Team:BIT/Proof Of Concept

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Proof Of Concept
Early screening and treatment of colorectal cancer(CRC) has been proved to be conducive to reducing the mortality rate of CRC. Currently, most diagnostic methods do not meet the requirements of rapid, sensitive and specific screening of CRC. Based on the selection of miRNA as our biomarker for its high sensitivity and specificity，we developed an ultrasensitive approach for miRNA detection based on a combination of Loop-mediated isothermal amplification(LAMP), CRISPR/Cas12a trans-cleavage and Slip-Chip. And in terms of hardware, in order to achieve rapid, portable and simple “sample-to-response” integrated detection of CRC, we constructed an intelligent interactive IVD device for large-scale early screening.
Our system has been tested to be simple to operate, for completing the process within 1h，and its sensitivity reaches to fM level. Furthermore, our system has the capacity to detect different miRNAs, giving the potential to build standardized cancer detection platforms.
We carried out verification experiments from biological and hardware aspects under laboratory conditions and combined the two together to produce a product object for verification. Our experimental results show that our project is feasible and can be applied to real environments.
Bio
Feasibility verification of miRNA detection in experimental environment
In order to verify the feasibility of LAMP + CRISPR amplification system, we choose the LAMP-CRISPR itself system and the system in the fetal bovine serum environment test results to analyze.
Verification of LAMP+CRISPR system
We designed the following experiments. First, ligation reaction is first performed to obtain the initial structure of the LAMP cycle amplification step of the neck loop structure. Then carry out LAMP amplification experiments. The obtained products are analyzed by agarose gel electrophoresis.
Agarose gel electrophoresis results show that LAMP amplification results are good. The following fluorescence curve is obtained by the next CRISPR specific detection test and there was no false positive. The fluorescence intensity of the experimental group is obvious, which proved that LAMP + CRISPR system is feasible.
At the same time, we also verified the necessity of LAMP through the control experiment. The results are as follows：
It can be seen that there is no significant fluorescence change in the group without LAMP reaction. But strong fluorescence is detected when the ligation product was added to the CRISPR/Cas12a system after LAMP amplification. According to the characteristics of the CRISPR/Cas12a system, it can recognize the double-stranded ligation product and the first step of the ligation product needs to undergo LAMP amplification reaction to produce double-stranded structure. This proves that the LAMP amplification reaction converts miRNA into a double-stranded structure recognizable by the CRISPR/Cas12a system while it can improve detection sensitivity.
Verification of Environmental anti-interference of fetal bovine serum
By simulating human blood sampling with fetal bovine serum, it is proved that miRNA can be specifically recognized in the interference system and can be successfully ligated and lamp amplified. Finally good fluorescence value can be detected in the final CRISPR/Cas fluorescence reaction.
In this step, we used fetal bovine serum to dilute miRNA to simulate the interference environment and set water to dilute miRNA at the same concentration for verification.
Experimental results and analysis:
1）Agarose gel electrophoresis results of LAMP
We can see from Images that our miRNAs can be specifically recognized and amplified in interference environments.
2）Results of Fluorescence Data Processing
From the fluorescence data processing diagram, it can be seen that in the case of other conditions are completely the same, water and fetal bovine serum diluted with the same concentration of miRNA, the fluorescence value of the detection effect is similar.
This experiment proved that in the interference environment, our experiment can still successfully identify and amplify miRNA and can obtain good fluorescence value.
Feasibility detection of system practical application
Feasibility detection of freeze-dried pellets instead of LAMP system
We can see our experiments have fantastic results under laboratory condition. But it can not meet all our needs. For realizing more convenient miRNA detection on our Slip-Chip, we pre-embed the freeze-dried ball to replace the traditional and complex system of configuration and sample addition process.
After miRNA linkage reaction, we use freeze-dried ball to construct a LAMP reaction system. React at 65°C for 30 minutes to obtain the following agarose gel electrophoresis.
The picture had shown that three groups: the standard positive group, the freeze-dried ball experiment group 1 and the freeze-dried ball experiment group 2 had positive bands; and there were no positive bands in that two groups: the standard negative group and the freeze-dried ball negative group. The experiments showed that the reaction system constructed by freeze-dried ball had a good performance.
Then we use the products to do the next CRISPR. React for 40 minutes at 37°C in a qPCR instrument and measure the fluorescence to obtain the following fluorescence curve.
We can see the Freeze-dried ball system can accomplish the tasks which can be accomplished by traditional methods in amplification efficiency and detection specificity.
Feasibility verification of miRNA detection based on chip
After determining that the miRNA amplification effect is good in the laboratory environment and the CRISPR experiment is successful, we conducted experiments on our chip to verify the feasibility of the experimental system applied to the chip.
1. Verify the feasibility of LAMP experiment on chip
We used a concentration of 50pM miRNA for experiments. We added sample to the chip for connection reaction. After LAMP reaction, the chip was placed under a fluorescence microscope. It was found that the positive group had obvious fluorescence, while the negative group had no fluorescence. It can show that:
1)LAMP reaction is effective in the chip system;
2)Positive group can not interfere with the negative group, which means there is no cross contamination between the chip holes.
2. Verify the feasibility of CRISPR experiment on chip
We used a concentration of 50pM miRNA for experiments. We added sample to the chip for connection reaction. After LAMP reaction and CRISPR reaction, positive samples were observed under fluorescence microscope, which indicated that the positive group produced better fluorescence.
Determination and optimization of detection limit of miRNA
Detection limit of miRNA
In order to find out the lowest concentration limit of miRNA that can produce fluorescence, we set different gradient miRNA to explore the factors affecting the detection limit of miRNA in this system.
The first miRNA concentration gradient (final concentration) is set to：A_50pm，B_5pm，C_500fm，D_50fm，E_5fm. Interval is 10 times.
Experiment results show that the LAMP amplification reaction system has good amplification performance.
Next we used the amplification products from the above experiments for further CRISPR reaction. Put them in qPCR instrument and react under 37℃ for 40 min, we get the following fluorescence curve.
It can be seen that the overall fluorescence intensity showed a downward trend according to the miRNA concentration gradient. It indicates that there is a linear relationship between miRNA concentration and fluorescence intensity. The higher the miRNA concentration is, the stronger the fluorescence intensity is.
Optimization of influencing factors of miRNA detection limit
Based on the results of the first experimental analysis, we performed the next miRNA concentration gradient experiment.
The second miRNA concentration gradient was gradually diluted from 500 fm to 0.05 fm. Gradient set to：A_500fm，B_50fm，C_5fm，D_0.5fm，E_0.05fm. Interval is 10 times.
The miRNA ligation product was obtained by linkagen reaction and then the LAMP amplification reaction was performed to obtain the product for electrophoresis. The results are shown in the following.
Each lane has the obvious band production, has the LAMP amplification product production.
Next we used the amplification products obtained from the above experiments for further CRISPR reaction. Put them in qPCR instrument and react under 37℃ for 40 min, we get the following fluorescence curve.
It can be obtained that the fluorescence intensity decreases as a whole according to the miRNA concentration gradient. The concentration of miRNA has a linear effect on fluorescence generation. The higher the miRNA concentration, the stronger the fluorescence intensity; the smaller the miRNA concentration, and the lower the fluorescence curve.
Standardization
We constructed a standardized plasmid containing sgRNA and provided the corresponding experimental manual to provide reference for other teams with similar needs.
We want to verify that the standardized plasmid can enter the experimental bacteria through the normal laboratory transformation steps and complete the corresponding amplification steps, so as to achieve the purpose of signal amplification.
Therefore, in this experiment, our experimental design is carried out according to the standard plasmid transformation steps, from PCR amplification, enzyme digestion, enzyme ligation to transformation into the experimental bacteria, and subsequent verification.
There is the DNA sequence we designed before the experiment：
T A A T A C G A C T C A C T A T A G G G G A A A T T A A T A C G A C T C A C T A T A G G G A A T T T C T A C T G T T G T A G A T T A G C T T A T C A G A C T G A T G T T G A T T.
Its length is 88 bp. We hope that this gene sequence can still be extracted after a series of biological operations such as inserting the fragment into the plasmid and transferring it into the experimental bacteria for amplification and culture.
We sent the results of the experiment to the company for DNA sequencing. The results showed that our experiment was successful. From the sequencing results, we can see that a small fragment with a length of about 88 bp has been produced, which proves that we have completed a series of transformation processes such as plasmid extraction, enzyme ligation and enzyme digestion.

Hardware & Software
1. SlipChip - Provide Bioreaction Platform
In order to verify that our sliding chip has good sealing performance, can ensure the normal progress of the biological reaction steps and has the ability of optical detection, we tested the sealing performance, push rod drive and on-chip experiments.
1.1 Tightness
In order to prevent the slip-chip leakage, we use a solid freeze-dried ball, effectively avoiding the chip in the sliding process of liquid leakage. We glued the slip-chip`s bottom and backplane at 170℃. And verified by experiments, there is no leakage problem.
We use two methods to verify the leakage. Firstly, we used a pipette to add blue pigment solution to the slip-chip, and slide the sliding layer of the chip by hand. It was observed that the pigment solution did not diffuse outside the main reaction chamber, indicating that the sliding chip had good sealing.
As we can see in Fig.3, we also added fluorescein sodium solution to the slip-chip for fluorescence detection. It was found that there was no fluorescence outside the main reaction chamber, indicating that the sliding chip was well sealed.
1.2 Radius-rod drive
Because PMMA material has a certain toughness, so the larger friction resistance may lead to micro electric push rod and slip-chip movement is not synchronous. This will reduce the accuracy of slip-chip sliding control, thus affecting the correct biological reaction steps. We connect the slip-chip with the electric push rod through the copper column to realize the push rod to drive the chip to slide.
1.3 On-chip biological reaction test
We used a concentration of 50pM miRNA for experiments. We added sample to the chip for connection reaction. After LAMP reaction and CRISPR reaction, positive samples were observed under fluorescence microscope, which indicated that the positive group produced better fluorescence.
2. Temperature Control - Provide Reaction Temperature for Biological Reactions
The temperature control module provides a constant temperature for each of the three reactions constructed by the biological part. This will help us achieve the goal of instrument miniaturization, low power consumption and high control accuracy. The principle verification platform of the temperature control module constructed by us is shown in the following figure. We tested the heating rate, module temperature control accuracy, temperature accuracy, temperature uniformity and LAMP reaction. We demonstrated that it can provide a suitable temperature for biological reactions
2.1 Heating rate
Open the software, observe and record the real-time temperature curve and data. Real-time temperature changes according to set temperature value and program setting. Set linkage reaction (85℃，2min；37℃，5min); LAMP reaction (67℃，15min) and CRISPR/Cas12a reaction (37℃，20min).
The room temperature is denoted as Ta and the target temperature is denoted as Tb. The time from Ta to Tb is denoted as t. The average heating rate is calculated according to Formula (15).
The heating rates were 2.26℃/s（85℃），0.36℃/s（37℃），1.68℃/s（67℃, respectively. And the condition of 85℃ and 67℃ can reach GB / T 1.1-2009 standard.
2.2 Module temperature control accuracy
Set the temperature control module to 67℃, constant temperature 10s, timing 30s and record the maximum temperature and minimum temperature. Half of the difference between the two is Δ. The maximum value of Δ (i = 1, 2... 6) in accordance with the difference between the measured value and the set temperature should not be greater than 0.5℃.
2.3 Temperature ac
Set the temperature control module to 67℃, constant temperature 10s, timing 60s and record the temperature. They are The absolute value of the difference between the average value and the set temperature should not be greater than 0.5 °C.
2.4 Using LAMP Reaction to Test Biological Function
Label LAMP reagent with fluorescent dye and put it into four chambers of slide chip center with negative control group. Then provide temperature for LAMP reaction.
The results of fluorescence microscopy are shown in Fig.26.It is found that obvious fluorescence could be observed in the chamber of LAMP reagent and the difference of fluorescence brightness is small, while there is no fluorescence in the chamber of negative control.
The fluorescence intensity is detected by enzyme-labeled instrument. As shown in Fig.27, it is found that the LAMP reagent could detect high fluorescence content in the cavity and the difference is small, while the negative control have no fluorescence in the cavity.

Fig.22.23 Observation of fluorescence microscope and the detection results of microplate reader

This shows that the temperature control module constructed for this project can meet the requirements of biological detection. The temperature control module provides a suitable reaction temperature for the biological reaction and can achieve the expected detection effect. At the same time, the reaction conditions (temperature) of biological reactions in different reaction chambers are basically the same, which can ensure the parallelism of biological experiments.
3 Fluorescence Detection - Detection of Biological Results
We construct a method for converting miRNA content into fluorescence signal based on CRISPR/Cas12a system in the biological part. The fluorescence detection module constructs a multi-channel, high detection accuracy, high repeatability and high linearity fluorescence detection method for slip-chip to realize the quantitative detection of miRNA. By constructing the standard curve, we can covert the fluorescence intensity to miRNA content.
In order to prove that our fluorescence detection module can achieve the expected goal, which is to achieve accurate fluorescence detection and obtain correct detection results, we carried out corresponding tests.
3.1Precision
Within the measuring range of the instrument, select 6 measuring chambers. We prepared calibration fluorescent dye solutions for each chamber and tested them. Each calibration dye with concentration of 0.1, 1, 10, 100 μmol/L was detected once. The optical system collected the data of the target channel and calculated the average value M and standard deviation SD of the measurement results of each concentration calibration dye. According to the formula, the coefficient of variation CV is not greater than 5%.
CV=SD/M×100%
CV is coefficient of variation, SD is standard deviation; M is the average value of the measurement results.
3.2 Different channel fluorescence interference and scanning
Fluorescence Interference of Different Channels and Scanning Test
The six chambers of the sliding chip were placed with different concentrations of fluorescent reagents of 0.2, 0.5, 0.8 μmol / L ) as shown in the table. The optical system was controlled by a self-sliding push rod to obtain the fluorescence intensity value, and the fluorescence value curve was plotted. The results are shown in the following figure.
It can be seen from the results that the fluorescence signal intensity between the hole 1 and 2, 3 and 4, 5 and 6 is basically similar, indicating that the crosstalk problem can be controlled to a certain extent. The fluorescence value of hole 1, 2 and 3, 4 and 5, 6 can be clearly distinguished, indicating that the instrument has the ability to detect different concentrations of fluorescent dyes.
The data curves before and after Kalman filtering are plotted respectively as shown in the following figure. The effect of the filtering algorithm on the fluorescence detection data can be clearly seen, and the peak detection is performed on the filtered algorithm. The results are shown in the following table, which is the fluorescence value of the material in the 6 chambers. List the peak detection results in the table and draw a histogram as follows.The fluorescence intensity signal can be effectively obtained by peak detection algorithm, indicating that the constructed multi-channel scanning system is basically correct in principle.
3.3 Linear Test and Construction of Standard Curve
The known concentration standard fluorescent dye was diluted to 0.1, 0.2,..., 0.9 μmol/L sodium fluorescein solution in gradient and different concentrations of sodium fluorescein solution were detected. Constructing fitting curve by mean value of concentration and fluorescence measurement and the calculated linear correlation coefficient r is 0.9976. The fitting curve formula is y = 0.1469x. These results show that the linearity is good and it can be used as a standard curve for the conversion between the concentration of fluorescein sodium solution and the fluorescence measurement value.

Fig.30.31 Operational process and Sodium fluorescein solution of the different concentration

3.4 Functional test of fluorescence detection module
After completing the construction of each part of the previous fluorescence detection module, we conduct the overall functional test to prove whether our fluorescence detection module can achieve the expected goal. The function detection of the fluorescence detection module includes: the use of CRISPR/Cas12 experiment on the slip-chip; the corresponding fluorescence value is obtained by fluorescence detection; the content value of miRNA is converted from the standard curve; the prognostic risk score model is constructed by the content value to obtain the risk classification. Our test steps and results are as follows：
1.First, the slip-chip was used for experiments to obtain the fluorescence results corresponding to different pore positions, as shown in the following figure. The four hole positions of the slip-chip detected as positive samples and negative samples respectively. The sample in the hole is shown in Fig.21. After the experiment was completed, the fluorescence detection was performed using the equipment constructed in this module to obtain the fluorescence content of the positive hole.
2.In the part of linear test and standard curve construction, we obtained the relationship curve between the concentration of fluorescein sodium and the fluorescence detection value. However, because of time, we did not do the relationship curve between miRNA and fluorescence value, here use the relationship curve between fluorescein sodium and fluorescence value as an alternative to illustrate the problem.
Four of them were selected as the fluorescence concentration of four miRNAs, and then the fluorescence concentration of four miRNAs was converted to the fluorescence content through the standard curve. The fluorescence concentrations of four positive holes from top to bottom were 0.2, 0.3, 0.4 and 0.6 so the corresponding sample contents were 0.088, 0.059, 0.044 and 0.029, respectively.
3.In the modeling part, we constructed a prognostic risk score model related to patient survival : Risk Score= -0.328×miR144_5p_1 + 0.496×miR193a_3p_1 + 0.578×miR126_5p_1 - 0.606×miR15b_3p_1 Substitute the resulting four miRNA levels into the scoring model and obtain the final risk score. We can get the Ris Score is 0.008. According to the scoring rules(Score≤0.34 belongs to low-risk group; 0.34＜score≤0.36 belongs to low-risk group; 0.36＜score≤0.44 belongs to middle-risk group; 0.44＜score≤0.46 belongs to high-risk group; score＞0.46 belongs to high-risk group）, this sample belongs to the low-risk group.
4. Support Structure-Integrated Hardware
4.1Assembly of instrument
We designed the corresponding support structure to integrate each module, The physical picture is shown as follows.

Fig.36.37.38 Inside,Structure and Appearence

4.2 Instrument operation
Users can complete the use of the instrument only by adding samples according to the operation tips of WeChat mini-app. As shown, pull out the main part of the reaction for reagent addition.
Software − Data interaction and control
In order to apply the end-user experience and meet the POCT test scenario, we adopted the WeChat mini-apps on the smartphone to deploy the system using the WeChat developer tool. The results are as follows:
We mainly realize the team introduction, Bluetooth control, temperature data visualization, fluorescence results display, as shown below.

Fig39.Team introduction Fig40. Bluetooth clntrol

Fig41. Temperature data visualization Fig.42 Fluorescence results

Summary
We have proved that our project is feasible through experiments. In this year 's project, we successfully developed a cancer detection platform for early screening of colorectal cancer:
In summary, we have successfully verified the feasibility of the project. The advantages of our project are as follows:














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