Electrochemical aptasensors for pathogenic detection toward point‐of‐care diagnostics

A biosensor system refers to a biomedical device, which detects biological, chemical, or biochemical components by converting those signals to an electrical signal by utilizing and uniting physical or chemical transducer with biorecognition elements. An electrochemical biosensor is generally based on the reaction of either production or consumption of electrons under a three‐electrode system. Biosensor systems are exploited in a wide range of areas, such as medicine, agriculture, husbandry, food, industry, environment protection, quality control, waste disposal, and the military. Pathogenic infections are the third leading cause of death worldwide after cardiovascular diseases and cancer. Therefore, there is an urgent need for effective diagnostic tools to control food, water, and soil contamination result in protecting human life and health. Aptamers are peptide or oligonucleotide‐based molecules that show very high affinity to their targets that are produced from large pools of random amino acid or oligonucleotide sequences. Generally, aptamers have been utilized for fundamental sciences and clinical implementations for their target‐specific affinity and have been intensely exploited for different kinds of biosensor applications for approximately 30 years. The convergence of aptamers with biosensor systems enabled the construction of voltammetric, amperometric, and impedimetric biosensors for the detection of specific pathogens. In this review, electrochemical aptamer biosensors were evaluated by discussing the definition, types, and production techniques of aptamers, the advantages of aptamers as a biological recognition element against their alternatives, and a wide range of aptasensor examples from literature in the detection of specific pathogens.

natives, and a wide range of aptasensor examples from literature in the detection of specific pathogens.

aptasensor, pathogenic microorganism detection, point-of-care devices INTRODUCTION
A biosensor is a biomedical device that detects a chemical substance via converging a biological component as the biorecognition element and a physicochemical detector as the transducer. 1 Biological recognition elements can be composed of enzymes, antibodies, cells, cell receptors, organelles, aptamers, nucleic acids, and microorganisms. The detector element is a transducer that enables converting biological signals to electrical signals by utilizing electrochemical, optical, gravimetric, thermal, acoustic, and electronic transducing elements. The analyte is the main target of the biosensor that can be an antigen, protein, nucleic acid, pathogen, toxin, organelle, and whole cell dependent on the aim of the biosensor, which is shown in Figure 1. On the other hand, point-of-care diagnosis represents the acquirement of specific clinical information of patients not only in clinical but also in resource-limited circumstances. It is a known fact that conventional clinical diagnostic tools and techniques need gradual progress with both time-consuming and high cost. 2 On top of all, it also requires expert technicians for operational execution and interpretation of results. Therefore, the convergence of biosensor devices with point-of-care diagnostics technology is a promising approach for a user-friendly, cost-effective, fast, and reliable test.
Biosensor devices demonstrate capabilities, such as being in small-size, portability, high specificity, high selectivity, low cost, and reproducibility. 3 Specifically, electrochemical biosensors (ECBs) are gaining more importance and attention day by day since it creates simple and inexpensive results, while demonstrating both accuracy and sensitivity. ECB consists of biological recognition element to an electrode transducer, which provides conversion of the biological recognition output into a significant and readable electrical signal. Recently, ECBs are designed in a point-of-care manner because of their aforementioned features and the development of micro and nanoscale fabrication technology, which enabled miniaturization and integration of the complex system into a portable and effective tool. 4 For example, conventional potentiostats are miniaturized to small as cellphone, while maintaining their task as being an electroanalytical tool to be used in the field without special training. 5 One of the distinct examples of this technology is glucose biosensor (glucometer), which gathers ECB with point-of-care diagnostics.
Yoo et al. reported the first glucometer in 1962 that was composed of a test strip based on screen-printed electrodes modified by utilizing glucose oxidase enzyme as a biorecognition element and miniaturized to a pocket-sized amperometric transducer resulting in commercialization with affordable prices. 6 Consequently, glucose biosensors took the majority of the market share of the diabetes monitoring market since then not only exploited by clinical professionals of laboratories but also by patients who are at home. This distinct example of success as an analytical tool led and encouraged to development of new electrochemical point-of-care biosensing devices, including cancer detection, toxin detection, and pathogen detection. Even though there have been continuous improvements and evolvement in electrochemical point-of-care biosensors with the contributions of advancements in material science, nanotechnology, molecular biology, and genetic engineering methodologies and applications, there are still major challenges to be overcome such as the design and development of new materials to enhance both specification and sensitivity of biorecognition reaction and new approaches to increase and maintain the stability of the biosensor. In addition, downscaling of these systems as in electrodes and biorecognition elements is also crucial to achieving portable, effective, and user-friendly products, Table 1 shows the biosensors based-on different parameters.
In this context, first, the main concept of biosensor technology for pathogen detection will be explained and then the necessity and the importance of point-of-care diagnostics will be analyzed. After that, the advantages and preferability of ECB in this technology will be discussed. After the brief introduction and illumination about aptamers in biosensor technology, aptamer-based ECB for pathogen detection will be reviewed thoroughly with the literature.

Biosensor technology
Based-on bioreceptors infectious microorganisms, which can cause mild to deadly diseases, such as bacteria, viruses, prions, protozoans, and fungi. There are different origin-based pathogens, such as foodborne, 7 waterborne, 8 and airborne 9 enter the body through various modes of infection, [10][11][12] which are main cause of more than millions of deaths worldwide. 13 The main concern of these pathogens is varying in different regards in terms of virulence, 16 contagiousness, 17 type of transmission, [18][19][20] and infection causing dose. 21 One of the most distinct examples can be considered COVID-19, which is being caused global pandemic rapidly and constantly evolving and increasing its virulence and transmission capabilities. 22,23 The methods utilized to detect these pathogens were limited in the laboratory, microbiology techniques specifically, which are still considered gold standard. However, it is a known fact that detecting the pathogens that were obtained from its original environment in the laboratory requires specific tools and trained technicians. For example, some fastidious yet deadly bacteria require specific media to grow and it can take more than a week to detect under the microscope 24 ; in the case of COVID-19, detection of the virus in the patient sample takes more than a day, which prevents and diminish the beginning and the process of the treatment. Hence, the urgent need for rapid, selective, sensitive, and reproducible pathogen detection reveals the biosensor technology and its undeniable value.
There are different kinds of biosensors based on biorecognition and transducer elements. Biosensing techniques of pathogenic detection are mainly based on deoxyribonucleic acid (DNA)/oligonucleotide biosensors or immunoassays (antibody biosensors). Specifically, antibodies can be utilized for direct or indirect detection of pathogens or their epitopes in biosensing technology, which makes it preferable for the detection process. On the other hand, DNA-based biosensors can be exploited in situations such as some infections that do not generate a significant level of antibody production or availability of limited antibodies. However, DNA-based biosensors need to be employed if the pathogen is present or has been recently present. The biorecognition elements are not limited to DNA or antibody that can be extended to aptamers and imprinted polymers, which enable the detection of target toxins, nucleic acids, viruses, cells, and oocytes.
Transducing element is one of the other components of the biosensor system vital for conversion of biochemical reactions to electronic signals successfully. Although there are different kinds of transducers sch as mechanical including cantilever biosensors or Abbreviations: 3D, three dimensional; AFM, atomic force microscopy; AEGIS, artificially expanded genetic information system; DNA, deoxyribonucleic acid; HTS, High-throughput sequencing; PCR, polymerase chain reaction; ssDNA, single-stranded DNA.
optical biosensors including surface plasmon resonance (SPR) biosensors, ECBs are mostly utilized for pathogen detection. 25,26 The transducer that is referred to as an electrode is mostly composed of conducting and semiconducting materials. Thus, the biochemical energy is generated due to the binding of target pathogens to the biorecognition element that is immobilized on the transducer/electrode, which is then converted into electrical energy by the electrochemical method consisting of an electrode and an electrolyte/sample solution. Because ECBs can be modulated depending on the target and infrastructure, the researchers have developed distinct sensor setups, such as no sample pre-preparation in the detection of pathogens, in situ detection of pathogens on various surfaces, utilization of low-cost setup and platforms for rapid pathogen detection, and multiple detection of pathogen in contaminated samples. Hence, ECB technology has enabled making scientific research on pathogen detection in food, water, air, and organic samples for environmental monitoring and both pathogen and pathogenic toxin detection for the threat of bioterrorism.
In addition, development technology in miniaturization promoted by micro and nanotechnology advancements is important and necessary for increment in portable biosensors for pathogen detection.

Importance of point-of-care diagnostic testing
It is a fact that the detection of an analyte on-site is crucial for the initiation of the right treatment. Therefore, pointof-care or on-site detection capable devices are important to make progress effectively. Because most of the conventional analysis tools and methods are conducted in a laboratory that is demanding and requires experienced personnel, it is essential to establish a more convenient but effective alternative solution. Point-of-care technology emerges as simple, user-friendly, cost-effective, affordable, and requiring no trained staff to perform analyses in contrast with conventional methods, whereas it provides real-time diagnostics directly to the patient. Hence, it enables diagnostics procedures more effective in terms of reduced cost analysis, saving time, and affordable worldwide even in developing countries with limited sources and nonprofessional users.
Point-of-care diagnostics tests are mainly distinguished as lateral flow assay, microfluidic systems, and plasmonic technologies, including SPR, localized SPR, surface-enhanced Raman scattering, and paper-based technologies. Although plasmonic technologies stand out as label free and real time monitoring, microfluidic technologies stand out as working with a very small amount of volume and portability. On the other hand, lateral flow assay and paper-based assay stand out as easy-to-use, low cost (especially fabrication process), rapid result, and disposable nature. Nevertheless, there are different kinds of technology utilization for pathogen detection in point-of-care diagnostics, such as fluorescence biosensors, colorimetric biosensors, chemiluminescence biosensors, magnetic biosensors, and ECB integrated with lab-on-a-disc devices, lab-on-a-chip devices, miniaturized polymerase chain reaction (PCR) devices, and isothermal nucleic acid amplification devices.

Aptamer concept and advantages over alternatives in biosensing technology
Aptamer term refers to single-stranded ribonucleic acid (RNA), DNA, and synthetic DNA or peptide-based molecules, which show a strong affinity to a specific target molecule by folding into tertiary structures. Aptamers are produced by in vitro molecular evolution method technique called systematic evolution of ligands by exponential enrichment (SELEX) and its variations as immunoprecipitation-coupled SELEX, capture-SELEX, cell-SELEX, capillary electrophoresis-SELEX, atomic force microscopy-SELEX, and artificially expanded genetic information system-SELEX, which are developed and modified not only for DNA but also for RNA to improve SELEX procedure in terms of immobilization matrix, changing of binding conditions, and library design. The method consists of incubating the target of interest with a pool of ∼10 16 single-stranded random oligonucleotides, comparison between the methods is illustrated in Table 2. After the binding of oligonucleotides to the target, the unbound one is separated from the environment. The bound DNA oligonucleotides are eluted and amplified by using a PCR. Processing of several rounds of selection reveals DNA or RNA sequences, which affinity and specification are enriched in the pool and sequenced. This procedure can be executed against a variety of targets, such as ligands, proteins, nanoparticles, ions, whole cells, and tissues. RNA SELEX procedure differs from DNA SELEX in terms of the requirement of protection of RNA from RNAses activities, amplification of RNA sequence by using T7 RNA polymerase enzyme, and conduction of reverse transcription reaction step before PCR.
Aptamers are suitable candidates for affinity-based purification methods and biosensing technology for replacing antibodies, which are utilized for wide range of areas such as food and water quality monitoring, clinical diagnostic agents, and therapeutic agents. Even though aptamers show similarity to antibodies in terms of recognizing and binding an analyte, they demonstrate some important superiorities, such as shorter production time, low cost of manufacturing, higher physical, chemical, and thermal stability, longer shelf life, no batchto-batch variability, and much higher target potential ranging from ions, nanoparticles to cells and tissues, which is compared in Table 3. Aptamers also demonstrate a variety of secondary structures, such as hairpin stem, hairpin loop, internal loop, bulge, duplex, multibranched loop, and G-quadruplex. 27 Aptamers are utilized wide range of areas due to their high selectivity, high specification, low-cost production, and capability of capture nearly any molecule or more complex structures. Manipulation and generation of aptamers for rapid and reliable cancer diagnosis and prognosis were introduced. There were some studies were conducted in order to detect a number of cancer-related biomarkers in diagnostics purposes such as MUC1 (mucin 1), 28 HER2 (human epidermal growth factor receptor 2), 29,30 estrogen receptor 31 that are multiple tumor related proteins or MCF-7 cells of breast cancer, 32 CCRF-CEM cells of leukemia 33 and tumor related soluble biomarkers such as carcinoembryonic antigen, 34 prostate-specific antigen, 35 which were successfully developed. Some other studies for exploiting aptamers is environmental monitoring of contaminations were conducted, such as chloramphenicol 36,37 and tetracycline 38,39 as pharmaceutic applications; endotoxins, 40,41 bisphenol A, 42,43 and ochratoxin A 44,45 as microorganism based toxins; mercury, 46 arsenic, 47 and copper 48 as heavy metal contamination; acetamiprid, 49 atrazine, 50 and carbendazim 51 as pesticides, herbicides, and insecticides. The concerts of concerns directed focuses and efforts to develop rapid and reliable biosensing systems as early and real-time detection platform. Therefore, biosensor systems were developed for pathogenic detection in order to prevent endemic and pandemic outbreaks alongside of aforementioned pollutants and toxins. Figure 2 shows the molecule-based aptamers and main aptamer targets.
Even though aptamers are stable, durable, and show high affinity against their targets, they have some drawbacks in the utilization of biosensor systems as biorecognition elements. For instance, aptamers may lose their three dimensional structures when they are immobilized on sensor systems. On the other hand, the effectivity of aptamers is associated with the performance of transducing elements, which requires superior conductivity and high area to volume ratio increases the overall cost, ultimately. 52 Therefore, researchers focused on development of sensitive, selective, and rapid electrochemical aptamer-based biosensor systems due to these features and have potential to be miniaturized, high durability, low cost, and rapid response. 53 On the other hand, since DNA-based aptamers have some advantages over RNAbased aptamers in terms of physical-chemical stability and durability against biodegradation, most of the studies were conducted on DNA-based ones. 54 In addition, aptamers have been exposed to chemical modifications associated with developing technology to increase overall stability and biological signal transfer quality, which are shown as examples of various chemical modifications in Figure 3.
The specific design and surface immobilization of aptamers are critical considerations in the development of aptasensors. Surface immobilization of aptamer provides adsorption on the electrode surface, whereas it dramatically reduces the background noise of the system. Adsorption of thiol tagged aptamer on the gold electrode surface is considered mostly utilized surface immobilization technique among physical and chemical conjugation methods 55 even though there are other immobilization methods such as the utilization of -COOH and -NH2 bonds to construct carbodiimide link 56 or utilization of biotin streptavidin affinity to create biotin-streptavidin complex between aptamers and electrode surfaces. 57 ECBs provide analytical information about the measurements via transducing biological signals resulting from the hybridization of aptamers with their target analyte in various forms, such as sandwich complex, TA B L E 3 A detailed comparison between aptamers and antibodies. [126][127][128]  Easy to lose functionality after a denaturation

Chemical modifications
Easy to modify with sugar ring, base, and functional groups from bot 5′ to 3′ ends

Ethical concern
Chemistry based production, no ethical concern Living organism-based production, ethical challenges present conformational change, and rearrangement. of the target molecule ( Figure 4).

ELECTROCHEMICAL APTASENSORS FOR PATHOGENIC POINT-OF-CARE DIAGNOSTICS
The convergence of aptamers with ECB opened a new path for the development of innovative, sensitive, rapid, and effective pathogen detection systems in environmental samples. The majority of aptamer-based biosensor comprises foodborne pathogens, especially Salmonella and E. coli, even though there are different kinds of pathogens rather than foodborne ones. Besides, there were aptamer-based biosensor studies based on detection of viruses, such as norovirus, 58 Salmonella is a rod-shaped, gram-negative, nonspore forming, motile, and one of the most known foodborne pathogen bacterium that is member of Enterobacteriaceae. 73 The bacteria is one of the major worldwide concerns intensified by nearly 95 million foodborne illness and 160,000 deaths per year. 74 Muniandy et al. developed reduced graphene oxide (rGO)-azophloxine-based nanocomposite aptasensor for detection of the foodborne pathogens that are Salmonella enterica and Salmonella typhimurium (ST). 75 Utilization of rGO was selected due to distinct advantages, such as superior conductivity, ultrahigh electron transfer ability, large surface volume ratio, and biocompatibility. They conducted analysis by monitoring differential pulse voltammetry (DPV), which demonstrated high selectivity and sensitivity against whole-cell of the target bacteria. The exhibition of linear range showed detection from 10 8 to 10 1 CFU/mL as R 2 = 0.98 with a detection limit of 10 1 CFU/mL. In addition, the developed aptasensor was also subjected to the detection of other foodborne pathogens that are E. coli, Shigella dysenteriae, Vibrio cholerae, and Klebsiella pneumonia, which resulted generation of redox current because of the presence of electroactive dye AP and conductivity of bacterial cell membranes below the detection limit of the biosensor. Figure 5 shows the result of the study. The sensitivity of the aptasensor was investigated for specific bacterial detection by using DPV in Zobell's solution in Figure 4A. DPV is a powerful electrochemical technique, which utilizes superimposition of small amplitudes and short pulses in a linear ramp. 76 Current is obtained before the implementation of the pulse and the difference between each pulse is calculated at the end of each pulse. Single-stranded DNA (ssDNA)-immobilized and rGOand titanium dioxide-modified glassy carbon electrodes were exposed to different concentrations of ST cells and DPV measurements were taken. The expectation was fulfilled by generation peak currents regarding target bacterial concentration, which significantly reduced when bacterial concentration was increasing from 10 1 to 10 8 CFU. The same group developed rGO and titanium dioxide nanocomposite electrochemical aptasensor for the detection of S. enterica serovar Typhimurium. 77 The label-free anti-Salmonella aptamer was immobilized on a composite matrix by electrostatic interactions. The main principle of the biosensor was the binding of aptamer to bacterial cells resulting in both generation of a physical barrier and inhibiting the electron transfer to the surface of the electrode. The DPV results demonstrated that the detection range of the biosensor was from 10 8 to 10 1 with the detection limit of 10 1 CFU/mL. The sensor system also showed great durability up to 21 days with a 10% decrease in signal after 30 days without use. In other studies, Li et al. reported aptasensor for direct detection of ST foodborne pathogens in a solution and a milk sample, which was a cloth-based super sandwich electrochemical aptasensor. 78 The electrodes were developed by carbon ink-based and wax-based screen-printing in order to generate clot-based electrodes and to create hydrophilic and hydrophobic regions for the sensing device. Methylene blue (MB) was employed as redox indicator and was inserted into two ssDNA complexes called DNA super sandwich in order to enhance the sensitivity of the sensor by amplifying the current signal. The working principle of the sensor system was binding of target-aptamers complex to the capture probe and DNA super sandwich and electron transfer from MB to electrode surface. The results from DPV showed that the linearity was found between 10 2 and 10 8 CFU/mL with a limit of detection of 16 CFU/mL. Appaturi et al. developed an aptasensor by utilizing rGO-carbon nanotube for the detection of S. enterica not only in culture method but also in real food samples. 79 As ultra-conductivity and superior area to volume ratio of carbon nanotubes were known facts, they employed it by using the hydrothermal method for building label-free ECB. The nanocomposite material was constructed on the glassy carbon electrode and then amino-modified DNA aptamer was immobilized on the surface. DPV measurements showed that the combination of rGO and carbon nanotube was stable and could detect ST in a linear range between 10 8 and 10 1 CFU/mL with a limit of detection of 10 1 CFU/mL. The reproducibility of the biosensor was stable after storage of 20 days in ultrapure water at 4 • C. Ranjbar et al. developed an electrochemical aptasensor for the detection of ST not only in solution but also in contaminated egg. 80 They utilized nanoporous gold structure that was electrochemically synthesized using gold-copper alloy at the surface of gold-glassy carbon electrode. The thiolated anti-Salmonella aptamers were immobilized on the surface of the electrode by exploiting self-assemble monolayer formations. The measurement results indicated that the wide linear dynamic range of the aptasensor was present from 6.5 × 10 2 to 6.5 × 10 8 CFU/mL with a limit of quantification of 6.5 × 10 1 CFU/mL and limit of detection of 1 CFU/mL. Dinshaw et al. reported an aptasensor guided for capturing of S. enterica serotype Typhimurium via utilizing electrochemically rGO-chitosan complex-composite system. 81 Outer membrane of the bacteria-specific aptamers was functionalized with a thiol group and immobilized on the composite sensor surface by using glutaraldehyde as the cross-linker. Cyclic voltammetry (CV) and DPV measurements demonstrated that the biosensor had a limit of detection of 10 1 CFU/mL, which was consistent even in artificially spiked raw chicken samples, whereas there was no significant voltage change in biosensor when it was worked against S. aureus, K. pneumonia, and E. coli bacteria.
E. coli is a gram-negative, rod-shaped, facultative anaerobic, and coliform bacteria that is a member of Enterobacteriaceae. 82 Although most of the strains of the bacteria live in the intestines without harm, various subtypes of the microorganism can cause diarrhea or extraintestinal diseases not only in immunocompromised individuals, especially elders but also in healthy individuals. 83 Kaur et al. reported a labelfree impedimetric aptasensor for the detection of E. coli O157:H7 by developing boron-carbon nanorods decorated by nickel nanoparticles nanostructured platform. 84 The nanorods were specifically designed to enhance electroactivity and sensitivity of the sensor system. DNA aptamer of anti-E. coli O157:H7 was selected via employing microtiter plate-based cell-SELEX technique. CV analysis demonstrated that the developed aptasensor successfully detected O157:H7 serotype as 10 CFU with a response range from 10 0 to 10 5 CFU in water, juice, and fecal samples. Wang et al. developed another impedimetric aptasensor for the detection of E. coli O157:H7 via exploiting coaxial capillary with immune magnetic nanoparticles (MNPs) to specifically separate the target bacteria in the sample. 85 Although the urease with urea was utilized for amplification of the impedance signals, the printed circuit board (PCB) gold electrode was used for analyzing the change in impedance. MNPs modified with streptavidin and conjugated with biotinylated polyclonal antibodies (PAbs) were formed the immune MNPs, which were then captured in the coaxial capillary in the presence of high gradient magnetic fields in order to separate the bacteria from the large volume of sample. The gold nanoparticles (GNPs) were modified with anti-E. coli and anti-urease aptamers and injected into the capillary to be reacted with the bacteria and form the MNP-PAb-bacteria-aptamer-GNPs-urease complexes. The complex was used to change the impedance via exploiting urease to catalyze the hydrolysis of urea into ammonium ions and carbonate ions in the capillary resulting in a decrease in the impedance of the catalyst that was F I G U R E 6 Main concept of working mechanism of aptasensor 85 : Magnetic nanoparticles (MNPs) are coated with streptavidin and then injected into the coaxial capillary system, which is filled with the line-up high-gradient magnetic fields to be confined uniformly. Thereafter, the biotinylated polyclonal antibodies (PAbs) are incubating injected to the capillary system to generate the immune MNPs by streptavidin-biotin binding interaction. In the following, the bacteria containing sample is transferred to the capillary, which are captured by MNPs generating the MNP-PAb-bacteria complexes, whereas the other impurities and components removed from the capillary system by flowing through. Anti-Escherichia coli aptamers and urease bound gold nanoparticles (GNPs) are injected into the capillary to enable creating the complex of the MNP-PAb-bacteria-aptamer-GNP-urease. In the end, urea is injected to the capillary to be filled and resulting hydrolyzing into ammonium ions and carbonate ions because of the catalysis of the urease on the final complex. It causes a decrease in the impedance of the catalyst that is measured and observed by the gold plating printed circuit board (PCB) electrode in order to detect the concentration of the bacteria in the sample. measured by the gold plating PCB electrode ( Figure 6). The detection limit of the aptasensor was 10 1 CFU/mL in a large volume of the sample as 10 mL for 3 h of measurement, whereas the mean recovery of the bacteria in the contaminated pasteurized milk was about 99%.
Another study on the detection of E. coli O157:H7 with aptasensor systems in solution and milk samples was reported by Bai et al. in 2020. 86 In this study, the researchers developed a cocoon-like DNA aptamer nanostructure as signal tags for the detection of the bacteria. Cocoon-like structures were synthesized by utilizing a rolling circle amplification reaction, which was loaded with hemin molecules as electrochemical signal tags to amplify the output signals. Biorecognition element immobilization was executed by immobilization of a DNA capture probe that was modified with a thiol group on a gold electrode and complementation of E. coli O157:H7 specific aptamer forming double-stranded DNA structures with the capture probe on the Au electrode. The binding reaction of aptamer with the bacteria caused the dissociation of some aptamer-capture probes, which resulted in releasing of capture probes and hybridization with DNA nanostructures through the complementary DNA (cDNA) sequence. DPV analysis demonstrated that the aptasensor had a signal proportional to the logarithm of the bacterial concentration between 10 8 and 10 1 CFU/mL with a detection limit of 10 1 CFU/mL. Another aptamer-based ECB system for E. coli O157:H7 detection was developed by Housaindokht et al. in 2018. 87 Single-wall carbon nanotube modification was applied on screen-printed electrodes to increase sensitivity of the sensor, and MB was used as the electrochemical probe. The results demonstrated that the sensor had a linear range from 1.1 × 10 7 CFU/mL to 1.7 × 10 1 with a limit of detection 1.7 × 10 1 CFU mL of concentration of E. coli O157:H7, whereas there was no significant voltage generation against S. aureus, Bacillus subtilis, Pseudomonas aeruginosa, E. coli O167, and ST concentration of 10 7 CFU/mL.
Vibrio parahaemolyticus (VP) is a gram-negative, curved, and rod-shaped bacterium that is a member of the Vibrionaceae family and it is mostly found in the sea and backwaters. 88 The bacteria is one of the main seafood borne pathogens, especially in Asian countries and can cause development of acute gastroenteritis that shows symptoms, such as diarrhea, headache, vomiting, nausea, and abdominal cramps. 89 Hu et al. developed a double stirring bars-based signal-amplified system for simultaneous electrochemical detection of VP and ST. They utilized MB and ferrocene labeled functionalized branched-chain DNA hybrid structure that was immobilized on magnetic microspheres to be used as encoded signal tags. 90 The tags were hybridized with the aptamer-embedded tetrahedral DNA nanostructures undertaking the assignment of capture probes on one of the gold stirring bars, which could capture both of the bacteria and the complex was released into the solution. Exonuclease immobilized on another gold stirring bar was digested and the complex resulted in improvement in magnetic tags for signal due to releasing of free bacteria from the former bar to the later bar. The tags were magnetically enriched on a screen-printed carbon electrode after a cycle of reaction that two distinct voltammetric peaks were obtained from MB and ferrocene. The double stirring bars-based signal amplification systems demonstrated high sensitivity and detection limit as low as 4 CFU/mL for VP and 7 CFU/mL for ST, whereas the detection range was detected between 10 8 and 10 1 CFU/mL. Figure 7 shows the results of the study. Square wave voltammetry (SWV) is one of the most sensitive voltammetric technique that is considered special type of DPV. 91 SWV utilizes combination of square wave and staircase potential to apply working electrode, which considered a large-amplitude differential technique. In Figure 7A, the aptamer-embedded TDNs containing probe demonstrated stronger electrochemical signal compared to single-stranded aptamer-based probe in the case of presence of the bacteria. In (a), there was no electrochemical signal since the target bacteria was absent. However, in (b) and (c) the electrochemical signal increased significantly because of addition of each target bacteria. TDNs could maintain their lateral spacing that resulted generation of space to provide interaction with the target bacteria, which concluded higher electrochemical signal (c). In Figure 7B, the feasibility of the double stirring bars-assisted signal amplification method was subjugated to test. Therefore, there was no observable electrochemical response as a signal peak (curve a) when there are no target pathogens. On the other hand, two current peaks were observed that refer to two target bacteria (VP and ST) using Fe 3 O 4 @Au@ssDNA as signal tags (curve b), and also the current signal peaks were remarkably increased when Fe 3 O 4 @Au@FBCHS was used as the signal tags (curve c). That was because of higher amount of MB or Fc's in Fe 3 O 4 @Au@FBCHS(II), because it could contain electroactive indicator from inside to outside in the hyper-branched DNA structure of the magnetic beads compared to Fe 3 O 4 @Au@ssDNA(I). Hu et al. reported another electrochemical aptasensor for the detection of VP for point-of-care testing in aquaculture water via utilizing antimicrobial peptide-labeled nano metal-organic framework (NMOF) signal tag. 92 ssDNA labeled MB was hybridized with aptamers on the electrode surface to build an electroactive DNA probe. On the other hand, NMOF modified antimicrobial peptide and ferrocene (NMOF@AMP-Fc) were prepared as the signal tag at the same time. Introduction of the bacteria to the aptasensor by incubating caused conjugation with the aptamers and releasing the ssDNA-MB into the supernatant, which then led to conjugation of (NMOF@AMP-Fc) signal tag with the captured bacteria to form a sandwich complex and resulting in the generation of an electrical signal from Fc. Analytical measurements demonstrated that the current signal of ssDNA-MB decreased, whereas the Fc current signal increased according to increase in the concentration of the bacteria as having a limit of detection of 4 CFU/mL. Jiang et al. developed an aptasensor for the detection of VP by utilizing a thread-based microfluidic system. 93 In the study, both microfluidic channels and electrodes for the sensor were fabricated on threads, which were composed of molybdenum disulfide nanosheets to increase the sensitivity of the electrochemical measurement. Molybdenum disulfide and polylysine conjugated aptamers were generated by exploiting electrostatic attraction, and both CV and DPV were used to analyze the change in voltage, which was shown a dynamic detection range from 10 6 to 10 1 with a limit of detection of 5.74 CFU/mL in 30 min.
S. aureus is a gram-positive, round-shaped, and facultative aerobe bacteria that is a member of Firmicutes and it is considered one of the microbiota members of the human body since it can be found mainly in the upper respiratory tract and on the skin. 94 Even though it is a commensal bacteria in the human body, it can turn into an opportunistic pathogen by causing skin infections such as abscesses or respiratory infections such as sinusitis and even food poisoning. 95 Ranjbar et al. developed an electrochemical aptasensor consisting of GNPs/carbon nanoparticles/cellulose nanofibers nanocomposite structures on the surface of the glassy carbon electrode for selective detection of S. aureus in human blood serum. 96 S. aureus-specific and thiolated aptamer was immobilized on nanocomposite electrode layer that was characterized with field emission scanning electron microscopy, energydispersive spectroscopy, dynamic light scattering, and ultraviolet (UV)-visible spectrophotometry techniques by self-assembly monolayer method. Impedimetric analyses showed that the aptasensor had a linear range from 1.2 × 10 8 to 1.2 × 10 1 CFU/mL with a limit of detection of 1 CFU/mL, which demonstrated both precise and accurate detection systems for real human samples. Kurt et al. reported an aptasensor for multiplex food pathogen detection by employing nanoparticles and quantum dots, which served as anti-Stokes and Stokes type excitation profiles. 97 The dual excitation system was based on preventing or minimizing cross-talk between the luminescent signals for the multiplexed detection of pathogens, while exploiting the down-conversion characteristic of carboxylic acid functionalized quantum dots and the upconversion characteristic of thulium-doped nanoparticles. They conjugated the aptamers specifically designed for S. aureus and ST with luminescent nanoparticles with the magnetic beads, which had a short cDNA sequence to the aptamer sequence to separate analyte-free conjugates for fluorescent measurements. Luminescent con-jugated aptamers were employed for model pathogens under UV excitation emission spectra obtained at 325 nm for quantum dots and near-infrared spectroscopy excitation at 980 nm for up-converting nanoparticles. The results showed that the limit of detection was 16 CFU/mL for S. aureus and 28 CFU/mL for ST. Sohouli et al. reported an innovative aptasensor construction by converging nitrogen-doped carbon nano-onions, GNPs, and thiol-terminated screen-printed carbon electrodes, which were functionalized and activated by covalent binding of anti-S. aureus aptamers. 98 The electrochemical impedance spectroscopy results demonstrated that the aptasensor had a linear range between 10 1 and 10 8 CFU/mL with a limit of detection of 3 CFU/mL within 15 min, whereas the measurements were obtained in both solution and human blood serum. There was a slight decrease in the R ct value when the aptasensor was subjected to E. coli, Shigella flexneri, and Salmon bacteria, whereas it was dramatically increased when it was subjected to S. aureus, which showed the high selectivity of the biosensor. Jia et al. developed an impedimetric aptasensor for S. aureus, which was equipped with rGO and GNPs containing nanocomposite material. 99 ssDNA as conjugated with rGO was on the glassy carbon electrode surface, and GNPs were on the rGO-and ssDNA-containing surfaces, whereas thiolated anti-S. aureus was covalently bound on the GNPs. The analytical applications were conducted on fresh fish tissue and contaminated water samples. The electrochemical impedance spectroscopy results showed that there was a linear range from 10 6 to 10 1 CFU/mL with a detection limit of 10 1 CFU/mL, which was completed within an hour. Reich et al. reported an impedimetric aptasensor for detection of S. aureus. 100 Thiol-modified aptamers were selected against the protein A domain of the bacteria, which was a surface bound virulence factor on a 6-mercapto-1-hexanol coated gold electrode. The working principle of the aptasensor was based on the inhibition of electron transfer between ferri-/ferrocyanide in solution and electrode due to the conjugation of aptamers to the target bacteria increasing in impedance, the main concept of the study was illustrated in Figure 8. Analytical measurements exhibited that the aptasensor had a limit of detection of 10 1 CFU/mL that was obtained within 15 min, whereas there was no change in impedance when the aptasensor was subjected to E. coli and Staphylococcus epidermidis showing high selectivity.
Peng et al. developed an aptasensor for Cronobacter sakazakii, 101 which was an opportunistic foodborne pathogen causing life-threatening infections with a high mortality rate up to 80% in children and elder people 102,103 and was reported that 94% of survivors of Cronobacter infections later suffered from not only neurological and developmental disorders but also damages on the organs. 104 In the study, graphene oxide was utilized via conjugated with MB as the redox mediator on the gold electrode surface. The working principle of the sensor was simple and based on either binding of C. sakazakii with its anti-aptamer causing hindrance of electrostatic adsorption of graphene oxide and MB that results in a decrease in the electrochemical signal of MB or the absence of the bacteria caused the binding of aptamer with graphene oxide by π-π interaction resulting in adsorption of MB through electrostatic adherence and increase in the electrochemical signal. The results exhibited that the aptasensor had linear range between 2 × 10 6 and 2 × 10 1 CFU/mL with a limit of detection of 7 CFU/mL. Zarei et al. developed a GNP modified impedimetric aptasensor for the detection of S. dysenteriae. 105 The GNPs were covered on a glassy carbon electrode by electrodeposition and then thiolated anti-S. dysenteriae aptamer was immobilized on gold surface via utilization of selfassembling method. The detection measurements were conducted by performing charge transfer resistance in both the presence and absence of the bacteria via exploiting hexacyanoferrate as an electrochemical probe, which binding of aptamer with outer-membrane proteins of S. dysenteriae caused increase in the charge transfer resistance. The distinct property of the biosensor was differentiating of alive S. dysenteriae from both dead S. dysenteriae and other pathogens in unpasteurized and pasteurized bacteria contaminated milk and water samples. The results showed that the linear range was formed between 10 6 and 10 1 CFU/mL with a limit of detection of 10 0 CFU/mL.
Pathogenic bacteria are one of the most concerned environmental pollutants and one of the leading causes of death in worldwide. Although advancing technology greatly contributes to the diagnosis and treatment of these bacteria, there is still an urgent need to develop an effective and rapid biosensor system for environmental monitoring and point-of-care diagnostics, which is a crucial step for identifying the target pathogen. Figure 9 shows the number of studies on pathogenic bacteria that were introduced in this review.

CONCLUSIONS
It is a known fact that advancement of biosensor technology associated with the development of nanotechnology and material science has been increasing year by year, significantly. 106 Development of new materials as transducers that is going to increase the conductivity along with complete biocompatibility and area-to-volume ratio, improving immobilization methods of biorecognition elements on transducer and progress of miniaturization of electrochemical aptasensors for not only pathogen detection but also other target analytes. Although the establishment of fabrication and modification technology for biosensors is going to be decreasing cost and ultimately labor, publishing of newly developed biosensor systems shall contribute to literature, which shall be enabling commercialization. On the other hand, improvements in aptamer screening and cloning methods shall reduce the time of finding target aptamer, whereas advancements in silico methods shall accelerate the prediction of secondary structures and final stance of aptamers. 107 It is a known fact that aptamers have wide range of area not only for biosensing technology. For instance, the aptamers are one of the not well-known components of qPCR mixture used for enhancement of DNA and primer binding during the annealing reaction. Aptamers are one of the most promising molecules in personalized medicine as some DNA-based aptamers were shown to some critical protein targeting to rehabilitate the diseases, such as nucleolin for providing healthy cell proliferation, tenascin-C for embryogenesis and oncogenesis, and Immunoglobulin heavy mu chain for inhibiting Burkitt's lymphoma development. 108 Therefore, advancements in molecular biology technology, improvement in the perspective of the utilization of a molecule in an unprojected ways shall open a new pathway for aptamers.

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare no conflict of interest.