A simple AuNPs-based colorimetric aptasensor for chlorpyrifos detection

Abstract

To develop a method, a target recognition unit is required. Aptamers, a class of single strand oligonucleic acids, are selected by systematic evolution of ligands by exponential enrichment (SELEX) [15 − 19], which possesses superior advantages over traditional recognition molecules, because of their nontoxicity, high speci city, high a nity, low cost and easier to synthesize and modify [20 − 23]. According to recent researches, highly speci city aptamers for diverse target substances (proteins [24], exsomes [25], cells [26,27], ions [28,29], bacterial [30], pesticides [31 − 33], etc.) have been screened. Hence, the aptamer based sensors (aptasensors) have been widely reported [34 − 36]. However, the aptasensors for small molecule targets need more challenge because of the small molecule size and low molecular weight, which is di culty to capture. Thus there are relatively few literature reports on small molecule aptasensors [37].
Gold nanoparticles (AuNPs), have many advantages in the eld of biochemical analysis because of their unique optical properties, high surface-to-volume ratio, high molar absorption coe cient, biocompatibility, non-toxicity and easy to prepare [38 − 40]. Among diverse AuNPs based biosensor system, colorimetric biosensing have been widely used for their simplicity, low cost, visible color changes [41 − 43]. The AuNPs could self-aggregate owing to high salt effects, the ssDNA can bind to negatively charged AuNPs through elecostatic force, thus served as the stabilizer of AuNPs [44]. Based on this mechanism, the AuNPs based colorimetric aptasensor become more popular [45 − 47].
In this article, we developed a label-free AuNPs-based colorimetric aptasensor for Chl detection, which is simple to operate. Figure 1 is the schematic illustration for Chl detection. The Chl-aptamer, which was selected using ssDNA library immobilized SELEX, could stabilize the AuNPs in an optimal concentration of salt solution. In the presence of Chl, the speci c Chl-aptamer will bind to Chl rst, thus the exposure to certain salt solution could cause the AuNPs self-aggregate and a color change from red to blue. Besides, the established colorimetric aptasensor was used to detect tap water, cucumber and cabbage samples to validate and evaluate the accuracy as well as practical application. The tested results were quilt satis ed demonstrating the potential use of the fabricated aptasensor to Chl capture and detection in aqueous solution and real samples.

Aptamer selection and characterization
In this work, we selected a highly binding Chl-aptamer. After eight selection rounds, a high enrichment ssDNA pool for Chl was obtained, the retention rates of each round were shown in Fig. S1. We can see that with the increase in screening pressure, the retention rate of positive selection reaches 4.49% after the eighth round. The secondary structure of the selected Chl-aptamer was shown in Fig. S2, which is composed of stem-loop and hairpin structures. The K d curve of the Chl-aptamer was shown in Fig. S3, showing that the Chl-aptamer has a low dissociation constant (K d = 58.59 ± 6.08 nM), which means highly binding to Chl. Characterization of AuNPs by aptamer, chlorpyrifos and NaCl AuNPs in different systems were characterized with UV-Vis spectrum and TEM images. As shown in Fig. 2, we can see that in the system of AuNPs in 70 mM NaCl, the A 520 hugely decreased and the A 650 increased, meaning the aggregation of AuNPs. Once the AuNPs were incubated with aptamer rst, the proper concentration of NaCl couldn't make the AuNPs aggregate. The Chl-aptamer could disperse AuNPs due to the electrostatic interaction between AuNPs and aptamers. In the presence of Chl, the strong interaction between Chl and aptamer could make the conformation of aptamer change, along with the aggregation of AuNPs. Thus, a decrease of A 520 and an increase of A 650 were observed, leading an increase in A 650 /A 520 . The corresponding TEM images were shown in Fig. 3.
Optimization of the aptasensor detection conditions Some experiment conditions are important for developing a sensitive colorimetric aptasensor, such as the concentration of NaCl, the incubation time between Chl and aptamer, and the incubation time of NaCl. As shown in Fig. 4A, the concentrations of NaCl from 20 mM to 100 mM were tested. The A 650 /A 520 value of AuNPs solution increased with the increasing in NaCl concentration, while the A 650 /A 520 value of AuNPs with aptamer solution almost keep the same during the concentration of NaCl increasing to 70 mM, which means the proper concentration of NaCl could not aggregate the aptamer stabilized AuNPs, but a higher concentration of NaCl could. Besides, for the AuNPs in the system with or without aptamer, the difference of A 650 /A 520 value reaches the maximum after incubating with 70 mM NaCl. Thus, we chose 70 mM NaCl for further study. In the incubation time optimization experiments, 1 µM Chl was used. From Sensitivity and selectivity of the aptasensor for chlorpyrifos detection Subsequently, the label-free AuNPs based colorimetric aptasensor was established for Chl standard solution (0 to 10 µM) analysis. The UV-Vis spectra were shown in Fig. 5A, we can see that with the increase in Chl concentration, the A 520 value kept decreasing, while the A 650 value also kept increasing.
The scatterplot of the A 650 /A 520 values with different concentrations of Chl was shown in Fig. 5B, it was shown that the A 650 /A 520 value increased signi cantly at the concentration of Chl from 0 to 5 µM, and slowed down after the Chl concentration kept increasing to 10 µM. It is worth mentioning that there are two linear relationships between A 650 /A 520 value and the concentration of Chl. At low concentration range of Chl (ranging from 50 nM to 200 nM), the linearization equation is y = 0.102 + 9.126 × 10 − 4 C (R 2 = 0.996) (Fig. 5C), while at high concentration range of Chl (ranging from 200 nM to 5000 nM), the Page 5/15 linearization equation is y = 0.266 + 1.124 × 10 − 4 C (R 2 = 0.997) (Fig. 5D). The limit of detection (LOD) is as low as 14.46 nM, which is given by the equation: LOD = 3×S B /b, (S B is the standard deviation of twenty independent blank samples and b is the sensitivity of the calibration graph.) The speci city of the fabricated colorimetric aptasensor was evaluated by comparing the A 650 /A 520 value of solutions containing 1 µM chlorpyrifos with other pesticides at concentrations 10 times higher, such as dimethoate, dichlorphos, carbofuran, malathion, and profenofos. A water solution was used as the blank. As shown in Fig. 6, a remarkable A 650 /A 520 value was obtained both in detecting the chlorpyrifos and the Mix, while a negligible change of A 650 /A 520 value in detecting other pesticides. The result indicates this aptasensor could speci cally detect chlorpyrifos among other pesticides.

Application in real samples
The sample detection results were shown in Table 1. It was con rmed that the developed colorimetric aptasensor displays excellent capability for the accurate detection of chlorpyrifos in tap water, cucumber and cabbage samples. The excellent recovery values with acceptable RSD values below 5% demonstrates that the proposed aptasensor could be applied to detecet chlorpyrifos in real samples.

Aptamer selection protocol and characterization
The details of aptamer selection protocol, sequencing and analysis of sequences, and dissociation constant K d measurement were shown in part 1.2 to part 1.4 of EIS.

Synthesis of AuNPs and characterization
All glasswares for preparation of AuNPs were dipped thoroughly in aquaregia (3:1 (v/v) HNO 3 -HCl) for 24 hours, then washed with double distilled water and dried for use. AuNPs with a diameter around 13.0 nm were synthesized by means of the classical citrate reduction method [48] with a slightly improvement.
Brie y, 25 mL of HAuCl 4 (0.01% (w/v)), 0.8 mL of 1% (w/v) fresh trisodium citrate were mixed thoroughly in the bottle. Subsequently, the bottle was put into the oven at 100 ℃ for 2 hours. Concentration of AuNPs was calculated using the following formula C=A/(ε·b), where C is the concentration of AuNPs, A is the UV-Vis absorbance of AuNPs at 520 nm, ε equals to 2.7 × 10 8 M −1 cm −1 , is the extinction coe cients of 13.0 nm AuNPs, b is the thickness of the measurement cuvette. In order to characterize the dispersion of AuNPs aqueous, UV-Vis spectra and transmission electron microscopy (TEM) images were acquired.
Fabrication of colorimetric aptasensor 25 μL (1 μM) of Chl-aptamer, 100 μL (3.1 nM) of AuNPs and Chl with different concentrations were evenly mixed and incubated for 30 min to let the target pesticide binding with aptamer completely. Later, 35 μL (500 mM) of NaCl was added to above mixture and further diluted with deionized water to 250 μL. After incubation for 15 min, the solutions were characterised by UV-Vis spectrum, and the absorbance wavelength was measured in 400-700 nm. The absorbances at 650 nm (A650) and 520 nm (A520) were recorded. The ratio of A 650 /A 520 was given. All the conditions were at room temperature.
Sensitivity and selectivity of the aptasensor A wide range of Chl concentration from 50 nM to 10 μM was used to test the sensitivity of the fabricated colorimetric aptasensor. The speci city of the aptasensor was determined with different pesticides (dimethoate, dichlorphos, carbofuran, malathion, profenofos), respectively. Besides, a mixture containing all other pesticides as well as Chl was also tested. The concentration of Chl was 1 μM, while the other pesticides were 10 μM.

Detection of chlorpyrifos in real sample
In order to validate and evaluate the accuracy as well as practical application of the constructed chlorpyrifos colorimetric aptasensor, the cucumber, cabbage and tap water samples were tested. Tap water was collected from our laboratory, the cucumber and cabbage were purchased from the local market. The pre-treatment procedure details are as follows: cucumber and cabbage were cut into small pieces and juiced rstly, then the juice and the tap water were ltered with a 0. 22 Figure 1 Schematic illustration of the fabricated colorimetric aptasensor for chlorpyrifos detection.