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Advanced Biosensors for Health Care Applications
Автор: Elsevier Science
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Advanced Biosensors for Health Care Applications highlights the different types of prognostic and diagnostic biomarkers associated with cancer, diabetes, Alzheimer's disease, brain and retinal diseases, cardiovascular diseases, bacterial infections, as well as various types of electrochemical biosensor techniques used for early detection of the potential biomarkers of these diseases. Many advanced nanomaterials have attracted intense interests with their unique optical and electrical properties, high stability, and good biocompatibility. Based on these properties, advanced nanoparticles have been used as biomolecular carriers, signal producers, and signal amplifiers in biosensor design. Recent studies reported that there are several diagnostic methods available, but the major issue is the sensitivity and selectivity of these approaches.
This book outlines the need of novel strategies for developing new systems to retrieve health information of patients in real time. It explores the potential of nano-multidisciplinary science in the design and development of smart sensing technology using micro-nanoelectrodes, novel sensing materials, integration with MEMS, miniaturized transduction systems, novel sensing strategy, that is, FET, CMOS, System-on-a-Chip (SoC), Diagnostic-on-a-Chip (DoC), and Lab-on-a-Chip (LOC), for diagnostics and personalized health-care monitoring. It is a useful handbook for specialists in biotechnology and biochemical engineering.
Describes advanced nanomaterials for biosensor applications Relates the properties of available nanomaterials to specific biomarkers applications Includes diagnosis and electrochemical studies based on biosensors Explores the potential of nano-multidisciplinary science to design and develop smart sensing technologies Describes novel strategies for developing a new class of assay systems to retrieve the desired health informationАктивность, связанная с книгой
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Advanced Biosensors for Health Care Applications
Автор: Elsevier Science
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Advanced Biosensors for Health Care Applications highlights the different types of prognostic and diagnostic biomarkers associated with cancer, diabetes, Alzheimer's disease, brain and retinal diseases, cardiovascular diseases, bacterial infections, as well as various types of electrochemical biosensor techniques used for early detection of the potential biomarkers of these diseases. Many advanced nanomaterials have attracted intense interests with their unique optical and electrical properties, high stability, and good biocompatibility. Based on these properties, advanced nanoparticles have been used as biomolecular carriers, signal producers, and signal amplifiers in biosensor design. Recent studies reported that there are several diagnostic methods available, but the major issue is the sensitivity and selectivity of these approaches.
This book outlines the need of novel strategies for developing new systems to retrieve health information of patients in real time. It explores the potential of nano-multidisciplinary science in the design and development of smart sensing technology using micro-nanoelectrodes, novel sensing materials, integration with MEMS, miniaturized transduction systems, novel sensing strategy, that is, FET, CMOS, System-on-a-Chip (SoC), Diagnostic-on-a-Chip (DoC), and Lab-on-a-Chip (LOC), for diagnostics and personalized health-care monitoring. It is a useful handbook for specialists in biotechnology and biochemical engineering.
Describes advanced nanomaterials for biosensor applications Relates the properties of available nanomaterials to specific biomarkers applications Includes diagnosis and electrochemical studies based on biosensors Explores the potential of nano-multidisciplinary science to design and develop smart sensing technologies Describes novel strategies for developing a new class of assay systems to retrieve the desired health informationОтрывок книги
Advanced Biosensors for Health Care Applications
China
Preface
Advanced materials nanotechnology is a promising and developing field that utilizes nanoparticles to encourage the treatment and/or diagnosis of different diseases, for example, cancer, diabetes, osteoarthritis, cerebrum, retinal and cardiovascular diseases, and bacterial infections. Advanced nanosensor technology improves everyday lifestyles via specially personalized health diagnostic and monitoring systems. Research communities from different fields including material science, physics, chemistry, and biology have met up to develop more modern, sophisticated, practically viable, and realistic diagnostic and monitoring devices. There are several diagnostic methods available to manage these devise’s major requirements such as sensitivity and selectivity. However, biosensors are important sensing tools that have received much interest due to their high sensitivity and selectivity and have inspired new thoughts for creating novel sensing materials, hardware gadgets, scaled-down transduction techniques, and gadget integration to create innovative future diagnostic systems. A biosensor is a small device designed to detect a chemical or biochemical target molecule. It is widely used as a powerful analytical tool in medical research, clinical diagnosis, environmental testing, agriculture, food quality control, bioprocess monitoring, and development of new pharmaceuticals, among others.
This book is an endeavor to portray the requirement for novel procedures for building up another class of assay system to retrieve the desired health information of the patient in real time. This book explores the potential of multidisciplinary science to design and develop smart-sensing technology using micro or nanoelectrodes, novel sensing materials, integration with microelectromechanical systems (MEMS), miniaturized transduction systems, novel sensing strategies such as field-effect transistor (FET), complementary metal–oxide–semiconductor (CMOS), system-on-a-chip (SoC), diagnostic-on-a-chip (DoC), and lab-on-a-chip (LOC) for diagnostics and personalized healthcare monitoring. These ongoing advancements on the nanoscale biomedical determination gadgets portrayed in this book will enable us to introduce these innovations into the market in the near future. Therefore we have highlighted different types of prognostic and diagnostic biomarkers associated with cancer, diabetes, Alzheimer’s disease, brain, retinal, and cardiovascular diseases, bacterial infections, and various types of electrochemical biosensor techniques used for early detection of the potential biomarkers of specific diseases.
There are 15 chapters written by the world’s foremost authors in this multidisciplinary subject. Some of the contributing authors are from India, Malaysia, Japan, Portugal, Greece, Spain, China, South Africa, Canada, Romania, Turkey, Germany, the United States, South Korea, Australia, and others, and are associated with academia, government, and industry. This book is planned with wide scope of gathering of people having an engineering or science background. This book will be valuable as a reference to researchers and engineers already experienced in the field or as a primer to researchers and graduate students just getting started in the art and science of electromechanical miniaturization. The applications of biosensors in the areas such as detection of foodborne pathogens, aptamer biosensor, genobiosensor, metal-based biomarker detection, glucose biosensor and breast cancer biomarkers, are discussed in details. The book is arranged according to these particular themes:
Chapter 1 discusses recent advances in biosensors for detecting foodborne pathogens found in food, beverages, and clinical samples.
Chapter 2 summarizes the various kinds of aptamer-based biosensors working on different transduction principles (e.g., optical, electrochemical, piezoelectrical, acoustic and cantilever sensors) for the detection of foodborne pathogens and their toxins.
Chapter 3 aims to provide insights into the current developments on breast cancer and the role of biosensors in its rapid detection, discussing the disease’s epidemiology, types, biomarkers, and current diagnosis. This chapter provides an overview of the different types of biosensors and their applicability in breast cancer detection.
Chapter 4 reviews the design and the use of electrochemical biosensors as diagnostic and decision-making tools in selected healthcare applications like the monitoring and quantification of various reactive oxygen species, assessment of the antioxidant capacity of plants and foods, and the electroanalysis of polyphenols in beverages. A discussion on these topics and the relationships between antioxidants and healthcare is also provided.
Chapter 5 discusses recent advances in the development of electrochemical immunosensors for quantification of clinically relevant protein biomarkers of breast cancer tumors. Details regarding multilabeled detection antibodies (Ab2) coupled with specific capture antibodies (Ab1) at sensor surfaces and electrochemical screening of biomarkers as antigens are discussed. Use of nanostructured surfaces, nanoparticle labels, enzyme labels, and magnetic beads are emphasized as signal amplification strategies. Possible opportunities for further improvement in the area of electrochemical immunoassays toward cancer diagnostics are also emphasized.
Chapter 6 discusses multifunctional, advanced, hybrid materials applied in optical biosensors design and development emphasizing the sensing methods based on fluorescence, chemiluminescence, electrochemiluminescence, photoelectrochemical materials, luminescent optical labels, surface plasmon resonance, and surface-enhanced Raman scattering applications.
Chapter 7 reports a short review of the current research in noninvasive sweat and gas sensors for healthcare. Recent studies on the detection of various sweat biomarkers such as glucose, lactose, and metal ions using wearable sensors are explored. Respiration sensing using humidity in breath to monitor sleep disorders as well as cardiovascular and pulmonary diseases are discussed. Selective VOC sensors and an array of sensors to detect specific diseases and discrimination of multiple diseases are reviewed. Finally, the use of ingestible sensors, an emerging and convenient technology, is described to diagnose various diseases using gas profiles in the gut. Further, the challenges and future scope of these diagnosing techniques are discussed.
Chapter 8 discusses the development of aptamer-based biosensor technologies that can surpass the conventional in vitro diagnostic for disease detection and health monitoring.
Chapter 9 discusses biosensing–drug delivery systems for in vivo applications. Different types of materials, devices, sensors, and case studies are discussed.
Chapter 10 deals with nanobodies and their in vivo applications. Nanobody characteristics and their binding to transmembrane proteins are highlighted. The applications of nanobodies in areas such as medical imaging, inflammatory diseases, and chronic respiratory diseases are discussed. The applications of nanobodies against viruses and bacteria are also highlighted. Finally, challenges with nanobodies and future directions are reviewed.
Chapter 11 presents recent advances of micro and nanotechnologies for electrochemical biosensor development, including microfluidics, graphene, graphitic carbon nitride, and quantum dots. The applications of quantum dots, graphene, and graphitic carbon nitride in electrochemical biosensors, such as enzyme, DNA, and immunosensing are summarized. The combination of microfluidics and electrochemical sensing in recent years are also introduced.
Chapter 12 highlights the cholesterol-enabled enzymatic and nonenzymatic sensors to understand the complex interface among cholesterol and bile acids absorptions. The main focus of this chapter is to briefly discuss the advances of cholesterol-based sensors and their applications.
Chapter 13 explores sensors for health and agricultural applications. The global market scenario for smart sensors and future prospects of sensors are also highlighted.
Chapter 14 reviews the recent progress in synthesis strategies and physicochemical properties of 3D graphene, and its hybrid materials functionalized with different inorganic metallic particles together with its applications for the rapid detection of various chemicals and biomolecules at low concentrations. The sensing mechanism and recyclability of sensing performance of graphene-based hybrid electrodes are also discussed.
Chapter 15 reviews the principles of nanosensors by describing their classification, principle of operation, and examples of various nanosensors. It also provides directions for research in this field and justifies their practical implementation in sensing by highlighting current challenges and future prospects.
This book is the consequence of the commendable cooperation of authors from various interdisciplinary fields of science. It thoroughly examines the most generous, start-to-finish, and forefront research and reviews. We are thankful to all the contributing authors and their coauthors for their commitment. We also thank all copyright holders, authors, and other individuals who agreed for us to use their figures, tables, and schemes. Although every effort has been made to secure copyright permissions from the individual copyright holders to use figures/tables/schemes we should need to offer our sincere proclamations of disappointment to them if, unintentionally, their benefit is being infringed.
Editors
Chapter 1
Advanced Nanoparticle-Based Biosensors for Diagnosing Foodborne Pathogens
Mohammad Lukman Yahaya¹, Rahmah Noordin² and Khairunisak Abdul Razak¹,²*, ¹School of Materials and Mineral Resources Engineering, Universiti Sains Malaysia, Penang, Malaysia, ²Nanobiotechnology Research and Innovation (NanoBRI), Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Penang, Malaysia
Abstract
As the world population increases, the demand for food supply increases. Thus food safety is a global concern. To ensure that the food supply is safe from pathogens, rapid, reliable, robust, sensitive, and specific diagnostic tools are required. Recently, biosensor technology was applied to develop these tools that offer more advantages than traditional and established methods. This chapter reviews recent advances in biosensors with nanoparticles for detecting foodborne pathogens found in foods, beverages, or clinical samples. The review briefly discusses established and traditional methods and types of biosensors, such as bioreceptors and transducers. Membrane-based biosensors are also addressed as a special type of biosensor. The discussion focuses on the principles, advantages, sensitivity, specificity, and drawbacks of these biosensors. The multiplexing potential of such biosensors is also explained in detail. In conclusion, nanoparticle-based biosensors with high sensitivity and specificity can be alternative tools for diagnosing foodborne pathogens to overcome the limitations of conventional methods.
Keywords
Biosensors; nanoparticle; foodborne pathogen; rapid diagnosis; multiplex
1.1 Introduction
The demand for pathogen-free food and beverages is increasing nowadays. Thus concern for disease transmission, which increases the potential for foodborne outbreaks and other associated health issues, has risen across boundaries. To solve this problem, all food and beverages must be screened to ensure that they are free from potential pathogens before entering the market. Some of these pathogens and their screening methods are discussed in the next section.
1.1.1 Typical Foodborne Pathogens
Bacterial pathogens have caused human illnesses over the past few decades through the consumption of undercooked or minimally processed ready-to-eat meats, dairy products, or fruits and vegetables [1]. The presence of pathogens in ready-to-eat products is a serious concern because these products generally do not receive any further treatment before being eaten. Animals and poultry are the most significant reservoir for many foodborne pathogens [2], although animal by-products, such as feed supplements, may also transmit pathogens to other animals. Seafood is another potential source of pathogens such as Vibrio, Listeria, Yersinia, Salmonella, Shigella, Clostridium, Campylobacter, and the hepatitis A virus [3].
One of the common foodborne pathogens is Escherichia coli. Pathogenic E. coli is composed of six main groups namely, enterohemorrhagic (EHEC), enterotoxigenic (ETEC), enteropathogenic (EPEC), enteroinvasive (EIEC), enteroaggregative (EAEC), and diffusely adherent E. coli (DAEC) [4]. Among them, E. coli of serogroup O157:H7 (E. coli O157:H7) is most frequently found to cause foodborne diseases that come from the EHEC group [5]. All members of EHEC, including E. coli O157:H7, can produce Shiga toxins (Stxs) that can cause hemorrhagic colitis, diarrhea associated with abdominal cramps, presentation of mild fever, thrombotic thrombocytopenic purpura, and life-threatening hemolytic uremic syndrome (HUS) in humans [5,6]. The United States Department of Agriculture and Food Safety and Inspection Service (USDA-FSIS) limits the detection of E. coli O157:H7 to one colony-forming unit (CFU) per 65 g of a sample of meat [7].
Salmonella spp. are Gram-negative, rod-shaped bacteria, and common foodborne pathogens. Salmonella enterica and Salmonella bongori are two species under the Salmonella genus of the Enterobacteriaceae family. This genus can be further divided into many serotypes, and all of the strains are potentially pathogenic to humans [8]. Salmonella spp. are usually found in dairy and farm products. Salmonella spp. can cause salmonellosis with symptoms of diarrhea, fever, and abdominal cramps. Furthermore, salmonellosis that infects other parts of the body other than the intestines can result in life-threatening and fatal infections [7,8]. However, Salmonella enteritidis and S. typhimurium, as well as S. enterica serovar Typhi, are epidemiologically important as the highest causative agents in human infections worldwide [7,9].
Another genus of the Enterobacteriaceae family that is responsible for foodborne disease is Shigella spp. This genus is composed of four species, namely, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, and Shigella boydii. Shigella spp. cannot be detected in animals because it only infects humans and other primates [10]. Disease transmission mainly occurs via food contaminated with human feces. When an individual becomes infected, Shigella spp. can invade the intestinal cells, causing inflammation and tissue damage [10,11]. Clinical manifestations vary among species. S. dysenteriae causes dysentery with complications, such as HUS, S. flexneri and S. boydii also cause dysentery but without these complications, whereas S. sonnei causes watery diarrhea [10].
Vibrio spp. are also responsible for foodborne diseases. The three species that commonly infect humans are Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus. Vibrio spp. usually contaminate water and seafood and transmit to humans via consumption. Ingestion of contaminated food with Vibrio spp. can cause gastroenteritis and septicemia [12,13]. Meanwhile, Campylobacter spp. are Gram-negative, spiral, and microaerophilic bacteria. A species that is clinically noteworthy as a human enteropathogen is Campylobacter jejuni, which infects poultry, dairy products, and milk. In addition, C. jejuni can attack the peripheral nervous system, causing partial paralysis [7,14].
Other foodborne bacteria that may infect humans are Gram-positive bacteria such as Bacillus cereus, Clostridium botulinum, Clostridium perfringens, and Staphylococcus aureus, and Gram-negative bacteria such as Arcobacter, Helicobacter, and Yersinia enterocolitica. Furthermore, bacterial toxins from Fusarium and fungi such as Aspergillus are also causative agents. Some viruses, such as the hepatitis A virus, and parasites also play a significant role in foodborne diseases [15,16].
1.1.2 Established and Traditional Diagnostic Tools
Established and traditional methods for bacterial testing rely on specific media to enumerate and isolate viable bacterial cells in food. These methods are extremely sensitive and inexpensive and offer both qualitative and quantitative information on the number and nature of microorganisms presence in a food sample [17]. Traditional methods for detecting bacteria involve some basic steps, namely pre-enrichment, selective plating, biochemical screening, and serological confirmation [18]. Hence a complete series of test is often required before any identification can be confirmed [19]. These traditional methods require several days to yield results because they rely on the ability of the organism to multiply into visible colonies [20]. Moreover culture medium preparation, inoculation of plates, and colony counting make these methods labor intensive. Traditional methods that are generally regarded as the golden standard often take days to completely identify viable pathogens. Any modification that reduces the analysis time can technically be called a rapid method.
Other established methods include molecular methods such as polymerase chain reaction (PCR) and sequencing analysis, enzyme-linked immunosorbent assay (ELISA), immunological techniques, and fluorescence-based assay using organic dye molecules [21]. These methods offer more rapid analyses compared to traditional methods. PCR is a powerful tool that allows species-specific detection of organisms based on nucleic acid amplification by using a small amount of target DNA. Different types of PCR are commonly used for detecting foodborne pathogens; specifically, real-time PCR uses fluorescent dyes to measure the amount of amplified product as amplification progresses [22,23], reverse transcriptase PCR (RT-PCR) is used to reverse-transcribe and amplify RNA to cDNA [24], and multiplex PCR simultaneously detects several pathogens in one reaction [25,26].
Although amplifying DNA derived from pure cultures is easy, problems arise if the sample is complex (such as food, soil, or biological waste) because PCR is easily inhibited by numerous substances, including humic acids, fats, and proteins [27]. Moreover PCR must be followed by agarose gel electrophoresis to view the results. This viewing method is tedious and uses hazardous chemicals, such as ethidium bromide. To overcome these disadvantages, a combination of PCR and other methods, such as biosensors is recommended [28].
Immunological techniques [29,30] and fluorescence-based assays involving organic dye molecules [31,32] are other established methods used for foodborne pathogen detection. These techniques are based on antigen–antibody bindings (immunodetection). Several methods have been developed using these techniques, which are commercially available and dependent on antibody type and format, such as ELISA, enzyme immunoassay (EIA), enzyme-linked fluorescent assay (ELFA), and bioluminescent enzyme immunoassay (BEIA) [21].
ELISA—including direct ELISAs, sandwich ELISAs, and competitive ELISAs—is the most common format used for the immunodetection of pathogens [33]. Many of these ELISA methods are available as commercial kits and approved by regulatory agencies. ELISA-based analyses can be directly applied for detecting foodborne pathogens. ELISAs combine the specificity of antibodies and the sensitivity of simple enzyme assays by using antibodies or antigens coupled to an easily assayed enzyme. However, a major drawback of these assay formats is their lengthy analysis time. Typical ELISAs comprise many steps such as blocking, washing, incubation, and substrate development. These steps can take several hours to complete and are understandably problematic in instances where rapid detection is required [34].
Although immunodetection-based methods are highly accurate, sensitive, and error proof, they are time- and labor-intensive and require skilled personnel, sophisticated equipment, and do not always provide the required detectability and specificity toward the target. Moreover these techniques require several enrichment steps and subsequent biochemical and serological identification; hence, the cost for analysis is high. Recent advances in immunodetection combine the established and traditional methods with biosensors as ideal tools for detecting foodborne pathogens [35–37]. Basic information on nanoparticles and biosensors is provided next.
1.1.3 Nanoparticles and Biosensors
Nanoparticles (NPs) are particles with diameters in the range of 1–100 nm according to the International Organization for Standardization (ISO) [38]. NPs possess several distinct physical and chemical properties that make them promising synthetic scaffolds to create novel chemical and biological detection systems. Over the past few years, nanostructured materials, such as noble metal NPs, quantum dots, and magnetic NPs, have been employed in a broad spectrum of highly innovative approaches for assays of metal ions, small molecules, proteins, and nucleic acid biomarkers. In addition, NPs can be fashioned with a wide range of small organic ligands and large biomacromolecules by using tools and techniques of surface modification. Each of these capabilities has allowed researchers to design novel diagnostic systems that offer significant advantages in terms of sensitivity, selectivity, reliability, and practicality [38,39].
A typical interaction between NPs and pathogens is via antibody–antigen recognition. For bacteria, many surface antigens are available for specific recognition by using antibody-conjugated NPs. NPs, such as gold (AuNPs), silver (AgNPs), quantum dots (QDs; e.g., CdSe–ZnS), magnetic beads (Fe3O4), up-converting phosphor NPs (UCPs), dye-doped NPs, fluorescent-silica NPs (FSNPs), and carbon nanotubes (CNTs) of various shapes, including nanoshells, can be used to construct NP-based assays [39]. Table 1.1 compares the most remarkable NPs used in NP-based lateral-flow biosensors [40].
Table 1.1
Biosensors involve any analytical device that incorporates biologically recognized molecules (bioreceptors) such as antibodies, aptamers, phages, nucleic acid (DNA), enzymes, or biomimics with a physical or chemical signaling device (transducer). Bioreceptors are the first element in biosensor systems that recognize targeted analyte in samples. The transducing mechanism converts the signal or recognition event from the bioreceptor into an interpretable result, either visually or as electrical signal to display and record data. Types of commonly used transducers in a biosensor system include electrochemical, optical, magnetic, thermometric, piezoelectric, or micromechanical transducers [7,21,33]. The types of bioreceptors and transducers are discussed in detail in Section 1.2.
In brief, the principle of biosensors recognizing pathogens in food is simple. The targeted pathogens in the sample are recognized or captured by a specific bioreceptor. This bioreceptor then responds and triggers the transducer, which is usually in contact with the bioreceptor. The transducer then responds to the input change and converts it into signal output. The signal can be visually recognized by the naked eye or be amplified on an electronic display. Finally, the results can be stored and analyzed [21].
Biosensors possess a number of characteristics, including rapid process, sensitive, specific, accurate, near real-time assay, reproducible, robust, and user friendly. Rapidity of the assay in biosensors is important to differentiate biosensors from conventional methods. Any diagnostic tool must have excellent sensitivity, specificity, and accuracy to avoid or minimize false-positive and false-negative results that can cause an expensive recall or loss of credibility. Moreover, desirable biosensors must provide reproducible measurements along an extended shelf life to ensure the accuracy of the test. The bioreceptor and transducer should be biochemically and mechanically stable, robust, and have a long shelf life. Finally, the device should be user friendly and the given results should be easy to interpret. These factors can avoid or reduce the need for high levels of training and skilled personnel [7,21].
1.2 Types of Bioreceptors
As mentioned, a bioreceptor is the most important element in biosensors because it is the first element that is in contact with the sample to be analyzed. Bioreceptors can recognize biological material and produce recognition events. Bioreceptors are later recognized by transducers because a transducer itself cannot recognize biological material. This section discusses the main bioreceptors such as antibodies, nucleic acids, enzymes, and aptamers used in biosensor devices.
1.2.1 Antibodies
In general, antibodies can be classified according to their production method, namely, polyclonal, monoclonal, and recombinant antibodies [34]. To produce specific antibodies for bacterial pathogens, living hosts are needed. The host can be immunized with bacterial cells, which may or may not be heat treated. Serum from that host is then collected. Polyclonal antibodies derived from different B cells recognize multiple epitopes on the same antigen. Each of these individual antibodies recognizes a unique epitope that is located on that antigen. Polyclonal antibodies are typically raised in rabbits, goats, or sheep hosts and they are frequently selected in immunosensor-based assays for pathogen detection. However, polyclonal antibodies have a high potential for cross-reactivity due to their ability to recognize multiple epitopes.
In cases where high specificity is required, monoclonal or recombinant antibodies are more applicable [34]. Monoclonal antibodies represent antibodies from a single antibody-producing B-cell, so they only bind with one unique epitope. Each individual antibody in a polyclonal mixture is technically a monoclonal antibody. However, this term generally refers to a process by which the actual B-cell is isolated and fused to an immortal hybridoma cell line, such that numerous identical antibodies can be generated.
Recombinant antibodies (rAbs) are monoclonal antibodies generated in vitro using synthetic genes. The main difference is that monoclonal antibodies are produced using hybridoma-based technologies. However, rAbs do not need hybridomas and animals in the production process. rAbs are produced in vitro using synthetic genes. The synthesis process involves recovering antibody genes from source cells and amplifying and cloning the genes into an appropriate phage vector. The vector is then introduced into a host and the expression of an adequate amount of functional antibody is achieved. Some advantages of using rAbs are animal-free technology, increased antibody production and control, and decreased production time and isotype of antibodies, which can easily be converted into a different species, isotype, or subtype [34,41].
Antibodies are usually immobilized on a substrate, either on a detector surface, carrier, or its vicinity, of the biosensor device [33]. Three ways are available for this immobilization process, namely, adsorption on gold, avidin–biotin system, and self-assembled monolayers (SAMs). In the adsorption process, a clean gold (Au) surface is immersed in an antibody solution and washed. A sample can then be added and the corresponding pathogen is detected through the antigen–antibody reaction. A drawback of this technique is that the orientation of the antibody binding site is uncontrollable because of the random attachment of antibodies on the Au substrate [21,33].
Moreover, the substrate’s surface can be coated with avidin to bind to biotinylated antibodies to create an avidin–biotin system. This method produces strong bonds that can allow for reuse of sensors through multiple washing [42]. Another immobilized method is the use of SAMs. Immobilization is achieved by forming a SAM when the substrate’s surface is immersed in an ethanol solution containing thiols or disulfides. The antibody is immobilized through 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide/N-hydroxysuccinimide (EDC/NHS) linker to the thiol end. Unattached antibodies are washed and the remaining site is blocked [33,43].
Antibodies can also be labeled by enzymes, biotins, fluorescent substances, or radioactive isotopes [21]. Antibodies are labeled to antigens in biosensor devices through two methods: direct and indirect. The direct method directly detects labeled antibodies to the targeted antigen. This method is convenient and inexpensive because it uses only one type of antibody. However, the sensitivity of the direct method is limited due to the insufficient labeling signal of the limited antibody. Meanwhile, the indirect method involves two types of antibodies, making it more expensive than the direct method [21,34]. The first antibody is unlabeled and used to capture a specific antigen. The second antibody is labeled and added to bind with the first antibody. Given that the indirect method involves two types of antibodies, signal generation is enhanced and sensitivity is increased.
1.2.2 Enzymes
Other bioreceptors that have been used to couple with a transducer are enzymes that are target specific. The catalytic enzyme with a suitable substrate produces electrons and then transfers to a transducer. Most of the enzymes are proteins with the exception of RNA enzymes [21].
For most applications, an enzyme is used as a label rather than as a bioreceptor alone. Enzymes can be conjugated with antibodies or aptamers. Enzymes as a label can increase sensitivity of antibodies or aptamers because the catalytic activity enhances the number of signals that can be detected by a transducer in biosensor devices. Examples of enzymes that are commonly used as labels are alkaline phosphatase, horseradish peroxidase (HRP), and beta-galactosidase. These enzymes are mostly applied in ELISA [21].
The advantages of using an enzyme as a label are high sensitivity, possible direct visualization, stability over time, and safety. Direct visualization can be achieved and the sensitivity for detection increases when combined with AuNPs because enzymes provide a colorimetric signal that further enhances visibility of AuNP in a lateral-flow assay [44–46]. Other labels such as fluorescent tags are unstable particularly when exposed to direct light, while the use of a radioisotope label can be a health hazard [21]. Details of the enzymatic reaction used in developing biosensors are discussed in the following section.
1.2.3 Nucleic acids
DNA is a genetic component that is unique for each organism. Given its unique sequence, nucleic acid bioreceptors can be designed to complementarily match with the DNA of the interested organism. These matching pairs can be detected by triggering the transducer of a biosensor. Nucleic acid, bioreceptor-based biosensors offer rapid processing and high specificity, and they are simple and inexpensive. They also match only with complementary DNA of the interested organism. However, DNA damage or mutation in an organism results in changes to the DNA structure and DNA replication is disrupted. This change can alter matching complementary sequences between nucleic acid bioreceptors and the targeted DNA. Thus sensitivity and specificity of the biosensor decrease [21].
Genosensor platforms are applied to develop nucleic acid–based biosensors. Biosensors were developed for detecting V. cholera [37] in which the dry reagent–based nucleic acid amplification assay was combined with a portable electrochemical genosensor. Fluorescein-tagged amplification reagent-targeted DNA of V. cholera caused corresponding single-stranded DNA (ssDNA) amplicons to be generated. These fluorescein-labeled ssDNA amplicons were then analyzed using capture probe-modified, screen-printed, gold, electrode biosensors produced via the SAM method. Antifluorescein-conjugated alkaline phosphatase produced electroactive α-naphthol through a catalytic reaction to form enzymatic amplification of the hybridization event. The developed biosensor could detect as low as 10 CFU/mL with 100% sensitivity and specificity using 168 spiked stool samples.
Other research groups developed excellent and efficient nucleic acid–based biosensors [28,39,47–49]. However, most of their work involved amplification to tag labels with the DNA of the interested organism. This approach requires pre-treatment of samples before they can be applied in the biosensor device. To overcome this limitation, Paniel et al. developed genosensors without amplification [50]. The screen-printed carbon electrode was immobilized with ssDNA, which was hybridized with a complementary DNA sample. Another ssDNA signal probe labeled with horseradish peroxidase enzyme was added for second hybridization. Unfortunately, LOD of this biosensor platform was 10²–10³ CFU/mL, which was lower than that of the pre-treatment biosensor by DNA amplification. Amplification multiplies the labeled probe compared with a single labeled probe without an amplification process.
Peptide nucleic acid (PNA) is an innovative probe in nucleic acid recognition for DNA-based biosensors. PNA is synthesized by substituting the sugar phosphate backbone of DNA with a pseudopeptide. PNA as a probe molecule has superior hybridization characteristics, detects single-based mismatches, and improves chemical and enzymatic stability. Moreover, developing label-free detection is possible with different molecular structures. Label-free detection contributes to the establishment of rapid, stable, and reliable biosensor devices. PNA-based nucleic acid biosensors can be used for detecting pathogens and offer more stable and increased affinity toward targeted sequences [51–53]. However, some of the disadvantages of PNA as a recognition element include their high synthesis cost, long aggregation-prone PNA oligomers, poor solubility in aqueous media, and difficulty to purify and characterize [21].
1.2.4 Aptamers
Aptamers can also be used as a bioreceptor in biosensor devices. Aptamers are a type of nucleic acid in which single-stranded nucleic acids fold together to form a well-defined 3D structure. The structure makes aptamers function like antibodies with high affinity, and they are only specific to their target molecules. Aptamers can be synthesized in the laboratory either chemically, via enzymatic procedures, or a combination of these methods. Aptamers can be both chemical and biological substances [54].
Systematic evolution of ligands by exponential enrichment (SELEX) is a procedure to engineer aptamers in vitro. This procedure involves several repeated cycles, which begins with selecting randomized ssDNA from the library and incubating the target molecule. After incubation, unbound and bound molecules are separated by partitioning. The target-bound sequences are amplified by PCR to produce an enriched pool as input for the next round of selection [55]. The cycle is usually repeated 8–15 times to obtain highly specific aptamers [56,57]. Fig. 1.1 summarizes the SELEX process.
Figure 1.1 SELEX cycle starts with selecting an aptamer library, followed by binding with the target, partitioning of bound and unbound targets, amplifying the bounded target, and producing an enriched pool of selected aptamers for subsequent cycles.
Considering the 3D structures of aptamers, these DNA ligands identify their target structurally and not by their sequence. These specific aptamers can recognize target proteins specific to some pathogen. Thus aptamers are suitable as bioreceptors to be combined with electrochemical, optical, or colorimetric transducers for biosensor devices [58]. Targeted molecules of a specific pathogen include some carbohydrates, such as lipopolysaccharides and teichoic acid, cell surface proteins, such as intimin of pathogenic E. coli or PilS of S. enterica type IVB pili, and pore-forming toxins, such as listeriolysin of Listeria monocytogenes [59,60].
Live and whole cells can also be used as a target in SELEX. This technique is suitable to design aptamers for microbial pathogens. Aptamers synthesized by this method offer increasing sensitivity of detection because they target several sites of molecules on the cell surface simultaneously. This method does not require labor-intensive steps of isolating and purifying complex targets. However, the technique involves several additional purification steps to ensure that family ligands are selected against the multiple targets, especially in complex mixtures [58]. SELEX was successfully used to identify S. enteritidis [61] and other pathogens [59].
1.2.5 Other Bioreceptors
Other bioreceptors used in biosensors are cellular bioreceptors, biomimetic receptors, and bacteriophages. Biomimetic receptors are artificial or synthetic bioreceptors that are designed and fabricated to mimic antibodies, enzymes, or nucleic acids as bioreceptors. Molecularly imprinted polymer (MIP) is one of the techniques in which a polymer is formed around a molecule as an artificial recognition site [62]. A colorimetric biomimetic, specifically polydiacetylene (PDA), produces visible color changes from blue to red when a bacterial target multiplies [63]. S. enterica, E. coli, and B. cereus are among the pathogens studied by this approach.
A cellular bioreceptor makes use of a whole cell or specific cellular component that can specifically bind to certain targets. The cellular bioreceptor can be classified into cellular systems, enzymes, and non-enzymatic proteins [21]. A cellular system is based on a whole cell recognition element. Two phases of transducers occur in this system. The whole cell acts as a transducer in the first phase where it produces a cellular response from the detected analyte. Another transducer is required to convert this response into an electrical signal to be processed and analyzed. A cellular bioreceptor has a low detection limit because of signal amplification, is sensitive to biochemical stimuli, and provides a useful assay for biochemical agents [21,64]. In addition, enzyme protein, as discussed in a previous section, is one of the cellular components that can specifically recognize target analytes. Non-enzymatic proteins, such as channel or carrier proteins, on the cellular surface are used to recognize molecules through active or potential sensitive sites. For example, lectins can be used as a bioreceptor for electrochemical transducer to bind with lipopolysaccharides on the cell surface for rapid identification of E. coli subspecies [65] and antibiotics to bind with E. coli and methicillin-resistant S. aureus (MRSA) for antibiotic susceptibility testing [66].
1.3 Types of Transducers
Bioreceptors cannot function alone as a biosensor and must be coupled with a transducer. The transducing mechanism converts the signal or recognition event from the bioreceptor into an electrical signal and is then processed or amplified. The interpretable result can be read either visually or as an electronic display. Transducers, such as electrochemical, optical, or mass-based types, are commonly used in biosensor systems and are discussed next.
1.3.1 Electrochemical Biosensors
In an electrochemical-based transducer, the interaction of samples with a bioreceptor produces detectable parameters, such as current, potential different, impedance, and conductance. On the basis of these parameters, electrochemical biosensors can be classified into amperometric, potentiometric, impedimetric, and conductometric types. Some advantages of electrochemical biosensors are favorable sensitivity, measurability in complex and turbid samples, potential for compact design, and low cost [7,21,33].
1.3.1.1 Amperometric
An amperometric biosensor measures the current produced by a catalytic enzyme against an analyte through the redox reaction. The current is directly proportional to the analyte concentration [33]. To measure current, the applied potential and time must be constant to allow electron transfer from the bioreceptor to transducer [21]. Typically, two electrodes are used for this reaction, namely, working and reference electrodes. Materials that are commonly used as working and reference electrodes are gold (Au), platinum (Pt), graphite, silver (Ag), carbon, and conducting polymers [7].
The working principle of amperometric biosensors for detecting pathogens is simple. The antibody against the target pathogen is immobilized on the working electrode surface. When the target pathogen presence in the sample, it binds to the antibody on the working electrode. This reaction produces a current signal. The secondary antibody against the target that is labeled with the enzyme can be used to enhance the first signal. By doing so, the redox reaction from the enzyme catalysis occurs to generate more electrons. Thus the electrical signal is enhanced and improves the sensitivity of the analysis [7]. Fig. 1.2A shows the working principle of amperometric biosensors.
Figure 1.2 Principle of electrochemical-based biosensor. (A) The schematic principle of an amperometric biosensor. The label (enzyme) accelerates the electrochemically active analyte in the solution to transfer electrons to the electrode. (B) The schematic principle of a potentiometric biosensor. The label changes the ion concentration, such as H+ and K+. The ion accumulates on the gate insulator, which produces a potential difference via current flows through the gate. (C) The schematic principle of an impedimetric biosensor. The impedance is increased because electron transfer is blocked upon the attachment of bacterial cells. Source: Reproduced with permission from Ref. M. Xu, R. Wang, Y. Li, Electrochemical biosensors for rapid detection of Escherichia coli O157:H7, Talanta 162 (2017) 511–522, doi:10.1016/j.talanta.2016.10.050.
NP-based amperometric biosensors have been recently used to diagnose foodborne pathogens. For example, Altintas et al. [67] developed a fully automated microfluidic-based amperometric biosensor for E. coli detection. They compared the LOD for standard and nanomaterial-amplified immunoassays (IAs) with LOD of 1.99×10⁴ and 50 CFU/mL, respectively. A specificity study was also conducted by using Salmonella spp., S. typhimurium, Shigella, and S. aureus and the results confirmed the high specificity of the developed amperometric biosensors. Furthermore, the sensor surface could be regenerated multiple times, significantly reducing the cost of the system.
In another study, bionanocomposite-modified pencil graphite electrode (PGE) was developed using polypyrrole (PPy)/AuNP/multiwalled carbon nanotubes (MWCNTs)/chitosan (Chi) [68]. This hybrid bionanocomposite platform was immobilized with E. coli O157:H7 monoclonal antibodies. The study reported selectivity to E. coli O157:H7 with LOD of ~30 CFU/mL in PBS buffer. Li et al. [69] also developed amperometric biosensors for detecting E. coli O157:H7. Another amperometric biosensor was developed for detecting L. monocytogenes [70] and Clostridium tetani [71].
1.3.1.2 Potentiometric
In potentiometric-based biosensors, ions in solution from the bioreceptor are converted to a potential signal. The electrical potential proportional to analyte activity is measured from samples by applying the Nernst relationship:
(1.1)
where E is the potential of measurement, E0 is the standard potential at a=1 mol/L, R is the gas constant at 8.314 J mol−1 K−1, T is the absolute temperature in K, n is the number of electrons that is transferred by electrode reaction, F is the Faraday constant, and a is the total number charges of ion based on the concentration at the anode (+) and cathode (–) [7,72]. From the Nernst relationship, potentiometry yields a logarithmic concentration response so it is highly sensitive toward extremely small concentration changes [7,21]. For potential measurement, the current is maintained near zero to allow for electromotive force (EMF) [21].
Similar to amperometric-based biosensors, two electrodes are used to measure ion potential change. One inert reference electrode and one working electrode must be in contact with the sample to be measured. An enzyme is used as a bioactive element surrounding the probe. The catalytic reaction consumes or produces chemical species [7,64]. It can be in the form of an ion concentration fluctuation or pH change, which generates potential that can be measured and directly read from a pH meter display [73]. Fig. 1.2B shows the working principle of potentiometric biosensors. Some advantages of potentiometric biosensors include their ability to measure pathogens under in situ conditions, portability, low cost, and high sensitivity. However, selectivity or specificity is poor in some samples.
Application of this principle toward pathogen detection is lesser compared with other types of electrochemical biosensors. The two major types of potentiometric biosensors are light-addressable potentiometric sensors (LAPSs) and ion-selective, field-effect transistors (ISFETs) [72]. In studies performed on ISFET-based biosensors, poor LODs and device stability have been encountered in detecting pathogens due to the incompatibility between most ISFET fabrication technology and biomolecular immobilization protocols [7,33,72]. The principle of ISFETs is based on the response of specific ions, such as H+, K+, and Cl−, by applying an electric field to create regions of excess charge to a semiconductor substrate. This process helps by using an ion-selective membrane to cover the gate insulator that only allows selective ions to pass [74].
Meanwhile, LAPS is based on inducing a transient photocurrent to an insulated p- or n-doped semiconductor thin film positioned to be in contact with an electrolyte via the transient illumination of an intensity-modulated light source, such as light-emitting diodes (LEDs). The induced photocurrent magnitude depends on the potential applied to the semiconductor plate [72]. LAPS is more feasible for detecting foodborne pathogens than ISFETs [75]. LAPS devices are also available commercially, such as the Threshold Immunoassay System, for the detection of E. coli O157:H7 in food samples [72]. Ercole et al. also reported the use of LAPs for E. coli detection [76]. Bisha and Brehm used the potentiometric alternating biosensing (PAB) system based on LAPs for detecting E. coli in vegetables [77]. Gehring et al. previously developed an immunoligand assay (ILA) combined with LAPs for the rapid detection of E. coli O157:H7 in buffered saline [78].
A potentiometric aptamer-based bioreceptor, which couples the DNA nanostructure-modified magnetic beads with a solid-contact polycation-sensitive membrane electrode, was developed for detecting Vibrio alginolyticus [79]. In this study, DNA nanostructure-modified magnetic beads were used to amplify the potential response and eliminate the interference effect in a complex sample matrix. The interaction between V. alginolyticus and the aptamer on the DNA nanostructures induced changes in the charge or DNA concentration on the magnetic beads. Chronopotentiometric detection was conducted on a solid-contact polycation-sensitive membrane electrode by using protamine as an indicator. This potentiometric aptasensing method offered LOD of 10 CFU/mL with acceptable specificity for the detection of V. alginolyticus.
Ding et al. reported label-free potentiometric aptasensor for rapid, selective, and sensitive detection of L. monocytogenes [80]. Aptamer as the bioreceptor binds specifically to internalin A present on the surface of L. monocytogenes. A polycation-sensitive membrane electrode is used to detect free protamine when target-binding events prevent the aptamer from electrostatically interacting with protamine. The LOD of this method is 10 CFU/mL with favorable recovery and high accuracy. Another label-free potentiometric aptasensor uses a network of single-walled carbon nanotubes (SWCNTs) as an ion-to-electron potentiometric transducer and aptamers as the bioreceptor for detecting S. aureus in real time [81] and living E. coli in complex matrices [82]. Hernández et al. [83] also reported a potentiometric aptasensor based on chemically modified graphene for detecting S. aureus in real
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