Resonance Rayleigh scattering detection of trace PDGF based on catalysis of an aptamer-modified nanogold probe in the Fehling reaction

Abstract

Gold nanoparticles (GN) were modified by a platelet-derived growth factor (PDGF) aptamer to obtain stable aptamer-nanogold probes (Apt-GN). The probes specifically combined with PDGF-AA to form Apt-GN–PDGF-AA clusters that exhibited a resonance Rayleigh scattering (RRS) peak at 550 nm. The RRS intensity ΔI_550nm was linear to the PDGF-AA concentration in the range of 0.33–40 ng mL⁻¹. The probes exhibit strong catalysis of the Fehling reagent–glucose Cu₂O particle reaction that can be monitored by the RRS technique at 610 nm, but the cluster is very weak. When PDGF-AA concentration increased, the Apt-GN decreased, and the RRS intensity at 610 nm decreased. The decreased RRS intensity ΔI_610nm was linear to PDGF-AA concentration in the range of 0.03–26.67 ng mL⁻¹. Accordingly, two new aptamer-nanogold RRS methods were established.

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Platelet-derived growth factor (PDGF) is a growth factor protein. There are at least five known PDGF isomers, including PDGF-AA, PDGF-BB, PDGF-AB, PDGF-CC and PDGF-DD. PDGF protein acts as a strong stimulator for cell proliferation, chemotaxis and the transformation of biological macromolecules, and plays an important role in the normal development of cells and the communication process of lesion.^1,2 Thus, the sensitive detection of PDGF is significant. At present, several analytical methods, including fluorescence spectrometry,^3–5 use of aptamers as sensors,⁶ electrochemistry,^7,8 colorimetry⁹ and chemiluminescence,¹⁰ have been proposed for the quantitative detection of PDGF. Among them, the electrochemical method is simple and convenient, but the sensitivity is not high enough. The fluorescence method is stable, simple and fast, but its selectivity is not satisfactory. The chemiluminescence method is very sensitive, but the result is unstable and the procedure is complicated.

Aptamer, also known as a chemosynthetic antibody, is a short single-stranded oligonucleotide containing 20–100 bases. It can specifically recognize proteins,^11,12 polypeptides, organic compounds, metal ions,^13,14 and various ligands, and has become one of the hot spots in analytical chemistry.¹⁵ Recently, few aptamer-based methods for detection of PDGF have been introduced. Ishii's research group reported that the release of an intercalating dye from the aptamer's stem structure results in fluorescence quenching during the deformation of the aptamer captured PDGF. The method is sensitive, with a detection limit of 1 pmol L⁻¹.¹⁶ Resonance Rayleigh scattering (RRS) spectroscopy is simple, rapid and sensitive, and has been used in several fields such as biochemistry, analytical chemistry, and nanomaterial research.^17–19 Gold nanoparticles have good biocompatibility and resonance Rayleigh scattering (RRS) effect, and have been used in the aptamer RRS methods for the determination of trace metal ions and organic compounds.^20–22 Based on our knowledge, there is no nanoparticle RRS method for PDGF using aptamer-modified nanogold. In this article, based on the high affinity between NG and PDGF-AA aptamer, a specific aptamer reaction of Apt-NG–PDGF-AA, and the catalysis of NG in the Fehling reagent–glucose particle reaction, a highly sensitive aptamer nanocatalytic RRS assay was proposed to detect PDGF-AA without centrifugation.

Experimental

Apparatus and reagents

A Cary Eclipse fluorescence spectrophotometer (Varian Company, USA), 79-1 Magnetic Heating Stirrer (Zhongda Instrumental Plant, Jiangsu, China), TU-1901 double beam UV-Vis spectrophotometer (Beijing Purkinje General Instrument Co., Ltd.), FEI Quanta 200 FEG scanning electron microscope (FEI Co., Holland), and J-180 circular dichroism spectrophotometer (Jasco Spectrophotometric Co., Ltd., Japan) were used.

A 100 nmol L⁻¹ PDGF-AA aptamer (Sangon Biotech Co., Ltd.) with a sequence of 5′-SH CAG GCT ACG GCA CGT AGA GCA TCA CCA TGA TCC TG-3′, 100 μg mL⁻¹ PDGF-AA solution (Biovision), 0.1% bovine serum albumin (BSA), 2.4 × 10⁻² mol L⁻¹ (1%) HAuCl₄ (National Pharmaceutical Group Chemical Reagents Company, China), 1% trisodium citrate, pH 7.2 Na₂HPO₄–NaH₂PO₄ (containing 0.2 mol L⁻¹ PO₄³⁻, 0.2 mol L⁻¹ NaCl), Fehling reagent that includes 0.2 mol L⁻¹ CuSO₄ and 1.23 mol L⁻¹ KNaC₄H₄O₆-6.25 mol L⁻¹ NaOH, and 5.56 mmol L⁻¹ glucose were used. All reagents were of analytical grade and the water was doubly distilled.

GN was synthesized through reduction of HAuCl₄ by trisodium citrate. Water (60 mL) was added into a flask, and heated to boil with stirring. Then, 0.50 mL of 1% HAuCl₄ and 3.0 mL of 1% trisodium citrate were added rapidly into the boiling solution successively. After 10 min, the color changed from colorless to wine red. The mixture was moved to cool naturally and stirring was continued up to room temperature, and then diluted to 50 mL with water. The size of NG is 15 nm and its concentration is 47.3 μg mL⁻¹ Au. Preparation of the Apt-GN probe is as follows: with stirring, 6.0 mL of 10 nmol L⁻¹ PDGF-AA aptamer solution was mixed with 4.0 mL of 47.3 μg mL⁻¹ NG solution to obtain a concentration of 18.9 μg mL⁻¹ Apt-GN that is denoted as GN. The probe was stored at 4 °C.

Procedure

120 μL of 18.9 μg mL⁻¹ Apt-GN, 10 μL of 0.1% BSA, 40 μL of pH 7.2 Na₂HPO₄–NaH₂PO₄, and a certain amount of PDGF-AA solution were added into a 5 mL marked test tube and mixed well. After 10 min, the mixture was diluted to 1.5 mL. The RRS spectra were recorded by synchronous scanning excited wavelength λ_ex and emission wavelength λ_em(λ_ex − λ_em = Δλ = 0), a PMT voltage of 500 V, both excited and emission slit width of 5 nm, and an emission filter of 1% T attenuator on a fluorescence spectrophotometer. The RRS intensity at 550 nm (I_550nm) and the blank value (I_550nm)_b without PDGF-AA were recorded. The value of ΔI_550nm = I_550nm − (I_550nm)_b was calculated.

In a 5 mL tube, 30 μL of 0.20 mol L⁻¹ CuSO₄, 0.15 mL of 1.23 mol L⁻¹ KNaC₄H₄O₆ solution, containing 6.25 mol L⁻¹ NaOH, 5 μL of the above Apt-GN reaction solution, and 0.20 mL of 5.56 mmol L⁻¹ glucose solution were added and diluted to 3.0 mL. Then, the mixture was heated at 70 °C for 7 min. The reaction was stopped by cooling under tap water. The RRS spectra were recorded by the synchronous scanning technique. The RRS intensity at 610 nm (I_610nm) and the blank value (I_610nm)_b without PDGF-AA were recorded. The value of ΔI_{610 nm} = (I_610nm)_b − I_610nm was calculated.

Results and discussion

In pH 7.2 Na₂HPO₄–NaH₂PO₄ buffer solution, containing 5.33 mmol L⁻¹ NaCl, the Apt-GN was stable, and the color was red that appeared as a weak RRS peak at 550 nm. When PDGF-AA was added, Apt-GN specifically combined with PDGF-AA to form an Apt-GN–PDGF-AA cluster (Fig. 1) and the color changed from red to purple, and the RRS peak was enhanced. When the PDGF-AA concentration increased, the RRS intensity at 550 nm increased linearly. Thus, the PDGF-AA can be determined directly by the Apt-GN RRS probe.

Fig. 1 Principle of Apt-GN catalytic RRS assay for PDGF-AA.

In the absence of a nanocatalyst, the reaction of glucose–Cu(II) is very slow with a low RRS signal. The GN and Apt-GN had a strong catalytic effect on the slow Cu₂O particle reaction of Fehling reagent–glucose because high density free electrons on the surface of GN enhanced the electron transfer that the adsorbed Cu(II) on the GN surface obtained a electron to form Cu(I). Note that the formed Cu₂O particles had a strong RRS effect at 610 nm, but the Apt-GN and GN aggregation had a weak catalytic effect. Thus, the aggregations cannot be separated from the aptamer reaction solution, making the operation inconvenient. When the concentration of PDGF-AA increased, the RRS peak at 610 nm decreased linearly due to the decrease of Apt-GN that caused the catalysis to weaken. Accordingly, a highly sensitive nanocatalytic RRS method was established for detection of PDGF-AA (Fig. 1).

RRS spectra

In pH 7.2 Na₂HPO₄–NaH₂PO₄ buffer solution and 5.33 mmol L⁻¹ NaCl, the Apt-NG showed a weak RRS signal. Upon addition of PDGF-AA, the Apt-GN specifically combined with PDGF-AA to form an Apt-GN–PDGF-AA cluster, leading to the enhancement of the RRS intensity. The RRS peak at 550 nm increased linearly with the increase in the concentration of PDGF-AA, and a wavelength of 550 nm was chosen to detect PDGF-AA (Fig. 2). The Apt-GN had a strong catalytic effect on the Cu₂O particle reaction of Fehling reagent–glucose, and the particle products had a strong RRS peak at 610 nm. The Apt-GN–PDGF-AA aggregations had a weak catalytic effect. When the concentration of PDGF-AA increased, the concentration of Apt-GN decreased, the Cu₂O particles decreased, and the RRS intensity at 610 nm decreased linearly (Fig. 3). Thus, a wavelength of 610 nm was selected for nanocatalytic determination of PDGF-AA.

Fig. 2 RRS spectra of the Apt-GN–PDGF-AA system. (a) 1.51 μg mL⁻¹ Apt-NG-6.67 μg mL⁻¹ BSA-pH 7.2 Na₂HPO₄–NaH₂PO₄-5.33 mmol L⁻¹ NaCl; (b) a-0.53 ng mL⁻¹ PDGF-AA; (c) a-6.67 ng mL⁻¹ PDGF-AA; (d) a-13.33 ng mL⁻¹ PDGF-AA.

Fig. 3 RRS spectra of the glucose–copper(II)–Apt-GN–PDGF-AA nanocatalytic system.

A 2.0 mmol L⁻¹ CuSO₄-61.5 mmol L⁻¹ KNaC₄H₄O₆-0.37 mmol L⁻¹ glucose-5 μL reaction solution containing (a) 0 ng mL⁻¹ PDGF-AA; (b) 0.03 ng mL⁻¹ PDGF-AA; (c) 0.33 ng mL⁻¹ PDGF-AA; (d) 0.67 ng mL⁻¹ PDGF-AA; (e) 13.33 ng mL⁻¹ PDGF-AA; (f) a-20.00 ng mL⁻¹ PDGF-AA.

Scanning electron microscope

Fig. 4a showed that Apt-GN particles disperse in solution, and stabilize in the pH 7.2 Na₂HPO₄–NaH₂PO₄ buffer solution and 5.33 mmol L⁻¹ NaCl. Upon addition of PDGF-AA, Apt-GN specifically combined with PDGF-AA to form an Apt-GN–PDGF-AA cluster (Fig. 4b). In the absence of PDGF-AA, the aptamer reaction solution exhibited the strongest catalytic effect on the Cu₂O particle reaction and the particles were cubic with an average length × width × height of 800 × 800 × 800 nm (Fig. 4c). In the presence of PDGF-AA, the concentration of Apt-GN decreased, the catalytic ability decreased, and the Cu₂O particles decreased.

Fig. 4 Scanning electron microscope images (a) 1.51 μg mL⁻¹ Apt-NG-6.67 μg mL⁻¹ BSA-pH 7.2 Na₂HPO₄–NaH₂PO₄-5.33 mmol L⁻¹ NaCl; (b) a-6.67 ng mL⁻¹ PDGF-AA; (c) 2.0 mmol L⁻¹ CuSO₄-61.5 mmol L⁻¹ KNaC₄H₄O₆-0.37 mmol L⁻¹ glucose-5 μL reaction solution (6.67 ng mL⁻¹ PDGF-AA).

Circular dichroism spectra

Circular dichroism (CD) is a commonly used method of analysing protein and peptide structures and their secondary structures in solution. In order to study the changing conformation of the Apt in the process of Apt-GN combining with PDGF-AA, the Apt reaction solution was considered by circular dichroism spectra. The results (Fig. 1S†) showed that upon addition of PDGF-AA, the position of the CD peak shifted, and the CD value of 200–240 nm decreased with the increasing concentration of PDGF-AA. This indicated that there is an interaction between PDGF-AA and the Apt, which resulted in the conformation change of Apt.

Optimization of the analytical conditions

The conditions of the Apt-GN aptamer reaction were examined. The effect of Na₂HPO₄–NaH₂PO₄ buffer solution on the ΔI_550nm was considered (Fig. 2S†). When pH was 7.2, the ΔI_550nm of the system was maximum. Moreover, the effect of the buffer solution volume was also tested. The results indicated that when the buffer solution volume was 40 μL, the ΔI_550nm was maximum. Thus, 40 μL of pH 7.2 Na₂HPO₄–NaH₂PO₄ was chosen for use, and 1.86 μg mL⁻¹ of Apt-NG, giving maximum ΔI_550nm (Fig. 3S†), was chosen for use. BSA is an effective substrate for passivating the Apt-NG surfaces, and thus minimizes any nonspecific adsorption of PDGF. When the BSA concentration was 6.67 μg mL⁻¹, the ΔI_550nm was maximum (Fig. 4S†); therefore, a 6.67 μg mL⁻¹ BSA was selected. The results showed that when the reaction time was 10 min, the ΔI_550nm was maximum; thus, a reaction time of 10 min was chosen for use.

Several nanocatalytic indicators, such as Cu(II)–glucose, NH₂OH–Cu(II), Ni(II)–Na₂PO₂, citrate–HAuCl₄ and HAuCl₄–vitamin C,^20,23–26 were considered to enhance the sensitivity. The results showed that the Cu₂O nanogold catalytic reaction is sensitive and reducible; therefore, it was chosen for use. The effect of nanocatalytic reaction conditions, including CuSO₄, KNaC₄H₄O₆–NaOH, glucose, reaction temperature and time, and the volume of aptamer reaction solution, on the ΔI_610nm were also investigated. When 2.0 mmol L⁻¹ CuSO₄, 61.5 mmol L⁻¹ KNaC₄H₄O₆–NaOH and 0.37 mmol L⁻¹ glucose reacted at 70 °C for 7 min and 5 μL aptamer reaction solution was added, ΔI_610nm was the maximum. Thus, the above conditions were chosen.

Analytical feature and application

Under the optimal conditions, different PDGF-AA concentrations were tested and the working curves were drawn according to the relationship between C_PDGF-AA and their corresponding ΔI_550nm values. The linear range (LR), regression equations, coefficients and detection limit (DL) of the Apt-GN–PDGF-AA system were 0.33–40 ng mL⁻¹, ΔI_550nm = 11.5C + 8.63, and 0.9850 and 0.1 ng mL⁻¹ PDGF-AA, respectively. This RRS method is very simple and convenient. The working curve of the Apt-NG catalytic system was also obtained to plot the PDGF-AA concentration C_PDGF-AA vs. ΔI_610nm. The LR, regression equations, coefficients and DL of the Apt-GN–PDGF-AA nanocatalytic system were 0.03–26.67 ng mL⁻¹, ΔI_610nm = 20.6C + 0.6, 0.9805 and 0.1 ng mL⁻¹ PDGF-AA, respectively. Compared to the reported assays^{3,6,7,9,10,16} (Table 1S†), this nanocatalytic RRS method was more simple, rapid, low cost, sensitive and selective, and the linear range was wider.

According to the procedure, the effect of foreign substances on the nanocatalytic determination of 1.0 ng mL⁻¹ PDGF-AA was tested. The results (Fig. 5S†) showed that 2.0 × 10⁻³ mol L⁻¹ Ca²⁺ and Mg²⁺, 1.3 × 10⁻³ mol L⁻¹ Cu²⁺, 2.0 × 10⁻³ mol L⁻¹ Zn²⁺, 6.7 × 10⁻⁴ mol L⁻¹ Fe³⁺ and K⁺, 0.60 μg mL⁻¹ L-valine (Val), 0.7 μg mL⁻¹ L-methionine (Me), 0.50 μg mL⁻¹ L-aspartic acid (Asp), and 0.75 μg mL⁻¹ IgG did not interfere in the determination with a relative error of ±10%. This showed that the RRS method had good selectivity due to the specific aptamer reaction.

Three serum samples were obtained from a hospital, and used to determine PDGF according to the procedure. The results are in agreement with the ELISA method (Table 2S†), in which the relative standard deviation (RSD) was in the range of 3.1–7.2% and the recovery was in the range of 96.3–98.1%.

Conclusions

A PDGF aptamer was used to modify GN to obtain a stable aptamer-nanogold probe (Apt-GN). Based on the Apt-GN probe, which specifically combined with PDGF-AA to form an Apt-GN–PDGF-AA cluster, a simple Apt-GN RRS assay for detection of PDGF-AA has been established. Because the Apt-GN had a strong catalytic effect on the particle reaction of Fehling reagent–glucose, the formed Cu₂O particles had a strong RRS peak at 610 nm, and a novel aptamer-modified nanogold catalytic RRS method for PDGF-AA has been established. Both methods have different features: the previous method is simple and fast, and the latter is sensitive and does not require centrifugation.

Acknowledgements

This work supported by the National Natural Science Foundation of China (no. 21267004, 21307017, 21367005, 21365011), the Research Funds of Guangxi Key Laboratory of Environmental Pollution Control Theory and Technology, and the Natural Science Foundation of Guangxi Province (no. 2013GXNSFFA019003, 2013GXNSFAA019046).

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra02857k

‡ These authors (X. H. Zhang and Y. H. Luo) contributed equally to this work.

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