Sensitive Electrochemical Detection of Tryptophan Using a Hemin/G-Quadruplex Aptasensor

: In this study, we design an electrochemical aptasensor with an enzyme-free amplification method to detect tryptophan (Trp). For the amplified electrochemical signal, the screen-printed electrode was modified with dendritic gold nanostructures (DGNs)/magnetic double-charged diazoniabicyclo [2.2.2] octane dichloride silica hybrid (Fe 3 O 4 @SiO 2 /DABCO) to increase the surface area as well as electrical conductivity, and the hemin/G-quadruplex aptamer was immobilized . The presence of Trp improved the catalytic characteristic of hemin/G-quadruplex structure, which resulted in the efficient catalysis of the H 2 O 2 reduction. As the concentration of Trp increased, the intensity of H 2 O 2 reduction signal increased, and Trp was measured in the range of 0.007 – 200 nM with a detection limit of 0.002 nM. Compared with previous models, our sensor displayed higher detection sensitivity and specificity for Trp. Furthermore, we demonstrated that the proposed aptasensor successfully determined Trp in human serum samples, thereby proving its practical applicability. five different aptasensors and measured the signal in the presence of Trp (7 nM). The new results show that the relative standard deviation is 2.8%, indicating that the fabricated sensor has an excellent reproducibility. Furthermore, the prepared aptasensors were stored at 4 °C in wet chamber for 3 weeks to investigate the stability of the developed aptasensor. As shown in Figure S3, 94% of the initial electrochemical signal in the presence of Trp (7 nM) was maintained at 21 days after the preparation of sensors, proving the high stability of the prepared aptasensor.


Introduction
Tryptophan (Trp), an essential amino acid for humans, serves as a precursor for some biological molecules and structural component in proteins [1,2]. So far, different methods have been reported for measuring Trp, such as high-performance liquid chromatography (HPLC) [3], spectroscopy [4,5], and chemiluminescence [6]. As alternatives, many innovative approaches have been developed. For example, Eser et al. used ultra-HPLC coupled to electrospray ionization triple−quadrupole mass spectrometry to detect Trp [7]. In another study, Hazra et al. used resonance energy transfer between Ce 3+ and Tb 3+ ions in Ce 3+ /Tb 3+ -doped CaMoO4 nanocrystals to detect Trp [8]. However, these methods have the limitations such as complicated sample preparation, long periods of time to acquire measurements, and the need for advanced and costly laboratory tools. Therefore, electrochemical methods are highly regarded as the simple and low-cost alternative that provides sensitive measurements of Trp in samples [9][10][11][12][13].
Recently, aptamers along with antibodies have been used to probe and selectively measure many biological and pharmaceutical compounds [14][15][16][17][18]. Aptamers have many advantages over antibodies. Namely, aptamers have high stability in the presence of different parameters such as pressure, temperature, pH, in vitro synthesis, and labeling with different groups during synthesis [19]. It has been shown that some aptamers catalyze reactions similar to peroxidases [20][21][22][23]. For example, the guanine-rich aptamer that forms a G-quadruplex structure in the presence of hemin catalyzes the reduction of H2O2, which has been used in a wide range of applications [24].
When generating new electrochemical sensors, great attention has been paid to using various nanomaterials to immobilize the aptamer on the electrode surface, which subsequently alters the effective surface area, the electron transfer kinetics, and the sensitivity of the sensor [25][26][27]. Notably, magnetic nanoparticles have been favorably considered in many studies because of their high surface area, super paramagnetic properties, biocompatibility, and low toxicity [28][29][30][31]. However, magnetic nanoparticles have a strong tendency to oxidize and aggregate when exposed to air, which requires them to be coated with other materials. Covering the magnetic nanoparticles with a silica layer increases the stability of the nanoparticles and facilitates surface modification with different functional groups [32][33][34][35].
In this study, we first prepared the magnetic nanocomposite as an ionic liquid core-shell structure by sol-gel method in order to improve its conductivity and make it a suitable modifier for the preparation of electrochemical biosensors [31]. Next, dendritic gold nanostructures (DGNs) were electrochemically synthesized on the magnetic double-charged diazoniabicyclo [2.2.2] octane dichloride silica hybrid (Fe3O4@SiO2/DABCO)-modified screen-printed electrode (SPE) to not only increase the effective surface area and electrical conductivity, but also immobilize guanine-rich Trp aptamer. The presence of Trp allowed Trp aptamer to form hemin/G-quadruplex structure, enabling the efficient catalysis of the H2O2 reduction. Because of the dual amplification strategy based on DGNs/Fe3O4@SiO2/DABCO and Trp aptamer-hemin-mediated catalysis of H2O2 reduction, our sensor showed higher sensitivity than other electrochemical sensors for determining Trp levels. Notably, we discovered that the Trp aptamer-hemin has significantly improved the peroxidase-mimicking activity after binding to Trp, which is successfully utilized for the detection of Trp levels even in human serum.

Synthesis of the Fe3O4@SiO2/DABCO
The Fe3O4@SiO2/DABCO nanocomposite was synthesized in three steps according to the method described by Bagheri et al. [36]. In the first step, magnetic nanoparticles (MNPs) were prepared by a co-precipitation method from the reaction of FeCl3·6H2O and FeCl2•4H2O, which were then dried under vacuum at 60 °C. In the second step, bis(n-propyl trimethoxysilane) octane chloride (BPTDABCOCl) was prepared by the reaction of 1,4-diazabicycle [2.2.2] octane (DABCO) and 3-chloropropyl trimethoxysilane (CPTMS) in DMF and the resulting product was dried in an electrical oven. Finally, Fe3O4@SiO2/DABCO nanocomposite was synthesized using MNPs, BPTDABCOCl, and tetraethyl orthosilicate (TEOS) by sol-gel method. Figure 1 shows a schematic of the different steps for constructing the Trp aptasensor. First, we synthesized Fe3O4@SiO2/DABCO as a biocompatible nanocomposite to modify the electrode surface as described in Section 2.2. Next, the dendritic gold nanostructures (DGNs) were prepared through the electrodeposition of gold solution (HAuCl4; 0.5 mM) on Fe3O4@SiO2/DABCO/SPE under the potential of −0.23 V for a duration of 300 s, and Trp aptamer (3.0 × 10 −6 M) was then immobilized on the electrode surface overnight at room temperature. Finally, the hemin solution (20 μM) was dropped onto the electrode, which was incubated for 15 min. The state was then ready for analysis of the samples containing Trp. After each step, the modified electrode was thoroughly washed using distilled water. The differential pulse voltammetry (DPV) was performed with the potential range from 0 to −0.6 V in HEPES buffer (20 mM HEPES, pH 7.4, 100 mM NaCl, and 50 mM KCl), and H2O2 (20 µM). The error bars in all of the figures indicate the standard deviation among three replicates.

Determination of Trp in Human Serum
To prevent protein adsorption on the electrode surface and the related interference with the measurement, the human serum was first de-proteinized using trichloroacetic acid and subjected to the centrifugation. After removing the precipitates, the supernatant was then diluted three-fold using 0.1 M PBS (pH 7.0) and different concentrations of Trp was spiked into diluted human serum. These samples were finally analyzed using the same procedures described in Section 2.3.

Characterization of Sensing Interface
The surface morphologies of the bare SPE, Fe3O4@SiO2/DABCO/SPE, and DGNs /Fe3O4@SiO2/DABCO/SPE were first characterized using FE-SEM ( Figure 2). As shown in Figure 2B, Fe3O4@SiO2/DABCO/SPE displayed a uniform spherical structure with an average size of 23 nm, which is different from the SPE (Figure 2A). In addition to the benefits of the ionic liquid framework, the presence of functional groups on magnetic nanocomposites leads to greater deposition of gold nanostructures and prevents the agglomeration of nanoparticles on the magnetic nanocomposite surface [36]. Furthermore, AuNPs with an average size of 13.5 nm were successfully deposited on the Fe3O4@SiO2/DABCO/SPE surface as demonstrated by a dendritic structure, which was achieved by the specific interaction with the functional groups of the magnetic nanocomposite ( Figure 2C). To prove that the dendritic structure is only formed with the help of magnetic nanocomposites, we obtained the SEM image of gold nanoparticles (AuNPs) electrodeposited on the surface of bare SPE without magnetic nanocomposites ( Figure S1). We assumed that the deposition of AuNPs with a dendritic structure enabled the signal amplification as well as the immobilization of thiolated Trp aptamer on the electrode surface. Next, EIS and CV were recorded to investigate the fabrication process of the Trp aptasensor. Recently, there has been an escalating interest in using EIS for biological measurements since this technique can be performed within a narrow range of small potentials and have no detrimental effects on biological interactions [37]. Figure 3A shows These results confirmed the formation of the Trp-aptamer complex at the electrode surface. The EIS results were also supported using the CV measurements. As shown in Figure 3B, the changes in CVs of the [Fe(CN)6] 3-/4-redox couple confirmed that each preparation step was successfully carried out on the electrodes surface, and the results are in good agreement with EIS experiments.

Aptamer Surface Density and Detection Feasibility of Trp Aptasensor
The aptamer surface density on the modified electrode surface was investigated using the Tarlov equation (Equation (1)) [38]: where Γapt is the aptamer surface density (molecules/cm 2 ), Γ0 is the quantity of adsorbed redox marker  Figure 4A, the Γapt was calculated to be 2.6 × 10 18 (±1.2%) molecules/cm 2 using the Tarlov equation. Next, to demonstrate the detection feasibility of Trp aptasensor, the CV method was used to measure reduction signals of H2O2 in the absence of Trp (a) and presence of Trp at 3 nM (b) and 100 nM (c) ( Figure 4B). The Trp aptamer with hemin was not strong enough to form an active G-quadruplex structure for the effective catalysis of H2O2 reduction as indicated by the weak H2O2 current intensity ( Figure 4B, a). On the other hand, the presence of Trp at 3 nM (b) and 100 nM (c) significantly increased the reduction signal of H2O2. These data confirm that the Trp aptamer forms a stable G-quadruplex structure only in the presence of Trp, and serves as an effective catalyst for the amplified electrochemical signal.

Optimization of the Proposed Aptasensor
To obtain the optimal condition for the proposed aptasensor, different concentrations of the aptamer (1.0-6.0 µM) were applied onto the surface of the modified electrode and the EIS spectra were recorded. As the concentration of the aptamer increased up to 3 µM, the Rct increased, whereas the aptamer concentration beyond 4 µM led to a decrease in the Rct (Figure S2A). At higher aptamer concentrations, the intermolecular hybridization of the complementary regions may lead to a decrease in the immobilization of the aptamer on the modified electrode surface. Additionally, we investigated the effect of Trp incubation time on the prepared sensor. EIS spectra results showed that the optimal time for trapping Trp molecules on the sensor surface is 40 min and incubating for longer did not have a significant effect ( Figure S2B).

Sensitivity of the Prepared Aptasensor
As the Trp aptamer is rich in guanine, we assumed that in the presence of Trp, a stable G-quadruplex structure could form to effectively catalyze H2O2 reduction [39] (QGRS Mapper software, Table S1) [40,41]. By measuring the reduction signals of H2O2 using DPV technique, the performance of the designed sensor to detect different concentrations of Trp was evaluated. Figure 5A shows the DPV signals of H2O2 in the absence or presence of different concentrations of Trp. Figure 5B shows the calibration plots of H2O2 reduction signals versus Trp concentrations in three linear ranges. We observed that with increasing Trp concentrations, H2O2 reduction was more efficiently catalyzed because of the formation of stable G-quadruplex structure on the electrode surface. It is assumed that Trp induces the conformational change of aptamer, mediating the formation of stable G-quadruplex, and accordingly improves the peroxidase-like activity of hemin/Gquadruplex structure. As seen in the calibration plot ( Figure 5B), the intensity of the current is proportional to the Trp concentration in the three ranges (0.007-0.1 nM, 0.1-10 nM, and 10-200 nM). The detection limit was estimated to be 0.002 nM (LOD = 3Sb/m, where Sb is the standard deviation of the blank signal (n = 7), and m is slope of the linear calibration range) [42,43], which is superior to that of other electrochemical detection methods (Table 1).

Selectivity, Real Sample, Reproducibility, and Stability
Finally, the selectivity of this sensor was examined by measuring the reduction signals of H2O2 using DPV technique in the presence of 7 nM of Trp (a), and 20 nM of either tyrosine (b), histidine (c), arginine (d), lysine (e), valine (f), or methionine (g), and a blank control (h) (Figure 6). The H2O2 reduction signal in the presence of Trp was higher than other amino acids that showed negligible responses comparable to the blank control. To demonstrate the potential clinical application for this sensor, three concentrations of Trp were spiked in diluted human blood serum (33%), which were analyzed using our proposed method. The results in Table S2 showed that the prepared aptasensor analyzes Trp in the human blood serum samples with excellent reproducibility and precision as evidenced by the coefficient of variation (<2%) and accuracy (<10%). Regarding the reproducibility between samples, we prepared the five different aptasensors and measured the signal in the presence of Trp (7 nM). The new results show that the relative standard deviation is 2.8%, indicating that the fabricated sensor has an excellent reproducibility. Furthermore, the prepared aptasensors were stored at 4 °C in wet chamber for 3 weeks to investigate the stability of the developed aptasensor. As shown in Figure S3, 94% of the initial electrochemical signal in the presence of Trp (7 nM) was maintained at 21 days after the preparation of sensors, proving the high stability of the prepared aptasensor.

Conclusions
We have developed a sensitive and selective electrochemical aptasensor for the detection of Trp using a hemin/G-quadruplex structure to amplify the H2O2 reduction signal. The fabricated aptasensor showed a low detection limit, wide linear range, and excellent reproducibility to measure Trp levels. The high sensitivity and reliable performance of this aptasensor are attributed to the dual approach for signal amplification. First, the modified electrode with DGNs/Fe3O4@SiO2/DABCO/SPE led to an increase in the effective surface area, electron transfer, and electrochemical signal. Second, the hemin/G-quadruplex structure served as a catalyst to enhance the H2O2 reduction signal. Furthermore, the prepared aptasensor exhibited high selectivity toward Trp and detected Trp in human blood serum with high levels of accuracy, which suggests the potential application of this sensor for clinical use.  Table S1: Analysis of Trp aptamer sequence by QGRS Mapper software, Table S2: Analysis of human serum samples with Trp at different concentrations.
Author Contributions: Carrying out experimental work, discussion of the results, and writing the text A.B.H.; editing of the text and supervision J.B.R. and K.S.P. All authors have read and agreed to the published version of the manuscript.