Nanoceria Particles As Catalytic Amplifiers for Alkaline Phosphatase

Sep 23, 2013 - We propose a novel system to enhance detection sensitivity of alkaline phosphatase (ALP) in electrochemical assays by using nanoceria p...
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Nanoceria Particles As Catalytic Amplifiers for Alkaline Phosphatase Assays Akhtar Hayat and Silvana Andreescu* Department of Chemistry and Biomolecular Science, Clarkson University, Potsdam, NY 13699, United States S Supporting Information *

ABSTRACT: We propose a novel system to enhance detection sensitivity of alkaline phosphatase (ALP) in electrochemical assays by using nanoceria particles as redox active catalytic amplifiers of ALP signals. The catalytic activity of nanoceria particles attributed to their dual oxidation state Ce4+/Ce3+ and high oxygen mobility enabled oxidation of the products of the ALP-catalyzed reaction. A suite of spectroscopic and electrochemical methods, including UV−vis spectroscopy, transmission electron microscopy (TEM), dynamic light scattering (DLS), and cyclic voltammetry (CV) were used to characterize the interaction of nanoceria with the ALPgenerated products. Spectrometric experiments demonstrate change in the oxidation state of nanoceria upon exposure to the hydrolytic products of ALP. Three enzymatically generated products of commonly used ALP substrates were detected at a screen printing electrode surface in the presence of nanoceria. Electrochemical experiments demonstrate signal amplification of the ALP activity assay by nanoceria for all three products, demonstrating remarkable sensitivity of this assay. The assay was optimized with respect to pH and buffer composition. Analytical characterization of the nanoceria-based ALP activity assay was established using a 1-naphthyl phosphate substrate. The proposed strategy can find widespread applications in sensing schemes involving ALP.

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Herein, we report studies that explore nanoceria as a catalytic amplifier of electrochemical signals in ALP assays. This approach provides a generic strategy for enhancing detection sensitivity of ALP assays by using a simple and inexpensive procedure based on earth abundant redox active inorganic nanoparticle catalysts. ALP is one of the most commonly used enzyme label in immuno- and aptamer-based assays, gene assays, histochemical staining, and related affinity methods for monitoring proteins, nucleic acid, drugs, enzymes, and other analytes due to its high turnover number and low cost.13 ALP is also present in human serum, and it is used as an indicator of hepatobiliary and bone disorders in routine clinical analysis.14 Therefore, high sensitive detection of ALP is needed for a wide range of analytical applications. ALP activity tests involve the use of ALP substrates with the monitoring of the enzymatically generated product by various detection techniques.15−18 Among these, electrochemical detection methods are the most promising for field monitoring and point-of-care diagnostic applications.19 Previously reported amplification strategies of ALP reactions include the use of multienzyme report probes, which are prepared by bioconjugating large amounts of enzyme,20 the use of p-aminophenol redox cycling by hydrazine,21 or multistep

igh sensitive detection of ultralow concentrations of analytes is of critical importance in the chemical laboratory and specialized areas of clinical diagnostic, food safety, and environmental monitoring.1 In an effort to increase detection sensitivity of analytical methodologies, a variety of strategies for signal amplification have been explored. These include the use of labels, polymerase chain reaction, mass spectrometry, and the integration of enzyme-assisted signal amplification processes,2 many of which involve timeconsuming derivatization, high cost, and require professional operation.3 With the advance of nanotechnology and nanoscience, nanomaterial-based signal amplification has gained increased importance in achieving high sensitivity and selectivity of analytes with added benefits for rapid analysis and easy miniaturization.4,5 Among the various types of nanoparticulate systems, cerium oxide or nanoceria particles have attained significant attention due to their catalytic and free radical scavenging properties. The catalytic nature of nanoceria has been used to modulate oxidative stress in biological systems,6 promote cell survival in oxidative conditions,7 provide oxygen to oxidase enzymes in enzyme-based biosensors, 8 function as a redox active colorimetric agent in bioanalytical assays,9 and act as oxidase,10 superoxide dismutase, and catalase enzyme mimetics.11 In spite of the many useful redox and catalytic properties of nanoceria, few analytical applications with these nanoparticles are reported.8,12 © XXXX American Chemical Society

Received: July 10, 2013 Accepted: September 22, 2013

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procedures.22 However, these assays require extensive preparative steps and added expensive and sensitive reagents which limit practical implementation. Table 1 in the Supporting Information provides a comparative analysis of the advantages and limitations of the nanoceria assay proposed in this work, as compared to previously reported ALP amplification procedures. Conventional electrochemical approaches for the detection of ALP involve enzymatic hydrolysis of esters of phosphoric acid substrates to various electroactive phenolate compounds.23 Here we show that nanoceria particles enhance oxidation of the ALP-generated products, resulting in a catalytically amplified signal, increasing sensitivity, and lowering detection limits. To demonstrate broad applicability of this method for analytical quantification purposes, the effect of nanoceria was examined on three commonly used ALP-hydrolyzed products, 1naphthol, p-nitrophenol and phenol, and one substrate, 1naphthyl phosphate. In all cases, addition of nanoceria resulted in a significant enhancement of the electrochemical signal of the ALP-catalyzed products. With the use of this strategy, the sensitivity of the sensor increased multiple folds. To our knowledge, the present report is the first to demonstrate the use of nanoceria particles as a signal amplifier for the electrochemical detection of ALP.

ALP product. For measurements with the nanoceria particles, 50 μL of buffer solution containing 5 μL ALP products in the concentration range (0.05−6.4 μM) were incubated on the electrode surface. Then 2 μL of nanoceria dispersion at a concentration of 1 × 10−3 ppm were added onto the electrode containing the ALP product. Electrochemical measurement of ALP activity was performed by incubating 50 μL of buffer solution containing varying units of enzyme on the SPCE surface to cover the three electrodes. Five microliters of 1-naphthyl acid phosphate solution prepared in ammonium buffer (pH 9) at a concentration of 4 mg/L were added to the three electrode surface containing the buffer. After a reaction period of 2 min, the enzymatically generated 1naphthol was oxidized at the electrode surface and the signal was recorded electrochemically. Signal amplification with nanoceria was obtained by adding 2 μL of nanoceria dispersion at a concentration of 1 × 10−3 ppm to the enzyme−substrate mixture after the substrate addition and the 2 min incubation time. The particles were let to react for 2 min before recording the electrochemical oxidation signals. Cyclic voltammetry measurements (CV) were performed with a scan rate of 0.1 V/s between the following potential ranges: 0.1−0.6 V for 1-naphthol, 0.1−1.2 V for p-nitrophenol, and 0.1−0.6 V for phenol. Differential pulse voltammetry (DPV) measurements were carried out under the following conditions: 0.2 s pulse width, 0.5 s pulse period, 0.1 V initial potential, and 0.6 V end potential.

2. EXPERIMENTAL SECTION 2.1. Chemicals and Biochemicals. Alkaline phosphatase (activity 31130.0 units/mL, protein concentration 19.10 mg/ mL, specific activity 1630.0 units/mg) from calf intestine was purchased from Calbiochem. 1-Naphthol was from Alfa Aesar. p-Nitrophenol standard solution (10 mM) and phenol (approximately 99%), stabilized with 0.15% H3PO4, were purchased from Sigma-Aldrich. 1-Naphthyl acid phosphate salt was from Santa Cruz Biotechnology. Ammonium chloride and ammonium hydroxide were from T.J. Baker. H2SO4 was purchased from Fisher, while sodium phosphate and potassium phosphate were from Acros and Sigma-Aldrich, respectively. 2.4. Instrumentation. A JEOL JSM-2010 instrument was used for transmission electron microscopy (TEM). UV−vis spectrophotmetric measurements were performed with a Schimadzu UV-2401PC spectrophotometer. Aggregation behavior of nanoceria particles in varying pH solutions was investigated using a Zeta PALS from Brookhaven Instrument. The electrochemical measurements were obtained using a CH 130B electrochemical analyzer (CH Instrument 1030 B, Houston, TX). Screen-printed carbon electrodes (SPCE C110) were purchased from DropSens (Spain). 2.5. Procedures. The experimental procedure to prepare nanoceria dispersion and buffer solutions is provided in the Supporting Information. Dynamic light scattering (DLS) measurements were performed on aqueous nanoceria particle dispersions with a concentration of 1 × 10−3 ppm in water, ammonium chloride/hydroxide buffer (pH 8.5−10) and phosphate buffer solution (PBS) (pH 7.5−9). UV−visible measurements spectra were recorded on samples prepared by incubating nanoceria dispersion (8.5 ppm) with 1 or 1.5 μM phenol, selected as a model ALP-catalyzed product. Prior to use, each electrode was subjected to electrochemical pretreatment by 5 cyclic potential scans between 0.0 and 1.5 V with 100 mV s−1 in 0.5 M H2SO4. Electrochemical measurements of the ALP-catalyzed products (1-naphthol, p-nitrophenol, and phenol) were performed by placing 45 μL of ammonium buffer or PBS to cover the surface of the three SPCE electrodes and adding 5 μL of the

3. RESULTS AND DISCUSSION 3.1. Reactivity of Nanoceria against ALP-Catalyzed Products. Details of the methodology along with the physiochemical characterization of particles are provided in Figures 1S and 2S of the Supporting Information. Spectrophotometric measurements were used to assess changes in the oxidation state and determine the reactivity of nanoceria upon exposure to the ALP-catalyzed products. Phenol was used in this experiment as a model because its absorbance does not overlap with the Ce4+ absorption band. Upon exposure to increasing concentrations of phenol, the characteristic absorbance peak of Ce4+ decreases (Figure 3S of the Supporting Information). This indicates surface reduction of nanoceria from Ce4+ to Ce3+. Unfortunately, 1-naphthol and pnitrophenol absorb strongly in the same spectral range as Ce3+/ Ce4+, which makes monitoring of their interaction in this region difficult. These preliminary findings demonstrate redox reactivity of these particles against the phenolic compounds associated with ALP reactions. 3.2. Electrochemical Oxidation of ALP-Catalyzed Products in the Presence of Nanoceria. 3.2.1. Optimization of Experimental Parameters: Effect of pH and Buffer Condition. ALP detection in our designed strategy involves three processes: the ALP activity toward substrate conversion, the electrochemical oxidation of the ALP-hydrolyzed products on SPCE, and the catalytic activity of nanoceria toward the ALP-catalyzed products. In addition, depending on the pH and buffer conditions, the particles can change their surface state and agregation behavior and, thus, the available surface area. Therefore, the first set of experiments was designed to allow for investigation of the pH effect on these processes and selection of the experimental variables for an optimum amplification effect. Two buffer conditions were selected, alkaline buffer (ammonium chloride/hydroxide) and PBS. 1-Naphthyl phosphate/1-naphthol was selected as model ALP substrate/ B

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Figure 1. Cyclic voltammograms of (A) p-nitrophenol (0.5 mM) and (B) phenol (0.5 mM) in ammonium chloride/hydroxide buffer at pH 9 in the (a) absence and (b) presence of nanoceria particles on SPCE.

Figure 2. Calibration curves of (A) 1-naphthol, (B) p-nitrophenol, and (C) phenol in the (b) presence and (a) absence of nanoceria particles on the SPCE surface, by employing differential pulse voltammetry in the concentration range from 0.05 to 6.5 μM (n = 3).

cerium phosphate.24 Cerium phosphate may inactivate the nanoceria surface, thus blocking the interconversion between the (+3) and (+4) oxidation states, limiting its redox properties and catalytic behavior. On the basis of these results, ammonium chloride/hydroxide buffer at pH 9 was selected as the medium to demonstrate the amplification strategy using the nanoceria particles for ALP-catalyzed products and determine the analytical performance of the method. To demonstrate a broad range of applicability of this approach to other ALP substrates, two additional ALPgenerated products were studied: p-nitrophenol and phenol. In the presence of nanoceria, the electrochemical oxidation signal increased in both cases, following a similar trend to that of 1-naphtol (Figure 1). The enhanced signal demonstrates the synergistic effect of nanoceria particles toward the oxidation of ALP-hydrolyzed products and provides evidence of potential implementation of this method as a generic amplification strategy for detection of phenolic-type ALP products. The shift of the oxidation peak to higher potential values could be attributed to the surface attachment of the oxidized quinones on the nanoceria particles. A similar surface attachment phenomenon with formation of highly conjugated charge transfer complexes was observed for nanoceria exposed to serotonin.25 Grafting of reactive quinones onto the surface of nanoceria could be responsible for the current enhancement and may explain the amplification mechanism.

product for this study. Both the enzyme hydrolysis and the electrochemical oxidation of the ALP products are favored at pH 9 in ammonium buffer and at pH 7.5 in PBS (experimental detail is discussed in Figures 4S and 5S of the Supporting Information). In addition, DLS measurements indicate that the particles have increased polydispersity (decreased aggregation) in the ammonium buffer at pH 9 (Table 2S of the Supporting Information). These conditions were further used to assess the oxidation of ALP products by nanoceria, using electrochemical means. 3.2.2. Signal Amplification with Nanoceria. To demonstrate the potential of nanoceria particles for signal amplification in electrochemical ALP assays, CV experiments were conducted to determine the oxidation of 1-naphthol, a common hydrolytic product of ALP. Optimization experiments were performed in the absence and presence of nanoceria in an ammonium buffer at pH 9 and in PBS at pH 7.5 at a SPCE surface (Figure 6S of the Supporting Information). The addition of nanoceria induces a considerable enhancement of the oxidation current in both ammonium and PBS buffers. This confirms the catalytic activity of these particles and demonstrates promising characteristics for uses in assays of ALP-related activity processes. The particles dispersed in ammonium buffer showed a significantly higher amplification signal as compared to those in PBS. The reduced activity in PBS could be explained by the formation of surface adsorbed C

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Figure 3. Proposed mechanism of the nanoceria-based ALP amplification assay.

3.3. Analytical Characterization of the NanoceriaBased ALP Assay. The analytical performance of the nanoceria assay was evaluated by DPV by measuring the current generated from the oxidation of ALP hydrolysis products. To assess the effect of nanoceria on the electrochemical signal, DPV voltammograms and calibration curves were recorded with SPCE electrodes in the presence and absence of particles. Figure 2 shows comparative calibration curves for 1-naphthol, p-nitrophenol, and phenol in the presence and absence of nanoceria. Use of nanoceria provided a multiple fold increase in sensitivity of the assay and smaller detection limits, in the low nanomolar concentration range for all three ALP-catalyzed products (Table S2 of the Supporting Information). The detection limits are lower than those reported previously with other electrochemical detection schemes of the three ALP-hydrolyzed products.26 These results demonstrate that nanoceria promotes oxidation of phenolic-like ALP-catalyzed products, further enhancing the sensitivity for the electrochemical detection of these compounds. To further demonstrate the feasibility of this approach as a viable platform for ALP activity assays, we have selected 1naphthyl phosphate, the most commonly used substrate for ALP measurements, as a model ALP substrate and have performed enzyme activity tests in the presence and absence of nanoceria. The reaction mechanism of the ALP assay with the proposed nanoceria amplification scheme is shown in Figure 3. Addition of the redox active nanoparticles to the ammonium buffer onto the SPCE electrode surface after substrate hydrolysis facilitates conversion of the enzymatically generated naphthol to naphthoquinone, producing a catalytically amplified signal. Figure 4 shows cyclic voltammograms of the ALP nanoceria assay and control electrodes, in the absence of enzyme and/or absence of particles. No oxidation peaks were observed in the CV of a blank electrode when only 1-naphthyl phosphate substrate was added. Similarly, there was no significant signal (other than the background capacitive current) when nanoceria particles were incubated with 1naphthyl phosphate. When 1-naphthyl phosphate was added to the buffer containing ALP on SPCE, a well-defined oxidation peak current was observed, corresponding to the oxidation of the enzymatically generated 1-naphthol. When nanoceria particles were added and incubated together with the ALP hydrolysis mixture, using the same experimental setup, a very strong oxidation peak was observed with a maximum current intensity at ∼0.42 V. The current enhancement demonstrates the conceptual validity of this strategy for achieving high detection sensitivity of ALP. Analytical performance characteristics of the method were determined with DPV with varying concentrations of ALP (5− 640 units/mL). Figure 5 shows the calibration curves for the

Figure 4. Cyclic voltammograms of (a) 1-naphthyl phosphate, (b) 1naphthyl phosphate in the presence of nanoceria, (c) 1-naphthyl phosphate with ALP, and (d) 1-naphthyl phosphate with ALP in the presence of nanoceria particles. Concentration of 1-naphthyl phosphate was 4 mg/mL. Concentration of ALP was 0.8 units/mL and that of nanoceria was one 10 × 10−3 ppm. Assay was performed in ammonium chloride/hydroxide buffer at pH 9.

Figure 5. Calibration curves for the detection of ALP activity in the (a) absence and (b) presence of nanoceria.

detection of ALP in the presence and absence of nanoceria. A dynamic linear increase in the electrochemical response was observed with varying ALP concentrations. The detection limit of the nanoceria assay was 0.02 units/mL, using the 3σ criteria with a sensitivity of 0.01387 mA/units/mL. In the absence of the particles, the detection limit was 0.35 units/mL with a 9fold decrease in sensitivity versus the nanoceria assay. This D

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clearly indicates the critical role of nanoceria in the assay and demonstrates the analytical potential of this method for fast and sensitive detection of ALP using an easy-to-use nanoparticle amplification strategy. The reproducibility of the sensor was within 3−5% for independently run experiments (calibration curves show the average current and standard deviation for n = 3 experiments with independently prepared electrodes).

4. CONCLUSION This study successfully demonstrated a new catalytic amplification scheme for increasing detection sensitivity of ALP assays using unique properties of redox-active nanoceria particles. The procedure has significant advantages over previously reported amplification strategies that involved uses of complex bienzymatic systems of ALP in conjunction with other enzymes, such as horseradish peroxidase,20 glucose oxidase,27 or polyphenol oxidase.28 Advantages of the nanoceria method include the use of a robust and highly stable amplification agent, simple and inexpensive procedure, and high sensitivity. Moreover, the procedure can be broadly applied as a generic approach for increasing sensitivity of other ALP activity assays that utilize phenolic-type substrates. The assay could be extended to other phosphatases and phosphatase substrates as well. In addition to enzyme activity studies demonstrated here, the method could be implemented as a general amplification strategy to ALP-labeled immuno and aptamer assays. Potential usage in studies of enzyme activity in living cells and gene diagnosis could also be envisaged. To the best of our knowledge, this is the first report to explore the catalytic properties of nanoceria particles toward phosphataseshydrolyzed products.

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ASSOCIATED CONTENT

S Supporting Information *

This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: (315) 268-2394. Fax: (315) 268-6610. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This material is based upon work supported by the National Science Foundation under Grant No. 0954919. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.

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REFERENCES

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