# Recent Advances in Optical Detection of Aminoglycosodies
## 2 Fluorescent Assays
### Turn-Off Fluorescent Analysis Strategies  
The turn-off sensing strategy means the presence of a target analyte induces a decrease in FL intensity. The FL signals are inversely proportional to the centration of the target analyte. DNA-binding dyes including double-strand DNA (dsDNA)-binding dyes (such as Synergy Brands, Inc. (SYBR) Green I) and G-quadruplex structure-binding dyes (such as Thioflavin T) have been employed to construct turn-off FL sensing methods for AGs [24; 25; 26]. It has been reported that anti-kanamycin-aptamers can form intermolecular G-quadruplex structures [27; 28]. Thus, G-quadruplex structure-sensitive dyes such as Thioflavin T, crystal violet, thiazole orange, N-methyl mesoporphyrin IX, etc., have been tested to develop FL sensing methods for kanamycin. Among them, Thioflavin T has the best performance and responds to the anti-kanamycin-aptamer specifically. The binding of kanamycin to the anti-kanamycin-aptamer disturbs the G-quadruplex structure, resulting in a decreased FL signal at 492 nm. For the detection of kanamycin, it took only 20 min and the limit of detection (LOD) was 0.3 nM [25]. SYBR Green I is a dsDNA-inserting FL molecule, which emits obvious FL in the presence of dsDNA. The hybridization of an aptamer with its complementary DNA forms a double-stranded helix. The binding of target aminoglycos to  
Figure 2: Illustration of optical detection methods for AGs. aptamers transforms the hybrid dsDNA into single-strand DNA (ssDNA), causing a decrease in the FL signal (Figure 3A). Based on this sensing scheme, [PERSON]'s group constructed a smartphone-based aptasensor for the point-of-care testing of streptomycin. A Touch Color application installed in a smartphone was employed to read the RGB values of the images, and the G value was used to quantify the streptomycin concentrations The POCT method was used to detect streptomycin in milk and the detection limit was 94 nM [26]. Almost all of the MIP-based sensing methods are turn-off mode FL sensors. MIPs are polymers processed using molecularly imprinted techniques that leave cavities in the polymer matrix and have an affinity for the template molecules. For the FL sensing of AGs, FL-emitting nanomaterials such as QDs [29; 30; 31] and FL-emitting metal-organic framework (MOF) NPs [32] have been employed as supports to construct MIPs. Typically, [PERSON] and [PERSON]'s group fabricated a MIP-coated europium metal-organic framework by using 4-vinylpyridine as the main monomer, ethylene glycol dimethacrylate as the cross-linking monomer, and limomycin as the template. The europium metal-organic framework has a long, persistent FL and the MIP layer provides specific binding sites for limomycin. The adsorption of limomycin into the cavities leads to a decrease in the FL intensity of the europium metal-organic framework. The LOD for limomycin was calculated to be 16.2 nM [32]. In addition, dual recognition MIPs have been constructed for highly specific sensing of AGs [29; 30]. For example, a dual-recognition MIP was constructed for the highly specific sensing of kanamycin by using CdSe QDs as supports, methacrylic acid, and thiol-modified aptamers as functional monomers. Through a thiol-ene click reaction, the aptamer was fixed into the polymer matrix. The prepared MIP had dual recognition functions from both aptamer and imprinted cavities, which improved the recognition ability of MIP toward kanamycin. The imprinting factor of the prepared MIPs was 3.51 for kanamycin. CdSe QDs have strong FL emissions around 530 nm; the binding of kanamycin to the MIP caused a decrease in the FL signal (Figure 3B). The MIP-based FL sensing platform showed a linear range from 85.7 nM to 17.16 \(\upmu\)M for the detection of kanamycin, and the LOD was 1.1 nM [29]. In addition to these sensing methods, [PERSON]'s group established a whole-cell-based bioassay for the detection of antibiotics based on the inhibitory effect of antibiotics on the growth of sensitive bacteria.