Team:BIT/SlipChip
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SlipChip
Overview
【Objective】For colorectal cancer related miRNAs，biology module has constructed a biosensing detection method that combines ligation reaction,
LAMP amplification reaction and CRISPR / cas12a reaction . In order to integrate the complex biological reaction process
mentioned above and save the trouble caused by multiple steps sampling in the detection process to achieve the ultimate goal of point-of-care testing，
we designed a portable SlipChip with simple operation and multi-channel detection.
【Methods】In order to make each step of the biology module efficiently implemented on the SlipChip, we designed and optimized the structure design of the SlipChip. We chose PMMA as the manufacturing material of the SlipChip. The reagent consumables is embed in the form of freeze-dried beads and store frozen. The chip drives the pre-embedded reagent by sliding the middle sliding layer, and the middle sliding layer of the SlipChip is driven by the micro electric push rod .
【Result】The operation steps of each stage of the biology module can be realized on the SlipChip. The mechanical, thermal and optical properties of PMMA materials meet the requirements of biological detection methods for materials. LAMP freeze-dried beads can move and fall according to a predetermined trajectory. The complete detection process can be realized on the SlipChip, and the liquid in each chamber will not leak and contaminate. The six chambers have little effect on crosstalk during fluorescence detection. When it is finally delivered to the user terminal, the user only needs to add samples according to the operation prompts of the WeChat Mini Program to complete the phased release and dissolution of the solid reagents, reducing errors and troubles caused by manual operations.
【Methods】In order to make each step of the biology module efficiently implemented on the SlipChip, we designed and optimized the structure design of the SlipChip. We chose PMMA as the manufacturing material of the SlipChip. The reagent consumables is embed in the form of freeze-dried beads and store frozen. The chip drives the pre-embedded reagent by sliding the middle sliding layer, and the middle sliding layer of the SlipChip is driven by the micro electric push rod .
【Result】The operation steps of each stage of the biology module can be realized on the SlipChip. The mechanical, thermal and optical properties of PMMA materials meet the requirements of biological detection methods for materials. LAMP freeze-dried beads can move and fall according to a predetermined trajectory. The complete detection process can be realized on the SlipChip, and the liquid in each chamber will not leak and contaminate. The six chambers have little effect on crosstalk during fluorescence detection. When it is finally delivered to the user terminal, the user only needs to add samples according to the operation prompts of the WeChat Mini Program to complete the phased release and dissolution of the solid reagents, reducing errors and troubles caused by manual operations.
Video. 1 SlipChip demo animation
Fig. 1 Physical image of SlipChip
Biological part reaction process and design constraints
Due to the multi-step-like process in the detection method of the biology module,
the SlipChip module integrates the reaction of the biology module through the SlipChip to
reduce the complexity and uncertainty caused by manual operation.
Investigation and Selection of Microfluidic Chips
At the beginning of the design, we investigated microfluidic chips with different driving methods,
the advantages and disadvantages are listed below.
Table. 1 Comparison of advantages and disadvantages of microfluidic chips with different driving methods
The SlipChip drives the pre-embedded reagents by the middle sliding layer to slide, and can complete multi-step and staged sample injection.
By comprehensively comparing the advantages and disadvantages of the SlipChip, microfluidic chip and centrifugal chip driving methods,
we finally chose the SlipChip as the experimental platform for biological reactions.
Reagent pre-embedding and production of freeze-dried beads
This project is dedicated to designing an integrated auxiliary diagnosis system that can be used for colorectal cancer,
solving the problems of high detection cost and complex detection process, and achieving the goal of instant detection.
Therefore, it is necessary to carry out reagent pre-embedding to avoid complicated sampling process.
We choose the freeze-dried beads to realize the pre-embedding of reagents .
Through research, we found that there were already LAMP freeze-dried beads on the market that have reached the level of commercialization.
So we purchased the Bst 4.0 Basic IsoAmp Lyophilized Bead model freeze-dried beads from HaiGene.
We can pre-embed the reagents in the reaction chamber of the SlipChip with freeze-dried beads technology which LAMP reaction required in advance ,
and directly add the sample to perform a series of reactions, eliminating the trouble of adding a large amount of reagents during detection.
This meets the requirements of instant detection.
Fig. 2＆3 LAMP freeze-dried beads picture
Because our project requires a large number of test experiments, considering the cost of LAMP freeze-dried beads,
we made our own freeze-dried beads simulate the experiment on the SlipChip during the SlipChip testing stage.
As Video2, we use a professional precision micro pump to pour the reagents into liquid nitrogen,
quickly freeze them into small solid spheres with uniform and regular shapes at low temperatures,
and then collect and put them into the freeze-drying equipment for freezing and storage.
We conducted a drive test on the homemade lyophilized beads on the SlipChip,
and the results showed that the SlipChip can control the movement and falling of the lyophilized beads.
In the final experimental stage, we used the purchased LAMP freeze-dried beads to conduct biological experiments on the SlipChip.
The results proved that the SlipChip can control the movement and falling of the LAMP freeze-dried beads.
Video. 2 Freeze-dried beads production
SlipChip
SlipChip material selection
The overall structural frame of the SlipChip is rectangular, and the material used is polymethylmethacrylate (PMMA). PMMA,
commonly known as acrylic or plexiglass, is a high-quality polymer material with a low melting point of about 130-140°C.
It can be bonded by thermal processing and is lower than the temperature required for our biological reactions.
PMMA has a light transmittance of 92%, which is higher than that of glass.
It is currently the most excellent polymer transparent material which can meet the requirements of fluorescence detection in our project.
In addition, PMMA has high mechanical strength and good comprehensive mechanical properties.
Considering the above factors, we choose PMMA as the manufacturing material of the SlipChip.
The structure design of the SlipChip
The total volume of the ligation reaction, the LAMP amplification reaction and the CRISPR/Cas12a reaction in the biology module of our project are 10ul,
10ul, and 20ul respectively. Therefore, we designed a cylindrical main reaction chamber with a volume of 4mm*4mm and a volume of about 50ul.
There are six parallel detection channels designed in the chip, and each channel can be designed with the same process structure to achieve
the purpose of parallel joint detection of multiple targets in a single sample and meet the functional requirements of high throughput.
In order to complete three-step continuous biological reactions to occur on the SlipChip, we designed the corresponding SlipChip.
The SlipChip consists of four parts: the top layer, the sliding layer, the sealing frame, the bottom layer and the back plate.
In the sliding operation steps of the chip, the top layer, the sealing frame,
the bottom layer and the back plate can be used as a closed overall structure and assist the sliding layer to make smoothly sliding motion to
realize solid reagent motion control. At the same time, the main functional chambers are constructed in some positions of the top layer,
the sliding layer and the bottom layer.
The six reaction chambers on the bottom layer are the main chamber for the overall detection reaction,
which completes the sample injection, ligation reaction, LAMP amplification reaction, and CRISPR/Cas12a reaction of the sample solution to be tested.
The Slipped layer has two parallel rows of circular structures: Amplification material chamber and Detection material chamber.
Amplification material chamber is pre-embedded with freeze-dried beads, containing LAMP reagents (Bst 3.0 DNA polymerase, FIP, BIP, dNTPs, etc.)
for nucleic acid amplification, and for adding sample reagents and sliding through the chip.
The detection material chamber is used to add the reagents required for the CRISPR reaction (Cas12a nuclease, NEBuffer 2.1, ssDNA reporter, etc.).
Fig. 4 SlipChip Design Drawing
Video. 3 Slipchip explosion animation
SlipChip working steps
ａ．The top layer is constructed with injection hole 1 and injection hole 2,
which respectively realize the pre-embedding of reagents and the injection of samples and reagents.
In the pre-embedding stage of the reagents, the SlipChip makes the injection hole 1 align with the original amplification chamber in turn,
and loads the LAMP lyophilized beads.
ｂ．The chip slides to seal the chamber, completes the pre-embedding of the amplified freeze-dried beads, and long-term storage at -4°C.
ｃ．Slide the Slipped layer until the amplification material chamber is aligned with the main chamber
to allow the lyophilized beads in the amplification material chamber to fall off.
ｄ．Add the reagents required for the first stage of the ligation reaction into the main chamber of
the bottom layer through the injection hole on the top layer.
ｅ．The chip slides into a sealed state, reacts at 85°C for 2 minutes and reacts at 37°C for 5 minutes.
ｆ．Add the reagents required for the second stage of the ligation reaction into
the bottom main chamber through the top injection hole.
ｇ．After the chip is slid and sealed, begin the reaction at 37°C for 15 minutes to complete the ligation reaction.
ｈ．Add deionized water to the main chamber to provide a solution for the LAMP process.
ｉ．Slide the Slipped layer to seal the main chamber. At the same time, the heating membrane and the temperature sensor start to work,
and heat at 65°C for 15 minutes to perform the LAMP reaction to achieve signal amplification and complete nucleic acid amplification.
ｊ．Slide the Slipped layer again until the detection material chamber is aligned with the main chamber,
and add the reagents required for the CRISPR reaction.
ｋ．Then slide the Slipped layer forward one step to seal the main chamber
and let it stand at 65°C for 10 minutes to achieve the final color reaction.
Fig. 5 Reaction flow chart
Slip chip machine driving design
We use miniature electric actuators to realize the machine driving of the SlipChip.
The micro electric push rod is a device that can be pushed at a constant speed after a stable voltage is applied.
We connect the miniature electric push rod with the sliding layer of the SlipChip so that the push rod can push the chip,
eliminating the need for the user to manually control the sliding of the chip, thus realizing the machine driving of the SlipChip,
preventing the trouble and uncertainty caused by manual operation.
Fig. 6 Slip chip machine driving design
Video. 4 Leak test video
SlipChip functional test and results
Bond the SlipChip production, as shown in the figure below：
Fig. 7 SlipChip physical image
SlipChip tightness test
In order to prevent the SlipChip from leaking, we used solid freeze-dried beads,
which effectively avoided the problem of liquid leakage during the chip's sliding process.
We also performed a hot-keying of the bottom and back of the SlipChip at 170°C.
Experiments verifies that there is no leakage problem.
We used two methods to verify the leakage. First, we use a pipette to add the blue pigment aqueous solution to the SlipChip,
and observe the sliding layer of the SlipChip by hand. We observed that the pigment solution does not diffuse out of the main reaction chamber,
which indicates that the SlipChip has good sealing properties.
Video. 5 Leak test video
As shown in Fig.8, we also added fluorescein sodium solution to the SlipChip for fluorescence detection,
and found that there was no fluorescence outside the main reaction chamber, indicating that the SlipChip was well sealed.
Fig. 8 SlipChip tightness test result image.
Friction resistance test of chip sliding
Because the PMMA material has a certain degree of toughness, greater frictional resistance may cause the movement of
the micro electric push rod and the SlipChip to be out of sync, reducing the accuracy of the sliding control of the SlipChip,
thereby affecting the correct progress of the biological reaction step. We connect the SlipChip with an electric push rod through a copper pillar,
so that the push rod drives the chip to slide. Therefore, the frictional resistance of the chip sliding is closely related to the length of the copper
pillar required for the connection. After testing and comparing the smoothness of sliding and the overall structure,
we chose a copper column with a total length of 45mm to connect the miniature electric push rod and the SlipChip.
Fluorescence detection crosstalk test
Place the fluorescent reagents of different concentrations of 0.2, 0.5, 0.8 μmol/L in the six chambers of the SlipChip.
As shown in the table, use the self-sliding push rod to control the optical system to scan to obtain the fluorescence
intensity value and draw the fluorescence value curve. The results are shown in the figure below.
It can be seen from the results that the fluorescence signal intensity between wells 1 and 2, 3 and 4, 5 and 6 are basically similar,
indicating that the crosstalk problem can be controlled in a certain extent.
The fluorescence values of the No. 1, 2 and 3, 4 and 5, 6 hole can be clearly distinguished,
indicating that the SlipChip module has the ability to support optical detection.
Table. 2 Chamber number and corresponding fluorescein concentration
Fig. 9 Schematic diagram of fluorescein placement
Fig. 10 Raw data of fluorescence detection signal graph
Fig. 11 Actual fluorescence detection image
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