Aptamer-Based Biosensors for Antibiotic Detection: A Review

Antibiotic resistance and, accordingly, their pollution because of uncontrolled usage has emerged as a serious problem in recent years. Hence, there is an increased demand to develop robust, easy, and sensitive methods for rapid evaluation of antibiotics and their residues. Among different analytical methods, the aptamer-based biosensors (aptasensors) have attracted considerable attention because of good selectivity, specificity, and sensitivity. This review gives an overview about recently-developed aptasensors for antibiotic detection. The use of various aptamer assays to determine different groups of antibiotics, like β-lactams, aminoglycosides, anthracyclines, chloramphenicol, (fluoro)quinolones, lincosamide, tetracyclines, and sulfonamides are presented in this paper.


Introduction
The increase of antibiotic-resistant germs is an acute challenge for consumer health protection and veterinary medicine. Inappropriate and prophylactic use of antibiotics (especially in the field of animal care) is common and associated with contamination of the environment with antibiotics and their metabolites. On one hand, this favors the development of antibiotic resistances of bacteria, while, on the other hand, this harms the environment, e.g., by uncontrolled disturbance of the ground flora [1][2][3]. To minimize the resistance towards antibiotics the use and the release of them into the environment must be first detected and, thereupon, can be limited [4].
In their original sense antibiotics are naturally-built low molecular weight metabolites of bacteria or fungi, which either kill or slow the growth of other microorganisms [5]. In the widest sense, partial synthetic derivates and chemically-synthesized compounds with antimicrobial effect belong to antibiotics. They are generally used for treating infections in modern healthcare [5]. According to their chemical structure and the resulting mode of action, antibiotics can be classified into different groups inter alia ß-lactams, aminoglycosides, anthracyclines, (fluoro)quinolones, tetracyclines, lincosamide, and sulfonamides.
Currently, residue levels of antibiotics in aqueous samples are mainly detected by high-performance liquid chromatography (HPLC) [6][7][8][9], gas chromatography-mass spectrometry (GC-MS) [10], and liquid chromatography-tandem mass spectrometry (LC-MS/MS) [11][12][13][14]. Despite their wide range of applications, these methods are usually time-consuming, require laborious pretreatment of samples, sophisticated instrumentation, and trained technical personnel. The use of biosensors circumvent these problems and could ensure fast on-site analysis. Biosensors are analytical  More methods for antibiotic detection based on the mentioned basic principles are described in detail in the appropriate sections of the paper. Additionally, further detailed information about the operating modes for aptamer-based biosensors can be read inter alia in the reported review papers [50].
This systematic and comprehensive review discusses the application of aptamers in the detection of different antibiotic groups. In this section, eight different groups of antibiotics, and the designed aptasensors for their detection, are discussed.

Aptasensors for Different Antibiotic Classes
The various currently-developed aptamer-based biosensors for antibiotic detection mentioned in the literature are ordered by their antibiotic class and discussed below.
To compare the performance of aptasensors the following parameters or characteristics are important.
Affinity is a measure of the tendency of molecules to bind to other molecules [51]. The higher the affinity, the greater the association constant K A . More common is the reciprocal value, the dissociation constant K D . The higher the affinity of a target to its ligand, the lower the K D of the complex, thus, low K D values are preferred.
Selectivity is the property to select multiple objects from a set of objects, while specificity is the property to select one object from a set of objects [52]. Thus, an analytical method is selective when different components of a mixture can be determined side by side and without interference. The method is specific when only one component of the mixture can be determined. Specificity tests are usually carried out by target detection in the simultaneous presence of the target and structurally similar substances. High specificity and selectivity is preferred.
The limit of detection (LOD) is the lowest quantity of a substance that can be distinguished from the absence of that substance (a blank value) within a stated confidence limit [53]. The maximum residue levels (MRL) is the highest concentration of an undesirable substance (impurity or pollutant), that is legally permitted in a food or commodity [54], defined by the European Union, e.g., in Council Directive 96/23/EC [54] for antibiotic residues in live animals and animal products. The aim in the development of a biosensor is to achieve a low sensitivity, such that the LOD is smaller than the MRL.
The reproducibility is the repeatability of scientific research results [55]. The recovery is determined by a standard addition method. Defined target concentrations are added to real samples and the recovery is detected. Furthermore, the results are compared to results with an alternative method, the enzyme-linked immunosorbent assay (ELISA).
The applicability of the proposed aptasensor for real-sample analysis is verified by the detection of the target in real samples, such as milk, honey, serum, water, and others.
The stability is the ability of the sensor to maintain its performance under the prevailing conditions for a certain period of time. It is tested by storing the sensor at defined conditions up to several weeks, comparing the analytical performance before and after storage.

β-Lactams
Due to their high efficacy, low toxicity, and the possibility to derivatize them by means of chemical and enzymatic methods, β-lactam antibiotics are considered to be the most important antibiotics in terms of quantity and value [56]. Their mechanism of action is based on the prevention of the formation of peptide cross-linking in the bacterial cell wall (murein) [5]. Therefore, they act specifically on prokaryotes with a mureous cell wall. They are characterized by their representative β-lactam ring (marked in red in Figure 2). The most important data of the discussed aptasensors, for the β-lactams, include the aptamer sequence, dissociation constant (K D ), limit of detection (LOD), real sample analysis (RSA), and applied sensor type, are summyarized in Tables 1 and 2

Ampicillin
The occurrence of penicillin-resistant strains has stimulated the search for new antibiotics, from which semi-synthetic penicillin, such as ampicillin, have been found [57]. Ampicillin (Figure 2a) is a widely used broad spectrum antibiotic in veterinary medicine for the treatment and prevention of primary respiratory, gastrointestinal, urogenital, and skin bacterial infections in food-producing animal and it has a low human toxicity [57].
Song et al. [58] reported the first aptasensor for ampicillin using AuNP-based dual fluorescence colorimetric methods. Ampicillin-specific aptamers were selected by magnetic bead-based SELEX, which was further used in other studies [59]. Another fluorescent aptasensor for ampicillin detection was reported by Luo et al. [60] using magnetic bead composites coated with AuNPs and a nicking enzyme, which showed more sensitive responses with the limit of detection of 0.07 ng mL −1 compared to the first proposed aptasensor by Song et al. Dapra et al. [59] designed an all-polymer impedimetric electrochemical microfluidic biosensor for the detection of ampicillin and kanamycin A. Cyclic olefin copolymer (Topas ® ) was used as a substrate on the top and bottom of the constructed chip. Due to their biocompatibility a conductive polymer bilayer consisting of tosylate-doped poly(3,4-ethylenedioxythiophene) (PEDOT) and the hydroxymethyl derivate was used as the electrode material. In a similar system Rosati et al. [61] optimized the geometry of the electrodes and the microchannels (e.g., thickness, width) and, therefore, the performance of the designed impedimetric aptasensor. Additionally, the ampicillinspecific aptamer was equipped with an appended poly(T)-poly(C) sequence which allowed a direct immobilization on the electrodes when UV irradiation was applied [62].
In order to enhance the aptasensor response to ampicillin and signal amplification, two electrochemical aptasensors were developed with the help of polymerase and nicking endonuclease [63], which was used for the first time as the target-aptamer binding triggered quadratic recycling amplification for electrochemical detection of antibiotics, and target-initiated T7 exonuclease in a homogeneous electrochemical sensing system [64].
There are many reports on electrochemical-based aptamer sensing assays for the detection of ampicillin [63][64][65][66][67][68], which are summarized in Table 1. Among all existing electrochemical sensor assays, the proposed aptasensor based on DNA-functionalized AuNPs and ssDNA binding protein (SSB) as the electrochemical signal inhibition reagent showed higher sensitivity with a detection limit of 0.38 pM [65].

Ampicillin
The occurrence of penicillin-resistant strains has stimulated the search for new antibiotics, from which semi-synthetic penicillin, such as ampicillin, have been found [57]. Ampicillin (Figure 2a) is a widely used broad spectrum antibiotic in veterinary medicine for the treatment and prevention of primary respiratory, gastrointestinal, urogenital, and skin bacterial infections in food-producing animal and it has a low human toxicity [57].
Song et al. [58] reported the first aptasensor for ampicillin using AuNP-based dual fluorescence colorimetric methods. Ampicillin-specific aptamers were selected by magnetic bead-based SELEX, which was further used in other studies [59]. Another fluorescent aptasensor for ampicillin detection was reported by Luo et al. [60] using magnetic bead composites coated with AuNPs and a nicking enzyme, which showed more sensitive responses with the limit of detection of 0.07 ng mL −1 compared to the first proposed aptasensor by Song et al.
Dapra et al. [59] designed an all-polymer impedimetric electrochemical microfluidic biosensor for the detection of ampicillin and kanamycin A. Cyclic olefin copolymer (Topas ® ) was used as a substrate on the top and bottom of the constructed chip. Due to their biocompatibility a conductive polymer bilayer consisting of tosylate-doped poly(3,4-ethylenedioxythiophene) (PEDOT) and the hydroxymethyl derivate was used as the electrode material. In a similar system Rosati et al. [61] optimized the geometry of the electrodes and the microchannels (e.g., thickness, width) and, therefore, the performance of the designed impedimetric aptasensor. Additionally, the ampicillin-specific aptamer was equipped with an appended poly(T)-poly(C) sequence which allowed a direct immobilization on the electrodes when UV irradiation was applied [62].
In order to enhance the aptasensor response to ampicillin and signal amplification, two electrochemical aptasensors were developed with the help of polymerase and nicking endonuclease [63], which was used for the first time as the target-aptamer binding triggered quadratic recycling amplification for electrochemical detection of antibiotics, and target-initiated T7 exonuclease in a homogeneous electrochemical sensing system [64].

Linker and Spacer
Aptamer Penicillin G (Figure 2b) is used for the production of 6-aminopenicillanic acid (6-APA), the main intermediate product for the synthesis of semi-synthetic penicillins and cephalosporins [56]. It is the most frequently used β-lactam antibiotic for the prevention and treatment of bacterial infections, like scarlet fever, diphtheria, gonorrhoea, angina, and tetanus. Penicillin is produced by the fungi Penicillium notatum and is hardly humanly toxic [57].
The first aptasensor for detection of penicillin was reported by Zhao et al. [69]. They developed an electrochemical aptasensor using a composite film consisting of a magnetic graphene nanocomposite (GR-Fe 3 O 4 NPs) and a poly(3,4-ethylenedioxythiophene)-gold nanoparticle composite (PEDOT-AuNPs) for the modification of the electrode to assemble the penicillin aptamer to it.
Paniel at al. [70] described the selection of aptamers selective to penicillin G using the capture-SELEX process. The process is based on the selection of DNA aptamers using the ssDNA fixed on a support, whereas the target is in solution. Selectivity tests showed that the aptamer was able to bind other β-lactam antibiotics, including amoxicillin and ampicillin, indeed with less affinity.
Lee et al. [71] identified ssDNA aptamers for the detection of penicillin G by reduced graphene oxide-SELEX (rGO-SELEX). rGO-SELEX is a method which uses the π-π stacking interaction between rGO and nucleic acids for an immobilization-free selection of aptamers. Furthermore, rGO is an effective fluorescence quencher through the FRET effect. Thus, the fluorescence recovery signal from the quenched FAM-labelled aptamer on the rGO surface can give information about the binding of a target to the aptamer.
The comparison between the different aptasensors for penicillin G are shown in Table 2. Although penicillin is an important and widely used antibiotic, only a few aptamer-based biosensors exist for its detection. Hence, there is still potential for research. Table 2. Aptamer sequence, dissociation constant (K D ), limit of detection (LOD), real sample analysis (RSA), and realized sensor type and measuring method for penicillin G, mentioned in the corresponding references (Ref). b = buffer, EIS = electrochemical impedance spectrometry, FAM = fluorescein amidite, FL = fluorometric, and IEC = impedimetric electrochemical.

Aminoglycosides
Aminoglycoside antibiotics are the most commonly used antibiotics worldwide, with a broad spectrum of activity-also against Gram-negative bacteria [56]. Despite their relatively high toxicity (especially on the ears and kidneys), they are the antibiotics of severe infections which, in turn, leads to an increase in resistance [56]. They exert their effect by binding to the 30S subunit of ribosomes, which leads to reading errors during translation and, ultimately, inhibition of protein biosynthesis [56]. The basic structure of most aminoglycoside antibiotics consists of an aminocyclitol ring which is linked glycosidically to other amino sugars ( Figure 3) [57]. The most important data of the discussed aptasensors, for the aminoglycosides including aptamer sequence, K D , LOD, RSA, and applied sensor type, are summarized in Tables 3-7.

Aminoglycosides
Aminoglycoside antibiotics are the most commonly used antibiotics worldwide, with a broad spectrum of activity-also against Gram-negative bacteria [56]. Despite their relatively high toxicity (especially on the ears and kidneys), they are the antibiotics of severe infections which, in turn, leads to an increase in resistance [56]. They exert their effect by binding to the 30S subunit of ribosomes, which leads to reading errors during translation and, ultimately, inhibition of protein biosynthesis [56]. The basic structure of most aminoglycoside antibiotics consists of an aminocyclitol ring which is linked glycosidically to other amino sugars ( Figure 3) [57]. The most important data of the discussed aptasensors, for the aminoglycosides including aptamer sequence, KD, LOD, RSA, and applied sensor type, are summarized in Tables 3-7.

Gentamicin
The alkaline aminoglycoside antibiotic gentamicin (Figure 3a), isolated in 1963, is a broadspectrum antibiotic and acts as a bactericide against a large number of Gram-positive and Gramnegative pathogens, such as E. coli and Pseudomonas [56]. It is used especially for severe wound infections and accidental injuries, as well as secondary infections after burns [56]. Therapeuticallyused gentamicin usually contains 70% of the C1 and 30% of the C2 component ( Figure 3a) [56]. Gentamicin is less toxic than kanamycin, neomycin, and streptomycin. However, ototoxic and nephrotoxic damage have been observed [56].
Rowe et al. [72] designed an electrochemical aptasensor based on RNA-aptamers and DNAaptamers for the detection of the aminoglycoside antibiotics gentamicin, tobramycin, and kanamycin

Gentamicin
The alkaline aminoglycoside antibiotic gentamicin (Figure 3a), isolated in 1963, is a broad-spectrum antibiotic and acts as a bactericide against a large number of Gram-positive and Gram-negative pathogens, such as E. coli and Pseudomonas [56]. It is used especially for severe wound infections and accidental injuries, as well as secondary infections after burns [56]. Therapeutically-used gentamicin usually contains 70% of the C 1 and 30% of the C 2 component (Figure 3a) [56]. Gentamicin is less toxic than kanamycin, neomycin, and streptomycin. However, ototoxic and nephrotoxic damage have been observed [56].
Rowe et al. [72] designed an electrochemical aptasensor based on RNA-aptamers and DNA-aptamers for the detection of the aminoglycoside antibiotics gentamicin, tobramycin, and kanamycin in blood samples in order to prevent overdosage and side effects. DNA aptamers were more stable, but the SWV experiments showed a lower sensitivity of the DNA-aptamer-based sensor to detect aminoglycosides in blood samples compared to the RNA-based sensor. The most important data of the developed sensor given in Table 3.
This work is the only aptasensor for gentamicin detection studied up to now in the literature, therefore, great potential exists for further research. Table 3. Aptamer sequence, dissociation constant (K D ), limit of detection (LOD), real sample analysis (RSA), and realized sensor type and measuring method for gentamicin, mentioned in the corresponding references (Ref). AEC = amperometric electrochemical, hs = human serum, MB = methylene blue, and SWV = square wave voltammetry. 1 When naming several references, the first always describes the realized sensor with associated LOD; aptamer sequence(s) and/or associated K D values are derived from the additional reference(s).

Kanamycin
Kanamycin ( Figure 3b) is a bactericidal antibiotic isolated from Streptomyces kanamyceticus, whose spectrum of activity comprises Gram-positive and Gram-negative bacteria [56]. It is widely used as a veterinary drug and as a second-line antibiotic to treat serious infections, such as pneumonia, septicemia, urinary tract infections, and intestinal infections [72]. If not explained in more detail, kanamycin stands for a mixture of kanamycin A, B, and C, in which kanamycin A has the largest share [57]. Since sensitive and selective methods to detect kanamycin residues for food safety and clinical diagnosis are of great interest, there are more reports in kanamycin aptamer-based sensors in comparison to other antibiotics.
Song et al. [74] discovered the kanamycin-specific aptamer, which was later used in a variety of other studies [75] (Table 4). They selected the aptamer in vitro by SELEX using affinity chromatography with kanamycin-immobilized sepharose beads. The specific aptamers were immobilized onto the AuNPs to fabricate a colorimetric-based aptasensor. In the presence of kanamycin, the addition of salt leads to an aggregation of the modified AuNPs, which results in a color change from red to purple.
Thus, different research groups have tried to design a sensitive and specific kanamycin aptasensor based on AgNPs and AuNPs for the possible application in food or clinical samples [76][77][78][79].
Wang et al. [80] designed a colorimetric aptasensor for the detection of kanamycin based on liquid crystal film. Amino-functionalized kanamycin-specific aptamers and N,N-dimethyl-N-(3-(trimethoxysilyl)propyl)-1-octadecanaminiuchloride (DMOAP) were co-immobilized onto the surface of a glass slide, resulting in a homeotropic orientation of the liquid crystal film. The addition of kanamycin resulted in the formation of G-quadruplex structures, which destroyed the oriented arrangement of the liquid crystals on the surface and caused a visible color change from pink to green.
Fluorescence, as one of the most common optical techniques, has been used in the fabrication of aptasensors for kanamycin detection [81][82][83][84][85][86][87][88][89][90]. Generally, the binding affinity of an aptamer towards its target decreases by tagging the aptamer with a fluorescent dye [83]. Therefore, it is needed to improve the sensitivity of the designed aptasensors. Regarding this fact, different strategies have been employed for amplification of fluorometric signals. Nanoparticles (NPs), due to their advantages, can be used as the modifier films and improve the sensitivity of fluorometric aptasensors. Different NPs have been employed in the construction of highly sensitive fluorometric aptasensors, like silica NP (SNPs) [81], amino-Fe 3 O 4 magnetic NPs [82], AuNPs [84], and UCNPs [86]. Immobilization of aptamers on the surface of reduced graphene oxide (rGO) [88] and carbon nanotubes (CNTs) [89] have showed great promise in the amplification of fluorometric signals in the detection of kanamycin. Moreover, Wang et al. [87] developed a fluorometric aptasensor, based on MoS 2 nanosheets and carbon dots. The quenching ability of layered MoS 2 was tested and compared to that of GO and AuNPs and showed comparable, or even better, values.
An impedimetric disposable and portable aptasensor for the detection of kanamycin was designed by Sharma et al. [99]. Amino-functionalized kanamycin-specific aptamers were immobilized onto the surface of the working screen-printed carbon electrode (SPCE) via NH 2 -COOH interaction. The interaction between the aptamers and kanamycin caused an inhibition in the Faradaic response and an increase in the electron transfer resistance.
Sometimes, in order to obtain high specificity and improve the sensitivity of aptasensors, the biocatalytic properties of enzymes are used to detect and amplify the analysis of targets with their aptamers. For example, horseradish peroxidase (HRP) [108] or glucose oxidase (GlO) [113], conjugated with AuNP-cDNA, have been used as biocatalysts for signal amplification for the detection of kanamycin.
Photoelectrochemical sensing is a novel method with high sensitivity and rapid response which combines the benefits of optical methods and electrochemical sensors [83]. Li et al. [96] developed a photoelectrochemical aptasensor for the detection of kanamycin based on the graphene-modified flour-doped SnO 2 electrode. In the absence of kanamycin, the generated photocurrent was low. When kanamycin was added, it was trapped by the aptamers on the surface of the electrode. The captured molecules were oxidized by photogenerated holes. The recombination of photogenerated holes and electrons was inhibited, resulting in an amplified photocurrent.
Several research efforts have been also made toward the design of aptasensors for detection of kanamycin A based on fluorometric [114,115] and electrochemical methods [59,116]. Nikolaus and Strehlitz [113] selected DNA-aptamers specific for binding of kanamycin A by capture SELEX according to the work of Stoltenburg's team [117] and further tested in bead-based or microplate-based assays by fluorescence detection of the 5 -FAM-labelled aptamers. By the way, Robati et al. [88] authored a review about aptasensors for quantitative detection of kanamycin and kanamycin A.
In summary, around half of all developed aptamer-based biosensors for the detection of kanamycin and kanamycin A are based on electrochemical sensor principles (either impedimetric or amperometric). Moreover, a comparatively large number of fluorometric aptasensors have been developed. The most important data of the discussed aptasensors are summarized in Table 4. The lowest LOD was reached with an amperometric aptasensor developed by Wang's team [110].

Neomycin
The spectrum of activity of neomycin ( Figure 3c) is mainly Gram-negative bacteria, including Salmonella and Shigella. Since it is hardly absorbed after oral administration, it is particularly suitable for combating infections of the digestive tract. It is also used for superficial skin and mucous membrane infections. A disadvantage is the high ear and kidney toxicity. In general, neomycin is an oligosaccharide mixture containing the three main components A, B, and C. Commercially available neomycin consist of about 90% neomycin B and 10% neomycin A and B [57].
In 1995 Wallis et al. [118] selected RNA-aptamers for neomycin B recognition by in vitro selection using SELEX, which was used in further studies to fabricate a fluorometric aptasensor based on AuNPs [119] and an impedimetric electrochemical aptasensor based on the immobilization of aptamers on the surface of a modified electrode with self-assembled monolayer (SAM) of mercaptopropionic acid [120].
In 2009 de-los-Santos-Alvarez et al. [121] studied how the modification of the RNA-aptamer influences the affinity of the interaction between the aptamer and neomycin B. In general, the fully 2 -O-methylization of the RNA-aptamer should prevent the degradation by endonuclease. They showed that this modification did not significantly alter the aptamer affinity towards neomycin B, but the proposed aptasensor was more sensitive towards neomycin B in comparison with other aptasensors ( Table 5).
The comparison of the obtained LODs (Table 5) showed that SPR [121] is more sensitive than the optical [119] and the electrochemical [120] method. Table 5. Aptamer sequence, dissociation constant (K D ), limit of detection (LOD), real sample analysis (RSA), and realized sensor type and measuring method for neomycin B, mentioned in the corresponding references (Ref). b = buffer, FAM = fluorescein amidite, FIS = Faradaic impedance spectroscopy, FL = fluorometric, IEC = impedimetric electrochemical, m = milk, and SPR = surface plasmon resonance spectroscopy.

Tobramycin
Tobramycin ( Figure 3d) is a semi-synthetic aminoglycoside antibiotic [56]. Its spectrum of activity comprises numerous Gram-negative pathogens, such as Escherichia coli, Klebsiella, Proteus, Pseudomonas, Salmonella, and Shigella, as well as Gram-positive Staphylococci and Enterococci [57]. It is therapeutically effective for infections of the respiratory and the urogenital tract, the skin, bones, the central nervous system (meningitis), and septicemia [57].
In 1995 Wang and Rando [73] selected RNA molecules that could specifically bind to the aminoglycoside antibiotic tobramycin by in vitro selection using SELEX and used in later studies [72].
Spiga et al. [122] introduced a DNA-based capture-SELEX coupled with in-stream direct-specificity monitoring via SPR. The aptamers were evaluated for their affinity to tobramycin via direct immobilization onto a SPR chip, which was used in further studies [123].
Han et al. [124] developed a magnetic bead-based SELEX to identify 37 ssDNA aptamers specific for tobramycin using a fluorescent method based on the reported principle by Ma's team [125], in which they developed a colorimetric aptasensor for the determination of tobramycin in milk and chicken eggs based on the adsorption of ssDNA aptamers on the surface of AuNPs. With the sensor of Ma et al. [125] the one reaching lowest LOD was developed (Table 6).
In order to detect drug concentration in patient samples, which are much more complex matrices than buffers, Cappi et al. [123] developed a portable, palm-sized transmission-localized SPR (TL-SPR) system for tobramycin detection. They used a setup based on aptamer-functionalized gold nanoislands (NIs) deposited on a glass slide covered with fluorine-doped tin oxide (FTO), which acts as a biosensor, and a complementary metal oxide semiconductor (CMOS) as a light detector. The sensitivity of the CMOS image sensor was matched to the localized plasmon resonance exhibited by the Au-NIs. For the first time it was shown that label-free direct detection and quantification of a small molecule can be reliably used in the complex matrix of filtered undiluted blood serum.
In an effort Gonzalez-Fernandez et al. [126] evaluated and compared the affinity and analytical characteristics of two partially and fully O-methylated modified RNA-aptamers for the design of electrochemical aptasensors for tobramycin detection in human serum. In addition to the higher endonuclease resistance, the fully O-methylated aptamer had a lower dissociation constant, as well as a lower LOD than the partially-methylated aptamer (Table 6), which was used in further experiments to develop the aptamer-based inhibition assays for detection of tobramycin [127,128].
Schoukroun-Barnes et al. [128] presented a systematic study of several approaches to develop an electrochemical RNA aptamer-based biosensor for the detection of aminoglycoside antibiotics, like tobramycin. They could design a highly sensitive aptasensor for tobramycin through the optimization of the electrochemical interrogation parameters and biomolecular engineering of the RNA aptamer-sequence (Table 6).
In summary, there are just a few aptasensors developed for tobramycin detection and with the exception of one, they are based on electrochemical principles. Almost all electrochemical sensors used RNA aptamer sequences for the specific tobramycin recognition. The loweset LOD and belonging K D value was determined with the RNA aptamer sequence II (Table 6), mentioned by Schoukroun-Barnes et al. [128]. An even higher affinity towards tobramycin was reached by Cappi et al. [123] by using ssDNA aptamer sequence (Table 6). Table 6. Aptamer sequence, dissociation constant (K D ), limit of detection (LOD), real sample analysis (RSA), and realized sensor type and measuring method for tobramycin, mentioned in the corresponding references (Ref). AEC = amperometric electrochemical, b = buffer, bs = blood serum, bsa = bovine serum, albumin, CA = chronoamperometry, ce = chicken egg, CO = colorimetric, DPV = differential pulse voltammetry, FIS = Faradaic impedance spectroscopy, h = honey, hs = human serum, IEC = impedimetric electrochemical, m = milk, MB = methylene blue, SPR = surface plasmon resonance, and SWV = square wave voltammetry.

Streptomycin
The discovery of streptomycin (Figure 3e) from Streptomyces griseus by Selmon Waksman (1943) allowed, for the first time, a therapy of the tuberculosis pathogen Mycobacterium tuberculosis. However, due to the renal and ear-harming properties of streptomycin, other antibiotics (e.g., rifampicin) are usually used today [56]. More frequently, it is used to combat penicillin-resistant strains of Neisseria gonorrhea infections and still used for the treatment of tuberculosis [57].
The first streptomycin-specific DNA-aptamers was screened by Zhou et al. [130] by affinity magnetic bead-based SELEX. Streptomycin was detected by using a label-free AuNP-based colorimetric method.
Liu et al. [131] developed an aptamer-based colorimetric sensor for the detection of streptomycin. Different streptomycin-specific aptamer sequences were obtained by SELEX. The selected aptamer was used for all further experiments.
Based on the interesting features of AuNPs for the construction of colorimetric aptasensor, there are several reports of such biosensors for streptomycin detection [130][131][132][133][134].
A colorimetric and fluorescence quenching aptasensor for streptomycin detection, based on the specific aptamer and its FAM-labelled complementary strand (cDNA) and aqueous AuNPs was reported by Emrani et al. [134]. Comparisons between the pure colorimetric and the pure fluorometric method showed higher sensitivity of the aptasensor by measuring with the fluorometric one. Consequently, a variety of studies have been carried out to fabricate the high-sensitivity aptasensors for streptomycin based on the fluorometric method [135][136][137].
Xu et al. [136] developed a photoelectrochemical aptasensor for streptomycin detection based on CdTe QDs single-walled carbon nanohorns, synthesized via the one-pot method, which acted as the photoactive species. These could inhibit electron-hole pair recombination, accelerate electron transfer, and improve the photocurrent intensity.
Ghanbari and Roushani [137] introduced an impedimetric electrochemical aptasensor for the detection of streptomycin based on the immobilization of streptomycin aptamers on the surface of a graphene QDs/AuNP nanocomposite. Upon addition of streptomycin, aptamer-target complexes were formed, causing an increase of the electrochemical signal.
Yin et al. [138][139][140] constructed three quite similar electrochemical aptasensors for the detection of streptomycin based on the immobilization of the aptamer on the surface of modified electrodes with different nanocomposites, including MWCNTs/copper oxide (CuO)/AuNPs [138], AuNPs/magnetic MWCNTs/nanoporous PtTi alloy [139], and graphene/Fe 3 O 4 /AuNPs [140]. Comparisons of the current responses of the aptasensors to streptomycin and to a mixture of streptomycin and interfering substances confirmed an excellent specificity and high sensitivity of the sensor based on an AuNPs/magnetic MWCNTs/nanoporous PtTi alloy modified electrode towards streptomycin.
Summarized, there are only a few papers dealing with aptasensing of streptomycin. According to the data of Table 7, more than half of them are are based on electrochemical measurements. The up to five orders of magnitude lower LOD than those reached with the other sensors, could be determined by Luan et al. [132] using a colorimetric assay and was followed by Yin et al. [139] with an amperometric aptasensor. Table 7. Aptamer sequence, dissociation constant (K D ), limit of detection (LOD), real sample analysis (RSA), and realized sensor type and measuring method for streptomycin, mentioned in the corresponding references (Ref). AEC = amperometric electrochemical, b = buffer, bs = blood serum, cap = capture probe, cDNA = complementary DNA, CO = colorimetric, DPV = differential pulse voltammetry, FAM = fluorescein amidite, FL = fluorometric, h = honey, IEC = impedimetric electrochemical, m = milk, PEC = photoelectrochemical, rs = rat serum, and SWV = square wave voltammetry.   -GGG GTC TGG TGT TCT GCT TTG TTC TGT CGG GTC

Anthracyclines
Anthracyclines inhibit the replication of DNA by intercalation and inhibition of topoisomerases. They are used clinically for the treatment of tumors, but they can cause heart damage in the long-term medication [144]]. The basic structure, which all anthracyclines exhibit, is marked in red in Figure 4.

Anthracyclines
Anthracyclines inhibit the replication of DNA by intercalation and inhibition of topoisomerases. They are used clinically for the treatment of tumors, but they can cause heart damage in the longterm medication [144]]. The basic structure, which all anthracyclines exhibit, is marked in red in Daunomycin Daunomycin (Figure 4), the first discovered anthracycline, produced naturally by Streptomyces peucetius, acts as an intercalator whereat the intercalation between DNA bases leads to a local structural change in the DNA and, thus, to an inhibition of DNA replication and transcription [145]. Therefore, daunomycin has a growth inhibitory effect on Gram-positive bacteria and fungi. Moreover, an antiviral effect by inhibiting viral DNA replication in the host cell was obtained. In 1963 an antileukemic activity was discovered [57]. Nowadays daunomycin is widely used for the treatment of breast tumors, lymphocytic and myeloid leukemia [146].
In 2008 Wochner et al. [147] selected ssDNA aptamers, specific for daunomycin and tetracycline, which were used in further studies for the fabrication of an aptasensor [148].
In the work of He et al. [146] a colorimetric aptasensor for daunomycin detection based on resonance scattering is described. A fluorescence spectrophotometer was used to record the resonance scattering intensity.
Chandra et al. [148] developed an electrochemical biosensor for daunomycin using the coimmobilization of the specific aptamers and phosphatidylserine on Au nanoparticle-deposited conducting polymer, which exhibited a higher sensitivity than the others (Table 8). Daunomycin Daunomycin (Figure 4), the first discovered anthracycline, produced naturally by Streptomyces peucetius, acts as an intercalator whereat the intercalation between DNA bases leads to a local structural change in the DNA and, thus, to an inhibition of DNA replication and transcription [145]. Therefore, daunomycin has a growth inhibitory effect on Gram-positive bacteria and fungi. Moreover, an antiviral effect by inhibiting viral DNA replication in the host cell was obtained. In 1963 an antileukemic activity was discovered [57]. Nowadays daunomycin is widely used for the treatment of breast tumors, lymphocytic and myeloid leukemia [146].
In 2008 Wochner et al. [147] selected ssDNA aptamers, specific for daunomycin and tetracycline, which were used in further studies for the fabrication of an aptasensor [148].
In the work of He et al. [146] a colorimetric aptasensor for daunomycin detection based on resonance scattering is described. A fluorescence spectrophotometer was used to record the resonance scattering intensity.
Chandra et al. [148] developed an electrochemical biosensor for daunomycin using the co-immobilization of the specific aptamers and phosphatidylserine on Au nanoparticle-deposited conducting polymer, which exhibited a higher sensitivity than the others (Table 8). Table 8. Aptamer sequence, dissociation constant (K D ), limit of detection (LOD), real sample analysis (RSA), and realized sensor type and measuring method daunomycin in the corresponding references (Ref). AEC = amperometric electrochemical, b = buffer, CO = colorimetric, DPV = differential pulse voltammetry, ELAA = enzyme-linked aptamer assay, FL = fluorometric, hu = human urine, and SPR = surface plasmon resonance.

Chloramphenicol
Chloramphenicol is an antibiotic class of its own [56]. The chemical structure is shown in Figure 5 [57]. It blocks the peptidyl transferase by binding to the 50S subunits of the 70S ribosomes [56]. It was isolated in 1950 from Streptomyces venezuelae, but nowadays it is exclusively produced synthetically [56]. It acts against Gram-positive and Gram-negative pathogens, as well as against Actinomycetes, Rickettsiae, and some large viruses [57]. Due to its serious side effects, such as leukemia, aplastic anemia, and grey baby syndrome, it is only a reserve antibiotic used to treat typhoid, shigellosis, and rickettsial infections [57,149].

Chloramphenicol
Chloramphenicol is an antibiotic class of its own [56]. The chemical structure is shown in Figure 5 [57]. It blocks the peptidyl transferase by binding to the 50S subunits of the 70S ribosomes [56]. It was isolated in 1950 from Streptomyces venezuelae, but nowadays it is exclusively produced synthetically [56]. It acts against Gram-positive and Gram-negative pathogens, as well as against Actinomycetes, Rickettsiae, and some large viruses [57]. Due to its serious side effects, such as leukemia, aplastic anemia, and grey baby syndrome, it is only a reserve antibiotic used to treat typhoid, shigellosis, and rickettsial infections [57,149]. In 2011 chloramphenicol-specific aptamers were selected and characterized by Mehta et al. [150] using the SELEX procedure, which was used in further studies for chloramphenicol detection [151,152].
In order to amplify the signals of colorimetric aptasensors, Miao's team utilized the enzymelinked polymer nanotracers labeled by a double-stranded DNA (ds-DNA) antibody. The aptamer was immobilized on Fe3O4/Au magnetic nanoparticles as a capture probe, and an enzyme-linked polymer nanotracer was fabricated by co-immobilization of HRP-labelled AuNPs and double stranded DNA (dsDNA) antibodies as signal tags on EnVision reagent, a kit containing about 100 HRPs and some anti-IgG [152]. The proposed aptasensor showed to sensitively respond down to 0.015 ng mL −1 towards chloramphenicol.
Based on magnetic aptamer-enzyme co-immobilization platinum nanoprobes and exonucleaseassisted target recycling, Miao's team [153] designed a triple amplification colorimetric aptasensor with a detection limit of 0.3 pg mL −1 towards chloramphenicol.
There are several reports in fluorometric aptasensors for chloramphenicol detection based on the immobilization of aptamers on different composites and using various prepared capture and signal probes [42,43,[155][156][157][158][159][160]. Their analytical features are summarized in Table 9. As seen, the proposed "off-on" fluorometric aptasensor by Miao et al. [42] using vesicle QD-Au colloid composite probes, showed higher sensitivity towards chloramphenicol. They used the vesicle nanotracer as a signal probe, consisting of liposome-CdSe/ZnS QD complex labelled with SSB. Aptamerfunctionalized AuNPs acted as the capture probe. The composite probe does not emit fluorescence signals, which represented the "off" state. Upon addition of chloramphenicol, the aptamer bound to it and the aptamer-target complex detached from the composite probe. The result is a fluorescence signal, which represents the "on" state.
Based on the sensor principle to develop a electrochemiluminescent aptasensor for the detection of chloramphenicol [149,161,162], a triple-amplification assay using polymer enzyme-linked nanotracers/Exonuclease-assisted target recycling method [154] and TiO2-based nanorod assay sensitized with Eu(III)-doped CdS QDs as the photoactive material [163] were designed with a detection limit of 0.034 and 0.36 pM towards chloramphenicol, respectively.
In order to amplify the signals of colorimetric aptasensors, Miao's team utilized the enzyme-linked polymer nanotracers labeled by a double-stranded DNA (ds-DNA) antibody. The aptamer was immobilized on Fe 3 O 4 /Au magnetic nanoparticles as a capture probe, and an enzyme-linked polymer nanotracer was fabricated by co-immobilization of HRP-labelled AuNPs and double stranded DNA (dsDNA) antibodies as signal tags on EnVision reagent, a kit containing about 100 HRPs and some anti-IgG [152]. The proposed aptasensor showed to sensitively respond down to 0.015 ng mL −1 towards chloramphenicol.
Based on magnetic aptamer-enzyme co-immobilization platinum nanoprobes and exonucleaseassisted target recycling, Miao's team [153] designed a triple amplification colorimetric aptasensor with a detection limit of 0.3 pg mL −1 towards chloramphenicol.
There are several reports in fluorometric aptasensors for chloramphenicol detection based on the immobilization of aptamers on different composites and using various prepared capture and signal probes [42,43,[155][156][157][158][159][160]. Their analytical features are summarized in Table 9. As seen, the proposed "off-on" fluorometric aptasensor by Miao et al. [42] using vesicle QD-Au colloid composite probes, showed higher sensitivity towards chloramphenicol. They used the vesicle nanotracer as a signal probe, consisting of liposome-CdSe/ZnS QD complex labelled with SSB. Aptamer-functionalized AuNPs acted as the capture probe. The composite probe does not emit fluorescence signals, which represented the "off" state. Upon addition of chloramphenicol, the aptamer bound to it and the aptamer-target complex detached from the composite probe. The result is a fluorescence signal, which represents the "on" state.
Based on the sensor principle to develop a electrochemiluminescent aptasensor for the detection of chloramphenicol [149,161,162], a triple-amplification assay using polymer enzyme-linked nanotracers/Exonuclease-assisted target recycling method [154] and TiO 2 -based nanorod assay sensitized with Eu(III)-doped CdS QDs as the photoactive material [163] were designed with a detection limit of 0.034 and 0.36 pM towards chloramphenicol, respectively.

(Fluoro)Quinolones
(Fluoro)Quinolones have a very broad spectrum of action, thus, they act against Gram-positives, Gram-negatives, Mycobacteria, Chlamydia, and anaerobes, and are just slightly toxic to humans. Their mechanism of action based on the inhibition of DNA-gyrase, which belongs to the group of topoisomerases II. Inter alia, DNA-gyrase is responsible for the derivatization of the DNA. Structurally, quinolones are derived from quinolone (marked in red in Figure 6) [56]. The efficacy of the quinolones was further enhanced by the introduction of an additional fluorine atom, resulting in a whole series of fluoroquinolones. and a circular strand-replacement DNA polymerization (CSRP) target triggered the amplification strategy. The proposed strategy exhibited a high sensitivity to chloramphenicol with a detection limit of 33 fM.

(Fluoro)Quinolones
(Fluoro)Quinolones have a very broad spectrum of action, thus, they act against Gram-positives, Gram-negatives, Mycobacteria, Chlamydia, and anaerobes, and are just slightly toxic to humans. Their mechanism of action based on the inhibition of DNA-gyrase, which belongs to the group of topoisomerases II. Inter alia, DNA-gyrase is responsible for the derivatization of the DNA. Structurally, quinolones are derived from quinolone (marked in red in Figure 6) [56]. The efficacy of the quinolones was further enhanced by the introduction of an additional fluorine atom, resulting in a whole series of fluoroquinolones.  The structure of quinolone is marked in red. Table 9. Aptamer sequence, dissociation constant (K D ), limit of detection (LOD), real sample analysis (RSA), and realized sensor type and measuring method for chloramphenicol, mentioned in the corresponding references (Ref). AEC = amperometric electrochemical, apt = aptamer, b = buffer, cap = capture probe, cDNA = complementary DNA, CO = colorimetric, d = drugs, DPV = differential pulse voltammetry, ECL = electrochemiluminescent, EIS = electrochemical impedance spectrometry, f = fish, FL = fluorometric, h = honey, hs = human serum, IEC = impedimetric electrochemical, LSV = linear sweep voltammetry, m = milk, PEC = photoelectrochemical, p = pork, rs = rat serum, SPR = surface plasmon resonance, u = urine, and w = water.

Linker and Spacer
Aptamer Sequence 5 →3       Ciprofloxacin (Figure 6a), a second-generation fluoroquinolone, which acts against Bacillus anthracis, the causative agent of anthrax, and is one of the most used quinolones nowadays [5,56].
There are just two reports of aptasensors for ciprofloxacin detection. In 2017 Lavee et al. [179] developed for the first time a colorimetric aptamer-based assay for the determination of ciprofloxacin using AuNPs. In another study an electrochemical aptasensor for ultrasensitive detection of fluoroquinolones, especially ciprofloxacin, based on a single-stranded DNA-binding protein, was presented [180].

Danofloxacin
Danofloxacin (Figure 6b) acts against Gram-positive and Gram-negative bacteria and is often used for the treatment of respiratory diseases of cattle and pigs [181]. It is exclusively used in animal husbandry, not least because of its toxicity to humans [181].
By the application of SELEX, Han et al. [181] selected specific and high-affinity RNA aptamers with 2 -fluoro-2 -deoxyribonucleotide-modified pyrimidine nucleotides bound to danofloxacin. As a consequence, they employed an optical aptasensor for the detection of danofloxacin in buffer. The most important data of the discussed aptasensor are given in Table 11.
There are no other reports about investigations for danofloxacin detection by an aptasensor mentioned in the literature.

Enrofloxacin
Enrofloxacin (Figure 6c) is a high-potency antibacterial agent which is widely employed for disease prevention and therapy in poultry and livestock breeding and aquaculture practice [182,183].
For the detection of enrofloxacin, Liu et al. [182] designed a fluorometric aptasensor based on the immobilization of aptamers on the surface of Yb, Er ion-pair doped magnetic Fe 3 O 4 UCNPs and amino-functionalized silica-modified (NH 2 -Si) UCNPs.
Moreover, Liu's group [183] developed a fluorometric "double recognition" aptasensor for the detection of enrofloxacin by integrating two antibiotic recognition elements, including aptamers and fully-synthetic molecularly-imprinted polymers (MIPs) The LOD of the proposed aptasensor was about five times lower than the previously presented "simple" one [182] (Table 12), which is presumably related to the improved recognition ability of the sensor by the use of aptamers in combination with MIPs.
The two described are the only papers which deal with the aptamer-based detection of enrofloxacin, so there exists potential for further research.

Ofloxacin
Ofloxacin (Figure 6d) is a second-generation fluoroquinolone, used in bacterial infections of the respiratory tract and the gastrointestinal tract [184].
Reinemann et al. [185] searched for aptamer sequences specific for ofloxacin and, furthermore, determined the dissociation constant (K D value) of the aptamer-target system.
In 2017 Pilehvar et al. [184] developed a rapid, stable, and sensitive label-free electrochemical aptasensor for ofloxacin detection based on the immobilization of the specific aptamer on AuNPs. The most important data of the two aptasensors are summarized in Table 13.
There are no more reports about aptasensors for ofloxacin detection. Table 10. Aptamer sequence, dissociation constant (K D ), limit of detection (LOD), real sample analysis (RSA), and realized sensor type and measuring method for ciprofloxacin, mentioned in the corresponding references (Ref). AEC = amperometric electrochemical, b = buffer, cDNA = complementary DNA, CO = colorimetric, DPV = differential pulse voltammetry, hs = human serum, m = milk, and sw = spiked water.

Lincosamide
Lincosamides, called acylaminopyranosides due to their chemical structure, bind to the 50S subunit of the bacterial ribosomes and block the enzyme peptidyltransferase, resulting in an interrupted chain elongation during protein biosynthesis [56]. Lincosamides are frequently used in the case of staphylococcal, streptococcal, and pneumococcal infections [57]. Three representatives exist: the natural lincomycin ( Figure 7) and two semi-synthetic derivates, clindamycin and pirlimycin [5]. The structure, which is common to all anthracyclines, is marked in red in Figure 7. Anthracyclines are applied especially if a penicillin allergy exists [57].

Lincosamide
Lincosamides, called acylaminopyranosides due to their chemical structure, bind to the 50S subunit of the bacterial ribosomes and block the enzyme peptidyltransferase, resulting in an interrupted chain elongation during protein biosynthesis [56]. Lincosamides are frequently used in the case of staphylococcal, streptococcal, and pneumococcal infections [57]. Three representatives exist: the natural lincomycin ( Figure 7) and two semi-synthetic derivates, clindamycin and pirlimycin [5]. The structure, which is common to all anthracyclines, is marked in red in Figure 7. Anthracyclines are applied especially if a penicillin allergy exists [57]. Lincomycin was the first discovered lincosamide, isolated from Streptomyces lincolnensis in a soil sample from Lincoln (Nebraska) [5,57]. It is preferable for the treatment of bone marrow inflammation and wound and respiratory infections [57].
To the best of our knowledge, there is just one report of sensor assay for lincomycin with a dual recognition system comprising a MIP and aptamers [186]. They used the AuNP-functionalized GO nanocomposite for signal amplification, and C-dots, which were modified onto the lincomycinspecific aptamers, serving as a signal indicator and exhibiting enhanced signal intensity in the absence of lincomycin. Electrogenerated chemiluminescence resonance energy transfer was observed between Au-GO and C-dots. After the C-dots accepted the energy, they acted as a signal indicator and exhibited enhanced signal intensity in the presence of the target lincomycin. The results confirmed that the combined characteristics of the specific molecular recognition properties of aptamers and MIPs enhance the recognition ability and cause a high specificity towards their target. The most important data of the discussed aptasensor are given below in Table 14. Table 14. Aptamer sequence, dissociation constant (KD), limit of detection (LOD), real sample analysis (RSA) and realized sensor type and measuring method for lincomycin, mentioned in the corresponding references (Ref). AC = alternating current, b = buffer, CV = cyclic voltammetry, ECL = electrochemiluminescent, and me = meat. 1 When naming several references, the first always describes the realized sensor with associated LOD; aptamer sequence(s) and/or associated KD values are derived from the additional reference(s). Lincomycin was the first discovered lincosamide, isolated from Streptomyces lincolnensis in a soil sample from Lincoln (Nebraska) [5,57]. It is preferable for the treatment of bone marrow inflammation and wound and respiratory infections [57].

5′
To the best of our knowledge, there is just one report of sensor assay for lincomycin with a dual recognition system comprising a MIP and aptamers [186]. They used the AuNP-functionalized GO nanocomposite for signal amplification, and C-dots, which were modified onto the lincomycin-specific aptamers, serving as a signal indicator and exhibiting enhanced signal intensity in the absence of lincomycin. Electrogenerated chemiluminescence resonance energy transfer was observed between Au-GO and C-dots. After the C-dots accepted the energy, they acted as a signal indicator and exhibited enhanced signal intensity in the presence of the target lincomycin. The results confirmed that the combined characteristics of the specific molecular recognition properties of aptamers and MIPs enhance the recognition ability and cause a high specificity towards their target. The most important data of the discussed aptasensor are given below in Table 14. Table 14. Aptamer sequence, dissociation constant (K D ), limit of detection (LOD), real sample analysis (RSA) and realized sensor type and measuring method for lincomycin, mentioned in the corresponding references (Ref). AC = alternating current, b = buffer, CV = cyclic voltammetry, ECL = electrochemiluminescent, and me = meat. 1 When naming several references, the first always describes the realized sensor with associated LOD; aptamer sequence(s) and/or associated K D values are derived from the additional reference(s).

Tetracyclines
Tetracyclines are the most widely used antibiotics besides penicillins which are of great economic importance due to their broad-spectrum activity (acting against Gram-positive, Gram-negative bacteria, Rickettsiae, Mycoplasmas, Leptospira, and some large viruses) and their low toxicity [56,57]. In some countries, they are widely used as nutritive antibiotics in poultry and pig fattening, which encourages resistance development. Tetracyclines inhibit protein biosynthesis by binding to the 50S subunit of the ribosomes. They are formed exclusively by Streptomyces. Their name derives from their basic structure, which consists of four linearly arranged six-rings (marked in red in Figure 8).

Tetracyclines
Tetracyclines are the most widely used antibiotics besides penicillins which are of great economic importance due to their broad-spectrum activity (acting against Gram-positive, Gramnegative bacteria, Rickettsiae, Mycoplasmas, Leptospira, and some large viruses) and their low toxicity [56,57]. In some countries, they are widely used as nutritive antibiotics in poultry and pig fattening, which encourages resistance development. Tetracyclines inhibit protein biosynthesis by binding to the 50S subunit of the ribosomes. They are formed exclusively by Streptomyces. Their name derives from their basic structure, which consists of four linearly arranged six-rings (marked in red in Figure 8).

Oxytetracycline
Oxytetracycline (Figure 8a) is the primary product in the formation of tetracyclines by Streptomyces [57].
Niazi et al. [187] selected oxytetracycline-specific ssDNA aptamers by Flu-Mag SELEX, which was later used in a variety of studies which deal with oxytetracycline detection [188][189][190][191][192]. In the Flu-Mag SELEX method fluorescent labels for DNA quantification and magnetic beads for target immobilization are used for aptamer selection [193]. In further investigation they selected ssDNA aptamers specific for tetracycline, oxytetracycline, and doxycycline [194].
Kwon et al. [195] truncated 76-mer ssDNA aptamers with high affinity and specificity for oxytetracycline, selected by SELEX, to a unique shortened 8-mer ssDNA, by selection of the nucleotide bases which exhibit high homogeneity in accordance with their conserved regions. By utilization of the shortened aptamer, an ultrasensitive (Table 15) colorimetric oxytetracycline detection based on unmodified AuNPs was possible. The truncated aptamer was used in other studies [196].
An aptamer-based cantilever array sensor for the detection of oxytetracycline at nanomolar concentrations was introduced by Hou et al. [197]. The sensing cantilevers were functionalized with SAMs of the specific aptamers while the reference cantilevers were modified with 6-mercapto-1hexanol (MCH) SAMs to eliminate the influence of environmental disturbances, such as temperature and non-specific adsorption.
Meng et al. [196] designed an ultrasensitive surface enhanced Raman scattering (SERS) aptasensor for the detection of oxytetracycline on the basis of the Raman hot spot between gold nanoparticles (AuNPs) (13 nm and 80 nm diameter respectively) linked by a DNA sequence. Advantages of SERS, a molecular fingerprint spectrum, are, amongst others, ultrasensitive and noninvasive probing, compatibility with aqueous solutions, minimal sample preparation, and label-free monitoring of analytes in complex matrices. Thiolated stem-loop DNA, containing the oxytetracycline specific aptamer, was immobilized onto the surface of 80 nm AuNPs and, subsequently, the 13 nm AuNPs were functionalized with the Raman reporter molecule 4mercaptobenzoic acid. Between the 80 nm AuNPs and the 13 nm AuNPs a SERS hot spot was formed, which is a highly-localized region of intense local field enhancement. In the presence of oxytetracycline, the aptamer preferentially bound to it, leading to a partial dehybridization of the

Oxytetracycline
Oxytetracycline (Figure 8a) is the primary product in the formation of tetracyclines by Streptomyces [57].
Niazi et al. [187] selected oxytetracycline-specific ssDNA aptamers by Flu-Mag SELEX, which was later used in a variety of studies which deal with oxytetracycline detection [188][189][190][191][192]. In the Flu-Mag SELEX method fluorescent labels for DNA quantification and magnetic beads for target immobilization are used for aptamer selection [193]. In further investigation they selected ssDNA aptamers specific for tetracycline, oxytetracycline, and doxycycline [194].
Kwon et al. [195] truncated 76-mer ssDNA aptamers with high affinity and specificity for oxytetracycline, selected by SELEX, to a unique shortened 8-mer ssDNA, by selection of the nucleotide bases which exhibit high homogeneity in accordance with their conserved regions. By utilization of the shortened aptamer, an ultrasensitive (Table 15) colorimetric oxytetracycline detection based on unmodified AuNPs was possible. The truncated aptamer was used in other studies [196].
An aptamer-based cantilever array sensor for the detection of oxytetracycline at nanomolar concentrations was introduced by Hou et al. [197]. The sensing cantilevers were functionalized with SAMs of the specific aptamers while the reference cantilevers were modified with 6-mercapto-1-hexanol (MCH) SAMs to eliminate the influence of environmental disturbances, such as temperature and non-specific adsorption.
Meng et al. [196] designed an ultrasensitive surface enhanced Raman scattering (SERS) aptasensor for the detection of oxytetracycline on the basis of the Raman hot spot between gold nanoparticles (AuNPs) (13 nm and 80 nm diameter respectively) linked by a DNA sequence. Advantages of SERS, a molecular fingerprint spectrum, are, amongst others, ultrasensitive and non-invasive probing, compatibility with aqueous solutions, minimal sample preparation, and label-free monitoring of analytes in complex matrices. Thiolated stem-loop DNA, containing the oxytetracycline specific aptamer, was immobilized onto the surface of 80 nm AuNPs and, subsequently, the 13 nm AuNPs were functionalized with the Raman reporter molecule 4-mercaptobenzoic acid. Between the 80 nm AuNPs and the 13 nm AuNPs a SERS hot spot was formed, which is a highly-localized region of intense local field enhancement. In the presence of oxytetracycline, the aptamer preferentially bound to it, leading to a partial dehybridization of the DNA. In consequence, the 13 nm AuNPs approach the 80 nm AuNPs more closely and the Raman intensity increased significantly.
Two colorimetric aptasensors for the detection of oxytetracycline based on the immobilization of specific aptamers onto AuNP surfaces were reported [190,198].
Based on quenching ability of rGO [34], GO sheets [199], and GO hydrogel [200], several fluorometric assays for oxytetracycline detection are reported, which are described in Table 15.

Tetracycline
Tetracycline (Figure 8b) is used in veterinary medicine and treatment, as well as the prevention of microbial infections, such as respiratory tract infections, arthritis, and severe acne [207]. In particular, it has been used as a feed additive to promote the growth of livestock in the agriculture sector [208].
Kwon et al. [195] truncated 76-mer ssDNA aptamers with high affinity and specificity for oxytetracycline to a unique shortened 8-mer ssDNA with selectivity to oxytetracycline, tetracycline, doxycycline, and chlortetracycline.
Aslipashaki et al. [207] developed an aptamer-based solid-phase extraction followed by electrospray ionization-ion mobility spectrometry (ESI-IMS) for tetracycline separation and detection in biological fluids via covalent binding of aptamers onto CNBr-activated sepharose.
Jeong and Rhee Paeng [210] introduced a competitive enzyme-linked aptamer assay (ELAA) for the determination of tetracycline residue in bovine milk using two different aptamers individually, one 76mer DNA and a 57mer RNA aptamer. The RNA aptamer featured a higher affinity to tetracycline than the DNA aptamer, and also the LODs obtained for the RNA aptamer were lower than the one for the DNA aptamer, as well as in buffer and in milk (Table 16).
Summarized, around half of all developed aptamer-based biosensors for the detection of tetracycline are based on electrochemical sensor principles (either impedimetric or amperometric). Colorimetric aptasensors are frequently used, mainly due to their simple handling and evaluation. In almost all of the mentioned studies, the same tetracycline-specific ssDNA aptamer was used, which was selected and investigated by Niazi et al. [194]. The summarized data in Table 16 shows, in comparison between the RNA aptamer [210] and the ssDNA aptamers applied for tetracycline detection, that the RNA aptamer featured a lower K D and, thus, a much higher affinity to tetracycline than the DNA aptamers [210]. By shortening the ssDNA sequence to the possible minimum with sufficient affinity, Kwon et al. [195] managed to achieve a similar high affinity of the ssDNA aptamer to the target as Jeong et al. [210] reached with the RNA aptamer. Table 16. Aptamer sequence, dissociation constant (K D ), limit of detection (LOD), real sample analysis (RSA), and realized sensor type and measuring method for tetracycline, mentioned in the corresponding references (Ref). AEC = amperometric electrochemical, apt = aptamer, b = buffer, cap = capture probe, cDNA = complementary DNA, CO = colorimetric, DPV = differential pulse voltammetry, EIS = electrochemical impedance spectrometry, ELAA = enzyme-linked aptamer assay, ESI-MS = electrospray ionization-ion mobility spectrometry, FAM = fluorescein amidite, FIS = Faradaic impedance spectroscopy, FL = fluorometric, h = honey, hp = human plasma, hs = human serum, hu = human urine, IEC = impedimetric electrochemical, m = milk, p = pork, PEC = photoelectrochemical, rs = rat serum, SERS = surface enhanced Raman scattering, SWV = square wave voltammetry, tw = tap water, u = urine, and uw = ultrapure water.

Sulfonamides
Sulfonamides were the first synthetic antibiotics [145]. As the name suggests they are characterized by their sulfonamide group (marked in red in Figure 9). As analogues of p-aminobenzoic acid they interfere with the synthesis of folic acid [5]. Sulfonamides act against enterobacteria, like Escherichia coli or Salmonella, and are mainly used to treat urinary tract infections and pneumonia.

Sulfonamides
Sulfonamides were the first synthetic antibiotics [145]. As the name suggests they are characterized by their sulfonamide group (marked in red in Figure 9). As analogues of p-aminobenzoic acid they interfere with the synthesis of folic acid [5]. Sulfonamides act against enterobacteria, like Escherichia coli or Salmonella, and are mainly used to treat urinary tract infections and pneumonia. Sulfadimethoxine Sulfadimethoxine ( Figure 9) is a cheap broad-spectrum antibiotic that is effective against bacterial and coccidial infections and used for treatment (and prevention) of poultry diseases [243,244].
Song et al. [243] selected aptamers specific for sulfadimethoxine by magnetic bead-based SELEX and identified the one with the highest affinity towards its target. The developed aptamer was utilized in various further work [244][245][246][247][248][249]. According to the data of Table 17, most of the developed aptasensors for sulfadimethoxine detection are colorimetric [75,[244][245][246][247]. However, there are some reports on the aptasensors based on fluorometric [248] and photoelectrochemical sensing assays [249]. As seen, the proposed photoelectrochemical aptasensing platform based on graphene-doped Bi2S3 nanorods, as photoactive materials, compared to other aptamer biosensing assays shows the higher sensitivity towards sulfadimethoxine with a detection limit of 0.55 nM [249]. Sulfadimethoxine Sulfadimethoxine ( Figure 9) is a cheap broad-spectrum antibiotic that is effective against bacterial and coccidial infections and used for treatment (and prevention) of poultry diseases [243,244].

Summary
In recent years widespread and uncontrolled usage of antibiotics and, accordingly, their resistance has emerged as a serious problem. Therefore, simple, sensitive, robust, and rapid methods for evaluation of antibiotics and their residues are needed for an on-site screening analysis. The most used conventional methods for antibiotic detection are instrumental ones, such as capillary electrophoresis (CE), gas chromatography (GC), and liquid chromatography (LC), or coupled with mass spectrometry (LC-MS). Despite their wide range of applications, these methods usually have limitations, such as expensive laboratory instruments, require skilled technicians, and require time consuming separation/sample preparation methodologies. Biosensors are considered as ideal alternatives to detect antibiotics in view of their superiority, such as rapid detection, high selectivity, and in situ applications. Therefore, the development of various biosensors and the design of several new signal transduction schemes is a rapidly growing field in biological, clinical, and environmental sciences.
Among different biosensors, the aptamer-based biosensors (aptasensors) are promising tools. Electrochemical aptamer biosensors compared to other developed aptasensors are the most common ones used for antibiotic detection, because of their operational simplicity, high sensitivity, portability, and low cost. Since antibiotics are most often not electrochemically active by themselves, redox tags like methylene blue, ferrocene, or the commonly used Fe 2+ /Fe 3+ system must be added. Target-induced strand displacement is one of the most widely used signal transduction strategies in aptamer-based biosensors for antibiotic detection. The challenge in replacement reactions is that the affinity of the aptamer towards the target must be stronger than to the complementary DNA (cDNA). The described indirect measuring methods, in which the target is immobilized, are usable for the proof of the function of the developed measurement method, but irrelevant for practical application.
Often a once-established aptamer sequence, specific for a target, is used for almost all further studies. In general, the 5 -end of the aptamer sequence is preferred for immobilization and the 3 -end for labeling (e.g., with FAM) of the aptamer. To evaluate the specificity of the used aptamer towards its target, structurally similar derivates and possible interfering substances are introduced into the sensing system and their influence onto the signal and the detection of the actual target are investigated. In most of the developed aptasensors DNA aptamers were used, and just a few of the mentioned papers dealt with RNA-aptamers. RNA aptamers featured lower K D values and, thus, a higher affinity to their target than DNA aptamers, but RNA is attacked and degraded faster. As shown in the tables, and also compared with the standard HPLC or ELISA methods, sensitivity and selectivity of many aptasensors are acceptable.
The most commonly investigated antibiotics detected by an aptasensor are kanamycin, chloramphenicol, tetracycline, and oxytetracycline. Therefore, a great potential for developing aptamers for other antibiotics with high affinity and specificity exists. Finally, regarding the advantages of aptamers over antibodies, aptamer-based sensors have the potential for clinical/commercial applications and point-of-care detection.
Author Contributions: Y.J. and A.M. contributed to the design and implementation of the research. A.M. wrote the manuscript and P.R. worked on the manuscript. All authors provided critical feedback and helped shape the research and manuscript.
Funding: This work was funded by the European Social Fund (ESF) in Germany.

Conflicts of Interest:
The authors declare no conflict of interest.