Zika virus (ZIKV) is a mosquito-borne virus that has caused several outbreaks in the recent years such as the epidemic in Brazil in 2015. These epidemics require rapid diagnosis for patient care and transmission control.  However, the recommended tests by the US Centers for Disease Control and Prevention (CDC), including nucleic acid test (NAT) and an IgM-based immunoassay are performed at a laboratory setting. Conventional nucleic acid preparation methods are also generally time-consuming and require laboratory instrumentation. To address these issues, microfluidic systems may be developed by integrating valves, mixers and filters.

We have developed a POC platform that integrates ball-based valves for the sequential delivery of reagents, a diffusion-based mixing unit, and a paper-based unit at the bottom of the device for collection of the RNA. This paper-based unit uses a cellulose chromatography paper that binds the RNA after releasing the lysis and binding buffers from the buffer unit into the next unit where they mix with the sample. Finally, we release two washing buffers to wash away all the particles collected in the paper besides RNA. After the sample preparation process, we use a commercially available coffee mug that has a microheater capable of maintaining a constant temperature needed for reverse transcription loop-mediated isothermal amplification (RT-LAMP). We finally add SYBR Green dye to the amplified solutions for colorimetric detection.  

Our platform is a valve-enabled lysis, paper-based RNA enrichment, and RNA amplification device (VLEAD). VLEAD  contains three parts, a 3-D printed buffer unit, a 3-D printed mixing unit, and a polycarbonate paper-based detection unit. The buffer unit contains fluid-control ball-based valves for storage and sequential delivery of reagents. The buffer contains four reservoirs with openings at the bottom to place one stainless steel ball in each, which protrudes slightly. Then, the different reagents are loaded in these wells of the buffer unit where the balls are already blocking the holes to contain the liquid. As we move the mixing unit, which contains a pivot that reaches the bottom surface of the buffer unit, it lifts the corresponding balls allowing the reagents to go down where they start mixing with the sample, or the corresponding buffers by diffusion. The detection unit containing the chromatography papers is inserted at the bottom of the mixing unit by protrusion so that the sample and reagents pass through it and the RNA is collected.  

After RNA enrichment and purification, the detection unit is taken apart from the mixing unit and inserted in a coffee mug that is commercially available (Ember Travel Mug) and provides a constant temperature for RT-LAMP, which was chosen due to its simplified thermal management, high specificity and sensitivity, and short incubation time.

Our real time RT-LAMP experiments show that 25-min incubation is long enough for amplification. As a result, the battery life in the Ember Travel Mug lasts more than three times than required for RT-LAMP while the constant temperature provided by the mug for amplification can vary from 55^o^C to 62.5^o^C. The calibration curve indicates that the temperature displayed on the coffee mug is in agreement with what was measured by a thermocouple. Detection of amplicons was carried out through color changes, observed by naked eye or smartphone camera and verified using gel electrophoresis

We have demonstrated the reproducible detection of ZIKV in human and saliva samples using an integrated POC platform using ball-based valves for sample preparation, and a microheater for RNA amplification Using this platform, we have achieved a detection limit of 0.5 PFU per device for human urine and saliva samples, and 0.1 PFU for water samples.

In summary, we have developed a highly sensitive and innovative POC testing platform, combining the VLEAD and Ember Travel mug, for ZIKV detection in human urine and saliva samples. It is foreseeable to apply this platform to other pathogens, including viruses (e.g. Dengue) and bacteria (e.g. *E. coli*).

 


Development of Ball-Enabled Miniaturized Valves for Sample Preparation and Microheaters for Pathogen Detection

Division / Sponsors: 
Heat Transfer Division (HTD), Fluids Engineering Division (FED)

Heat Transfer Summer Conference
Paper Proceedings: https://asmedigitalcollection.asme.org/HT/HT2020/volume/83709

18th International Conference on Nanochannels, Microchannels, and Minichannels
Paper Proceedings: https://asmedigitalcollection.asme.org/ICNMM/ICNMM2020/volume/83693

Fluids Engineering Division Summer Meeting
Paper Proceedings: https://asmedigitalcollection.asme.org/FEDSM/FEDSM2020/volume/83716

HT-ICNMM2020 - Virtual Conference, SHTC - Virtual Conference, FEDSM2020 - Virtual Conference

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