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May 13, 2015 - Alcohol Dehydrogenase-Catalyzed Gold Nanoparticle Seed-Mediated. Growth Allows Reliable Detection of Disease Biomarkers with the...
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Alcohol Dehydrogenase-Catalyzed Gold Nanoparticle Seed-Mediated Growth Allows Reliable Detection of Disease Biomarkers with the Naked Eye Mao-Pan Peng, Wei Ma,* and Yi-Tao Long* Key Laboratory for Advanced Materials & Institute of Fine Chemicals, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, P. R. China S Supporting Information *

ABSTRACT: Here, we reported a strategy-based plasmonic enzymelinked immunosorbent assay (ELISA) using alcohol dehydrogenasecatalyzed gold nanoparticle seed-mediated growth to serve as a colorimetric signal generation method for detecting disease biomarkers with the naked eye. This system possesses the advantages of outstanding robustness, sensitivity, and universality. By using this strategy, we investigated the hepatitis B surface antigen (HBsAg) and α-fetoprotein (AFP) with the lowest concentration of naked-eye detection down to 1.0 × 10−12 g mL−1. Experiments with real serum samples from HBsAginfected patients are presented, demonstrating the potential for clinical analysis. Our method eliminates the need for sophisticated instruments and high detection expenses, making it possible to be a reliable alternative in resource-constrained regions.

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n recent decades, detection of infectious1,2 [e.g., human immunodeficiency virus (HIV), hepatitis B surface antigen (HBsAg)] and noncommunicable3,4 [e.g., prostate specific antigen (PSA), α-fetoprotein (AFP)] disease biomarkers has been a major global health challenge, particularly in developing countries.5 Although currently plenty of approaches have been developed for disease biomarkers detection, they require bulky instruments, complex reagents, and skilled operating personnel.6,7 The general goal is to develop affordable detection methods at ultralow concentrations that can be commonly applied around the world, especially in resource-constrained regions. For this purpose, enzyme-linked immunosorbent assay (ELISA) with naked-eye readout detection is becoming one of the most widely used immunoassay formats and is performed routinely in laboratories around the world owing to its ease of operation, without the need for advanced equipment.8 With the rapid development of nanotechnology in recent years, nanomaterials have drawn much attention for their utility as labels for probe molecules in various signal-generation systems for immunoassays.9−13 Among these systems, gold nanoparticle (AuNP)-based colorimetric assays combined with ELISA have been widely applied in bioanalysis because of the convenient readout.14−17 Presently, the respective red-to-blue color change of AuNP-based colorimetric assays is attributable to the monodisperse or aggregated state of AuNPs, which can be used for the detection of target-induced molecular events.18,19 However, external factors in complicated application environments (e.g., the existence of other impurities, extreme temperature, or a high ionic strength) may induce © XXXX American Chemical Society

undesirable aggregation of AuNPs, resulting in unreliable detection probability.20 Herein, we adopted a strategy that the AuNP seeds-mediated growth generates colored solutions based on plasmonic ELISA, thus providing convenient and effective detection for disease biomarkers (Scheme 1). This kind of signal-generated method using AuNP seed-mediated growth in our experiment is much more reliable and specific than the aggregated colorimetric signal method. In order to increase the detection sensitivity and Scheme 1. Schematic Representation of the Sandwich Plasmonic ELISA and the Signal-Generation Method

Received: October 16, 2014 Accepted: May 13, 2015

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DOI: 10.1021/acs.analchem.5b00287 Anal. Chem. XXXX, XXX, XXX−XXX

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mL H2O and stirred vigorously for 1 min before the addition of 10 mg sodium citrate. After 1 min, 42 μL of the newly prepared 11.4 mg mL−1 NaBH4 was added quickly to the previous solution and stirred for another 5 min. UV−vis spectra and transmission electron microscopy (TEM) were used to characterize the size of prepared 5 nm gold nanoparticles. The concentration of the final solution was approximately 3.34 × 10−8 M according to the UV−vis spectra (Figure S1 of the Supporting Information).26 The complete reduced solution was stored at 4 °C for conservation and further use. Verification of Streptavidin-ADH Conjugate. First, 96well polystyrene plates were modified with biotinylated antimouse IgG(H+L) and non-biotinylated anti-mouse IgG(H+L), respectively (100 μL), diluted 1:300 in PBS for 1 h at room temperature. After three washes with PBS, the plates were blocked with blocking buffer (1 mg mL−1 BSA in PBS, pH = 7.0, 0.2 M) for 1 h at room temperature. Next, the plates were washed three times with PBS, and the streptavidin-ADH conjugate was added (100 μL), at different concentrations (6.7 × 10−9, 2.0 × 10−8, 4.0 × 10−8, 6.7 × 10−8, 2.0 × 10−7, 4.0 × 10−7, 6.7 × 10−7, and 2.0 × 10−6 M) in blocking buffer for 1 h at room temperature. Then, the plates were washed three times, and NAD+ (100 μL, 10 mg mL−1) in PBS buffer (pH 8.0, 0.2 M) and ethanol (6 μL) were added to each well of the plate at 37 °C. After 1 h, freshly prepared HAuCl4·3H2O solution (4 μL, 1 wt %) and 5 nm AuNPs (50 μL) in CTAB solution (100 μL) were added to each well at 37 °C. The absorbance value at 550 nm was collected by UV−vis spectra to make the plot. Error bars represent the standard deviations for measurements taken from three independent experiments. Sandwich Assay for HBsAg. First, 96-well polystyrene plates were modified with goat IgG (100 μL) diluted 1:300 in PBS for 1 h at room temperature. After washing the plates three times with PBS, the plates were blocked with blocking buffer (1 mg mL−1 BSA in PBS) for 1 h at room temperature. Subsequently, the plates were washed three times with PBS, and HBsAg (100 μL) was added to the desired final concentration by diluting a stock solution with PBS. After 1 h, the plates were washed three times with PBS, and mouse IgM (100 μL) diluted 1:300 in blocking buffer was added for 1 h at room temperature. The plates were then washed three times, and biotinylated anti-mouse IgG(H+L) diluted 1:300 in blocking buffer was added (100 μL) for 1 h at room temperature. After washing three times, the streptavidin-ADH conjugate diluted 1:50 in blocking buffer was added (100 μL) for 1 h at room temperature. Then, the plates were washed three times, and NAD+ (100 μL, 10 mg mL−1) in PBS buffer (pH 8.0, 0.2 M) and ethanol (6 μL) were added to each well of the plate at 37 °C. After 1 h, the solution was detected by UV− vis spectra and showed the presence of produced NADH (the characteristic absorbance value at 340 nm). Then, freshly prepared HAuCl4·3H2O solution (4 μL, 1 wt %) and 5 nm AuNPs (50 μL) in CTAB solution (100 μL) were added to each well at 37 °C. The final solution can be observed with the naked eye. Moreover, UV−vis spectra and transmission electron microscopy (TEM) were used to characterize the growth of gold nanoparticles. The absorbance values at 550 nm were collected and plotted. Error bars represent the standard deviations for measurements taken from three independent experiments. For detailed procedures of “naked-eye detection of HBsAg without adding AuNPs seeds” and “blank experiment of sandwich assay”, see the Supporting Information.

lower the experimental cost of our assay, we adopted the secondary antibody. It is notable that in this proposed method, an ADH-labeled secondary antibody, via a biotin−streptavidin linkage, is used as a probe as well as for signal amplification. In this work, goat IgG and mouse IgM were employed as the capture antibody and primary antibody, respectively, to model the sandwich ELISA with HBsAg by immunoreaction. Meanwhile, we conjugated streptavidin (SA) to alcohol dehydrogenase (ADH) by a 24-unit ethylene glycol functionalized with succinimidyl and maleimido ends [SM(PEG)24] linker. By introducing the biotinylated anti-mouse IgG(H+L) as the secondary antibody, we attached streptavidin-ADH to biotinylated secondary antibodies easily and firmly via a biotin− streptavidin linkage, demonstrating the biocatalytic cycle of the labeled enzyme in our ELISA assay. In the presence of antigen, the labeled ADH catalyzed the reaction between NAD+ and ethanol to generate NADH and acetaldehyde.21−23 NADH reduces HAuCl4, which resulted in the enlargement of AuNP seeds.24,25 Therefore, the solution color changed from yellow to purple, which was identified by the naked eye.



EXPERIMENTAL SECTION Reagents. All reagents were of analytical grade for all experiments and used without further purification. Gold chloride trihydrate (HAuCl 4 ·3H 2 O, >99.0%), ethanol (C2H5OH, 99.9%), dimethyl sulfoxide (DMSO), hexadecyl trimethyl ammonium bromide (CTAB), sodium phosphate monobasic (NaH2PO4, 99%), and sodium phosphate dibasic (Na2HPO4, 99%) were purchased from Aladdin. Sodium citrate (98%), sodium borohydride (NaBH4, 98%), and glycerol (99%) were obtained from J&K Scientific Ltd. (China). Nicotinamide adenine dinucleotide (NAD+), albumin from bovine serum (BSA), and alcohol dehydrogenase (ADH) were purchased from Sigma-Aldrich. 24-Unit ethylene glycol functionalized with succinimidyl and maleimido ends [SM(PEG)24] and desalting columns were obtained from Thermo Scientific. Goat IgG, HBsAg, mouse IgG, mouse IgM were purchased from Shanghai Yemin Biotech Inc. Streptavidin and biotinylated anti-mouse IgG(H+L) were purchased from Jackson ImmunoResearch Laboratories, Inc. Polyclonal Rabbit AFP was obtained from GeneTex while monoclonal mouse AFP was obtained from HyTest. AFP was purchased from the National Institute for Biological Standards and Control (NIBSC). Goat anti-mouse IgG(H+L) was obtained from Abcam. 96-Well polystyrene plates were obtained from Corning Incorporated. Conventional HBsAg ELISA Kit was purchased from Shanghai MLBIO Biotechnology Co. Ltd. Ultrapure water (18.2 MΩ cm) was produced from a Millipore system and used for the preparation of all solutions. Conjugation of Streptavidin to Alcohol Dehydrogenase. The conjugation proceeded as follows: 4 μL of 24-unit ethylene glycol functionalized with succinimidyl and maleimido ends (250 mM in dry DMSO) was added to 1 mL of streptavidin (1 mg mL−1) in PBS (0.2 M, pH = 7.5) for 30 min at room temperature. The excess cross-linker was removed with a desalting column. Next, 5 mg of alcohol dehydrogenase was added, and the solution was kept at 4 °C overnight. Finally, the streptavidin-ADH conjugate was aliquoted and stored at 4 °C until used. The concentration was approximately 2.0 × 10−5 M for the streptavidin-ADH solution before dilution. Preparation of 5 nm AuNPs. A solution of approximately 5 nm diameter AuNPs was prepared via citrate reduction. Briefly, 0.5 mL of 1% HAuCl4·3H2O solution was added to 50 B

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Analytical Chemistry Sandwich Assay for AFP. The procedure of adding each reagent in our assay was the same as for the Sandwich Assay for HBsAg in the previous section. However, in order to detect AFP antigen, we replaced “goat IgG′” with “polyclonal rabbit AFP” as the capture antibody. The primary antibody was also changed from “mouse IgM” to “monoclonal mouse AFP”. Availability of the Plasmonic ELISA System. Well A1 represented a standard procedure of ELISA in our method, which was identical to the Sandwich Assay for HBsAg in the previous paragraph. Well A2 to A8 was deliberately omitted one or more steps of our ELISA procedure. Well A2 had no ADH and well A3 lacked the secondary antibody. The primary antibody was not added into well A4 while A5 had no primary antibody and secondary antibody. The assay in A6 was not blocked by BSA. A7 was prepared without the capture antibody or the primary antibody, while A8 had no capture antibody, primary antibody, or secondary antibody. The specific procedure followed for each well is described in the chart. Real Sample Detection of HBsAg. The HBsAg samples were obtained from the Second Military Medical University. The samples were centrifuged, aliquoted, and stored at −20 °C for further use. The real sample detection procedure was almost identical to the standard sandwich assay procedure. The only difference was that the HBsAg was replaced by real serum HBsAg samples from different patients. All serum samples were diluted 10 times before adding to the plates. The absorbance values at 550 nm were collected to fill in the chart which represented the average absorbance value for measurements taken from three independent experiments. For detailed procedures of “conventional ELISA kit” and “clinical detection”, see the Supporting Information.

Figure 1. Plates modified with (a) biotinylated anti-mouse IgG(H+L) or (b) non-biotinylated anti-mouse IgG(H+L) after incubation with the streptavidin-ADH conjugate at different concentrations from 6.7 × 10−9 to 2.0 × 10−6 M. (a,b) Photographs showing various color changes of the solution mixed with 5 nm AuNPs. (c) The absorbance value of AuNPs collected at 550 nm at different concentrations.



biomarkers (e.g., HBsAg), we first tested the necessity of every step in our method to exclude other possible factors that would adversely change the solution color. As shown in Figure 2, we

RESULTS AND DISCUSSION Verification of the Streptavidin-ADH Conjugate. To verify the success of the conjugation reaction and the efficiency of the streptavidin-ADH conjugate, ELISA plates were incubated with biotinylated anti-mouse IgG(H+L) overnight. After washing and blocking the plates with 1% bovine serum albumin (BSA)-PBS solution, the streptavidin-ADH conjugate was added at different concentrations. The resulting color of each solution is shown in Figure 1a. It can be seen that the solutions turned purple at higher concentrations from 2.0 × 10−7 to 2.0 × 10−6 M, whereas the solutions remained a light yellow color at lower concentrations. The wells shown in Figure 1b were set as a control by incubating non-biotinylated antimouse IgG(H+L) on the plate. The color of each solution remained yellow regardless of the concentration of the conjugate they contained. Furthermore, the ability to detect the color change with the naked eye can be verified by measuring the absorbance of the enlarged AuNP solution as shown in Figure 1c. It shows that the absorbance value decreased at 550 nm as the concentration decreased when the plates were modified with biotinylated anti-mouse IgG(H+L) (red dots), indicating a concentration-dependent behavior. However, a control experiment was performed with nonbiotinylated anti-mouse IgG(H+L) (black dots), which showed no color change at all. The result confirmed that streptavidin and ADH were successfully conjugated and that a suitable concentration was determined. Unless otherwise stated, the streptavidin-ADH conjugate was used at a concentration of 4.0 × 10−7 M for further experiments. Availability of Plasmonic ELISA System. To confirm the availability of our proposed model system for detecting disease

Figure 2. Integrated effect of the model plasmonic ELISA procedure. Photographs demonstrate the color change in the absence of certain experimental steps. The reagents added to each well are indicated below the photos.

deliberately omitted one or more steps of our ELISA procedure in wells A2 to A8 while a complete ELISA procedure was set as the control on well A1. The photo indicated that only A1 and A6 underwent a significant color change. Well A1 was prepared using a standard procedure for ELISA in our method, which could immobilize the ADH-labeled secondary antibody in the well by the immunoreaction. In the well A6, the excessive ADH in the solution was nonspecifically adsorbed because the well was not blocked with BSA, thus resulting in a deep purple solution. Well A2 did not contain any ADH to catalyze the reaction with NAD+ to produce NADH, whereas wells A3 and C

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Analytical Chemistry A5 lacked the secondary antibody to make the streptavidinADH conjugate bind to the well via the biotin−streptavidin linkage. Well A4 was negative because the secondary antibody could not remain on the well in the absence of the primary antibody. Wells A7 and A8 had BSA blocked at the beginning to prevent all the antibodies from adsorbing on the well. TEM images, shown in Figure S2 of the Supporting Information, were taken to characterize the sizes of AuNPs in solutions with different colors from A1 to A8. The sizes of AuNPs in red solutions, such as A1 and A6 grew to approximately 50 nm, whereas those in yellow solutions remained in its original sizes. As a control, we also replaced goat IgG with mouse IgG as the capture antibody and repeated the above experiments, which are shown in Figure S3 of the Supporting Information. Compared to A4, well B4 became positive because antimouse IgG(H+L) was directly attached to the mouse IgG, which showed a false positive. Moreover, the solution also turned purple without adding HBsAg to confirm the case when the well was modified with mouse IgG (Figure S4 of the Supporting Information). Therefore, we used goat IgG as the capture antibody to exclude possible false positives and ensure that any color variation was properly attributed to the specific binding of disease biomarkers and antibody. Naked-Eye Detection of Disease Biomarkers Using Plasmonic ELISA. After demonstrating the efficiency of streptavidin-ADH and ensuring a specific color variation response to HBsAg concentration, we adapted this conjugate as the signal-generation substrate for the plasmonic ELISA assay. The goat IgG was modified in the 96-well plate as a capture antibody, and the wells were blocked with 1% BSA-PBS solution. HBsAg of serially diluted concentrations from 10−6 to 10−13 g mL−1 was added to the wells. After adding mouse IgM and biotinylated anti-mouse IgG(H+L) in sequence as primary and secondary antibodies, the conjugate could be easily targeted via biotin−streptavidin linkage. Finally, the generated NADH was mixed with AuNP seeds (5 nm in diameter) and HAuCl4. Theoretically, AuNPs with smaller diameters have lower absorption efficiencies and extinction coefficient, indicating that there is no color interference from the addition of AuNP seeds.26−28 Figure 3a (top) showed the results for the detection of HBsAg, and the lowest concentration of naked-eye detection defined as the lowest concentration of HBsAg, was 1.0 × 10−12 g mL−1. We also performed the ELISA assay without adding AuNP seeds in Figure S5 of the Supporting Information, indicating that the detection sensitivity could be enhanced in the presence of AuNP seeds. Control experiments, carried out by spiking the solution with BSA instead of HBsAg (Figure 3a bottom), led to a yellow-colored solution because the antibodies could not specifically recognize this unrelated globular protein. The blank experiment, which left out the antigen, was also performed as shown in Figure S6 of the Supporting Information and the solution color remained in yellow. Three sets of control experiments were also illustrated in Figure S7 of the Supporting Information. We used AFP to replace HBsAg in the first set, mouse AFP antibody to replace HBsAg mouse IgM in the second set, and non-biotinylated anti-mouse IgG(H+L) to replace biotinylated anti-mouse IgG(H+L) in the third set, which all exhibited no color change, demonstrating the great specificity of our assay. Another set of antibodies for detecting AFP was also utilized in the assay (Figure 3a middle), and the lowest concentration of naked-eye detection was also approximately 1.0 × 10−12 g mL−1,

Figure 3. Naked-eye detection of disease biomarkers with plasmonic ELISA. (a). Photographs illustrating the growth of AuNP seeds with various concentrations of HBsAg analyte, AFP analyte, and BSA from 10−6 to 10−13 g mL−1, respectively. (b) The absorbance value of AuNPs generated at 550 nm in the presence of HBsAg (black ■), AFP (blue ▲) and BSA (red ●) at various concentrations. (c,d) Transmission electron microscopy (TEM) images of AuNPs in purple (c) and yellow (d) solution.

supporting the claim that our assay was a universal tool for antigen detection. The calculated detection limit for concentrations of HBsAg and AFP was approximately 10−12 g mL−1, which was the same order of magnitude as the naked eye detection limit (For detailed calculation, see the Supporting Information). The observation of a distinguishable color change was attributed to the biocatalytic action of ADH, which enabled the production of NADH for enlarging AuNPs by HAuCl4 reduction. Figure S8 of the Supporting Information shows the UV−vis absorbance value of catalytic NADH at 340 nm decreased when the amount of HBsAg was decreased. The growth of AuNPs leading to the purple solutions can be confirmed by TEM, whereas the AuNPs in the yellow solutions remained at their original sizes, which are shown in Figure 3 (panels c and d, respectively). It was noteworthy that the size of the AuNPs increased as the concentration of HBsAg was elevated, consistent with the deepening of the purple color of the solution (TEM shown in Figure S9 of the Supporting Information). Furthermore, we measured the absorbance value at 550 nm to monitor the growth of AuNPs, as shown in Figure D

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method for the detection of HBsAg and AFP in developing countries. Moreover, this proposed method could potentially be a versatile tool for the detection of any other infectious and communicable disease biomarkers by the naked eye as long as the antibody directly against it were available. Taken together, our approach provides a different vision for developing a versatile and sensitive detection method by ELISA.

3b. Taken together, the results in Figure 3 indicated that the ELISA assay we developed could be used for a sensitive detection of disease biomarkers. Real Sample Detection of HBsAg. Considering the excellent sensitivity of our proposed colorimetric assay, we demonstrated the robustness of the analytical procedure against possible interferences present in real sera before implementing this assay in resource-constrained countries. Four independent HBsAg-positive samples collected from clinical patients who carried hepatitis B virus and four other HBsAg-negative samples were used as control. In Figure 4, wells 1 to 4 contained serum



ASSOCIATED CONTENT

* Supporting Information S

Experiment details. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b00287.



AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS This research was supported by the 973 Program (Grants 2013CB733700 and 2014CB748500), National Natural Science Foundation of China (Grants 21125522 and 21305045), Fundamental Research Funds for the Central Universities (Grant 222201313004) and Postdoctoral Science Special Foundation of China (Grant 2014T70398).

Figure 4. Detection of HBsAg in sera from clinical patients. Patient nos. 15, 20, 26, and 109 are HBsAg-positive, while patient nos. 121, 122, 125, and 126 are HBsAg-negative. The detection procedure is identical to the HBsAg model experiment. A550 is presented as the absorbance of AuNPs at 550 nm, which was due to the enlargement of AuNP seeds by generated NADH. The results of conventional ELISA and clinical detection are also shown in the chart as the confirmatory test.



samples of different HBsAg-positive patient nos. 15, 20, 26, and 109, whereas HBsAg-negative patients nos. 121, 122, 125, and 126 were added to wells 5 to 8. The other conditions were identical to those for the HBsAg experiment. Clearly, wells 1 to 4 showed color change while the color in wells 5 to 8 did not change. Thus, we can distinguish the positive blood serum from the negative by the naked eye. The absorbance values of AuNPs at 550 nm (A550), which were produced via the enlargement of AuNP seeds by generated NADH, was measured. Confirmatory tests were also performed by using conventional ELISA and clinical detection methods (chemiluminescence microparticle immunoassays). The data are shown in the chart of Figure 4 (For details of procedures, see the Supporting Information). On the basis of the criteria set by clinical detection protocols, it could be considered positive when the concentration of HBsAg was higher than 0.2 ng mL−1, which was equivalent to an absorbance value of 0.030 in our plasmonic ELISA. Therefore, the result can be consistent with the result from clinical detection and conventional ELISA tests which was demonstrated in the chart below. All results indicated that our approach could be utilized for the real samples detection.

REFERENCES

(1) Damhorst, G. L.; Watkins, N. N.; Bashir, R. IEEE Trans. Biomed. Eng. 2013, 60, 715−726. (2) Maity, S.; Nandi, S.; Biswas, S.; Sadhukhan, S. K.; Saha, M. K. Virol. J. 2012, 9, 290. (3) Liu, D.; Huang, X.; Wang, Z.; Jin, A.; Sun, X.; Zhu, L.; Wang, F.; Ma, Y.; Niu, G.; Hight Walker, A. R.; Chen, X. ACS Nano 2013, 7, 5568−5576. (4) Warren, A. D.; Kwong, G. A.; Wood, D. K.; Lin, K. Y.; Bhatia, S. N. Proc. Natl. Acad. Sci. U.S.A. 2014, 111, 3671−3676. (5) Patton, J. C.; Coovadia, A. H.; Meyers, T. M.; Sherman, G. G. Clin. Vaccine Immunol. 2008, 15, 388−391. (6) Chen, S.; Svedendahl, M.; Duyne, R. P.; Kall, M. Nano Lett. 2011, 11, 1826−1830. (7) Oh, B.-R.; Huang, N.-T.; Chen, W.; Seo, J. H.; Chen, P.; Cornell, T. T.; Shanley, T. P.; Fu, J.; Kurabayashi, K. ACS Nano 2014, 8, 2667− 2676. (8) de la Rica, R.; Stevens, M. M. Nat. Protoc. 2013, 8, 1759−1764. (9) Gao, Z.; Xu, M.; Hou, L.; Chen, G.; Tang, D. Anal. Chem. 2013, 85, 6945−6952. (10) Howes, P. D.; Rana, S.; Stevens, M. M. Chem. Soc. Rev. 2014, 43, 3835−3853. (11) Rodríguez-Lorenzo, L.; de la Rica, R.; Á lvarez-Puebla, R. A.; LizMarzán, L. M.; Stevens, M. M. Nat. Mater. 2012, 11, 604−607. (12) Li, D.; Wieckowska, A.; Willner, I. Angew. Chem., Int. Ed. 2008, 47, 3927−3931. (13) Liu, D.; Chen, W.; Sun, K.; Deng, K.; Zhang, W.; Wang, Z.; Jiang, X. Angew. Chem., Int. Ed. 2011, 50, 4103−4107. (14) Ambrosi, A.; Airò, F.; Merkoçi, A. Anal. Chem. 2010, 82, 1151− 1156. (15) Cecchcin, D.; de la Rica, R.; Bain, R. E. S.; Finnis, M.; Stevens, M. M.; Battaglia, G. Nanoscale 2014, 6, 9559−9562. (16) de la Rica, R.; Stevens, M. M. Nat. Nanotechnol. 2012, 7, 821− 824. (17) Lee, J. S.; Han, M. S.; Mirkin, C. A. Angew. Chem., Int. Ed. 2007, 46, 4093−4096.



CONCLUSIONS We have developed a sensitive colorimetric assay for the detection of disease biomarkers using ADH-catalyzed gold nanoparticle seed-mediated growth. Compared to other sophisticated detection methods, such as chemiluminescence microparticle immunoassays, our assay could be easily performed without trained operating personnel or sophisticated equipment, which could save the cost of purchasing and maintaining advanced instruments, making it an effective E

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Analytical Chemistry (18) Liu, D.; Wang, Z.; Jin, A.; Huang, X.; Sun, X.; Wang, F.; Yan, Q.; Ge, S.; Xia, N.; Niu, G.; Liu, G.; Hight Walker, A. R.; Chen, X. Angew. Chem., Int. Ed. 2013, 52, 14065−14069. (19) Qu, W.; Liu, Y.; Liu, D.; Wang, Z.; Jiang, X. Angew. Chem., Int. Ed. 2011, 50, 3442−3445. (20) Zhang, M.; Qing, G.; Xiong, C.; Cui, R.; Pang, D. W.; Sun, T. Adv. Mater. 2013, 25, 749−754. (21) Xiao, Y.; Shlyahovsky, B.; Popov, I.; Pavlov, V.; Willner, I. Langmuir 2005, 21, 5659−5662. (22) Zhang, L.; Li, Y.; Li, D.-W.; Jing, C.; Chen, X.; Lv, M.; Huang, Q.; Long, Y.-T.; Willner, I. Angew. Chem., Int. Ed. 2011, 50, 6789− 6792. (23) Freeman, R.; Gill, R.; Shweky, I.; Kotler, M.; Banin, U.; Willner, I. Angew. Chem., Int. Ed. 2009, 48, 309−313. (24) Willner, I.; Baron, R.; Willner, B. Adv. Mater. 2006, 18, 1109− 1120. (25) Xiao, Y.; Pavlov, V.; Levine, S.; Niazov, T.; Markovitch, G.; Willner, I. Angew. Chem. 2004, 116, 4619−4622. (26) Haiss, W.; Thanh, N. T. K.; Aveyard, J.; Fernig, D. G. Anal. Chem. 2007, 79, 4215−4221. (27) Khlebtsov, N. G. Anal. Chem. 2008, 80, 6620−6625. (28) Li, Y.; Jing, C.; Zhang, L.; Long, Y.-T. Chem. Soc. Rev. 2012, 41, 632−642.

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DOI: 10.1021/acs.analchem.5b00287 Anal. Chem. XXXX, XXX, XXX−XXX