Electrochemiluminescence Biosensor for MMP-2 Analysis Using CRISPR/Cas13a and EXPAR Amplification: A Novel Approach for Anti-Aging Research

Electrochemiluminescence Biosensor for MMP-2 Analysis Using CRISPR/Cas13a and EXPAR Amplification: A Novel Approach for Anti-Aging Research | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Electrochemiluminescence Biosensor for MMP-2 Analysis Using CRISPR/Cas13a and EXPAR Amplification: A Novel Approach for Anti-Aging Research Qiang Tang, Jie Wang, Jiayi Zhang, Hongyu Zeng, Zhixue Su, Xiying Zhu, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4689418/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 14 Oct, 2024 Read the published version in Microchimica Acta → Version 1 posted 9 You are reading this latest preprint version Abstract Matrix metalloproteinase-2 (MMP-2) plays a pivotal role in anti-aging research. Developing advanced detection platforms for MMP-2 with high specificity, sensitivity, and accessibility is crucial. This study introduces a novel electrochemiluminescence (ECL) biosensor for MMP-2 analysis, leveraging the CRISPR/Cas13a system and Exponential Amplification Reaction (EXPAR). The biosensor operates by utilizing the T7 RNA polymerase to transcribe RNA from a DNA template upon MMP-2 interaction. This RNA activates Cas13a, leading to signal amplification and ECL detection. The incorporation of the "photoswitch" molecule [Ru(phen) 2 dppz] 2+ streamlines the process by eliminating the need for extensive electrode modification and cleaning. Under optimized conditions, the biosensor achieved an impressive detection limit of 12.8 aM for MMP-2. The platform demonstrated excellent selectivity, reproducibility, and stability, making it highly suitable for detecting MMP-2 in complex biological samples. This innovative approach shows great potential for applications in molecular diagnostics and anti-aging research. CRISPR/Cas13a MMP-2 T7 RNA Electrochemiluminescence Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction MMP-2, or matrix metalloproteinase 2, plays a pivotal role in anti-aging research. [ 1 , 2 ]Throughout the aging process, the disruption of MMP-2 contributes to the breakdown of the extracellular matrix (ECM), leading to wrinkles, sagging skin, and reduced elasticity. [ 3 – 5 ] Scientists investigate the signaling pathways and factors that regulate MMP-2 expression to identify potential intervention points. Research aimed at MMP-2 focuses on restoring ECM balance and mitigating skin aging. Both natural compounds and synthetic inhibitors, as well as various interventions, are used to regulate MMP-2's expression and activity. Given its role in degrading crucial ECM components, controlling MMP-2 is essential for maintaining skin health. [ 4 – 6 ] Thus, detecting MMP-2 is significant in anti-aging studies. [ 7 ] Currently, there are multiple methods available to detect MMP-2, including ELISA, [ 8 ] gelatin zymography, [ 9 ]Western blotting, [ 10 , 11 ]and immunohistochemistry.[ 12 ] However, each technique has its limitations regarding sensitivity, specificity, and reliability, with potential for false positives or negatives. Additionally, sample preparation and handling can impact results, and some methods require specialized equipment and expertise. The choice of detection method should depend on the sample type, the research question, and the resources available, with careful consideration of each method's limitations to ensure accurate and reliable results. The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas (CRISPR-associated) system originated from the adaptive immune system of prokaryotes, providing defense against invading nucleic acids. [ 13 – 15 ] In recent years, the CRISPR-Cas system has garnered significant attention from biological researchers as a highly effective gene editing tool. [ 13 , 15 – 18 ] Broadly, CRISPR/Cas systems can be categorized into Class I and Class II, based on whether they use single or multiple effectors. Class II systems, such as Cas9, Cas12, and Cas13, are particularly notable for their broad applications due to their simpler composition, consisting of a single effector protein and a programmable guide RNA. [ 19 – 25 ] Recent advancements in gene editing have led to notable discoveries, including new genome editing techniques. Specifically, the trans-cleavage activity discovered in Cas12a, Cas13a, and Cas14a has positioned the CRISPR/Cas system as a promising candidate for next-generation diagnostic biosensing platforms. [ 26 – 32 ] Among these, the CRISPR/Cas13a system, part of Class II type VI-A, stands out as the only RNA-targeted CRISPR effector with exceptional signal amplification capabilities. [ 14 , 16 , 23 , 33 ]Cas13a, along with its corresponding CRISPR RNA (crRNA), can precisely recognize and cleave target RNA based on crRNA-target complementarity. This recognition triggers its trans-cleavage activity, which further cleaves fluorophore-quencher-labeled RNA probes with remarkable turnover efficiency (approximately 4854), leading to amplified fluorescence detection signals. [ 24 , 26 , 34 , 35 ] To enhance sensitivity, various amplification strategies, such as recombinase polymerase amplification (RPA), have been integrated into the CRISPR/Cas system. Consequently, developing a new detection strategy that combines the high specificity and signal amplification capabilities of the CRISPR/Cas system with the efficiency of exponential nucleic acid amplification is highly valuable. [ 16 , 23 , 24 , 33 ] In addition, T7 RNA polymerase is an important tool for creating biosensors. The enzyme is an excellent tool for creating RNA probes to detect target analytes in biosensors because of its ability to efficiently and specifically convert DNA to RNA. Due to the specificity of the T7 RNA polymerase, RNA probes corresponding to the target analytes can be transcribed, resulting in accurate and sensitive detection.[ 16 , 36 – 38 ] T7 RNA polymerase can also be used to enhance RNA signaling in biosensors, resulting in T7 RNA polymerase has a variety of uses in molecular biology and biotechnology, but is not limited to the manufacture of biosensors. In addition, the use of T7 RNA polymerase allows for the synthesis of RNA vaccines, which synthesize RNA encoding target antigens to elicit an immune response, as well as RNA standards for qPCR testing.[ 39 – 41 ] In summary, T7 RNA polymerase is a flexible tool with multiple uses in molecular biology and biotechnology, including the construction of biosensors. Due to its selectivity, potency and ability to amplify RNA signals, T7 RNA polymerase can be used to create very precise and sensitive biosensors. Electrochemiluminescence (ECL) is a chemiluminescence technique that utilizes electrochemical stimulation by applying a voltage or electric current to the surface of an electrode or an electrolyte to trigger a exothermic redox reaction in a substance, resulting in the production of molecules or free radicals in the excited state and the release of photons as they return to their ground state.[ 22 , 28 , 42 ] This process has a number of advantages. First, the background noise is very low because only substances in which exothermic redox reactions are present can emit light, excluding the influence of other interfering substances. Secondly, electrochemiluminescence has a wide dynamic range and the intensity of light can be controlled by adjusting electrical parameters, thus enabling the detection of substances at different concentrations.[ 16 , 19 , 43 – 46 ] In addition, it has high sensitivity, and the luminescence efficiency can be enhanced by co-reactants or co-catalysts to realize the detection of very low concentration substances. Most importantly, it is simple to operate, requiring only a simple electrochemical cell and a photodetector for fast, convenient and cost-effective analysis.[ 30 , 47 – 54 ] In this study, we integrated T7 RNA polymerase into a CRISPR/Cas13a amplification-based electrochemiluminescence (ECL) biosensing platform for the ultrasensitive and specific detection of MMP-2. The method involves an MMP-2-interacting probe that, in the presence of T7 RNA polymerase, amplifies a substantial amount of RNA. This amplified RNA activates Cas13a, which precisely identifies miRNA targets and acts as a primer for subsequent exponential amplification. Unlike earlier Cas13a-based RNA detection approaches, our method harnesses the full potential of Cas13a's RNA recognition and signal amplification capabilities. Both the trans-cleavage activity and isothermal exponential amplification (EXPAR) of Cas13a enhance the assay's sensitivity. Furthermore, the RNA amplification mediated by T7 RNA polymerase upon MMP-2 interaction allows for highly specific MMP-2 detection.Additionally, incorporating the photoswitch [Ru(phen) 2 dppz] 2+ into the detection platform eliminates the need for complex electrode modification and cleaning procedures. [Ru(phen) 2 dppz] 2+ shows a significant luminescence increase when it interacts with double-stranded DNA (dsDNA). This enhancement occurs because the planar phenazine ligand of [Ru(phen) 2 dppz] 2+ binds with the base pairs in the major groove of dsDNA, protecting the nitrogen atoms of phenazine and boosting the luminescent state population.[ 34 ] This property is crucial for amplifying the luminescence signal, which is essential for detecting the presence of the target. This innovation simplifies the experimental workflow, reduces detection costs, and improves reproducibility. We also evaluated the sensing system's applicability for detecting MMP-2 in serum samples. 2. Experimental section 2.1 Materials and chemicals All oligonucleotides utilized in this study were sourced from Genscript Biotechnology Co., Ltd (Nanjing, China), and their respective sequences are documented in Table S1 . The PNA (Peptide Nucleic Acid) employed in this research was provided by TAHE-PNA Co., Ltd. (Hangzhou, China). All other chemicals, which met reagent-grade standards, were used without further purification. Solutions were prepared using ultrapure water that underwent purification through the Milli-Q purification system (Branstead), resulting in a specific resistivity exceeding 18.2 MΩ·cm. 2.2 Instruments and measurements The detection of the electrochemiluminescence (ECL) signal was performed using a specialized ECL instrument, which was generously provided by the State Key Laboratory of Analytical Chemistry for Life Sciences at Nanjing University. For the ECL measurements, a three-electrode system was utilized. This configuration included a glassy carbon working electrode (GCE), which served as the primary site for electrochemical reactions, an Ag/AgCl reference electrode to maintain a stable reference potential, and a platinum counter electrode to complete the circuit and facilitate the overall electrochemical process. 2.3 MMP-2 proteolytic cleavage, enzymatic amplification mediated by T7 RNA polymerase, and activation of CRISPR/Cas13a To create the PNA/T7 promoter/DNA template complex, the T7 promoter, DNA template, and PNA were combined in an HES buffer with 1 U µL⁻¹ of RNase inhibitor, at a ratio of 1:1:1.2. This mixture was incubated at 41.5°C for 15 minutes, then gradually cooled to room temperature and kept at 4°C overnight, resulting in the successful synthesis of the PNA/Bandage complex. For the release of the DNA template/T7 promoter duplex triggered by MMP-2, recombinant human MMP-2 was activated in an AC buffer (50 mM HEPES, 10 mM CaCl₂, 150 mM NaCl, 0.05% (w/v) Brij 35, pH 7.0) at 37°C for 2 hours. After activation, different amounts of MMP-2 were added to the PNA/T7 promoter/DNA template mixture and incubated for 30 minutes. Subsequently, 50 µL of this mixture was added to an amplification reaction system to a final volume of 100 µL. This system contained 40 µM NTPs, 30 U T7 RNA polymerase, 1 µM DNA template, 20 U RNase inhibitor, and 2 µL of a 10× RNAPol reaction buffer. The mixture was incubated at 37°C for 40 minutes to enable transcription amplification. Post-transcription, the solution was incubated at room temperature for 20 minutes with 10 nM CRISPR/Cas13a and 15 nM gRNA in 1× NEBuffer (NEB). The biosensor was then immersed in the activated CRISPR/Cas13a solution for 20 minutes to cleave the pre-trigger DNA. 2.4 EXPAR amplification The Exponential Amplification Reaction (EXPAR) system required the preparation of two solutions, Solution A and Solution B. Solution A consisted of 0.1 µM template, 250 µM dNTP mix, and 1.2 µL of cleaved products from previous steps. Solution B contained 0.4 U/µL NEase Nt.BstNBI, 0.05 U/µL Vent (exo-) DNA polymerase, 1× Thermopol buffer, and 1× NEBuffer 3.1. These solutions were promptly mixed to form a final volume of 10 µL and incubated at 55°C for 30 minutes to facilitate exponential amplification. This process involved cleaved products generated through the activation and interaction of MMP-2 with the PNA/T7 promoter/DNA template complex. The mixture was incubated at 55°C for 30 minutes, ensuring efficient exponential amplification of target sequences. This protocol was essential for achieving the high sensitivity and specificity needed for MMP-2 detection in the developed ECL biosensing platform, significantly enhancing the accuracy and reliability of the detection method. 2.5 C haracterization of EXPAR Amplification and Cas13a Cutting Performance using Polyacrylamide Gel Electrophoresis (PAGE) To verify the experimental process of the characterization of EXPAR amplification and the cutting performance of Cas13a, we conducted polyacrylamide gel electrophoresis (PAGE). The samples, including the T7 promoter, DNA template, DNA template/T7 promoter duplex, products after T7 promoter amplification reaction based on DNA template/T7 promoter duplex, Pre-Trigger DNA, and CRISPR/Cas13a treated Pre-Trigger DNA, were loaded onto a 20% native polyacrylamide gel in 0.5× Tris-borate-EDTA (TBE). The gels were run at room temperature for 1 hour at 120 V. After electrophoresis, the gels were stained with a SYBR Green I mixture and photographed using a Bio-Rad digital imaging system. 2.6 ECL assay: A 25 µL solution was meticulously prepared, combining 15 µL of EXPAR products, 1 µL of a 0.5 × 10 − 3 M [Ru(phen) 2 dppz] 2+ solution, and 9 µL 70 × 10 − 3 M TPrA solution. This solution was meticulously crafted in a 0.01 M phosphate-buffered saline (PBS) with a pH of 7.4. Following this precise formulation, the resulting mixture was then expertly applied to the electrode. ECL signal detection was carried out using an advanced ECL instrument, provided by the State Key Laboratory of Analytical Chemistry for Life Sciences at Nanjing University. The three-electrode system employed consisted of a glassy carbon working electrode (GCE), an Ag/AgCl reference electrode, and a platinum counter electrode. This setup facilitated precise and efficient ECL measurements, contributing to the robustness and sensitivity of the MMP-2 detection platform. For the modification of the glassy carbon electrode (GCE), we followed a standard procedure to ensure optimal performance. The detailed process involves the following steps: The GCE surface was first polished to achieve a mirror-like finish, which is crucial for removing any surface impurities and ensuring a smooth, conductive surface. Alumina slurries of different particle sizes (e.g., 1.0 µm, 0.3 µm, and 0.05 µm) were used sequentially for polishing. The electrode was placed on a polishing cloth, and the alumina slurry was applied. The GCE was then polished in a figure-eight motion for about 5 minutes with each slurry, starting with the coarsest (1.0 µm) and moving to the finest (0.05 µm). This process ensures a smooth and clean electrode surface, essential for reproducible and reliable electrochemical measurements. After polishing, the electrode was thoroughly cleaned to remove any residual polishing particles and contaminants. The polished GCE was rinsed with distilled water to remove the bulk of the alumina particles, then sonicated in ethanol for 5 minutes, followed by sonication in deionized water for another 5 minutes to ensure that any remaining polishing debris or organic contaminants were removed from the electrode surface. 3. Results and discussion 3.1 Principle of the proposed ECL biosensor Scheme 1 provides an illustration of the platform and its operational principles. In this context, MMP-2 was chosen as the model target for the proof-of-concept. The Cas13a enzyme employed in this investigation is derived from Leptotrichia buccalis (LbuCas13a). The crRNA consists of a 30 nucleotide repeat region facilitating interaction with Cas13a, and a 20 nucleotide programmable guide region, often referred to as the spacer, designed for the recognition of target RNA. Notably, the trans cleavage activity of LbuCas13a exhibits a preference for cleaving the ribose-phosphodiester bond flanked by uracil. To leverage this preference, a Pre-trigger DNA was designed, incorporating two uracil ribonucleotides (rU). The 5′ end of this Pre-trigger DNA comprises 16 complementary bases with the amplification template and five random bases at the opposite end. The amplification template features a central nicking endonuclease (NEase) cleavage site and two separate repeat sequences, both of which are complementary to the 5′ end of the Pre-trigger DNA. Additionally, both the Pre-trigger DNA and the amplification template were equipped with a C3 spacer at their 3′-end to prevent target-independent polymerase amplification. A specific PNA sequence was designed, containing a peptide sequence (Gly-Pro-Leu-Gly-Val-Arg-Gly) that can be precisely cleaved by MMP-2. It is essential to note that PNA is a synthetic nucleic acid analog characterized by a peptide backbone rather than a sugar-phosphate backbone. In the context of the assay reaction, the process unfolds as follows: Initially, the PNA/T7 promoter/DNA template complex, featuring a double-stranded DNA template connected by PNA and T7 promoter, which encompasses a peptide bond cleavable by MMP-2. In the absence of MMP-2 within the system, the PNA/DNA template duplex inhibits the T7 promoter amplification reaction (as depicted in Scheme 1 A). However, with the introduction of MMP-2 into the system (as presented in Scheme 1 B), the cleavage of the peptide bond is triggered, leading to the release of the single-stranded DNA template/T7 promoter duplex. This released duplex assumes the role of a substrate for T7 promoter-aided transcription amplification. Subsequent transcription amplification generates a substantial quantity of single-stranded RNA transcripts, encompassing the target sequence that is identifiable and bindable by gRNA. Following the binding of gRNA to the transcript, it activates the nuclease activity of CRISPR/Cas13a, thereby enabling the cleavage of any single-stranded RNA molecules. The assembled Cas13a/crRNA system is designed to specifically recognize and cleave target RNA sequences. Upon recognition, Cas13a's two conserved higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domains reposition to form a new RNase active site, activating its trans-cleavage activity. This leads to the cleavage of the Pre-trigger DNA into two fragments. The 5′ fragment of the cleaved Pre-trigger DNA can hybridize with an amplification template, initiating a polymerization reaction in the presence of T4 polynucleotide kinase (PNK) and Vent DNA polymerase. The newly synthesized double-stranded DNA is then recognized and nicked by nicking endonuclease (NEase). This process involves a continuous cycle of nicking, strand extension, and displacement, resulting in the generation of substantial amounts of double-stranded DNA products. Notably, the trans-cleavage activity of Cas13a remains inactive without the presence of the specific portion of the Pre-trigger DNA. If the Pre-trigger DNA is not cleaved, it cannot be extended by DNA polymerase, thereby failing to initiate the downstream exponential amplification reaction (EXPAR). This mechanism ensures that only in the presence of the target RNA does the system proceed, thereby providing high specificity and sensitivity in detecting the target sequence. This feature is critical for applications requiring precise molecular detection, such as the developed ECL biosensing platform for MMP-2 detection. Subsequently, the complex [Ru(phen)2dppz]2 + is incorporated into the Cas13a-enhanced EXPAR (CAS-EXPAR) system. In its free state, this complex fails to emit luminescence due to nitrogen protonation in aqueous environments. However, when it binds to the double-stranded DNA products produced by EXPAR, there is a significant increase in luminescence. This boost in signal is linked to the planar phenazine ligand of [Ru(phen) 2 dppz] 2+ engaging with the base pairs in the major groove of the DNA. This interaction shelters the phenazine's nitrogen atoms, promoting a state conducive to luminescence. Following this, the [Ru(phen) 2 dppz] 2+ supplemented amplification system and excess TPrA are introduced into the biosensor platform. The electrochemiluminescence (ECL) signal, which serves as an indicator of MMP-2 activity, is detected through a photomultiplier tube (PMT, Hamamatsu R928 PMT). The reaction mechanism is as follows: [Ru(phen) 2 dppz] 2+ /DNA-e − →[Ru(phen) 2 dppz] 3+ +H + TPrA-e − → TPrA∙ + → TPrA∙ + H + [Ru(phen) 2 dppz] 3+ -DNA + TPrA∙ → [Ru(phen) 2 dppz] 2+∗ -DNA + products [Ru(phen) 2 dppz] 2+ ∗-DNA → [Ru(phen) 2 dppz] 2+ -DNA + hν 3.2 Feasibility study The CEISPR/Cas13a-based amplification system was subjected to thorough examination with regards to its electrochemiluminescence (ECL) performance. The results revealed a substantial enhancement in the ECL signal when the system was employed for detecting as low as 1 pM of MMP-2 (curve c). In stark contrast, the ECL signals observed were notably diminished in the absence of MMP-2 (curve a) or in the presence of a substantially lower concentration of MMP-2 (100 aM, curve b), as illustrated in Fig. 1 . These findings underscore the crucial role of the target MMP-2 in initiating the biosensor's activation. In response to the need for characterization of the EXPAR amplification and the cutting performance of Cas13a, we have conducted the necessary experiments. The results are detailed in Figures S1 and S2. Figure S1 presents the electrophoresis identification of the T7 RNA polymerase transcription-mediated amplification process, confirming the feasibility of the EXPAR amplification. Additionally, Figure S2 illustrates the cutting performance of Cas13a, demonstrating its efficiency in the process. These characterizations verify the reliability and effectiveness of the amplification and cleavage mechanisms used in our study. 3.3 Optimization of experimental parameters In our pursuit of enhancing the performance of the biosensor, we meticulously conducted a comprehensive optimization process, focusing on three pivotal experimental parameters. Employing ECL, we conducted an in-depth examination of the biosensor's current response under various conditions and compared it to a control group (as illustrated in Fig. 2 ). The parameters that underwent meticulous optimization included: Optimizing T7 RNA Polymerase Transcription Duration: As revealed in Fig. 2 A, the ECL signal achieved stability after 30 minutes. This observation was the result of a progressive sequence from curve a to curve g, with time intervals set at 5, 10, 20, 30, 40, 50, and 60 minutes. This analysis pinpointed the ideal duration for T7 RNA polymerase transcription, which was determined to be 30 minutes. Fine-Tuning CRISPR/Cas13a Reaction Time: As portrayed in Fig. 2 B, the ECL signal exhibited a gradual decline from 5 minutes to 30 minutes, corresponding to time intervals of 5, 10, 15, 20, 25, and 30 minutes. Ultimately, stability was reached after 25 minutes, underscoring the optimal 25-minute CRISPR/Cas13a reaction time that consistently yielded the most favorable results. Optimizing Cas13a/crRNA Concentration: The concentration of Cas13a/crRNA holds direct sway over its intrinsic cis-cleavage efficiency, consequently impacting the subsequent isothermal amplification efficiency. We introduced differing concentrations of Cas13a/crRNA (2.5 nM, 5 nM, 10 nM, and 20 nM) into the sensing system in a 1:1 ratio and subjected them to ECL analysis. Figure 2 C clearly indicates that the sensor treated with 10 nM Cas13a/crRNA exhibited the highest signal. However, once the concentration of Cas13a/crRNA was elevated to 20 nM, the ECL signal ceased to increase, signifying that an excess of Cas13a/crRNA failed to further enhance the ECL signal. Consequently, for the ensuing research, we opted for a 10 nM concentration of Cas13a/crRNA. This meticulous series of optimization steps not only served to amplify the biosensor's performance but also significantly bolstered its precision in the detection and quantification of MMP-2. 3.4 Detection of MMP-2 with the biosensor To quantify MMP-2 biomarkers accurately, we established a robust correlation between the peak intensities of the Electrochemiluminescence (ECL) signal, denoted as E Ru , as depicted in Fig. 3 A and B (Time vs. ECL intensity and Potential vs. ECL intensity respectively), and the MMP-2 concentration, labeled as C MMP−2 (Fig. 3 C). The observed linear correlation between E Ru and C MMP−2 spans concentrations ranging from 10 aM to 100 pM. From the data, we formulated a regression equation: E Ru (a. u.) = 0.091 × lgC MMP−2 + 0.040 (R² = 0.9976, n = 5). The linear regression equation in Fig. 3 C is established using data from both Fig. 3 A and Fig. 3 B. The signal values in Figs. 3 A and 3 B are consistent, with the only difference being that the horizontal axis in Fig. 3 A corresponds to Time, while in Fig. 3 B it corresponds to Potential. This equation not only facilitates precise logarithmic quantification of MMP-2 in biological samples but also confirms the biosensor’s high precision and reliability. The detection limit (LOD), a critical metric of the biosensor’s sensitivity, was determined using the 3σ method, resulting in an LOD of 12.8 aM. This LOD notably outperforms those of many existing methodologies, highlighting our biosensor’s superior sensitivity and its ability to detect extremely low concentrations of MMP-2. To provide a broader context, Table 1 contrasts our newly developed methodology with existing MMP-2 quantification techniques. The comparative analysis demonstrates that our approach provides comparable sensitivity and a linear detection range similar to current methods but with a slightly more advantageous detection limit. Such findings suggest that our biosensor not only meets but potentially exceeds current MMP-2 analysis standards, making it a promising alternative for anti-aging research applications, especially in diagnostic and monitoring scenarios. Table 1 Comparison of different methods for MMP-2 assay. Method LOD Linear Range Reference Fluorescent Nanoprobe 32 pM 0.1–20 nM [ 55 ] Silicon Nanowire-Based Biosensor 0.1 pM 100 fM-10 nM [ 5 ] CRISPR Cas13a based biosensor 62.05 fM 150–2000 fM [ 33 ] Bipedal walking robot 12.8 aM 0.01–100 pM This work 3.5 Specificity and reproducibility of the strategy Specificity is crucial in developing enzymatic methodologies, especially when employing Peptide Nucleic Acid (PNA) as a substrate to detect matrix metalloproteinase 2 (MMP-2). Ensuring that MMP-2 selectively cleaves the PNA substrate is essential for reducing unintended off-target interactions and increasing the accuracy of the detection method. To ascertain the specificity of this technique, we identified several proteins unlikely to interfere with PNA, including Esterase, Matrix Metalloproteinase 1 (MMP1), Thrombin, Bovine Serum Albumin (BSA), Alpha-fetoprotein (AFP), and Carcinoembryonic Antigen (CEA), used as reference proteins (illustrated in Fig. 4 A). We measured their electrochemiluminescent (ECL) responses using the CRISPR/Cas13a amplification method. As demonstrated in Fig. 4 A, the ECL readings for these non-target proteins were compared against those for MMP-2 (concentration of 500 fM). The findings clearly showed that MMP-2's ECL signal underwent the most notable change compared to the baseline, whereas the signals from the other proteins remained largely consistent with the blank controls. In conclusion, the effectiveness of using PNA as a substrate for MMP-2 detection heavily depends on the specificity of the interaction. By carefully selecting reference proteins and fine-tuning the PNA sequence to specifically recognize a target site within MMP-2, we can significantly enhance the method's specificity and minimize the likelihood of off-target effects. The electrochemiluminescent biosensor developed via the CRISPR/Cas13a amplification method showcases exceptional selectivity, positioning it as an effective tool for various applications in biotechnology and medical diagnostics. Subsequently, we conducted an assessment of the stability of the biosensing platform. Figure 4 B provides a detailed representation of the detection stability within this Electrochemiluminescence (ECL) detection platform. This figure depicts the detection signal generated by the biosensing platform when detecting 1 pM MMP-2 as the target analyte. Remarkably, as depicted in Fig. 4 B, the detection signal for 1 pM MMP-2 remains consistently stable, even after subjecting it to ten consecutive scans, each spaced an hour apart (RSD = 1.34%). There is no substantial decline in the signal's intensity. These observations solidify the remarkable stability of our sensing platform. Furthermore, we carried out an evaluation of the biosensor's shelf life. The biosensors, containing DNA/[Ru(phen) 2 dppz] 2+ complexes, were stored in a sealed container at a constant temperature of 4°C, shielded from light, following the detection of 1 pM MMP-2. After each test, the ECL signal of the biosensor was measured, and the relative standard deviation (RSD) of the signal over the course of 14 days was calculated (as shown in Fig. 4 C). Notably, the functionality and sensitivity of the CRISPR/Cas13a-based biosensor remained consistent throughout the entire testing period, exhibiting no noticeable performance degradation. The RSD value of the ECL signal was a mere 0.88%, underscoring the biosensor's high stability and repeatability. 3.6 Recovery studies of the biosensor in real samples Biosensors based on electrochemiluminescence, engineered through the CRISPR/Cas13a isothermal amplification method, display an impressive blend of heightened sensitivity and precision. These qualities render them highly effective for detecting MMP-2 activity within complex biological matrices. Owing to the prevalent surge in MMP-2 activity across a variety of human cancers and tissues, targeting MMP-2 has become a focal point in the realms of cancer diagnostics and treatment. Faced with the challenge of scarce aging-related cell samples, this study utilized supernatants from HepG2 and LO2 cell cultures to evaluate the biosensor's efficacy. As depicted in Fig. 5 , the examination of these supernatants indicated a negligible variation in electrogenerated chemiluminescence (ECL) signals among the LO2 cell samples. In contrast, the HepG2 cell samples exhibited a significant surge in ECL signals, marking a 6.73-fold increase relative to the LO2 samples. This significant increase is attributed to the heightened expression of MMP-2 in cancerous cells. To ensure the specificity of our electrochemiluminescence sensor, we performed a targeted pre-treatment experiment involving HepG2 cells and a known MMP-2 inhibitor, ARP 100. This intervention significantly reduced the ECL signal change to levels comparable with those of the control group, nearly zero. Such a result strongly indicates that the ECL response is directly attributable to MMP-2's specific activity on the PNA substrate. This validation highlights the exceptional potential of electrochemiluminescence sensors utilizing the bipedal walker isothermal amplification method for detecting MMP-2 activity in clinical settings. These findings are crucial as they confirm the sensor's high specificity and sensitivity, demonstrating its significant advantages for practical applications in the medical field, particularly in cancer diagnostics and therapy development. The technology’s precise detection capabilities enable it to identify MMP-2 activity accurately, making it a valuable tool for developing targeted treatment strategies and improving patient outcomes in oncology. Subsequently, we measured the content and recovery rate of matrix metalloproteinase-2 (MMP-2) by correlating it with the spectrum of electrochemiluminescence (ECL) signals derived from LO2 cell culture supernatants. These signal levels fluctuated between 99.8% and 105.0%, as outlined in Table 2 . This significant finding highlights the biosensor's outstanding ability to withstand external disturbances, making it an excellent candidate for reducing CRISPR/Cas13a activity, especially under complex conditions. The findings from these tests conclusively demonstrate the superior analytical capabilities of our approach in detecting MMP-2. The ECL signal levels we obtained play a crucial role in deciphering both the concentration and recovery of MMP-2, offering a reliable method for its assessment and quantification across various sample types. The wide range of recovery rates demonstrates the robust stability of our biosensors, even in the face of potential disruptions caused by background noise, contaminants, or other biological elements prevalent in real-world specimens. This resistance to interference is particularly valuable in practical scenarios, where dealing with complex biological matrices demands high precision and specificity in detection. Table 2 Recovery results for the assay of MMP-2 in cell culture supernatants of LO2. Sample number Added (aM) Found (fM) Recovery (%) RSD (%, n = 3) 1 0 2.6 3 100 103.2 100.6 3.73 4 1000 1052.6 105.0 4.65 5 10000 9979.8 99.8 4.62 6 100000 100159.8 100.2 3.63 4. Conclusions This paper presents a novel electrochemiluminescence (ECL) biosensor for the detection of matrix metalloproteinase 2 (MMP-2), a key enzyme involved in anti-aging research. The biosensor integrates the high specificity and signal amplification of the CRISPR/Cas13a system with the high efficiency and simplicity of the exponential amplification reaction (EXPAR) and the “light switch” [Ru(phen)2dppz]2 + probe. The main findings and contributions of this paper are summarized as follows: The biosensor exhibits a low detection limit of 12.8 aM and a wide linear range of 10 aM to 100 pM for MMP-2, surpassing or comparable to many existing methods. The biosensor also demonstrates excellent selectivity, reproducibility, and stability, as well as applicability in real samples such as cell culture supernatants. The biosensor provides a promising tool for MMP-2 analysis in anti-aging research, as well as cancer diagnosis and therapy. The biosensor also showcases the potential of integrating CRISPR/Cas13a with other amplification and detection strategies for creating sensitive and specific biosensors for various targets. The biosensor employs a novel design that combines PNA, T7 promoter, DNA template, and Cas13a to achieve transcription amplification, trans-cleavage activation, and EXPAR amplification. The biosensor also utilizes the “photoswitch” molecule [Ru(phen) 2 dppz] 2+ to avoid the cumbersome electrode modification and cleaning process, simplifying the experimental procedures and reducing the testing cost. The biosensor is based on a solid theoretical foundation and rigorous experimental validation. The paper provides a comprehensive explanation of the principles, mechanisms, and optimization of the biosensor. The paper also presents detailed experimental results, data analysis, and discussion to support the claims and conclusions. In conclusion, this paper reports a novel ECL biosensor for MMP-2 detection that is highly sensitive, specific, simple, and cost-effective. The biosensor is expected to have broad applications in anti-aging research and other fields that require MMP-2 analysis. The paper also contributes to the advancement of CRISPR/Cas13a-based biosensing technologies and opens new avenues for future research. Declarations Supplementary data Supplementary data can be found in the online version. Acknowledgment We would like to thank the Startup Foundation for Introducing Talent of NUIST (2023r129), the financial support of the National Natural Science Foundation of China (21964018), the Guangxi key laboratory of basic and translational research of Bone and joint Degenerative Disease (21-220-06-202205), the State Key Laboratory of Analytical Chemistry for Life Science (SKLACLS2314). Conflict of Interest All authors disclosed no relevant relationships. Ethical Approval not applicable Funding We would like to thank the Startup Foundation for Introducing Talent of NUIST (2023r129), the financial support of the National Natural Science Foundation of China (21964018), the Guangxi key laboratory of basic and translational research of Bone and joint Degenerative Disease (21-220-06-202205), the State Key Laboratory of Analytical Chemistry for Life Science (SKLACLS2314). 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Biosens Bioelectron.178:113015. doi: https://doi.org/10.1016/j.bios.2021.113015. Fan Z, Yao B, Ding Y, Xu D, Zhao J, Zhang K. (2022) Rational engineering the DNA tetrahedrons of dual wavelength ratiometric electrochemiluminescence biosensor for high efficient detection of SARS-CoV-2 RdRp gene by using entropy-driven and bipedal DNA walker amplification strategy. Chem Eng J.427:131686. doi: https://doi.org/10.1016/j.cej.2021.131686. Wang Z, Li X, Feng D, Li L, Shi W, Ma H. (2014) Poly(m-phenylenediamine)-Based Fluorescent Nanoprobe for Ultrasensitive Detection of Matrix Metalloproteinase 2. Anal Chem.86(15):7719-25. doi: 10.1021/ac5016563. Scheme 1 Scheme 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Scheme1.png Scheme 1. Schematic diagram of the CAS-EXPAR platform for MMP-2 detection. (A) In the absence of MMP-2, the PNA/T7 promoter/DNA template complex inhibits the transcription amplification. (B) In the presence of MMP-2, the peptide bond in the PNA is cleaved, releasing the DNA template/T7 promoter duplex, which serves as the substrate for transcription amplification. The transcription amplification produces a large amount of dsRNA transcripts, which are recognized and bound by gRNA, activating the CRISPR/Cas13a system. The activated Cas13a cleaves the Pre-trigger DNA, initiating the EXPAR amplification. The EXPAR amplification produces a large amount of dsDNA products, which interact with [Ru(phen) 2 dppz] 2+ , generating a luminescence signal. 5Cas13aT7SI.docx Cite Share Download PDF Status: Published Journal Publication published 14 Oct, 2024 Read the published version in Microchimica Acta → Version 1 posted Editorial decision: Revision requested 07 Aug, 2024 Reviews received at journal 05 Aug, 2024 Reviewers agreed at journal 29 Jul, 2024 Reviews received at journal 23 Jul, 2024 Reviewers agreed at journal 15 Jul, 2024 Reviewers invited by journal 10 Jul, 2024 Editor assigned by journal 05 Jul, 2024 Submission checks completed at journal 05 Jul, 2024 First submitted to journal 04 Jul, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Electrochemiluminescence (ECL) performance of the CEISPR/Cas13a-based amplification system for detecting MMP-2. The system shows a substantial enhancement in the ECL signal when detecting as low as 1 pM of MMP-2 (curve c), while the ECL signals observed were notably diminished in the absence of MMP-2 (curve a) or in the presence of a substantially lower concentration of MMP-2 (100 aM, curve b).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4689418/v1/a7f61183335ce27023fc5409.png"},{"id":61426754,"identity":"984a0f16-69db-4cbb-92cc-370b06a6742e","added_by":"auto","created_at":"2024-07-30 14:55:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":185779,"visible":true,"origin":"","legend":"\u003cp\u003eOptimization of experimental parameters for the biosensor. The biosensor's current response was measured by ECL under various conditions and compared to a control group. The optimized parameters were: A) T7 RNA polymerase transcription duration of 30 minutes, B) CRISPR/Cas13a reaction time of 25 minutes, and C) Cas13a/crRNA concentration.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4689418/v1/7603ac31d6b13accc34f634c.png"},{"id":61426750,"identity":"3ebefe15-9741-4304-a938-414826e51724","added_by":"auto","created_at":"2024-07-30 14:55:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":137687,"visible":true,"origin":"","legend":"\u003cp\u003eQuantification of MMP-2 biomarkers by the biosensor. (A) Time vs. ECL intensity (a to i: 0 aM, 10 aM, 100 aM, 1.0 fM, 10 fM, 100 fM, 1.0 pM, 10 pM, 100 pM), (B) Potential vs. ECL intensity (a to i: 0 aM, 10 aM, 100 aM, 1.0 fM, 10 fM, 100 fM, 1.0 pM, 10 pM, 100 pM), and (C) MMP-2 concentration vs. ECL intensity. The linear relationship between E\u003csub\u003eRu\u003c/sub\u003e and logarithm of CMMP-2 is expressed by the equation E\u003csub\u003eRu\u003c/sub\u003e.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4689418/v1/9788d46c099b8f8b7ee63197.png"},{"id":61426752,"identity":"57c921d6-c0f1-4703-9908-22a1040f28a0","added_by":"auto","created_at":"2024-07-30 14:55:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":193760,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Specificity of the ECL biosensor for MMP-2 detection. The ECL signals of non-specific proteins and target MMP-2 (500 fM) are compared. (B) Stability of the ECL biosensor. The ECL signal of 1 pM MMP-2 is measured after ten consecutive scans with one-hour intervals. (C) Shelf life of the ECL biosensor. The ECL signal of 1 pM MMP-2 is measured after 14 days of storage at 4 °C.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4689418/v1/908bdbe32c1365ee135a4f0b.png"},{"id":61426756,"identity":"c8684d20-78f6-4d4a-a20a-d25139b281aa","added_by":"auto","created_at":"2024-07-30 14:55:28","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":63618,"visible":true,"origin":"","legend":"\u003cp\u003eElectrochemiluminescence (ECL) intensity of CRISPR/Cas13a-based sensors in the detection of MMP-2 activity. The sensors were tested using supernatants from LO2 and HepG2 cell cultures, with HepG2 showing a significantly higher ECL signal, indicative of MMP-2 overexpression. Pre-treatment with MMP-2 inhibitor ARP 100 in HepG2 cells reduced the ECL intensity to baseline levels, demonstrating the sensor’s specificity for MMP-2 detection. This suggests a promising application in cancer diagnostics, due to the enhanced sensitivity and specificity of the biosensor for MMP-2, a key marker in cancer progression.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-4689418/v1/5a89c856a98b3cf9317d3436.png"},{"id":67149602,"identity":"2fa8d04f-a449-4f99-8573-dec12e8d9482","added_by":"auto","created_at":"2024-10-21 16:13:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2195218,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4689418/v1/e458ce31-4da9-4a60-9b57-3ca02f7aaeac.pdf"},{"id":61426755,"identity":"23ce431c-e8f6-48ae-b538-251a721b1970","added_by":"auto","created_at":"2024-07-30 14:55:28","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":150006,"visible":true,"origin":"","legend":"\u003cp\u003eScheme 1. Schematic diagram of the CAS-EXPAR platform for MMP-2 detection. (A) In the absence of MMP-2, the PNA/T7 promoter/DNA template complex inhibits the transcription amplification. (B) In the presence of MMP-2, the peptide bond in the PNA is cleaved, releasing the DNA template/T7 promoter duplex, which serves as the substrate for transcription amplification. The transcription amplification produces a large amount of dsRNA transcripts, which are recognized and bound by gRNA, activating the CRISPR/Cas13a system. The activated Cas13a cleaves the Pre-trigger DNA, initiating the EXPAR amplification. The EXPAR amplification produces a large amount of dsDNA products, which interact with [Ru(phen)\u003csub\u003e2\u003c/sub\u003edppz]\u003csup\u003e2+\u003c/sup\u003e, generating a luminescence signal.\u003c/p\u003e","description":"","filename":"Scheme1.png","url":"https://assets-eu.researchsquare.com/files/rs-4689418/v1/23e7f2ab81bfd3fdef50de50.png"},{"id":61426759,"identity":"be672295-c013-4df7-853d-a10ccc5f598c","added_by":"auto","created_at":"2024-07-30 14:55:30","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1952911,"visible":true,"origin":"","legend":"","description":"","filename":"5Cas13aT7SI.docx","url":"https://assets-eu.researchsquare.com/files/rs-4689418/v1/052ff2d4b961df260dafdc40.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Electrochemiluminescence Biosensor for MMP-2 Analysis Using CRISPR/Cas13a and EXPAR Amplification: A Novel Approach for Anti-Aging Research","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eMMP-2, or matrix metalloproteinase 2, plays a pivotal role in anti-aging research. [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]Throughout the aging process, the disruption of MMP-2 contributes to the breakdown of the extracellular matrix (ECM), leading to wrinkles, sagging skin, and reduced elasticity. [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] Scientists investigate the signaling pathways and factors that regulate MMP-2 expression to identify potential intervention points. Research aimed at MMP-2 focuses on restoring ECM balance and mitigating skin aging. Both natural compounds and synthetic inhibitors, as well as various interventions, are used to regulate MMP-2's expression and activity. Given its role in degrading crucial ECM components, controlling MMP-2 is essential for maintaining skin health. [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] Thus, detecting MMP-2 is significant in anti-aging studies. [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] Currently, there are multiple methods available to detect MMP-2, including ELISA, [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] gelatin zymography, [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]Western blotting, [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]and immunohistochemistry.[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] However, each technique has its limitations regarding sensitivity, specificity, and reliability, with potential for false positives or negatives. Additionally, sample preparation and handling can impact results, and some methods require specialized equipment and expertise. The choice of detection method should depend on the sample type, the research question, and the resources available, with careful consideration of each method's limitations to ensure accurate and reliable results.\u003c/p\u003e \u003cp\u003eThe CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas (CRISPR-associated) system originated from the adaptive immune system of prokaryotes, providing defense against invading nucleic acids. [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] In recent years, the CRISPR-Cas system has garnered significant attention from biological researchers as a highly effective gene editing tool. [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan additionalcitationids=\"CR16 CR17\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] Broadly, CRISPR/Cas systems can be categorized into Class I and Class II, based on whether they use single or multiple effectors. Class II systems, such as Cas9, Cas12, and Cas13, are particularly notable for their broad applications due to their simpler composition, consisting of a single effector protein and a programmable guide RNA. [\u003cspan additionalcitationids=\"CR20 CR21 CR22 CR23 CR24\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] Recent advancements in gene editing have led to notable discoveries, including new genome editing techniques. Specifically, the trans-cleavage activity discovered in Cas12a, Cas13a, and Cas14a has positioned the CRISPR/Cas system as a promising candidate for next-generation diagnostic biosensing platforms. [\u003cspan additionalcitationids=\"CR27 CR28 CR29 CR30 CR31\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] Among these, the CRISPR/Cas13a system, part of Class II type VI-A, stands out as the only RNA-targeted CRISPR effector with exceptional signal amplification capabilities. [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]Cas13a, along with its corresponding CRISPR RNA (crRNA), can precisely recognize and cleave target RNA based on crRNA-target complementarity. This recognition triggers its trans-cleavage activity, which further cleaves fluorophore-quencher-labeled RNA probes with remarkable turnover efficiency (approximately 4854), leading to amplified fluorescence detection signals. [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] To enhance sensitivity, various amplification strategies, such as recombinase polymerase amplification (RPA), have been integrated into the CRISPR/Cas system. Consequently, developing a new detection strategy that combines the high specificity and signal amplification capabilities of the CRISPR/Cas system with the efficiency of exponential nucleic acid amplification is highly valuable. [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eIn addition, T7 RNA polymerase is an important tool for creating biosensors. The enzyme is an excellent tool for creating RNA probes to detect target analytes in biosensors because of its ability to efficiently and specifically convert DNA to RNA. Due to the specificity of the T7 RNA polymerase, RNA probes corresponding to the target analytes can be transcribed, resulting in accurate and sensitive detection.[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] T7 RNA polymerase can also be used to enhance RNA signaling in biosensors, resulting in T7 RNA polymerase has a variety of uses in molecular biology and biotechnology, but is not limited to the manufacture of biosensors. In addition, the use of T7 RNA polymerase allows for the synthesis of RNA vaccines, which synthesize RNA encoding target antigens to elicit an immune response, as well as RNA standards for qPCR testing.[\u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] In summary, T7 RNA polymerase is a flexible tool with multiple uses in molecular biology and biotechnology, including the construction of biosensors. Due to its selectivity, potency and ability to amplify RNA signals, T7 RNA polymerase can be used to create very precise and sensitive biosensors.\u003c/p\u003e \u003cp\u003eElectrochemiluminescence (ECL) is a chemiluminescence technique that utilizes electrochemical stimulation by applying a voltage or electric current to the surface of an electrode or an electrolyte to trigger a exothermic redox reaction in a substance, resulting in the production of molecules or free radicals in the excited state and the release of photons as they return to their ground state.[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] This process has a number of advantages. First, the background noise is very low because only substances in which exothermic redox reactions are present can emit light, excluding the influence of other interfering substances. Secondly, electrochemiluminescence has a wide dynamic range and the intensity of light can be controlled by adjusting electrical parameters, thus enabling the detection of substances at different concentrations.[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan additionalcitationids=\"CR44 CR45\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] In addition, it has high sensitivity, and the luminescence efficiency can be enhanced by co-reactants or co-catalysts to realize the detection of very low concentration substances. Most importantly, it is simple to operate, requiring only a simple electrochemical cell and a photodetector for fast, convenient and cost-effective analysis.[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan additionalcitationids=\"CR48 CR49 CR50 CR51 CR52 CR53\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eIn this study, we integrated T7 RNA polymerase into a CRISPR/Cas13a amplification-based electrochemiluminescence (ECL) biosensing platform for the ultrasensitive and specific detection of MMP-2. The method involves an MMP-2-interacting probe that, in the presence of T7 RNA polymerase, amplifies a substantial amount of RNA. This amplified RNA activates Cas13a, which precisely identifies miRNA targets and acts as a primer for subsequent exponential amplification. Unlike earlier Cas13a-based RNA detection approaches, our method harnesses the full potential of Cas13a's RNA recognition and signal amplification capabilities. Both the trans-cleavage activity and isothermal exponential amplification (EXPAR) of Cas13a enhance the assay's sensitivity. Furthermore, the RNA amplification mediated by T7 RNA polymerase upon MMP-2 interaction allows for highly specific MMP-2 detection.Additionally, incorporating the photoswitch [Ru(phen)\u003csub\u003e2\u003c/sub\u003edppz]\u003csup\u003e2+\u003c/sup\u003e into the detection platform eliminates the need for complex electrode modification and cleaning procedures. [Ru(phen)\u003csub\u003e2\u003c/sub\u003edppz]\u003csup\u003e2+\u003c/sup\u003e shows a significant luminescence increase when it interacts with double-stranded DNA (dsDNA). This enhancement occurs because the planar phenazine ligand of [Ru(phen)\u003csub\u003e2\u003c/sub\u003edppz]\u003csup\u003e2+\u003c/sup\u003e binds with the base pairs in the major groove of dsDNA, protecting the nitrogen atoms of phenazine and boosting the luminescent state population.[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] This property is crucial for amplifying the luminescence signal, which is essential for detecting the presence of the target. This innovation simplifies the experimental workflow, reduces detection costs, and improves reproducibility. We also evaluated the sensing system's applicability for detecting MMP-2 in serum samples.\u003c/p\u003e"},{"header":"2. Experimental section","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials and chemicals\u003c/h2\u003e \u003cp\u003eAll oligonucleotides utilized in this study were sourced from Genscript Biotechnology Co., Ltd (Nanjing, China), and their respective sequences are documented in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The PNA (Peptide Nucleic Acid) employed in this research was provided by TAHE-PNA Co., Ltd. (Hangzhou, China). All other chemicals, which met reagent-grade standards, were used without further purification. Solutions were prepared using ultrapure water that underwent purification through the Milli-Q purification system (Branstead), resulting in a specific resistivity exceeding 18.2 MΩ\u0026middot;cm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Instruments and measurements\u003c/h2\u003e \u003cp\u003eThe detection of the electrochemiluminescence (ECL) signal was performed using a specialized ECL instrument, which was generously provided by the State Key Laboratory of Analytical Chemistry for Life Sciences at Nanjing University. For the ECL measurements, a three-electrode system was utilized. This configuration included a glassy carbon working electrode (GCE), which served as the primary site for electrochemical reactions, an Ag/AgCl reference electrode to maintain a stable reference potential, and a platinum counter electrode to complete the circuit and facilitate the overall electrochemical process.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 MMP-2 proteolytic cleavage, enzymatic amplification mediated by T7 RNA polymerase, and activation of CRISPR/Cas13a\u003c/h2\u003e \u003cp\u003eTo create the PNA/T7 promoter/DNA template complex, the T7 promoter, DNA template, and PNA were combined in an HES buffer with 1 U \u0026micro;L⁻\u0026sup1; of RNase inhibitor, at a ratio of 1:1:1.2. This mixture was incubated at 41.5\u0026deg;C for 15 minutes, then gradually cooled to room temperature and kept at 4\u0026deg;C overnight, resulting in the successful synthesis of the PNA/Bandage complex.\u003c/p\u003e \u003cp\u003eFor the release of the DNA template/T7 promoter duplex triggered by MMP-2, recombinant human MMP-2 was activated in an AC buffer (50 mM HEPES, 10 mM CaCl₂, 150 mM NaCl, 0.05% (w/v) Brij 35, pH 7.0) at 37\u0026deg;C for 2 hours. After activation, different amounts of MMP-2 were added to the PNA/T7 promoter/DNA template mixture and incubated for 30 minutes.\u003c/p\u003e \u003cp\u003eSubsequently, 50 \u0026micro;L of this mixture was added to an amplification reaction system to a final volume of 100 \u0026micro;L. This system contained 40 \u0026micro;M NTPs, 30 U T7 RNA polymerase, 1 \u0026micro;M DNA template, 20 U RNase inhibitor, and 2 \u0026micro;L of a 10\u0026times; RNAPol reaction buffer. The mixture was incubated at 37\u0026deg;C for 40 minutes to enable transcription amplification.\u003c/p\u003e \u003cp\u003ePost-transcription, the solution was incubated at room temperature for 20 minutes with 10 nM CRISPR/Cas13a and 15 nM gRNA in 1\u0026times; NEBuffer (NEB). The biosensor was then immersed in the activated CRISPR/Cas13a solution for 20 minutes to cleave the pre-trigger DNA.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 EXPAR amplification\u003c/h2\u003e \u003cp\u003eThe Exponential Amplification Reaction (EXPAR) system required the preparation of two solutions, Solution A and Solution B. Solution A consisted of 0.1 \u0026micro;M template, 250 \u0026micro;M dNTP mix, and 1.2 \u0026micro;L of cleaved products from previous steps. Solution B contained 0.4 U/\u0026micro;L NEase Nt.BstNBI, 0.05 U/\u0026micro;L Vent (exo-) DNA polymerase, 1\u0026times; Thermopol buffer, and 1\u0026times; NEBuffer 3.1. These solutions were promptly mixed to form a final volume of 10 \u0026micro;L and incubated at 55\u0026deg;C for 30 minutes to facilitate exponential amplification. This process involved cleaved products generated through the activation and interaction of MMP-2 with the PNA/T7 promoter/DNA template complex. The mixture was incubated at 55\u0026deg;C for 30 minutes, ensuring efficient exponential amplification of target sequences. This protocol was essential for achieving the high sensitivity and specificity needed for MMP-2 detection in the developed ECL biosensing platform, significantly enhancing the accuracy and reliability of the detection method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 C\u003cem\u003eharacterization of EXPAR Amplification and Cas13a Cutting Performance using Polyacrylamide Gel Electrophoresis (PAGE)\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eTo verify the experimental process of the characterization of EXPAR amplification and the cutting performance of Cas13a, we conducted polyacrylamide gel electrophoresis (PAGE). The samples, including the T7 promoter, DNA template, DNA template/T7 promoter duplex, products after T7 promoter amplification reaction based on DNA template/T7 promoter duplex, Pre-Trigger DNA, and CRISPR/Cas13a treated Pre-Trigger DNA, were loaded onto a 20% native polyacrylamide gel in 0.5\u0026times; Tris-borate-EDTA (TBE). The gels were run at room temperature for 1 hour at 120 V. After electrophoresis, the gels were stained with a SYBR Green I mixture and photographed using a Bio-Rad digital imaging system.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 ECL assay:\u003c/h2\u003e \u003cp\u003eA 25 \u0026micro;L solution was meticulously prepared, combining 15 \u0026micro;L of EXPAR products, 1 \u0026micro;L of a 0.5 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e M [Ru(phen)\u003csub\u003e2\u003c/sub\u003edppz]\u003csup\u003e2+\u003c/sup\u003e solution, and 9 \u0026micro;L 70 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e M TPrA solution. This solution was meticulously crafted in a 0.01 M phosphate-buffered saline (PBS) with a pH of 7.4. Following this precise formulation, the resulting mixture was then expertly applied to the electrode.\u003c/p\u003e \u003cp\u003eECL signal detection was carried out using an advanced ECL instrument, provided by the State Key Laboratory of Analytical Chemistry for Life Sciences at Nanjing University. The three-electrode system employed consisted of a glassy carbon working electrode (GCE), an Ag/AgCl reference electrode, and a platinum counter electrode. This setup facilitated precise and efficient ECL measurements, contributing to the robustness and sensitivity of the MMP-2 detection platform.\u003c/p\u003e \u003cp\u003eFor the modification of the glassy carbon electrode (GCE), we followed a standard procedure to ensure optimal performance. The detailed process involves the following steps: The GCE surface was first polished to achieve a mirror-like finish, which is crucial for removing any surface impurities and ensuring a smooth, conductive surface. Alumina slurries of different particle sizes (e.g., 1.0 \u0026micro;m, 0.3 \u0026micro;m, and 0.05 \u0026micro;m) were used sequentially for polishing. The electrode was placed on a polishing cloth, and the alumina slurry was applied. The GCE was then polished in a figure-eight motion for about 5 minutes with each slurry, starting with the coarsest (1.0 \u0026micro;m) and moving to the finest (0.05 \u0026micro;m). This process ensures a smooth and clean electrode surface, essential for reproducible and reliable electrochemical measurements. After polishing, the electrode was thoroughly cleaned to remove any residual polishing particles and contaminants. The polished GCE was rinsed with distilled water to remove the bulk of the alumina particles, then sonicated in ethanol for 5 minutes, followed by sonication in deionized water for another 5 minutes to ensure that any remaining polishing debris or organic contaminants were removed from the electrode surface.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Principle of the proposed ECL biosensor\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eScheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e provides an illustration of the platform and its operational principles. In this context, MMP-2 was chosen as the model target for the proof-of-concept. The Cas13a enzyme employed in this investigation is derived from Leptotrichia buccalis (LbuCas13a). The crRNA consists of a 30 nucleotide repeat region facilitating interaction with Cas13a, and a 20 nucleotide programmable guide region, often referred to as the spacer, designed for the recognition of target RNA. Notably, the trans cleavage activity of LbuCas13a exhibits a preference for cleaving the ribose-phosphodiester bond flanked by uracil. To leverage this preference, a Pre-trigger DNA was designed, incorporating two uracil ribonucleotides (rU). The 5\u0026prime; end of this Pre-trigger DNA comprises 16 complementary bases with the amplification template and five random bases at the opposite end. The amplification template features a central nicking endonuclease (NEase) cleavage site and two separate repeat sequences, both of which are complementary to the 5\u0026prime; end of the Pre-trigger DNA. Additionally, both the Pre-trigger DNA and the amplification template were equipped with a C3 spacer at their 3\u0026prime;-end to prevent target-independent polymerase amplification. A specific PNA sequence was designed, containing a peptide sequence (Gly-Pro-Leu-Gly-Val-Arg-Gly) that can be precisely cleaved by MMP-2. It is essential to note that PNA is a synthetic nucleic acid analog characterized by a peptide backbone rather than a sugar-phosphate backbone.\u003c/p\u003e \u003cp\u003eIn the context of the assay reaction, the process unfolds as follows: Initially, the PNA/T7 promoter/DNA template complex, featuring a double-stranded DNA template connected by PNA and T7 promoter, which encompasses a peptide bond cleavable by MMP-2. In the absence of MMP-2 within the system, the PNA/DNA template duplex inhibits the T7 promoter amplification reaction (as depicted in Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eHowever, with the introduction of MMP-2 into the system (as presented in Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), the cleavage of the peptide bond is triggered, leading to the release of the single-stranded DNA template/T7 promoter duplex. This released duplex assumes the role of a substrate for T7 promoter-aided transcription amplification. Subsequent transcription amplification generates a substantial quantity of single-stranded RNA transcripts, encompassing the target sequence that is identifiable and bindable by gRNA. Following the binding of gRNA to the transcript, it activates the nuclease activity of CRISPR/Cas13a, thereby enabling the cleavage of any single-stranded RNA molecules.\u003c/p\u003e \u003cp\u003eThe assembled Cas13a/crRNA system is designed to specifically recognize and cleave target RNA sequences. Upon recognition, Cas13a's two conserved higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domains reposition to form a new RNase active site, activating its trans-cleavage activity. This leads to the cleavage of the Pre-trigger DNA into two fragments. The 5\u0026prime; fragment of the cleaved Pre-trigger DNA can hybridize with an amplification template, initiating a polymerization reaction in the presence of T4 polynucleotide kinase (PNK) and Vent DNA polymerase. The newly synthesized double-stranded DNA is then recognized and nicked by nicking endonuclease (NEase). This process involves a continuous cycle of nicking, strand extension, and displacement, resulting in the generation of substantial amounts of double-stranded DNA products. Notably, the trans-cleavage activity of Cas13a remains inactive without the presence of the specific portion of the Pre-trigger DNA. If the Pre-trigger DNA is not cleaved, it cannot be extended by DNA polymerase, thereby failing to initiate the downstream exponential amplification reaction (EXPAR). This mechanism ensures that only in the presence of the target RNA does the system proceed, thereby providing high specificity and sensitivity in detecting the target sequence. This feature is critical for applications requiring precise molecular detection, such as the developed ECL biosensing platform for MMP-2 detection.\u003c/p\u003e \u003cp\u003eSubsequently, the complex [Ru(phen)2dppz]2\u0026thinsp;+\u0026thinsp;is incorporated into the Cas13a-enhanced EXPAR (CAS-EXPAR) system. In its free state, this complex fails to emit luminescence due to nitrogen protonation in aqueous environments. However, when it binds to the double-stranded DNA products produced by EXPAR, there is a significant increase in luminescence. This boost in signal is linked to the planar phenazine ligand of [Ru(phen)\u003csub\u003e2\u003c/sub\u003edppz]\u003csup\u003e2+\u003c/sup\u003e engaging with the base pairs in the major groove of the DNA. This interaction shelters the phenazine's nitrogen atoms, promoting a state conducive to luminescence.\u003c/p\u003e \u003cp\u003eFollowing this, the [Ru(phen)\u003csub\u003e2\u003c/sub\u003edppz]\u003csup\u003e2+\u003c/sup\u003e supplemented amplification system and excess TPrA are introduced into the biosensor platform. The electrochemiluminescence (ECL) signal, which serves as an indicator of MMP-2 activity, is detected through a photomultiplier tube (PMT, Hamamatsu R928 PMT). The reaction mechanism is as follows:\u003c/p\u003e \u003cp\u003e[Ru(phen)\u003csub\u003e2\u003c/sub\u003edppz]\u003csup\u003e2+\u003c/sup\u003e/DNA-e\u003csup\u003e\u0026minus;\u003c/sup\u003e \u0026rarr;[Ru(phen)\u003csub\u003e2\u003c/sub\u003edppz]\u003csup\u003e3+\u003c/sup\u003e+H\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eTPrA-e\u003csup\u003e\u0026minus;\u003c/sup\u003e \u0026rarr; TPrA∙\u003csup\u003e+\u003c/sup\u003e \u0026rarr; TPrA∙ + H\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e[Ru(phen)\u003csub\u003e2\u003c/sub\u003edppz]\u003csup\u003e3+\u003c/sup\u003e-DNA\u0026thinsp;+\u0026thinsp;TPrA∙ \u0026rarr; [Ru(phen)\u003csub\u003e2\u003c/sub\u003edppz]\u003csup\u003e2+\u0026lowast;\u003c/sup\u003e-DNA\u0026thinsp;+\u0026thinsp;products\u003c/p\u003e \u003cp\u003e[Ru(phen)\u003csub\u003e2\u003c/sub\u003edppz]\u003csup\u003e2+\u003c/sup\u003e\u0026lowast;-DNA \u0026rarr; [Ru(phen)\u003csub\u003e2\u003c/sub\u003edppz]\u003csup\u003e2+\u003c/sup\u003e-DNA\u0026thinsp;+\u0026thinsp;hν\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Feasibility study\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe CEISPR/Cas13a-based amplification system was subjected to thorough examination with regards to its electrochemiluminescence (ECL) performance. The results revealed a substantial enhancement in the ECL signal when the system was employed for detecting as low as 1 pM of MMP-2 (curve c). In stark contrast, the ECL signals observed were notably diminished in the absence of MMP-2 (curve a) or in the presence of a substantially lower concentration of MMP-2 (100 aM, curve b), as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. These findings underscore the crucial role of the target MMP-2 in initiating the biosensor's activation.\u003c/p\u003e \u003cp\u003eIn response to the need for characterization of the EXPAR amplification and the cutting performance of Cas13a, we have conducted the necessary experiments. The results are detailed in Figures \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and S2. Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e presents the electrophoresis identification of the T7 RNA polymerase transcription-mediated amplification process, confirming the feasibility of the EXPAR amplification. Additionally, Figure S2 illustrates the cutting performance of Cas13a, demonstrating its efficiency in the process. These characterizations verify the reliability and effectiveness of the amplification and cleavage mechanisms used in our study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Optimization of experimental parameters\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn our pursuit of enhancing the performance of the biosensor, we meticulously conducted a comprehensive optimization process, focusing on three pivotal experimental parameters. Employing ECL, we conducted an in-depth examination of the biosensor's current response under various conditions and compared it to a control group (as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The parameters that underwent meticulous optimization included:\u003c/p\u003e \u003cp\u003eOptimizing T7 RNA Polymerase Transcription Duration: As revealed in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, the ECL signal achieved stability after 30 minutes. This observation was the result of a progressive sequence from curve a to curve g, with time intervals set at 5, 10, 20, 30, 40, 50, and 60 minutes. This analysis pinpointed the ideal duration for T7 RNA polymerase transcription, which was determined to be 30 minutes.\u003c/p\u003e \u003cp\u003eFine-Tuning CRISPR/Cas13a Reaction Time: As portrayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, the ECL signal exhibited a gradual decline from 5 minutes to 30 minutes, corresponding to time intervals of 5, 10, 15, 20, 25, and 30 minutes. Ultimately, stability was reached after 25 minutes, underscoring the optimal 25-minute CRISPR/Cas13a reaction time that consistently yielded the most favorable results.\u003c/p\u003e \u003cp\u003eOptimizing Cas13a/crRNA Concentration: The concentration of Cas13a/crRNA holds direct sway over its intrinsic cis-cleavage efficiency, consequently impacting the subsequent isothermal amplification efficiency. We introduced differing concentrations of Cas13a/crRNA (2.5 nM, 5 nM, 10 nM, and 20 nM) into the sensing system in a 1:1 ratio and subjected them to ECL analysis. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC clearly indicates that the sensor treated with 10 nM Cas13a/crRNA exhibited the highest signal. However, once the concentration of Cas13a/crRNA was elevated to 20 nM, the ECL signal ceased to increase, signifying that an excess of Cas13a/crRNA failed to further enhance the ECL signal. Consequently, for the ensuing research, we opted for a 10 nM concentration of Cas13a/crRNA.\u003c/p\u003e \u003cp\u003eThis meticulous series of optimization steps not only served to amplify the biosensor's performance but also significantly bolstered its precision in the detection and quantification of MMP-2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Detection of MMP-2 with the biosensor\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo quantify MMP-2 biomarkers accurately, we established a robust correlation between the peak intensities of the Electrochemiluminescence (ECL) signal, denoted as E\u003csub\u003eRu\u003c/sub\u003e, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and B (Time vs. ECL intensity and Potential vs. ECL intensity respectively), and the MMP-2 concentration, labeled as C\u003csub\u003eMMP\u0026minus;2\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). The observed linear correlation between E\u003csub\u003eRu\u003c/sub\u003e and C\u003csub\u003eMMP\u0026minus;2\u003c/sub\u003e spans concentrations ranging from 10 aM to 100 pM. From the data, we formulated a regression equation: E\u003csub\u003eRu\u003c/sub\u003e (a. u.)\u0026thinsp;=\u0026thinsp;0.091 \u0026times; lgC\u003csub\u003eMMP\u0026minus;2\u003c/sub\u003e + 0.040 (R\u0026sup2; = 0.9976, n\u0026thinsp;=\u0026thinsp;5). The linear regression equation in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC is established using data from both Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB. The signal values in Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB are consistent, with the only difference being that the horizontal axis in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA corresponds to Time, while in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB it corresponds to Potential. This equation not only facilitates precise logarithmic quantification of MMP-2 in biological samples but also confirms the biosensor\u0026rsquo;s high precision and reliability. The detection limit (LOD), a critical metric of the biosensor\u0026rsquo;s sensitivity, was determined using the 3σ method, resulting in an LOD of 12.8 aM. This LOD notably outperforms those of many existing methodologies, highlighting our biosensor\u0026rsquo;s superior sensitivity and its ability to detect extremely low concentrations of MMP-2.\u003c/p\u003e \u003cp\u003eTo provide a broader context, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e contrasts our newly developed methodology with existing MMP-2 quantification techniques. The comparative analysis demonstrates that our approach provides comparable sensitivity and a linear detection range similar to current methods but with a slightly more advantageous detection limit. Such findings suggest that our biosensor not only meets but potentially exceeds current MMP-2 analysis standards, making it a promising alternative for anti-aging research applications, especially in diagnostic and monitoring scenarios.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of different methods for MMP-2 assay.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMethod\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLOD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eLinear Range\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eReference\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFluorescent Nanoprobe\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e32 pM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.1\u0026ndash;20 nM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSilicon Nanowire-Based Biosensor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e0.1 pM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100 fM-10 nM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCRISPR Cas13a based biosensor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e62.05 fM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e150\u0026ndash;2000 fM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBipedal walking robot\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e12.8 aM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.01\u0026ndash;100 pM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThis work\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Specificity and reproducibility of the strategy\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSpecificity is crucial in developing enzymatic methodologies, especially when employing Peptide Nucleic Acid (PNA) as a substrate to detect matrix metalloproteinase 2 (MMP-2). Ensuring that MMP-2 selectively cleaves the PNA substrate is essential for reducing unintended off-target interactions and increasing the accuracy of the detection method. To ascertain the specificity of this technique, we identified several proteins unlikely to interfere with PNA, including Esterase, Matrix Metalloproteinase 1 (MMP1), Thrombin, Bovine Serum Albumin (BSA), Alpha-fetoprotein (AFP), and Carcinoembryonic Antigen (CEA), used as reference proteins (illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). We measured their electrochemiluminescent (ECL) responses using the CRISPR/Cas13a amplification method. As demonstrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, the ECL readings for these non-target proteins were compared against those for MMP-2 (concentration of 500 fM). The findings clearly showed that MMP-2's ECL signal underwent the most notable change compared to the baseline, whereas the signals from the other proteins remained largely consistent with the blank controls. In conclusion, the effectiveness of using PNA as a substrate for MMP-2 detection heavily depends on the specificity of the interaction. By carefully selecting reference proteins and fine-tuning the PNA sequence to specifically recognize a target site within MMP-2, we can significantly enhance the method's specificity and minimize the likelihood of off-target effects. The electrochemiluminescent biosensor developed via the CRISPR/Cas13a amplification method showcases exceptional selectivity, positioning it as an effective tool for various applications in biotechnology and medical diagnostics.\u003c/p\u003e \u003cp\u003eSubsequently, we conducted an assessment of the stability of the biosensing platform. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB provides a detailed representation of the detection stability within this Electrochemiluminescence (ECL) detection platform. This figure depicts the detection signal generated by the biosensing platform when detecting 1 pM MMP-2 as the target analyte. Remarkably, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, the detection signal for 1 pM MMP-2 remains consistently stable, even after subjecting it to ten consecutive scans, each spaced an hour apart (RSD\u0026thinsp;=\u0026thinsp;1.34%). There is no substantial decline in the signal's intensity. These observations solidify the remarkable stability of our sensing platform.\u003c/p\u003e \u003cp\u003eFurthermore, we carried out an evaluation of the biosensor's shelf life. The biosensors, containing DNA/[Ru(phen)\u003csub\u003e2\u003c/sub\u003edppz]\u003csup\u003e2+\u003c/sup\u003e complexes, were stored in a sealed container at a constant temperature of 4\u0026deg;C, shielded from light, following the detection of 1 pM MMP-2. After each test, the ECL signal of the biosensor was measured, and the relative standard deviation (RSD) of the signal over the course of 14 days was calculated (as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Notably, the functionality and sensitivity of the CRISPR/Cas13a-based biosensor remained consistent throughout the entire testing period, exhibiting no noticeable performance degradation. The RSD value of the ECL signal was a mere 0.88%, underscoring the biosensor's high stability and repeatability.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Recovery studies of the biosensor in real samples\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBiosensors based on electrochemiluminescence, engineered through the CRISPR/Cas13a isothermal amplification method, display an impressive blend of heightened sensitivity and precision. These qualities render them highly effective for detecting MMP-2 activity within complex biological matrices. Owing to the prevalent surge in MMP-2 activity across a variety of human cancers and tissues, targeting MMP-2 has become a focal point in the realms of cancer diagnostics and treatment. Faced with the challenge of scarce aging-related cell samples, this study utilized supernatants from HepG2 and LO2 cell cultures to evaluate the biosensor's efficacy. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, the examination of these supernatants indicated a negligible variation in electrogenerated chemiluminescence (ECL) signals among the LO2 cell samples. In contrast, the HepG2 cell samples exhibited a significant surge in ECL signals, marking a 6.73-fold increase relative to the LO2 samples. This significant increase is attributed to the heightened expression of MMP-2 in cancerous cells.\u003c/p\u003e \u003cp\u003eTo ensure the specificity of our electrochemiluminescence sensor, we performed a targeted pre-treatment experiment involving HepG2 cells and a known MMP-2 inhibitor, ARP 100. This intervention significantly reduced the ECL signal change to levels comparable with those of the control group, nearly zero. Such a result strongly indicates that the ECL response is directly attributable to MMP-2's specific activity on the PNA substrate. This validation highlights the exceptional potential of electrochemiluminescence sensors utilizing the bipedal walker isothermal amplification method for detecting MMP-2 activity in clinical settings. These findings are crucial as they confirm the sensor's high specificity and sensitivity, demonstrating its significant advantages for practical applications in the medical field, particularly in cancer diagnostics and therapy development. The technology\u0026rsquo;s precise detection capabilities enable it to identify MMP-2 activity accurately, making it a valuable tool for developing targeted treatment strategies and improving patient outcomes in oncology.\u003c/p\u003e \u003cp\u003eSubsequently, we measured the content and recovery rate of matrix metalloproteinase-2 (MMP-2) by correlating it with the spectrum of electrochemiluminescence (ECL) signals derived from LO2 cell culture supernatants. These signal levels fluctuated between 99.8% and 105.0%, as outlined in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. This significant finding highlights the biosensor's outstanding ability to withstand external disturbances, making it an excellent candidate for reducing CRISPR/Cas13a activity, especially under complex conditions. The findings from these tests conclusively demonstrate the superior analytical capabilities of our approach in detecting MMP-2.\u003c/p\u003e \u003cp\u003eThe ECL signal levels we obtained play a crucial role in deciphering both the concentration and recovery of MMP-2, offering a reliable method for its assessment and quantification across various sample types. The wide range of recovery rates demonstrates the robust stability of our biosensors, even in the face of potential disruptions caused by background noise, contaminants, or other biological elements prevalent in real-world specimens. This resistance to interference is particularly valuable in practical scenarios, where dealing with complex biological matrices demands high precision and specificity in detection.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eRecovery results for the assay of MMP-2 in cell culture supernatants of LO2.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample number\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eAdded (aM)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eFound (fM)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRecovery (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eRSD (%, n\u0026thinsp;=\u0026thinsp;3)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e2.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e103.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e100.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.73\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e1052.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e105.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4.65\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e9979.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e99.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4.62\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e100159.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e100.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.63\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eThis paper presents a novel electrochemiluminescence (ECL) biosensor for the detection of matrix metalloproteinase 2 (MMP-2), a key enzyme involved in anti-aging research. The biosensor integrates the high specificity and signal amplification of the CRISPR/Cas13a system with the high efficiency and simplicity of the exponential amplification reaction (EXPAR) and the \u0026ldquo;light switch\u0026rdquo; [Ru(phen)2dppz]2\u0026thinsp;+\u0026thinsp;probe. The main findings and contributions of this paper are summarized as follows:\u003c/p\u003e \u003cp\u003eThe biosensor exhibits a low detection limit of 12.8 aM and a wide linear range of 10 aM to 100 pM for MMP-2, surpassing or comparable to many existing methods. The biosensor also demonstrates excellent selectivity, reproducibility, and stability, as well as applicability in real samples such as cell culture supernatants. The biosensor provides a promising tool for MMP-2 analysis in anti-aging research, as well as cancer diagnosis and therapy. The biosensor also showcases the potential of integrating CRISPR/Cas13a with other amplification and detection strategies for creating sensitive and specific biosensors for various targets.\u003c/p\u003e \u003cp\u003eThe biosensor employs a novel design that combines PNA, T7 promoter, DNA template, and Cas13a to achieve transcription amplification, trans-cleavage activation, and EXPAR amplification. The biosensor also utilizes the \u0026ldquo;photoswitch\u0026rdquo; molecule [Ru(phen)\u003csub\u003e2\u003c/sub\u003edppz]\u003csup\u003e2+\u003c/sup\u003e to avoid the cumbersome electrode modification and cleaning process, simplifying the experimental procedures and reducing the testing cost.\u003c/p\u003e \u003cp\u003eThe biosensor is based on a solid theoretical foundation and rigorous experimental validation. The paper provides a comprehensive explanation of the principles, mechanisms, and optimization of the biosensor. The paper also presents detailed experimental results, data analysis, and discussion to support the claims and conclusions.\u003c/p\u003e \u003cp\u003eIn conclusion, this paper reports a novel ECL biosensor for MMP-2 detection that is highly sensitive, specific, simple, and cost-effective. The biosensor is expected to have broad applications in anti-aging research and other fields that require MMP-2 analysis. The paper also contributes to the advancement of CRISPR/Cas13a-based biosensing technologies and opens new avenues for future research.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch3\u003eSupplementary data\u003c/h3\u003e\n\u003cp\u003eSupplementary data can be found in the online version.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Acknowledgment\u003c/p\u003e\n\u003cp\u003eWe would like to thank the Startup Foundation for Introducing Talent of NUIST (2023r129), the financial support of the National Natural Science Foundation of China (21964018), the Guangxi key laboratory of basic and translational research of Bone and joint Degenerative Disease (21-220-06-202205), the State Key Laboratory of Analytical Chemistry for Life Science (SKLACLS2314).\u003c/p\u003e\n\u003ch3\u003e\u0026nbsp;\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eAll authors disclosed no relevant relationships.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003enot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank the Startup Foundation for Introducing Talent of NUIST (2023r129), the financial support of the National Natural Science Foundation of China (21964018), the Guangxi key laboratory of basic and translational research of Bone and joint Degenerative Disease (21-220-06-202205), the State Key Laboratory of Analytical Chemistry for Life Science (SKLACLS2314).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding authors upon reasonable request.\u003c/p\u003e "},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePientaweeratch S, Panapisal V, Tansirikongkol A. 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Anal Chem.86(15):7719-25. doi: 10.1021/ac5016563.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Scheme 1","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"microchimica-acta","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"miac","sideBox":"Learn more about [Microchimica Acta](https://link.springer.com/journal/604)","snPcode":"604","submissionUrl":"https://submission.springernature.com/new-submission/604/3","title":"Microchimica Acta","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"CRISPR/Cas13a, MMP-2, T7 RNA, Electrochemiluminescence","lastPublishedDoi":"10.21203/rs.3.rs-4689418/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4689418/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMatrix metalloproteinase-2 (MMP-2) plays a pivotal role in anti-aging research. Developing advanced detection platforms for MMP-2 with high specificity, sensitivity, and accessibility is crucial. This study introduces a novel electrochemiluminescence (ECL) biosensor for MMP-2 analysis, leveraging the CRISPR/Cas13a system and Exponential Amplification Reaction (EXPAR). The biosensor operates by utilizing the T7 RNA polymerase to transcribe RNA from a DNA template upon MMP-2 interaction. This RNA activates Cas13a, leading to signal amplification and ECL detection. The incorporation of the \"photoswitch\" molecule [Ru(phen)\u003csub\u003e2\u003c/sub\u003edppz]\u003csup\u003e2+\u003c/sup\u003e streamlines the process by eliminating the need for extensive electrode modification and cleaning. Under optimized conditions, the biosensor achieved an impressive detection limit of 12.8 aM for MMP-2. The platform demonstrated excellent selectivity, reproducibility, and stability, making it highly suitable for detecting MMP-2 in complex biological samples. This innovative approach shows great potential for applications in molecular diagnostics and anti-aging research.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e","manuscriptTitle":"Electrochemiluminescence Biosensor for MMP-2 Analysis Using CRISPR/Cas13a and EXPAR Amplification: A Novel Approach for Anti-Aging Research","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-30 14:55:23","doi":"10.21203/rs.3.rs-4689418/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-08-07T06:18:49+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-05T20:32:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"159267614788538972171259953433297943637","date":"2024-07-29T15:10:44+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-23T17:08:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"333007481106056834489539606998795638769","date":"2024-07-15T10:07:03+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-10T06:06:13+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-05T08:35:05+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-05T08:34:30+00:00","index":"","fulltext":""},{"type":"submitted","content":"Microchimica Acta","date":"2024-07-05T03:46:28+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"microchimica-acta","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"miac","sideBox":"Learn more about [Microchimica Acta](https://link.springer.com/journal/604)","snPcode":"604","submissionUrl":"https://submission.springernature.com/new-submission/604/3","title":"Microchimica Acta","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"12901851-ea61-4ba0-90dd-aac6148d6900","owner":[],"postedDate":"July 30th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-10-21T16:09:14+00:00","versionOfRecord":{"articleIdentity":"rs-4689418","link":"https://doi.org/10.1007/s00604-024-06707-4","journal":{"identity":"microchimica-acta","isVorOnly":false,"title":"Microchimica Acta"},"publishedOn":"2024-10-14 15:58:05","publishedOnDateReadable":"October 14th, 2024"},"versionCreatedAt":"2024-07-30 14:55:23","video":"","vorDoi":"10.1007/s00604-024-06707-4","vorDoiUrl":"https://doi.org/10.1007/s00604-024-06707-4","workflowStages":[]},"version":"v1","identity":"rs-4689418","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4689418","identity":"rs-4689418","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}