Team:ZJU-China/Human Practices
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OVERVIEW
Lianjun Lin, Director of the IVDs department of MDST.
Is our diagnostic device biosafe? Is it good for our community? Does it meet the demands of public? Can it be truly applied to our truly life? To complete the investigation of the big background where our ideas were born, we visited experts in related area and performed questionnaires in order to evaluate the public acceptance of our device.
A Public investigation concerning about the public acceptance of our device:
• Biosecurity & Ethics Consideration
Biosecurity is the most important and a basic consideration of our product since we chose to develop an in-vitro medical device. MDST (Zhejiang Institute of Medical Device Testing) is a supervisor department of government responsible for regulating medical device. Hence, we thought the suggestions from this department would make a great difference to our final product. Here, we applied a filed visit to their lab and interview the department charge, Lianjun Lin to consult his advice and opinions about the biosecurity and our medical device. We summarize his points of view which do make great impact on our project designing and list them below:
• Questionaries
Q: How to promise the security of a medical diagnostic device?
A: It’s hard to define the word “security” while it related to many other concepts. The first thing you should concern about you device security is the national standard of in-vitro medical device. It represents the minimum level of safety that a product can be allowed to produce. Particularly for your device, I recommended you to focus on the basic security concerning about the Cell-Free Biosensor.
Q: How long can a medical device be reached out and scalable produced? How can we be more quickly to transform technology?
A: It depends. Usually, the process of acquiring the permission of scalable producing takes 2-3 years, including the product research period, which is according to my working experiences. But this situation is for biotech company, not for a scientific research and innovation team like you. To transform your ideas, I recommend you to think about cooperating with biotech company involving with in-vitro diagnostic device. Additionally, you should take it seriously to protect your patent right while you are sharing your ideas to others.
Q: What are your suggestions in ethics while we are designing our device?
A: Ethics consideration is as important as the security of a medical device. Nowadays, the mode of Internet plus Hospital is increasing with the concerning on the privacy disclosure. So, my suggestion here is data security while you have a further development in your device which combines with big data and internet.
Q: What do you think of our product if it can be worked out? Can you picture out your ideal in-vitro diagnostic device?
A: I think what you’re doing is great which directs me to a different way to develop in-vitro diagnostic device. If you ask me the ideal IVD from my perspective, I would love to tell you that I want to have everyone have their family physical examination at home. I think your design will possibly give out a solution for this since it is able to use types of enzymes to detect different indicators. Keep doing!
Meet with CCiCTime: 2018.8.27-2018.8.31
Place: ShanghaiTech University
• Overview
As an 8-year-old team, ZJU-iGEM absolutely won’t miss this biggest iGEM conference in China! This year, CCiC attracted more than 80 teams to share their projects and exchange their valuable experiences.
• Details
Also, we presented our project and drew much attention. Then, during panel session, Xuan Wang, an experienced iGEMer, post doctor in Tsinghua University, told us that especially for the New Application Track, if we want to stand out, we must convince others by showing them a workable device. Since we were trying to construct a user-defined sensor, we considered to write a universal and detailed instruction which made it possible for everyone to construct his or her own sensor.
Particularly this year, we found that there were 2 teams trying to construct cell-free systems. And we exchanged our experience on how to construct cell-free systems and shared our ideas on how to make a more distinctive ‘cell-free’ system. We all agreed that there doesn’t exist a clear definition about what is cell-free. So, we decided to have further understandings and exploring how to construct CFPS (cell-free protein synthesis) in a better way.
What’s more, our inspirations burst out when we communicated with other iGEM teams. How to predict our protein docking model was one of the biggest barriers toward our project’s completion, but after talking to Peking-iGEM, we surmised that their theoretical modeling can help us out! We were also worried about lacking negative control in Curli-Fibers Expression . Fortunately, UTSC-iGEM gave us a suggestion that we can use Acetobacter xylinus to express Curli-Fibers without SpyTag so that we could set it as our negative control when demonstrating the specific binding for our reconstructed Curli-Fibers.
In iGEM HQ face-to-face session, we sit around Mrs Meagan and discussed how to make iGEM projects better and lead iGEM competition to a more influential competition. We shared suggestions one by one. Also, through this session, we understood the significance of after-iGEM, and we knew how to break a complex project into parts and accomplish them one by one. We could take just several parts with breakthroughs to Boston for the competition and finish rest parts during following years. It also helped our team to inherit team culture.
All in all, CCiC gives us a great platform to communicate, to exchange experiences and to explore ideas we haven’t come up with. Thanks, CCiC!
Expert interview• Uartar Seker, Assistant Professor, Synthetic Biology Group, UNAW
To ensure our curli fiber have a better conductivity, we choose to have our curli fibers enhanced with nanogold particles. But we were confused on how to choose the best concentration and the best size of nanogold particles for our experiments. So, we connected to Professor Uartar and below is his reply:"5nm gold nanoparticles should be fine. Additionally, try to do gold enhancement protocol to fill up the gaps between the particles. Actually, to increase the conductivity you my try to add cytochromes by fusion and extracellular secretion."
• Chao Zhong, Ph.D., Assistant Professor, Integrative Bio-inspired Molecular Engineering (IBME) Group, ShanghaiTech University
Our project aims to detect the current at the downstream of our enzymes’ reactions. But at that time, the problem was that the current was so weak that we could not detect them in a convenient way which definitely didn’t reach our expectations. So, after we searched papers on how to enhance Curli-Fibers’ conductivity, we turned to Professor Timothy and Professor Zhong for advice.
Professor Zhong advised us to fill up all the gaps above our electrodes. He said it would be much better if we filled up with nanogold particles and he also offered us his E.coli strain (PIN4) whose endogenous CsgA was knocked off. And finally, to help us catch up with more ideas, he suggested us to take a glance at his newest paper[2].
• Christopher Whitford, ZJU-China & Julius-Maximilians Universität Würzburg Mentor in After iGEM
Mr. Chris and ZJU-iGEM were firstly connected by After iGEM. He is an experienced iGEMer and such an earnest man. We had our first meetup in 8/8/2018, during which Mr. Chris pointed out that iGEM concerns not only the main body of your project but also the demonstration of the significance of your project. iGEMer should realize that it would never be useless to show your comprehensive considerations of your project’s Human Practice.
In the second meetup, we were confused by how to ensure the biosafety of our cell-free biosensor. He provided us his recent paper talking about how to apply the biosecurity of iGEM projects.
The third meetup was mainly talking about the Human Practice Part of iGEM project. “The more Human Practice for your project, the more competitive you project is.” We realized it would be a promising Human Practice activity for us to keep frequent communication with Acon-Biotech and have more cooperation with Sir Run Run Shaw Hospital.
We sincerely thanks for his selfless denote!
Single Enzyme TestAt the very beginning, we used a single enzyme sensor to detect the actual function of A Detector Ⅰ. We measured the function of three enzymes separately (glucose oxidase, horseradish peroxidase and lactate dehydrogenase, which are components of designed logical gate). The characteristics of each enzyme provided useful information for further experiments.
Test results were quite satisfactory. This indicated that there was still a large degree of retention of the enzyme activity after modification of the Tag/Catcher system. Figure 4, Figure 5, and Figure 6 illustrate that these three enzyme electrodes worked well.
Fig. 3
The activity test of the Glucose Oxidase (GOx）sensor. (A) The Cyclic Voltammetry Curve of glucose oxidase had a distinct oxidation peak around 0.3-0.4V, and its height increased with increasing substrate concentrations. (B) Picture B is a partial enlargement of Picture A. The relationship between the height of the peak and the concentration of the substrates could be observed more clearly on the Fig B. (C) Fig. C shows the linear relationship between substrate concentrations and peak currents. The correlation coefficient of 0.9966 indicates that the sensor had a fairly good capability within this concentration range.
Fig. 4
The activity test of the Horseradish Peroxidase (HRP) sensor. (A) Similar to the glucose oxidase sensor, the horseradish peroxidase sensor had a distinct oxidation peak (around 1.0-1.3V) that can be used as an “indicator” of substrate concentrations. (B) Picture B is a partial enlargement of Picture A. (C) Picture C shows a high correlation between peak currents and substrate concentrations, thus it is a satisfactory standard curve.
Fig. 5
The activity test of the Lactate Dehydrogenase(LDH) (A) The oxidation peak of the LDH sensor was not very obvious, but after calculating, we found that its peak current values still had a satisfactory relationship with the substrate concentrations. (B) Picture B is a partial enlargement of Picture A. Although this oxidation peak was relatively inconspicuous, we could still see it. (C) The relatively high correlation coefficient indicates that our sensor had good detection capability in the concentration range of 0.5-6mmol.
Enzyme Complex (Logical Gate) test
As we explained in the multi-enzyme complex part, part, glucose oxidase and horseradish peroxidase can be used together to construct an AND gate, while lactate dehydrogenase, glucose oxidase and horseradish peroxidase can be used to construct an XOR gate. Figure 7 and 8 show that both have been successfully constructed. Our project ideas have been implemented.
Fig. 6
Current-time diagram (I-T) of the AND gate. (A) Picture A shows the I-T curve for different input conditions (0/0, 0/1, 1/0, 1/1). The current was stable at about 80s. In theory, the 1/1 input had the highest current output, and the actual result met this expectation. (B) Picture B is a partial enlargement of Picture A. We could find that the sensor clearly distinguished between different input conditions. The 1/1 input had the highest current output, followed by the 0/1 input, and the other two inputs lower. Different inputs were clearly distinguished, indicating that the AND gate met our expectations.
Fig. 7
Current-time diagram (I-T) of the XOR gate. (A) Figure A shows the I-T curve for different input conditions (0/0, 0/1, 1/0, 1/1). The current was stable at about 90s. (B) Picture B is a partial enlargement of Picture A. The output of 0/1 and 1/0 were significantly different from (higher than) those of 1/1 and 0/0, which was in line with the characteristics of the XOR gate.
Future Plan
We were satisfactory with the results from the first generation of products, A Detector I. However, we cannot DIY our hardware, and the electrodes themselves need to rely on the huge electrochemical workstation, which let us think about the possibility of exploring the next portable generation of products----A Detector II
References[1] Barnhart, Michelle M; Chapman, Matthew R (2006). "Curli Biogenesis and Function". Annual Review of Microbiology. 60: 131-47. doi:10.1146/annurev.micro.60.080805.142106. PMC 2838481. PMID 16704339
[2] Wang J, Polsky R, Xu D (2001). "Silver-Enhanced Colloidal Gold Electrochemical Stripping Detection of DNA Hybridization". Langmuir. 17 (19): 5739. doi:10.1021/la011002f.
[3] Wang J, Xu D, Polsky R (April 2002). "Magnetically-induced solid-state electrochemical detection of DNA hybridization". Journal of the American Chemical Society. 124 (16): 4208-9. doi:10.1021/ja0255709. PMID 11960439.
[4] Daniel MC, Astruc D (January 2004). "Gold nanoparticles: assembly, supramolecular chemistry, quantum-size-related properties, and applications toward biology, catalysis, and nanotechnology". Chemical Reviews. 104 (1): 293-346. doi:10.1021/cr030698. PMID 14719978.
[5] Hu M, Chen J, Li ZY, Au L, Hartland GV, Li X, Marquez M, Xia Y (November 2006). "Gold nanostructures: engineering their plasmonic properties for biomedical applications". Chemical Society Reviews. 35 (11): 1084-94. doi:10.1039/b517615h. PMID 17057837.
[6] Heitner-Wirguin, C. (1996). "Recent advances in perfluorinated ionomer membranes: structure, properties and applications". Journal of Membrane Science. 120: 1-33. doi:10.1016/0376-7388(96)00155-X.
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