Overview
In order to achieve the integration of biological reactions and liquid flow control, we finally chose paper-based chips as our reaction platform to realize the final detection by convenient injection.
We designed the stacking structure of the paper and selected reasonable materials and processing methods for different layers to achieve our goals. We tested its performance and the integration capability of the biological reaction to verify the feasibility of our design. The demonstration video of the paper-based chip is shown below.
Target Analysis
To achieve the overall goals of amplification and visual detection of target miRNAs, and integration of HCR and CRISPR reactions, we developed a fast and convenient reaction platform. The platform is capable of controlling the directional flow of liquid and cooperating with other modules to accomplish the detection task.
Paper-based chips have natural capillary forces and do not require external forces to control liquid flow, and they can also be combined with hydrophobic materials to set up multiple channel structures. Therefore, the design of patterned hydrophobic channels and the stacking of paper can be used to achieve our goal. In addition, the advantages of simple manufacturing, portability, and low cost of paper-based chips make our devices easier to process and use.
In order to achieve the specific goals of fluid flow control, HCR and CRISPR reactions, and fluorescence output as well as detection, we select specific paper-based membrane materials and build specific structures, all of which were verified by experiments for feasibility.
Design
The fluid tends to flow more horizontally on the small pore side and vertically on the large pore side of the asymmetric membrane, and the PES membrane is equipped with hydrophobic channels. Therefore, the fluid can flow from the asymmetric membrane to the PES membrane and pass through the fluid channels. Thus, we can achieve fluid flow control.
The final design of the paper chip structure is divided into five layers, from bottom to top: a sample transfer pad cut from asymmetric polysulfone membrane, a fluidic channel pad consisting of two hydrophobic PES membranes together with a layer of non-hydrophobic PES membrane, and four glass-fiber reaction pads. The large pore side of the asymmetric polysulfone membrane is in contact with the PES membrane. The reaction pads hold the HCR probes and integrate biological reactions. The fluidic channel pads and transfer pads are used to control the fluid flow.
The stacking method, size, and reaction process diagram are shown below.
Processing
We cut the corresponding size of asymmetric polysulfone film, PES film, and glass fiber paper according to the design above.
For the choice of the hydrophobic method of PES film, we try to find a simpler method of crayon coating considering that wax printing, photoresist, and other technologies have disadvantages such as high requirements for processing equipment, high price, the difficulty of processing double layers and solubility. We conducted a test by treating the PES film in different ways, such as dipping wax, paraffin wax application, and crayon application. The results are compared as follows, and we can see that only the crayon-applied PES film has good hydrophobic performance. So, we finally chose the simple crayon-applied method for hydrophobic treatment.
After hydrophobic treatment by crayon, the hydrophobic and hydrophilic PES films are stacked to form the fluidic channel pad. Then we stack all layers together according to the design to form a complete paper-based chip.
The complete processing flow of the paper-based chip is shown in the figure below. In order to facilitate the processing of other users or iGEM teams, we put the specific processing flow in contribution.
For more details, please click>>contribution<<
Experimental testing
We validate the hardware functionality through a series of experiments, from testing the performance of the hardware itself to its ability to integrate biological reactions and fix HCR probes, and finally, we validate the fluorescence output. The specific flow chart is as follows.
Chip Performance Test
We first tested the ability of the paper-based chip to control the flow of liquid.
By dropping sodium fluorescein solution on the paper-based chip, we tested whether the paper-based chip can control the liquid flow into the reaction pad correctly without leakage. The test result is shown below. It shows that no fluorescence appears except at the four reaction pads and the inlet holes, which proves our paper-based chip does not leak liquid.
Then we determine the injection volume. The weights of the reaction pad, asymmetric polysulfone membrane, and monolayer hydrophilic PES membrane were tested before and after water immersion to determine the injection volume of the paper-based chip. The injection volume of the paper-based chip was calculated to be approximately 60μL. We used this as a benchmark for fine-tuning the actual injection volume in our experiments.
In order to verify that the liquid could flow uniformly into the reaction pad, we added an appropriate volume of sodium fluorescein solution and observed the result with a fluorescence microscope. The fluorescence microscope observation graph is shown below. The fluorescence at the reaction pad is relatively uniform, thus the ability of the paper-based chip to control the uniformity of the injection is verified.
Paper substrate compatibility testing
We performed the reaction on a wet reaction pad to confirm that the reaction could be performed normally on the reaction pad.
After performing the HCR reaction in the laboratory and on a wet glass fiber reaction pad respectively, the electrophoresis results are shown below. It can be seen that in the laboratory as well as on the wet reaction pad, the bands appeared only when both the probes and miRNA were present. And the results all had a clear linear phenomenon, the higher the miRNA concentration, the more obvious the bands.
The electrophoresis results show that reaction on the wet reaction pad is not significantly different from those in the laboratory. Thus, the HCR system on a wet reaction pad can go on normally and the HCR reaction can tolerate the reaction pad environment.
HCR reagent fixation test
In order to achieve HCR and CRISPR reactions, we fixed the HCR probes and then tested HCR reactions under dry conditions.
We first added a buffer containing the HCR probes to the reaction pad. Then we placed the reaction pad on the lab bench for air-drying. The sample solution was added after a while. The electrophoresis results are shown in the figure below.
It can be seen that the bands appear only when both the probes and miRNA are present. The results of the dry reaction pad were more complete and the bands were clearer than those in the laboratory environment. The HCR system on the dry reaction pad could react normally and the results were linear. Therefore, the HCR reaction could tolerate the dry reaction pad environment.
HCR+CRISPR overall reaction test
In order to ensure that the complete reaction could go on and the fluorescence could output normally, we performed the complete reaction on our paper-based chip. After fixing the HCR reaction system on the reaction pad, the miRNA target and the CRISPR reagent are injected into the paper-based chip for the two-step reaction of HCR and CRISPR.
The results are shown in the figure below, in which one of the reaction pads is without the HCR probe fixed and a paper-based chip with ultrapure water injection was used as control. It can be seen that the fluorescence is as expected when the probe is normally fixed and injected with the corresponding reagent. No fluorescence is detected when the HCR probe was not fixed or when ultrapure water was injected, and the pictures of both are the same. This proves that the HCR and CRISPR reactions can go on normally on the paper-based chip and output fluorescence signals.
