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Core-shell red silica nanoparticles based immunochromatographic assay for detection of Escherichia coli O157:H7
Analytica Chimica Acta 1038 (2018) 97e104
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Core-shell red silica nanoparticles based immunochromatographic assay for detection of Escherichia coli O157:H7 Chunjie Zhu, Guangying Zhao, Wenchao Dou* Food Safety Key Laboratory of Zhejiang Province, College of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou, 310018, China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
The colored SiNPs were first used as label to develop immunochromatographic assay strip for pathogenic bacteria. Core-shell red SiO2NPs based ICA was successfully developed for E. coli O157:H7 detection. The synthesis and functionalization procedures of core-shell red SiO2NPs were presented in detail. Two real samples with simple pretreatment spiked E. coli O157:H7 were tested by the novel ICA strip in this work.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 April 2018 Received in revised form 23 May 2018 Accepted 2 July 2018 Available online 9 July 2018
In this paper, a new type immunochromatographic assay (ICA) based on core-shell red silica nanoparticles (core-shell red SiO2NPs) was proposed and used to detect Escherichia coli O157:H7 (E. coli O157:H7). This is the first report of qualitative ICA for detecting E. coli O157:H7 in phosphate buffer saline (PBS) and food sample using core-shell red SiO2NPs. Monodispersed red SiO2NPs were synthesized in the aqueous solution by modifying amino silane and C.I Reactive Red 136 on unmodified silica nanoparticles. The limit of detection (LOD) of this core-shell red SiO2NPs based ICA for E. coli O157:H7 was 4.5 105 CFU/mL in sterile PBS within 20 min. The LOD of this ICA strip for E. coli O157:H7 in milk and pork samples both were 4.5 106 CFU/mL. The core-shell red SiO2NPs based ICA for detection of E. coli O157:H7 has no cross activity with other bacteria. All these results show that this new kind of core-shell colored SiO2NPs is promising for the practical applications in ICA and other rapid detection fields. © 2018 Elsevier B.V. All rights reserved.
Keywords: Immunochromatographic assay C.I reactive red 136 Sandwich immunoassay E. coli O157:H7 Carboxyethylsilanetriol sodium salt
1. Introduction Immunochromatographic assay (ICA) is a immunochromatographic technology with several advantages, such as procedure simplicity, rapid operation, immediate results, low cost, and no requirement for skilled technicians or expensive equipment [1]. ICA
* Corresponding author. E-mail address: [email protected] (W. Dou). https://doi.org/10.1016/j.aca.2018.07.003 0003-2670/© 2018 Elsevier B.V. All rights reserved.
is suitable for on-site detection and real-time inspection [2], it has been widely used in various biological detections, including a variety of pathogenic microorganisms. Probe materials are very important to the detection effect of ICA. Many colored materials like gold nanoparticles (AuNPs) [3], colored latex beads [4], and carbon nanomaterial [5,6] have been used as ICA labels. For the ICA, the most widely used probe material is AuNPs. The AuNPs are easy to prepare and label antibody, but AuNPs are substantially affected by various factors such as pH,
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temperature, salt concentration [7,8]. Besides, AuNPs and carbon nanomaterial both display a single color, which limits their use in the multiple testing. Until to now, colored latex beads are the only labels used in ICA, which can display different colors. As labeling agents, colored latex beads are low cost and rich color, but the dye of which is very easy to leak [9,10], which affects their application. Colored silica nanoparticles (colored SiO2NPs) are kinds of organic dye doped monodispersed silica nanoparticles with bright color. SiO2NPs are amenable to incorporating colored dye molecules, and the optical properties of colored SiO2NPs are depending on the color and amount of dye molecules contained. The dye is general bound on the silica core via the sulfonic acid-or carbonic acid-aminosi1ane bond [11]. Since the sulfonic acid group is used in many dyes to improve their water solubility and reactivity, there is a large variety of compounds which can be used, in principle, to produce colored SiO2NPs of nearly every color. Each colored SiO2NP contains a large number of photochemically active dyes allowing bright color. Colored SiO2NPs form rigid, robust structures, are chemically inert and easy to fabricate. Colored SiO2NPs are synthesized by modifying SiO2NPs with organic reactive dyes which have good stability, and don't fade in the harsh conditions [12]. Colored SiO2NPs are good candidate to be used as ICA probe, due to their inherent advantages, such as rich color, excellent stability, high hydrophilicity, good dispensability in water and easy surface modification for bioconjugation. In previous work, our team has proved that monodispersed colored SiO2NPs are kinds of good optical labels, which can be used in immune assay regarded as markers to amplify the response signal for detection of pathogenic bacteria [13e16]. Shiga toxin-producing Escherichia coli is an important human pathogen producing a similar clinical spectrum of disease [17]. Escherichia coli O157:H7 (E. coli O157:H7) is of special note, as it has been strongly associated with both food and water contamination [18,19]. To decrease bacterial infections, rapid detection of the E. coli O157:H7 is urgently needed in all health and safety related areas, especially in food safety. In this study, we report, for the first time, on the development of a specific, sensitive, and economical core-shell red SiO2NPs based ICA for diagnosis of E. coli O157:H7. The dye of C.I. Reactive Red 136 was introduced onto SiO2NPs and to form the red SiO2NPs which €ber system to produce core-shell then were introduced into a Sto red SiO2NPs with desirable color property and good stability. Due to the advantages of core-shell red SiO2NPs, a sensitive and specific ICA strip was developed for E. coli O157:H7 detection. Experimental results demonstrated that core-shell red SiO2NPs based ICA had a good ability for detection of E. coli O157:H7.
brought from Thermo Fisher Scientific Co., Ltd (Shanghai, China). The above mentioned reagents were all of analytical reagents. All reagents were used as received without further purification. The water used was pure water. The sample pad (glass fiber membrane) and release pad (glass fiber membrane) were provided by Ahl€ (Stockholm, Sweden). The nitrocellulose (NC) strom-Munksjo membrane (pore size is 15 mm) was provided by Sartorius Stedim Plastics GmbH (Goettingen, Germany). The absorbent pad, poly (vinylchloride) adhesive plate support board (PVC board) and plastic cassette were obtained from Shanghai Jieyi Biotechnology Co., Ltd. (Shanghai, China). The E. coli O157:H7 and two types of mouse anti-E. coli O157:H7 monoclonal antibodies for labeling (mAb1) and capturing (mAb2) were purchased from Shanghai HuiYun biological technology Co., Ltd (Shanghai, China). Cronobacter sakazakii (C. sakazakii, ATCC 43864), Escherichia coli (E. coli, ATCC 8739), Staphylococcus aureus (S. aureus, ATCC 27217), Bacillus cereus (B. cereus, ATCC 10987), Citrobacter freundii (C. freundii, ATCC 43864), Bacillus subtilis (B. subtilis, ATCC 11060) were purchased from China Center of Industrial Culture Collection (CICC) (Beijing, China), and conserved in our laboratory. Hitachi SU8010 scanning electron microscopy (SEM) was purchased from Hitachi Inc. (Tokyo, Japan); Malvern Nano ZS potential laser particle analyzer was provided by Malvern Instruments Co., Ltd. (Worcestershire, UK); Multiskan spectrum was purchased from Thermo Fisher Scientific Inc. (Waltham, USA); Solution sprayer (HGS510) equipped with a motion controller for spray coating was supplied by Hangzhou Autokun Technology Co., Ltd. (Hangzhou, China); Bio-vinostech strip cutter (HGS201) was purchased from Shanghai Hangan Electronic Technology Co., Ltd. (Hangzhou, China).
2.2. Synthesis of monodispersed red SiO2NPs € ber method with Monodispersed SiO2NPs were prepared by Sto slight modification [20,21]. In a typical reaction process, 2.7 mL of TEOS in 27.3 mL of ethanol was rapidly added into a mixture containing 10.3 mL of ethanol, 18 mL of H2O, and 1.7 mL of ammonia. The reaction mixture was stirred for 5 h. The resulting SiO2NPs were isolated by centrifugation, washed with ethanol and water. 0.1 g SiO2NPs were dispersed in 15 mL of water and treated with ultrasonic for 30 s, 15 mL of 3APTMS and 80 mL C.I. Reactive Red 136 aqueous solution (0.1 g/mL) were added, and kept at room temperature for 3 h under magnetic stirring. The resulting red SiO2NPs were collected by centrifugation and washed with water and ethanol, finally dispersed in 2.5 mL of ethanol for further usage.
2. Experimental section 2.1. Chemicals and apparatus
2.3. Synthesis of the core-shell red SiO2NPs and carboxyl-modified core-shell red SiO2NPs
Tetraethyl orthosilicate (TEOS), 2-(N-morpholino)ethanesulfonic acid (MES) and Polyvinyl Pyrrolidone (PVP) were obtained from Aladdin Industrial Inc. (Shanghai, China); 3-[2-(2aminoethylamino)ethylamino]propyl-trimethoxysilane (3APTMS) was purchased from Tianjin Heowns Biochemical Technology Co., Ltd. (Tianjin, China); ethanol and ammonia (25e28 wt%) were purchased from Xilong Sientific Co., Ltd (Guangdong, China); carboxyethylsilanetriol sodium salt 25 wt% in water (CES) was obtained from J&K Scientific Co., Ltd (Shanghai, China); N-(3Dimethylaminopropyl)-N0 -ethylcarbodiimide hydrochloride crystalline (EDC) and N-Hydroxysuccinimide (NHS) were brought from Sigma-Aldrich (Shanghai, China); bovine serum albumin (BSA) and goat anti-mouse IgG were purchased from Shanghai Sangon Biotech Co., Ltd (Shanghai, China); Pierce BCA Protein Assay Kit was
€ber system containing 10 mL ethanol, 0.8 mL H2O, 0.2 mL A Sto TEOS and 0.1 mL NH3$H2O (25e28%) was hydrolyzed for 30 min. 2.5 mL of above prepared red SiO2NPs ethanol dispersion solution €ber system and react for was added into the pre-hydrolyzed Sto 12 h at room temperature under magnetic stirring. The core-shell red SiO2NPs were isolated by centrifugation and washed several times with ethanol and water. Carboxyl-modified core-shell red SiO2NPs were synthesized in the same system, before the stop of the reaction, 25 mL of CES was € ber system to react for another 24 h. The added into the above Sto resulting core-shell red SiO2NPs were covered with protective layer and carboxyl group. The final carboxyl-modified core-shell red SiO2NPs were isolated by centrifugation and washed several times with ethanol and water.
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2.4. Preparation of antibody-modified core-shell red SiO2NPs conjugates The antibodies were immobilized on the surface of carboxylmodified core-shell red SiO2NPs through carbodiimide chemistry with slight modification [22], briefly described in following procedures: 1 mg carboxyl-modified core-shell red SiO2NPs were diluted in MES (0.05 M, pH 6.1) with a total volume of 1 mL. 1 mg EDC and 1.5 mg NHS were added to the particle dispersion and allowed to activate for 15 min with gentle mixing. The activated carboxyl-modified core-shell red SiO2NPs were washed twice to remove unreacted chemicals and reconstituted in 1 mL of phosphate buffer (PB, 0.02 M, pH 7.2). 30 mg mAb1 was added into the activated particles and mixed gently for 2.5 h at room temperature. The conjugates were centrifuged and resuspended in 1 mL of 2% BSA (w/v) in PB to block unreacted sites for 1 h at room temperature. The blocked conjugates were centrifuged and resuspended in phosphate buffered saline (PBS, 0.01 M) containing 1% BSA (w/v), 5% sucrose (w/v), 3% trehalose (w/v) at a final concentration of 10 mg/mL and stored at 4 C before use. 2.5. Bacterial strains and preparation E. coli O157:H7 used as the detection antigen was grown in Lysogeny broth (LB) medium with shaking at 37 C. Cells were harvested in late exponential growth phase by centrifugation at 6000 rpm at 4 C for 10 min and washed in triplicate using physiological saline aqueous solution and dispersed in 5 mL physiological saline aqueous solution. Concentration of the bacteria was confirmed by the colony counting (CFU/mL). The enriched bacteria were inactivated with 0.4% formaldehyde and stored at 4 C before use. 2.6. Fabrication of immunochromatographic strip ICA test strips were composed of 4 components including sample pad, release pad, NC membrane and absorbent pad. A sample pad and release pad made from glass fiber were pre-treated with PBS buffer (0.01 M, pH 7.4) containing 1% BSA, 0.5% Tween-20, 3% sucrose (w/v), 1% trehalose (w/v), 0.5% PVP, further dried at 37 C overnight. The mAb2 (1.2 mg/mL) and goat anti-mouse IgG (1.0 mg/ mL) were sprayed onto the test (T) and control (C) lines on the NC membrane by solution sprayer. They were both sprayed on the NC membrane at the speed of 1 mL/cm and then dried at 37 C for 1 h. The NC membrane, absorption pad, pre-treated release pad and sample pad were assembled on a PVC board sequentially with a 1e2 mm overlap to form the final test strip (the fabrication steps are shown in Fig. 1s in the supplemental information), cut into 4 mm width by Bio-vinostech strip cutter and to package into the plastic cassette.
Fig. 1. SEM photograph of SiO2NPs (a), red SiO2NPs (b) and carboxyl-modified coreshell red SiO2NPs (c), inset: physical image of carboxyl-modified core-shell red SiO2NPs. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
2.7. Fabrication of core-shell red SiO2NPs-based ICA and analytical procedure
2.8. Detection of E. coli O157:H7 in pork and milk samples by coreshell red SiO2NPs-based ICA
To assess the performance of detection, the E. coli O157:H7 culture was diluted into different concentrations from 4.5 103 to 4.5 108 CFU/mL in sterile PBS, which were then mixed with the core-shell red SiO2NPs-antibody conjugate and wash buffer (0.01 M PBS containing 0.5% BSA, 1% Tween-20, 3% sucrose (w/v) and 1% trehalose (w/v). The ratio of above mixture consisted of 35 mL sample, 10 mL core-shell red SiO2NPs-antibody conjugate and 35 mL wash buffer, respectively. The detection was performed by applying 80 mL of the mixture on a sample pad of the test strip to react for 15 min. All the concentrations were performed in triplicate.
The pork and fresh milk were purchased from a local supermarket (Hangzhou, China). For pork tissue samples, 5 g of pork muscle homogenate was weighed and mixed with 10 mL PBS buffer in a sterile bag. The sample was centrifuged for 5 min at 10,000 rpm after extraction under homogeneity about 5 min. The supernatant was collected and fortified with different concentration of E. coli O157:H7 solution for ICA detection. And the fresh milk samples were diluted in PBS buffer by 10 fold, and spiked with different concentration of E. coli O157:H7. Spiked milk samples were directly detected without any further sample preparation. Every sample
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was performed in triplicate. 3. Results and discussion 3.1. Synthesis and characterization of core-shell red SiO2NPs The core-shell red SiO2NPs were synthesized by multistep method, the schematic diagram is presented in part A of Scheme 1. € ber system. Then Firstly, bare SiO2NPs were synthesized by Sto 3APTMS and C.I. Reactive Red 136 solution were added sequentially into the SiO2NPs aqueous dispersion to graft the dye molecules onto the surface of SiO2NPs. The hydrolysis and condensation of 3APTMS and dye coupling were preceded at the same time because hydrolysis and condensation of 3APTMS was a quick procedure in the presence of water. The water would increase the surface density of amine [23], thus, leading coloration of a great deal of dye molecules on the surface of SiO2NPs and showing a bright red color. To prevent the dye from shedding, the red SiO2NPs were introduced €ber system to carry out regrowth of the into a pre-hydrolyzed Sto € ber system, a small amount of NH3$H2O silica layer. In this Sto
catalyzed TEOS to hydrolyze, produce soluble silica covered with a large number of negative charges. The amino groups on the surface of red SiO2NPs were not fully occupied by the dye molecules, thus soluble silica can be absorbed onto the red SiO2NPs due to electrostatic interactions. With the catalysis of TEOS, the silica layer was formed through Si-O-Si bond between the soluble silica on the red SiO2NPs, to protect the dye on the particles, as well as the morphology and dispersity. The synthesis of core-shell red SiO2NPs and modification of carboxyl group were conducted by one step. The carboxyl-modified core-shell red SiO2NPs were directly produced by adding CES in the procedure of regrowth of silica protection layer. CES is a kind of silane coupling agent that can be grafted to the surface of SiO2NPs by through Si-O-Si bond in alkaline conditions. With the exist of a small amount of NH3$H2O, the CES was hydrolyzed gradually, eventually to modified carboxyl group on the core-shell red SiO2NPs. Conventionally, modification of carboxyl group on SiO2NPs was carried out by multistep functionalization [24]. Our synthesis method is simpler and cheaper than former modification method. Fig. 1 shows the SEM pictures of the SiO2NPs, red SiO2NPs
Scheme 1. Schematic representation of core-shell red SiO2NPs based ICA. (A) The procedure of synthesis and carboxyl modification of the core-shell red SiO2NPs; (B) The formation of mAb1-modified red SiO2NPs probes; (C) The dimensions of the core-shell red SiO2NPs based ICA; (D) Assay principle of core-shell red SiO2NPs-based ICA, the appearance of two lines indicates a positive result, whereas a valid negative test produces only the control line.
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and carboxyl-modified core-shell red SiO2NPs, all of them exhibit regular spherical morphology and good dispersity, demonstrating that there is almost no change in morphology after coloration and silica shell regrowth. Especially, carboxyl-modified core-shell red SiO2NPs show better dispersity compared with SiO2NPs and red SiO2NPs due to the surface of core-shell red SiO2NPs rich in carboxyl group. And they are perfectly spherical with an average diameter of about 216 nm. Zeta potential of core-shell red SiO2NPs and carboxyl-modified core-shell red SiO2NPs were also measured. As shown in Fig. 2, the core-shell red SiO2NPs present an isoelectric point around 7, while the carboxyl-modified core-shell red SiO2NPs are negatively charged in the range of pH 3e12. The isoelectric point of carboxylmodified core-shell red SiO2NPs shifts to much lower pH values suggesting that the density of carboxyl group on the core-shell red SiO2NPs surface is high on account of the introduction of CES. Compared with the AuNPs, the core-shell red SiO2NPs have good stability and don't fade in the harsh conditions such as pH and salt. Fig. 3 exhibits the influence of pH on AuNPs and core-shell red SiO2NPs. From Fig. 3a, the pH values of samples adjusted by NaOH and HCl solutions (0.1 M) are 2e13 from the left to right. The coreshell red SiO2NPs (bottom) show good stability and bright color, while AuNPs are easily aggregated, when the pH value is lower than pH 5 or higher than pH 10. Besides, we examined the influence of salt concentration on stability of core-shell red SiO2NPs and AuNPs. As shown in Fig. 3b, AuNPs begin degeneration even the salt concentration is as low as 0.1%. Compared with the AuNPs, core-shell red SiO2NPs remain monodispersed when the salt concentration is as high as 0.4%. Actually, core-shell red SiO2NPs can be dispersed without flocculation in the solution containing higher concentration of salt. Based on these advantages, core-shell red SiO2NPs have been selected to be used as label of ICA in this work.
3.2. Characterization of antibody-modified core-shell red SiO2NPs The mAb1 was immobilized on the surface of carboxyl-modified core-shell red SiO2NPs through carbodiimide chemistry. Fig. 4 shows the particle size distribution curves of carboxyl-modified core-shell red SiO2NPs and antibody-modified core-shell red SiO2NPs. The size distribution curve of antibody-modified coreshell red SiO2NPs moves slightly to the right compared with
Fig. 2. Zeta potentials of unmodified core-shell red SiO2NPs and carboxyl-modified core-shell red SiO2NPs dispersed in water with different pH. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
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carboxyl-modified core-shell red SiO2NPs. In accordance with the particle size distribution curve, the average diameter of carboxylmodified core-shell red SiO2NPs is 216 nm, and that of the antibody-modified core-shell red SiO2NPs is determined to be 236 nm, which proves the immobilization of antibodies on these particles. Besides, Pierce BCA Protein Assay Kit was used to examine the effect of mAb1 immobilization, and the rate of mAb1 that modified on the core-shell red SiO2NPs was more than 70% (More experimental details and results are shown in Tables S1 and S2 in the supplemental information, respectively). ICA detection was also employed to validate the antibodymodified core-shell red SiO2NPs conjugates. Goat anti-mouse IgG was coated on NC membrane at C line. The carboxyl-modified coreshell red SiO2NPs and antibody-modified core-shell red SiO2NPs conjugates were sprayed onto the release pad and dried, respectively. The sample pad, release pad, NC membrane and absorbent pad were integrated in accordance with part of 2.7. Then 80 mL of PBS was loaded on the sample pad and reacted about 10 min. The results were observed by naked eye and presented in Fig. 4 inset. It is observed that the antibody-modified core-shell red SiO2NPs conjugates are captured by goat anti-mouse IgG and show strong red color intensity at C line. On the contrary, the core-shell red SiO2NPs without antibody modification cannot be captured by IgG, the red color band is not observed. The combined results of particle size distribution curve, Pierce BCA Protein Assay and ICA detection demonstrate that mouse anti-E. coli O157:H7 mAb1 is successfully modified on the surface of carboxyl-modified core-shell red SiO2NPs. 3.3. Assay principle of core-shell red SiO2NPs-based ICA In the conventional sandwich ICA strip, the target analytes interact with the antibody-labeled particles in the conjugate pad, and flow laterally through the test strip. The complexes are then captured by the immobilized capture antibodies on the T line of the strip and form the sandwich immunocomplex which leads to a darkening color of the T line, which can be observed by naked eyes. Unlike the conventional ICA strip, a separated incubation of E. coli O157:H7 and mAb1-modified core-shell red SiO2NPs was proposed for this core-shell red SiO2NPs based ICA strip. Scheme 1 exhibits the schematically configuration principle of this core-shell red SiO2NPs based ICA system. As shown in part C of Scheme 1, E. coli O157:H7 and mAb1-modified core-shell red SiO2NPs form immune complexes. And the immune complexes are added onto sample pad of the test strip and flow laterally through the strip under capillary force. The complexes are then captured by the immobilized mAb2 and form a sandwich immunocomplexes, resulting in a red band on the T line. For the detection of non-E. coli O157:H7 analysts, there is no red band on the T line. The extra antibody-modified core-shell red SiO2NPs bound to goat antimouse IgG on C line regardless of the presence of E. coli O157:H7. The mAb1-modified core-shell red SiO2NPs form a red-colored band indicates validity of test result. This kind of ICA own some advantages, such as elimination of the antibody-particles conjugate pad, making the fabrication of the strip easier [25]. And the separated incubation of E. coli O157:H7 allows easier control of the reaction time, resulting in the complete interaction between E. coli O157:H7 and mAb1-modified core-shell red SiO2NPs. This effective incubation of E. coli O157:H7 with mAblabelled core-shell red SiO2NPs gives a high detection performance. 3.4. Sensitivity and specificity of core-shell red SiO2NPs-based ICA system To demonstrate the performance of this core-shell red SiO2NPs
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Fig. 3. The influences of pH value and salt on the core-shell red SiO2NPs and AuNPs. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 5. The sensitivity of the core-shell red SiO2NPs based ICA, E. coli O157:H7 was 10fold diluted from 4.5 108 to 4.5 103 CFU/mL (from left to right) by PBS, the rightmost strip was a negative control. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 4. The diameter distributions of the carboxyl-modified core-shell red SiO2NPs and antibody-modified core-shell red SiO2NPs, inset: the results of ICA detection of carboxyl-modified core-shell red SiO2NPs (up) and antibody-modified purple SiO2NPs (bottom). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
based ICA system, the color intensity of T line was observed within 15 min after adding the immune complexes. The range of detection was determined with different E. coli O157: H7 concentrations from 4.5 103 to 4.5 108 CFU/mL and with negative sample. The detection procedure was repeated three times and one of typical picture was obtained to show the detection efficiency. The ICA detection results of E. coli O157:H7 samples are shown in Fig. 5. As increasing of E. coli O157:H7 concentration in samples, the color at the T line is proportionally increased. In this present study, the visual inspection limit was defined as the minimum analyte concentration required for showing no obvious visual color on the T line. Following this definition, the color band of T lines was not visible when the bacteria concentration below 4.5 104 CFU/mL. Thus, 4.5 105 CFU/mL was considered to be the visual limit of detection (LOD) of this ICA strip for the detection of E. coli O157:H7, which is comparable to the detection performance with naked eyes of former reported other nanomaterials based ICA [25e27].
To assess the specificity and selectivity of core-shell red SiO2NPs-based ICA, we challenged the system against E. coli O157:H7 and other pathogen (E. coli, C. sakazakii, S. aureus, B. cereus, C. freundii and B. subtilis) at the bacteria concentration of 4.5 108 CFU/mL. After applying mixture solutions containing bacterium and mAb1-core-shell red SiO2NPs on the sample pad, the color intensity was observed within 15 min. The same detection process was repeated three times for each bacterium sample. As shown in Fig. 6, strong red signal of E. coli O157: H7 is observed,
Fig. 6. The cross reaction results of the core-shell red SiO2NPs based ICA, from the left to right: E. coli O157:H7, E. coli, C. sakazakii, S. aureus, B. cereus, C. freundii, B. subtilis respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
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immunomagnetic nanoparticles to isolate and enrich E. coli O157:H7 [29]. In future research, if the selective improved medium or immunomagnetic enrichment method can be used for milk and meat, the sensitivity will be improved. In this study, the development of colored silica nanoparticles based ICA is in the preliminary stage. 4. Conclusion
Fig. 7. Detection of E. coli O157:H7 in milk (a) and pork (b) food samples by the coreshell red SiO2NPs based ICA. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
while the other bacteria show no signal on T line. The good specificity of core-shell red SiO2NPs based ICA is because of the specificity reaction between the antigen and antibody. The monoclonal antibody can specifically identify antigen site on E. coli O157, but does not recognize other bacteria. In the specific experiment, other bacteria model cannot form sandwich structure with mAb1-coreshell red SiO2NPs, so cannot produce the red band on the T line. These results demonstrate high specificity of this ICA strip against other nonspecific bacteria used in this work. From these results both in sensitivity and specificity detection, it is also found that the intensities of the red color at C line are nearly constant for all strips.
In this work, we used the core-shell red SiO2NPs as novel visual probes in ICA for the detection of E. coli O157:H7. A model system comprising a target E. coli O157:H7 analyte and a pair of antibody probes were used to demonstrate the proof-of-concept. Under optimal conditions, the visual qualitative inspection limits (the red color of T line) significantly observed when the concentration of E. coli O157:H7 was as low as 105 CFU/mL and 106 CFU/mL in sterile PBS and real samples, respectively. This core-shell red SiO2NPs based immunochromatographic sensor was simple, fast and without complicated sample preparation process, it is suitable for field detection. The immunoassay system in this work also has acceptable specificity and no cross activity with six other bacteria. In addition, the optimized newly core-shell colored SiO2NPs based ICA can be extended in food quality control, environmental monitoring and clinical analysis. In the future there is still a lot of work can be done to explore the improvement and application of colored SiO2NPs based ICA. Such as, combine colored SiO2NPs probes based ICA and strip reader, more sensitive and quantitative detection of E. coli O157:H7 can be obtained partially in a wide concentration range with good repeatability. We will also develop a sensitive and quantitative sandwich ICA for rapid detection of hepatitis B surface antigen based on red SiNPs in our next work. By synthesizing and using blue SiO2NPs and red SiO2NPs, a multiplex ICA can be developed for the simultaneous detection of ractopamine and clenbuterol hydrochloride. Compliance with ethical standards
3.5. Application in milk and pork samples by core-shell red SiO2NPs-based ICA Based on the above mentioned detection conditions, E. coli O157:H7 cultures at different concentrations in pork and milk samples were detected with this core-shell red SiO2NPs based ICA, each sample was examined in triplicate. The typical results for pork and milk samples are shown in Fig. 7a and Fig. 7b, respectively. It is demonstrated that E. coli O157:H7 of 4.5 106 CFU/mL can be detected in both milk and pork samples using this detection system. When the ICA strip was used to detect E. coli O157:H7, the detection limit of ICA increased from 4.5 105 to 4.5 106. The reason is ascribed to the fat and protein may influence the detection of E. coli O157:H7 in real sample. As the E. coli O157:H7, it is forbidden to exist in any food matrix [28]. For rapid detection methods of E. coli O157:H7, they rely on enrichment to make it reaches an enough concentration, such as immunological assays, serological tests, PCR-based method and DNA probes. For example, the selective media are one of the most commonly used methods for detection of E. coli O157:H7. As to different matrix, like milk or pork sample, if we use selective improved medium to culture E. coli O157:H7 for 8e12 h to enough concentration, they could be detected. And the time of 8e12 h is much shorter than that of culture based methods, which often require 4e7 days to obtain a confirmed result for E. coli O157:H7. The sensitivity of the ICA strip can also be improved by immunomagnetic nanoparticles. The ICA strips have been used to detect E. coli O157:H7 in real samples using
The authors declare that they have no conflict of interest. This article does not contain any studies with animals performed by any of the authors. Informed consent was obtained from all individual participants included in the study. Acknowledgement This work was financially supported by a grant from National Natural Science Foundation of Zhejiang Province (LY17C200003), the Food and Engineering most important discipline of Zhejiang province (2017SIAR210, JYTSP20141062), Zhejiang public Innovation Platform Analysis and testing project (2018C37056), Zhejiang Xinmiao Talents Program (1110KZN0217054G, 1110KZN0217053G), open fund of State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, the First Affiliated Hospital of Medical College, Zhejiang University (2017KF02). Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.aca.2018.07.003. References [1] D. Zhang, P. Li, Q. Zhang, W. Zhang, Ultrasensitive nanogold probe-based immunochromatographic assay for simultaneous detection of total aflatoxins in peanuts, Biosens. Bioelectron. 26 (2011) 2877e2882. [2] G.A. Posthumatrumpie, J. Korf, A. Van Amerongen, Lateral flow (immuno) assay, Anal. Bioanal. Chem. 393 (2015) 569e582.
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[3] D. Zhang, P. Li, Q. Zhang, W. Zhang, Ultrasensitive nanogold probe-based immunochromatographic assay for simultaneous detection of total aflatoxins in peanuts, Biosens. Bioelectron. 26 (2011) 2877e2882. [4] X. Mao, W. Wang, T.E. Du, Dry-reagent nucleic acid biosensor based on blue dye doped latex beads and lateral flow strip, Talanta 114 (2013) 248e253. n, J. Wichers, A. Abad-Somovilla, A.A. Van, A. Abad-Fuentes, [5] C. Su arez-Pantaleo Development of an immunochromatographic assay based on carbon nanoparticles for the determination of the phytoregulator forchlorfenuron, Biosens. Bioelectron. 42 (2013) 170e176. [6] W. Qiu, H. Xu, S. Takalkar, A.S. Gurung, B. Liu, Y. Zheng, Z. Guo, M. Baloda, K. Baryeh, G. Liu, Carbon nanotube-based lateral flow biosensor for sensitive and rapid detection of DNA sequence, Biosens. Bioelectron. 64 (2015) 367e372. [7] J. Joo, C. Yim, D. Kwon, J. Lee, H.H. Shin, H.J. Cha, S. Jeon, A facile and sensitive detection of pathogenic bacteria using magnetic nanoparticles and optical nanocrystal probes, Analyst (Cambridge, U.K.) 137 (2012) 3609e3612. [8] K. Rajesh, S. Chandrak, K. Suchitra, S. Rajeshwarp, B. Rajk, K. Devendran, Development of nanocolloidal gold based immunochromatographic assay for rapid detection of transgenic vegetative insecticidal protein in genetically modified crops, Food Chem. 122 (2010) 1298e1303. [9] R.P. Bagwe, C. Yang, L.R.H. And, W. Tan, Optimization of dye-doped silica nanoparticles prepared using a reverse microemulsion method, Journal of Surfaces & Colloids 20 (2004) 8336. [10] B. Li, J. Shen, R. Liang, W. Ji, C. Kan, Synthesis and characterization of covalently colored polymer latex based on new polymerizable anthraquinone dyes, Colloid Polym. Sci. 290 (2012) 1893e1900. [11] H. Giesche, E. Matijevi, Well-defined pigments: I. Monodispersed silica-acid dyes systems, Dyes Pigments 17 (1991) 323e340. [12] F.M. Winnik, B. Keoshkerian, J.R. Fuller, P.G. Hofstra, New water-dispersible silica-based pigments: synthesis and characterization, Dyes Pigments 14 (1990) 101e112. [13] H. Yu, G. Zhao, W. Dou, Simultaneous detection of pathogenic bacteria using agglutination test based on colored silica nanoparticles, Curr. Pharmaceut. Biotechnol. 16 (2015) 716e723. [14] Q. Sun, G. Zhao, W. Dou, A nonenzymatic optical immunoassay strategy for detection of Salmonella infection based on blue silica nanoparticles, Anal. Chim. Acta 898 (2015) 109e115. [15] Q. Sun, G. Zhao, W. Dou, Blue silica nanoparticle-based colorimetric immunoassay for detection of Salmonella pullorum, Analytical Methods 7 (2015) 8647e8654. [16] Q. Sun, G. Zhao, W. Dou, An optical and rapid sandwich immunoassay method for detection of Salmonella pullorum and Salmonella gallinarum based on immune blue silica nanoparticles and magnetic nanoparticles, Sensor. Actuator. B Chem. 226 (2016) 69e75.
[17] J.K. Varma, K.D. Greene, M.E. Reller, S.M. Delong, J. Trottier, S.F. Nowicki, M. Diorio, E.M. Koch, T.L. Bannerman, S.T. York, An outbreak of Escherichia coli O157 infection following exposure to a contaminated building, JAMA, J. Am. Med. Assoc. 290 (2003) 2709e2712. [18] R.S. Quilliam, A.P. Williams, L.M. Avery, S.K. Malham, D.L. Jones, Unearthing human pathogens at the agriculturaleenvironment interface: a review of current methods for the detection of Escherichia coli O157 in freshwater ecosystems, Agri. Ecosyst. Environ. 140 (2011) 354e360. [19] S. Shan, D. Liu, Q. Guo, S. Wu, R. Chen, K. Luo, L. Hu, Y. Xiong, W. Lai, Sensitive detection of Escherichia coli O157:H7 based on cascade signal amplification in ELISA, J. Dairy Sci. 99 (2016) 7025e7032. [20] T. Zhang, Q. Zhang, J. Ge, J. Goebl, M. Sun, Y. Yan, Y. Liu, C. Chang, J. Guo, Y. Yin, A self-templated route to hollow silica microspheres, J. Phys. Chem. C 113 (2009) 3168e3175. [21] W. Wei, B. Gu, L. Liang, W. Hamilton, Fabrication of two- and threedimensional silica nanocolloidal particle arrays, J. Phys. Chem. B 107 (2003) 3400e3404. [22] S. Bamrungsap, C. Apiwat, W. Chantima, T. Dharakul, N. Wiriyachaiporn, Rapid and sensitive lateral flow immunoassay for influenza antigen using fluorescently-doped silica nanoparticles, Microchimica Acta 181 (2013) 223e230. [23] P.J.E. Harlick, A. Sayari, Applications of pore-expanded mesoporous silica. 5. Triamine grafted material with exceptional CO2 dynamic and equilibrium adsorption performance, Ind. Eng. Chem. Res. 46 (2007) 446e458. [24] X. Zhao, L.R. Hilliard, S.J. Mechery, Y. Wang, R.P. Bagwe, S. Jin, W. Tan, A rapid bioassay for single bacterial cell quantitation using bioconjugated nanoparticles, Proc. Natl. Acad. Sci. U.S.A. 101 (2004) 15027. [25] L. Zhang, Y. Huang, J. Wang, Y. Rong, W. Lai, J. Zhang, T. Chen, Hierarchical flowerlike gold nanoparticles labeled immunochromatography test strip for highly sensitive detection of Escherichia coli O157:H7, Langmuir 31 (2015) 5537e5544. [26] H. Qi, Z. Zhong, H.X. Zhou, C.Y. Deng, H. Zhu, J.F. Li, X.L. Wang, F.R. Li, A rapid and highly sensitive protocol for the detection of Escherichia coli O157:H7 based on immunochromatography assay combined with the enrichment technique of immunomagnetic nanoparticles, Int. J. Nanomed. 6 (2011) 3033e3039. [27] B.Y. Jung, S.C. Jung, C.H. Kweon, Development of a rapid immunochromatographic strip for detection of Escherichia coli O157, J. Food Protect. 68 (2005) 2140e2143. [28] Y. Yoon, S. Lee, K.H. Choi, Microbial benefits and risks of raw milk cheese, Food Contr. 63 (2016) 201e215. [29] Q. Xiong, X. Cui, J.K. Saini, D. Liu, S. Shan, Y. Jin, W. Lai, Development of an immunomagnetic separation method for efficient enrichment of Escherichia coli O157:H7, Food Contr. 37 (2014) 41e45.
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Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
Core-shell red silica nanoparticles based immunochromatographic assay for detection of Escherichia coli O157:H7 Chunjie Zhu, Guangying Zhao, Wenchao Dou* Food Safety Key Laboratory of Zhejiang Province, College of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou, 310018, China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
The colored SiNPs were first used as label to develop immunochromatographic assay strip for pathogenic bacteria. Core-shell red SiO2NPs based ICA was successfully developed for E. coli O157:H7 detection. The synthesis and functionalization procedures of core-shell red SiO2NPs were presented in detail. Two real samples with simple pretreatment spiked E. coli O157:H7 were tested by the novel ICA strip in this work.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 April 2018 Received in revised form 23 May 2018 Accepted 2 July 2018 Available online 9 July 2018
In this paper, a new type immunochromatographic assay (ICA) based on core-shell red silica nanoparticles (core-shell red SiO2NPs) was proposed and used to detect Escherichia coli O157:H7 (E. coli O157:H7). This is the first report of qualitative ICA for detecting E. coli O157:H7 in phosphate buffer saline (PBS) and food sample using core-shell red SiO2NPs. Monodispersed red SiO2NPs were synthesized in the aqueous solution by modifying amino silane and C.I Reactive Red 136 on unmodified silica nanoparticles. The limit of detection (LOD) of this core-shell red SiO2NPs based ICA for E. coli O157:H7 was 4.5 105 CFU/mL in sterile PBS within 20 min. The LOD of this ICA strip for E. coli O157:H7 in milk and pork samples both were 4.5 106 CFU/mL. The core-shell red SiO2NPs based ICA for detection of E. coli O157:H7 has no cross activity with other bacteria. All these results show that this new kind of core-shell colored SiO2NPs is promising for the practical applications in ICA and other rapid detection fields. © 2018 Elsevier B.V. All rights reserved.
Keywords: Immunochromatographic assay C.I reactive red 136 Sandwich immunoassay E. coli O157:H7 Carboxyethylsilanetriol sodium salt
1. Introduction Immunochromatographic assay (ICA) is a immunochromatographic technology with several advantages, such as procedure simplicity, rapid operation, immediate results, low cost, and no requirement for skilled technicians or expensive equipment [1]. ICA
* Corresponding author. E-mail address: [email protected] (W. Dou). https://doi.org/10.1016/j.aca.2018.07.003 0003-2670/© 2018 Elsevier B.V. All rights reserved.
is suitable for on-site detection and real-time inspection [2], it has been widely used in various biological detections, including a variety of pathogenic microorganisms. Probe materials are very important to the detection effect of ICA. Many colored materials like gold nanoparticles (AuNPs) [3], colored latex beads [4], and carbon nanomaterial [5,6] have been used as ICA labels. For the ICA, the most widely used probe material is AuNPs. The AuNPs are easy to prepare and label antibody, but AuNPs are substantially affected by various factors such as pH,
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temperature, salt concentration [7,8]. Besides, AuNPs and carbon nanomaterial both display a single color, which limits their use in the multiple testing. Until to now, colored latex beads are the only labels used in ICA, which can display different colors. As labeling agents, colored latex beads are low cost and rich color, but the dye of which is very easy to leak [9,10], which affects their application. Colored silica nanoparticles (colored SiO2NPs) are kinds of organic dye doped monodispersed silica nanoparticles with bright color. SiO2NPs are amenable to incorporating colored dye molecules, and the optical properties of colored SiO2NPs are depending on the color and amount of dye molecules contained. The dye is general bound on the silica core via the sulfonic acid-or carbonic acid-aminosi1ane bond [11]. Since the sulfonic acid group is used in many dyes to improve their water solubility and reactivity, there is a large variety of compounds which can be used, in principle, to produce colored SiO2NPs of nearly every color. Each colored SiO2NP contains a large number of photochemically active dyes allowing bright color. Colored SiO2NPs form rigid, robust structures, are chemically inert and easy to fabricate. Colored SiO2NPs are synthesized by modifying SiO2NPs with organic reactive dyes which have good stability, and don't fade in the harsh conditions [12]. Colored SiO2NPs are good candidate to be used as ICA probe, due to their inherent advantages, such as rich color, excellent stability, high hydrophilicity, good dispensability in water and easy surface modification for bioconjugation. In previous work, our team has proved that monodispersed colored SiO2NPs are kinds of good optical labels, which can be used in immune assay regarded as markers to amplify the response signal for detection of pathogenic bacteria [13e16]. Shiga toxin-producing Escherichia coli is an important human pathogen producing a similar clinical spectrum of disease [17]. Escherichia coli O157:H7 (E. coli O157:H7) is of special note, as it has been strongly associated with both food and water contamination [18,19]. To decrease bacterial infections, rapid detection of the E. coli O157:H7 is urgently needed in all health and safety related areas, especially in food safety. In this study, we report, for the first time, on the development of a specific, sensitive, and economical core-shell red SiO2NPs based ICA for diagnosis of E. coli O157:H7. The dye of C.I. Reactive Red 136 was introduced onto SiO2NPs and to form the red SiO2NPs which €ber system to produce core-shell then were introduced into a Sto red SiO2NPs with desirable color property and good stability. Due to the advantages of core-shell red SiO2NPs, a sensitive and specific ICA strip was developed for E. coli O157:H7 detection. Experimental results demonstrated that core-shell red SiO2NPs based ICA had a good ability for detection of E. coli O157:H7.
brought from Thermo Fisher Scientific Co., Ltd (Shanghai, China). The above mentioned reagents were all of analytical reagents. All reagents were used as received without further purification. The water used was pure water. The sample pad (glass fiber membrane) and release pad (glass fiber membrane) were provided by Ahl€ (Stockholm, Sweden). The nitrocellulose (NC) strom-Munksjo membrane (pore size is 15 mm) was provided by Sartorius Stedim Plastics GmbH (Goettingen, Germany). The absorbent pad, poly (vinylchloride) adhesive plate support board (PVC board) and plastic cassette were obtained from Shanghai Jieyi Biotechnology Co., Ltd. (Shanghai, China). The E. coli O157:H7 and two types of mouse anti-E. coli O157:H7 monoclonal antibodies for labeling (mAb1) and capturing (mAb2) were purchased from Shanghai HuiYun biological technology Co., Ltd (Shanghai, China). Cronobacter sakazakii (C. sakazakii, ATCC 43864), Escherichia coli (E. coli, ATCC 8739), Staphylococcus aureus (S. aureus, ATCC 27217), Bacillus cereus (B. cereus, ATCC 10987), Citrobacter freundii (C. freundii, ATCC 43864), Bacillus subtilis (B. subtilis, ATCC 11060) were purchased from China Center of Industrial Culture Collection (CICC) (Beijing, China), and conserved in our laboratory. Hitachi SU8010 scanning electron microscopy (SEM) was purchased from Hitachi Inc. (Tokyo, Japan); Malvern Nano ZS potential laser particle analyzer was provided by Malvern Instruments Co., Ltd. (Worcestershire, UK); Multiskan spectrum was purchased from Thermo Fisher Scientific Inc. (Waltham, USA); Solution sprayer (HGS510) equipped with a motion controller for spray coating was supplied by Hangzhou Autokun Technology Co., Ltd. (Hangzhou, China); Bio-vinostech strip cutter (HGS201) was purchased from Shanghai Hangan Electronic Technology Co., Ltd. (Hangzhou, China).
2.2. Synthesis of monodispersed red SiO2NPs € ber method with Monodispersed SiO2NPs were prepared by Sto slight modification [20,21]. In a typical reaction process, 2.7 mL of TEOS in 27.3 mL of ethanol was rapidly added into a mixture containing 10.3 mL of ethanol, 18 mL of H2O, and 1.7 mL of ammonia. The reaction mixture was stirred for 5 h. The resulting SiO2NPs were isolated by centrifugation, washed with ethanol and water. 0.1 g SiO2NPs were dispersed in 15 mL of water and treated with ultrasonic for 30 s, 15 mL of 3APTMS and 80 mL C.I. Reactive Red 136 aqueous solution (0.1 g/mL) were added, and kept at room temperature for 3 h under magnetic stirring. The resulting red SiO2NPs were collected by centrifugation and washed with water and ethanol, finally dispersed in 2.5 mL of ethanol for further usage.
2. Experimental section 2.1. Chemicals and apparatus
2.3. Synthesis of the core-shell red SiO2NPs and carboxyl-modified core-shell red SiO2NPs
Tetraethyl orthosilicate (TEOS), 2-(N-morpholino)ethanesulfonic acid (MES) and Polyvinyl Pyrrolidone (PVP) were obtained from Aladdin Industrial Inc. (Shanghai, China); 3-[2-(2aminoethylamino)ethylamino]propyl-trimethoxysilane (3APTMS) was purchased from Tianjin Heowns Biochemical Technology Co., Ltd. (Tianjin, China); ethanol and ammonia (25e28 wt%) were purchased from Xilong Sientific Co., Ltd (Guangdong, China); carboxyethylsilanetriol sodium salt 25 wt% in water (CES) was obtained from J&K Scientific Co., Ltd (Shanghai, China); N-(3Dimethylaminopropyl)-N0 -ethylcarbodiimide hydrochloride crystalline (EDC) and N-Hydroxysuccinimide (NHS) were brought from Sigma-Aldrich (Shanghai, China); bovine serum albumin (BSA) and goat anti-mouse IgG were purchased from Shanghai Sangon Biotech Co., Ltd (Shanghai, China); Pierce BCA Protein Assay Kit was
€ber system containing 10 mL ethanol, 0.8 mL H2O, 0.2 mL A Sto TEOS and 0.1 mL NH3$H2O (25e28%) was hydrolyzed for 30 min. 2.5 mL of above prepared red SiO2NPs ethanol dispersion solution €ber system and react for was added into the pre-hydrolyzed Sto 12 h at room temperature under magnetic stirring. The core-shell red SiO2NPs were isolated by centrifugation and washed several times with ethanol and water. Carboxyl-modified core-shell red SiO2NPs were synthesized in the same system, before the stop of the reaction, 25 mL of CES was € ber system to react for another 24 h. The added into the above Sto resulting core-shell red SiO2NPs were covered with protective layer and carboxyl group. The final carboxyl-modified core-shell red SiO2NPs were isolated by centrifugation and washed several times with ethanol and water.
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2.4. Preparation of antibody-modified core-shell red SiO2NPs conjugates The antibodies were immobilized on the surface of carboxylmodified core-shell red SiO2NPs through carbodiimide chemistry with slight modification [22], briefly described in following procedures: 1 mg carboxyl-modified core-shell red SiO2NPs were diluted in MES (0.05 M, pH 6.1) with a total volume of 1 mL. 1 mg EDC and 1.5 mg NHS were added to the particle dispersion and allowed to activate for 15 min with gentle mixing. The activated carboxyl-modified core-shell red SiO2NPs were washed twice to remove unreacted chemicals and reconstituted in 1 mL of phosphate buffer (PB, 0.02 M, pH 7.2). 30 mg mAb1 was added into the activated particles and mixed gently for 2.5 h at room temperature. The conjugates were centrifuged and resuspended in 1 mL of 2% BSA (w/v) in PB to block unreacted sites for 1 h at room temperature. The blocked conjugates were centrifuged and resuspended in phosphate buffered saline (PBS, 0.01 M) containing 1% BSA (w/v), 5% sucrose (w/v), 3% trehalose (w/v) at a final concentration of 10 mg/mL and stored at 4 C before use. 2.5. Bacterial strains and preparation E. coli O157:H7 used as the detection antigen was grown in Lysogeny broth (LB) medium with shaking at 37 C. Cells were harvested in late exponential growth phase by centrifugation at 6000 rpm at 4 C for 10 min and washed in triplicate using physiological saline aqueous solution and dispersed in 5 mL physiological saline aqueous solution. Concentration of the bacteria was confirmed by the colony counting (CFU/mL). The enriched bacteria were inactivated with 0.4% formaldehyde and stored at 4 C before use. 2.6. Fabrication of immunochromatographic strip ICA test strips were composed of 4 components including sample pad, release pad, NC membrane and absorbent pad. A sample pad and release pad made from glass fiber were pre-treated with PBS buffer (0.01 M, pH 7.4) containing 1% BSA, 0.5% Tween-20, 3% sucrose (w/v), 1% trehalose (w/v), 0.5% PVP, further dried at 37 C overnight. The mAb2 (1.2 mg/mL) and goat anti-mouse IgG (1.0 mg/ mL) were sprayed onto the test (T) and control (C) lines on the NC membrane by solution sprayer. They were both sprayed on the NC membrane at the speed of 1 mL/cm and then dried at 37 C for 1 h. The NC membrane, absorption pad, pre-treated release pad and sample pad were assembled on a PVC board sequentially with a 1e2 mm overlap to form the final test strip (the fabrication steps are shown in Fig. 1s in the supplemental information), cut into 4 mm width by Bio-vinostech strip cutter and to package into the plastic cassette.
Fig. 1. SEM photograph of SiO2NPs (a), red SiO2NPs (b) and carboxyl-modified coreshell red SiO2NPs (c), inset: physical image of carboxyl-modified core-shell red SiO2NPs. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
2.7. Fabrication of core-shell red SiO2NPs-based ICA and analytical procedure
2.8. Detection of E. coli O157:H7 in pork and milk samples by coreshell red SiO2NPs-based ICA
To assess the performance of detection, the E. coli O157:H7 culture was diluted into different concentrations from 4.5 103 to 4.5 108 CFU/mL in sterile PBS, which were then mixed with the core-shell red SiO2NPs-antibody conjugate and wash buffer (0.01 M PBS containing 0.5% BSA, 1% Tween-20, 3% sucrose (w/v) and 1% trehalose (w/v). The ratio of above mixture consisted of 35 mL sample, 10 mL core-shell red SiO2NPs-antibody conjugate and 35 mL wash buffer, respectively. The detection was performed by applying 80 mL of the mixture on a sample pad of the test strip to react for 15 min. All the concentrations were performed in triplicate.
The pork and fresh milk were purchased from a local supermarket (Hangzhou, China). For pork tissue samples, 5 g of pork muscle homogenate was weighed and mixed with 10 mL PBS buffer in a sterile bag. The sample was centrifuged for 5 min at 10,000 rpm after extraction under homogeneity about 5 min. The supernatant was collected and fortified with different concentration of E. coli O157:H7 solution for ICA detection. And the fresh milk samples were diluted in PBS buffer by 10 fold, and spiked with different concentration of E. coli O157:H7. Spiked milk samples were directly detected without any further sample preparation. Every sample
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was performed in triplicate. 3. Results and discussion 3.1. Synthesis and characterization of core-shell red SiO2NPs The core-shell red SiO2NPs were synthesized by multistep method, the schematic diagram is presented in part A of Scheme 1. € ber system. Then Firstly, bare SiO2NPs were synthesized by Sto 3APTMS and C.I. Reactive Red 136 solution were added sequentially into the SiO2NPs aqueous dispersion to graft the dye molecules onto the surface of SiO2NPs. The hydrolysis and condensation of 3APTMS and dye coupling were preceded at the same time because hydrolysis and condensation of 3APTMS was a quick procedure in the presence of water. The water would increase the surface density of amine [23], thus, leading coloration of a great deal of dye molecules on the surface of SiO2NPs and showing a bright red color. To prevent the dye from shedding, the red SiO2NPs were introduced €ber system to carry out regrowth of the into a pre-hydrolyzed Sto € ber system, a small amount of NH3$H2O silica layer. In this Sto
catalyzed TEOS to hydrolyze, produce soluble silica covered with a large number of negative charges. The amino groups on the surface of red SiO2NPs were not fully occupied by the dye molecules, thus soluble silica can be absorbed onto the red SiO2NPs due to electrostatic interactions. With the catalysis of TEOS, the silica layer was formed through Si-O-Si bond between the soluble silica on the red SiO2NPs, to protect the dye on the particles, as well as the morphology and dispersity. The synthesis of core-shell red SiO2NPs and modification of carboxyl group were conducted by one step. The carboxyl-modified core-shell red SiO2NPs were directly produced by adding CES in the procedure of regrowth of silica protection layer. CES is a kind of silane coupling agent that can be grafted to the surface of SiO2NPs by through Si-O-Si bond in alkaline conditions. With the exist of a small amount of NH3$H2O, the CES was hydrolyzed gradually, eventually to modified carboxyl group on the core-shell red SiO2NPs. Conventionally, modification of carboxyl group on SiO2NPs was carried out by multistep functionalization [24]. Our synthesis method is simpler and cheaper than former modification method. Fig. 1 shows the SEM pictures of the SiO2NPs, red SiO2NPs
Scheme 1. Schematic representation of core-shell red SiO2NPs based ICA. (A) The procedure of synthesis and carboxyl modification of the core-shell red SiO2NPs; (B) The formation of mAb1-modified red SiO2NPs probes; (C) The dimensions of the core-shell red SiO2NPs based ICA; (D) Assay principle of core-shell red SiO2NPs-based ICA, the appearance of two lines indicates a positive result, whereas a valid negative test produces only the control line.
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and carboxyl-modified core-shell red SiO2NPs, all of them exhibit regular spherical morphology and good dispersity, demonstrating that there is almost no change in morphology after coloration and silica shell regrowth. Especially, carboxyl-modified core-shell red SiO2NPs show better dispersity compared with SiO2NPs and red SiO2NPs due to the surface of core-shell red SiO2NPs rich in carboxyl group. And they are perfectly spherical with an average diameter of about 216 nm. Zeta potential of core-shell red SiO2NPs and carboxyl-modified core-shell red SiO2NPs were also measured. As shown in Fig. 2, the core-shell red SiO2NPs present an isoelectric point around 7, while the carboxyl-modified core-shell red SiO2NPs are negatively charged in the range of pH 3e12. The isoelectric point of carboxylmodified core-shell red SiO2NPs shifts to much lower pH values suggesting that the density of carboxyl group on the core-shell red SiO2NPs surface is high on account of the introduction of CES. Compared with the AuNPs, the core-shell red SiO2NPs have good stability and don't fade in the harsh conditions such as pH and salt. Fig. 3 exhibits the influence of pH on AuNPs and core-shell red SiO2NPs. From Fig. 3a, the pH values of samples adjusted by NaOH and HCl solutions (0.1 M) are 2e13 from the left to right. The coreshell red SiO2NPs (bottom) show good stability and bright color, while AuNPs are easily aggregated, when the pH value is lower than pH 5 or higher than pH 10. Besides, we examined the influence of salt concentration on stability of core-shell red SiO2NPs and AuNPs. As shown in Fig. 3b, AuNPs begin degeneration even the salt concentration is as low as 0.1%. Compared with the AuNPs, core-shell red SiO2NPs remain monodispersed when the salt concentration is as high as 0.4%. Actually, core-shell red SiO2NPs can be dispersed without flocculation in the solution containing higher concentration of salt. Based on these advantages, core-shell red SiO2NPs have been selected to be used as label of ICA in this work.
3.2. Characterization of antibody-modified core-shell red SiO2NPs The mAb1 was immobilized on the surface of carboxyl-modified core-shell red SiO2NPs through carbodiimide chemistry. Fig. 4 shows the particle size distribution curves of carboxyl-modified core-shell red SiO2NPs and antibody-modified core-shell red SiO2NPs. The size distribution curve of antibody-modified coreshell red SiO2NPs moves slightly to the right compared with
Fig. 2. Zeta potentials of unmodified core-shell red SiO2NPs and carboxyl-modified core-shell red SiO2NPs dispersed in water with different pH. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
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carboxyl-modified core-shell red SiO2NPs. In accordance with the particle size distribution curve, the average diameter of carboxylmodified core-shell red SiO2NPs is 216 nm, and that of the antibody-modified core-shell red SiO2NPs is determined to be 236 nm, which proves the immobilization of antibodies on these particles. Besides, Pierce BCA Protein Assay Kit was used to examine the effect of mAb1 immobilization, and the rate of mAb1 that modified on the core-shell red SiO2NPs was more than 70% (More experimental details and results are shown in Tables S1 and S2 in the supplemental information, respectively). ICA detection was also employed to validate the antibodymodified core-shell red SiO2NPs conjugates. Goat anti-mouse IgG was coated on NC membrane at C line. The carboxyl-modified coreshell red SiO2NPs and antibody-modified core-shell red SiO2NPs conjugates were sprayed onto the release pad and dried, respectively. The sample pad, release pad, NC membrane and absorbent pad were integrated in accordance with part of 2.7. Then 80 mL of PBS was loaded on the sample pad and reacted about 10 min. The results were observed by naked eye and presented in Fig. 4 inset. It is observed that the antibody-modified core-shell red SiO2NPs conjugates are captured by goat anti-mouse IgG and show strong red color intensity at C line. On the contrary, the core-shell red SiO2NPs without antibody modification cannot be captured by IgG, the red color band is not observed. The combined results of particle size distribution curve, Pierce BCA Protein Assay and ICA detection demonstrate that mouse anti-E. coli O157:H7 mAb1 is successfully modified on the surface of carboxyl-modified core-shell red SiO2NPs. 3.3. Assay principle of core-shell red SiO2NPs-based ICA In the conventional sandwich ICA strip, the target analytes interact with the antibody-labeled particles in the conjugate pad, and flow laterally through the test strip. The complexes are then captured by the immobilized capture antibodies on the T line of the strip and form the sandwich immunocomplex which leads to a darkening color of the T line, which can be observed by naked eyes. Unlike the conventional ICA strip, a separated incubation of E. coli O157:H7 and mAb1-modified core-shell red SiO2NPs was proposed for this core-shell red SiO2NPs based ICA strip. Scheme 1 exhibits the schematically configuration principle of this core-shell red SiO2NPs based ICA system. As shown in part C of Scheme 1, E. coli O157:H7 and mAb1-modified core-shell red SiO2NPs form immune complexes. And the immune complexes are added onto sample pad of the test strip and flow laterally through the strip under capillary force. The complexes are then captured by the immobilized mAb2 and form a sandwich immunocomplexes, resulting in a red band on the T line. For the detection of non-E. coli O157:H7 analysts, there is no red band on the T line. The extra antibody-modified core-shell red SiO2NPs bound to goat antimouse IgG on C line regardless of the presence of E. coli O157:H7. The mAb1-modified core-shell red SiO2NPs form a red-colored band indicates validity of test result. This kind of ICA own some advantages, such as elimination of the antibody-particles conjugate pad, making the fabrication of the strip easier [25]. And the separated incubation of E. coli O157:H7 allows easier control of the reaction time, resulting in the complete interaction between E. coli O157:H7 and mAb1-modified core-shell red SiO2NPs. This effective incubation of E. coli O157:H7 with mAblabelled core-shell red SiO2NPs gives a high detection performance. 3.4. Sensitivity and specificity of core-shell red SiO2NPs-based ICA system To demonstrate the performance of this core-shell red SiO2NPs
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Fig. 3. The influences of pH value and salt on the core-shell red SiO2NPs and AuNPs. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 5. The sensitivity of the core-shell red SiO2NPs based ICA, E. coli O157:H7 was 10fold diluted from 4.5 108 to 4.5 103 CFU/mL (from left to right) by PBS, the rightmost strip was a negative control. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 4. The diameter distributions of the carboxyl-modified core-shell red SiO2NPs and antibody-modified core-shell red SiO2NPs, inset: the results of ICA detection of carboxyl-modified core-shell red SiO2NPs (up) and antibody-modified purple SiO2NPs (bottom). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
based ICA system, the color intensity of T line was observed within 15 min after adding the immune complexes. The range of detection was determined with different E. coli O157: H7 concentrations from 4.5 103 to 4.5 108 CFU/mL and with negative sample. The detection procedure was repeated three times and one of typical picture was obtained to show the detection efficiency. The ICA detection results of E. coli O157:H7 samples are shown in Fig. 5. As increasing of E. coli O157:H7 concentration in samples, the color at the T line is proportionally increased. In this present study, the visual inspection limit was defined as the minimum analyte concentration required for showing no obvious visual color on the T line. Following this definition, the color band of T lines was not visible when the bacteria concentration below 4.5 104 CFU/mL. Thus, 4.5 105 CFU/mL was considered to be the visual limit of detection (LOD) of this ICA strip for the detection of E. coli O157:H7, which is comparable to the detection performance with naked eyes of former reported other nanomaterials based ICA [25e27].
To assess the specificity and selectivity of core-shell red SiO2NPs-based ICA, we challenged the system against E. coli O157:H7 and other pathogen (E. coli, C. sakazakii, S. aureus, B. cereus, C. freundii and B. subtilis) at the bacteria concentration of 4.5 108 CFU/mL. After applying mixture solutions containing bacterium and mAb1-core-shell red SiO2NPs on the sample pad, the color intensity was observed within 15 min. The same detection process was repeated three times for each bacterium sample. As shown in Fig. 6, strong red signal of E. coli O157: H7 is observed,
Fig. 6. The cross reaction results of the core-shell red SiO2NPs based ICA, from the left to right: E. coli O157:H7, E. coli, C. sakazakii, S. aureus, B. cereus, C. freundii, B. subtilis respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
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immunomagnetic nanoparticles to isolate and enrich E. coli O157:H7 [29]. In future research, if the selective improved medium or immunomagnetic enrichment method can be used for milk and meat, the sensitivity will be improved. In this study, the development of colored silica nanoparticles based ICA is in the preliminary stage. 4. Conclusion
Fig. 7. Detection of E. coli O157:H7 in milk (a) and pork (b) food samples by the coreshell red SiO2NPs based ICA. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
while the other bacteria show no signal on T line. The good specificity of core-shell red SiO2NPs based ICA is because of the specificity reaction between the antigen and antibody. The monoclonal antibody can specifically identify antigen site on E. coli O157, but does not recognize other bacteria. In the specific experiment, other bacteria model cannot form sandwich structure with mAb1-coreshell red SiO2NPs, so cannot produce the red band on the T line. These results demonstrate high specificity of this ICA strip against other nonspecific bacteria used in this work. From these results both in sensitivity and specificity detection, it is also found that the intensities of the red color at C line are nearly constant for all strips.
In this work, we used the core-shell red SiO2NPs as novel visual probes in ICA for the detection of E. coli O157:H7. A model system comprising a target E. coli O157:H7 analyte and a pair of antibody probes were used to demonstrate the proof-of-concept. Under optimal conditions, the visual qualitative inspection limits (the red color of T line) significantly observed when the concentration of E. coli O157:H7 was as low as 105 CFU/mL and 106 CFU/mL in sterile PBS and real samples, respectively. This core-shell red SiO2NPs based immunochromatographic sensor was simple, fast and without complicated sample preparation process, it is suitable for field detection. The immunoassay system in this work also has acceptable specificity and no cross activity with six other bacteria. In addition, the optimized newly core-shell colored SiO2NPs based ICA can be extended in food quality control, environmental monitoring and clinical analysis. In the future there is still a lot of work can be done to explore the improvement and application of colored SiO2NPs based ICA. Such as, combine colored SiO2NPs probes based ICA and strip reader, more sensitive and quantitative detection of E. coli O157:H7 can be obtained partially in a wide concentration range with good repeatability. We will also develop a sensitive and quantitative sandwich ICA for rapid detection of hepatitis B surface antigen based on red SiNPs in our next work. By synthesizing and using blue SiO2NPs and red SiO2NPs, a multiplex ICA can be developed for the simultaneous detection of ractopamine and clenbuterol hydrochloride. Compliance with ethical standards
3.5. Application in milk and pork samples by core-shell red SiO2NPs-based ICA Based on the above mentioned detection conditions, E. coli O157:H7 cultures at different concentrations in pork and milk samples were detected with this core-shell red SiO2NPs based ICA, each sample was examined in triplicate. The typical results for pork and milk samples are shown in Fig. 7a and Fig. 7b, respectively. It is demonstrated that E. coli O157:H7 of 4.5 106 CFU/mL can be detected in both milk and pork samples using this detection system. When the ICA strip was used to detect E. coli O157:H7, the detection limit of ICA increased from 4.5 105 to 4.5 106. The reason is ascribed to the fat and protein may influence the detection of E. coli O157:H7 in real sample. As the E. coli O157:H7, it is forbidden to exist in any food matrix [28]. For rapid detection methods of E. coli O157:H7, they rely on enrichment to make it reaches an enough concentration, such as immunological assays, serological tests, PCR-based method and DNA probes. For example, the selective media are one of the most commonly used methods for detection of E. coli O157:H7. As to different matrix, like milk or pork sample, if we use selective improved medium to culture E. coli O157:H7 for 8e12 h to enough concentration, they could be detected. And the time of 8e12 h is much shorter than that of culture based methods, which often require 4e7 days to obtain a confirmed result for E. coli O157:H7. The sensitivity of the ICA strip can also be improved by immunomagnetic nanoparticles. The ICA strips have been used to detect E. coli O157:H7 in real samples using
The authors declare that they have no conflict of interest. This article does not contain any studies with animals performed by any of the authors. Informed consent was obtained from all individual participants included in the study. Acknowledgement This work was financially supported by a grant from National Natural Science Foundation of Zhejiang Province (LY17C200003), the Food and Engineering most important discipline of Zhejiang province (2017SIAR210, JYTSP20141062), Zhejiang public Innovation Platform Analysis and testing project (2018C37056), Zhejiang Xinmiao Talents Program (1110KZN0217054G, 1110KZN0217053G), open fund of State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, the First Affiliated Hospital of Medical College, Zhejiang University (2017KF02). Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.aca.2018.07.003. References [1] D. Zhang, P. Li, Q. Zhang, W. Zhang, Ultrasensitive nanogold probe-based immunochromatographic assay for simultaneous detection of total aflatoxins in peanuts, Biosens. Bioelectron. 26 (2011) 2877e2882. [2] G.A. Posthumatrumpie, J. Korf, A. Van Amerongen, Lateral flow (immuno) assay, Anal. Bioanal. Chem. 393 (2015) 569e582.
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[3] D. Zhang, P. Li, Q. Zhang, W. Zhang, Ultrasensitive nanogold probe-based immunochromatographic assay for simultaneous detection of total aflatoxins in peanuts, Biosens. Bioelectron. 26 (2011) 2877e2882. [4] X. Mao, W. Wang, T.E. Du, Dry-reagent nucleic acid biosensor based on blue dye doped latex beads and lateral flow strip, Talanta 114 (2013) 248e253. n, J. Wichers, A. Abad-Somovilla, A.A. Van, A. Abad-Fuentes, [5] C. Su arez-Pantaleo Development of an immunochromatographic assay based on carbon nanoparticles for the determination of the phytoregulator forchlorfenuron, Biosens. Bioelectron. 42 (2013) 170e176. [6] W. Qiu, H. Xu, S. Takalkar, A.S. Gurung, B. Liu, Y. Zheng, Z. Guo, M. Baloda, K. Baryeh, G. Liu, Carbon nanotube-based lateral flow biosensor for sensitive and rapid detection of DNA sequence, Biosens. Bioelectron. 64 (2015) 367e372. [7] J. Joo, C. Yim, D. Kwon, J. Lee, H.H. Shin, H.J. Cha, S. Jeon, A facile and sensitive detection of pathogenic bacteria using magnetic nanoparticles and optical nanocrystal probes, Analyst (Cambridge, U.K.) 137 (2012) 3609e3612. [8] K. Rajesh, S. Chandrak, K. Suchitra, S. Rajeshwarp, B. Rajk, K. Devendran, Development of nanocolloidal gold based immunochromatographic assay for rapid detection of transgenic vegetative insecticidal protein in genetically modified crops, Food Chem. 122 (2010) 1298e1303. [9] R.P. Bagwe, C. Yang, L.R.H. And, W. Tan, Optimization of dye-doped silica nanoparticles prepared using a reverse microemulsion method, Journal of Surfaces & Colloids 20 (2004) 8336. [10] B. Li, J. Shen, R. Liang, W. Ji, C. Kan, Synthesis and characterization of covalently colored polymer latex based on new polymerizable anthraquinone dyes, Colloid Polym. Sci. 290 (2012) 1893e1900. [11] H. Giesche, E. Matijevi, Well-defined pigments: I. Monodispersed silica-acid dyes systems, Dyes Pigments 17 (1991) 323e340. [12] F.M. Winnik, B. Keoshkerian, J.R. Fuller, P.G. Hofstra, New water-dispersible silica-based pigments: synthesis and characterization, Dyes Pigments 14 (1990) 101e112. [13] H. Yu, G. Zhao, W. Dou, Simultaneous detection of pathogenic bacteria using agglutination test based on colored silica nanoparticles, Curr. Pharmaceut. Biotechnol. 16 (2015) 716e723. [14] Q. Sun, G. Zhao, W. Dou, A nonenzymatic optical immunoassay strategy for detection of Salmonella infection based on blue silica nanoparticles, Anal. Chim. Acta 898 (2015) 109e115. [15] Q. Sun, G. Zhao, W. Dou, Blue silica nanoparticle-based colorimetric immunoassay for detection of Salmonella pullorum, Analytical Methods 7 (2015) 8647e8654. [16] Q. Sun, G. Zhao, W. Dou, An optical and rapid sandwich immunoassay method for detection of Salmonella pullorum and Salmonella gallinarum based on immune blue silica nanoparticles and magnetic nanoparticles, Sensor. Actuator. B Chem. 226 (2016) 69e75.
[17] J.K. Varma, K.D. Greene, M.E. Reller, S.M. Delong, J. Trottier, S.F. Nowicki, M. Diorio, E.M. Koch, T.L. Bannerman, S.T. York, An outbreak of Escherichia coli O157 infection following exposure to a contaminated building, JAMA, J. Am. Med. Assoc. 290 (2003) 2709e2712. [18] R.S. Quilliam, A.P. Williams, L.M. Avery, S.K. Malham, D.L. Jones, Unearthing human pathogens at the agriculturaleenvironment interface: a review of current methods for the detection of Escherichia coli O157 in freshwater ecosystems, Agri. Ecosyst. Environ. 140 (2011) 354e360. [19] S. Shan, D. Liu, Q. Guo, S. Wu, R. Chen, K. Luo, L. Hu, Y. Xiong, W. Lai, Sensitive detection of Escherichia coli O157:H7 based on cascade signal amplification in ELISA, J. Dairy Sci. 99 (2016) 7025e7032. [20] T. Zhang, Q. Zhang, J. Ge, J. Goebl, M. Sun, Y. Yan, Y. Liu, C. Chang, J. Guo, Y. Yin, A self-templated route to hollow silica microspheres, J. Phys. Chem. C 113 (2009) 3168e3175. [21] W. Wei, B. Gu, L. Liang, W. Hamilton, Fabrication of two- and threedimensional silica nanocolloidal particle arrays, J. Phys. Chem. B 107 (2003) 3400e3404. [22] S. Bamrungsap, C. Apiwat, W. Chantima, T. Dharakul, N. Wiriyachaiporn, Rapid and sensitive lateral flow immunoassay for influenza antigen using fluorescently-doped silica nanoparticles, Microchimica Acta 181 (2013) 223e230. [23] P.J.E. Harlick, A. Sayari, Applications of pore-expanded mesoporous silica. 5. Triamine grafted material with exceptional CO2 dynamic and equilibrium adsorption performance, Ind. Eng. Chem. Res. 46 (2007) 446e458. [24] X. Zhao, L.R. Hilliard, S.J. Mechery, Y. Wang, R.P. Bagwe, S. Jin, W. Tan, A rapid bioassay for single bacterial cell quantitation using bioconjugated nanoparticles, Proc. Natl. Acad. Sci. U.S.A. 101 (2004) 15027. [25] L. Zhang, Y. Huang, J. Wang, Y. Rong, W. Lai, J. Zhang, T. Chen, Hierarchical flowerlike gold nanoparticles labeled immunochromatography test strip for highly sensitive detection of Escherichia coli O157:H7, Langmuir 31 (2015) 5537e5544. [26] H. Qi, Z. Zhong, H.X. Zhou, C.Y. Deng, H. Zhu, J.F. Li, X.L. Wang, F.R. Li, A rapid and highly sensitive protocol for the detection of Escherichia coli O157:H7 based on immunochromatography assay combined with the enrichment technique of immunomagnetic nanoparticles, Int. J. Nanomed. 6 (2011) 3033e3039. [27] B.Y. Jung, S.C. Jung, C.H. Kweon, Development of a rapid immunochromatographic strip for detection of Escherichia coli O157, J. Food Protect. 68 (2005) 2140e2143. [28] Y. Yoon, S. Lee, K.H. Choi, Microbial benefits and risks of raw milk cheese, Food Contr. 63 (2016) 201e215. [29] Q. Xiong, X. Cui, J.K. Saini, D. Liu, S. Shan, Y. Jin, W. Lai, Development of an immunomagnetic separation method for efficient enrichment of Escherichia coli O157:H7, Food Contr. 37 (2014) 41e45.