Team:Mingdao/Hardware
Mingdao
Purpose
Imagine an application of real Salmonella diagnosis in a food or drink. A contaminated sample collected in a large volume may find 1-100 CFU/ml of bacteria. Large volume and low bacterial density are key parameters to affect the result of Salmonella detection with our product. To overcome the large volume of a sample, we were inspired by Nickel column purification, in which His-tagged phi29 DNA polymerase were bound. Therefore, we think this method may enrich the His-phi29 DNA polymerase from the sample of large volume.
We searched and found out that no currently existing attachment, from Luer connectors to syringe adapter, fits our needs. To achieve this, we decided to design a Luer Locker-like attachment with an embedded mini nickel column that connects an easily available syringe and a disposable pipette tip.
Schematic Diagram
Materials and Methods
To create a syringe attachment, we utilized 3D printing. We chose this method because of its several advantages. With the uncommon diameter of the mini nickel column and the need for customized structure, 3D printing provides us the ability to produce almost any structure that can be 3D modeled. Its rather cheap price (not considering the 3D printer) is advantageous in production. It is also easily accessible for the team as in recent years the school has installed multiple 3D printers for the students.
However, after several trials with the normal 3D printer that utilizes PLA filaments, we have discovered a significant weakness of 3D printing: a limitation to its preciseness. With the nozzle of the 3D printers being 0.6 mm in diameter, there is an end to its precision. After some discussion with the school’s 3D printing teacher, we had been suggested to use the photo-curing 3D printer. This kind of 3D printer has a longer production time and slightly more expensive material but has greater precision as well as strength. We ultimately adopted this method as our solution.
For the 3D modeling of the attachment, Blender was used. Blender is a free 3D software more commonly known for making models in video games. It is powerful nonetheless and includes the ability to output as STL files, a standard file type for 3D printing. With one of the hardware group members experienced with it, it is our best choice.
Structure Design and Appearance
The structure of the attachment consists of 3 sections: the upper tube that connects to the standard syringe, the middle, chamber-like section that contains the membrane, and the bottom section that connects to the P200 tip. The attachment is normally split into two parts as the figure shown. When in use, the user puts the membrane in the chamber and closes it by pushing them together. We have applied a mechanism called annular snap fit that makes sure the two parts are tightly combined. The following is the appearance of 3D-printed Luer lock adapter connected a standard syringe.
Demonstration Video
How to Use
To use deSALMONEtor and its corresponding hardware correctly, the user must follow the steps presented in the diagram. First, the user needs to collect the sample and put it into the syringe WITHOUT the attachment. The user then needs to draw the engineered reporter phages into the syringe. After an incubation of 25 minutes, attach the attachment containing the Ni Column and pass the mixture through the attachment. RCA can then be performed by adding the reagent with circular DNAs and primers on the column. Lastly, the user must elute the amplified ssDNA products into a 96 well plate containing EvaGreen®and read data through a microplate reader with a setting at Ex/Em=500/530 nm.
*Important: The kit is for single use only. Do not under any circumstances reuse the syringe attachment, the syringe itself, or any other part of the kit.
*Warning: since the product contains genetically modified phage, please make sure all waste and used parts are decontaminated before disposal and should refer to each lab’s SOP of the disposal of genetically modified organisms.
User Feedback
To know the user-experience of our hardware, we found several laboratory personnel with bio engineering experiences to it when we visited college professors or biotech company experts. We then interviewed them for feedback and advice. A common feedback of the interviewees is that to combine the two parts of the luer-connector attachment requires quite some force. This is due to the material that we used, which is quite hard and difficult to bend. However, this is essential because of the precision needed and to prevent leakage. Other than that, most of others found the hardware quite comfortable to use.
We did receive extra advice that suggested us not to assemble the connector to a P200 tip. One of the interviewees noticed that due to the P200 tip, the amount of RCA reagent needed is greatly increased because it must pass through the tip before reaching the connector with Ni-column installed. This is an issue that we want to improve in the future that may possibly further reduce the production price, as well as increase the efficiency of our hardware.
Proof of Concept
To examine the feasibility, we made a serial dilution of Salmonella from 107 to 103 cells in a beaker of 500ml water and prepared the water without bacteria as a control. The water were mixed with Salmonella phage #ST1::Ф29 at the concentration of 105 PFU/500ml at room temperature for 25 min. The 3D-printed Luer locker embedded a mini Ni-column was assembled onto a syringe, followed by repeatedly drawing up and pushing back the water in order to pass through the Ni-column. Then, the RCA materials were drawn onto the Ni-column. If His-phi29 DNA polymerase is present, the RCA reaction may be turned on. After 30 min incubation for RCA reaction, the mixtures were push back into a well of a 96-well black plate containing EvaGreen Dye in a total volume of 50 μl. The fluorescence signals were measured at Ex/Em=500/530 nm. Significant RCA signals began to appear in 2x102 bacterial cells/ml (Fig. 1). 20 cells/ml can be detected with a slight enhanced signal that is able to be distinguished from the background. However, we can’t measure the cell density under 10 cells/ml of a liquid to be examined.
Figure 1 | Salmonella test at various concentrations between 2-2x104 cells/ml in 500ml water with engineered Salmonella phage carrying His-phi29 DNA polymerase gene at the concentration of 200 PFU/ml. RCA was performed on the embedded Ni-column in a 3D-printerd Luer lock adapter. The amplified DNAs were stained with EvaGreen Dye and measured at Ex/Em=500/530 nm in a microplate reader (BioTek Synergy H1).
Compared to traditional Salmonella tests and published engineered reporter phage, we think our product is better than traditional method in term of test time, and our product is comparable and competitive in terms of test limit and time to the current designer phages carrying reporter genes1,2,3 (Table 1).
Conclusion
Hardware is an essential part of our project and greatly improves the user experience. We aim to make our product easily accessible and doesn’t require too much former knowledge. With low price production of hardware, easy-to-utilize parts, and a clear instruction manual, we hope our product will ease the inconvenience of Salmonella detection and contribute to public food safety.
Reference
1. Smartt AE, Ripp S. Bacteriophage reporter technology for sensing and detecting microbial targets. Anal Bioanal Chem. 2011 May;400(4):991-1007. doi: 10.1007/s00216-010-4561-3
2. Vinay M, Franche N, Grégori G, Fantino JR, Pouillot F, Ansaldi M. Phage-Based Fluorescent Biosensor Prototypes to Specifically Detect Enteric Bacteria Such as E. coli and Salmonella enterica Typhimurium. PLoS One. 2015 Jul 17;10(7):e0131466. doi: 10.1371/journal.pone.0131466.
3. Kim S, Kim M, Ryu S. Development of an engineered bioluminescent reporter phage for the sensitive detection of viable Salmonella typhimurium. Anal Chem. 2014 Jun 17;86(12):5858-64. doi: 10.1021/ac500645c.