*2.1. Enzymes-Based Probes*

Enzymes are robust signal amplification systems in bioassays and biosensors. They can be used in both optical and electrochemical sensing strategies. The principle of the enzymebased colorimetric assays is to detect target analyte through the enzymatic conversion of a chromogenic substrate into a colored product. The produced color can be detected by the naked eye (qualitative methods) and through spectrophotometry or colorimetric analysis software (semi-quantitative or quantitative methods). The three most common types of enzyme-based colorimetric probes—including enzyme horseradish peroxidase (HRP), G-quadruplex sequences or DNAzymes, and acetylcholinesterase (AChE)—have been applied for colorimetric detection of mycotoxins and are hence discussed here.

HRP, found in the roots of horseradish plant, is the most popular enzymatic marker in bioassays due to ability to be conjugated with antibodies or other recognition elements, while preserving its activity, low-cost, and versatility. HRP can catalyze the reaction of hydrogen peroxide with certain organic, electron-donating substrates to yield highly colored products. An extensive range of electron-donating dye substrates are commercially available for use as HRP detection reagents. Some of them can be employed to form soluble colored products suitable for use in spectrophotometric detection methods, while other substrates form insoluble products that are mainly appropriate for staining techniques. Among them, 3,30 ,5,50 -Tetramethylbenzidine or TMB is widely used as a soluble chromogenic substrate for colorimetric detection in enzyme-linked immunosorbent assay (ELISA) and other bioassays. However, some HRP-based bioassays suffer from limited sensitivity due to small amount of enzyme (i.e., HRP) that catalyzes chromogenic substrate. To address this issue, Lin et al. presented a method to combine the analyte-recognition element complex with a large number of enzymes [11]. They developed a liposome-based colorimetric aptasensor for ochratoxin A (OTA) detection in a TMB-H2O<sup>2</sup> reaction medium. In this context, liposome as a sphere-shaped vesicle with hydrophobic and hydrophilic character was used for encapsulation of HRP. The main component of the detection system was a dumbbell-shaped probe including magnetic beads (MBs), double-stranded DNA (dsDNA), and HRP-encapsulated liposome (Figure 1a). The dsDNA was formed by the hybridization between OTA aptamer and its complementary probes ssDNA-1 and ssDNA-2. ssDNA-2 was conjugated with liposome and used as detection probe. In the presence of OTA, the aptamer combined with OTA to form G-quadruplex, resulting in the release of the ssDNA-2 and the HRP-encapsulated liposome. Each liposome containing a large quantity of HRP was lysed by adding the mixed solution of TMB and H2O2. HRP catalyzed H2O2-mediated oxidation of TMB and resulted in color change from colorless to blue. The assay was highly sensitive due to the signal amplification caused by the large amount of HRP embedded in liposome. The limit of detection (LOD) was obtained 0.023 ng·mL−<sup>1</sup> . The assay was simple, low-cost, highly selective and reliable for the analysis of real samples. However, the reaction time for the G-quadruplex formation (40 min) and TMB oxidation (20 min) was too long. The aptasensor was also applied for OTA detection in corn samples.

Haem peroxidases such as HRP use protein scaffolds that activate heme to react with H2O<sup>2</sup> [12]. There is extensive information on the reaction mechanism and properties of protein-based peroxidases. It was recently revealed that certain nucleic acid sequences have the ability to catalyze reactions similar to those carried out by heme. These nucleic acid sequences are non-canonical Guanine-rich structures with stacked G-tetrads assembled by Hoogsteen hydrogen-bonding. These sequences, named as G-quadruplex (G4), are able to bind hemin (iron (III)-protoporphyrin IX) to form a unique type of G4 DNAzyme or RNAzyme with powerful peroxidase-mimicking activity [13]. In comparison with natural protein peroxidases, G4 DNAzymes/RNAzymes show several advantages such as

small size, easy synthesis, more stability, and facile manipulation, which make them good candidates in biosensing [13]. However, they suffer from relatively low catalytic activity compared to protein peroxidases which restricts their further development and application [14]. To overcome this limitation, several strategies have been developed to improve the catalytic efficiency of G4 DNAzymes/RNAzymes. These include (1) addition of polycationic amines such as spermine, spermidine, and putrescine; (2) addition of the nucleotide ATP to DNAzyme reactions; (3) conjugation of hemin with the G4-quadruplex moiety through covalent linkage or with cationic peptides; and (4) flanking adenine or cytosine nucleotides on G-quadruplex activities [12]. Incorporation of aptamers and DNAzymes as functional nucleic acids results in simple detection of target analyte by visual color development. bled by Hoogsteen hydrogen-bonding. These sequences, named as G-quadruplex (G4), are able to bind hemin (iron (III)-protoporphyrin IX) to form a unique type of G4 DNAzyme or RNAzyme with powerful peroxidase-mimicking activity [13]. In comparison with natural protein peroxidases, G4 DNAzymes/RNAzymes show several advantages such as small size, easy synthesis, more stability, and facile manipulation, which make them good candidates in biosensing [13]. However, they suffer from relatively low catalytic activity compared to protein peroxidases which restricts their further development and application [14]. To overcome this limitation, several strategies have been developed to improve the catalytic efficiency of G4 DNAzymes/RNAzymes. These include (1) addition of polycationic amines such as spermine, spermidine, and putrescine; (2) addition of the nucleotide ATP to DNAzyme reactions; (3) conjugation of hemin with the G4-quadruplex moiety through covalent linkage or with cationic peptides; and (4) flanking adenine or cytosine nucleotides on G-quadruplex activities [12]. Incorporation of aptamers and DNAzymes as functional nucleic acids results in simple detection of target

acid sequences are non-canonical Guanine-rich structures with stacked G-tetrads assem-

analyte by visual color development.

**Figure 1.** (**a**) Schematic illustration of the colorimetric aptasensor for OTA detection based on HRPencapsulated liposome; (**b**) developed aptasensor for AFB1 detection using G-quadruplex as the signal reporter. Domain a is complementary to domain a\*. Domain b is the caged G-rich sequence. Exo III performs the cyclic cleavage reactions in Cycles I and II; (**c**) aptasensor for OTA detection, based on rolling circle amplification and an auto-catalytic DNAzyme structure. Reproduced with permission from [11,15,16], respectively. Using G-quadruplex as the signal reporter, a colorimetric aptasensor was developed for AFB1 detection [15]. The aptasensor was fabricated by the combination of an ingenious **Figure 1.** (**a**) Schematic illustration of the colorimetric aptasensor for OTA detection based on HRPencapsulated liposome; (**b**) developed aptasensor for AFB1 detection using G-quadruplex as the signal reporter. Domain a is complementary to domain a\*. Domain b is the caged G-rich sequence. Exo III performs the cyclic cleavage reactions in Cycles I and II; (**c**) aptasensor for OTA detection, based on rolling circle amplification and an auto-catalytic DNAzyme structure. Reproduced with permission from [11,15,16], respectively.

hairpin DNA probe with exonuclease III (Exo III)-assisted signal amplification. The hairpin DNA probe contained a 3′-protruding segment (domain a) as the recognition unit, the stem zone (domains a and a\*), and a caged G-rich sequence located in the loop region (domain b). The presence of the AFB1 activated the continuous cleavage reactions by Exo Using G-quadruplex as the signal reporter, a colorimetric aptasensor was developed for AFB1 detection [15]. The aptasensor was fabricated by the combination of an ingenious hairpin DNA probe with exonuclease III (Exo III)-assisted signal amplification. The hairpin DNA probe contained a 30 -protruding segment (domain a) as the recognition unit, the stem zone (domains a and a\*), and a caged G-rich sequence located in the loop region (domain b). The presence of the AFB1 activated the continuous cleavage reactions by Exo III toward a hairpin probe, resulting in the autonomous accumulation of numerous free G-quadruplex sequences, which catalyzed the oxidation of TMB by H2O<sup>2</sup> to generate a colorimetric signal (Figure 1b). The aptasensor represented many advantages including high sensitivity (LOD of 1 pM), good selectivity, simple operation, wash-free, label-free format, low-cost, naked-eye detection, and applicability to samples with complex matrices. However, the assay time was long (incubation time 40 min). The assay was used for AFB1 detection in peanut samples.

Detection with the aid of magnetic beads-based separation has emerged as a rapid, simple, reliable, and efficient alternative to conventional immobilization methods. In

this regard, a colorimetric aptasensor based on apta-magnetic separation assisted with DNAzyme was developed for AFB1 detection [17]. The procedure consisted of one-step separation of AFB1 by biotinylated aptamer conjugated to streptavidin magnetic beads which was followed by the addition of DNAzyme modified aptamer in the presence hemin and TMB/H2O<sup>2</sup> to produce a colorimetric signal. The aptasensor was able to detect as low as 40 ppb and 22.6 ppb OTA visually and by spectrophotometer, respectively. The developed assay was selective, reliable, inexpensive, and rapid (incubation time 15 min). However, the incubation time of DNAzyme was long (30 min). The aptasensor was able to detect AFB1 in food samples.

Sensitivity of DNA-based biosensors can be significantly increased using a technique known as rolling circle amplification (RCA). RCA is an isothermal enzymatic amplification process of DNA where a short DNA or RNA primer is amplified using circular DNA template. With proper application of this technique, it is possible to synthesize large quantities of any type of nucleic acid strand. In this context, a highly sensitive aptasensor based on RCA and an auto-catalytic DNAzyme structure was designed for OTA detection [16]. In this work, a capture aptamer was linked to paramagnetic beads for specific capturing of OTA while a second aptamer was applied for OTA detection. The detection aptamer contained a DNAzyme producing sequence and an RCA priming sequence for the isothermal DNA amplification triggered by a circular ssDNA. When OTA was captured, the circular DNA was amplified, generating a single-stranded and tandem repeated long homologous copy of its sequence. In the DNA strand, a self-catalytic structure was formed with hemin as the catalytic core causing a blue color in the presence of 2,20 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and H2O<sup>2</sup> (Figure 1c). A low LOD of 1.09 <sup>×</sup> <sup>10</sup>−<sup>12</sup> ng·mL−<sup>1</sup> was obtained. Although the aptasensor was highly sensitive and selective, it suffered from long incubation time and complicated operation with multiple steps of washing and separation. The aptasensor was used for OTA detection in urine samples.

There are various enzymes—such as cholinesterase, urease, glucose oxidase, etc. which have been employed in mycotoxin detection methods based on enzymatic inhibition. AChE (obtained from electric eel) is the most commonly used enzyme due to its susceptibility toward mycotoxin [7]. It can be used for the detection of aflatoxin B1 (AFB1) due to the inhibitory effect of AFB1 to AChE enzymatic activity [18]. It has been proven that AChE is inhibited by the AFB1 due to non-covalently binding of toxin at the external site, which is placed on the active site gorge entrance (located at the tryptophan residue) [7]. Based on AChE inhibition, AFB1 was determined by a colorimetric method (Ellman's method) developed on chromatography paper [19]. In this work, genipin cross-linked chitosan was used for AChE immobilization. For the colorimetric detection of AFB1 on microfluidic paper-based analytical device (µPAD), AChE immobilized on cross-linked chitosan was loaded on the edges of the flower-shaped µPAD. Then, AFB1 solution and 5,5-dithiobis-2-nitrobenzoic acid (DTNB, Ell-man's reagent) solution were applied at the center of flower-shaped µPAD. After 3-min incubation, acetylthiocholine iodide (ATCh) solution was also added at the center, and incubated for 5 min. The Ellman's colorimetric assay is based on the reaction of thiocholine (a product of enzymatic hydrolysis of ATCh) with DTNB to form a colored product. In the presence of AFB1, the AChE activity on ATCh substrate is inhibited resulting in failure to form a colored product. Cross-linking of chitosan resulted in a colorimetric signal enhancement. The assay was simple, low-cost, rapid (detection time ≈ 8 min), and fairly selective. However, the sensitivity of the assay was not reported. The assay was used for the detection of AFB1 in spiked corn samples.

AChE is considered very stable but lack of selectivity towards many toxins such as carbamates, organophosphate pesticides, anatoxin-a (a natural neurotoxic), and mycotoxins, which restrict its applicability. To address this issue, many efforts have been made to produce mutants of AChEs to improve the selectivity of enzyme against a specific toxin. Genetic modification of enzyme can also improve its stability and the assay sensitivity [20].

Representative examples of recent developed enzyme-based probes for the colorimetric detection of mycotoxins are reported in Table 1.
