Chemically Regulated ROS Generation from Gold Nanoparticles for

Subscriber access provided by UNIV OF SCIENCES PHILADELPHIA

Chemically regulated ROS generation from gold nanoparticles for enzyme-free electrochemiluminescent immunosensing Yui Higashi, Joyotu Mazumder, Hiroyuki Yoshikawa, Masato Saito, and Eiichi Tamiya Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00118 • Publication Date (Web): 11 Apr 2018 Downloaded from http://pubs.acs.org on April 11, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

Chemically regulated ROS generation from gold nanoparticles for enzyme-free electrochemiluminescent immunosensing Yui Higashi,† Joyotu Mazumder, † Hiroyuki Yoshikawa, † Masato Saito, †‡ Eiichi Tamiya*† Phone: +81-6-6879-4087 E-mail: [email protected] † Department of Applied Physics, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871 Japan ‡ Advanced Photonics and Biosensing Open Innovation Laboratory, AIST-Osaka University, Photonics Center Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871, Japan ABSTRACT: In the present work, we report on an enzyme-free electrochemiluminescent (ECL) immunosensing scheme utilizing the catalytic generation of reactive oxygen species (ROS) from gold nanoparticles (AuNPs) (diameter ≥ 5 nm) dispersed in aqueous solutions of trishydroxymethylaminomethane (Tris). First, to examine this catalytic pathway in detail, the effects of various factors such as the AuNP size and concentration, dispersant type and concentration, and dissolved oxygen were investigated using the electrochemiluminescence (ECL) of luminol. It was found that the catalytic generation of ROS from AuNPs can be regulated chemically by altering conditions such as the type, concentration and pH of the solution that the AuNPs are dispersed in. Under the best conditions studied in this work, the AuNPs displayed high catalytic activity towards ROS generation, with an estimated apparent turnover number per AuNP of 0.1 s-1, comparable to those of several common peroxide-producing enzymes. Following these studies, this phenomenon was applied to develop a one-step enzyme-free ECL immunosensor based on sandwiching the target analyte using antibody-conjugated magnetic beads (MB) and AuNPs. Using IgA as a model analyte, the developed immunosensor was able to detect the target in the range of 1 ng/mL ~ 10 µg/mL, with the lower detection limit being comparable to those of commercial assays for the same target. Altering the antibodies used to modify the MB and AuNPs could further improve the detection limit as well as expand the applicability of this immunoassay to the detection of other analytes.

Electrochemiluminescence (ECL) is an analytical technique closely related to chemiluminescence (CL), in which light emission from an excited luminophore is detected, except that the reaction leading to luminescence is initiated not by the mixing of reagents in a tube but by the application of an electric potential at an electrode surface. This makes ECL a robust and simple-to-use technique, in addition to having low background signals, high sensitivity and a wide quantitative range. Owing to these advantages, ECL has emerged relatively recently as a powerful tool for the detection of biological analytes in a variety of fields ranging from, for instance, the measurement of the antioxidative capacity of beverages, to detecting glycated albumin in human serum.1–3 On the other hand, magnetic beads (MBs) have been used in the development of many biosensing technologies to improve the detection sensitivity and to simplify the assay procedure. For instance, MBs have high surface areas and can be easily modified with biomolecular linkers such as streptavidin and antibodies.4,5 In addition, using MBs in sandwich immunoassays with a second probe allows for facile and selective collection of the sandwiched analytes, as well as signal enhancement due to the confinement and concentration of the MB/analyte/probe conjugates within finite volumes close to the sensing surface.6,7 Moreover, these MB-based sandwich immunoassays could be modified to develop wash-free one step immunoassays with very simple handling, with the appropriate selection of probes and antibodies.8 In particular, enzyme-free immunoassays using ECL with inorganic catalysts as probes is

expected to find more practical applications because of the simplicity of the required instrumentation and because it is less constrained by factors such as pH and temperature.9 It is well known that gold nanoparticles (AuNPs) possess high catalytic activity towards various chemical reactions, unlike its bulk form. For instance, in some of the earliest works on such activity, Haruta et al. reported on the low-temperature oxidation of carbon monoxide using AuNPs on transition metal supports, while Hutchings et al. studied the hydrochlorination of acetylene using AuNPs supported on carbon.10,11 Since then, many other works on catalysis of oxidation reactions, such as those of benzyl alcohol and glucose, by supported and unsupported AuNPs have also been published.12,13 On the other hand, catalysis of reduction reactions using AuNPs has also been reported. The AuNP-assisted reduction of aromatic nitro compounds such as p-nitrophenol and p-nitroaniline by sodium borohydride, as well as the reduction of oxygen on gold/graphene nanocomposites, have each been investigated in a number of studies.14–19 These activities are thought to be attributed to a number of factors including size effects, and the presence of active sites at low-coordination Au atoms and at the Au/support junctions.20–22 In our study, we report on an enzyme-free electrochemiluminescent (ECL) immunosensing scheme utilizing a unique catalytic pathway involving unsupported AuNPs (with sizes of 5 nm or more). In this pathway, AuNPs

ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 1. Proposed ECL-based immunoassay using AuNP-catalyzed ROS generation conducted on SPE with portable ECL setup.

dispersed in aqueous solutions of tris(hydroxymethyl)aminomethane (Tris) were found to spontaneously generate reactive oxygen species (ROS), which can react with luminol to enhance its CL/ECL signal. We first studied the mechanism of ROS generation from AuNPs and examined conditions to chemically regulate and efficiently generate ROS, using ECL to indirectly measure the generated ROS. In several past studies, Au nanoclusters (~ 100 atoms) have been used as ECL luminophores that emit light in the near infrared region,23–25 but in this case luminol was used as the luminophore, since its oxidized form reacts with ROS to emit blue luminescence. We then used these results to apply AuNPs to an ECL-based enzyme-free sandwich immunoassay with MBs, conducted on disposable screen-printed electrode (SPE) chips using a portable ECL setup (Figure 1). AuNPs can be modified easily with biomolecules such as antibodies, and as such they are used for many biosensing applications as supports to immobilize a large number of these biomolecules.26–28 Our group has also previously reported on modifying AuNPs with luminol derivatives and immobilizing them on graphene oxide nanoribbons (GONRs) for enhanced ECL on screen printed electrodes, and on its application to the detection of urea.29,30 In our current work, we employ AuNPs both as supports for attaching antibodies and also as ECL probes to enhance the luminescent signal by generating ROS. As a proof of concept, our proposed immunoassay was used for the detection of IgA, a common biomarker for various diseases.

EXPERIMENTAL SECTION Reagents and apparatus. All materials used are of analytical grade. Luminol was purchased from Sigma-Aldrich (MO, USA). TAPS ([tris(hydroxymethyl) methylamino]propanesulfonic acid), Tricine (N(Tri(hydroxymethyl)methyl)glycine), tert-butylamine and trimethylolethane were purchased from Tokyo Chemical Industry (Tokyo, Japan). Tris, NaCl, Na2HPO4・12H2O, KCl, KH2PO4, H3BO3, Na2B4O7 and bovine serum albumin (BSA) were purchased from Wako (Tokyo, Japan). NHS activated magnetic beads were kindly provided by Tamagawa Seiki (Nagano, Japan). Tris solutions were adjusted to pH 6.0～12.0 with HCl and NaOH. A stock solution of luminol was prepared by dissolving luminol powder in 0.1 M NaOH and stirring until the powder was completely dissolved. The stock solution was stored at -20°C until use. Gold nanoparticles were purchased from TANAKA Precious Metals (Tokyo, Japan) and SigmaAldrich (MO, USA). IgA antigen and anti-IgA antibodies were

purchased from Bethyl Laboratories, Inc. (TX, USA). Ultrapure water was used to dissolve and dilute all reagents and samples. The ECL monitoring system (BDTeCLP100, Bio Device Technology Ltd., Ishikawa, Japan) comprises a photon detection unit (C969212; Hamamatsu Photonics K.K., Hamamatsu, Japan) and an USB powered hand-held potentiostat (Bio Device Technology Ltd., Ishikawa, Japan). The potentiostat has a system that sends a trigger signal to the photon detection unit to coordinate the electrochemical and the ECL measurements. SPEs consisted of carbon working and counter electrodes, and an Ag/AgCl reference electrode. The electrode chip (type EP-P) used with the ECL monitoring system has dimensions of 12.5×4 mm with the working electrode having an area of 2.64 mm2. For the following experiments, a new SPE was used for every measurement. General ECL measurement procedure. For ECL measurements, a 0.2 mM luminol was first prepared by diluting the 10 mM luminol-NaOH stock solution in 200 mM Tris-HCl (pH 8). Next, AuNPs adjusted to a given concentration was mixed with a given solution of interest (e.g. 200 mM Tris-HCl, pH 8) in a 1:1 volume ratio. This mixed AuNP solution was then left stationary, and at given time intervals following this step, aliquots of the mixed AuNP solution were mixed with 0.2 mM luminol, again in a 1:1 volume ratio, to complete the sample preparation. 20 µL of this sample was then pipetted on an SPE, which was inserted into the monitoring device. The ECL intensity from the sample was measured with an integration time of 500 ms while linear sweep voltammetry (LSV) was conducted on the SPE (0~700 mV, scan rate 50 mV/s). Detailed procedures for measuring the effects of various factors are included in the Supporting Information. Preparation of antibody/MB and antibody/AuNP conjugates. MBs were modified with anti-IgA antibodies according to a protocol from previous research.31 In brief, 200 nm MBs activated with N-hydroxysuccinimide (NHS) were first washed with methanol by centrifugation for 5 min at 15,000 rpm and 4°C. After discarding the supernatant, antibodies were allowed to react with the NHS groups on the MBs for 1 day at 4°C.The modiﬁed MBs were collected and dispersed in aminoethanol overnight to mask unreacted NHS groups. After this step, the MBs were washed three times with buffer containing 200 mM PBS-NaOH (pH 7.40), re-dispersed in the same buffer and stored at 4°C until use. AuNPs were also modified with anti-IgA antibodies according to a protocol from previous research.31 100 µL antiIgA was added to 10 mL of AuNPs dispersed in PBS under


Page 2 of 8

Page 3 of 8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry stirring for 24 h at 4°C. The resulting anti-IgA/AuNP conjugates were obtained by centrifuging (15,000 rpm, 30 min) and washing with 200 mM PBS (pH 7.40) three times. Afterwards, the anti-IgA/AuNP conjugates were dispersed in PBS (pH 7.40) and stored at 4°C until use. Enzyme-free ECL immunoassay for IgA. 90 µL of the antiIgA/MB conjugates were first mixed with 10 µL of PBS spiked with various concentrations of IgA. The mixture was then incubated under mild agitation at room temperature for about 1.5 h until reaction equilibrium between the IgA and the antiIgA/MB conjugates was reached. Afterwards, these mixtures containing different IgA concentrations were incubated with 100 µL of anti-IgA/AuNP conjugates for 30 min. As a result, magnetic sandwich-type conjugates formed between the IgA and the antibodies coated on the two nanomaterials respectively, through antigen-antibody recognition. Finally, the conjugates were collected by a magnet, and used for ECL detection by dispersing in 200 mM Tris-NaOH (pH 12), leaving for 15 minutes and then mixing with 0.2 mM luminol-NaOH diluted in 200 mM borate buffer (pH 9).

RESULTS AND DISCUSSION Effect of Tris concentration. To investigate the role of Tris in AuNP-catalyzed ROS emission, ECL responses were measured for 15 nm AuNPs dispersed in several concentrations of Tris buffer (Figure 2). As shown in Figure 2, the amount of emitted luminescence increased with increasing concentration of Tris buffer. The generation of ROS from AuNPs mixed with Tris and with several other reagents was additionally affirmed by taking UV-vis absorption spectra of Tris after mixing with AuNPs and of H2O2 dissolved in Tris (Figure S-1),32,33 and by conducting a fluorimetric peroxidase assay using Amplite™ Red as the fluorescent reporter (Figure S-2).

Figure 2. Time change in ECL intensities of 0.2 mM luminol mixed with 15 nm AuNPs (0.04 mg/mL) dispersed in different concentrations of Tris-HCl (pH 8).

Effects of AuNP size and concentration. Next, ECL was used to look at the influences of the concentration and size of AuNPs on AuNP-catalyzed ROS generation. The ECL intensity was observed to increase with increasing concentration of AuNP (Figure 3A), and in general, with decreasing AuNP size (Figure 3B). It has been observed in previous studies of catalysis by supported AuNPs that the size of the Au catalysts has a significant effect on their catalytic activity.34,35 However, the results obtained for 15 nm AuNPs shows a large deviation

from this tendency, with the ECL intensity being much higher than those of AuNPs with other sizes. For the 15 nm AuNPs, unlike the other sizes, an instantaneous color change of the nanoparticle was observed after the addition of Tris, which indicates significant nanoparticle aggregation. This suggests that aggregation of the AuNPs, in some manner, played a role in enhancing their catalytic activity. This aggregation is observed, to a degree, for AuNPs with other sizes as well, as investigated by nanoparticle tracking analysis (NTA) (Figure S3) and UV-vis absorption spectroscopy (Figure S-4). In investigating the relationship between nanoparticle aggregation and their catalytic activity, Liao et al. have reported that introduction of Pb2+ cations induced aggregation of glutathionecapped Au nanoclusters, which in turn enhanced their peroxidase-mimetic catalytic activity.36 In this case as well as those pertaining to AuNP size dependence, these previous studies have focused on Au nanoclusters, which are comprised of a very small number of atoms (< 100). At this scale, differences in a few atoms could possibly alter the unique electronic structure of the AuNPs, in addition to their geometric properties e.g. the number and density of sites with lowcoordinated Au atoms. The AuNPs used in this study are markedly larger than those used in the aforementioned studies, which suggests that the observed differences in catalytic activity could perhaps be attributed more to geometric factors than to those pertaining to the particles’ electronic structures. Whether the enhancement of catalytic activity observed for the 15 nm AuNPs happened through a pathway similar to that reported by Liao et al., and why this occurred significantly for this size only, are yet unclear, but could be potential topics for further studies in the future. Effect of dispersant molecular structure. Following this, the influence of the type of dispersant on AuNP-catalyzed ROS generation was explored. Luminescence was observed only when AuNPs were dispersed in solutions that contain species with both hydroxyl and amino groups (Figure 4B). The influence of these functional groups was further examined by comparing the ECL intensity for AuNPs dispersed in Tris with those of AuNPs dispersed in tert-butylamine, trimethylolethane and a mixture of the two. These molecules are structurally analogous to Tris, except that the amino group of Tris has been replaced by a methyl group in the former, whereas in the latter methyl groups are present in place of the hydroxyl groups on Tris (Figure 4A). The results in Figure 4C show that luminescence was observed from AuNPs dispersed in Tris buffer, but not for the other two, nor for the mixture. Effects of dispersant pH, dissolved oxygen and surface blocking of AuNPs. The effect of dispersant pH was investigated by taking ECL measurements for AuNP (15 nm) dispersed in Tris adjusted to various pH values. Luminescence was shown to increase with increasing solvent pH (Figure 5A). While other studies have also indicated that the solution pH plays a role in the catalytic activity of AuNPs in the liquid phase, these studies each look at different systems and have come up with conflicting tendencies: some have reported on increasing activity with increasing pH, while others have come across the opposite tendency, and some have found a volcano-shaped pH dependence, all of which reflect the complexity of the systems and reactions involved.37–40 A number of theories for this pH dependence have been posited so far, including the pH’s effects on the oxidation state of Au and the coordination environment of Au to the support material, but conclusive results are yet to be obtained. It is possible that investigating the oxidation states



Figure 3. Time change in ECL intensities of 0.2 mM luminol mixed with 15 nm AuNPs of varying concentrations dispersed in 200 mM Tris-HCl (pH 8) (A). ECL intensities of 0.2 mM luminol mixed with AuNPs of varying sizes (0.04 mg/mL) dispersed in 200 mM Tris-HCl (pH 8) (B).

present before and after the experiments using XPS could provide more information on the factors at play. Next, the effect of dissolved oxygen on ROS generation from AuNPs was studied by sparging the AuNPs dispersed in Tris with nitrogen gas. ECL measurements using the resulting solution and the same solution not treated by nitrogen bubbling showed that only the latter exhibited appreciable luminescence (Figure 5B). This result indicates that ROS is generated from the reaction of oxygen dissolved in solution on the surface of AuNPs. In addition, to determine whether ROS generation was occurring on or away from the surface of AuNPs, BSA was used to coat AuNP surfaces prior to ECL measurements. The ECL intensity was observed to decrease with increasing concentrations of BSA (Figure 5C). This suggests that ROS generation from dissolved molecular oxygen is enabled by the binding of Tris to the AuNP surface. Mechanism for AuNP-catalyzed ROS generation. These results were then interpreted to come up with a hypothesis for the mechanism of AuNP-catalyzed ROS generation observed in our studies. Chang et al. have previously studied the activation of molecular oxygen on Au cluster surfaces for the catalysis of various oxidation and hydrogenation reactions, and suggested that O2 could be activated by the abstraction of H atoms from H-containing species including water and various

Page 4 of 8

hydrocarbons.41 On the other hand, Tsunoyama et al. have studied the use of AuNPs stabilized by poly(N-vinyl-2pyrrolidone) (PVP) as catalysts for aerobic oxidation of alcohols, and have proposed that their high catalytic activity is attributed to electron donation from PVP to the Au core. The donated electrons can then be further transferred to molecular oxygen adsorbed on the AuNP surface to generate highly reactive superoxo- or peroxo-like species.42 To investigate whether the pathway involved in this study is related to those suggested in the above studies, Raman spectra of the AuNPs dispersed in Tris and in NaCl were recorded and compared against each other (Figure S-5). For the AuNPs dispersed in Tris, peaks are present at 870 cm-1 and 1649 cm-1, which are likely attributed to vibrational modes of H2O2 and O2 respectively.43,44 This suggests that O2 adsorption to the AuNP surface and subsequent activation by some form of electron transfer is in fact a plausible pathway in our system. However, the results shown earlier in Figures 4B and 4D indicate that the presence of species with amino and hydroxyl groups is also important. Based on this information, it was hypothesized that ROS generation could occur by 1) oxidation of the amine- and hydroxyl-containing species on the AuNP surface, followed by 2) activation of adsorbed O2 by electron transfer through the AuNP, and finally 3) reduction of the activated O2 to form and release H2O2, in a manner resembling those of oxidase-type enzymes. Further investigation, for example using operando spectroscopic analyses, is necessary to elucidate the exact nature of the reaction(s) involved and their mechanisms. The presence and types of surface protecting groups on the AuNPs alter their surface properties and are thus expected to affect their catalytic activity. As mentioned earlier, in Tsunoyama et al.’s studies it has been posited that electron donation from the PVP protecting groups to the Au core is important to their high catalytic activity towards aerobic alcohol oxidation.42 They have also noted, however, that the protected Au nanoclusters used in their studies behave similarly to metalligand complexes with discrete energy levels, and that larger nanoparticles such as those used in the current study behave more closely to metals or semiconductors, where the state of the Au core becomes dominant over effects from the protecting groups. On the other hand, Wang et al. have studied the effect of protecting groups with different charges on the peroxidasemimetic activity of larger (10 – 30 nm) AuNPs, and observed that the protecting groups electrostatically affect the AuNPs’ activity by altering their affinity towards the substrate, rather than by direct electron donation or abstraction.45 Since the sizes of the AuNPs used in this study are closer in scale to those used by Wang et al., it reasonably follows that effects of the protecting groups will likely also be similar. Further studies using AuNPs with different capping groups will be necessary to Table 1. Comparison of turnover numbers of AuNPs and common H2O2-producing enzymes Enzyme Glucose oxidase46–48 Cholesterol oxidase Alcohol oxidase

49–51

52,53

AuNPs (this study) *

Substrate

Turnover number (s-1)

Glucose

0.03 ~ 2000

Cholesterol

0.001 ~ 350

Methanol

15 ~ 60

O2

~ 0.1

* Apparent turnover number per individual AuNP.


Page 5 of 8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

Figure 4. Molecular structures of Tris (1), Tricine (2), TAPS (3), trimethylolethane (4) and tert-butylamine (5) (A). Time change in ECL intensity of 0.2 mM luminol mixed with 15 nm AuNPs (0.04 mg/mL) dispersed in various solutions (including 1, 2 and 3) (B). Time change in ECL intensity of 0.2 mM luminol mixed with 15 nm AuNPs (0.04 mg/mL) dispersed in 1, 4, 5, and an equimolar mixture of 4 and 5 (all concentrations 1 mM) (C).

ascertain how exactly the groups will affect the AuNPs’ reaction with molecules such as Tris. Additionally, the data obtained in these experiments, along with ECL responses of luminol mixed with known concentrations of H2O2 (data not shown), were used to estimate the apparent turnover number of the 5 nm AuNPs. This figure was then compared with those of several other peroxideproducing enzymes, as summarized in Table 1. As can be seen in the table, while the AuNPs’ catalytic activity towards H2O2 production is not exceptionally high, it is still comparable to those of some of these enzymes. It is possible that optimization of experimental conditions could lead to further improvements in this figure. Enzyme-free ECL immunoassay for IgA. The applicability of AuNP-catalyzed ROS generation to ECL-based immunoassays was studied by conducting sandwich immunoassays for IgA (0-100 µg/mL) using anti-IgAconjugated magnetic beads and 5nm AuNPs, followed by ECL measurements of the collected AuNPs with IgA bound to them. As seen in Figure 6A, the ECL intensity increased with the IgA concentration. In addition, the immunoassay displayed a linear response over a wide range of 1 ng/mL to 10 µg/mL, with the lower limit comparable to those of commercial kits for the same target. In addition, the selectivity of this immunoassay was tested by measuring the output ECL intensities from samples containing IgA, C reactive protein (CRP, another protein present in blood), and a mixture of IgA and CRP (Figure 6B).

As shown in the figure, the sample containing only CRP showed signals comparable to the blank sample (without any proteins). On the other hand, the samples containing IgA and the mixture of IgA and CRP showed ECL intensities that were comparable with each other and significantly higher compared to that of the Table 2. Comparison of proposed immunoassay with other ECL immunoassays utilizing AuNPs Target

Role of AuNPs

IgA (this study)

Enhancement of luminol ECL through catalytic ROS generation

1 ng/mL ~ 10 µg/mL

Carcinoembryonic antigen (CEA)54

Enhancement of ECL of CeO2 nanoparticles

0.05 ~ 100 ng/mL

β-trophin55

Catalysis of luminol oxidation

0.5 ng/mL ~ 1.1 µg/mL

Prostate specific antigen (PSA)56

Electron transfer enhancement

1 pg/mL ~ 10 ng/mL

Acceleration of radical formation necessary for ECL of CdS quantum dots

0.2 ~ ng/mL

Morphine

57


Detection range

180


Figure 5. ECL intensities of 0.2 mM luminol mixed with 15 nm AuNPs (0.04 mg/mL) dispersed in 100 mM Tris adjusted to various pH (6~12) and left for 10 minutes (A). Time change in ECL intensity of 0.2 mM luminol mixed with 15 nm AuNPs (0.04 mg/mL) dispersed in 200 mM Tris-HCl (pH 8) with and without continuous nitrogen gas sparging (B). Time change in ECL intensity of 0.2 mM luminol mixed with 15 nm AuNPs (0.04 mg/mL) dispersed in 200 mM Tris-HCl (pH 8) after being coated with different concentrations of BSA (C).

blank sample, which indicate the selectivity of the immunoassay towards IgA as well as a lack of interference from CRP. These results point to the potential applicability of this immunoassay for various biosensing applications. The analytical performance of the immunoassay proposed in this study was compared with those of other ECL-based immunoassays that utilize AuNPs in some fashion (Table 2). As summarized in the table, while the detection range of the immunoassay proposed here is lacking compared to those in other studies, it is unique in that the AuNPs here enhance the ECL signal by the enzyme mimetic generation of the coreactant species, and not through indirect means such as the enhancement of electron transfer. A possible method to further improve the sensitivity of this assay would be to use electrodes that have a large number of miniscule chambers to each isolate small numbers of MB/antigen/AuNP sandwich conjugates. Assuming that luminol and the substrates from which ROS are produced are present in excess in the chambers, confining the AuNPs in smaller volumes should result in an increase in the luminescence per AuNP present proportional to the decrease in volume. In the future, this type of signal enhancement strategy could possibly be applied to develop digital assays that can, for instance, detect catalytic activity from single AuNPs. Digital assays are analytical techniques that have been implemented in other methods such as the enzyme-linked immunosorbent assay (ELISA)58,59 and polymerase chain reaction (PCR).60,61 In this

approach, target molecules, probes and other necessary reagents are confined in and react in numerous compartments of minute size, much like the method described earlier with microchamber electrodes, and the presence or absence of the target in a given compartment results in a discrete “on” or “off” signal from the compartment. At such low volumes and concentrations, the percentage of compartments that will return “on” signals follows a Poisson distribution. Reading out and counting the “on” signals then allows for absolute quantitation of extremely small numbers of the target molecules without the need for calibration curves or additional calibrants. In a related study, our group has previously reported on using MBs modified with catalase, an enzyme that digests H2O2, along with microchamber SPEs and imaging of ECL to achieve highly sensitive detection of the enzyme.62 While part of the efficacy of this approach lies in the fact that catalase is an extremely efficient enzyme with a turnover rate of approximately 107 molecules s-1, 63 it is quite possible that employment of a similar strategy with AuNPs along with further optimization of conditions for efficient catalysis of ROS generation and sufficient incubation times could allow for similarly sensitive ECL-based digital detection of AuNPs and of biomolecules using AuNPs as the ECL probe.

CONCLUSIONS In this work, the AuNP-catalyzed in situ generation of ROS was studied using ECL and applied to an immunoassay for the


Page 6 of 8

Page 7 of 8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry Corresponding Author * Phone: +81-6-6879-4087 E-mail: [email protected].

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by KAKENHI Grant-in-Aid for Scientific Research(S) Grant 15H05769 and CREST, JST. In addition, the authors would like to thank H. Yamashita from the Department of Materials and Manufacturing Science at the Graduate School of Engineering, Osaka University, for his discussions and insight in the hypothesis of the catalysis mechanism.

REFERENCES (1) (2) (3) (4) (5) (6) (7) (8) (9)

Figure 6. Detection results for IgA using proposed ECL-based immunoassay with MBs and AuNPs (A). Selectivity of proposed immunoassay (B).

detection of IgA. It was found that ROS was generated only from AuNPs dispersed in solutions that contain species with both hydroxyl and amino groups. The obtained results indicate that ROS generation at the AuNP surface is enabled by the binding of such species to the AuNP surface, and that altering the type and concentration of these species, among other factors, can regulate ROS generation. Furthermore, ECL immunoassays for IgA using this phenomenon showed that the luminescence intensity increased with increasing IgA concentration in the range of 1 ng/mL ~ 10 µg/mL. It is expected that further improvement of this immunoassay could enable its use in other biosensing applications.

(10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22)

ASSOCIATED CONTENT

(23)

Supporting Information

(24) (25)

The Supporting Information is available free of charge on the ACS Publications website. Detailed experimental procedures; UV-vis absorption spectra of H2O2 and of supernatant obtained after mixing AuNPs with Tris and centrifuging the mixture; Amplite™ Red fluorescent reporter assay results for various solutions, AuNPs, and AuNPs mixed with solutions; Raman spectra of AuNPs mixed with NaCl and with Tris (PDF)

AUTHOR INFORMATION

(26) (27) (28) (29) (30)

Richter, M. M. Chem. Rev. 2004, 104, 3003–3036. Nagatani, N.; Inoue, Y.; Araki, A.; Ushijima, H.; Hattori, G.; Sakurai, Y.; Ogidou, Y.; Saito, M.; Tamiya, E. Electrochim. Acta 2016, 222, 580–586. Inoue, Y.; Inoue, M.; Saito, M.; Yoshikawa, H.; Tamiya, E. Anal. Chem. 2017, 89, 5909–5915. Ambrosi, A.; Castañeda, M. T.; Killard, A. J.; Smyth, M. R.; Alegret, S.; Merkoçi, A. Anal. Chem. 2007, 79, 5232–5240. Campuzano, S.; de Ávila, B. E. F.; Yuste, J.; Pedrero, M.; García, J. L.; García, P.; García, E.; Pingarrón, J. M. Biosens. Bioelectron. 2010, 26, 1225–1230. S. Piermarini, G. Volpe, L. Micheli, D. Moscone, G. P. Food Control 2009, 20, 371–375. Biscay, J.; García, M. B. G.; García, A. C. Sensors Actuators, B Chem. 2014, 205, 426–432. Tsaloglou, M. N.; Jacobs, A.; Morgan, H. Anal. Bioanal. Chem. 2014, 406, 5967–5976. Zhang, X.; Zhang, B.; Miao, W.; Zou, G. Anal. Chem. 2016, 88, 5482–5488. Haruta, M.; Kobayashi, T.; Sano, H.; Yamada, N. Chem. Lett. 1987, 16, 405–408. Hutchings, G. J. J. Catal. 1985, 96, 292–295. Miedziak, P.; Sankar, M.; Dimitratos, N.; Lopez-Sanchez, J. A.; Carley, A. F.; Knight, D. W.; Taylor, S. H.; Kiely, C. J.; Hutchings, G. J. Catal. Today 2011, 164, 315–319. Comotti, M.; Della Pina, C.; Matarrese, R.; Rossi, M. Angew. Chemie - Int. Ed. 2004, 43, 5812–5815. Pradhan, N.; Pal, A.; Pal, T. Langmuir 2001, 17, 1800–1802. Gangula, A.; Podila, R.; M, R.; Karanam, L.; Janardhana, C.; Rao, A. M. Langmuir 2011, 27, 15268–15274. Yin, H.; Tang, H.; Wang, D.; Gao, Y.; Tang, Z. ACS Nano 2012, 6, 8288–8297. Xu, S.; Yong, L.; Wu, P. ACS Appl. Mater. Interfaces 2013, 5, 654–662. Wang, F.-B.; Wang, J.; Shao, L.; Zhao, Y.; Xia, X.-H. Electrochem. commun. 2014, 38, 82–85. Govindhan, M.; Chen, A. J. Power Sources 2015, 274, 928–936. Valden, M. Science (80-. ). 1998, 281, 1647–1650. Mason, M. Phys. Rev. B 1983, 27, 748–762. Remediakis, I. N.; Lopez, N.; Nørskov, J. K. Angew. Chemie Int. Ed. 2005, 44, 1824–1826. Swanick, K. N.; Hesari, M.; Workentin, M. S.; Ding, Z. J. Am. Chem. Soc. 2012, 134, 15205–15208. Hesari, M.; Ding, Z. Acc. Chem. Res. 2017, 50, 218–230. Hesari, M.; Workentin, M. S.; Ding, Z. ACS Nano 2014, 8, 8543–8553. Niu, H.; Yuan, R.; Chai, Y.; Mao, L.; Yuan, Y.; Zhuo, Y.; Yuan, S.; Yang, X. Biosens. Bioelectron. 2011, 26, 3175–3180. Jie, G.; Li, L.; Chen, C.; Xuan, J.; Zhu, J. J. Biosens. Bioelectron. 2009, 24, 3352–3358. Mao, L.; Yuan, R.; Chai, Y.; Zhuo, Y.; Xiang, Y. Biosens. Bioelectron. 2011, 26, 4204–4208. Ismail, N. S.; Le, Q. H.; Hasan, Q.; Yoshikawa, H.; Saito, M.; Tamiya, E. Electrochim. Acta 2015, 180, 409–418. Ismail, N. S.; Hoa, L. Q.; Huong, V. T.; Inoue, Y.; Yoshikawa, H.; Saito, M.; Tamiya, E. Electroanalysis 2017, 29, 938–943.


Analytical Chemistry (31)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(32) (33) (34) (35) (36) (37) (38) (39) (40) (41) (42) (43) (44) (45) (46) (47) (48) (49) (50) (51)

Huang, X.; Li, Y.; Huang, X.; Xie, X.; Xu, Y.; Chen, Y.; Gao, W. Anal. Bioanal. Chem. 2016, 408, 619–627. Ferreira, S. M.; Lerner, S. F.; Brunzini, R.; Evelson, P. A.; Llesuy, S. F. Am. J. Ophthalmol. 2004, 137, 62–69. Ueno, S.; Iwasaka, M. J. Appl. Phys. 1996, 79, 4705–4707. Hartadi, Y.; Widmann, D.; Behm, R. J. ChemSusChem 2015, 8, 456–465. Liu, Y.; Tsunoyama, H.; Akita, T.; Xie, S.; Tsukuda, T. ACS Catal. 2011, 1, 2–6. Liao, H.; Liu, G.; Liu, Y.; Li, R.; Fu, W.; Hu, L. Chem. Commun. 2017, 53, 10160–10163. Gao, Q.; Xing, Y.; Peng, M.; Liu, Y.; Luo, Z.; Jin, Y.; Fan, H.; Li, K.; Chen, C.; Cui, Y. Chinese J. Chem. 2017, 35, 1431– 1436. Gutiérrez, L. F.; Hamoudi, S.; Belkacemi, K. Appl. Catal. A Gen. 2012, 425–426, 213–223. Yin, H.; Zhou, C.; Xu, C.; Liu, P.; Xu, X.; Ding, Y. J. Phys. Chem. C 2008, 112, 9673–9678. El-Sayed, R.; Ye, F.; Asem, H.; Ashour, R.; Zheng, W.; Muhammed, M.; Hassan, M. Biochem. Biophys. Res. Commun. 2017, 491, 15–18. Chang, C. R.; Huang, Z. Q.; Li, J. Nano Res. 2015, 8, 3737– 3748. Tsunoyama, H.; Ichikuni, N.; Sakurai, H.; Tsukuda, T. J. Am. Chem. Soc. 2009, 131, 7086–7093. Li, X.; Gewirth, A. A. J. Am. Chem. Soc. 2003, 125, 7086–7099. Kong, X.; Sun, Z.; Chen, Q. Phys. Chem. Chem. Phys. 2012, 14, 13564. Wang, S.; Chen, W.; Liu, A. L.; Hong, L.; Deng, H. H.; Lin, X. H. ChemPhysChem 2012, 13, 1199–1204. Witt, S.; Wohlfahrt, G.; Schomburg, D.; Hecht, H. J.; Kalisz, H. M. Biochem. J. 2000, 347, 553–559. Holland, J. T.; Lau, C.; Brozik, S.; Atanassov, P.; Banta, S. J. Am. Chem. Soc. 2011, 133, 19262–19265. Witt, S.; Singh, M.; Kalisz, H. M. Appl. Environ. Microbiol. 1998, 64, 1405–1411. Yin, Y.; Liu, P.; Anderson, R. G. W.; Sampson, N. S. Arch. Biochem. Biophys. 2002, 402, 235–242. Chen, L.; Lyubimov, A. Y.; Brammer, L.; Vrielink, A.; Sampson, N. S. Biochemistry 2008, 47, 5368–5377. Pollegioni, L.; Wels, G.; Pilone, M. S.; Ghisla, S. Eur J Biochem 1999, 264, 140–151.

(52) (53) (54) (55) (56) (57) (58)

(59)

(60)

(61) (62) (63)

Page 8 of 8 Daniel, G.; Volc, J.; Filonova, L.; Plíhal, O.; Kubátová, E.; Halada, P. Appl. Environ. Microbiol. 2007, 73, 6241–6253. Shleev, S. V; Shumakovich, G. P.; Nikitina, O. V; Morozova, O. V; Pavlishko, H. M.; Gayda, G. Z.; Gonchar, M. V. Biochemistry. (Mosc). 2006, 71, 245–250. Pang, X.; Li, J.; Zhao, Y.; Wu, D.; Zhang, Y.; Du, B.; Ma, H.; Wei, Q. ACS Appl. Mater. Interfaces 2015, 7, 19260–19267. Zheng, L.; Fang, C.; Yan, J.; Li, H.; Tu, Y. Sci. Rep. 2017, 7, 1– 9. Zhao, Y.; Wang, Q.; Li, J.; Ma, H.; Zhang, Y.; Wu, D. J. Mater. Chem. B 2016, 4, 2963–2971. Fei, W.; Chen, F.; Sun, L.; Li, Q.; Yang, J.; Wu, Y. Microchim. Acta 2014, 181, 419–425. Rissin, D. M.; Kan, C. W.; Campbell, T. G.; Howes, S. C.; Fournier, D. R.; Song, L.; Piech, T.; Patel, P. P.; Chang, L.; Rivnak, A. J.; Ferrell, E. P.; Randall, J. D.; Provuncher, G. K.; Walt, D. R.; Duffy, D. C. Nat. Biotechnol. 2010, 28, 595–599. Rissin, D. M.; Fournier, D. R.; Piech, T.; Kan, C. W.; Campbell, T. G.; Song, L.; Chang, L.; Rivnak, A. J.; Patel, P. P.; Provuncher, G. K.; Ferrell, E. P.; Howes, S. C.; Pink, B. A.; Minnehan, K. A.; Wilson, D. H.; Duffy, D. C. Anal. Chem. 2011, 83, 2279–2285. Hindson, B. J.; Ness, K. D.; Masquelier, D. A.; Belgrader, P.; Heredia, N. J.; Makarewicz, A. J.; Bright, I. J.; Lucero, M. Y.; Hiddessen, A. L.; Legler, T. C.; Kitano, T. K.; Hodel, M. R.; Petersen, J. F.; Wyatt, P. W.; Steenblock, E. R.; Shah, P. H.; Bousse, L. J.; Troup, C. B.; Mellen, J. C.; Wittmann, D. K.; Erndt, N. G.; Cauley, T. H.; Koehler, R. T.; So, A. P.; Dube, S.; Rose, K. A.; Montesclaros, L.; Wang, S.; Stumbo, D. P.; Hodges, S. P.; Romine, S.; Milanovich, F. P.; White, H. E.; Regan, J. F.; Karlin-Neumann, G. A.; Hindson, C. M.; Saxonov, S.; Colston, B. W. Anal. Chem. 2011, 83, 8604–8610. Pinheiro, L. B.; Coleman, V. A.; Hindson, C. M.; Herrmann, J.; Hindson, B. J.; Bhat, S.; Emslie, K. R. Anal. Chem. 2012, 84, 1003–1011. Inoue, Y.; Saito, M.; Yoshikawa, H.; Tamiya, E. Electrochim. Acta 2017, 240, 447–455. Augusto Vicente Seixas, F.; Ribeiro da Silva, M.; Tyago Murakami, M.; Tosqui, P.; Francisco Colombo, M. Protein Pept. Lett. 2011, 18, 879–885.

For TOC only


Chemically Regulated ROS Generation from Gold Nanoparticles for

Recommend Documents