Intracellular Imaging Research Articles

Recent Advances in DNA Systems for In Situ Telomerase Activity Detection and Imaging

Telomeres play a key role in maintaining chromosome stability and cellular aging. They consist of repetitive DNA sequences that protect chromosome ends and regulate cell division. Telomerase is a reverse transcriptase enzyme counteracts the natural shortening of telomeres during cell division by extending them. Its activity is pivotal in stem cells and cancer cells but absent in most normal somatic cells. Recent advances in biosensor technologies have facilitated the in situ detection of telomerase activity, which is essential for understanding its role in aging and cancer. Techniques such as fluorescence, electrochemistry, and DNA nanotechnology are now being employed to monitor telomerase activity in living cells, providing real-time insights into cellular processes. DNA-based biosensors, especially those incorporating molecular beacons, DNA walkers, and logic gates, have shown promise for enhancing sensitivity and specificity in telomerase imaging. These approaches also facilitate the simultaneous analysis of related cellular pathways, offering potential applications in early cancer detection and precision therapies. This review explores recent developments in intracellular telomerase imaging, highlighting innovative approaches such as DNA-functionalized nanoparticles and multi-channel logic systems, which offer non-invasive, real-time detection of telomerase activity in complex cellular environments.

Visualizing Endoplasmic Reticulum Stress and Autophagy in Alzheimer's Model Cells by a Peroxynitrite-Responsive AIEgen Fluorescent Probe.

Endoplasmic reticulum (ER) stress and autophagy (ER-phagy) occurring in nerve cells are crucial physiological processes closely associated with Alzheimer's disease (AD). Visualizing the two processes is paramount to advance our understanding of AD pathologies. Among the biomarkers identified, peroxynitrite (ONOO-) emerges as a key molecule in the initiation and aggravation of ER stress and ER-phagy, highlighting its significance in the underlying mechanisms of the two processes. In this work, we designed and synthesized an innovative ONOO--responsive AIEgen-based fluorescent probe (DHQM) with the ability to monitor ER stress and ER-phagy in AD model cells. DHQM demonstrated excellent aggregation-induced emission (AIE) properties, endowing it with outstanding ability for washing-free intracellular imaging. Meanwhile, it exhibited high sensitivity, remarkable selectivity to ONOO-, and exceptional ER-targeting ability. The probe was successfully applied for fluorescence imaging of ER ONOO- fluctuations to assess the ER stress status in aluminum-induced AD model cells. Our findings revealed that aluminum-induced ferroptosis, a regulated cell death process, was pivotal in the excessive ONOO- production, which in turn activated and exacerbated ER stress. Furthermore, the aluminum-stimulated ER-phagy was observed utilizing DHQM, which might be crucial in inhibiting ferroptosis and mitigating aberrant ER stress. Overall, this study not only offers valuable insights into the pathological mechanisms of AD at the ER level but also opens new potential therapeutic avenues targeting these pathways.

Regulation of MAP2, GFAP, and calcium in the CA3 Region Following Kainic Acid Exposure to organotypic hippocampal slice culture.

Neurodegeneration due to neurotoxicity is one of the phenomena in temporal lobe epilepsy. Experimentally, hippocampal excitotoxicity process can occur due to kainic acid exposure, especially in the CA3 area. Neuronal death, astrocyte reactivity and increased calcium also occur in hippocampal excitotoxicity, but few studies have investigated immediate effect after kainic acid exposure. The organotypic hippocampal slice culture (OHSC) is a useful model for studying the neurodegeneration process, but there are still many protocol differences. In this study, minor modifications were made in the OHSC protocol. OHSC was obtained from three healthy wild type Wistar rats aged P10. Healthy culture slices were obtained and lasted up to 10 days in vitro (DIV 10). Bath application of kainic acid for 48 hours in DIV 10 followed by observation of its initial effects on neurons, astrocytes, and calcium via the expression of MAP2, GFAP, and intracellular calcium imaging, subsequently. After 48 h of kainic acid administration, there was a significant increase in intracellular calcium intensity (p = 0.006 < α), accompanied by a significant decrease in MAP2 (p = 0.003 < α) and GFAP (p = 0.010 < α) expression. These findings suggest early neuronal and astrocyte damage at the initial onset of hippocampal injury. This implies that astrocyte damage occurs early before an increase in GFAP that characterizes reactive astrogliosis found in other studies. Damage to neurons and astrocytes may be associated with increased intracellular calcium. It is necessary to develop further research regarding regulation of calcium, MAP2, and GFAP at a spatial time after exposure to kainic acid and strategies to reduce damage caused by excitotoxicity.

A review on quantum dots and their applications

Quantum dots (QDs) are nanosized semiconductor crystals with unique chemical and physical properties, enabling them to emit bright, photobleaching-resistant light. They are classified into three categories: core type QDs, core-shell QDs, and alloyed QDs. Core-type QDs are composed of a single substance, while core-shell QDs have a semiconductor core surrounded by a shell of another semiconductor material. Alloyed QDs are manufactured using high-temperature or wet-chemical processes, while silicon-based QDs display unusual optical and electrical features due to quantum confinement forces. QDs have exceptional optoelectronic capabilities, producing photoluminescence quantum yields of up to 90%. They are useful for various applications, including optoelectronics and photocatalysis. There are three main types of QDs: colloidal, magnetic, and fluorescent. Colloidal synthesis is a widely used technique for creating QDs, offering applications in visible and near-infrared wavelengths. Magnetic QDs display unique magnetic characteristics and can be synthesized using various methods. Fluorescent QDs have high fluorescence quantum yields and are resistant to photobleaching, making them useful in imaging, sensing, and therapies. QDs are being developed to enhance biological imaging capabilities by increasing cellular uptake, reducing toxicity, and enhancing stability. They can be used for intracellular imaging, deep tissue imaging, single-molecule tracking, super-resolution imaging, targeting ligands, selective imaging of mitochondria, pH-responsive fluorescence, responding to specific cellular signals, and self-illuminating systems. However, they have limitations such as toxicity, structural instability, and deterioration due to UV light or aquatic environments.

Stimuli-Responsive DNA Nanomachines for Intracellular Targeted Electrochemiluminescence Imaging in Single Cells.

Electrochemiluminescence (ECL) microscopy has emerged as a powerful technique for single-cell imaging owing to its unparalleled background-free imaging advantages. However, controlled intracellular ECL imaging remains challenging. Here, we developed a stimuli-responsive self-assembled DNA nanomachine that enables the ECL imaging of intracellular target biomolecules in single cells. The ECL nanoprobe consists of an ECL nanoemitter constructed from Ru(bpy)32+-doped metal-organic framework as the nanoreactor core, with a DNA polymer hydrogel shell (DNAgel) acting as the stimuli-gated layer. The outer functionalized DNAgel of the ECL nanoprobe was specifically designed to block ECL generation and to dissociate by ATP molecules, thereby enabling selective recovery of ECL emission capability. Such an engineered stimuli-responsive nanomachine successfully achieved the targeted ECL imaging of intracellular ATP distribution with spatial resolution. In addition, ECL imaging of various intracellular biomolecules should be generalizable by simply changing the switching DNA sequence of the probe. Our research provided a reliable strategy for ECL microscopy within cells, which will broaden the application of ECL in single-cell and single-molecule profiling.

Sequentially amplified integration of catalytic DNA circuits for high-performance intracellular imaging of miRNA and interpretation of mRNA-miRNA signalling pathway

The cascaded catalytic circuits are viable tools for improving the signal gain of biosensors, yet their sensing performance is still limited by the signal leakage from complex biological environment and unsatisfying reaction efficiency from inter-reactants steric hindrance. Herein, we proposed a catalytically localized DNA (CLD) circuit for the accurate and high-efficiency imaging of microRNA (miRNA) in living cells by virtue of the sequentially and successively amplified integration of catalytic DNA circuits. The compact CLD circuit was constructed by integrating two elemental catalytic circuits, cell-responsive EDR module and analyte-sensing CHA module, where CHA module was initially caged in EDR module for eliminating the unwanted off-site and off-target signal leakage. Only by cell-specific messenger RNA (mRNA)-activated EDR operation then the elemental CHA circuit could be successively connected to facilitate the highly efficient intramolecular reaction with low steric hindrance, thus leading to accelerated reaction efficiency for miRNA analyte. The multiple molecular recognition and the spatial self-confinement of the smart CLD circuit enable the accurate and high-efficiency imaging of intracellular miRNA. The interaction network of mRNA and miRNA was then investigated in situ through our CLD circuit, which provides a powerful tool for discovering the underlying signal pathways between these different RNAs in living cells.

Rational design of a ratiometric indicator-displacement assay for the monitoring of chlortetracycline and intracellular imaging

Chlortetracycline (CTC), one of the tetracyclic antibiotics, has been widely employed for the inhibition of bacteria. Due to the potential threat to human health posed by the residual CTC, the development of ratiometric fluorescent probes that can achieve accurate measurement and in-situ imaging is urgently needed but remains challenged. Here, a bovine serum albumin (BSA)-involved host-guest system was designed as the novel ratiometric probe (C3@BSA) for CTC based on the indicator-displacement assay principle. Significantly, the fluorescence difference between the CTC@BSA complex and other tetracyclic@BSA complexes ensured high selectivity of this probe. The indicator (C3) was rationally derived from a simple chalcone dye with weak binding affinity to BSA (1.7 × 104 M−1) compared with CTC (2.2 × 104 M−1). The sensing process can finish in 10 s with sensitive response (detection limit: 0.23 μM) and anti-interference capacities over common ions and amino acids. On this basis, the probe was successfully used to detect CTC in HeLa cells and B16 cells, where the distributions of CTC were displayed under a dual-channel imaging mode for the first time. Our research provided a novel ratiometric probe for CTC and an effective strategy for designing ratiometric fluorescent indicator-displacement assays.

Splitting and Aggregation of Carbon Dots: Wavelength-Shifted and Ratiometric Fluorescence Sensing of Peroxynitrite.

Peroxynitrite (ONOO-) is a short-term reactive biological oxidant and plays an important role in cellular signal transduction and homeostatic regulation. However, excess ONOO- is associated with neurodegenerative and cardiovascular diseases. Therefore, rapid, sensitive, and accurate assays for ONOO- detection are essential for exploring its physiological and pathological function. In this work, a wavelength-shifted and ratiometric fluorescent sensing platform for ONOO- is constructed by splitting green fluorescent carbon dots (G-CDs) and aggregating orange fluorescent carbon dots (O-CDs). The mixed G-CDs and O-CDs (M-CDs) show a fast and precise response to ONOO- in the range of 0-250 μM, with a detection limit of 10 nM. In the linearity range within 3 μM ONOO-, an obvious wavelength shift of G-CDs from 495 to 475 nm is observed owing to the oxidation and nitration of ONOO- to the surface-state fluorescence of G-CDs, accompanied by the splitting of G-CDs. In the linearity range of 3-250 μM ONOO-, the fluorescence of G-CDs remains constant, while the molecular-state fluorescence of O-CDs gradually quenches by the oxidation and nitration of ONOO- through the fluorescence static process and induces their aggregation. Additionally, M-CDs show favorable intracellular imaging of endogenous and exogenous ONOO-. This study not only presents a new fluorescence wavelength shift mechanism for ONOO- sensing but also provides insights into CDs' fluorescence mechanism by exploring their morphology and structure via reacting with reactive oxygen species (ROS).

Live-Cell RNA Imaging with a DNA-Functionalized Metal-Organic Framework-Based Fluorescent Probe.

Live-cell fluorescence imaging of tumor-associated miRNAs is significant for understanding the cancer onset and progression and the diagnosis and prognosis of clinical diseases. In this protocol, we describe the construction of a DNA-functionalized metal-organic framework-based fluorescent probe and demonstrate that the probe can be used to detect miRNA in vitro and live-cell imaging. The formed DNA-MOF probe can specifically target miRNA with high sensitivity and specificity. The detection limit of the extracellular assay is as low as the picomolar level. In addition, we found that the probe could permeate cells and can be successfully applied in intracellular miRNA imaging, even achieving the distinction of cancer cells and normal cells, as well as the cancer cell lines with different miRNA expression levels.

A Catalytic Hairpin Assembly-Based Approach for RNA Imaging in Living Cells.

The advancement of nucleic acid nanotechnology has resulted in broad applications of DNA- and RNA-based molecular sensors for bioanalysis. Catalytic hairpin assembly is such a type of programmable and enzyme-free nucleic acid circuit that has been popularly used in developing biosensors. Genetically encodable fluorogenic RNA-based devices have recently gained a lot of attentions as a powerful tool for intracellular imaging. Combining these two techniques together, we have developed a genetically encodable RNA-based catalytic hairpin assembly circuit for the highly sensitive detection of low-abundance RNAs in living cells. In this system, the binding of one target RNA can catalytically trigger the generation of tens-to-hundreds of fluorogenic RNA reporters and produce a significantly enhanced fluorescence signal. Here, we will introduce the detailed design, optimization, and imaging protocol of this new type of powerful imaging tools.

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

Recent Advances in Calixarene-Based Fluorescent Sensors for Biological Applications.

Due to their structural features, macrocyclic compounds such as calixarenes, conjugated with a variety of fluorophores have led to the development of fluorescent probes for numerous applications. This review covers the recent advances (from 2009 to date) made in calixarene-based fluorescent sensors and their biological applications. In addition to the fluorescence mechanisms used to signal the analyte binding, this article focuses mainly on the detection of biological relevant ions, on the selective sensing of biomolecules, such as amino acids, enzymes, drugs and other organic compounds, and on intracellular imaging. Calixarene-containing fluorescent nanoparticles and nanoaggregates for imaging and drug delivery are also described. Finally, this review presents some conclusions and future perspectives in this field.

CoOOH nanoflake as supporter of CRISPR-Cas12a system for facilitated imaging of miRNA-21 in cells

The aberrant expression of miRNA-21 in cells is frequently associated with the occurrence and metastasis of tumors. The CRISPR-cas12a system has been developed for in vitro detection of miRNA due to its distinctive signal amplification characteristics. But its intricate composition and challenges in cellular delivery have limited its application in cell imaging. In this study, we designed an electrostatic adsorption-based probe by combining positively charged cobalt hydroxyl oxide (CoOOH) nanoflakes with negatively charged CRISPR-cas12a system which allows for detection and intracellular imaging of miRNA-21. This method involves using ascorbic acid to reduce CoOOH nanoflakes into Co2+, which subsequently releases the active form of CRISPR-Cas12 for detecting miRNA-21. The probe has a linear detection range for miRNA-21 from 0.5 pM-50 pM and 100 pM-20 nM, with a detection limit of 0.32 pM. It also shows good selectivity, accuracy, good reproducibility and biocompatibility for miRNA-21 analysis. In subsequent cell experiments, the probe exhibits low cytotoxicity and remarkable cell imaging capabilities. Therefore, this probe offers an innovative approach for miRNA-21 analysis.

AIE active hydroxycoumarin-anthranilic acid coupled enamine: Sequential detection of Cu2+/S2− ions and live cell imaging application

In this contribution, we designed and developed 7-diethylamino-4-hydroxycoumarin coupled anthranilic acid-based enamine (DHCBA) and its Cu2+ complex (DHCBA-Cu)via simple synthetic strategy. DHCBA shows solvatochromic effect, aggregation induced emission feature at 80 % methanol–water mixture and colorimetric as well as fluorescence turn-off sensing features towards Cu2+ ions. The DHCBA-Cu2+ ensemble has been utilized as a turn-on fluorescence sensor for the detection of S2− through a decomplexation approach. The formation of a 1:1 DHCBA-Cu2+ ensemble was further supported by 1H NMR studies, HRMS analysis and Density Functional Theory (DFT) calculation. The detection limit for Cu2+ was found to be 6.5 nM and then the binding affinity and binding stoichiometry of the probe towards Cu2+ ion was calculated using the online BindFit v0.5 supramolecular software. Moreover, the 1:1 stoichiometric ratio between DHCBA and Cu2+ ions were confirmed via single crystal X-ray analysis. The structural analysis of DHCBA-Cu2+ indicates that it forms an interesting one-dimensional coordination polymeric chain-like structure, further transformed into a two-dimensional polymeric network through hydrogen bonding interactions. The reversible on–off–on emission character of the probe, DHCBA was successfully applied for the construction of the IMPLICATION logic gate as well as on-site detection applications using paper strip and cotton-swab tests. Furthermore, the sensing feature of the enamine against Cu2+/S2− ions were validated by intracellular imaging applications.

In vivo imaging of alkaline phosphatase in lipid metabolic diseases with a photoacoustic probe

Lipid metabolic diseases have become an important challenge to global public health. Along with lifestyle changes, the incidence of obesity, diabetes and other metabolic syndromes is on the rise, and the number of patients with fatty liver disease is also increasing. Therefore, it is particularly important to develop effective lipid imaging strategies to monitor and manage fatty liver disease. Herein, based on the essential role of alkaline phosphatase (ALP) in both AS and OB, in vivo imaging of ALP was achieved in two lipid metabolic diseases models with a photoacoustic (PA) probe phosphorylated hemicyanine (P-Hcy). After being triggered by ALP, P-Hcy responded in different modalities including absorbance, fluorescence and, most significantly, PA-reporting. Notably, the PA signal showed the reliable linear correlation to the ALP level within the range of 0–800 U/L. The probe P-Hcy exhibited the advantages including high sensitivity, high selectivity, and steadiness in required biological conditions. The intracellular imaging results ensured that P-Hcy could visualize the ALP level in the foam cells induced from mouse mononuclear macrophages. In the healthy and lipid metabolic diseases models, P-Hcy was able to distinguish well between a lipid metabolic disease model and a healthy mouse model by photoacoustic imaging. By combining the ALP detection with P-Hcy in PA/fluorescence modality and traditional techniques such as blood biochemical testing and immunohistochemically staining, more potential strategy to accurately diagnose lipid metabolic diseases in the pre-clinical trials might be developed in future.

Aggregation-induced emission luminogens for intracellular bacteria imaging and elimination

Intracellular bacterial infections are a serious threat to human health due to their ability to escape immunity and develop drug resistance. Recent attention has been devoted to identifying and ablating intracellular bacteria with fluorescence probes. Aggregation-induced emission luminogens (AIEgens) photosensitizers as fluorescence probes possess excellent photostability and rapid response, which have emerged as powerful fluorescent tools for intracellular bacterial detection and antibacterial therapy. This review is intended to highlight the current advances in AIEgens on intracellular bacteria imaging and elimination, which covers topics from intracellular AIE mechanism, intracellular bacteria imaging of AIEgens to the elimination of intracellular bacteria with AIEgens. AIEgens utilized different interactions to detect intracellular bacteria, emitting bright light due to restricted intramolecular movement to visualize intracellular bacteria. Photosensitive AIEgens generate reactive oxygen species (ROS) in the aggregate state to elimate intracellular bacteria. Moreover, the prospects and application of AIEgens in intracellular bacteria imaging and elimination are also discussed, which provides insights for the development of AIE-based diagnostic and therapeutic materials and technologies.

Selective Recognition of the Dimeric NG16 Parallel G-Quadruplex Structure Using Synthetic Turn-On Red Fluorescent Protein Chromophore.

Red fluorescent protein (RFP)-based fluorescent probes that can selectively interact with specific nucleic acids are of great importance for therapeutic and bioimaging applications. Herein, we have reported the synthesis of RFP chromophores for selective recognition of G-quadruplex nucleic acids in vitro and ex vivo. We identified DFHBI-DM as a fluorescent turn-on probe that binds to the dimeric NG16 parallel quadruplex with superior selectivity and sensitivity over various parallel, antiparallel, and hybrid topologies. The binding of DFHBI-DM to NG16 exhibited excellent photophysical properties, including high binding affinity, large Stokes shift, high photostability, and quantum yield. The MD simulation study supports the 1:1 binding stoichiometry. It confirms the planar conformation of DFHBI-DM, which makes strong binding interactions with a flat quartet of NG16 compared to other antiparallel and hybrid topologies. The cell imaging and MTT assays revealed that DFHBI-DM is a biocompatible and efficient fluorescent probe for intracellular imaging of NG16. Overall, these results demonstrated that DFHBI-DM could be an effective fluorescent G4-stabilizing agent for the dimeric NG16 parallel quadruplex, and it could be a promising candidate for further exploration of bioimaging and therapeutic applications.

Nanomolar Hg2+ detection via phenothiazine fluorogenic chemosensor: Applications in water analysis and intracellular imaging

A Knoevenagel condensation reaction paved the way for the development of the PTZ-BCN probe namely (z)-2-(1H-benzo[d]imidazole-2-yl)-3-(10-ethyl-10H-phenothiazin-3-yl) acrylonitrile. PTZ-BCN’s spectral characteristics were verified through the application of IR, 1H NMR, 13C NMR, and HRMS techniques. The PTZ-BCN probe showed a high selectivity and sensitivity toward Hg2+ ions over other interfering competing metal ions in CH3CN: HEPES buffer (9:1, v/v) system. The addition of Hg2+ results in a notable redshift and fluorescence quenching of the PTZ-BCN probe. An examination of the interaction between the PTZ-BCN probe and Hg2+ involved recoding 1H NMR titration spectra, HRMS, DFT analysis, and Job’s plot respectively. Quantification of the lowest detectable Hg2+ concentration at 2.3 nM, exhibiting a correlation coefficient of R2 = 0.9985. PTZ-BCN probe has proven effective in quantitatively determining the existence of Hg2+ in real-time water samples, test strips, solid state, and Hela living cells.

Mitochondria-Targeted Ratiometric KLA-Tweezers for Fluorescence Imaging of Potassium Ions.

Intracellular potassium ions play a crucial role in cellular homeostasis associated with physiological and pathological processes. It is still challenging but definitely crucial to precisely and dynamically image subcellular K+. Herein, the first mitochondria-targeted DNA tweezers (KLA-tweezers) were developed for the fluorescence imaging of mitochondrial K+ with high selectivity and accuracy. The proposed KLA-tweezers consisted of two DNA double-crossover (DX) motifs, a K+-aptamer, and a mitochondria-targeted peptide (KLA) which was conjugated at the rear of DX arms via complementary base pairing. Upon contact with K+, the K+-aptamer experienced a conformational change from an open-chain G-rich sequence to a G-quadruplex with a compact conformation, which was reflected by the Förster resonance energy transfer process between Cy3 and Cy5 labeled at the end of the DX arms. The open and closed states of the tweezers before and after KLA modification were confirmed by gel electrophoresis and fluorescence spectra together with atomic force microscopy (AFM) and transmission electron microscopy (TEM) images, suggesting the KLA assembly had no effect on the morphology change. The proposed tweezers and KLA-tweezers showed low cytotoxicity, excellent selectivity, and good reversibility upon alternating addition of 18-crown-6 and K+. Besides, the KLA-tweezers exhibited outstanding stability and accurate mitochondria location ability. Upon stimulation of ATP or nigericin, intracellular fluorescence imaging of mitochondrial K+ dynamics was successfully achieved. This strategy has broad prospects as a general optical sensing platform for other metal ions or organelles in living cells.

On-Site Multiply Stimulated Self-Confinement of an Integrated DNA Cascade Circuit for Highly Reliable Intracellular Imaging of miRNA and In Situ Interrogation of the Relevant Regulatory Pathway.

Artificial DNA circuits represent a versatile yet promising toolbox for in situ monitoring and concomitant regulation of diverse biological events within live cells. Nonetheless, their performance is significantly impeded by the diffusion-dominated slow reaction kinetics and the uncontrollable off-target activation. Herein, a self-localized cascade (SLC) circuit is reported for the robust and efficient microRNA (miRNA) analysis in living cells. The SLC circuit consists of the cell-specific localization module and the analyte-specific signal amplification module. By integrating the reaction probes of these two modules, the complexity of the system is reduced to realize the responsive co-localization of circuitry probes and the simultaneous cascade signal amplification. Taking advantage of the specifically activated, self-localized, and cascade design, the SLC circuit successfully achieves the robust miRNA-21 (miR-21) imaging and the accurate cells differentiation. Moreover, the reverse regulation mechanism is successfully explored between messenger RNA (mRNA) and miRNA through the engineered SLC circuit and further elucidates the underlying signaling pathways between them. Therefore, the SLC circuit provides a powerful tool for the sensitive detection of intracellular biomolecules and the study of the corresponding cell regulatory mechanisms.