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1,4-Benzenediboronic-Acid-Induced Aggregation of Gold Nanoparticles: Application to Hydrogen Peroxide Detection and Biotin-Avidin-Mediated Immunoassay with Naked-Eye Detection Ya-Chun Yang, and Wei-Lung Tseng Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b00668 • Publication Date (Web): 19 Apr 2016 Downloaded from http://pubs.acs.org on April 19, 2016

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Analytical Chemistry

1,4-Benzenediboronic-Acid-Induced Aggregation of Gold Nanoparticles: Application to Hydrogen Peroxide Detection and Biotin-Avidin-Mediated Immunoassay with Naked-Eye Detection

Ya-Chun Yang,a and Wei-Lung Tseng*a,b,c a

Department of Chemistry, National Sun Yat-sen University, Taiwan

b

School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Taiwan

c

Center for Nanoscience and Nanotechnology, National Sun Yat-sen University,

Taiwan.

Correspondence: Dr. Wei-Lung Tseng, Department of Chemistry, National Sun Yat-sen University, 70, Lien-hai Road, Kaohsiung, Taiwan 804. E-mail: [email protected] Fax: 011-886-7-3684046.

1 ACS Paragon Plus Environment


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ABSTRACT Hydrogen-peroxide (H2O2)-induced growth of small-sized gold nanoparticles (AuNPs) is often implemented for H2O2 sensing and plasmonic immunoassay. In contrast, there is little-to-no information in the literature regarding the application of H2O2-inhibited aggregation of citrate-capped AuNPs. This study discloses that benzene-1,4-diboronic acid (BDBA) was effective in driving the aggregation of citrate-capped AuNPs through an interaction between α-hydroxycarboxylate of citrate and boronic acids of BDBA. The H2O2-mediated oxidation of BDBA resulted in the conversion of boronic acid groups to phenol groups. The oxidized BDBA was incapable of triggering the aggregation of citrate-capped AuNPs. Thus, the presence of H2O2 prohibited BDBA-induced aggregation of citrate-capped AuNPs. The BDBA-induced aggregation of citrate-capped AuNPs can be paired with the glucose oxidase (GOx)–glucose system to design a colorimetric probe for glucose. Moreover, a H2O2⋅BDBA⋅AuNP probe was integrated with sandwich immunoassay, biotinylated antibody, and avidin-conjugated GOx for the selective naked-eye detection of rabbit immunoglobulin G (IgG) and human-prostate-specific antigen (PSA). The lowest detectable concentrations of rabbit IgG and human PSA by naked eye were down to 0.1 and 4 ng/mL, respectively. More importantly, the proposed plasmonic immunoassay allowed the naked-eye quantification of 0–10 ng/mL PSA at an interval of 2 ng/mL in plasma samples.


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INTRODUCTION In the past decade, gold-nanoparticle (AuNP)-based colorimetric assay was extensively used in chemical and biomolecular sensing. Compared to other nanomaterials used as biosensors, AuNPs offer distinct advantages such as facile synthesis,1 size- and shape-dependent optical properties,2 naked-eye visibility,3-5 and easy surface modification.6,7 The most extensively studied aspects in AuNP-based colorimetric assays are target-analyte-stimulated aggregation or growth of AuNPs. The binding of a target analyte to recognition ligands on a nanoparticle surface can trigger the aggregation of AuNPs accompanying color change, leading to electromagnetic coupling among the localized surface plasmon resonance (SPR) of nearby nanoparticles.4,8 The aggregation-induced color change of AuNPs caused a red-shifted and broadened SPR peak. The binding types of nanoparticles with the target analyte aptamer–analyte

include antibody–antigen complex,11,12

host–guest

recognition,9

DNA

interaction,13

hybridization,10

metal

ion–ligand

chelation,14,15 electrostatic attraction,16,17 and covalent bond formation.18 The aggregation of AuNPs can be driven by the direct attachment of the target analyte on the nanoparticle surface through the removal of a nanoparticle stabilizer19,20 and change in surface charges of nanoparticles. This type of nanoparticle aggregation was implemented for optical sensing of thiols,21 cyanide,22 thiol-product-related enzyme reactions,23 and adenosine-related enzyme systems.24 In addition to the aggregation phenomena of AuNPs, their target-analyte-mediated growth was utilized to develop a sensing platform through the target-analyte-directed reduction of AuCl4– to Au0 on the surface of AuNP seeds. Examples of analytes for use in developing such sensing



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methods include hydrogen peroxide (H2O2),25,26 catecholamine,27 and phenol analogs.28 Recently, plasmonic immunoassays were developed by combining enzyme-linked immunosorbent assay (ELISA) with AuNP-based colorimetric assay.26,29,30 Compared to other detection methods used in ELISA, plasmonic immunoassay enables the naked-eye detection of a target analyte without the use of sophisticated instruments. To perform this combination method, an enzyme is commonly labeled as either a detection antibody or streptavidin. After immunoreaction, the labeled enzyme catalyzes its substrate to generate the product, which triggers the aggregation or enlargement of AuNPs.8,29,31,32 In general, the red-to-blue color change caused by nanoparticle aggregation is more easily recognizable than the light-red-to-deep-red color change induced by nanoparticle growth. In addition, Stevens and co-workers combined the H2O2-based growth of AuNPs with catalase-catalyzed decomposition of H2O2 for an ultrasensitive immunoassay of target proteins when a biotinylated detection antibody and streptavidin-conjugated catalase were used in a plasmonic immunoassay.33,34 Although most abovementioned AuNP-based immunoassays offer high sensitivity and large linear dynamic range for a target protein, they were insensitive to a slight change in the concentration of the target protein. Therefore, these methods are difficult to use in clinical practice. For example, only a small dynamic range is required for the determination of human-prostate-specific antigen (PSA) in human blood because 25%–35% of men with a total PSA concentration of 4–10 ng/mL suffer from prostate cancer, and 50% of men have prostate cancer at a total PSA concentration greater than 10 ng/mL.35 Additionally, the antibody conjugated to glucose oxidase (GOx),32,36 alkaline phosphatase (ALP),29,30 or horseradish peroxidase (HRP)37,38 is typically used to detect the capture antigen in


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sandwich ELISA systems. However, only a few plasmonic immunoassays employed these enzymes as an antibody label for the detection of a target protein.39,40 29,41,42 To circumvent these problems, this study reports a simple, sensitive naked-eye immunoassay for an accurate quantification of immunoglobulin G (IgG) and PSA in plasma samples. Figure 1 illustrates that the proposed plasmonic immunoassay mainly consists of sandwich ELISA with avidin-biotin detection, the binding of avidin-conjugated GOx to biotinylated antibody, GOx-mediated oxidation of glucose, H2O2-induced

conversion

of

benzene-1,4-diboronic

acid

(BDBA),

and

BDBA-triggered aggregation of citrate-capped AuNPs. After the operation of capture antibody immobilization and antibody–antigen binding, the biotinylated detection antibody is linked to avidin-conjugated GOx. GOx in a 96-well microplate catalyzes the oxidation of glucose to generate gluconic acid and H2O2. We find that BDBA drove the aggregation of citrate-capped AuNPs through the complexation of citrate and boronic acids. The produced H2O2 induced the oxidation of boronic groups to phenol groups in BDBA, retaining the dispersion of citrate-capped AuNPs. Therefore, the blue-to-red color change of AuNPs reflects the amount of the produced H2O2, which is directly dependent on the concentration of the target protein. To prove its practicality, the present plasmonic immunoassay was applied to the naked-eye detection of human PSA in plasma samples.

EXPERIMENTAL SECTION Chemicals. 30% hydrogen peroxide were bought from SHOWA (Tokyo, Japan). BDBA and 4-hydroxyphenylboronic acid were ordered from Alfa Aesar (Ward Hill, Massachusetts). Na2CO3, NaHCO3, NaH2PO4, Na2HPO4, Na3PO4, Tween 20, hydroquinone,

4-Hydroxyphenylboronic

acid,

bovine


serum

albumin,


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(R,R)-(+)-sodium tartrate dibasic dihydrate, glucose, fructose, galactose, arabinose, mannose, xylose, maltose, raffinose, glucose oxidase (from Aspergillus Niger), beta-casein, cytochrome c (from bovine heart), ribonuclease A (from bovine pancreas), fibrinogen (from bovine plasma), hemoglobin (from human), human serum albumin, lysozyme (from chicken egg white), IgG (from rabbit serum), anti-Rabbit IgG developed in goat were bought from Sigma-Aldrich (St. Louis, MO). PSA (from human seminal fluid) were obtained from Aviva systems biology (San Diego, CA). Avidin-glucose oxidase (from egg white and Aspergillus Niger) were ordered from US Biological (MA, USA). Biotin-SP-conjugated AffiniPure Goat anti-rabbit IgG (H+L) were bought from Jackson IR (PA, USA). Biotin-conjugated PSA antibody produced in mouse and human PSA antibody produced from sheep were ordered from Thermo Scientific (MA, USA). Water used in all experiments was doubly distilled and purified by Milili-Q system (Millipore, Milford, MA, USA). Apparatus. The extinction spectra of AuNPs were measured by a double-beam UV-visible spectrophotometer (Cintra 10e; GBC, Victoria, Australia). JEM-2100 transmission electron microscopy (JEOL, Japan) was used to record the distribution images of nanoparticles operating at a 200 kV accelerating voltage. The hydrodynamic diameter of the AuNPs was measured by dynamic light scattering (N5 Submicrometer Particle Size Analyzer, Beckman Coulter Inc., USA). Synthesis of AuNPs. (a) citrate-capped AuNPs. The synthesis of citrate-capped AuNPs was performed via citrate-induced reduction of gold ion precursor. Briefly, 200 mL of1mM of HAuCl4 was heated to boil under the vigorous stirring. After refluxing for 5 min, we rapidly added 38.8 mM sodium citrate to the heated solution. This heating continued for an additional 15 min. During this time, the color of the mixture solution changed from a pale yellow to a dark red, indicating the formation of


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citrate-capped AuNPs. The size of citrate-capped AuNPs was estimated to be 13 ± 1 nm (extinction coefficient = 2.7 × 108) and their SPR peak was located at 518 nm. The particle concentration of 13 nm-sized AuNPs was calculated to be 11 nM by Beer’s law. (b) tartrate-capped AuNPs. Tartrate-capped AuNPs were synthesized according to the modification of the reported method

43

. Briefly, 10 mL of 1 mM

HAuCl4 was heated to boil under reflux. Subsequently, we rapidly introduced 6 mL of 38.8 mM sodium tartrate to the boiled solution under the vigorous stirring. The color of the resulting solution changed to a dark red during the reaction time of 15 min. The SPR peak of the formed AuNPs was observed to be 525 nm (extinction coefficient = 1.98× 109) and their particle concentration was estimated to be 0.24 nM. (c) Glutamic acid- and aspartic acid-capped AuNPs. Amino acid-capped AuNPs were prepared via the reduction of gold ion precursor with amino acid based on the previously reported method.44,45 For the synthesis of glutamic acid-capped AuNPs, 5.3 mL of 4 mM glutamic acid was heated to boil for 5 min and then vigorously stirred upon the addition of 1 mL of 0.2 mM HAuCl4. After 5 min, the obtained solution had a purple red color, reflecting the formation of the AuNPs. Similarly, 5.3 mL of 1.5 mM aspartic acid was kept stirring and heated to boil. After 5 min of boiling, 1 mL of 0.2 mM HAuCl4 was added to the heated solution and reacted for an additional 5 min. The SPR peaks of glutamic acid- and aspartic acid-capped AuNPs were found to be 533 nm (extinction coefficient = 1.37 × 1010) and 525 nm (1.98 × 109), respectively. The particles concentrations of glutamic acid- and aspartic acid-capped AuNPs were calculated to be 0.07 and 0.57 nM, respectively. Colorimetric Detection of H2O2 and Glucose. For H2O2 sensing, different concentrations of H2O2 (0−75 µM, 800 µL) reacted with BDBA (0−1.6 mM, 100 µL) in 10 mM phosphate buffer (pH 2.0−10.0) at ambient temperature for 30 s. The 7 ACS Paragon Plus Environment


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resulting solutions were incubated to citrate-capped AuNPs (11 nM, 100 µL) at ambient temperature for 15 min before the measurement of their extinction spectra. For glucose sensing, a series of concentration of glucose (0−120 µM, 500 µL) reacted with GOx (0.6 units/mL, 100 µL) at 50 oC for 40 min. The obtained products were incubated with a mixture of BDBA (1 mM, 100 µL) and phosphate (50 mM, 200 µL; pH 10.0) at ambient temperature for 30 s. We equilibrated the resulting solutions with citrate-capped AuNPs (11 nM, 100 µL) at ambient temperature for 15 min and collected their extinction spectra. Plasmonic Immunoassay. (a) Sensing of rabbit IgG. Anti-Rabbit IgG was prepared in 25 mM sodium carbonate (pH 9.0). Rabbit IgG, biotinylated anti-Rabbit IgG, and avidin-conjugated GOx were dissolved in the blocking buffer (1% w/v BSA, 0.05% v/v Tween 20, and 10mM phosphate, pH 7.4). Each well in the 96-well polystyrene plate was coated with anti-Rabbit IgG (5 µg/mL, 100 µL) and stored overnight at 4 oC. The blocking buffer (200 µL) was then added to each well and incubated at ambient temperature for 1 h. After washing three times with washing buffer (0.05% v/v Tween 20, and 10 mM phosphate, pH 7.4; 200 µL), each well was filled with rabbit IgG (0.1−10ng/mL, 200 µL) or other proteins (100 ng/mL, 200 µL). The plates were maintained at ambient temperature for 1 h on a plate shaker, followed by washing three times with washing buffer. Each well was incubated with biotinylated anti-Rabbit IgG (1 mg/mL, 200 µL) at ambient temperature for 1 h and then washed for three runs. A solution of avidin-conjugated GOx (0.5 mg/mL, 200 µL) reacted with biotinylated anti-Rabbit IgG at ambient temperature for 1 h. The plate was washed three times with washing buffer. A solution of glucose (10 mM, 200 µL) was transferred to each well and incubated at 50 oC for 1 h. Subsequently, BDBA (1 mM, 40 µL) was added to each well followed by the addition of phosphate buffer 8 ACS Paragon Plus Environment

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(10 mM, 320 µL; pH 10.0). After 30 s, citrate-capped AuNPs (11 nM, 40 µL) was transferred to each well. The extinction spectra and photographs of the resulting solutions were collected 15 min later. (b) Sensing of human PSA. Anti-PSA, PSA, biotinylated anti-PSA, and avidin-conjugated GOx were prepared in the same buffers as used in the plasmonic immunoassay of rabbit IgG. The following steps, including the immobilization of anti-PSA (10 µg/mL, 100 µL), the washing of coated plate, the bindings of anti-PSA to human PSA (1−40 ng/mL, 200 µL), human PSA to biotinylated anti-PSA, and biotinylated anti-PSA to avidin-conjugated GOx, and the sensing of H2O2, were the same as the method mentioned in the detection of rabbit IgG. (c) Determination of human PSA in plasma samples. Blood samples were collected from four healthy adult males. The plasma samples were immediately (within 2 h) separated from the cells by the treatment of whole blood samples with centrifugation at 3000 rpm for 10 min at 4 °C. The obtained plasma samples (100 µL) were spiked with standard solutions of human PSA (0−20 ng/mL, 100 µL). The spiked samples were diluted to 2-fold throughout the sample preparation process. To remove large-sized proteins, the spiked samples were filtered with 300 kDa Nanosep centrifugal device (Pall Co., East Hills, NY) at 3000 rpm for 5 min at 4 °C. The obtained samples containing different concentrations of human PSA was detected by the proposed system.

RESULTS AND DISCUSSION BDBA-induced aggregation of citrate-capped AuNPs for H2O2 sensing. To conduct the aforementioned strategy, this study first demonstrated that BDBA is capable of triggering the aggregation of citrate-capped AuNPs. Compared to the extinction spectrum of citrate-capped AuNPs in a 10 mM phosphate buffer at pH 10.0 9 ACS Paragon Plus Environment


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(curve a in Figure 2A), the presence of 0.1-mM BDBA caused a decrease in the strength of the surface plasmon resonance (SPR) band at 520 nm, production of a new red-shift band, and blue-to-red color change, which were characteristic of nanoparticle aggregation (curve b in Figure 2A). The driving force for this nanoparticle aggregation could arise from the binding of citrate to BDBA. Once 0.1 mM H2O2 was incubated with 0.1 mM BDBA for 30 s, resulting products did not drive any change in the extinction spectrum and solution color of citrate-capped AuNPs (curve c in Figure 2A). This result clearly reflects that the H2O2-treated BDBA cannot stimulate nanoparticle aggregation. This is attributed to the fact that H2O2 can react with boronic groups to form phenol groups in boronate-derivatized organic compounds.46,47 Note that the presence of H2O2 was incapable of disassembling the aggregation of citrate-capped AuNPs induced by BDBA (Figure S1, Supporting Information). To support the abovementioned finding, dynamic light scattering (DLS) and transmission electron microscopy (TEM) were used to monitor the degree of nanoparticle aggregation in the presence of BDBA and H2O2-treated BDBA. Figure 2B shows that the average hydrodynamic diameter of citrate-capped AuNPs remarkably enlarged after the addition of BDBA; however, their hydrodynamic diameter remained almost constant in the presence of H2O2-treated BDBA. The TEM images show that the separate addition of BDBA and H2O2-treated BDBA to a solution of citrate-capped AuNPs resulted in aggregated and dispersed AuNPs (Figure 2C). These results are consistent with the fact that H2O2 is efficient in oxidizing BDBA, suppressing the BDBA-mediated aggregation of citrate-capped AuNPs. To verify the mechanism of the BDBA-induced aggregation of citrate-capped AuNPs, 4-hydroxylpheylboronic acid (one boronic acid group and one hydroxyl group) and hydroquinone (two hydroxyl groups) were used in place of BDBA (two boronic acid groups) under the


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same conditions. Figure S2 (see Supporting Information) reveals that both 4-hydroxylpheylboronic acid and hydroquinone are incapable of inducing the aggregation of citrate-capped AuNPs, signifying that two boronic acid groups of BDBA serve as a bridge to link individual AuNPs together. According to the strong interaction of boronic acids and α-hydroxycarboxylic acids such as citrate and tartrate,48-50 we propose that the mechanism for nanoparticle aggregation originates from the complexation of α-hydroxycarboxylate of citrate and boronic acids of BDBA (Figure S3, Supporting Information). To examine this hypothesis, the synthesis of AuNPs with different capping ligands was performed via the reduction of gold-ion precursor with citrate analogs, including tartrate, aspartic acid, and glutamic acid. Note that aspartic and glutamic acids are not classified as α-hydroxycarboxylic acids. When the extinction and DLS spectra of three types of AuNPs were obtained on the addition of BDBA and H2O2-treated BDBA, only tartrate-capped AuNPs were found to exhibit similar behavior to citrate-capped AuNPs (Figure S4, Supporting Information). Evidently, only α-hydroxycarboxylic acids adsorbed on the surface of AuNPs can bind to boronic acids of BDBA. Additionally, tartrate-capped AuNPs is less sensitive to BDBA than citrate-capped AuNPs. This could be attributed to the differences in the concentration and particle size between citrate-capped AuNPs (13 nm; 11 nM) and tartrate-capped AuNPs (0.24 nM; 25 nM). Figure S5 (Supporting Information) showed that the degree of the BDBA-induced aggregation of citrate-capped AuNPs was highly sensitivity to their concentration, supporting this hypothesis.

These results are in agreement with those of previous studies

48-50

and

our hypothesis regarding the mechanism of the BDBA-induced aggregation of citrate-capped AuNPs.



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To optimize conditions for H2O2 detection, the effects of solution pH and BDBA concentration on detection sensitivity were explored based on the comparison of the molar ratio of aggregated and dispersed AuNPs, which are expressed by the ratio of the extinction value (Ex) at 650 nm to that at 520 nm (Ex650

nm/Ex520 nm).When

citrate-capped AuNPs were incubated with 0.1 mM BDBA at different pH conditions, the Ex650 nm/Ex520 nm value increased with rising solution pH (Figure S6, Supporting Information). This result indicates that the binding of citrate by BDBA is favored above pH 4.0. In the pH range of 2.0–7.0, the Ex650 nm/Ex520 nm value of a solution containing citrate-capped AuNPs and 0.1-mM BDBA remained almost unchanged on the addition of 0.1 mM H2O2. In contrast, the Ex650 nm/Ex520 nm value of the same solution sharply decreased with an increase in solution pH. This result clearly reflects that an alkaline condition promotes the oxidation of BDBA by H2O2; this finding is in line with that of previous studies.51 When citrate-capped AuNPs reacted with various concentrations of BDBA at pH 10.0, the Ex650 nm/Ex520 nm value of citrate-capped gold nanoparticles reached its saturation level above 0.1 mM BDBA (Figure S7, Supporting Information). Thus, the concentration of BDBA was optimized to be 0.1 mM. To quantify the concentration of H2O2, 0.1 mM BDBA was reacted with different concentrations of H2O2 in 10 mM phosphate buffer (pH 10.0) for a fixed time interval of 30 s, and subsequently, products obtained were incubated with citrate-capped AuNPs for 15 min. As the concentration of H2O2 increased, the intensities of the SPR bands at 650 and 520 nm gradually decreased and increased, respectively (Figure 3A). Thus, the degree of nanoparticle aggregation can be easily visualized via the color change of citrate-capped AuNPs. The linear relationship (R2 = 0.9960) of the Ex650 nm/Ex520 nm

value versus the H2O2 concentration was obtained in the range of 0.6–6


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µM (inset of Figure 3A). The limit of detection (LOD) at a signal-to-noise ratio of 3 for H2O2 was estimated to be 200 nM, which is lower than LODs measured from the H2O2-mediated inhibition of cysteine-induced AuNPs aggregation,8 H2O2-stimulated synthesis of AuNPs,26,52 and H2O2-induced enlargement of small-sized AuNPs.27,32 The H2O2-induced oxidation of BDBA coupled to the BDBA-stimulated aggregation of AuNPs might be suitable for detecting H2O2 product-related enzyme reactions such as

GOx-catalyzed

oxidation

of

glucose32

and

acetylcholine

esterase/choline-oxidase-mediated hydrolysis and oxidation of acetylcholine.23,31,53 For example, the H2O2⋅BDBA⋅AuNP probe was used to detect glucose in the presence of GOx. Because the activity of GOx can be adjusted with the temperature,43 the optimal temperature condition for GOx-mediated oxidation of glucose was found to be 50 oC. As the glucose concentration increased at a fixed concentration of GOx, the intensity of the SPR band at 650 nm and the Ex650 nm/Ex520 nm value progressively reduced (Figure 3B). By plotting the Ex650

nm/Ex520 nm

value against the glucose

concentration, a linear calibration was built up from 1 to 10 µM. This probe enabled the detection of glucose with a LOD corresponding to 0.3 µM. Because GOx specifically catalyzes the oxidation of glucose, this probe is highly selective to glucose over other saccharides (Figure. S8, Supporting Information). Plasmonic immunoassay. We further combined the H2O2⋅BDBA⋅AuNP probe, GOx-mediated oxidation of glucose, and sandwich immunoassay for the naked-eye detection of rabbit immunoglobulin (IgG). In the sandwich immunoassay, capture antibody, antigen, and detection antibody corresponded to anti-rabbit IgG, rabbit IgG, and biotinylated anti-rabbit IgG, respectively. Similar to ALP- and HRP-conjugated antibodies, biotinylated antibodies are currently available in ELISA.29,54,55 Commercial antibody providers such as Abcam, AnaSpec, Sigma-Aldrich, and Santa 13 ACS Paragon Plus Environment


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Cruz Biotechnology offer numerous biotinylated antibodies, enabling the proposed system to be adapted to the traditional ELISA. After the binding of biotinylated anti-rabbit IgG to avidin-labeled GOx, a fixed concentration of glucose was added to react with GOx. The produced H2O2 inhibited the aggregation of citrate-capped AuNPs by BDBA, whereas the unreacted BDBA led to their aggregation. As the concentration of rabbit IgG varied from 0.1 to 8 ng/mL, the Ex650 nm/Ex520 nm value of the H2O2⋅BDBA⋅AuNP probe progressively decreased, accompanied by a gradual color change from blue to red (Figure 4A). Figure S9 (Supporting Information) shows their corresponding spectra. Interestingly, the proposed system not only allowed the naked-eye readout of rabbit IgG values as low as 0.4 ng/mL but also facilitated the detection of a slight change in the concentration of rabbit IgG. For example, this system is capable of discriminating between 0.4 and 0.6 ng/mL rabbit IgG with naked-eye detection. For the naked eye, the sensitivity of the proposed system for rabbit IgG is superior to that of azide/alkyne-functionalized AuNPs coupled

to

ALP-related

immunoassay

(80

ng/mL),29

p-nitrophenyl

phosphate/ALP-related immunoassay (1000 ng/mL),29 cysteine-mediated AuNP aggregation

combined

with

HRP-linked

immunoassay

(1

ng/mL),41

and

tetramethylbenzidine/HRP-related immunoassay (20 ng/mL).41 By plotting the Ex650 nm/Ex520 nm

value versus the logarithm of the IgG concentration, the plot was found to

be linear (R2 = 0.9945) in the range of 0.1–1 ng/mL. The lowest detection concentration of rabbit IgG was observed to be 0.1 ng/mL. To examine the selectivity of the proposed system for rabbit IgG, other proteins were used instead of rabbit IgG. Figure S10 (Supporting information) shows that only 10 ng/mL rabbit IgG induced a noticeable decrease in the Ex650 nm/Ex520 nm value, whereas other proteins (100 ng/mL) exhibited a similar Ex650 nm/Ex520 nm value in comparison to that of the absence of 14 ACS Paragon Plus Environment

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rabbit IgG. This result reflects that the proposed system offers excellent selectivity to rabbit IgG because anti-rabbit IgG only recognizes rabbit IgG. Moreover, the proposed system was used for the quantification of human PSA when anti-PSA and biotinylated anti-PSA were selected as the capture antibody and detection antibody, respectively. Note that PSA is approved by the U.S. Food and Drug Administration as a biomarker for prostate cancer diagnosis.56 As the concentration of PSA was raised from 0 to 40 ng/mL, the color of the H2O2⋅BDBA⋅AuNP probe progressively varied from purple to red in the PSA concentration range of 1–10 ng/mL (Figure 4B). Their corresponding spectra are shown in Figure S11 (Supporting Information). The lowest concentration of PSA by naked-eye detection was 4 ng/mL. Because the concentration of total PSA in normal serum is lower than 4 ng/mL,57 we suggest that the color change of the proposed system falls within the range of clinical interest for prostate cancer diagnosis. Meanwhile, the Ex650 nm/Ex520 nm value of the H2O2⋅BDBA⋅AuNP probe steadily decreased and leveled off above 10 ng/mL. On plotting the Ex650 nm/Ex520 nm

value against the logarithm of the PSA concentration, a linear relationship

(R2 = 0.9972) for PSA quantification was obtained in the range of 1–8 ng/mL. With high naked-eye sensitivity and selectivity, the feasibility of the proposed system was validated by quantifying PSA in four normal human plasma samples (denoted as samples 1, 2, 3, and 4). A series of PSA concentrations ranging from 0 to 10 ng/mL was spiked into each of the four human plasma samples. Prior to the sandwich immunoassay, the PSA-spiked plasma samples were pretreated with a 300-kDa Nanosep centrifugal ultrafiltration device to remove large-sized proteins (molecular weight greater than 900 kDa). Because the molecular weights of free PSA and α1-antichymotrypsin-PSA complex are approximately 30 and 90 kDa, respectively, they can pass through the membrane of the centrifugal ultrafiltration 15 ACS Paragon Plus Environment


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device into the filtrate receiver. In the sandwich immunoassay, anti-PSA capture antibodies immobilized onto the microwell surface were used to selectively bind to total PSA (free PSA and α1-antichymotrypsin-PSA complex). On the addition of PSA-spiked samples collected from the centrifugal ultrafiltration device, the sandwich complex is formed between the anti-PSA capture antibody and biotinylated anti-PSA antibody. The following steps are similar to those performed on the detection of standard PSA. As shown in Figure 5A, the color of the H2O2⋅BDBA⋅AuNP probe gradually changed from purple to red on increasing the spiked PSA concentration in human plasma samples 1–4 over the range of 2–10 ng/mL. Progressive decreases in the degree of nanoparticle aggregation were also observed after human plasma samples 1–4 were spiked with different PSA concentrations (Figure S12, Supporting Information). When the Ex650 nm/Ex520 nm values were plotted against the spiked PSA concentrations, four linear calibration curves were constructed for the quantification of PSA in human plasma samples 1–4 (Figure 5B). The slope of the calibration curve obtained from sample 1 was similar to those obtained from samples 2–4, suggesting that the proposed system revealed a similar response for the determination of PSA in the four different spiked plasma samples. These results indicate that the combination of our proposed system and a centrifugal ultrafiltration device can be well suited to detect PSA in real samples without a matrix effect.

3. Conclusion This study demonstrated that the combination of the BDBA-induced aggregation of citrate-capped AuNPs and H2O2-mediated oxidation of BDBA can be utilized for sensitive and selective detection of H2O2 and H2O2-related enzyme systems. Moreover,

the

H2O2⋅BDBA⋅AuNP

probe

was

well


integrated

into

the

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avidin-biotin-mediated immunosorbent assay, enabling the naked-eye detection of rabbit IgG and human PSA. Compared to other reported AuNP-based ELISA methods,26,29,31,32,34,36,53 the proposed plasmonic method offers numerous advantages: (1) unmodified AuNPs are directly used to detect H2O2 without further modification, making the proposed method easier and cheaper to operate; (2) it is well suited to be integrated with conventional ELISA platforms because biotin-labeled antibodies are commercially available; (3) even a slight change in the concentration of a target protein causes a remarkable color change of AuNPs; (4) it can detect relatively much lower concentrations of rabbit IgG with the naked eye; and (5) it can quantify 1–10 ng/mL human PSA at an interval of 2 ng/mL in plasma samples, which lie within the range of clinical interest for prostate cancer diagnosis. These findings suggest that the proposed plasmonic immunoassay has wide-ranging applications in clinical diagnosis.

ASSOCIATED CONTENT Supporting Information Additional detailed information as noted in the text. The Supporting Information is available free of charge on the ACS Publications website. AUTHOR INFORMATION Corresponding Author *E-Mail: [email protected]. Fax: 011-886-7-3684046. Present Addresses †Department of Chemistry, National Sun Yat-sen University, 70, Lien-hai Road, Kaohsiung, 804, Taiwan.

Author Contributions 17 ACS Paragon Plus Environment


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† These authors contributed equally. (match statement to author names with a symbol)

Notes Any additional relevant notes should be placed here.

ACKNOWLEDGMENT We would like to thank the Ministry of Science and Technology (NSC104-2113-M-110-004-MY3) and for the financial support of this study.

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Figure 1. Naked-eye readout of plasmonic immunoassays. Detection of target protein via the combination of sandwich immunoassay, avidin-biotin interaction, GOx-mediated the oxidation of glucose, H2O2-induced oxidation of BDBA, and BDBA-triggered aggregation of citrate-capped AuNPs.



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Figure 2. BDBA-mediated aggregation of citrate-capped AuNPs for H2O2 sensing. (A) UV-vis absorption spectra and optical photographs, (B) DLS spectra, and (C) TEM images of the AuNP solution (a) before and (b, c) after the addition of (b) BDBA and (c) H2O2-treated BDBA. A solution of citrate-capped AuNPs (1.1 nM) was incubated with (b) 0.1 mM BDBA and (c) a mixture of 0.1 mM BDBA and 100 µM H2O2 in 10 mM phosphate buffer (pH 10.0) at ambient temperature for 10 min. BDBA reacted with H2O2 at ambient temperature for 30 s.


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Figure 3. Quantification of H2O2 and glucose by the H2O2⋅BDBA⋅AuNP probe.(A) UV-vis absorption spectra of the AuNP solution in the presence of increased H2O2 concentration. Inset: Plot of the Ex650nm/Ex520nmvalue versus the H2O2 concentration. (B) UV-vis absorption spectra of the AuNP solution upon analyzing different glucose concentrations in the presence of 0.06 units/mL GOx. GOx reacted with glucose at 50 o C for 40 min. The produced H2O2 was detected by the H2O2⋅BDBA⋅AuNPs probe. Inset: Plot of the Ex650nm/Ex520nm value versus the glucose concentration. The error bars represent the standard deviation based on three independent measurements. See text for more detailed information.



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Figure 4. Naked-eye colorimetric detection of target protein by the proposed plasmonic immunoassay. Plot of the Ex650nm/Ex520nmvalue versus the concentration of (A) rabbit IgG and (B) human PSA. The arrow indicates the lowest detectable concentration of target protein with the naked eye. Inset: Optical photographs of the AuNP solution upon analyzing different concentrations of (A) rabbit IgG and (B) human PSA. The error bars represent the standard deviation based on three independent measurements. See text for more detailed information.


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Figure 5. Naked-eye colorimetric detection of PSA in four plasma samples by the proposed plasmonic immunoassay. (A) Optical photographs of the AuNP solution upon analyzing different spiked concentrations of PSA in four plasma samples. (B) Four calibration curves for the quantification of PSA in human plasma samples 1−4. Error bars represent the standard deviation in three independent measurements.



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