versatile modality in the field of
biotechnology, especially biochemical assays and affinity purification,
over four decades.
Biotin and analogues
• Biotin is a vitamin also known as vitamin H, vitamin B7 or co-enzyme R.
Biotin is composed of a tetrahydrothiophene ring fused to a
tetrahydroimidizalone (ureido) ring. It plays a key role in cell signaling and
acts as a cellular growth promoter. Biotin receptor (sodium-dependent
multivitamin transporter and high-affinity biotin transporter) is widely
expressed in nearly all living cells.
• Moreover, its expression in dividing cancer cells is higher than in normal
cells, making biotin a potential targeting moiety for cancer therapeutics.
• Extensive effort has therefore been made to develop biotin-based
platforms for tumor targeting and diagnosis.
• The functional groups of biotin have been chemically modified to
synthesize biotin analogues, such as iminobiotin, ethylbiotin,
desthiobiotin, biotin-carbamate, and biotin-carbonate for various
Avidin-Biotin: Nature’s superglue
• Exhibit highest known affinity in nature between a ligand and a protein-
• Arises from hydrophobic interactions between biotin and aromatic
amino acids arranged inside the avidin biding pockets
• Avidin-biotin bonds are very difficult to break- pH 2-13, 9M Urea
• Strongest non-covalent bond in nature
• The avidin-biotin is highly specific and strong binding, the avidin-biotin
system is widely used in a variety of biochemical applications
Immobilizing a protein onto a biosensor using
Uses of avidin-biotin system
• Applied to every type of biosensor and nanoparticle surface
• Detection of BIOTIN in an analyte solution using a sensor immobilized with
AVIDIN is a standard test to simulate detection of PROTEIN-DRUG interactions
High MW immobilized ligands
Low MW analyte
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