Bioconjugated nanomaterials for monitoring food contamination

Maintaining food safety and hygiene standards is top priority and challenge for farmers, food industries, governments and food technologists working in the food supply chain. Pesticides, toxins, veterinary drug residues, foodborne pathogens and many other harmful chemicals that may be present in a vast array of food products, due to various stages of their production like packaging and transport, constitute a global health problem that requires powerful and innovative technologies allowing constant and accurate detection of food products from production to consumption. Recent progress in generation of specific synthetic oligonucleotides against food contaminants has provided a new insight into the current sensor technologies, where these functional synthetic oligonucleotides, so-called aptamers, have been successfully combined with nanomaterials for rapid and cost-effective detection of several substances related to the food contamination, such as antibiotics, mycotoxins, heavy metals, carcinogenic dyes, pesticides, pathogens and other plastic products used for food packaging. Unique characteristics of aptamers over antibodies, such as in vitro selection, chemical and thermal stability, small size and ease of labeling have laid the solid foundation for exploring aptamers further in multiplexed food monitoring systems. In this chapter, we reviewed the application of aptamer-conjugated nanomaterials in food safety surveillance as well as the conventional techniques used for food safety monitoring in order to provide a comprehensive and comparative approach

Identification and Biosensing Application of Molecular Recognition Elements

Molecular recognition elements (MREs) are biomolecules such as single-stranded DNA (ssDNA), RNA, small peptides and antibody fragments that can bind to user defined targets with high affinities and specificities. This binding property allows MREs to have a wide range of applications, including therapeutic, diagnostic, and biosensor applications. The identification of MREs can be achieved by using the process called Systematic Evolution of Ligands by Exponential Enrichment (SELEX). This process begins with a large library of 109 to 1015 different random molecules, molecules that bind to the user defined target or positive target are enriched in the process. Subsequently, this process can be modified and tailored to direct the enriched library away from binding to related targets or negative targets, and thus increasing the specificity. Single-stranded DNA (ssDNA) MREs are particularly favorable for biosening applications due to their relative stability, reusability and low cost in production. This work investigated the identification and application of ssDNA MREs to detect different bacterial toxins and pesticide.;In Chapter 1, it begins by reviewing recent discovery and advancement in the SELEX technique for the identification and biosensing application of ssDNA MREs specific for bacteria, viruses, their related biomolecules, and selected environmental toxins. It is then followed by a brief discussion on major biosensing principles based upon ssDNA MREs. In Chapter 2, the pilot project of this work, ssDNA MRE specific for Pseudomonas aeruginosa exotoxin A was identified. In this chapter, a novel variation of SELEX called Decoy-SELEX, previously developed by our laboratory is described in greater detail. Additionally, the development of a ssDNA MRE modified enzyme-linked immunosorbent assay (ELISA) for the exotoxin A detection is also discussed. In Chapter 3, similar methodology was applied to identify a ssDNA MRE specific for the second target, Clostridium difficile toxin B. Subsequently, similar ssDNA MRE modified ELISA was developed for target detection in clinically relevant samples. In Chapter 4, ssDNA MRE specific for alpha toxin of Staphylococcus aureus was identified, and it was applied for sensitive detection of the target in clinically relevant samples. In Chapter 5, the overall conclusion and potential future studies as a result from this work is discussed. Lastly, in Appendix, the project of identifying and potential future application of ssDNA MREs specific for a pesticide, Fipronil is described.;Overall, this work has shown the proof-of-principle of using ssDNA MREs in biosensing application for target detections in clinically relevant samples. The work will be useful in the development of potential point-of-care diagnostic tools for rapid diagnosis of bacterial infections

Rapid and Sensitive Detection of Escherichia coli O157:H7 Using a QCM Sensor based on Aptamers Selected by Whole-Bacterium SELEX and a Multivalent Aptamer System

Escherichia coli O157:H7 is one of the top five pathogens contributing to foodborne diseases, causing an estimated 2,138 cases of hospitalization in the US each year. The extremely low infectious dose demands for more rapid and sensitive methods to detect E. coli O157:H7. The objective of this study is to select aptamers specifically binding to E. coli O157:H7 using whole-bacterium SELEX (Systematic Evolution of Ligands by Exponential Enrichment) and to create a multivalent aptamer system by rolling circle amplification (RCA) with the selected aptamer sequence for sensitive detection of E. coli O157:H7 using a quartz crystal microbalance (QCM) sensor. Briefly, A total of 19 rounds of selection against live E. coli O157:H7 and 6 rounds of counter selection were performed for SELEX. One sequence S1 that appeared 16 (out of 20) times was characterized and a dissociation constant (Kd) of 10.30 nM was obtained. Using phi29 DNA polymerase, RCA reaction was performed, which produced a long ssDNA strand composed of thousands of repetitive aptamer sequences, termed as a multivalent aptamer system, on the electrode. The QCM sensor based on a multivalent aptamer system was able to quantitatively detect E. coli O157:H7. The limit of detection (LOD) of the QCM sensor was determined to be 34 CFU/ml, respectively, with the whole detection procedure in less than 40 min. The QCM sensor also showed high specificity for E. coli O157:H7 when it was cross-tested with five non-target bacteria. The QCM aptasensor in this study provided a common platform for detection of different foodborne pathogens

Advances, applications, and limitations of portable and rapid detection technologies for routinely encountered foodborne pathogens

Traditional foodborne pathogen detection methods are highly dependent on pre-treatment of samples and selective microbiological plating to reliably screen target microorganisms. Inherent limitations of conventional methods include longer turnaround time and high costs, use of bulky equipment, and the need for trained staff in centralized laboratory settings. Researchers have developed stable, reliable, sensitive, and selective, rapid foodborne pathogens detection assays to work around these limitations. Recent advances in rapid diagnostic technologies have shifted to on-site testing, which offers flexibility and ease-of-use, a significant improvement from traditional methods’ rigid and cumbersome steps. This comprehensive review aims to thoroughly discuss the recent advances, applications, and limitations of portable and rapid biosensors for routinely encountered foodborne pathogens. It discusses the major differences between biosensing systems based on the molecular interactions of target analytes and biorecognition agents. Though detection limits and costs still need further improvement, reviewed technologies have high potential to assist the food industry in the on-site detection of biological hazards such as foodborne pathogens and toxins to maintain safe and healthy foods. Finally, this review offers targeted recommendations for future development and commercialization of diagnostic technologies specifically for emerging and re-emerging foodborne pathogens

Development of a Bacteriophage-based Portable Biosensor for the Detection of Shiga-toxin Producing Escherichia coli (STEC) Strains in Food and Environmental Matrices

A fast and reliable on-site foodborne pathogens screening can reduce the incidence of foodborne illnesses, hospitalizations and economic loss. It can also circumvent conventional laboratory-based tests with minimal sample treatments and shorter turnaround time. Rapid detection of biological hazards has been largely dependent on immunological agents (ie antibodies). Antibodies are expensive to manufacture and experience cross-reactivity, instability with shorter shelf life. Our aim was to improve the screening process of Shiga-toxin producing Escherichia coli (STEC) strains in food and environmental matrices by developing a novel, inexpensive handheld bacteriophage-based amperometric biosensor that can directly detect live STEC cells.This biosensor development began by isolating STEC-specific bacteriophages from natural environmental samples (ie cow manure and surface water) hence, constructing a comprehensive bacteriophage isolates collection targeting an array of significant STEC serogroups. As an alternative to antibodies, purified bacteriophages could be easily and inexpensively propagated in a standard laboratory. Isolated bacteriophages were morphologically characterized while its physiologic behavior and specific host interactions were also investigated. The results indicated that majority of STEC-specific bacteriophages belong to Myoviridae and Siphoviridae families. Suitable bacteriophages for biosensor purposes were selected on the basis of the presence of head and tail and absence of virulence genes (stx1/stx2). Chemical modification via site-specific biotinylation of bacteriophage heads was performed prior to its biosensor incorporation. The results showed that biotinylation of bacteriophages did not reduce biofunctionality. Representative STEC O26, O157, and O179-specific biotinylated bacteriophages were immobilized onto the surface of streptavidin-modified screen-printed carbon electrodes (SPCE) to capture their target STEC cells. After STEC cells were bound to the capture elements, another set of biotinylated bacteriophages labeled with streptavidivin-horseradish peroxidase were added forming stable binding complexes which were then subjected to amperometric detection. The sandwich-type bacteriophage-based detection approach allowed live STEC cells rapid detection in microvolume samples (50 µL) via amperometric readouts (∆ current) between target and non-target bacteria in pure culture setup and complex matrices. With its simplicity and reliability, this technology can immensely assist the food industry and regulatory inspectors to efficiently maintain food safety in a fraction of the cost of traditional method

Aptamers for Diagnostics with Applications for Infectious Diseases

Aptamers are in vitro selected oligonucleotides (DNA, RNA, oligos with modified nucleotides) that can have high affinity and specificity for a broad range of potential targets with high affinity and specificity. Here we focus on their applications as biosensors in the diagnostic field, although they can also be used as therapeutic agents. A small number of peptide aptamers have also been identified. In analytical settings, aptamers have the potential to extend the limit of current techniques as they offer many advantages over antibodies and can be used for real-time biomarker detection, cancer clinical testing, and detection of infectious microorganisms and viruses. Once optimized and validated, aptasensor technologies are expected to be highly beneficial to clinicians by providing a larger range and more rapid output of diagnostic readings than current technologies and support personalized medicine and faster implementation of optimal treatments

Application of aptamers in diagnostics, drug-delivery and imaging

Aptamers are small, single-stranded oligonucleotides (DNA or RNA) that bind to their target with high specificity and affinity. Although aptamers are analogous to antibodies for a wide range of target recognition and variety of applications, they have significant advantages over antibodies. Since aptamers have recently emerged as a class of biomolecules with an application in a wide array of fields, we need to summarize the latest developments herein. In this review we will discuss about the latest developments in using aptamers in diagnostics, drug delivery and imaging. We begin with diagnostics, discussing the application of aptamers for the detection of infective agents itself, antigens/toxins (bacteria), biomarkers (cancer), or a combination. The ease of conjugation and labelling of aptamers makes them a potential tool for diagnostics. Also, due to the reduced off-target effects of aptamers, their use as a potential drug delivery tool is emerging rapidly. Hence, we discuss their use in targeted delivery in conjugation with siRNAs, nanoparticles, liposomes, drugs and antibodies. Finally, we discuss about the conjugation strategies applicable for RNA and DNA aptamers for imaging. Their stability and self-assembly after heating makes them superior over protein-based binding molecules in terms of labelling and conjugation strategies.Peer reviewe

Moving towards personalized medicine - the broad use of aptamers for targeted theranostic

Aptamers are short, single-stranded oligonucleotides synthesized in vitro from a randomized oligonucleotide library against a specific target. These molecules are capable of binding to a wide range of biological targets with high specificity and affinity. They present great advantages over antibodies with potential applications in research, diagnosis, and therapeutics. Specifically for tumors with late-stage identification and poor prognosis, like pancreatic cancer, the study of novel aptamers holds tremendous potential for cancer diagnosis and treatment. Along with cancer treatment, aptamers have also shown high potential in regulating the immune response and modulating several critical steps of signaling cascades, such as in immune checkpoints. In the context of microbiota and infection, aptamers are being studied to identify microbes and their metabolites. This assessment has the potential to improve the detection and management of infectious diseases while assisting us in better understanding health risks and treatment outcomes by tracking changes in the microbiota. In this review, the potential of aptamers is explored regarding their applications in cancer, immune, and microbiota therapy.Pilar Baylina (P.B.) acknowledges on the behalf of the authors the support of Fundação para a Ciência e Tecnologia (FCT), Portuguese Government, under the Strategic Project Reference: UID/BIM/04293/2013. Pilar Baylina (P.B.) and R.F. was also supported by FEDER/02/SAICT/2020/072560. Ana Cláudia Pereira (A.C.P.) acknowledges the support of Fundação para a Ciência e Tecnologia (FCT), Portuguese Government, under the Strategic Project Reference: 2022.09032.PTDC. André P. Sousa acknowledges FCT for the PhD grant 2022.12441.BD. Ana C. Rocha acknowledges FCT for the PhD grant 2021.06521.BD. Patrick P. Pais acknowledges FCT for the PhD grant 2021.09498.BD. Susana Ramalho acknowledges LaBMI for the PhD grant LABMI/BI/2021/01 (NEURO4COVID).info:eu-repo/semantics/publishedVersio

Simultaneous Colorimetric Detection of a Variety of Salmonella spp. in Food and Environmental Samples by Optical Biosensing Using Oligonucleotide-Gold Nanoparticles

Optical biosensors for rapid detection of significant foodborne pathogens are steadily gaining popularity due to its simplicity and sensitivity. While nanomaterials such as gold nanoparticles (AuNPs) are commonly used as signal amplifiers for optical biosensors, AuNPs can also be utilized as a robust biosensing platform. Many reported optical biosensors were designed for individual pathogen detection in a single assay and have high detection limit (DL). Salmonella spp. is one of the major causative agents of foodborne sickness, hospitalization and deaths. Unfortunately, there are around 2,000 serotypes of Salmonella worldwide, and rapid and simultaneous detection of multiple strains in a single assay is lacking. In this study, a comprehensive and highly sensitive simultaneous colorimetric detection of nineteen (19) environmental and outbreak Salmonella spp. strains was achieved by a novel optical biosensing platform using oligonucleotide-functionalized AuNPs. A pair of newly designed single stranded oligonucleotides (30-mer) was displayed onto the surface of AuNPs (13 nm) as detection probes to hybridize with a conserved genomic region (192-bases) of ttrRSBCA found on a broad range of Salmonella spp. strains. The sandwich hybridization (30 min, 55°C) resulted in a structural formation of highly stable oligonucleotide/AuNPs-DNA complexes which remained undisturbed even after subjecting to an increased salt concentration (2 M, final), thus allowing a direct discrimination via color change of target (red color) from non-target (purplish-blue color) reaction mixtures by direct observation using the naked eye. In food matrices (blueberries and chicken meat), nineteen different Salmonella spp. strains were concentrated using immunomagnetic separation and then simultaneously detected in a 96-well microplate by oligonucleotide-functionalized AuNPs after DNA preparation. Successful oligonucleotide/AuNPs-DNA hybridization was confirmed by gel electrophoresis while AuNPs aggregation in non-target and control reaction mixtures was verified by both spectrophotometric analysis and TEM images. Results showed that the optical AuNP biosensing platform can simultaneously screen nineteen (19) viable Salmonella spp. strains tested with 100% specificity and a superior detection limit of <10 CFU/mL or g for both pure culture and complex matrices setups. The highly sensitive colorimetric detection system can significantly improve the screening and detection of viable Salmonella spp. strains present in complex food and environmental matrices, therefore reducing the risks of contamination and incidence of foodborne diseases