Design general description
To address the problem of chronic musculoskeletal pain (CMP) classification, we developed a programmable quantitative detection device for multiple miRNAs based on the HCR+CRISPR system and paper-based chips. Based on the designed biosensing method, the system uses a portable paper chip and an incubator to integrate all aspects of the detection and realizes interaction and visualization through a smartphone.
Technical background
Since the target detection object of this project, miRNA, is short and its concentration in the sample is very small, the detection of miRNAs often requires the use of nucleic acid amplification technology to achieve signal amplification. At present, the commonly used nucleic acid amplification technology is mainly divided into two categories: PCR (Polymerase Chain Reaction) and nucleic acid isothermal amplification technology. Since PCR technology requires a thermal cycler to provide temperature changes, to realize POCT detection, we chose the appropriate amplification method from the isothermal amplification technology, which is not limited by the use of instruments. Among them, the enzyme-free nucleic acid isothermal amplification technology only has the DNA hybridization process and does not require the reaction of cyclic enzyme digestion. Compared with the enzyme-mediated nucleic acid isothermal amplification technology, the enzyme-free nucleic acid isothermal amplification technology avoids the restrictions brought by the use of enzymes on the transportation and preservation of reagents, which is more in line with the application requirements of this project. Hybridization chain reaction(HCR) is a commonly used technique for enzyme-free isothermal amplification of nucleic acids, which is a triggered non-enzymatic process of self-assembly in which single nucleotide inputs initiate a cascade of hybridization to form long-strand DNA outputs with notches [1]. HCR has been widely used due to its advantages such as rapid isothermal reaction without enzymes.
The CRISPR/Cas12a system can nonspecifically cleave single-stranded DNA after specific recognition of the target DNA, showing great potential for molecular diagnostics. In the detection of target molecules, CRISPR/Cas12a system can improve the specificity and sensitivity of detection, realize the transduction of target signals, and facilitate the visualization of results.
Biosensor design
Overview
In this system, miRNA was used as the detection target. Due to the small size of miRNAs and difficulty to detect, amplification technology is often used in miRNA detection to achieve signal amplification. HCR technology is an enzyme-free nucleic acid polymerization reaction with high compatibility and throughput and can amplify several targets to detectable levels within 30 minutes under isothermal conditions. To realize POCT, HCR technology was used to perform isothermal amplification of target miRNAs. On this basis, the CRISPR/Cas12a detection system was introduced to assist in the specific detection of HCR amplification products.
Biomarker
At present, the literature[2] has confirmed the feasibility of using miRNAs as a biomarker to determine the origin of chronic pain, and 14 miRNAs related to pain classification were screened out in this study. Based on the above research, this project designed a variety of algorithms including ROC, logistic regression, and decision tree, and selected two highly correlated c-miRNAs— has-miR-7d-5p and has-miR-98-5p, as biomarkers for pain classification. This combination has a reasonably good predictive value for distinguishing between neuropathic and nociceptive origins of pain.
Description of the steps in our design
- The blood of patients was collected for extraction to obtain samples that may contain the miRNAs to be tested
- Perform hybrid chain reaction (HCR) isothermal amplification on the miRNAs to be tested
- Identify and cleave specific miRNA sequences using CRISPR/Cas-based detection systems
- The POCT device utilizes paper chips combined with incubation devices and smartphones to provide visualized results of miRNA concentrations for pain classification based on mathematical models.
Amplification
In this project, the enzyme-free nucleic acid polymerization reaction (HCR) was used to perform isothermal amplification of the miRNA signals in the samples. HCR only initiates a cascade reaction of several self-stable DNA stem-loop structures by the target molecule to produce a long DNA structure with an incision. The HCR reaction process does not require the assistance of enzymes to avoid the influence of non-specific amplification on the analysis results. The reaction conditions are mild and easy to control. Under isothermal conditions, the amplification of short-strand DNA can be achieved in a one-step reaction and does not require complex instruments and equipment, so it is easy to realize the detection process with high sensitivity and low operation difficulty. The traditional HCR mechanism uses two hairpins, H1 and H2. Each starting target molecule can trigger a hybrid chain reaction to form an ultra-long DNA strand, and the signal of the target molecule can be amplified by the hybrid chain reaction. We optimized the traditional HCR system based on the study of Hai-Yan Jia[3] et al. . The new system contains three hairpins, an H1 variable hairpin, and H2 and H3 universal hairpins. The H1 variable hairpin greatly improves the programmability of the HCR system and enables the system to detect a variety of different miRNAs, which is helpful to realize the pain classification process based on multiple miRNA detection.
Visible readout
In this project, the CRISPR/Cas system was introduced downstream of the biological route to assist the specific detection of HCR amplification products, and then fluorescence signals were generated to realize signal transduction.
In this CRISPR system, crRNA can specifically recognize the amplified sequence of the target miRNA and then activate the side-chain cleavage activity of the Cas12a protein [4] [5]. The Cas12a protein can specifically recognize the fluorescent probe and nonspecifically cleave nucleic acid-labeled fluorophore quenchers (FQ), achieving specific fluorescence detection of target nucleic acids. The introduction of the CRISPR/Cas system reduced false positive results in detection, improved the detection limit of reaction, enhanced the sensitivity and specificity of detection, and realized signal transduction.
In this project, through HCR, the isothermal amplification of the miRNAs to be tested is realized, and the downstream CRISPR/Cas12a system recognizes and cleaves HCR amplification products to generate fluorescence signals. The relationship between fluorescence intensity and miRNA concentration was established to achieve quantitative detection. The design of the biosensor makes full use of the advantages of HCR enzyme-free isothermal amplification and the high specificities of CRISPR and combines the two to form HCR-CRISPR/Cas system, and the programmability of the system was improved by optimizing the HCR system, which could quantitatively detect multiple target miRNAs with high sensitivity and specificity.
[1] Dirks RM, Pierce NA. Triggered amplification by hybridization chain reaction[J]. Proc Natl Acad Sci U S A. 2004 Oct 26;101(43):15275-8.
[2] Dayer CF, Luthi F, Le Carré J, et al. Differences in the miRNA signatures of chronic musculoskeletal pain patients from neuropathic or nociceptive origins[J]. Plos one, 2019, 14(7): e0219311.
[3] Jia HY, Zhao HL, Wang T, Chen PR, Yin BC, Ye BC. A programmable and sensitive CRISPR/Cas12a-based MicroRNA detection platform combined with hybridization chain reaction[J]. Biosens Bioelectron. 2022 Sep 1;211:114382.
[4] SWARTS DC, JINEK M. Mechanistic insights into the cis- and trans-acting DNase activities of Cas12a [J]. Mol Cell, 2019, 73: 1‒12.
[5] STRECKER J, JONES S, KOOPAL B, et al. Engineering of CRISPR-Cas12b for human genome editing [J]. Nat Commun, 2019, 10(1): 212.