The Role of Ladder-Branch HCR in the Development of Precision Biosensors for MMP-2 Quantification

doi:10.21203/rs.3.rs-4939093/v1

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Research Article

The Role of Ladder-Branch HCR in the Development of Precision Biosensors for MMP-2 Quantification

https://doi.org/10.21203/rs.3.rs-4939093/v1

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This study successfully developed an advanced Electrochemiluminescence (ECL) biosensor that integrates T7 RNA polymerase amplification, ladder-branch hybridization chain reaction (HCR), and the precise targeting capabilities of CRISPR/Cas13a technology. This innovative biosensor addresses the critical need for sensitive and specific detection of matrix metalloproteinase-2 (MMP-2), a key biomarker in cancer diagnostics. Through meticulous optimization of amplification and reaction conditions, the biosensor demonstrated remarkable sensitivity and specificity, achieving a detection limit as low as 6.34 aM, surpassing existing methodologies. The biosensor also exhibited excellent stability and reproducibility across multiple scans and maintained consistent functionality over an extended period, highlighting its reliability for practical applications. The effectiveness of the biosensor was validated using real samples, demonstrating its capability to accurately quantify MMP-2 in complex biological matrices with high recovery rates and minimal interference. The integration of isothermal amplification and CRISPR/Cas13a within the ECL biosensor platform represents a significant advancement in molecular diagnostics, offering a powerful tool for real-time monitoring of MMP-2. This biosensor holds substantial promise for revolutionizing cancer diagnostics and facilitating personalized treatment strategies, ultimately aiming to improve patient outcomes in cancer care. Future research may explore further enhancements and applications of this biosensor in various clinical and environmental settings.

MMP-2

CRISPR/Cas13a

HCR

Electrochemiluminescence biosensor

In the expansive field of molecular biology and disease diagnostics, matrix metalloproteinases (MMPs), specifically MMP-2, have garnered significant attention due to their pivotal role in extracellular matrix degradation, influencing critical biological processes such as wound healing, angiogenesis, and tissue remodeling.[1–4] The enzyme's profound implications in cancer progression, by facilitating tumor invasion and metastasis, underscore the urgent need for highly sensitive, specific, and efficient MMP-2 detection methods.[5] These methods are crucial for the early diagnosis of cancer and the monitoring of treatment responses, thereby presenting a significant leap forward in oncology diagnostics and personalized medicine.

Electrochemiluminescence (ECL) biosensors emerge as a beacon of innovation in this context, promising to transcend the limitations of conventional MMP-2 detection methods.[6–10] ECL biosensors, renowned for their remarkable sensitivity, specificity, and the potential for miniaturization, represent a groundbreaking approach in the real-time monitoring of MMP-2 within clinical specimens.[11–14] The integration of T7 RNA polymerase within these biosensors marks a transformative advancement, amplifying low-concentration biomarkers essential for the early detection of cancer. This novel biosensor paradigm utilizes a hairpin probe mechanism, offering cycle-based signal amplification that drastically enhances both sensitivity and specificity in MMP-2 detection, thus heralding a new era in cancer diagnostics.

Furthering the technological frontier, isothermal amplification techniques, paired with the revolutionary CRISPR cas13a system, have set the stage for the next generation of biosensing platforms.[15–19] Unlike traditional polymerase chain reaction (PCR) methodologies, isothermal amplification facilitates nucleic acid amplification at a constant temperature, greatly simplifying the detection apparatus and making it apt for point-of-care applications. The CRISPR cas13a system, repurposed from its original function in bacterial adaptive immunity, offers unprecedented precision in biosensing. Programmed with CRISPR RNA (crRNA), cas13a targets specific RNA sequences, exhibiting collateral activity by indiscriminately cleaving nearby non-targeted RNA.[20–25] This unique characteristic is ingeniously exploited in biosensor development, enabling significant signal amplification and creating highly sensitive and specific platforms for the detection of nucleic acid biomarkers directly from clinical samples.

This study introduces an avant-garde ECL biosensor that ingeniously integrates T7 RNA polymerase amplification, ladder-branch hybridization chain reaction (HCR),[26] and the precision of CRISPR cas13a. This biosensor leverages the robust signal amplification capabilities of T7 RNA polymerase and the specificity of CRISPR cas13a, targeting MMP-2 with unparalleled sensitivity and specificity. By employing ECL for detection, this biosensor offers quantifiable results, thereby significantly enhancing its practical applications in clinical diagnostics and environmental monitoring. This innovative biosensor not only simplifies the detection process but also broadens the horizons of advanced molecular diagnostics, paving the way for enhanced disease management and therapeutic interventions.

2.1 Materials and chemical reagents

The oligonucleotides for this investigation were acquired from Genscript Biotechnology Co., Ltd, located in Nanjing, China, and their sequences are detailed in Supplementary Table S1. Peptide Nucleic Acid (PNA), crucial for this study, was sourced from TAHE-PNA Co., Ltd in Hangzhou, China. The research also utilized essential reagents such as Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and 6-mercaptohexanol (MCH), obtained from Sigma-Aldrich, based in St. Louis, MO, USA. Other chemicals employed were of reagent-grade quality and were used as obtained. Solution preparations were conducted with ultrapure water, purified using the Milli-Q system by Branstead, which achieved a resistivity higher than 18.2 MΩ·cm.

2.2 Instruments and Measurements

The detection of the ECL signal was performed using an ECL detection instrument, kindly provided by the State Key Laboratory of Analytical Chemistry for Life Sciences, located at Nanjing University. The setup utilized a three-electrode system, comprising a glassy carbon electrode (GCE) as the working electrode, an Ag/AgCl electrode as the reference, and a platinum electrode serving as the counter electrode.

2.3 Proteolytic cleavage by MMP-2, enzymatic amplification facilitated through T7 RNA polymerase, and the activation process of CRISPR/Cas13a system

The construction of the PNA/T7 promoter/DNA template began with mixing the T7 promoter, DNA template, and PNA in HES buffer containing 1 U/µL RNase inhibitor, in proportions of 1:1:1.2, respectively. This mixture was then incubated at 41.5°C for 15 minutes, subsequently cooled to ambient temperature, and left to stabilize at 4°C for an overnight period, leading to the successful formation of the PNA/Bandage complex.

To proceed with the MMP-2-induced release of the DNA template/T7 promoter complex, recombinant human MMP-2 was first activated in AC buffer (which includes 50 mM HEPES, 10 mM CaCl2, 150 mM NaCl, and 0.05% (w/v) Brij 35, at a pH of 7.0) at 37°C over 2 hours. After this activation, different concentrations of MMP-2 were incorporated into the PNA/T7 promoter/DNA template blend and incubated for 30 minutes.

Subsequently, 50 µL of this mixture was added to an amplification reaction mix, resulting in a total volume of 100 µL. This amplification mix contained 40 µM NTPs, 30 U T7 RNA polymerase, 1 µM DNA template, 20 U RNase inhibitor, and 2 µL of a 10x RNAPol reaction buffer. To achieve transcription amplification, the reaction was incubated at 37°C for 40 minutes.

Subsequent to the transcription stage, the mixture was incubated at room temperature for 20 minutes, integrating 50 nM hybrid DNA (HDNA), 10 nM CRISPR/Cas13a, and 15 nM guide RNA (gRNA) into a 1x NEBuffer solution provided by New England Biolabs (NEB). Post-incubation, the prepared biosensor was immersed in the CRISPR/Cas13a activated solution for a 20-minute duration to enable the cleavage of the previously activated DNA. Following this cleavage, 0.2 units of T4 polynucleotide kinase (T4 PNK) along with the T4 PNK reaction buffer were introduced to phosphorylate the 3′-end of the cleaved HDNA, thereby setting the stage for the subsequent amplification phase, which was conducted at 37°C for 30 minutes.

2.4 Ladder-branch hybridization chain reaction

The electrode, treated with gold electrode-CP and hybridization probes H1, H2, H3, H4, H5, and H6, was incubated at room temperature for 90 minutes. Subsequently, the modified electrode was rinsed five times with PBS. After that, it was mixed with 10 µL of a 0.5 × 10^− 3 M solution of [Ru(phen)₂dppz]²⁺ and 90 µL of a 70 × 10^− 3 M TPrA solution for 30 minutes. Following this preparation step, the measurement of the ECL signal was conducted.

3.1 The working principle

Scheme 1 illustrates the conceptual framework and operational mechanics of the proposed platform. For this experimental demonstration, MMP-2 was selected as the representative biomarker. A unique Peptide Nucleic Acid (PNA) sequence was engineered, incorporating a peptide chain (Gly-Pro-Leu-Gly-Val-Arg-Gly) that is specifically cleavable by MMP-2. It’s crucial to recognize that PNA is a synthetic mimic of nucleic acids, distinguished by its peptide-based backbone in place of the traditional sugar-phosphate structure.

The initial configuration consists of a PNA/T7 promoter/DNA template assembly, where a double-helical DNA template is bridged by PNA and a T7 promoter, incorporating a peptide linkage susceptible to MMP-2 cleavage. In scenarios devoid of MMP-2, the PNA/DNA complex forms a blockade, preventing the amplification cascade triggered by the T7 promoter, as depicted in Scheme 1A.

Upon the integration of MMP-2 into the system, as depicted in Scheme 1B, the enzymatic action on the peptide linkage is initiated. This enzymatic activity results in the liberation of the single-stranded DNA template paired with the T7 promoter. The freed complex then functions as a template for the T7 promoter-driven transcriptional amplification process. The amplification yields a significant number of single-stranded RNA transcripts, which contain sequences that are specifically recognized and bound by guide RNA (gRNA).

The interaction of gRNA with these transcripts activates the CRISPR/Cas13a system’s nuclease function, leading to the cleavage of targeted single-stranded RNA. The Cas13a/crRNA complex is engineered for precise identification and severing of the designated RNA. This interaction initiates the trans-cleavage activity, where the two HEPN domains of Cas13a reposition to create an active RNase site. Following this, Cas13a executes trans-cleavage on the -U-U-modified Hairpin DNA (H-U), splitting it into two distinct segments.

These segments are then recognized by DNA (DNA1) affixed to the Glassy Carbon Electrode (GCE) surface, and hybridization occurs with sequences H1, H2, H3, H4, H5 and H6. This triggers a ladder-branch HCR, resulting in a complex DNA nanostructure. The next step involves introducing the “light switch” [Ru(phen)₂dppz]²⁺ molecule into the Cas13a-facilitated ladder-branch HCR system. In its free state, [Ru(phen)₂dppz]²⁺ does not emit light due to the protonation of its nitrogen atoms in an aqueous environment. However, when it encounters the double-helical DNA from the ladder-branch HCR reaction, a marked increase in luminescence is observed. This boost is due to the interaction of the planar phenazine ligand of [Ru(phen)2dppz]2 + with the base pairs within the major groove of the DNA, which protects the nitrogen atoms and promotes the luminescent state.

Subsequently, the biosensor platform is supplemented with the [Ru(phen)₂dppz]²⁺ enhanced amplification system and an excess of Tripropylamine (TPrA). The resulting electrochemiluminescence (ECL) signal, indicative of MMP-2 activity, is captured by a photomultiplier tube (PMT). The detailed reaction mechanism is outlined as follows:

[Ru(phen)₂dppz]²⁺/DNA-e⁻ →[Ru(phen)₂dppz]³⁺+H⁺

TPrA-e⁻ → TPrA∙⁺ → TPrA∙ + H⁺

[Ru(phen)₂dppz]³⁺-DNA + TPrA∙ → [Ru(phen)₂dppz]^2+∗-DNA + products

[Ru(phen)₂dppz]²⁺∗-DNA → [Ru(phen)₂dppz]²⁺-DNA + hν

3.2 Electrochemical characterization of the stepwise process for the biosensor

The functionality and mechanism of the biosensor were elucidated through detailed analyses of cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) data. As shown in Fig. 1A, the CV curve for the pristine gold electrode (curve a) exhibits prominent oxidation-reduction peaks, indicative of efficient electron transfer under unmodified conditions. Upon sequential modification of the electrode with the DNA1 probe (curve b) and 6-Mercapto-1-hexanol (MCH, curve c), a marked decrease in these peaks is observed, signaling restricted electron flow due to the insulating effects of the DNA and MCH layers.

Further modification with hybridized single-stranded DNA, resulting from the cleavage of DNA1 and Hairpin DNA, demonstrates a continued reduction in current (curve d). This indicates an enhanced impediment to electron transfer, which is exacerbated when multiple DNA segments (H1, H2, H3, H4, H5, and H6) hybridize on the electrode surface, leading to a further decrease in the electrochemical signal (curve e).

Figure 1B illustrates the corresponding impedance characteristics of the biosensor. The inherent impedance of the unaltered gold electrode is low, as depicted by curve a, which signifies minimal resistance to electron flow. The application of DNA1 and MCH increases the impedance, as evidenced by the expanded semicircular profiles in the Nyquist plots (curves b and c, respectively). Post-hybridization impedance further increases, with a notable expansion of the semicircle (curve d), indicative of elevated barriers to electron transport due to the presence of hybridized DNA segments. The introduction of multiple DNA segments further increases the impedance, significantly enlarging the semicircle in the Nyquist plot (curve e), thus confirming substantial opposition to electron transfer.

The consistent trends observed in both the CV and EIS data affirm the precise engineering and functional integrity of the electrochemical biosensor. These modifications in the biosensor design validate its capacity to transduce biomolecular interactions into quantifiable electrochemical signals, thereby providing a robust platform for the sensitive detection of MMP-2.

3.3 Feasibility study

Evaluations using Electrochemiluminescence (ECL) were performed to verify the performance of a newly devised ECL biosensor, which was engineered specifically for the detection of matrix metalloproteinase-2 (MMP-2). The outcomes, illustrated in Fig. 1C, shed light on its efficacy. When the gold electrode, modified with DNA and treated with [Ru(phen)₂dppz]²⁺, was the sole component present, the ECL response was notably subdued, as demonstrated in curve a. This suggested a scant adsorption of [Ru(phen)₂dppz]²⁺ on the electrode's surface. Contrasting this, curve b exhibits a significant ECL intensification post the application of CRISPR/Cas13 directed enzymatic cleavage and dual signal amplification methods (100 fM MMP-2). The signal surge is attributed to the amplification via T7 RNA polymerase and the subsequent integration of [Ru(phen)₂dppz]²⁺ within the double-stranded DNA on the electrode surface, catalyzed by the ladder-branch hybridization chain reaction (HCR), resulting in the observed ECL signal elevation.

3.4 Optimization of analytical conditions

In the effort to enhance the biosensor’s performance, a comprehensive optimization study was conducted, focusing on four key experimental variables. Using Electrochemiluminescence (ECL), the biosensor’s electrical response was thoroughly evaluated under various conditions, as shown in Fig. 2. The optimization process meticulously addressed the following parameters:

Optimization of T7 RNA Polymerase Transcription Time

As illustrated in Fig. 2A, the ECL signal increased with time, reaching a plateau after 30 minutes. This conclusion was drawn from a systematic investigation over a period ranging from 0 to 50 minutes. Consequently, the optimal transcription time for T7 RNA polymerase was determined to be 30 minutes.

Adjusting the CRISPR/Cas13a Reaction Interval

Figure 2B shows an increase in the ECL signal from 0 to 50 minutes, measured at intervals of 5, 10, 15, 20, 25, and 30 minutes. Equilibrium was reached after 25 minutes, indicating that this interval is the most effective reaction time for optimal CRISPR/Cas13a outcomes.

Adjusting Cas13a/crRNA Amounts

The ratio of Cas13a to crRNA significantly impacts the cis-cleavage activity and the efficiency of the isothermal amplification process. Various concentrations of Cas13a/crRNA (2.5 nM, 5 nM, 7.5 nM, 10 nM, 15 nM, and 20 nM) were tested using ECL, as shown in Fig. 2C. The optimal performance was achieved with 10 nM of Cas13a/crRNA. Increasing the concentration to 20 nM did not enhance the ECL signal, suggesting that higher concentrations do not lead to better signal amplification. Therefore, future experiments will utilize a 10 nM Cas13a/crRNA concentration.

Ladder-branch HCR Reaction Time

A detailed analysis of the ladder-branch HCR reaction duration revealed that the ECL intensity gradually increased from 10 to 60 minutes, as depicted in Fig. 2D, and then plateaued. This trend indicates that the reaction approached completion around the 60-minute mark. Beyond this point, the ECL signal's growth plateaued, suggesting that the reaction had reached saturation. Therefore, 60 minutes is identified as the optimal reaction time for the ladder-branch HCR to maximize the ECL signal and ensure the reaction's completion.

3.5 Detection of MMP-2 with the biosensor

To ascertain the levels of MMP-2 biomarkers, a solid correlation was delineated between the peak Electrochemiluminescence (ECL) signal intensity, denoted as E_Ru (illustrated in Fig. 3A showcasing Time vs. ECL intensity), and the corresponding logarithm of the concentration of MMP-2, labeled as CMMP-2 (presented in Fig. 3B). The linear correlation between E_Ru and C_MMP−2 was discernible across a span from 10 aM to 100 fM. The regression formula derived from data fitting is: E_{Ru (a. u.)} = 0.00188 + 0.0042lgC_{MMP − 2} with an R² value of 0.9962. This formula not only provides a precise method for quantifying MMP-2 logarithmic concentrations in biological specimens but also affirms the biosensor’s outstanding accuracy and dependability.

The detection threshold, or limit of detection (LOD), is a crucial metric indicative of the biosensor’s acuity. Through the application of the 3σ approach, the LOD was calculated to be 6.34 aM, markedly exceeding the LOD of numerous existing methodologies, thereby highlighting the biosensor’s extraordinary sensitivity and its proficiency in detecting minuscule concentrations of MMP-2.

For an expanded view, Table 1 juxtaposes the methodological approach of this study with those currently in use for MMP-2 quantification. The findings clearly demonstrate that our approach yields sensitivity on par with, a linear range comparable to, and a slightly more refined detection threshold than, certain existing methods. This implies that the biosensor formulated in this research not only meets but also enhances the prevailing benchmarks for MMP-2 quantification. Consequently, it holds promise as an instrumental alternative for MMP-2 related anti-aging research applications, especially in diagnostic and monitoring scenarios.

Table 1

Comparison of different methods for MMP-2 assay.
Method	LOD		Reference
Fluorescent Nanoprobe	32 pM	0.1–20 nM	[27]
Silicon Nanowire-Based Biosensor	0.1 pM	100 fM-10 nM	[28]
CRISPR Cas13a based biosensor	62.05 fM	150–2000 fM	[19]
CRISPR/Cas 13a aidded Ladder-branch HCR	6.34 aM	10 aM to 100 fM	This work

3.6 Specificity and reproducibility of the strategy

The essence of specificity is crucial in enzymatic strategy development, especially when employing peptide nucleic acid (PNA) as a substrate for matrix metalloproteinase 2 (MMP-2). The targeted cleavage of PNA by MMP-2 is essential to reduce off-target interactions and enhance the method’s precision. To ascertain this specificity, proteins unlikely to interact with PNA were used as controls. These included Esterase, Matrix Metalloproteinase 1 (MMP1), Thrombin, Bovine Serum Albumin (BSA), Alpha-fetoprotein (AFP), and Carcinoembryonic Antigen (CEA), as shown in Fig. 4A. Their electrochemiluminescent (ECL) signals were measured using the CRISPR/Cas13a amplification method. The ECL readings for these reference proteins, compared with MMP-2 (100 fM), confirmed that only MMP-2’s signal notably differed from the baseline, while the others remained close to the blank controls. Thus, the specificity of using PNA as a substrate for MMP-2 is affirmed, which is pivotal for the method’s success. By selecting suitable reference proteins and fine-tuning the PNA sequence for MMP-2 recognition, specificity is maximized, reducing off-target effects. The developed electrochemiluminescent biosensor, utilizing CRISPR/Cas13a amplification, exhibited high selectivity, promising for diverse biotechnological and medical uses.

The stability of the biosensing platform was also evaluated. Figure 4B details the detection stability of the Electrochemiluminescence (ECL) platform when targeting 100 fM MMP-2. The detection signal remained stable across ten consecutive scans, spaced 16 min apart, with a relative standard deviation (RSD) of 0.46%, indicating no significant signal reduction, thus confirming the platform’s stability.

An assessment of the biosensor’s shelf life was conducted. Post 100 fM MMP-2 detection, the biosensors, with DNA/[Ru(phen)₂dppz]²⁺ complexes, were stored at 4°C, protected from light. Subsequent ECL signal measurements over 14 days showed a minimal RSD of 0.251%, as depicted in Fig. 4C, demonstrating consistent functionality and sensitivity, highlighting the biosensor’s durability and reliability.

3.7 Applicability of the biosensor in real samples

Electrochemiluminescence (ECL) biosensors, crafted via the CRISPR/Cas13a isothermal amplification technique, exhibit remarkable sensitivity and precision. These attributes make them exceptionally suitable for detecting MMP-2 activity within intricate biological environments. Given the widespread increase in MMP-2 activity in various cancers and tissues, MMP-2 has emerged as a key target in cancer diagnostics and therapeutic strategies. The content and recovery rate of matrix metalloproteinase-2 (MMP-2) were measured by correlating them with the spectrum of ECL signals from LO2 cell culture supernatants, with signal levels ranging between 99.98% and 102.56%, as detailed in Table 2. This underscores the biosensor’s exceptional resilience to external perturbations, positioning it as an ideal tool for diminishing CRISPR/Cas13a activity, particularly in challenging conditions. These results affirm the superior analytical performance of our method in MMP-2 detection.

The obtained ECL signal levels are instrumental in determining MMP-2 concentration and recovery, providing a dependable means for its evaluation and quantification across diverse sample matrices. The broad spectrum of recovery rates attests to the biosensors’ robust stability, capable of countering potential interferences from background noise, contaminants, or other biological factors common in real samples. This interference resistance is invaluable in practical applications, where navigating complex biological matrices necessitates 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)			RSD (%, n = 3)
1	0	9.8
3	100	112.36	102.56	3.56
4	1000	1011.56	100.18	4.23
5	10000	10007.59	99.98	4.16
6	100000	100026.35	100.02	3.28

This research has successfully developed an advanced Electrochemiluminescence (ECL) biosensor that combines T7 RNA polymerase amplification, ladder-branch hybridization chain reaction (HCR), and the precise targeting capabilities of CRISPR/Cas13a technology. This innovative biosensor addresses the critical need for sensitive and specific detection of matrix metalloproteinase-2 (MMP-2), a key biomarker in cancer diagnostics. The biosensor's design was meticulously optimized, ensuring maximum performance through fine-tuning of amplification and reaction conditions.

Key findings of this study include:

Enhanced Sensitivity and Specificity: The biosensor demonstrates remarkable sensitivity and specificity in detecting MMP-2, with a detection limit as low as 6.34 aM. This performance surpasses existing methodologies, making it a highly reliable tool for early cancer diagnosis.

Robust Stability and Reproducibility: The biosensor exhibits excellent stability across multiple scans and maintains consistent functionality over an extended period, highlighting its reliability for practical applications.

Broad Applicability: The biosensor's effectiveness was validated using real samples, demonstrating its capability to accurately quantify MMP-2 in complex biological matrices with high recovery rates and minimal interference.

The integration of isothermal amplification and CRISPR/Cas13a within the ECL biosensor platform marks a significant advancement in molecular diagnostics, offering a powerful tool for real-time monitoring of MMP-2. This biosensor holds substantial promise for revolutionizing cancer diagnostics and facilitating personalized treatment strategies, ultimately aiming to improve patient outcomes in cancer care. Future research may explore further enhancements and applications of this biosensor in various clinical and environmental settings.

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

Clinical trial number: not applicable.

Funding

Availability of data and materials

The data that support the findings of this study are available from the corresponding authors upon reasonable request.

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Scheme 1 is available in the Supplementary Files section.

No competing interests reported.

11LadderbranchHCRSI.docx
floatimage2.png
Scheme 1. Schematic of CRISPR/Cas13a and ladder-branch HCR biosensor platform detecting MMP-2. A) shows the initial configuration where the PNA/DNA complex forms a blockade in the absence of MMP-2, preventing transcriptional amplification. B) depicts the system upon MMP-2 integration, where the enzymatic action on the peptide linkage allows the transcriptional amplification to proceed, leading to the activation of the CRISPR/Cas13a system and the subsequent signal generation steps.

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The Role of Ladder-Branch HCR in the Development of Precision Biosensors for MMP-2 Quantification

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials and Method

2.1 Materials and chemical reagents

2.2 Instruments and Measurements

2.3 Proteolytic cleavage by MMP-2, enzymatic amplification facilitated through T7 RNA polymerase, and the activation process of CRISPR/Cas13a system

2.4 Ladder-branch hybridization chain reaction

3. Results and discussion

3.1 The working principle

3.2 Electrochemical characterization of the stepwise process for the biosensor

3.3 Feasibility study

3.4 Optimization of analytical conditions

3.5 Detection of MMP-2 with the biosensor

3.6 Specificity and reproducibility of the strategy

3.7 Applicability of the biosensor in real samples

4. Conclusions

Declarations

Supplementary data

Acknowledgment

Conflict of interest

Ethical Approval

Funding

Availability of data and materials

References

Scheme

Additional Declarations

Supplementary Files

Status:

Version 1