Development and characterization of a portable electrochemical aptasensor for IsdA protein and Staphylococcus aureus detection.

Staphylococcus aureus (S. aureus) is recognized as one of the most common causes of gastroenteritis worldwide. This pathogen is a major foodborne pathogen that can cause many different types of various infections, from minor skin infections to lethal blood infectious diseases. Iron-regulated surface determinant protein A (IsdA) is an important protein on the S. aureus surface. It is responsible for iron scavenging via interaction with hemoglobin, haptoglobin, and hemoglobin-haptoglobin complexes. This study develops a portable aptasensor for IsdA and S. aureus detection using aptamer-modified gold nanoparticles (AuNPs) integrated into screen-printed carbon electrodes (SPCEs). The electrode system was made of three parts, including a carbon counter electrode, an AuNPs/carbon working electrode, and a silver reference electrode. The aptamer by Au-S bonding was conjugated on the electrode surface to create the aptasensor platform. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were utilized to investigate the binding interactions between the aptasensor and the IsdA protein. CV studies showed a linear correlation between varying S. aureus concentrations within the range of 101 to 106 CFU/mL, resulting in a limit of detection (LOD) of 0.2 CFU/mL. The results demonstrated strong reproducibility, selectivity, and sensitivity of the aptasensor for enhanced detection of IsdA, along with about 93% performance stability after 30 days. The capability of the aptasensor to directly detect S. aureus via the IsdA surface protein binding was further investigated in a food matrix. Overall, the aptasensor device showed the potential for rapid detection of S. aureus, serving as a robust approach to developing real-time aptasensors to identify an extensive range of targets of foodborne pathogens and beyond.

Multimodal Biosensing of Foodborne Pathogens.

Microbial foodborne pathogens present significant challenges to public health and the food industry, requiring rapid and accurate detection methods to prevent infections and ensure food safety. Conventional single biosensing techniques often exhibit limitations in terms of sensitivity, specificity, and rapidity. In response, there has been a growing interest in multimodal biosensing approaches that combine multiple sensing techniques to enhance the efficacy, accuracy, and precision in detecting these pathogens. This review investigates the current state of multimodal biosensing technologies and their potential applications within the food industry. Various multimodal biosensing platforms, such as opto-electrochemical, optical nanomaterial, multiple nanomaterial-based systems, hybrid biosensing microfluidics, and microfabrication techniques are discussed. The review provides an in-depth analysis of the advantages, challenges, and future prospects of multimodal biosensing for foodborne pathogens, emphasizing its transformative potential for food safety and public health. This comprehensive analysis aims to contribute to the development of innovative strategies for combating foodborne infections and ensuring the reliability of the global food supply chain.

Advances in aptamer-based biosensors for monitoring foodborne pathogens.

Biosensors are analytical devices for detecting a wide range of targets, including cells, proteins, DNA, enzymes, and chemical and biological compounds. They mostly rely on using bioprobes with a high binding affinity to the target for specific detection. However, low specificity and effectiveness of the conventional biosensors has led to the search for novel materials, that can specifically detect biomolecules. Aptamers are a group of single-stranded DNA or RNA oligonucleotides, that can bind to their targets with high specificity and serve as effective bioprobes for developing aptamer-based biosensors. Aptamers have a shorter production time, high stability, compared to traditional bioprobes, and possess ability to develop them for specific target molecules for tailored applications. Thus, various aptasensing approaches, including electrochemical, optical, surface plasmon resonance and chip-dependent approaches, have been investigated in recent times for various biological targets, including foodborne pathogens. Hence, this article is an overview of various conventional foodborne pathogen detection methods, their limitations and the ability of aptamer-based biosensors to overcome those limitations and replace them. In addition, the current status and advances in aptamer-based biosensors for the detection of foodborne pathogens to ensure food safety were also discussed. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-023-05889-8.

FRET-Based Single-Molecule Detection of Pathogen Protein IsdA Using Computationally Selected Aptamers.

Iron-regulated surface determinant protein A (IsdA) is a key surface protein found in the foodborne bacteria─Staphylococcus aureus (S. aureus)─which is known to be critical for bacterial survival and colonization. S. aureus is pathogenic and has been linked to foodborne diseases; thus, early detection is critical to prevent diseases caused by this bacterium. Despite IsdA being a specific marker for S. aureus and several detection methods have been developed for sensitive detection of this bacteria such as cell culture, nucleic acids amplification, and other colorimetric and electrochemical methods, the detection of S. aureus through IsdA is underdeveloped. Here, by combining computational generation of target-guided aptamers and fluorescence resonance energy transfer (FRET)-based single-molecule analysis, we presented a widely applicable and robust detection method for IsdA. Three different RNA aptamers specific to the IsdA protein were identified and their ability to switch a FRET construct to a high-FRET state in the presence of protein was verified. The presented approach demonstrated the detection of IsdA down to picomolar levels (×10-12 M, equivalent to ∼1.1 femtomoles IsdA) with a dynamic range extending to ∼40 nM. The FRET-based single-molecule technique that we reported here is capable of detecting the foodborne pathogen protein IsdA with high sensitivity and specificity and has a broader application in the food industry and aptamer-based sensing field by enabling quantitative detection of a wide range of pathogen proteins.

Editorial to the IJMS Special Issue "Aptamer-Mediated Cancer Theranostics".

Aptamers have emerged as a new generation of bioaffinity probes with enhanced target binding specificity and selectivity [...].

Neuroblastoma GD2 Expression and Computational Analysis of Aptamer-Based Bioaffinity Targeting.

Neuroblastoma (NB) is a neuroectodermal embryonic cancer that originates from primordial neural crest cells, and amongst pediatric cancers with high mortality rates. NB is categorized into high-, intermediate-, and low-risk cases. A significant proportion of high-risk patients who achieve remission have a minimal residual disease (MRD) that causes relapse. Whilst there exists a myriad of advanced treatment options for NB, it is still characterized by a high relapse rate, resulting in a reduced chance of survival. Disialoganglioside (GD2) is a lipo-ganglioside containing a fatty acid derivative of sphingosine that is coupled to a monosaccharide and a sialic acid. Amongst pediatric solid tumors, NB tumor cells are known to express GD2; hence, it represents a unique antigen for subclinical NB MRD detection and analysis with implications in determining a response for treatment. This article discusses NB MRD expression and analytical assays for GD2 detection and quantification as well as computational approaches for GD2 characterization based on high-throughput image processing and genomic data analysis.

Probing the characteristics and biofunctional effects of disease-affected cells and drug response via machine learning applications.

Drug-induced transformations in disease characteristics at the cellular and molecular level offers the opportunity to predict and evaluate the efficacy of pharmaceutical ingredients whilst enabling the optimal design of new and improved drugs with enhanced pharmacokinetics and pharmacodynamics. Machine learning is a promising in-silico tool used to simulate cells with specific disease properties and to determine their response toward drug uptake. Differences in the properties of normal and infected cells, including biophysical, biochemical and physiological characteristics, plays a key role in developing fundamental cellular probing platforms for machine learning applications. Cellular features can be extracted periodically from both the drug treated, infected, and normal cells via image segmentations in order to probe dynamic differences in cell behavior. Cellular segmentation can be evaluated to reflect the levels of drug effect on a distinct cell or group of cells via probability scoring. This article provides an account for the use of machine learning methods to probe differences in the biophysical, biochemical and physiological characteristics of infected cells in response to pharmacokinetics uptake of drug ingredients for application in cancer, diabetes and neurodegenerative disease therapies.

Prospects in the use of aptamers for characterizing the structure and stability of bioactive proteins and peptides in food.

Food-derived bioactive proteins and peptides have gained acceptance among researchers, food manufacturers and consumers as health-enhancing functional food components that also serve as natural alternatives for disease prevention and/or management. Bioactivity in food proteins and peptides is determined by their conformations and binding characteristics, which in turn depend on their primary and secondary structures. To maintain their bioactivities, the molecular integrity of bioactive peptides must remain intact, and this warrants the study of peptide form and structure, ideally with robust, highly specific and sensitive techniques. Short single-stranded nucleic acids (i.e. aptamers) are known to have high affinity for cognate targets such as proteins and peptides. Aptamers can be produced cost-effectively and chemically derivatized to increase their stability and shelf life. Their improved binding characteristics and minimal modification of the target molecular signature suggests their suitability for real-time detection of conformational changes in both proteins and peptides. This review discusses the developmental progress of systematic evolution of ligands by exponential enrichment (SELEX), an iterative technology for generating cost-effective aptamers with low dissociation constants (K d) for monitoring the form and structure of bioactive proteins and peptides. The review also presents case studies of this technique in monitoring the structural stability of bioactive peptide formulations to encourage applications in functional foods. The challenges and potential of aptamers in this research field are also discussed. Graphical abstract Advancing bioactive proteins and peptide functionality via aptameric ligands.

Deploying aptameric sensing technology for rapid pandemic monitoring.

The genome of virulent strains may possess the ability to mutate by means of antigenic shift and/or antigenic drift as well as being resistant to antibiotics with time. The outbreak and spread of these virulent diseases including avian influenza (H1N1), severe acute respiratory syndrome (SARS-Corona virus), cholera (Vibrio cholera), tuberculosis (Mycobacterium tuberculosis), Ebola hemorrhagic fever (Ebola Virus) and AIDS (HIV-1) necessitate urgent attention to develop diagnostic protocols and assays for rapid detection and screening. Rapid and accurate detection of first cases with certainty will contribute significantly in preventing disease transmission and escalation to pandemic levels. As a result, there is a need to develop technologies that can meet the heavy demand of an all-embedded, inexpensive, specific and fast biosensing for the detection and screening of pathogens in active or latent forms to offer quick diagnosis and early treatments in order to avoid disease aggravation and unnecessary late treatment costs. Nucleic acid aptamers are short, single-stranded RNA or DNA sequences that can selectively bind to specific cellular and biomolecular targets. Aptamers, as new-age bioaffinity probes, have the necessary biophysical characteristics for improved pathogen detection. This article seeks to review global pandemic situations in relation to advances in pathogen detection systems. It particularly discusses aptameric biosensing and establishes application opportunities for effective pandemic monitoring. Insights into the application of continuous polymeric supports as the synthetic base for aptamer coupling to provide the needed convective mass transport for rapid screening is also presented.

Emerging Biohybrids of Aptamer-Based Nano-Biosensing Technologies for Effective Early Cancer Detection.

Cancer is a leading global cause of mortality, which underscores the imperative of early detection for improved patient outcomes. Biorecognition molecules, especially aptamers, have emerged as highly effective tools for early and accurate cancer cell identification. Aptamers, with superior versatility in synthesis and modification, offer enhanced binding specificity and stability compared with conventional antibodies. Hence, this article reviews diagnostic strategies employing aptamer-based biohybrid nano-biosensing technologies, focusing on their utility in detecting cancer biomarkers and abnormal cells. Recent developments include the synthesis of nano-aptamers using diverse nanomaterials, such as metallic nanoparticles, metal oxide nanoparticles, carbon-derived substances, and biohybrid nanostructures. The integration of these nanomaterials with aptamers significantly enhances sensitivity and specificity, promising innovative and efficient approaches for cancer diagnosis. This convergence of nanotechnology with aptamer research holds the potential to revolutionize cancer treatment through rapid, accurate, and non-invasive diagnostic methods.

Surface plasmon resonance aptasensing and computational analysis of Staphylococcus aureus IsdA surface protein.

Staphylococcus aureus (S. aureus), a common foodborne pathogen, poses significant public health challenges due to its association with various infectious diseases. A key player in its pathogenicity, which is the IsdA protein, is an essential virulence factor in S. aureus infections. In this work, we present an integrated in-silico and experimental approach using MD simulations and surface plasmon resonance (SPR)-based aptasensing measurements to investigate S. aureus biorecognition via IsdA surface protein binding. SPR, a powerful real-time and label-free technique, was utilized to characterize interaction dynamics between the aptamer and IsdA protein, and MD simulations was used to characterize the stable and dynamic binding regions. By characterizing and optimizing pivotal parameters such as aptamer concentration and buffer conditions, we determined the aptamer's binding performance. Under optimal conditions of pH 7.4 and 150 mM NaCl concentration, the kinetic parameters were determined; ka = 3.789 × 104/Ms, kd = 1.798 × 103/s, and KD = 4.745 × 10-8 M. The simulations revealed regions of interest in the IsdA-aptamer complex. Region I, which includes interactions between amino acid residues H106 and R107 and nucleotide residues 9G, 10U, 11G and 12U of the aptamer, had the strongest interaction, based on ΔG and B-factor values, and hence contributed the most to the stability of the interaction. Region II, which covers residue 37A reflects the dynamic nature of the interaction due to frequent contacts. The approach presents a rigorous characterization of aptamer-IsdA binding behavior, supporting the potential application of the IsdA-binding aptamer system for S. aureus biosensing.

Bioaffinity Nanoprobes for Foodborne Pathogen Sensing.

Bioaffinity nanoprobes are a type of biosensor that utilize the specific binding properties of biological molecules, such as antibodies, enzymes, and nucleic acids, for the detection of foodborne pathogens. These probes serve as nanosensors and can provide highly specific and sensitive detection of pathogens in food samples, making them an attractive option for food safety testing. The advantages of bioaffinity nanoprobes include their ability to detect low levels of pathogens, rapid analysis time, and cost-effectiveness. However, limitations include the need for specialized equipment and the potential for cross-reactivity with other biological molecules. Current research efforts focus on optimizing the performance of bioaffinity probes and expanding their application in the food industry. This article discusses relevant analytical methods, such as surface plasmon resonance (SPR) analysis, Fluorescence Resonance Energy Transfer (FRET) measurements, circular dichroism, and flow cytometry, that are used to evaluate the efficacy of bioaffinity nanoprobes. Additionally, it discusses advances in the development and application of biosensors in monitoring foodborne pathogens.

Magnetic Nanoparticles for Protein Separation and Purification.

Proteins are essential for various functions such as brain activity and muscle contraction in humans. Even though food is a source of proteins, the bioavailability of proteins in most foods is usually limited due to matrix interaction with other biomolecules. Thus, it is essential to extract these proteins and provide them as a nutraceutical supplement to maintain protein levels and avoid protein deficiency. Hence, protein purification and extraction from natural sources are highly significant in biomedical applications. Chromatography, crude mechanical disruption, use of extractive chemicals, and electrophoresis are some of the methods applied to isolate specific proteins. Even though these methods possess several advantages, they are unable to extract specific proteins with high purity. A suitable alternative is the use of nanoparticles, which can be beneficial in protein purification and extraction. Notably, magnetic iron and iron-based nanoparticles have been employed in protein extraction processes and can be reused via demagnetization due to their magnetic property, smaller size, morphology, high surface-to-volume ratio, and surface charge-mediated property. This chapter is a summary of various magnetic nanoparticles (MNPs) that can be used for the biomolecular separation of proteins.

Development of Janus Particles as Potential Drug Delivery Systems for Diabetes Treatment and Antimicrobial Applications.

Janus particles have emerged as a novel and smart material that could improve pharmaceutical formulation, drug delivery, and theranostics. Janus particles have two distinct compartments that differ in functionality, physicochemical properties, and morphological characteristics, among other conventional particles. Recently, Janus particles have attracted considerable attention as effective particulate drug delivery systems as they can accommodate two opposing pharmaceutical agents that can be engineered at the molecular level to achieve better target affinity, lower drug dosage to achieve a therapeutic effect, and controlled drug release with improved pharmacokinetics and pharmacodynamics. This article discusses the development of Janus particles for tailored and improved delivery of pharmaceutical agents for diabetes treatment and antimicrobial applications. It provides an account of advances in the synthesis of Janus particles from various materials using different approaches. It appraises Janus particles as a promising particulate system with the potential to improve conventional delivery systems, providing a better loading capacity and targeting specificity whilst promoting multi-drugs loading and single-dose-drug administration.

Target-Specific Delivery and Bioavailability of Pharmaceuticals via Janus and Dendrimer Particles.

Nanosized Janus and dendrimer particles have emerged as promising nanocarriers for the target-specific delivery and improved bioavailability of pharmaceuticals. Janus particles, with two distinct regions exhibiting different physical and chemical properties, provide a unique platform for the simultaneous delivery of multiple drugs or tissue-specific targeting. Conversely, dendrimers are branched, nanoscale polymers with well-defined surface functionalities that can be designed for improved drug targeting and release. Both Janus particles and dendrimers have demonstrated their potential to improve the solubility and stability of poorly water-soluble drugs, increase the intracellular uptake of drugs, and reduce their toxicity by controlling the release rate. The surface functionalities of these nanocarriers can be tailored to specific targets, such as overexpressed receptors on cancer cells, leading to enhanced drug efficacy The design of these nanocarriers can be optimized by tuning the size, shape, and surface functionalities, among other parameters. The incorporation of Janus and dendrimer particles into composite materials to create hybrid systems for enhancing drug delivery, leveraging the unique properties and functionalities of both materials, can offer promising outcomes. Nanosized Janus and dendrimer particles hold great promise for the delivery and improved bioavailability of pharmaceuticals. Further research is required to optimize these nanocarriers and bring them to the clinical setting to treat various diseases. This article discusses various nanosized Janus and dendrimer particles for target-specific delivery and bioavailability of pharmaceuticals. In addition, the development of Janus-dendrimer hybrid nanoparticles to address some limitations of standalone nanosized Janus and dendrimer particles is discussed.

Computational generation and characterization of IsdA-binding aptamers with single-molecule FRET analysis.

Staphylococcus aureus is a major foodborne bacterial pathogen. Early detection of S. aureus is crucial to prevent infections and ensure food quality. The iron-regulated surface determinant protein A (IsdA) of S. aureus is a unique surface protein necessary for sourcing vital iron from host cells for the survival and colonization of the bacteria. The function, structure, and location of the IsdA protein make it an important protein for biosensing applications relating to the pathogen. Here, we report an in-silico approach to develop and validate high-affinity binding aptamers for the IsdA protein detection using custom-designed in-silico tools and single-molecule Fluorescence Resonance Energy Transfer (smFRET) measurements. We utilized in-silico oligonucleotide screening methods and metadynamics-based methods to generate 10 aptamer candidates and characterized them based on the Dissociation Free Energy (DFE) of the IsdA-aptamer complexes. Three of the aptamer candidates were shortlisted for smFRET experimental analysis of binding properties. Limits of detection in the low picomolar range were observed for the aptamers, and the results correlated well with the DFE calculations, indicating the potential of the in-silico approach to support aptamer discovery. This study showcases a computational SELEX method in combination with single-molecule binding studies deciphering effective aptamers against S. aureus IsdA, protein. The established approach demonstrates the ability to expedite aptamer discovery that has the potential to cut costs and predict binding efficacy. The application can be extended to designing aptamers for various protein targets, enhancing molecular recognition, and facilitating the development of high-affinity aptamers for multiple uses.

Novel Concepts for Graphene-Based Nanomaterials Synthesis for Phenol Removal from Palm Oil Mill Effluent (POME).

In recent years, the global population has increased significantly, resulting in elevated levels of pollution in waterways. Organic pollutants are a major source of water pollution in various parts of the world, with phenolic compounds being the most common hazardous pollutant. These compounds are released from industrial effluents, such as palm oil milling effluent (POME), and cause several environmental issues. Adsorption is known to be an efficient method for mitigating water contaminants, with the ability to eliminate phenolic contaminants even at low concentrations. Carbon-based materials have been reported to be effective composite adsorbents for phenol removal due to their excellent surface features and impressive sorption capability. However, the development of novel sorbents with higher specific sorption capabilities and faster contaminant removal rates is necessary. Graphene possesses exceptionally attractive chemical, thermal, mechanical, and optical properties, including higher chemical stability, thermal conductivity, current density, optical transmittance, and surface area. The unique features of graphene and its derivatives have gained significant attention in the application of sorbents for water decontamination. Recently, the emergence of graphene-based adsorbents with large surface areas and active surfaces has been proposed as a potential alternative to conventional sorbents. The aim of this article is to discuss novel synthesis approaches for producing graphene-based nanomaterials for the adsorptive uptake of organic pollutants from water, with a special focus on phenols associated with POME. Furthermore, this article explores adsorptive properties, experimental parameters for nanomaterial synthesis, isotherms and kinetic models, mechanisms of nanomaterial formation, and the ability of graphene-based materials as adsorbents of specific contaminants.

Selective adsorption of organic dyes from aqueous environment using fermented maize extract-enhanced graphene oxide-durian shell derived activated carbon composite.

A secure aquatic environment is essential for both aquatic and terrestrial life. However, rising populations and the industrial revolution have had a significant impact on the quality of the water environment. Despite the implementation of strong and adapted environmental policies for water treatment worldwide, the issue of organic dyes in wastewater remains challenging. Thus, this study aimed to develop an efficient, cost-effective, and sustainable material to treat methylene blue (MB) in an aqueous environment. In this research, maize extract solution (MES) was utilized as a green cross-linker to induce precipitation, conjugation, and enhance the adsorption performance of graphene oxide (GO) cross-linked with durian shell activated carbon (DSAC), resulting in the formation of a GO@DSAC composite. The composite was investigated for its adsorptive performance toward MB in aqueous media. The physicochemical characterization demonstrated that the cross-linking method significantly influenced the porous structure and surface chemistry of GO@DSAC. BET analysis revealed that the GO@DSAC exhibited dominant mesopores with a surface area of 803.67 m2/g. EDX and XPS measurements confirmed the successful cross-linking of GO with DSAC. The adsorption experiments were well described by the Harkin-Jura model and they followed pseudo-second order kinetics. The maximum adsorption capacity reached 666.67 mg/g at 318 K. Thermodynamic evaluation indicated a spontaneous, feasible, and endothermic in nature. Regenerability and reusability investigations demonstrated that the GO@DSAC composite could be reused for up to 10 desorption-adsorption cycles with a removal efficiency of 81.78%. The selective adsorptive performance of GO@DSAC was examined in a binary system containing Rhodamine B (RhB) and methylene orange (MO). The results showed a separation efficiency (α) of 98.89% for MB/MO and 93.66% for MB/RhB mixtures, underscoring outstanding separation capabilities of the GO@DSAC composite. Overall, the GO@DSAC composite displayed promising potential for the effective removal of cationic dyes from wastewater.

Removal of endotoxins from plasmid DNA: analysis of aggregative interaction of mobile divalent metal cations with endotoxins and plasmid DNA.

Endotoxin lipopolysaccharide removal from plasmid DNA-based vaccine remains a very challenging task for bioprocess engineers. This paper examined the potential use and advantages of divalent cation (Zn(2+), Ca(2+), Mg(2+)) induced aggregation as a plasmid DNA purification method for lipopolysaccharide removal. Analysis of zeta potential, hydrodynamic size, percentage of aggregation; UV-Vis spectroscopy and electron microscopy were performed to determine the optimal cation for preferential aggregation of lipopolysaccharide over plasmid DNA. The results from the hydrodynamic size analysis showed that the addition of Zn(2+) resulted in the maximum theoretical number of lipopolysaccharide molecules per aggregate particle. Dynamic light scattering analysis showed that plasmid DNA aggregates formed a larger maximum hydrodynamic size when it was treated with Ca(2+) than the other two cations. The K(m) value for lipopolysaccharide-Zn(2+) was substantially low (0.28 M) and considerably large (>2 M) for plasmid DNA-Zn(2+). Scatchard plots for plasmid DNA cations showed positive slopes indicating that there was a minimum concentration of plasmid DNA or cations before a significant aggregation occurred. This work concluded that Zn(2+) had the most preferential aggregative interaction with lipopolysaccharide compared to Mg(2+) and Ca(2+).

Multivalent ACE2 engineering-A promising pathway for advanced coronavirus nanomedicine development.

The spread of coronavirus diseases has resulted in a clarion call to develop potent drugs and vaccines even as different strains appear beyond human prediction. An initial step that is integral to the viral entry into host cells results from an active-targeted interaction of the viral spike (S) proteins and the cell surface receptor, called angiotensin-converting enzyme 2 (ACE2). Thus, engineered ACE2 has been an interesting decoy inhibitor against emerging coronavirus infestation. This article discusses promising innovative ACE2 engineering pathways for current and emerging coronavirus therapeutic development. First, we provide a brief discussion of some ACE2-associated human coronaviruses and their cell invasion mechanism. Then, we describe and contrast the individual spike proteins and ACE2 receptor interactions, highlighting crucial hotspots across the ACE2-associated coronaviruses. Lastly, we address the importance of multivalency in ACE2 nanomedicine engineering and discuss novel approaches to develop and achieve multivalent therapeutic outcomes. Beyond coronaviruses, these approaches will serve as a paradigm to develop new and improved treatment technologies against pathogens that use ACE2 receptor for invasion.