Nonantibody-based recognition: alternative molecules for detection of pathogens

Immunoassays have been well established for many years as the cornerstone of detection technologies. These assays are sensitive, selective and, in general, highly resistant to interference from complex sample matrices when compared with nucleic acid-based tests. However, both antibody- and nucleic acid-based detection systems require a priori knowledge of the target and development of specific reagents; multiplexed assays can become increasingly problematic when attempting to detect a plethora of different targets, the identities of which are unknown. In an effort to circumvent many of the limitations inherent in these conventional assays, other recognition reagents are being explored as alternatives, or indeed as adjuncts, to antibodies for pathogen and toxin detection. This article will review a number of different recognition systems ranging in complexity from small molecules, such as nucleic-acid aptamers, carbohydrates and peptides, to systems as highly complicated as whole cells and organisms. All of these alternative systems have tremendous potential to achieve superior sensitivity, selectivity, and stability, but are also subject to their own limitations, which are also discussed. In short, while in its infancy, this field holds great promise for the development of rapid, fieldable assays that are highly complementary to existing antibody- and nucleic acid-based technologies.     

Advances towards integrated biodetection systems for environmental molecular microbiology

To overcome many of the limitations associated with indirect detection methods, new techniques for the sensitive, specific, and direct detection of nucleic acids are required in order to accurately and quantitatively ascribe phenotype/function to uncultivated microorganisms. However, if advanced diagnostic and detection systems are going to be applied in environmental microbiology, future "biodetection" technologies and systems must be developed not from the point of view of the detector, but from the unique aspects of the environmental sample and the entire analytical process. This article highlights recent advances in nucleic acid-based technologies, and looks towards future advances that may address the broad needs and conditions imposed by environmental molecular microbiology.     

Developing nucleic acid-based electrical detection systems

Development of nucleic acid-based detection systems is the main focus of many research groups and high technology companies. The enormous work done in this field is particularly due to the broad versatility and variety of these sensing devices. From optical to electrical systems, from label-dependent to label-free approaches, from single to multi-analyte and array formats, this wide range of possibilities makes the research field very diversified and competitive. New challenges and requirements for an ideal detector suitable for nucleic acid analysis include high sensitivity and high specificity protocol that can be completed in a relatively short time offering at the same time low detection limit. Moreover, systems that can be miniaturized and automated present a significant advantage over conventional technology, especially if detection is needed in the field. Electrical system technology for nucleic acid-based detection is an enabling mode for making miniaturized to micro- and nanometer scale bio-monitoring devices via the fusion of modern micro- and nanofabrication technology and molecular biotechnology. The electrical biosensors that rely on the conversion of the Watson-Crick base-pair recognition event into a useful electrical signal are advancing rapidly, and recently are receiving much attention as a valuable tool for microbial pathogen detection. Pathogens may pose a serious threat to humans, animal and plants, thus their detection and analysis is a significant element of public health. Although different conventional methods for detection of pathogenic microorganisms and their toxins exist and are currently being applied, improvements of molecular-based detection methodologies have changed these traditional detection techniques and introduced a new era of rapid, miniaturized and automated electrical chip detection technologies into pathogen identification sector. In this review some developments and current directions in nucleic acid-based electrical detection are discussed.     

Rapid diagnostic assays in the genomic biology era: detection and identification of infectious disease and biological weapon agents

In this special section of BioTechniques, we examine the role of rapid molecular technologies in the detection and identification of agents of infectious disease (ID) and biological weapons (BWs). Besides the threat posed by the global proliferation of BW technologies, there are numerous emerging and reemerging ID agents with significant public health consequences. Further compounding this already complicated situation are the estimated 600 million international tourists annually, many with the potential to the spread disease globally in a matter of hours. While clinical laboratories have key roles in the detection and identification of potential ID/BW agents, most staff are unfamiliar with these agents because of their rarity and the often laborious conventional methodologies needed to identify them. To meet this challenge, a vast array of rapid assay strategies has been developed for use in clinical diagnostics and environmental detection. Technologies have been developed or adapted to the challenges posed by these agents, permitting detection and identification in several minutes to hours. In particular, the development of improved reagents and detection systems has led to dramatic improvements in the sensitivity and specificity of immunological and nucleic acid-based systems, allowing an ever-increasing range of analytes to be identified and quantitated. In the accompanying articles, we have brought together experts from the many overlapping aspects of this arena in order to present a comprehensive and critical analysis of these technologies.     

A review of molecular recognition technologies for detection of biological threat agents

The present review summarizes the state of the art in molecular recognition of biowarfare agents and other pathogens and emphasizes the advantages of using particular types of reagents for a given target (e.g. detection of bacteria using antibodies versus nucleic acid probes). It is difficult to draw firm conclusions as to type of biorecognition molecule to use for a given analyte. However, the detection method and reagents are generally target-driven and the user must decide on what level (genetic versus phenotypic) the detection should be performed. In general, nucleic acid-based detection is more specific and sensitive than immunological-based detection, while the latter is faster and more robust. This review also points out the challenges faced by military and civilian defense components in the rapid and accurate detection and identification of harmful agents in the field. Although new and improved sensors will continue to be developed, the more crucial need in any biosensor may be the molecular recognition component (e.g. antibody, aptamer, enzyme, nucleic acid, receptor, etc.). Improvements in the affinity, specificity and mass production of the molecular recognition components may ultimately dictate the success or failure of detection technologies in both a technical and commercial sense. Achieving the ultimate goal of giving the individual soldier on the battlefield or civilian responders to an urban biological attack or epidemic, a miniature, sensitive and accurate biosensor may depend as much on molecular biology and molecular engineering as on hardware engineering. Fortunately, as this review illustrates, a great deal of scientific attention has and is currently being given to the area of molecular recognition components. Highly sensitive and specific detection of pathogenic bacteria and viruses has increased with the proliferation of nucleic acid and immuno-based detection technologies. If recent scientific progress is a fair indicator, the future promises remarkable new developments in molecular recognition elements for use in biosensors with a vast array of applications.     

Current and future hepatitis C virus diagnostic testing: problems and advancements

Serological antibody assays used in hepatitis C virus diagnosis have improved in sensitivity and specificity. However, detection of active viremia or monitoring levels of virus during or after patient treatment is most commonly undertaken using nucleic acid-based technologies. Advancements in diagnostic technologies and implications for managing patients with hepatitis C in various clinical settings are discussed.     

Semi-synthetic nucleic acid-protein conjugates: applications in life sciences and nanobiotechnology

Semi-synthetic conjugates of nucleic acids and proteins can be generated by either covalent coupling chemistry, or else by non-covalent biomolecular recognition systems, such as receptor-ligands of complementary nucleic acids. These nucleic acid-protein conjugates are versatile molecular tools which can be applied, for instance, in the self-assembly of high-affinity reagents for immunological detection assays, the fabrication of laterally microstructured biochips containing functional biological groups, and the biomimetic 'bottom-up' synthesis of nanostructured supramolecular devices. This review summarizes the current state-of-the-art synthesis and characterization methods of artificial nucleic acid-protein conjugates, as well as applications and perspectives for future developments of such hybrid biomolecular components in life sciences and nanobiotechnology.     

A field-deployable method for single and multiplex detection of DNA or RNA from pathogens using Cas12 and Cas13

For some Cas nucleases, trans-cleavage activity triggered by CRISPR/Cas-mediated cis-cleavage upon target nucleic acid recognition has been explored for diagnostic detection. Portable single and multiplex nucleic acid-based detection is needed for crop pathogen management in agriculture. Here, we harnessed and characterized RfxCas13d as an additional CRISPR/Cas nucleic acid detection tool. We systematically characterized AsCas12a, LbCas12a, LwaCas13a, and RfxCas13d combined with isothermal amplification to develop a CRISPR/Cas nucleic acid-based tool for single or multiplex pathogen detection. Our data indicated that sufficient detection sensitivity was achieved with just a few copies of DNA/RNA targets as input. Using this tool, we successfully detected DNA from Fusarium graminearum and Fusarium verticillioides and RNA from rice black-streaked dwarf virus in crude extracts prepared in the field. Our method, from sample preparation to result readout, could be rapidly and easily deployed in the field. This system could be extended to other crop pathogens, including those that currently lack a detection method and have metabolite profiles that make detection challenging. This nucleic acid detection system could also be used for single-nucleotide polymorphism genotyping, transgene detection, and qualitative detection of gene expression in the field.

     

Selection and characterization of peptide memitopes binding to ricin

A combinatorial random peptide display library expressed in E. coli was employed to identify short, linear peptide sequences that showed affinity for ricin and could be used as reagents for detection and identification of ricin. One peptide, P3, from a collection of four short peptides showed specific binding to ricin. The kinetic analysis of this peptide binding to the ricin showed lower equilibrium binding constants for the peptide P3 than monoclonal antibody. This is attributed due to both slower association and faster dissociation rates for the peptide P3. The random ricin peptide P3 binds to ricin with a K_D of 1 M versus the antibody's K_D of 14 nM. This particular peptide memitope P3 against ricin showed specific binding to ricin without any significant cross-reactivity against other proteins such as bovine serum albumin (BSA), lysozyme and natural bacterial toxins such as Staphylococcal enterotoxins A and B. The results provided proof-of-principal that peptide memitopes are another choice of reagents due to ease in production to be used for the detection of highly toxic bio-threat or biowarfare agents such as ricin.     

In vitro selection of high-affinity nucleic acid ligands to parasite target molecules

The logic of using nucleic acids as pharmaceutical reagents is in part based on their capacity to interact with high affinity and specificity with other biological components. Considerable progress has been made over the past 10 years in the development of nucleic acid-based drug molecules using a variety of different technologies. One approach is a combinatorial technology that involves an iterative Darwinian-type in vitro evolution process, which has been termed SELEX for 'systematic evolution of ligands by exponential enrichment'. The procedure is a highly efficient method of identifying rare ligands from combinatorial nucleic acid libraries of very high complexity. It allows the selection of nucleic acid molecules with desired functions and it has been instrumental in the identification of a number of synthetic DNA and RNA molecules, so-called aptamers that recognise ligands of different chemical origin. The method is fast, it does not require special equipment and the selected aptamers typically bind their target with high affinity and high specificity. Here we summarise the recent examples of the SELEX technique within the context of identifying high-affinity ligands against parasite target molecules.     

In vitro selection of nucleic acids for diagnostic applications

In vitro selection methods have proven to be extraordinarily adept at generating a wide variety of nucleic acid-binding species (aptamers) and catalysts (ribozymes). To date, selected nucleic acids have primarily been of academic interest. However, just as antibodies have proven utility as ‘universal receptors’ that can be crafted against a huge variety of ligands and can be readily adapted to diagnostic assays, aptamers may yet find application in assays. A new class of research reagents, aptazymes, are not mere mimics of antibodies but in fact allow the direct transduction of molecular recognition to catalysis. Aptamers and aptazymes may prove to be uniquely useful for the development of chip arrays for the detection and quantitation of a wide range of molecules in organismal proteomes and metabolomes.     

蛋白激酶的生化活性检测方法研究进展

蛋白激酶在真核细胞信号转导中起重要作用,影响了生长、发育、迁移以及凋亡等各个细胞过程。其表达水平或活性异常时,就有可能导致癌症、心血管疾病以及其他各种疾病,因此蛋白激酶是治疗这些疾病很好的分子靶点。迄今为止,美国食品药品监督管理局已经批准了28个蛋白激酶的抑制剂作为上市药物,用于相应的临床治疗。目前存在着各种检测激酶活性的方法,激酶生化检测方法尤为众多,比较经典的有放射性同位素的方法,也有一些非均相非放射性同位素的方法,诸如酶联免疫法、反相高效液相色谱法、核磁共振分析法等等。而各种均相非放射性同位素的检测方法,由于其污染小、操作便捷,逐渐成为激酶抑制剂筛选的首选。本文综述了各种激酶生化检测方法及其发展历史,并介绍了一些新的趋势,如激酶酶谱筛选。     

Lipid-based systems for the intracellular delivery of genetic drugs

Currently available delivery systems for genetic drugs have limited utility for systemic applications. Cationic liposome/ plasmid DNA or oligonucleotide complexes are rapidly cleared from circulation, and the highest levels of activity are observed in `first pass' organs, such as the lungs, spleen and liver. Engineered viruses can generate an immune response, which compromises transfection resulting from subsequent injections and lack target specificity. A carrier, which can accumulate at sites of diseases such as infections, inflammations and tumours, has to be a small, neutral and highly serum-stable particle, which is not readily recognized by the fixed and free macrophages of the reticuloendothelial system (RES). This review summarizes lipid-based technologies for the delivery of nucleic acid-based drugs and introduces a new class of carrier systems, which solve, at least in part, the conflicting demands of circulation longevity and intracellular delivery. Plasmid DNA and oligonucleotides are entrapped into lipid particles that contain small amounts of a positively charged lipid and are stabilized by the presence of a polyethylene glycol (PEG) coating. These carriers protect nucleic acid-based drugs from degradation by nucleases, are on average 70 nm in diameter, achieve long circulation lifetimes and are capable of transfecting cells.     

核酸自组装纳米递送载体的研究进展

随着核酸纳米技术的飞速发展,核酸自组装纳米载体已成为药物递送领域的研究热点。针对核酸自组装纳米载体在药物递送中的应用进展进行了系统综述,讨论了不同的核酸自组装策略,阐述了多种靶向递送和药物控制释放方法,同时,总结了核酸自组装纳米递送载体在蛋白质药物、核酸药物、小分子药物和纳米药物递送中的应用,并针对该领域的挑战和未来发展趋势进行了总结和展望,以期为药物递送领域和新型药物系统研究提供参考。     

单核细胞增生李斯特菌的检测技术进展

单核细胞增生李斯特菌（Listeria monocytogenes）是一类人畜共患的食源性致病菌。近年来其检测技术取得了迅猛的发展,本文对目前使用的基于培养、免疫学和分子生物学技术的三大类单核细胞增生李斯特菌检测方法进行了综述,同时对单核细胞增生李斯特菌检测的新策略进行了展望。     

Advances in Candida detection platforms for clinical and point-of-care applications

Invasive candidiasis remains one of the most serious community and healthcare-acquired infections worldwide. Conventional Candida detection methods based on blood and plate culture are time-consuming and require at least 2–4 days to identify various Candida species. Despite considerable advances for candidiasis detection, the development of simple, compact and portable point-of-care diagnostics for rapid and precise testing that automatically performs cell lysis, nucleic acid extraction, purification and detection still remains a challenge. Here, we systematically review most prominent conventional and nonconventional techniques for the detection of various Candida species, including Candida staining, blood culture, serological testing and nucleic acid-based analysis. We also discuss the most advanced lab on a chip devices for candida detection.     

Better together: Strategies based on magnetic particles and quantum dots for improved biosensing

Novel technologies and strategies for sensitive detection of biological responses in healthcare, food and environmental monitoring continue to be a priority. The present review focuses on bioassay development based on the simultaneous use of quantum dots and magnetic beads. Due to the outstanding characteristics of both particles for biosensing applications and the large number of publications using a combined approach, we aim to provide a comprehensive overview of the literature on different bioassays, the most recent advances and innovative strategies on the topic, together with an analysis of the main drawbacks encountered and potential solutions offered, with a special emphasis on the requirements that the transfer of technologies from the laboratory to the market will demand for future commercialization of biodevices. Several procedures used in immunoassays and nucleic acid-based bioassays for the detection of pathogens and biomarkers are discussed. The improvement of current approaches together with novel multiplex detection systems and nanomaterials-based research, including the use of multimodal nanoparticles, will contribute to simpler and more sensitive bioanalyses.