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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

chain reaction Single stranded DNA production Electrochemical detection

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


There are several characteristics of small scale fluid flow



Easy to predict the flow patterns


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



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


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 Overall Process

Controlling the IMED


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).


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

from a more complex original

Integrated Signal

on chip mixing

on detection

Starting From a Complex Initial Sample (Blood)


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


Top view

Top view

Signal on Detection

Primer 5 is phosphorolated
Post PCR product


Signal on Detection
Before Hybridization: After Hybridization:

Au Electrode

Small 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

Scott Ferguson Kuangwen Hsieh Jonathan Adams Professor Tom Soh

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

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


On Chip PCR


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