Enzyme-Induced Metallization as a Signal Amplification Strategy for

Article pubs.acs.org/ac

Enzyme-Induced Metallization as a Signal Amplification Strategy for Highly Sensitive Colorimetric Detection of Avian Influenza Virus Particles Chuan-Hua Zhou, Jing-Ya Zhao, Dai-Wen Pang, and Zhi-Ling Zhang* Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, State Key Laboratory of Virology, Wuhan University, Wuhan 430072, P. R. China Wuhan Institute of Biotechnology, Wuhan 430075, P. R. China S Supporting Information *

ABSTRACT: A novel colorimetric assay method based on enzyme-induced metallization has been proposed for detection of alkaline phosphatase (ALP), and it was further applied to highly sensitive detection of avian influenza virus particles coupled with immunomagnetic separation. The enzymeinduced metallization-based color change strategy combined the amplification of the enzymatic reaction with the unique optical properties of metal nanoparticles (NPs), which could lead to a great enhancement in optical signal. The detection limit for ALP detection was 0.6 amol/50 μL which was 4−6 orders of magnitude more sensitive than other metal NP-based colorimetric methods. Moreover, this technique was successfully employed to a colorimetric viral immunosensor, which could be applied to complex samples without complicated sample pretreatment and sophisticated instruments, and a detection limit as low as 17.5 pg mL−1 was achieved. This work not only provides a simple and sensitive sensing approach for ALP and virus detection but also offers an effective signal enhancement strategy for development of a highly sensitive nonaggregation metal NP-based colorimetric assay method.

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lead to a significant increase in sensitivity without the background that was typical in the case of the conventional metal enhancement process.20−23 This approach also applied to the liquid-crystal biosensor for signal amplification24 or controllable synthesis of nanowires.25 This strategy related to the possible influence of the enzyme activity by silver deposition.26 As for optical detection application, the enzymeinduced growth of gold nanoparticles (AuNPs) was used for determination of small biological molecules27−29 (such as glucose, neurotransmitters, lactate, and so on), inhibitors of enzymes,30 and the activity of the related enzymes.28 However, HAuCl4 is a strong oxidizing agent and it tends to be reduced even in a weak reducing condition (such as glucose solution).31 As a result, the assay methods based on the growth of AuNPs were easy to be influenced, which would restrict their practical applications. In addition, some of these methods related to the preparation of AuNP seed-functionalized glass slides, and the sensitivity of these methods for enzyme detection was also limited.

arly and accurate diagnosis is the key to provide immediate, appropriate, and effective clinical treatment of diseases.1 Consequently, simple and rapid detection methods suitable for point-of-care (POC) testing are very important in clinical diagnosis. Colorimetric detection by the naked eye has been widely accepted for its simplicity and practicality.2,3 Especially, metal nanoparticle (NP)-based colorimetric assays have received considerable attention due to the low cost, simplicity, and convenience of these methods, as well as the unique optical properties of the metal NPs.2,4 Many metal NPbased colorimetric biosensors have been developed on the basis of interparticle cross-linking or a destabilization-induced aggregation mechanism for determination of DNA,5−7 metal ions,8−11 cancer cells,12,13 enzymes,14−17 melamine,18,19 and so on. However, the practical application of the aggregation-based colorimetric method suffered from several drawbacks. For example, the aggregation of metal NPs is susceptible to external factors, such as high ionic strength or other impurities in complicated application environments. There is thus significant interest in developing strategies for the metal NP-based colorimetric assay to circumvent the problems of detection sensitivity and anti-interference ability. The enzyme-induced silver deposition, which enables highly specific silver deposition at the presence of enzyme, was innovatively applied to electrochemical detection and could © 2014 American Chemical Society

Received: December 23, 2013 Accepted: January 29, 2014 Published: January 29, 2014 2752

dx.doi.org/10.1021/ac404177c | Anal. Chem. 2014, 86, 2752−2759

Analytical Chemistry

Article

AuNP Synthesis. The GSH-capped AuNP of about 6 nm in diameter was prepared following our previous report.34 Briefly, 1 mL of the 1% HAuCl4 solution (24 μmol) was mixed with 7.5 mg of GSH (24 μmol) under vigorous stirring. Then, the pH value of the solution was quickly adjusted to 2.5−3.0 with 10 M NaOH, and a pale-yellow precipitate appeared. The precipitate was collected by centrifugation and then dissolved in 1 mL of 5 mM NaOH. The pH value of the solution was adjusted to 5.5. Then, 4 mg of NADPH and about 2 units of GR in 9 mL H2O were added to the solution under stirring at room temperature (25 °C) for 2 h. The product was purified using an Amicon centrifugal filter unit (MWCO = 30 kDa) and then dispersed in water and stored at 4 °C. The particle concentration was measured by UV−vis spectroscopy using the molar extinction coefficient at the wavelength of the maximum absorption.35 The resulting AuNPs showed good stability in aqueous solution even with high salt concentration and could be stored at room temperature for months without any aggregation. Colorimetric Detection of ALP. Different amounts of ALP were added into the detection solution, which consisted of AuNPs, AgNO3, and p-APP in diethanolamine buffer (DEA; 500 mM, pH 9.8, containing 1 mM MgSO4). Then the UV−vis absorption spectra of these suspensions were recorded directly after incubating these mixtures at 37 °C for a designated time.To examine the influence of incubation time on the colorimetric analysis, enzymatic reactions incubated for different reaction times were also investigated. To examine the specificity of ALP catalytic silver deposition, glucose, urea, ascorbic acid, cytochrome c, and thrombin were investigated instead of ALP, under other conditions identical to those used for ALP detection. Inhibition Assay of ALP. For the inhibition assay of enzyme activity, the final concentration of ALP was fixed to 5 pM. The protocols were as follows: first, 20 μL of ALP and 20 μL of various concentrations of Na3VO4 were mixed and incubated at 37 °C for 10 min; second, 40 μL of the detection solution was added to the mixture, and the resulting mixed solution was incubated at 37 °C for 5 min; finally, the absorbance intensity of the suspension at 370 nm (A370) was recorded immediately. Detection of H9N2 AIV. The immunomagnetic beads (IMBs) and biotinylated-pAb (B-pAb) were prepared following our previous reports.36,37 The detection protocol was outlined in Figure 5A. (1) IMBs (20 μL, about 40 μg solid content) were added in 1 mL 5% chicken serum, which contained different concentrations of H9N2 AIV, and then incubated for 20 min at 37 °C with gentle shaking (200 rpm). The bead− virus composites were separated with a magnetic scaffold to remove the suspension and then washed with 400 μL rising buffer (RB), which consisted of 0.1% skim milk, 0.05% Tween 20 in phosphate buffer (0.1 M, pH 7.2). (2) After the composites were incubated with B-pAb (2.5 μg mL−1) in 200 μL binding buffer (BB; consisted of 0.3% skim milk, 0.05% Tween 20 in 0.1 M, pH 7.2 phosphate buffer), for 20 min at 37 °C, the composites of IMB/virus/B-pAb were washed twice with 400 μL RB. (3) These composites were suspended in 200 μL of 5 μg mL−1 SA-ALP in BB and incubated at 37 °C for 10 min. Then, the composites (IMB/H9N2/B-pAb/SA-ALP) were washed with RB for three times. (4) Subsequently, 76 μL of p-APP (8 mM) in DEA (500 mM, pH 9.8, containing 1 mM MgSO4) was added in the centrifuge tube to redisperse the immunocomplex-coated magnetic beads and then incubated at 37 °C for 30 min. (5) Finally, the suspension was separated

Herein, we propose a new nonaggregation metal NP-based colorimetric assay with ultrahigh sensitivity and anti-interference ability based on the integration of enzyme-induced metallization with the high sensitive AuNPs induced silver deposition reaction, which first demonstrates that the enzymatic metallization shows great promise for constructing a highly sensitive nonaggregation metal NP-based colorimetric sensor. The principle of this colorimetric strategy is that the related enzyme could induce a silver deposition process on the surface of AuNPs, which can result in an obvious color change of the detection solution. The presence of AuNPs can greatly enhance the enzyme-induced silver deposition reaction, and the inactivation of the enzyme can also be avoided. To demonstrate the concept of the biometallization-based colorimetric assay, alkaline phosphatase (ALP) was used as a model enzyme due to its significant importance in clinical diagnosis as an important biomarker. The level of ALP is connected to several diseases, such as adynamic bone disease, hepatitis, prostatic cancer, and so on.32,33 For the high molar extinction coefficient of silver nanoparticles (AgNPs) and the high amplification and specificity of the enzymatic catalytic reaction, this biometallization-based colorimetric assay owns high sensitivity and specificity and can be applied to complex samples. The application of this system can be further expanded to immunoassays by using ALP as a signal tag. Taking whole virus particles as a model, the concentration of ALP is directly related to the concentration of virus particles through immunoreaction, and as a result, the presence of the target virus is expected to yield a color change by means of enzyme-induced silver deposition on the surface of AuNPs. The biometallizationbased colorimetric assay not only provides a simple platform for enzyme and pathogen detection, which has a broad prospect in clinical diagnosis applications, but also initiates a new kind of nonaggregation metal NP-based colorimetric sensor.

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EXPERIMENTAL SECTION Materials and Reagents. Calf intestine alkaline phosphatase (ALP), N-(3-dimethylamino-propyl)-N′-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), and thrombin were purchased from Sigma-Aldrich (St. Louis, MO). p-Aminophenyl phosphate monohydrate (p-APP) was purchased from Santa Cruz Biotechnology, Inc. Alkalinephosphatase-labeled streptavidin (SA-ALP) was purchased from Vector (Burlingame, CA). Inactivated H9N2 avian influenza virus (AIV), H5N1 AIV, H1N1 AIV, and newcastle disease virus (NDV) were obtained from Wuhan Institute of Virology, Chinese Academy of Sciences. Anti-influenza A H9N2 hemagglutinin (HA) mouse monoclonal antibody (mAb) and rabbit polyclonal antibody (pAb) were purchased from Sino Biological, Inc. (Beijing, China). Superparamagnetic magnetic beads (500 nm in diameter) were purchased from Ademtech SA (Pessac, France). EZ-link sulfo-NHS-LCbiotinylation kit was purchased from Pierce Biotechnology (Rockford, IL). Skim milk was received from Becton, Dickinson and Company. Reduced glutathione (GSH) was purchased from Amresco. NADPH was purchased from Biomol. Yeast glutathione reductase (GR) was purchased from Calbiochem. Amicon centrifugal filter units were purchased from Millipore. Cytochrome c was purchased from Sangon Biotech Co., Ltd. (Shanghai, China). All other chemical reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 2753



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Figure 1. (A) Sensing mechanism for the enzyme-induced metallization-based colorimetric assay. AuNPs are well-dispersed in the detection solution which displays red color, and the presence of ALP will induce a two-step biometallization process which results in a significant color change of solution from red to yellow. (B) Photograph for the color change of the detection solutions after incubation with different concentrations of ALP for 30 min. (C) Typical absorption spectra for ALP at different concentrations: 0, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, and 1 pM. The UV−vis spectra were recorded after 30 min of incubation.

Figure 2. TEM images of (A) the AuNPs and (C and E) the resulting Au/AgNPs after the biometallization process at the presence of different concentrations of ALP. Their corresponding absorption spectra and photographs were displayed under their TEM images (B,D,F).

Experimental Instrumentation. The UV−vis spectra were recorded using an UV-2550 UV−vis spectrophotometer (Shimadzu, Tokyo, Japan). The transmission electron microscopy (TEM) images were obtained using a Hitachi H-7000FA transmission electron microscope at 75 kV.

from the IMBs with a magnetic scaffold and then added to a new centrifuge tube which contained 2 μL AgNO3 and 2 μL AuNPs. The AuNPs solution would undergo a quick color change within 2 min, and H9N2 AIV could be detected by the naked eye. The concentration of the virus could also be quantified by a UV−vis spectrophotometer. The time consumed for the immunoassay was about 1.5 h. Detection of H9N2 AIV in Complex Biological Samples. Fresh heart and fecal material from healthy chickens were crushed, and the supernatants were separated by centrifuging. Then H9N2 AIV was added in these supernatants or chicken serum to form the synthetic complex samples. These samples were then detected using the protocol described above. The control experiments were performed with the same supernatants and chicken serum in the absence of H9N2 AIV.

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RESULTS AND DISSUSION Sensing Mechanism of the Biometallization-Based Colorimetric Assay. The principle of the biometallizationbased colorimetric assay was showed in Figure 1A. In the absence of ALP, the unreacted p-APP could not react with Ag+, thus the color of the detection solution remained red (color of AuNPs). Whereas in the presence of ALP, p-APP was enzymatically converted into a reducing agent p-aminophenol (p-AP) that reduced Ag+ to Ag0 in the presence of AuNPs, 2754



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Figure 3. (A) and (B) are time-dependent plots of the A370 of the detection solution after adding varying concentrations of ALP. (C) A370 versus ALP concentration at the 30 min time point (inset: its corresponding linear relationship). (D) Plot of the absorption change per minute at 370 nm versus ALP concentration.

Sodium orthovanadate (Na3VO4), a well-known inhibitor for ALP,39 was used to demonstrate the enzyme inhibition (under investigated conditions, Na3VO4 itself does not influence the reduction reaction of Ag+, as shown in Figure S3 in Supporting Information). When the activity of ALP was inhibited by Na3VO4, the dephosphorylation of p-APP was blocked, which would hamper the enzyme-induced silver deposition reaction. A sigmoidal profile was obtained (Figure S4, Supporting Information) when A370 at the 5 min time points versus the Na3VO4 concentration were plotted. A370 decreased gradually with the increasing concentration of Na3VO4, which indicated the activity of ALP was crucial for the biometallization reaction. Influence Parameters for the Enzyme-Induced Silver Deposition Reaction. The enzyme-induced silver deposition reaction could be described by the following equations.

resulting in a color change of the suspension. To demonstrate our system was applicable for ALP sensing, different amounts of ALP were added in the detection solution (p-APP, AgNO3, and AuNPs in DEA) and incubated at 37 °C to observe the color change. As shown in Figure 1B, the color of the detection solution remained red (color of AuNPs) in the absence of ALP, which also indicated that the GSH-capped AuNPs had very good colloidal stability even in 0.5 M buffer solution. While in the presence of ALP, the color of the detection solution changed from red to yellow very quickly. This phenomenon contributed to the biometallization process in which ALP catalyzed silver deposition on the surface of AuNP seeds (Figure 2C), and AgNPs exhibit much higher extinction coefficient than AuNPs.4 The color of the detection solution would turn to brown or even black (Figure 1B), which contributed to the further growth of the Ag shell when longer incubation time or higher ALP concentration was employed (Figure 2E). As a consequence, the presence of ALP could be directly observed with the naked eye through this biometallization-induced color change phenomenon, realizing the detection of ALP in a very convenient way. Figure 1C displayed the typical UV−vis absorption spectra of detection solution in the presence of different concentrations of ALP. The control group (curve a) displayed a well-defined surface plasmon resonance (SPR) absorption of AuNPs around 520 nm. As a result of adding ALP into the p-APP-Ag+/AuNP solution, the absorbance band at 370 nm attributed to the SPR absorption of AgNPs was observed, and this SPR absorption increased dramatically with the increased ALP concentration, which indicated a higher content of Ag0 deposited on AuNPs. In addition, the blue shift of AuNP SPR absorption also demonstrated the deposition of Ag0 on the surface of AuNPs.38 The growth of the Ag shell on the surface of AuNPs was further confirmed by the TEM results (Figure 2C,E). The mechanism of the biometallization-based colorimetric assay was further investigated by the enzymatic inhibition.

ALP

p‐APP ⎯⎯⎯→ p‐AP + PO34−

(1)

AuNPs

p‐AP + 2Ag + ⎯⎯⎯⎯⎯⎯→ p‐QI + 2Ag 0 + 2H+

(2)

It is clear that the colorimetric signal originated from the deposition of silver. The A370 depended on the reduction of Ag+ by p-AP intermediate which generated by enzymatic hydrolysis of p-APP substrate. Therefore, higher concentration of p-APP and ALP could accelerate the enzymatic hydrolysis reaction, and as a result, a high SPR absorption of AgNPs at λ = 370 nm could be obtained (Figure S5A, Supporting Information). Higher concentration of Ag+ could accelerate the reduction reaction of Ag+, and more deposited Ag could also be obtained (Figure S5B, Supporting Information). In addition, the presence of AuNPs could greatly enhance the enzyme-induced silver deposition reaction (Figure S2, Supporting Information). It also acted as seeds for silver deposition, thus the inactivation of ALP which was induced by Ag deposition on the enzyme could be avoided. 2755



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The solvent also influenced the enzyme-induced silver deposition reaction greatly. First of all, the acidity of the buffer solution could greatly affect the enzyme activity, and the ALPcatalyzed reaction could be accelerated under its optimal alkaline condition. Second, the reduction reaction of Ag+ (eq 2) refers to a two electron and two proton transfer process, and the alkaline condition was beneficial to this reaction. The presence of DEA not only offered the alkaline buffer solution but also formed complexes with Ag+ to prevent the formation of the AgOH precipitate in alkaline condition. As a result, a relatively high concentration of DEA solution in the alkaline condition was beneficial to the silver deposition reaction (Figures S5C and S5D, Supporting Information). The reaction temperature could also influence the enzymeinduced silver deposition reaction. As shown in Figure S5E in Supporting Information, A370 of detection solution increased with the increasing reaction temperature up to 37 °C. A higher reaction temperature could accelerate the reduction reaction of Ag+, but the thermal deactivation of the enzyme might also take place. As a result, a moderate reaction temperature was suitable for the metallization reaction. As a consequence, the detection solution with 8 mM p-APP, 2 mM AgNO3, and 15 nM AuNPs in 0.5 M pH 9.8 DEA buffer and the reaction temperature of 37 °C were adopted in the subsequent study. Application for Colorimetric Detection of ALP. The A370 changes with time were recorded after adding different concentrations of ALP under the optimized conditions. As shown in Figure 3A,B, A370 increased immediately after mixing ALP with detection solution, while the negative controls could not cause changes in absorbance with time. The kinetics of silver deposition was observed to be faster with higher concentration of ALP. The A370 versus the concentration of ALP at the 30 min time point was plotted in Figure 3C. It was clearly observed that the absorbance value increased gradually with the increasing concentration of ALP. The detection limit was 12 fM (∼0.6 amol/50 μL, or 1.2 × 10−5 U mL−1) at a signal-to-noise ratio of 3, which was 4−6 orders of magnitude more sensitive than other metal NP-based colorimetric methods14−16 and fluorescence assay methods40−43 for ALP detection proposed in the literature (Table S2 in Supporting Information). More particularly, this method was about 6 orders of magnitude more sensitive than the enzyme-induced growth of AuNP based colorimetric assay for tyrosinase detection (detection limit: 10 units).28 Furthermore, the lower detection limit is possible to be achieved when a longer reaction time is employed. Due to the extraordinary sensitivity of this method, the A370 easily exceeded the detection range of the UV−vis spectrophotometer, which resulted in a relatively narrow linear range (0.05−0.3 pM) (Figure 3C, inset). However, the presence of AuNP seeds could avoid the inactivation of ALP induced by Ag deposition on the enzyme, thus the absorbance intensity had a linear relationship with the reaction time (Figure 3A,B). As a result, a wider linear range (0.05−20 pM) could be obtained when the absorbance change per minute at 370 nm was used (Figure 3D). To test the selectivity of the developed method for ALP detection, the control experiments were taken using glucose, urea, ascorbic acid, cytochrome c, and thrombin as negative controls. As shown in Figure 4, about 6−9 orders of magnitude more concentrated interfering substances, such as glucose (10 mM), urea (10 mM), ascorbic acid (10 μM), cytochrome c (5

Figure 4. Histogram for the specificity of this method for ALP detection. The corresponding photograph was displayed underneath the histogram. Inset: the typical absorption spectra.

μM), and thrombin (1 μM), did not exhibit any response, whereas 4 pM ALP could induce a significant signal in 5 min. Thus, the proposed biometallization-based colorimetric approach showed good selectivity toward ALP detection. Additionally, the proposed method was used to detect ALP in human serum samples. The results for the human serum samples spiked with 0.5−1 pM ALP were given in Table S3 in the Supporting Information, which were in good agreement with those added with the quantitative recoveries from 95.0% to 106.8%, demonstrating the potential clinical applicability of the proposed method. Application for Colorimetric Detection of H9N2 AIV. We also demonstrated that the application of the biometallization-based colorimetric assay could be expanded to immunoassay. The H9N2 virus particle was used as a model analyte, because fast and sensitive viral detection techniques are crucial to control future epidemics and the spread of viruses. However, a sensitive colorimetric assay for virus particles is rarely reported. This colorimetric viral assay method combined the advantages of the high-efficiency immunomagnetic separation and the highly sensitive biometallization-based colorimetric assay. The detection process could be divided into two independent parts: the immunoreaction process and the signal-generation process as illustrated in Figure 5A. The IMBs, which were modified with the monoclonal antibody of target virus, were used to capture and separate H9N2 virus particles from complex samples without any pretreatment. Then biotinylated antibody and SA-ALP were used as signal tags to form a sandwich-type immunocomplex with virus particles on the surface of IMBs. The fast reaction kinetics between the IMBs and target virus ensured the rapid detection of virus particles,44 and the use of magnetic beads as reaction carrier could simplify the pretreatment procedures, reduce the immunoreaction time, and amplify the detection signal due to the excellent properties of magnetic beads.36 After the immunoreaction process, the substrate of ALP was added to the centrifuge tube, which could be enzymatically converted into a reducing agent that could induce the deposition of silver on the surface of AuNPs, resulting in an obvious color change of the detection solution. As a result, H9N2 AIV could be determined conveniently by the naked eye. The time consumed for the immunoassay was about 1.5 h (see the Experimental Section). Figure 5B shows a photograph for semiquantitative detection of H9N2 virus in chicken serum samples by the naked eye. In 2756



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Figure 5. (A) Illustration of the protocol for the colorimetric magnetoimmunoassay of H9N2 AIV: (i) the immunoreaction process using IMBs as reaction carrier; (ii) the signal-generation process by using enzyme-induced silver deposition for signal amplification. (B) Visualization photo of semiquantitative determination of H9N2 AIV. (C) Typical absorption spectra and (D) linear curve for H9N2 virus detection.

the presence of target virus, the color of the AuNP colloidal solution turned from red to yellow or brown, which depended on the concentration of H9N2 AIV. However, no appreciable color change was obtained for the negative controls. Spectroscopically, the absorbance band at 370 nm increased linearly with the increase of the H9N2 AIV concentration, as shown in Figure 5C,D. The detection range of this method was from 0.02 to 1 ng mL−1 (Figure 5D, inset), and amounts as low as 17.5 pg mL−1 H9N2 AIV could be detected at a signal-tonoise ratio of 3, which indicated that this immunosensor could successfully detect H9N2 AIV with a high sensitivity and a low detection limit. The specificity of this method was evaluated by using other viruses such as inactivated H5N1 AIV, H1N1, and Newcastle disease virus (NDV) as negative controls (Figure 6). The negative samples still displayed the red color of AuNP colloidal solution, and the color change was only observed in the presence of the target virus, which demonstrated the specificity of the method. The concentration of ALP was related to the concentration of analyte through the highly specific interaction between the antibody and the target virus, which ensured the specificity of the method. It is reported that AIV could replicate in the organs of infected human or animals,45 so the detection methods should possess strong anti-interference ability which could be applied to complex samples such as tissues and feces. Complex biological samples including chicken serum, fresh chicken heart, and chicken fecal material were used to investigate the feasibility of this method. As shown in Figure 7, all positive samples displayed obvious color change, the negative controls

Figure 6. Histogram for the specificity of this method by using H9N2 samples and other viruses, such as inactivated H1N1, H5N1, and NDV as negative samples. The corresponding photograph was displayed underneath the histogram. Inset: the typical absorption spectra.

displayed no appreciable color change, and H9N2 virus could be detected in complex samples with high signal-to-noise ratio. These results demonstrated that this method could be applied to complex biological samples directly, and the effective immunomagnetic separation ensured the strong anti-interference ability of this method. The sensitivity of the proposed method is competitive with the most sensitive viral immunosensor (Table S4 in Supporting Information). Moreover, the proposed method does not require sophisticated instruments to achieve rapid determination of the target virus by the naked eye. This strategy for virus detection is very attractive due to these important features: First of all, taking advantages of the amplification 2757



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ACKNOWLEDGMENTS This work was supported by the National Basic Research Program of China (2011CB933600), the 863 Program (2013AA032204), the Science Fund for Creative Research Groups of NSFC (20921062), the National Natural Science Foundation of China (21175100), the Program for New Century Excellent Talents in University (NCET-10-0656), and the 111 Project (111-2-10).

■ Figure 7. (A) Photograph and (B) histogram of this method for H9N2 AIV detection in complex biological samples.

effect of the enzymatic metallization and AuNP-induced silver deposition, as well as the extraordinarily high molar extinction coefficient of AgNPs, the metallization-based colorimetric strategy exhibits ultrahigh sensitivity. Second, the use of magnetic beads also amplifies the signal because of the increased surface area and the magnetic enrichment effect. Third, the use of magnetic beads as reaction carrier reduces the immunoreaction time, and the proposed method can be used to complex samples directly due to the effective immunomagnetic separation.46

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CONCLUSIONS In summary, a simple and highly sensitive biometallizationbased colorimetric assay method for ALP detection was first developed by coupling the highly specific enzyme-induced metallization with the highly sensitive AuNP-induced silver deposition reaction. Taking advantages of the amplification effect of the enzymatic reaction and the extraordinarily high molar extinction coefficient of AgNPs, ALP can be detected with an ultrahigh sensitivity, which is 4−6 orders of magnitude more sensitive than other metal NP-based colorimetric methods. Furthermore, the proposed method was applied to highly sensitive colorimetric viral detection by coupling with a magnetic bead-based sandwich immunoassay. Amounts as low as 17.5 pg mL−1 H9N2 AIV could be detected in about 1.5 h as a result of the high sensitivity of the biometallization-based colorimetric assay coupled with the magnetic enrichment. More inspiringly, the proposed methods for ALP and H9N2 virus detection can be applied to complex samples without complicated sample pretreatment and sophisticated instruments, which is well-suited for POC diagnosis. Therefore, the proposed method has a broad prospect in clinical diagnosis applications.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in the text. 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]. Fax: 0086-27-68754067. Tel.: 0086-27-68756759. Notes

The authors declare no competing financial interest. 2758



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