Team:NYU Abu Dhabi/Engineering

E N G I N E E R I N G S U C C E S S

Introduction Design Biology Methodology Engineering Methodology Build Test Learn

Introduction

Our Engineering Success submission revolves around our efforts to improve the efficiency of target DNA extraction in our study while following the guidelines of the Engineering Design Cycle (Design → Build → Test → Learn→ Design...). For this section of our study, our experimentation focuses on determining the efficiency of our prototype microfluidic chip in extracting DNA from Bsal bacterial cultures.

Design

First, a design for an experiment to test the extraction efficiency was laid out. Inoculations of Batrachochytrium salamandrivorans (Bsal) were mixed and the Optical Density(OD) was measured on the spectrophotometer (OD=0.842A) Converted OD to cell number (Number of cells/ml = 6.74E-08). Then, the Bsal mixture (24 ml) was separated into 6 different tubes of 4 ml for 5 tubes and 2ml for 1 tube, each tube with the same number of bacterial cells per milliliter.

Three tubes were sent to the biology team for DNA extraction using a QIAprep Spin Miniprep Kit, a current tool in the market used to extract high-quality DNA at large concentrations. The 3 other tubes were sent to the engineering team where our prototype microfluidic chips were used to extract DNA from the Bsal Samples. Both final concentrations were obtained and compared. The experiments were modeled as shown below:

Biology Methodology

Miniprep of inoculations was performed (3 Bsal tubes of 4 ml each) which resulted in 50 μL from each tube. (Note: optional LyseBlue reagent was not added.)
Following the QIAprep Spin Miniprep Kit protocol, all centrifugation steps were carried out at 13000rpm in a table-top microcentrifuge. The bacterial overnight culture was centrifuged at 7830 rpm for 3 minutes at room temp. The liquid broth was removed so that only the pellet was left in the tube.
The pellet was resuspended in 250 µl Buffer P1 and was transferred into a microcentrifuge tube. 250 µl of Buffer P2 was added and the tube was inverted 6 times to mix the solution. 350µl of Buffer N3 was added and mixed immediately by inverting the tube 6 times.
The resulting solution was then centrifuged for 10 minutes at 13000rpm. 800µl of supernatant was applied to the QIAprep 2.0 spin column, centrifuged for 30 seconds, and the flow-through was discarded.
The QIAprep 2.0 spin column was washed by adding 0.5 ml of Buffer PB, centrifuged for 30 seconds, and had the flow-through discarded. The QIAprep 2.0 spin column was washed again by adding 0.75 ml of Buffer PE, centrifuged for 30 seconds, and the flow-through was discarded.
The QIAprep spin column was then transferred to the collection tube and centrifuged for 1 minute to remove residual wash buffer. It was then placed in a clean 1.5 ml microcentrifuge tube.
The DNA was eluted with 50 µl of Buffer EB to the center of the spin column and was left to stand for 1 minute then centrifuged for 1 minute.
2µl of each sample was used on the Nanodrop 2000 Spectrophotometer to measure the concentration of the extracted DNA.

Engineering Methodology

First, a prototype microfluidic chip was designed and built using a Silhouette Cameo cutting machine.
The chip was then passed through a plasma purification chamber to remove all impurities on the surfaces of the chip. To functionalize the chip, a solution of 60 µl of the APTES stock and 240 µl of distilled DNA-free water was then passed at 100 µl/min through the chip, but not allowed to flow out.
The microfluidic chip was then left to rest for one hour at a temperature of 65 C. After this, the chip was removed and the APTES stock solution was rinsed out using 900 µl of distilled DNA-free water flowing at 300 µl/min. Functionalization of the chip served the purpose of improving the binding capability of DNA to the inner walls of the chip.
The extraction process began after the functionalization of the chip. 3 tubes, each filled with 400 µl of Bsal sample, were centrifuged at 8000 rpm for 5 minutes. The bacterial broth was removed and 400 µl of Tris-EDTA buffer was added to each tube to lyse the bacterial cells and separate DNA from the dead cells.
300 µl of this DNA solution from each tube was allowed to flow through the functionalized microfluidic chip at 100 µl/min. The flow was stopped once the fluid had reached the end of the microfluidic chip. The openings of the chip were then closed off to prevent contamination from air particles and the DNA samples were then left to incubate at room temperature for 20 minutes.
After this incubation period, the waste liquid in the chip was washed out using Phosphate Buffered Saline (PBS) solution, while the DNA clung to the inner surface of the microfluidic chip.
Sodium bicarbonate (NaHCO3) was then used to extract the target DNA into a thoroughly cleaned syringe at a rate of 100 µl/min.
The concentration of the extracted DNA was then measured using the Nanodrop 2000 Spectrophotometer and compared against the concentrations derived from the QIAprep Spin Miniprep Kit.

Build

The build of the experiment can be seen in the video below, walking us through the process:

Test

Using the Nanodrop 2000 Spectrophotometer, the respective DNA concentrations from both tests were recovered.

Table 1: DNA Extraction Results from QIAprep Spin Miniprep Kit protocol

Sample	Concentration (ng/µl.)
Bsal	106.0
Bsal	116.17
Bsal	127.0

Table 2: DNA Extraction Results from Prototype Microfluidic Chip

Sample	Concentration (ng/µl.)
Bsal	6.5
Bsal	7.1
Bsal	7.9

The average concentration from the QIAprep Spin Miniprep Kit protocol was observed to be 116.6 ng/µl. The average concentration from the Prototype Microfluidic Chip was observed to be 7.2 ng/µl.

Learn

Successful approach

From the test results, it is observed that improvements can be made in our project. Such improvements are shown below.

The extraction medium can be changed from Sodium bicarbonate. Another possible alternative is Tris-EDTA Buffer which has the attribute to attract DNA from lysed cells.
Literature review suggests that DMP (dimethyl pimelimidate) can be used as a linker in the DNA solution to strengthen the ability of the target DNA to cling to the inner walls of the microfluidic chip. As such, tests with the addition of DMP (dimethyl pimelimidate) have commenced.

Unsuccessful approach

After our initial results, we took inspiration from the EPFL 2019 igem team and tried to create a microneedle patch to perform extraction of DNA. We created the following patch using their dimensions and 3D printed it using a BMF S240 3D printer. Next, this was used to create a PDMS mold in which Polyvinyl alcohol (PVA) solution was added to attempt to create a microneedle patch. We were not able to proceed with this approach as the microneedles were not able to extract DNA from a meat-like material.