Article pubs.acs.org/Langmuir
Hierarchical Flowerlike Gold Nanoparticles Labeled Immunochromatography Test Strip for Highly Sensitive Detection of Escherichia coli O157:H7 Lei Zhang,† Youju Huang,*,† Jingyun Wang,†,‡ Yun Rong,† Weihua Lai,*,‡ Jiawei Zhang,† and Tao Chen*,† †
Division of Polymer and Composite Materials, Ningbo Institute of Material Technology and Engineering, Chinese Academy of Science, No. 1219 Zhongguan West Road, Zhenhai District, Ningbo 315201, China ‡ State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China S Supporting Information *
ABSTRACT: Gold nanoparticles (AuNPs) labeled lateral-flow test strip immunoassay (LFTS) has been widely used in biomedical, feed/food, and environmental analysis fields. Conventional ILFS assay usually uses spherical AuNPs as labeled probes and shows low detection sensitivity, which further limits its widespread practical application. Unlike spherical AuNP used as labeled probe in conventional ILFS, in our present study, a hierarchical flowerlike AuNP specific probe was designed for LFTS and further used to detect Escherichia coli O157:H7 (E. coli O157:H7). Three types of hierarchical flowerlike AuNPs, such as tipped flowerlike, popcornlike, and large-sized flowerlike AuNPs were synthesized in a one-step method. Compared with other two kinds of Au particles, tipped flowerlike AuNPs probes for LFTS particularly exhibited highly sensitive detection of E. coli O157:H7. The remarkable improvement of detection sensitivity of tipped flowerlike AuNPs probes can be achieved even as low as 103 colonyforming units (CFU)/mL by taking advantages of its appropriate size and hierarchical structures, which is superior over the detection performance of conventional LFTS. Using this novel tipped flower AuNPs probes, quantitative detection of E. coli O157:H7 can be obtained partially in a wide concentration range with good repeatability. This hierarchical tipped flower-shaped AuNPs probe for LFTS is promising for the practical applications in widespread analysis fields. properties of component materials.1 Compared with other two ways, preparing appropriate probe materials is a simple and effective mean to improve the detection sensitivity.6,7 The unique physicochemical properties of AuNPs depend closely on their morphology and size.8 At present, spherical AuNPs labeled probes are used widely in the conventional LFTS, but their light scattering properties9 and conjugation ability with antibody are not optimal for LFTS because of their low dimension and ordinary shapes.6,10 On the other hand, hierarchical Au structures with rough surface and large surface-to-volume ratio are more favorable for the strong absorption of antibodies than low-dimensional spherical AuNPs,11−14 and the hierarchical flowerlike Au nanostructures with tipped surface exhibit strong optical signal sensitivity.15−17 These two advantages allow hierarchical Au structures suit particularly to be desirable test probes. The previous work had shown the excellent performance of
1. INTRODUCTION Immunochromatography assay integrated with chromatography and immunochemical reactions has been applied in many different fields because of its advantages of rapid and handy implementation.1,2 At present, the widespread immunochromatographic system is gold nanoparticles (AuNPs) labeled lateral-flow test strip immunoassay (LFTS),3 which is widely used in the detection of pathogens, drugs, hormones, and metabolites in biomedical, feed/food, and environmental settings.4 A typical AuNP-LFTS consists of several plain porous carriers including sample pad, conjugate release pad, and absorbent pad. When the sample fluid flow laterally through the test strip, the antibody-labeled AuNPs interact with the target analysts in the fluid and aggregate subsequently at the test line (T line) due to the specific interaction between analyst and antibody, which leads to color darkening of the T line.5 The light scattering signal of AuNPs aggregation at the T line is recorded to determine the content of target analyst. According to the testing process and principle, the detection sensitivity of LFTS can be promoted by designing sensitive reader instrument, optimizing test procedure and improving the © XXXX American Chemical Society
Received: February 13, 2015 Revised: April 27, 2015
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DOI: 10.1021/acs.langmuir.5b00592 Langmuir XXXX, XXX, XXX−XXX
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Scheme 1. (A) Surface Functionalization of Tipped Flowerlike AuNPs with Antibody I (Murine Anti-E. coli O157:H7 mAb) and BSA; (B) Schematic Illustration of LFTS Detection of E. coli O157:H7
Ltd. (Shanghai). Immunochromatography test strip consists of nitrocellulose reaction membrane, sample pad, conjugate pad, and absorbent pad. These materials were supplied by Schleicher & Schuell BioScience GmbH (Dassel, Germany). The antibody pairs, namely, murine anti-E. coli O157:H7 monoclonal antibody (mAb) and goat anti-E. coli O157:H7 polyclonal antibodies (pAb) (applied to the test line), and donkey anti-mouse IgG pAb (applied to control line) were obtained from Meridian Life Science, Inc. (Memphis, TN). According to previous work,26 E. coli O157:H7 strain (ATCC 43888) used in this study was cultured in Luria−Bertani medium (Oxoid, Basing stoke, UK) at 37 °C for 20 h before using. Phosphate buffer saline (PBS, pH = 7.4) is purchased from Sigma Chemical Company (St. Louis, MO) and is used to dilute the E. coli O157:H7 stock solution (4.6 × 108 CFU/mL) to analyte samples with different concentrations (from 103 to 107 CFU/mL). Bovine serum albumin (BSA, 0.1 g/mL) is obtained from Beijing Xinjingke Biotechnology Co., Ltd. All other reagents were used as purchased without further purification. The E. coli O157:H7 were cultured strictly according to pathogens culturing regulation. All procedures involving the manipulation of E. coli O157:H7 were conducted within biological safety cabinet. The morphologies of AuNPs were observed by an S-4800 (Hitachi, Japan) field emission scanning electron microscope (SEM) at an acceleration voltage of 8 kV. Transmission electron microscopy (TEM) was performed on a JEOL JEM 2010 electron microscope operating at 2.0 kV. UV/vis absorption spectra were recorded by virtue of TU-1810 UV/vis spectrophotometer provided by Purkinje General Instrument Co. Ltd. SkanFlexi BioAssay reader for test strip immnoassay was supplied by Skannex Biotech Co., Ltd. (Oslo, Norway) and used to record the optical density on the T line. 2.2. Synthesis of Spherical AuNPs. The spherical AuNP were prepared according to the Frens method.27 Briefly, 100 mL of HAuCl4 aqueous solution (2.5 × 10−4 M) was heated to 120 °C in an oil bath under vigorous stirring. Subsequently, 10 mL of sodium citrate solution (1 wt %) was added into above solution with continuous
hierarchical AuNP in the sensory detections. For example, Soleymani and co-workers18 prepared hierarchical nanotextured Au microelectrodes to be used for rapid and highly sensitive bacterial detection. Xia and co-workers19 reported the high catalysis activity of hierarchical nanodendric Pd−Pt bimetallic to oxygen reduction. Wang and co-workers20 reported high SERS-active gold nanoflower tags for in vivo detection. Although previous reports indicated that hierarchical flowerlike AuNPs can improve remarkably the detection performance in the sensors based on electrochemistry and surface-enhanced Raman spectroscopy,21−24 there is no report on hierarchical flowerlike AuNP used as labeled probes for LFTS assay.25 Herein, we selected polyelectrolyte poly(diallyldimethylammonium chloride) as capping agent to synthesize efficiently hierarchical flowerlike Au particles with different sizes and morphologies. The resultant flowerlike AuNPs were labeled with specific antibody for the rapid detection of E. coli O157:H7 by LFTS assay. Compared with popcornlike and large-sized flowerlike, the tipped flowerlike AuNP labeled probes showed higher sensitivity and reduced the detection limit to 103 CFU/mL, which would be beneficial for developing novel LFTS with high sensitivity and widespread practical applications in various fields.
2. MATERIALS AND METHODS 2.1. Materials and Instruments. Poly(diallylmethylammonium chloride) (PDDA, 35 wt % aqueous, Mw
