Team:ECUST China/Poster

Poster:ECUST_China

Poster Title

Team: ECUST_China
Title: D-E-tector for SARS-CoV-2
Abstract:
SARS-CoV-2 is highly transmissible and pathogenic. In addition to the nature of RNA virus with high mutation rate, antigenic drift is the main evolutionary mechanism for SARS-CoV-2. Its wide-spreading has currently resulted in the evolutionary variants different from the originate isolate. Therefore, to curb this pandemic, effective surveillance of circulating viruses is much more urgent. We developed a high throughput POCT platform for the SARS-CoV-2 detection with the help of DMF. A nanomachine called DNA Walker can amplify the region-specific sequence of the virus genome in the specimen. And then it will be read out by E-CRISPR, which can output an electric signal by a Cas9 nickase and corresponding electrode. We are targeting not only the detection of COVID-19 viruses with alarming dangerous mutations, but also differentiating other related viruses and pathogens. This is not only for managing today’s pandemic, but also for the potential outbreaks of other coronaviruses.
Authors: Detector team
Affiliated Institution: School of bioengineering, East China University of science and technology
Special thanks: Thanks to Nurse Wang from the Affiliated Hospital of Jining Medical College, who was interviewed by us. Thanks to Jianghai Primary School and Xuhui District Youth Activity Center, which provided us with opportunities for offline education.

Introduction

D-E-Tector is a POCT  device, which combines the advantages of nanomachines, electrochemical, and digital microfluidics, to achieve an automatic and high-throughput detection. In our design, the sample input flows on the DMF platform in the form of droplets. Each droplet firstly reaches the DNA Walker reaction zone for signal amplification and then arrived at the E-CRISPR reaction zone to complete a conversion from a chemical signal to an electrical one. The electrochemical workstation module will transmit the signal to the testers’ cell phone to realize the visualization of the signal.
The device is expected to complete accurate, automatic nucleic acid detection and return visual electronic results in less than an hour. After replacing specific sequences in DNA Walker and E-CRISPR modules within the device, the device can detect different targets and viruses.
Due to the advantages of digital microfluidics, D-E-tector can also be designed as multi-channel simultaneous version, which we call D-E-tector plus. By detecting different mutations in the same target through different channels, the device can easily detect the possible mutations in the target sample. It is also suitable for simultaneous detection of different targets and viruses.

Inspiration

One of our team member was misdiagnosed with COVID-19 during the outbreak, which led to his dissatisfaction with conventional nucleic acid detection technology. He decided to develop a new efficient and convenient detection method, which was supported by other team members.

Problem

We have done a lot of investigation and found that the nucleic detection is significance in restrain of epidemic, but the conventional nucleic acid detection is time-consuming with poor portability and low accuracy. We also found that there are frequent mutations in the genome of the SARS-CoV-2, which is unfavorable for its detection and prevention. However, the traditional detection methods can not detect the occurrence of mutation. Thus we need a new efficient and convenient detection method, and it is better to realize mutation detection.

Idea

In order to achieve the purpose of efficient and convenient detection of viruses, we designed D-E-tec tor. D-E-tector is made up of three modules: DNA Walker, E-CRISPR and digital microfluidic.
Among them, DNA Walker is a nano-machine that skillfully converts input RNA signal to double-stranded DNA output driven by entropy increase, which can realize target recognition at the pM level and respond to changes of one base or more. It was used to achieve RNA-DNA replacement and primary amplification of the sample.
E-CRISPR is a technology arose in recent years. It applies the properties of CRISPR specifically identifying target sequences to the detection field and realizes the output of electrical signals with the help of electrochemical platform.
The way we used to connect DNA Walker and E-CRISPR was digital microfluidic. DMF is a technology to control droplet flow through the voltage imbalance on the electrode. It can easily realize droplet drive, hold, split and other behaviors. We combine DNA Walker module and E-CRISPR module with DMF device to form an integrated detection device: D-E-tector, which perfectly suit the need of efficiency and convenience.

The advantage of DMF makes the device can realize multi-channel detection at the same time. Therefore, we design a multi-channel version: D-E-tector plus, to suit the need of mutation detection.

Engineering

For DNA Walker:
When the machine encounters a target, it will bind the target to the W chain, making W a self-walking molecule. The W chain will undergo a chain substitution reaction with the surrounding ABC trihybrid chain, which we call orbitals. Displacement occurs at the end of the orbit, toehold 1, which causes the B chain to leave the machine and enter the extracellular environment, at which point the exposed toehold 2 will be recognized by the F chain in the environment, thus making a second displacement. The ultimate result of the displacement is that the next CF double strand is released, and W strand will be free again and continue to complete the displacement of other orbitals. As the W chain walks, it releases large amounts of CF double chains step by step, which is why it's called the Walker. For E-Crispr:
the single strand of DNA coupled to the electrode, known as the G strand, has a methylene blue group attached to its 3 'end, which causes the electrode to produce a steady current that can be detected. In the CF double strand output by DNA Walker, the C strand is paired with the G strand, and the G strand thus competes with the F strand to pair with the C strand to form the CG double strand. According to our design, the G chain will be recognized and bound by sgRNA, causing Cas9 Nickase to cut on the G chain, while the C chain remains intact. The G-beta chain carrying methylene blue then moves away from the electrode, causing the current on the electrode to drop. Due to the re-displacement of F, the displacement of the surrounding G chain, and the entropy enhancement effect, the C chain will leave the remaining G-beta chain to combine with other G chains, causing more cutting and greater electric signal, which is the secondary amplification of the signal. After the reaction for a certain time, the electrode was eluted to remove the F chain, Cas9 protein, free G-beta chain, and other components on the surface of the electrode, and the total current intensity change was calculated to determine the intensity of the input signal. Compared with the control, if the variation of current intensity reaches a certain threshold, CF double-chain input is indicated, indicating that there is a target sequence in the original sample.

Human Practice

Investigation:

In the early stage of the project, we investigated the time it took for the nucleic acid test takers to get the results through questionnaires, and found that more than half of them needed to get the results after more than one day, and some even waited for more than four days. We also surveyed people's acceptance of post-based devices and found that many people are willing to try them, but need to consider factors such as price. Further figures show that the widely accepted expectation is below $30.

Interview:

We had the honor to interview Nurse Wang, who has experience in fighting against the epidemic, and learned about the importance of testing kits and what should be paid attention to with new testing methods.

meet up:

On July 11th, we participated in the Eastern China Meetup hosted by Jiangnan University. At the meeting, we received many useful suggestions from Ambassador Zhang Nan on Human Practices. We have invited Pro. Wu, PI of NCKU_Tainan to comment on our work, and received many valuable suggestions. We held a seminar with ZJUT_China and NEU_China, whose project also aim to detect SARS-CoV-2. We learned about each other’s projects and exchanged our opinions, and created a chat group and shared some of our survey results. The data provided by ZJUT_China solved our problem of insufficient sample size of the lower and higher age groups. We conducted several online conferences with the team JACOXH_China. We provided them some theoretical guidance on modeling.And Jiangnan_China provided us with the opportunity to publicize our project on their monthly magazine, which is being passed around among the youngsters.

Publicity:

We attach great importance to public education and publicity in this year’s IGEM process. Knowing that most residents do not understand the principle of covid19 detection, we made a popular science video on the principle of COVID-19 nucleic acid detection and put it on various video platforms and WeChat public accounts, which received a considerable amount of feedback. Besides, we also produced many issues of scientific articles, which were distributed in the form of a WeChat public account and they received good results.

Model

Our modeling work is mainly focused on the DNA Walker module, which is divided into a dynamic part and an optimization part. In the kinetics part, we use the W chain as a catalyst and simplify the chain reaction into a two-stage confrontation reaction. Only by monitoring the amount of CF complex produced in real time, the reaction equation can be calculated. In the optimization part, the number of assembly of the L chain should be as small as possible and efficient because the content of the T chain is unknown. We regard the two composite structures of ABC and LTW as rigid long chains, and use solid geometric calculations and linear programming to obtain the upper and lower limits of the ratio of L chain and ABC composite structure assembly in the same specification of magnetic bead suspension.

Hardware

We’d glad to introduce our work, 3D printing design of D-E-tector shell. This shell was completed by our team member Zhang Liangkun under the guidance of Dr Gu Zhen. The DMF equipment and electrochemical workstations used in D-E-Tector come from the Intelligent Perception and Robotics Research Group from East China University of Science and Technology. And we combine all the parts together to detect SARS-CoV-2.

Results and Conclusions

We carry out qualitative verification of DNA Walker and E-CRISPR respectively. And fortunately, the qualitative verification of these two parts has been successful. In addition, the competition of the C/G/F triple chain, which is a series of two parts, has also been preliminarily verified.
For the DNA Walker module, lane 6 in the Figure1 is the double-stranded signal amplified by using the positive reference, which indicates that DNA Walker can indeed amplify the single-stranded signal to a certain extent. But a negative control in Lane 4 showed a degree of leakage, or nonspecific signal output, from DNA Walker. We are trying to optimize this situation in subsequent experiments and modeling.

For the E-CRISPR module, lane 7, 8, 9 in Figure 2 shows that Cas9 can specifically recognize and cut target sequences when reacting in the PCR tube. Figure 3 shows that when line G is modified on the electrode, cutting can occur and lead to a decrease in the current signal. During the experiment, we found that the quality of electrode modification would greatly affect the output signal. In subsequent experiments, we replaced electrodes with better quality and conducted a cutting time gradient experiment to find the best reaction time.
Figure4 demonstrated that line G can pair with line C instead of line F to some extent both in PCR tube and on electrode plate. However, Cas9 nickase can cut line G in PCR tube when line F is absent, it doesn’t work well when we added line F to the system. We detected that line F might promote the formation of a trihybrid of CFG thus inactivated the Cas 9 nickase.
Overall, we preliminarily verified the usability of DNA Walker and E-CRISPR, as well as the feasibility of linking the two modules together. In the future, we will continue to optimize the experimental process in order to achieve the original intention of D-E-Tector design.

References and Acknowledgements

[1] Chu D K W , Pan Y , Cheng S M S , et al. Molecular Diagnosis of a Novel Coronavirus (2019-nCoV) Causing an Outbreak of Pneumonia[J]. Clinical Chemistry, 2020, 66(4).
[2] Xiaolu T , Changcheng W , Xiang L , et al. On the origin and continuing evolution of SARS-CoV-2[J]. National Science Review(6):6.
[3] Maclean O A , Orton R J , Singer J B , et al. No evidence for distinct types in the evolution of SARS-CoV-2[J]. Virus Evolution(1):1.
[4] Forster P , Forster L , Renfrew C , et al. Phylogenetic network analysis of SARS-CoV-2 genomes[J]. Proceedings of the National Academy of ences, 2020, 117(17):202004999.
[5] Liang C P , Ma P Q , Liu H , et al. Rational Engineering of a Dynamic, Entropy‐Driven DNA Nanomachine for Intracellular MicroRNA Imaging[J]. Angewandte Chemie, 2017.
[6] Dai Y , Somoza R A , Wang L , et al. Exploring the Trans‐Cleavage Activity of CRISPR‐Cas12a (cpf1) for the Development of a Universal Electrochemical Biosensor[J]. Angewandte Chemie International Edition, 2019, 58(48).
[7] Xu W , Jin T , Dai Y , et al. Surpassing the detection limit and accuracy of the electrochemical DNA sensor through the application of CRISPR Cas systems[J]. Biosensors & Bioelectronics, 2020, 155:112100.