Novel detection techniques for Protein microarrays


Welcome to the MOOC course on interactomics
In last lectures we have discussed various conventional detection methods employed for
protein microarrays In todays lecture we will discuss about various advanced detection methods
We have to talk about few methods in more detail such as SERS based Nanoparticles quantum
dots dye doped Nanoparticles and bio barcodes We will first start with surface enhanced
Raman scattering or SERS based methods Light which is incident on an atom or molecule it
is scattered back the same energy and wavelength This is a general phenomenon however Raman
effect prevails in a small fraction of photons where energy of scattered photo is different
from the incident photons Therefore the improved optical properties are obtained because of
enhanced electromagnetic field at the surface of the particle which is detected by using
spectroscopic methods such as SERS The surface enhancing agents include gold and silver as
well as functionalization with target molecules which enhances the sensitivity of Raman spectroscopy
Let us now look at how this system has been employed for the detection methods for microarrays The Raman dye labeling involves coating of
antibodies on the array surface like gold by using the Raman dye directly on the gold
surface which are in nanoparticles probes The visualization can be carried out by staining
with silver enhancement solution as well as hydroquinom Spot can be detected by Raman
spectrometry coupled with fiber optic microscopy As compared to the fluorescence based detection
methods the Raman dye labeled gold Nano probes offers several advantages which include high
sensitivity flexibility due to non overlapping probes sharp scattering peaks and cost effectiveness
of the assays However the certain demerits of using Raman dye labeled nanoparticles these
drawbacks include the complexity in synthesis of nanoparticles and the lack of uniformity The macromolecular single walled Nano tubes
SWNTs functionalized with a specific Raman dye neighbor antibodies are used for multiplexed
detection target proteins bound on the microarrays slide which is gold coated As you can see
this slide SWNT functionalized with Raman dye labeled antibodies are used for multiplexed
detection of target proteins which are bound on the gold coated microarray surface The SWNTs offer several advantages such as
high sensitivity the multiplex detection capability of proteins minimum background signal due
to sharp scattering peaks and high signal to noise ratio you also offer resistance to
photobleaching therefore SWNTs have several advantages However they also possess some
limitations such as metal impurities during preparation of these nanotubes that can be
interfere with the activity They are insoluble in biological buffers and there is also difficulty
in determining how pure your preparation is The degree of purity is one of the measure
limitations here Let us discuss gold Nanoparticles GNPs The
excitation of coherent electron oscillations that exist on interface of 2 materials is
known as surface plasmon resonance which forms the basis for the use of gold nanoparticles
as detection system The proportion of light absorption to a scattering depends on the
size of the nanoparticle Enlarge nanoparticles can be used for biological imaging due to
the need for high scattering cross section The GNP labeled with a suitable capture molecules
exhibit change in the emission spectrum of scattered light upon binding to the analyte
of interest from a protein mixture due to specific bimolecular interactions As we can
see here in this slide the change in the emission spectrum of scattered light directed upon
binding of gold nanoparticle which is conjugated with antibody to the analyte of interest The gold nanoparticles offer several advantages
it has been used for several applications for sensitive detection of standard proteins
they provide improved optical property superior quantum efficiency so compatibility with wide
range of wavelengths and chemical stability against photobleaching However there are certain
limitations of using GNPs which are similar to other Nano techniques such as biocompatibility
and no cellular toxicity The systematic cytotoxicity study should be performed if you want use
these GNPs for different protein microarray based applications The high cost cytotoxicity
and non uniform size and shape of the nanoparticles are some of the limitations of using GNPs
as sensitive detection platform for microarray experiments Let us now discuss Quantum dots Quantum dots
are nanometer size crystals composed of semiconductor fluorescence core coated with another semiconductor
shell having large spectral band gap which is the stable light scattering or emitting
properties In quantum dots the formation of excitons takes place when light of higher
energy in that of the band gap of composite semiconductor is incident on the quantum dots
When these excitons return to their energy level in fact low energy level then emission
of narrow symmetric energy takes place as we can see in the slide the change in the
optical properties because of the formation of a excitons upon binding of quantum dot
conjugated antibody to the target analyte can be used as a method for detection of microarray
based method Key advantages of quantum dots compare to
the organic dyes includes its brighter fluorescence excellent photostability multicolour fluorescent
excitation and higher quantum yield Despite of several benefits and applications in a
variety of biological sample and demerits includes toxicity Therefore quantum dots have
shown various applications but the still they have certain limitations We will talk some
of these applications during the course of interactomics molecules Changes in the emission wavelength upon binding
to the antibody conjugated quantum dot are recorded by the microchip and used for detection
of various biomolecules Quantum dots are capable of detecting molecules down to femtomolar
levels and provide significant advantages over conventionally used organic fluorophores
In this interaction we will see how quantum dots work The inorganic fluorophores known
as quantum dots have been developed that can conjugate with several biomolecules and they
use for protein microarrays signal detection They are made up of semiconductor devices
which form excitons up on absorption of light There is emission of a narrow energy band
when these excitons are return to their lower energy level Let us click on these quantum
dots to view how they work So as you can see upon binding of the target
protein to the antibody Now these changes are plotted on the wavelength verses florescence
intensity graph So these quantum dots can detect molecules with very high sensitivity
in femtomolar range Now let discuss about Dye doped silica nanoparticles
The silica based nanomaterial have large quantity of fluorescent dye packed inside the silica
matrix which possess ability to selectively tag a wide variety of biological important
targets such as cancer cells bacteria and many other biological samples as shown in
this in this slide here the silica based nanoparticles have large quantity of fluorescent dye packed
inside the matrix and it can be used for selectively labeling of protein molecules for detection
of bimolecular interactions Dye doped silica nanoparticles application
have been used for a variety of investigations Application of various functionalized silica
nanoparticles have been demonstrated in diversified fields .. such as bimolecular discovery drug
delivery multiplex signaling in biomolecules Its various merits include biocompatibility
high sensitivity minimal aggregation and dye leakage photostability and high capacity The
demerits of dye doped silica nanoparticles include it is complex synthesis process We will now talk about bio barcodes The nanoparticle
probes encoded with DNA unique to the protein of interest and suitable antibodies capture
the magnetic micro particle probes known as MMPs having antibodies for the target analytes
thereby sandwiching the target proteins which is shown in this slide These are magnetically
separated oligonucleotides dehybridized and then sequenced to identify the protein of
interest The nanoparticle based bio barcode have increase the detection limits down to
attomolar range The liberated oligonucleotide barcodes can be identified on microarray surface
by scanometric detection as well as within conventional fluorophores The nanoparticle based bio barcodes offer
various advantages The merits include high sensitivity less detection time and it can
be easily adapted to multiple protein targets The demerits of this method is that it can
only be used with known antibodies therefore a number of antibodies as well as good quality
of antibodies is one of the limiting factor for performing the nanoparticle based bio
barcode assays In fact the same is also true for many applications in proteomics which
also required antibodies To summarize all the various type of detection
techniques which we have discussed today The advancement in the microarray technology have
let to the development of sensitive and reliable detection systems There are different label
based detection techniques which have been employed to study high throughput ways of
analyzing thousands of proteins as well as studying their interactions and function by
using protein microarray platform These various novel detection techniques which we have discussed
today have facilitated sensitive specific high throughput as well as rapid analysis
for many proteomics based applications The label based detection systems have been
taken rapid strides to satisfy the demands of proteomic applications with significant
improvement in sensitivity multiplexing capability and reproducibility So we have discussed variety
of label based methods although fluorescence based method is one of the most commonly used
method for various protein microarray based application but there is an increasing demand
and need to try out new labels so that one could achieve ideal system which can be applied
for microarrays and also provide good detection system with high specificity sensitivity and
large dynamic range So we have until now discussed various traditional
and novel detection techniques used in protein microarrays In next lecture we would talk
about recombination cloning and it is application in protein microarrays Thank you