3.1. Principles of STR detection by the aptamer/AuNF-PEI-MIL-101(Cr)/GE
The working principle of the label-free electrochemical aptasensor is illustrated in Scheme 1. The bare GE surface was first modified by AuNF-PEI-MIL-101(Cr) nanomaterials. Then, the aptamer was self-assembled on the AuNF-PEI-MIL-101(Cr)/GE via electrostatic adsorption and Au-S bond. Upon introducing the STR, specific binding was performed based on the high recognition ability between the aptamer and the STR. At the same time, the combination between STR and aptamer creates steric hindrance, making it difficult for [Fe(CN)63−/4−] anions to reach the sensing interface, and thereby reducing current signals. As a result, the concentration of target STR can be determined based on the change in current signal caused by the specific recognition and binding via DPV measurements.
3.2 Morphology, structure, composition, and size of nanomaterials
SEM and TEM are used to investigate the surface morphology, microstructure, and size of these nanomaterials. As shown in Figs. 1A-1B, the MIL-101(Cr) has a relatively smooth surface and an octahedral morphology with a size of about 500‒600 nm. After MIL-101(Cr) functionalization with PEI (Figs. 1C-1D), PEI-MIL-101(Cr) did not undergo any significant size or morphology change. Figure 1E shows the TEM image of the AuNF, which exhibits a flower-like structure with a uniform particle size and a diameter of about 50 nm. Figures 1F-1G depict the TEM and SEM of AuNF-PEI-MIL-101(Cr). It can be observed that AuNF was successfully loaded on the surface of PEI-MIL-101(Cr). In addition, the elemental composition of the AuNF-PEI-MIL-101(Cr) nanocomposites was further investigated by high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM). As shown in Figs. 1H-1J, the Au element is located on the outside of the Cr element, indiciating the successful synthesis of AuNF-PEI/MIL-101(Cr) nanohybrid.
Powder X-ray diffraction (XRD) is employed to characterize the crystalline structure of nanomaterials. The XRD patterns of MIL-101(Cr), PEI-MIL-101(Cr), and AuNF-PEI-MIL-101(Cr) are shown in Fig. 2A. MIL-101(Cr) showed diffraction peaks at 2θ° of 5.56°, 8.98°, 16.42°, and 24.72° (black line), which were consistent with previous literature (Pourreza, Askari, Rashidi, Seif, & Kooti, 2019). After being functionalized by PEI (red line), PEI-MIL-101(Cr) exhibited similar diffraction peaks to MIL-101(Cr), indicating the crystal structure has not changed. While the intensity of diffraction peaks were reduced. This was attributed to the filling of MIL-101(Cr) pores by PEI, which decorated the pore properties, such as pore size and polarity (Xin et al., 2015). In addition, the AuNF-PEI-MIL-101(Cr) not only showed all the diffraction peaks of the PEI-MIL-101(Cr), but also four additional diffraction peaks at 38.06, 44.18, 64.64, and 77.42, corresponding to (111), (200), (220), and (311) faces of Au (Lu et al., 2014).
FT-IR is used to identify the functional groups in MIL-101(Cr), PEI-MIL-101(Cr), and AuNF-PEI-MIL-101(Cr). As shown in Fig. 2B, the FTIR spectrum of MIL-101(Cr) displayed obvious vibration peaks at 589.62, 748.24, 1014.89, 1405.37, 1615.09, and 3340.10 cm− 1. The peak at 589.62 cm− 1 belonged to the vibration of Cr‒O bond (Bayazit et al., 2017), and the vibration peak at 748.24 cm− 1 assigned to the deviational vibration of carboxylate groups. At 1014.89 cm− 1, in-plane bending vibration of C‒H on the benzene ring was observed. The two peaks at 1405.37 cm − 1 and 1615.09 cm − 1 may be due to both the symmetric stretching and asymmetric tensile vibration of the carboxyl (− COO−) group, respectively (Wan, Li, Jiang, Lin, & Yin, 2021; Zhang et al., 2021). After modification of MIL-101(Cr) with PEI, the spectrum of the PEI-MIL-101(Cr) exhibited some changes. The peak at 1671.98 cm‒1 disappeared. The peak at 3412.90 cm − 1 can be attributed to N − H bond in the secondary amine group, indicating that the PEI had been successfully grafted onto the MIL-101(Cr) (Wan, Li, Jiang, Lin, & Yin, 2021). However, compared with PEI-MIL-101(Cr), the absorption peak shape and peak intensity of AuNF-PEI-MIL-101(Cr) are changed (decreased or increased), indicating the composition of hybrid AuNF-PEI-MIL-101(Cr).
X-ray photoelectron spectroscopy (XPS) is employed to analyze the chemical states and elemental composition of AuNF-PEI-MIL-101(Cr). Figure 2C shows the XPS survey spectrum of the AuNF-PEI-MIL-101(Cr) nanocomposites, in which the characteristic peaks of N 1s, Cr 2p, and Au 4f could be all clearly observed. For the high-resolution Cr 2p spectrum (Fig. 2D), the two peaks at 577.25 eV and 586.75 eV can be assigned to Cr 2p3/2 and Cr 2p1/2, revealing the existence of Cr‒O bond (Cheng et al., 2020). The high-resolution N 1s spectrum (Fig. 2E) can be divided into three peaks. The main peak at 399.8 eV is attributed to secondary amine (− NH−), and the peaks at 398.89 eV and 400.64 eV belong to tertiary (‒N‒) and primary (− NH2) amine groups, respectively (Shen, Wu, & Meng, 2021). These nitrogen species are typical chemical compositions of PEI, indicating that PEI has been successfully modified to the surface of MIL−101(Cr). The high-resolution Au 4f spectrum (Fig. 2F) exhibits two peaks at 84.05 eV and 87.65 eV which were assigned to the Au 4f 7/2 and the Au 4f 5/2, respectively, proving the Au (0) is present in AuNF-PEI-MIL-101(Cr). These experimental results well demonstrated that AuNF-PEI-MIL-101(Cr) nanocomposites were successfully synthesized.
3.3 Electrochemical behavior of AuNF-PEI-MIL-101(Cr)/GE
The CV curves of bare GE and AuNF-PEI-MIL-101(Cr) modified GE at different scan rates (20‒100 mV s-1) are shown in Figs. 3A and 3B. Both bare GE and AuNF-PEI-MIL-101(Cr)/GE showed a pair of well-defined redox peaks. Figures 3C and 3D show the relationship between the redox peak current (Ipa/Ipc) versus the square root of the scan rate (v1/2). It is observed that Ipa and Ipc are linearly proportional to v1/2 in the range of 20‒100 mV s − 1, suggesting that a diffusion controlled process occurred on the bare GE and AuNF-PEI-MIL-101(Cr)/GE. Thus, their electrochemically active surface area can be estimated according to the Randles-Sevcik equation (E q (1)).
Ip = 2.69×105AD1/2n3/2v1/2C (1)
In the formula, Ip is the peak current (µA), A is the electroactive surface area (cm2), D is the diffusion coefficient of [Fe(CN)6]3-/4- (D = 7.6×10 − 6 cm2 s-1), n is the number of transfer electrons (n = 1), v is the scan rate (V s-1), and C is the redox medium concentration (C = 5.0 mol cm-3). The calculated active surface areas of the bare GE and AuNF-PEI-MIL-101(Cr)/GE were 0.0512 cm2 and 0.0664 cm2, respectively. The electroactive area of the AuNF-PEI-MIL-101(Cr)/GE was 1.30 times that of the bare GE, demonstrating AuNF-PEI-MIL-101(Cr)/GE has a faster electron transfer rate.
3.4 Electrochemical characterization of the Stepwise Modification Process
The EIS measurements were carried out during the construction process of the electrochemical aptasensor using [Fe(CN)6]3-/4-as a redox probe. In the EIS spectrum, the semicircle diameter equals the electron transfer resistance (Ret). As shown in Fig. 4A, the bare GE possessed a low electron transfer resistance (curve a), indicating superior conductive property. When the AuNF-PEI-MIL-101(Cr) was modified on the surface of GE, the Ret decreased because the nanocomposites accelerated the electron transfer of [Fe(CN)6]3-/4-on the GE surface (curve b). However, with the immobilization of thiolated Apts on the AuNF-PEI-MIL-101(Cr)/GE via Au-S bond, the Ret increased (curve c). This is because negatively charged phosphate backbone of the aptamer made [Fe(CN)63-/4-] anions inaccessible to the electrode surface. In the presence of STR, a decrease in Ret is observed, which is caused by the target being specifically bound with the aptamer on the surface of the aptamer/AuNF-PEI-MIL-101(Cr)/GE.
CV is another effective approach used to monitor the aptasensor assembly. As shown in Fig. 4B, the bare GE exhibited clearly separated redox peaks of [Fe(CN)6]3-/4- in the range of -0.2V‒0.6V (curve a'). When AuNF-PEI-MIL-101(Cr) were modified onto the GE, the peak current was obviously increased relative to the bare GE. This change is attributed to the good conductivity of AuNF-PEI-MIL-101(Cr) (curve b'). After the STR aptamer immobilization onto the AuNF-PEI-MIL-101(Cr)/GE, the current signal significantly diminished (curve c'). When the STR appeared, the current signal further decreased (curve d'), suggesting that the formation of the aptamer-STR complex hindered the interfacial electron transfer to the modified electrode surface. The CV results were in accordance with EIS. These results greatly demonstrate that the electrochemical aptasensor was successfully constructed.
3.5 Optimization of the electrochemical sensing conditions
In order to achieve the best analytical performance of the aptasensor, the experimental conditions were optimized, namely the concentration of aptamer, the incubation time of aptamer, and the incubation time of STR.
To investigate the effect of the aptamer concentration on the changes in the current response (ΔI = I-I0, where I is the current signal of the aptasensor toward STR, and I0 is the background signal), aptamer with different concentrations (0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 µM) were fixed to the surface of the modified electrode during the construction of the aptasensor and the current signals were recorded. As shown in Fig. S1A, the ΔI (the difference between the DPV peak current in the presence of the STR and that of the blank) increased gradually with the increase of aptamer concentration from 0.5 to 2.0 µM, and then remained almost constant, indicating that the aptamer was saturated at the electrode surface. Therefore, 2.0 µM was selected as the optimum concentration of aptamer in subsequent experimental assays.
The aptamer incubation time is an essential factor affecting the performance of the aptasensor. If the incubation time of the aptamer is too short, the aptamer will not be completely immoblized on the AuNF-PEI-MIL-101(Cr)/GE. Vice versa, this process may take too much time. The working electrodes were separately placed in 2.0 µM of aptamer solution for different times (40, 50, 60, 70, 80, and 90 min). As shown in Fig. S1B, with the increasing incubation time from 40 to 60 min, the ΔI increases. When the incubation time was greater than 60 min, there was no obvious change in the ΔI. Thus, 60 min was selected as the best aptamer incubation time for the aptamer/AuNF-PEI-MIL-101(Cr)/GE.
The incubation time of STR with the aptamer was also investigated. The aptamer/AuNF-PEI-MIL-101(Cr)/GE was separately incubated with the STR (200 nM) for different times (40, 50, 60, 70, 80, and 90 min). As shown in Fig. S1C, the maximum ΔI was obtained at 60 min of incubation time, but it subsequently exhibited a downward trend. Hence, 60 min was selected as the optimal incubation time for STR.
3.6 Analytical performance of the electrochemical sensing method
Under the optimal experimental conditions, the aptamer/AuNF-PEI-MIL-101(Cr)/GEs were placed in different concentrations of STR (0.01 nM, 0.1 nM, 1 nM, 10 nM, 50 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM), and their DPV peaks current were recorded. As shown in Fig. 5A, the signals decreased with increasing the STR concentration within the range of 0.1‒400 nM. Figure 5B exhibited the linear relationship between the current response difference (ΔI) and STR concentrations (ΔI = I-I0, where I is the current signal of the aptasensor toward STR, and I0 is the background signal). The linear equation was ΔI = 0.5705CSTR + 0.9955 (R2 = 0.9988), and the limit of detection (LOD) was calculated to be 0.003 nM based on 3S/N (where S is the standard deviation of blank samples and N is the slope of the calibration plot). Compared with previously reported methods for detection of STR (Table S1) (Chen, Zhang, Lin, Ge, & Qiu, 2012; Luo et al., 2020; Taghdisi, Danesh, Nameghi, Ramezani, & Abnous, 2016; Xu et al., 2017; Yin et al., 2017), the developed electrochemical aptasensor has a wider detection range and lower detection limit. The excellent performance could be attributed to the following two aspects. (ⅰ) AuNF-PEI-MIL-101(Cr) nanocomposites that act as the substrate material significantly increase the aptamer loading efficiency, further enhancing the sensitivity of the aptasensor. (ⅱ) The STR aptamer, used as recognition molecules, are not only easy to synthesize, preserve, and modify, but also have good specificity and high affinity for the target STR.
3.7 Reproducibility, stability, and selectivity of the electrochemical sensing method
In order to investigate the reproducibility of the aptasensor, six independently prepared aptamer/AuNF-PEI-MIL-101(Cr)/GEs were used to detect STR of 50 nM at room temperature. As shown in Fig. S1A, there was little difference in the DPV peaks current, and the relative standard deviation (RSD) was less than 2%, suggesting that the aptasensor has good reproducibility.
In addition, the storage stability of the proposed aptasensor was also studied. The STR/aptamer/AuNF-PEI-MIL-101(Cr)/GEs were stored in a refrigerator (4℃) for 21 days (The concentration of STR is 50 nM), followed by periodical measurement of the current signals of electrodes. As shown in Fig. S1B, the current signals slowly declined with increasing storage time. After 21 days, the current signals of STR/aptamer/AuNF-PEI-MIL-101(Cr)/GEs were 94.29% of their original current signals. It indicated that the electrochemical aptasensor possessed desirable stability.
The specificity of an aptasensor is an essential indicator to achieve accurate detection. Eight other antibiotics, including neomycin, oxytetracycline, tetracycline, penicillin, chloramphenicol, kanamycin, erythromycin, and cephalexin hydrate, were selected as interfering antibiotics for detection. The concentration of STR was 50 nM, and that of interfering substances was 100 nM. As shown in Fig. S1C, compared with the blank, only STR caused a distinct change in current signals, whereas other antibiotics, even at 2 times higher concentrations, showed no clear change. The results show that the aptasensor exhibited high specificity.
3.8 The STR detection in real samples
In order to demonstrate the applicability of this method in actual analysis, the developed aptasensor was employed to detect STR in cow milk, sheep milk, and goat milk powder spiked with different STR concentrations (1, 10, and 50 nM). As shown in Table 1, the recovery values were in the range of 96.0%-109.4% and the relative standard deviation were between 0.55% and 1.41%, indicating that this aptasensor showed great application potential for rapid detection of STR in real food samples.