Fluorescence-Based Bioassays for the Detection and Evaluation of Food Materials

We summarize here the recent progress in fluorescence-based bioassays for the detection and evaluation of food materials by focusing on fluorescent dyes used in bioassays and applications of these assays for food safety, quality and efficacy. Fluorescent dyes have been used in various bioassays, such as biosensing, cell assay, energy transfer-based assay, probing, protein/immunological assay and microarray/biochip assay. Among the arrays used in microarray/biochip assay, fluorescence-based microarrays/biochips, such as antibody/protein microarrays, bead/suspension arrays, capillary/sensor arrays, DNA microarrays/polymerase chain reaction (PCR)-based arrays, glycan/lectin arrays, immunoassay/enzyme-linked immunosorbent assay (ELISA)-based arrays, microfluidic chips and tissue arrays, have been developed and used for the assessment of allergy/poisoning/toxicity, contamination and efficacy/mechanism, and quality control/safety. DNA microarray assays have been used widely for food safety and quality as well as searches for active components. DNA microarray-based gene expression profiling may be useful for such purposes due to its advantages in the evaluation of pathway-based intracellular signaling in response to food materials.


Introduction
Fluorescent dyes or fluorophores have been widely used as probes (for physical and structural parameters), indicators (e.g., for molecular concentrations) or labels/tracers (e.g., for visualization and localization of biomolecules) in various bioassays [1]. While the development of fluorescent dyes has a history many centuries long, their importance has increased due to the recent advancement of new fluorescent dyes [2], which have been developed along with the development of new biotechnological tools and devices. For example, Laurdan, a naphthalene-based amphiphilic fluorescent dye having as characteristics the ability to penetrate membranes and a large Stokes shift, was developed to study membrane fluidity and dynamics, and its usage was made quite effective by the development of two-photon fluorescent microscopy, a microscope system with two-photon excitation, which enables the detection of signals with less background, less photodamage and more depth discrimination [3][4][5].
Therefore, the development of fluorescent dyes has had quite an impact when accompanied by the development of suitable devices and their applications. One of the most important currently emerging research fields is the development and application of technologies for new functional foods and quality control and safety of its production. Along with technological innovations, the effective usage of gene/genome information in the pathway-based evaluation of materials is crucial. We summarize here recent progress in fluorescence-based bioassays, including genomic and transcriptomic assays, by focusing on their applications in the study of food safety, quality and efficacy.

Overview of Fluorescent Dyes
Fluorescent dyes are generally polyaromatic or heterocyclic hydrocarbons, which undergo a three-stage process of fluorescence: excitation, excited-state lifetime and fluorescence emission [6]. Fluorescent dyes are characterized by key properties, such as those revealed by the absorption maximum (λmax), the emission maximum (λem), the extinction coefficient (ε) and the fluorescence quantum yield (Φ) [2]. For example, the "Stokes shift", defined by the difference between λmax and λem, is an important property of a fluorescent dye, and a large Stokes shift helps to avoid the reabsorption of emitted photons, giving higher contrast in fluorescent imaging [7].
New technologies, materials and devices have been developed for the efficient detection and utilization of the fluorescence signals in a biological specimen. For example, fluorescence-activated cell sorting (FACS) is an example of the successful application of fluorescence technologies for flow cytometry, and is now used in basic as well as industrial fields of life science [8,9]. Flow cytometry is a technique used for cell counting, cell sorting and biomarker detection, by passing a cell suspension in a stream of fluid through an electronic detection apparatus, allowing simultaneous multiparametric analyses of many thousands of micrometer-sized particles per second. Its applications include food study, such as water testing, milk analysis, brewing/wine production and food microbiology [10]. Meanwhile, fluorescence in situ hybridization (FISH) is a cytogenetic technique in which fluorescently labeled probes are hybridized with parts of DNA on chromosomes or specific RNA targets (e.g., mRNA and miRNA), and signals are detected by fluorescence microscopy. After a 30-year history, the original FISH protocol has been diversified into a number of new protocols with improved sensitivity, specificity and resolution [11]. For example, chromosome orientation-FISH, or CO-FISH, can detect strand-specific target DNA, and thus is useful to detect chromosomal abnormalities, such as Robertsonian translocations, chromosomal inversion and telomeric alterations [12].
A number of fluorescent techniques utilize Förster resonance energy transfer (FRET), a mechanism of energy transfer from a donor dye to a different acceptor dye, which is used to analyze conformations, interactions and concentrations of proteins and nucleic acids [6]. Protein-protein interactions can be detected by other fluorescent techniques, such as bioluminescence resonance energy transfer (BRET) assay, a modification of FRET, and biomolecular fluorescence complementation (BiFC) assay. BiFC assay is based on structural complementation between two non-fluorescent N-and C-terminal fragments of a fluorescent protein, and has contrasting advantages and disadvantages compared with FRET [13,14].
Other than aromatic hydrocarbons, several unique materials have also been utilized for fluorescence applications. Quantum dots are fluorescent semiconductor nanoparticles that have potential in biology, such as specific labeling of cells and tissues, long-term imaging, lack of cytotoxicity, in vivo multicolor imaging and FRET-based sensing [15]. A variety of fluorescent colors are available, depending on the size and shape of the particles. Additionally, some lanthanide ions are useful for bioassays due to their superior characteristics, such as long fluorescent lifetimes, large Stokes shifts and sharp emission profiles [16]. These materials have been used to study food safety, quality and efficacy (see Section 2).

Fluorescent Dyes for Bioassays
Fluorescent probes are required to match certain conditions for experiments, such as wavelength range, Stokes shift and spectral bandwidth, which are partly imposed by the instrumentation and the requirements of multicolor labeling experiments [6]. To design fluorescent experiments, the fluorescent output of a dye judged by the extinction coefficient and the fluorescence quantum yield needs to be considered. Additionally, under high-intensity illumination conditions, the irreversible destruction or photobleaching of fluorescent dyes is an important factor. Polyaromatic fluorescent dyes with extended π-conjugated systems could thus be ideal for designing dyes with longer Stokes shifts [7], which may improve the performance of fluorescent dyes. Here, we summarize the fluorescent dyes frequently used for bioassays.
Since its first synthesis in 1871, fluorescein, along with its derivatives, has been used as a powerful tool in various fields of life science [17]. Fluorescein is composed of two parts of xanthene, the chromophore part, and benzene, and exhibits excitation at 490 nm and emission at 514 nm (λmax/λem = 490/514 nm), with fluorescent properties of ε = 9.3 × 10 4 M −1 ·cm −1 and Φ = 0.95 [2]. A variety of fluorescein derivatives have been synthesized to improve its chemical, fluorescent and biological properties, and its stability, such as Oregon Green, fluorescein isothiocyanate (FITC), fluorescein diacetate and carboxyfluorescein (FAM). These dyes and fluorescein have been used in various bioassays/biomaterials, such as cell assays (flow cytometry, suspension arrays, fluorescent microscopy, fluorescent cell assay and fluorescent cytomics), FRET-based assays, probing (CO-FISH, probing (fluorescent caspase assay and fluorescent reporter assay), protein/immunological assays (BiFC) and microarray/biochip assays (see Section 2.1).

Fluorescent Dyes Used in DNA Microarray Assay
Fluorescent dyes play important roles in DNA microarray assays due to their detectivity, speed and increased safety [28]. Fluorescent dyes frequently used in DNA microarray assays are phycoerythrin, Alexa Fluor dyes and cyanines. They have been used either as a single dye, such as phycoerythrin, or as two-color fluorescent probes, such as Cy3/Cy5 and Alexa Fluor 555/647. Owing to their superior reliability, Cy3 and Cy5 have been frequently used in gene expression profiling by means of DNA microarray assays since the early days [19]. Cyanines are, however, suggested to have low photostability and to be destabilized by their negative charges [32], as well as being affected by atmospheric ozone in the laboratory [33] and fluorescence quenching [34]. Alexa Fluor dyes, on the other hand, show greater brightness and photostability than cyanines [6]. Phycoerythrin is used in Affymetrix GeneChip assay as a streptavidin-conjugated form to detect biotinylated target cRNA hybridized with the probes on the platform. However, a significant decrease in fluorescent intensity was observed for phycoerythrin [35]. Mitsubishi Rayon developed a hollow fiber array, Genopal, in which fibers are filled with hydrogels attached to oligonucleotide probes and Cy5-labeled target cDNA is hybridized with the probes [36]. Although cyanines were generally used to label probes in the DNA microarray assay developed by GE Healthcare, a fluorescent dye, Amersham HyPer5, was developed and used to label target DNA [37]. Fluorescence-based microarray/biochip assays are summarized below (see Sections 2.3 and 3.1).

Application of Fluorescence-Based Bioassays
Fluorescence-based bioassays have been applied in biotechnology and various fields in life science. For example, various fluorescently labeled antibodies have been used to detect specific organelles, cellular activities (e.g., cell morphology, viability and functions) and cellular processes (e.g., transportation, endocytosis and receptor function) [38]. Clinical and pharmacological applications of fluorescent probes have been explored to diagnose leukemia and other cancers [39]. These applications are supported by basic characteristics of fluorophores, such as structural and environmental effects on fluorescence emission, fluorescence polarization and FRET, which are applied for spectrofluorometry, fluorescent microscopy and fluorescence-based chemical sensing to trace and image biological objects [1]. In this section, we summarize first fluorescence-based bioassays and then their applications by discussing representative literature.

Fluorescence-Based Bioassays
Fluorescence-based bioassays, classified into biosensing, cell assays, energy transfer-based assays, probing, protein/immunological assays and microarray/biochip assays, are summarized in Table 1. Biosensing, such as fluorescent molecular biosensor, fluorometric high-performance liquid chromatography (HPLC) and magnetic modulation biosensing, has been used to detect intermolecular interactions and targets at low concentrations, or to analyze nitrite/nitrate, where fluorescent dyes, such as Alexa Fluor 488, GFP and 2,3-naphthotriazole, have been used. Cell assays, such as flow cytometry (fluorescence-activated cell sorting: FACS), fluorescent cytomics, fluorescence microscopy, fluorescent reporter-gene assay and live-cell imaging, have been used in particle-based flow cytometric assay, drug delivery research and applications of RNA/DNA aptamers to measure cell fluorescence, to screen hormonally active compounds and to examine gene expression/protein interaction, where fluorescent dyes, such as fluoresceins (including FAM and FITC), GFP/GFP-family proteins, lanthanides, phycoerythrin and rhodamines (TMR-C5), have been used. Meanwhile, energy transfer-based assays have been used in live-cell imaging and to analyze protein structure, where fluorescent dyes, such as BFP/GFP, FITC and phycoerythrin, have been used.
A number of technologies have been developed for various types of probe, such as fluorescent calcium indicators, fluorescent caspase substrates, fluorescent nanoparticles, fluorescent nucleic acids, quantum dots and small-molecule fluorochromes, which have been used in fluorescent hybridization (e.g., FISH), reporter-gene assay and fluorescent spectroscopy, often used in combination with FRET, to examine chromosome aberrations/segregations, gene-gene/DNA-protein interactions, calcium signaling and ion channeling; to evaluate fluorescence bioassays, imaging/labeling/sensing, immunoassays/microarray assays, quantitative structure-activity relationship (QSAR), antimycobacterial susceptibility, biological enzymatic reactions, G-protein-coupled receptor (GPCR) ligands and reactive oxygen species; and to screen bladder and other tumor markers, antagonists of GPCRs and anticancer drugs, by using fluorescent dyes, such as Alexa Fluor 546, Cy3/Cy5, FDA, FITC, Fluo-4, fluorescein, FuraRed, GFP/RFP, hydroethidine/hydrocyanines, quantum dots, rhodamine, SpectrumGold/SpectrumOrange, TRITC and Texas Red.  Microarray/Biochip Assay (see Table 2) Protein/immunological assays, such as BiFC, chemifluorescent enzyme-linked immunosorbent assay (ELISA), fluorescent dye-based protein assay, immuno-detection (FACTT; see Table 1), lanthanide-doped fluorescent assay, lanthanide fluorescent immunoassay, real-time immuno-PCR and sandwich fluoroimmunoassay, have been used to examine bioassay/therapy, kinase activity, protein interaction/modification and time-resolved fluorescence bioassay, to screen/identify rare blood biomarkers and some toxins, to study prion diseases and to identify viral antigens/pathogens, for which fluorescent dyes, such as BEBO (a cyanine dye), Cy5, GFP/YFP, lanthanides (e.g., Eu 3+ , Sm 3+ , Tb 3+ and Dy 3+ ), NanoOrange, RiboGreen and SYBR Green I, have been used. Microarray/biochip assays are discussed below (see Section 2.3). GFP has been used quite often as reporter conjugates in cell assay. For example, estrogen activity was detected by a reporter construct of the human ERα gene fused to yeast enhanced GFP (yEGFP), which was used as a rapid yeast bioassay to screen estrogen activity in calf urine [83]. This construct was validated as a bioassay for hormonal substances in feed [84], and concomitantly improved by the combination with mass-spectrometry techniques [85,86] and by the use of various test samples [87,88], and combined with androgen assay [89], to attain to a level of a standardized multi-hormonal bioassay system.

Antibody/Protein Microarray
Protein microarray assay is a high-throughput method used to study biochemical activities of proteins, by measuring their binding affinities, specificities and quantities [90]. The array has a support surface, such as a slide glass, a nitrocellulose membrane, a bead and a microtiter plate, to which the captured protein is bound as an array, and probe molecules, typically labeled with a fluorescent dye or conjugated with enzymes for chemiluminescent or colorimetric assays, are added to the array. In a fluorescent assay, the reaction between the fluorescence-labeled probe and the immobilized protein causes the emission of a fluorescent signal at a specific position, which is detected by a laser scanner. There are three types of protein microarray used to study the biochemical activities of proteins: analytical microarrays, functional protein microarrays and reverse-phase protein microarrays [90]. Antibody microarrays belong to the category of analytical microarrays, and sometimes use a sandwich format consisting of capture antibodies (e.g., biotinylated antibodies), analytes (e.g., toxins) and reporter molecules (e.g., avidin-conjugated nanoparticles and fluorophore-conjugated secondary antibodies). They have been used to screen foodborne pathogens such as Escherichia coli O157:H7 and Salmonella spp.

Bead/Suspension Array
The detection of bacterial/plant toxins [93], mycotoxins [94] and pesticides [95] in food has been carried out by using bead/suspension array technology, in which fluorescent dye-labeled microspheres/beads are often used. Appropriate molecules or receptors, such as DNA (oligonucleotides), and antibodies and other proteins, are attached to the microspheres differently labeled with fluorescent dyes, for example. Beads are readily suspendable in solution and are used for hybridization between receptors and corresponding reactive biomolecules. Bead arrays have advantages over flat arrays in the array preparation (containing millions of particles per milliliter) and density (containing hundreds of thousands of array elements per microliter), enabling multiparameter detection and high-throughput processing [96]. Since the optical property of each bead is known, target biomolecules hybridized/bound to the beads can be easily differentiated, and quantification can be achieved by comparing the relative intensity of targets in a set of beads with that of markers in another set of beads using fluorescence detection apparatuses, such as a flow cytometer.

Capillary/Sensor Array
A sensor array typically consists of a recognition component, a transducer component and an electronic detection system. The recognition component uses biomolecules to interact with the analyte of interest. This interaction is measured by biotransducers, such as an optical transducer, which outputs a measurable signal proportional to the presence of the target analyte in the sample. Meanwhile, biomolecules are separated first by capillary electrophoresis in an array and then detected by appropriate sensors in capillary arrays. There have been cases of the application of capillary/sensor arrays for food analysis, such as detecting pathogens and toxins, and fluorescent substances are commonly used in their detection systems. Recently, researchers have performed successful analyses of food using improved sensor arrays, such as those with dendritic fluorophores [97] and a fluorescent indicator-displacement sensor array using titania as a host material [98].

DNA Microarray/PCR-Based Array
Using DNA microarray technology, multiple genes can be characterized simultaneously in a single assay. It has been used widely for the analysis of gene expression, but it can also be used for the analysis of microbial pathogens for food safety and environmental applications. A DNA microarray involves the immobilization of numerous probes, such as cDNA and oligonucleotide probes, at a high density on a solid matrix, such as glass, to which fluorescence-labeled PCR-amplified target DNA fragments can be hybridized. The signal generated by the bound labeled targets on the microarray allows identification based on the known locations of the probes on the array. Applications of DNA microarray technology for the detection of pathogens contaminating food have been reported (detailed in Section 3).

Glycan/Lectin Array
The use of glycan microarrays, comprising multiple different glycans on a single platform, is a technique for the analysis of glycosylation patterns and the screening of a number of glycan-binding proteins for investigation of their roles in biological systems. Recently, a shotgun glycan microarray prepared from isolated human milk glycans was reported, where viruses, antibodies and glycan-binding proteins including lectins were detected in order to examine the diverse recognition functions of human milk glycans [99]. In addition, a lectin microarray, based on the specific affinity of a lectin to a specific glycan, is another useful platform for glycan analysis. Recently, a bead-based multiplex lectin array was developed, where respective lectins were coupled to differentially fluorescent dye-coated microbeads [100]. These beads were incubated with biotin-labeled glycoproteins in suspension, with visualization using the interaction between biotin and streptavidin-R-phycoerythrin. This microarray was applied for glycosylation profiling of hepatocellular carcinoma-associated immunoglobulin G in a rapid, sensitive and reproducible manner.

Immunoassay/ELISA-Based Array
An immunoassay is a test that relies on the inherent ability of an antibody to recognize and bind to a specific antigen, which might exist in a complex mixture, to measure the presence and/or concentration of the antigen. In life science research, immunoassays are often used in studies of the biological functions of proteins, while, in industry, immunoassays are used in various applications, such as to detect contaminants in food and water and to monitor and assess specific molecules during food processing. In immunoassays, antibodies or antigens are conjugated or coupled with fluorescent dyes, or labeled with other materials, such as biotin and horseradish peroxidase, to produce measurable fluorescent, chemiluminescent or chromogenic signals for detection. One of the most popular immunoassays is ELISA, in which antigens in a sample are first attached to the surface of the platform (e.g., a 96-well microtiter plate), which are then detected with a specific antibody linked to an enzyme (for enzymatic reactions) or a fluorescently labeled secondary antibody. In recent years, fluorogenic labels, such as cyanines and phycoerythrin, have been used in immunoassays to detect mycotoxins for food safety [101,102].

Microfluidic Chip
Microfluidic chips have been used in many biological fields, such as drug screening and the monitoring of food processing. A microfluidic chip is a set of microchannels molded into a material like glass, silicon or polymer. The microchannels are connected together forming a network, which is connected to the outside by transporting inputs and outputs through the chip platform. The surface patterning of bonded or sealed microchannels in a microfluidic chip can be achieved by technologies such as laminar flow and capillarity, photolithography, microplasmas and electrochemical biolithography [103]. Microfluidic chips have advantages over conventional devices, such as that the assay can be performed on a small scale and thus requires less time and smaller amounts of samples and reagents, and can be performed automatically with high reproducibility [104]. Thus, microfluidic chips have been combined with other systems, such as capillary electrophoresis, PCR and flow cytometry. For example, a simple microfluidic chip system combined with a probe-immobilized fluorescent bead assay was developed for the rapid detection of bacteria associated with food poisoning [105]. Meanwhile, a microfluidic chip system combined with a BRET-based biosensor was developed for real-time, continuous detection with superior sensitivity of maltose in water or beer [106].

Tissue Array
The assay using a tissue array is a high-throughput analysis that utilizes hundreds or up to a thousand separate tissue samples on a single platform. Using this method, tissue samples can be rapidly analyzed by histological analyses, such as immunohistochemistry and FISH, in order to screen genetic or protein markers, or to detect tissues infected with pathogenic/toxigenic factors. Since most dyes currently used for microbial fluorescent staining are toxic or carcinogenic, a tissue array system using brilliant blue FCF, which is a food dye and thus has no toxic effects, was developed and applied for microbial cell fluorescence staining of pathogenic/toxigenic and beneficial fungi and bacteria [107].

Application of Fluorescence-Based Microarrays/Biochips for Food Study
Fluorescence-based microarrays/biochips for food study are summarized in Table 2. Antibody/protein microarrays have been applied to detect/screen foodborne pathogens and toxins, where fluorescent dyes, such as Cy3, fluorescein and RuBpy, have been used. Bead/suspension arrays, such as cytometric bead arrays, liquid/magnetic bead arrays and suspension arrays, have been used to detect/quantify mycotoxins, pathogens, genetically modified maize, pesticides and bacterial/plant toxins, where fluorescent dyes, such as Alexa Fluor 532, Cy3, FITC and phycoerythrin, have been used. Capillary/sensor arrays, such as capillary arrays, chemical sensor arrays and fluorescent sensor arrays, have been used to analyze carbohydrates, fresh fruit juices and various food materials, where fluorescent dyes, such as sulforhodamine B, lissamine rhodamine B and synthetic dendritic fluorophores, have been used. DNA microarrays/PCR-based arrays, such as direct RNA hybridization/microarrays, DNA/PNA microarrays, laser microdissection/microarrays, oligonucleotide microarrays, PCR/bead arrays, PCR/microarrays (mutant analysis by PCR and restriction enzyme cleavage or MAPREC assay, and nucleic acid sequence-based amplification implemented microarray analysis or NAIMA; see Table 2) and PCR/single-base extension-tag arrays, have been used to detect mycoplasmas, pathogenic bacteria, grapevine viruses, genetically modified cotton, pathogenic Vibrio spp., genetically modified soybean and seafood-borne pathogens, to screen hypoxia-inducible genes and recombinant flavivirus vaccine strains, to examine genotypes of beef/chicken and gene expression profiles of fungi, and to evaluate the authenticity of ginseng drugs, along with fluorescent dyes, such as Alexa Fluor 546/647, Cy3/Cy5, phycoerythrin, PolyAn-Green/PolyAn-Red, AmCyan1, NIR Dye 700/800, Oyster-550 and quantum dots.
Glycan/lectin arrays have been used for functional glycomic analysis or glycosylation profiling, where Alexa Fluor 488, Cy5 and phycoerythrin have been used as fluorescent dyes. Immunoassay/ELISA-based arrays, such as ELISA chips and immunoassay microarrays, or those used in competitive immunoassay, fluoroimmunoassay and sandwich fluoroimmunoassay, have been used to detect/quantify food allergens, mycotoxins, ochratoxin A, pathogens/toxins, staphylococcal enterotoxin B or to assess food safety, where fluorescent dyes, such as Alexa Fluor 647, Cy3/Cy5, fluorescein, FluoSpheres, phycoerythrin and RuBpy, have been used. Microfluidic chips have been used to detect food poisoning bacteria or single-base mismatches, or to monitor food processing, along with fluorescent dyes, such as Alexa Fluor 647, FAM and GFP. Tissue arrays have also been used to stain microbial cells using brilliant blue FCF.
Fluorescence-based microarrays/biochips can be categorized by the number of target chemicals; either the characterization of a single chemical, or the screening of multiple chemicals from a number of samples or mixtures of chemicals. Among the assays shown in Table 2, antibody/protein microarrays, DNA microarrays, glycan/lectin arrays and tissue arrays are advantageous for profiling and analyzing a single chemical due to the ability of multiple probing, while bead/suspension arrays, capillary/sensor arrays and immunoassay/ELISA-based arrays, microfluidic chips and PCR-based arrays are useful for screening because of their high-throughput processing ability.

DNA Microarray-Based Assay for Food Study
DNA microarray-based assay for food study has been compared with other technologies. For example, foodborne diseases are a major issue among global public health problems and the development of rapid detection methods is crucial for their prevention and treatment. Law et al. summarized rapid methods for the detection of foodborne bacterial pathogens, such as PCR-based methods, PCR-independent methods, DNA microarray assay, biosensor-based assays and immunological methods, and discussed their principles, applications, advantages and limitations [135]. Nucleic acid-based methods generally give high sensitivity, although they require trained personnel and specialized instruments. Biosensor-based assays, on the other hand, can be used without sample pre-enrichment, although they need improvements for on-site detection. Immunological methods, such as ELISA and flow immunoassay, are currently widely used, but have difficulties when interfering molecules are included in the samples. Josefsen et al. compared assays for the rapid monitoring of Campylobacter bacteria in poultry production, and real-time PCR is currently closest to a realistic monitoring system, although other methods, such as microarray PCR, miniaturized biosensors, chromatographic techniques and DNA sequencing, could be considered in the future when cost-effective on-site/at-line monitoring capability is achieved [136]. Gui and Patel discussed the merits of DNA testing, such as DNA microarray assay and next-generation sequencing, to detect Yersinia and other foodborne pathogens [137]. DNA testing is generally a high-sensitivity and high-throughput assay, allowing the detection of a single molecule in multiple reactions to be performed at once, thus allowing a range of characteristics to be rapidly and simultaneously determined. However, improvements in sample preparation, data analysis and molecular detection techniques are still needed. Lauri and Mariani compared potentials and limitations among four molecular diagnostic methods: PCR, nucleic acid sequence-based amplification (NASBA), oligonucleotide DNA microarray and ligation detection reaction (LDR), in food safety assessment [138]. While DNA microarrays can be used to detect quite a number of DNA species simultaneously, they are expensive and need more time for processing. DNA-based technologies have been used to assess the safety and quality of food, animal feed and environmental samples, by providing traceability to prevent foodborne diseases and markers to monitor genetically modified organisms [139].

DNA Microarray Assay Protocols
Among microarrays and biochips, DNA microarrays have been developed most extensively and some have already been used to diagnose cancer and other diseases or symptoms [140]. While the traditional solid-phase microarrays contain specific DNA probes attached to the surface of glass, plastic or silicon chips, other types have been developed, which include bead, fiber and electric arrays, where DNA is attached on the surface of latex or polystyrene beads (bead arrays) or attached to gels within plastic hollow fibers (fiber arrays), or an electrical current is generated by redox recycling upon target/probe hybridization (electric arrays). While a variety of DNA microarray assays have been developed, they can be classified into two major types: those for genotyping (e.g., for comparative genomic hybridization, identifying mutations and single-nucleotide polymorphisms and chromatin-immunoprecipitation on a chip) or gene expression analyses (e.g., for gene expression profiling, screening expression marker genes and identifying splice variants). Genotyping is used to detect the contamination of microbes in food, to identify pathogenic/toxic microbial strains/subtypes and to examine the authenticity of plants or the presence of genetically modified organisms by using 16S rRNA genes and/or genomic DNA markers specific to the microbes or the plants. Gene expression profiling, on the other hand, has been used to identify contaminated pathogenic/toxic bacterial strains, to detect specific stress responses and to examine the efficacy of food materials or components by examining the expression of pathogenic/toxic genes, stress-responsive genes and disease/metabolism-associated genes.
Fluorescent dyes, such as cyanines (Cy3 and Cy5), fluoresceins (including FITC and FAM) and Alexa Fluor dyes, have been used in DNA microarray assays. New fluorescent dyes, Fluolid dyes, which have characteristics of higher light/temperature resistance and longer Stokes shifts, have been developed and applied for DNA microarray assays [26]. These fluorescent dyes are used to label target DNA either by direct labeling, where fluorescent dyes directly attached to nucleotides (e.g., deoxyuridine 5′-triphosphate or dUTP) are used to label DNA by nick translation or primer extension, or by indirect labeling, where small nucleotides, such as aminoallyl nucleotides, are used to label DNA first, and the primary amino group attached to DNA is subjected to a reaction with the N-hydroxysuccinimide ester group attached to a fluorescent dye. Alternatively, small nucleotides, such as biotinylated or digoxigenin-labeled ones, are used to label DNA first, and the labeled DNA is then detected by secondary molecules, such as fluorescently labeled streptavidin or anti-digoxigenin antibodies, respectively. Biotinylated or digoxigenin-labeled DNA can alternatively be detected by non-fluorescent assays, such as colorimetric and chemiluminescent ones by using chromogens, such as Seramun Green, Silverquant and True Blue, or chemiluminescent substrates, such as chloro-5-substituted adamantyl-1,2-dioxetane phosphate (CSPD) and luminol (see below).

Application of DNA Microarray Assay for Food Study
DNA microarrays used for food study are summarized in Table 3. DNA microarrays have been used to examine the following subjects: allergies such as latex and/or vegetable food allergy; poisoning by microbes, such as Bacillus cereus, Clostridium botulinum, Campylobacter spp., Clostridium perfringens, Escherichia coli, Salmonella enterica and Staphylococcus aureus; toxic effects of cadmium, mycotoxins, silver-nanoparticles and tetrodotoxin; contamination of microbes, such as Alicyclobacillus spp., Arcobacter butzleri, Bacillus anthracis, Lactobacillus spp., Listeria monocytogenes, Yersinia enterocolitica and Yersinia pestis; the efficacy of food and food materials, such as that in the absorption of phenolic acids, suppressing cancer, the plasma triglyceride-lowering effect, the lipid consumption in skeletal muscle and the improvement of diabetic symptoms and osteoporosis; mechanisms such as those involved in the response to drought stress, immune stress, inflammation, mucosal IgA antibodies and oxidative stress/DNA damage; and quality control and safety of food, such as the authenticity of food, food safety assessment and the identification of genetically modified organisms.
The food/food materials analyzed by DNA microarray assays include the following: bovine milk, cheese, fish, horseradish, meat (pork and chicken), pancake with chicken, pufferfish, rice and vegetables for the study of allergy, poisoning or toxicity; alfalfa, bread (whole-grain and fiber-rich), cantaloupe, cilantro, egg, fish, juice, maize, meat (beef, pork and poultry meat), milk, mung bean, potato, rice, sausage (Thai Nham) and water for the study of food contamination; beverage, cassava, chitooligosaccharide, dairy, herbs (e.g., licorice and those used in Hochuekkito), high-cholesterol/fat diet, imbibed soybean, phenolic preservatives, pineapple, polyunsaturated fatty acids, psyllium, quercetin, skim milk, sweet corn, tea and xanthan gum for study of their efficacy and mechanisms; and canola, cereal (e.g., barley, oat, rice and wheat), citrinin, cotton, crop, food additives, ginseng, Kothala himbutu (a medicinal plant), maize, olive, potato, royal jelly and soybean for the study of food quality control and safety. Other materials besides food are composts, digestates and waste for the study of food contamination.
The types of DNA microarray used can be classified into those for genotyping and gene expression analyses ( Table 3). The sources of microarrays are either custom arrays or microarrays supplied by companies, such as Affymetrix (USA), Agilent Technologies (USA), Alere Technologies (Clondiag Chip Technologies, Germany), Amersham/GE Healthcare (USA), GeneSystems (France), Mitsubishi Rayon (Japan) and Pathogen Functional Genomic Research Center (USA).

Merits of DNA Microarray Assay
Applications and potentials of DNA microarray technologies (e.g., DNA, cDNA and oligonucleotide microarray assays) for the detection and identification of microbial pathogens, such as antibiotic resistance genes, virulence factors and strain subtypes, have been discussed by comparison with other DNA-based methods, including PCR [224][225][226][227][228]. While PCR-based methods are normally limited to the analysis of a single or a small number of pathogens, microarray technology can analyze a significant number of pathogens simultaneously, and thus it has potential for use in basic research and industrial applications, such as food safety assessment. Gene expression profiling by DNA microarray assay has an advantage of examining the expression of large numbers of genes in a single experiment, and thus has been widely used to analyze food samples and materials. DNA microarray technologies have also been applied to monitor genetically modified food [225,229] and traditional Chinese medicine [230,231], and to evaluate drug safety [232]. Degenkolbe et al. discussed how quality control was examined for the procedures in DNA microarray assay, such as mRNA preparation, cDNA synthesis, fluorescent dye-labeling, hybridization/imaging and data analysis, using plant leaf tissue as a source of mRNA [233].
While DNA microarray assay has been considered to be effective and sensitive for assaying microbial spoilage of food, it is expensive and requires technical expertise. Therefore, several alternative methods were developed to explore cost-effective but still high-throughput assay systems. Böhme et al. developed an efficient method for bacterial identification based on detection of the 16S rRNA gene by flow-through hybridization on membranes, coupled to ligation detection reaction, which may provide an alternative to a DNA microarray assay for the rapid, accurate and cost-effective identification of bacterial species in order to assess food quality and safety [234]. Atanasova and Druzhinina discussed the Phenotype MicroArray, which tests cell respiration as a reporter system to characterize the metabolism of food spoilage pathogens, including conidial fungi [235]. However, the number of probes in these alternative assays is generally up to 100, and DNA microarray assays would be more useful when the number is over 100.
One of the merits of a DNA microarray assay is that it provides information about pathway-based intracellular signaling, which is important to evaluate the efficacy and mechanism of action of food materials. For example, a variety of signaling pathways have been identified by DNA microarray assay for traditional herbal medicine, such as traditional Chinese medicine (TCM) and traditional Japanese medicine (Kampo), which are associated with effects on cell functions and diseases, such as anti-adipogenesis, anti-atherosclerosis, anti-carcinogenesis, anti-inflammation, apoptosis, chemoprevention, circulation disorder and neuroprotection [231]. For example, the mitogen-activated protein kinase (MAPK) signaling pathway was shown to be associated with the apoptotic effect of Inchin-ko-to (Kampo) [236] and the anti-carcinogenic effect of Juzen-taiho-to (Kampo) [237], while TGF-β1/Smad and IGF-1 signaling pathways were associated with the inhibitory effect of Kangxianling (TCM) on renal fibrosis [238] and the immune response against viral infection induced by VI-28 (TCM) [239], respectively. The pathways associated with environmental estrogens, such as phytoestrogens, which are also important food materials, include a variety of signaling pathways related to apoptosis, carcinogenesis, cell growth/proliferation, differentiation/development and inflammation [240]. Therefore, the information about pathway-based intracellular signaling provided by DNA microarray assays will add variability and sensitivity to the assay system.

Conclusions
We have here summarized recent progress in fluorescence-based bioassays developed and applied for the detection and evaluation of food materials. A comprehensive list of fluorescent dyes used in recent bioassays includes those in biosensing, cell assay, energy transfer-based assay, probing, protein/immunological assay and microarray/biochip assay. Among these technologies, fluorescence-based microarrays/biochips, such as antibody/protein microarrays, bead/suspension arrays, capillary/sensor arrays, DNA microarrays/PCR-based arrays, glycan/lectin arrays, immunoassay/ELISA-based arrays, microfluidic chips and tissue arrays, have been developed and used widely for food safety and quality as well as the search for effective components. Applications of DNA microarray assay were discussed for important issues, such as allergy/poisoning/toxicity, contamination, efficacy/mechanism and quality control/safety, based on a comprehensive list of references showing these cases. The merits of DNA microarray assays were discussed by pointing to their advantages over other technologies in terms of features such as the sensitivity and efficiency, the number of probes to be analyzed rapidly and simultaneously, and the quality and quantity of information about pathway-based intracellular signaling in response to food materials.