Electrochemical sensor
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Transcript of Electrochemical sensor
The detection of specific DNA sequences provides the basis for detecting a
wide variety of infectious and inherited diseases.
Traditional methods for DNA sequencing, based on the coupling of
electrophoretic separations and radioisotopic detection.
These are labor intensive and time consuming, and are thus not well suited
for routine and rapid medical analysis.
Electrochemical hybridization biosensors (genosensors) for the detection of
DNA sequences may greatly reduce the assay time and simplify its protocol.
Such fast on-site monitoring schemes are required for quick preventive action
and early diagnosis.
Therefore, genosensors have recently been the subject of extensive research
activities.
Introduction
1.Label based
a) Hybridization indicators
– metal complexes
– organic dye molecules
b) Labelled probe
- Metal label (Au or Ag-nanoparticles,)
- oligonucleotide containing -SH, -NH2 groups.
2. Label free
– Electrochemical signals of DNA purine bases guanine, (Inosine), adenine
Electrochemical DNA Hybridization Sensing Strategies
Ruthenium bipyridine
Methylene blue
Cobalt
phenanthroline
HYBRIDIZATION INDICATORSHYBRIDIZATION INDICATORS
Examples for commonly used indicators in DNA biosensors
Ruthenium bipyridine : A strong optical absorbance in the visible region of
the spectrum and a long lived emissive excited state.
Methylene blue (MB), an electroactive indicator, upon the hybridization of
immobilized ssDNA with cDNA absorb light at particular wavelength.
Inosine is an electro-inactive analogue of guanine,
which can also bind to cytosine by forming two hydrogen bonds.
Guanine and inosine are similar molecules. There are one difference each other which is amino group of guanine is at
second carbon atom.
Guanine, and cytosine make a three hydrogen bounds but inosine and cytosine make two hydrogen bounds.
In the experiments,which the inosine is used instead of guanine base,it isn’t observed the peak of guanine which is +1.0 V.
Electrochemically active DNA ligands
Electrochemically active DNA ligands,
which can discriminate between single- and
double-stranded DNA….. it is helpful for DNA
sensing coupled with a probe DNA-
immobilized electrode.
Such a type of DNA sensor is useful in
clinical areas with respect to sensitivity,
quickness and cost.
This system can be extended to multi-
electrodes, i. e., an electrochemical array,
ECA, as a DNA microarray of the next
generation.
DNA-Chip technologyHoechst-33342 and Hoechst-33258
Guanine, Adenine Inosine, Adenine
Oxidation signal of DNA bases measured by differential pulse voltammetry (DPV)
The electrochemical DNA detection procedure based on oxidation signals of
guanine and Au nanoparticles to detect an inherited disease.
When hybridization was occured between probe and target on carbon pasteelectrode (CPE) surface, a guanine oxidation signal at ~+1.00 V wasappeared.
The YES / NO system was established for the electrochemical detection ofallele – specific mutation on Factor V.
Experimental Procedure
Factor V Leiden Mutation using polymerase chain reaction (PCR) amplicons and synthetic oligonucleotides.
Designated as 1691 G > A or……… R506Q, is the major heritable risk factor for venous thromboembolism.
This mutation in the coagulation factor V gene results in the resistance of Factor V to inactivation by activated protein C(APC).
If the coagulation Factor V cannot be inactivated, blood coagulates in venums.
Sequences
Wild-type (WT) capture probe :
5’ – AAT ACC TIT ATT CCT CIC CTI TC – 3’
Wild-type target :
5’ – GAC AGG CGA GGA ATA CAG GTA TT – 3’
Mutant (MT) capture probe :
5’ – AAT ACC TIT ATT CCT TIC CTI TC – 3’
Mutant target :
5’ – GAC AGG CAA GGA ATA CAG GTA TT – 3’
Deep vein thrombosis (DVT) and pulmonary, embolism (PE).
I this case, vessel wall damage, venous stasis, and increased activation of clotting factors.
The Factor V Leiden mutation
An electrochemical DNA biosensor used for the detection of Factor V Leiden
mutation and the discrimination of mutation type using the oxidation signal of
guanine in connection with differential pulse voltammetry (DPV) for the first
time.
DNA-Directed Attachment of Carbon Nanotubes for Enhanced
Label-Free Electrochemical Detection of DNA HybridizationCarbon paste
electrode(CPE)
It is made from a mixture of conducting graphite
powder and a pasting liquid.
These electrodes are simple to make and offer an
easily renewable surface for electron exchange.
Carbon paste electrodes belong to a special group
of heterogeneous carbon electrodes.
These electrodes are widely used mainly for
voltammetric measurements; however, carbon
paste-based sensors are also applicable in
coulometry (both amperometry and potentiometry).
Covalent immobilization of Oligonucleotide onto graphite
Ethylene Dichloride N-Hydroxysuccinimide
Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J.
Nature 1996, 382, 607.
Gold nano-particles have been an attractive material in research for a long time …
The visible color shift and aggregation of oligonucleotide modified Au nanoparticlesupon binding to target DNA is a well-described event.
Elghanian, R.; Storhoff, J. J.; Mucic, R. C.; Letsinger, R. L.; Mirkin, C. A. "Selective
Colorimetric Detection of Polynucleotides Based on the Distance-Dependent Optical
Properties of Gold Nanoparticles," Science, 1997, 277, 1078-1080.
Color shift is only
observed from the
hybridization with the
target DNA.
Hybridization forms a self-assembly of Au nanoparticles in the nanogap
between two nanoelectrodes.
Silver precipitation on Au nanoparticles facilitates the electrical flow from one
electrode to the other.
Park, S.-J.; Taton, T. A.; Mirkin, C. A. "Array-Based Electrical Detection of DNA Using
Nanoparticle Probes," Science, 2002, 295, 1503-1506.
Nanoelectrodes with nanoparticles
Electrochemical Coding of Single-Nucleotide
Polymorphisms By Monobase-modified Gold Nanoparticles
Schematic representation of the principle for the electrochemical identification of SNP by using monobase Au naonoparticle
Which bases are involved in an unknown SNP can be identified by comparing the volumetric signal obtained from the four different monobasic- modified Au Nanoparticles.
SWV: Square-Wave Voltammetry Electrochemical oxidation
signal of gold nanoparticle
Nano Bio Products under development/approval stages in the commercial sector
DDS: drug delivery systemNDA: New Drug Application IND: Investigational New Drug, its part of the NDA.
United States and Europe lead the way in applying nanotechnology for medical
applications.
The funding from federal governments, extensive collaborations and
opportunities for spin-offs are the key reasons for the leadership of these
countries in the field.
Asian countries serve as hubs for contract research for American and European
Conglomerates.
The revenue generated from such outsourcing has slowly begun to boost the
Asian economy toward investing heavily in indigenous research and application.
SUMMARY OF CURRENT TRENDS
FUTURE TRENDS IN NANOBIOTECHNOLOGY
It is an artificial device extension that repairing or replacing body part.
TIMELINE FOR COMMERCIALIZATION OF NANO-BIO APPLICATIONS
Implantable cardioverter defibrillators, cardiac resynchronization therapy
devices, tissue and spinal orthopedic implants and hip replacements will be
among the top sellers.
Current medical implants, such as orthopaedic implants and heart valves,
are made of titanium and stainless steel alloys, primarily because they are
biocompatible..
FUTURE GLOBAL NANOBIOTECHNOLOGY ACTIVITY DISTRIBUTION BY 2015
Asian and European countries shall increase their foothold on Nanobiotechnology
In 2015In 2010
SAMPLERS OF FUTURE APPLICATIONS OF NANOTECHNOLOGY
IN MEDICAL SCIENCE
Delivery of nanoparticle based drugs through the blood brain barrier-Nanovic, Australia
Development of “biocompatible quantum dots” for in vivo imaging-IBN,Singapore
Optical microchips to aid vision in patients affected with eye disorders such as retinitis
pigmentosa-Photobionics, USA
Development of Nanocoatings for implants such as nano hydroxyapatite to withstand
stress and promote better osteointegration-Inframed, USA
Development of bone scaffolds using nanostructured porous silicon-pSivida ,Australia
Nanoencapsulation technology for drug delivery-LBL Technology,Germany
Silicon microneedles for improved transdermal drug delivery- NanoPass, Israel
Development of diagnostics on a nanoscale for diseases such as cervical cancer –
University of Queensland, Australia
INDIAN SCENARIO IN THE NANO-BIO-MEDICO SECTOR
Nanobiotechnology in India is at a nascent stage.
The funding toward nanotechnology applications is largely from DST,DSIR and
DBT.
The total funding by these funding agencies amounted to <20 million until
2003/04 out of an annual R&D expenditure of $3.03 billion.
During the last five years the total investment in nanotechnology has been $50
million.
Few companies in the field of nanotechnology-focus on nanomaterials and
nanobiotechnology,sensors and semiconductors.
Some companies with a focus on nanobiotechnology include- Dabur,
VelNanobiotech , Bharat Biotechnology, Nanocet
Developments limited to research institutes such as IISc and IIT, University of
Delhi, Institute of Nuclear Medicine to name a few.
Strengths, Weaknesses, Opportunities, and Threats (SWOT)
SWOT ANALYSIS OF THE INDIAN NANO-BIO-MEDICO SECTOR
O T
Commercialization Opportunities for India in NanoBioMedicine
Dabur has launched Nanoxel a drug delivery system for the anticancer drug
Paclitaxel.
Typhoid detection kit developed by DRDE Gwalior using a nanosensor
developed at the IISc,Bangalore
The Department of Chemistry, University of Delhi has developed nanopartices
to encapsulate steroidal drugs for delivery to the eye-an invention that has
been transferred for commercialization to Panacea Biotech, Chandigarh
Bhaskar Center for Innovation and Scientific Research, Chennai is developing
an antimicrobial spray using silver nanoparticles and herbal extracts
The Central Scientific Instruments Organization is developing a nanotech
based kit for tuberculosis.