Infections by the tapeworm T. solium are currently diagnosed by detecting tapeworm eggs in a patient’s stool sample by microscopic examination. In rural areas, there is not only lack of microscopes, but also lack of trained pathologists that can perform such examinations. We have developed a molecular sensor that can detect RNA from T. solium and create a color change upon detection.
By discovering the first promising nano-sensors and conducting first-hand investigation about the disease in India, we were able to: (i) built a device that allows purification of T. solium eggs in field settings, (ii) immobilize our detection system on paper strips, and (iii) lyophilize the detection paper strip to make it easy to store. We integrated user feedback and prototyping into our project design from the very beginning, in order to identify challenges that may slow down the translation of our molecular sensor to a real product.
Developing the purEgg concentrator
On our field trip we learnt that eggs are only present at a low concentration in stool samples and that the microenvironment introduced by the human gut may disturb our test. For this reason, we developed the purEgg concentrator; a microfluidic device that allows us to concentrate eggs from several grams of stool and to separate these eggs from other stool components that may inhibit efficient detection.
The separation of T. solium eggs from other stool components is based on the egg’s distinct diameter of 25 to 35 µm. We used this property to catch the eggs between a set of filters and pool them in a narrow space between a pair of filters for further processing.
Design & Prototyping:
To keep the costs of device production below 1$, we vastly used commercially available products. Since the filter mesh with the desired micron size (≤ 25µm and ≥ 50µm) was available in Polyamid as well as stainless steel, we ordered and evaluated both materials.
Our first thought for designing a prototype was 3D-printing. After constructing a CAD-Model and presenting it to 3D-printing experts for evaluation, we realised the existence of several problems. The first problem was that the thin walls and small overall size of the model was difficult to 3D-print. Secondly, the standard 3D-printing material (PLA) is not very water resistant, which would lead to short-lived prototypes.
For this reason, we opted for the construction of a prototype, which was made of water-resistant acryl, and which was manufactured using simple precision mechanics. We added ISO-standard Luer-lock fittings as connectors for the input and output of biological samples. The standardised Luer-lock allows for any kind of syringe to be used in conjunction with the device.
To evaluate the efficacy of the device, we used fluorescent beads (UVPMS-BR-0.995, Cospheric LLC) in a size-range of 10-150µm. Beads were brought into an aqueous solution according to the manufacturer’s instructions. A 20ml syringe was used to inject the bead-solution through the prototype. For each filter set, we examined the exclusion of particles smaller than 25um and larger than 50um under a fluorescence microscope.
The testing showed that Polyamid filter mesh performed better than stainless steel mesh, as it let through more of the beads of the desired size range. Field tests with real stool samples are required to confirm this efficacy. Our application for proceeding onto a clinical study, included testing devices for purification and concentration of tapeworm eggs. The application has been approved by the Institutional Review Board.
Immobilising our detection system on cellulose membranes
The function of Toehold Switches relies on a specific secondary structure of RNA molecules. Attaching Toehold Switches to solid support matrices, imposes the risk of disturbing RNA secondary structure and compromising the function of the Toehold Switches.
When loading Toehold Switches on cellulose membranes for the first time, we were basing our approach of blocking the membranes with 5% BSA , on a published example of maintaining Toehold function on charged supports like cellulose. In our experiments we could not reach sufficient color changes when blocking membranes with BSA and suspected that in our setup, toehold sensors were still binding to the cellulose.
A deeper patent search, led us to the idea of adding amphiphilic components like tween, in order to block the membranes and thereby reduce interactions between Toehold switch and cellulose. For our setup, this turned out to be the critical step to successfully immobilize toehold switches to membranes.
We discovered that about 1/3 of color intensity was lost on membranes. This issue was reversed by adding the double amount of sensor molecule to the system. Finally, we began to examine whether freeze drying can improve the storage properties of our membranes. We freeze dried the membranes at -20°C overnight. The next morning we rehydrated the membranes and examined whether our sensor reactions still exhibit color reactions upon incubation with a specific sensor. Preliminary data suggest this approach was successful.