Team:ECUST China/Design
D-E-tector is a brilliant detector who can detect virus like SARS-CoV-2 efficiently and accuracy, using nano DNA machine and electrochemical techniques. It combined with two parts, DNA Walker and E-CRISPR, which are in series connected by digital microfluidics.
In D-E-tector, we designed an enzymatic cleavage area to treat samples, just entering the equipment. Enzymatic cleavage is a commonly used method to treat and detect samples, and its function is to remove the virus shells that may exist in the samples and expose the DNA.
DNA Walker is a kind of nano machine driven by entropy, which consists of magnetic beads and oligonucleotides. Oligo ssDNA lineA and lineL connected to the magnetic beads through the biotin-streptavidin system, while lineB and lineC form a three-stranded substrate complex with lineA by base pairing. When need to start, add lineW and lineF to the system, both of which carry complementary sequences with lineC. When there is target sequence exist in system, it can combine with lineL and lineW, form another three-stranded substrate complex. Then lineB will replace lineB from toehold1, the single chain part of lineC/B complex. This will expose toehold2, the single chain part of lineA/C. Then line C will be replaced to form dsDNA C:F with lineF. Thus lineW can attack another toehold1, realizing cyclic chain replacement. In theory, the input of a target will result in a 10 to 500 fold C:F chain output, the amplification multiple can be adjusted by design.
If there is no target in system, lineW will be in a stable secondary structure to avoid nonspecific chain substitution. When DNA Walker working with target, lineW connected with magnetic beads. The chain substitutions that lineW creates are all intramolecular reactions, while the lineB and lineC/F replaced will no longer belong to the original molecules. This ensures that the whole machine is driven by entropy without additional energy supply and has high efficiency. Since we get C:F chain as a output, what will we do next?
CRISPR could be a powerful biosensing medical device when combine with electrochemical platform. That’s what we called E-CRISPR. Similar to how CRISPR is a specialized protein designed to cut out harmful mutations within DNA, E-CRISPR relies on the same mechanism to recognize cut specialized site. However, the oligonucleotides to be cut will be modified on the electrode and connected with an electrical group at the end. When oligonucleotides be cut, the electrical group will be far away from the electrode surface, resulting in the change of current. We modified ssDNA lineG on the electrode surface by Au-S-bond, with a methylene blue molecular on 3’ end. LineG and lineC are complementary, while PAM is located on lineC. If there is no lineC, Cas9 protein cannot recognize and cut lineG. When lineC existed, Cas9 will bind dsDNA C:G via sgRNA. We choose D10A mutant rather than regular Cas9, as the former can only cut the complementary strand of sgRNA because of the HNH activity losing. In this way, when the lineG is cleaved, the lineC can combine with another lineG, resulting in multiple cuts and a higher output. Considered DNA Walker output lineC/F, lineG is designed to replace lineF from lineC. In this process, we combine the effects of enthalpy and entropy to make the displacement more efficient. The generation of E-CRISPR electrical signal depends on electrochemical workstation. We obtained a portable electrochemical workstation from Dr. Gu, and you can see its details on the hardware page.
Microfluidics has long been used in the production of detection kits, it roughly divided into two types: channel-based microfluidic and digital microfluidic (DMF).
Channel-based devices offer high precision and low cost at scale. These devices move liquids through a fixed set of channels, so they are single-purpose by nature. Considering the requirements of fluid volume and flexibility, we chosen DMF.
DMF technology offers flexibility at small size and potentially at low cost. DMF devices manipulate individual droplets of liquids on a grid of electrodes, taking advantage of a phenomenon called electrowetting on dielectric. Activating electrodes in certain patterns can move, mix, or split droplets anywhere on the chip. Figure 3 shows how our DMF device, moves droplets by activating electrodes in sequence. The droplets can move through either an oil or air medium.
Electrodes (yellow) sit at the top of the PCB (purple) in a grid. The PCB is topped with a dielectric and hydrophobic layer. The top plate sits on risers and is also hydrophobic and conductive. Droplets are attracted to the activated electrodes (shown with diagonal lines). Activating the neighboring electrode will move a droplet. We got a DMF equipment provided by Dr. Gu to realize the assembly of D-E-tector, you can see how it work in hardware.
We aim to combine DNA Walker and E-CRISPR with DMF, building a amazing detect machine: D-E-tector. The collected samples will entry the machine as droplets. After enzymatic cleavage treatment, the droplets will flow through DNA Walker part and E-CRISPR part. If target strand is existed in droplets, it will converted into C:F in DNA Walker part, and the magnetic beads will remain in place due to magnetic adsorption. Then droplets containing C:F arrived electrode surface of E-CRISPR, promoting cleavage of lineG. The generation and display of electrical signals are completed by the existing electrochemical platform. In addition to the above parts, a complete D-E-tector also includes the shell and supporting software. This will make the detection of the virus like SARS-CoV-2 simple to the citizen can complete it at home. See our hardware for details of the 3D print shell.
Obviously, the function of D-E-tector can't be fully exerted by simple detection. Therefore, in addition to the original version designed to detect single virus in the community, we also designed the plus version for D-E-tector. We have described that DMF can conveniently move droplets, so it can realize multi-channel simultaneous detection by virtue of its characteristics. In other words, we will sacrifice the portability of the original version of D-E-tector, and connect multiple DMF devices in parallel to realize the simultaneous detection of multiple channels. In this high throughput situation, each channel can be designed differently, so that different channels can detect different viruses or mutants. In this way, D-E-tector plus can realize rapid detection of mutations through simultaneous detection of multiple channels. This equipment can be used in laboratories to meet the needs of researchers for rapid detection of mutation of strains.