Recent Progress on the Electrochemical Biosensing of Escherichia coli O157:H7: Material and Methods Overview

Escherichia coli O157:H7 (E. coli O157:H7) is a pathogenic strain of Escherichia coli which has issued as a public health threat because of fatal contamination of food and water. Therefore, accurate detection of pathogenic E. coli is important in environmental and food quality monitoring. In spite of their advantages and high acceptance, culture-based methods, enzyme-linked immunosorbent assays (ELISAs), polymerase chain reaction (PCR), flow cytometry, ATP bioluminescence, and solid-phase cytometry have various drawbacks, including being time-consuming, requiring trained technicians and/or specific equipment, and producing biological waste. Therefore, there is necessity for affordable, rapid, and simple approaches. Electrochemical biosensors have shown great promise for rapid food- and water-borne pathogen detection. Over the last decade, various attempts have been made to develop techniques for the rapid quantification of E. coli O157:H7. This review covers the importance of E. coli O157:H7 and recent progress (from 2015 to 2020) in the development of the sensitivity and selectivity of electrochemical sensors developed for E. coli O157:H7 using different nanomaterials, labels, and electrochemical transducers.


Introduction
The rapid spread of pathogenic bacteria, as well as their rapid development of antibiotic resistance, has caused worldwide concern as they are a major source of both foodborne and waterborne illnesses [1][2][3]. Pathogenic strains of bacteria are the main concern for environmental biology, hospitals, water supplies, and the food industry because of the diverse illnesses that microbial infection can cause, some of which can lead to death [4]. Contamination of food resources has led to the occurrence of certain diseases, placing a heavy responsibility on food distributors to restrict outbreaks [2,5,6]. More important, the majority of water sources are contaminated with pathogenic bacterial strains, such as Salmonella, Staphylococcus, and Escherichia coli, resulting in typhoid fever, gastroenteritis, cholera, and several diarrheal responses [2,7,8]. According to the 2016 report of the World Health Organization (WHO), 829,000 annual deaths from diarrhea occurred due to bacterial water contamination [9]. Around 600 million-nearly 1 in 10 people in the world-become ill due to the consumption of contaminated food, resulting in 420,000 deaths every year and the loss of 33 million healthy life years (disability adjusted life years; DALYs) [10]. E. coli is a fecal coliform bacterium found in the human gut and other warm blooded animals, and is typically harmless to humans [1,11]. However, pathogenic groups of E. coli strains can cause diarrheal illnesses. Pathogenic E.coli can be categorized into six groups,

Electrochemical Biosensors for the Detection of E. coli O157:H7
Electrochemical biosensors are frequently designed and have been widely used for the detection of food-borne and water-borne pathogens due to the possibility of miniaturization and construction of disposable, flexible, and cheap sensing systems. As reported by the International Union of Pure and Applied Chemistry (IUPAC), an electrochemical biosensor is an independently integrated system using a bioreceptor in contact with an electrochemical transduction part that provides specific quantitative or semiquantitative analytical data [41,42]. The current produced by oxidation and reduction reactions related to the presence of the electroactive species or its rate of production/consumption is measured by an electrochemical biosensor. The produced electrical signal is proportional to the target's concentration [43,44]. Electrochemical biosensors are categorized into four classes: impedance, amperometric, conductometric, and potentiometric, according to the nature of the electrochemical changes detected via the biorecognition reaction [38]. Simplicity and speed are the key advantages of electrochemical biosensors. Low-cost electrodes incorporated with simple electronics allow rapid detection in easy-to-use, miniaturized portable devices. For environmental monitoring, the capability to detect the target concentration within a complex sample at the point-of-care and in real time is particularly interesting [38,45]. The number of papers reported for the detection of E. coli O157:H7 using an electrochemical transducer from 2015 to 2020 is large. Table 2 summarizes the studies related to using an electrochemical transducer for the detection of E. coli O157:H7. As shown in Table 2, many of the studies are devoted to genosensors and immunosensors. Genosensors use DNA sequencing analysis for bacterial detection. Nucleic acid hybridization is based on the immobilization of a single-stranded DNA sequence on a specific substrate. An obtained electrical current signal is the result of the binding of a complementary DNA sequence to the probe DNA. Detection of a specific DNA sequence provides a rapid, simple, cost-effective, and physically small assay that can be operated by nonprofessional users [46,47]. Electrochemical immunosensors rely on an electrochemical signal resulting from stable antigen-antibody complex formation, allowing highly sensitive detection. A label or marker attached to an antibody (Ab) or an antigen (Ag) is required for labeled electrochemical immunosensors to achieve electron transfer. The detected amount of the label corresponds to the target analyte's concentration. In sandwich-based immunosensors, two specific antibodies are used to capture the target cell. One of the antibodies is immobilized on the surface of the electrode and the other one is labeled with an electroactive marker or a label which can produce an electroactive product [48,49]. For detection of pathogenic bacteria, different kinds of nanomaterials have been integrated into the biosensors, yielding improvements in terms of stability, sensitivity, selectivity, and speed of the electrochemical biosensors. As shown in Table 2, gold nanoparticles and nanostructures have gained considerable attention for the detection of E. coli O157:H7. Providing a stable biomolecule immobilization while retaining their bioactivity is the major advantage of using gold nanoparticles in electrochemical biosensors. Application of gold nanomaterials in an electrochemical biosensor offers improvements in signal amplification, electron transfer, and electrocatalytic activity. The unique properties of gold nanoparticles, including their inert nature in biological fluids, biocompatibility, presence of functional groups for binding ligands, high surface to volume ratio, etc., make their use promising in the construction of electrochemical biosensors [50]. In this review, an attempt has been made to organize the recently reported studies using electrochemical transducers (Table 2) for the detection of E. coli O 157:H7. The current challenges and future directions are discussed.

Voltammetric-Based Biosensors
Voltammetric measurement is based on the principle of measuring the flowing current produced through the working electrode dipped in a solution containing an electroactive species. The easy recognition of the target via its voltammetric peak potential qualifies voltammetry as a strong electrochemical technique in biosensing [44,87]. Cyclic voltammetry (CV), square wave voltammetry (SWV), and differential pulse voltammetry (DPV) are the frequently applied techniques in voltammetric biosensors. In the past few years, numerous approaches using aptamers, enzymes, and nanomaterials have been successfully incorporated into voltammetric biosensors for the detection of E. coli O 157:H7. Zhong and coworkers proposed a new electrochemical biosensor for the detection of E. coli O157:H7 ( Figure 1). As signal-amplifying tags for the determination, cadmium sulfide quantum dots (CdS QDs) and encapsulated zeolite imidazolate framework-8 (ZIF-8) particles were used. In the presence of CdS QDs, the growth of ZIF-8, CdS@ZIF-8 muticore-shell particles on the sample was achieved. In order to introduce amino groups on the surface, CdS@ZIF-8 particles were coated via polyethyleneimine, followed by an anti-E. coli O157:H7 antibody modification on the surface for the selective detection of E. coli O157:H7. CdS@ZIF-8 particles, as signal tags, were used for preparing a sandwich-based sensor. By HCl leaching, Cd (II) ions were released from CdS@ZIF-8, leading to E. coli O157:H7 detection by differential pulse voltammetry. The linear range of 10 to 10 8 colony forming units (CFU)/mL and 3 CFU/mL was achieved by the fabricated immunosensor which also showed good sensitivity and selectivity of E. coli O157:H7 in milk samples. The proposed biosensor can be expanded to be used for detection of other pathogenic bacteria [59]. Very recently, Yan li and coworkers [60] used multiple amplification strategies via 3D DNA walker, rolling circle amplification (RCA), and hybridization chain reaction (HCR) to develop a sensitive and selective electrochemical biosensor for the accurate determination of E. coli O157:H7 ( Figure 2). The target sequence of the E. coli O157:H7 was extracted, transformed, and amplified. After that, in order to generate an enhanced electrochemical signal, a large sequence of double-stranded DNA as a result of HCR progress, immobilized electrochemical indicators. Based on the proposed strategy the detection limit was 7 CFU/mL for E. coli O157:H7 with a linear range of 10 to 10 × 10 4 CFU/mL. The proposed multiple amplification strategy-based biosensor can be readily used for determination of different microorganisms, allowing a novel approach for early diagnosis of malignancies and monitoring the therapy responses [60]. The advantage of the voltammetric technique is that it provides highly sensitive measurements and the possibility of simultaneous detection of multiple analytes. This technique can provide low LODs of 2 CFU/mL using an electrochemical aptasensor detection strategy based on amino-functionalized metal-organic frameworks. Despite the low LOD achievements in some of the studies, testing the fabricated biosensors in real and complex samples remains to be done. Moreover, the detection time in most of the conducted studies was long, which needs to be improved. Research towards the simultaneous detection of E. coli O157:H7 in complex and real samples is also a major requirement.   [60].

Impedimetric Based Biosensors
Impedimetric biosensors are one of the earliest approaches developed for rapid pathogen detection [88]. The main difference between this technique and other electrochemical techniques is conductivity detection [38]. Impedimetric biosensors work by analyzing the electron transfer at the electrode surface or measuring the solution/medium conductivity, which can be read as an impedance response [38]. The most frequently used technique for impedimetric biosensors is

Impedimetric Based Biosensors
Impedimetric biosensors are one of the earliest approaches developed for rapid pathogen detection [88]. The main difference between this technique and other electrochemical techniques is conductivity detection [38]. Impedimetric biosensors work by analyzing the electron transfer at the electrode surface or measuring the solution/medium conductivity, which can be read as an impedance response [38]. The most frequently used technique for impedimetric biosensors is electrochemical impedance spectroscopy (EIS). This technique scans the detection volume and uses an electrical frequency sweep in the range of 10 KHz to 10 MHz [35,38]. Electrochemical impedance spectroscopy (EIS) is an easily operated, simple, straightforward, and sensitive technique which has attracted substantial interest for E. coli O157:H7 determination. Barreiros de Santos and colleagues developed an indium tin oxide (ITO)-based impedimetric biosensor by using a robust, simple, and direct approach for the detection of E. coli O157:H7 (Figure 3). Immobilization of anti-E. coli antibodies onto ITO electrodes was done, and epoxy silane on the surface of ITO was attached covalently, as shown by atomic force microscopy and cyclic voltammetry. By using optical waveguide light mode spectroscopy (OWLS), antibody immobilization on the epoxy silane layer was quantified and a mass variation of 12 ng cm −2 (0.08 pmol cm −2 ) was achieved. The selectivity of the antibodies and functionalization procedure's efficiency were confirmed by achieving a ratio of 1:500 Salmonella typhimurium/E. coli O157:H7. The proposed ITO-based immunosensor was evaluated by electrochemical impedance spectroscopy. A very low limit of detection of 1 CFU mL −1 with a large linear working range of 10-10 6 CFU mL −1 was achieved by using electrochemical impedance spectroscopy. The 20% detection of nonspecific bacteria, made up of E. coli K12 and S. typhimurium, showed the specificity of the impedimetric immunosensor, meaning that ITO is highly selective and sensitive [62]. Lan Yao and coworkers developed a microfluidic impedance biosensor for sensitive, rapid, and continuous E. coli O157:H7 detection by applying immune magnetic nanoparticles. For biological signal amplification, urease was used. In order to make the immune magnetic nanoparticles (MNPs), streptavidin-modified MNPs conjugated with biotinylated polyclonal antibodies were used. To make the MNP-bacteria complexes, the target is separated by the MNPs. Afterwards, to form the MNP-bacteria-gold nanoparticles (GNP)-urease complex, the gold nanoparticles modified with the urease and aptamers with the MNP-bacteria were conjugated. Then, hydrolysis of urea into ammonium carbonate led to impedance decrease. A low detection limit of 12 CFU/mL was obtained by online impedance measurement [64]. Recently, Martina Cimafonte and coworkers developed an electrochemical impedance immunosensor based on a screen-printed gold electrode by immobilizing anti-E. coli antibodies onto the gold surface covalently by the photochemical immobilization strategy for fast E. coli determination in water ( Figure 4). In this study, in order to develop an "on-off" electrochemical impedimetric immunosensor, photochemical immobilization technique (PIT) was used for the first time in the functionalization of commercial gold electrodes using Fe (CN) 6 3− /Fe (CN) 6 4− as a redox probe. E. coli in drinking water was selectively and sensitively detected with a limit of detection of 3 × 10 CFU/mL. The proposed biosensor needed no preconcentration or pre-enrichment steps for the detection process [73]. The ability to perform label-free detection is the most important advantage of impedimetric electrochemical biosensors; however, in some of the conducted studies, labels have been used for signal amplification. As shown in Table 2, by using an impedimetric sensing strategy, label-free direct detection of E. coli O157:H7 with a LOD of 1 CFU/mL was achieved. However, substantial time was required for the patterning of anti-E. coli O157 antibodies. Moreover, a low limit of quantification and testing of the fabricated biosensors in real and complex samples in most of the studies have not been achieved.     immunosensor. The black line shows the redox reaction intensity, which is inhibited as the surface covering grows. Its thickness reduction is related to decrease of the "effective" area available for the electrolyte current, which is measured through an increase of the charge transfer resistance. (I: functionalization of the surface with antibodies, II: blocking the free remaining spaces on the gold electrode by BSA, III: reaction of immobilized antibodies and the E. coli cells, IV: conveying a fresh anti-E. coli Abs solution to the circuit) [73]. immunosensor. The black line shows the redox reaction intensity, which is inhibited as the surface covering grows. Its thickness reduction is related to decrease of the "effective" area available for the electrolyte current, which is measured through an increase of the charge transfer resistance.

Amperometric-Based Biosensors
As a class of electrochemical biosensors, amperometric biosensors transduce the biological recognition reactions caused by electroactive agents at the electrode surface into a current signal to determine the target molecule within a sample matrix. They can be integrated with nucleic acids, enzymes, and antibody recognition elements, and are applicable for environmental monitoring [38,44]. Differential pulse voltammetry, cyclic voltammetry, and square wave voltammetry are different amperometric methods which are applied in biosensors. The false current reading because of the electroactive interference present in the sample matrix is the limitation of this technique, and can be solved by various methods such as changing the analyte, diluting the sample, etc. [38,44]. Ahmet Guner and coworkers developed a highly sensitive sandwich assay electrochemical immunosensor based on a Py, Pyrrole/gold nanoparticles/multiwalled carbon nanotube/chitosan (PPy/AuNP/MWCNT/Chi) hybrid nanobiocomposite-modified pencil graphite electrode (PGE) for E. coli O157:H7 detection ( Figure 5). The hybrid bionanocomposite platform was modified with anti-E. coli O157:H7 monoclonal antibodies and the product was characterized by using cyclic voltammetry. A detection limit of 30 CFU/mL in PBS buffer with a linear range of 3 × 10 to 3 × 10 7 CFU/mL was achieved. For application in food quality and safety control, the produced sensor showed high stability and reproducibility [77]. In'another study, Lingxian Ye et al. proposed a sensitive point-of-care testing (POCT) with Au-Pt bimetallic nanoparticle (Au@Pt)-functionalized silica nanoparticles (SiO 2 NPs) and Fe 3 O 4 magnetic nanoparticles (Fe 3 O 4 NPs) for E. coli O157:H7 determination ( Figure 6). As a negatively charged polyelectrolyte, poly-(4-styrenesulfonic acid-co-maleic acid) (PSSMA) coated on the amino group modified the SiO 2 NPs surface, conferring electrostatic force. The PSSMA applied to connect the negatively charged Au@Pt NPs to the SiO 2 NPs led to the formation of Au@Pt/SiO 2 NPs. As signal labels, antibody-and invertase-conjugated Au@Pt/SiO 2 NPs were used. In order to enrich and capture the target in a positive sample, monoclonal antibody-functionalized magnetic nanoparticles (Ab-Fe 3 O 4 @SiO 2 NPs) were used. For the quantitative readout by the PGM, the invertase in the proposed sandwich assay catalyzed the hydrolysis of sucrose to generate a large amount of glucose. A low detection limit of 1.83 × 10 2 CFU/mL was achieved [76]. Electrochemical biosensors based on amperometry have the advantages of high sensitivity, rapid, low cost, and robustness with the possibility of portability. Focusing of these aspects, more studies need to be performed in order to provide a portable sensitive biosensor for the detection of E. coli O157. Multiplexing detection and assessing the reproducibility are other parameters which should be focused on while conducting future studies based on amperometry. As the limit of quantification is rarely calculated in the reviewed studies, it also should be included to enable comparison between the reported studies.

Amperometric-Based Biosensors
As a class of electrochemical biosensors, amperometric biosensors transduce the biological recognition reactions caused by electroactive agents at the electrode surface into a current signal to determine the target molecule within a sample matrix. They can be integrated with nucleic acids, enzymes, and antibody recognition elements, and are applicable for environmental monitoring [38,44]. Differential pulse voltammetry, cyclic voltammetry, and square wave voltammetry are different amperometric methods which are applied in biosensors. The false current reading because of the electroactive interference present in the sample matrix is the limitation of this technique, and can be solved by various methods such as changing the analyte, diluting the sample, etc. [38,44]. Ahmet Guner and coworkers developed a highly sensitive sandwich assay electrochemical immunosensor based on a Py, Pyrrole/gold nanoparticles/multiwalled carbon nanotube/chitosan (PPy/AuNP/MWCNT/Chi) hybrid nanobiocomposite-modified pencil graphite electrode (PGE) for E. coli O157:H7 detection ( Figure 5). The hybrid bionanocomposite platform was modified with anti-E. coli O157:H7 monoclonal antibodies and the product was characterized by using cyclic voltammetry. A detection limit of 30 CFU/mL in PBS buffer with a linear range of 3 × 10 to 3 × 10 7 CFU/mL was achieved. For application in food quality and safety control, the produced sensor showed high stability and reproducibility [77]. In another study, Lingxian Ye et al. proposed a sensitive point-of-care testing (POCT) with Au-Pt bimetallic nanoparticle (Au@Pt)-functionalized silica nanoparticles (SiO2 NPs) and Fe3O4 magnetic nanoparticles (Fe3O4 NPs) for E. coli O157:H7 determination ( Figure 6). As a negatively charged polyelectrolyte, poly-(4-styrenesulfonic acid-comaleic acid) (PSSMA) coated on the amino group modified the SiO2 NPs surface, conferring electrostatic force. The PSSMA applied to connect the negatively charged Au@Pt NPs to the SiO2 NPs led to the formation of Au@Pt/SiO2 NPs. As signal labels, antibody-and invertase-conjugated Au@Pt/SiO2 NPs were used. In order to enrich and capture the target in a positive sample, monoclonal antibody-functionalized magnetic nanoparticles (Ab-Fe3O4@SiO2 NPs) were used. For the quantitative readout by the PGM, the invertase in the proposed sandwich assay catalyzed the hydrolysis of sucrose to generate a large amount of glucose. A low detection limit of 1.83 × 10 2 CFU/mL was achieved [76]. Electrochemical biosensors based on amperometry have the advantages of high sensitivity, rapid, low cost, and robustness with the possibility of portability. Focusing of these aspects, more studies need to be performed in order to provide a portable sensitive biosensor for the detection of E. coli O157. Multiplexing detection and assessing the reproducibility are other parameters which should be focused on while conducting future studies based on amperometry. As the limit of quantification is rarely calculated in the reviewed studies, it also should be included to enable comparison between the reported studies.

Potentiometric-Based Portable Baiosensors
Recently, efforts have been focused on the design of portable sensors for low-cost, on-site, and fast E. coli O157:H7 detection due to the zero-tolerance policy concerning its presence in food. The portability of electrochemical biosensors is of critical importance to realize in-field determination of foodborne and waterborne microorganisms [35]. Few electrochemical lab-on-a-chip and portable biosensors have been made for E. coli determination. Potentiometric biosensors, as low-cost, small, and highly sensitive and selective sensors, apply an ion-selective electrode and ion-sensitive field effect transistor to acquire analytical data [38]. Recently, Parmiss Mojir Shaibani and coworkers (Figure 7) reported a paper for the detection of E. coli in orange juice using a portable nanofiber-light addressable potentiometric sensor (NF-LAPS). As the sensitive layer, electrospun pH-sensitive poly (vinyl alcohol)/poly(acrylic acid) (PVA/PAA) hydrogel NFs was chosen. A limit of detection of 100 CFU/mL was obtained selectively in less than one hour by using NF-LAPS [86].

Potentiometric-Based Portable Baiosensors
Recently, efforts have been focused on the design of portable sensors for low-cost, on-site, and fast E. coli O157:H7 detection due to the zero-tolerance policy concerning its presence in food. The portability of electrochemical biosensors is of critical importance to realize in-field determination of foodborne and waterborne microorganisms [35]. Few electrochemical lab-on-a-chip and portable biosensors have been made for E. coli determination. Potentiometric biosensors, as low-cost, small, and highly sensitive and selective sensors, apply an ion-selective electrode and ion-sensitive field effect transistor to acquire analytical data [38]. Recently, Parmiss Mojir Shaibani and coworkers ( Figure 7) reported a paper for the detection of E. coli in orange juice using a portable nanofiber-light addressable potentiometric sensor (NF-LAPS). As the sensitive layer, electrospun pH-sensitive poly (vinyl alcohol)/poly(acrylic acid) (PVA/PAA) hydrogel NFs was chosen. A limit of detection of 100 CFU/mL was obtained selectively in less than one hour by using NF-LAPS [86]. sensor comprising a three-electrode system [86].

Nanoimpact Method
During the last decade, a powerful new electrochemical technique named the "nanoimpact method" has been developed for single bacterial cell characterization and detection. The method is based on the Faradaic charge transfer following particle collision. Diffusional Brownian motion causes particle movement, and due to the interaction of suspended particles with the electrode, under an oxidizing potential, a short current burst results from the interaction between the particles and the electrode [89]. The detection of the nanoimpact is performed using the change in diffusion current. Lee et al. [90] has reported a fast electrochemical label-free approach through current blockages caused by collision events for E. coli single cell detection on an ultramicroelectrode. The ferrocyanideferricyanide redox couple was used in this study. This methodology has the capability to be used to study other pathogenic bacteria and different target molecules [90]. The problem with the surface blockage detection strategy is the lack of selectivity between various bacterial species and dead and alive cells. In another study, Couto and coworkers [91] applied a carbon microelectrode for fast redox mediated detection of E. coli using impact electrochemistry. N,N,N′,N′-tetramethyl-1,4phenylenediamine (TMPD) was used as a redox mediator to interact with cytochrome c oxidases of bacteria and obtain an electrochemical current, or "on" signals. The advantage of this system is the

Nanoimpact Method
During the last decade, a powerful new electrochemical technique named the "nanoimpact method" has been developed for single bacterial cell characterization and detection. The method is based on the Faradaic charge transfer following particle collision. Diffusional Brownian motion causes particle movement, and due to the interaction of suspended particles with the electrode, under an oxidizing potential, a short current burst results from the interaction between the particles and the electrode [89]. The detection of the nanoimpact is performed using the change in diffusion current. Lee et al. [90] has reported a fast electrochemical label-free approach through current blockages caused by collision events for E. coli single cell detection on an ultramicroelectrode. The ferrocyanide-ferricyanide redox couple was used in this study. This methodology has the capability to be used to study other pathogenic bacteria and different target molecules [90]. The problem with the surface blockage detection strategy is the lack of selectivity between various bacterial species and dead and alive cells. In another study, Couto and coworkers [91] applied a carbon microelectrode for fast redox mediated detection of E. coli using impact electrochemistry. N,N,N ,N -tetramethyl-1,4-phenylenediamine (TMPD) was used as a redox mediator to interact with cytochrome c oxidases of bacteria and obtain an electrochemical current, or "on" signals. The advantage of this system is the minimization of false-positive signals.
The integration of the reported study with microfluidic devices may lead to the low concentration detection of bacteria [91].

Conclusions and Future Perspectives
Bacterial detection is very important in water monitoring, environmental assessment, and the food industry. E. coli O157:H7 has caused several outbreaks worldwide since its first recognition. Thus, the detection of E. coli O157:H7 is one of the major challenges to prevent severe outbreaks. Although currently available methods are highly sensitive, they require a long time to perform and are labor-intensive; therefore, there is a demand for a rapid, simple, sensitive, and low-cost alternative. Among the different types of transducers, electrochemical biosensors, owing to their fast response, selectivity, low cost, sensitivity, possibility of miniaturization, and capability of being integrated into one device, are extensively studied and well developed for the detection of Escherichia coli O157:H7. In this review, the recent developments of label and label-free electrochemical biosensors for the detection of Escherichia coli O157:H7 have been summarized. Although label-free methodologies have the advantages of direct and simple detection with a relatively low detection time and possibility of integration into one test chip, the lack of additional signal amplification and the incubation of the target bacteria with the electrodes are the main disadvantages. To overcome the interference of nontarget molecules in label-free biosensors, appropriate selection of more specific bioreceptors, such as aptamers, etc., is necessary. As illustrated in Table 2, most of the recent chemical recognition approaches involve affinity sensing strategies using aptamers and antibodies as bioreceptors. Aptamers with similar affinity and specificity are chemically stable, small, and they have a simple development process, high target potential, and less production time and cost compared to antibodies. Despite some advantages that aptamers over antibodies, some of the recent studies overviewed in this manuscript are immunosensors based on immobilized antibodies using different kinds of linkages, such as gold nanoparticles. This may be because antibodies are an established technology in all labs, whereas aptamer commercialization has not occurred as quickly as expected. However, the appropriate orientation of antibodies is the most important factor for the improvement of the performance of antibody-based immunosensors in terms of specificity and sensitivity, and the appropriate selection of nanomaterials could overcome this problem. Nanotechnology is an emerging field in science and different nanomaterials, especially gold nanoparticles, have been integrated in the development of electrochemical biosensors for E. coli O157:H7 detection. Nanomaterials are highly important for the immobilization of bioaffinity agents for label-free strategies, and further research should be conducted on the improvement of novel nano-scale materials for effective electron transduction. Current progress in nanotechnology is growing; further studies regarding nanomaterial stability and toxicity in aqueous environments and further progress of smart nanomaterials with useful functions with low cost are expected to solve the improve the sensitivity of electrochemical biosensors towards E. coli O157:H7 detection. Because of the relatively large size of whole bacteria compared to typical biological targets and the existence of different epitopes on the surface of bacteria that can lead to nonspecificity of the approach, a product for real sample applications and the commercial market is yet to be successfully developed. Although impact electrochemistry, as a promising and sensitive technique, has gained attention for E. coli bacteria sensing at the single cell scale, selectivity is the main challenge of this technique. Digital microfluidics, as portable and stable platforms with the power of automation, have the capability to overcome the limitations of current analytical methods in real-time applications, but remain challenging. Overall, sensitivity, specificity, stability, detection time, sample processing, size, ability to perform in different conditions, and no special training requirement are the key features of a biosensor. In addressing all these issues, electrochemical biosensors have a long way to go, but collaboration between academia and industry can pave the way for developing a desirable, portable product.