Current optical biosensors are based on different optical principles, such as fluorescence, colorimetry, (chemi) luminescence (CL), surface plasmon resonance (SPR), and surface-enhanced Raman scattering (SERS).
3.1. Fluorescent Aptasensors
The main advantages of optical biosensors, like fluorescence and colorimetric aptasensors, are the relatively simple detection procedures and the minimal usage of analytical instruments. Fluorescent aptasensors are one of the most common optical aptasensors developed over the past ten years for the detection of antimicrobial residues in food products [71
]. Fluorescent aptasensors are quick, sensitive, easy to manipulate, and cost-effective. Applications of fluorescent aptasensors to the detection of antimicrobial residues in animal-derived food products are presented in Table 1
Label-free fluorescence aptasensors can be based on DNA intercalating dyes (e.g., Ru (phen)2
(dppz))2+, SYBR Green I [96
], thiazole orange (TO) [101
], metal nanoparticles (NPs) [109
]). The detection is based on the fluorescence signal of intercalating dyes (e.g., TO) that is low in solutions, but increases when the dye intercalates into dsDNA (Figure 2
). When the target is absent, the dye TO intercalates into the aptamer structure, and the emitted fluorescence is strong. When the target is present, the aptamer binds to the target, and so the intercalating dye is released into the solution, and the fluorescence decreases.
New fluorescent probe curcumin has been tested to improve the sensitivity of fluorescent aptasensors compared to classical synthetic dyes (e.g., SYBR Green I) that bind strongly to the aptamer sequence and result in low detection limits [110
]. The aptasensor based on curcumin dye was at least six orders of magnitude more sensitive than sensor constructed with the synthetic dye SYBR Green I. Curcumin is a groove binding ligand (i.e., insertion of curcumin molecule in the voids within the double helix of the ssDNA), while SYBR Green I is an intercalator agent (i.e., intercalates between base pairs). Furthermore, curcumin weakly binds to the aptamer, while SYBR Green I strongly binds to the aptamer. These two parameters could explain the lower sensitivity obtained with curcumin. It was applied for the detection of Vitamin A and Bisphenol A as proofs of concept. It showed that the increase of sensitivity is not linked to only one aptamer and its specific sequence. Therefore, this new probe could also be exploited to develop novel aptasensors for the detection of antimicrobial residues.
The most common format of aptasensor is the use of a fluorescence-labeled aptamer that is a combination of one aptamer labeled with a fluorophore and a quencher (Figure 3
). The fluorescence emitted by the fluorophore (e.g., fluorescein, FAM, cyanine) is quenched in the presence of nanomaterials, such as carbon nanotubes (CNTs), carbon nanoparticles (CNPs), graphene oxide (GO), MoS2
nanostructures, gold nanoparticles (AuNPs), quantum dots (QDs), or composite materials.
Fluorescence resonance energy transfer (FRET) assay is based on the energy transfer between two fluorescent molecules (a donor and an acceptor, such as fluorophore and quencher, respectively). The consequence is a strong fluorescence quenching. When the quencher is far from the fluorescent probe, the fluorescence is increased. On the contrary, when the quencher is close to the fluorescent probe, the fluorescence is quenched. The advantages of FRET-based aptasensors are their rapidity, high sensitivity, good selectivity, and little or no pollution [80
Two major types of fluorescent detection use metal nanoclusters for quenching purposes: “turn-off” mode (fluorescence quench induced by the presence of the target) and “turn-on” mode (fluorescence increases in the presence of the target). For instance, aptamers with a hairpin structure can be labeled with a fluorescent compound and a quenching dye at the 5′ and 3′ end of the aptamer (named aptabeacon), respectively (Figure 4
). Quencher and fluorophore are in close vicinity in such configuration. Therefore, the fluorescence is quenched. The change of conformation of the aptamer in the presence of the target will tend to increase the distance between the fluorophore and the quencher, and, as a consequence, the fluorescence will tend to increase. This is a “turn-on” aptasensor.
In recent years, triple-helix molecular switch (THMS)-based aptasensors have been used for food analysis [111
]. For instance, Tu et al. (2020) developed a THMS-based aptasensor for the detection of chloramphenicol in honey samples (Figure 3
]. This method is based on the formation of the triple-helix molecular switch (THMS) between a signal transduction probe (STP) and a label-free hairpin-shaped aptamer with two arms segments acting as a recognition probe. The STP is a dual-labeled oligonucleotide (labeled with a fluorophore and a quencher at the 3′ and 5′-end). When the STP and the aptamer form the THMS structure, the fluorescence can be emitted. Therefore, when the target analyte is absent, the THMS structure is formed, and the fluorescence increases. On the contrary, the structure cannot be formed when the target analyte is present. In this case, the fluorescence is quenched because the fluorophore and the quencher are in close vicinity in the dual-labeled oligonucleotide. This technique is interesting when compared to the molecular beacon and double-helix molecular switches because, in this case, the aptamer is not labeled, and, therefore, its affinity and specificity are preserved. Furthermore, the THMS structure is very stable.
The use of nanomaterials has led to great improvements in the performance of aptasensors in terms of sensitivity [112
]. Typical nanomaterials, such as carbon dots (CDs) and gold nanoparticles (AuNPs), are often employed as quenchers in FRET-based aptasensors [77
]. More recently, metal-organic frameworks (MOFs) have been exploited because of their highly efficient quenching properties [85
]. Upconversion nanoparticles (UCNPs) are a class of optical nanomaterials doped with lanthanide ions as an activator. UCNPs can be used as highly sensitive label reagents. Wu et al. (2015) combined cDNA to UCNPs, emitting a fluorescent signal for the detection of CAP in milk [99
]. When the target is absent, cDNA binds to the aptamer, and the fluorescence emitted by UNCPs is maximal. When the target is present, the complex UCNPs-cDNA is released from the aptamer-conjugated magnetic nanoparticles. Therefore, the fluorescence decreases. MNPs are often used for both recognition and concentration of the target analyte. The authors have reported a detection limit as low as 0.01 ng/mL.
Quantum dots (QDs) are innovative nanocrystalline semiconductors (10 to 100 Å) (e.g., cadmium telluride quantum dots (CdTe QDs), nitrogen-doped graphene quantum dots (N-GQDs) [114
]). They are constituted of a semiconductor core coated by a semiconductor polymer shell [115
]. Stanisavljevic et al. (2015) presented QDs as the new fluorophores because of their interesting optical properties (e.g., higher fluorescence, photostability, multicolor QDs) [116
]. QDs have been used directly or in conjugation with a biosensing element (i.e., aptamer) for the detection of pesticides and veterinary drug residues [117
]. QDs have been used several times as quenchers in FRET-based aptasensors for the detection of antimicrobial residues in food products [83
]. Wu et al. (2017) developed an aptasensor for the detection of streptomycin in milk [95
]. The signal can be amplified, and so the sensitivity increases, by using exonuclease-assisted target recycling technique (Figure 5
). The aptamer binds first to the single-strand DNA binding protein (SSB) grafted on quantum dots (QDs). QDs tend to aggregate in solution, which leads to the self-quenching effect of QDs. However, in the presence of the target, the aptamer preferentially binds to the target, resulting in the release of QDs and the increase of fluorescence signal. Further use of Exonuclease Exo I to digest the complex aptamer-target will result in the release of the target in the solution for a new binding cycle with other aptamers.
The multiplex detection of authorized antimicrobials in food products is of great importance. Youn et al. (2019) developed a multiplex FRET-based aptasensor for the multiplex detection of three antimicrobials (sulfadimethoxine, kanamycin, and ampicillin) in milk (Figure 6
]. The multiplexing strategy is based on the combined use of three different aptamers, specific for sulfadimethoxine, kanamycin, and ampicillin, labeled with three different fluorophores (cyanine 3 (Cy3), 6-carboxyfluorescein (FAM), and cyanine 5 (Cy5)), quenched by graphene oxides (GO). Furthermore, to improve the sensitivity of the aptasensor, a DNase I-assisted cyclic enzymatic signal amplification (CESA) method is used. DNase I is a non-specific endonuclease that cleaves single-stranded DNA. Graphene oxides are protective elements towards DNase I. The fluorophore-labeled aptamers are non-covalently bound to GO. Therefore, when the antibiotic is added and binds to the aptamer, the fluorophore-labeled aptamers are released in solution and digested by DNase I. The digestion leads to the recycling of antibiotics that can again bind to another fluorophore-labeled aptamer. The recycling of antibiotics allows the amplification of the fluorescence signal and improves the sensitivity of the aptasensor.
Liu et al. (2015) developed a fluorescent aptasensor for the detection of oxytetracycline and kanamycin, based on two aptamers and two signal labels (i.e., two fluorophores: fluorescein amidite or carboxyfluorescein (FAM) and carboxy-X-rhodamine (ROX)) for multiplexing purposes [105
]. Both aptamers are immobilized onto magnetic nanoparticles (MNPs). Two different cDNA are conjugated, each one with a different fluorophore, one with green FAM (520 nm), and one with yellow ROX (608 nm). When both targets (oxytetracycline and kanamycin) are missing, both cDNA can bind to the dual aptamers-MNPs. Therefore, the fluorescent signal reaches maximum intensity at the two wavelengths (520 and 608 nm). When one of the targets is present, it binds with its specific aptamer. Therefore, fluorophore-labeled cDNA is released. The fluorescence signal intensity emitted by this fluorophore declines. With such molecular configuration, it is possible to discriminate between both antimicrobials and to conclude which one is present in a sample. Limits of detection have been set as low as of 0.85 ng/mL and 0.92 ng/mL for kanamycin and oxytetracycline (OTC), respectively.
3.2. Colorimetric Aptasensors
The second type of aptasensors that was developed during the 10 past years for the detection of antimicrobial residues are colorimetric aptasensors. Their advantages are their low cost and the possibility to read the results visually sometimes (simply using the naked eye) or with a smartphone (easy to export the data). Naked eye detection, as well as reading with smartphones, presents a great advantage to develop simple, quick, and cheap aptasensors for field-testing, on-site applications, and self-control in food industries. Wu et al. (2020) developed a colorimetric aptasensor that uses a smartphone for reading (Figure 7
]. The detection limits in the buffer are calculated as low as 7.0 nM and 32.9 for tetracycline (TET) and CAP, respectively.
In the past, colorimetric assays often lacked sensitivity. The use of advanced nanomaterials (e.g., gold nanoparticles (AuNPs), magnetic beads (MBs)) has improved their sensitivity [118
Different types of colorimetric aptasensors have been designed based on Horseradish peroxidase (HRP) or HRP-mimicking DNAzyme and some others based on induced-aggregation of AuNPs [109
]. Examples of colorimetric aptasensors developed for the detection of antimicrobial residues in animal-derived food products are presented in Table 2
• Colorimetric aptasensors based on natural HRP:
Yan et al. (2018) designed a simple colorimetric aptasensor for the detection of chloramphenicol (CAP) in honey and fish, based on HRP covalently bound to a specific aptamer pre-immobilized on a microtiter plate by hybridization with a complementary DNA (cDNA) (Figure 8
]. When CAP interacts with the specific aptamer, the aptamer-HRP conjugate is released from the plate and removed by washings. The enzyme HRP catalyzes the conversion of the chromogenic substrate (e.g., 3,3′,5,5′-tetramethylbenzidine (TMB)) into colored compounds in the presence of hydrogen peroxide (H2
). The detection limit is calculated at 0.0031 ng/mL. Xu et al. (2018) developed a multiplex colorimetric aptasensor for the detection of oxytetracycline and kanamycin in milk combining HRP for colorimetric detection and AuNPs for signal amplification [119
]. The aptamers are hybridized with cDNA (complementary probe) immobilized on magnetic beads (MBs). In the presence of one target, the aptamer is released, and so the cDNA is free to hybridize with the HRP-signal probe immobilized on AuNPs. In this case, HRP can catalyze the conversion of substrates. Two different substrates (TMB and o-phenylenediamine (OPD)) are used to simultaneously detect OTC and kanamycin and discriminate one from the other (when OTC is added, it changes from transparent to deep blue; when kanamycin is added, it changes from transparent to deep yellow).
The polymer reagent (EnVision™ (EV)) that contains 100 HRP per polymer chain can be used to amplify the enzyme-linked chromogenic reaction to improve the sensitivity of aptasensors. Miao et al. (2015) developed an aptasensor, employing EV reagent immobilized onto magnetic nanoparticles (AuMNPs) (Figure 9
]. A double-strand (ds)-DNA antibody is co-immobilized with the enzymes. In the absence of the target, the CAP aptamer is hybridized with its complementary DNA (cDNA) to form the ds-DNA, which is recognized by the ds-DNA antibody. The EV reagent also contains a secondary antibody, which is able to recognize the ds-DNA antibody. The signal amplification is linked to both a high number of enzymes in EV reagent and a large surface of immobilization of MNPs. Similarly, signal amplification can also be obtained using the reagent PowerVision™ (PV) as a nano tracer because PV also contains many HRP enzymes [121
Luan et al. (2017) combined two ways of signal amplification: the use of PV reagent and exonuclease-assisted target recycling (Figure 10
]. The aptamer is bound to HRP (PV reagent) and metallic NPs (AuNPs) (Apt-Au-PV). When the target is present in solution, the aptamer-PV conjugate preferentially binds to the target, and AuNPs are released. Exonuclease I is added, which digests the aptamer, and so releases the target and PV reagent that are ready for a new cycle of reaction with aptamers and AuNPs. These repeated cycles lead to signal amplification. Due to the amplification systems (a combination of PV and exonuclease-assisted target recycling), the sensitivity is improved. Streptomycin can be quantified in milk samples using the linear relationship between streptomycin concentrations and UV absorbance intensity at 650 nm (Figure 11
A,B). The detection limit is as low as 1 pg/mL with the instrumental reading. Furthermore, the color differences between different streptomycin concentrations can be distinguished with the naked eye (Figure 11
• Colorimetric aptasensors based on HRP-mimicking DNAzymes:
As an effective alternative to the natural enzyme, the use of HRP-mimicking DNAzymes (i.e., nucleic acids with catalytic properties) is now getting more interest. The peroxidase-like activity of DNAzymes can be detected with a colorimetric output signal. One big advantage of DNAzymes is that there is no need to produce HRP-conjugates (i.e., antibody-HRP or target-HRP conjugates) that can be easily altered. Therefore, the hemin/G-quadruplex DNAzymes are extensively used in optical and electrochemical biosensors to detect a broad range of targets [144
]. The principle of detection of DNAzyme-based aptasensors relies on the activity of a DNAzyme that is blocked by a cDNA. When the target is present, the aptamer binds to the target and its conformation changes. Then, the cDNA is released from the DNAzyme, which recovers its enzymatic activity. The oxidization of the substrate by the DNAzyme produces the colorimetric signal. Cui et al. (2018) developed a colorimetric aptasensor based on HRP-mimicking DNAzyme (hemin/G-quadruplex) for the detection of kanamycin in milk [123
]. The G-quadruplex structure bound with hemin (ferric chloride heme) becomes an active HRP-mimicking DNAzyme, in the presence of alkali metal cations (e.g., potassium). The formation of the G-rich structure is modulated by the target. Huang et al. (2019) developed a colorimetric aptasensor for the detection of CAP in milk powder based on the aptamer-conjugated magnetic beads for the recognition of the target and the combination of gold nanoparticles and hemin/G-quadruplex DNAzymes for the detection (Figure 12
]. Then, the colorimetric signal is amplified due to the high surface of AuNPs for the immobilization of many hemin/G quadruplex DNAzymes.
Luan et al. (2018) developed a colorimetric aptasensor for the detection of CAP in milk, based on a novel tag, DNAzyme-labeled Fe-MIL-88-Pt (Figure 13
]. Firstly, hairpin cDNA containing the aptamer sequence is immobilized onto magnetic beads (MBs). Secondly, the signal tag is composed of three elements, single strand DNAzyme (G-quadruplex/hemin structure), platinum nanoparticles, and MIL-88 that all together contribute to the catalysis of the substrate tetramethylbenzidine (TMB) in the presence of H2
The catalytic activity is amplified because the three elements possess a peroxidase-like activity. In the absence of the target, the DNAzyme-labeled Pt-MIL88 can hybridize with its cDNA contained in the hairpin DNA-labeled MBs. In this case, the DNAzyme is not free to catalyze the oxidation of the substrate TMB. There is no change in color. Conversely, when the target (CAP) is present, the DNAzymes-labeled Pt-MIL-88 are released, followed by the TMB substrate oxidation and the generation of a blue color. Furthermore, target-triggered circular strand-displacement polymerization (CSDP) has been used for signal amplification [145
]. CSDP is based on the lengthening of an oligonucleotide primer based on Bst DNA polymerase, to replace the target (CAP), and thus release it to initiate a new cycle.
• Colorimetric aptasensors based on peroxidase-like activity of Au nanoparticles (or nanozymes):
More recently, AuNPs are the most widely used nanomaterials in colorimetric aptasensors. Different characteristics of NPs are exploited to develop AuNPs-based optical aptasensors, but with different modes of detection. The first exploited characteristic is the intrinsic peroxidase-like activity of AuNPs. Zhang et al. (2020) developed a very simple colorimetric aptasensor for the detection of tetracycline in milk [138
] based on the intrinsic peroxidase-like activity of gold nanoclusters (AuNCs). The aptamer is combined with AuNCs for two main reasons. Firstly, the aptamer is specific for the detection of tetracycline. Secondly, the aptamer that could bind onto the surface of AuNCs tends to improve their catalytic activity. When tetracycline is absent, the enhanced peroxidase-like activity of aptamer-labeled AuNCs (Apt-AuNCs) oxidizes the substrate TMB to form an intense blue product. When tetracycline is present in solution, the aptamer that binds to the target will alter its conformation. As a consequence, the interaction between the aptamer and AuNCs is modified, the capacity of oxidation is reduced, which leads to a lighter blue color. The signal is inversely proportional to the target concentration. On the contrary, Zhao et al. (2017) developed a colorimetric aptasensor for the detection of streptomycin in milk, based on the inhibition of oxidative capacity (i.e., peroxidase-like activity) of gold nanoparticles when the aptamer is adsorbed onto their surface [125
]. Therefore, in the absence of the target, the aptamer is adsorbed onto the surface of AuNPs, which inhibits the catalytic activity of AuNPs and causes limited substrate oxidation (Figure 14
). When the target is present, the aptamer binds to the target instead of AuNPs, which, in turn, recover their oxidative capacity. The substrate 2,20-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) is usually oxidized to generate a green product. The absorbance value (at 733 nm) is proportional to the streptomycin concentration. Finally, it is noteworthy that the complex streptomycin-aptamer tends to enhance the oxidative capacity of AuNPs, which leads to signal amplification.
• Colorimetric aptasensors based on size-dependent colors of AuNPs:
In this case, the principle of colorimetric detection is based on AuNPs characteristics. The second interesting characteristic of AuNPs is size-dependent colors (dispersed AuNPs: red color, aggregated AuNPs: blue/purple color), high extinction coefficients (>3 orders of magnitude times larger than those of organic dyes [139
]). This change of color is visible by the naked eye. Two different types of aptasensors based on AuNPs can be developed: (i) label-free and (ii) labeled aptasensors.
Label-free aptasensors can be based on target-specific and salt-induced aggregation of unmodified AuNPs. However, aptasensors based on the aggregation of NPs in saline solution are sensitive to environmental conditions (e.g., acidity, ionic strength). Therefore, they are more likely to give false-positive results. In label-free aptasensors, aptamers are physically adsorbed onto unmodified AuNPs, preventing the aggregation of AuNPs. In the presence of the target, the aptamers are released from the AuNPs that are no more protected, and so the AuNPs aggregation leads to a change of color (i.e., from red to purple/blue). Luo et al. (2015) developed a label-free colorimetric aptasensor for the detection of tetracycline in milk [139
]. Cysteamine-stabilized gold nanoparticles (CS-AuNPs) are positively charged, while aptamers (polyanionic DNA) are negatively charged. The attraction between aptamers and NPs leads to the aggregation of NPs; there is no salt-induced aggregation in this case. However, the interaction between the aptamer and its target is stronger than the electrostatic interaction of the aptamer with CS-AuNPs. Therefore, when the target is present in solution, the aptamer binds to its target and prevents the aggregation of the released CS-AuNCs. The color change is ranging from purple/blue to red.
As it was previously seen in fluorescence aptasensors, triple-helix molecular switch (THMS) is also used with colorimetric detection for signal amplification. Ramezani et al. (2015) developed a label-free colorimetric aptasensor that uses gold nanoparticles (AuNPs) for the colorimetric detection associated with THMS as a signal amplifier (Figure 15
]. The THMS structure is formed by the aptamer hybridized with the signal transduction probe (STP) (i.e., label-free STP). The THMS structure is stable when the target is absent. So, the salt-induced (i.e., NaCl) AuNPs aggregation can occur (blue color). In the presence of the target, the STP is released because the aptamer binds to its target, followed by its adsorption onto AuNPs, leading to their dispersion (red color).
Wu et al. (2020) developed a label-free colorimetric aptasensor for the multiplex detection of CAP and oxytetracycline (OTC) in chicken meat and milk, based on salt-induced gold nanoparticles (AuNPs) aggregation (Figure 7
]. In this setting, the capture probe is a multifunctional aptamer formed of two specific aptamers, one for each target, adsorbed onto AuNPs. When the target is absent, the aptamers will tend to adsorb onto the surface of AuNPs, and the aggregation of nanoparticles will be prevented. When one of the targets is present, it binds to its specific aptamer and causes the release from the AuNPs. It is noteworthy that the salt-induced aggregation of AuNPs can occur. Because the two aptamers for CAP and OTC are formed of fragments of different lengths (i.e., the length fragment for OTC is a fifth of that for CAP), their binding affinities to nanoparticles can be different (i.e., shorter sequence (OTC), stronger affinity). Consequently, different color changes can be observed, depending on the nature of the target that is present in the solution. Label-free aptasensors are easy to prepare, cost-effective, and time-saving.
Labeled aptasensors based on AuNPs modified with aptamers or their specific probes are developed [126
]. The aptamer can be first covalently bound onto the AuNPs. Then, a further step is to purify modified AuNPs by separating them from unmodified AuNPs and free aptamers. This extra step is time-consuming, increases the cost, and can modify the affinity of the aptamer towards its target. In the presence of the target, the target binds to the AuNPs-labeled aptamer, and so the aggregated AuNPs are dispersed, changing the color from purple to red. Lateral flow devices (LFD) (i.e., dipsticks) are simple immunoassay systems intended to detect the presence of a target analyte in a sample. This principle can be applied to the fabrication of dipsticks, by the immobilization of aptamer-linked NPs aggregates onto the pad (i.e., cellulose membrane) [31
]. Aptamer-linked NP aggregates are spotted on the conjugation pad, while streptavidin is spotted as a thin line. When the target analyte is present, it binds to the aptamer, and so the NP aggregates disassemble. Dispersed biotinylated nanoparticles can migrate along the membrane and be captured by streptavidin. Then, a red line appears. LFD is much more convenient and more sensitive aptasensors than colorimetric assays in solution, especially for point of care (POC) testing. It can be applicable to the detection of antimicrobial residues in foodstuffs. When an aptamer binds covalently to AuNPs, a conformational change of the aptamer occurs in the presence of the target, it then induces the aggregation of AuNPs in a salt solution, and subsequently causes a change of color from red to purple [126
]. Abnous et al. (2018) used a further approach when they have developed an aptasensor based on aptamers that bind to AuNPs and specific DNA probes [147
]. In such settings, aptamers would hybridize with their complementary ssDNAs, and the newly formed dsDNA would stabilize the AuNPs that cannot aggregate anymore, and hence the color would stay red. In the presence of the target, the DNA probe and the aptamer would be released because the aptamer tends to bind preferentially to its specific target. Consequently, the AuNPs would aggregate, and the color would turn to blue.
Instead of using NPs, aptamers can bind to the surface of a conducting colored polymer (polydiacetylene (PDA)) liposomes. The PDA liposomes change their color by external stimuli, including ligand interaction, temperature, solvent, and pH. When the aptamer binds to the target, the color of liposomes changes [31
Song et al. developed a dual-detection method for the detection of ampicillin in milk [143
]. Two modes of detection—colorimetric and fluorimetric—are simultaneously employed because AuNPs are both used as a quencher for the fluorescent reagent (FAM) and as a color signal probe due to the salt-induced aggregation/dispersion phenomenon of AuNPs. When ampicillin is absent in solution, the FAM-aptamer is adsorbed onto the AuNPs, and the fluorescence is quenched by AuNPs. The AuNPs are then dispersed, producing a red color signal. In the presence of ampicillin and salt (NaCl), the aptamer binds to its target and is desorbed from AuNPs. As a consequence, the fluorescence signal is recovered, and simultaneously AuNPs can aggregate to produce a color transition from red to purple/blue. The detection limits of fluorescence and colorimetric aptasensors range from 2 ng/mL to 10 ng/mL, respectively, for ampicillin in milk. Emrani et al. (2016) also proposed a dual-detection aptasensor for streptomycin in milk (Figure 16
]. The limits of detection for FRET and colorimetric aptasensors are equal to 47.6 and 73.1 nM, respectively. These two studies are in favor of using fluorescence aptasensors than colorimetric aptasensors because of lower detection limits.