Team:Sheffield/Hardware
Introduction
Much of the current lab equipment commonly used is very expensive. Due to this, some labs that have a smaller budget are limited to the amount of equipment they are able to provide, and as the equipment is very expensive to replace, labs that do have many pieces of equipment rarely update this machinery resulting in outdated hardware and software.
Our team wanted to devise a solution for this problem, and thus we started investigated the different types of methods currently employed for the detection of bacterial growth. A simple experiment, which we believe everyone should be able to perform without the need of expensive lab equipment. This will then allow future iGEM team who perhaps do not have access to these technologies will be able to build and design from our open source manual and allow them to perform essential experiment for the development of their projects, without the need of a large sum of money.
We focused on detection methods that may be adjusted to make automated measurements within a warm room, as this was the most applicable for our desired applications so, for example, we could use this device for an overnight experiment, allows us to run many more experiments with the bacteria day to day.
Detection Methods
Detection methods that we considered are listed in the table below: To read a brief description about each detection method, click on their title in the table:
| Impedance microbiology | Capacitance | Opacity | Light microscopy | pH reading | |
|---|---|---|---|---|---|
| Advantages | Can use simple electronic components. | Can use simple electronic components. | Well known principle, used frequently in current labs. | Allows for the imaging of cells. | Can provide very sensitive information regarding the growth of bacteria. |
| Disadvantages | For a well in a 96 well plate the change would be too small to read. (explained in detail below) | For a well in a 96 well plate the change would be too small to read. (explained in detail below) | More complex circuitry required. | Does not provide quantitative information about the bacterial growth and requires sensitive camera equipment to achieve realistic magnification. | pH sensors can be very expensive. |
Impedance Microbiology:
One of the methods we considered using to measure the bacterial growth was by measuring the change of the impedance, or the electrical resistance, across the bacterial sample. As bacteria grow they release soluble ionic compounds, known as electrolytes, into their growth medium. As the concentration of the electrolytes in the solution increases the impedance of the solution decreases. As a result, this decrease in impedance has a direct correlation to the increase in the number of bacterial and can be quite sensitive [1]. Seeing as electronic components can be relatively cheap to source our team investigated whether the construction of a low cost impedance microbiology kit would be feasible to implement.
One of the first questions we needed to answer in order to understand whether this idea was feasible was: exactly what change of impedance would we expect to measure? This will then give us a good idea about the sensitivity of the components required and therefore the cost that would be roughly expected to build the device. In order to answer this question, we set up a simple model to investigate how much change in impedance we could expect across a standard well in a 48 plate.
*INPUT MODEL INFORMATION HERE*
Once we realised that the change in impedance that we wanted to measure was miniscule the circuitry required to measure this change would have to be highly sensitive and therefore this add extra cost which we are trying to avoid with this project
Capacitance:
After realising that measuring the change in impedance would not be feasible, we could use a similar equipment to measure the capacitance across the bacterial solution instead. Capacitance occurs when two conductor plates are separated by an insulator, and a current is applied to the conductors, creating an electric field between the plates with positive charge collecting on one plate and negative charge collecting on the other plate [2].
*INPUT IMAGE/DIAGRAM HERE*
When electrodes are placed into an electrolyte solution a charge separation occurs across the interface of the electrodes, which is analogous to that of a capacitor. This is where charge separation, and therefore the value of capacitance is directly proportional to the electric potential across the solution [3]. *peter sending me paper on how to describe measuring the capacitance* Again to determine whether this would be feasible, a simple model based on literature was constructed as shown below:
*INPUT MODEL INFORMATION HERE*
Opacity:
One other method of measurement that we looked out was opacity, or rather, the transmittance of light through a sample. As bacteria grow, the turbidity of the medium solution increases. We can measure this change by measuring the transmittance of light through the solution. This principle is used in both plate readers and spectrophotometer.
As we wanted a device to make measurements from our well plate, we will focus on the discussion of a standard well plate reader vs. our device. Plate readers have an automated x,y stage which moves a plate, well-by-well, over a single sensor, usually a silicon photodiode. This is placed directly underneath a light source, which is usually a tungsten halogen lamp or a xenon flash lamp, as both these light sources are able to produce a high-quality range of wavelengths.
*INPUT MODEL INFORMATION HERE*
References:
[1] http://www.sciencedirect.com/science/article/pii/S0956566308000481?via%3Dihub
[2] http://oatao.univ-toulouse.fr/4813/1/Miller_4813.pdf
[3]
Light Source
In order to achieve a reliable and affordable light source we investigated multiple options. These included Light Emitting Diodes (LEDs), an Electroluminescent (EL) Panel and a standard halogen lamp. The advantages and disadvantages are shown in the table below.
| LEDs | EL Panel | Halogen Lamp | |
|---|---|---|---|
| Advantages | Cheap (£2.88 or $3.82 for 96) Small - uniform coverage can be provided in an array | Uniform coverage | Cheap (£0.80-£2 or $1.06-$2.65) |
| Disadvantages | Expensive (~£20 or $~26) Dim | No uniform coverage |
All light sources considered were capable of emitting white light. However, for detecting the concentration of bacteria in a sample, a light of wavelength 600nm is required. At this length, the waves get scattered by the bacteria in the solution, causing a much greater change in light detected by the sensors under the wells. As such, the chosen light source was provided with a plastic “orange” filter that only allowed 600nm waves to filter through.
Alternatively, plate readers allow for the measurement of a multitude of types of biological experiments such as protein and enzyme assays. Our device then requires different filters for alternative wavelengths to allow for use as an affordable plate reader. Fortunately, these plastic filters with specific designated wavelengths can be purchased from … for under £10 ($~13). For laboratories wishing to use the plate reader at multiple wavelengths, they can purchase as many filters as required, although a standard range of 400-650nm should be sufficient and cost-effective. Within the device the filters are easily exchangeable, simply slotting into place.
Tests were completed in order to investigate whether light was being lost from the device. Preventing excess light from entering the wells would allow the results to be more accurate. As shown in the figures below, we completed tests in an open lab, in a Styrofoam box, and in an opaque, black Perspex box.
We also tested the brightness of the individual LEDs in order to calibrate them.
Wells
One of the assets of our device is that the design is based on standard well plates. Instead of reinventing the wheel we decided to choose a more commercially available solution. However there are a wide variety of well plates on the market, the design of which differs in three key aspects:
Number of wells
Well cross section
Well Depth
Number of wells:
Common well plates exist in 24, 48 and 96 well formats. We knew we wanted our device to fit one of these configurations so as to keep the barrier to wider adoption of our product as low as possible. After speaking to various teaching and diagnostics labs we decided on the 96 well configuration. This has a variety of advantages, for example, in our AMR case study ideally we will be able to test up to 10 types of antibiotic with 2 controls at various concentrations (12*8) requiring 96 wells.
Well cross section:
The shape of the cross section of the well was important to consider for a variety of reasons. We decided to go for circular wells as when designing the 3D printed construct realised they would provide the best solution with regards to the structural integrity of the device and light leakage between wells. Having circular wells allowed us to add dividers that separate out wells so as to remove any light leakage between wells leading to inaccuracies in our data.
Well depth:
After speaking to various academics it was highlighted to us that to increase the accuracy of our device the volume of liquid in each well is irrelevant, what is more impactful is the path length of the light rays (the length of light that travels through the well). We decided to derive and simulate a simple monte carlo model showing the difference in sensitivity of our device at different attenuation lengths using Beer Lambert's law. Here is the graph that our simulation produced showing absorbance at different concentrations for various well depths. The well depth increases as we move up the graph so wells with a greater depth have a greater gradient.
*INPUT GRAPH HERE*
In order to increase and show the maximum potential accuracy we utilised deep well plates and contacted a company called eppendorf to get their advice and test various sample before settling on a specific well plate.
Sensors
One of the assets of our device is that the design is based on standard well plates. Instead of reinventing the wheel we decided to choose a more commercially available solution. However there are a wide variety of well plates on the market, the design of which differs in three key aspects:
Number of wells
Well cross section
Well Depth
| Impedance microbiology | Capacitance | Opacity | Light microscopy | pH reading | |
|---|---|---|---|---|---|
| Advantages | Can use simple electronic components. | Can use simple electronic components. | Well known principle, used frequently in current labs. | Allows for the imaging of cells. | Can provide very sensitive information regarding the growth of bacteria. |
| Disadvantages | For a well in a 96 well plate the change would be too small to read. (explained in detail below) | For a well in a 96 well plate the change would be too small to read. (explained in detail below) | More complex circuitry required. | Does not provide quantitative information about the bacterial growth and requires sensitive camera equipment to achieve realistic magnification. | pH sensors can be very expensive. |
Capacitance:
Common well plates exist in 24, 48 and 96 well formats. We knew we wanted our device to fit one of these configurations so as to keep the barrier to wider adoption of our product as low as possible. After speaking to various teaching and diagnostics labs we decided on the 96 well configuration. This has a variety of advantages, for example, in our AMR case study ideally we will be able to test up to 10 types of antibiotic with 2 controls at various concentrations (12*8) requiring 96 wells.
Capacitance:
The shape of the cross section of the well was important to consider for a variety of reasons. We decided to go for circular wells as when designing the 3D printed construct realised they would provide the best solution with regards to the structural integrity of the device and light leakage between wells. Having circular wells allowed us to add dividers that separate out wells so as to remove any light leakage between wells leading to inaccuracies in our data.
Capacitance:
After speaking to various academics it was highlighted to us that to increase the accuracy of our device the volume of liquid in each well is irrelevant, what is more impactful is the path length of the light rays (the length of light that travels through the well). We decided to derive and simulate a simple monte carlo model showing the difference in sensitivity of our device at different attenuation lengths using Beer Lambert's law. Here is the graph that our simulation produced showing absorbance at different concentrations for various well depths. The well depth increases as we move up the graph so wells with a greater depth have a greater gradient.
*INPUT GRAPH HERE*
In order to increase and show the maximum potential accuracy we utilised deep well plates and contacted a company called eppendorf to get their advice and test various sample before settling on a specific well plate.
Casing
Construction:
As our device is being designed to aid in providing research, teaching and diagnostic labs with a cheap, DIY, cloud-integrated plate reader, our design criteria include:
Easily obtainable materials
Simplistic build
Simple operation
We decided to use a standard 96 well-plate to design our device around as it’s the most commonly available well-plate in the average microbiology lab.
LED and Photodiode Casings:
Due to the specifications of this well-plate (Fig. 1 – specs of well-plate), we developed a casing for the LEDs and photodiodes in an array that perfectly complements the plate. These were designed using Autodesk Fusion 360 Software (Fig. 2,3 – Images of arrays on software). Exporting of the files from CAD to .step files, followed by conversion to an .stl file resulted in an ability to print the casings in blue and red PLA, using an Ultimaker 3D-printer (Fig. 4 – picture of 3D-printer printing our casings).
The links below provide access to the .stl files for downloading. This should allow any teams access for future attempts to build the device. We’ve also included files for LED and photodiode casings for both 24 and 48 well plates due to their prevalence within biology laboratories.
When complete, these casings (Fig. 5,6) containing the photodiodes and LEDs were able to simply slide in and out of the lower and upper layers, respectively, of the outer box. The upper casing, containing the LEDs (Fig. 7 – 3D-printed LED case) appears slightly different to the lower one for the photodiodes as the photodiodes required the well-plate to retain its position directly above them for the duration of the readings (Fig. 8 – 3D-printed photodiode case).
The box for the device (Fig. 9 – laser cut box) had parts individually laser-cut from an opaque, black Perspex acrylic sheet, sourced from a local company in Sheffield, that aided in addressing previous light-escaping issue with the design. This took 2-3 hours to assemble, simply allowing for the glue to dry.
The figures below (Fig. 10, 11 – casings in box), shows the overall layout of the device casings both as initially developed on Autocad software and a photo of the end result.
