Analytical biosensors for the pathogenic microorganisms determination
Julio Raba1,*, Martín A. Fernández-Baldo1, Sirley V. Pereira1, Germán A. Messina1, Franco A. Bertolino1, Santiago Tosetti2 and María I. Sanz Ferramola3 1INQUISAL-CONICET, Área de Química Analítica, Departamento de Química, Facultad de Química, Bioquímica y
Farmacia, Universidad Nacional de San Luis. Chacabuco 917, D5700BWS, San Luis, Argentina 2INAUT-CONICET, Facultad de Ingeniería, Universidad Nacional de San Juan, Av. Libertador 1109 (O), J5400, San
Juan, Argentina 3Área de Tecnología Química y Biotecnología, Departamento de Química, Facultad de Química Bioquímica y Farmacia,
Universidad Nacional de San Luis. Ejército de los Andes 950, D5700BWS, San Luis, Argentina *Corresponding author
Detection and quantification of microbial pathogens are usually the first procedure before the application of any strategy for combating them. In the last years, analytical biosensors provide a promising alternative for direct pathogenic microorganisms determination. These sensors are analytical devices composed of a recognition element of biological origin and a physico-chemical transducer. Biosensors technology in general seeks to improve analytical performance by reducing the consumption of reagents, decreasing the analysis time, increasing reliability and sensitivity through automation, and integrating multiple processes in a single device. The present chapter focuses applications of analytical biosensors to microbiological diagnostics in areas such as diagnosis of diseases and food quality control including fabrication techniques as well as future perspectives.
Classical methods such as isolation on selective media are useful for the pathogenic microorganisms detection but subject to limitations due to the fact that many pathogens are masked by overgrowth of faster growing microorganisms [1, 2]. Furthermore, these methods present the drawbacks of tedious procedure, need for trained personnel and long time to yield results [1, 2]. Moreover, quantitative nucleic acid-based procedures have been developed, but these methods are expensive and difficult to perform as routine assays [1, 2]. On the other hand, some bacteria, toxins, fungi and mycotoxins has been detected by enzyme-linked immunosorbent assay (ELISA) [3-6], but unfortunately, this technique requires highly qualified personnel and consumes a lot of time. Development of antibody-based ELISA and DNA-based PCR techniques helped in improving the time required to yield results [4, 6]. The selectivity and sensitivity of ELISA depends on the binding strength of the antibody to its antigen and they work well for samples without interfering molecules such as other non-target cells, proteins and DNA [5, 6]. Moreover, PCR has high selectivity, good sensitivity and takes a shorter time than culture based techniques whose requirement of trained personnel and expensive instruments limits it use in practical environment [2, 4]. Using these techniques for detection of pathogens present in biological or food matrixes requires an enrichment step for concentrating the pathogens from the complex media and dispensing them in buffer for its detection [2-6]. Due to the disadvantages of the above mentioned techniques the development of new analysis methodologies with high sensitivity and specificity for direct pathogenic microorganisms determination are highly desirable [2-6]. In the last years, analytical biosensors provide a promising alternative for direct pathogenic microorganisms determination in areas such as clinical diagnostics, food analysis, bioprocess and environmental monitoring [7-28]. A biosensor is an analytical device that integrates a biological element on a solid-state surface which enables a reversible biospecific interaction with the analyte, and a signal transducer [29]. The biological element is a layer composed of molecules qualified for biorecognition, such as enzymes, receptors, peptides, single-stranded DNA, even living cells are applicable [29]. If antibodies or antibody fragments are applied as biological element the device is called immunosensor [29]. Compared to conventional analytical instruments, biosensors are characterized by an integrated structure of these two components [29]. Many devices are connected with a flow-through cell, enabling a flow-injection analysis (FIA) mode of operation [29]. Biosensors combine high analytical specificity with the processing power of modern electronics components to achieve highly sensitive detection systems [29]. There are two different types of biosensors: biocatalytic and bioaffinity-based biosensors [29]. The biocatalytic biosensor uses mainly enzymes as the biological compound, catalyzing a signaling biochemical reaction [29]. The bioaffinity-based biosensor, designed to monitor the binding event itself, uses specific binding proteins, lectins, receptors, nucleic acids, membranes, whole cells or antibodies for biomolecular recognition. Biosensors technology seeks to improve analytical performance by reducing the consumption of reagents, decreasing the analysis time, increasing reliability and sensitivity through automation, and integrating multiple processes in a single device [29]. Recently, some biosensors for pathogenic microorganisms, toxins and mycotoxins determination have been fabricated using microfluidic technology and nanotechnology [25-28]. These kinds of novel devices have high speed of
Microbial pathogens and strategies for combating them: science, technology and education (A. Méndez-Vilas, Ed.)
response, accuracy, lower cost and less operator intervention [25-28]. Others advantages of these methods are the reduction of the amount of solvents and reagents required in sample pre-treatment as well as in the measurement steps. These benefits are consequence of the automation and miniaturization which reduced the adverse environmental impact of analytical methodologies [25-28]. Besides, the use of some nanomaterials (quantum dots, carbon nanotubes, magnetic and metallic nanoparticles) as a bio-affinity platform for the immobilization of biomolecules had permitted the development of analytical biosensors with enhanced sensitivities and improved response times [25-28, 30]. The present chapter focuses on the applications of analytical biosensors to microbiological diagnostics in areas such as diagnosis of diseases and food quality control, by providing methodologies with rapid detection, high sensitivity and specificity, including fabrication techniques as well as future perspectives.
2. Analytical biosensors
2.1. What is an analytical biosensor?
The concept biosensor appeared in the scientific literature in the late 1970s; however, the basic term and even the commercialization of biosensors predate this. The first biosensor, known as the enzyme electrode, was designed by Clark and Lyons [31] in 1962, when they coupled the enzyme glucose oxidase to an amperometric electrode. The enzyme-catalyzed oxidation of glucose lowered PO2 in the test solution. The lowering of PO2 was sensed by the electrode and shown to be proportional to the concentration of glucose in the sample. In the years that followed, enzyme electrodes for a variety of other clinically important substances were developed by coupling the appropriate enzymes to electrochemical sensors. Then, in 1977 Rechnitz et al. [32] immobilized living microorganisms on the surface of an ammonia gas-sensing electrode to construct a selective electrode for the amino acid arginine, which they described as a bio–selective sensor. The authors claimed that leaving an enzyme in its native environment would optimize its biological activity and improve sensor characteristics. Bio-selective sensor was later shortened to biosensor and has remained the popular term for the marriage between a material of biological origin and a physical transducer. The designs and applications of biosensors in various fields of analytical chemistry have continued to grow since then. According to a document of the International Union of Pure and Applied Chemistry (IUPAC) [33] a biosensor is defined as a specific type of chemical sensor comprising a biological recognition element (BRE) and a physico-chemical transducer. The biological element is capable of recognizing the presence, activity or concentration of a specific analyte in solution. The recognition may be either a binding process (affinity ligand-based biosensor, when the recognition element is, for example, an antibody, DNA segment or cell receptor) or a biocatalytic reaction (enzyme-based biosensor). The interaction of the recognition element with a target analyte results in a measurable change in a solution property, such as formation of a product. The transducer converts the change in solution property into a quantifiable electrical signal. The mode of transduction may be one of several approaches, including electrochemical, optical and the measurement of mass or heat.
2.2. Classification
Biosensors may be classified according to the biological specificity conferring mechanism, or to the mode of signal transduction or, alternatively, a combination of the two. These might also be described as amperometric, potentiometric, field-effect or conductivity sensors. Alternatively, they could be termed, for example, as amperometric enzyme sensors [34]. As an example, the former biosensors may be considered as enzyme- or immunosensors.
2.3. BREs such as specific receptors
2.3.1. Biocatalytic recognition element
The biosensor operation is based on a reaction catalyzed by macromolecules, which are present in their original biological environment, and have been isolated previously or have been manufactured. Thus, a continuous consumption of substrate(s) is achieved by the immobilized biocatalyst incorporated into the sensor: transient or steady-state responses are monitored by the integrated detector. Three types of biocatalyst are commonly used: 1. Enzyme (mono- or multi-enzyme), the most common and well developed recognition system, 2. Whole cells (micro-organisms, such as bacteria, fungi, eukaryotic cells or yeast) or cell organelles or particles (mitochondria, cell walls), 3. Tissue (plant or animal tissue slice). The biocatalytic-based biosensors are the best known and studied and have been the most frequently applied to biological matrices since the pioneering work of Clark et al., 1962. One or more analytes, usually named substrates S
Microbial pathogens and strategies for combating them: science, technology and education (A. Méndez-Vilas, Ed.)
and S’, react in the presence of enzyme(s), whole cells or tissue culture and yield one or several products, P and P’, according to the general reaction scheme:
There are four strategies that use adjacent transducers for monitoring the analyte S consumption by this biocatalyzed reaction: 1. Detection of the co-substrate S’ consumption, e.g., oxygen depleted by oxidase, bacteria or yeast reacting layers, and the corresponding signal decrease from its initial value. 2. Recycling of P, one of the reaction products, e.g., hydrogen peroxide, H+, CO2, NH3, etc. production by oxidoreductase, hydrolase, lyase, etc., and corresponding signal increase. 3. Detection of the state of the biocatalyst redox active centre, cofactor, prosthetic group evolution in the presence of substrate S, using an immobilized mediator which reacts sufficiently rapidly with the biocatalyst and is easily detected by the transducer; various ferrocene derivatives organic salt, quinones, quinoid dyes, Ru or Os complexes in a polymer matrix, have been used [35]. 4. Direct electron transfer between the active site of a redox enzyme and the electrochemical transducer. When several enzymes are immobilized within the same reaction layer, several strategies for improving biosensor performance can be developed. Three following possibilities have been most frequently proposed: 1. Several enzymes facilitate the biological recognition by sequentially converting the product of a series of enzymatic reactions into the final electroactive specie: this configuration allows a much wider range of possible biosensor [36]. 2. Multiple enzymes applied in series may regenerate the first enzyme co-substrate and a real amplification of the biosensor output signal may be achieved by efficient regeneration of another co-substrate of the first enzyme. 3. Multiple enzymes, applied in parallel, may improve the biosensor selectivity by decreasing the local concentration of electrochemical interfering substance: this configuration is an alternative to the use of either a permselective membrane or a differential set-up, i.e., subtraction of the output signal generated by the biosensor and by a reference sensor having no biological recognition element [37].
2.3.2. Biocomplexing or bioaffinity recognition element
In this case, the biosensor is based on interaction of the analyte with macromolecules or organized molecular assemblies that have either been isolated from their original biological environment or engineered [38]. Thus, equilibrium is usually reached and there is no further net consumption of the analyte by the immobilized biocomplexing agent. These equilibrium responses are monitored by the integrated detector. In some cases, this biocomplexing reaction is itself monitored using a complementary biocatalytic reaction. Steady-state or transient signals are then monitored by the integrated detector. 1. Antibody–antigen interaction. The most developed examples of biosensors using biocomplexing receptors are based on immunochemical reactions, i.e. binding of an antigen (Ag) to a specific antibody (Ab). Formation of such Ab-Ag complexes has to be detected under conditions where non-specific interactions are minimized. Each Ag determination requires the production of a particular Ab, its isolation and, usually, its purification. Several studies have been described involving direct monitoring of the Ab-Ag complex formation on ion-sensitive-field effect transistors (ISFETs). In order to increase the sensitivity of immunosensors, enzyme labels are frequently coupled to Ab or Ag, thus requiring additional chemical synthesis steps. Even in the case of the enzyme-labelled Ab, these biosensors will essentially operate at equilibrium, and the enzymatic activity being there only to quantify the amount of complex produced. As the binding or affinity constant is usually very large, such systems are either irreversible (single-use biosensors) or placed within an FIA environment where Ab may be regenerated by dissociation of complexes by chaotropic agents, such as glycine–HCl buffer at pH 2.5. 2. BRE/antagonist/agonist. More recently, at tempts have been made to use ion channels, membrane receptors or binding proteins as molecular recognition systems in conductometric, ISFET or optical sensors [39]. For example, the transport of protein lactose permease (LP) may be incorporated into liposome bilayers thus allowing coupling of sugar proton transport with a stoichiometric ratio of 1:1, as demonstrated with the fluorescent pH-probe pyranine entrapped in
Microbial pathogens and strategies for combating them: science, technology and education (A. Méndez-Vilas, Ed.)
these liposomes [40]. These LP-containing liposomes have been incorporated within planar lipid bilayer coatings of an ISFET gate sensitive to pH.
3. Analytical biosensors applications
3.1. Biosensors for diagnosis of diseases
In this section, we will discuss current biosensors for pathogenic microorganisms, such as virus, protozoa, and bacteria for diagnosis and monitoring of infectious diseases, such as HIV/AIDS, malaria, tuberculosis and more. Each year they have caused great harm to the health of humans, animals and plants, leading to huge economic losses [41]. The detection and quantification of viruses are the basis for various fields of applications from food production/sanitation to diagnostics and therapeutics [42].Conventional diagnostic methods for HIV infection are blood tests such as enzyme immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA), and Western blot test. Guo et al. (2013) have proposed a label-free electrochemical DNA biosensor for HIV related gene based on the interaction of hybrid double-stranded DNA and protein [43]. In the presence of target DNA, the target/probe hybridization encountered the NF-kB protein. Upon addition of HRP, they formed the sandwich-like DNA sensor. The formed sandwich-like ds/NF-kB/HRP complex effectively catalyzed the H2O2-mediated oxidation of TMB. A label-free immunosensor for the detection of HIV-1 based on localized surface plasmon resonance was proposed for Lee et al. (2013) [44]. Uniform nanopattern of circular Au-dots on indium tin oxide was modified with HIV-1 neutralizing gp120 monoclonal antibody fragments. The modified substrate was employed to measure various concentrations of HIV-1 particles quantitatively based on the shift of longitudinal wavelength in the UV–Vis spectrum which results from the changes of local refractive index induced by specific antigen-antibody recognition events. Hepatitis B virus (HBV) is one of the causative agents of viral hepatitis. Infection of HBV is a public health problem of worldwide importance with acute and chronic clinical consequences. Acute HBV infection may lead to liver failure or may progress to chronic liver disease [45]. Biosensors based on nucleic acid hybridization processes are rapidly being developed towards the goal of rapid and inexpensive diagnosis of genetic and infectious diseases [45]. Rolling circle amplification (RCA) is an isothermal amplification technique for small, circular DNA templates with the unique property of the product localization. A RCA-based quartz crystal microbalance (QCM) biosensor was proposed for Yao et al. (2013) for direct detection of HBV genomic DNA from clinical samples [45]. The covalent bonding between the capture probes and the gold electrode surface guarantees that the linkage between the capture probes and the amplified RCA products is maintained during the assay. In this study, 104 copies/mL HBV genomic DNA can be detected. A peptide nucleic acid (PNA) piezoelectric biosensor for real-time monitoring of hybridization of HBV genomic DNA was developed for Yao et al. (2008) [46]. The PNA probe was designed and immobilized on the surface of the biosensor to substitute the conventional DNA probe for direct detection of HBV genomic DNA without previous amplification by PCR. The PNA probe was able to distinguish sequences that differ only in one base. Hepatitis C virus (HCV) infection, which presents as persistent infection in up to 85% of all infected individuals, is a global health problem. Once HCV-infected patients develop cirrhosis or hepatocellular carcinoma, low cure rates and serious side effects shall be expected. That is why accurate and sensitive diagnosis of HCV in blood samples during the early stages of infection is so crucial [47]. Resonant microcantilever arrays are developed for the purpose of label-free and real-time analyte monitoring and biomolecule detection. MEMS cantilevers made of electroplated nickel were functionalized with Hepatitis antibodies by Timurdoganet al. (2013) [47]. Actuation is achieved using an electromagnet and the interferometric optical sensing is achieved using laser illumination and embedded diffraction gratings at the tip of each cantilever. This biosensor was applied for detecting both Hepatitis A and HCV antigens and their negative controls in undiluted serum. HCV genotype is one of the most significant baseline predictors of response to HCV antiviral therapy. Sam et al. (2013) evaluate an HCV genotyping method that targets the 5′-untranslated region (UTR) to detect genotypes/subtypes using the GenMark eSensor® XT-8 system [48]. The HCV amplicon of major genotypes/subtypes from the Roche TaqMan® HCV assay served as a template for the nested PCR followed by a direct analysis on the XT-8 detection system. An electrochemical immunosensor was developed for Ma et al. (2013) for the detection of HCV core antigen [49]. The immunosensor consisted of graphitized mesoporous carbon–methylene blue (GMCs–MB) nanocomposite as an electrode modified material with Au nanoparticles electro-deposited on to the electrode to immobilize the captured antibodies and a horseradish peroxidase-DNA-coated carboxyl multi-wall carbon nanotubes (CMWNTs) as a secondary antibody layer. The bridging probe and secondary antibodies linked to the CMWNTs, and DNA concatemers were obtained by hybridization of the biotin-tagged signal and auxiliary probes. Finally, streptavidin-horseradish peroxidases (HRP) were labeled on the secondary antibody layer via biotin-streptavidin system. Hydatidosis is a common disease of humans and animals, resulting from infection with the larval stage or metacestode of Echinococcus granulosus [50]. This disease has a worldwide distribution, with a considerable impact in both human and animal health, causing important socioeconomic consequences in endemic areas. For these reasons, an early and fast diagnostic can provide improvements in the management and treatment of the E. granulosus infection [50]. Pereira et al. (2011) described an automated microfluidic immunosensor, for the detection of IgG antibodies
Microbial pathogens and strategies for combating them: science, technology and education (A. Méndez-Vilas, Ed.)
specific to E. granulosus in human serum samples, E. granulosus antigens were immobilized on gold electrode modified with gold nanoparticles [50]. Which was allowed to react with IgG anti-E.granulosus antibodies in samples, and these were quantified by HRP enzyme-labeled secondary antibodies specific to human IgG using catechol (Q) as enzymatic mediator. Chagas disease or American trypanosomiasis is caused by the hemoflagellate Trypanosoma cruzi and is one of the greatest health problems faced by Latin American countries [51]. The high incidence of the infection and the enormous amount of people at risk encouraged us to continue searching for better analytical tools to provide a reliable chagasic infection diagnosis. In the chronic phase the detection of anti-T.cruzi antibodies is the method of choice for the etiological diagnosis of Chagas disease and is the best tool for blood bank screening [51]. Pereira et al. (2011) reported an integrated microfluidic system coupled to a screen printed carbon electrode (SPCE) modified by electrodeposition of gold nanoparticles (AuNPs) and functionalized with T.cruzi proteins from epimastigote membranes, which was applied in the detection of specific IgG anti-T. cruzi antibodies, which react immunologically with immobilized T. cruzi antigen [51]. After that, labelled antibodies were quantified through the HRP enzyme-labeled secondary antibodies specific to human IgG, using 4-tert-butylcatechol (4-TBC) as enzymatic mediator.
(2)
(3)
(1)
SAMPLE HRP-ENZYMECONJUGATED
H2O2+ 4-TBC
4-TBOQ SPCE
4-TBC
SSS
AuNP 3-mercaptopropionic acidT. Cruzi antigens
+ Au(III)Electrodep.
SPCE
(1) Modification procedure, (2) principle of immune reaction and (3) schematic representation of microfluidic immunosensor.
(3)
Fig. 1 Schematic representation of modification procedure, immune reaction and microfluidic device for the quantitative determination of IgG anti-T.cruzi antibodies. From Pereira et al, 2011 [51] - Reproduced by permission of The Royal Society of Chemistry. Belluzo et al. (2011) designed an immunosensor to perform indirect immunoassays with amperometric detection using tailor-made chimeric receptors to react with the analyte, specific anti-T. cruzi immunoglobulin G (IgG) [52]. Recombinant chimeras were designed to favor their oriented covalent attachment. This allows the chimeras to properly expose their epitopes, to efficiently capture the analyte. By further binding the secondary antibody, HRP-labeled anti-human IgG, in the presence of the soluble mediator and the enzyme substrate, a current that increased with the analyte concentration was measured. Other immunosensor was proposed for Salinas et al. (2005) composed for a rotating bioreactor with electrochemical detection mediated by [Os(bpy)2Cl(pyCOOH)]Cl for the detection of T. cruzi, antibodies in the serum sample are allowed to react immunologically with whole homogenates of the parasite as antigen that are immobilized on a rotating disk [53]. The bound antibodies are quantified by HRP enzyme labeled second antibodies specific to human IgG in presence of hydrogen peroxide using an osmium complex [Os(bpy)2Cl(pyCOOH)]Cl as enzymatic mediators. On the other hand, Diniz et al. (2003) produced a polypeptide chain formed by recombinant antigens, cytoplasmic repetitive antigen (CRA) and flagellar repetitive antigen (FRA) of T. cruzi, which was adsorbed on gold and platinum electrodes and investigated their interaction with sera from chronic chagasic patients by electrochemical impedance spectroscopy [54]. Malaria is a major infectious disease widely spread in tropical and subtropical regions. In humans, malaria is caused by four different protozoan species of the genus Plasmodium. The malarial parasite Plasmodium falciparum causes the most severe illness and is prevalent in sub-Saharan Africa, while Plasmodium vivax contributes significantly to malaria morbidity in Africa, Asia, and Latin America [55]. In malaria diagnosis, specific gene identification is required in cases with subclinical infection or cases with mixed infection [55]. This study applied the biosensor technology based on
Microbial pathogens and strategies for combating them: science, technology and education (A. Méndez-Vilas, Ed.)
quartz crystal microbalance (QCM) to differentially diagnose of P. falciparum and P. vivax [55]. The QCM surface was immobilized with malaria biotinylated probe. Specific DNA fragments of malaria-infected blood were amplified and the hybridization between the amplified products and the immobilized probe resulted in quartz frequency shifts. Jeon et al. (2013) proposed a method based on the interaction among the Plasmodium lactate dehydrogenase (pLDH), and pL1 aptamer against P. vivax lactate dehydrogenase (PvLDH) and P. falciparum lactate dehydrogenase (PfLDH) [56]. In addition, the cationic polymers, poly(diallyldimethylammonium chloride) (PDDA) and poly(allylamine hydrochloride) (PAH), aggregate AuNPs that should be possible to observe the change in color from red to blue, which depends on the concentration of pLDH. A disposable amperometric immunosensor was developed for the detection of P. falciparum histidine-rich protein 2 (PfHRP-2) in human serum with P. falciparum malaria [57]. For this purpose, Sharmaet al. (2008) used disposable SPEs, which were modified with MWCNTs, AuNPs and rabbit anti-PfHRP-2 antibody [57]. Further, the electrode was exposed to a mouse anti-PfHRP-2 antibody from a serum sample, followed by a rabbit anti-mouse immunoglobulin G-alkaline phosphatase conjugate. Tuberculosis (TB) is a deadly infectious disease caused by Mycobacterium tuberculosis (Mtb), and its early diagnosis is essential for the proper treatment of patients and for the prevention of further spread of the pathogen [58]. Early diagnosis of active tuberculosis (TB) remains an elusive challenge, especially in individuals with disseminated TB and HIV co-infection. Recent studies have shown a promise for the direct detection of pathogen-specific biomarkers such as lipoarabinomannan (LAM) for the diagnosis of TB in HIV-positive individuals. Currently, traditional immunoassay platforms that suffer from poor sensitivity and high nonspecific interactions are used for the detection of such biomarkers [58]. Mukundan et al. (2012) developed a sandwich immunoassays for the direct detection of three TB-specific biomarkers, namely LAM, early secretory antigenic target 6 (ESAT6) and antigen 85 complex (Ag85), using a waveguide-based optical biosensor platform [58]. Combining detection within the evanescent field of a planar optical waveguide with functional surfaces that reduce non-specific interactions in complex patient samples (urine, serum) within a short time. In addition, a QCM immunosensor was employed by Hiatt et al. (2012) to screen for both whole Mtb bacilli and aMtb surface antigen, LAM [59]. Between Mtb DNA-based biosensors, Bernacka-Wojcik et al. (2013) fabricated a microfluidic platform applied to the DNA detection of Mtb that makes use of an optical colorimetric detection method based on gold nanoparticles [60]. The platform was fabricated using replica moulding technology in PDMS patterned by high-aspect-ratio SU-8 moulds. Rapid and accurate detection of causative pathogen is essential in determining the choice of treatment in acute-care settings. Cholera is an acute infectious disease characterized by rapid onset of severe secretory diarrhea with the production of “rice water” stools. Without immediate rehydration treatment, death by fluid loss can occur within hours or days [681]. Low et al. (2013) presented a thermostabilized electrochemical genosensing assay for the detection and quantification of Vibrio cholera lolB gene single-stranded asymmetric PCR amplicons [61]. The asymmetric PCR amplicons were hybridized to a magnetic bead-functionalized capture probe and a fluorescein-labeled detection probe followed by tagging with gold nanoparticles. Electrochemical detection of the chemically dissolved gold nanoparticles was performed using the differential pulse anodic stripping voltammetry method. Another genosensor was proposed for Chua et al. (2011) for V. cholerae O1 determination [62]. They reports the development of a glass fibre-based lateral flow DNA biosensor that contains a test line which captures biotin labelled DNA, an internal amplification control (IC) line which digoxigeninlabelled DNA and a control line which acts as membrane control. The detector reagent recognizes the fluorescein haptens of the amplified DNA and produces visual red lines. In light of the need for rapid diagnosis, Yu et al. (2011) developed a direct one-step lateral flow biosensor for the simultaneous detection of both V. cholerae O1 and O139 serogroups using serogroup specific monoclonal antibodies raised against lipopolysaccharides (LPS) were used to functionalize colloidal AuNPs applying an immunochromatographic principle [63]. In addition, Chen et al. (2010) developed a piezoelectric biosensor for cholera toxin (CT), where the gold electrode was modified by a GM1-functionalized supported lipid membrane [64]. In presence of CT, the GM1-incorporated liposomes specifically agglutinate at the electrode surface, this results in an enormous mass loading on the piezoelectric crystal. The Gram-negative bacterium Helicobacter pylori is the most important etiological agent of chronic active type B gastritis and peptic ulcer diseases. The infection produced by this microorganism is a risk factor in the development of gastric mucosa associated with lymphoid tissue lymphoma and adenocarcinoma [65]. Conventional diagnostic methods such as gastric biopsy, ELISA and culture, require a long time for the determination of H. pylori infections. Regarding the immunoassay field, various microfluidic devices have been used to adopt the conventional (ELISA) at microscale [65]. A portable microfluidic immunosensor coupled to laser-induced fluorescence (LIF) detection system for the determination of IgG antibodies against H. pylori in human serum samples were proposed for Seia et al. (2012) [65]. The device has a central channel (CC) with packed H. pylori antigen immobilized on 3-aminopropyl-modified controlled pore glass (AP-CPG). Antibodies in serum samples reacted immunologically with the immobilized antigen and then, they were determined using alkaline phosphatase (AP) enzyme-labeled second antibodies specific to human IgG. The 4-methylumbelliferyl phosphate (4-MUP), employed as enzymatic substrate. Pereira et al. (2010) developed a microfluidic magnetic immunosensor coupled to a gold electrode using a non-competitive immunoassay based on the use of purified H. pylori antigens immobilized on magnetic microspheres injected into microchannel devices and manipulated for an external removable magnet [66]. The IgG antibodies in human serum sample were quantified by AP enzyme-labeled second antibodies specific to human IgG. The p-aminophenyl phosphate (p-APP) was converted to p-
Microbial pathogens and strategies for combating them: science, technology and education (A. Méndez-Vilas, Ed.)
aminophenol (p-AP) by AP and the electroactive product was detected on gold layer electrode at 0.250 V. Stege et al. (2010) developed an online entirely automatized immunoaffinity assay-CE to determine the concentration of anti-H. pylori IgG using magnetic nanobeads as a support of the immunological affinity ligands and an LIF as a detector [67]. The separation was performed in 0.1M glycine1–HCl, pH 2, as the background electrolyte. In addition, Molina et al. (2008) reported an human serum IgG antibodies to H. pylori quantitation procedure based on the multiple use of an immobilized H. pylori antigen on an immuno-column incorporated into an a flow-injection (FI) analytical system [68]. The immuno-adsorbent column was prepared by packing APCPG covalently linking H. pylori antigens in a 3-cm of teflon tubing (0.5 i.d.). Antibodies in the serum sample were quantified by AP enzyme-labeled second antibodies specific to human IgG.
3.2. Biosensors for food quality control
A wide range of plant species, including economically important crops such as vegetables and fundamentally fruits, can be affected by gray mold caused by the fungal pathogen Botrytis cinerea [26, 27]. Moreover, some phytopathogenic fungi produce secondary metabolites called mycotoxins as such as ochratoxin A [28]. Furthermore, an interesting aspect is that very few biosensors applied for food quality control have been reported for fungi determination to date. On the other hand, the most common foodborne infections are those caused by the bacteria Escherichia coli O157:H7, Salmonellae (S. enterica and S. typhimurium), Listeria monocytogenes and Campylobacter jejuni [21]. In this section, we will focus on some biosensors applied to food quality control reported in the last years for these pathogenic microorganisms. Botrytis cinerea is a phytopathogenic fungus responsible for the disease known as gray mold, which causes substantial losses of fruits at postharvest. Fruit infections often remain dormant until the fruit ripens, when symptoms of the disease appear [26, 27]. Fernández-Baldo et al. (2009) developed an immunosensor coupled to carbon-based screen-printed electrodes (SPCE) modified with multi-walled carbon nanotubes (CNTs), which show a rapid and sensitive determination of B. cinerea in apple tissues (Red-delicious) using a competitive immunoassay method [26]. The detection of B. cinerea was carried out using a competitive immunoassay method based on the use of purified B. cinerea antigens that are immobilized on a rotating disk. The B. cinerea purified antigens or plant tissue sample (prepared in buffer PBS) and the B. cinerea-specific monoclonal antibody (BC-12.CA4) are allowed to react immunologically with the immobilized antigens, and the bound antibodies are quantified by a horseradish peroxidase (HRP) enzyme labeled second antibodies specific to mouse IgG, using 4-tertbutylcatechol (4-TBC) as enzymatic mediators. HRP in the presence of hydrogen peroxide (H2O2) catalyses the oxidation of 4-TBC to 4-tertbutyl o-benzoquinone (4-TBBQ). The electrochemical reduction back to 4-TBC is detected on SPCE-CNT at -0.15 V. The response current is inversely proportional to the amount of the B. cinerea antigens present in the fruit sample. This electrochemical immunosensor promises to be usefully suited to the detection and quantification of B. cinerea in apparently healthy fruit prior to the development of the symptoms. Later, Fernández-Baldo et al. (2010) developed a microfluidic immunosensor with micromagnetic beads (MMBs) coupled to carbon-based screen-printed electrodes (SPCEs) for the rapid and sensitive quantification of B. cinerea in apple (Red Delicious), table grape (pink Moscatel), and pear (William’s) tissues, before and after the accurrence of rot symptoms. [27]. This immunosensor was based on a competitive reaction by the specific monoclonal antibody (BC-12.CA4) between the free antigen (present in the fruit tissue sample or as purified antigen) and the purified B. cinerea antigen immobilized on 3-aminopropyl modified MMBs. MMBs were injected into microchannels and were manipulated with an external removable magnet. Then, the bound antibodies were quantified by using a second antibody specific to mouse IgG labeled with horseradish peroxidase (HRP). 4-TBC was used as enzymatic mediators. When hydrogen peroxide (H2O2) is present, HRP catalyzes the oxidation of 4-TBC to 4-TBBQ. The electrochemical reduction back to 4-TBC was detected on SPCEs at -0.15 V. The response current was inversely proportional to the amount of B. cinerea antigens present in the fruit sample. In the present immunosensor, which was developed with a microfluidic system, all reactions and washing procedures were performed using a syringe pump. Thus, the present device has the potential to answer the need for inexpensive, sensitive, and portable automation. As mentioned, the purified antigens of B. cinerea were immobilized on modified MMBs, which increased significantly the immunoreactive surface area; as a consequence, the limit of detection (LOD) was lower than the previous immunosensor reported by Fernández-Baldo et al. (2009) by about 10 times. An important advantage of this microfluidic immunosensor is that LOD achieved allowed us to detect the presence of B. cinerea even if the fruits did not show visible rot. Ochratoxin A (OTA) is a secondary metabolite produced by many species of moulds contaminating food, such as Penicillium verrucosum, Aspergillus ochraceus and Aspergillus niger [28]. In humans, it has been related to the Balkan endemic nephropathy (BEN) and the outbreak of urinary tract tumours [28]. IARC (International Agency on Cancer Research) has classified OTA as ‘‘possible human carcinogen (Group 2B)’’, and also teratogenic and carcinogenic effects have been described in some animal species. This mycotoxin has been found mainly in food derived from plants, such as cereals, coffee, nuts, spices, and wine. It has also been reported to occur in fruits (apples, oranges, plums, grapes and dried fruits) and fruit juices (apple juice, grape juice, and orange juice). Fernández-Baldo et al. (2011) developed, characterized and applied an immunoassay methodology comprised of magnetic nanoparticles (MNPs) as platform for immobilizing bioactive materials incorporated into a microfluidic system for rapid and sensitive quantification of OTA
Microbial pathogens and strategies for combating them: science, technology and education (A. Méndez-Vilas, Ed.)
in apples (Red Delicious) contaminated with Aspergillus ochraceus [28]. In this work, detection of OTA was carried out using a competitive indirect immunoassay method based on the use of anti-OTA monoclonal antibodies immobilized on 3-aminopropyl-modified MNPs. The MNPs were injected into microchannel devices and manipulated by external magnets. The OTA present in apple sample competes immunologically with OTA-HPR conjugate for the immobilized anti-OTA specific to OTA. After washing, enzyme HRP, in the presence of H2O2, catalyzes the oxidation of 4-TBC, whose back electrochemical reduction was detected on the gold electrode at 0.0 V. The response current obtained from the enzymatic product is directly proportional to the amount of enzyme bound to the antibody which is inversely proportional to the amount of OTA bound to the surface of the microfluidic immunosensor of interest. Moreover, this immunosensor has the advantages of low LOD, speed and simplicity as well as its utility to detect the presence of OTA even when the fruits did not show visible rot produced by A. ochraceus (fruits apparently healthy). The authors proposed this microfluidic immunosensor as the first developed for OTA determination in apples. It also can be a very promising analytical tool for the determination of this mycotoxin in real samples and for its application in the agricultural industry.
Fig. 2 Schematic representation of the biochemical immunoreaction and microfluidic immunosensor for the quantitative determination of OTA. From Fernández-Baldo, et al, 2011 [28] - Reproduced by permission of The Royal Society of Chemistry. Escherichia coli O157:H7 is a bacterial pathogen that is commonly found in the intestinal tracts in cattle and is carried over to the consumers via ground beef. Ingestion of the bacteria causes severe and bloody diarrhea and painful abdominal cramps [21]. In a small number of cases, a complication called hemolytic uremic syndrome (HUS) can occur which cause profuse bleeding, and kidney failure [21]. Wang et al. (2013) reported a novel surface plasmon resonance (SPR) biosensor using lectin as bioreceptor was for the rapid detection of E. coli O157:H7 in real food samples [69]. In this method, the selective interaction of lectins with carbohydrate components from bacterial cells surface was used as the recognition principle for the detection. Five types of lectins from Triticum vulgaris, Canavailia ensiformis, Ulex europaeus, Arachis hypogaea, and Maackia amurensis, were employed to evaluate the selectivity of the approach for binding E. coli O157:H7 effectively. Moreover, a gold interdigitated microelectrode (IME) impedance biosensor was fabricated for the detection of viable E. coli O157:H7. This sensor was fabricated using lithography techniques [70]. The surface of the electrode was immobilized with anti-E. coli IgG antibodies. This approach is different from other studies where the change in impedance is measured in terms of growth of bacteria on the electrode, rather than the antibody/antigen bonding. The impedance values were recorded for frequency ranges between 100 Hz and 10 MHz. In addition, a new and simple method for label free, rapid and inexpensive impedimetric sensing of E. coli O157:H7 using antibody–antigen binding method based on covalently linked antibody on a conducting polyaniline film surface has been reported [71]. Electrochemical impedance spectroscopy was used to test the sensitivity and effectiveness of the sensor electrode by measuring the change in impedance values of electrodes before and after incubation with different concentrations of bacteria. An equivalent electrical circuit model has also been proposed to explain the sensing mechanism. Also, Waswa et al. (2007) reported a surface plasmon resonance (SPR)-based biosensor for E. coli O157:H7 determination in spiked samples such as milk, apple juice and ground beef extract using specific antibodies [72]. In this biosensor light from an LED is reflected off a gold surface, and the angle and intensity corresponding to the SPR minimum is measured and represented as a refractive index (RI) change corresponding to the antigen–antibody coupling at the sensor surface. Salmonella is a bacterium that is often found in the intestines of chickens. It also affects the ovaries of healthy-looking hens which lead to presence of this microbe in raw eggs [21]. The illness caused is salmonellosis and manifests as fever, diarrhea and abdominal cramps. Salmonellosis can spread from the intestines to the blood stream and then to other parts of the body resulting in life-threatening infections in patients who are in poor health or weakened immune systems, and has been known to be fatal [21]. Among the over 2000 serovars that have been identified and
Microbial pathogens and strategies for combating them: science, technology and education (A. Méndez-Vilas, Ed.)
characterized, S. enterica and S. typhimurium are the epidemiologically the most important ones because they are the causative agent in 80% of all human infections reported word wide [21]. Lan et al. (2008) reported an optical Surface Plasmon Resonance (SPR) biosensor for S. typhimurium determination in chicken carcass [73]. In this work, a taste sensor like electronic tongue or biosensors was used to basically “taste” the object and differentiated one object from the other with different taste sensor signatures. The present biosensor has potential for use in rapid, real-time detection and identification of bacteria, and to study the interaction of organisms with different antisera or other molecular species. Also, a method based on surface plasmon resonance (SPR) DNA biosensor has been developed for label-free and high-sensitive detection of Salmonella [74]. A biotinylated single-stranded oligonucleotide probe was designed to target a specific sequence in the invA gene of Salmonella and then immobilized onto a streptavidin coated dextran sensor surface. The invA gene was isolated from bacterial cultures and amplified using a modified semi-nested asymmetric polymerase chain reaction (PCR) technique. In order to investigate the hybridization detection, experiments with different concentration of synthetic target DNA sequences have been performed. This proposed method was applied successfully to the detection of single-stranded invA amplicons from three serovars of Salmonella, i.e., typhimurium, enterica and derby. On the other hand, a ME biosensor method was evaluated by comparison with TaqMan-based quantitative PCR (qPCR) for the detection of S. typhimurium on spinach leaves [75]. This study demonstrated that the ME biosensor method was competitive and promising as an on-site and in-field detection method for the detection of pathogens. Moreover, a sensitive and stable label-free electrochemical impedance immunosensor for the detection of S. typhimurium in milk samples was developed by immobilizing anti-Salmonella antibodies onto the gold nanoparticles and poly(amidoamine)-multiwalled carbon nanotubes-chitosan nanocomposite film modified glassy carbon electrode [76]. Listeria monocytogenes is an emerging bacterial foodborne pathogen responsible for listeriosis, an illness characterized by meningitis, encephalitis, and septicaemia [21]. Less commonly, infection can result in cutaneous lesions and flu-like symptoms. In pregnant women, the pathogen can cause bacteraemia, and stillbirth or premature birth of the fetus [21]. In foods, it has been found in raw or processed food samples including dairy products as such milk, meat, vegetables and seafood [21]. Sim et al. (2012) developed a biosensor system integrated with a microfluidic channel, enabling label-free live cell detection of L. monocytogenes [77]. It consists of a waveguide sensor platform to determine the immune reaction and a birefringence measurement system that measures the phase difference between two orthogonal polarizations, arising from the refractive index change of the immune reaction in the channel of the sensor platform. Fabrication of the waveguide sensor for this pathogen detection system is compatible with conventional integrated circuit processes. The waveguide platform is overlaid with TiO2 films of different thicknesses to optimize sensitivity. In this work, the authors experimentally confirmed a method for controlling the length of the TiO2 film to efficiently increase the detection sensitivity in our waveguide composition. Sim et al. (2012) also demonstrated the proof of concept of our approach with a milk buffer to see similar detection results in a complex food matrix. Also, Sharma et al. (2013) reported a novel piezoelectric cantilever sensor for L. monocytogenes determination in milk samples [78]. In this work, L. monocytogenes determination was demonstrated using a novel asymmetrically anchored cantilever sensor and a commercially available antibody. Sensor responses were confirmed using a secondary antibody binding step, similar to the sandwich ELISA assays, as a means of signal amplification that also reduced the occurrence of false negatives. Moreover, Park et al. (2012) reported a dithiobis-succinimidyl propionate-modified immunosensor platform to detect L. monocytogenes in chicken skin [79]. In this work, the authors demonstrated the potential feasibility of a surface modified gold-coated immunosensor platform combined with light microscopic imaging system (LMIS) to detect L. monocytogenesin chicken. The reactivity of custom prepared antibodies (P-pAbs) was significantly higher than the reactivity of commercial antibodies and the P-pAbs were highly specific with L. monocytogenes strains. A gold-coated sensor platform modified with dithiobis-succinimidyl propionate (DSP) exhibited a 40% increase in binding efficiency, compared to the gold-coated sensor platform. Furthermore, Ohk et al. (2013) developed a fiber optic sensor for simultaneous detection of L. monocytogenes, E. coli O157:H7 and S. enteric from food [80]. The streptavidin coated optical waveguides were immobilized with biotinylated polyclonal antibodies and exposed to the bacterial suspensions or enriched food samples for 2 h. In this work, pathogens were detected after reacting with Alexa-Fluor 647-labeled monoclonal antibodies. Campylobacter jejuni is a gram-negative, spiral, microaerophilic bacteria. It has now been identified as one of the main causes of bacterial foodborne diseases [21]. C. jejuni cause fever, diarrhea, and abdominal cramps and gastroenteritis in humans [21]. This bacterium can lead to the development of Guillian-Barre Syndrome (GBS), a disorder of the peripheral nervous system often leading to partial paralysis. The food matrixes that act as carriers for C. jejuni are poultry, meat and milk [21]. Campylobacter is thermo-tolerant and can survive and grow at 40ºC. This bacterium lives in the intestines of chicken, and is often carried over with raw poultry. Eating undercooked chicken is the most frequent source of infection [21]. Wei et al. (2007) developed a biosensor based on surface plasmon resonance (SPR) for the rapid identification of C. jejuni in broiler samples [81]. Wei et al. (2007) examined the specificity and sensitivity of commercial antibodies against C. jejuni with six Campylobacter strains and six non-Campylobacter bacterial strains.
Microbial pathogens and strategies for combating them: science, technology and education (A. Méndez-Vilas, Ed.)
Rapid detection of pathogenic microorganisms is important for reducing diseases. Current methods rely on conventional culture-based techniques, antibody-based ELISA or DNA-based PCR techniques which are time-wise lengthy, need expensive instrumentation, require trained personnel, and are not effective for on-site use. On the other hand, analytical biosensors can detect pathogens in a much shorter time with high sensitivity, selectivity and offer the possibility to perform multiple analyses. These devices can work with fairly complex target samples without the need for sample enrichment and without a major compromise on either sensitivity or selectivity. Since they are low-cost and high performance devices and do not require trained personnel, they potentially can be used as stand-alone devices for on-site monitoring. The future holds much promise to apply analytical biosensors for the rapid detection of pathogenic microorganisms in several sectors such as food, agriculture and horticulture, medical and veterinary diagnostics and environmental field.
Acknowledgements The support by the Universidad Nacional de San Luis, the Agencia Nacional de Promoción Científica y Tecnológica, and the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) is gratefully acknowledged.
References [1] Sachse K, Frey J, eds. Methods in Molecular Biology, PCR Detection of Microbial Pathogens: Methods and Protocols. 216, 1st
ed. Totowa, NJ: Humana Press Inc, Springer; 2002:352. [2] Diguta CF, Rousseaux S, Weidmann S, Bretin N, Vincent B, Guilloux-Benatier M, Alexandre H. Development of a qPCR assay
for specific quantification of Botrytis cinerea on grapes. FEMS Microbiology Letters. 2010;313:81-87. [3] Thacker JD, Casale ES, Tucker CM. Immunoassays (ELISA) for rapid, quantitative analysis in the food-processing industry.
Journal Agricultural and Food Chemistry. 1996;44:2680-2685. [4] Iqbal SS, Mayo MW, Bruno JG, Bronk BV, Batt CA, Chambers P. A review of molecular recognition technologies for
detection of biological threat agents. Biosensors and Bioelectronics. 2000;15:549-578. [5] Andreotti PE, Ludwig GV, Peruski AH, Tuite JJ, SS Morse, Peruski LF. Immunoassay of infectious agents. BioTechniques.
2003;35:850-859. [6] Peruski AH, Peruski LF. Immunological methods for detection and identification of infectious disease and biological warfare
agents. Clinical and Diagnostic Laboratory Immunology. 2003;10:506-513. [7] Nayak M, Kotian A, Marathe S, Chakravortty D. Detection of microorganisms using biosensors-A smarter way towards
detection techniques. Biosensors and Bioelectronics. 2009;25:661-667. [8] Skládal P, Kovář D, Krajíček V, Šišková P, Přibyl J, Švábenská E. Electrochemical immunosensors for detection of
microorganisms. International Journal of Electrochemical Science. 2013;8:1635-1649. [9] D’Orazio P. Biosensors in clinical chemistry. Clinica Chimica Acta. 2003;334:41-69. [10] Cruz HJ, Rosa CC, Oliva AG. Immunosensors for diagnostic applications. Parasitology Research. 2002;88:S4-S7. [11] Luppa PB, Sokoll LJ, Chan DW. Immunosensors-principles and applications to clinical chemistry. Clinica Chimica Acta.
2001;314:1-26. [12] Lazcka O, Del Campo FJ, Muñoz FX. Pathogen detection: A perspective of traditional methods and biosensors. Biosensors and
Bioelectronics. 2007;22:1205-1217. [13] Deisingh AK, Thompson M. Biosensors for the detection of bacteria. Canadian Journal of Microbiology. 2004;50:69-77. [14] Hall RH.Biosensor technologies for detecting microbiological foodborne hazards. Microbes and Infection. 2002;4:425-432. [15] Ivnitski DI, Abdel-Hamid P, Atanasov A, Wilkins E. Biosensors for detection of pathogenic bacteria. Biosensors and
Bioelectronics. 1999;14:599-624. [16] Aberl F, Kößlinger C. Biosensor-based methods in clinical diagnosis. Reischl U, ed. In Molecular diagnosis of infectious
diseases.13, Totowa, NJ: Humana Press Inc, Springer; 1998:503-517. [17] Mittelmann AS, Ron EZ, Rishpon J. Amperometric quantification of total coliforms and specific detection of Escherichia coli.
Analytical Chemistry. 2002;74:903-907. [18] Leonarda P, Heartya S, Brennana J, Dunnea L, Quinna J, Chakrabortyc T, O’Kennedya R. Advances in biosensors for detection
of pathogens in food and water. Enzyme and Microbial Technology. 2003;32:3-13. [19] Warriner K, Namvar A. 4.54 Biosensors for Foodborne Pathogen Detection. Agricultural and Related Biotechnologies.
[20] Bhunia AK. Biosensors and Bio‐Based Methods for the Separation and Detection of Foodborne Pathogens. Advances in Food and Nutrition Research. 2008;54:1-44.
[21] Sharma CH, Mutharasan R. Review of Biosensors for Foodborne Pathogens and Toxins. Sensors and Actuators B: Chemical. 2013;183:535-549.
[22] Arora P, Sindhu A, Dilbaghi N, Chaudhury A. Biosensors as innovative tools for the detection of food borne pathogens. Biosensors and Bioelectronics. 2011;28:1-12.
[23] Tothill IE. Biosensors developments and potential applications in the agricultural diagnosis sector. Computers and Electronics in Agriculture. 2001;30:205-218.
[24] Mello LD, Kubota LT. Review of the use of biosensors as analytical tools in the food and drink industries. Food Chemistry. 2002;77:237-256.
[25] Garcia-Aljaro C, Cell LN, Shirale DJ, Park M, Muñoz FJ, Yates MV, Mulchandani A. Carbon nanotubes-based chemiresistive biosensors for detection of Microorganisms. Biosensors and Bioelectronics. 2010;26:1437-1441.
Microbial pathogens and strategies for combating them: science, technology and education (A. Méndez-Vilas, Ed.)
[26] Fernández-Baldo MA, Messina GA, Sanz MI, Raba J. Screen-printed immunosensor modified with carbon nanotubes in a continuous flow system for the Botrytis cinerea determination in apple tissues. Talanta. 2009;79:681-686.
[27] Fernández-Baldo MA, Messina GA, Sanz MI, Raba J. Microfluidic Immunosensor with Micromagnetic Beads Coupled to Carbon-Based Screen-Printed Electrodes (SPCEs) for Determination of Botrytis cinerea in Tissue of Fruits. Journal of Agricultural and Food Chemistry. 2010;58:1120-1120.
[28] Fernández-Baldo MA, Bertolino FA, Fernández G, Messina GA, Sanz MI, Raba J. Determination of Ochratoxin A in apples contaminated with Aspergillus ochraceus by using a microfluidic competitive immunosensor with magnetic nanoparticles. Analyst. 2011;136:2756-2762.
[29] Tothill IE, Turner APF. Biosensors. Encyclopedia of Food Sciences and Nutrition.2nd ed. Amsterdam: Elsevier; 2003:489-499. [30] Gilmartina N, O’Kennedya R. Nanobiotechnologies for the detection and reduction of pathogens. Enzyme and Microbial
Technology. 2012;50:87-95. [31] Clark Jr LC, Lyons C. Electrode systems for continuous monitoring in cardiovascular surgery. Annals of the New York Academy
of Sciences. 1962;102:29-45. [32] Rechnitz GA, Kobos RK, Riechel SJ, Gebauer CR. A bioselective membrane electrode prepared with living bacterial cells.
Analytica Chimica Acta. 1977;94:357-365. [33] Thevenot DR, Toth K, Durst RA, Wilson GS. Electrochemical biosensors: recommended definitions and classifications.
Biosensensors and Bioelectronics. 2001;16:121-131. [34] Inczedy J, Lengyel T, Ure A.M, eds. IUPAC Compendium of Analytical Nomenclature. 3rd ed. Blackwell Science, Oxford;
1998:964. [35] Bartlett PN, Tebbutt P, Whitaker RG. Kinetic aspects of the use of modified electrodes and mediators in bioelectrochemistry.
Progress in Reaction Kinetics and Mechanism. 1991;16:55-155. [36] Wollenberger U, Schubert F, Pfeiffer D, Scheller FW. Enhancing biosensor performance using multienzyme systems. Trends
determination of glucose. Analytical Chemistry. 1979;51:96-100. [38] Aizawa M. Principles and applications of electrochemical and optical biosensors. Analytica Chimica Acta. 1991;250:249-256. [39] Sugawara M, Sato H, Ozawa T, Umezawa Y, Scheller FW, Scubert F, Fedrowitz J, eds. Receptor Based Chemical Sensing.
Frontiers in Biosensorics, I, Fundamental Aspects. Birkhäuser Verlag, Basel/Switzerland; 1997:121-131. [40] Kiefer H, Klee B, John E, Stierhof YD, Jähnig F. Biosensors based on membrane transport proteins. Biosensensor and
Bioelectronics. 1991;6:233-237. [41] Nayak M, Kotian A, Marathe S, Chakravortty D. Detection of microorganisms using biosensors-A smarter way towards
detection techniques. Biosensors and Bioelectronics. 2009;25:661-667. [42] Rodríguez-Lázaro D, Cook N, Ruggeri FM, Sellwood J, Nasser A, Nascimento MSJ, D'Agostino M, Santos R, Saiz JC,
Rzeżutka A, Bosch A, Gironés R, Carducci A, Muscillo M, Kovač K,Diez-Valcarce M, Vantarakis A, von Bonsdorff C-H, Roda Husman AM, Hernández M, van der Poel WHM. Virus hazards from food, water and other contaminated environments. FEMS Microbiology Reviews. 2012;36:786-814.
[43] Guo Y, Chen JH, Guonan C. A label-free electrochemical biosensor for detection of HIV related gene based on interaction between DNA and protein. Sensors and Actuators B: Chemical. 2013;184:113-117.
[44] Lee JH, Kim BC, Oh BK, Choi JW. Highly sensitive localized surface plasmon resonance immunosensor for label-free detection of HIV-1. Nanomedicine: Nanotechnology, Biology, and Medicine. 2013;DOI:10.1016/j.nano.2013.03.005.
[45] Yao C, Xiang Y, Deng K, Xia H, Fu W. Sensitive and specific HBV genomic DNA detection using RCA-based QCM biosensor. Sensors and Actuators B: Chemical. 2013;181:382-387.
[46] Yao C, Zhu T, Tang J, Wu R, Chen Q, Chen M, Zhang B, Huang J, Fu W. Hybridization assay of hepatitis B virus by QCM peptide nucleic acid biosensor. Biosensors and Bioelectronics. 2008;23:879-885.
[47] Timurdogan E, Alaca BE, Kavakli IH, Urey H. MEMS biosensor for detection of Hepatitis A and C viruses in serum. Biosensors and Bioelectronics. 2011;28:189-194.
[48] Sam SS, Steinmetz HB, Tsongalis GJ, Tafe LJ, Lefferts JA. Validation of a solid-phase electrochemical array for genotyping hepatitis C virus. Experimental and Molecular Pathology. 2013;95:18-22.
[49] MultisHRP-DNA-coated CMWNTs as signal labels for an ultrasensitive hepatitis C virus core antigen electrochemical immunosensor. Ma C, Liang M, Wang L, Xiang H, Jiang Y, Li Y, Xie G. Biosensors and Bioelectronics. 2013;47:467-474.
[50] Pereira SV, Bertolino FA, Messina GA, Raba J. Microfluidic immunosensor with gold nanoparticle platform for the determination of immunoglobulin G anti-Echinococcus granulosus antibodies. Analytical Biochemistry. 2011;409:98-104.
[51] Pereira SV, Bertolino FA, Fernandez-Baldo MA, Messina GA, Salinas E, Sanz MI, Raba J. A microfluidic device based on a screen-printed carbon electrode with electrodeposited gold nanoparticles for the detection of IgG anti-Trypanosoma cruzi antibodies. Analyst. 2011;136:4745-4751.
[52] Belluzo MS, Ribone ME, Camussone C, Marcipar IS, Lagier CM. Favorably orienting recombinant proteins to develop amperometric biosensors to diagnose Chagas’ disease. Analytical Biochemistry. 2011;408:86-94.
[53] Salinas E, Torriero AAJ, Battaglini F, Sanz MI, Olsina R, Raba J. Continuous-flow/stopped-flow system for enzyme immunoassay using a rotating bioreactor: determination of Chagas disease. Biosensors and Bioelectronics. 2005;21:313-321.
[54] Diniz FB, Ueta RR, Pedrosa AMC, Areias MC, Pereira VRA, Silva ED, da Silva JG, Ferreira AGP, Gomes YM. Impedimetric evaluation for diagnosis of Chagas’ disease: antigen-antibody interactions on metallic electrodes. Biosensors and Bioelectronics. 2003;19:79-84.
[55] Ittarat W, Chomean S, Sanchomphu C, Wangmaung N, Promptmas C, Ngrenngarmlert W. Biosensor as a molecular malaria differential diagnosis. Clinica Chimica Acta. 2013;419:47-51.
[56] Jeon W, Lee S, DH M, Ban C. A colorimetric aptasensor for the diagnosis of malaria based on cationic polymers and gold nanoparticles. Analytical Biochemistry. 2013;439:11-16.
Microbial pathogens and strategies for combating them: science, technology and education (A. Méndez-Vilas, Ed.)
[57] Sharma MK, Rao VK, Agarwal GS, Rai GP, Gopalan N, Prakash S, Sharma SK, Vijayaraghavan R. Highly Sensitive Amperometric Immunosensor for Detection of Plasmodium falciparum Histidine-Rich Protein 2 in Serum of Human with Malaria: Comparison with a Commercial Kit. Journal of Clinical Microbiology. 2008;46:3759-3765.
[58] Mukundan H, Kumar S, Price DN, Ray SM, Lee Y-J, Min S, Eum S, Kubicek-Sutherland J, Resnick JM, Grace WK, Anderson AS, Hwang SH, Cho SN, Via LE, Barry C 3rd, Sakamuri R, Swanson BI. Rapid detection of Mycobacterium tuberculosis biomarkers in a sandwich immunoassay format using a waveguide-based optical biosensor. Tuberculosis. 2012;92:407-416.
[59] Hiatt LA, Cliffel DE. Real-time recognition of Mycobacterium tuberculosis and lipoarabinomannan using the quartz crystal microbalance. Sensors and Actuators B: Chemical. 2012;174:245-252.
[60] Bernacka-Wojcik I, Lopes P, Catarina Vaz A, Veigas B, Jerzy Wojcik P, Simões P, Barata D, Fortunato E, Viana Baptista P, Aguas H, Martins R. Bio-microfluidic platform for gold nanoprobe based DNA detection-application to Mycobacterium tuberculosis. Biosensors and Bioelectronics. 2013;48:87-93.
[61] Low K-F, Karimah A, Yean CY. A thermo stabilized magnetogenosensing assay for DNA sequence-specific detection and quantification of Vibrio cholera. Biosensors and Bioelectronics. 2013;47:38-44.
[62] Chua A, Yean CY, Ravichandran M, Lim B, Lalitha P.A rapid DNA biosensor for the molecular diagnosis of infectious disease. Biosensors and Bioelectronics. 2011;26:3825-3831.
[63] Yu CY, Ang GY, Chua AL, Tan EH, Lee SY, Falero-Diaz G, Otero O, Rodríguez I, Reyes F, Acosta A, Sarmiento ME, Ghosh S, Ramamurthy T, Yean Yean C, Lalitha P, Ravichandran M. Dry-reagent gold nanoparticle-based lateral flow biosensor for the simultaneous detection of Vibrio cholera serogroups O1 and O139. Journal of Microbiological Methods. 2011;86:277-282.
[64] Chen H, Hu Q-Y, Yue-Zheng, Jiang J-H, Shen G-L, Yu R-Q. Construction of supported lipid membrane modified piezoelectric biosensor for sensitive assay of cholera toxin based on surface-agglutination of ganglioside-bearing liposomes. Analytica Chimica Acta. 2010;657:204-209.
[65] Seia MA, Pereira SV, Fontan CA, De Vito IE, Messina GA, Raba J. Laser-induced fluorescence integrated in a microfluidic immunosensor for quantification of human serum IgG antibodies to Helicobacter pylori. Sensors and Actuators B: Chemical. 2012;168:297-302.
[66] Pereira SV, Messina GA, Raba J. Integrated microfluidic magnetic immunosensor for quantification of human serum IgG antibodies to Helicobacter pylori. Journal of Chromatography B. 2010;878:253-257.
[67] Stege PW, Raba J, Messina GA. Online immunoaffinity assay-CE using magnetic nanobeads for the determination of anti-Helicobacter pylori IgG in human serum. Electrophoresis. 2010;31:3475-3481.
[68] Molina L, Messina GA, Stege PW, Salinas E, Raba J. Immuno-column for on-line quantification of human serum IgG antibodies to Helicobacter pylori in human serum samples. Talanta. 2008;76:1077-1082.
[69] Wang Y, Ye Z, Si C, Ying Y. Monitoring of Escherichia coli O157:H7 in food samples using lectin based surface plasmon resonance biosensor. Food Chemistry. 2013;136:1303-1308.
[70] Dweik M,Stringer RC, Dastider SG,Wu Y, Almasri M, Barizuddin S. Specific and targeted detection of viable Escherichia coli O157:H7 using a sensitive and reusable impedance biosensor with dose and time response studies. Talanta. 2012;94:84-89.
[71] Chowdhury AD, De A, Chaudhuri CR, Bandyopadhyay K, Sen P. Label free polyaniline based impedimetric biosensor for detection of E. coli O157:H7 Bacteria. Sensors and Actuators B: Chemical. 2012;171:916-923.
[72] Waswa J, Irudayaraj J, DebRoy C. Direct detection of E. Coli O157:H7 in selected food systems by a surface plasmon resonance biosensor. LWT - Food Science and Technology. 2007;40:187-192.
[73] Lan Y, Wang Z, Yin Y, Hoffmann C, Zheng X. Using a Surface Plasmon Resonance Biosensor for Rapid Detection of Salmonella Typhimurium in Chicken Carcass. Journal of Bionic Engineering. 2008;5:239-246.
[74] Zhang D, Yan Y, Li Q, Yu T, Cheng W, Wang L, Ju H, Ding S. Label-free and high-sensitive detection of Salmonella using a surface plasmon resonance DNA-based biosensor. Journal of Biotechnology. 2012;160:123-128.
[75] Park MK, Park JW, Wikle HC, Chin BA. Evaluation of phage-based magnetoelastic biosensors for direct detection of Salmonella typhimurium on spinach leaves. Sensors and Actuators B: Chemical. 2013;176:1134-1140.
[76] Dong J, Zhao H, Xu M, Ma Q, Ai S. A Label-free Electrochemical Impedance Immunosensor Based on AuNPs/PAMAM-MWCNT-Chi nanocomposite Modified Glassy Carbon Electrode for Detection of Salmonella typhimuriumin milk. Food Chemistry. 2013;DOI:dx.doi.org/10.1016/j.foodchem.2013.04.098.
[77] Sim JH, Kwak YH, Choi CH, Paek S, Park SS, Seo S. A birefringent waveguide biosensor platform for label-free live cell detection of Listeria monocytogenes. Sensors and Actuators B: Chemical. 2012;173:752-759.
[78] Sharma H, Mutharasan R. Rapid and sensitive immunodetection of Listeria monocytogenesin milk using a novel piezoelectric cantilever sensor. Biosensors and Bioelectronics. 2013;45:158-162.
[79] Park MK, Park JW, Oh JH. Optimization and application of a dithiobis-succinimidyl propionate-modified immunosensor platform to detect Listeria monocytogenes in chicken skin. Sensors and Actuators B: Chemical. 2012;171-172:323-331.
[80] Ohk SH, Bhunia AK. Multiplex fiber optic biosensor for detection of Listeria monocytogenes, Escherichia coli O157:H7 and Salmonella enteric from ready-to-eat meat samples. Food Microbiology. 2013;33:166-171.
[81] Wei D, Oyarzabal OA, Huang TS, Balasubramanian S, Sista S, Simonian AL. Development of a surface plasmon resonance biosensor for the identification of Campylobacter jejuni. Journal of Microbiological Methods. 2007;69:78-85.
Microbial pathogens and strategies for combating them: science, technology and education (A. Méndez-Vilas, Ed.)
