Design of an innovative aptasensor for the detection of chemotherapeutic drug Fludarabine phosphate

ssDNA selection and characterization of aptamers against Fludarabine phosphate

The binding of Fludara drug to the beads in SELEX is a crucial step that enables the separation of the bound and unbound DNA. Fludara was immobilized on NHS-activated Sepharose beads through its terminal amine groups. The NH₂ groups on the drug molecules were activated and then used to react with the terminal carboxyl groups on the beads through amide bond formation. After the covalent binding of Fludara molecules to the sepharose beads, 0.1 M of Tris was used to block the remaining unreacted amine groups on the beads. This step would minimize the non-specific electrostatic attraction of the DNA to sepharose beads. The prepared Fludara-conjugated sepharose beads were used for all the aptamer selection cycles. Each cycle consisted of two main steps: selection and amplification. For selection, the beads were incubated with the DNA library composed of 10¹⁵ random 40 nucleotide sequences followed by partitioning the bound and unbound DNA via binding buffer washing. Then, the ssDNA candidates specifically bound to the Fludara-conjugated sepharose beads were eluted, PCR amplified and purified to obtain a new enriched DNA pool to start the subsequent SELEX cycle.

The DNA recovery after each cycle was monitored by measuring the fluorescence of the eluted DNA. A gradual increase in the recovered DNA was observed after each cycle. Eight rounds of positive selection were performed, after which a counter selection, consisting of incubating the DNA collected from the eighth round with negative unconjugated sepharose beads, was carried out. Negative selection is a crucial step to avoid the selection of aptamers exhibiting an affinity for the immobilization support. It has been demonstrated that the elimination of these non-specific candidates improves the affinity of the generated aptamers ten times²². Fig. 1 shows the fluorescence intensities obtained from the DNA measurements of aptamers eluted from each cycle.

As it can be seen from the histogram, the fluorescence intensity increased with successive SELEX cycles. After the counter selection, a significant decrease in the fluorescence intensity was noted at rounds 9 and 10, indicating the removal of the DNA that was non-specifically bound to the beads. Then, a dramatic increase in the fluorescence intensity was observed at rounds 10 and 11, implying the enrichment of the DNA pool with the specific sequences to Fludara. Therefore, the selection process was stopped after 11 rounds, and the recovered DNA was cloned into E.coli competent cells. The selected colonies were then picked up for amplification and sequencing. Finally, the identified sequences were grouped using the multiple sequence alignment software PRALINE (Supporting information).

Binding affinity studies of the selected aptamers towards Fludara

A total of ten sequences was generated from the aptamer’s selection process. These sequences underwent a binding affinity study, to identify the most specific aptamers to our targeted analyte; Fludarabine phosphate drug. To enable the DNA quantification, the selected sequences were labeled with fluorescein. The study was carried out by incubating a fixed number of Fludarabine-conjugated beads with increasing concentrations (0 to 400 nM) of each fluorescently-labelled sequence, individually. Then, the unbound sequences were eliminated by washing and the bound sequences were eluted, and their fluorescence intensities were quantified at 515 nm. Afterwards, the binding curves were plotted for each aptamer sequence as the fluorescence intensity versus the DNA concentration. The dissociation constants (Kds) of the various aptamer-target complexes were calculated by non-linear regression analysis of the binding curves. The aptamers sequences and dissociation constants are listed in Table 1.

The obtained Kd values range from 18.86 to 373.7 nM showing the success of the aptamer selection process towards Fludara with high affinity. As it can be seen from the table, Flu-3 exhibited the highest affinity with a Kd as low as 18.86 nM. Therefore, it was selected to pursue the following experiments.

Electrochemical biosensing of Fludara drug using the selected aptamers

Design of the apatasensor for Fludara detection

Flu-3 was employed in the fabrication of the electrochemical aptasensor for Fludarabine detection. The integration of the selected aptamer into an electrochemical platform was achieved through the covalent linking of SH groups labelling the ssDNA with the gold surface of the SPGE. This immobilization strategy was chosen for different reasons. First, it is a simple technique that doesn’t require many steps or reagents. More importantly, the spontaneous adsorption of sulfur groups onto metal surfaces generates uniform layers of well oriented bioreceptors. In parallel, the inert properties of gold and its ability to form well-defined crystal structures influence strongly the generation of self-assembled monolayers leading to high density and excellent degree of regularity²³. The surface modification process is illustrated on Fig. 2.

Aptasensor design for Fludara drug detection.

To confirm the successful linking of our aptamer onto the gold surface, electrochemical characterization was proceeded. For that, cyclic voltammetry measurements were conducted in redox solution (5mM ferri/ferrocyanide solution prepared in PBS buffer pH 7.4) within a potential range of (-0.6 to 0.6 V) and a scan rate of 100 mV/s. Figure 3A displays the cyclic voltammograms recorded on the bare gold surface (a), the aptamer functionalized surface (b) and the SPGE after MCH blocking step (c). As it can be seen from curve a and b, the chemisorption of thiols to gold resulted in a remarkable drop in the oxydo/reduction peaks. This decrease is certainly due to the formation of the aptamers monolayer which is based on three mains steps; diffusion-controlled physisorption and chemisorption of the thiolated-DNA, followed by the crystallization process²⁴. Then, after the aptamer immobilization, MCH was used to displace the non-specific adsorption of DNA via nitrogen atoms thus enhancing the accessibility of aptamer molecules for Fludarabine. The corresponding cyclic voltammogram shows a decrease in the peak currents due to the repulsion between negatively charged DNA backbones and the negative dipole of the MCH alcohol terminus^25,26.

(A) Cyclic voltammetry, (B) electrochemical impedance spectroscopy of bare gold electrode, Flu-3 aptamer modified electrode and after blocking with MCH. The CV and EIS measurements were recorded in the presence of Fe(CN₆)^3–/4– as the redox probe, within a potential range of (-0.6 to 0.6 V) and a scan rate of 100 mV/s for CV and frequency range between 100 kHz and 0.1 Hz for EIS.

To improve the investigation of surface modification through various steps, electrochemical impedance spectroscopy was conducted under consistent conditions as shown in Fig. 3B. Initially, a very small semicircular domain with R_CT= 1.34 Kῼ was recorded, indicating high surface conductivity. Following the biosensor modification with the aptamer, the R_CT ​ value increased to 1.89 Kῼ, and further increased to 4.67 Kῼ after the addition of the blocking agent. This increase in electron transfer resistance confirms the successful immobilization of both the aptamer and MCH on the gold surface. The results obtained demonstrate good concordance between the electrochemical techniques used. To better control the surface morphology during the different modification steps, the subsequent section will elucidate TO surface analysis using SEM techniques.

Electrocatalytic study of the aptamer-modified surface

To better quantify the amount of aptamer covering the electrode surface, we calculated the surface area before and after modification with aptamer/SAM. To achieve this objective, we recorded the cyclic voltammetry (CV) voltammograms of the bare gold electrode and the gold electrode modified with aptamer and MCH, in response to varying scan rates using a redox probe. The obtained voltammograms are illustrated in Fig. 4A.

To enhance visualization of the CV variations, the oxidation and reduction currents versus the square root of the scan rate for the bare gold electrode and the modified electrode were plotted in Fig. 4B and C, respectively.

(A) Cyclic voltammograms recorded at different scan rates (10, 20, 50, 100, 200, 300, 500 mV/s) in an equimolecular solution of 5 mM ferro/ferricyanide ([Fe (CN)6]^4−/3−), Plots of the peak current (Ip) versus the square root of the potential scan rate(v^1/2) (B) on bare SPGE and (C) Flu-3- aptamer/ SPGE.

In both representations, a linear relationship between the oxidation and reduction peak current (ip) versus in visualized, revealing a diffusion-controlled oxidation process, permitting therefore the estimation of the diffusion coefficient of ferricyanide cation on bare gold (Fig. 4B). The D was found in the order of 24.62 × 10^− 6 cm².s^− 1. By applying the Randles–Sevcik equation (Eq. (1), noted below), we have determined the surface area after modification (Table 2).

Where I_p.a. refers to the anodic peak current, n is the number of electrons transferred, A is the dynamic surface area of electrode (cm²), D₀ is the diffusion coefficient (cm².s^− 1), v is the scan rate (V.S^− 1), and C₀ is the concentration of K₃Fe(CN)₆ (mol.cm^− 3).

The results obtained in Table 2 demonstrate a decrease in the geometric surface about only 7.7% compared the bare gold. Such variation reveals the effectiveness of the immobilization of biological layer (Flu-3 aptamer) on the surface of SPGE.

Electrochemical detection of Fludarabine

The principle of the proposed aptasensor is based on monitoring the electrochemical behavior resulting from the specific binding between the aptamer and its target, Fludarabine phosphate. To enhance the analytical performance of the sensor, several parameters must be controlled and optimized, including the aptamer concentration, the aptamer immobilization time, and the analyte incubation time. Based on our previous investigations, we selected an aptamer concentration of 1 µM and an overnight incubation time for immobilization²⁷.

In parallel, to investigate the most appropriate time of Fludarabine incubation on the modified surface, the normalized variation of the output signal ((i⁰– i) / i⁰%) is followed versus different incubation time ranging from 5 to 30 min, where i is the peak current obtained after incubating the aptasensor with Fludara (100 pg/mL) and i⁰ is the peak current of the control. The obtained results are illustrated on Fig. 5.

Optimization of time incubation of aptamer incubation on the electrode surface.

The normalized current variation shows an increase versus time incubation from 5 to 25 min, until reaching a stability between 25 and 30 min. The results indicate that the appropriate time for a maximal binding and recognition between the aptamer and the molecule is in the order of 25 min.

Once the sensor performances are well optimized, the surface was incubated with increasing concentrations of Fludarabine, prepared in binding buffer, in the range of 1 to 150 pg/mL. The electrochemical measurements were carried out by SWV in the redox solution of 5mM ferri/ferrocyanide prepared in PBS buffer pH 7.4 in the potential range of (-0.2 to 0.5 V), frequency 25 Hz, interval time 0.04 s, step potential − 5 mV, scan rate 125 mV/s and amplitude 20 mV. In parallel, a control experiment was realized by incubating the aptasensors with 10 µL of binding buffer (0 pg/mL of Fludarabine). Figure 6A shows the obtained SWV voltammograms after incubation of the gold electrode with the different concentrations of Fludarabine (1 to 150 pg/mL). From the figure, we observe that the peak current decreases by increasing the Fludarabine phosphate concentration. This decrease is mainly due to the formation of the complex aptamer-Fludarabine phosphate with high affinity and specificity. Therefore, this binding hampers the electron transfer to the gold surface resulting in decreasing current peaks. The electrochemical response of our aptasensor towards Fludara was subsequently determined based on the obtained peak currents. This response was then used to plot the calibration curve shown in Fig. 6B. As it can be observed from the figure, a good linearity was obtained for Fludara detection within the range of concentrations; 1 to 150 pg/mL.

(A) SWV voltammograms of the aptamer-functionalized electrode incubated with increasing concentrations of Fludarabine phosphate (0.5 to 150 pg/mL), (B) Calibration curve of Fludara: Plot of the aptasensor response ((i⁰-i/i⁰) %) versus Fludara concentration (pg/mL). SWV was performed in 5mM ferri/ferrocyanide solution prepared in PBS buffer pH 7.4, potential range of (-0.2 to 0.5 V).

The linear regression in the range of 1–150 pg/mL was as follows: ((i⁰-i)/i⁰%) = 18.25 + 0.20 Fludara concentration (pg/mL) with R² equal to 0.99. The limit of detection limit as well as the limit of quantification were calculated as LOD = 3.3σ / S, and LOQ = 10σ / S respectively, where σ is the standard deviation of the blank and S is the slope. LOD and LOQ were found in the order of 0.11 pg/mL (0.31 fM) and 0.39 pg/mL (1.06 fM) respectively.

As mentioned in the introduction, electrochemical detection of Fludarabine using sensors is rarely reported in the literature. To highlight the analytical performance of the prepared sensor, particularly in term the limit of detection, we present a comparative analysis in Table 3. This table includes data from conventional techniques as well as sensors developed in silico for Fludarabine detection.

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Table 3 illustrates that the present work, characterized by simplicity in preparation, rapidity, and low cost, demonstrates excellent performance with a very low limit of detection compared to conventional techniques and even to other designed electrochemical sensors.

Investigation of the analytical performance of the elaborated sensor

Reproducibility and repeatability

Given that reproducibility is fundamental to the successful design, implementation, and adoption of sensors in healthcare, this parameter was investigated by following the electrochemical response of triplicate aptasensors prepared under the same conditions. The results demonstrated good reproducibility with an RSD of 4.5%, indicating the robustness of the prepared aptamers. Similarly, sensor stability over time is essential for ensuring reliable performance, reducing costs, meeting regulatory standards, and maintaining user trust. Therefore, stability was examined by recording the SWV measurements after 1, 7, and 15 days. The measurements demonstrated stability with deviations of 2%, 4%, and 8%, respectively. These results suggest the potential for repetitive sensor use, underscoring the stability of the aptamer designed using the SELEX method. Additionally, the repeatability of the proposed aptasensor was evaluated by recording five successive current signals, yielding an RSD of 1.99%.

Selectivity measurements

Aiming to validate the clinical applicability of the selected aptamers as well as the fabricated aptasensor for Fludara monitoring, it was necessary to realize a cross-reactivity study. To achieve that, the Flu-3 functionalized SPGEs were incubated with different drugs including chemotherapeutics and antibiotics, separately. The analytical procedure was performed in the same experimental conditions and according to the protocol previously described for Fludara detection. In Fig. 7, a histogram comparing the aptasensor response towards Fludara with that of the potential interfering drugs: 5-fluorouracil, docetaxel, amoxicillin, penicillin and doxycycline.

Cross-reactivity study of the electrochemical Fludara aptasensor.

From the histogram, we note that all the incubation of the non-specific drugs (100 pg/mL) with the functionalized gold surface didn’t induce a peak decrease in the SWV measurements. Therefore, the corresponding responses based on the calculated ((i⁰-i)/i⁰%) were all in negative order. Therefore, these findings confirm the excellent selectivity of our aptasensor to Fludarabine detection.

Real sample investigation

In parallel, to demonstrate that the designed aptamer-based strategy could be applied in blood patients’ samples without matrix effects, the aptasensor was tested with serum samples diluted (1: 100 in buffer solution). The Flu-3 modified SPGE were incubated with serum samples spiked with different concentrations of Fludarabine phosphate within the analytical range. Then, square wave voltammograms were recorded by using the same experimental conditions described above.

Finally, the recovery percentage was calculated by comparing the analytical response of the aptasensor in binding buffer with that in serum samples. Table 4 presents the added concentrations to each serum sample, the calculated concentrations, and the recovery percentages. The results showed excellent recovery percentages ranging from 96, indicating that the developed Fudara aptasensor can be successfully applied in biological fluids without interferences or matrix effects.