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 µl/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 <1% change in the

faradaic current (red) compared to the baseline (blue). Probe

regeneration with guanidine hydrochloride reset the sensor to within

98% of its initial state (green).

(B) The 100 aM sample produced a52% signal change, and (C) the

10 aM sample produced a 12% signal change, with respect to the

baseline (red, blue). Each detection was validated with sensor

regeneration, which returned the probe current to >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 <10 aM was demonstrated by the authors.

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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, 6503–6508

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