Team:SJTU-BioX-Shanghai/Loader

Loader

Introduction

We first seek for a suitable loader for our engineered bacteria, after we had the plan to make a product. In order to make bacteria fixed and to present a clear color for photography and color analysis, we considered three possible loaders. We had three potentials evaluated. Finally, we decided to take glass fiber filter paper as our loader.

Chip

We got the idea of combining chip with our project when communicating with Prof. Xiang Chen from Micro/Nano Research Institute of SJTU. Chip provided by their lab is a flexible carrier with a micro chamber which is made of PDMS material. The micro chamber has a height of approximately 80-100 μm and can be filled with 2-3 μL of solution to the maximum.

Chip is a small-sized and low-cost carrier. It can be combined with a box holding a smart phone. The box can further be applied to photo analysis with the chip（Kanakasabapathy, M. K., et.al，2017）. We planned to have a combination with smart phone since smart phone enjoys greater popularity and most products have a tendency to be mobilized. Meanwhile, chip is light and small, whose characteristics exactly fit our need for making a portable tool.

To demonstrate the feasibility of the chip, we carried out experiment. We added 2μL mutant RFP solution into the chip chamber and observed the color. As is shown in Fig.1, color is indistinguishable wherever observed, under naked eye or in picture. We then tried to have centrifuged hoping the color will be more distinctive. However, the color is still hard to tell (Fig.2).

Fig.1 （a）Chip photographed by mobile phone. Solution is added into the labelled chamber on the right. (b) The color of 1mL solution inside an EP tube

Fig.2 (a) Chip photographed by mobile phone. Solution is added into the labelled chamber on the left. (b) The color of the centrifugal solution inside an EP tube

We supposed that the reason why color was not distinctive was the small size of the chamber. So we put our chip under microscope to take a look. It is shown in Fig.3 that it is difficult to distinguish red color from the picture. Though red color can be observed on the wall of the chamber after centrifugation of 4k rpm, chip is not suitable for photo analysis.

Besides weak color presentation, another negative characteristic of the chip is tough condition for long-term culture of bacteria. The reaction period of our engineered bacteria will last several hours, so the culture medium inside the chip may not be able to support the normal production of bacteria.

Fig.3 （a）RFP solution observed under microscope without centrifugation（b）RFP solution observed under microscope after centrifugation of 2k rpm（c）RFP solution observed under microscope after centrifugation of 4k rpm

Picture（a）,（b）,（c）is exposed for 4ms, at 10x magnification.

Microdroplet

When communicating with our advisor Prof. Chen, we learned that Microdroplet is an extending area of microfluidics field.

We found it specially beneficial in providing nutrition to bacteria. Microdroplet is made of agarose in which we can add some nutrients. If we wrap the bacteria with nutritious agarose, we can culture bacteria in a microdroplet. So microdroplet is better than chip considering the aspect of culturing.

However, we learned from a senior that microdroplet is not suitable for our project. He told us that microdroplet is so small in size that it will suspend on the liquid, resulting in the number of bacteria in each region uneven. Thus, the color taken from photographing will have a difference if the shooting position changes, leading to a greater deviation of the results of color analysis. So we gave up the idea of applying microdroplet.

Glass Fiber Filter Paper

Ostrov et. al. (2017), presented another method of fixing bacteria in their article. They used glass fiber filter papers as the carrier of the bacteria. Because the pore size of glass fiber filters is 0.7μm, which is close to the diameter of E. coli. Thus, E. coli can be attached to the paper without leakage.

We applied the method of vacuum filtration, with a mold controlling the range of the bacteria to have a circle of 5mm diameter. The bacteria will present a uniform colored round. In order to test whether the paper can effectively show clear color and even the difference between colors, we carried out the following experiment. A serial dilution of amilCP solution was carried out. We had 1x, 2x, 4x, 8x, 16x, 32x dilution in row. After filtration, naked eyes can clearly observe the gradient of color changes.

Fig.4 Colors shown on the paper after filtration

Fig.5 Color comparison between solution and papers

We then tried to analyze our color using our app version No.1 to get the statistics about grayscale. Statistics shown in Table.1.

Concentration ratio	1	1/2	1/4	1/8	1/16	1/32
Statistics from app analysis	76	82	102	123	125	Unable to distinguish

Table1

Fig.6 Graph of the relationship between concentration ratio and statistics from app analysis

Comprehensive evaluation

Table.2 A comprehensive score table of three possible loaders（Out of 5 points）

	Chip	Microdroplet	Paper
Price	4	4	5
Technical Realization	3	2	5
Obvious Color	1	-	5
Portable	5	5	5
Bacteria fixation	4	5	3
Even Color	3	1	4
Culture Environment	1	5	3
Average	3.0	3.7	4.3

Table2

Fig.7 Radar Graph for three potential loaders

Reference

Kanakasabapathy, M. K., Sadasivam, M., Singh, A., Preston, C., Thirumalaraju, P., & Venkataraman, M., et al. (2017). An automated smartphone-based diagnostic assay for point-of-care semen analysis. Science Translational Medicine, 9(382).

Ostrov, N., Jimenez, M., Billerbeck, S., Brisbois, J., Matragrano, J., & Ager, A., et al. (2017). A modular yeast biosensor for low-cost point-of-care pathogen detection. Science Advances, 3(6), e1603221.