Functional Nucleic Acid Based Biosensor for Microorganism Detection
Food safety especially the problems of microorganism pollution is always a nonnegotiable attribute in food trade, sales, and consumption. It is significant to detect the microorganisms including themselves, their crude excretion or their toxin, and so on that are possible to decrease the quality of food and increase the food safety risks. With the contributions of the progress in analytical chemistry and molecular biology, many kinds of technology satisfy the rapid detections of microorganisms with high specification and sensitivity. However, nucleic acid is a typical factor in these hazard exposures. In this review, we have reached up to a comprehensive representation of functional nucleic acid biosensors for detecting microorganism. Functional nucleic acid is one of the most vital biological micromolecules, indispensable for almost every life events of microorganisms and rich in all organisms. As for the research idea, highlight, and superiority of the functional nucleic acid biosensor for microorganisms, the sequence of nucleic acid is the important part where the information is taken from. From another point of view, DNA can be utilized as recognizing element and enzyme upon the specific structure to detect microorganisms. Therefore, it is shown obviously that functional nucleic acid biosensors can be efficient for detecting microorganisms, and research on it is becoming profound in microorganism detection. And this chapter will be nearly the most comprehensive description about functional nucleic acid-based biosensor for the microorganism detection.
KeywordsFunctional nucleic acid Biosensor microorganisms Detection
Foods can be affected by many kinds of microorganisms, such as harmful microorganisms to people in the programs of production, packing, as well as transportation. The microorganisms can easily make people feel uncomfortable, catch disease, and even die. It is also an important factor which causes sudden food safety accident. As we know, waterborne diseases are that caused by waterborne pathogens . In developing countries, waterborne diseases cause thousands of people’s death every year . In addition, drinking water polluted by pathogens may also be harmful to various organisms including animals and plants . It can be seen that infectious diseases affected by microorganisms have been becoming increasingly severe all around the world. Therefore, to control the issue of pathogenic infection, there is an expanding need to detect the microorganisms related to health of people in the air, water, and soil rapidly and accurately.
Isolating and culturing techniques are basic ways of traditional methods for microorganism detecting. After that, microorganisms will be identified by biochemical method or direct microscopy which is accurate. However, there are many disadvantages at the same time, such as that it cannot detect microorganisms in a short while.
To make the detection of microorganism more convenient, instrumental analytical methods are developed such as HPLC and GC which are utilized for analysis of the biochemical composition of various bacteria. So, the microorganisms can be identified [4, 5]. The mass-spectrometric technique and capillary electrophoresis (CE) are also used to identify microorganisms [6, 7]. In addition, the gas of various microorganisms is also a typical signal for assay of microorganisms, which is the basic principle of electronic nose . In conclusion, the instrumental analytic technique holds some advantages such as simple, convenient, and easy to learn, but it also has many shortcomings including high cost and low efficiency which makes it hard to be adopted to food safety detection widely.
Immunological methods are techniques that have high specificity because antigens and antibodies can bind each other with high specificity which can be utilized to qualitatively or quantitatively detection of microorganisms. A great variety of immunological methods are developed [9, 10, 11, 12, 13, 14]. There are a lot of strong points of immunological methods including relatively simple machines, easily storing of samples, high specificity, and quantitative detection. But, some shortcomings of this type of method cannot be ignored such as the fact that it is hard to detect several types of microorganisms, false-positive result, and limited sensitivity related to molecular biology methods. Thus, it is obvious that immunological methods are not perfect enough for microorganism detection.
Functional nucleic acid based biosensors have been extensively adopted in microbe assay. This method can detect microorganism in short time specifically and sensitively. Besides, it provides a rapid and simple method to differentiate viable and nonviable microbes. The biggest advantage over antibodies is probably that aptamers are amenable to SELEX. Unlike the isolation of antibodies, aptamer selection can be carried out at designated conditions, and counter selections can be performed to remove cross activity, which is difficult to achieve in antibodies. Functional nucleic acid based biosensors overcome the disadvantages of microorganism detections by the general molecular methods.
Functional nucleic acids (FNAs) describe a family of molecules whose function goes beyond the recognitions of complementary nucleic acids . We can define FNAs in microorganisms assay from two major classes in microorganism detection. The first class is FNAs for signal transduction in microorganisms assay. Using FNAs for signal transduction means that FNAs can transduct microorganism signal to nucleic acids signal. For non-culturable method, which means that nucleic acids of microorganisms can be detected directly, the most common FNAs are probes. For non-extraction method, in which the microorganisms will be recognized by specific binding in whole cells, the aptamers are adopted in general. The second class of FNAs is used for signal amplification. For example, RCA, LAMP, SDP, and HCR are all signal amplification methods based on FNAs. In addition, FNAs combined with nanometer materials or fluorophores can be used for signal output.
In this review, we introduce the FNA-based biosensors for microorganisms. In next part, the construction of functional nucleic acid biosensors (FNABs) for microorganism detection and its technological elements will be reviewed to supplying basic information of biosensors. Then, the various biosensors will be introduced in aspects of principles, correlation technique, related applications, and properties in third part. Finally, future perspectives on FNABs for microorganism detection with rapid, accurate, and multiplexing capability are provided.
2.2 The Construction of Functional Nucleic Acid Based Biosensors for Microorganism Detection and Its Technological Element
Functional nucleic acid based biosensor detecting microorganism consists of signal recognition, transduction and signal amplification components. Many kinds of targets can be identified by target recognition elements. Molecular recognition events can be converted into other signals easy to detect by transduction elements. There are different structural DNA motifs such as fluorescence probe, hairpin, quadruplex, crossover, DNAzyme, aptamer–substrate complex, which make detection specific, sensitive, and rapid. Signal amplification elements are dependent for some amplification technologies such as PCR, RT-PCR, Real-Time PCR, LAMP, RCA, EXPAR.
As an analytical tool, biosensor has been utilized in many fields such as environmental monitoring, medical detections, safety testing of food, and disease diagnosis. The adoption of biosensors for microorganism assay is of great significance. FNABs have advantages over other classes of biosensors because of high selectivity and sensitivity including the fact that the FNAs are easy to immobilize, prepare and label different signals. In the following parts, various elements of functional nucleic acids will be introduced as well as their applications. Novel synthetic probes (e.g., PNAs, aptamers) that are low costing and flexible fabrication have been adopted to make point-of-care FNABs for rapid and credible detection of microorganism.
There are many kinds of NABs including DNA, RNA, PNA, and aptamers. The working principle of NABs except for aptamers is Chargaff’s rules of base pairing, and the combinations of aptamers and target such as proteins, cells, and small organic molecules are similar to the reaction between antibodies and antigens.
There are some chemical methods to compound DNA probe, such as PCR. Differently, RNA probe is obtained using the reverse transcription (RT) of messenger RNA (mRNA) or the approach of utilizing the order of amino acids of relative protein to forecast nucleotide sequence. This method has some shortcomings because of codon degeneracy although its feasibility is proved. Typical nucleic acids hybridization methods need more labor force and time than hybridization procedure of a physical biosensor.
FNABs are physicochemical transducer because its carrier is immobilized by nucleic acids . There are a large number of methods that can be utilized for immobilization. What is more, the process can be promoted by immobilization tenor .
2.2.1 Signal Recognization and Transduction of Functional Nucleic Acid Based Biosensor
A DNAzyme with Peroxidase Activity
A DNAzyme with Cleavage Activity
Triplex DNA Structures
2.2.2 Signal Transduction Elements
Fluorescence Resonance Energy Transfer (FRET)
When two chromophores are close to each other, the energy of a chromophore called donor can transfer to the other called accepter, which is FRET. And the distance must range from 10 to 100 A. In addition to the distance, effective FRET also needs an adequate overlap of the emission spectrum of the donor and the excitation spectrum of the accepter [33, 34]. FRET increases the fluorescence intensity of accepter chromophore and decreases the energy of donor chromophore at the same time.
Gold nanoparticles (AuNPs) are the most widely used nanoparticles in the detection of microorganism. For example, gold nanoparticles can be utilized in localized surface plasmon resonance (LSPR), and extinction coefficients of them are much higher than those of organic chromophores . Due to the good features of AuNPs, they are extensively adopted as signal transduction elements . When the distances between AuNPs change, the color will change. This principle can be used in the DNA detection because the DNA can change the distance. The combination of DNA and AuNPs makes the detection simpler and easier to observe with no need of advanced instruments .
2.2.3 Signal Amplification of Functional Nucleic Acid Based Biosensor
DNA can act as transduction as well due to the amplification method. In general, the amplifications of nucleic acids are classified into two groups: thermocycling and isothermal amplification methods. The most important difference of the two groups is the temperature: the temperature of thermocycling is changing in the reaction process, but the temperature of isothermal amplification is same during the process. There are many advantages of thermocycling amplification such as the high efficiency of amplification, but the drawbacks exist as well, such as high probability of sequence mismatches, high cost, and susceptibility to contamination.
Isothermal Amplification Technology
Rolling Circle Amplification (RCA)
Loop-Mediated Isothermal Amplification (LAMP)
Strand Displacement Amplification (SDA)
SDA is another isothermal amplification. In the process of SDA, the DNA was nicked at the recognition site at first. Then, with the presence of DNA polymerase, the 3′ end of DNA was extended, and the downstream DNA strand was displaced. The SDA can amplify target DNA in exponential amplification due to the displaced DNA strand that acts as a template for an antisense reaction . In the reported studies, SDA is often combined with HRP-like DNAzymes.
Hybridization Chain Reaction (HCR)
Thermal Amplification Technology
There are several advantages of PCR which is effective, specific, and widely used in sample with complex and shortcomings, for example, qualitative detection cannot be satisfied using traditional PCR. To overcome this shortcoming, the real-time PCR is developed, which consists of two types: fluorescence probes and specific nucleic acid dye system. Due to the advantages of real-time PCR, a lot of researchers utilized it into microorganism detection and analysis [44, 45].
In addition to the traditional PCR and RT-PCR, digital PCR was also developed and studied as the third generation of PCR technology . In digital PCR, the reaction volume was divided into thousands of reaction cells at first, and the copy number of target can be taken from dilution percentage and Poisson distribution. Digital PCR was designed in the 1990s for the first time, and after that, it has been developed to several fields. Due to the function of quantitative detection, digital PCR is regarding as the improved technology of real-time PCR . Now, Bio-Rad QX100 digital PCR system made by Bio-Rad Company has been developed since about one decade ago and been regarded as the best digital PCR system which is accurate, stable, and cost-effective, which has been utilized in bacteria detection .
2.2.4 Signal Output Systems of Functional Nucleic Acid Based Biosensor
Another strategy is modified aptamers by organic fluorophores in conformationally labile regions of aptamers. When the target combines to the aptamer, the structure of aptamer will change and the fluorescence characteristics such as intensity and anisotropy are changed as well. This kind of signaling aptamer has been built by modified DNA aptamers with a fluorophore adjacent. And the addition of fluorophore makes the Kd much higher than the aptamers without modification. However, signaling aptamers that based on the conformational transduction lose the affinity which is the common shortcoming of this method. The features and binding site can affect the signal as well, and the ways of combinations of aptamers and labels are various. In addition, the changes of local environment also change the signal.
AuNP-Based Colorimetric Signal
The combination of nanomaterial science and biology can bring a variety of advantages of both technologies and promote the development of bionanotechnology. And the typical example is the combination of gold nanoparticles and DNA. In solution, the distance between AuNPs can change the surface plasmon properties and large extinction coefficients, and if the AuNPs are close to each other, the color is blue, and if not, the color will turn red. There are two kinds of colorimetric functional nucleic acids with the function of gold nanoparticles.
DNAzyme-Based Colorimetric Signal
The most commonly used DNAzyme is that with peroxide activity in bioanalytical chemistry. To guarantee the high peroxide activity, a hemin is needed to combine with oligonucleotides in order to form G-quadruplex structure.
When aptamers bind to the target molecular, they will be folded, and the structure of aptamer will turn to three-dimensional (3D) shapes. If we labeled the aptamers on the conductive support, the part of redox-active moieties can be tethered to the aptamers in 3D structure. Thus, the presence of target can be analyzed via analyzing the change of electron transfer features of the redox moieties. Until now, many biosensors based on this theory have been developed by researchers.
2.3 Functional Nucleic Acid Based Biosensors for Microorganism Detection
2.3.1 Aptamer Based Biosensors for Microorganism Detection
Aptamer Selection Strategies
In general, there are two classes of selection (SELEX strategies and SELEX variant strategies) which will be introduced in this part.
Ellington and Szostak designed the SELEX process for aptamers selection for the first time . The basic process of general SELEX is constructing random oligonucleotide libraries, separating the target nucleic acid complex and amplification by RT-PCR (for RNA selection) or PCR (for DNA selection). The first aptamers selected by SELEX are against organic dyes and T4 DNA polymerase. After that, many other biomolecules are also targeted which can be found in the aptamer database written by Ellington et al. Except for this database, some other publications summarized the kinds of target molecules as well [28, 56]. Many aptamers isolated by SELEX can combine to whole cell, crude extracellular mixture, intracellular proteins, and purified molecules.
The SELEX experiment will be hard to program utilizing a purified, soluble target if it needs the cell membrane or a coreceptor to fold properly. The most typical advantage of complex-target SELEX is that it can be utilized in complex protein mixtures. Besides, although the information about the cell membrane is not known, aptamers can identify and bind the target [60, 61].
Properties and Categories of Aptamers
The Application of Aptamer Biosensors for Microorganism Detection
After obtaining aptamers with high binding affinity and specificity, the next step is to design a signaling mechanism so that a sensor can be produced. Aptamers can be combined to deferent signal transduction technologies to construct biosensors to detect microorganisms, such as fluorescent biosensor, flow cytometry, electrochemical sensor.
2.3.2 Functional Nucleic Acid Based Colorimetric Biosensors for Microorganism Detection
Gold Nanoparticles-Functional Nucleic Acid Based Colorimetric Biosensors for Microorganism Detection
The intersection between molecular and nanomaterials science offers fertile ground to advance the development of versatile biomaterials and bionanotechnology . A successful example is the interaction between gold nanoparticles (AuNPs) and DNA. AuNPs are typical optical materials that display distance-dependent surface plasmon properties, resulting in strong color changes that rival or even exceed the most intense organic dyes . Nucleic acid has been used as a programmable molecule to tune the distance between AuNPs. In addition to tunable properties from conventional hybridization between one single-stranded sequence and its complementary sequence, functional nucleic acids that can perform specific binding features with conformational changes or catalytic reactions in the presence of specific non-DNA molecules [72, 73], bacteria , cells , or even viruses  have been reported.
G-Quadruplex-Functional Nucleic Acid Based Colorimetric DNAzyme Biosensors for Microorganism Detection
There are some short, single-stranded DNA molecules called DNAzymes, which consist of a special G-quadruplex structure within an intercalated hemin. This kind of DNAzymes can oxidize the ABTS2− by H2O2 to cause a green-colored radical ion (ABTS•+, = 3.6 × 104 M−1 cm−1) . Because of the great colorimetric property, DNAzymes perform a significant role in the analytical assays as a simple colorimetric format, and they also will be great molecular tools in the biosensors and nanodevices design. In addition, DNAzymes have a lot of advantages, such as low cost, high stability against heat , and easy labeling, and can be utilized to detect different targets microorganisms DNA .
Nano-polymer-Based Functional Nucleic Acid Based Colorimetric Biosensor for Microorganism Detection
Streptavidin-horseradish peroxidase modified hybridization chain reaction (HCR-HRP) nanocomposites is another signal amplifier and colorimetric signal conversion element, which catalyzed hydrogen peroxide (H2O2) via TMB to generate an obvious green color and turned yellow after sulfuric acid termination with optical absorption at 450 nm. Bulb-like triplex turn-on switch (BTTS) acts as a novel selective molecular recognition and signal transduction element and was designed as bulb-like and was composed of a bulb-like microorganisms aptamer (BLA) in the center to capture target microorganisms flanked by mirror sequences to hybridize with the bridge probe (BP) to form a triplex nucleic acid stem by Watson-Crick base pairing and Hoogsteen base pairing.
2.3.3 Lateral Flow Nucleic Acid Based Biosensors (LFNABs) for Microorganism Detection
The Development of Lateral Flow Biosensor
The Structures of Lateral Flow Biosensors
The sample pad acted as a platform to ensure the best analytical status of preparation and delivery sample as well as buffer salts, proteins, detergents, and viscosity enhancers. Thus, the materials of sample pad are porous materials in general including cellulose fiber or glass fiber . The porous of materials can isolate the coarse molecular and whole cells.
Biorecognition molecules, e.g., aptamer, are in the conjugate pad. Conjugate pad would better to liberate recognition molecules rapidly to promote the reaction of liquid sample and molecules. The lack of preparation of a labeled conjugate may bring the bad effect to the susceptibility of the test.
Nitrocellulose membrane is the most important part of LFA because the test line and control line are there. It is better that the nitrocellulose membrane can bind the seized molecules but does not bind the molecules that are detected. In addition to nitrocellulose membrane, many other types of membranes are also utilized .
The adsorbent pad is used to supply traveling power to guarantee the liquid sample traveling through the strip in suitable flow rate. Absorbent capacity is important because it affects the background of results. Besides, cellulose filter is wildly utilized.
The function of backing pad is supporting the strip and making the test easier. In addition, its materials are not strict.
Signal Amplification, Recognition, and Output Elements of Lateral Flow Biosensor
Signal Amplification Systems
Amplification can increase the sensitivity greatly and let down the detection limit. PCR and isothermal amplification such as NASBA, HDA, RPA, LAMP, and SDA are commonly used in this process. Recently, in order to decrease the detection time, the amplification process is canceled sometimes.
Signal Recognition Systems
Sandwich reaction is utilized in lateral flow biosensor to recognize targets. There are three formats of recognition principles of LFNABs: binding of antibodies and antigens, hybridization, and FNA-based reaction.
Signal Output Systems
There are many kinds of reporter materials that can be utilized in lateral flow biosensor for signal output, such as AuNPs, fluorophores, quantum dots, and so on.
Different Signaling Systems of LFNABs for Microorganism Detection
There are a series of labels in LFNAB including textile dyes, carbon nanoparticles, gold nanoparticles, selenium nanoparticles, colored latex beads, liposomes, p-converting phosphors, magnetic particles, quantum dots, organic fluorophores, and so on. Any material which is utilized in the detection should maintain its superiority upon compound with biorecognition molecules. A good signal label should have some great character such as high affinity with biomolecules. Recently, several reviews whose focus is signal systems which are applied in the LFB were reported [87, 88, 89, 90].
2.3.4 PCR-Functional Nucleic Acid Based Biosensors for Microorganisms Quantitative Detection
RT-PCR-Based Functional Nucleic Acid Based Biosensors for Microorganisms Quantitative Detection
SYBR Green I which is most extensively utilized among fluorescent dye can link to double-stranded DNA . In addition to fluorescent dye, detection probes that are modified by fluorophore can be used in PCR progress. The principle of it is FRET: when there is overlap of excitation spectrum of a fluorophore and that of quencher, the quencher can quench the fluorophore, and if not, fluorophore will emit low fluorescence but high-density fluorescence . In order to make specificity higher, specific probe will be not closed during the PCR progress which makes it can be captured by products of PCR [93, 94, 95, 96].
Digital-PCR-Functional Nucleic Acid Based Biosensors for Microorganisms Quantitative Detection
“Digital PCR” was reported in 1999 by Kinzler and Vogelstein . Digital PCR (dPCR) reported in 1999 is the PCR type that achieves quantification detection without reference material.
The analysis of target locus of individual molecules is the key point of digital PCR. At first, the sample was separated into a variety of droplets, and some of them have at least one target that is “positive,” but other droplets are “negative.” Then, PCR will measure the number of positive aliquots.
Digital PCR has been applied extensively over a wide range of fields to detect microorganisms and developed to be more efficient. For example, using Escherichia coli as a target, Dong-Ku Kang demonstrates that the IC3D can achieve quantification detection of both stock and clinical isolates of E. coli in spiked blood .
Droplet digital PCR is the third generation of PCR techniques, which achieves the absolute quantification of molecular target without the utilization of standard curves, due to the recent advent of compartmentalization. Droplet digital PCR (ddPCR) is an efficient technique for quantitative detection of microorganisms. Here, Davide Porcellato reported a new ddPCR assay for the quantitative detection of the Bacillus cereus group in milk. The main advantage of ddPCR is low detection limit compared to dPCR. The new ddPCR technique is a promising method for the quantification of target bacteria in low concentration in milk .
Aurélie Hennebique developed digital PCR (dPCR) assays allowing rapid and accurate detection and quantification of these resistant mutants in respiratory samples, especially when the proportion of mutants in a wild-type background is low. There are three dPCR gyrA assays designed to detect and differentiate the wild-type and one of the three gyrA mutations previously described as associated with FQ resistance in L. pneumophila: 248C>T (T83I), 259G>A (D87N), and 259G>C (D87H). These results demonstrate that dPCR is a highly sensitive alternative to quantify FQ resistance in L. pneumophila, and it could be used in clinical practice to detect patients that could be at higher risk of therapeutic failure .
In digital PCR, any targets will be detected when the efficiency of the reaction is high enough which is different from real-time PCR. Thus, it is not an important thing that whether a response is more effective than another, because the target can be detected if they are fully amplified.
2.3.5 Isothermal Amplification-Functional Nucleic Acid Based Biosensors for Microorganism Detection
LAMP-Based Functional Nucleic Acid Based Biosensors for Microorganism Detection
SDA-Based Functional Nucleic Acid Based Biosensors for Microorganism Detection
RCA-Based Functional Nucleic Acid Based Biosensors for Microorganism Detection
NASBA-Based Functional Nucleic Acid Based Biosensors for Microorganism Detection
HDA-Based Functional Nucleic Acid Based Biosensors for Microorganism Detection
NEMA-Based Functional Nucleic Acid Based Biosensors for Microorganism Detection
NEMA has multiple advantages and has been developed in wide fields. This is an amplification technology which is based on the theory of strand displacement. In the reaction process, one strand of duplex DNA can be first cleaved only, and then the amplicons will be amplified greatly through a nicking endonuclease activity. NEMA attracts more interests from scientists because it just needs only two pairs of general primers and the simple need can easy the design work of primer . Besides, NEMA produces less pollution by aerosol than LAMP. In addition, NEMA can produce approximately 400 bp product, which makes it more universal than other isothermal amplification strategies [113, 116, 119]. Kong et al. developed the detection of M. tuberculosis, which is a typical example of NEME application .
2.3.6 Functional Nucleic Acid Based High-Throughput Biosensors for Microorganism Detection
Multiplex PCR-Based Functional Nucleic Acid Based Biosensors for Microorganism Detection
Multiplex PCR (M-PCR) is similar to traditional PCR because the principles of them are same to each other, but there is more than one pair of primers in multiple PCR. Because of that, there are more than one DNA templates that are combined by primers, and more than one DNA fragments are amplified in one progress .
M-PCR has a variety of advantages, but there are several disadvantages of it including the primers’ inhibition, various efficiencies of different templates, and so on. Because of these disadvantages, M-PCR cannot be developed and adopted to wider uses, especially in high-throughput detection.
Universal Primer-Multiplex PCR-Functional Nucleic Acid Based Biosensors for Microorganism Detection
In order to overcome the shortcomings of traditional M-PCR, universal primer (UP) was designed in the reaction . UP is the most important element in the progress. In this method, the primers consist of complementary sequence of templates and UP sequence. Thus, the amplicons of primers can be amplified by UP.
Multiplex LAMP and Multiplex Lateral Flow Nucleic Acid Biosensor
Multiplex Fluorescence-Functional Nucleic Acid Based Biosensors for Microorganism Detection
Adapting different fluorescence elements to detect various microorganisms in a progress is a convenient and rapid method. A variety of molecular targets can be detected in one reaction which is the typical advantage and make high-throughput analysis of multiple samples from large study groups or longitudinal studies technically feasible.
Gene Chip-Functional Nucleic Acid Based Biosensors for Microorganism Detection
There are two types of methods which are developed for arraying a variety of DNA molecules in a very small space. One type is cDNA-sized fragments which are the production of PCR and spotted onto poly lysine-coated glass slides . The other one is short (∼25 nucleotide) oligonucleotides that are modified on a glass surface . This kind of production of both methods is called “chips.” In order to increase specificity, it is important that each gene has to be represented by several (typically 20) different oligonucleotides. What is more, there is only one different central base between oligonucleotide and partner adjacent on the chips.
Multiplex Ligation-Dependent Probe Amplification-Functional Nucleic Acid Based Biosensors for Microorganism Detection
It is difficult for multiplex PCR to achieve high specificity which is really significant. So, multiplex ligation-dependent probe amplification (MLPA) was developed by Schouten et al. to make up for deficiencies . Capillary electrophoresis (CE) was always adopted to analyze the products according to the small differences among the lengths of the amplicons. In addition, fluorescence is often labeled to the ligation probes in order to achieve quantitation. MLPA was extensively utilized in medical diagnostics and clinical applications since the development of it [136, 137].
Multiplex Digital PCR-Functional Nucleic Acid Based Biosensors for Microorganism Detection
Multiplex digital PCR is an innovative PCR technology developed in the 2000s, based in the partition of the sample to be analyzed in thousands to millions of individual PCR reaction. A major advantage of it is an increased sensitivity for detection of a few mutants mixed with wild-type DNA sequences. Digital PCR has wide clinical applications in the oncology field and ongoing applications in noninvasive prenatal diagnosis and organ transplant rejection monitoring.
The Second-Generation Sequencing Technology-Functional Nucleic Acid Based Biosensors for Microorganism Detection
TaqMan-Functional Nucleic Acid Based Biosensors for Microorganism Detection
Fluorogenic PCR-based (TaqMan) technology has been reported to detect quantities of organisms, such as clinical bacteria [143, 144, 145] and plant pathogenic potato leaf roll virus . Besides, a TaqMan assay for detection of the potato ring rot bacterium, Clavibacter michiganensis subsp. sepedonicus, is shown, too . TaqMan PCR exploits the 59 nuclease activity of Taq DNA polymerase  in conjunction with fluorogenic DNA probes . There are one fluorescent reporter dye and one quencher dye that are labeled on probe which hybridized specifically to the target PCR product. In PCR amplification process, the reporter fluorescence is increasing because fluorescent reporter dyes and quencher dyes are separated by Taq DNA polymerase. Repeated PCR cycles result in exponential amplification of the PCR product and a corresponding increase in fluorescence intensity.
S. A. Weller et al. reported the design of a fluorogenic PCR-based assay which utilizes a probe-primer set (RS) to detect all known strains of R. solanacearum and another set (B2) specific for the biovar 2A genotype . Because each probe is labeled with a different reporter dye, both tests can be achieved in a single tube.
2.3.7 Functional Nucleic Acid Based Biosensor for Living or Dead Bacteria Detection
The Development of Functional Nucleic Acid Based Biosensor for Living or Dead Bacteria Detection
There are advantages and shortcomings of culture, immunology, and nucleic acid-based approaches. As for culturing method, it can analyze living microorganism only, but the detection time is about 3–7 days . Although the detection time of immunological diagnostic method is short, it cannot identify the living and dead microorganisms . Nucleic acid-based approaches have been utilized to microbe tests . Due to the development of molecular methods, the detection of microorganisms has become more sensitive, specific, and rapid. Unfortunately, these approaches cannot be the rapid and simplistic measure to distinguish viable and nonviable bacteria. Viable bacteria can cause food corruption and pathogenicity rather than nonviable bacteria. Thus, distinguishing living and dead microorganisms is important, which is a challenge.
Functional Nucleic Acid Based Biosensor for Living or Dead Bacteria Detection
Reverse Transcription PCR-Functional Nucleic Acid Based Biosensor for Living or Dead Bacteria Detection
According to the studies, mRNA is an excellent referent of animate cells. And it is used in reverse transcription-PCR (RT-PCR) to analyze the condition of gene expression in cells. RT-PCR was adopted to detect living Legionella pneumophila and Vibrio cholerae based on that mRNA only exists in the sample that contains living microorganisms.
In addition, real-time RT-PCR (RRT-PCR) method was also designed. The common feature of these two types of methods is that they both need purified mRNA which is difficult to obtain because the half-lives of most mRNAs are 1.5–2 min. At the same time, there are also some problems that are to extract and preserve intact RNA in the physiological or the environmental condition.
Viability PCR-Functional Nucleic Acid Based Biosensors for Living or Dead Bacteria Detection
There are many viability dyes that have been utilized in microorganism detection combined with qPCR in general, which is called v-PCR. DNA-binding dyes such as ethidium monoazide (EMA) or propidium monoazide (PMA) can only get through membrane of dead cell which is damaged and combined to DNA which makes it unable to be amplified by qPCR. And this is the pretreatment of qPCR.
The membrane integrity is one of the most commonly used differences between living and dead cells. Upon adding DNA-binding dyes, the dead cells can be easily infiltrated, but living cells are not. EMA-qPCR is a simple approach to analyze living and dead cells. Besides, PMA is also utilized in this field because PMA can enter the dead cells and bind to DNA irreversibly.
Nuclease PCR-Based Functional Nucleic Acid Biosensors for Living or Dead Bacteria Detection
Another sample pretreatment for distinguish viable and nonviable cells is utilizing nuclease to nick the exposed DNA. The membranes of living cells are stable to protect nucleic acid. But the DNA of dead cells cannot be protected due to damaged membrane and can be affected easily by outside contamination. Deoxyribonuclease I (DNase I) can cleave ssDNA and dsDNA. Thus, it is usually adopted to remove DNA in sample for analyzing RNA. Due to the membrane of dead cells that cannot protect the DNA inside, the DNA exists only in living cells with the adding of DNase. In Villarreal’s research, it was reported about a DNase I- and Proteinase K-based treatment protocol developed and optimized for the detection, characterization, and analysis of live populations of bacteria present in drinking water biofilms.
Nanoparticles and Spectroscopy Technology-Functional Nucleic Acid Based Biosensors for Living or Dead Bacteria Detection
It is an important challenge to characterize whether the cell is alive or not especially in severe conditions. The nucleic acid dyes can be utilized to enter cells with imperfect membrane to solve this problem. Nevertheless, the problem still exists. Thus, nanoparticles and spectroscopy technology is adapted to solve this problem.
Fourier transform infrared (FTIR) and Raman spectroscopy are the technologies that can analyze the reaction on the surface. The Raman micro-spectrometer is utilized in situ rapid discriminating the viable cells as well. 58S substrates, 45S5 Bioglass, and bioinert silica can be labeled to the viable microorganisms, which makes them able to be detected. These two methods can distinguish microorganisms with high specification. However, nanostructures are needed to combine with SERS to achieve high sensitivity. According to the report, the silver nanoparticles are used to characterize the living and dead microorganisms.
In recent years, studies had shown the strongpoints of laser-induced breakdown spectroscopy (LIBS) in differentiating microorganism. Escherichia coli are characterized, which have divalent cation in the outer membrane. With its function of characterizing microorganisms, it can also be utilized to analyze living and dead cells.
Dielectrophoresis Technology-Functional Nucleic Acid Based Biosensors for Living or Dead Bacteria Detection
Dielectrophoresis (DEP) had been utilized to differentiate living and dead microorganisms by nonuniform AC electric fields based on the principle that frequency responses of cells on the different states are different. Several novel types of DEP have been developed such as iDEP, cEDP, and rEDP. For example, the biggest difference of iDEP is the utilization of insulators to overcome the problems of electrodes. Although the living and dead microorganisms cannot be differentiated using electrokinetic mobility, it can be differentiated via analyzing dielectrophoretic mobility based on the principle that dielectrophoretic mobility of viable microorganism is higher than that of nonviable microorganism.
2.3.8 Artificial Nucleic Acid Based Biosensors for Microorganism Detection
The artificial nucleic acid, peptide nucleic acid (PNA), which is a special nucleic acid with a peptide-like backbone, can also be utilized in nucleic acid hybridization. Compared to DNA, PNA has similar structure with DNA but different backbone . This difference between DNA and PNA makes the PNA more stable and easier to combine with DNA. In the utilization of PNA, fluorescent dye is commonly used.
2.3.9 DNAzyme Based Biosensors for Microorganism Detection
DNAzymes are short synthetic oligonucleotide molecules, which are also called deoxyribozymes, catalytic DNAs, or DNA enzymes. They have catalytic activity which can be isolated in a kind of in vitro method called SELEX. After it was first found, DNAzymes have been utilized for the many kinds of biosensors development. Herein, we developed a colorimetric G-quadruplex LAMP sensor that combine the isothermal amplification and the DNAzyme for the ultrasensitive detection of Salmonella . This is an example of functional nucleic acid colorimetric biosensor for microorganism detection. First of the research, there are primers within a signal inner primer of a 17-nt DNAzyme complementary sequence (a signal precursor), which were designed for the amplification process and colorimetric detection. The target DNA can initiate LAMP amplification, and the amplification results conclude a larger number of DNAzyme sequences. After adding the hemin, the free DNAzyme fragments combined with each other and formed G-quadruplex-hemin conjugates which perform as colorimetric signal readouts for the naked eye observation. The novel colorimetric strategy does not need any forges or other apparatus, and the detection limit can achieve less than 0.5 pg. Moreover, the reported sensor showed high foreground in the DNA visual detection and may even pave the way for other amplification-based colorimetric detection and the point-of-care determination.
Recently, DNAzyme has been applied in various chemiluminescent or colorimetric determinations [159, 160]. For example, Willner and his coworkers reported a method based on this DNAzyme for single-stranded DNA and telomerase activity detection . They also explored the same DNAzyme-containing primer to detect PCR product detection . However, there are false-positive signals because of primer dimer formation in this method like all other primer probes. More recently, Feng Du and Zhuo Tang reported a facile technology based on the advantage of the 5′-nuclease activity of Taq DNA polymerase to free a DNAzyme inserted in the sensor for PCR product colorimetric analysis . This DNAzyme, discharged as a byproduct of a specific DNA target PCR, can fold up and form G-quadruplex structure with hemin and then oxidize ABTS into a green condition in the presence of hydrogen peroxide. In this research, the detection limit could reach as low as 100 A. hydrophila cells.
2.3.10 Gold Nanoparticle (AuNP)-Functional Nucleic Acid Based Biosensors for Microorganism Detection
AuNPs perform a significant role as excellent labels in many diagnosis because of their great properties including easy functionalization, biocompatibility, good stability, a characteristic surface plasmon resonance, a strong red color, easy manipulation, and prominent enhancement of signal in nanoscale morphology. The gold nanoparticles can make the detection result observed by naked eyes, and they have been applied into many areas consisting of the analysis of nucleic acid, proteins, small molecules, ions, and even cancer cells. The size and shape change causes the optical properties of gold nanoparticles, which varies from 2 to 150 nm, but 15–40 nm sizes were used generally. However, AuNPs still have some drawbacks: the sensitivity is dependent on the amount of the targets, and amplification of signal is costly.
We developed a gold nanoparticles visual strategy for P. aeruginosa detection as well as its toxin genes. This approach can recognize the internal standard gene ecfX and toxin genes (ExoS and ExoU) in P. aeruginosa by modifying primers using different labels in multiple LAMP including hex, FITC, and digoxin. In the presence of primers with different labels consist of FITC (hex, digoxin) and biotin, Bst DNA polymerase and dntps, the mLAMP can start and generate a lot of duplex DNA products with the label of biotin and FITC (hex, digoxin). Then the lateral flow biosensor recognizes the label of the product by the different antibody fixed on the strip. Anti-biotin antibodies were labeled on the AuNP. Anti-FITC (hex, digoxin) antibodies were labeled on the test line of lateral flow biosensor. The accumulation of AuNP generated a significant red band, enabling visual detection of P. aeruginosa and its toxin genes by naked eyes. After systematic optimization of LFNAB preparation and detecting conditions, the limit sensitivity detection of current approach can reach as low as 20 CFU/mL P. aeruginosa and its toxin. At the meantime the limit time of the detection can be achieved in 50 min, which is more sensitive than PCR. Therefore, this strategy offered a fast, easy-operating, sensitive, low-cost, and pollution-free method for the determination of P. aeruginosa and its toxin genes. Except for the above, many other chemical substances were also synthesized to detect microorganism.
AuNPs were also prepared for synthetic DNA and applied into bacterial 16S rRNA detection of Escherichia coli (DH5α) in cell cultures . AuNP–DNA molecular beacon conjugates produced a sensitivity provident by three orders of magnitude, and the detection limit achieved 100 CFU/mL of E. coli within 1 h, which is much more sensitive than molecular beacons alone. In another example, Donmez et al. reported a nanosensor based on Tb3+ and Eu3+ chelated AuNP to detect dipicolinic acid, which is a unique biomarker of bacterial spores . Also, another strategy based on gold nanoparticles has been demonstrated to detect E. coli in real water or food samples by Jin et al. This method established platform based on FRET and used the conjunction of lanthanide-doped up conversion nanoparticles functionalized with complementary DNA and aptamer functionalized gold nanoparticles .
2.3.11 Silver Nanoparticle (AgNP)-Functional Nucleic Acid Based Biosensors for Microorganism Detection
Nano-biotechnology is a novel technology that developed in recent years and has potential in wide fields. In the area of metal nanoparticles, silver nanoparticles are widely utilized in microorganisms due to the toxicity against a broad spectrum of pathogenic microbes. There are some typical applications of silver nanoparticles as follow.
2.3.12 Nanozyme-Functional Nucleic Acid Based Biosensors for Microorganism Detection
In order to meet the need of the detection, nanozyme-strip has attracted significant attention in recent years because of its stability and ability to be reused. The nanozyme-strip is based on the peroxidase-like activity of MNPs and has been successfully applied to biomedical detection of Ebola and environmental analysis. The main objective of this study was to develop a continual cascade nanozyme biosensor for the detection of viable ES. The ompA gene of ES was determined using FITC-modified and BIO-modified primers in LAMP process. In the presence of BIO-and FITC-modified primers and Bst DNA polymerase large fragments, the LAMP produced 109 dual-labeled DNA products. LAMP combined with PMA treatment was applied for differentiation from viable and dead state of ES. Then, by using Fe3O4 magnetic as a nanozyme probe, a MNP-based immunochromatographic strip (nanozyme strip) was further employed for amplified signal to allow visual detection and quantification by strip reader. Owing to the catalytic properties of the probe, the detection sensitivity was improved compared with colloidal gold strip. And the 10 CFU/mL lower limit of biosensor is improved compared with previously reported techniques and is much faster (within 1 h) and simpler (without specialist facilities). Hence, the developed continual cascade nanozyme biosensor provides a rapid, ultrasensitive, and simple tool for on-site detection of viable ES.
There are some other research on the nanozyme-based biosensor for microorganism detection. Demin Duan et al. generated a novel strip test based on nanozyme for the glycoprotein (GP) of EBOV (EBOV-GP) detection and the sensitivity limit reached at 1 ng/mL by the naked eye, which is more than 100-fold higher the step based on colloidal gold . This method can make it true that as low as 240 pfu/mL, pseudo-EBOV can be rapidly detected within 30 min. Demin duan et al. previous focused on the intrinsic peroxidase-like activity of MNPs which is fundamental in this research. In this research, they let the nanozyme take place of colloidal gold nanoparticles in the lateral flow biosensor because of the catalyzing peroxidase activity of the nanozyme which can produce an obvious color reaction. This novel probe can recognize, separate, and visualize EBOV on the strip after labeling with anti-EBOV antibody. Owing to the catalytic properties of the probe, the detection sensitivity of the nanozyme-strip has been improved significantly without any need of special equipment. This novel assay represents a suitable technology for Ebola-stricken areas due to the high sensitivity and simplicity. In addition, the nanozyme also has been applied in the cancer therapy. ShiyanFu did a research on the structural effect of Fe3O4 nanoparticles on peroxidase-like activity for cancer therapy . More recently, the nanozyme is applied into the molecule detection, such as malathion , glucose and antioxidant , mercury(II) ions , multiple DNAs , and so on.
2.3.13 Magnetic Nanoparticle (MNP)-Functional Nucleic Acid Based Biosensors for Microorganism Detection
Nanoparticle-based platforms have been applied into many detection areas so as to reach real-time detection for better foodborne pathogen monitor. Magnetic nanoparticles have performed a significant role in much diagnosis because of their great properties including easy functionalization, biocompatibility, and high stability to reach fast, accurate, simple, and cheap analysis of detection. There are some typical applications of magnetic nanoparticles in biology detection.
2.3.14 Functional Nucleic Acid Based Fluorescence Biosensors for Microorganism Detection
In general, the fluorophore and quencher are mostly used in biosensor to result fluorescence because it is convenient and easy to be modified to other substances. Because the fluorophore and quencher are positioned right next to each other, the sensor background is extremely low, leading to a large signal enhancement in the presence of the target.
The principle of fluorophore is that when they absorb energy from light, they will transfer this energy and emit it as light of a characteristic wavelength. Briefly, after absorpting the energy from the light, a fluorophore at ground state will be raised to a higher vibrational level of an excited singlet state, which takes about one femtosecond (10−15 s). In the next process, the fluorophore will return to the lowest vibrational level of an excited singlet state because some energy is lost as heat. And about 1 ps (10−12 s) will be taken in this process. And because of the energy is lost, the energy of the absorbed light is higher than the energy of the emitted fluorescence light, and therefore emission occurs at a longer wavelength than absorption. Fluorescence is the light that is emitted from the excited singlet state .
If the fluorophore react with other fluorophore or nonfluorescent molecules and produce a nonfluorescent complex, the fluorophore is quenched. This is the principle of contact quenching, static quenching, or ground-state complex formation. In contact quenching, two molecules interact by proton-coupled electron transfer through the formation of hydrogen bonds. When the complex absorbs energy from light, the excited state immediately does not emit a photon, and the molecules do not emit fluorescent light and just return to the ground state. The key point of contact quenching is that the complex of two molecules changes in the absorption spectra.
“Collisional quenching” or “dynamic quenching” is another kind of decrease the fluorescence intensity. When a fluorophore at excited state contact with another molecule in the one solution, the fluorophore is deactivated and that is called collisional quenching. After contraction, the fluorophore returns to the ground state without emission of fluorescence light. The features and structures of the fluorophore and the manner of its interaction with the other molecule can all affect the extent of quenching. Oxygen, halogens, and amines can quench the fluorophore in collisional quenching.
QDs have a variety of advantages over the organic fluorophore and fluorescent proteins, which are extensively adopted in biological labeling : broad excitation spectra and narrow emission spectra can be excited and detected at the same time. These features make it possible that the multiple targets can be detected at the same time utilizing multicolor QD systems [188, 189]. Compared with other fluorescent dyes, QDs are more stable which can be utilized in real-time monitor .
Other Fluorescent Labels
2.3.15 Functional Nucleic Acid Based Electrochemical Biosensors for Microorganism Detection
Functional Nucleic Acid Based Electrochemical Biosensors for Virus Detection
Although avian influenza virus shows rapid evolutionary dynamics, consistent with a high background mutation rate and rapid replication , electrochemical biosensors for its detection are highly recommended. Such strategy allows for making simple and rapid changes of specific recognition elements and solves the problem associated with high mutation rate of the avian influenza virus .
Functional Nucleic Acid Based Electrochemical Biosensors for Pathogen Detection
Pathogen detection is another field of electrochemical biosensors applications which is rapid method for microorganism detection and is researched in the past two decades.
Moreover, the electrochemical biosensors can be made into a simple device for low cost because of the presented state-of-the-art techniques utilized in the fabrication of electronics [193, 199, 200, 201, 202]. However, some disadvantages restrict electrochemical biosensors to pathogens detection which are needed to be overcome. Particularly, the complex food sample is the most concerned factor which causes the most difficulties because the bacteria are highly unlikely distributed in/on foods in the pattern of uniformity. Thus, collections and pretreatments of sample are needed to satisfy the requirement of direct use of electrochemical biosensors .
Functional Nucleic Acid Based Electrochemical Biosensors for Toxin Detection
In recent years, the nanotechnology has been extensively utilized in bioanalytical devices. Moreover, in the detection of toxins, it also shows a lot of advantages for food safety and environmental applications. Because of high sensitivity and design versatility of electrochemical biosensors, the toxins of low levels and the small size can be detected by electrochemical biosensors. The nanomaterials will be developed further to higher sensitivity and short detection time .
2.3.16 Surface-Enhanced Raman Spectroscopy-Functional Nucleic Acid Based Biosensors for Microorganism Detection
Principles of Surface-Enhanced Raman Spectroscopy
Surface (S): SERS is a surface spectroscopy technique, and the molecules must be on (or close to) the surface which is an important factor in adoptions of SERS. Before the utilization of SERS, it is must be guaranteed that the molecules can attach to (or at least be very close to) the surface of the metal substrate.
Enhanced (E): The signal enhancement is provided by plasmon resonances in the metal substrate. In fact, the term “plasmon resonances” means a family of effects in related to the interaction of electromagnetic radiation with metals.
Raman (R): The technique consists in measuring the Raman signals of molecules (the SERS probes or analytes). Raman spectroscopy is the study of inelastic light scattering, and, when applied to molecules, it provides an insight into their chemical structure (in particular their vibrational structure).
The Application of Surface-Enhanced Raman Spectroscopy-Functional Nucleic Acid Based Biosensorsfor Microorganism Detection
Surface-enhanced Raman spectroscopy as a sensitive and specific method has been utilized in many fields of biology. Besides, the SERS technique can analyze molecules even in trace amounts . Resonance Raman at different excitation wavelengths can be widely used to analyze different microorganisms. Gold nanoparticles, silver nanoparticles, and other heavy metal nanomaterials and various core–shell nanoparticles are widely utilized as SERS-enhanced substrates [209, 210, 211].
L. Zeiri developed a SERS-based biosensor for the quantitative detection of S. typhimurium and S. aureus simultaneously using aptamers and nanoparticles . The signal probe consists of AuNPs, Raman signal molecule, and aptamers. And the combinations of GNPs and aptamers were utilized to capture. In the range of 102–107 cfu mL−1, S. typhimurium and S. aureus concentration exhibited a good linear relationship, and the detection limit was 35 cfu mL−1 for S. aureus and 15 cfu mL−1 for S. typhimurium. This method is sensitive, selective, and rapid.
In 2011, Sandeep P. Ravindranath demonstrated a cross-platform method to detect three different pathogens at the same time utilizing Raman and UV–vis absorption spectroscopy. Gold (Au), silver (Ag), and Ag–Au core–shell nanoparticles were modified with anti-Salmonella typhimurium aptamers, anti-Staphylococcus aureus, and anti-Escherichia coli O157:H7 antibodies. In order to signal output, Raman reporter molecules were also labeled to them. A microfiltration step was utilized to achieve a detection platform with high selection and good specification, with total detection time under 45 min for both species (E. coli O157:H7 vs. S. typhimurium) and strain (E. coli O157:H7 vs. E. coli K12) level sensing at a limit of a detection ranging between 102 and 103 CFU/ml .
The multiple target detection method using SER technology was also developed by Hui Zhang . The Raman signal probes are built using AuNPs labeled by Raman molecules (mercaptobenzoic acid and 5, 5′-Dithiobis (2-nitrobenzoic acid)) and aptamer. This method with short detection time, high sensitivity, and specificity was widely utilized to analyze the microorganisms in actual samples.
2.3.17 Surface Plasmon Resonance (SPR)-Functional Nucleic Acid Based Biosensors for Microorganism Detection
Principles and Advantages of SPR
According to the studies, surface plasmon resonance (SPR) is an important technology in monitoring the real-time reaction. Two mechanisms have been considered to explain the SERS effect. The main contribution arises from a huge enhancement of the local electromagnetic field close to surface roughness, due to the excitation of a localized surface plasmon, while a further enhancement can be observed for molecules adsorbed onto specific sites when resonant charge transfer occurs. If there are any molecules that bonds to the conducting surface, the oscillations will be changed with high sensitivity .
The Application of Surface Plasmon Resonance (SPR)-Functional Nucleic Acid Based Biosensors for Microorganism Detection
In 2005, Lee et al. developed a sensor that combined the DNA microarrays and surface plasmon resonance (SPR) to measure the single-stranded DNA (ssDNA) . When ExoIII and target DNA are modified to a 3′-terminated ssDNA microarray, hybridization adsorption of the target ssDNA leads to the direction-dependent ExoIII hydrolysis of probe ssDNA strands and the release of the intact target ssDNA back into the solution. The targets bonded to probe and it caused the change of SPR signal. The detection limit is 10–100 pM.
Tan Tai Nguyen developed surface plasmon resonance (SPR) optical fiber sensor to analyze PCR amplification without fluorophore . The integrated device was comprised of the microfluidic PCR reactor and the optical fiber SPR sensor with bimetallic (Ag/Al) coating. This sensor can amplify the DNA of Salmonella spp. within 30 min. Besides, the SPR device can measure the DNA amplicon. Thus, it is an all-in-one device that can serve as a DNA amplification-to-detection instrument.
2.3.18 Flow Cytometry-Functional Nucleic Acid Based Biosensors for Microorganism Detection
Flow cytometry (FCM) can analyze the single cell according to the size and granularity by using light-scattering features when the cell flows through a measuring device .
The main components of flow cytometers and cell sorters are basically fluidics, optics (excitation and collection), an electronic network (detectors), and a computer. The fluidics is responsible for directing liquid containing particles to the focused light source. The excitation optic focuses the light source on the cells/particles, while collection optics transmits the light scatter or fluorescent light of the particle to an electronic network. The electronic network detects the signal and converts the signals to a digital data that is proportional to light intensity, and the computer is also required to analyze data [219, 220] .
Flow cytometry is used in various applications based on the detection of the membrane and cytoplasmic and nuclear antigens. Additionally, whole cells and cellular components such as organelles, DNA, RNA, chromosomes, cytokines, hormones, and protein content can also be investigated by flow cytometry. Analysis of cell proliferation and cell cycle and measurements of calcium flux and membrane potentials are the commonly used examples of methods developed for flow cytometry .
Flow cytometry was first used in the 1970s as a common method for cellular biology. In recent years, it is utilized in microorganisms although they are hardly characterized because of small size. In addition, FCM can achieve high-throughput detection as well as single cell distinguishment. Nucleic acid-binding dyes especially SYTO dyes and PI have been extensively adopted to characterize cell viability. If DNA of living and dead microorganisms is binding to DNA dyes, it is easy to distinguish by UVA. In addition, EMA, annexin V, and amine reactive viability dyes (ViDs) are dead cell dyes in general. FCM can be adopted for the live/dead microorganism detection. According to the report, FCM has been utilized to detect E. coli, S. enterica serovar Typhimurium, Shigella flexneri, and a community of freshwater bacteria .
FCM-FACS enables us to isolate single cells directly from water samples prior to incubation in a medium. This single-cell isolation reflects bacterial population diversity. This is in contrast to most of the conventional isolation procedures, in which cells are grown in a medium prior to isolation. However, FCM-FACS has not been used to isolate pathogens that are present in low concentrations in environmental samples without preincubation. Therefore, Ozawa S. developed FCM-FACS method to detect and isolate pathogens that are present in low concentrations in water environment. As a result, specific isolation was achieved even when the target was present at 0.01% of the total population in pure culture study and when the target was present at 10 cells/mL in spiked water sample. As a result of comparison with conventional methods, the bacterial proportion was almost preserved by FCM-FACS method better than the result of by dynabeads separation technique .
2.3.19 Gene Chip-Functional Nucleic Acid Based Biosensors for Microorganism Detection
Detection Techniques Based on Solid Arrays
A DNA microarray, also called DNA chip or biochip, has so many microscopic DNA that modified a solid surface. In order to make sure that the PCR primers were modified onto substrates, the chemical bonds that connect the oligonucleotides and the substrates cannot be damaged by thermocycling conditions, especially the high temperatures of 95 °C.
Hoffmann et al.  summarized a universal protocol for grafting PCR primers onto solid body for solid-phase PCR. The primers are labeled by using PCR-compatible method. The DNA microarrays can be integrated into microfluidic lab-on-a-chip cartridges of various materials by immobilization and SP-PCR protocols that have been reported. What is more, covering the inner space with PCR primers makes the generated PCR products recovered in digital PCR.
Detection Techniques Based on Liquid Arrays
Kopp et al. designed continuous-flow PCR chip for the first time in 1998, which is widely used for microorganism detection . Wang et al. utilized liquid chips to detect Staphylococcus aureus rapidly with the detection limit of 103 CFU/ml and high specification. Another advantage of this method is that it can be used for 200 food samples . What is more, multiple detections of seven microorganisms can be achieved using this technology which is developed by Lü et al. This developed method can detect 140 strains of bacteria and can be utilized for 56 food samples with high specification and a detection limit of 1–100 pg and 105–106 CFU/ml .
A miniaturized, disposable microbial culture chip has been fabricated by Colin J. Ingham group with up to one million growth compartments . This chip can be utilized for Escherichia coli detection based on expression of the lacZ reporter gene and high-throughput screening.
2.3.20 Functional Nucleic Acid Based Biosensors for Microorganisms Diversity Analysis
16S rDNA and 18S rDNA
Restriction Fragment Length Polymorphism (RFLP)
Random Amplified Polymorphic DNA (RAPD)
DGGE and TGGE
High-throughput sequencing is a rapid method because it can analyze a variety of different DNA sequences at one time. There are so many advantages of it, including high sensitivity, high specificity, low-cost and short detection time, which makes it much better than traditional methods. In microbial diversity research, people often utilize amplicon sequencing and whole-metagenome shotgun sequencing.
2.4 Conclusion and Prospects
This review is focused on the construction, basic principles, amplifications and recent development in functional nucleic acid based biosensor for microorganism detection. In few years, the microorganism detection technologies are developed from traditional approaches such as microorganism cultivation, physiological and biochemical testing, instrument analysis, and immunology, to molecular biological detection methods especially functional nucleic acid detection technology. Because of the development of it, the detecting of microorganisms is becoming quicker, more specific, sensitive and accurate in recent years.
There have been series of functional nucleic acid based biosensors for microorganism detection including functional nucleic acid-aptamer biosensor, functional nucleic acids colorimetric biosensor, new nanomaterial-based biosensor, lateral flow biosensor, high-throughput biosensor, and so on. However, the sensitivity, the specificity and the species of the functional nucleic acid based biosensors still need to be developed. For an instance, we have done a lot of research on the redox activity of the DNAzyme in the detection, while the other activity of the DNAzyme still needs to be developed further. For another example, as we know, the nanomaterials can facilitate the sensitivity and specificity of functional nucleic acid based biosensor in the detection of microorganisms, and a lot of them including the gold-nanoparticles, silver-nanoparticles, QD-nanoparticles, and so on have been applied into the functional nucleic acid based biosensors for microorganism detection. However, there are still some nanomaterials with good property which have not been utilized and developed. In addition, there is another research direction that is combining the good signal recognition technology, signal transduction technology, signal application technology, and signal output technology to develop more sensitive, rapid, easy operation functional nucleic acid based biosensor for microorganism detection.
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