Distinguish Cancer Cells Research Articles

A novel NIR-activatable and photodegradable mitochondria-targeted nano-photosensitizer for superior photodynamic therapy

Photodynamic therapy (PDT) has garnered significant interest as a promising approach for cancer treatment due to its effectiveness, versatility, and non-invasiveness. However, its clinical application is hindered by several factors, such as suboptimal efficacy, poor targeting capabilities, shallow penetration depth, and safety concerns. Herein, we introduce B-SQ, a novel nano-photosensitizer that addresses these challenges. B-SQ is a self-assembling, cancer mitochondria-targeting agent that is both activatable and photodegradable, designed by strategically modifying a near-infrared fluorescent squaraine dye. We extensively assessed the PDT potential of B-SQ in various cancer cell models and explored the molecular mechanisms of PDT-induced cell death. Importantly, B-SQ demonstrated high specificity for cancer cells, and its PDT efficiency was activatable, with near-infrared fluorescence to distinguish cancer cells from normal cells. This work provides the first evidence of B-SQ inducing the intrinsic mitochondrial apoptosis pathway and cell cycle arresting in cancer cells via PDT. Additionally, its photodegradable nature is likely to minimize side effects after treatment. This research introduces an innovative class of nano-photosensitizers that are activatable, targeting cancer mitochondria, and are photodegradable with near-infrared fluorescence. These qualities present new possibilities for safer and more effective clinical cancer treatments in the future.

Lysosome-Targeted Bifunctional Fluorescent Probe and Type I/II Photosensitizer for Viscosity Imaging and Cancer Photodynamic Therapy.

Abnormal lysosomal viscosity is closely associated with cancer progression, underscoring the need for bifunctional fluorescent probes and photosensitizers (PSs) that can both monitor viscosity and facilitate imaging-guided therapy for simultaneous cancer diagnosis and treatment. Despite advances in lysosome-targeted PSs development, few have demonstrated the ability to generate both Type I and Type II reactive oxygen species (ROS). In this study, we present BTTPA, a lysosome-targeted fluorescent probe and photosensitizer, designed to integrate cancer diagnosis via viscosity imaging and cancer treatment through photodynamic therapy (PDT). Our findings reveal that BTTPA selectively targets lysosomes, enabling dynamic monitoring of cellular viscosity and distinguishing cancer cells from normal cells. Upon light activation, BTTPA efficiently generates both Type I and Type II ROS. Apoptosis assays further confirm BTTPA's effectiveness in inducing cancer cell apoptosis, highlighting its potential as a powerful tool for cancer diagnosis and therapy.

CRISPR-based molecule-regulatory expression platform for specific immunotherapy of cancer.

Cancer is still a major challenge of human health. The abnormality of intracellular cancer-related signal pathways is an important mechanism for the occurrence of cancer. We used a molecular-senor to act on the endogenous signal molecules within the cell to redirect the abnormal signal flows in the cell to treat cancer. Based on CRISPR-dCas12f procedures, we combined aptamers and ribozymes to construct riboswitches, which served as molecular switches to reprogram sgRNAs, so that CRISPR-dCas12f redirected the intracellular anti-cancer signal flows after sensing specific input signal molecules. In addition, the activated molecular sensors and the inhibitory molecular sensors were constructed by combining transcription factors (VP64) and transcription inhibitors (KRAB) to specifically activate and inhibit target genes of interest. Our experimental results showed that the molecular sensors that we designed and constructed specifically sensed the endogenous signal molecules and then redirect the cancer related signal networks of cancer cells. In addition, corresponding logic gates were constructed to distinguish cancer cells from normal cells and redirect anticancer signal flows to trigger specific cancer immunotherapy. The constructed molecular sensors constructed specifically recognized the signal molecules within the cell and redirected the endogenous signal pathway to reprogram the fate of cancer cells.

The Acid-Stimulated Self-Assembled DNA Nanonetwork for Sensitive Detection and Living Cancer Cell Imaging of MicroRNA-221.

Herein, a novel functional DNA structure, acid-stimulated self-assembly DNA nanonetwork (ASDN), was proposed for miRNA-221 sensitive detection and high-resolution living cancer cell imaging. Significantly, the self-assembly of ASDN only occurred in the acidic extracellular environment of cancer cells, which could be endocytosed by cancer cells to eliminate the interference of noncancer cells and deliver the ASDN into cancer cells. Subsequently, endogenous miRNA-221 could trigger the catalytic hairpin assembly within ASDN, resulting in the separation of the fluorophore Cy5 and the quencher BHQ2 to recover the substantial Cy5 fluorescence signals, thus achieving signal amplification for sensitive detection of miRNA-221 with a detection limit of 5.5 pM, as well as facilitating high-resolution and low-background imaging of miRNA-221 in cancer cells. In consequence, this strategy provides an innovative DNA nanonetwork to distinguish cancer cells from other cells for sensitive detection of biomarkers, offering a meaningful reference for the application of DNA nanostructure self-assembly technology in relevant fundamental research and disease diagnosis.

A coumarin-based fluorescent probe for imaging H2S and distinguishing breast cancer cells from normal ones

Hydrogen sulfide (H2S) is a crucial endogenous gas signal molecule involved in several physiological functions. Elevation of H2S concentrations is considered a cancer biomarker because it may be connected to the growth of tumors. A thorough comprehension of measuring H2S in living cells and differentiating between malignant and normal cells is crucial. Herein, a multi-responsive probe (C-DNP) was constructed to detect H2S and observe the intramolecular charge transfer “turn-off” sensing mechanism caused by the intervention of the irradiative intramolecular charge transfer between C and DNP. Given that DNP recognizes H2S, a reaction between H2S and the probe-attached DNP could eliminate DNP via ether bond breakage and increased emission. Moreover, 1H NMR titrations, HR-MS, and density-functional theory (DFT) calculations confirmed the proposed sensing mechanism. The probe effectively monitored H2S in different water samples, food samples, paper sensor trips, and living cells. Surprisingly, the probe distinguished tumor-bearing cells from normal cells by expressing the variations in H2S both exogenously and endogenously in normal and breast cancer cells. Therefore, it is anticipated to be an efficient method for monitoring H2S in real samples and biosystems.

Optical Based Surface Plasmon Resonance Sensor for the Detection of the Various Kind of Cancerous Cell.

This research explores a novel biosensor design that exhibits much higher sensitivity compared to conventional biosensors. The biosensor's uniqueness originated from its innovative structure, which incorporates N-FK51A/Ag/AlON/BlueP materials, as well as its cutting-edge fabrication method. The refractive index component was considered when designing the SPR biosensor, which was developed from the angular analysis of the attenuated total reflection (ATR) approach for cancer detection. For instance, the resonance angle shifts by 15.57 deg when the refractive index changes from 1.360 to 1.401, demonstrating the sensor's responsiveness to variation in the refractive index. The sensitivities for skin (basal), cervical (HeLa), blood (Jurkat), adrenal gland (PC12), and breast (MDA-MB-231 and MCF-7) cancer cells were 197.65, 243.66, 255.36, 302.71, 372.57, and 416.85 deg/RIU, respectively. Also, the detection accuracy (DA), the figure of merit (FoM), and the quality factor were 0.37/deg, 155.94 (deg/RIU), and 26.71 RIU-1. We also examine the effects of substituting the noble, dielectric, 2D material layer with conventional biosensor materials for six cancers. Each time, the Ag/AION/BlueP layered structure performed best in distinguishing cancer cells from healthy cells. We also study the prism effects. The proposed biosensor, with a RI of 1.29-1.40, has a linear regression coefficient of R2 of 0.96094.

Bidirectional Polymerization-Transcription Amplification-Encoded Dual-Color Fluorescent Biosensor for Label-Free and Primer-Free Detection of Multiple piRNAs.

PIWI-interacting RNAs (piRNAs) are a type of endogenous noncoding RNAs with a length of 24-31 nucleotides, and they can specifically bind with PIWI proteins to form the piRNA/PIWI complexes for regulating multiple physiological and pathological processes. Herein, we develop a bidirectional polymerization-transcription amplification-encoded dual-color fluorescent biosensor for label-free and primer-free measurements of multiple piRNAs. The designed hairpin probe contains a palindromic tail, and it can serve as the target recognition unit, polymerization primer, and transcription template. In the presence of target piRNAs, the hairpin probes are opened to expose a palindromic sequence that can trigger bidirectional polymerization and transcription reaction with the assistance of KF polymerase and T7 RNA polymerase for the production of numerous RNA aptamers. The aptamers subsequently bind with the corresponding fluorophores (DFHBI-1T/MG) to form the RNA aptamer-fluorophore complexes for the generation of enhanced fluorescence signals. This biosensor can sensitively detect piR-36026 with a limit of detection (LOD) of 82.08 aM and piR-36743 with a LOD of 44.44 aM. Moreover, it can quantify cellular piRNAs with single-cell sensitivity and distinguish cancer cells from normal cells. Furthermore, it has the capability of distinguishing the expression of piRNAs in the tissues of breast cancer patients and healthy individuals. By simply altering the target recognition site of the hairpin probe, this biosensor can be extended to detect various piRNAs, offering a powerful platform for piRNA-related clinical diagnostics and therapeutics.

Integrating high mannose-binding aptamer and CRISPR/Cas12a machinery for the remote detection of cell surface high mannose

High mannose is overexpressed on the cell surface during carcinogenesis; however, there are currently no efficient techniques for its detection. Herein, we propose an assay called RADAR (remote high mannose detection on cell surface with CRISPR) for the quantification of high mannose, leveraging the integration of high mannose-binding aptamer and CRISPR technique. The high mannose-binding aptamer is hybridized with a locker, which is released upon specific binding to high mannose. The released locker then activates CRISPR/Cas12a to produce a fluorescence signal. The "remote" concept in RADAR refers to the recognition of high mannose occurring on the cell surface, while signal generation takes place in a solution separate from the target. This feature makes RADAR suitable for the detection of suspended bio-samples without the need for sophisticated imaging. RADAR can detect high mannose Man9 with a limit of detection of 2.5 nM, distinguish cancer cells from non-cancerous cells, and profile high mannose level in human serum for potential non-small cell lung cancer (NSCLC) diagnosis. This work represents the first attempt to integrate CRISPR technology into the aptasensing of high mannose, which may serve as a synthetic biology-enabled tool in glycochemistry and glycobiology.

Persistent autonomous molecular motion of DNA walker along a single-molecule nano-track for intracellular MicroRNA imaging

While the intracellular imaging of miRNA biomarkers is of significant importance for the diagnosis and treatment of human cancers, DNA assembled nanoprobe has recently attracted considerable attention for imaging intracellular biomolecules. However, the complex construction process, intrinsic vulnerability to nuclease degradation and the limited signal transduction efficiency hamper its widespread application. In this contribution, based on persistent autonomous molecular motion of DNAzyme walker along a nano-substrate track, a DNA nanosphere probe (PNLD) is developed for the sensitive intracellular miR-21 imaging. Specifically, DNA nanosphere (called PN, single-molecule nano-track) is assembled from only one palindromic substrate, into which the locking strand-silenced DNAzymes (LD) are installed in a controlled manner. PNLD (made of PN and LD) can protect all DNA components against nuclease attack and maintain its structural integrity in serum solution over 24 h. Upon the activation by target miRNA, DNAzyme walker can move on the substrate scattered within PNLD (or on the surface) and between different PNLD objects and cleave many DNA substrates, generating an amplified signal. As a result, miR-21 can be detected down to 6.83 pM without the detectable interference from co-existing nontarget miRNAs. Moreover, PNLD system can accurately screen the different expression levels of miR-21 within the same type of cells and different types of cells, which is consistent with gold standard polymerase chain reaction (PCR) assay. Via changing the target recognition sequence, the PNLD system can be suitable for the intracellular imaging of miR-155, exhibiting the desirable universality. In addition, the DNAzyme walker-based PNLD system can be used to distinguish cancer cells from healthy cells, implying the potential application in cancer diagnosis and prognosis.

Optimized Two-Photon Imaging by Stimuli-Responsive Peptide Self-Assembly Facilitates Self-Assisted Counteraction of Cisplatin-Resistance in Cancer Cells.

Accurate diagnosis and effective treatment of tumors remain significant clinical challenges. While fluorescence imaging is essential for tumor detection, it has limitations in terms of specificity, penetration depth, and emission wavelength. Here, we report a novel glutathione (GSH)-responsive peptide self-assembly excimer probe (pSE) that optimizes two-photon tumor imaging and self-assisted counteraction of the cisplatin resistance in cancer cells. The GSH-responsive self-assembly of pSE induces a monomer-excimer transition of coumarin, promoting a near-infrared redshift of fluorescence emission under two-photon excitation. This process enhances penetration depth and minimizes interference from biological autofluorescence. Moreover, the intracellular self-assembly of pSE impacts GSH homeostasis, modulates relevant signaling pathways, and significantly reduces GSTP1 expression, resulting in decreased cisplatin efflux in cisplatin-resistant cancer cells. The proposed self-assembled excimer probe not only distinguishes cancer cells from normal cells but also enhances the efficacy of cisplatin chemotherapy, offering significant potential in tumor diagnosis and overcoming cisplatin-resistant tumors.

Synergistic powering of DNA walker movement by endogenous dual enzymes for constructing dual-mode biosensors

To achieve highly sensitive and reliable detection of apurinic/apyrimidinic endonuclease 1 (APE1), a critical cancer diagnostic biomarker, we designed a DNA walker-based dual-mode biosensor, utilizing cellular endogenous dual enzymes (APE 1 and Flap endonuclease 1 (FEN 1)) to collaborate in activating and propelling DNA walker motion on DNA-functionalized Au nanoparticles. Incorporating both fluorescence and electrochemical detection modes, this system leverages signal amplification from DNA walker movement and cascade amplification through tandem hybridization chain reactions (HCR), achieving highly sensitive detection of APE 1. In the fluorescence mode, continuous DNA walker movement, initiated by APE1 and driven by FEN1, generates a robust signal response within a concentration range of 0.01–500 U mL−1, presenting a good linearity in the concentration range of 0.01–10 U mL−1, with a detection limit of 0.01 U mL−1. In the electrochemical detection module, the cascade upstream DNA walker and downstream HCR dual signal amplification strategy further enhances the sensitivity of APE1 detection, extending the linear range to 0.01–50 U mL−1 and reducing the detection limit to 0.002 U mL−1. Rigorous validation demonstrates the biosensor's specificity and anti-interference capability against multiple enzymes. Moreover, it effectively distinguishes cancer cells from normal cell lysates, exhibiting excellent stability and consistency in the dual-modes. Overall, our findings underscore the efficacy of the developed dual-mode biosensor for detecting APE1 in serum and cell lysates samples, indicating its potential for clinical applications in disease diagnosis.

PH-sensitive phosphorescence in penicillamine-coated Au22 nanoclusters: Theoretical and experimental insights

Penicillamine-coated Au22 nanoclusters have an intensified yet pH-adjustable red to near infrared-I (NIR-I) phosphorescence due to their unique electronic structure, which endues them a high potential for tumor imaging and detecting in nanotheranostics. This study reports a sort of engineered Au22(D-pen)18 nanoclusters with a high quantum yield 7.39% as well as pH-conditioned phosphorescence intensity and lifetime that were addressed experimentally and theoretically. Through spin-orbital coupling calculation based on time-dependent density functional theory (TD-DFT), this study ascertains that the deprotonation of carboxylic groups on the D-penicillamine gives rise to a series of changes in the electronic structure of Au22(D-pen)18 nanocluster, leading to an ensuing pH-conditioned phosphorescence from pH-dependent spin–orbit coupling matrix element during inter-system crossing. The deprotonation also alters the bonding and interaction between Au-S core of the Au22(D-pen)18 nanocluster, which compromises the structural stability and potentially generates non-radiative decay with a quenched phosphorescence. The unique in-vitro and in vivo tumor-imagingadvantage of Au22(D-pen)18 nanocluster qualifies it as an efficient tumor-targeting phosphorescence sensor with pH-sensitivity capability for bright contrast to distinguish cancer cells from normal ones. This study provides a comprehensive view on the preparation and characterization of Au22(D-pen)18 nanoclusters, as well as the underlying mechanism of pH-conditioned phosphorescence and application prospects beside uniqueness in methodology, shedding light on further investigation for rational design and engineering of surface of ligand-coated nanoclusters.

A fluorescence probe with targeted mitochondria was developed for detecting H2O2 in vitro and vivo

We designed and developed the probe P-1 for detecting H2O2. The probe can selectively detect H2O2 with colorimetric change. In addition, the combination of portable test strips with a smartphone platform provided great convenience for on-site and visual detection of H2O2 with satisfactory sensitivity and reliability. The P-1 can image exogenous and endogenous H2O2 in cells and distinguish cancer cells and normal cells by fluorescence image of H2O2 and flow cytometry. Meanwhile, the P-1 probe has been successfully applied to monitor H2O2 levels of tumor in mice, and the results showed the level of H2O2 in tumor tissue increased significantly. Therefore, P-1 provided a potential chemical tool to understand pathological and physiological mechanisms of diseases by imaging H2O2.

Near-infrared fluorescent probe based on carboxylesterase 2 and viscosity cascade response for early diagnosis of thyroid cancer

Carboxylesterase 2 (CES2) plays a critical role in the metabolism of ester- and amide-containing drugs and thus has an impact on drug efficacy and toxicity. This study introduces QVJ-CES2, a near-infrared fluorescent probe designed to enhance thyroid cancer diagnosis through CES2 enzyme activity and distinct cellular viscosity of thyroid cells. The probe's dual-response mechanism significantly amplified fluorescence in high-viscosity environments, thus had high sensitivity. Our findings revealed that QVJ-CES2 could detect CES2 activity with a limit of detection as low as 1.9 ng/mL. In vitro experiments confirmed the rapid and selective response of QVJ-CES2 to CES2 and the ability to distinguish cancer cells from their normal cells with high specificity. Most importantly, the fluorescence intensity difference between cancerous and normal cells after incubation with the probe was as high as 7-fold, attributable to the synergistic enzyme and viscosity cascade response. Further, in vivo studies demonstrated that the probe was effective in accurately identifying thyroid cancer tissues, showing that it is a promising tool for early cancer detection and precise surgical interventions.

An enzymatically activated AND-gate DNA logic circuit for tumor cells recognition via multi-microRNAs detection

The DNA-based logic circuit, constructed to mimic biochemical reaction networks, is highly significant in detecting biomarkers at the molecular level. The differences in the expression levels of microRNAs (miRNAs) within different types of cells provide hope for distinguishing cell subtypes. However, reliance on a single miRNA often leads to unreliable results. Herein, we constructed an enzyme-triggered cascade logic circuit based on the AND gate, which is capable of generating corresponding fluorescence signals in the presence of target miRNAs. The introduction of apurinic/apyrimidinic (AP) sites effectively reduces the likelihood of false signal generation. Amplification of the fluorescence signal relies on the catalytic hairpin assembly and the repetitive reuse of the multicomponent nucleic acid enzyme (MNAzyme). We demonstrated that the logic circuit can not only distinguish cancer cells from normal cells but also identify different types of cancer cells. The programmability of the logic circuits and the simplicity of the assay system allow us to modify the functional sequences to recognize different types of biomarkers, thus providing a reference for the identification of various cell subtypes.

Self-Feedback DNAzyme Motor for Cascade-Amplified Imaging of mRNA in Live Cells and In Vivo.

DNA motors have attracted extensive interest in biosensing and bioimaging. However, the amplification capacity of the existing DNA motor systems is limited since the products from the walking process are unable to feedback into the original DNA motor systems. As a result, the sensitivities of such systems are limited in the contexts of biosensing and bioimaging. In this study, we report a novel self-feedback DNAzyme motor for the sensitive imaging of tumor-related mRNA in live cells and in vivo with cascade signal amplification capacity. Gold nanoparticles (AuNPs) are modified with hairpin-locked DNAzyme walker and track strands formed by hybridizing Cy5-labeled DNA trigger-incorporated substrate strands with assistant strands. Hybridization of the target mRNA with the hairpin strands activates DNAzyme and promotes the autonomous walking of DNAzyme on AuNPs through DNAzyme-catalyzed substrate cleavage, resulting in the release of many Cy5-labeled substrate segments containing DNA triggers and the generation of an amplified fluorescence signal. Moreover, each released DNA trigger can also bind with the hairpin strand to activate and operate the original motor system, which induces further signal amplification via a feedback mechanism. This motor exhibits a 102-fold improvement in detection sensitivity over conventional DNAzyme motors and high selectivity for target mRNA. It has been successfully applied to distinguish cancer cells from normal cells and diagnose tumors in vivo based on mRNA imaging. The proposed DNAzyme motor provides a promising paradigm for the amplified detection and sensitive imaging of low-abundance biomolecules in vivo.

Intrinsic dual emissive insulin capped Au/Ag nanoclusters as single ratiometric nanoprobe for reversible detection of pH and temperature and cell imaging

pH and temperature are two important characteristics in cells and the environment. These, not only in the well-done regulation of cell functions but also in diagnosis and treatment, have a key role. Protein-protected bimetallic nanoclusters are abundantly used in the building of biosensors. However, insulin-stabilized Au–Ag nanoclusters with dual intrinsic emission have not been investigated yet. In this work, Dual emissive insulin templated Au–Ag nanocluster (Ins(Au/Ag)NCs) were first synthesized in a simple and green one-put manner. The two emission wavelengths of, as-prepared NCs centered at 410 and 630 nm, excited in one excitation wavelength (330 nm). These two emission peaks were assigned to the di-Tyrosine cross-linked formation and bimetallic nanoclusters respectively. Further analysis displayed that each emission band of Ins(Au/Ag)NCs responded to one variable whilst another peak remained constant; For blue and red emission wavelengths, pH dependency and thermo-responsibility were observed respectively. As-prepared nanoprobe with the intrinsic dual emissive feature was used for ratiometric determination of these parameters, each with a discrete response from another. The linear range of 6.0–9.0 for pH and 1 to 71 °C for temperature was obtained, which comprises the physiological range of pH and temperature and afforded intracellular sensing and imaging capability. As-prepared NCs probe show excellent biocompatibility and cell membrane permeability, and so were successfully applied as direct ratiometric pH and temperature probes in HeLa and HFF cells. More interestingly, this dual emissive nanoprobe is capable of distinguishing cancer cells from normal ones.

Nanocomposite hydrogels in skin cancer medicine.

Skin cancer is one of the most common malignancies in white populations. The therapy strategy is important in skin cancer treatment, depending on several criteria such as stage, size, and localization. Removal of cancerous tissue following anticancer therapeutic administration is considered as gold standard in skin cancer treatment. However, annually rising drug resistance, local inflammation, and ineffective treatment result in a reduction in the effectiveness of the patient's treatment. Nanotechnology has emerged as a prospective in the field of skin cancer medicine, offering innovative, promising solutions for therapeutic procedures and targeted drug delivery. Different nanomaterials are investigated for their potential in skin cancer treatment. Nanohydrogels as a hybrid material, have gained considerable attention due to their unique biomedical and pharmaceutical properties, such as biocompatibility, high water content, and tunable physicochemical characteristics. The principal problem with common skin melanoma chemotherapy is the strong side effects because therapeutics used for treatment do not distinguish cancer cells from healthy cells. Nanohydrogels, as a new-generation, versatile system with the possession of dual characteristics of hydrogels and nanoparticles have shown great potential in targeted delivery in cancer therapy thanks to the possibility of their various modifications, and by that overcome problems with side effects of treatment. This scientific review provides an analysis of the current state of research on nanohydrogels in skin cancer medicine, highlighting their design principles, synthesis methods, and applications in drug delivery, imaging, and combination therapies.

DeepHLApan: A Deep Learning Approach for the Prediction of Peptide-HLA Binding and Immunogenicity.

Neoantigens are crucial in distinguishing cancer cells from normal ones and play a significant role in cancer immunotherapy. The field of bioinformatics prediction for tumor neoantigens has rapidly developed, focusing on the prediction of peptide-HLA binding affinity. In this chapter, we introduce a user-friendly tool named DeepHLApan, which utilizes deep learning techniques to predict neoantigens by considering both peptide-HLA binding affinity and immunogenicity. We provide the application of DeepHLApan, along with the source code, docker version, and web-server. These resources are freely available at https://github.com/zjupgx/deephlapan and http://pgx.zju.edu.cn/deephlapan/ .

A label-free activatable biosensor for in situ detection of exosomal microRNAs based on DNA-AgNCs and hairpin type nucleic acid probes.

Exosomal microRNA (miRNA) is a potential biomarker for cancer diagnosis, metastasis, and treatment. In situ detection of exosomal miRNA is an attractive option due to its simplicity and high accuracy. However, in situ exosomal miRNA detection has encountered challenges because of the low target abundance of targets and limited probe permeability. Herein, a label-free and activatable biosensor was developed for in situ exosomal miRNA assays by utilizing hairpin-shaped nucleic acid probes and DNA-hosted silver nanoclusters (DNA-AgNCs). The probe is directly internalized into the exosomes, and then hybridized with the target miRNA-21. Subsequently, the DNA-AgNCs are pulled closer to the G-rich sequence, ultimately leading to in situ red fluorescence activation. The biosensor not only can detect exosomal miRNA-21 but also distinguish cancer cells from normal cells. Under optimal reaction conditions, the detection limit (LOD) of exosomal miRNA-21 is 1.53 × 107 particles per mL. Furthermore, DNA-AgNCs are used as label-free signal elements for in situ detection of exosomal miRNAs for the first time, expanding the application of nanomaterials in this field. This strategy does not require tedious RNA extraction steps and expensive instruments, and may develop into a non-invasive diagnostic tool for ovarian cancer.