DNA Biosensors based on electrochemical

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.

Carbon Nanotubes(CNT)

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

Electrochemical Genosensor based on colloidal gold nanoparticles

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

Current status of nanobiotechnology

Current Areas of Global focus on Nanobiotechnology

Current Global Scenario of Activity in Nanbiotechnology and associated medical applications

Organizations

Global Nanobiotechnology activity distribution by organizational setup

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 PERSPECTIVE OF NANOBIOTECHNOLOGY

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

HURDLES IN APPLYING NANOTECHNOLOGY TO MEDICINE

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.

FUTURE DIRECTIONS FOR INDIA TO MAKE AN IMPACT

DNA biosensor scheme

GOX glucose oxidase

-D-glucose + O2 + H2O Gluconolactone + H2O2

Transducer

Analytical signal

Basic principle of a glucose biosensor