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Integrated Microfluidic Electrochemical DNA Sensor (IMED)

Steven Buchsbaum

Point of Care Diagnostics

Point of Care (POC) refers to the ability to make a diagnosis at or near the patients location POC diagnostics has many advantages

Quicker treatment for patients Military use Food testing Environmental monitoring

It is challenging to create a POC diagnostic device that is both portable, sensitive and reliable

The IMED as a POC Tool

The IMED will be able to detect a specific target from a very low initial concentration This will be done by combining three processes on a microfluidic chip
Polymerase

chain reaction Single stranded DNA production Electrochemical detection

The IMED is designed so that it is easy to change the target to almost anything

Microfluidics

There are several characteristics of small scale fluid flow


Laminar

flow

Easy to predict the flow patterns

Very

little diffusion volumes fluid control

This can make mixing difficult Dont need to waste expensive reagents By using pumps it is easy to automate fluid handling

Small

Easy

Polymerase Chain Reaction

Polymerase Chain Reaction (PCR) is the process of exponentially duplicating DNA First described around 1971 by Kjell Kleppe but did not receive much attention In 1976 the discovery of Taq polymerase, which can withstand high temperatures, made it possible to easily automate PCR First put into practice in 1983 by Kary Mullis He received the Nobel Prize in Chemistry in 1993

Polymerase Chain Reaction


1. Denaturation Temp is raised to 95 degrees to separate DNA strands 2. Annealing Primers attach to DNA because temp is lowered to ~55 degrees 3. Elongation Polymerase extends the primers making a copy of the DNA

Voltammetry

1. Working electrode this is where the reaction of interest is occurring, electrons flow into this electrode as reaction occurs 2. Auxiliary electrode (counter) electrons flow out of this electrode to replace leaving electrons 3. Reference electrode used to keep track of what the applied voltage

Single Stranded DNA (ssDNA)


ssDNA is important because it is easy to detect It is produced using an enzyme that will digest a strand of DNA if a phosphorus is attached to the end Forward primer 5 is phosphorolated
P Replase

Detecting single stranded DNA

Blue circle is methalyne blue, a redox indicator which transfers an electron to the gold surface when a potential is applied

The IMED

The Overall Process

Controlling the IMED

Data

There is a very clear signal drop for the sample with an initial concentration of 100aM of genomic salmonella DNA while the negative control shows no signal drop. Limit of detection is around 10 aM or about 300 copies of DNA template (data now shown).

Conclusion
The
It

IMED is a large step towards a POC sensor


has high sensitivity It shows potential for portability It is relatively cheap to make It has high versatility

Continued Work
Starting

from a more complex original

sample
Integrated Signal

on chip mixing

on detection

Starting From a Complex Initial Sample (Blood)


The

idea is to lyse (break apart) all cells in the blood to expose all DNA DNA is then captured using negatively charged magnetic beads These beads are then captured with a magnet DNA is removed from beads

DNA Purification

The DNA will be extracted using Invitrogen Charge Switch beads.


Cellular Lysis: Cells are lysed and beads are added Contamination Removal: Non-specific binding objects are washed away Bind DNA: Beads acquire positive charge in a low ph solution (<6.5) DNA Elution: Beads lose charge in high ph solution (>8.5)
Diagram from Invitrogen web site

On Chip Mixing

Remember that because of laminar flow mixing is challenging


T- Junction Inlet

Top of channel

Bottom of channel

Saw-tooth

Top view

Top view

Signal on Detection
Forward

Primer 5 is phosphorolated
Post PCR product

Replase

Signal on Detection
Before Hybridization: After Hybridization:

Au Electrode

Small signal:

Signal:

Note: the redox label may end up to be methylene blue

One Idea for Chip Design


Replase and MgCl2 inlet Bead capture Valve Sample inlet Valve Mechanism Chamber 2 Waste outlet Edna inlet

NO fluid flow

YES fluid flow

Acknowledgments
Scott Ferguson Kuangwen Hsieh Jonathan Adams Professor Tom Soh

Sources
Chemical Principals, Steven Zumdahl, 2005 Some Redox Indicators, L. Michaels and H. Eagle, 1930 Wikipedia, PCR diagram Invitrogen website, chargeswitch bead technology Wikipedia, Electrochemistry diagram

Questions
1) What is the polymerase chain reaction (PCR)?
a) A mixing method used when dealing with laminar flow b) A process that exponentially duplicates a DNA sequence c) The reaction that takes place during voltammetry d) A chemical reaction used to heat the IMED chip

2) The small scale of the IMED results in laminar fluid flow which makes which of the following challenging?
a) On chip fluid handling b) Predicting fluid flow patterns c) Observing the fluids d) On chip fluid mixing

Fabrication

On Chip PCR

Terms
Plasma

enhanced chemical vapor deposition (pecvd) Self assembled monolayer (SAM)