By Andrew Hurst

1
2005 Cornell University, NBTC Summer Research
Experience for Undergrads

• By Andrew Hurst
• Towson University
• Professor Dr Carl A. Batt
• Co Arthur Michael Abraham
• Cornell University

2
Introduction

• The focus of this project was to measure how much
  colorimetric precipitation was being produced by
  running homogenous solutions of Horseradish
  Peroxidase (HRP), Peroxide, and
  3,3'-diaminobenzidine (DAB) with Cobalt through
  the channels of a microfluidic device
• The Michealis-Menten equation was used to find
  the reaction rate of the enzyme and determine how
  much substrate was considered necessary to
  produce a product

3

• The microfluidic platform provide ways of
  reducing the reagents, time and cost needed to
  perform an analysis
• The size of the laboratory is reduce to the size
  of a device

4
The Reactions Chemistry

• How does the reaction of HRP, H2O2 DAB/Co
  produce a dark precipitate?
• HPR enzyme binds to two peroxide molecules and
  catalyzes the breakdown into oxygen and water
• The oxygen molecules oxidize the DAB/Co complex
  and produces a black/blue precipitate

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Closer Look into the Chemistry

• When oxidized the
• DAB/Co polymerizes and
• forms long chains

• The Cobalt goes to its highest transition state
  from Co2 Co3

6
Michaelis-Menten Equation

• Michaelis-Menten equation gives an expression for
  the rate of an enzyme reaction and how an enzyme
  reacts on its substrate
• The Michaelis-Menten kinetics applies to many
  enzymes under steady-state conditions

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Michaelis-Menten Equation

• v the velocity of the reaction
• Vmax maximum velocity of the reaction
• S the concentration of substrate
• Km Michalis-Mentens constant

The goal was to find v and when S equal
E, which is the Km or Vmax/2
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Procedure

• Find the wavelength at which the reaction mixture
  of HRP, H2O2, DAB/Co has the highest absorbance
  using a UV-Vis spectrum
• At that wavelength measure the absorbance vs.
  time by adding the H2O2 right before running the
  UV-Vis Spectrum (do this at several
  concentrations)
• Find the slope at this parameter, which is the
  reaction rate at that concentration, and graph
  the results in the form of Michealis-Menten graph

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Graphical Results

• The graph of DAB substrate diluted 4X
• The plot represents absorbance vs. time

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Graphical Results

• The graph of Opti 4-CN diluted 1X
• The plot represents absorbance vs. time

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Graphical Results

• The graph of Opti 4-CN diluted 2X
• The plot represents absorbance vs. time

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Final Kinetic Graphical Results
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Graphical Discussions

• Why didnt the Michealis-Menten graphs turn out
  as expected?

14
Enzyme Fluidics

• The chemicals were put through the microfluidic
  channels to see if the reaction would happen
  under laminar flow
• If the reaction did happen, how much product was
  being produce?

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What Happens in the Microfluidic Device
44.5 of product was produce in a cross-sectional
area
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What Happens in the Microfluidic Device
Control
0 of product was produce in a cross-sectional
area
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Conclusion

• Neither the rate of reaction nor the minimum
  amount of substrate could be found due to the
  unusual consistent results
• In the microfluidic device, the reaction did take
  place as long as both H2O2 HRP were present
• Because of the data produced from the
  Michealis-Menten graphs, it was impracticable to
  go further in the goal of the project

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Acknowledgments

• Dr Carl Batt
• Michael Abraham
• Batts Lab
• Anna Waldron
• National Science Foundation