All-in-one fabrication of a ratiometric electrochemical aptasensor with tetrahedral DNA nanostructure for fumonisin B1 detection

Abstract

Here, we develop an all-in-one strategy for efficient assembly of an electrochemical aptasensor. A multifunctional structure based on a tetrahedral DNA nanostructure (TDN) was synthesized via a one-step annealing process, providing DNA fixation, target recognition, signal amplification and space regulation. Based on the integration of this multifunctional structure, the sensing interface was assembled in one step. A ratiometric aptasensor was constructed by anchoring methylene blue (MB) to the TDN and ferrocene (Fc) on the cDNA. Using the ratio of the currents obtained from Fc and MB as a measure, the developed aptasensor shows excellent analytical performance for fumonisin B1 detection. This strategy is universal and could simplify the fabrication of aptasensors.

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Due to the specific recognition properties of aptamers (Apts) and the ability to design specific DNA chains, electrochemical aptasensors have significant advantages and have become an ideal strategy for various quantifying analyses.^1–4 In conventional electrochemical sensing strategies, aptasensors can be functionalized to improve analytical performance by introducing functional units into their self-assembled layers.^5–10 In these strategies, the assembly of complicated DNA structures requires sophisticated techniques,^11,12 such as layer-by-layer assembly methods. The layer-by-layer method commonly suffers from several potential problems, including the crowding effect at the sensing interface, cross-interference from the introduction of new interfaces, interfacial consistency issues, and the introduction of contaminants resulting from the multi-step assembly process. Ultimately, these limitations restrict the potential applications of aptasensors and must be overcome. In previous studies, researchers ingeniously designed a palindromic molecular beacon to integrate multifunctional elements, which can reduce the complexity of the sensing system.^13,14

Motivated by the integrated model concept, we envisioned the possibility of directly modifying the sensing interface on a substrate electrode through the design of a multifunctional structure, enabling the all-in-one fabrication of an aptasensor without compromising the sensing performance. In this work, we synthesized a novel multifunctional structure, including complementary DNA (cDNA), Apt, and a tetrahedral DNA nanostructure (TDN) through a one-step annealing process (Scheme 1). Each of the three components played a different function. Specifically, the cDNA functionalized with sulfhydryl groups could immobilize the multifunctional structure at the sensor interface.¹⁵ The Apt enabled specific recognition of the target through its specific structural and chemical properties,¹⁶ and the TDN was used for signal amplification, space regulation, and programmable design. The abundant binding sites of the tetrahedron could enhance the payloads of the signaling molecules to improve sensing performance.^17–19 The tetrahedron could also preserve spatial separation between the units for structural stability and mechanical rigidity, thus avoiding unnecessary interactions.^20–22 Additionally, different functional units can be assembled in the TDN based on rational design of complementary sequences to facilitate the integration of functions. According to the integration of multifunctional structures, an aptasensor was constructed by attaching it directly to the electrode surface and closing the non-specific binding site at the sensing interface with 6-mercapto-1-hexanol (MCH). This strategy addressed potential problems in the process of layer-by-layer assembly of the functional components.

Scheme 1 Scheme showing all-in-one fabrication process, including the all-in-one synthesis, all-in-one function, and all-in-one assembly of the ratiometric electrochemical aptasensor.

Methylene blue (MB) adsorption on the TDN and the functionalization of ferrocene (Fc) on the cDNA created the output currents of MB (I_MB) and Fc (I_Fc) which served as reference and response signals, used as a ratiometric strategy to improve accuracy. Fumonisin B1 (FB1) was selected as the model analyte to validate the applicability of the strategy. In the presence of FB1, the hybridization of Apt with FB1 induced the release of the TDN, which led to reduced steric hindrance and decreased the number of MB units present. Thus, I_Fc increased while I_MB decreased in the presence of FB1, which in turn enhanced the I_Fc/I_MB ratio. This strategy was not only simple in design, but also highly accurate due to the ratiometric strategy, and demonstrated a promising electrochemical detection capacity.

First, the designed multifunctional structure composed of cDNA, Apt, and TDN was prepared via a one-step annealing process, and the synthesis of the multifunctional structure was assessed using agarose gel electrophoresis. The leftmost lane served as the marker, while lanes 1, 2, 3, 4, and 5 corresponded to the S1, S1 + S2, S1 + S2 + S3, TDN, and multifunctional structures, respectively (Fig. 1A). Compared to other lanes, lane 5 indicated the lowest velocity migration, which was due to the large molecular weight.²³ Next, atomic force microscopy (AFM) was employed to characterize the multifunctional structure. As shown in Fig. S1 (ESI†), the AFM diagram reveals that the multifunctional structures have a stereo structure with a height of 7.1–8.3 nm and have no obvious aggregates.

Fig. 1 (A) Agarose gel analysis of the multifunctional structure: lane M, 20 bp ladder; lane 1, S1; lane 2, S1 + S2; lane 3, S1 + S2 + S3; lane 4, TDN; lane 5, multifunctional structure. (B) Chronoelectric curves for MCH/AuE, and MCH/multifunctional structure/AuE.

Next, the multifunctional structure was directly fixed on the electrode surface, and the density of the immobilized multifunctional structure was estimated from chrono-coulometric (CC) measurements using RuHex as the cation redox indicator. Increased CC charge was observed after assembling the multifunctional structure on the gold electrode (AuE) (Fig. 1B). The assembly density of the probes was deduced from the Cottrell equation and the quantitative relationship between the ruthenium ion and phosphate backbone.

According to the Cottrell equation (eqn (1)), the quantity of ruthenium ion adsorption at the AuE surface can be calculated as follows:²⁴

Q = 2nFAC(D/Π)t^1/2 + Q_c + nFAΓ_Ru, (1)

where C is the bulk concentration of the redox probe (mol cm⁻³), Γ_Ru is the quantity of the adsorbed redox probe (mol cm⁻²), A is the electrode area (cm²), D denotes the diffusion coefficient (cm² s⁻¹), Q_c is the capacitive charge (C), F denotes the Faraday constant (96487C/equiv.), t is the time (s), and n is the number of transferred electrons.

According to eqn (2), the assembly density of DNA at the AuE can be converted based on the quantity of ruthenium ion adsorption as follows:²⁴

Γ_DNA = Γ_Ru(z/m)N_A, (2)

where N_A denotes Avogadro's number, m is the number of nucleotides in the DNA strand, and z is the charge of the redox probe.

The assembled density of the multifunctional structure was calculated as 3.22 × 10¹² cm⁻². The presence of the TDN could enable the modulation of the probe density, improving the detection sensitivity of the aptasensor. The active electrode area after modification was estimated from cyclic voltammetry measurements at different scan rates (Fig. S2, ESI†).²⁵ The active electrode area was calculated to be 9 mm² using the Randles–Sevcik equation.

Fabrication of the electrochemical aptasensor was monitored using electrochemical impedance spectroscopy (EIS) measurements,^26,27 and a Randles equivalent circuit was used to fit the experimental data, as shown in Fig. 2A. Here, R_s represents the solution resistance, C_dl represents the double-layer capacitance, R_et indicates the electron-transfer resistance, and Z_w represents the Warburg impedance. The diameter of the semicircle indicates the R_et. As shown in Fig. 2A, the R_et of the AuE was small (curve a, R_et = 97.84 Ω) due to the high conductivity of the AuE. With the modification of the multifunctional structure, R_et (curve b, R_et = 2286 Ω) increased due to the blocking effect of DNA. Furthermore, after connecting with MCH, R_et increased to 3250 Ω (curve c), due to a further increase in the hindrance of electron transfer. After incubating FB1 on the modified electrode, a decreased R_et (curve d, R_et = 2640 Ω) was observed due to the stripping of the TDN. Next, a slight increase was observed in R_et after the adsorption of MB (curve e, 2803 Ω). The EIS results confirmed that the electrochemical aptasensor was successfully fabricated.

Fig. 2 (A) Nyquist plots of (a) the bare AuE, (b) multifunctional structure/AuE, (c) MCH/multifunctional structure/AuE, (d) FB1/MCH/multifunctional structure/AuE, and (e) MB/FB1/MCH/multifunctional structure/AuE. The inset is the Randles equivalent circuit. (B) SWVs of the developed aptasensor with and without FB1.

Different modified electrodes were investigated using square wave voltammetry (SWV) (Fig. 2B) to verify the feasibility of the electrochemical aptasensor for FB1 detection. Without FB1, I_MB was 325 nA due to the adsorption of MB on the TDN, and I_Fc was 55 nA because of the steric effect of the TDN. I_MB decreased by 57.1% and I_Fc increased by 39.4% in the presence of FB1, which was attributed to the stripping of the TDN. The above results verified that the ratiometric electrochemical aptasensor was usable for the detection of FB1.

A multifunctional structure concentration of 2 μM, a multifunctional structure incubation time of 12 h, and a FB1 binding time of 40 min were used for the following experiments. The analytical performance of the electrochemical aptasensor for the detection of FB1 was then investigated. As shown in Fig. 3A, when the FB1 concentration increased from 0.5 to 500 pg mL⁻¹, I_Fc increased while I_MB decreased. As shown in Fig. 3B, the variation in I_Fc/I_MB displayed a good linear relationship with the logarithmic value of FB1 concentration (lg C_FB1), in the range from 0.5 to 500 pg mL⁻¹, with a fitted equation of I_Fc/I_MB = 0.180 lg C_FB1 + 0.268 (R² = 0.994). Ratiometric measurements provided a broader linear range than the single-signal measurements (Fig. 3C). Compared with the reported methods, the developed method exhibited a lower LoD than that of liquid chromatography-tandem mass spectrometry, surface enhanced Raman scattering, or colorimetry (Table S1, ESI†). Meanwhile, this aptasensor shows a comparable LoD to analytical methods based on electrochemiluminescence or electrochemistry, but the dual-signal output mode and one-step assembly could allow for better reliability.

Fig. 3 (A) SWV responses of the modified electrodes at various concentrations of FB1 (0.5, 1, 5, 10, 50, 100, and 500 pg mL⁻¹). (B) Corresponding calibration curve of the relationship between I_Fc/I_MB and lg C_FB1. (C) Changes in I_Fc (orange) and I_MB (blue) as a function of lg C_FB1. (D) Electrochemical responses of the aptasensor with different interferents (AFB1, AFB2, OTA, and ZEN). (E) Reproducibility of aptasensor over 6 parallel experiments. (F) Stability analysis of the aptasensor after 7 days storage with FB1. The number of measurements is 3.

To assess the selectivity of the electrochemical aptasensor, the effects of common interfering substances, including aflatoxin B2 (AFB2), aflatoxin B1 (AFB1), zearalenone (ZEN), and ochratoxin (OTA), on the sensing performance were investigated. Fig. 3D shows that the developed aptasensor had a significant response to FB1 or a mixture containing FB1. The developed aptasensor could only identify FB1 due to the specificity of Apt to FB1, indicating the selectivity of the aptasensor.

The reproducibility of the electrochemical aptasensor for the detection of FB1 was evaluated, indicating that the relative standard deviation (RSD) of six parallel electrodes for the detection of 100 pg mL⁻¹ FB1 was 5.56%. This demonstrated that the developed aptasensor exhibited good reproducibility (Fig. 3E).

Moreover, the stability of the aptasensor was evaluated by recording the electrochemical response to 100 pg mL⁻¹ of FB1 for a week. As shown in Fig. 3F, the I_Fc/I_MB value of the aptasensor remained at 86.7% of the initial response after one week, indicating the stability of the aptasensor.

To investigate the applicability of the electrochemical aptasensor, an FB1 standard was added to obtain sample solutions of 100, 500, and 1000 ng mL⁻¹. As shown in Table S2 (ESI†), the sample solution diluted to 1/10 000 was assessed 3 times, and the recoveries were in the range of 91.0–99.7% with a RSD of less than 5.9%. In addition, as a control, UPLC-MS/MS was used to determine the FB1 quantity in the samples. The results of the two methods were consistent, indicating that the aptasensor had great potential for detecting FB1 in real samples.

In summary, we designed a multifunctional structure and developed an all-in-one fabrication strategy for an electrochemical aptasensor. The different functions, including DNA fixation, FB1 recognition, signal amplification, and space regulation, were integrated into a single multifunctional structure. The good performance of the aptasensor was attributed to the designed multifunctional structure, which favors efficient FB1 recognition and signal amplification. The all-in-one strategy could simplify the fabrication of the aptasensor, and aid the sensor portability. The dual-signal output mode improved the accuracy of the aptasensor. This approach may be universal and could be applied to the detection of other targets by changing the aptamer or adjusting the size of the TDN.

Na Dong: investigation, data curation, writing – original draft. Shuda Liu: resources, software. Yuye Li: resources, software. Shuyun Meng: resources, software. Yifan Liu: resources. Xia Li: resources. Dong Liu: supervision, methodology, conceptualization, formal analysis, writing – review & editing. Tianyan You: supervision, conceptualization, writing – review & editing.

This work was supported by the National Natural Science Foundation of China (No. 22074055, and 22374061), Natural Science Foundation of Jiangsu Province (No. BK20200104), Postgraduate Research & Practice Innovation Program of Jiangsu Province (No. KYCX23_3663).

Conflicts of interest

There are no conflicts to declare.

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Footnote

† Electronic supplementary information (ESI) available: Experimental details and supplementary figures. See DOI: https://doi.org/10.1039/d3cc04991d

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