Assembly of multifunctionalized gold nanoparticles with

Subscriber access provided by UNIVERSITY OF THE SUNSHINE COAST

Biological and Medical Applications of Materials and Interfaces

Assembly of multifunctionalized gold nanoparticles with chemiluminescent, catalytic and immune activity for label-free immunoassays Yao Huang, Lingfeng Gao, and Hua Cui ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b02521 • Publication Date (Web): 04 May 2018 Downloaded from http://pubs.acs.org on May 5, 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 25 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

ACS Applied Materials & Interfaces

Assembly of multifunctionalized gold nanoparticles with chemiluminescent, catalytic and immune activity for label-free immunoassays Yao Huang, Lingfeng Gao and Hua Cui* CAS Key Laboratory of Soft Matter Chemistry, iChEM (Collaborative Innovation Center of Chemistry for Energy Materials), Department of Chemistry, University of Science and Technology of China, Hefei, Anhui 230026, P.R. China

ACS Paragon Plus Environment

1

ACS Applied Materials & Interfaces 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

Page 2 of 25

ABSTRACT

In this study, we report a universal label-free immunoassay to detect antigen based on multifunctionalized gold nanoparticles (MF-GNPs), which were obtained by successive assembly of N-aminobutyl-N-ethylisoluminol functionalized gold nanoparticles (ABEI-GNPs) with antibody, bovine serum albumin (BSA) and Co2+. MF-GNPs exhibited excellent chemiluminescent (CL), catalytic and immune activity. It was demonstrated that the CL signal of MF-GNPs decreased in the presence of antigens via antigen-antibody specific binding using human immunoglobulin G (hIgG) and corresponding antibody goat-anti-human IgG (anti-hIgG) as a model system, due to that immunoreaction led to the aggregation of GNPs. According to the decreased CL intensity, hIgG could be determined in the range of 1.0 fM to 1.0 nM with a low detection limit of 0.13 fM. Furthermore, this CL strategy was also confirmed to be a general one by replacing hIgG with heart-type fatty acid-binding protein (H-FABP), which is a biomarker of early Acute Myocardial infarction (AMI). The CL strategy could be employed to detect H-FABP ranging from 10.0 fM to 10.0 nM, and the detection limit is 7.8 fM. The CL strategy also showed good selectivity. It might be extended to detect other antigens if their corresponding antibodies are available.

KEYWORDS: gold nanoparticles, multifunctionalization, chemiluminescence, label-free, immunoassay


2

Page 3 of 25 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


1. INTRODUCTION Gold nanoparticles (GNPs) have attracted great interests in bioassays due to their unique physical and chemical properties, excellent stability, ease of functionalization, and good biocompatibility.1-3 Chemiluminescence is a highly sensitive detection technology for bioassays. Recently, much attention has been paid to chemiluminescent (CL) functionalized nanomaterials, especially gold nanoparticles, in bioassays due to their good CL property, ease of self-assembly and good biocompatibility.4-7 At the beginning, luminol-type CL reagent functionalized gold nanoparticles (CF-GNPs) were synthesized using direct reduction methods.8-9 CL reagent molecules were used to reduce HAuCl4 to obtain GNPs and acted as stabilizers to be coated on the surfaces of GNPs. CF-GNPs with good CL activities as analytical probes and interfaces have been successfully applied to immunoassays and DNA assays.10-12 Afterwards, in order to develop CF-GNPs with high CL efficiency, catalysts metal ions were further coated onto the surfaces of GNPs with the help of chelators to form gold nanoparticles bifunctionalized by CL molecules and metal complexes (BF-GNPs) with excellent CL efficiency.13-14 However, it is very challengeable to connect BF-GNPs with recognition elements like antibody, aptamer and DNA owing to the high surface coverage fraction of BF-GNPs, limiting their applications in bioassays. Therefore, we wonder if it is possible to directly assemble catalysts metal ions onto the surfaces of antibody conjugated CF-GNPs to obtain an ideal analytical interface with high CL efficiency during the fabrication of immunosensors by virtue of blocker bovine serum albumin (BSA), since BSA contains amino and carboxyl groups and is ready to coordinate with metal ions, avoiding the use of additional chelators. Herein, a general assembly strategy for multifunctionalized gold nanoparticles (MF-GNPs) with CL, catalytic and immune activity was developed, which could be directly used for CL


3


Page 4 of 25

immumoassays. Antibody was firstly grafted on the surfaces of N-aminobutyl-N-ethylisoluminol functionalized gold nanoparticles (ABEI-GNPs), and then BSA was used to block the non-specific

adsorption

of

proteins

on

the

surfaces

of

GNPs

to

form

BSA/anti-hIgG/ABEI-GNPs. Finally, Co2+ was further coated onto BSA/anti-hIgG/ABEI-GNPs through the coordination of the amino and carboxyl groups of BSA to form MF-GNPs, which exhibited excellent CL and immune activities. Moreover, the CL signal of MF-GNPs decreased with increasing antigen concentration due to antigen-antibody specific binding. The CL inhibition mechanism was explored. Based on the decreased CL intensity, using human immunoglobulin G (hIgG) as a model target, a label-free CL immunosensor with high sensitivity and selectivity was established for antigen detection. Experimental parameters were optimized and analytical performances were investigated. Finally, the generalization of this strategy for other antigens was also explored by determining the early Acute Myocardial infarction (AMI) biomarker, heart-type fatty acid-binding protein (H-FABP). 2. EXPERIMENTAL PROCEDURES. 2.1. Synthesis of Co2+-BSA/anti-hIgG/ABEI-GNPs nanocomposites. Firstly, ABEI-GNPs were synthesized by reducing HAuCl4 with ABEI through the seed-growth method9 and stored at 4 ℃. Then 25 µL anti-hIgG (1.0 mg/mL) was introduced to 1.0 mL ABEI-GNPs colloid. The mixed solution was incubated at 27 ℃ for 30 min to obtain anti-hIgG/ABEI-GNPs. 250 µL BSA solution (5% w/w) was subsequently added to a final concentration of 1% by vigorously stirring for 5 min. Next, 100 µL of 1 mM cobalt chloride solution was added by stirring for another 5 min. In order to remove the unreacted reagents, the colloidal solution was centrifuged to obtain Co2+-BSA/anti-hIgG/ABEI-GNPs nanocomposites.


4

Page 5 of 25 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


The red precipitates were resuspended in phosphate-buffered saline (PBS) (20 mM, pH = 7.4) with 1% (w/w) BSA and stored at 4 ℃. 2.2. Characterization of Co2+-BSA/anti-hIgG/ABEI-GNPs nanocomposites. The morphologies of ABEI-GNPs and Co2+-BSA/anti-hIgG/ABEI-GNPs nanocomposites were characterized by high resolution transmission electron microscope (HRTEM, JEM-2100F, Hitachi, Japan). Circular dichroism (CD) spectra was taken to confirm the coating of anti-hIgG and BSA onto ABEI-GNPs by a J-1500 CD Spectrometer (Jasco, Japan). The conjugation of ABEI-GNPs with anti-hIgG and BSA was also confirmed by agarose gel electrophoresis on migration properties. The as-obtained nanocomposites were loaded on a 0.5% agarose gel and ran in PBS (20 mM, pH = 7.4) at 6 V/cm for 20 min. The agarose gels featured red-colored electrophoresis bands benefiting from the intense surface-plasmon resonance at ∼520 nm of GNPs. Agarose gels were transferred onto a white light transilluminator and digital photos were taken on a D7200 Nikon. Co content in Co2+-BSA/anti-hIgG/ABEI-GNPs nanocomposites was determined by inductively coupled plasma atomic emission spectrometry (ICP-AES) on an Optima 7300 DV plasma atomic emission spectrometry (PerkinElmer, USA). The Zeta potentials of the nanocomposites were measured on a 90 Plus PALS Zeta Potential Analyzer (Brookhaven, USA). 2.3. CL Detection of Antigen. Typically, 100 µL Co2+-BSA/anti-hIgG/ABEI-GNPs nanocomposites solution and 100 µL antigen in PBS (20 mM, pH = 7.4) were mixed in a microwell and incubated at 37 ℃ for one hour. The temperature of incubations was controlled by a MB 100-4A Thermo Shaker Incubator


5


Page 6 of 25

(Allsheng, China). After that, when 100 µL 10 mM H2O2 (pH=13.0) was injected into each well, the CL signal was generated and recorded. A Centro LB 960 microplate luminometer (Berthold, Germany) was utilized to measure the CL intensity. An F-7000 FL spectrometer (Hitachi, Japan) was employed to measure the CL spectrum when operated with the lamp off. 3. RESULTS AND DISCUSSION 3.1. Assembly and Characterization of Co2+-BSA/anti-hIgG/ABEI-GNPs nanocomposites. Figure 1 schematically depicted the proposed strategy for the assembly of MF-GNPs. Taking hIgG as a model of antigen, corresponding antibody anti-hIgG was used as the recognition element. Initially, ABEI-GNPs were prepared by using ABEI to reduce HAuCl4 through the seed growth method as previously described.9 Then anti-hIgG was attached to ABEI-GNPs to obtain anti-hIgG/ABEI-GNPs via electrostatic, hydrophobic and weak covalent interactions.15 BSA was used to block nonspecific bindings of proteins on the surfaces of GNPs to form BSA/anti-hIgG/ABEI-GNPs. Finally, catalyst Co2+ was mixed with BSA/anti-hIgG/ABEI-GNPs and assembled on the surfaces to obtain Co2+-BSA/anti-hIgG/ABEI-GNPs.

Figure 1. A schematic for fabrication of Co2+-BSA/anti-hIgG/ABEI-GNPs nanocomposites.


6

Page 7 of 25 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


The morphologies of ABEI-GNPs and Co2+-BSA/anti-hIgG/ABEI-GNPs nanocomposites were characterized by HRTEM (Figure 2). ABEI-GNPs (Figure 2a) showed assembled chain structure of sphere GNPs with average diameter of 14.3 nm, which was due to hydrogen bonding between the amide groups in ABEI molecules and the carboxyl groups in the oxidized molecules of ABEI on adjacent GNPs.9 Co2+-BSA/anti-hIgG/ABEI-GNPs nanocomposites (Figure 2b) demonstrated monodisperse sphericity with an average diameter of 14.1 nm. The diameter of GNPs before and after coating with proteins and metal ions remained identical but the morphology obviously changed. The change in morphology from assembled chain of sphere ABEI-GNPs to monodisperse sphericity of Co2+-BSA/anti-hIgG/ABEI-GNPs might be due to that the coating of proteins anti-hIgG and BSA destroyed the hydrogen bonding of adjacent GNPs and increased the distance of interparticles.

Figure 2. TEM of (a) ABEI-GNPs and (b) Co2+-BSA/anti-hIgG/ABEI-GNPs nanocomposites. The coating of anti-hIgG, BSA and Co2+ onto ABEI-GNPs was confirmed by using CD spectroscopy and ICP-AES. As shown in Figure 3, anti-hIgG and BSA had absorption bands in CD spectrum because biomacromolecules were generally chiral. ABEI-GNPs were nanomaterials without chirality and no absorption band was observed in CD spectrum. When anti-hIgG and BSA were successively coated onto ABEI-GNPs, corresponding absorption bands


7


Page 8 of 25

were observed, demonstrating that anti-hIgG and BSA were successfully assembled onto the surfaces of ABEI-GNPs. ICP-AES analysis demonstrated the existence of Co2+ in Co2+-BSA/anti-hIgG/ABEI-GNPs

nanocomposites.

Co2+

concentration

on

Co2+-BSA/anti-hIgG/ABEI-GNPs was calculated to be 3.35×10-6 mol/L and 4.63% Co2+ added was grafted onto the nanocomposites.

Figure 3. CD spectra of (a) ABEI-GNPs, anti-hIgG, anti-hIgG/ABEI-GNPs and (b) ABEI-GNPs, BSA, BSA/anti-hIgG/ABEI-GNPs, Co2+-BSA/anti-hIgG/ABEI-GNPs. Zeta potentials of nanocomposites at different assembly stages were measured as shown in Table S1. The zeta potential of ABEI-GNPs showed a net negative charge of -14.0 mV. The pI value of anti-hIgG and BSA was 5.5 and 4.7, respectively. Both of them were negatively charged in PBS (pH = 7.4) solution. When anti-hIgG was conjugated to ABEI-GNPs, the zeta potential of anti-hIgG/ABEI-GNPs was negatively shifted by 3.9 mV from -14.0 to -17.9 mV. Likewise, when

BSA

was

conjugated

with

anti-hIgG/ABEI-GNPs,

the

zeta

potential

of

BSA/anti-hIgG/ABEI-GNPs was further negatively shifted by 3.8 mV from -17.9 to -21.7 mV. Finally, when Co2+ was grafted onto BSA/anti-hIgG/ABEI-GNPs, the zeta potential of Co2+-BSA/anti-hIgG/ABEI-GNPs was positively shifted from -21.7 to -19.4 mV. The changes in


8

Page 9 of 25 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


zeta potential after the successive assembly of anti-hIgG, BSA and Co2+ were reasonable, implying successful assembly of these substances. Furthermore, agarose gel electrophoresis was employed to verify the conjugation of anti-hIgG and BSA with ABEI-GNPs on migration properties. The relative mobility of different nanoparticles in gel electrophoresis depended on both the size and the net charge. As shown in Figure 4, most of ABEI-GNPs aggregated and a few ABEI-GNPs with small size migrated towards the anode, which may be due to low charge of ABEI-GNPs. When anti-hIgG was conjugated to ABEI-GNPs, the formed anti-hIgG/ABEI-GNPs became purple due to the increase in distance of interparticles. A few anti-hIgG/ABEI-GNPs migrated towards the anode despite of the increased negative charge, which may be due to lower concentration of anti-hIgG. BSA/anti-hIgG/ABEI-GNPs moved more slowly and migrate closer than anti-hIgG/ABEI-GNPs owing to the larger size. However, a lot of BSA/anti-hIgG/ABEI-GNPs migrated due to high concentration of BSA. When Co2+ was assembled to BSA/anti-hIgG/ABEI-GNPs, the nanocomposites in agarose gel electrophoresis moved more slowly due to the reduced negative charge. Furthermore, anti-hIgG and BSA resulted in relatively stable and well-defined bands in the gel electrophoresis. This phenomenon may be due to the stabilization of anti-hIgG and BSA on GNPs, which maintained high negative surface charge of GNPs, increased the distance between GNPs and prevented GNPs from aggregating during gel electrophoresis.


9


Page 10 of 25

Figure 4. Agarose gel electrophoresis of different nanocomposites. Lane 1-4 correspond to ABEI-GNPs,

anti-hIgG/ABEI-GNPs,

BSA/anti-hIgG/ABEI-GNPs,

Co2+-BSA/anti-hIgG/ABEI-GNPs, respectively. Reaction conditions: nanocomposites are centrifuged and concentrated in 100 µL PBS (20 mM, pH = 7.4). 3.2. CL behavior of nanocomposites at different assembly stages The CL behavior of nanocomposites at different assembly stages was studied. All the nanocomposites were centrifuged and resuspended in PBS solution (20 mM, pH = 7.4) in order to remove the unconjugated reactants in the liquid phase. As shown in Figure 5, no CL emission was observed in blank (curve a) and the nanocomposites including ABEI-GNPs, anti-hIgG/ABEI-GNPs, BSA/anti-hIgG/ABEI-GNPs (curve b-d) demonstrated weak CL emission. In contrast, Co2+-BSA/anti-hIgG/ABEI-GNPs showed excellent CL activity (curve e). Moreover, the CL spectrum of Co2+-BSA/ABEI-GNPs-H2O2 reaction had an emission peak at 433 nm (Figure 5 inset), which indicated that the CL was from the CL reaction of ABEI with H2O2.


10

Page 11 of 25 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


Compared with ABEI-GNPs, anti-hIgG/ABEI-GNPs nanocomposites did not show significant change

in

CL

intensity,

whereas

BSA/anti-hIgG/ABEI-GNPs

and

Co2+/BSA/anti-hIgG/ABEI-GNPs (curves d-e) was almost 6 and 300 times the CL intensity of ABEI-GNPs, respectively. Moreover, Co2+/BSA/anti-hIgG/ABEI-GNPs even showed better emission performance compared with DTDTPA/Co2+-ABEI-GNPs with additional chelator DTDTPA (Figure S4), which was bifunctionalized gold nanomaterials with the highest CL intensity previously reported by our group. Co2+/BSA/anti-hIgG/ABEI-GNPs exhibited excellent intensity because Co2+ could catalyze the decomposition of H2O2, facilitating the formation of OH●- and O2●-, accelerating the CL reaction.13 As was reported, proteins could enhance CL because proteins with –COO- reacted with O2●-, producing –CO4●2-, which further accelerated the CL reaction.12 BSA had much stronger enhancement than anti-hIgG because the concentration of BSA was an order of magnitude higher than that of anti-hIgG. According to our earlier studies, gold nanoparticles could not only immobilize a large number of CL molecules for signal amplification, but also promote radical generation and electron transfer in CL reactions.13 Most importantly, the decomposition of H2O2 catalyzed by Co2+ could be greatly enhanced by complexation and heterogenization of Co2+, leading to strong light emission.7 Accordingly, the excellent CL efficiency of Co2+-BSA/anti-hIgG/ABEI-GNPs nanocomposites resulted from the synergetic effect of BSA, Co2+, and gold nanoparticles on the ABEI-H2O2 reaction.


11


Page 12 of 25

Figure 5. CL kinetic curves of (a) PBS, (b) ABEI-GNPs, (c) anti-hIgG/ABEI-GNPs, (d) BSA/anti-hIgG/ABEI-GNPs, (e) Co2+-BSA/anti-hIgG/ABEI-GNPs reacting with H2O2. Inset shows magnification of curves a-d and CL spectrum of Co2+/BSA/anti-hIgG/ABEI-GNPs-H2O2 reaction. Reaction conditions: (b-e) are centrifuged-dispersed, 100 µL 10 mM H2O2 (pH=13.0) was injected into 100 µL GNPs dispersion in a microwell, temperature: 16 ℃. 3.3. Optimization of experiment conditions. It is known that the CL reactions of luminol-type CL reagents with H2O2 can be catalyzed by various metals and Co2+ is the best one.7,13 The effects of Co2+ and other metal ions Cu2+, Hg2+, Cr3+, Ce3+, Mn2+, Cd2+, Fe2+, Ni2+, Pb2+ and Fe3+ on the CL intensity have been further studied and compared in Supplementary Figure S3. Although other metal ions showed enhancement on CL intensity to some degree, the CL intensity with Co2+ was 1-2 orders of magnitude higher than that with other tested metal ions. Therefore, Co2+ is the optimal metal ion for the MF-GNPs, which is consistent with that in literatures. The related reaction conditions, including the concentration and volume of BSA, anti-hIgG and ABEI-GNPs, were chosen according to earlier work.16-17 In this work, the effect of Co2+ concentration on synthesis of Co2+-BSA/anti-hIgG/ABEI-GNPs nanocomposites was optimized.


12

Page 13 of 25 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


As shown in Figure S1, Co2+-BSA/anti-hIgG/ABEI-GNPs synthesized with 5 mM Co2+ exhibited relatively high CL intensity. As shown in Figure S2, nanocomposites synthesized with 0.1 mM, 0.5 mM and 1 mM Co2+ were wine red, which were stable with good dispersibility. However, nanocomposites using higher Co2+ concentrations became purple and even aggregated. Considering aforementioned two factors, 1 mM Co2+ was eventually adopted. 3.4. CL response of Co2+-BSA/anti-hIgG/ABEI-GNPs to hIgG. As shown in Figure 6a, the CL response of Co2+-BSA/anti-hIgG/ABEI-GNPs to hIgG was explored. In the absence of hIgG, strong CL emission was observed. However, CL intensity decreased in the presence of hIgG. Compared with Figure 6b, TEM image (Figure 6c) demonstrated that specific binding between anti-hIgG and hIgG led to the aggregation of MF-GNPs and a subsequent decrease in CL intensity. It was reported that aggregated GNPs induced by immunoreaction showed the enhanced catalytic effect on luminol CL reactions when luminol was in a solution because of low activation energy for electron transfer, proper surface area, and high electron density and oscillation effect in the conduction bands.18 Other than the previously reported work, in this case, CL reagent ABEI was directly bonded onto GNPs by the Au–N covalent interaction. The intra- and interparticle quenching effects of GNPs on the fluorescence of the oxidation products of ABEI molecules on their surface were observed,19 which were relatively small when CL molecules were in a solution. The quenching effects were usually caused by the electron/energy transfer from the excited-state of FL molecules to the metal nanoparticles. Metal particles could cause an enhancement or quenching of the fluorescence depending on the distance between the fluorescence molecule and the metal NPs surface.20 The critical distance is usually around more than ten or even tens of nm. In this case, the aggregation of MF-GNPs decreased the distance of interparticles, which led to strong


13


Page 14 of 25

quenching effects on CL emission. The quenching effects dominated the CL reaction after immunoreaction.

Figure 6. (a) CL response of Co2+-BSA/anti-hIgG/ABEI-GNPs to hIgG. (b)-(c): TEM images of Co2+-BSA/anti-hIgG/ABEI-GNPs in absence and presence of hIgG. (d) Schematic diagram of the proposed label-free CL immunosensor. 3.5. Analytical Performance for Detecting hIgG. Based on the decreased CL signal, a label-free CL immunosensor for hIgG detection with high sensitivity and selectivity was established as presented in Figure 6d. The relative CL intensity ∆I was used for the quantitative analysis (∆I=I0 – I), in which I0 and I respectively represented the CL intensity of Co2+-BSA/anti-hIgG/ABEI-GNPs before and after adding hIgG. For the purpose of getting optimal analysis performance, the concentration of Co2+ and H2O2, and pH of H2O2 solution were optimized. As shown in Figure S5, the highest ∆I was obtained under the synthesis condition of 1 mM Co2+ and the detection conditions of 10 mM H2O2 (pH=13.0), which were


14

Page 15 of 25 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


chosen for further experiments. Under the optimized experiment conditions, the developed immunosensor was applied to hIgG detection and the working curve is presented in Figure 7a. The CL intensity decreased with the increase of hIgG concentration and the immunosensor exhibited a wide linear range from 1.0 fM to 1.0 nM. The regression equation was ∆I = 189206.0 + 11203.8 × logC (R2 = 0.994) and the detection limit (LOD) for hIgG was 0.13 fM (S/N = 3). The relative standard deviations (RSD) of seven replicate detections within a day (n=7) and in different days (n=7) were 3.30 % and 5.69 % respectively, which indicated good stability of the nanocomposites and good reproducibility of the proposed immunosensor. Moreover, the sensitivity of this immunosensor is superior to most existing label-free methods (Table 1). 15,21,22 Therefore, the proposed MF-GNPs could capture antigen effectively and respond to antigen in different levels, which exhibited excellent immune activity; whereas previously reported DTDTPA/Co2+-ABEI-GNPs were very difficult to connect with antibody because of the high coverage surface fraction of the BF-GNPs.


15


Page 16 of 25

Table 1. A comparison of existing label-free immunosensors for hIgG and H-FABP detection with the proposed label-free CL immunosensor

Methods

Materials

Detection limit (fM)

Ref.

ECLa

nano-Au-ERGOc

8.13

21

ABEI-GCd

0.31

15

ECL

P-GR-CdSee

0.031

22

CL

MF-GNPs

0.13

This work

Microscope

Double GNPs Probes and 4267 Protein Chip

25

MUA mSAMf electrode

26

Detection of ECL hIgG

Detection of EISb H-FABP CL

gold 780

MF-GNPs

7.8

This work

a

ECL, electrochemiluminescence. b EIS, electrochemical impedance spectroscopy.

c

ERGO, electrochemically reduced graphene oxide. d GC, graphene composite.

e

P-GR-CdSe, poly (diallyldimethylammonium chloride)-protected graphene-CdSe.

f

MUA mSAM, 11 mercaptoundecanoic acid mixed self-assembled monolayer.

To study the selectivity of the proposed label-free immunosensor, myoglobin (Mb), human serum albumin (HSA), and streptavidin (SA) that possibly interfere with the hIgG sensing, were used for immunoassays. As shown in Figure 7a, although the concentration of interfering species was 10-fold over that of hIgG, only hIgG exhibited strong CL signal and other proteins demonstrated very weak CL signals, revealing that the immunosensor could distinguish hIgG from other species investigated. These results demonstrated that the fabricated label-free CL immunosensor was highly specific towards hIgG.


16

Page 17 of 25 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 7. (a) Calibration curve for hIgG. The inset shows a comparison of CL signal ∆I of hIgG (0.1 nM) with those of other interferents (1.0 nM). (b) Calibration curve for H-FABP. The inset shows a comparison of CL signal ∆I of H-FABP (1.0 nM) with those of other interferents (10 nM). Reaction conditions: 100 µL 10 mM H2O2 (pH=13.0) was injected into 100 µL MF-GNPs dispersion mixed with 100 µL antigen in a microwell. The clinical applicability of the proposed immunoassay was investigated by detecting hIgG in real human serum samples. The samples were progressively diluted before the assay to be in the linear range of the proposed strategy. Table 2 shows that the recoveries were in the range of 95%–101%. Thus, the proposed label-free CL immunoassay was reliable and could be employed to detect hIgG in human serum samples.


17


Page 18 of 25

Table 2. hIgG detection in human serum samplesa measured by proposed immunoassay. Total hIgG hIgG found Samples

a

hIgG added

Recovery detected

(10-11 mol/L)

(10-11 mol/L)

1

4.3±0.1

10

14.4±0.3

101

2

5.2±0.2

10

15.0±0.4

98

3

10.9±0.3

10

20.4±0.7

95

(10-11 mol/L)

(%)

Mean Value ± SD of three independent experiments, n = 3.

3.6. Generalization of the CL Strategy to detect other antigens. With the view of investigating whether the CL strategy is a general and effective method for determining other antigens, antibody of H-FABP (anti-H-FABP) was used to fabricate MF-GNPs nanocomposites to detect H-FABP. Figure 7b shows the working curve for detecting H-FABP based on the proposed label-free CL immunosensor. The regression equation is ∆I = 22043.2 + 1458.5 × logC and linear range is from 10.0 fM to 10.0 nM. The limit detection is 7.8 fM. In healthy humans the normal range of H-FABP in serum was reported to vary between 0.0 and 0.37 nM and a value of 0.47 nM was used for an AMI cutoff.23-24 The LOD of this immunoassay is superior to existing label-free immunoassays for H-FABP detection (Table 1).25-26 Selectivity of this proposed immunoassay is also good. These results demonstrated that the CL sensing strategy could be used to detect H-FABP and may provide promising approaches to the determination of other antigens. 4. CONCLUSION In summary, a universal strategy for the assembly of MF-GNPs with CL, catalytic and immune activity for label-free immunoassays has been reported. The MF-GNPs were fabricated by


18

Page 19 of 25 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


successive assembly of ABEI-GNPs with antibody, BSA and Co2+. BSA was usually used as blocker to block the nonspecific bindings of proteins on the surfaces of nanomaterials. In this work, BSA was for the first time used as both of blocker and chelator, which could sequester and entrap Co2+ for signal amplification. The proposed MF-GNPs exhibited excellent CL and immune activity. It was demonstrated that the MF-GNPs could respond to antigens in different levels via antigen-antibody specific binding and showed decreased CL intensity with the increase of the antigen concentration, due to that immunoreaction led to the aggregation of GNPs. The decreased CL signal could be used for the detection of antigens including hIgG and H-FABP. Compared with existing methods, this label-free immunoassay strategy was reagentless, time-saving and avoided additional chelator and complicated assembly procedures, showing good sensitivity, selectivity, stability and repeatability. Also, these developed immunoassays presented a promising application potential in quantitative analysis of other disease-related proteins. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Additional information are as noted in text, including Reagents and Materials, Centrifugation conditions, Zeta potential of different nanocomposites (Table S1), Effect of Co2+ concentration on the CL intensity of Co2+-BSA/anti-hIgG/ABEI-GNPs nanocomposites (Figure S1), Images of Co2+-BSA/anti-hIgG/ABEI-GNPs nanocomposites using different Co2+ concentrations (Figure S2), CL kinetic curves of reactions of metal ion-BSA/anti-hIgG/ABEI-GNPs with H2O2 using different metal ions (Figure S3), CL kinetic curves of Co2+-BSA/anti-hIgG/ABEI-GNPs and DTDTPA/Co2+-ABEI-GNPs reacting with H2O2 (Figure S4), Effects of Co2+ concentration, H2O2 concentration and pH of H2O2 on CL signal of the immunosensor (Figure S5).


19


Page 20 of 25

AUTHOR INFORMATION Corresponding Author Tel: +86-551-63600730. Fax: +86-551-63600730. Email: [email protected]. ORCID Hua Cui: 0000-0003-4769-9464 Author Contributions All authors have contributed to the manuscript and approved the final version of the manuscript. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT The support of this research by the National Key Research and Development Program of China (Grant No. 2016YFA0201300) and the National Natural Science Foundation of China (Grant Nos. 21475120 and 21527807) are gratefully acknowledged. REFERENCES (1) Song, S. P.; Qin, Y.; He, Y.; Huang, Q.; Fan, C. H.; Chen, H. Y. Functional Nanoprobes for Ultrasensitive Detection of Biomolecules. Chem. Soc. Rev. 2010, 39 (11), 4234-4243.


20

Page 21 of 25 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


(2)

Peterle,

T.;

Ringler,

P.;

Mayor,

M.

Gold

Nanoparticles

Stabilized

by

Acetylene-Functionalized Multidentate Thioether Ligands: Building Blocks for Nanoparticle Superstructures. Adv. Funct. Mater. 2009, 19 (21), 3497-3506. (3) Bi, S.; Zhou, H.; Zhang, S. S. A Novel Synergistic Enhanced Chemiluminescence Achieved

by a Multiplex

Nanoprobe for Biological

Applications Combined with

Dual-Amplification of Magnetic Nanoparticles. Chem. Sci. 2010, 1 (6), 681-687. (4) Li, Q.; Zhang, L.; Li, J.; Lu, C. Nanomaterial-Amplified Chemiluminescence Systems and their Applications in Bioassays. TrAC, Trends Anal. Chem. 2011, 30 (2), 401-413. (5) Li, N.; Liu, D. Q.; Cui, H. Metal-Nanoparticle-Involved Chemiluminescence and its Applications in Bioassays. Anal. Bioanal. Chem. 2014, 406 (23), 5561-5571. (6) Ding, C. F.; Zhang, W.; Wang, W.; Chen, Y. Y.; Li, X. Q. Amplification Strategies Using Electrochemiluminescence Biosensors for the Detection of DNA, Bioactive Molecules and Cancer Biomarkers. TrAC, Trends Anal. Chem. 2015, 65, 137-150. (7) Liu, Y. T.; Shen, W.; Li, Q.; Shu, J. N.; Gao, L. F.; Ma, M. M.; Wang, W.; Cui, H. Firefly-Mimicking Intensive and Long-Lasting Chemiluminescence Hydrogels. Nat. Commun. 2017, 8 (1), 1003. (8) Cui, H.; Wang, W.; Duan, C. F.; Dong, Y. P.; Guo, J. Z. Synthesis, Characterization, and Electrochemiluminescence of Luminol-Reduced Gold Nanoparticles and their Application in a Hydrogen Peroxide Sensor. Chem. - Eur. J. 2007, 13 (24), 6975-6984.


21


(9)

Tian,

D.

Y.;

Zhang,

H.

L.;

Chai,

Y.;

Page 22 of 25

Cui,

H.

Synthesis

of

N-(aminobutyl)-N-(ethylisoluminol) Functionalized Gold Nanomaterials for Chemiluminescent Bio-Probe. Chem. Commun. 2011, 47 (17), 4959-4961. (10) Jiang, J.; He, Y.; Yu, X. X.; Zhao, J. Y.; Cui, H. A Homogeneous Hemin/G-Quadruplex DNAzyme Based Turn-On Chemiluminescence Aptasensor for Interferon-Gamma Detection via In-Situ Assembly of Luminol Functionalized Gold Nanoparticles, Deoxyribonucleic Acid, Interferon-Gamma and Hemin. Anal. Chim. Acta 2013, 791, 60-64. (11) Li, S. Y.; Chen, D. Y.; Zhou, Q. T.; Wang, W.; Gao, L. F.; Jiang, J.; Liang, H. J.; Liu, Y. Z.; Liang, G. L.; Cui, H. A General Chemiluminescence Strategy for Measuring Aptamer-Target Binding and Target Concentration. Anal. Chem. 2014, 86 (11), 5559-5566. (12) Gao, L. F.; Ju, L.; Cui, H. Determination of the Binding Constant of Specific Interactions and Binding Target Concentration Simultaneously Using a General Chemiluminescence Method. RSC Adv. 2016, 6 (7), 5305-5311. (13) Liu, M. X.; Zhang, H. L.; Shu, J. N.; Liu, X. Y.; Li, F.; Cui, H. Gold Nanoparticles Bifunctionalized by Chemiluminescence Reagent and Catalyst Metal Complexes: Synthesis and Unique Chemiluminescence Property. Anal. Chem. 2014, 86 (6), 2857-2861. (14) Li, F.; Liu, Y. T.; Zhuang, M.; Zhang, H. L.; Liu, X. Y.; Cui, H. Biothiols as Chelators for Preparation of N-(aminobutyl)-N-(ethylisoluminol)/Cu(2+) Complexes Bifunctionalized Gold Nanoparticles and Sensitive Sensing of Pyrophosphate Ion. ACS Appl. Mater. Interfaces 2014, 6 (20), 18104-18111.


22

Page 23 of 25 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


(15) Shu, J. N.; Shen, W.; Cui, H. Ultrasensitive Label-Free Electrochemiluminescence Immunosensor

Based

on

N-(4-aminobutyl)-N-ethylisoluminol-Functionalized

Graphene

Composite. Sci. China Chem. 2015, 58 (3), 425-432. (16) Duan, C. F.; Yu, Y. Q.; Cui, H. Gold Nanoparticle-Based Immunoassay by Using Non-Stripping Chemiluminescence Detection. Analyst 2008, 133 (9), 1250-1255. (17) Tian, D. Y.; Duan, C. F.; Wang, W.; Cui, H. Ultrasensitive Electrochemiluminescence Immunosensor Based on Luminol Functionalized Gold Nanoparticle Labeling. Biosens. Bioelectron. 2010, 25 (10), 2290-2295. (18) Qi, Y. Y.; Xiu, F. R.; Li, B. X. One-Step Homogeneous Non-Stripping Chemiluminescence Metal Immunoassay Based on Catalytic Activity of Gold Nanoparticles. Anal. Biochem. 2014, 449, 1-8. (19) Wei, W.; Tao, X.; Hua, C. Fluorescence and Electrochemiluminescence of Luminol-Reduced Gold Nanoparticles: Photostability and Platform Effect. Langmuir 2008, 24, 2826-2833. (20) Qin, H. Y.; Ma, D.; Du, J. X. Distance Dependent Fluorescence Quenching and Enhancement of Gold Nanoclusters by Gold Nanoparticles. Spectrochim. Acta, Part A 2018, 189, 161-166. (21) Peng, S. S.; Zou, G. Z.; Zhang, X. L. Nanocomposite of Electrochemically Reduced Graphene Oxide and Gold Nanoparticles Enhanced Electrochemilunescence of Peroxydisulfate and its Immunosensing Abililty towards Human IgG. J. Electroanal. Chem. 2012, 686, 25-31.


23


Page 24 of 25

(22) Li, L. L.; Liu, K. P.; Yang, G. H.; Wang, C. M.; Zhang, J. R.; Zhu, J. J. Fabrication of Graphene–Quantum Dots Composites for Sensitive Electrogenerated Chemiluminescence Immunosensing. Adv. Funct. Mater. 2011, 21, 869-878. (23) Abir, J.; Sondes, S.; Rania, E.; Latifa, K.; Mokhles, B. D.; Nedia, B.; Manel, B. H. M.; Souhir, K.; Hejer, G.; Salima, F.; Abdelhedi, M. Serum Heart Type Fatty Acid Binding Protein (H-FABP) Levels in Metabolic Syndrome. Int. J. Pharm. Sci. Res. 2017, 8(3), 1441-1448. (24) Ishii, J.; Ozaki, Y.; Lu, J. C.; Kitagawa, F.; Kuno, T.; Nakano, T.; Nakamura, Y.; Naruse, H.; Mori, Y.; Matsui, S.; Oshima, H.; Nomura, M.; Ezaki, K.; Hishida, H. Prognostic Value of Serum Concentration of Heart-Type Fatty Acid–Binding Protein Relative to Cardiac Troponin T on Admission in the Early Hours of Acute Coronary Syndrome. Clin. Chem. 2005, 51, 1397-1404. (25) Huang, L. J.; Zhang, L. H.; Mao, H. J.; Wang, P.; Jiang Y. X.; Li, P. P.; Jin, Q. H.; Zhao, J. L. Detection of Myocardial Injury Markers Based on Double Gold Nanoparticles Probes and Protein Chip. Chem. J. Chin. Univ. 2015, 36, 1687-1693. (26) Stan, D.; Mihailescu, C. M.; Iosub, R.; Moldovan, C.; Savin, M.; Baciu, I. Electrochemical Studies of Homogeneous Self-Assembled Monolayers versus Mixed Self-Assembled Monolayers on Gold Electrode for “Label Free” Detection of Heart Fatty Acid Binding Protein. Thin Solid Films 2012, 526, 143-149.


24

Page 25 of 25 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


For TOC only


25

Assembly of multifunctionalized gold nanoparticles with

Recommend Documents