Alabama A&M UniversitySchool of Engineering and TechnologyDepartment of Mechanical Engineering

ME 470: Senior Design

Automated Washing Process for Microarray Plates

Submitted by:Jose Long

Ezban MorissetteDarneshia Williams

Cameron Alexander (Team Leader)

Date Submitted: January 21, 2014

Instructor:

Dr. Mohamed Seif Table of Contents

Abstract..............................................................................................................................................4

I. Introduction....................................................................................................................................5

II. Project Description........................................................................................................................5

II. a Design Constraints and Design Variable................................................................................7II. b Automation.............................................................................................................................8

III. Results and Discussion................................................................................................................9

IV. Conclusion...................................................................................................................................9

References.........................................................................................................................................9

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Automated Washing Process for Microarray Plates

Abstract

The purpose of the following research is to build an automated system that will be used in

facilitating the washing process of microarray plates. The current process of manually washing

microarray plates, developed for the immobilization of DNA molecules, can become a tedious and

time-consuming task. The critical issue is that with the current automated methods plates can only

process a small number of plates. This is a very costly and time consuming problem considering

there are anywhere from 100-300 plates processed in a week. The project will concentrate on

developing several conceptual designs with the plan to implement a program in order to automate

the process. The process will concentrate on using LabView, a visual programming language. We

chose to use this simply for the fact it is what our corporate sponsor uses and it is now an industry

standard. After a comparison of several design concepts, one design was deemed feasible and

suitable for the needed applications, pending further investigation. This is helpful because a

starting point has been provided in regards to how this device will be constructed and a building

block is set in place for future research.

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I. Introduction

The microarray plate we are using to develop an automated washing process is called a

Nexterion® Plate E MTP 96 (Figure 1). It was developed for the immobilization of DNA

molecules onto a coated microplate sized glass allowing high sample throughout with

reproducibility and higher sensitivity. The plate is manufactured from high quality glass and laser

cutting technology is used to obtain defect and particle free plate surfaces. They are cleaned and

coated under a strict process to control all the fabrication step endures coating uniformity and

reproducibility from batch to batch.

Currently, the process for washing and drying microarray plates to remove unbound molecules are

mostly done manually. Because of the time and expenses involved, this process can become

problematic to a company. The objective of this project is to automate the process in order to

improve the time and cost efficiency.

The current process is as follows:

1. Arrays are printed and DNA is immobilized being incubated at room temperature for 30

minutes

2. Plates put through following washing process:

a. Rinsing plates once for 5 minutes in

Triton® X-100 solution at room

temperature .

b. Rinsing plates twice for 2 minutes in

Hydrochloric acid solution at room

temperature .

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Figure 1

c. Rinsing once, for 10 min in Potassium chloride solution at room temperature.

d. Rinsing once, for 1 minute in de-ionized water at room temperature.

e. Rinsing once, for 15 min in ethanolamine solution at room temperature.

f. Rinsing a minimum of four times for 1 minute in de-ionized water at room

temperature.

g. Drying the plates in an oil-free air or nitrogen stream to avoid any water stains on

the plate surface.

3. Entire process conducted by hand, using beakers and an platform shaker.

The Triton® X-100 contains 100 micro liters Triton® X-100 and 100 milliliters de-ionized

water warmed to 37° C (before adding Triton® X-100). The Hydrochloric acid solution

contains10 micro liters in Hydrochloric acid and 100 milliliters de-ionized water at room

temperature. The Potassium chloride solution contains 0.58 grams Potassium chloride and 100

milliliters de-ionized water at room temperature. The ethanolamine solution contains 310 micro

liters ethanolamine: 100 milliliters blocking solution (warmed to 50° C).

II. Project Description

The main objective of this project is to build an automated system that will be used to wash DNA

covered microarray plates. This automated system will include several sub-systems, including, but

not limited to:

1. Racking System

2. The Agitator System

3. The Automation Programming

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II. a Design Constraints and Design Variable

The process in which we use must first accommodate the Nexterion® Plate (110 x 74 x 1.0

mm (± 0.1 thickness and weight: 18 g (± 0.5 g) plate type Schott) and 25 x 75 mm slides as well.

Next we have to incorporate a minimum of four wash buffers at appropriate temperatures. It must

alert user when going into block wash then pause the process and require user intervention to

continue. At this step in the process addition of ethanolamine will be required. The materials

cannot interact with reagents. Lastly, it must safely & effectively dry plates. The process will be

ergonomically friendly with weight of handling no more than 20lbs, and about waist high (for a

5’2 female).

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The Constraint The Associated Design Variables

Accommodate the plate’s dimensions (110 x 74 x 1.0 mm), and thickness (± 0.1 thickness).

Design a rack that will give the plates a safe fit.

The plates’ weight can range from 18 g (± 0.5 g) to 21 g (± 0.5 g), depending on supplier used.

Design a rack with the appropriate material that can withstand the weight of the plates.

Incorporate a minimum of four wash buffers at appropriate temperatures.

Use previously designed food grade soda canisters for wash buffers.

Alert user when going into block wash. Create program using LabView to alert user after wash sequences.

Pause the process and require user intervention to continue.

Create program using LabView to run vs. stop/pause, and abort sequence to safely terminate the procedure by draining all liquids and drying plates/slides.

Materials cannot interact with reagents. Select appropriate material that will not react to buffers.

Weight of handling no more than 20lbs. Select appropriate material(s) that will allow device to be over 20lbs.

Bench top device useable by a for a 5’2 female.

Design bench top device at appropriate height.

Over the course of the fall semester, three conceptual designs were presented. The first

was inspired by a conventional washing machine; it included circular racks that could be stacked

in order to maximize washer capacity (Figure 2). A second design was inspired by a dish washer

and included a square rack that would slide horizontally into the device (Figure 3). A third and

final concept was inspired by a shoe box, the bottom half included the shaker table where the

plates would be sterilized (Figure 4a) and the top portion is used as the drying mechanism (Figure

4b).

Upon further deliberation, it was decided that the design that was inspired by a

conventional washing machine would be best for our applications. A further description of this

design includes an agitator to gently wash the microarray plates and possible drying capabilities.

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With this design we have the option on sling drying with the agitator or using a gas such as

nitrogen to dry the plates.

The purpose of using the agitator is to gently wash the plates using a specially designed set

of racks that will lock into the agitator. The agitator will encompass a slow spin cycle once the

plates are submerged into each buffer. After the device purges each buffer into the system, the

agitator will slowly spin, washing the plates in a manner that will clean each plate while going as

a speed that will not damage them.

Figure 2 Figure 3

Figure 4a Figure 4b8

Further analysis is scheduled for the research of various concepts surrounding our chosen design.

Planned analysis includes a finite element analysis to test maximum durability and performance

and to ensure the agitator concept does not damage any of the microarray plates. With the washing

machine concept we have the option on sling drying with the agitator or using a gas such as

nitrogen to dry the plates. An investigation into what substances will be used in order to dry the

microarray plates will be conducted. Another factor to consider will be what material would be

most efficient for the device itself, something sturdy but not too heavy and still within design

constraints.

III. Automation

For our design we will implement LabView since it is the program our corporate sponsors

Microarrays use. The program created will be programmable to process using alternate times

and/or iterations, log temperature & humidity readings during the processing event(s), LED

indicator – running vs. stopped/paused, and abort sequence to safely terminate the procedure by

draining all liquids and drying plates/slides.

1. Program#1: Purge/Prime sequences if necessary

2. Program#2: Full processing event

3. Program#3: Partial processing event

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IV. Results and Discussion

Thus far, our project has been following the timeline we set in order last semester. We researched

current process and brainstorm design considerations, conducted trade studies on project

alternatives, did conceptual development of project, and drafted CAD designs of the project with

two alternatives. As of late, we came upon an issue. When trying to test the drafted CAD model

with ANSYS we discovered our school’s license was expired. Hopefully this will be resolved in a

very timely manner, but it is out of our control.

V. Conclusion

After a comparison of several design concepts, one design was deemed feasible and suitable for

the needed applications. What material would be most efficient for the device itself, and the rack

inside the device is still to be determined. We will work on the design details and analysis of the

rack system. More over, the agitator system will be designed and analyzed. In the meantime, we

will be conducting trade studies on the automation process that will be best implemented in our

system.

Acknowledgements

Jerry C. Collins, Ph.D.Fellow, BMES; Chair, Ethics Committee.Fellow, AIMBE; CURM Committee.Board of Directors, AEMB.Adjunct Faculty, Alabama A & M University.Adjunct Faculty, Lipscomb University.

Maria del Carmen BrownMicro Arrays, Operations Manager.

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Joel Peek, Ph.D.Micro Arrays, CEO

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