miRNA Detection Research Articles

Binding-driven forward tearing protospacer activated CRISPR-Cas12a system and applications for microRNA detection

CRISPR-Cas12a system, characterized by its precise sequence recognition and cleavage activity, has emerged as a powerful and programmable tool for molecular diagnostics. However, current CRISPR-Cas12a-based nucleic acid detection methods, particularly microRNA (miRNA) detection, necessitate additional bio-engineering strategies to exert control over Cas12a activity. Herein, we propose an engineered target-responsive hairpin DNA activator (TRHDA) to mediate forward tearing protospacer activated CRISPR-Cas12a system, which enables direct miRNA detection with high specificity and sensitivity. Target miRNA specifically binding to hairpin DNA can drive forward tearing protospacer in the stem sequence of hairpin structure, facilitating the complementarity between crRNA spacer and protospacer to activate Cas12a. Upon the hairpin DNA as input-responsive activator of Cas12a, a universal biosensing method enables the multiple miRNAs (miR-21, let-7a, miR-30a) detection and also has exceptional capability in identifying single-base mismatches and distinguishing homologous let-7/miR-30 family members. Besides, TRHDA-mediated Cas12a-powered biosensing has realized the evaluation of miR-21 expression levels in diverse cellular contexts by intracellular imaging. Considering the easy programmability of hairpin DNA in responsive region, this strategy could expand for the other target molecules detection (e.g., proteins, micromolecules, peptides, exosomes), which offers significant implications for biomarkers diagnostics utilizing the CRISPR-Cas12a system toolbox.Graphical abstract

Chain extension-mediated colorimetric and sensitive detection of osteosarcoma-related miRNA based on CHA-cooperated self-priming

The accurate and dynamic monitoring of osteosarcoma, one of the most prevalent cancers, has a substantial impact on the quality of life of numerous individuals. This study proposes a sample, sensitive, and portable approach to miRNA detection that integrates a catalytic hairpin assembly (CHA) circuit with self-priming mediated chain extension. This method employs CHA as a protein enzyme-free isothermal amplification technique to amplify miRNA signals and induce self-priming through a specially designed hairpin probe. The pyrophosphate sensing probe (pp probe) was employed, which enables dual high-efficiency and stable colorimetric signaling, in contrast to traditional colorimetric modes. This novel system for the colorimetric detection of target miRNA is operated with high sensitivity (LOD of 3.5 fM) and selectivity, with the entire detection procedure requiring approximately 60 min. Additionally, this system could detect miRNA from constructed serum samples with high stability and recovery. This system is unique, extremely simple, and rapid, and it is designed to detect osteosarcoma-related miRNA through a highly sensitive color change. It is likely to be useful in applications that necessitate point-of-care detection of osteosarcoma.

Catalytic hairpin assembly-triggered endonuclease-mediated cascade signal recycling for sensitive and colorimetric miRNA detection

AbstractThe catalytic hairpin assembly (CHA)-based miRNA detection methods have garnered significant attention due to their simplicity and acceptable amplification efficiency. However, these methods necessitate improved sensitivity. In this work, we present a colorimetric and ultrasensitive approach for the detection of cancer-related miRNAs which is initiated by CHA-mediated nicking endonuclease-assisted signals recycling. The target initiates the CHA process to expose the functional section in the P2 probe. This section can activate cascaded recycling cycles to produce numerous linker sequences by Nt.AlwI endonuclease-assisted cleavage of two hairpin signal probes. The 3,3’,5,5’-tetramethylbenzidine sulfate (TMB)/H2O2-based color reaction is induced by the fixation of the cDNA-HRP on the surface of magnetic beads, which is mediated by the linker sequence. The proposed method demonstrates a sensitivity that is either comparable to or superior to that of previous colorimetric miRNA detection methods, as a result of this design. Furthermore, the method demonstrated a promising potential for clinical applications and a high selectivity to target miRNA. Consequently, it provides a colorimetric assay that is both ultrasensitive and dependable for the visual detection of miRNA, which has the potential to revolutionize the early diagnosis of cancer.

Enzyme-Assisted Fluorescence Biosensor Based on Circular Single-Stranded DNA Without Group Modification for MicroRNA Detection

Fluorescent biosensor, which has the characteristics of high sensitivity, specificity, and low cost, can be directly detected in physiological fluids such as blood and serum. Therefore, the development of fluorescence sensor platforms for miRNA detection has a positive effect on the prevention and treatment of various diseases. In this paper, miR-34a was selected as a biological indicator of Alzheimer’s disease (AD). We designed a circular single-stranded DNA (CSSD) biosensor, which uses two unmodified single-stranded DNA (ssDNA) with complementary ends, DNAa and DNAb, to form CSSD by DNA sequence pairing to improve thermal stability and achieve signal amplification. At the same time, CSSD can react with miR-34a, and then the DNA of the DNA–RNA chain is hydrolyzed by duplex-specific nuclease (DSN enzyme). Finally, miR-34a is released to partake in the subsequent step, thus realizing cycle amplification. By evaluating the change in fluorescence signal under the optimized conditions, we discovered that this approach exhibits impressive sensitivity, with a detection threshold reaching as low as 0.36 nM. This surpasses the performance of numerous preceding miRNA detection biosensors. Furthermore, the system displays excellent detection capabilities even in intricate settings like serum, showcasing a strong ability to differentiate and choose effectively. In summary, this is a signal-off fluorescent biosensor, which realizes the purpose of double amplification of biosensor signal by using CSSD and enzyme assistance so that it can be used as a valuable tool for early diagnosis of diseases.

Construction of an efficient microRNA sensing platform based on terminal deoxynucleotidyl transferase-mediated synthesis of copper nanoclusters

A conceptual innovative electrochemical sensing platform was constructed for microRNA (miRNA) detection based on terminal deoxynucleotidyl transferase (TdT)-mediated synthesis of copper nanoclusters. In principle, the substrate strand immobilized on working electrode acted as the template for the TdT-mediated DNA extension reaction, while the DNAzyme probe would be activated in the absence of miRNA 21, which led to the cleavage of the substrate strand to produce the 5’ phosphate terminus and inhibited the TdT-mediated DNA extension reaction. On the other hand, the presence of miRNA 21 initiated the toehold-mediated strand displacement reaction, causing the disassembly of the DNAzyme structure and preventing the substrate strand from being cleaved. This enabled the recycle amplification of target miRNA 21, and TdT catalyzed the DNA extension reaction on the 3’ hydroxyl terminus of the substrate strand, producing a large number of poly thymine (polyT) sequences. These sequences bound with copper ions for in-situ synthesis of copper nanoclusters on the sensing electrode, and the increased electrochemical signal of copper was recorded by differential pulse stripping voltammetry (DPSV), which paved the way for sensitive determination of miRNA 21 extracted from cancer cells with a detection limit of 36 aM. Due to its high sensitivity, user-friendly operation, and cost-effectiveness, this method is helpful for the fundamental research of sensing mechanism as well as the diagnosis of related diseases.

Detecting Dirofilaria immitis: Current Practices and Novel Diagnostic Methods

The nematode Dirofilaria immitis is responsible for a vector-borne disease affecting canines and humans worldwide, known as cardiopulmonary dirofilariasis. An accurate and early diagnosis is of the utmost importance for effective disease management. While traditional microscopy-based methods remain invaluable, they have inherent limitations. Serological tests, in particular ELISA and immunochromatographic tests, are employed due to their capacity to detect D. immitis antigens, offering ease of use and diagnostic accuracy. The advent of molecular methods has the potential to enhance routine diagnostic approaches, with polymerase chain reaction (PCR) and real-time PCR (qPCR) becoming the most prevalent techniques. Despite not yet being integrated into routine diagnostics, which are predominantly based on the Knott’s test and serological methods, these techniques offer significant benefits in the context of scientific research. This article proceeds to examine the potential of advanced techniques, such as high-resolution melting qPCR (HRM-qPCR), loop-mediated isothermal amplification (LAMP), droplet digital PCR (ddPCR), and microRNA (miRNA) detection, which are capable of enhanced sensitivity and early detection. The following work provides an in-depth analysis of the various diagnostic methods, emphasising the necessity of the continuous improvement and adaptation of these tools to effectively combat D. immitis. The findings underscore the importance of integrating these advanced methods into routine practice to improve detection rates and outcomes for infected animals.

Description Generation Using Variational Auto-Encoders for Precursor microRNA

Micro RNAs (miRNA) are a type of non-coding RNA involved in gene regulation and can be associated with diseases such as cancer, cardiovascular, and neurological diseases. As such, identifying the entire genome of miRNA can be of great relevance. Since experimental methods for novel precursor miRNA (pre-miRNA) detection are complex and expensive, computational detection using Machine Learning (ML) could be useful. Existing ML methods are often complex black boxes that do not create an interpretable structural description of pre-miRNA. In this paper, we propose a novel framework that makes use of generative modeling through Variational Auto-Encoders to uncover the generative factors of pre-miRNA. After training the VAE, the pre-miRNA description is developed using a decision tree on the lower dimensional latent space. Applying the framework to miRNA classification, we obtain a high reconstruction and classification performance while also developing an accurate miRNA description.

Exploring the diagnostic potential of miRNA signatures in the Fabry disease serum: A comparative study of automated and manual sample isolations.

Fabry disease, an X-linked lysosomal storage disorder caused by galactosidase α (GLA) gene mutations, exhibits diverse clinical manifestations, and poses significant diagnostic challenges. Early diagnosis and treatment are crucial for improved patient outcomes, pressing the need for reliable biomarkers. In this study, we aimed to identify miRNA candidates as potential biomarkers for Fabry disease using the KingFisher™ automated isolation method and NanoString nCounter® miRNA detection assay. Clinical serum samples were collected from both healthy subjects and Fabry disease patients. RNA extraction from the samples was performed using the KingFisher™ automated isolation method with the MagMAX mirVanaTM kit or manually using the Qiagen miRNeasy kit. The subsequent NanoString nCounter® miRNA detection assay showed consistent performance and no correlation between RNA input concentration and raw count, ensuring reliable and reproducible results. Interestingly, the detection range and highly differential miRNA between the control and disease groups were found to be distinct depending on the isolation method employed. Nevertheless, enrichment analysis of miRNA-targeting genes consistently revealed significant associations with angiogenesis pathways in both isolation methods. Additionally, our investigation into the impact of enzyme replacement therapy on miRNA expression indicated that some differential miRNAs may be sensitive to treatment. Our study provides valuable insights to identify miRNA biomarkers for Fabry disease. While different isolation methods yielded various detection ranges and highly differential miRNAs, the consistent association with angiogenesis pathways suggests their significance in disease progression. These findings lay the groundwork for further investigations and validation studies, ultimately leading to the development of non-invasive and reliable biomarkers to aid in early diagnosis and treatment monitoring for Fabry disease.

Split G-Quadruplex Programmed Recyclable AIE-Biosensor for Label-Free Detection of miRNA in Acute Kidney Injury.

We herein rationally designed a target-recyclable AIE-biosensor based on a split G-quadruplex for label-free detection of miRNA in acute kidney injury. Initially, the PG was in an "OFF" state, and the two split segments (G4-a and G4-b) of G4 were tethered at the two terminals of P1 and far away from each other due to the rigid duplex structure formed by the partially complementary intermediate sequences of P2 and P1, bringing MG with quenched fluorescence. In the presence of target, the 5'-PO4 P2 was displaced from PG probe and competitively hybridized with target, which led to G4-a and G4-b tending to form an intact intermolecular G-quadruplex, providing sites for MG intercalation, thus generating an activated "ON" fluorescence signal due to the restriction of intermolecular motion. Successively, relying on the λ-exonuclease (λ-Exo) cleavage reaction-assisted target recycling, more amounts of targets will be liberated, accompanied by forming more G-quadruplex and binding more MG, resulting in a strong fluorescence signal, further realizing the sensitive detection of the targets. As a proof of concept, miRNA-21 was chosen as the model target. Endowing with the precise target recognition and efficient cleavage activity of λ-Exo, the AIE-biosensor exhibited excellent detection sensitivity and specificity, which could quantitatively detect miRNA-21 down to 10.36 fM with a single mismatch specificity. The results revealed that the distinctive attributes of noninvasive, simple, and efficient in this G-quadruplex-based AIE-biosensor offered promising prospects for extensive applications in AKI screening and early clinical diagnosis.

Pump-free SERS microfluidic chip based on an identification-competition strategy for ultrasensitive and efficient simultaneous detection of liver cancer-related microRNAs

In this study, we present a pump-free SERS microfluidic chip capable of detecting liver cancer-related miR-21 and miR-155 concurrently with ultra-sensitivity and high efficiency. We employed a Fe3O4@cDNA-AuNPs@Raman reporter@H composite structure and a recognition competition strategy. When the target miRNAs (miR-21 and miR-155) are present in the test liquid, they specifically compete with the nucleic acid complementary strand(H) of Fe3O4@cDNA-AuNPs@Raman reporter@H, causing AuNPs to competitively detach from the surface of Fe3O4, resulting in a decrease in the SERS signal. Consequently, this pump-free SERS microfluidic chip enables the detection of the target miRNAs more rapidly and accurately in complex environments. This method offers an approach for the simultaneous and efficient detection of miRNAs and holds promising applications in the early diagnosis of liver cancer.

Target-assisted self-cleavage DNAzyme electrochemical biosensor for MicroRNA detection with signal amplification.

In this work, we reported an electrochemical biosensor with target-assisted self-cleavage DNAzyme function for signal amplified detection of miRNA. The target-recycling amplification led to significant signal enhancement and thus offers high detection sensitivity.

Recent Advances in DNA-Based Nanoprobes for In vivo MiRNA Imaging.

As a post transcriptional regulator of gene expression, microRNAs (miRNA) is closely related to many major human diseases, especially cancer. Therefore, its precise detection is very important for disease diagnosis and treatment. With the advancement of fluorescent dye and imaging technology, the focus has shifted from in vitro miRNA detection to in vivo miRNA imaging. This concept review summarizes signal amplification strategies including DNAzyme catalytic reaction, hybrid chain reaction (HCR), catalytic hairpin assembly (CHA) to enhance detection signal of lowly expressed miRNAs; external stimuli of ultraviolet (UV) light or near-infrared region (NIR) light, and internal stimuli such as adenosine triphosphate (ATP), glutathione (GSH), protease and cell membrane protein to prevent nonspecific activation for the avoidance of false positive signal; and the development of fluorescent probes with emission in NIR for in vivo miRNA imaging; as well as rare earth nanoparticle based the second near-infrared window (NIR-II) nanoprobes with excellent tissue penetration and depth for in vivo miRNA imaging. The concept review also indicated current challenges for in vivo miRNA imaging including the dynamic monitoring of miRNA expression change and simultaneous in vivo imaging of multiple miRNAs.

Efficient Multidriven Strand Displacement Reaction for Biosensing.

A key challenge for achieving high-efficient DNA strand displacement reaction (SDR) with existing technologies is the inferior kinetic performance due to the alternately cumbersome conjunction and dissociation of dsDNA. In this work, a novel multidriven SDR collaborated by toehold initiator, strand towing, and click chemistry is engineered. The invasion strand (O) endows the hybridization with a basal strand (M) in dsDNA for releasing a displacement strand (P), which can be significantly boosted by the towing of a helper strand and impetus from the click reaction. Accordingly, the hybridization rate and dissociation extent of P can be largely improved and showed a desiring displacement rate close to 6-fold compared with the traditional method, providing a newly high-efficient SDR strategy for potential application in biosensing, clinical diagnostics, and DNA nanotechnology. In view of this, a practical biosensing platform by combining the multidriven SDR (MSDR) with waste-free DNA multi-cycle amplification is constructed for the rapid and ultrasensitive electrochemical detection of cancer-related miRNA-21. The substantial output DNA as an invasion strand (O) from target-triggered waste-free DNA multicycle can high-efficiently release a signal probe (Fc)-labeled displacement strand (P) on an electrode by using the proposed MSDR, obtaining a low detection limit below 106.8 aM.

Minimally Invasive Real-Time Monitoring for Rapid and Sensitive Diagnosis of Spinal Cord Injury.

Spinal cord injury (SCI) is a serious neurological injury that is currently extremely difficult to cure clinically. SCI involves numerous pathophysiological processes, and microRNAs (miRNAs) play an important role in these processes. Meanwhile, miRNAs have received a lot of attention for their role in other diseases as well. Therefore, the detection of disease-related miRNAs is important for the study of disease development, treatment, and prognosis. With the rapid development of molecular biology, the traditional detection methods of miRNA can no longer meet the needs of experiments. Electrochemical detection methods are widely used because of their excellent detection performance. Here, we designed an electrochemical sensor prepared using borosilicate glass microneedle electrodes for real-time monitoring of miR-21-5p expression in vivo after SCI. The sensor showed a good linear relationship between the oxidation peak current value and the concentration of miR-21-5p in the concentration range 0-2 fM (Y = 12.025X + 90.396, R2 = 0.98). The limit of detection (LOD) of the sensor was 0.3667 fM. The experimental results showed that the borosilicate glass microneedle electrochemical sensor achieved fast, accurate, highly sensitive, highly specific, highly stable, and reproducible monitoring of miR-21-5p. More importantly, the electrochemical sensor has a better clinical translation prospect, which is important for the research of clinical diseases.

PCR- and wash-free detection of serum miRNA via signaling probe hybridization.

Detection of microRNAs (miRNAs) in the serum is an effective liquid biopsy technique for cancer diagnosis. However, conventional diagnostic methods are time-consuming and complex. Therefore, in this study, we established a signaling probe-based DNA microarray system for miRNA detection. PCR, fluorescence labeling, and washing are not necessary for signaling probes. Four probes were designed using different miRNAs as diagnostic cancer markers. The developed system is useful for various miRNAs, regardless of their target lengths (18-26-mer) and GC content (36%-89%). Here, all the assays were performed within 40 min. Overall, our signaling probe-based DNA hybridization system facilitates the simple and rapid detection of serum miRNAs without the need for gene amplification, fluorescence labeling and washing.

Machine learning-aided microRNA discovery for olive oil quality.

MicroRNAs (miRNAs) are key regulators of gene expression in plants, influencing various biological processes such as oil quality and seed development. Although, our knowledge about miRNAs in olive (Olea europaea L.) is progressing, with several miRNAs being identified in previous studies, but most of these reported miRNAs have been predicted without the aid of a reference genome, primarily due to limited genome accessibility at the time. However, significant knowledge gaps still need to be improved in this area. This study addresses the complexities of miRNA detection in olive, using a high quality reference genome and a combination of genomics and machine learning-based methods. By leveraging random forest and support vector machine algorithms, we successfully identified 56 novel miRNAs in olive, surpassing the limitations of conventional homology-based methods. Our subsequent analysis revealed that some of these miRNAs are implicated in the regulation of key genes involved in oil quality. Within the context of oil biosynthesis pathways, the novel miRNA Oeu124369 regulates fatty acid biosynthesis by targeting acetyl-CoA acyltransferase 1 and palmitoyl-protein thioesterase, thereby influencing the production of acetyl-CoA and palmitic acid, respectively. These findings underscore the power of machine learning in unraveling the complex miRNA regulatory network in olive and provide a high quality miRNA resource for future research aimed at improving olive oil production by exploring the target genes of the identified miRNAs to understand their role and their biological processes.

Just-in-Time Generation of Nanolabels via In Situ Biomineralization of ZIF-8 Enabling Ultrasensitive MicroRNA Detection on Unmodified Electrodes.

Nanolabels can enhance the detection performance of electrochemical biosensing methods, yet their practical application is hindered by complex preparation, batch-to-batch variability, and poor long-term storage stability. Herein, we present a novel electrochemical method for miRNA detection based on the just-in-time generation of zeolitic imidazolate framework-8 (ZIF-8) nanolabels initiated by nucleic acids. In this design, the target miRNA-21 is captured with magnetic beads and polyadenylated by Escherichia coli Poly(A) polymerase (EPP), producing miRNA-21 molecules with poly(A) tails (miR-21-poly(A)). These molecules are then adsorbed onto a bare gold electrode (AuE) surface via adenine-gold affinity interactions, serving as nucleation sites for the rapid in situ formation of ZIF-8 nanoparticles. The ZIF-8 nanoparticles function as signal labels, impeding electron transfer at the electrode interfaces and thereby generating a notable electrochemical signal. The developed method demonstrated exceptional sensitivity, with a detection limit (LOD) as low as 2.3 aM and a linear detection range from 10 aM to 1000 fM. The practical application of the developed method was validated by using it to evaluate miRNA-21 expression levels in various biological samples, including cell lines, tumor tissues, and clinical blood samples from non-small cell lung cancer (NSCLC) patients. This approach simplifies the detection process by eliminating the need for presynthesized nanomaterials and premodified electrodes. Its simplicity and high sensitivity make this method a promising tool for point-of-care testing and a wide range of biomedical research applications.

Silver Nanoclusters-Decorated Porous Microneedles Coupling Duplex-Specific Nuclease-Assisted Signal Amplification for Sampling and Detection of MicroRNA in Interstitial Fluid.

MicroRNAs (miRNAs) in dermal interstitial fluid (ISF) have recently been recognized as clinically promising biomarkers for the diagnosis and prognosis of cancer. However, the detection poses significant challenges, primarily due to the low abundance of miRNAs and the limitations of current sampling techniques. To address this issue, we develop novel porous microneedles (PMNs) array-based sensor composed of poly(vinyl alcohol) porous hydrogel and DNA-templated silver nanoclusters (AgNCs) to facilitate the enrichment and highly sensitive detection of ISF miRNA. Leveraging the capillary action facilitated by its unique porous structure and the swelling properties of the hydrogel, the PMNs array can efficiently extract 2.7 ± 0.3 mg of ISF within 5 min. Additionally, the interconnected pores within the PMNs array contribute to an increased specific surface area, thereby offering a convenient platform for the decoration of DNA-templated AgNCs. The immobilized large amount of AgNCs effectively capture the target miRNA from the extracted ISF, resulting in miRNA-induced fluorescence quenching of AgNCs. Subsequently, the introduction of the duplex-specific nuclease leads to the cleavage of DNA in DNA-RNA heteroduplexes, which release miRNA to interact with other AgNCs. This process of target recycling triggers a further reduction in fluorescence intensity, thereby enabling sensitive detection of the low-abundant miRNA down to 1.6 pM. Both in vitro and in vivo experiments validate the efficacy of the AgNCs immobilized PMNs array for the detection of miRNA biomarkers in ISF within minutes. These results indicate that the proposed PMNs array-based sensor holds great potential for the development of noninvasive personalized diagnostic strategies.

On-Site Visualization Assay for Tumor-Associated miRNAs: Using Ru@TiO2 as a Peroxidase-like Nanozyme.

Accurate diagnosis of highly aggressive and deadly tumors is essential for effective treatment and improved patient outcomes, and microRNAs (miRNAs) have emerged as crucial biomarkers for their roles in tumor initiation, progression, and metastasis. Herein, we present an on-site visualization colorimetric assay for tumor-associated miRNAs using ruthenium nanoparticle decorated titanium dioxide nanoribbon (Ru@TiO2) as a peroxidase-like (POD) nanozyme. Remarkably, the Ru@TiO2 nanozyme can catalyze the oxidation of chromogenic substrates through its POD-like activity, which is effectively inhibited by pyrophosphate generated during the rolling circle amplification process, thereby enabling miRNA detection through a visible colorimetric readout. This approach provides a highly sensitive and specificity assay for miRNAs in diluted human serum with a detection limit of 100 pM. It shows great potential for clinical diagnostics and biological research, offering a promising tool for early cancer diagnosis and molecular diagnostics.

Activating Two-Photon Silica Nanoamplifier-Based CHA and FRET for Accurate Ratiometric Bioimaging of Intracellular MicroRNA.

In situ visualization of microRNA (miRNA) in cancer cells and diseased tissues is essential for advancing our comprehension of the onset and progression of associated diseases. Two-photon (TP) imaging, as an imaging technology with high spatiotemporal resolution, deep tissue penetration, and accurate target quantification, has distinctive advantages over single-photon imaging and has attracted increasing attention. Extensive research has been conducted on two-photon dye-doped silica nanoparticles, which exhibit a large two-photon absorption (TPA) cross-section, high fluorescence quantum yield, and excellent biocompatibility. However, the low abundance of RNA in tumor cells leads to insufficient signal output. Based on functional nucleic acid, a catalyzed hairpin self-assembly (CHA) signal amplification strategy, which has simplicity, robustness, and nonenzymatic characteristics, can achieve the amplification of DNA or RNA signals. Here, a two-photon silica nanoamplifier (TP-SNA) utilizing TP dye-doped silica nanoparticles (SiNPs) and functional nucleic acid was constructed, employing triggering catalyzed hairpin self-assembly and fluorescence resonance energy transfer (FRET) for highly sensitive detection and precise TP imaging of endogenous miRNAs in tumor cells and tissues at varying depths. The TP-SNA demonstrated the capability to detect miR-203 with a detection limit of 33 pM. The maximum two-photon tissue penetration depth of the two-photon nanoamplifier was 210 μm. The two-photon nanoamplifier developed in this study makes full use of the advantages of accurate TP ratiometric bioimaging and the CHA signal amplification strategy, which shows good application value for future transformation into clinical diagnosis.