Complex Biological Environments Research Articles

Alginate Formulation for Wound Healing Applications.

Significance: Alginate, sourced from seaweed, holds significant importance in industrial and biomedical domains due to its versatile properties. Its chemical composition, primarily comprising β-D-mannuronic acid and α-L-guluronic acid, governs its physical and biological attributes. This polysaccharide, extracted from brown algae and bacteria, offers diverse compositions impacting key factors such as molecular weight, flexibility, solubility, and stability. Recent Advances: Commercial extraction methods yield soluble sodium alginate essential for various biomedical applications. Extraction processes involve chemical treatments converting insoluble alginic acid salts into soluble forms. While biosynthesis pathways in bacteria and algae share similarities, differences in enzyme utilization and product characteristics are noted. Critical Issues: Despite its widespread applicability, challenges persist regarding alginate's stability, biodegradability, and bioactivity. Further understanding of its interactions in complex biological environments and the optimization of extraction and synthesis processes are imperative. Additionally, concerns regarding immune responses to alginate-based implants necessitate thorough investigation. Future Directions: Future research endeavors aim to enhance alginate's stability and bioactivity, facilitating its broader utilization in regenerative medicine and therapeutic interventions. Novel approaches focusing on tailored hydrogel formations, advanced drug delivery systems, and optimized cellular encapsulation techniques hold promise. Continued exploration of alginate's potential in tissue engineering and wound healing, alongside efforts to address critical issues, will drive advancements in biomedical applications.

Dental Stem Cell Sphere Formation and Potential for Neural Regeneration: A Scoping Review

BackgroundDental stem cells with neurosphere-forming abilities are a promising cell source for the treatment of neural diseases and injuries. This scoping review aimed to systematically map the existing literature on dental sphere formation assays and their characteristics associated with neural regeneration potential. MethodsThe Web of Science, EMBASE, SCOPUS, and PubMed databases were systematically searched for in vitro, animal, and clinical studies and reviews focusing on stem cells isolated from the oral cavity, subsequently cultured as spheres with neural regeneration potential. Data were extracted and evidence was synthesized according to the predetermined variables in the registered protocol. ResultsA total of 35 articles (31 in vitro, 1 combined in vitro and in vivo, and 3 reviews) were included. The predominant method utilized for sphere formation was low-attachment culture. Spheres were characterized using assessment of neural marker expression via confocal microscopy, immunohistochemistry, RT-qPCR, or western blotting. Overall, the synthesized results indicate a lack of in vivo studies investigating the utility of dental neurospheres for neural regeneration, with dental pulp stem cells being the most investigated for their neural regenerative potential. ConclusionDental stem cell spheres demonstrate significant potential for neural regeneration. Several assays and characterizations have been performed to characterized the mechanisms underlying dental sphere formation. Furthermore, in vivo studies are imperative to deduce the neural regenerative potential of stem cells in complex biological environments.

Sensitive detection of K-ras gene by a dual-mode “on-off-on” sensor based on bipyridine ruthenium-MOF and bis-enzymatic cleavage technology

This study developed a dual-mode “on-off-on” sensor based on a bipyridine ruthenium metal–organic framework (Ru-MOF) and dual enzyme cleavage technology for the sensitive detection of the K-ras gene. The sensor combines electrogenerated chemiluminescence (ECL) and fluorescence (FL) detection modes, achieving high sensitivity and specificity in detecting the K-ras gene through catalytic hairpin assembly (CHA) and dual enzyme cleavage reactions. Experimental results showed that the detection limits for the K-ras gene were 0.044 fM (ECL) and 0.16 fM (FL), demonstrating excellent selectivity and stability during detection. Through testing actual samples, the sensor has shown potential for application in complex biological environments. This method offers an efficient and reliable new tool for cancer diagnosis and treatment.

A Nonfouling Electrochemical Biosensor for Protein Analysis in Complex Body Fluids Based on Multifunctional Peptide Conjugated with PEGlyated Phospholipid.

Developing antifouling biosensors capable of performing robustly in complex human body fluids is crucial for biomarker diagnosis and health monitoring. Herein, an antifouling and highly sensitive and stable biosensor was constructed through the self-assembly of the designed conjugates composed of a multifunctional peptide (MP) and PEGylated distearoylphosphatidylethanolamine (DSPE-PEG). The self-assembly capability of the DSPE-PEG-MP was demonstrated clearly through coarse-grained molecular dynamics simulation and transmission electron microscopy, and it can be effectively self-assembled onto the electrode surface modified with gold nanoparticles. The MP was designed to be antifouling and contained a peptide sequence that can specifically bind the target protein Annexin A1 (ANXA1), and the D-type amino acid composition of MP can enhance its resistance to enzymatic hydrolysis. The unique design of MP, in conjugation with the self-assembly capability of the PEGylated phospholipid DSPE-PEG, enabled the biosensor to exhibit excellent antifouling capability and stability in various complex human body fluids. The biosensor was capable of sensitively and selectively quantifying ANXA1 and achieved a limit of detection down to 0.12 pg mL-1. More importantly, the biosensor demonstrated satisfactory accuracy for ANXA1 detection in clinical serum samples, as verified by the enzyme linked immunosorbent assay (ELISA) kits. It is expected that various antifouling biosensors suitable for application in complex biological environments can be constructed by utilizing the strategy of designing similar DSPE-PEG-MP conjugates.

Enhanced Sensitivity of Cell Identification in Complex Environments Using Chirally Inverted L-DNA-Based Logic Devices.

Accurate identification and isolation of target cells are crucial for precision diagnosis and treatment. DNA aptamer-based logic devices provide a distinct advantage in this context, as they can logically analyze multiple cell surface markers with high efficiency. However, the susceptibility of natural DNA (D-DNA) to degradation can compromise the sensitivity and specificity of these devices, potentially leading to false-positive and false-negative results, particularly in complex biological environments. To address this issue, dual- and triple-aptamer-based cell-surface logic devices are designed and developed using mirror-image L-DNA, a chiral molecule of D-DNA with high biostability. These devices allow for simultaneous analysis of multiple cell surface proteins, achieving greater specificity in cell identification and isolation than D-DNA-based logic devices. The L-DNA probes realized 98.7% and 70.5% sensitivities in FBS buffer with dual- and triple-aptamer-based logic devices for target cell identification, while D-DNA probes only showed 27.9% and 0.1%. It is believed that the high stability of L-DNA and the high efficiency of the devices for labeling cell subpopulations will have broad applications in the life sciences, biomedical engineering, and personalized medicine.

Atomically dispersed recognition unit for selective in vivo photoelectrochemical medicine detection

Continuous and long-term therapeutic monitoring of medicine molecules in biological systems will revolutionize healthcare by offering personalized pharmacokinetic reports. However, the extremely complex biological environment brings great challenges for in vivo molecule detection in living organisms. Here we introduce an in vivo photoelectrochemical biosensor following a reverse design strategy with single atoms as molecular recognition units. Atomic dispersion of Cu single atoms on TiO2-x substrate create synergistic anchoring triple-site for efficiently and selectively capturing of dual-carbonyl group and neighboring dual-hydroxyl group of tetracycline molecules. The photoelectrode is encapsulated with antibiofouling layer and implanted into the vein of living mouse to enable long-term in vivo monitoring of tetracycline in real biological environments. It is important to note that our approach was exclusively tested in male mice, and therefore, the findings may not be generalizable to female mice or other species without further research. The rationally designed biological-components-free in vivo biosensor with excellent selectivity, robustness, and stability endows possibility for enabling personalized medicine guidance through real-time feedbacking information and providing direct and authentic medicine molecular analysis.

Micro/Nanorobots for Advanced Light‐Based Biosensing and Imaging

AbstractSensing and imaging of biomolecules are crucial to disease diagnosis, prognosis, and therapy where optical techniques have essential utility. Untethered and remotely controlled micro/nanorobots have shown promising sensing and imaging capabilities, especially in complex biological environments. In this review, how micro/nanorobots are used for optical biosensing and imaging while highlighting the significant developments in the field is discussed. Starting is done by exploring colorimetric biosensing methods enabled by micro/nanorobots. Significant advancements in surface‐enhanced Raman spectroscopy‐integrated micro/nanorobots are reviewed. Further, state‐of‐the‐art optical bio‐imaging applications by micro/nanorobots at in vitro intracellular level are highlighted. Novel in vivo bio‐imaging assisted by optical micro/nanorobot sensors is examined. Furthermore, innovations in micro/nanorobots are assessed where motion augmentation is used as a detection mechanism, with applications in point‐of‐care molecular diagnostics. Finally, the challenges associated with micro/nanorobots‐assisted advanced optical biosensing and imaging while discussing insights about potential research directions for this rapidly progressing field are summarized.

Fungal communities in three root herbs: Insights and implications

The roots of Astragali Radix (AM), Dioscoreae Rhizome (DR), and Codonopsis Radix (CR) are frequently used in classical tonic formulations and dietary supplements. Given the extended exposure of rhizomes to complex biological environments, it is necessary to investigate the fungal composition of their surface. In this study, the fungal communities in the three root herbs were analyzed by DNA metabarcoding, and an extensive comparison of the fungal diversity at each taxonomic level was carried out. Furthermore, we examined the effects of species, collection site, and processing method on the fungal community. In Astragali Radix and Codonopsis Radix samples, Cladosporium was the predominant genus, with relative abundances of 1.98 %-76.81 % and 1.69 %-85.59 %, respectively. Aspergillus (0.08 %–99.92 %) was the prevailing genus in Dioscoreae Rhizome samples. Meanwhile, a total of 12 potential toxigenic fungi were identified, including Aspergillus restrictus, Fusarium oxysporum, and Penicillium citrinum. Moreover, the variations in fungal diversity and community composition from different collection sites and processing approaches were observed. Linear discriminant analysis effect size indicated significant differences in the relative abundance of genera among the three root herbs. Gibberella and Mucor genera were significantly enriched in Astragali Radix samples, while Yarrowia and Cladosporium genera exhibited significant enrichment in Dioscoreae Rhizome and Codonopsis Radix samples, respectively (p≤0.001). This study presents novel insights into the fungal profiles of three root herbs, thereby providing references for their safe utilization and quality improvement.

Distinguishing Protein Corona from Nanoparticle Aggregate Formation in Complex Biological Media Using X-ray Photon Correlation Spectroscopy.

In biological systems, nanoparticles interact with biomolecules, which may undergo protein corona formation that can result in noncontrolled aggregation. Therefore, comprehending the behavior and evolution of nanoparticles in the presence of biological fluids is paramount in nanomedicine. However, traditional lab-based colloid methods characterize diluted suspensions in low-complexity media, which hinders in-depth studies in complex biological environments. Here, we apply X-ray photon correlation spectroscopy (XPCS) to investigate silica nanoparticles (SiO2) in various environments, ranging from low to high complex biological media. Interestingly, SiO2 revealed Brownian motion behavior, irrespective of the complexity of the chosen media. Moreover, the SiO2 surface and media composition were tailored to underline the differences between a corona-free system from protein corona and aggregates formation. Our results highlighted XPCS potential for real-time nanoparticle analysis in biological media, surpassing the limitations of conventional techniques and offering deeper insights into colloidal behavior in complex environments.

A non-enzymatic hydrogen peroxide biosensor based on cerium metal-organic frameworks, hemin, and graphene oxide composite

This study presents the development of a novel non-enzymatic electrochemical biosensor for the real-time detection of hydrogen peroxide (H2O2) based on a composite of cerium metal–organic frameworks (Ce-MOFs), hemin, and graphene oxide (GO). The Ce-MOFs served as an efficient matrix for hemin encapsulation, while GO enhanced the conductivity of the composite. Characterization techniques including scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier-transform infrared (FTIR) spectroscopy, UV–vis spectroscopy, and thermogravimetric analysis (TGA) confirmed the successful integration of hemin into the Ce-MOFs. The Ce-MOFs@hemin/GO-modified sensor demonstrated sensitive H2O2 detection due to the exceptional electrocatalytic activity of Ce-MOFs@hemin and the high conductivity of GO. This biosensor exhibited a linear response to H2O2 concentrations from 0.05 to 10 mM with a limit of detection (LOD) of 9.3 μM. The capability of the biosensor to detect H2O2 released from human prostate carcinoma cells was demonstrated, highlighting its potential for real-time monitoring of cellular oxidative stress in complex biological environments. To further assess its practical applicability, the sensor was tested in human serum samples, yielding promising results with recovery values ranging from 94.50 % to 103.29 %. In addition, the sensor showed excellent selectivity against common interfering compounds due to the outstanding peroxidase-like activity of the composite.

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

AbstractMarine 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.

Controlled Fabrication of Unimolecular Micelles as Versatile Nanoplatform for Multifunctional Applications.

Unimolecular micelles (UMs) are nano-sized structures that are composed of single molecules with precise composition. Compared to self-assembled polymeric micelles, UMs possess ultra-stable property even in complex biological environment. With the development of controllable polymerization and coupling chemistry, the preparation of narrowly monodispersed UMs with precise morphology and size has been realized, which further facilitates their multifunctional applications. After brief introduction, state-of-the-art advances in the synthesis and applications of UMs are discussed with an emphasis on their bioapplications. It is believed that these UMs have great potential in future fabrication of multifunctional nanoplatforms.

The effect of fluence rate and wavelength on the formation of protoporphyrin IX photoproducts

Photodynamic diagnosis and therapy (PDD and PDT) are emerging techniques for diagnosing and treating tumors and malignant diseases. Photoproducts of protoporphyrin IX (PpIX) used in PDD and PDT may be used in the diagnosis and treatment, making a detailed analysis of the photoproduct formation under various treatment and diagnosis conditions important.Spectroscopic and mass spectrometric analysis of photoproduct formation from PpIX dissolved in dimethyl sulfoxide were performed under commonly used irradiation conditions for PDD and PDT, i.e., wavelengths of 405 and 635 nm and fluence rates of 10 and 100 mW/cm2. Irradiation resulted in the formation of hydroxyaldehyde photoproduct (photoprotoporphyrin; Ppp) and formyl photoproduct (product II; Pp II) existing in different quantities with the irradiation wavelength and fluence rate. Ppp was dominant under 635 nm irradiation of PpIX, with a fluorescence peak at 673 nm and a protonated monoisotopic peak at m/z 595.3. PpIX irradiation with 405 nm yielded more Pp II, with a fluorescence peak at 654 nm. A higher photoproduct formation was observed at a low fluence rate for irradiation with 635 nm, while irradiation with 405 nm indicated a higher photoproduct formation at a higher fluence rate.The photoproduct formation with the irradiation conditions can be exploited for dosimetry estimation and may be used as an additional photosensitizer to improve the diagnostics and treatment efficacies of PDD and PDT. Differences in environmental conditions of the present study from that of a biological environment may result in a variation in the photoproduct formation rate and may limit their clinical utilization in PDD and PDT. Thus, further investigation of photoproduct formation rates in more complex biological environments, including in vivo, is necessary. However, the results obtained in this study will serve as a basis for understanding reaction processes in such biological environments.Graphical abstract

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.

Martini 3 OliGo̅mers: A Scalable Approach for Multimers and Fibrils in GROMACS.

Martini 3 is a widely used coarse-grained simulation method for large-scale biomolecular simulations. It can be combined with a Go̅ model to realistically describe higher-order protein structures while allowing the folding and unfolding events. However, as of today, this method has largely been used only for individual monomers. In this article, we describe how the Go̅ model can be implemented within the framework of Martini 3 for a multimer system, taking into account both intramolecular and intermolecular interactions in an oligomeric protein system. We demonstrate the method by showing how it can be applied to both structural stability maintenance and assembly/disassembly of protein oligomers, using aquaporin tetramer, insulin dimer, and amyloid-β fibril as examples. We find that addition of intermolecular Go̅ potentials stabilizes the quaternary structure of proteins. The strength of the Go̅ potentials can be tuned so that the internal fluctuations of proteins match the behavior of atomistic simulation models, however, the results also show that the use of too strong intermolecular Go̅ potentials weakens the chemical specificity of oligomerization. The Martini-Go̅ model presented here enables the use of Go̅ potentials in oligomeric molecular systems in a computationally efficient and parallelizable manner, especially in the case of homopolymers, where the number of identical protein monomers is high. This paves the way for coarse-grained simulations of large protein complexes, such as viral protein capsids and prion fibrils, in complex biological environments.

Redox-Activated Substrates for Enhancing Activatable Cyclopropene Bioorthogonal Reactions.

Bioorthogonal chemistry has become a mainstay in chemical biology and is making inroads in the clinic with recent advances in protein targeting and drug release. Since the field's beginning, a major focus has been on designing bioorthogonal reagents with good selectivity, reactivity, and stability in complex biological environments. More recently, chemists have imbued reagents with new functionalities like click-and-release or light/enzyme-controllable reactivity. We have previously developed a controllable cyclopropene-based bioorthogonal ligation, which has excellent stability in physiological conditions and can be triggered to react with tetrazines by exposure to enzymes, biologically significant small molecules, or light spanning the visual spectrum. Here, to improve reactivity and gain a better understanding of this system, we screened diene reaction partners for the cyclopropene. We found that a cyclopropene-quinone pair is 26 times faster than reactions with 1,2,4,5-tetrazines. Additionally, we showed that the reaction of the cyclopropene-quinone pair can be activated by two orthogonal mechanisms, caging group removal on the cyclopropene and oxidation/reduction of the quinone. Finally, we demonstrated that this caged cyclopropene-quinone can be used as a bioorthogonal imaging tool to label the membranes of fixed, cultured cells.

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.

Photoinduced Electron-Transfer-Mediated Differential Recognition of Proteins on Plasmonic Surfaces.

Photoinduced enhanced Raman spectroscopy (PIERS) has emerged as an efficient technique for enhancing the vibrational modes of analyte molecules adsorbed on a plasmonic nanoparticle-semiconductor hybrid material through chemical enhancement governed by electron transfer from the semiconductor to the plasmonic nanoparticles under an additional ultraviolet (UV) preirradiation step. The increase in chemical enhancement is imperative in analyzing and detecting pharmaceutically important moieties, such as amino acids and proteins, with a low Raman scattering cross section, even in complex biological environments. Herein, we demonstrate that UV preirradiation induced the creation of additional oxygen vacancies by introducing a low concentration (≈1%) of Ni as a dopant in the 2D platelike morphology of the BiOCl semiconductor; i.e., defect states in the semiconductor can induce charge transfer from the semiconductor to the plasmonic nanoparticles. This phenomenon facilitates electron transfer to the adsorbed analyte on the plasmonic surface. Additionally, we have shown the usefulness of this method in protein immobilization on the substrate surface, followed by the identification of a specific protein in the mixture of proteins. Proteins containing cysteine residues capture these electrons to form a surface-bound thiol group via a transient disulfide electron adduct radical. This allows differential binding of the protein molecules to the semiconductor plasmonic hybrid depending on the concentration of surface cysteine residues in proteins. Through PIERS and principal component analysis, we demonstrate the possibility of probing and distinguishing biomolecules based on their surface composition and secondary structure components even in their mixtures, thus paving the way for efficient analysis of complex biological systems.

Mirror-Image DNA Nanobox for Enhancing Environment Resistance of Nucleic Acid Probes.

Degradation and interference of the nucleic acid probes in complex biological environments like cytoplasm or body fluid can cause obvious false-positive signals and inefficient bioregulation in biosensing and biomedicine. To solve this problem, here, we proposed a universal strategy, termed L-DNA assembly mirror-image box-based environment resistance (L-AMBER), to protect nucleic acid probes from degradation and maintain their responsive activity in complex biological environments. Strand displacement reaction (SDR), aptamer, or DNAzyme-based D-DNA probes were encapsulated into an L-DNA box by using an L-D-L block DNA carrier strand to construct different kinds of L-AMBER probes. We proved that the L-DNA box could effectively protect the encapsulated D-DNA probes by shielding the interference of complex biological environments and only allowing small target molecules to enter for recognition. Compared with the D-AMBER probes, the L-AMBER probes can realize DNase I-assisted amplification detection of biological samples, low false-positive bioimaging, and highly efficient miRNA silence in living cells. Therefore, L-AMBER provided a universal and effective strategy for enhancing the resistance to environmental interference of nucleic acid probes in biosensing and biomedicine applications.