Label-Free Fluorescent Aptasensor for Ochratoxin—A Detection Based on CdTe Quantum Dots and (N-Methyl-4-pyridyl) Porphyrin

With the widespread contamination of ochratoxin A (OTA), it is of significant importance for detecting OTA in foods and traditional Chinese medicine (TCM). In this study, a novel label-free fluorescent aptasensor utilizing the interaction between OTA-triggered antiparallel G-quadruplex and (N-methyl-4-pyridy) porphyrin (TMPyP) for the rapid and sensitive determination of OTA was established. The fluorescence of CdTe quantum dots (QDs) could be quenched by TMPyP. In the presence of analyte (OTA), the aptamer could recognize OTA and transform from a random coil to the antiparallel G-quadruplex. The interaction between G-quadruplex and TMPyP could release CdTe QDs from TMPyP, and thus recover the fluorescence of CdTe QDs. Under optimized conditions, the detection limit of the designed aptasensor was 0.16 ng mL−1, with a linear range of 0.2 to 20 ng mL−1. Furthermore, this aptasensor showed high selectivity toward OTA against other structural analogs and other mycotoxins, and was successfully applied in Astragalus membranaceus samples. The presented aptasensor for OTA detection could be a promising tool for the field monitoring of food and TCM.


Introduction
Ochratoxin A (OTA) is a toxic metabolite derived mainly from Aspergillus ochraceus and Penicillium verrucosum [1,2], which are widely present as contaminants in a variety of products, including wines, corn, coffee, milk, and traditional Chinese medicine (TCM) [3,4]. OTA is found to cause severe toxic effects, such as those that are teratogenic, embryotoxic, genotoxic, neurotoxic, immunosuppressive, carcinogenic, and nephrotoxic to humans [5]. Since OTA presents a serious threat to the health and safety of humans, it was classified as being possibly carcinogenic to humans (Group 2B) by the International Agency for Research on Cancer (IARC) [6]. Astragalus membranaceus, as a major medicinal herb, has been broadly applied in clinical settings for treating diseases and promoting health [7]. It is especially important to ensure the safety of TCM, which has been frequently contaminated by mycotoxins during growth, collection, transportation, and storage [8,9].

Design Strategy for OTA Detection
Scheme 1 shows the design strategy of a label-free fluorescent aptasensor for OTA detection. The fluorescence intensity of CdTe QDs was effectively quenched by TMPyP through PET [27]. In the presence of the analyte (OTA), the aptamers recognized the target and were induced to conformationally transform into the antiparallel G-quadruplex structure [28]. As TMPyP is a distinguished G-quadruplex ligand [29], it could interact with G-quadruplexes by outside groove binding and terminal π-π stacking interactions [30]. Since TMPyP occupied by G-quadruplexes was buried inside the structures, the interaction force between the CdTe QDs and the TMPyP became very weak, resulting in a significantly enhancement in the fluorescence intensity. Therefore, this restoration of fluorescence was interpreted as an analytical signal to evaluate the amount of OTA. To further validate the feasibility of the established method, corresponding control experiments were carried out. In the absence of TMPyP, the fluorescence intensity of the CdTe QDs/aptamer was highly overlapping with or without OTA ( Figure 1A). However, when TMPyP was included in the determination steps, the sample containing 20 ng mL −1 of OTA showed significantly higher fluorescence intensity than the blank sample ( Figure 1B). This phenomenon proved that the fluorescence recovery of CdTe QDs was indeed caused by the combination of the TMPyP and Gquadruplex.

Characterization of G-Quadruplex Formation
To verify the formation of the antiparallel G-quadruplex, the circular dichroism (CD) spectrum was employed. Before the introduction of OTA, the aptamer presented the characteristic of randomcoil DNA (a in Figure 2). After the addition of OTA (5 μg mL −1 ), the CD spectrum exhibited an evident enhancement at about 265 nm for the negative band and at 290 nm for the positive band (b in Figure  2). This signal change in the CD spectra could be due to the interaction of OTA with the aptamer, which is a notable characteristic of antiparallel G-quadruplex formation [31]. To further validate the feasibility of the established method, corresponding control experiments were carried out. In the absence of TMPyP, the fluorescence intensity of the CdTe QDs/aptamer was highly overlapping with or without OTA ( Figure 1A). However, when TMPyP was included in the determination steps, the sample containing 20 ng mL −1 of OTA showed significantly higher fluorescence intensity than the blank sample ( Figure 1B). This phenomenon proved that the fluorescence recovery of CdTe QDs was indeed caused by the combination of the TMPyP and G-quadruplex. To further validate the feasibility of the established method, corresponding control experiments were carried out. In the absence of TMPyP, the fluorescence intensity of the CdTe QDs/aptamer was highly overlapping with or without OTA ( Figure 1A). However, when TMPyP was included in the determination steps, the sample containing 20 ng mL −1 of OTA showed significantly higher fluorescence intensity than the blank sample ( Figure 1B). This phenomenon proved that the fluorescence recovery of CdTe QDs was indeed caused by the combination of the TMPyP and Gquadruplex.

Characterization of G-Quadruplex Formation
To verify the formation of the antiparallel G-quadruplex, the circular dichroism (CD) spectrum was employed. Before the introduction of OTA, the aptamer presented the characteristic of randomcoil DNA (a in Figure 2). After the addition of OTA (5 μg mL −1 ), the CD spectrum exhibited an evident enhancement at about 265 nm for the negative band and at 290 nm for the positive band (b in Figure  2). This signal change in the CD spectra could be due to the interaction of OTA with the aptamer, which is a notable characteristic of antiparallel G-quadruplex formation [31].

Characterization of G-Quadruplex Formation
To verify the formation of the antiparallel G-quadruplex, the circular dichroism (CD) spectrum was employed. Before the introduction of OTA, the aptamer presented the characteristic of random-coil DNA (a in Figure 2). After the addition of OTA (5 µg mL −1 ), the CD spectrum exhibited an evident enhancement at about 265 nm for the negative band and at 290 nm for the positive band (b in Figure 2). This signal change in the CD spectra could be due to the interaction of OTA with the aptamer, which is a notable characteristic of antiparallel G-quadruplex formation [31].

Optimization of the Concentration of Porphyrin and Incubation Time
To achieve high sensitivity for the label-free aptasensor, we optimized the concentration of TMPyP and incubation time. A higher concentration of TMPyP can largely quench the fluorescence intensity of CdTe QDs, and at a sufficient concentration of TMPyP, the fluorescence intensity of CdTe QDs can be completely quenched. However, an excess of quencher will inhibit the fluorescence recovery of CdTe QDs after the addition of an OTA aptamer, which is possibly due to the interaction between the G-quadruplex and "free" TMPyP. On the other hand, TMPyP deficiency does not effectively quench the fluorescence intensity of CdTe QDs, which may affect the detection of OTA. The value, I, was introduced to describe relative fluorescence recovery rates: where F0 and F1 are the fluorescence intensity of CdTe QDs with and without TMPyP, respectively, and F2 represents the fluorescence intensity in the presence of the OTA aptamer. As shown in Figure  3, excessive or insufficient TMPyP is not advantageous to the fluorescence recovery of CdTe QDs. When the concentration of TMPyP was 0.175 μmol L −1 , the fluorescence of QDs was quenched to 39.67%, and recovered to 86.90% of the original fluorescence of CdTe QDs after being exposed to OTA (20 ng mL −1 ). Based on this result, the concentration of TMPyP was fixed as 0.175 μmol L −1 . As shown in Figure 3C, the incubation time was optimized, the fluorescence recovery reaches a plateau at 5 min. Therefore, the five minutes was chosen as the incubation time.

Optimization of the Concentration of Porphyrin and Incubation Time
To achieve high sensitivity for the label-free aptasensor, we optimized the concentration of TMPyP and incubation time. A higher concentration of TMPyP can largely quench the fluorescence intensity of CdTe QDs, and at a sufficient concentration of TMPyP, the fluorescence intensity of CdTe QDs can be completely quenched. However, an excess of quencher will inhibit the fluorescence recovery of CdTe QDs after the addition of an OTA aptamer, which is possibly due to the interaction between the G-quadruplex and "free" TMPyP. On the other hand, TMPyP deficiency does not effectively quench the fluorescence intensity of CdTe QDs, which may affect the detection of OTA. The value, I, was introduced to describe relative fluorescence recovery rates: where F 0 and F 1 are the fluorescence intensity of CdTe QDs with and without TMPyP, respectively, and F 2 represents the fluorescence intensity in the presence of the OTA aptamer. As shown in Figure 3, excessive or insufficient TMPyP is not advantageous to the fluorescence recovery of CdTe QDs. When the concentration of TMPyP was 0.175 µmol L −1 , the fluorescence of QDs was quenched to 39.67%, and recovered to 86.90% of the original fluorescence of CdTe QDs after being exposed to OTA (20 ng mL −1 ). Based on this result, the concentration of TMPyP was fixed as 0.175 µmol L −1 . As shown in Figure 3C, the incubation time was optimized, the fluorescence recovery reaches a plateau at 5 min. Therefore, the five minutes was chosen as the incubation time.

Quantitative Analysis of OTA
Under the optimized conditions, we investigated the fluorescence spectrum after the addition of OTA. As shown in Figure 4A, a gradual increase in the fluorescence intensity at 555 nm was observed following an increase in OTA concentration from 0.2 to 20 ng mL −1 . Figure 4B shows the linear relationship between the fluorescence intensity and the concentration of OTA (0.2 to 20 ng mL −1 ) (R 2 = 0.994). The limit of detection (LOD) for OTA was calculated to be 0.16 ng mL −1 by three times the standard deviation of the background (3σ). As shown in Table 1, the linear range and LOD of the fluorescent aptasensor were compared with the current methods, and it was concluded that the current fluorescent aptasensor for the detection and quantification of OTA has a satisfactory linear dynamic range and detection limit. The repeatability was obtained by measuring the fluorescent intensity of the same sample (15 ng mL −1 , OTA) six times, in which the coefficient of variation (CV) was 2% and the reproducibility was measured in different days, with a CV less than 2%, showing the good repeatability and reproducibility of this method.

Quantitative Analysis of OTA
Under the optimized conditions, we investigated the fluorescence spectrum after the addition of OTA. As shown in Figure 4A, a gradual increase in the fluorescence intensity at 555 nm was observed following an increase in OTA concentration from 0.2 to 20 ng mL −1 . Figure 4B shows the linear relationship between the fluorescence intensity and the concentration of OTA (0.2 to 20 ng mL −1 ) (R 2 = 0.994). The limit of detection (LOD) for OTA was calculated to be 0.16 ng mL −1 by three times the standard deviation of the background (3σ). As shown in Table 1, the linear range and LOD of the fluorescent aptasensor were compared with the current methods, and it was concluded that the current fluorescent aptasensor for the detection and quantification of OTA has a satisfactory linear dynamic range and detection limit. The repeatability was obtained by measuring the fluorescent intensity of the same sample (15 ng mL −1 , OTA) six times, in which the coefficient of variation (CV) was 2% and the reproducibility was measured in different days, with a CV less than 2%, showing the good repeatability and reproducibility of this method.

Specificity of the OTA Aptasensor
To confirm that the fluorescence intensity signal was caused by the unique recognition between aptamers and OTA, it was critical to test the specificity of the fluorescent aptasensor against other structure analogs of OTA (ochratoxin B (OTB) and ochratoxin C (OTC)) and other mycotoxins (zearalenone (ZEA) and HT-2 toxin (HT-2)). The concentration of OTA was 15 ng mL −1 , whereas the concentration of the other toxins was 25 ng mL −1 in the specificity experiment. As shown in Figure 5, the fluorescence intensity displayed no evident change in the presence of HT-2, ZEN, OTB, and OTC. Considering that a sample may contain multiple mycotoxins, we also investigated the recognition performance of OTA in the presence of other mycotoxins. The signal responses did not change significantly when other mycotoxins were introduced to the aptasensor. These results clearly demonstrated that this fluorescent aptasensor for the determination of OTA exhibited an excellent specificity against different kinds of mycotoxins. The high specificity of the reported sensing platform was mainly due to the high affinity of the OTA aptamer.

Specificity of the OTA Aptasensor
To confirm that the fluorescence intensity signal was caused by the unique recognition between aptamers and OTA, it was critical to test the specificity of the fluorescent aptasensor against other structure analogs of OTA (ochratoxin B (OTB) and ochratoxin C (OTC)) and other mycotoxins (zearalenone (ZEA) and HT-2 toxin (HT-2)). The concentration of OTA was 15 ng mL −1 , whereas the concentration of the other toxins was 25 ng mL −1 in the specificity experiment. As shown in Figure 5, the fluorescence intensity displayed no evident change in the presence of HT-2, ZEN, OTB, and OTC. Considering that a sample may contain multiple mycotoxins, we also investigated the recognition performance of OTA in the presence of other mycotoxins. The signal responses did not change significantly when other mycotoxins were introduced to the aptasensor. These results clearly demonstrated that this fluorescent aptasensor for the determination of OTA exhibited an excellent specificity against different kinds of mycotoxins. The high specificity of the reported sensing platform was mainly due to the high affinity of the OTA aptamer.

Detection of OTA in Real Samples
To evaluate the practicability and reliability of the presented aptasensor, we challenged our aptasensor with Astragalus membranaceus samples. As expressed in Table 2, the recoveries of OTAspiked samples ranged from 98.9% to 102.2%, suggesting that this label-free aptasensor can be applied for the detection of OTA analysis in Astragalus membranaceus. However, CdTe QDs and the TMPyP based sensing system have limitations in detecting OTA in complex environmental samples, which may frequently contain nickel ions, Hg (II) ions, and pesticides. These coexistences can quench the fluorescence intensity of CdTe QDs and then affect the sensing ability of OTA detection.

Conclusions
In summary, a label-free fluorescent aptasensor based on the TMPyP/G-quadruplex for OTA detection was reported. Benefiting from a high specificity and affinity between the OTA and aptamer, the aptamer was switched into G-quadruplex, which could combine with TMPyP. This detection strategy avoids the laborious and expensive process of DNA modification. The designed sensing platform exhibits a superior range of detection and potential selectivity. The linear dynamic range and detection limits were 0.2 to 20 ng mL −1 and 0.16 ng mL −1 , respectively. The application of the aptasensor has been verified in Astragalus membranaceus, with relative recovery values of 98.9% to 102.2%. This work provides a brand-new strategy for the OTA aptasensor based on the Gquadruplex-aptamer and TMPyP, and has great potential for applications for OTA analysis in food and TCM contamination detection.

Detection of OTA in Real Samples
To evaluate the practicability and reliability of the presented aptasensor, we challenged our aptasensor with Astragalus membranaceus samples. As expressed in Table 2, the recoveries of OTA-spiked samples ranged from 98.9% to 102.2%, suggesting that this label-free aptasensor can be applied for the detection of OTA analysis in Astragalus membranaceus. However, CdTe QDs and the TMPyP based sensing system have limitations in detecting OTA in complex environmental samples, which may frequently contain nickel ions, Hg (II) ions, and pesticides. These coexistences can quench the fluorescence intensity of CdTe QDs and then affect the sensing ability of OTA detection.

Conclusions
In summary, a label-free fluorescent aptasensor based on the TMPyP/G-quadruplex for OTA detection was reported. Benefiting from a high specificity and affinity between the OTA and aptamer, the aptamer was switched into G-quadruplex, which could combine with TMPyP. This detection strategy avoids the laborious and expensive process of DNA modification. The designed sensing platform exhibits a superior range of detection and potential selectivity. The linear dynamic range and detection limits were 0.2 to 20 ng mL −1 and 0.16 ng mL −1 , respectively. The application of the aptasensor has been verified in Astragalus membranaceus, with relative recovery values of 98.9% to 102.2%. This work provides a brand-new strategy for the OTA aptasensor based on the G-quadruplex-aptamer and TMPyP, and has great potential for applications for OTA analysis in food and TCM contamination detection.

Instrumentation
CD spectra were measured on a JASCO J-1500 spectropolarimeter (Tokyo, Japan) in 10 mM of binding buffer. The fluorescence spectra were acquired by a Hitachi F-2700 fluorescence spectrophotometer (Tokyo, Japan). The experimental parameters were set as follows: emission wavelength (λem), 490 to 630 nm; excitation wavelength (λex), 360 nm; emission slit, 10 nm; excitation slit, 10 nm. The deionized water used throughout the study was prepared by a Milli-Q Reference water purification system (Merck Millipore, Billerica, MA, USA).

Measurement of Aptamer Conformation with CD
The aptamer conformation was measured by a CD spectropolarimeter. The DNA aptamer (5 µM) and DNA aptamer (5 µM) with OTA (5 µg mL −1 ) were placed in an optical chamber (1-cm path length, 400 µL volume), which was deoxygenated with dry purified nitrogen (99.99%) prior to analysis and also insulated by a nitrogen atmosphere. The CD spectrum was run in triplicate with a 200-nm/min scan speed, 1-nm bandwidth, and 1-s time constant. The data were measured in a range of 230 to 340 nm at 0.1-nm intervals. The buffer solution was used as a blank to correct the background.

Determination of OTA by Fluorescence Aptasensors
For the quantitative determination of OTA, different concentrations of OTA were mixed with 250-nM aptamers in binding buffer and heated at 95 • C for 5 min, and were then cooled down to room temperature for upcoming experiments. After that, samples containing 175 µL of TMPyP (1.75 × 10 −7 mol L −1 ), 80 µL of CdTe QDs (4.2 × 10 −9 mol L −1 ), and 50 µL of OTA aptamer were made up to 1 mL. After equilibrating at room temperature for 5 min, the fluorescence emission spectra were measured at a wavelength range from 490 to 630 nm.

Determination of OTA in Astragalus membranaceus
To verify the feasibility of this aptasensor, Astragalus membranaceus was chosen and finely grounded with a disintegrator. Then, samples were passed through a 0.22-mm aperture test sieve. A total of 1 g of the samples was spiked into different concentrations of OTA, and the mixtures were introduced into a 5-mL extracting solution (acetonitrile/water, 80:20, v/v). The samples were ultrasonicated for 10 min and then centrifuged at 4000 rpm for 10 min. The supernatant underwent a fivefold dilution with deionized water to minimize the matrix effect [37]. Additionally, subsequent manipulation was conducted according to the reported method.