Dipstick Experiment

Dipstick DNA Extraction Method

We are using a dipstick DNA extraction kit for our project. The dipstick is a rapid (30-seconds), cost-efficient (0.35$ per extraction), equipment-free method to extract and purify DNA samples. It is also great for PCR assays, sequencing, and DNA barcoding. Based on the three-step DNA extraction method with limited tool requirements, the kit can also be easily used on site.

To prove that this method works on related fungal species. We conducted a test using the Dipstick DNA extraction method on the Zancudomyces culisetae (fungal species) at Yan Wang’s lab at the University of Toronto Scarborough.

Materials used:

DNA Dipstick Extraction kit, Zancudomyces culisetae sample (from Yan Wang’s lab), inoculation loops, 200 µl PCR tubes, Platinum™ II Hot-Start PCR Master Mix, GeneRuler 100 bp Plus DNA Ladder, PCR machine, centrifuge, blueGel electrophoresis with built-in transilluminator (from Minipcr bio™)

DNA Extraction, PCR, and Gel Electrophoresis Experiment Protocol:

1. Pipette 100 µL Extraction Buffer into a 1.5 mL Eppendorf tube (test1)

2. Pipette 500 µL Extraction Buffer into a 1.5 mL Eppendorf tube (test2, replicate of the first experiment with the total volume of 500 µL)

3. Sterilize inoculation loops

4. Homogenize the fungus sample using a plastic pestle

5. Inoculate 1mm^3 of fungus sample to test 1 and test 2

6. Add a one mm^3 fungus sample to each tube respectively.

7. Add 400 µL Extraction Buffer to test 1 for a total volume of 500 µL

8. Pipette 1 mL of Wash Buffer into an empty 1.5 mL tube and close the lid. Repeat this another time.

9. Take a dipstick and dip it into the Extraction buffer three times

10. Then dip it in the wash buffer tube by five times

11. Dip the dipstick into the PCR mix up to 15 times to release the DNA

12. Add Platinum™ II Hot-Start PCR Master Mix with the samples. Transfer 6 µl mixing solution into PCR tube.

13. Place tubes containing DNA extraction inside the PCR machine and run them for 2 hours.

14. Gel Electrophoresis with PCR products and ladder

Results

Through the PCR and gel electrophoresis, we saw the bands (indicating PCR products) on the gel indicated below:

Figure 1: gel electrophoresis result: Test 1 (left) and Test 2 (right)

Where Test1 is the 100 µL Extraction Buffer and it is added first, then 400 µL is added after inserting the sample, and Test2 is the total amount of 500 µL Extraction Buffer, which is added before inserting the sample.

The result indicates successful amplification of extracted DNA. No negative control was included this time.

Test 1 and Test 2 have the same band, which indicates that adding an Extraction buffer all at once has no impact on DNA extraction. This makes sense of our hardware design that mixes the full volume of the Extraction buffer with sapwood tree samples.

Contribution Members and Experiment photos

Special Thanks to the members of Wang’s lab for helping us.

(left to right: Professor Yan Wang, Miranda Shou, Tuo Xin, and Huimei Yang)

(Tuo Xin conducts dipstick methods on the bench)

(blueGel by Minipcrbio™ we used for gel electrophoresis)

DNA Extraction

Early Detection

The reason we use wood instead of leaves and soil is to have early detection of oak wilt.

• From our interview and meeting with stakeholders (practitioners and researchers), they indicate that the open cut or wound of oak will most likely infect oak wilt by beetles

• Sapwood was chosen as the preliminary sampling region owing to the nature of the vectors (beetles) spreading the fungi through cracks in the bark. Moreover, by the time the leaf shows signs of the disease, neighbouring trees have been infected.

In this paper, the research shows that most fungal community structures were observed in sapwood compartments. The sapwood layer is also easily identifiable and accessible for sample extraction.

In our design, it is crucial that the device is able to access the sapwood, the outermost portion of a woody stem or branch.

Figure 2: Sapwood compartment highlighted in the tree structure

Reference: https://www.fs.usda.gov/learn/trees/anatomy-of-tree

Before coming up with the prototype, the team evaluated different potential mechanisms, such as drilling, scrapping, and digging. However, the dipstick required a fine granular sample for successful DNA extraction. Drilling is a perfect candidate for getting powder out of hard material.

Extraction Tool Design:

Inspiration

We first analyzed the tools that have similar mechanisms to our design. While this mechanism is perfect for extracting powder from soft materials, the applied force to the area of the application ratio is low.

Figure 3: Pineapple cutter https://www.leevalley.com/en-ca/shop/kitchen/kitchen-tools/corers/75880-ratcheting-pineapple-corer-and-slicer?item=EV527

The idea for the iteration of the design came from the cork remover, where users rotate the handle to drill into the wood sample. The new design is better at applying force when drilling compared to the last design, as there is more area for users to apply force and prevent the drill bit from moving.

Figure 4: Cork Remover (https://www.recipetips.com/glossary-term/t--35273/cork-remover.asp)

From this inspiration, we found that the conventional screwdriver mechanism has a much larger force-to-area ratio, which makes it a perfect candidate for the design.

We tested a 3D extraction design on an oak tree in a local park and it showed functional results.

Figure 5: 3D prototype, testing in the field and sampling from the oak tree

In this testing, we found that the wood dust from drilling is more granular and easier to process for the DNA extraction process. As oak tree wood is softer than others, it can be easily grounded with a drilling tool.

However, we found that it was slightly difficult to apply force by hand. Thus we started our design iterations.

Design Iteration 1:

The team noticed the cap that holds the drill bit can be replaced with a drill handle (similar to the corkscrew). The drill handle allows the user to choose different ways of drilling and drilling with more power.

Figure 6: 3D design iteration 1 on the left and drilling demonstration on the right

Design Iteration 2:

The previous iteration of the drill handle was deemed too small to support the fingers properly. In this iteration of the design, the drill handle size is increased and finger slots are added. This iteration of the design is much more effective.

Liquid Handling and Mixing

Previous Designs:

Idea Generation Step

Preliminary design of liquid mixing

The design consists of three parts: the interior connecting to the rotator, the outer container, and the cap on the top.

The overall idea of the design is to both crush the sample and perform liquid mixing at the same time. The user will rotate the handlebar clockwise, translating the rotational motion into translational movement using the Scotch Yoke Mechanism. This leads to the control of the up and down movement of the cylinder, which assists in providing force and crushing the sample, but at the same time, mixing the liquid, as the liquid level will go up as the interior tube goes down and vice versa. This liquid can then be used for the DNA extraction step.

The first CAD design below is the interior cylinder connecting to the rotator. Between two washers, the connector to the cylinder and the container are filled in the circular gap hole in the second picture, which connects the interior and exterior of the model. Two washers are placed, one outside the container and one inside the container, to prevent liquid leakage during the liquid mixing step.

The second picture below is of the container, where on the left, the circular gap is connected with the spin handle part. The two tubes on the right of the container are the tubes provided to place the buffer solution. Inside the two tubes, there are threads to screw in the buffer solution tube. The top piece is the cap to secure the design and prevent liquid from escaping when the interior tube moves up and down.

When users complete the steps of crushing samples and liquid mixing, they will unscrew the cap, access the buffer liquid/sample, and perform the dipstick method for DNA extraction. After the sanitization step, the design can be reused.

Why we are using our current design?

June 30th: First Design: based on a garden hose valve with an opening valve inside. A 3D printer could not print it. This design also has no foil cutter.

July 25th: Second Design: A foil cutter was added as a needle. Rubber bands and rubber band slots hold together the three-piece design. There was a ramp in the non-foil cutter tube to overcome surface tension. The back of each tube had a block that opened and closed the tube via the user spinning the tube.

Aug 8th: Third Design: The foil cutter was redesigned because the needle was not easily printable. A medicine bottle foil cutter was adopted.

Aug 26th: Fourth Design: The connector tubes were redesigned for a smaller liquid container (however, the dimensions were still off). The foil cutter’s size was changed as well. The rubber band design was removed because the tube held together well enough and the rubber band complicated the design.

Sept 6th: Fifth Design: The design (including the threads) now fits the liquid container.

Sept 8th: Sixth Design: A new cutter design was used because of the smaller liquid containers. However, they were too small to print.

Our current design:

Sept 12th: Seventh Design: The cutter was enlarged. The aligning mechanism was also redesigned to make sure it was printed properly.

To validate the liquid mixing mechanism we used a pipette to control the input and output amounts of water liquid. After collecting 6 sample points, we observed 14.3% error on average with standard deviation of 1.2%.

Initial Amount	Added Amount	Final Amount	Error %
100ul	400ul	440ul	12%
100ul	400ul	420ul	16%
100ul	400ul	430ul	14%
200ul	300ul	433ul	14.4%
200ul	300ul	427ul	14.6%
200ul	300ul	425ul	15%

Heating Mechanism

Based on the requirement to perform the LAMP reaction on site, we attempted to design a portable heating unit that can heat up to 71°C (+ or -1°C) consistently for 30 minutes. We currently have a prototype that is portable and can sustain heat up to 100°C. We also found a suitable thermistor that measures the temperature with a one-degree fluctuation. The main components we used included a PTC heating module, a 10k thermistor, an Arduino nano, a rotary encoder, and an OLED screen for readings. For the next step, we plan to work on a consistent heating function for a low-cost and portable heating unit. We also aim to design a PID control algorithm for precision, a portable battery or alternative battery options for forestry practitioners and researchers to work on site.

Heating Element

We tested Peltier, PTC, and coiled heaters and selected the PTC heater as it consumes the least amount of energy with room-efficient build for portable on-site heating purposes.

Thermistor

We tested a 1K NTC thermistor, a 10K NTC thermistor, an IR temperature sensor, and a 5V 10A temperature control relay. The 10K NTC thermistor has the most accurate temperature reading since it employs contact sensing.

Then, we tested with the temperature module. We needed more control of the interface and indicators for easier use by practitioners.

To address this issue, we designed a LCD screen that can display input reading.

We also have designed the heating plate made of aluminum for the LAMP reaction

(Version 1.0 on the left and version 2.0 on the right)

Circuit Diagram

Decomposition of the Portable Heater

Components	Type	Purpose
Push Switch Rotary Encoder	User Input Device	• The rotary knob can be used to input temperature, time segments and other analog values for checkpoints in the thermal cycle  • The push switch can be used for registration of the value pointed at by  the rotary knob
OLED Display	User Output Device	• The OLED displays the input variables (rotary knob selection), state variables (time elapsed in the cycle), and output variables (heating module temperature).  • OLED uses I2C communication protocol and does not have backlighting saving energy and reducing wires for communication, unlike our previous LCD screen.
10kOhms Thermistor	State Input Device	• Thermistor is used for contact temperature measurement of the heating module. (Thermistor attached to the heating module via a polyimide film) • Thermistor has a time constant which affects the accuracy of the real-time temperature. We tried addressing this issue using an OpAmp setup to directly measure the resistance of the heating module.  • The thermistor is connected in series with a 10kOhms resistor for 1:1 voltage divider configuration.  • The node voltage (node where the resistor and thermistor are connected) is logged by the Arduino to determine the resistance of the thermistor and hence its sensed temperature. • In the future, a Wheatstone bridge can be used for Temperature error correction
Operational Amplifier (U4741CP)	State Device	• The Operational Amplifier configuration provides a virtual GND for the Heating Module.  • This ensures its current draw does not affect the Arduino and other connected circuit components • The OpAmp is used in order to calculate the current drawn by the heating module and through that determine its resistance and provide feedback to the Arduino to adjust the digital signal for controlling  its temperature.  • Voltage at the inverting input was not exactly zero when measured with a Voltmeter due to non-ideal conditions which caused the circuit to not function as expected.
Resistors	State Device	• Several values are used for several reasons from redistricting excess current draw to voltage division for sensing purposed
Transistor (IRF150)	State Device	• In an amplifier configuration with the base connected to the digital output of the Arduino for Signal Amplification  (Input digital from the Arduino for the heater and amplified output signal using the help of the 5V ideal voltage source to the heating module).  • NPN transistor was used as seen from the schematics
Arduino NANO	Microcontroller	• Chosen for the size while bearing the powerful atmega328P microcontroller.  • The number of I/O pins was sufficient for the application.  • In the future, boards with onboard power management such as the Adafruit Feather Series can be used for convenient power management.  • Over-current prevention was ensured using the OpAmp with the output pin connected to GND

Circuit Analysis

Let current in the IR3 be the current drawn by the Heating Module R3.

Assuming Ideal OpAmp Linear Mode Operation:

IR3 = IR1 (KCL at the inverting input node).

(Vn - Vout)/R1 = IR1 = IR3

But, IR3 = (5 - Vn)/R3

⇒ (5 - Vn)/R3 = (Vn - Vout )/R1

But, Vn = 0V (virtual GND)

⇒ R3 (Resistance of the Heating Module) = 5 * R1 / Vout

The circuit is probed at Vout by Arduino analog input pin.

Hence,

Resistance of the Heating Module can be determined by only knowing the instantaneous Vout and chosen R1 value.

Note: R2 is chosen as large as possible for preventing over current draw.

Reflections and future considerations:

• The OpAmp used in the circuit was assumed to be ideal but testing showed unexpected Vn values > 0V hence the heating module did not have enough operating potential across its terminals.

• We experimented with a MLX90614 IR temperature sensor for real time temperature measurement; however, unknown emissivity value of the Aluminum surface of the heating module resulted in inaccurate and inconsistent temperature measurement.

• Future sensing systems should use as hybridized model of the OpAmp powered resistance measurement circuit and another dedicated sensor for reliable temperature reading.

• Current Digital signal input for controlling the heater temperature can be made accurate with PID control. Since, there are three error functions as part of the closed loop control as opposed to the present single error function driven system.