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DNA SORTERS -A Review

DNA sensors

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  • DNA SORTERS-A Review

  • CONTENTS

    INTRODUCTION

    Variousmethods

    ONCHIP DETECTION OF DNA USING GOLD NANORINGS

    INTEGRATED MICROFLUDIC BASED ELECTOCHEMICAL DNA SENSOR

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  • INTRODUCTION

    DNA sorter refers to detection of DNA in a bio fluid (Blood)

    Separation of specific DNA strand from a fluid

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  • GOLD NANORING AS A SENSITIVE PLASMONIC BIOSENSOR FOR ON-CHIP DNA

    DETECTIONLocalized Surface Plasmon Resonance (LSPR)

    LSPR are unique and sensitive to RI change.

    Previous methods using various nanostructures such as nanodiscs,nanorods,nanospheres and Nano holes.

    In this paper , the authors investigated the LSPR based DNA detector to detect target DNA Sequence.

    They probe immobilize the ssDNA on the sensor and detect the complimentary cDNA.

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  • FABRICATION OF GOLD NANORINGS

    The gold nanorings were fabricated using nanosphere lithography.

    The substrate used was Quartz

    Typical size of the nanorings are 50nm and the space between the nanorings are 200nm

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  • CHARACTERIZATION OF NANORINGS

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  • WORKING PRINCIPLE OF THE BIO SENSOR

    The bio sensor measures the change in Refractive index due to the introduction of external biomolecules.

    The sensitivity of the biochip is explored by injecting various glycerol/water mixture on the biochip and corresponding LSPR spectra were analyzed.

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  • LSPR PROFILE OF VARIOUS MIXTURES OF GLYCEROL/WATER

    Linear regression analysis of LSPR spectra indicates the RI sensitivity of 350 nm/RIU . (Previous results were around 60nm/RIU).

    When compared to a similar chip fabricated by E-Beam Lithography, which has RI sensitivity around 333nm/RIU.

    FoM values nearly 3.1,(FoM=RI Sensitivity/FWHM)

    Higher value than previous values which indicates high capability of the sensor.

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  • ANALYSIS OFDNA SENSIBILITY

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  • METHODOLOGYTo demonstrate the DNA detection, the LSPR spectra were monitored upon

    the ssDNA probe immobilization on the nanosensor surface and the subsequent hybridization with complementary target DNA.

    A 25-nucleotides long thiolated ssDNA probes (MW: 8 kDa) dissolved in an immobilization buffer were injected into for 45 min with a flow rate of 20 l/min. After ssDNA probe immobilization on the Nano sensor via a thiol-gold bond, the sensor surface was rinsed with 1mM 11-mercapto-1-undercanol for 15 min to eliminate the possible non-specific DNA adherence on the nanosensor.

    Complementary DNA hybridization was performed by injecting 100nM complementary DNA (25-nucleotides long) in a hybridization buffer into the sensor-chip for 45 min.

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  • ANALYSIS OF DNA SENSING RESULTSAfter the ssDNA probe immobilization,

    the LSPR peak position red-shifted 8.2 nm . More interestingly, after the hybridization with the complementary target DNA, the LSPR peak position further red-shifted 3.1 nm.

    In contrast, a non complementary target DNA injection for the same time only caused 0.19 nm shift, indicating a good specificity of the nanosensor. In this research, the DNA probe immobilization and the hybridization procedures were both performed for 45 min

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  • CONCLUSION OF PAPER

    Nanoparticles based sensors tends to aggregate and forms loosens from the substrates. (Because of exposure of ionic buffer solution)

    Nanoring structure shows good stability in the exposure of ionic solution.

    Thin layer of Ti was deposited for improve the adhesion

    The results was discussed in the paper with measurement of LSPR spectra of the sensor with different concentration of ionic solution concentration and various flow rate.

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  • INTEGRATED MICROFLUIDIC ELECTROCHEMICAL DNA

    SENSORThe Integrated Microfluidic Electrochemical DNA (IMED) sensor, which

    combines three key biochemical functionalities symmetric PCR, enzymatic single-stranded DNA generation, and sequence-specific electrochemical detections in a disposable, monolithic chip.

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  • DEVICE FABRICATION

    Device size:73 X 13mm Six Fluidics inlet/outlets Detection chamber Gold

    electrodes of area19mm2

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  • WORKING PRINCIPLE

    DNA of Interest

    Sample

    PCR with a primer

    Amplification

    Exonucleotide based digestion

    SSDNA

    Sequence specific

    DNA Detection

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  • OVERVIEW OF PAPER

    As a model the authors attempted to detect Gry B gene of Salmonella enterica serovar Typhimurium

    They demonstrated detection of specific gene of concentration as low as 10aM. (Atto molar)

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  • METHODOLOGY

    (A) Template DNA is added to a PCR The

    template is PCR amplified. (C)

    (D) Lambda exonuclease is mixed.

    (E) MgCl2 is added to the IMED chip to adjust the

    salt concentration

    from 1.5 mM to 50 mM to optimize the

    hybridization conditions.

    (F) a baseline redox current is measured via ACV.

    The ssDNA product hybridizes with the E-DNA

    probe modulating the redox current signal.

    Finally, the E-DNA probe is regenerated to verify

    the hybridization event.

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  • DEMONSTRATION OF ON-CHIP PCR AND SSDNA GENERATION.

    Lane 1, 100 base-pair ladder;

    lane 2, positive control from bench top thermal cycler;

    lane 3, negative control from the IMED chip without template DNA;

    lane 4, IMED output with template DNA, which showed similar efficiency to the bench top thermal cycler; and

    lane 5, IMED output after ssDNA generation. The lower band is ssDNA and upper band indicates incompletely digested double-stranded DNA.

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  • CHANGE IN FARADAIC CURRENT

    The no-template negative control yielded 96% of the

    baseline (green). Signals in panels B and C were

    also compared against externally prepared zero-template negative

    controls, which resulted in drops of 1% and 0%, respectively

    (purple).

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  • EDNA SENSOR RESPONSE

    E-DNA sensor response as a function of

    concentration.

    The E-DNA sensor signal, as represented

    by the percent change in

    peak current between the baseline and

    after incubation with synthetic

    DNA target for 30 min. The standard

    deviation for each point was

    calculated from three measurements from

    three separate chips.

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  • CONCLUSION

    The integrated Microfluidic Electrochemical sensor of system represents a completely integrated electrochemical DNA detection architecture with a limit of detection of

  • REFERENCE PAPER 1

    Gold nanoring as a sensitive plasmonic biosensor for on-chip DNA Detection

    Authors: Chengjun Huang, Jian Ye, Shuo Wang, Tim Stakenborg, and LiesbetLagae

    Journal: Appl. Phys. Lett. 100, 173114 (2012); doi: 10.1063/1.4707382

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  • REFERENCE PAPER 2

    Integrated Microfluidic Electrochemical DNA Sensor

    Brian S. Ferguson, Steven F. Buchsbaum, James S. Swensen,, KuangwenHsieh, Xinhui Lou,and H. Tom Soh*,,

    Department of Mechanical Engineering, University of California, Santa Barbara, California 93106, College of Creative Studies, Physics, University of California, Santa Barbara, California 93106, and Department of Materials, University of California, Santa Barbara, California 93106

    Journal:Anal. Chem. 2009, 81, 65036508

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