Complex Biological Environments Research Articles

Toward Accurate Photoluminescence Nanothermometry Using Rare-Earth Doped Nanoparticles for Biomedical Applications.

ConspectusPhotoluminescence nanothermometry can detect the local temperature at the submicrometer scale with minimal contact with the object under investigation. Owing to its high spatial resolution, this technique shows great potential in biomedicine in both fundamental studies as well as preclinical research. Photoluminescence nanothermometry exploits the temperature-dependent optical properties of various nanoscale optical probes including organic fluorophores, quantum dots, and carbon nanostructures. At the vanguard of these diverse optical probes, rare-earth doped nanoparticles (RENPs) have demonstrated remarkable capabilities in photoluminescence nanothermometry. They distinguish themselves from other luminescent nanoprobes owning to their unparalleled and versatile optical properties that include narrow emission bandwidths, high photostability, tunable lifetimes from microseconds to milliseconds, multicolor emissions spanning the ultraviolet, visible, and near-infrared (NIR) regions, and the ability to undergo upconversion, all with excitation of a single, biologically friendly NIR wavelength. Recent advancements in the design of novel RENPs have led to new fundamental breakthroughs in photoluminescence nanothermometry. Moreover, driven by their excellent biocompatibility, both in vitro and in vivo, their implementation in biomedical applications has also gained significant traction. However, these nanoprobes face limitations caused by the complex biological environments, including absorption and scattering of various biomolecules as well as interference from different tissues, which limit the spatial resolution and detection sensitivity in RENP temperature sensing.Among existing approaches in RENP photoluminescence nanothermometry, the most prevalent implemented mechanisms either leverage the changes in the relative intensity ratio of two emission bands or exploit the lifetimes of various excited states. Photoluminescence intensity ratio (PLIR) nanothermometry has been the mainstream method owing to the readily available spectrometers for photoluminescence acquisition. Despite offering high temperature sensitivity and spatial resolution, this technique is restricted by tedious calibration and undesirable fluctuation in photoluminescence intensity ascribed to factors such as probe concentration, excitation power density, and biochemical surroundings. Lifetime-based nanothermometry uses the lifetime of a specific transition as the contrast mechanism to infer the temperature. This modality is less susceptible to various experimental factors and is compatible with a broader range of photoluminescence nanoprobes. However, due to relatively expensive and complex instrumentation, long data acquisition, and sophisticated data analysis, lifetime-based nanothermometry is still breaking ground with recently emerging techniques lightening its path.In this Account, we provide an overview of RENP nanothermometry and their applications in biomedicine. The architectures and luminescence mechanisms of RENPs are examined, followed by the principles of PLIR and lifetime-based nanothermometry. The in-depth description of each approach starts with its basic principle of accurate temperature sensing, followed by a critical discussion of the representative techniques, applications as well as their strengths and limitations. Special emphasis is given to the emerging modality of lifetime-based nanothermometry in light of the important new developments in the field. Finally, a summary and an outlook are provided to conclude this Account.

Tetrazine Amino Acid Encoding for Rapid and Complete Protein Bioconjugation.

Generating protein conjugates using the bioorthogonal ligation between tetrazines and trans-cyclooctene groups avoids the need to manipulate cysteine amino acids; this ligation is rapid, site-specific, and stoichiometric and allows for labeling of proteins in complex biological environments. Here, we provide a protocol for the expression of conjugation-ready proteins at high yields in Escherichia coli with greater than 95% encoding and labeling fidelity. This protocol focuses on installing the Tet2 tetrazine amino acid using an optimized genetic code expansion (GCE) machinery system, Tet2 pAJE-E7, to direct Tet2 encoding at TAG stop codons in BL21 E. coli strains, enabling reproducible expression of Tet2-proteins that quantitatively react with trans-cyclooctene (TCO) groups within 5 min at room temperature and physiological pH. The use of the BL21 derivative B95(DE3) minimizes premature truncation byproducts caused by incomplete suppression of TAG stop codons, which makes it possible to use more diverse protein construct designs. Here, using a superfolder green fluorescent protein construct as an example protein, we describe in detail a four-day process for encoding Tet2 with yields of ~200 mg per liter of culture. Additionally, a simple and fast diagnostic gel electrophoretic mobility shift assay is described to confirm Tet2-Et encoding and reactivity. Finally, strategies are discussed to adapt the protocol to alternative proteins of interest and optimize expression yields and reactivity for that protein. Key features • Protocol describes site-specific encoding of the tetrazine amino acid Tet2-Et into proteins for bioorthogonal, quantitative, and rapid attachment of trans-cyclooctene-containing labels. • Protocol uses auto-induction methods for the production Tet2-Et protein in E. coli. • This protocol focuses on Tet-protein expressions in BL21(DE3) and B95(DE3) strains, which take approximately 4 days to complete. • SDS-PAGE mobility shift assay using a strained TCO-PEG5000 (sTCO-PEG5000) reagent provides a simple, generalizable method for testing Tet-protein reactivity.

A lipid droplet-targeted NIR fluorescent probe for viscosity detection in Parkinson's disease

Investigating lipid droplets (LDs) is crucial for elucidating their role in biological function and the pathogenesis of related diseases, including Parkinson's disease (PD). Therefore, developing a fluorescent probe to measure LDs viscosity is crucial. To address the challenges of complex biological environments, a chemical toolbox, F-675, was introduced based on the twisted - intramolecular charge transfer mechanism. F-675 featured a large Stokes shift of 158 nm and emitted near-infrared emission at 675 nm, facilitating LDs detection with minimal biological fluorescent background. Additionally, F-675 exhibited excellent selectivity and sensitivity to changes of LDs. Furthermore, F-675 effectively detected LDs changes in PD models, ranging from cells, C. elegans and Parkin null Drosophila. These findings indicate the potential of F-675 as an effective tool for PD diagnosis.

Triangle-toothed gear occlude-guided universal nanotechnology constructs 3D symmetric DNA polyhedra with high assembly efficiency for precision cancer therapy

Chemotherapy is commonly used to treat malignant tumors. However, conventional chemotherapeutic drugs often cannot distinguish between tumor and healthy cells, resulting in adverse effects and reduced therapeutic efficacy. Therefore, zigzag-shaped gear-occlude-guided cymbal-closing (ZGC) DNA nanotechnology was developed based on the mirror-symmetry principle to efficiently construct symmetric DNA polyhedra. This nanotechnology employed simple mixing steps for efficient sequence design and assembly. A targeting aptamer was installed at a user-defined position using an octahedron as a model structure. Chemotherapeutic drug-loaded polyhedral objects were subsequently delivered into tumor cells. Furthermore, anticancer drug-loaded DNA octahedra were intravenously injected into a HeLa tumor-bearing mouse model. Assembly efficiency was almost 100 %, with no residual building blocks identified. Moreover, this nanotechnology required a few DNA oligonucleotides, even for complex polyhedrons. Symmetric DNA polyhedrons retained their structural integrity for 24 h in complex biological environments, guaranteeing prolonged circulation without drug leakage in the bloodstream and promoting efficient accumulation in tumor tissues. In addition, DNA octahedra were cleared relatively slowly from tumor tissues. Similarly, tumor growth was significantly inhibited in vivo, and a therapeutic outcome comparable to that of conventional gene-chemo combination therapy was observed. Moreover, no systemic toxicity was detected. These findings indicate the potential application of ZGC DNA nanotechnology in precision medicine.

Semisynthetic ferritin-based nanoparticles with high magnetic anisotropy for spatial magnetic manipulation and inductive heating.

The human iron storage protein ferritin represents an appealing template to obtain a semisynthetic magnetic nanoparticle (MNP) for spatial manipulation or inductive heating applications on a nanoscale. Ferritin consists of a protein cage of well-defined size (12 nm), which is genetically modifiable and biocompatible, and into which a magnetic core is synthesised. Here, we probed the magnetic response and hence the MNP's suitability for (bio-)nanotechnological or nanomedical applications when the core is doped with 7% cobalt or 7% zinc in comparison with the undoped iron oxide MNP. The samples exhibit almost identical core and hydrodynamic sizes, along with their tunable magnetic core characteristics as verified by structural and magnetic characterisation. Cobalt doping significantly increased the MNP's anisotropy and hence the heating power in comparison with other magnetic cores with potential application as a mild heat mediator. Spatial magnetic manipulation was performed with MNPs inside droplets, the cell cytoplasm, or the cell nucleus, where the MNP surface conjugation with mEGFP and poly(ethylene glycol) gave rise to excellent intracellular stability and traceability within the complex biological environment. A magnetic stimulus (smaller than fN forces) results in the quick and reversible redistribution of the MNPs. The obtained data suggest that semisynthetic ferritin MNPs are highly versatile nanoagents and promising candidates for theranostic or (bio-)nanotechnological applications.

An electrochemical biosensor with enhanced antifouling properties enabled by peptide self-assembly via robust Pt-S interactions

Designing effective biosensing interfaces requires robust biomolecule immobilization strategies. Traditional gold-sulfur bonds (Au-S) have low affinity and are prone to ligand substitution reactions in complex biological environments. In this study, we introduce an innovative approach utilizing a trifunctional branched-cyclopeptide (TBCP) for self-assembly onto platinum nanoparticles (PtNP)-modified electrodes. TBCP immobilization on PtNP via Pt-S interaction proves significantly more stable than Au-S bonding. In addition, TBCP peptides assembled through Pt-S interactions exhibit low susceptibility to displacement by small molecules like glutathione, making them ideal for constructing robust biosensors with high stability in biological fluids. Leveraging peptide multifunctionality and strong Pt-S interactions, TBCP-modified electrodes excel in various biological fluids, detecting the breast cancer marker ErbB2 in human serum. The high sensitivity of the TBCP/PtNP-based biosensor distinguishes serum samples from breast cancer patients and healthy individuals. This self-assembly strategy represents a promising advance in constructing biosensors with robust operation in complex biological media.

Redox Concomitant Formation Method for Fabrication of Cu(I)-MOF/Polymer Composites with Antifouling Properties.

Marine biofouling, which is one of the technical challenges hindering the growth of the marine economy, has been controlled using cuprous oxide (Cu2O) nanoparticles due to the exceptional antifouling properties of Cu(I) ions. However, Cu2O nanoparticles have encountered bottlenecks due to explosive releases of Cu+ ions, high toxicity at elevated doses, and long-term instability. Here, we present a novel method called Redox Concomitant Formation (RCF) for fabricating a hierarchical Cu(I) metal-organic framework polypyrrole (Cu(I)-MOF/PPy) composite. This method enables in-situ phase transition via successive redox reactions that change the chemical valence state and coordination mode of Cu(II)-MOF, resulting in a new structure of Cu(I)-MOF while creating a PPy layer surrounded by the hierarchical structure. Owing to the steady release of Cu+ ions from the Cu(I) sites and photothermal properties of PPy, Cu(I)-MOF/PPy exhibits superior and broad-spectrum resistance to marine bacteria, algae, and surface-adhered biofilms in complex biological environments, as well as long-term stability, resulting in 100% eradication efficiency under solar-driven heating. Mechanistic insights into successive structural redox reactions and formation using the RCF method are provided in detail, enabling the fabrication of novel MOFs with the desired composition and structure for a wide range of potential applications.

Redox Concomitant Formation Method for Fabrication of Cu(I)‐MOF/Polymer Composites with Antifouling Properties

Marine biofouling, which is one of the technical challenges hindering the growth of the marine economy, has been controlled using cuprous oxide (Cu2O) nanoparticles due to the exceptional antifouling properties of Cu(I) ions. However, Cu2O nanoparticles have encountered bottlenecks due to explosive releases of Cu+ ions, high toxicity at elevated doses, and long‐term instability. Here, we present a novel method called Redox Concomitant Formation (RCF) for fabricating a hierarchical Cu(I) metal‐organic framework polypyrrole (Cu(I)‐MOF/PPy) composite. This method enables in‐situ phase transition via successive redox reactions that change the chemical valence state and coordination mode of Cu(II)‐MOF, resulting in a new structure of Cu(I)‐MOF while creating a PPy layer surrounded by the hierarchical structure. Owing to the steady release of Cu+ ions from the Cu(I) sites and photothermal properties of PPy, Cu(I)‐MOF/PPy exhibits superior and broad‐spectrum resistance to marine bacteria, algae, and surface‐adhered biofilms in complex biological environments, as well as long‐term stability, resulting in 100% eradication efficiency under solar‐driven heating. Mechanistic insights into successive structural redox reactions and formation using the RCF method are provided in detail, enabling the fabrication of novel MOFs with the desired composition and structure for a wide range of potential applications.

Selective Activation of Cascade Assembly Amplification for DNA Methyltransferase Detection Using a Double-Loop Interlocked DNA Circuit.

Precise and reliable monitoring of DNA adenine methyltransferase (Dam) activity is essential for disease diagnosis and biological analysis. However, existing techniques for detecting Dam activity often rely on specific DNA recognition probes that are susceptible to DNA degradation and exhibit limited target sensitivity and specificity. In this study, we designed and engineered a stable and dynamic DNA nanodevice called the double-loop interlocked DNA circuit (DOOR) that enables the sensitive and selective monitoring of Dam activity in complex biological environments. The DOOR incorporates two interlocked specialized sequences: a palindromic sequence for Dam identification and an initiator sequence for signal amplification. In the presence of Dam, the DOOR is cleaved by double-stranded DNA phosphodiesterase I endonuclease, generating massive double-stranded DNA (dsDNA) units. These units can self-assemble into a long dsDNA scaffold, thereby enhancing the subsequent reaction kinetics. The dsDNA scaffold further triggers a hyperbranched hybrid chain reaction to produce a fluorescent 3D DNA nanonet, enabling more precise monitoring of the Dam activity. The DOOR device exhibits excellent sensitivity, specificity, and stability, rendering it a powerful tool for studying DNA methylation in various biological processes and diseases.

Copolymer-Coated Gold Nanoparticles: Enhanced Stability and Customizable Functionalization for Biological Assays.

Gold nanoparticles (AuNPs) play a vital role in biotechnology, medicine, and diagnostics due to their unique optical properties. Their conjugation with antibodies, antigens, proteins, or nucleic acids enables precise targeting and enhances biosensing capabilities. Functionalized AuNPs, however, may experience reduced stability, leading to aggregation or loss of functionality, especially in complex biological environments. Additionally, they can show non-specific binding to unintended targets, impairing assay specificity. Within this work, citrate-stabilized and silica-coated AuNPs (GNPs and SiGNPs, respectively) have been coated using N,N-dimethylacrylamide-based copolymers to increase their stability and enable their functionalization with biomolecules. AuNP stability after modification has been assessed by a combination of techniques including spectrophotometric characterization, nanoparticle tracking analysis, transmission electron microscopy and functional microarray tests. Two different copolymers were identified to provide a stable coating of AuNPs while enabling further modification through click chemistry reactions, due to the presence of azide groups in the polymers. Following this experimental design, AuNPs decorated with ssDNA and streptavidin were synthesized and successfully used in a biological assay. In conclusion, a functionalization scheme for AuNPs has been developed that offers ease of modification, often requiring single steps and short incubation time. The obtained functionalized AuNPs offer considerable flexibility, as the functionalization protocol can be personalized to match requirements of multiple assays.

Nanofiber enhanced carbon cloth based flexible Miniaturized electrodes for Real-Time and specific insulin detection in blood serum

The development of sensitive and specific biosensors for real-time insulin monitoring is crucial for advancing diabetes management and improving patient outcomes. This study reports the fabrication of flexible electrodes based on titanium dioxide (TiO2) nanofiber-enhanced carbon cloth (CC) for the real-time, selective picomolar range of detection of insulin in blood serum. TiO2 nanofibers significantly increase the electrodes surface area and electron transfer capabilities, enhancing sensitivity and enabling rapid response times essential for real-time monitoring. The flexibility and mechanical stability of the carbon cloth substrate make it ideal for wearable and implantable biosensors, addressing limitations of conventional sensors, such as inadequate sensitivity, delayed response times, rigidity, and non-specific binding. The enhanced properties of the TiO2 nanofiber-modified carbon cloth composite facilitate the detection of low insulin concentrations, essential for early diagnosis and continuous monitoring in diabetes care. The robust TiO2 nanofibers and durable carbon cloth ensure sustained performance in complex biological environments. Chronoamperometry (CA) and cyclic voltammetry (CV) techniques are used to examine the electrocatalytic activity of the fabricated sensor, assessing parameters like pH, electrode resistance, concentration, and scan rate. While fasting, the normal range of insulin ranges from 12 to 150 pM. This sensor detects insulin in the 7 pM to 1000 pM range, with a detection limit of 2.7 pM and a quantification limit of 8.7 pM. Portability is validated using a handheld smartphone-based potentiostat. This developed miniaturized sensor provides a selective, interference-mitigated response for insulin detection in human serum, demonstrating significant advancements in flexible electrochemical sensor technology with crucial implications for diabetes care.

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.