OD/F device

Measuring Optical Density (OD) is a central element in microbiological work and synthetic biology. One question that has to be answered often is how many cells are in a suspension. Here, the OD can give you a hint. Unfortunately, commercially available OD meters cost several hundred dollars ([http://www.laboratory-equipment.com/laboratory-equipment/cell-density-meter.php OD meter]), and can limit the spread of synthetic biology.

Therefore, we wanted to devenlop an alternative for measuring OD, specifically designed for BioHack-Spaces, DIY and community laboratories and schools. With our OD/F device, we want to enable many people to do good, precise and inexpensive science research.

Especially for the Interlab Study fluorescence, too, has been of importance. One aim of this study was to measure the correlation between OD and fluorescence. Since the taks of measuring OD and fluorescence are often performed at the same time, we want to present a device that can measure both fluorescence and OD with just some easy adjustments. This way, we can measure how much fluorescence there is per amount of cells.

In fact, you can find some DIY posts for turbidity meters such as [http://www.thingiverse.com/thing:74415 Turbidity sensors]. However, a proper assessment of their linearity as well as a calculated OD-value are missing.

Regarding fluorescence, we are of course not re-inventing the wheel (well, not totally). The 2010 iGEM Cambridge team actually built a very similar device, the E.glometer. However, there's no data available showing an actual comparison of the data from their device and some proven commercial system to, for example, to assess linearity of the measurement.

Development

Building the OD/F device has been an interesting task. On the one hand, this device has been developed mainly by the IT division of our team. On the other hand, we got assistance from the biologists suffering from color-blindness, yet eager to help selecting the best color filters for the LEDs. For the next year, you really have to select carefully who's going to help with which task!

The essential part of this device is the cuvette holder which has been created in an incremental development approach. First, a 3 mL volume was considered, and an optimal height of 1.5 cm from ground was determined as optimal. Indeed, this worked quite well for the large cuvettes, but unfortunately delivers random^[1] results on semi-micro cuvettes. We could determine that the cause of this problem originates from refractions on the sample surface in the cuvette which coincides with the measurement height. Being naive, the computer guys first attached the sensor to the cuvette holder at approximately 1.5 cm, which was perfectly suited for 3 mL cuvettes. However, those are not widely used anymore, and we switched to 1.6 mL cuvettes. As it turns out, if the sensor sits at 1.5 cm, it just hits the position where the cuvette enlarges again and - most importantly - where the meniscus of the sample solution is located. This results in diffraction on the surface of the sample, and finally renders any measurement results into - from a computer scientific point of view - a perfect random number generator.

In summary, we had to overcome a dilemma created by the need for an optimal height for the sensor:

• A too low sensor position bears problems with sedimentation as well as diffraction from the bottom of the cuvette
• The sensor has to be as close as possible to the bottom so that enough light shines through for the fluorescence measurement.

As a compromise, we place the sensor in 0.75 cm height, which, as it turned out later, is very close to one of the standard heights (0.2 cm, 0.8 cm, 1.2 cm) of OD meters. It is important to note that despite the minimal fill height of the 1.6 mL cuvettes of 1.2&nbspmL, our device also works with filling volumens of just 1 ml, which comes closer to reality in the lab.

The final cuvette holder design was rendered in a stl-file shown below:

Once the cuvette holder was finished, finding good filters was a tough challenge. The overall goal has been to choose easily available parts which are also inexpensive. So choosing Schott glasses as filters unfortunately could not be considered. Instead, filters used for illumination of theaters seemed to be ideal solution.

Especially for the fluorescence measurements of GFP this has been a big problem. [http://parts.igem.org/Part:BBa_E0040 GFPmut3b] has a peak excitation at 501 nm and a peak emission at 511 nm - too close together for our low-cost filters. Thus, we chose to excite at around 485 nm reduce false positive results below 500 nm. However, no adequate filter for these settings could be found. Eventually, using the dark greenish Twickenham Green filter only little amounts of light shorter than 500 nm got through, reducing any bias excitation illumination significantly. Unfortunately, the transmission rate of this filter is quite bad, at 20 %, for the target emission wavelength. Also for the OD measurement we had similar problems. The solution to this problem is presented in the F device section.

^{1. Quite a good random number generator from a computer-scientific perspective!↩}

Combined Device

Even though evaluation of the measurements have been performed in two separate device, it is fairly well possible to put everything into one casing. All you need to do is choosing another lid, and connect a second light to frequency sensor to your Arduino. Right at the bottom we present you the differences in wiring things up.

Measurement Setup

As for any scientifc device it is crucial to question the results one gets from the device. To ensure that our device actually works, we performed a set of measurements which are presented below.

Linearity

It is crucial that the selected hardware is mapping reality into the digital world of our $\mu$-Controller. In order to sense reality our setup uses a light to frequency sensor, TSL235R-LF. The light to frequency sensor resembles the most to a photo transistor and thus is less sensible to temperature than a light dependant resistor. Additionally counting a frequency using interrupts seems to be easier and more accurate than using the analog to digital converter.

Using a dilution series of purified [ iLOV] we could determine the characteristic curve for the light sensor. Finally we can conclude that the sensor is linear as expected and shown in the datasheet.

Interlab Study Results Our measurements of fluorescence and optical density of the three genetic devices and a negative control.

OD device

From Transmittance to True Optical Density

True Optical Density

Transmittance to TrueOD

Hint: Building it

Our novel biosensor approach Expression of the TEV protease is induced by HSL. The protease cleaves the GFP-REACh fusion protein to elecit a fluorescence response.

If you want to build the OD device, make sure to use the following secret ingredients:

• Filter: [http://www.leefilters.com/lighting/colour-details.html#019 Fire 019]
• LED:

F device

• filter selection
• led selection
• resolution
• correlation to platereader

Hint: Building it

Our novel biosensor approach Expression of the TEV protease is induced by HSL. The protease cleaves the GFP-REACh fusion protein to elecit a fluorescence response.

If you want to build the OD device, make sure to use the following secret ingredients:

• Filter: [http://www.leefilters.com/lighting/colour-details.html#736 Twickenham Green 736]
• LED:

Economical View

TODO:

• check prices
• what does the market offer, what does the market not offer
• what is the closest available device to ours and what does it cost? where is it possibly better? where is ours better?
• how easy is it to get the parts?

Table 2: Needed number of pieces, components and prices for creating your own OD or F device

Building your own OD/F device

While the casing and the cuvette holder are custom made, most of the parts are pre-made and only need to be bought. The previous section Economical View lists all needed parts.

Please find our custom parts for download below^[1]. Despite being custom parts, these are quite inenxpensive - so feel free to give our OD/F device a test :) !

• cuvette holder STL file
• casing single device [ SVG file]
• casing combined device [ SVG file]
• Arduino Code for single device Sketchbook (.ino)
• Arduino Code for combined device Sketchbook (.ino)

You will need a special [http://www.exp-tech.de/images/product_images/description%20images/YWRobot/1602/LiquidCrystal_I2C1602V2.rar library] for the display, which can not be uploaded for legal reasons.

Build you own device

	First we want to assemble the casing. Once you have all the cut parts, you can start to assemble them. For cutting, we really recommend using a laser cutter.
	Attach the cuvette-holder holders such that the cuvette holder is placed directly under the opening hole.
	Next build the lid of the device. At this stage you can already mount the button. We recommend to glue any parts.
	Your lid finally should look like this.
	Next we want to assemble the cuvette holders. On the side with the square hole attach the light-to-frequency sensor with glue. For the OD case place the orange LED opposite, or for fluorescence, the LED in the hole in the bottom. Make sure to close any remaining open hole!
	Your final assembly should then look like this. Now place the correct filter into the cuvette holder, directly in front of the sensor. Make sure that the filter does not degrade due to the glue!
	As the case can be used for both, fluorescence and OD measurement, we use a combined plug. Just three header rows (7 pins) and connect them as we did.
	Now we're doing the wiring. Connect the Arduino 5V and GND such that you have one 5V and one GND line on your breadboard.
	Then connect the button to 5V on the one side, and to GND via a resistor on the other side. Connect this side also to port __ on your Arduino. This will sense the blank. Next connect the display to the Arduino and our connector. See the Fritzing diagram at the bottom for a detailed information.
	Now put everything into the case and ...
	... also place the cuvette holder into the device. Attach the display to the device lid and close the casing.
	Congratulations! You have finished constructing your own OD/F device!

^{1. iGEM really does not make it easy to distribute non-common files!↩}

Building the combined device

Table 1: Needed number of pieces, components and costs for building your own OD/F device