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Polymer-encapsulated dye nanoparticle sensors are a valuable approach to achieving in situ analyte measurements with luminescence; however, typical emulsion-based nanosensors are poorly suited for large-scale biological samples due to limitations of synthesis scalability and stability. Branched polyethylenimine (PEI) is a versatile polymer scaffold ideal for constructing nanoparticles with various covalently conjugated moieties due to their high density of reactive primary amines, high water solubility, and biological stability. In this work, we used branched polyethylenimine as a scaffold-based approach for making a stable and scalable ratiometric oxygen sensor. Pt (II) tetracarboxyporphine was used as an oxygen-sensing dye and coumarin 343 as a reference dye, all covalently linked to the PEI scaffold producing a product that could withstand sterilization procedures and easily be scaled. To minimize toxicity from the PEI scaffold, we conjugated it with 2000 MW PEG. The applicability of the sensors was demonstrated in a 200 mL Saccharomyces cerevisiae yeast culture, using orthogonal luminescent and electrochemical oxygen measurements to validate sensor response and measure the metabolic activity of the yeast in our culture. This approach was able to match the sensitivity of our electrochemical measurements while improving upon drawbacks of other luminescent methods of oxygen detection, demonstrating effective monitoring for at least 20 h. Our scaffold-based approach is a modular and easily translatable technology that could be useful in various biotechnological applications. [ABSTRACT FROM AUTHOR]

Enterotoxigenic Escherichia coli (ETEC) strains, which produce the heat‐stable enterotoxin (ST) either alone or in combination with the heat‐labile enterotoxin, contribute to the bulk of the burden of child diarrheal disease in resource‐limited countries and are associated with mortality. Developing an effective vaccine targeting ST presents challenges due to its potent enterotoxicity, non‐immunogenicity, and the risk of autoimmune reaction stemming from its structural similarity to the human endogenous ligands, guanylin, and uroguanylin. This study aimed to assess a novel synthetic vaccine carrier platform employing a single chemical coupling step for making human ST (STh) immunogenic. Specifically, the method involved cross‐linking STh to an 8‐arm N‐hydroxysuccinimide (NHS) ester‐activated PEG cross‐linker. A conjugate of STh with 8‐arm structure was prepared, and its formation was confirmed through immunoblotting analysis. The impact of conjugation on STh epitopes was assessed using ELISAs with polyclonal and monoclonal antibodies targeting various epitopes of STh. Immunization of mice with the conjugate induced the production of anti‐STh antibodies, exhibiting neutralizing activity against STh. [ABSTRACT FROM AUTHOR]

Biocatalysts for fluorination are rare, and thus of great interest for artificial enzyme design. Biohybrid catalysts including Cu-based DNAzymes and dinucleotide catalysts can catalyse enantioselective electrophilic fluorination of β-ketoesters. Here we report the investigation of Cu-based artificial metalloenzymes as catalysts for electrophilic fluorination reactions. A library of artificial copper proteins was prepared by bioconjugation of bidentate and tridentate nitrogen ligands to cysteine variants of the Sterol Carrier Protein 2 L (SCP-2 L) and subsequent addition of Cu(II) salts. The resulting copper proteins were screened for activity for the fluorination of β-ketoesters using Selectfluor. Under aqueous acidic conditions it was observed that the designed catalysts did not outcompete the uncatalysed background reaction. This work highlights that careful consideration of substrate reactivity and background reactions is needed when considering potential reactions for artificial metalloenzyme catalysis. Artificial SCP-2 L variants were engineered with nitrogen-based copper-binding ligands via cysteine bioconjugation to serve as Lewis acid catalysts for the fluorination of β-ketoesters. The artificial metalloenzymes showed no increase in yield over the background reaction which is promoted in mildly acidic buffers compared to other solvents. [Display omitted] • Twenty-one modified SCP-2 L proteins containing nitrogen-ligands were prepared. • Successful Cu-binding was observed. • Cu-SCP-2 L ArMs do not outcompete the background reaction for the fluorination of β-ketoesters under aqueous conditions. [ABSTRACT FROM AUTHOR]

Due to the increasing crop losses caused by common and newly emerging phytopathogens, there is a pressing need for the development of rapid and reliable methods for phytopathogen detection and analysis. Leveraging advancements in biochemical engineering technologies and nanomaterial sciences, researchers have put considerable efforts on utilizing biofunctionalized magnetic micro- and nanoparticles (MPs) to develop rapid and reliable systems for phytopathogen detection. MPs facilitate the rapid, high-throughput analysis and in-field applications, while the biomacromolecules, which play key roles in the biorecognitions, interactions and signal amplification, determine the specificity, sensitivity, reliability, and portability of pathogen detection systems. The integration of MPs and biomacromolecules provides dimensionality- and composition-dependent properties, representing a novel approach to develop phytopathogen detection systems. In this review, we summarize and discuss the general properties, synthesis and characterization of MPs, and focus on biomacromolecule-functionalized MPs as well as their representative applications for phytopathogen detection and analysis reported over the past decade. Extensively studied bioreceptors, such as antibodies, phages and phage proteins, nucleic acids, and glycans that are involved in the recognitions and interactions, are covered and discussed. Additionally, the integration of MPs-based detection system with portable microfluidic devices to facilitate their in-field applications is also discussed. Overall, this review focuses on biomacromolecule-functionalized MPs and their applications for phytopathogen detection, aiming to highlight their potential in developing advanced biosensing systems for effective plant protection. Particularly attractive: This paper provides a comprehensive review of magnetic particles (MPs) functionalized with biomacromolecules for pathogen detections purposes, with a particular focus on their representative applications in phytopathogen detection and analysis. [Display omitted] • The properties, synthesis and characterization of magnetic particles (MPs) are introduced. • Biomacromolecules functionalized MPs used for phytopathogen detection are reviewed. • The biorecognitions mediated by proteins, nucleic acids and glycans are presented. • The integration of biofunctionalized MPs with portable devices is discussed. [ABSTRACT FROM AUTHOR]

Klebsiella pneumoniae is a leading cause of hospital-acquired infections as well as the leading cause of neonatal sepsis worldwide. Further, increasing antibiotic resistance in this pathogen makes K. pneumoniae troublesome to treat. Despite its clinical importance, there is not yet an approved K. pneumoniae vaccine available. Here we tested antibody durability and long-term functionality of two previously reported bioconjugate vaccines targeting the K. pneumoniae capsular type K2 and O-antigen type O1v1. We demonstrate that both antibodies are durable in mice for up to six months with significant IgG titers. However, only the K2 antibodies exhibit functionality out to six months as evidenced by serum bactericidal activity and survival in a murine bacteremia challenge model. These results are another promising step towards demonstrating the clinical capacity of bioconjugate vaccines and their induction of durable antibody responses. [ABSTRACT FROM AUTHOR]

The functionalization of gold nanoparticle (AuNP) is the key procedure for the biochemical and biomedical application. The conventional salt-aging method requires the stepwise additions of NaCl and excessive thiolated DNA, mainly due to the poor tolerance of the DNA/AuNP mixture toward NaCl. Herein, we found that NaF is capable of improving the stability for the modification of AuNP with different bases of DNA sequences (poly A/T/C/G), and allows for adding up with a high concentration of 200 mM at one time, which greatly reduces the total modification time to 0.5–1 h. Intriguingly, the introduction of NaBr effectively increases the DNA loading capacity. Besides the advantages of NaF and NaBr , the modification performance is improved via the introduction of the oligo A/T spacer for the G -rich DNA sequences. Furthermore, this method shows the superiority to another two methods (pH 3-based and salt-aging) in terms of the loading capacity or sequence components. [Display omitted] [ABSTRACT FROM AUTHOR]

This study focuses on the surface functionalization of citrate-stabilized gold nanoparticles (AuNPs) with disease-specific aptamers to enhance the detection of multifactorial disease (MD) biomarkers. MDs, characterized by complex pathophysiology involving multiple genetic and environmental factors, present significant diagnostic challenges. Aptamers, which are short, single-stranded oligonucleotides with high specificity and affinity for target molecules, have emerged as promising tools for biomarker detection. By utilizing response surface methodology (RSM) and face-centered central composite design (FCCCD), this research systematically optimized the bioconjugation process of AuNPs with different aptamer sequences, focusing on parameters such as AuNP size and aptamer concentration. The developed protocol in this study demonstrated that aptamer-functionalized AuNPs can be optimized for high yield, bioconjugation efficiency, stability, and surface coverage, making them suitable for diagnostic applications, particularly in surface-enhanced Raman spectroscopy (SERS). The findings provide a foundation for the development of customizable nanoprobes that can be adapted for the detection of various biomarkers associated with MDs, potentially improving early diagnosis and therapeutic outcomes.

In histology, immunostaining of biological samples is a gold standard for studying cellular processes, such as the expression of cell surface markers or the cellular uptake of proteins and drug molecules. Immuno-gold labeling is a commonly used technique to achieve nanometer spatial resolution, but simultaneous visualization of multiple antigens in parallel is an unresolved challenge. Herein, we demonstrate a DNA nanotechnology-based approach to label antigens in transmission electron microscopy images of tissue sections with high contrast patterns. For this, we attached gold nanoparticles to designated binding positions on DNA origami structures that act as visual "barcodes." These barcodes are then hybridized to complementary strands of DNA-modified antibodies that are bound to their respective antigens on ultrathin tissue resin sections. As a proof of concept, we demonstrate several types of barcodes and two different antibody labeling techniques that will expand the multiplexing abilities of immunostaining in a highly modular way.

RNA ligands of retinoic acid-inducible gene I (RIG-I) are a promising class of oligonucleotide therapeutics with broad potential as antiviral agents, vaccine adjuvants, and cancer immunotherapies. However, their translation has been limited by major drug delivery barriers, including poor cellular uptake, nuclease degradation, and an inability to access the cytosol where RIG-I is localized. Here this challenge is addressed by engineering nanoparticles that harness covalent conjugation of 5'-triphospate RNA (3pRNA) to endosome-destabilizing polymers. Compared to 3pRNA loaded into analogous nanoparticles via electrostatic interactions, it is found that covalent conjugation of 3pRNA improves loading efficiency, enhances immunostimulatory activity, protects against nuclease degradation, and improves serum stability. Additionally, it is found that 3pRNA could be conjugated via either a disulfide or thioether linkage, but that the latter is only permissible if conjugated distal to the 5'-triphosphate group. Finally, administration of 3pRNA-polymer conjugates to mice significantly increases type-I interferon levels relative to analogous carriers that use electrostatic 3pRNA loading. Collectively, these studies have yielded a next-generation polymeric carrier for in vivo delivery of 3pRNA, while also elucidating new chemical design principles for covalent conjugation of 3pRNA with potential to inform the further development of therapeutics and delivery technologies for pharmacological activation of RIG-I., (© 2024 The Authors. Advanced Healthcare Materials published by Wiley‐VCH GmbH.)

Antibody-drug conjugates (ADCs) are a growing class of chemotherapeutic agents that have yielded striking clinical successes. However, the efficacy of ADCs often suffers from issues associated with tumor heterogeneity and resistance. To overcome these problems, a new generation of ADCs comprising a single monoclonal antibody with multiple different payloads attached, termed multi-payload ADCs, have been developed. Here we deploy multiple orthogonal site-specific protein modification strategies to generate highly homogeneous multi-functionalised antibody conjugates comprising up to four different functionalities installed at four unique sites on the antibody. This work, which includes the use of a site-specific cyclopropenone (CPO)-based reagent, represents the first example of a homogeneous multi-payload ADC with a payload count greater than two, and thereby facilitates the development of the next generation of ADCs., (© 2024 The Author(s). Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

The devastating impact of malaria includes significant mortality and illness worldwide. Increasing resistance of the causative parasite, Plasmodium, to existing antimalarial drugs underscores a need for additional compounds with distinct modes of action in the therapeutic development pipeline. Here we showcase peptide-drug conjugates (PDCs) as an attractive compound class, in which therapeutic or lead antimalarials are chemically conjugated to cell-penetrating peptides. This approach aims to enhance selective uptake into Plasmodium-infected red blood cells and impart additional cytotoxic actions on the intraerythrocytic parasite, thereby enabling targeted drug delivery and dual modes of action. We describe the development of PDCs featuring four compounds with antimalarial activity-primaquine, artesunate, tafenoquine and methotrexate-conjugated to three cell-penetrating peptide scaffolds with varied antiplasmodial activity, including active and inactive analogues of platelet factor 4 derived internalization peptide (PDIP), and a cyclic polyarginine peptide. Development of this diverse set of PDCs featured distinct and adaptable conjugation strategies, to produce conjugates with in vitro antiplasmodial activities ranging from low nanomolar to low micromolar potencies according to the drug cargo and bioactivity of the partner peptide. Overall, this study establishes a strategic and methodological framework for the further development of dual mode of action peptide-drug antimalarial therapeutics., (© 2024 Wiley-VCH GmbH.)

Cancer patients suffer greatly from the severe off-target side effects of small molecule drugs, chemotherapy, and radiotherapy─therapies that offer little protection following remission. Engineered immunotherapies─including cytokines, immune checkpoint blockade, monoclonal antibodies, and CAR-T cells─provide better targeting and future tumor growth prevention. Still, issues such as ineffective activation, immunogenicity, and off-target effects remain primary concerns. "Prodrug" therapies─classified as therapies administered as inactive and then selectively activated to control the time and area of release─hold significant promise in overcoming these concerns. Bioconjugation techniques (e.g., natural linker conjugation, bioorthogonal reactions, and noncanonical amino acid incorporation) enable the rapid and homogeneous synthesis of prodrugs and offer selective loading of immunotherapeutic agents to carrier molecules and protecting groups to prevent off-target effects after administration. Several prodrug activation mechanisms have been highlighted for cancer therapeutics, including endogenous activation by hypoxic or acidic conditions common in tumors, exogenous activation by targeted bioorthogonal cleavage, or stimuli-responsive light activation, and dual-stimuli activation, which adds specificity by combining these mechanisms. This review will explore modern prodrug conjugation and activation options, focusing on how these strategies can enhance immunotherapy responses and improve patient outcomes. We will also discuss the implications of computational methodology for therapy design and recommend procedures to determine how and where to conjugate carrier systems and "prodrug" groups onto therapeutic agents to enhance the safety and control of these delivery platforms.