Overview
In this project, a biosensing system based on HCR-CRISPR was constructed to realize the recognition and amplification of the target miRNA molecular signal and to output the amplified molecular signal in the form of fluorescence ( as shown in Fig.1 ). At the same time, the project builds a supportive device module based on paper-based chips and smart phones, and combines it with biosensing systems to ultimately achieve the integration and portable detection of miRNA. In order to realize the system, we divide the verification experiment into three parts: feasibility verification and optimization of HCR, feasibility verification and detection of HCR + CRISPR, and feasibility verification based on paper-based chip. We designed corresponding H1 probes for two miRNAs ( miR-98-5p and miR-7d-5p ) and carried out experiments respectively. Next, we describe the results of miR-98-5p detection.
HCR reaction
Feasibility verification of HCR reaction
In order to verify the feasibility of HCR detection of miRNA, we carried out HCR reaction experiments based on miR-98-5p.
CF triggers HCR reaction
The trigger chain CF was added to 1μM H2 and H3 according to the concentration gradient of 100nM, 300nM, 600nM, 1μM ( The trigger chain CF refers to the sequence that triggers the downstream HCR reaction after the H1-98-5p stem-loop structure is opened by miR-98-5p in Fig.2 ).
The reaction is carried out at room temperature for 30 min and the products are electrophoresed on gel. The results are shown in Fig. 2. It can be seen that Lane 1, Lane 2, Lane 3 and Lane 4 all have tailing phenomenon, which means that there are different sequences of relative molecular mass in the corresponding HCR results of this lane. Lane 5 does not have tailing phenomenon, which means that HCR reaction cannot be triggered in the absence of trigger chain CF. Therefore, it is proved that the HCR reaction is triggered by the trigger chain CF, and different concentrations of trigger chain CF can trigger the HCR reaction.
1: CF(100nM)+ H2+H3;
2: CF(300nM) +H2+H3;
3: CF (600nM) +H2+H3;
4: CF(1μM)+ H2+H3;
5: H2+H3;
6: D2000
miR-98-5p triggers HCR reaction
In the HCR system, the reaction is carried out as shown on the right side of the following diagram.The reaction is carried out at room temperature for 30 min and the products are electrophoresed on gel, the results are shown in Fig. 3. HCR occurred in Lane 1, Lane 2 and Lane 3, corresponding to the HCR reaction triggered by the trigger chain CF, Lane 4 is a negative control, In Lane 5,miR-98-5p opened H1-98-5p,but the reaction lacked H3 did not trigger HCR reaction, Lane 6 and Lane 7 did not respond; no band was produced in Lane 8 with a single-stranded nucleic acid.
The results showed that miR-98-5p could open the circular H1-98-5p probe and trigger the HCR reaction.
2: miR-98-5p(400nM)+H1+H2+H3;
3: miR-98-5p(200nM)+H1+H2+H3;
4: H1+H2+H3;
5: miR-98-5p(1μM)+H1+H2;
6: miR-98-5p(1μM)+H1；
7: H1;
8: miR-98-5p(1μM);
9: D2000.
optimization
To screen for better reaction conditions and provide a better environment for the HCR + CRISPR reaction, we optimized several aspects of the HCR system.
H1 stem-loop length
In order to optimize the experimental system, we adjusted the stem-loop length of H1-98-5p, and constructed H1-98-5p probes with stem lengths of 8, 11, and 13, which referred to as H1-8, H1-11 and H1-13. The results of the reaction between HCR system and miR-98-5p were obtained by experiments. The gel electrophoresis results of H1-98-5p are shown in Fig. 4.
It can be seen from Fig.4 that the length of H1-98-5p stem-loop is different, and the degree of HCR reaction is different. The more sufficient the HCR reaction, the more obvious the bands. Among them, Lane 1 is H1-13, with weak a HCR reaction ; Lane 3 is H1-11, HCR reaction is better than H1-13 trigger effect ; Lane 5 is H1-8, and the HCR reaction works best. When miR-98-5p was not added, HCR reaction could not be triggered.
Therefore,we chose the H1-8 probe.
2: H1-13+H2+H3;
3: H1-11+H2+H3+miR-98-5p(300nM);
4: H1-11+H2+H3;
5: H1-8+H2+H3+miR-98-5p(300nM);
6: H1-8+H2+H3;
7: D2000
H1 Probe ratio
The results of the reaction between HCR system and miR-98-5p were tested when the concentration of H1-98-5p was 0.1μM, 0.2μM, 0.4μM, 0.6μM, 0.8μM and 1.0μM, respectively. The results of gel electrophoresis are shown in Fig. 5.
Significant HCR response was triggered at lane 1-3, and the degree of HCR reaction was different with the concentration of H1-98-5p. The more HCR reaction goes on, the more obvious the band, and the best reaction was 0.6μM and 0.8μM. Considering the saving of raw materials, we chose 0.6μM.
2: miR-98-5p+H1-98-5p（0.8μM）+ H2+H3+Na+；
3: miR-98-5p+H1-98-5p（0.6μM）+H2+H3+Na+；
4: miR-98-5p +H1-98-5p（0.4μM）+H2 +H3+Na+;
5: miR-98-5p +H1-98-5p（0.2μM）+H2+H3+Na+；
6: miR-98-5p +H1-98-5p（0.1μM）+ H2+H3+Na+
7: H1-98-5p+H2+H3+Na+；
8：D2000.
Na + concentration
The results of the reaction between HCR system and miR-98-5p were tested when the concentration of Na + was 95mM, 140mM, 350mM, 550mM, 750mM. The sodium ion concentration optimization results are shown in Fig.6.
It can be seen that there is no miR-98-5p in the Lane 6 HCR system, which can not trigger the HCR reaction, and there is no false positive ; Lane 1-5 can trigger HCR reaction. The degree of HCR reaction is different with different sodium ion concentration. The more HCR reaction goes on, the more obvious the band. It can be seen that the reaction result of 350mM Na + is the best.
1:miR-98-5p+H1-98-5p+H2+H3+Na+(750mM);
2:miR-98-5p+H1-98-5p+H2+H3+Na+(550mM);
3:miR-98-5p+H1-98-5p+H2+H3+Na+(350mM);
4:miR-98-5p+H1-98-5p+H2+H3+Na+(140mM);
5:miR-98-5p+H1-98-5p+H2+H3+Na+(95mM);
6:H1-98-5p+H2+H3+Na+(350mM);
7:D2000.
HCR + CRIPSR system
Feasibility
Positive template triggers HCR response
First, we used positive templates to preliminarily prove that our HCR + CRISPR reaction is feasible. The positive template is a double-stranded DNA synthesized with fragments of H2 and H3 that can be recognized by crRNA. We used different concentrations of positive templates to trigger the CRISPR reaction, and the image of its fluorescence intensity changing with time is shown in Fig.7. From the diagram, it can be seen that compared with the blank group, the addition of the positive template can significantly increase the fluorescence intensity. And in the concentration range involved in the experiment, the higher the concentration of positive template, the higher the fluorescence intensity.
Blank indicated that no positive template was added. The concentrations of 0.5μM, 1.25μM and 2.5μM represent the concentration of the added positive template, respectively.
Verification of HCR + CRISPR reaction based on miR-98-5p
The miR-98-5p was added for CRISPR reaction, and a blank control group without miR-98-5p was set up. The fluorescence intensity obtained by analysis is shown in Fig. 10. When miR-98-5p was added, it showed strong fluorescence intensity ; when miR-98-5p was not added, the fluorescence intensity was weak, which was significantly different from the other group, and the false positive was weak.
Blank represents the control group without miR-98-5p.
optimization
HCR + CRISPR Reaction Based on Different H1 Probe Stem Lengths
HCR + CRISPR reactions based on probes with different stem lengths H1-8, H1-11, and H1-13 were performed. Blank indicated a control group without miRNA. The results are shown in Fig.9. It can be seen from the figure that H1-8 and H1-11 showed strong fluorescence intensity when miRNA was added. When miRNA was not added, the fluorescence intensity corresponding to H1-8-Blank was weak, which was significantly different from H1-8. Therefore, we believe that the H1-8 reaction works best.
H1-8, H1-11, and H1-13 represent the length of the stem-loop exposed at the end of the H1-98-5p probe, respectively. Blank represents the control group without miR-98-5p.
HCR + CRISPR Reaction Based on Different Concentrations of Fluorescent Probes
For HCR + CRISPR reactions based on different concentrations of fluorescent probes, Blank represents the control group without miR-98-5p. The results are shown in Fig.10. As can be seen from the figure, at different fluorescent probe concentration,the effects of 5μM fluorescent probe (reporter) and 10μM are not much different, so we select the 5μM fluorescent probe.
Reporter-2.5μM indicated that the concentration of the fluorescent probe was 2.5μM. Similarly, Reporter-5μM, 7.5μM, and 10μM indicated that the concentration of the fluorescent probe was 5μM, 7.5μM, and 10μM, respectively. Blank represents the control group without miR-98-5p.
Specificity test
Different kinds of miRNA were added to the HCR + CRISPR system corresponding to miR-98-5p for CRISPR reaction. The blank control group without miRNA ( Blank ) was set up, and the fluorescence intensity was analyzed. The results are shown in Fig.11. The miR-98-5p can trigger HCR + CRISPR reaction,the fluorescence effect is obvious. Other miRNAs do not trigger the HCR + CRISPR reaction.The above results prove that the HCR + CRISPR reaction system has recognition specificity for target miRNA.
Blank represents the control group without miRNA.The three groups of miR-98-5p, miR-320a, miR-7b-5p represent the results obtained by adding miR-98-5p, miR-320a, miR-7d-5p, respectively.
Detection limit determination
We detected the fluorescence intensity of Cas12a activation by HCR results triggered by different concentrations of miR-98-5p. The obtained fluorescence results are shown in Fig.12 ( A ), and the results are linearly fitted, as shown in Fig.12 ( B ).
The results showed that the fluorescence intensity of miR-98-5p at different concentrations was linearly related to the cutting time. The dynamic detection range of miR-98-5p is 10 pM to 300 nM as shown in Fig.12 ( A ). In addition, the fluorescence intensity increased linearly with the logarithm of miR-98-5p concentration in the range of 300nM to 10pM ( R2 = 0.9605 ). The LOD is 10 pM. From 10 pM to 100 nM, the fluorescence intensity was linearly related to the logarithm of miR-98-5p. As shown in Fig.12 ( B ).
(A)Fluorescence Intensity of Cas12a Activation by HCR Results Triggered by Different Concentrations of miR-98-5p.(B)Linear fitting of fluorescence intensity versus logarithm of miR-98-5p concentration.
Hardware-related experiments
compatibility test
In order to test the compatibility of hardware and biological reaction, we first carried out HCR reaction experiments in solution systems with different carriers, including HCR reaction in pure biological process ( laboratory environment ), HCR reaction on reaction pad in a solution system, and HCR reaction on paper-based chip in solution system.
HCR reaction in laboratory environment
In the laboratory environment, different concentrations of miR-98-5p were added to the new HCR system and reacted at 37 ° C and pH 7.5 for 30 min to obtain gel electrophoresis results, as shown in Fig.13.
It can be seen that when miR-98-5p is added to the HCR system (Lane 1-3), the HCR reaction can be triggered. The higher the miR-98-5p concentration, the more complete the HCR reaction and the more obvious the bands. When there was miR-98-5p without H1-98-5p in HCR system (Lane 4) or H1-98-5p without miR-98-5p in HCR system (Lane 5), HCR reaction could not be triggered, and there was no false positive.
1: miR-98-5p (50nM)+H1-98-5p+ H2+H3；
2: miR-98-5p (200nM)+H1-98-5p+H2+H3
3: miR-98-5p(600nM)+H1-98-5p +H2+H3；
4: miR-98-5p (600nM))+ H2 +H3;
5: H1-98-5p+H2+H3；
6: D2000
HCR Reaction on Reaction Pad in Solution System ( Wet Paper Base )
The gel electrophoresis results obtained by repeating the previous experiment with wet paper as the carrier are shown in Fig.14. There was no miR-98-5p in H1-98-5p in the HCR system shown in Lane 2, which could not trigger the HCR reaction, and the false positive was not obvious. In the HCR system shown in Lane 3,there exists miR-98-5p has no H1-98-5p, so the HCR reaction cannot be triggered , and there is no false positive ;
Lane 4 ~ 6 trigger HCR reaction, and according to the order of Lane 6, Lane 5, Lane 4, the higher the miR-98-5p concentration, the more HCR reaction, and more obvious the bands.
In general, the reaction results are not much different from the laboratory environmental reaction results. It can be seen that the HCR system on the wet paper can react normally, that is, the HCR system can tolerate the paper-based reaction environment.
1: D2000；
2: H1-98-5p+H2+H3；
3: miR-98-5p (600nM))+ H2 +H3；
4: miR-98-5p (600nM)+H1-98-5p +H2+H3；
5: miR-98-5p (200nM)+H1-98-5p +H2+H3；
6: miR-98-5p (50nM)+H1-98-5p+ H2+H3.
tolerance test
In order to meet the application requirements in non-solution environments, we need to fix the reaction system in a paper-based chip. We chose to use a dry method to fix the system and performed a tolerance test-reaction on a dry reaction pad.
HCR reaction on dry reaction mat
In addition, we use dry paper as the carrier, with different concentrations of trigger chain to trigger the HCR reaction, the results are shown in Fig. 15. The results further prove that the HCR system on dry paper can react normally, and the reaction results are more complete than the laboratory environmental reaction results.
1: D2000；
2: H2+H3；
3: CF(1μM)+ H2+H3 ；
4: CF(600nM) +H2+H3；
5: CF(300nM)+ H2+H3
HCR + CRISPR reaction on paper-based chips
On a paper-based chip, we performed an overall verification of the HCR + CRISPR two-step reaction. It has been verified that the paper-based chip can occur normally and output fluorescence normally. The results are shown in Fig.16. It can be seen that the fluorescence generated when the probe is normally fixed and the corresponding reagent is injected is in line with expectations. No fluorescence was detected when the reaction pad of the unfixed HCR probe and the water was injected, and the pictures were basically the same. It is proved that the paper-based chip can normally generate and output fluorescence.
It can be seen that when the reaction pad normally fixes the probe and injects the corresponding reagent, the fluorescence generated is in line with expectations, and no fluorescence is detected when the reaction pad of the unfixed HCR probe and the water is injected. It is proved that the paper-based chip can normally produce the reaction and output fluorescence.