Interface-constrained catalytic hairpin assembly permits highly sensitive SERS signaling of miRNA.

The rapid and precise monitoring of peripheral blood miRNA levels holds paramount importance for disease diagnosis and treatment monitoring. In this study, we propose an innovative research strategy that combines the catalytic hairpin assembly reaction with SERS signal congregation and enhancement. This combination can significantly enhance the stability of SERS detection, enabling stable and efficient detection of miRNA. Specifically, our paper-based SERS detection platform incorporates a streptavidin-modified substrate, biotin-labeled catalytic hairpin assembly reaction probes, 4-ATP, and primer-co-modified gold nanoparticles. In the presence of miRNA, the 4-ATP and primer-co-modified gold nanoparticles can specifically recognize the miRNA and interact with the biotin-labeled CHA probes to initiate an interfacial catalytic hairpin assembly reaction. This enzyme-free high-efficiency catalytic process can accumulate a large amount of biotin on the gold nanoparticles, which then bind to the streptavidin on the substrate with the assistance of the driving liquid, forming red gold nanoparticle stripes. These provide a multitude of hotspots for SERS, enabling enhanced signal detection. This innovative design achieves a low detection limit of 3.47 fM while maintaining excellent stability and repeatability. This conceptually innovative detection platform offers new technological possibilities and solutions for clinical miRNA detection.

Controlled In Situ Self-Assembly of Biotinylated Trans-Cyclooctene Nanoparticles for Orthogonal Dual-Pretargeted Near-Infrared Fluorescence and Magnetic Resonance Imaging.

A pretargeted strategy that decouples targeting vectors from radionuclides has shown promise for nuclear imaging and/or therapy in vivo. However, the current pretargeted approach relies on the use of antibodies or nanoparticles as the targeting vectors, which may be compromised by poor tissue penetration and limited accumulation of targeting vectors in the tumor tissues. Herein, we present an orthogonal dual-pretargeted approach by combining stimuli-triggered in situ self-assembly strategy with fast inverse electron demand Diels-Alder (IEDDA) reaction and strong biotin-streptavidin (SA) interaction for near-infrared fluorescence (NIR FL) and magnetic resonance (MR) imaging of tumors. This approach uses a small-molecule probe (P-Cy-TCO&Bio) containing both biotin and trans-cyclooctene (TCO) as a tumor-targeting vector. P-Cy-TCO&Bio can efficiently penetrate subcutaneous HeLa tumors through biotin-assisted targeted delivery and undergo in situ self-assembly to form biotinylated TCO-bearing nanoparticles (Cy-TCO&Bio NPs) on tumor cell membranes. Cy-TCO&Bio NPs exhibited an "off-on" NIR FL and retained in the tumors, offering a high density of TCO and biotin groups for the concurrent capture of Gd-chelate-labeled tetrazine (Tz-Gd) and IR780-labeled SA (SA-780) via the orthogonal IEDDA reaction and SA-biotin interaction. Moreover, Cy-TCO&Bio NPs offered multiple-valent binding modes toward SA, which additionally regulated the cross-linking of Cy-Gd&Bio NPs into microparticles (Cy-Gd&Bio/SA MPs). This process could significantly (1) increase r1 relaxivity and (2) enhance the accumulation of Tz-Gd and SA-780 in the tumors, resulting in strong NIR FL, bright MR contrast, and an extended time window for the clear and precise imaging of HeLa tumors.

Combining the Benefits of Biotin-Streptavidin Aptamer Immobilization with the Versatility of Ni-NTA Regeneration Strategies for SPR.

The high affinity of the biotin-streptavidin interaction has made this non-covalent coupling an indispensable strategy for the immobilization and enrichment of biomolecular affinity reagents. However, the irreversible nature of the biotin-streptavidin bond renders surfaces functionalized using this strategy permanently modified and not amenable to regeneration strategies that could increase assay reusability and throughput. To increase the utility of biotinylated targets, we here introduce a method for reversibly immobilizing biotinylated thrombin-binding aptamers onto a Ni-nitrilotriacetic acid (Ni-NTA) sensor chip using 6xHis-tagged streptavidin as a regenerable capture ligand. This approach enabled the reproducible immobilization of aptamers and measurements of aptamer-protein interaction in a surface plasmon resonance assay. The immobilized aptamer surface was stable during five experiments over two days, despite the reversible attachment of 6xHis-streptavidin to the Ni-NTA surface. In addition, we demonstrate the reproducibility of this immobilization method and the affinity assays performed using it. Finally, we verify the specificity of the biotin tag-streptavidin interaction and assess the efficiency of a straightforward method to regenerate and reuse the surface. The method described here will allow researchers to leverage the versatility and stability of the biotin-streptavidin interaction while increasing throughput and improving assay efficiency.

Self-assembly of protein-DNA hybrids dedicated to an accelerated and self-primed strand displacement amplification for reinforced serum microRNA probing.

BACKGROUND: High-efficiency and highly reliable analysis of microRNAs (miRNAs) in bodily fluids highlights its significance to be extensively utilized as candidates for non-invasive "liquid biopsy" approaches. DNA biosensors based on strand displacement amplification (SDA) methods have been successfully designed to detect miRNAs given the efficiently amplified and recycled of the target sequences. However, the unpredictable DNA framework and heavy reliance on free diffusion or random reactant collisions in existing approaches lead to delayed reaction kinetics and inadequate amplification. Thus, it is crucial to create a modular probe with a controlled structure, high local concentration, and ease of synthesis. RESULTS: Inspired by the natural spatial-confinement effect based on a well-known streptavidin-biotin interaction, we constructed a protein-DNA hybrid, named protein-scaffolded DNA tetrads (PDT), which consists of four biotinylated Y-shaped DNA (Y-DNA) surrounding a streptavidin protein center via a streptavidin-biotin bridge. The streptavidin-biotin recognition system significantly increased the local concentration and intermolecular distance of the probes to achieve enhanced reaction efficiency and kinetics. The PDT-based assay starts with the target miRNA binding to Y-DNA, which disassembles the Y-DNA structures into three types of hairpin-shaped structures via self-primed strand displacement amplification (SPSDA) and generates remarkable fluorescence signal that is proportional to the miRNA concentration. Results demonstrated that PDT enabled a more efficient detection of miRNA-21 with a sensitivity of 1 fM. Moreover, it was proven reliable for the detection of clinical serum samples, suggesting great potential for advancing the development of rapid and robust signal amplification technologies for early diagnosis. SIGNIFICANCE: This simple yet robust system contributes to the early diagnosis of miR-21 with satisfactory sensitivity and specificity, and display a significantly improved nuclease resistance owing to their unique structure. The results suggested that the strategy is expected to provide a promising potential platform for tumor diagnosis, prognosis and therapy.

A Facile Method to Append a Bio-ID Tag to Endogenous Mutant Kras Alleles.

KRAS mutations occur in approximately ~50% of colorectal cancers (CRCs) and are associated with poor prognosis and resistance to therapy. While these most common mutations found at amino acids G12, G13, Q61, and A146 have long been considered oncogenic drivers of CRC, emerging clinical data suggest that each mutation may possess different biological functions, resulting in varying consequences in oncogenesis. Currently, the mechanistic underpinnings associated with each allelic variation remain unclear. Elucidating the unique effectors of each KRAS mutant could both increase the understanding of KRAS biology and provide a basis for allele-specific therapeutic opportunities. Biotinylation identification (BioID) is a method to label and identify proteins located in proximity of a protein of interest. These proteins are captured through the strong interaction between the biotin label and streptavidin bead and subsequently identified by mass spectrometry. Here, we developed a protocol using CRISPR-mediated gene editing to generate endogenous BioID2-tagged KrasG12D and KrasG12V isogenic murine colon epithelial cell lines to identify unique protein proximity partners by BioID.

Surface-Activated Ti3C2Tx Adsorption of Acetylene Black Coupled with Polyaniline as a Signal Tag for the Detection of the ESAT-6 Antigen of Mycobacterium tuberculosis.

Early secretory antigenic target-6 (ESAT-6) is regarded as the most immunogenic protein produced by Mycobacterium tuberculosis, whose detection is of great clinical significance for tuberculosis diagnosis. However, the detection of the ESAT-6 antigen has been hampered by the expensive cost and complex experimental procedures, resulting in low sensitivity. Herein, we developed a titanium carbide (Ti3C2Tx)-based aptasensor for ESAT-6 detection utilizing a triple-signal amplification strategy. First, acetylene black (AB) was immobilized on Ti3C2Tx through a cross-linking reaction to form the Ti3C2Tx-AB-PAn nanocomposite. Meanwhile, AB served as a conductive bridge, and Ti3C2Tx can synergistically promote the electron transfer of PAn. Ti3C2Tx-AB-PAn exhibited outstanding conductivity, high electrochemical signals, and abundant sites for the loading of ESAT-6 binding aptamer II (EBA II) to form a novel signal tag. Second, N-CNTs were adsorbed on NiMn layered double hydride (NiMn LDH) nanoflowers to obtain NiMn LDH/N-CNTs, exhibiting excellent conductivity and preeminent stability to be used as electrode modification materials. Third, the biotinylated EBA (EBA I) was immobilized onto a streptavidin-coated sensing interface, forming an amplification platform for further signal enhancement. More importantly, as a result of the synergistic effect of the triple-signal amplification platform, the aptasensor exhibited a wide detection linear range from 10 fg mL-1 to 100 ng mL-1 and a detection limit of 4.07 fg mL-1 for ESAT-6. We envision that our aptasensor provides a way for the detection of ESAT-6 to assist in the diagnosis of tuberculosis.

Redox-Responsive "Catch and Release" Cryogels: A Versatile Platform for Capture and Release of Proteins and Cells.

Macroporous cryogels are attractive scaffolds for biomedical applications, such as biomolecular immobilization, diagnostic sensing, and tissue engineering. In this study, thiol-reactive redox-responsive cryogels with a porous structure are prepared using photopolymerization of a pyridyl disulfide poly(ethylene glycol) methacrylate (PDS-PEG-MA) monomer. Reactive cryogels are produced using PDS-PEG-MA and hydrophilic poly(ethylene glycol) methyl ether methacrylate (PEGMEMA) monomers, along with a PEG-based cross-linker and photoinitiator. Functionalization of cryogels using a fluorescent dye via the disulfide-thiol exchange reactions is demonstrated, followed by release under reducing conditions. For ligand-mediated protein immobilization, first, thiol-containing biotin or mannose is conjugated onto the cryogels. Subsequently, fluorescent dye-labeled proteins streptavidin and concanavalin A (ConA) are immobilized via ligand-mediated conjugation. Furthermore, we demonstrate that the mannose-decorated cryogel could capture ConA selectively from a mixture of lectins. The efficiency of protein immobilization could be easily tuned by changing the ratio of the thiol-sensitive moiety in the scaffold. Finally, an integrin-binding cell adhesive peptide is attached to cryogels to achieve successful attachment, and the on-demand detachment of integrin-receptor-rich fibroblast cells is demonstrated. Redox-responsive cryogels can serve as potential scaffolds for a variety of biomedical applications because of their facile synthesis and modification.

A strategy to enhance the water solubility of luminescent ß-diketonate-Europium(III) complexes for time-gated luminescence bioassays.

Luminescent ß-diketonate-europium(III) complexes have been found a wide range of applications in time-gated luminescence (TGL) bioassays, but their poor water solubility is a main problem that limits their effective uses. In this work we propose a simple and general strategy to enhance the water solubility of luminescent ß-diketonate-europium(III) complexes that permits facile synthesis and purification. By introducing the fluorinated carboxylic acid group into the structures of ß-diketone ligands, two highly water-soluble and luminescent Eu3+ complexes, PBBHD-Eu3+ and CPBBHD-Eu3+, were designed and synthesized. An excellent solubility exceeding 20 mg/mL for PBBHD-Eu3+ was found in a pure aqueous buffer, while it also displayed strong and long-lived luminescence (quantum yield φ = 26%, lifetime τ = 0.49 ms). After the carboxyl groups of PBBHD-Eu3+ were activated, the PBBHD-Eu3+-labeled streptavidin-bovine serum albumin (SA-BSA) conjugate was prepared, and successfully used for the immunoassay of human α-fetoprotein (AFP) and the imaging of an environmental pathogen Giardia lamblia under TGL mode, which demonstrated the practicability of PBBHD-Eu3+ for highly sensitive TGL bioassays. The carboxyl groups of PBBHD can also be easily derivatized with other reactive chemical groups, which enables PBBHD-Eu3+ to meet diverse requirements of biolabeling technique, to provide new opportunities for developing functional europium(III) complex biolabels serving for TGL bioassays.

Development and validation of streptavidin-biotin-based double antibody sandwich ELISA for ricin diagnosis.

BACKGROUND: Ricin is a potential biowarfare agent. It is a phytotoxin isolated from castor seeds. At present there is no antidote available for ricin poisoning, patients only get supportive treatment based on their symptoms. This highlights the importance of early detection to avoid severity of accidents and reduce the risk factor. Considering this, our study aimed to develop a highly sensitive and specific sandwich ELISA for the detection of ricin. METHODS: Ricin was purified from castor seeds. Anti-ricin polyclonal and monoclonal antibodies were generated from rabbit antisera and hybridoma cell (1H6F1) supernatant using a protein A/G column. Antibody titer estimation was done using Indirect ELISA. A streptavidin-biotin-based sandwich ELISA was developed and the limit of detection (LOD), linear range, intra and inter-assay coefficient of variation (CV), and cross-reactivity with other similar toxins were determined. Interference of human plasma samples spiked with ricin was also checked. RESULTS: The LOD of the ELISA was found to be 0.45 ng/ml, with a linear range of 0.90-62 ng/ml, intra and inter-assay CV ranged from 3.34 % to 5 % and 5.17 % to 10.80 % respectively. The assay was not cross-reactive with other similar ribosome-inactivating protein (RIP) toxins. Ricin was detected in spiked plasma samples. CONCLUSION: The developed assay is highly sensitive and specific for detecting ricin and is not cross-reactive with other similar types of toxins. The assay can detect ricin in spiked plasma samples, so it has the potential to be used for the analysis of clinical samples after ricin poisoning.

Modified streptavidin-biotin based lateral flow test strip for rapid detection of SARS-CoV-2 S1 antigen in saliva samples.

Compared to other infectious diseases, for which LFT development can take years, SARS-CoV-2 antigen LFTS were developed and deployed within months. LFTS for antigen detection were adopted on an unprecedented scale during the COVID-19 pandemic, but many of them lack the sensitivity especially for samples with low viral load. In our previous work, we developed an enhanced signal strip for detection of SARS CoV-2 SI antigens in saliva. Here we introduce some modification to improve the sensitivity, and specificity, and to lower the cost of the strip, by using biotin streptavidin (BS) system. In the modified BS strip, gold-streptavidin and biotinylated Nanobodies (Nbs) against S1 antigen were externally mixed with the tested samples (saliva or nasopharyngeal swab) before their application on the sample pad of the test strip containing angiotensin converting enzyme (ACE-2), as the capturing probe. The study included 320 individuals, with 180 being positively confirmed by RT-PCR and 140 confirmed negative, as well as, 45 health care workers, who were responsible for screening and handling of surgical cases in General Surgery Department and COVID clinic of TBRI. Our results proved that modified BS strip improved the overall sensitivity and specificity of S1antigen detection in saliva samples (95.21% and 99.29% respectively) compared to our previously developed enhanced LFTS (91.66% and 98.57% respectively). Also, the sensitivity of cases with Ct ≤ 30, Ct ≤ 35, and Ct ≤ 40 using the modified BS strip showed higher values (98.54%, 95.38%, and 88.89% respectively), compared to the corresponding results of our previously developed enhanced LFTS (95.86%, 92.31%, and 82.22% respectively). There were no cross-reactions with either Middle East respiratory syndrome corona virus MERS-CoV or SARS-CoV antigens. Furthermore, we found that the lower viral detection limit (LVD) of BS strip was obviously lower than our previous LVD limit of the enhanced LFTS (0.2 × 104 copies/ml vs. 0.4 × 104 copies/ml, respectively). Our developed BS strip showed that saliva samples gave better results than nasopharyngeal swabs of the same patients. The fact of using smaller amounts of Nbs, and ACE2, as well as the dispensing off of conjugate pad when applying BS strip modifications, justified the expected reduction in the costs of the strip. The implementation of BS strips on saliva samples of 45 health co-workers, who were tested 4 and 6 days after exposure to infection, showed an increase in the sensitivity, starting from the 4th day and reaching its highest level on the 6th day in both high risk and paramedic groups (90.9%, and 80.0%, respectively). This study provides evidence that employment of the modified BS system could increase the sensitivity of the strips, lower their cost, and render them an effective screening tool for early detection of the virus in saliva of suspected Covid-19 patients.

Steric trapping strategy for studying the folding of helical membrane proteins.

Elucidating the folding energy landscape of membrane proteins is essential to the understanding of the proteins' stabilizing forces, folding mechanisms, biogenesis, and quality control. This is not a trivial task because the reversible control of folding is inherently difficult in a lipid bilayer environment. Recently, novel methods have been developed, each of which has a unique strength in investigating specific aspects of membrane protein folding. Among such methods, steric trapping is a versatile strategy allowing a reversible control of membrane protein folding with minimal perturbation of native protein-water and protein-lipid interactions. In a nutshell, steric trapping exploits the coupling of spontaneous denaturation of a doubly biotinylated protein to the simultaneous binding of bulky monovalent streptavidin molecules. This strategy has been evolved to investigate key elements of membrane protein folding such as thermodynamic stability, spontaneous denaturation rates, conformational features of the denatured states, and cooperativity of stabilizing interactions. In this review, we describe the critical methodological advancement, limitation, and outlook of the steric trapping strategy.

Exploring Options for Proximity-Dependent Biotinylation Experiments: Comparative Analysis of Labeling Enzymes and Affinity Purification Resins.

Proximity-dependent biotinylation (PDB) techniques provide information about the molecular neighborhood of a protein of interest, yielding insights into its function and localization. Here, we assessed how different labeling enzymes and streptavidin resins influence PDB results. We compared the high-confidence interactors of the DNA/RNA-binding protein transactive response DNA-binding protein 43 kDa (TDP-43) identified using either miniTurbo (biotin ligase) or APEX2 (peroxidase) enzymes. We also evaluated two commercial affinity resins for purification of biotinylated proteins: conventional streptavidin sepharose versus a new trypsin-resistant streptavidin conjugated to magnetic resin, which significantly reduces the level of contamination by streptavidin peptides following on-bead trypsin digestion. Downstream analyses involved liquid chromatography coupled to mass spectrometry in data-dependent acquisition mode, database searching, and statistical analysis of high-confidence interactors using SAINTexpress. The APEX2-TDP-43 experiment identified more interactors than miniTurbo-TDP-43, although miniTurbo provided greater overlap with previously documented TDP-43 interactors. Purifications on sepharose resin yielded more interactors than magnetic resin in small-scale experiments using a range of magnetic resin volumes. We suggest that resin-specific background protein binding profiles and different lysate-to-resin ratios cumulatively affect the distributions of prey protein abundance in experimental and control samples, which impact statistical confidence scores. Overall, we highlight key experimental variables to consider for the empirical optimization of PDB experiments.

An array of femtoliter wells for sensitive detection of copper using click chemistry.

Sensitive detection of copper ion (Cu2+), which is of great importance for environmental pollution and human health, is crucial. In this study, we present a highly sensitive method for measuring Cu2+ in an array of femtoliter wells. In brief, magnetic beads (MBs) modified with alkyne groups were bound to the azide groups of biotin-PEG3-azide (bio-PEG-N3) via Cu+-catalyzed click chemistry. Cu+ in the click chemistry reaction was generated by reducing Cu2+ with sodium ascorbate. Following the ligation, the surface of the MBs was modified with biotin, which could be labeled with streptavidin-ß-galactosidase (SßG). The MBs complex was then suspended in ß-galactosidase substrate fluorescein-di-ß-d-galactopyranoside (FDG), and loaded into the array of femtoliter wells. The MBs sank into the wells due to gravity, and the resulting fluorescent product, generated from the reaction between SßG on the surface of the MBs and FDG, was confined within the wells. The number of fluorescent wells increased with higher Cu2+ concentrations. The bright-field and fluorescent images of the wells were acquired using an inverted fluorescent microscope. The detection limit of this assay for Cu2+ was 1 nM without signal amplification, which was 103 times lower than that of traditional fluorescence detection assays.

Improving the Performance of Selective Solid-State Nanopore Sensing Using a Polyhistidine-Tagged Monovalent Streptavidin.

Solid-state (SS-) nanopore sensing has gained tremendous attention in recent years, but it has been constrained by its intrinsic lack of selectivity. To address this, we previously established a novel SS-nanopore assay that produces translocation signals only when a target biotinylated nucleic acid fragment binds to monovalent streptavidin (MS), a protein variant with a single high-affinity biotin-binding domain. While this approach has enabled selective quantification of diverse nucleic acid biomarkers, sensitivity enhancements are needed to improve the detection of low-abundance translational targets. Because the translocation dynamics that determine assay efficacy are largely governed by constituent charge characteristics, we here incorporate a polyhistidine-tagged MS (hMS) to alter the component detectability. We investigate the effects of buffer pH, salt concentration, and SS-nanopore diameter on the performance with the alternate reagent, achieve significant improvements in measurement sensitivity and selectivity, and expand the range of device dimensions viable for the assay. We used this improvement to detect as little as 1 nM miRNA spiked into human plasma. Overall, our findings improve the potential for broader applications of SS-nanopores in the quantitative analyses of molecular biomarkers.

Metabolic labeling-mediated visualization, capture, and inactivation of Gram-positive bacteria via biotin-streptavidin interactions.

We introduce a biotinylated D-amino acid probe capable of metabolically incorporating into bacterial PG. Leveraging the robust affinity between biotin and streptavidin, the probe has demonstrated efficacy in imaging, capture, and targeted inactivation of Gram-positive bacteria through synergistic pairings with commercially available streptavidin-modified fluorescent dyes and nanomaterials. The versatility of the probe is underscored by its compatibility with a variety of commercially available streptavidin-modified reagents. This adaptability allows the probe to be applied across diverse scenarios by integrating with these commercial reagents.

A Biotinylated cpFIT-PNA Platform for the Facile Detection of Drug Resistance to Artemisinin in Plasmodium falciparum.

The evolution of drug resistance to many antimalarial drugs in the lethal strain of malaria (Plasmodium falciparum) has been a great concern over the past 50 years. Among these drugs, artemisinin has become less effective for treating malaria. Indeed, several P. falciparum variants have become resistant to this drug, as elucidated by specific mutations in the pfK13 gene. This study presents the development of a diagnostic kit for the detection of a common point mutation in the pfK13 gene of P. falciparum, namely, the C580Y point mutation. FIT-PNAs (forced-intercalation peptide nucleic acid) are DNA mimics that serve as RNA sensors that fluoresce upon hybridization to their complementary RNA. Herein, FIT-PNAs were designed to sense the C580Y single nucleotide polymorphism (SNP) and were conjugated to biotin in order to bind these molecules to streptavidin-coated plates. Initial studies with synthetic RNA were conducted to optimize the sensing system. In addition, cyclopentane-modified PNA monomers (cpPNAs) were introduced to improve FIT-PNA sensing. Lastly, total RNA was isolated from red blood cells infected with P. falciparum (WT strain - NF54-WT or mutant strain - NF54-C580Y). Streptavidin plates loaded with either FIT-PNA or cpFIT-PNA were incubated with the total RNA. A significant difference in fluorescence for mutant vs WT total RNA was found only for the cpFIT-PNA probe. In summary, this study paves the way for a simple diagnostic kit for monitoring artemisinin drug resistance that may be easily adapted to malaria endemic regions.

Drug-Loaded Bacillus Calmette-Guérin Bacteria for Immuno-Chemo Combo Therapy in Bladder Cancer.

Intravesical Bacillus Calmette-Guérin (BCG) is a well-established strategy for managing high-risk nonmuscle-invasive bladder cancer (NMIBC); however, over half of patients still experience disease recurrence or progression. Although the combined intravesical instillation of various chemotherapeutic drugs is implemented in clinical trials to enhance the BCG therapy, the outcome is far from satisfying due to severe irritative effects and treatment intolerance at high doses. Therefore, it is adopted the "biotin-streptavidin strategy" to doxorubicin (DOX)-encapsulated nanoparticles within live BCG bacteria (DOX@BCG) to improve treatment outcomes. Adherence of BCG to the bladder epithelium helps precisely target DOX@BCG to the local tumor cells and simultaneously increases intratumoral transport of therapeutic drugs. DOX@BCG effectively inhibits cancer progression and prolongs the survival of rats/mice with orthotopic bladder cancer owing to synergism between BCG-immunotherapy, DOX-chemotherapy, and DOX-induced immunogenic tumor cell death; furthermore, it exhibits improved tolerance and biosafety, and establishes antitumor immunity in the tumor microenvironment. Therefore, the drug-loaded live BCG bacterial delivery system holds considerable potential for clinical translation in the intravesical treatment of bladder cancer.

Streptavidin-biotin system-mediated immobilization of a bivalent nanobody onto magnetosomes for separation and analysis of 3-phenoxybenzoic acid in urine.

The compound 3-phenoxybenzoic acid (3-PBA) is frequently utilized as a biomarker to detect exposure to various pyrethroids. In this study, a bivalent nanobody (Nb2) specifically targeting 3-PBA was biotinylated and immobilized onto streptavidin (SA)-modified bacterial magnetic nanoparticles (BMPs), resulting in the formation of BMP-SA-Biotin-Nb2 complexes. These complexes demonstrated remarkable stability when exposed to strongly acidic solutions (4 M HCl), methanol (80%), and high ionic strength (1.37 M NaCl). An immunoassay was subsequently developed utilizing BMP-SA-Biotin-Nb2 as the capture agent and 3-PBA-horseradish peroxidase as the detection probe. The immunoassay exhibited an IC50 value (half-maximum signal inhibition concentration) of 1.11 ng mL-1 for 3-PBA. To evaluate the accuracy of the assay, spiked sheep and cow urine samples (ranging from 3.0 to 240 ng mL-1) were analyzed. The quantitative recoveries ranged from 82.5% to 113.1%, which agreed well with the findings obtained using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Overall, the BMP-SA-Biotin-Nb2-based immunoassay holds great promise for rapid monitoring of 3-PBA following acid dissociation.

Intracellular Delivery of Functional Proteins with DNA-Protein Nanogels-Lipids Complex.

Using functional proteins for therapeutic purposes due to their high selectivity and/or catalytic properties can enable the control of various cellular processes; however, the transport of active proteins inside living cells remains a major challenge. In contrast, intracellular delivery of nucleic acids has become a routine method for a number of applications in gene therapy, genome editing, or immunization. Here we report a functionalizable platform constituting of DNA-protein nanogel carriers cross-linked through streptavidin-biotin or streptactin-biotin interactions and demonstrate its applicability for intracellular delivery of active proteins. We show that the nanogels can be loaded with proteins bearing either biotin, streptavidin, or strep-tag, and the resulting functionalized nanogels can be delivered into living cells after complexation with cationic lipid vectors. We use this approach for delivery of alkaline phosphatase enzyme, which is shown to keep its catalytic activity after internalization by mouse melanoma B16 cells, as demonstrated by the DDAO-phosphate assay. The resulting functionalized nanogels have dimensions on the order of 100 nm, contain around 100 enzyme molecules, and are shown to be transfectable at low lipid concentrations (charge ratio R± = 0.75). This ensures the low toxicity of our system, which in combination with high local enzyme concentration (∼100 µM) underlines potential interest of this nanoplatform for biomedical applications.

Development of gold nanocluster complex for the detection of tumor necrosis factor-alpha based on immunoassay.

Tumor necrosis factor-alpha, TNF-α, a cytokine recognized as a key regulator of inflammatory responses, is primarily produced by activated monocytes and macrophages. Measuring TNF-α levels serves as a valuable indicator for tracking several diseases and pathological states. Gold nanotechnology has been identified as a highly effective catalyst with unique properties for measuring inflammatory cytokines. This study aimed to synthesize gold nanoclusters (AuNCs) and the AuNCs-streptavidin system, along with their characterizations and spherical morphology. The detection of TNF-α antigen with AuNCs was determined, and a new immunoassay-based AuNCs analytical platform was studied. In this study, it was demonstrated that the synthesized AuNCs and AuNCs-streptavidin showed a bright-yellow appearance with absorption peaks at A600 and A610 nm, respectively. The approximately spherical shape was observed by TEM analysis. The AuNCs demonstrated a sensitivity limit for the detection of the TNF-α antigen, with a linear dose-dependent detection range of less than 1.25 ng/mL. The products of the band sizes and band intensities were proportional to the amount of TNF-α in the range of ∼80 kDa, ∼55 kDa, and âˆ¼ 25 kDa in western blot analysis. The TNF-α in cell lysate was successfully detected using an immunoassay after the activation of RAW264.7 cells with lipopolysaccharide (LPS). This assay may serve as a viable alternative for TNF-α detection with high speed, sensitivity, and qualities, ensuring its broad applications.