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. 2026 Aug 19;6(5):1489–1495. doi: 10.1021/acsmeasuresciau.6c00254

Robust Electrochemical Aptasensing of VEGF Based on Poly-adenine Mediated DNA Self-Assembly and Triple Labeling of Silver Nanoparticles

Zhenming Zhang †,‡, Hua Chai ‡,§, Xin Ma †,*, Peng Miao §,∥,*
PMCID: PMC13693276  PMID: 42921454

Abstract

Vascular endothelial growth factor (VEGF) is a cell-secreted signaling protein that is regarded as the biomarker and therapy target for certain diseases. In order to provide an accurate and robust sensing method, we herein construct a cost-effective electrochemical interface based on polyadenine-mediated DNA self-assembly and tetrahedral DNA (TDNA). The three-dimensional DNA nanostructure possesses multiple sites to immobilize silver nanoparticles (AgNPs) for strong electrochemical response, which also contains aptamer sequence against target VEGF. Thus, the density of AgNPs can be modulated by VEGF and the recorded stripping voltammetric signal can sensitively reflect the level of target protein. The electrode modification technique and recognition element improved the practical utility of this method. It is also challenged with human serum samples, and the results are consistent with the standard method. This sensor provides a feasible way to identify VEGF for potential clinical applications.

Keywords: VEGF, Electrode Modification, Aptamer, Silver nanoparticles, DNA tetrahedron

graphic file with name tg6c00254_0007.webp

graphic file with name tg6c00254_0006.webp

Vascular endothelial growth factor (VEGF) stimulates certain enzymes in endothelial cells and regulates the formation of new blood vessels, which plays a pivotal role in angiogenesis and vascular homeostasis. , Meanwhile, it is critical to maintain the balance of tissue oxygen supply and metabolism. , Abnormal expression of VEGF affects physiological functions in embryonic vascular development, tissue repair, and regeneration, thereby inducing the occurrence and progression of certain diseases such as malignant tumors, retinal lesions, inflammatory disorders, and orthopedic diseases. − For example, VEGF165, the dominant isoform within the VEGF family, is frequently overexpressed by tumor cells and tumor-associated macrophages through autocrine and paracrine pathways. , It triggers the formation of disordered neovascularization for the exponential growth of tumor cells and assists tumor immune escape. Therefore, the level of VEGF is clinically associated with tumor staging and prognosis. The development of VEGF-targeted drugs is also an important strategy for antitumor therapy, further highlighting the significance of the detection of VEGF.

Traditional VEGF detection methods include enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC), and Western blot, in which ELISA serves as the gold standard. Despite their widespread applications, some inherent limitations exist, which restrict their further clinical translation for the diagnosis of diseases such as cancers. For example, cumbersome operations and long reaction times are always required, making them difficult to apply for rapid or on-site detection. To meet the high demand for VEGF assay in modern clinical scenarios, a variety of other techniques have been employed including time-resolved fluorescence immunoassay, microfluidic platform, surface plasmon resonance (SPR), as well as electrochemical sensors. Nevertheless, most of these methods still face challenges of complex probe labeling, professional operations, or expensive chips, limiting their popularization. Although electrochemical techniques exhibit the advantages of high sensitivity, rapid detection speed, simple operation, and low cost, several critical problems still need to be addressed. First, nonspecific adsorption of complex biological samples may occur on the electrode surface, which leads to undesired background interference. Second, the immobilization efficiency of the antibody as a target recognition element on the electrode surface is insufficient. Third, electrochemical protein assays always lack powerful signal amplification compared to electrochemical nucleic acid sensors.

DNA nanotechnology emerges as a powerful tool for the construction of rationally designed nanostructures, which benefit improved electrochemical sensing applications. − For instance, three-dimensional DNA frameworks offer confine affinity recognition and resist nonspecific adsorption. Herein, we devise an electrochemical aptasensor for VEGF by coupling poly-adenine mediated DNA modification and triple labeling of silver nanoparticles (AgNPs) on a single tetrahedral DNA (TDNA). TDNA is a typical nanoscale DNA framework with high rigidity and biostability. , It was first constructed by annealing four appropriately designed oligonucleotides together in Turberfield’s Lab. Currently, TDNA has been widely explored for developing electrochemical sensors. − However, most previous strategies were accomplished by anchoring TDNA to the working electrode via triple gold–sulfur interactions, lacking the advantages of cost-effectiveness and versatility. In this work, poly-adenine sequence is utilized to assemble the DNA nanostructures on the electrode surface with high affinity, endowing TDNA more sites for the signal labels. Besides, aptamer sequence is embedded in TDNA, which helps the translation of protein into nucleic acid information. Currently, AgNPs are extensively applied for electrochemical sensing due to their unique properties. , In our previous studies, we took good advantage of the highly characteristic solid-state Ag/AgCl reaction of AgNPs for electrochemical sensing applications. , In this work, the triple labeling of AgNPs provides sufficient stripping voltammetric responses for trace VEGF detection.

Results and Discussion

Working Mechanism

Detailed sensing principle of this electrochemical method is illustrated in Figure . VEGF165 is utilized as the target, hereafter abbreviated as VEGF. First, four single-stranded DNA probes (T1, T2, T3, and T4) are blended to form the three-dimensional TDNA nanostructure. All sequences can be found in Table S1. Three probes are modified with amino groups at the 5′ end, which are located at three vertexes of TDNA after DNA assembly. In addition, a pendant single-stranded region is located at the fourth vertex, which contains the aptamer sequence (26-base) of target VEGF (Figure S1). To immobilize TDNA nanostructures at the electrode, we introduce poly-adenine sequence instead of modifying thiol groups. Generally, A30 tailed sequence (Probe 1) is used to incubate with the bare gold electrode. This strategy allows high affinity comparable to Au–S chemistry and well-organized upright conformation of single strand region. Since it contains a 15-base region which is complementary with the aptamer sequence, Probe 1 can capture TDNA via hybridization reaction. Furthermore, silver plays the role as a proton acceptor and form nonconventional H-bonds (N–H···Ag). Thus, AgNPs can be attached to the nitrogen of the amino group on the three vertexes of TDNA. Solid-state Ag/AgCl reaction of AgNPs can thus provide sharp stripping signals. On the other hand, the pendant aptamer sequence (26-base) on TDNA has significant affinity for the heparin-binding domain of VEGF with K d value of 0.5 nM. Therefore, in the presence of VEGF, the metastable duplex (15 base pairs) formed by T1 and Probe 1 is disrupted. The aptamer tends to bind well with target protein, releasing the single-stranded 15-base region of Probe 1. Triple AgNPs anchored to TDNA are thus kept away from the electrode interface. By analyzing the decreased electrochemical response, highly sensitive analysis of VEGF can be achieved.

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Illustration of the VEGF-responsive electrochemical sensor based on poly-adenine mediated electrode immobilization and triple AgNPs labeling.

Characterizations of AgNPs and DNA Assembly Events

The successful synthesis of AgNPs is first confirmed by dynamic light scattering (DLS), transmission electron microscopy (TEM), and UV–vis absorbance spectroscopy. An average size of about 12 nm is obtained by DLS analysis, which is slightly larger than the TEM conclusion (Figure S2).This is due to the electrical double layer surrounding the AgNPs. In a typical TEM image, sphere-structured nanoparticles are observed with good dispersibility, and the size is about 6 nm (Figure S3a). The characteristic peak around 400 nm appears in the UV–vis absorbance spectrum, which is further evidence of successful synthesis of AgNPs (Figure S3b).

The formation of TDNA is confirmed by the results of polyacrylamide gel electrophoresis (PAGE) image (Figure S4). The fuels strands of T2, T3, T4, and T1 were mixed with different combinations. With the increase of the number of TDNA fuel strands (from 1 to 4), the sizes of the hybridized products exhibit larger molecule weights (lanes a–d), demonstrating the successful hybridization reactions. In the presence of the four strands, the band with the largest molecule weight is ascribed to the formed TDNA nanostructure. The three-dimensional structure of TDNA formed by the annealing of four fuel strands was already demonstrated in our previous study. With further addition of Probe 1, the position of the resulting band runs slower (lane e). To convince the hybridization between TDNA and Probe 1, we have further compared the bands of T1, Probe 1, and their product. It is clearly observed that after mixing the two strands, a new band with an obvious larger molecule weight appears, demonstrating successful formation of the duplex (lanes f, g, and h).

Electrochemical Characterizations

To further study the DNA assembly process at the electrode surface, cyclic voltammograms (CV) and electrochemical impedance spectroscopy (EIS) were performed after each step of the experiments using the classic electrochemical species: [Fe­(CN)6]3–/4–. As shown in Figure a, well-defined oxidation and reduction peaks are observed for the bare electrode, demonstrating excellent electric conductivity of the substrate working gold electrode. The immobilization of Probe 1 with poly-adenine tail introduces a negatively charged molecular layer on the electrode, which repels [Fe­(CN)6]3‑/4‑. Therefore, the peak currents become smaller. After further loading of mercaptohexanol (MCH) as a spacer to modulate Probe 1, the peak currents are even smaller. Meanwhile, the peak potential distance is also enlarged. Next, TDNA with a pendant aptamer sequence can hybridize with Probe 1 and further block electron transfer, which is reflected by the significantly declined peak currents (Figure b). EIS curves were then recorded to further verify the process. In a typical Nyquist plot, a semicircle portion at higher frequencies relating to the electron-transfer-limited process and a linear part at lower frequencies corresponding to diffusion can be observed. As depicted in Figure c, only a straight line is shown for the bare electrode. After subsequent modifications of Probe 1, MCH, and TDNA, the diameter of the semicircle portions is increased gradually, demonstrating the increase of the interfacial charge transfer resistance. Impedance data can be quantitatively calculated by fitting to an equivalent circuit (Table S2). R ct values during each modification step are compared in Figure d. The results indicate that the modification of DNA or hybridization brings higher density of negative charges, which hinder the approach of [Fe­(CN)6]3–/4–.

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(a) Cyclic voltammograms of bare electrode, Probe 1 modified electrode, after MCH treatment and subsequent TDNA incubation. (b) Corresponding differences between oxidation and reduction peaks. (c) Nyquist diagrams of bare electrode, Probe 1 modified electrode, after MCH treatment and subsequent TDNA incubation. Inset is the equivalent circuit. (d) Corresponding R ct values. Statistical significances were defined as (*) p < 0.05, (***) p < 0.001, (****) p < 0.0001.

Silver Stripping Voltammetric Responses

The quantitative signal of this electrochemical sensor is from solid-state Ag/AgCl reaction of immobilized AgNPs. Thus, the density of AgNPs on the surface is critical to the electrochemical signals. We have introduced three single-stranded DNA probes (T2–2, T3–2, T4–2) with the same sequences as T2, T3 and T4. The differences are the deficiency of amino groups at the 5′ terminals. We thus synthesized four different TDNA nanostructures with 0, 1, 2, and 3 amino groups to capture AgNPs (Figure a). These TDNA nanostructures were incubated with Probe 1 modified gold electrode, which were further subject to linear sweep voltammetry (LSV) measurements. As shown in Figure b, TDNA without labeling AgNPs exhibit no stripping response, which is as expected. In the presence of amino groups on TDNA, AgNPs can be localized and a stripping peak around 0.1 V is observed. With the increase of the number of AgNPs on a single TDNA, the peak intensity becomes significantly larger (Figure c). Therefore, we choose TDNA with triple labeling of AgNPs in the following experiments. The stripping signal is extremely sharp compared with commonly used electrochemical species, which is quite suitable for quantitative analysis.

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(a) Illustration of zero, one, two, and three AgNPs labeled TDNA localized at the electrode. (b) Linear sweep voltammograms of the electrode modified with different forms of TDNA nanostructures. (c) Corresponding current intensities at 0.1 V. (d) Analysis results of peak current intensities using varied concentrations of Probe 1 for electrode modifications. (e) Optimization of VEGF reaction time using different modified electrodes. Statistical significance was defined as (****) p < 0.0001.

The density of Probe 1 on the electrode determines the cap of AgNPs density. However, too crowded environment may prevent efficient hybridization. Therefore, we prepared a series of concentrations of Probe 1 (0.01, 0.05, 0.1, 0.2, 0.5 μM) for electrode modification. With the increase of concentration from 0.01 to 0.1 μM, more AgNPs can be captured by the immobilized Probe 1 strands, which provide intense silver stripping current. However, with further increase of Probe 1 density, the strands are not favorable to load AgNPs and the LSV peak current declines. Therefore, 0.1 μM is chosen as the optimal concentration (Figure d). The stability and reproducibility of Probe 1/TDNA modified electrodes are also investigated. Three independent electrodes were first modified and then stored at 4 °C for 1, 3, 5, and 7 days before LSV scans. The recorded peak intensities are nearly the same as freshly prepared electrodes, indicating excellent batch-to-batch reproducibility and high stability of silver-amino chemistry during 7 days (Figure S5).

The reaction time of VEGF was also tested. Probe 1/TDNA-modified electrodes were immersed in solutions of VEGF for 5, 10, 20, 30, 40, and 50 min. After being carefully rinsed, these electrodes were measured with the LSV technique. As shown in Figure e, the peak intensity becomes smaller from 5 to 30 min. With a longer incubation time, the values barely change. Therefore, 30 min was selected as the optimized reaction time. Besides, we also introduced Probe 1* with an A30 tailed sequence and a random sequence that was not complementary with the aptamer of VEGF. T1* was also designed to hybridize with probe 1* instead. Probe 1*/TDNA*-modified electrodes were then treated with VEGF solution for different times. As expected, the LSV peak intensity barely changes during 50 min, demonstrating that target VEGF cannot bind with the DNA sequence, and the duplex is not disturbed, verifying the high selectivity of the strategy.

Quantification of VEGF

Under optimized experimental conditions, the analytical performance of this aptasensor for VEGF assay was investigated in detail. The prepared TDNA modified electrodes were incubated with a series of concentrations of VEGF for 30 min. Upon careful rinsing, linear sweep voltammograms were recorded and compared in Figure a. With the increase of VEGF, the LSV peak intensity exhibits a progressive decline, which demonstrates more TDNA nanostructures are conjugated with VEGF and kept away from the electrode interface. The decreased density of immobilized AgNPs thus leads to the changes of stripping signal. To analyze the quantitative relationship, the peak intensities are calculated and compared. As shown in Figure b, a linear relationship is established between the peak current and the logarithm of VEGF level in the range from 10 to 104 pg/mL. The equation is as follows:

y=−3.424x+18.32⁣(R2=0.98,n=4)

in which y stands for the peak current intensity (μA), while x is logarithmic concentration of VEGF (pg mL−1). The limit of detection (LOD) is estimated to be 6.5 pg/mL (S/N = 3). The analytical characteristics of this sensor are then compared with previously reported methods (Table S3). As illustrated, this sensor demonstrates superiority in broad linear range and low LOD. Meanwhile, the costs of sensing interface modification, recognition and signal elements are significantly reduced. These excellent performances are mainly ascribed to the combination of poly-adenine mediated DNA self-assembly and triple labeling of AgNPs on TDNA.

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(a) Linear sweep voltammograms for the analysis of VEGF of concentrations from 0 to 10 ng mL–1. (b) Calibration curve reflecting the linear relationship between the peak current intensity and the logarithm of VEGF concentration. (c) Peak currents intensities in the presence of VEGF and potentially interfering species. (d) LSV responses for the analysis of VEGF spiked in PBS, cell medium and serum samples. Statistical significance was defined as (****) p < 0.0001.

To evaluate the selectivity of this VEGF sensor, we first selected bovine serum albumin (BSA), platelet-derived growth factor (PDGF), trypsin, tumor necrosis factor-α (TNF-α), hemoglobin and thrombin as potential interferents (1 ng mL–1), which were tested under optimized experimental conditions. The LSV peak intensities were recorded and compared with that of VEGF (100 pg mL–1) case. The results demonstrate a remarkable decrease in LSV response for VEGF, while the peak intensities for these potential interferents are nearly the same with TDNA-modified electrode (Figure c). This finding highlights the good selectivity of the VEGF sensor, which is originated from the specific recognition ability of aptamer sequence. To further check the antijamming capability of this method, different levels of standard VEGF solutions (20, 100, 500 pg mL–1) were first spiked into biological samples of fresh cell medium and fetal bovine serum without pretreatments. Afterward, these samples were incubated with Probe 1/TDNA modified electrodes as mentioned above. The reacted electrodes were subjected to the electrochemical sensing. The obtained peak current intensities are summarized in Figure d. Compared with the values obtained in phosphate buffered solution (PBS), the electrochemical responses in biological matrixes are similar, demonstrating practical potential of this sensor for VEGF in real samples.

Real Sample Analysis

Encouraged by the good performance, we further evaluated the feasibility of this sensor in clinical applications. Serum samples from nine cervical cancer patients and nine healthy individuals were collected and diluted with PBS for 10 folds. VEGF levels in the solutions were then determined by this aptamer-based electrochemical sensor and a commercial ELISA kit with antibody as the recognition element, respectively. The calculated values listed in Figure a show that the two methods provide consistent VEGF levels across the 18 clinical samples, demonstrating robustness and practical utility of the proposed sensor for future applications. In addition, different expression levels of VEGF from two groups of clinical samples (healthy individuals’ and patients’ samples) can be directly revealed by the heat map (Figure b). The average concentration is elevated in cervical cancer patients, averaging 0.675 ± 0.321 ng mL–1 compared to 0.166 ± 0.065 ng mL–1 in healthy controls. The significant difference proves that the electrochemical method has diagnostic potential for cervical cancer (Figure c). The receiver operating characteristic (ROC) curve analysis is also established to quantitatively assess the analytical performance in clinical samples (Figure d). The calculated AUC value is 0.988, which verifies good sensitivity and specificity to assist the diagnosis of certain diseases.

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(a) Comparison of the detected results obtained by ELISA and the proposed electrochemical method. (b) Heat map of the electrochemical response of VEGF across samples from 9 healthy individuals and 9 cancer patients. H1–3 means the samples from H1, H2, H3, and so on. (c) Box plot of the calculated results of the two groups. (d) ROC curve analysis of the electrochemical method for the detection of 18 clinical samples. Statistical significance is defined as (***) p < 0.001.

Conclusion

In summary, we have systematically evaluated a highly sensitive and facile electrochemical aptasensor based on poly-adenine-mediated DNA self-assembly. The electrode modification process is convenient and cheap, compared with the thiol-based method. The as-fabricated TDNA layer on the electrode can be displaced by target VEGF, leading to significant LSV signal decline, demonstrating superior analytical performance. This electrochemical sensor for VEGF exhibits robust practicality, which not only possesses excellent specificity against potential protein interferents or any biological matrix but also provides comparable results with commercial ELISA. It provides a versatile strategy for accurate and economic protein assays, which can assist the study of biological mechanisms and disease diagnosis.

Supplementary Material

tg6c00254_si_001.pdf (804.8KB, pdf)

Acknowledgments

This work is supported by the Project of Shandong Provincial Laboratory (SYS202207) and the Basic Research Program of Suzhou (SSD2024017).

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmeasuresciau.6c00254.

  • Experimental details; illustration of the structure of TDNA; DLS analysis of AgNPs; TEM image and UV–vis absorbance spectrum of AgNPs; PAGE analysis of DNA assembly events; reproducibility and stability investigations of Probe 1/TDNA/AgNPs modified electrode; DNA sequences with corresponding labels; fitting parameters of the equivalent circuit of Nyquist diagrams; comparison of the analytical performances of recent VEGF assays (PDF)

The authors declare no competing financial interest.

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