A novel label-free aptasensor based on target-induced structure switching of aptamer-functionalized mesoporous silica nanoparticles

Abstract

In this study, a sensitive protocol for the high-performance liquid chromatography (HPLC) detection of adenosine triphosphate (ATP) based on mesoporous silica nanoparticles (MSN) functionalized with an aptamer as a cap has been designed. Aminopyrine (AP) is sealed in the inner pores of MSN with single-stranded DNA. In the presence of ATP, an ATP aptamer combines with ATP and escapes from a pore and thus opens the DNA biogate to release AP. The released signal tags can be easily read out by HPLC. The designed protocol provides sensitive HPLC detection of ATP down to 0.41 nM with a linear range of four orders of magnitude (from 1.0 nM to 10 μM) and has high selectivity toward its target ATP, which can be attributed to the large loading capacity and highly ordered pore structure of mesoporous silica nanoparticles, as well as the selective binding of the aptamer with ATP. This proof of concept might promote the application of ATP-responsive devices and also provide ideas for designing various target-responsive systems using other aptamers as caps.

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1. Introduction

Adenosine triphosphate (ATP), which is a major cellular energy source, plays an important role in the regulation of cellular metabolism and supplies energy for various biochemical reactions in almost every organism.^1,2 ATP is involved in the regulation of DNA replication, biosynthesis, membrane ion-channel pumps, and hormonal and neuronal activities and their effects in every organism.^3–5 An abnormal concentration of ATP has been reported to be closely associated with cardiovascular, Parkinson's and Alzheimer's diseases, and it has also been widely used as an indicator in the diagnosis of diseases related to cell viability and injury.^6,7 In addition, ATP is often used as an indicator of microbial contamination, in which the content of ATP corresponds to the degree of contamination. Therefore, the accurate detection and quantification of ATP is important for biochemical, clinical, and food hygiene applications.^8–10

ATP is usually detected using chromatographic,^11,12 mass spectrometric,¹³ chemiluminometric, and bioluminometric methods.^14,15 Mass spectrometry is accurate but requires expensive equipment and both bioluminescence and chemiluminescence methods involve chemical reactions among multiple components; in particular, the enzymes used in bioluminescence are costly and unstable. High-performance liquid chromatography (HPLC) is one of the most suitable techniques for the simultaneous analysis of different compounds at analytical (or preparative) levels because of its high efficiency, speed, reproducibility, and wide range of applications.^16,17 However, most of these reported methods lack a process of signal amplification, so their sensitivities are relatively lower than those of biosensors. In addition, the use of antibodies as affinity stationary phases has constraints such as low surface loading and batch-to-batch reproducibility. Low surface loading capability can be remedied by the suitable construction of conjugated antibodies.

In recent years, aptamer-based strategies for sensing ATP have attracted significant interest because of the specific binding between a selected aptamer and ATP.^18,19 Aptamers are single-stranded nucleic acids isolated from DNA/RNA libraries of random sequences by an in vitro selection process, which is termed as the systematic evolution of ligands by exponential enrichment (SELEX).^20,21 Compared with antibodies or enzymes, aptamers are promising molecular probes for bioanalytical applications because of their simple synthesis, easy labeling, high stability, and high affinity and specificity for various types of targets.^22,23 Aptamers have in particular undergone substantial progress in bioanalytical fields.^24–26 Many different methods of chemical modification of aptamers have been developed to functionalize them with various functional groups. Because aptamers can be selected for any target molecule and because of their endless opportunities for chemical modifications, they can be considered to be very attractive detecting and diagnostic tools in various bioassays.^27,28 To date, many different aptamer-based sensors (aptasensors) for various types of targets, including small molecules, pharmaceutical drugs, biological macromolecules, and even whole cells have been successfully fabricated.^29,30 To the best of our knowledge, no report has focused on target-responsive controlled release of cargo from mesoporous silica for the development of high-performance liquid chromatography until now.

Mesoporous silica nanoparticles (MSN) contain hundreds of channels (mesopores) arranged in a 3D network with a honeycomb-like porous structure.^31–33 Owing to their highly ordered pore structure, biocompatibility, large loading capacity, adjustable pore size, and ease of functionalization, MSNs have attracted substantial research attention in the fields of biotechnology and nanomedicine.^34–36 In particular, some signal molecules can be trapped in the mesopores of MSN and then sealed with different gatekeepers such as nanoparticles, organic molecules, supramolecules, and biomolecules to construct stimuli-responsive MS nanoprobes. Various stimuli, such as heat, magnetic fields, pH, target molecules, enzymes, or even light, have been employed to trigger the opening of the pores and controllable release of the encapsulated substrates.^37–43 These stimuli-responsive MSNs are frequently used as delivery vehicles and sensing nanoprobes. For example, a fluorescent dye has been trapped within a MSN for a fluorescence assay of ATP, but all these need labeling of the probes for attachment on Au nanoparticles or MSN surfaces, which basically increases the complexity and cost of ATP monitoring.^27,28 In this study, we report a novel label-free ATP aptasensor based on target-induced structure switching of aptamer-functionalized MSN.

This study made use of the flexible binding properties of negatively charged DNA for the electrostatic adsorption of positively charged amine-functionalized MSNs to seal aminopyrine (AP) in the mesopores, which prevented the steric hindrance of AP release by the complex that formed on MSN and simplified the sealing procedure of AP. The aptamer DNA strand could conveniently detach from the MSN surface upon its hybridization with target ATP. The assay is carried out based on the target-responsive controlled release of AP from an aptamer-gated mesoporous silica nanocontainer. Initially, AP is loaded into the pores of mesoporous silica and the pores are then capped with the aptamer. Upon introduction of the target, the molecular gate is opened, resulting in the release of cargo from the pores. The released AP can be quantitatively monitored by HPLC. By monitoring the shift in HPLC intensity, we could quantitatively determine the content of target ATP in a sample.

2. Experimental

2.1 Reagents and materials

Adenosine 5′-triphosphate (ATP), guanosine 5′-triphosphate (GTP), cytidine 5′-triphosphate (CTP), uridine 5′-triphosphate (UTP), tetraethyl orthosilicate (TEOS, 28%) and 3-aminopropyltriethoxysilane (APTES) were purchased from Sigma-Aldrich (USA). Milli-Q water (resistance > 18 MΩ cm) was used in all experiments. The hybridization buffer (HB, pH 7.4) contained 10 mM Tris–HCl, 50 mM NaCl, and 10 mM MgCl₂. The washing buffer was PBS (0.1 M, pH 7.4) containing 0.05% (w/v) Tween-20. ATP aptamer was obtained from Takara (Dalian, China). The oligonucleotide was as follows: 5′-CACCTGGGGGAGTATTGCGGAGGAAGGTT-3′.²⁸

2.2 Apparatus

HPLC analysis was performed on a Shimadzu LC-20AD system. Separation was performed on a Diamonsil C18 column (200 × 4.6 mm, 5 μm). The mobile phases used for elution were H₂O : CH₃OH = 35 : 65 (v/v). The injection volume was 20 μL, the flow rate was 1 mL min⁻¹ and detection was carried out at a wavelength of 259 nm. Transmission electron microscopy (TEM) images were obtained on a JEM-2100 transmission electron microscope (JEOL Ltd., Japan). Nitrogen adsorption/desorption measurements were carried out with a porosimeter (ASAP 2020, Micromeritics, USA). Dynamic light scattering (DLS) was observed with 90 Plus/BI-MAS equipment (Brookhaven, USA). Analysis of zeta potential was performed on a Zetasizer (Nano-Z, Malvern, UK).

2.3 Preparation of MSN

MSNs were first synthesized by the following procedure.⁴⁷ First, 1.75 mL NaOH (2.00 M) was added to 240 mL CTABr (2 mg mL⁻¹) and heated to 95 °C. Under continuous stirring, 2.5 mL TEOS was added dropwise. The mixture was stirred for 3 h to obtain a white precipitate. Then, the solid product was centrifuged, washed with deionized water and ethanol, and dried at 60 °C overnight. The obtained white powder was finally calcined at 550 °C using an oxidizing atmosphere for 5 h to remove the template phase. Next, 0.5 g calcined MSNs and 0.5 mL APTES were suspended in 50 mL anhydrous ethanol inside a round-bottom flask. After the mixture was stirred continuously for 6 h at 36 °C, it was filtered, washed with ethanol, and dried at 60 °C to obtain amine-functionalized MSNs.

2.4 AP loading and capping

Then, 1 mg amine-functionalized nanoparticles was dispersed in 1 mL HB containing 35 mg mL⁻¹ AP. The mixture was shaken overnight in the dark at room temperature and then was centrifuged and washed with ultrapure water to obtain AP-loaded MSNs, which were further suspended in 1 mL HB containing 0.04 mM DNA (aptamer) and shaken for 60 min at room temperature. The resulting solids were isolated by centrifugation and washed with hybridization buffer to obtain MSNs.

2.5 AP release

Detection was performed by mixing 100 μg MSN with 10 μL of various concentrations of ATP and the reaction solution was mixed with 100 μL HB for incubation at 37 °C for 60 min. Subsequently, 0.1 mL supernatant was taken periodically from the suspension at 25 °C followed by centrifugation (15 000 rpm, 10 min). The release of AP from the pore voids to the buffer solution was determined by HPLC.

3. Results and discussion

3.1 Principle of the proposed method

The working principle of the controlled-release system that is responsive to aptamer–target interaction is illustrated in Scheme 1. In this study, MSN could be utilized as building blocks for the encapsulation of AP. Solid MSN were first functionalized with APTES to attach aminopropyl groups on the walls of mesopores. The functionalized MSN were then loaded with AP. After modification by APTES, MSN became positively charged; positively charged MSN were demonstrated to be beneficial for electrostatic adsorption of the negatively charged aptamer. Because the flexibility of the single-stranded DNA enabled good coverage of the MSN pores, the MSNs remained “capped” at this stage. In the absence of ATP, the pores of MSN were blocked and the release of guest molecules was inhibited. In the presence of ATP, a competitive reaction occurred due to the higher affinity and tighter binding of the ATP aptamer with ATP, which resulted in the opening of pores and release of guest molecules. The released AP can obtain an enhanced HPLC signal. Consequently, the increase in the HPLC signal directly depends on the concentration of the target in the sample, which results in improved sensitivity for the detection of ATP at low abundance.

Scheme 1 Schematic of high-performance liquid chromatography detection of ATP based on target-induced structure switching of aptamer-functionalized mesoporous silica nanoparticles.

3.2 Characterization of MSN

The morphology of MSN was characterized by TEM (Fig. 1A), which showed a uniform porous structure of MSN with a diameter of around 120 nm. The size of MSN was confirmed by DLS measurements (Fig. 1B). In addition, the DLS results also indicated the good dispersibility of MSNs in an aqueous medium, which is essential for biological applications. The nitrogen adsorption–desorption isotherm of MSNs showed an average pore diameter of 2.5 nm (Fig. 1C), which was in agreement with the porosity shown in the TEM image. The total pore volume and total specific surface area of MSNs were calculated to be 0.76 cm³ g⁻¹ and 860.5 m² g⁻¹ using the BJH and BET models on the adsorption branch of the isotherm, respectively. Analysis of zeta potential was used to characterize the preparation of MSN (Fig. 1D). After functionalization with APTES, MSNs became positively charged, which indicated that the amine groups were successfully functionalized on the surface of MSNs (column b). The trapping of AP slightly increased the positive charge of MSNs (column c). The positively charged amine-functionalized MSNs were beneficial for the electrostatic adsorption of negatively charged DNA, which resulted in negatively charged MSNs (column d). This result indicated the successful attachment of DNA on the surface of MSNs. The amount of DNA that was attached on MSNs was determined to be 0.0364 mmol per gram of MSNs.⁴⁴

Fig. 1 (A) TEM image, (B) DLS characterization, and (C) nitrogen absorption–desorption isotherm of MSNs. Inset in (C): pore size distribution. (D) Zeta potential of MS nanoparticles (a), amine-functionalized MS nanoparticles (b), AP-MS nanoparticles (c), and DNA-loaded MSN (d).

3.3 Feasibility of the proposed method

To validate the design, the feasibility of the developed strategy was confirmed by HPLC detection. As shown in Fig. 2, in the absence of target ATP, the signal intensity of HPLC at 2.5 min (curve b) was the same as that of the blank (curve a), which resulted from the HB solvent. The signal intensity of HPLC at 6 min was substantially lower. This may be attributed to desorption of residual AP from surface domains on the exterior of the pores or slow release of AP from incompletely blocked pores. In the presence of 10 μM ATP, the incubation of MSN with ATP led to much higher HPLC signal intensity (Fig. 2, curve c), which indicated recognition of target ATP by the aptamer to form a complex; this led to dissociation of the aptamer from the surface of MSN by hybridization of the complex with ATP, which resulted in opening of the pores. Moreover, the peak increased with time (curve d), which indicated that more AP was released from the aptamer–MSN system. To provide evidence of adsorption of the ATP–aptamer complex onto the solid phase in HPLC, hybridization tests using water as the solvent were performed. As shown in Fig. S1,† in the absence of target ATP, the signal intensity of HPLC at 2.5 min (curve b) exhibited no peak and was the same as that of the blank (curve a). The signal intensity of HPLC at 6 min was substantially lower (curve b), which can be attributed to the slow release of AP from incompletely blocked pores. In the presence of 10 μM ATP, the incubation of MSN with ATP led to much higher HPLC signal intensity at 6 min (Fig. S1,† curve c) and a new peak appeared at 2.5 min, which can be attributed to adsorption of the ATP–aptamer complex onto the solid phase in HPLC; this indicated the recognition of target ATP by the aptamer to form a complex, which led to dissociation of the aptamer from the surface of MSN. This result showed that ATP could easily reverse the adsorption of the aptamer on the MSN system and form a very stable aptamer–ATP structure at room temperature, which indicates that ATP is a good agent for uncapping MSN pores and releasing trapped AP molecules.

Fig. 2 HPLC curves of (a) blank (HB); (b) MSN; (c) ATP and MSN for 10 min; (d) ATP and MSN for 60 min.

3.4 Optimization of detection conditions

Because the HPLC signal resulted from the release of AP, the amount of AP in the mesopores, which depended on the concentration that was used for preparation of MSN, was first optimized. With an increase in the concentration of AP from 0 to 40 mg mL⁻¹, the HPLC signal of the released AP increased and reached a plateau at 35 mg mL⁻¹ (Fig. 3A). Therefore, 35 mg mL⁻¹ of AP was chosen for the preparation of MSN. To investigate the effect of the concentration of the aptamer on the detection system, the aptamer at different concentrations ranging from 0 to 60 μM was optimized. As shown in Fig. 3B, the signal intensity of the system decreased as the concentration of the aptamer increased. Furthermore, the optimum concentration of the aptamer that was used in this system was 40 μM. The reaction time was another important parameter that affected analytical performance. It was clear that the signal response increased with an increase in reaction time and tended to a maximum value at 60 min (Fig. 3C). To minimize non-specific release of the dye molecule from the particles, 60 min was chosen as the detection time point. Because adsorption of the aptamer on MSN is based on electrostatic interaction, the pH of the reaction solution should be important for the analytical performance of the aptamer–ATP complex. In this case, aptamer–MSN was initially prepared in a HB system of pH 7.4 and then the as-prepared MSN-aptamer was redispersed into HB solutions of various pH values in the absence of target ATP. The resulting supernatant was monitored by the abovementioned method. As indicated in Fig. 3D, the signal intensity almost tended to level off when the pH of HB was higher than 6. In contrast, the signal intensity increased with a decrease in the pH of PBS from 6 to 3.5. The reason is a consequence of the fact that parts of the aptamer–MSN conjugates were separated from each other owing to the formation of MSN-APTES with negative charge or without charge when the pH of HB was ≤6, thereby resulting in the release of trapped AP from the pores. Considering the bioactivity of conjugated ATP and the capping efficiency of aptamer–MSN for AP with APTES, however, HB with a pH of 7.4 was used as the reaction solution for the detection of ATP.

Fig. 3 Dependence of HPLC signal intensity on (A) AP concentration for preparation of MSN, (B) concentration of aptamer, (C) reaction time in the absence of target (a) or in the presence of target (b), and (D) pH. When one parameter changes, the others are at their optimal values.

3.5 Performance of the ATP aptasensor

Under the optimal conditions, the developed method was employed for quantifying ATP standards with various concentrations based on the target-responsive controlled release of AP from aptamer-gated MSN nanocontainers. As shown in Fig. 4, the HPLC signal increased with the concentration of ATP ranging from 1 nM to 10 μM. The maximum release was observed at 10 μM ATP, which suggests that the ATP aptamer combined with ATP thoroughly at this ATP concentration. The corresponding calibration plots are presented in the inset and it was found that the increased HPLC peak area exhibited a good linear relationship with the concentration of ATP. The linear equation was fitted as I = 13 477 log c + 127 025 (R = 0.9889). The limit of detection (LOD) was 0.41 nM at a signal-to-noise ratio of 3σ (where σ is the standard deviation of a blank solution). To confirm that the high sensitivity of the current strategy was a consequence of target-induced structure switching of aptamer-functionalized mesoporous silica nanoparticles, control experiments at different target concentrations were conducted by HPLC. As shown in Fig. S2,† the intensity decreased with an increase in the concentration of the target in the range from 100 nM to 1 mM. The limit of detection was 31 nM. In addition, the detection performance of the proposed method was compared with that of other approaches for ATP detection and the results are shown in Table 1. This comparison indicated that the proposed aptasensor exhibited much higher sensitivity, which provided powerful evidence of our strategy for the sensitive detection of ATP. The abovementioned results indicate the successful achievement of amplified ATP detection by HPLC, which should be attributed to these factors: the key advantage of this stimulus-release system is the signal amplification process. Because of the unique hollow structure and large inner space of MSN, a large number of dye molecules can be trapped, but only a small concentration of the “key” molecule (ATP in this paper) is required to uncap an MSN pore and release a large amount of dye molecules to generate a strong HPLC signal. Therefore, the results confirm that this signal amplification method was efficient for the sensitive detection of ATP by HPLC.

Fig. 4 (A) HPLC responses of ATP at concentrations of 1.0 nM to 10 μM (from a to e) and (B) calibration curve. Error bars represent standard deviations of three parallel experiments.

Table 1 Comparison of the proposed method with ATP assays using an aptamer as a recognition molecule

Analytical method	Detection limit	Linear range	Refs
Fluorescence	1 mM	1 mM to 8 mM	Ref. 27
Fluorescence	1 mM	1 mM to 20 mM	Ref. 28
Fluorescence	5.2 nM	10 nM to 100 μM	Ref. 29
Electrochemical	1 μM	1 μM to 3 mM	Ref. 45
Electrochemical	10 nM	10 nM to 1 mM	Ref. 46
HPLC	0.41 nM	1 nM to 10 μM	This work

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3.6 Specificity and practicability of the aptasensor

To investigate the specific response of the biosensor to ATP, control experiments were performed by incubating the biosensors in several aqueous solutions containing 0.1 μM ATP, UTP, CTP and GTP, respectively. A HB solution was used as a blank. It is shown in Fig. 5 that only ATP samples gave obvious changes in HPLC intensity, whereas UTP, CTP and GTP samples delivered the same intensity as the blank sample. This proves that the ATP-binding aptamer sequence is very specific to ATP. The cross-sensitivity of the sensor in a mixture of three different nucleotides containing ATP was also examined. The signal that was obtained from the mixture was similar to that obtained from ATP only, which further indicated that the HPLC biosensor was very specific for the determination of ATP with negligible responses to UTP, CTP and GTP. Furthermore, this approach has limitations in distinguishing ATP from its analogues, such as adenosine, AMP, and ADP (Fig. S3†), because the ATP aptamer recognizes the adenine and ribose moieties, not the phosphate moiety. To solve this problem, further study is in progress in our lab. The low CVs indicate the possibility of batch preparation of aptamer–MSN. When the as-prepared aptamer–MSN was not in use, it was stored in HB with a pH of 7.4 at 4 °C. No clear change in the signal was observed after storage for 7 days, but a 5% decrease in the current was noticed at the 14^th day.

Fig. 5 Histograms of the selectivity of the aptamer–MSN system, which was examined by being incubated in the following samples under the same experimental conditions: (a) blank; (b) GTP, (c) CTP, (d) UTP, (e) ATP, and (f) GTP + CTP + UTP + ATP; the concentrations of ATP, CTP, UTP, and GTP were 0.1 μM, respectively.

3.7 Analysis of ATP in human serum

To further evaluate the possible application of the developed aptasensor for the analysis of real samples, four ATP standards, including 1.0 μM, 100 nM, 10 nM and 1.0 nM ATP, were spiked into human serum samples. Then, these samples were tested using the newly prepared aptasensor. The assay results were calculated according to the abovementioned linear regression equation. As shown in Table 2, the recoveries of the added ATP fall in the range from 97.6% to 105%, which indicates that the proposed ATP sensing method can be applied for real samples. Therefore, the developed aptasensor could be utilized for the detection of target ATP in real samples.

Table 2 Analysis of ATP in human serum samples

Sample	Added ATP	Our proposed method	RSD (%)	Recovery (%)
1	1 μM	1.02 μM	3.3	102
2	100 nM	97.6 nM	4.1	97.6
3	10 nM	10.4 nM	2.8	104
4	1.0 nM	1.05 nM	3.2	105

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4. Conclusion

In summary, we have developed a simple and sensitive HPLC aptasensor for the detection of ATP based on a target-induced displacement reaction accompanying the release of cargo from aptamer-gated mesoporous silica nanocontainers. The results demonstrated that the system had a high loading amount of the guest compound and good release behavior in the presence of ATP. The proposed assay exhibited a wide detection range, low limit of detection, and acceptable accuracy. Compared with conventional HPLC, the dye molecules released when triggered by the target can provide a strong HPLC signal. Significantly, the assay can be easily varied for use with other toxins relevant to food by changing the target antibody that is used, and thus represents a versatile detection method.

Acknowledgements

This study was supported by the National Natural Science Foundation of China (21405130), the Excellent Talents of Xuzhou Medical College (D2014007), and State Key Laboratory of Analytical Chemistry for Life Science (SKLACLS1405).

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra18918g

‡ These authors contributed equally to this work.

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