Horseradish Peroxidase-Mediated, Iodide-Catalyzed Cascade

Oct 13, 2015 - This report outlines an enzymatic cascade reaction for signal .... A novel strategy for amplified probing versatile biomolecules throug...
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Horseradish Peroxidase-Mediated, Iodide-Catalyzed Cascade Reaction for Plasmonic Immunoassays Yunlei Xianyu,†,‡ Yiping Chen,† and Xingyu Jiang*,† †

Beijing Engineering Research Center for BioNanotechnology & Key Lab for Biological Effects of Nanomaterials and Nanosafety, National Center for NanoScience and Technology, Beijing 100190, China ‡ University of Chinese Academy of Sciences, Beijing 100049, China S Supporting Information *

ABSTRACT: This report outlines an enzymatic cascade reaction for signal transduction and amplification for plasmonic immunoassays by using horseradish peroxidase (HRP)-mediated aggregation of gold nanoparticles (AuNPs). HRP-catalyzed oxidation of iodide and iodide-catalyzed oxidation of cysteine is employed to modulate the plasmonic signals of AuNPs. It agrees well with the current immunoassay platforms and allows naked-eye readout with enhanced sensitivity, which holds great promise for applications in resource-constrained settings.

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CuAAC between azide and alkyne to aggregate azide/alkynefunctionalized AuNPs, allowing for naked-eye readout of immunoassays.14 Although CuAAC proves to be an elegant sensing platform, a limitation is that Cu(I) inhibits the enzymatic activity of HRP.15 Due to this reason, HRP is beyond the CuAAC-based sensors and we have to resort to more powerful chemical strategies to design HRP-triggered plasmonic sensors for immunoassays. Iodide-catalyzed reaction provides such a platform to develop HRP-triggered sensors. Iodide can catalyze the oxidation of thiol compounds (such as cysteine and glutathione) to form disulfide compounds (such as cystine and glutathione disulfide) in the presence of H2O2 (Scheme S1). Previous studies have revealed that cysteine can assemble on the surface of AuNPs through the Au−S bond and stimulate the aggregation of AuNPs by means of zwitterionic electrostatic interactions.16,17 In contrast, disulfide cystine does not cause the aggregation of AuNPs due to the absence of free cysteine thiol groups and the increased steric hindrance.18 On the basis of these facts, we design a HRP-mediated, iodide-catalyzed cascade reaction that can modulate the dispersion/aggregation of AuNPs so as to achieve plasmonic immunoassays. Iodide can be oxidized to elemental iodine in the presence of H2O2 where HRP acts as the catalyst. By means of HRP-catalyzed oxidation of iodide and iodide-catalyzed oxidation of cysteine, we design a HRPmediated enzymatic cascade reaction to modulate the AuNPs which provides a feasible approach for plasmonic immuno-

urrent diagnostic technology for the detection of disease biomarkers often requires skilled technicians or sophisticated instrumentation. To develop a simple methodology that allows for straightforward readout of biomedical diagnostics is still a challenge for healthcare infrastructure in resourceconstrained areas.1 Immunoassays such as the enzyme-linked immunosorbent assay (ELISA) are currently commonly used methods to detect disease biomarkers in clinical practice.2,3 ELISA is one particular format of immunoassay that simultaneously makes use of the specific antibody−antigen interactions and the catalytic capability of an enzyme. Conventional ELISA typically employs horseradish peroxidases (HRP) or alkaline phosphatase (ALP) as the labeling enzymes due to their good stability, high efficiency, and commercial availability.4 With the emergence and advancement in nanotechnology, the extraordinary properties of nanomaterials enable plasmonic sensors that can detect different species of analytes.5−11 Intelligent strategies have been proposed to design plasmonic immunoassays that permit convenient readout by means of enzyme-mediated modulation of plasmonic nanoparticles.12,13 However, a hurdle against practical applications is their incompatibilities with conventional immunoassay platforms because they additionally require the introduction and conjugation of an enzyme. Therefore, the primary challenge to develop a plasmonic immunoassay is to put forward a straightforward methodology that is compatible with the current immunoassay platform and can be directly applicable without resorting to other enzymes. To resolve this issue, we previously exploited Cu(I)-catalyzed azide/alkyne cycloaddition (CuAAC) and gold nanoparticles (AuNPs) to design ALPtriggered CuAAC for plasmonic immunoassays. ALP triggers © XXXX American Chemical Society

Received: August 12, 2015 Accepted: October 12, 2015

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DOI: 10.1021/acs.analchem.5b03522 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry

aggregation process at room temperature which makes this assay compatible with conventional immunoassays. The A650/ A520 value reaches a plateau after incubation for 60 min; we therefore employ this time interval in the experiments. Control experiments show that neither iodide alone nor H2O2 alone stimulates the aggregation of AuNPs. Cysteine alone or the coexistence of cysteine and H2O2 aggregates the AuNPs due to the cysteine-mediated aggregation of AuNPs (Figure 1). In

assays (Scheme 1). In this strategy, HRP enables the oxidation of iodide to iodine which eliminates the amount of iodide that Scheme 1. Plasmonic Immunoassay Based on HRPMediated Modulation of AuNPs That Enables Naked-Eye Readouta

a In the presence of H2O2, HRP-catalyzed oxidation of iodide and iodide-catalyzed oxidation of cysteine can modulate the dispersion/ aggregation of AuNPs.

can be used as the catalyst in the transformation of cysteine to cystine. In the absence of HRP, iodide catalyzes the oxidation of cysteine to yield cystine that retains the dispersion of AuNPs to present a red color. In contrast, the presence of HRP results in the consumption of iodide, so the oxidation of cysteine catalyzed by iodide is limited, leading to the cysteine-stimulated aggregation of AuNPs. The enzyme-triggered cascade reaction in this strategy can not only achieve naked-eye readout but also enhance the sensitivity of immunoassays due to the two-round amplifications. In the first round, one molecule of HRP can consume many molecules of iodide due to the catalysis of the enzyme. In the second round, iodide as a catalyst can dramatically accelerate the generation of cystine to amplify the analytical signals. This cascade reaction makes use of the catalytic ability of both the enzyme and iodide, contributing to the signal amplification of the plasmonic immunoassays. The overall sensitivity is expected to be remarkably enhanced after the two-round amplifications, and in the presence of HRP, it shows a “red-to-blue” change of the color that enables the naked-eye readout of immunoassays. We first demonstrate that iodide-catalyzed oxidation of cysteine to cystine can be implemented for iodide sensing. Owing to the strong Au−S interaction, cysteine assembles on the surface of AuNPs and stimulates the aggregation of AuNPs via electrostatic interactions. The aggregation of AuNPs brings a “red-to-blue” change in color, making possible the naked-eye readout. This dynamic aggregation process can be quantitatively reflected by the ultraviolet/visible (UV/vis) spectrum of the AuNPs because of their surface plasmon resonance (SPR) absorption. The characteristic SPR absorption of dispersed AuNPs is 520 nm, while that of aggregated AuNPs is 650 nm. Cysteine-mediated aggregation of AuNPs is a dynamic process, and the time-dependent UV/vis spectra show a decreased absorption at 520 nm and an increased absorption at 650 nm (Figure S1). We use the ratio between the absorbance at 650 nm and that at 520 nm (A650/A520) to quantitatively evaluate the degrees of aggregation of AuNPs. Cysteine-mediated aggregation of AuNPs at different temperatures shows that increasing the temperature can accelerate the aggregation process (Figure S2). For convenience, we carry out this

Figure 1. Iodide-catalyzed oxidation of cysteine to modulate AuNPs. (a) Schematic iodide-catalyzed oxidation of cysteine. Cysteine is oxidized to disulfide cystine under the catalysis of iodide which prevents AuNPs from cysteine-stimulated aggregation. (b and c) UV/ vis spectra and corresponding photographs of the AuNPs solution when cysteine, H2O2, and iodide are present.

contrast, iodide catalyzes the reaction between cysteine and H2O2 to generate disulfide cystine, which prevents the aggregation of AuNPs due to the lack of free thiol group and increased steric hindrance of cystine compared to cysteine. This iodide-catalyzed reaction prevents the AuNPs from cysteinestimulated aggregation, making possible the naked-eye readout of iodide sensing. We utilized both transmission electron microscopy (TEM) and dynamic light scattering (DLS) to characterize the AuNPs. TEM images show that cysteine stimulates the aggregation of initially dispersed AuNPs, and the iodide-catalyzed reaction prevents the aggregation of AuNPs (Figure S3). Accordingly, DLS analyses suggest that the hydrodynamic diameter of AuNPs increases due to cysteinestimulated aggregation through electrostatic interaction (Figure S4). In contrast, the hydrodynamic diameter is nearly the same as the initially dispersed AuNPs after the iodide-catalyzed reaction, suggesting that cystine does not aggregate the AuNPs within the time interval in this study. In addition, both AuNPs and AuNPs after the iodide-catalyzed reaction show a zeta potential of −33 mV (Figure S5). In contrast, the zeta potential of AuNPs increases to −21 mV after cysteine-induced aggregation of AuNPs, suggesting that cysteine can assemble on the surface of AuNPs and aggregate them. We then evaluate the sensitivity of this chemical strategy for iodide sensing. Iodide, which acts as a catalyst in the oxidation of cysteine by H2O2, has a dose-dependent effect on the reaction and can be exploited to modulate the AuNPs through cysteine-stimulated aggregation. We employ different concentrations of iodide to catalyze the reaction and investigate the dispersion/aggregation of AuNPs by means of the color and B

DOI: 10.1021/acs.analchem.5b03522 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry UV/vis spectrometry (Figure S6). The color of the solution changes from blue to red when an increased amount of iodide is introduced. In the absence of iodide, cysteine cannot transform into cystine, inducing the cysteine-stimulated aggregation of AuNPs. The UV/vis spectra show a characteristic SPR absorption at 650 nm due to the aggregated AuNPs. When iodide is present, it catalyzes the oxidation reaction to eliminate cysteine and generate cystine that prevents the aggregation of AuNPs. We use the UV/vis spectrometry to quantitatively characterize the AuNPs, which indicates that the SPR absorption at 650 nm gradually decreases with an increased amount of iodide due to the elimination of cysteine. The ΔA650/A520 value represents the difference between the A650/A520 value of the blank sample (without iodide) and that added with iodide. The ΔA650/A520 value rises as the concentration of iodide increases, accompanied by a color change of the solution from blue to red that can be read by the naked eye. The lowest concentration that can be sensed with the naked eye is 200 nM, and 10 nM iodide can be distinguished by the UV/vis spectrometer. The good sensitivity suggests that this iodide-catalyzed reaction can be further implemented to design iodide-based sensors. To develop a HRP-mediated plasmonic immunoassay, a main challenge is how to make use of HRP to generate the signal and transduce it to a molecular event that can modulate AuNPs for the plasmonic readout. To achieve this goal, we introduce the HRP-catalyzed oxidation of iodide to consume iodide. Iodide acts as the catalyst in the transformation of cysteine to cystine which can modulate the dispersion/ aggregation of AuNPs (Figure 2a). Without HRP, iodide helps catalyze the reaction to eliminate cysteine that prevents the aggregation of AuNPs to display a red color. In contrast, the presence of HRP results in the consumption and absence of iodide, so cysteine cannot transform to disulfide cystine, leading to the cysteine-stimulated aggregation of AuNPs as a result of electrostatic interaction. Accordingly, the solution of AuNPs shows a blue color when HRP exists while it displays a red color in the absence of HRP. This signal generation and transduction strategy transform the “aggregation-to-dispersion” state of the AuNPs for iodide sensing to a “dispersion-to-aggregation” change for HRP sensing that allows a straightforward and convenient readout. We test the sensitivity and specificity of the HRP-catalyzed oxidation of iodide toward HRP sensing. We choose 20 μM iodide for the plasmonic immunoassay, considering that 20 μM iodide can prevent the AuNPs from aggregation, and it shows an obvious color change from red to purple after the consumption of iodide which brings convenience for the naked-eye readout (Figure S6). We investigate HRP-catalyzed oxidation of iodide under different pH values which shows that this reaction works well at acidic conditions (Figure S7). As the concentration of HRP increases, more iodide can be consumed which leads to an increased concentration of cysteine and increased level of cysteine-stimulated aggregation of AuNPs. The UV/vis spectra show that the absorbance at 650 nm gradually increases as a result of HRP-induced aggregation of AuNPs. Final concentrations of HRP as low as 0.16 ng/mL can be distinguished by the naked eye (Figure 2b,c). We compare this iodide-based approach with the conventional method that employs tetramethyl benzidine (TMB) for the colorimetric sensing of HRP. The conventional method is based on the HRP-catalyzed oxidation of TMB into a blue-colored product, which only allows for a naked-eye readout when the

Figure 2. HRP-mediated cascade reaction for modulating the AuNPs. (a) Schematic HRP-catalyzed oxidation of iodide and iodide-catalyzed oxidation of cysteine to modulate the dispersion/aggregation of AuNPs. (b) A comparison of the color of solution between the conventional TMB-based method and the AuNPs-based method for assaying HRP-conjugated antibody (HRP-Ab). (c) UV/vis spectra of the AuNPs-based method to detect HRP-Ab. (d) UV/vis spectra of the TMB-based method to detect HRP-Ab.

concentration of HRP is between 0.64 and 1.6 ng/mL (Figure 2b,d). In addition, the iodide-based approach is more stable and user-friendly than the TMB-based method because of the short shelf life and the potential toxicity of TMB. We also investigate the specificity of this iodide-based sensor for HRP sensing. The elemental iodine is insoluble in water; thus, it interferes little with the color of AuNPs in aqueous solution. A panel of potentially interfering proteins and enzymes are tested on HRP-catalyzed oxidation of cysteine. Only HRP catalyzes the reaction to consume iodide that remarkably stimulates the aggregation of AuNPs (Figure S8). Other proteins or enzymes are incapable of consuming iodide, and they can be washed off to avoid interference in the immunoassays to result in the dispersion of AuNPs. Both the sensitivity and selectivity suggest that the iodide-based approach enables HRP sensing with convenient readout and little interference, which holds great promise for immunoassays. To test the application of the plasmonic immunoassay, we use this iodide-based approach to detect a model protein. We use an indirect ELISA to assay rabbit antihuman IgG, in which human IgG acts as the antigen, rabbit antihuman IgG acts as the antibody, and HRP-conjugated goat antirabbit IgG acts as the enzyme-conjugated secondary antibody. This iodide-based approach is fully compatible and comparable with the current ELISA platform. HRP-conjugated secondary antibodies are most prevalently used in current ELISA due to their commercial availability and good stability, which is of great significance for this HRP-mediated plasmonic immunoassay. The procedures of this plasmonic immunoassay are nearly the same as the traditional immunoassay, with the only difference C

DOI: 10.1021/acs.analchem.5b03522 Anal. Chem. XXXX, XXX, XXX−XXX

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liver cancer.19 Detecting the anti-HCV antibodies in serum can identify whether or not individuals have been infected with HCV.20 In clinical diagnosis, HCV-positive indicates that the measured signal is above the cutoff value, whereas that below the cutoff value indicates HCV-negative. The serum from the patients that contains anti-HCV antibodies is diluted to react with HCV antigen that is previously coated on the 96-well plate, followed by the addition of goat antihuman IgG labeled with HRP. Iodide-mediated plasmonic immunoassay is implemented to achieve the naked-eye readout of diagnosis (Scheme S3). Positive serum that contains anti-HCV antibodies can bind HRP-conjugated antibody to catalyze the cascade reaction that stimulates the aggregation of AuNPs (Figure 3c). In contrast, neither blank control nor negative serum can trigger the reaction to aggregate the AuNPs due to the lack of antibody−antigen interaction. For comparison, we use conventional TMB-based ELISA to detect the anti-HCV antibodies in 500-fold-diluted clinical samples. Only 2 out of the 8 positive samples (detection rate: 25%) can be distinguished through the naked eyes by the conventional immunoassay, while this plasmonic immunoassay enables a naked-eye diagnosis of all the positive samples (detection rate: 100%) (Figures 3d and S9). This plasmonic immunoassay shows good specificity and allows naked-eye readout in clinical diagnosis, which has great potential for biomedical diagnostics to improve the healthcare infrastructure in resource-constrained areas. In conclusion, we employ HRP-catalyzed oxidation of iodide and iodide-catalyzed oxidation of cysteine to modulate the plasmonic signals of AuNPs for immunoassays. This plasmonic immunoassay directly uses HRP as the enzyme to initiate the cascade reaction that is fully compatible with current immunoassay platforms. It remarkably enhances the sensitivity and allows a straightforward readout of immunoassays with the naked eye. This cascade reaction provides a promising means to design plasmonic nanosensors. Since HRP is a widely used enzyme in many fields, we envisage that this approach can become useful in a myriad of applications ranging from enzyme-mediated organic synthesis to point-of-care biomedical diagnostics.21,22

in that iodide and H2O2 rather than TMB is introduced into the well plate, followed by the addition of cysteine and AuNPs for the naked-eye readout. Rabbit antihuman IgG with a range of concentrations is investigated, which shows that the iodidebased approach enables a naked-eye readout of the immunoassay when the concentration of the target is 1 ng/mL (Figure 3a). For comparison, we employ the conventional TMB-based

Figure 3. Plasmonic immunoassay and conventional TMB-based immunoassay for detection of model protein and anti-HCV antibodies. UV/vis spectra and photographs of the (a) AuNPs-based method and (b) TMB-based method for detection of rabbit antihuman IgG. (c) Detection of human anti-HCV IgG using plasmonic ELISA. Positive serum that contains anti-HCV antibodies can bind HRP-conjugated antibody to catalyze the cascade reaction that stimulates the aggregation of AuNPs. (d) A comparison between AuNPs-based ELISA and conventional TMB-based ELISA for detection of anti-HCV antibodies in real clinical samples. P1 to P8 indicate different HCVinfected patients that are clinically diagnosed to be positive.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b03522. Experimental details including materials and methods, and additional schemes and figures (PDF)

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method to detect the same target, which shows that it can only sense 20 ng/mL of rabbit antihuman IgG through the naked eye (Figure 3b). It suggests a 20-fold enhancement in sensitivity for the naked-eye readout. The conventional TMBbased immunoassay relies on a readout of the brightness of the blue-colored product, while the AuNPs-based plasmonic immunoassay enables a more convenient readout with a “redto-blue” change in color (Scheme S2). This naked-eye readout is extremely important in resource-limited settings where instrumentation is unavailable. To further demonstrate the utility of this plasmonic immunoassay in real clinical diagnosis, we test the antibody against hepatitis C virus (HCV) in the serum of HCV-infected patients. HCV infection is prevalent in East Asia, and it is associated with life-threatening diseases such as cirrhosis and

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: (+86)10-82545631. Phone: (+86)10-82545558. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Ministry of Science and Technology of China (2011CB933201, 2013AA032204), the National Science Foundation of China (21025520, 21105018, 21222502, 91213305), and Chinese Academy of Sciences (XDA09030305) for financial support. D

DOI: 10.1021/acs.analchem.5b03522 Anal. Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.analchem.5b03522 Anal. Chem. XXXX, XXX, XXX−XXX