The purpose of our hardware is to produce a simple, effective sputum sample collection device. Combined with our universal virus detection kit, our device can increase the practicality of at-home testing as it makes sputum sample collection and purification more accessible. The device comes prefilled with a solution at pH 10 composed of lysis buffer, ADEs, HIs, and protector RNase inhibitor. A rubber stopper prevents the solution from leaking out the tip, and a plunger prevents the solution from leaking out the barrel. Users begin sample collection by removing the plunger and transferring sputum directly into the barrel through the funnel. Users then recap the barrel with the plunger and thoroughly mix the sample with the solution. During this step, pathogens are lysed, and nucleic acids are attached to the surface of ADEs through electrostatic interactions assisted by HIs. After 15 minutes, users replace the rubber stopper with a 1.0 micron syringe filter and pass the solution through, trapping the ADE-nucleic acid complexes. Users then detach the filter and reattach it after drawing up an elution buffer at pH 10. The increased alkalinity will separate the nucleic acid from ADEs and HIs, allowing it to be collected in the eluent.
Team:TAS Taipei/Hardware
Hardware
Purpose of Hardware
Struture
The sputum sample collection device has a simple and accessible design. It can be broken down into three components: an ergonomic collection funnel, a luer-lock syringe barrel, and a rubber stopper, which can be replaced with a 1.0 micron syringe filter (Figure 1). The funnel allows users to produce sputum samples in a clean and comfortable manner, the syringe barrel allows for immediate purification of the samples, and the rubber stopper prevents the solution from leaking. During purification, the 1.0 micron syringe filter is replaced with the rubber stopper to isolate nucleic acid from pathogen within the sputum sample.
Figure 1: An isometric view of the sputum sample collection and purification device. The device is based on a luer-loc syringe, with an ergonomic saliva collection funnel at the top and a rubber stopper at the bottom. The device would be prefilled with a solution composed of lysis buffer, ADE, His, and protector RNase inhibitor, set to a pH of 8. The stopper prevents the solution from leaking during package transport and sample collection, and it can be replaced with a filter during purification.
Theory
Positively charged amine-functionalized diatomaceous earth (ADE) is capable of enriching negatively charged pathogens; attaching homobifunctional imidoesters (HIs), which attach positively charged groups, allows an enhanced enrichment of pathogens. In the purification method developed by Zhao et al., dimethyl suberimidate (DMS), which provides additional amidine bonds, is used as the HI reagent.
For sample collection, users directly produce sputum into the syringe barrel, which is prefilled with a solution at pH 8 composed of lysis buffer, ADEs, HIs, and protector RNase inhibitor. The pathogens within the sample are lysed, releasing nucleic acid which are attached to the surface of ADEs through electrostatic interactions assisted by HIs. For sample collection, users replace the rubber stopper with a 1.0 micron syringe filter and push the solution through with the plunger. The filter traps the ADE-nucleic acid complexes and allows the rest of the solution to flow out. Users then fill the syringe barrel with an elution buffer at pH 10 and push it through the filter with the ADE-nucleic acid complexes. The increase in alkalinity causes the HIs to detach from both ADEs and nucleic acid, allowing nucleic acid to be collected in the eluent.
Figure 2: A diagram for the collection and purification of a sputum sample. Users first spit into the barrel through the funnel, allowing a solution at pH 8 composed of ADEs, HIs, and lysis buffer to isolate nucleic acid from pathogens. The solution is then passed through a 1 micron filter, trapping the ADE-nucleic acid complexes. This first solution is not collected. The barrel is then refilled with an elution buffer at pH 10, which is passed through the same filter. The increased alkalinity releases nucleic acid from ADEs and HIs, allowing it to be collected in the eluent.
References
Zhao, F., Lee, E. Y., Noh, G. S., Shin, J., Liu, H., Qiao, Z., & Shin, Y. (2019). A robust, hand-powered, instrument-free sample preparation system for point-of-care pathogen detection. Scientific Reports, 9(1), 16374. https://doi.org/10.1038/s41598-019-52922-y