Team:DTU-Denmark/Hardware
Hardware - Spectrophotometer
The creation of a pocket-sized spectrophotometer allows for more applications. A spectrophotometer is now available to more people in developing countries, due to its low cost, size, and convenient design. The spectrophotometer utilizes standard cuvettes and attaches to a smartphone’s flash and camera, making it more accessible.
Our 3D printed spectrophotometer can be attached to the camera and flash of a Sony M4A phone. The spectrophotometer is created with a cuvette holder and a bandpass filter (region: 335-610 nm). This allows only blue light to be sent through the sample that consists of blood plasma.
The spectrophotometer works by utilizing the flash on the phone as a lightsource. Silver surfaces are used to reflect the light back in the direction of the camera lens, where the light passes through a filter that removes all but the blue colour of the spectra. The remaining light passes through the sample before reflecting back to the camera lens where a picture is taken. The picture is then analyzed by the SV Detector app on the phone (see more under software).
The spectrophotometer’s 3D-model can be easily modified to fit most smartphones as well as any optical filter. The fact that it only needs the smartphone and does not rely on an arduino or other external devices, nor needs any external power source, means that it is very portable.
The three substrates that were used in the app should at least give a dilution of ~50% at 30 minutes (the time that the samples are supposed to be incubated for, see Incubation Chamber), making the accuracy at 20% more than appropriate.
Mechanism of Action
Our proposed solution utilizes the Scavidin present in the composite parts by having a chamber (see Incubation Chamber) with biotin beads that BioBricks BBa_k2355313 and BBa_k2355302 could bind to. After possible cleavage, the chromophore or enzyme would be released from the chamber and would be allowed to flow to a second chamber through a filtering system. The sample would then be removed from the second chamber and placed in a standard cuvette that is then placed in the spectrophotometer and analysed via the app.
Testing of the Spectrometer
The only available resin was white. However, this problem can be solved by printing the spectrophotometer in black resin. The effect of white resin was mitigated during testing by covering the spectrophotometer in black putty.
The spectrophotometer was tested both without an optical filter and with one. The raw pictures are shown in figures 3a-3d.
Figures 3a-d show raw pictures that are captured by the phone’s camera. It is possible, with software and post processing, to only focus on the centre of the images. This creates a more uniform environment, especially as the ray of light is relatively directed because the “tunnel” for the flash is so precise.
Figures 3a-b show that there is quite a difference between the raw pictures despite not including an optical filter. We have further tested the spectrophotometer and can detect differences between concentrations of 0%, 25%, 50%, 75%, and 100%. Figures 3c-d demonstrate that the higher the concentration of amilCP the more is absorbed by the sample.
It is evident that there is a difference, even to the naked eye, in the dilution of amilCP from figures 3a-d. This is however subject to change, especially if the app is to be expanded in the future by including more snakes.
When the dilution is close to 15% it is barely visible to the naked eye, especially in poor conditions. This is even more useful if our approach is to be expanded to more snakes. It is possible that some substrates will result in a lower amount of amilCP, thus making a more quantifiable approach beneficial, adding to the necessity of the spectrophotometer.
The App
The SV Detector app is developed to work with the spectrophotometer and is capable of analysing pictures of the samples. The app captures the spectrophotometer’s output that is then analysed in order to give the operator feedback on the type of snake that the victim has been envenomed by, if envenomed.
A more detailed description of the app can be read in the software section.
Incubation Chamber
The incubation chamber is created for the incubation of the serum and/or plasma with the biotin beads. The eluate from the incubation chamber is transferred to the cuvette of the spectrophotometer. The chamber is designed to work with standard medical equipment through luer locks. The incubation chamber has two inner rings for two filters in order to keep the biotin beads in place.
The chamber has two male luer locks so that it can be attached to a syringe on both sides. The workflow would be:
1. Attaching a syringe with the centrifuged blood to the larger end of the chamber.
2. Attaching another syringe on the lower end.
This would create a vacuum and thus one would be able to pull the sample into the incubation chamber by pulling the plunger on the second syringe. We decided to design the chamber with a vacuum based system as it is safer since there is no risk of the chamber coming apart and exposing the operator to the fluids inside. Once the liquid is in the second syringe it can be easily put into a standard cuvette for testing.
Handheld Centrifuge
Our handheld 3D printed centrifuge is based on the paperfuge created by Manu Prakash
Our centrifuge is created to fit 1.5mL Eppendorf tubes and secures the tubes to the print. After centrifugation with our 3D printed centrifuge, the resulting plasma is added to the incubation chamber.
Our hope is that the miniature manpowered centrifuge allows for use in tough and inaccessible environments. The centrifuge can handle Eppendorf tubes, thus making it intuitive to operate. The disc’s design eliminates sharp edges and secures the tubes thus ensuring the operator’s safety.
Testing of the centrifuge
We unfortunately had no opportunity to test the centrifuge. Considering it working, it would enable a technician to centrifuge 100x more blood at once than the amount that the paperfuge already developed was able to. Another benefit is the fact that our design provides more sufficient handling of blood thanks to the Eppendorf tubes.
The one worrying factor that might limit the centrifuge’s capabilities is the danger of hemolysis. The vigorous shaking of the eppendorf tube could cause hemolysis of the blood, thus making the centrifugation and the separation of plasma from the blood unsuccessful [1]. The gentle handling of the blood, during the centrifugation is really crucial, and the fact that the centrifuge would rotate around the horizontal axis might cause some problems.
One solution to the abovementioned problem would be to modify our current design so it would spin around the vertical axis and the tubes would be put in at an angle - thus making the process more robust for the technician. This might reduce the RPMs of the centrifuge however even after 5 mins at 4000 RPM a 1 ml sample of blood in an Eppendorf tube is fairly split.
Attribution
All of the models were 3D printed in white resin on a Form 2 printer from Formlabs with help from Associate Professor Martin Dufva at DTU Nanotech. The 3D-models were designed in SolidWorks 2017.
References
[1] Carraro, Paolo, Giuseppe Servidio, and Mario Plebani. "Hemolyzed specimens: a reason for rejection or a clinical challenge?." Clinical Chemistry 46.2 (2000): 306-307.
