The use of microfluidic chambers would allow for multiplexing and rapid high-throughput screening. Microfluidic technology allows the device to be smaller and inexpensive while retaining an acceptable level of precision. The device we developed makes use of microfluidic cartridges that are manufactured from double-coated spacer tapes, hydrophilic films and acrylic. These materials are used to create layers - of which the bottom acrylic half serves as a reservoir to store reagents and the top half serves as a microfluidic distribution and diffusion channel. The figure below illustrates the design of one of our prototypes specifically designed to carry out an RPA + CRISPR-Cas9 reaction. Most microfluidic chips involve significant macro-accessories such as pumps and external fluid handling systems which oftentimes limit flexibility of the devices. Our innovative chip avoids these hurdles by the use a distribution microfluidic layer whose multiplexing is controlled by air flow from a built-in chamber. The air chamber is actuated by a simple vertically moving module which is integrated in the device and is capable of carrying out the same process for multiple chips simultaneously. Figure 1, and Figure 2 below illustrate chips capable of dispensing different reagents at different times while reaction is and proceeding the chip used with Volatect that dispenses CRISPR reagents to the RPA reservoir after an incubation and amplification phase. In our testing jig from, a modified chip layout seen in Figure 2 was used to deliver 5 μl of fluid of CRISPR reagents into the RPA reagent reservoir.
Figure 1: Microfluidics of testing chip
Figure 2: Final chip design (40mm x 90mm)
In the engineering testing labs, the chips were tested using a combination of TE Buffer and food coloring to simulate the flow of fluids. A successful interaction between two different colored fluids was noticed indicated a successful diffusion area was achieved. Fluorescence was later tested by running an RPA reaction with SYBR Green I. Even though, the white spacer tube that was used to manufacture the chip flourcesed significantly at the excitation wavelength, a careful manufacturing of the chassis inside the device ensured all areas except the reservoirs were covered (black polymethyl methacrylate) sensors were not receiving any readout from the other background sources.
The team strongly encourages the use of excitation and emission filters if this method was to be replicated as a test carried out with Quencher on an RPA-CRISPR reaction yielded more accurate results and indicates a potential for quantification and further intensity analysis.
TMP36 thermoelectric heater was chosen as ideal sensors for our minimum viable product for it has a robust way of determining temperature. It works under the principle of increase in temperature decreases the voltage between the base and the emitter. By amplifying that difference as a transistor, a signal directly proportional to the temperature can be generated.
The team employed the use of a thermal tape which significantly increased the heat conduction between the TEC and the TMP36, and further reduced the time it took the TMP36 sensor to give a good approximate of the temperature of the TEC. Temperature calibration was then implemented by measuring the temperature of the TEC using a high precision IR temperature reader, and calibrating accordingly to achieve the required temperature on the chip.
Inline with iGEM values, the team was highly keen on developing a unique synthetic biological-related hardware. The team explored ways in which synthetic biology products can be brought to the field to be used by trained and untrained professionals alike. Particularly in our target market, airports and immigration health check services we had close contact with indicated that a product needs to inexpensive and not require high level training for it be practicable. As such, the NYUAD iGEM team conceived a fully automated diagnostic device capable of automating the entire process of detecting diseases by multiplexing fluids on our proprietary microfluidic chip, read the data using affordable sensors, and send the data for further processing. By reducing the number of interactions between the end-user and diagnostic device the team insured Volatect would be able to enter the travel sphere and provide a solution for current well-being and epidemiological risks associated with an increasingly interconnected world.
The NYUAD iGEM 2019 team strongly believes in the potential of synthetic biological hardware devices - if you are interested in learning more about our process, please consult our lab notebooks or reach out to us via email (nyuad.igem2019@nyu.edu).
Actuation is achieved by rotating a precise threaded rod attached to an acrylic platform. Upon rotation, the platform moves either up or down, providing the necessary pressure to move the reagents through the microfluidics chip and to bring the light source and sensors close to the wells. Given that the microfluidic chips are manufactured from hydrophilic materials, the position of fluids in retained within a channel unless an external force is applied. This property, coupled with the precision in the dispensing, gives the device the ability of dispensing more than one reagent into a single well by loading them in two different areas. Vector files used to manufacture the 3mm layers which are stacked together and assembled can be accessed here: https://2019.igem.org/File:T--NYU_Abu_Dhabi--chas.ai.zip. The assembled structure is able to receive and test three microfluidic cartridges and manage the actuation and sensing unit.
Detection of fluorescence is achieved by measuring the value of the lux (read by the TEMT6000) of the control measurement and comparing it to the value of the lux after the reaction. The control measurements are taken from each chamber after five minutes and this value is compared to the lux value at the end of the reaction.
Figure 4: Sensor schematic for sensor, 1 chip
Flourecense Sensing
The figure below illustrates a comparison between different values read from the sensing unit of a PCR reaction of GPPA at various concentrations of DNA. A comparison between the team’s sensing unit and a more precise digital microscope reveals that while the sensors are not ideal for intensity analysis, there exists a clear difference between flourishing runs and negative controls.
Figure 1: Sensor output from Volatect’s fluorometry setup.
Figure 2: ImageJ processing of output from DinoLight blue light microscope
For fluorescence to be confirmed as positive, the difference between the control lux measurement and the lux measurement taken at the end of the reaction must be greater than or equal to 50 lux units (candela).
Volatect made use of axiomatic design principles to ensure that any function it has is directly involved in reaching its main objective of fast and reliable diagnosis. Volatect is stripped down to its most bare necessities, there are no extraneous features that extract from the final result, maximizing the straightest path towards properly providing our advertised function. In essence the device is made to clearly highlight two aspects: the status LEDs and the chip slots. As the device is designed to be in a sparsely occupied and clean room we decided that the clarity and ease of accessibility provided by the open slot superseded the reasons for applying any slot cover. The interface is solely managed by our proprietary database and API. Details about the data acquisition and transmission can be found on the software page.
Our team was able to leverage our international background to conduct several meetings with related stakeholders from all over the world. Besides taking all the valuable feedback into consideration, the team ensured the device is able to fulfil the World Health Organization's point of care criteria - ASSURRE - that outlines that a diagnostic product would have to be affordable, sensitive, specific, user-friendly, rapid, robust, equipment-free, and deliverable.
