Colorimetric aptasensor for Listeria monocytogenes detection using dual functional Fe3O4@MIL-100(Fe) with magnetic separation and oxidase-like activities in food samples.

A novel colorimetric aptasensor assay based on the excellent magnetic responsiveness and oxidase-like activity of Fe3O4@MIL-100(Fe) was developed. Fe3O4@MIL-100(Fe) absorbed with aptamer and blocked by BSA served as capture probe for selective isolation and enrichment of Listeria monocytogenes one of the most common and dangerous foodborne pathogenic bacteria. The aptamer absorbed on Fe3O4@MIL-100(Fe) was further used as signal probe that specifically binds with target bacteria conjugation of capture probe for colorimetric detection of Listeria monocytogenes, taking advantages of its oxidase-like activity. The linear range of the detection of Listeria monocytogenes was from 102 to 107 CFU mL-1, with the limit of detection as low as 14 CFU mL-1. The approach also showed good feasibility for detection of Listeria monocytogenes in milk and meat samples. The spiked recoveries were in the range 81-114% with relative standard deviations ranging from 1.28 to 5.19%. Thus, this work provides an efficient, convenient, and practical tool for selective isolation and colorimetric detection of Listeria monocytogenes in food.

Controllable Construction of Aptamer-Modified Fe3O4@SiO2-Au Core-Shell-Satellite Nanocomposites with Surface-Enhanced Raman Scattering and Photothermal Properties and Their Effective Capture, Detection, and Elimination of Staphylococcus aureus.

The early monitoring and inactivation of bacteria are of crucial importance in preventing the further spread of foodborne pathogens. Staphylococcus aureus (S. aureus), a prototypical foodborne pathogen, is widely present in the natural environment and has the capability to trigger a range of diseases at low concentrations. In this work, we designed Fe3O4@SiO2-Au core-shell-satellite nanocomposites (NCs) modified with aptamer for efficient capture, high-sensitivity surface-enhanced Raman scattering (SERS) detection, and photothermal therapy (PTT) against S. aureus. Fe3O4@SiO2-Au NCs with tunable Au nanocrystal nanogaps were prepared. By combining the finite-difference time-domain (FDTD) method and experimental results, we studied the electric field distribution of Fe3O4@SiO2-Au under different Au nanogaps and ultimately obtained the optimal SERS substrate FSA-60. The modification of aptamer on the surfaces of FSA-60 could be used for the specific capture and selective detection of S. aureus, achieving a detection limit of as low as 50 cfu/mL. Furthermore, Apt-FSA-60 possessed excellent photothermal properties, demonstrating the strong photothermal killing ability against S. aureus. Therefore, Apt-FSA-60 is a promising high-sensitivity SERS substrate and efficient photothermal agent and is expected to be widely applied and promoted in future disease prevention and treatment.

A multifunctional N-GO/PtCo nanocomposite bridged carbon fiber interface for the electrochemical aptasensing of CA15-3 oncomarker.

The development of integrated analytical devices is crucial for advancing next-generation point-of-care platforms. Herein, we describe a facile synthesis of a strongly catalytic and durable Nitrogen-doped graphene oxide decorated platinum cobalt (NGO-PtCo) nanocomposite that is conjugated with target-specific DNA aptamer (i-e. MUC1) and grown on carbon fiber. Benefitting from the combined features of the high electrochemical surface area of N-doped GO, high capacitance and stabilization by Co, and high kinetic performance by Pt, a robust, multifunctional, and flexible nanotransducer surface was created. The designed platform was applied for the specific detection of a blood-based oncomarker, CA15-3. The electrochemical characterization proved that nanosurface provides a highly conductive and proficient immobilization support with a strong bio-affinity towards MUC1 aptamer. The specific interaction between CA15-3 and the aptamer alters the surface properties of the aptasensor and the electroactive signal probe generated a remarkable increase in signal intensity. The sensor exhibited a wide dynamic range of 5.0 × 10-2 -200 U mL-1, a low limit of detection (LOD) of 4.1 × 10-2 U mL-1, and good reproducibility. The analysis of spiked serum samples revealed outstanding recoveries of up to 100.03 %, by the proposed aptasensor. The aptasensor design opens new revelations in the reliable detection of tumor biomarkers for timely cancer diagnosis.

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.

Phage-induced "one-to-many" FRET sensor for highly sensitive detection of Escherichia coli O157:H7.

As a foodborne pathogen capable of causing severe illnesses, early detection of Escherichia coli O157:H7 (E. coli O157:H7) is crucial for ensuring food safety. While Förster resonance energy transfer (FRET) is an efficient and precise detection technique, there remains a need for amplification strategies to detect low concentrations of E. coli O157:H7. In this study, we presented a phage (M13)-induced "one to many" FRET platform for sensitively detecting E. coli O157:H7. The aptamers, which specifically recognize E. coli O157:H7 were attached to magnetic beads as capture probes for separating E. coli O157:H7 from food samples. The peptide O157S, which specifically targets E. coli O157:H7, and streptavidin binding peptide (SBP), which binds to streptavidin (SA), were displayed on the P3 and P8 proteins of M13, respectively, to construct the O157S-M13K07-SBP phage as a detection probe for signal output. Due to the precise distance (≈3.2 nm) between two neighboring N-terminus of P8 protein, the SA-labeled FRET donor and acceptor can be fixed at the Förster distance on the surface of O157S-M13K07-SBP via the binding of SA and SBP, inducing FRET. Moreover, the P8 protein, with ≈2700 copies, enabled multiple FRET (≈605) occurrences, amplifying FRET in each E. coli O157:H7 recognition event. The O157S-M13K07-SBP-based FRET sensor can detect E. coli O157:H7 at concentration as low as 6 CFU/mL and demonstrates excellent performance in terms of selectivity, detection time (≈3 h), accuracy, precision, practical application, and storage stability. In summary, we have developed a powerful tool for detecting various targets in food safety, environmental monitoring, and medical diagnosis.

A sensitive tobramycin electrochemical aptasensor based on multiple signal amplification cascades.

The residue of tobramycin, a broad spectrum antibiotic commonly used in animal husbandry, has evitable impact on human health, which may cause kidney damage, respiratory paralysis, neuromuscular blockade and cross-allergy in humans. Sensitive monitoring of tobramycin in animal-derived food products is therefore of great importance. Herein, a new aptamer electrochemical biosensor for sensing tobramycin with high sensitivity is demonstrated via exonuclease III (Exo III) and metal ion-dependent DNAzyme recycling and hybridization chain reaction (HCR) signal amplification cascades. Tobramycin analyte binds aptamer-containing hairpin probe to switch its conformation to expose the toehold sequence, which triggers Exo III-based catalytic digestion of the secondary hairpin to release many DNAzyme strands. The substrate hairpins immobilized on the Au electrode (AuE) are then cyclically cleaved by the DNAzymes to form ssDNAs, which further initiate HCR formation of lots of long methylene blue (MB)-tagged dsDNA polymers on the AuE. Subsequently electro-oxidation of these MB labels thus exhibit highly enhanced currents for sensing tobramycin within the 5-1000 nM concentration range with an impressive detection limit of 3.51 nM. Furthermore, this strategy has high selectivity for detecting tobramycin in milk and shows promising potential for detect other antibiotics for food safety monitoring.

Highly sensitive SERS nanoplatform based on aptamer and vancomycin for detection of S. aureus and its clinical application.

Staphylococcus aureus (S. aureus) is the most common pathogen in human purulent infections, which can cause local purulent infections, as well as pneumonia, pseudomembranous enteritis, pericarditis, and even systemic infections. The conventional methods including bacteria colony counting, polymerase chain reaction and enzyme-linked immunosorbent assay can't fully meet the requirement of highly sensitive detection of S. aureus due to their own disadvantages. Therefore, it's an urgent need to develop new platform to detect S. aureus in the early infection stage. In this study, a new surface-enhanced Raman scattering (SERS)-based nanoplatform based on dual-recognition of aptamer (Apt) and vancomycin (Van) was developed for the highly sensitive detection of S. aureus. The SERS nanoplatform consisted of two functional parts: aptamer-conjugated Fe3O4 magnetic nanoparticles (Fe3O4-Apt MNPs) for bacteria enrichment and vancomycin modified-Au nanoparticles (Van-Au NPs) as the SERS probes for S. aureus quantitative detection. Upon the target bacteria enrichment, the SERS signals of the supernatant after magnetic separation could be obtained and analyzed under different concentrations of S. aureus. The limit of detection of the proposed assay was found to be 3.27 CFU/mL. We believe that the proposed SERS-based nanoplatform has great potential as a powerful tool in the early detection of specific bacteria.

Enhanced SELEX Platforms for Aptamer Selection with Improved Characteristics: A Review.

This review delves into the advancements in molecular recognition through enhanced SELEX (Systematic Evolution of Ligands by Exponential Enrichment) platforms and post-aptamer modifications. Aptamers, with their superior specificity and affinity compared to antibodies, are central to this discussion. Despite the advantages of the SELEX process-encompassing stages like ssDNA library preparation, incubation, separation, and PCR amplification-it faces challenges, such as nuclease susceptibility. To address these issues and propel aptamer technology forward, we examine next-generation SELEX platforms, including microfluidic-based SELEX, capillary electrophoresis SELEX, cell-based aptamer selection, counter-SELEX, in vivo SELEX, and high-throughput sequencing SELEX, highlighting their respective merits and innovations. Furthermore, this article underscores the significance of post-aptamer modifications, particularly chemical strategies that enhance aptamer stability, reduce renal filtration, and expand their target range, thereby broadening their utility in diagnostics, therapeutics, and nanotechnology. By synthesizing these advanced SELEX platforms and modifications, this review illuminates the dynamic progress in aptamer research and outlines the ongoing efforts to surmount existing challenges and enhance their clinical applicability, charting a path for future breakthroughs in this evolving field.

Pyoverdine binding aptamers and label-free electrochemical detection of pseudomonads.

Pyoverdines are iron-chelating siderophores employed by various pseudomonads to promote their growth in iron-limited environments, facilitating both beneficial and detrimental interactions with co-inhabiting microbes or hosts, including plants and animals. The fluorescent pseudomonads produce fluorescent pyoverdines comprised of a conserved central chromophore and a unique strain-specific peptidic side chain produced by non-ribosomal peptide synthetases. Pyoverdine Pf5 (PVD-Pf5) is produced by Pseudomonas protegens Pf-5, a species known for supporting plant growth and its involvement in plant pathogen control. To develop a means of exploring the dynamics of P. protegens activity in soil and in the rhizosphere, we selected DNA aptamers that specifically recognize PVD-Pf5 with high affinities. Two selected aptamers with only 16% identity in sequence were examined for structure and function. We found evidence that both aptamers form structures in their apo-forms and one aptamer has structural features suggesting the presence of a G-quadruplex. Although their tertiary structures are predicted to be different, both aptamers bind the target PVD-Pf5 with similar affinities and do not bind other siderophores, including the related pyoverdine, pseudobactin, produced by Pseudomonas sp. B10. One aptamer binds the pyoverdine peptide component and may also interact with the chromophore. This aptamer was integrated into a nanoporous aluminum oxide biosensor and demonstrated to successfully detect PVD-Pf5 and not to detect other siderophores that do not bind to the aptamer when evaluated in solution. This sensor provides a future opportunity to track the locations of P. protegens around plant roots and to monitor PVD-Pf5 production and movement through the soil.

Super-Resolved Protein Imaging Using Bifunctional Light-Up Aptamers.

Efficient labeling methods for protein visualization with minimal tag size and appropriate photophysical properties are required for single-molecule localization microscopy (SMLM), providing insights into the organization and interactions of biomolecules in cells at the molecular level. Among the fluorescent light-up aptamers (FLAPs) originally developed for RNA imaging, RhoBAST stands out due to its remarkable brightness, photostability, fluorogenicity, and rapid exchange kinetics, enabling super-resolved imaging with high localization precision. Here, we expand the applicability of RhoBAST to protein imaging by fusing it to protein-binding aptamers. The versatility of such bifunctional aptamers is demonstrated by employing a variety of protein-binding aptamers and different FLAPs. Moreover, fusing RhoBAST with the GFP-binding aptamer AP3 facilitates high- and super-resolution imaging of GFP-tagged proteins, which is particularly valuable in view of the widespread availability of plasmids and stable cell lines expressing proteins fused to GFP. The bifunctional aptamers compare favorably with standard antibody-based immunofluorescence protocols, as they are 7-fold smaller than antibody conjugates and exhibit higher bleaching-resistance. We demonstrate the effectiveness of our approach in super-resolution microscopy in secondary mammalian cell lines and primary neurons by RhoBAST-PAINT, an SMLM protein imaging technique that leverages the transient binding of the fluorogenic rhodamine dye SpyRho to RhoBAST.

Microfluidic device: A versatile biosensor platform to multiplex aptamer-based detection of malaria biomarkers.

Plasmodium falciparum malaria remains a dominant infectious disease that affects Africa than the rest of the world, considering its associated cases and death rates. It's a febrile illness that produces several reliable biomarkers, for example, P. falciparum lactate dehydrogenase (PfLDH), P. falciparum Plasmodium glutamate dehydrogenase (PfGDH), and P. falciparum histidine-rich proteins (HRP-II) in blood circulatory system that can easily be employed as targets in rapid diagnostic tests (RDTs). In recent times, several DNA aptamers have been developed via SELEX technology to detect some specific malaria biomarkers (PfLDH, PvLDH, HRP-II, PfGDH) in a biosensor mode with good binding affinity properties to overcome the trend of cross-reactivity, limited sensitivity and stability problems that have been observed with immunodiagnostics. In this review, we summarized existing diagnostic methods and relevant biomarkers to suggest promising approaches to develop sensitive and species-specific multiplexed diagnostic devices enabling effective detection of malaria in complex biological matrices and surveillance in the endemic region.

The effects of conjugating anti-MUC1 aptamers on gold nanobipyramids and nanostars for photothermal cancer ablation.

Aim: To ascertain the impact of shape and surface modification of anisotropic nanoparticles on the toxicity and photothermal efficiency toward cancerous cell lines. Methods: Gold nanobipyramids and nanostars surface modified with MUC1 aptamer were used in the current study to explore the toxicity and photothermal efficiency on MCF7 breast cancer cell lines via MTT assay. Results: Surface functionalization with MUC1 aptamer showed significant reduction in % cytotoxicity and increase in % specific internalization of nanostructures into MCF7 cell lines. Further, the photothermal studies accomplished at IC50 concentration for 6 h of treatment and laser exposure for 15 min reported that aptamer-conjugated nanobipyramids were more effective and specific toward MCF7 cell lines than aptamer-conjugated nanostars. Conclusion: This work establishes a platform for the development of tailored photoablation based gold nanostructures for in vivo studies.

[Box: see text].

Design and application of an ultrasensitive and selective tobromycin electrochemiluminescence aptasensor using MXene /Ni/Sm-LDH-based nanocomposite.

Using a chemiluminescence reaction between luminol and H2O2 in basic solution, an ultrasensitive electrochemiluminescence (ECL) aptasensor was developed for the determination of tobramycin (TOB), as an aminoglycoside antibiotic. Ti3C2/Ni/Sm-LDH-based nanocomposite effectively catalyzes the oxidation of luminol and decomposition of H2O2, leading to the formation of different reactive oxygen species (ROSs), thus amplifying the ECL signal intensity of luminol, which can be used for the determination of TOB concentration. To evaluate the performance of the electrochemiluminescence aptasensor and synthesized nanocomposite, different methods such as cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) analyses were performed. The considerable specific area, large number of active sites, and enhanced electron transfer reaction on this nanocomposite led to the development of an ECL aptasensor with high sensitivity and electrocatalytic activity. After optimizing the preparation method and analysis conditions, the aptasensor revealed a wide linear response ranging from 1.0 pM to 1.0 µM with a detection limit of 18 pM, displaying outstanding accuracy, specificity, and response stability. The developed ECL sensor was found to be applicable to the determination of TOB in human serum samples and is anticipated to possess excellent clinical potentials for detecting other antibiotics, as well.

Colorimetric aptasensor based on self-screened aptamers and cascaded catalytic reaction for the detection of quarantine plant bacteria.

Quarantine plant bacteria (QPB) are significant component of invasive alien species that result in substantial economic losses and serious environmental damage. Herein, a colorimetric aptasensor has been proposed based on the sandwich structure and the cascaded catalytic strategy for on-site detecting Xanthomonas hyacinthi, a type of QPB, in natural environments. The self-screened aptamer obtained through SELEX can bind to specific sites on the surface of viable organism with high affinity and specificity, which guarantees the selectivity of aptasensor. As an important part of the aptasensor, MIL-88-NH2(Fe) not only acts as a multifunctional carrier for both aptamers and glucose oxidase, but also catalyzes enzyme-like reaction because of specific surface area, amino and peroxidase-like activity. The present of Xanthomonas hyacinthi can trigger the formation of a sandwich structure and the occurrence of cascade catalytic reaction, enabling the detection with UV-Vis spectra and naked eyes. The proposed aptasensor presents a low detection limit of 2 cfu/mL and a wide linear range of 10 -107 cfu/mL. Compared to traditional detection methods for QPB, the reasonable design, high selectivity and convenience significantly improve the detection efficiency and contribute to environmental protection.

Drop-by-Drop Addition of Reagents to a Double Emulsion.

Developments in droplet microfluidics have facilitated an era of high-throughput, sensitive single-cell, or single-molecule measurements capable of tackling the heterogeneity present in biological systems. Relying on single emulsion (SE) compartments, droplet assays achieve absolute quantification of nucleic acids, massively parallel single-cell profiling, and more. Double emulsions (DEs) have seen recent interest for their potential to build upon SE techniques. DEs are compatible with flow cytometry enabling high-throughput multi-parameter drop screening and eliminate content mixing due to coalescence during lengthy workflows. Despite these strengths, DEs lack important technical functions that exist in SEs such as methods for adding reagents to droplets on demand. Consequently, DEs cannot be used for multistep workflows which has limited their adoption in assay development. Here, strategies to enable reagent addition and other active manipulations on DEs are reported by converting DE inputs to SEs on chip. After conversion, drops are manipulated using existing SE techniques, including reagent addition, before reforming a DE at the outlet. Device designs and operation conditions achieving drop-by-drop reagent addition to DEs are identified and used as part of a multi-step aptamer screening assay performed entirely in DE drops. This work enables the further development of multistep DE droplet assays.

Computational and experimental investigations of a novel aptamer targeting oxidized low-density lipoprotein.

Oxidized low-density lipoprotein (oxLDL) induces the formation of atherosclerotic plaques. Apolipoprotein B100 (apoB100) is a crucial protein component in low-density lipoprotein (LDL), which includes oxLDL. The oxidation of amino acids and subsequent alterations in their structure generate oxLDL, which is a significant biomarker for the initial phases of coronary artery disease. This study employed molecular docking and molecular dynamics utilizing the MM/GBSA method to identify aptamers with a strong affinity for oxidized apoB100. Molecular docking and molecular dynamics were performed on two sequences of the aptamer candidates (aptamer no.11 (AP11: 5'-CTTCGATGTAGTTTTTGTATGGGGTGCCCTGGTTCCTGCA-3') and aptamer no.26 (AP26: 5'-GCGAACTCGCGAATCCAGAACGGGCTCGGTCCCGGGTCGA-3')), yielding respective binding free energies of -149.08 kcal/mol and -139.86 kcal/mol. Interaction modeling of the simulation revealed a strong hydrogen bond between the AP11-oxidized apoB100 complexes. In an aptamer-based gold nanoparticle (AuNP) aggregation assay, AP11 exhibits a color shift from red to purple with the highest absorbance ratio, and shows strong binding affinity to oxLDL, correlating with the simulation model results. AP11 demonstrated the potential for application as a novel recognition element in diagnostic methodologies and may also contribute to future advancements in preventive therapies for coronary artery disease.

Assembly of a multivalent aptamer for efficient inhibition of thermostable direct hemolysin toxicity induced by Vibrio parahaemolyticus.

Thermostable direct hemolysin (TDH) is a key virulence factor of Vibrio parahaemolyticus, capable of causing seafood-mediated outbreaks of gastroenteritis, posing a threat to the aquatic environment and global public health. In the present study, we explored a multivalent aptamer-mediated inhibition strategy to mitigate TDH toxicity. Based on the characteristic structure of TDH, a stable multivalent aptamer, Ap3-5, was rationally designed by truncation, key fragment evolution, and end fixation. Ap3-5 exhibited strong affinity (Kd=39.24 nM), and thermal (Tm=57.6 °C) and enzymatic stability. In silico studies also revealed that Ap3-5 occupied more active sites of TDH and covered its central pore, indicating its potential as a blocking agent for inhibiting TDH toxicity. In the hemolysis assay, Ap3-5 significantly suppressed the hemolytic effect of TDH. A cellular study revealed a substantial (∼80 %) reduction in TDH cytotoxicity. Supporting these findings, in vivo trials confirmed the inhibitory action of Ap3-5 on both the acute and intestinal toxicity of TDH. Overall, benefiting from the strong binding affinity, high stability, and multisite occupation of the multivalent aptamer with TDH, Ap3-5 displayed robust potential against TDH toxicity by inhibiting membrane pore formation, providing a new approach for alleviating bacterial infections.

Integrating CdIn2S4 semiconductors with S-vacancy MoS2 nanosheets to fabricate a multi-channel aptasensing chip for photoelectrochemical detection of multiple mycotoxins.

BACKGROUND: The importance of multi-target simultaneous detection lies in its ability to significantly boost detection efficiency, making it invaluable for rapid and cost-effective testing. Photoelectrochemical (PEC) sensors have emerged as promising candidates for detecting harmful substances and biomarkers, attributable to their unparalleled sensitivity, minimal background signal, cost-effectiveness, equipment simplicity, and outstanding repeatability. However, designing an effective multi-target detection strategy remains a challenging task in the PEC sensing field. Consequently, there is a pressing need to address the development of PEC sensors capable of simultaneously detecting multiple targets. RESULTS: CdIn2S4/V-MoS2 heterojunctions were successfully prepared via a hydrothermal method. These heterojunctions exhibited a high photocurrent intensity, representing a 1.53-fold enhancement compared to CdIn2S4 alone. Next, we designed a multi-channel aptasensing chip using ITO as the substrate. Three working electrodes were created via laser etching and subsequently modified with CdIn2S4/V-MoS2 heterojunctions. Thiolated aptamers were then self-assembled onto the CdIn2S4/V-MoS2 heterojunctions via covalent bonds, serving as recognition tool. By empolying the CdIn2S4/V-MoS2 heterojunctions as the sensing platform and aptamers as recognition tool, we successfully developed a disposable aptasensing chip for the simultaneous PEC detection of three typical mycotoxins (aflatoxin B1 (AFB1), ochratoxin A (OTA), and zearalenone (ZEN)). This aptasensing chip exhibited wide detection range for AFB1 (0.05-50 ng/mL), OTA (0.05-500 ng/mL), and ZEN (0.1-250 ng/mL). Furthermore, it demonstrated ultra-low detection limits of 0.017 ng/mL for AFB1, 0.016 ng/mL for OTA, and 0.033 ng/mL for ZEN. SIGNIFICANCE AND NOVELTY: The aptasensing chip stands out for its cost-effectiveness, simplicity of fabrication, and multi-channel capabilities. The versatility and practicality enable it to serve as a powerful platform for designing multi-channel PEC aptasensors. With its ability to detect multiple targets with high sensitivity and specificity, the aptasensing chip holds immense potential for applications across diverse fields, such as environmental monitoring, clinical diagnostics, and food safety monitoring, where multi-target detection is crucial.

Development of Aptamer-Functionalized Gold Nanoparticles as Probes in Point-of-Care Diagnostic Device for Rapid Detection of Multidrug-Resistant Bacteria in Bombyx mori L. .

The sericulture industry suffers severe crop losses due to various silkworm diseases, necessitating the development of further technologies for rapid pathogen detection. Here, we report an all-in-one portable biosensor that combines conjugated gold nanoparticles (Au NPs) with an aptamer-based lateral flow assay (LFA) platform for the real-time analysis of Mammaliicoccus sp. and Pseudomonas sp. Our platform enables sample-to-answer naked eye detection within 5 min without any cross-reactivity with other representatives of the silkworm pathogenic bacterial group. This assay was based on the sandwich-type format using a bacteria-specific primary aptamer (Apt1) conjugated with 23 nm ± 1.27 nm Au NPs as a signal probe and another bacteria-specific secondary aptamer (Apt2)-coated nitrocellulose membrane as a capture probe. The hybridization between the signal probe and the capture probe in the presence of bacteria develops a red band in the test line, whose intensity is directly proportional to the bacterial concentration. Under the optimal experimental conditions, the visual limit of detection of the strip for Mammaliicoccus sp. and Pseudomonas sp. was 1.5 × 104 CFU/mL and 1.5 × 103 CFU/mL, respectively. Additionally, the performance of the LFA device was validated by using a colorimetric assay, and the results from the colorimetric assay are consistent with those obtained from the LFA. Our findings indicate that the developed point-of-care diagnostic device has significant potential for providing a cost-effective, scalable alternative for the rapid detection of silkworm pathogens.

EpCAM-targeted betulinic acid analogue nanotherapy improves therapeutic efficacy and induces anti-tumorigenic immune response in colorectal cancer tumor microenvironment.

BACKGROUND: Betulinic acid (BA) has been well investigated for its antiproliferative and mitochondrial pathway-mediated apoptosis-inducing effects on various cancers. However, its poor solubility and off-target activity have limited its utility in clinical trials. Additionally, the immune modulatory role of betulinic acid analogue in the tumor microenvironment (TME) is largely unknown. Here, we designed a potential nanotherapy for colorectal cancer (CRC) with a lead betulinic acid analogue, named as 2c, carrying a 1,2,3-triazole-moiety attached to BA through a linker, found more effective than BA for inhibiting CRC cell lines, and was chosen here for this investigation. Epithelial cell adhesion molecule (EpCAM) is highly overexpressed on the CRC cell membrane. A single-stranded short oligonucleotide sequence, aptamer (Apt), that folds into a 3D-defined architecture can be used as a targeting ligand for its specific binding to a target protein. EpCAM targeting aptamer was designed for site-specific homing of aptamer-conjugated-2c-loaded nanoparticles (Apt-2cNP) at the CRC tumor site to enhance therapeutic potential and reduce off-target toxicity in normal cells. We investigated the in vitro and in vivo therapeutic efficacy and anti-tumorigenic immune response of aptamer conjugated nanotherapy in CRC-TME. METHODS: After the characterization of nanoengineered aptamer conjugated betulinic acid nanotherapy, we evaluated therapeutic efficacy, tumor targeting efficiency, and anti-tumorigenic immune response using cell-based assays and mouse and rat models. RESULTS: We found that Apt-2cNP improved drug bioavailability, enhanced its biological half-life, improved antiproliferative activity, and minimized off-target cytotoxicity. Importantly, in an in vivo TME, Apt-2cNP showed promising signs of anti-tumorigenic immune response (increased mDC/pDC ratio, enhanced M1 macrophage population, and CD8 T-cells). Furthermore, in vivo upregulation of pro-apoptotic while downregulation of anti-apoptotic genes and significant healing efficacy on cancer tissue histopathology suggest that Apt-2cNP had predominantly greater therapeutic potential than the non-aptamer-conjugated nanoparticles and free drug. Moreover, we observed greater tumor accumulation of the radiolabeled Apt-2cNP by live imaging in the CRC rat model. CONCLUSIONS: Enhanced therapeutic efficacy and robust anti-tumorigenic immune response of Apt-2cNP in the CRC-TME are promising indicators of its potential as a prospective therapeutic agent for managing CRC. However, further studies are warranted.