Complex Biological Environments Research Articles

Detection of Sulfoquinovosidase Activity in Cell Lysates Using Activity‐Based Probes

AbstractThe sulfolipid sulfoquinovosyl diacylglycerol (SQDG), produced by plants, algae, and cyanobacteria, constitutes a major sulfur reserve in the biosphere. Microbial breakdown of SQDG is critical for the biological utilization of its sulfur. This commences through release of the parent sugar, sulfoquinovose (SQ), catalyzed by sulfoquinovosidases (SQases). These vanguard enzymes are encoded in gene clusters that code for diverse SQ catabolic pathways. To identify, visualize and isolate glycoside hydrolase CAZY‐family 31 (GH31) SQases in complex biological environments, we introduce SQ cyclophellitol‐aziridine activity‐based probes (ABPs). These ABPs label the active site nucleophile of this enzyme family, consistent with specific recognition of the SQ cyclophellitol‐aziridine in the active site, as evidenced in the 3D structure of Bacillus megaterium SQase. A fluorescent Cy5‐probe enables visualization of SQases in crude cell lysates from bacteria harbouring different SQ breakdown pathways, whilst a biotin‐probe enables SQase capture and identification by proteomics. The Cy5‐probe facilitates monitoring of active SQase levels during different stages of bacterial growth which show great contrast to more traditional mRNA analysis obtained by RT‐qPCR. Given the importance of SQases in global sulfur cycling and in human microbiota, these SQase ABPs provide a new tool with which to study SQase occurrence, activity and stability.

Detection of Sulfoquinovosidase Activity in Cell Lysates Using Activity-Based Probes.

The sulfolipid sulfoquinovosyl diacylglycerol (SQDG), produced by plants, algae, and cyanobacteria, constitutes a major sulfur reserve in the biosphere. Microbial breakdown of SQDG is critical for the biological utilization of its sulfur. This commences through release of the parent sugar, sulfoquinovose (SQ), catalyzed by sulfoquinovosidases (SQases). These vanguard enzymes are encoded in gene clusters that code for diverse SQ catabolic pathways. To identify, visualize and isolate glycoside hydrolase CAZY-family 31 (GH31) SQases in complex biological environments, we introduce SQ cyclophellitol-aziridine activity-based probes (ABPs). These ABPs label the active site nucleophile of this enzyme family, consistent with specific recognition of the SQ cyclophellitol-aziridine in the active site, as evidenced in the 3D structure of Bacillus megaterium SQase. A fluorescent Cy5-probe enables visualization of SQases in crude cell lysates from bacteria harbouring different SQ breakdown pathways, whilst a biotin-probe enables SQase capture and identification by proteomics. The Cy5-probe facilitates monitoring of active SQase levels during different stages of bacterial growth which show great contrast to more traditional mRNA analysis obtained by RT-qPCR. Given the importance of SQases in global sulfur cycling and in human microbiota, these SQase ABPs provide a new tool with which to study SQase occurrence, activity and stability.

Hydrogel/MOF Dual-Modified Photoelectrochemical Biosensor for Antibiofouling and Biocompatible Dopamine Detection.

For accurate in vivo detection, nonspecific adsorption of biomacromolecules such as proteins and cells is a severe issue. The adsorption leads to electrode passivation, significantly compromising both the sensitivity and precision of sensing. Meanwhile, common antibiofouling modifications, such as polymer coatings, still grapple with issues related to biocompatibility, electrode passivation, and miniaturization. Herein, we propose a composite antibiofouling coating strategy based on zwitterionic metal-organic frameworks (Z-MOFs) and a combination of acrylamide hydrogels. On a well-designed TiO2/Z-MOF/hydrogel photoelectrode, we achieve highly sensitive and selective detection of dopamine in complex biological environments. The hydrogel's three-dimensional porous structure combined with unique microporous architecture of Z-MOF ensures effective sieving of interfering macromolecules while preserving efficient small molecules and electron transport. This innovative approach paves the way for constructing miniature, in vivo antibiofouling sensors for molecule monitoring in living organisms with complicated chemical environments.

Genomic and biological characterization of bacteriophages against Enterobacter cloacae, a high-priority pathogen

Enterobacter cloacae is a clinically significant pathogen due to its multi-resistance to antibiotics, presenting a challenge in the treatment of infections. As concerns over antibiotic resistance escalate, novel therapeutic approaches have been explored. Bacteriophages, characterized by their remarkable specificity and ability to self-replicate within target bacteria, are emerging as a promising alternative therapy. In this study, we isolated and partially characterized nine lytic bacteriophages targeting E. cloacae, with two selected for comprehensive genomic analysis based on their host range and bacteriolytic activity. All identified phages exhibited a narrow host range, demonstrated stability within a temperature range of 30–60 °C, displayed pH tolerance from 3 to 10, and showed an excellent bacteriolytic capacity for up to 18 h. Notably, the fully characterized phage genomes revealed an absence of lysogenic, virulence, or antibiotic-resistance genes, positioning them as promising candidates for therapeutic intervention against E. cloacae-related diseases. Nonetheless, translating this knowledge into practical therapeutic applications mandates a deeper understanding of bacteriophage interactions within complex biological environments.

Predicting Solvatochromism of Chromophores in Proteins through QM/MM and Machine Learning.

Solvatochromism occurs in both homogeneous solvents and more complex biological environments, such as proteins. While in both cases the solvatochromic effects report on the surroundings of the chromophore, their interpretation in proteins becomes more complicated not only because of structural effects induced by the protein pocket but also because the protein environment is highly anisotropic. This is particularly evident for highly conjugated and flexible molecules such as carotenoids, whose excitation energy is strongly dependent on both the geometry and the electrostatics of the environment. Here, we introduce a machine learning (ML) strategy trained on quantum mechanics/molecular mechanics calculations of geometrical and electrochromic contributions to carotenoids' excitation energies. We employ this strategy to compare solvatochromism in protein and solvent environments. Despite the important specifities of the protein, ML models trained on solvents can faithfully predict excitation energies in the protein environment, demonstrating the robustness of the chosen descriptors.

A low-fouling electrochemical biosensor based on BSA hydrogel doped with carbon black for the detection of cortisol in human serum

Electrochemical biosensors with high sensitivity can detect low concentrations of biomarkers, but their practical detection applications in complex biological environments such as human serum and sweat are severely limited by the biofouling. Herein, a conductive hydrogel based on bovine serum albumin (BSA) and conductive carbon black (CCB) was prepared for the construction of an antifouling biosensor. The BSA hydrogel (BSAG) was doped with CCB, and the prepared composite hydrogel exhibited good conductivity originated from the CCB and antifouling capability owing to the BSA hydrogel. An antifouling biosensor for the sensitive detection of cortisol was fabricated by drop-coating the conductive hydrogel onto a poly(3,4-ethylenedioxythiophene) (PEDOT) modified electrode and further immobilizing the cortisol aptamer. The constructed biosensor showed a linear range of 100 pg mL−1 - 10 μg mL−1 and a limit of detection of 26.0 pg mL−1 for the detection of cortisol, and it was capable of assaying cortisol accurately in complex human serum. This strategy of preparing antifouling and conductive hydrogels provides an effective way to develop robust electrochemical biosensors for biomarker detection in complex biological media.

MOF-based ratiometric fluorescent nanoprobe for quantitative detection of hydrogen sulfide in acute pancreatitis

The timely detection of acute pancreatitis (AP) is crucial for mitigating its severe complications, including pancreatic necrosis and multi-system organ failure. In this work, we developed LMOF-BR, an innovative luminescent metal–organic framework (LMOF)-based ratiometric fluorescent nanoprobe, specifically designed for the sensitive and selective detection of hydrogen sulfide (H2S) in AP. This nanoprobe employs a synthesized LMOF to provide a stable reference signal, and a fluorescent dye (BR) as the target recognizer and signal output unit, enabling H2S detection through single-excitation dual fluorescence emission. The LMOF-BR nanoprobe demonstrates significant advantages, including improved water solubility, superior photostability, a high signal-background ratio, and a low limit of detection (LOD), all of which are crucial for accurate detection in complex biological environments. Successful quantification of H2S in serum samples and AP mouse plasma has been achieved by LMOF-BR nanoprobe, showcasing notable accuracy and precision. The LMOF-BR nanoprobe holds great promise in advancing biosensing technology, particularly for the timely and reliable diagnosis of AP.

Micro robot as the feature of robotic in healthcare approach from design to application: the State of art and challenges

Micro robots, miniature robotic devices typically ranging from micrometers to a few millimeters in size, hold immense potential in various fields, particularly healthcare. Their diminutive stature enables access to intricate anatomical regions previously unreachable, facilitating targeted drug delivery, localized treatment, and precise monitoring. These robots offer numerous advantages, including enhanced maneuverability, reduced invasiveness, and minimized tissue damage. By navigating through complex biological environments, micro robots can deliver therapies with unprecedented precision, improving treatment efficacy and patient outcomes. Additionally, their small size allows for minimally invasive procedures, reducing recovery times and enhancing patient comfort. Overall, micro robots represent a groundbreaking technological advancement with the potential to revolutionize healthcare delivery and significantly benefit human well-being.Their small size enables access to intricate anatomical regions for targeted drug delivery, localized treatment, and precise monitoring. Despite challenges like size constraints and navigation complexities, innovative solutions and interdisciplinary collaboration are driving their advancement in improving healthcare outcomes.

Fluorescence lifetime imaging and phasor analysis of intracellular porphyrinic photosensitizers applied with different polymeric formulations

The fluorescence lifetime of a porphyrinic photosensitizer (PS) is an important parameter to assess the aggregation state of the PS even in complex biological environments. Aggregation-induced quenching of the PS can significantly reduce the yield of singlet oxygen generation and thus its efficiency as a medical drug in photodynamic therapy (PDT) of diseased tissues. Hydrophobicity and the tendency to form aggregates pose challenges on the development of efficient PSs and often require carrier systems. A systematic study was performed to probe the impact of PS structure and encapsulation into polymeric carriers on the fluorescence lifetime in solution and in the intracellular environment. Five different porphyrinic PSs including chlorin e6 (Ce6) derivatives and tetrakis(m-hydroxyphenyl)-porphyrin and -chlorin were studied in free form and combined with polyvinylpyrrolidone (PVP) or micelles composed of triblock-copolymers or Cremophor. Following incubation of HeLa cells with these systems, fluorescence lifetime imaging combined with phasor analysis and image segmentation was applied to study the lifetime distribution in the intracellular surrounding. The data suggest that for free PSs, the structure-dependent cell uptake pathways determine their state and emission lifetimes. PS localization in the plasma membrane yielded mostly monomers with long fluorescence lifetimes whereas the endocytic pathway with subsequent lysosomal deposition adds a short-lived component for hydrophilic anionic PSs. Prolonged incubation times led to increasing contributions from short-lived components that derive from aggregates mainly localized in the cytoplasm. Encapsulation of PSs into polymeric carriers led to monomerization and mostly fluorescence emission decays with long fluorescence lifetimes in solution. However, the efficiency depended on the binding strength that was most pronounced for PVP. In the cellular environment, PVP was able to maintain monomeric long-lived species over prolonged incubation times. This was most pronounced for Ce6 derivatives with a logP value around 4.5. Micellar encapsulation led to faster release of the PSs resulting in multiple components with long and short fluorescence lifetimes. The hydrophilic hardly aggregating PS exhibited a mostly stable invariant lifetime distribution over time with both carriers. The presented data are expected to contribute to optimized PDT treatment protocols and improved PS-carrier design for preventing intracellular fluorescence quenching. In conclusion, amphiphilic and concurrent hydrophobic PSs with high membrane affinity as well as strong binding to the carrier have best prospects to maintain their photophysical properties in vivo and serve thus as efficient photodynamic diagnosis and PDT drugs.

Protein Aggregation on Metal Oxides Governs Catalytic Activity and Cellular Uptake.

Engineering of catalytically active inorganic nanomaterials holds promising prospects for biomedicine. Catalytically active metal oxides show applications in enhancing wound healing but have also been employed to induce cell death in photodynamic or radiation therapy. Upon introduction into a biological system, nanomaterials are exposed to complex fluids, causing interaction and adsorption of ions and proteins. While protein corona formation on nanomaterials is acknowledged, its modulation of nanomaterial catalytic efficacy is less understood. In this study, proteomic analyses and nano-analytic methodologies quantify and characterize adsorbed proteins, correlating this protein layer with metal oxide catalytic activity in vitro and in vivo. The protein corona comprises up to 280 different proteins, constituting up to 38% by weight. Enhanced complement factors and other opsonins on nanocatalyst surfaces lead to their uptake into macrophages when applied topically, localizing >99% of the nanomaterials in tissue-resident macrophages. Initially, the formation of the protein corona significantly reduces the nanocatalysts' activity, but this activity can be partially recovered in endosomal conditions due to the proteolytic degradation of the corona. Overall, the research reveals the complex relationship between physisorbed proteins and the catalytic characteristics of specific metal oxide nanoparticles, providing design parameters for optimizing nanocatalysts in complex biological environments.

Understanding the role of π-π stacking from red carbon for selective, high stable and anti-external interference detecting ferric ions and cellular imaging

The subcarboxylated carbon (C3O2) is a unique molecule that spontaneously polymerizes into highly conjugated fluorescence structures p(C3O2)n, named “red carbon”. This unique property, combined with its role as aggregation-induced emission luminogens, has allowed for a deeper exploration of their emission mechanism. Herein, both density functional theory calculations and experimental approaches to elucidate the crystal structure of π–π stacking in red carbon, thereby enhancing the fluorescence properties of p(C3O2)n polymer in its aggregated state. The time-dependent density functional theory calculations and experimental observations indicate that the fluorescence of red carbon can be quenched by ferric ions (Fe3+) and subsequently restored by ethylenediaminetetraaceticacid (EDTA). This quenching process follows first-order reaction kinetics. On the basis of the coordination to metal ions from abundant heteroatoms on red carbon, the high sensitivity, quick responsive (1 min), sensitive detection (the lowest detection limit is 0.97 μM) for the Fe3+ were realized in red carbon, which provides inspirations for the design of new turn-on probes with high stability in detecting the Fe3+ in aqueous solution. Crucially, the red carbon as a light-up fluorescence probe can be employed for the Fe3+ sensing in living cells, which enlighten a new perspective for designing non-conjugated fluorescent polymers. This innovative approach opens up new avenues for developing probes that can detect Fe3+ with high precision and reliability in complex biological environments.

Building a stable and robust anti-interference DNA dissipation system by eliminating the accumulation of systemic specified errors

BackgroundThe emergence of DNA nanotechnology has enabled the systematic design of diverse bionic dissipative behaviors under the precise control of nucleic acid nanodevices. Nevertheless, when compared to the dissipation observed in robust living systems, it is highly desirable to enhance the anti-interference for artificial DNA dissipation to withstand perturbations and facilitate repairs within the complex biological environments. ResultsIn this study, we introduce strategically designed “trash cans” to facilitate kinetic control over interferences, transforming the stochastic binding of individual components within a homogeneous solution into a competitive binding process. This approach effectively eliminates incorrect binding and the accumulation of systemic interferences while ensuring a consistent pattern of energy fluctuation from response to silence. Remarkably, even in the presence of numerous interferences differing by only one base, we successfully achieve complete system reset through multiple cycles, effectively restoring the energy level to a minimum. SignificanceThe system was able to operate stably without any adverse effect under conditions of irregular interference, high-abundance interference, and even multiplex interferences including DNA and RNA crosstalk. This work not only provides an effective paradigm for constructing robust DNA dissipation systems but also greatly broadens the potential of DNA dissipation for applications in high-precision molecular recognition and complex biological reaction networks.

Roadmap for Clinical Translation of Mobile Microrobotics.

Medical microrobotics is an emerging field to revolutionize clinical applications in diagnostics and therapeutics of various diseases. On the other hand, the mobile microrobotics field has important obstacles to pass before clinical translation. This article focuses on these challenges and provides a roadmap of medical microrobots to enable their clinical use. From the concept of a "magic bullet" to the physicochemical interactions of microrobots in complex biological environments in medical applications, there are several translational steps to consider. Clinical translation of mobile microrobots is only possible with a close collaboration between clinical experts and microrobotics researchers to address the technical challenges in microfabrication, safety, and imaging. The clinical application potential can be materialized by designing microrobots that can solve the current main challenges, such as actuation limitations, material stability, and imaging constraints. The strengths and weaknesses of the current progress in the microrobotics field are discussed and a roadmap for their clinical applications in the near future isoutlined.

Virtual Reality‐Enabled Intuitive Magnetic Manipulation of Microrobots and Nanoparticles

Magnetic microrobots and nanoparticles offer unique capabilities for medical applications by granting unprecedented access to intricate and delicate anatomical structures. Nevertheless, an intuitive manipulation approach remains a challenge due to their small size and limited feedback. This study presents a new method for magnetic microrobot and nanoparticle control that employs virtual reality (VR), creating an immersive and realistic view of the bodily anatomy and the microrobot within. This study compares three manipulation modes: a traditional mode using 2D displays, VR mode using a VR headset and touch controllers, and VR autonomous programmed maneuvering. It is shown that VR‐assisted modes reduce the manipulation time by improving spatial awareness. Nanoparticles in a VR environment are also manipulated. The proposed method will find applications in terms of intuitive, immersive microrobots or magnetic nanoparticle control in complex biological environments, thus in many medical procedures and for drug and cell delivery. The immersive nature of this approach enhances the ability of the user to perceive and understand complex anatomical structures, facilitating better navigation within delicate environments; both dexterity and spatial awareness are improved. Thus, it is shown how microrobots and magnetic nanoparticles can be controlled using immersive simulations to improve both visualization and manipulation.

Clustering of chemically propelled nanomotors in chemically active environments.

Synthetic nanomotors powered by chemical reactions have been designed to act as vehicles for active cargo transport, drug delivery, and a variety of other uses. Collections of such motors, acting in consort, can self-assemble to form swarms or clusters, providing opportunities for applications on various length scales. While such collective behavior has been studied when the motors move in a chemically inactive fluid environment, when the medium in which they move is a chemical network that supports complex spatial and temporal patterns, through simulation and theoretical analysis we show that collective behavior changes. Spatial patterns in the environment can guide and control motor collective states, and interactions of the motors with their environment can give rise to distinctive spatiotemporal motor patterns. The results are illustrated by studies of the motor dynamics in systems that support Turing patterns and spiral waves. This work is relevant for potential applications that involve many active nanomotors moving in complex chemical or biological environments.

Multiplexed near infrared fluorescence lifetime imaging in turbid media.

Fluorescence lifetime imaging (FLI) plays a pivotal role in enhancing our understanding of biological systems, providing a valuable tool for non-invasive exploration of biomolecular and cellular dynamics, both in vitro and in vivo. Its ability to selectively target and multiplex various entities, alongside heightened sensitivity and specificity, offers rapid and cost-effective insights. Our aim is to investigate the multiplexing capabilities of near-infrared (NIR) FLI within a scattering medium that mimics biological tissues. We strive to develop a comprehensive understanding of FLI's potential for multiplexing diverse targets within a complex, tissue-like environment. We introduce an innovative Monte Carlo (MC) simulation approach that accurately describes the scattering behavior of fluorescent photons within turbid media. Applying phasor analyses, we enable the multiplexing of distinct targets within a single FLI image. Leveraging the state-of-the-art single-photon avalanche diode (SPAD) time-gated camera, SPAD512S, we conduct experimental wide-field FLI in the NIR regime. Our study demonstrates the successful multiplexing of dual targets within a single FLI image, reaching a depth of 1cm within tissue-like phantoms. Through our novel MC simulation approach and phasor analyses, we showcase the effectiveness of our methodology in overcoming the challenges posed by scattering media. This research underscores the potential of NIR FLI for multiplexing applications in complex biological environments. By combining advanced simulation techniques with cutting-edge experimental tools, we introduce significant results in the non-invasive exploration of biomolecular dynamics, to advance the field of FLI research.

Three-dimensional DNA biomimetic networks (B-3D Net)-based ratiometric fluorescence platform for cancer-related gene biosensing

Efficient detection of cancer-related nucleic acids is pivotal for early cancer diagnosis. This study introduces a target induced three-dimensional DNA biomimetic networks (B-3D Net)-based ratiometric fluorescence platform using manganese dioxide nanosheets (MnO2 NS)/o-phenylenediamine in combination with hybridization chain reaction to detect cancer-related genes (p53 gene). The incorporation of multiple signals within the B-3D networks can significantly enhance catalytic activity and amplify the output signals, enabling a high sensitivity. Compared with traditional ratio fluorescence platforms, there is no demand to synthesize fluorescent nanoprobes due to the in-situ formation of fluorescence species, which is simple and cost-effective. The corresponding assay demonstrated exceptional sensitivity (with a detection limit as low as 2 fM), selectivity, reproducibility, and accuracy, which mitigates disturbances caused by instrument errors, an inaccurate probe count, and the microenvironment. Furthermore, the ease and straightforwardness of discerning changes in fluorescent brightness and colour by the naked eye are evident. Using the relevant software, a linear relationship between fluorescent images using a smartphone and target concentration was obtained. Hence, the novel ratiometric sensing system will demonstrate new opportunities on determination of target DNA samples in complex biological environments.

Novel SERS Signal Amplification Strategy for Ultrasensitive and Specific Detection of Spinal Cord Injury-Related miRNA.

The expression of microRNA (miRNA) changes in many diseases plays an important role in the diagnosis, treatment, and prognosis of diseases. Spinal cord injury (SCI) is a serious disease of the central nervous system, accompanied by inflammation, cell apoptosis, neuronal necrosis, axonal rupture, demyelination, and other pathological processes, resulting in impaired sensory and motor functions of patients. Studies have shown that miRNA expression has changed after SCI, and miRNAs participate in the pathophysiological process and treatment of SCI. Therefore, quantitative analysis and monitoring of the expression of miRNA were of great significance for the diagnosis and treatment of SCI. Through the SCI-related miRNA chord plot, we screened out miRNA-21-5p and miRNA-let-7a with a higher correlation. However, for traditional detection strategies, it is still a great challenge to achieve a fast, accurate, and sensitive detection of miRNA in complex biological environments. The most frequently used method for detecting miRNAs is polymerase chain reaction (PCR), but it has disadvantages such as being time-consuming and cumbersome. In this paper, a novel SERS sensor for the quantitative detection of miRNA-21-5p and miRNA-let-7a in serum and cerebrospinal fluid (CSF) was developed. The SERS probe eventually formed a sandwich-like structure of Fe3O4@hpDNA@miRNA@hpDNA@GNCs with target miRNAs, which had high specificity and stability. This SERS sensor achieved a wide range of detection from 1 fM to 1 nM and had a good linear relationship. The limits of detection (LOD) for miRNA-21-5p and miRNA-let-7a were 0.015 and 0.011 fM, respectively. This new strategy realized quantitative detection and long-term monitoring of miRNA-21-5p and miRNA-let-7a in vivo. It is expected to become a powerful biomolecule analysis tool and will provide ideas for the diagnosis and treatment of many diseases.

Micrometer-thick and porous nanocomposite coating for electrochemical sensors with exceptional antifouling and electroconducting properties

Development of coating technologies for electrochemical sensors that consistently exhibit antifouling activities in diverse and complex biological environments over extended time is vital for effective medical devices and diagnostics. Here, we describe a micrometer-thick, porous nanocomposite coating with both antifouling and electroconducting properties that enhances the sensitivity of electrochemical sensors. Nozzle printing of oil-in-water emulsion is used to create a 1 micrometer thick coating composed of cross-linked albumin with interconnected pores and gold nanowires. The layer resists biofouling and maintains rapid electron transfer kinetics for over one month when exposed directly to complex biological fluids, including serum and nasopharyngeal secretions. Compared to a thinner (nanometer thick) antifouling coating made with drop casting or a spin coating of the same thickness, the thick porous nanocomposite sensor exhibits sensitivities that are enhanced by 3.75- to 17-fold when three different target biomolecules are tested. As a result, emulsion-coated, multiplexed electrochemical sensors can carry out simultaneous detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid, antigen, and host antibody in clinical specimens with high sensitivity and specificity. This thick porous emulsion coating technology holds promise in addressing hurdles currently restricting the application of electrochemical sensors for point-of-care diagnostics, implantable devices, and other healthcare monitoring systems.

A cysteine/lipid droplets sequentially activated dual-locked fluorescent probe for accurate bioimaging of tumor tissues

The previous activatable probe toward one analyte can still produce imprecise signal as a result of the challenge cased by accurate identification in complex biological environment. As smarter molecules, dual-locked probes are able to respond to two different analyte with one or more output signals, which display significantly reduced background interference and increased spatial resolution. In this study, a smart cysteine/lipid droplets sequence-activated dual-locked fluorescent probe CN–NO2 has been designed and synthesized for the detection of tumor tissues. The recognition group of CN–NO2 removed after reacting with cysteine (Cys) and undergoing intramolecular rearrangement to generate complete fluorophores. This structure had a strong solvent effect; it could recognize lipid droplets (LDs) in cells, thus exhibiting fluorescence without secondary molecular adjustment. The fluorescence of CN–NO2 was enhanced 1752-fold after double unlocking. Importantly, the unlocking process only takes 10 min. Furthermore, the fluorescence of CN–NO2 was amplified remarkable and anchored at LDs after reacting with Cys in A549 cells. Therefore, CN–NO2 clearly indicated the site of mouse xenograft tumor in vivo. Such sequence-activated dual-locked fluorescent probes are envisioned to execute more analysis and diagnostic tasks giving these unique characteristics.