New Functional TracerâTwo-Dimensional Nanosheet-Based

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New functional tracers-two dimensional nanosheets based immunochromatographic assay for Salmonella enteritidis detection Tong Bu, Jianlong Wang, Lunjie Huang, Leina Dou, Bingxin Zhao, Tao Li, and Daohong Zhang J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 22 May 2019 Downloaded from http://pubs.acs.org on May 25, 2019

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Journal of Agricultural and Food Chemistry

New functional tracers—two dimensional nanosheets based

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immunochromatographic assay for Salmonella enteritidis detection

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Tong Bu,‡a Jianlong Wang,‡a Lunjie Huang,b Leina Dou,a Bingxin Zhao,a Tao Lic and

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Daohong Zhanga*

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a

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712100, Shaanxi, China

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b

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Guangzhou 510641, China

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c Shaanxi

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College of Food Science and Engineering, Northwest A&F University, Yangling

School of Food Science and Engineering, South China University of Technology,

Institute for Food and Drug Control, Xi'an, 710065, China

*Corresponding author. E-mail: [email protected];

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Fax: +86 29-8709-2275;

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Tel: +86 29-8709-2275

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‡ These authors contributed equally to this work

ACS Paragon Plus Environment


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Abstract

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The rapid monitoring of foodborne pathogens by monoclonal antibody (McAb) based

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immunochromatographic tests (ICTs) is desirable but highly challenging due to the

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screening obstacle for a superior performance probe which will greatly determine the

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capture efficiency of targets and the sensitivity of immunoassay. In this work, based

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on two dimensional (2D) nanosheets (including MoS2 and Graphene) as the

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extraordinary capture probe and signal indicator, we fabricated a label-free ICT

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method for Salmonella enteritidis (S. enteritidis) detection. Especially, without the

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customarily labeled Ab probe, these 2D versatile probes presented strong capture

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ability toward bacteria by directly assembling onto the surface of bacteria. An ideal

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analytical performance with high sensitivity and specificity was achieved by virtue of

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the novel nanosheets-bacteria-McAb sandwich format. Based on MoS2 2D nanosheets

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as a fabulous probe element, the developed ICT exhibited a lowest detectable

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concentration of 103 CFU/mL for S. enteritidis and could be well applied in drinking

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water and watermelon juice samples. By the smart design, this work gets rid of series

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conditionality issues of traditional double antibody sandwich based ICTs, and can

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give a new application direction for 2D nanosheet materials in the rapid detection

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field.

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Keywords: 2D nanosheets; Label-free; Immunochromatographic test; Salmonella

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enteritidis

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Introduction

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Bacterial contaminations and infections have always been a major threat to public

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health and human life.1 The recent outbreaks of diseases continue to highlight the

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need for diagnostic tools that are affordable, sensitive, simple, and rapid for pathogens

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detection under challenging circumstances. These demands have led to the popularity

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of paper-based ICTs as the most prominent rapid point-of-care (POC) diagnostic

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tests.2-4 It is generally known that the efficient capture ability of recognition agents

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toward targets is an essential prerequisite for all immunoassay-based methods. To

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achieve this, in the conventional sandwich ICTs both the signal nanomaterials and the

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test (T)-line must be decorated with antibodies (typical affinity ligands) that are able

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to separately bind two discrete regions of the target.5 In this sandwich format, the

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binding affinity or interaction strength between a specific antigen and antibody can

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intrinsically determine the detection performance.6 Unfortunately, the practical

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applications of ICTs are often limited by some uncontrollable factors of antibodies: (1)

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complicated cross-linking procedures between antibodies and nanomaterials are still

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required for probes preparation;7 (2) the binding ability of antibody is likely to be

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affected during the labeling process; (3) the labeled antibody’s capacity of resistance

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to rough chemical conditions is a key issue must to be considered because the

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antibody often needs to face unsatisfactory circumstances such as organic extracts or

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complex sample matrices when detecting targets.8

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Taking into account of the above issues, the preparation of advanced label-free

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materials, which can be directly used as efficient adsorbents to bacteria is

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advantageous and urgently needed. Very recently 2D materials, e.g., graphene,

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layered boron nitride, and transition metal dichalcogenides (TMDs), have attracted

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great attention owing to their unique large surface area, strong surface adsorb ability

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and low cost.9-14 The huge surface of 2D nanosheets can non-selectively interact with

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bacteria via van der Waals forces, as well as electrostatic and hydrophobic

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interactions etc.15 Simultaneously, these outstanding materials have been explored as

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tools to kill pathogenic microbes through direct contaction with the bacteria, sharp

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edges of nanosheets may induce membrane stress by puncturing or penetrating into

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the cell membranes, resulting in the morphological destruction of bacterial cells and

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leakage of intracellular components, such as proteins, phospholipids, RNA, and

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DNA.16,

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(including graphene-based materials and TMD nanosheets) may have potentials as

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label-free bacteria adsorbents to replace traditional probes in the ICT.

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Therefore, the admirable characteristics suggest that these 2D materials

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Herein, on the basis of the above mentioned thought, with S. enteritidis as a model

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bacteria, 2D materials, including MoS2 and graphene were synthesized as

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cross-linking agents for bacteria capture and applied in the ICT method. This strategy

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exempted the use of traditional label materials and probes, opened a new application

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field for 2D materials. Taking MoS2 for example, the S. enteritidis in the sample were

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first captured by MoS2, and then the MoS2-S. enteritidis complex was selectively

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immunocaptured by the detection McAb coated on T-line. The introduction of 2D

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materials as adsorbents in immunoassays for pathogen detection not only got rid of a

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series of troublesome marking steps and inherent disadvantages of the capture

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antibody probe providing an unexpected alternative to traditional probes, but also

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avoid the use of paired McAbs obtained by time-consuming screening process. The

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novel ICT format developed here allows the test of S. enteritidis in spiked drinking

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water and watermelon juice samples, and can push the application of 2D nanosheet

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materials in POC tests.

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Materials and methods

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Reagents and chemicals. The McAb against target S. enteritidis flagellin was

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prepared in our laboratory as previously reported.18 MoS2 (bulk particle size < 2 µm),

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and expanded graphite were acquired from Sigma Aldrich. Ethanol was obtained from

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Sinopharm Chemical Reagent Co., Ltd. Drinking water and watermelon juice were

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provided from a supermarket. Nitrocellulose (NC) reaction membrane, conjugate pad,

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sample pad, and absorbent pad were supplied by Shanghai Kingdiag-biotech CO., Ltd.

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Luria-Bertani (LB) medium was purchased from Beijing Land Bridge Technology

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Co., China. All solvents and other chemicals used in this work were of the best degree

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and used as received.

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Bacterial strains. In this study, S. enteritidis (ATCC 13076) was used as the model

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target bacteria for the proof of concept, and Salmonella typhimurium (S. typhimurium)

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(ATCC 14028), Salmonella paratyphi B (S. paratyphi B) (ATCC 10719),

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Salmonella hadar (S. hadar) (ATCC 51956), Salmonella london (S. london) (ATCC

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8389), Staphylococcus aureus (S. aureus) (ATCC 25923), Candida albicans (C.

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albicans) (ATCC 96268), Escherichia coli O157 (E. coli O157) (ATCC 43889),

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Listeria monocytogenes (L. monocytogenes) (ATCC 19114) and Campylobacter coli

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(C. coli) (ATCC 43461) were used as the non-target interfering bacteria for the

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specificity evaluation. The strains were cultured in LB liquid medium at 37 C for

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18–24 h under shaking at 150 rpm, respectively. Then, the bacteria stock solution was

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diluted with the sterile phosphate buffered solution (PBS) to 108 CFU/mL as the

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testing concentration.

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Preparation of MoS2 and Graphene nanosheets. MoS2 nanosheets were prepared

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according to mixed-solvent exfoliation method with little modifications.19 Herein, 50

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mg of MoS2 powder was added to 100 mL of ethanol-water (45% v/v) solution. The

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mixture was sonicated for 48 h, and the resulting colloidal dispersion was then

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purified by three centrifugation/redispersion cycles at 3,000 rpm for 10 min, the

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supernatant was replaced with fresh deionized water each time. Graphene nanosheets

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were prepared using the same procedures as aforementioned.

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Characterization of the nanosheets. Scanning electron microscopy (SEM) using a

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Hitachi S-4800 was employed for the morphological characterization of nanosheets.

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For transmission electron microscopy (TEM) characterization, samples were

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drop-casted onto carbon-coated copper grids (Electron Microscopy Sciences), and

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TEM imaging was performed using a JEOL 2100F at 80 kV. Fourier transform

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infrared (FTIR) spectra were performed using Perkin-Elmer spectrometer in the

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frequency range of 4000–400 cm−1 with a resolution of 4 cm−1. Absorbance spectra

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were obtained with an UV-vis spectrophotometer.

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Assembly of the immunochromatographic test strip. The ICT strip from top to

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bottom was composed of an absorbent pad, reaction membrane, conjugate pad and

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sample pad as illustrated in Scheme 1B. The absorbent pad was used as received

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without further modification. The T-line on NC membrane was drawn with detection

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McAb solution (1 mg/mL dispersed in PBS buffer, pH 7.4) at a speed of 1 μL/cm.

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The distance between the T-line and the bottom of the strip was 7 mm. Then, the NC

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membrane was dried for 1 h at 37 °C and stored under dry conditions at 4 °C until use.

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Sample and conjugate pads were blocked by soaking them into the immunobuffer (2%

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BSA in PBS), followed by drying overnight at 37 C. The polyvinyl chloride (PVC)

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sheet was used as the backing plate of the test strip. After that, all of the parts

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mentioned above were assembled onto the PVC adhesive backing card, then the

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assembled card was cut into 3 mm wide strips and stored at a desiccator.

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Immunoassay procedure and performance evaluation of the developed ICT. S.

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enteritidis of different concentrations ranging from 0 to 108 CFU/mL in 100 μL PBS

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buffer was separately incubated with 1.5 mg/mL of MoS2 (30 μL) or Graphene (20 μL)

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for 10 s. After addition onto the bottom of strip, the mixture migrated from sample

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pad to the absorbent pad under the force of capillary action. After 10 minutes, the

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result of the lateral flow test was recorded by a camera and analyzed with Image J

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software to obtain the signal intensity of T-line.20, 21

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To investigate the specificity of the ICT for S. enteritidis detection, other common

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strains, including S. london, S. typhimurium, S. paratyphi B and S. hadar, and 5

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non-Salmonella strains of S. aureus, E. coli O157, C. albicans, L. monocytogenes and

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C. coli, all with a concentration of 108 CFU/mL, were tested along with S. enteritidis

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of a same concentration.

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Detection of S. enteritidis in food samples. Drinking water and watermelon juice

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were employed as matrices for the detection of S. enteritidis in food samples. After

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high-temperature sterilization and filtration (through 0.22 µm sterile filters), the

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samples were confirmed to be free of any bacteria by plate counting method.22 To

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minimize the matrix effects, 1 mL of each sample solution was diluted with 9 mL

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PBS buffer. Then different concentrations (0 to 108 CFU/mL) of S. enteritidis were

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inoculated to the diluted sample solution.23 Finally, the solution was tested by the 2D

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nanosheets based ICT.

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Results and discussion

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Relevant interpretation on the 2D nanosheets based ICT system. 2D materials

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provide an ideal platform for target capture by virtue of their large surface area which

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is retained independent of the common recognition molecule of antibody. The

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versatile nanosheets capture reagents were synthesized by simple sonication as

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revealed in Scheme 1A. The schematics in Scheme 1B-1D highlight the essential

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components and detection process of the nanosheets based ICT. During the assay, 2D

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materials were firstly added into the sample solution and incubated for a period of

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time. 2D nanosheets could interact with bacteria rapidly owing to their huge

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adsorption surface (Scheme 1C). Next, the nanosheets-bacteria complex solution was

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dropped onto the sample pad of the lateral flow strip, and immediately migrated

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toward the detection antibody bearing T-line by capillary action. For a positive

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sample, S. enteritidis in the solution could then be sandwiched by the 2D nanosheets

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and detection antibody in the T-line, forming a clear black-gray line that was visible

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with naked eyes due to the intrinsic absorbance of nanosheets bound at the T-line. On

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the other hand, for a negative sample, no visible band would appear on T-line.

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Because of the unambiguous colored conjugates of nanosheets-bacteria-McAb

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deposited at the T-line, the assay result could be easily readout by naked eyes or

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recorded by a camera for quantitative determination (Scheme 1D).

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Characterization of exfoliated nanosheets and nanosheets-bacteria conjugate.

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The typical morphologies, structures, and dispersivity of the nanosheets were

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characterized by TEM and SEM. As shown in Fig. 1A-1D, MoS2 and graphene

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nanosheets have a well-defined laminar morphology with a uniform size of around

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200 nm and 500 nm, respectively. Besides, they render as wrinkled sheets and are

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highly dispersed rather than large-size aggregated bulks, which are consistent with

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what are usually observed for exfoliated layered compounds.24 FT-IR spectra of MoS2

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and Graphene were recorded between 4000 and 400 cm-1 (Fig. S1). These results

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demonstrated the 2D nanosheets were successfully prepared. As observed from Fig.

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1E and 1F, the UV-vis spectra of MoS2 and Graphene locate around 624 nm, 683 nm

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and 270 nm, which are in good agreement with dispersed, layered MoS2 and

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Graphene obtained from previously reported results.19,

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peaks for bacteria in the full-wave band,26 while the UV-vis spectra of MoS2-bacteria

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and Graphene-bacteria composites all exhibit the same typical characteristic

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absorption bands of two nanosheets, demonstrating that they had been adsorbed onto

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the bacteria surface. The huge surface of 2D nanosheets can non-selectively interact

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with bacteria via van der Waals forces, as well as electrostatic and hydrophobic

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There are no absorption


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interactions etc.15 Taking this advantage, the formation of the nanosheets-bacteria

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conjugate was investigated. With MoS2-bacteria as an example, once MoS2

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nanosheets were added to the bacteria solution, obvious aggregation and microbial

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arrest occurred immediately. To further explore the adsorption ability of 2D

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nanosheets, SEM was employed to directly observe the morphological changes of the

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bacteria and nanosheets. Figure 1G and 1H show the outer morphologies of S.

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enteritidis treated with MoS2 and Graphene. It can be seen that massive nanosheets

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adhered to the surface of bacteria (red arrows). To evaluate the antibacterial ability of

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MoS2 and Graphene nanosheets against S. enteritidis, bacteria in the agar plates were

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incubated with 1.5 mg/mL of MoS2 and Graphene at 37 °C for 12 h, with PBS as the

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blank control. As shown in Fig. 1I, the small white spots stand for the living bacteria

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on the culture plates. In comparison, many small white spots can be seen with only

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PBS buffer, however, the number of small white spots decreased after incubation with

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2D nanosheets, indicating that the MoS2 and Graphene have prominent antibacterial

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activity by combining with bacteria. In addition, after conjugation with bacteria, a

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marked change in zeta potential of the MoS2 had taken place, take MoS2 for instance,

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after combining with S. enteritidis, the zeta potential changed from -18.3 mV to -9.6

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mV (Fig. S2). These results were all attributed to the strong interactions between 2D

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nanosheets and bacteria cell walls.

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Optimization of the assay conditions. As key parameters, some assay conditions,

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including the concentration and volume of MoS2 and Graphene, the incubation time

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of 2D nanosheets and S. enteritidis, and the pH value of bacterial suspension were

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investigated to achieve ideal analytical performances of the MoS2-ICT and

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Graphene-ICT. Coloration assays were conducted to observe and compare the T-line

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intensity of ICT strips under different experimental conditions. The amounts of 2D

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materials played an important role in the detection performance of the novel strip

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platform, especially in assay cost and sensitivity. In detail, 0.5, 1, 1.5, and 2 mg/mL of

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MoS2 and Graphene solutions were collected by serial dilution with ultra-pure water.

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The optimization results were shown in Fig. 2A, with the increase of MoS2 quantity,

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the color intensity on T-line enhanced gradually until the MoS2 concentration raised to

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1.5 mg/mL, and thereafter kept at an almost constant level. In addition, we observed

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that the T-line color of Graphene-ICT deepened with the increase of Graphene

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between 0.5–1.5 mg/mL and kept almost consistent during 1.5–2 mg/mL. Thereby,

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1.5 mg/mL was considered to be the optimal concentration of both MoS2 and

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Graphene. Then, the color development on T-line of the strip was examined under

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different volumes (5, 10, 15, 20, 30, 40 μL) of MoS2 and Graphene respectively

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mixing with 100 μL of 108 CFU/mL bacteria solution. As shown in Fig. 2B, the T-line

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signal increased obviously with the increasing volume of both nanosheets, and almost

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reached to the maximal value at 30 μL and 20 μL separately for MoS2 and Graphene,

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suggesting that the binding between 2D nanosheets and S. enteritidis had tended to be

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saturated under that condition. Thus, 30 μL and 20 μL of 1.5 mg/mL MoS2 and

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Graphene was screened out for the further experiment, respectively. Simultaneously,

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the influence of incubation time on the color development of strip’s T-line was also

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examined. The mixture of 2D nanosheets and bacteria was allowed to incubate for 10,

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60, 120, 180, 240 and 300 s, respectively. As shown in Fig. 2C, the color intensity on

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T-line shows an obvious downward tendency with the increasing of incubation time.

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Maybe the long reaction time between absorbent and bacteria could make the

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resultant conjugate sink, resulting in a hard chromatographic movement of the

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reaction solution. Thereby, 10 s of incubation time was the best condition. Moreover,

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the pH value of the tested solution has a great impact on the protein-protein

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interaction.27 So, we also studied the pH influence of bacterial solution. As shown in

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Fig. 2D, 108 CFU/mL S. enteritidis solution of different pH was respectively mixed

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with a fixed concentration of MoS2 and Graphene; the color intensity of T-line

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obviously varied with the changing of the solution pH value. When the pH was in the

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range of 5 to 10, the color of T-line was extremely deep. When the pH value reached

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7, the highest color signal on T-line was obtained. Therefore, pH 7.0 was selected for

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the following experiments.

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Sensitivity and specificity evaluations of the 2D nanosheets based ICT. The

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primary assay performances of sensitivity and specificity of the lateral flow assay for

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S. enteritidis detection were evaluated by qualitative and quantitative analyzing

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different concentrations (0–108 CFU/mL) of standard S. enteritidis solutions. Under

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optimized conditions, the experiments were performed according to the general

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procedure as described in the previous section. As can be seen from Fig. 3A and 3C, a

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dramatic increase of color intensity on the T-line of MoS2-ICT is observed as the S.

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enteritidis concentration increases from 103 to 108 CFU/mL. The calibration curve for

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S. enteritidis detection was shown in Fig. 3E, and a dynamic range from 104 to 108

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CFU/mL for S. enteritidis was achieved with a correlation coefficient (R2) of 0.9905.

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Moreover, Fig. 3B and 3D show the corresponding results of the Graphene-ICT, a

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weakest gray-black T-line can be observed by naked eyes at 104 CFU/mL of S.

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enteritidis. The calibration curve (Fig. 3F), constructed after image analysis,

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demonstrated a linear relationship during the S. enteritidis concentration of 104–108

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CFU/mL. The linear regression from the calibration curve presented a correlation

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coefficient of 0.989. In addition, after characterization by enzyme linked

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immunosorbent assay (ELISA), the sensitivity of McAb for S. enteritidis detection

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was 103 CFU/mL (Fig. S3). And we also summarized the recently published results

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for Salmonella detection by ICTs based on different signals (Table 1). The

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comparison result revealed that our 2D nanosheets based ICT device could rival and

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surpass these reports in LOD in the absence of capture antibody, which was of great

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benefit to the low-cost application in both resource-rich and resource-limited settings.

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A reliable specificity is extremely important for the further application of any

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immunoassay biosensor. To evaluate the specificity of this novel lateral flow strip test,

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the disturbance intensity of nine kinds non-target foodborne pathogens on the

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detection was investigated at a same bacteria concentration of 108 CFU/mL. As

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shown in Fig. 4, 108 CFU/mL of S. enteritidis can bring strong color development,

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while other non-target bacteria can not induce any obvious color on T-lines,

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indicating that our ICT biosensor exhibited a high selectivity to S. enteritidis, which

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was consistent with the evaluation results of ELISA (Fig. S4). The excellent results

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should be attributed to the specific McAb which was elicited by Salmonella flagellin

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in the animals.28

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Application in of the nanosheets based ICT in food samples. As a point-of-care

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testing or on-site detection method, its acceptability highly depends on the assay

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performance and applicability.33 Samples inoculated with different concentrations of S.

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enteritidis were analyzed by the nanosheets based ICT according to the

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aforementioned procedure. As shown in Fig. 5A and 5C, the color intensity of strip’s

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T-line keeps strengthening with the gradual increase of S. enteritidis concentration

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from 103 to 108 CFU/mL and 104 to 108 CFU/mL separately for the MoS2-ICT and

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Graphene-ICT in drinking water, which corresponded to the results in PBS buffer. On

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the other side, the color signal changes slightly in strips for the watermelon juice tests

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compared with that in standard bacterial solution (Fig. 5B and 5D), which might be

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ascribed to the potential matrix effect of watermelon juice.34 In order to further certify

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the accuracy and precision of results, recovery studies were conducted by analyzing

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drinking water and watermelon juice spiked with 106, 107 and 108 CFU/mL of S.

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enteritidis. The analysis was carried out with three replicates for each addition

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concentration (Table 2). The recovery rates of S. enteritidis in the two kinds samples

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displayed in Table 2 were 88.75–101.48% and 85.5–100.55%, respectively, with all

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relative standard deviation (RSD) values lower than 5.6%, verifying the reliability of

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the proposed ICT for S. enteritidis detection in practical applications.

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In summary, the label-free 2D nanosheets probe based immunochromatographic

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test, developed in this research was superior to the conventional antibody labeled

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probe based ICTs, and was well validated by two kinds of nanosheet materials with S.

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enteritidis as a model analyte. The commendable 2D nanosheets exhibited strong

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adsorption binding capacity toward bacteria and showed intense color development on

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strip’s T-line even at a low concentration. The introduction of the label-free,

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non-protein and colored nanosheets probe could not only avoid the use of paired

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McAbs obtained by time-consuming screening process, but also get rid of series

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troublesome issues in the probe preparation. Due to the superior adsorption ability of

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2D nanosheets and the tremendous selectively of anti-S. enteritidis McAb coated on

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T-line, high efficiency, sensitivity and specificity performances were achieved by

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means of this ICT biosensor. Under this novel sandwich detection format, low

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concentrations of S. enteritidis in drinking water (103 CFU/mL) and watermelon juice

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(104 CFU/mL) could be easily detected by naked-eyes in 10 min. This work has

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further extended the application field of 2D nanosheet materials. This rapid, sensitive

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and cost-effective lateral chromatography platform is of great value in the pathogens

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detection and has broad application potential in disease diagnosis, food inspection and

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environmental monitoring, especially suitable for POC tests.

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ASSOCIATED CONTENT

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Supporting Information

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FTIR spectra of the MoS2 and Graphene; Zeta potentials of the MoS2 and MoS2-S.

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enteritidis complex; Sensitivity analysis curve of the McAb for Salmonella detection;

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Specificity test results of anti-S. enteritidis McAb with different pathogens as

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interfering bacteria. The Supporting Information is available free of charge on the

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ACS Publications website.

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AUTHOR INFORMATION

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Corresponding Author

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*Daohong Zhang. Phone/fax: +86 29-8709-2275. E-mail: [email protected]

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ORCID

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Jianlong Wang: 0000-0002-2879-9489

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Daohong Zhang: 0000-0003-2989-9761

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Funding

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This work was supported by the National Natural Science Foundation of China

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(21675127), the Natural Science Foundation of Qinghai Province (No.2019-ZJ-904),

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Development Project of Qinghai Key Laboratory (No. 2017-ZJ-Y10), Capacity

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Building Project of Engineering Research Center of Qinghai Province (2017-GX-G03)

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and the Shaanxi Provincial Science Fund for Distinguished Young Scholars

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(2018JC-011).

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Notes

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The authors declare no competing financial interest.

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ABBREVIATIONS USED

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McAb, monoclonal antibody; ICTs, immunochromatographic tests; 2D, two

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dimensional; S. enteritidis, Salmonella enteritidis; POC, point-of-care; T-line, test line;

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TMDs, transition metal dichalcogenides; NC, nitrocellulose; LB, Luria-Bertani; S.

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typhimurium, Salmonella typhimurium; S. paratyphi B, Salmonella paratyphi B;

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hadar, Salmonella hadar; S. London, Salmonella London; S. aureus, Staphylococcus

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aureus; C. albicans, Candida albicans; E. coli O157, Escherichia coli O157; L.

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S.

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monocytogenes, Listeria monocytogenes; C. coli, Campylobacter coli; PBS,

343

phosphate buffered solution; SEM, scanning electron microscopy; TEM, transmission

344

electron microscopy; FTIR, fourier transform infrared; PVC, polyvinyl chloride;

345

ELISA, enzyme linked immunosorbent assay; LOD, limit of detection; RSD, relative

346

standard deviation; GMBNs, gold magnetic bifunctional nanobeads; Ab, antibody;

347

GNPs, gold nanoparticles; HRP, horseradish peroxidase.

348

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molecular light switch complex. Analytical Chemistry 2004, 76, 5230-5235.

21


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459

Figure captions

460

Scheme 1. Relevant schematic illustrations of the 2D nanosheets based ICT system.

461

(A) Sonication-induced liquid phase exfoliation of 2D nanosheets in ethanol aqueous

462

solution. (B) Structure of the test strip. (C) Combining process of 2D nanosheets with

463

S. enteritidis. (D) Working principle of the 2D nanosheets based ICT for rapid

464

detection of S. enteritidis.

465

Figure 1. Characterization results of the 2D nanosheets and nanosheets-bacteria

466

complex. (A)-(D), images of the MoS2 and Graphene separately scanned by TEM (A,

467

C) and SEM (B, D). (E) and (F), UV-vis absorption spectra of MoS2, S. enteritidis,

468

MoS2-S. enteritidis (E) and Graphene, S. enteritidis, Graphene-S. enteritidis (F),

469

respectively. (G) and (H), SEM images of MoS2 (blue arrows) (G) and Graphene

470

(blue arrows) (H) conjugated with S. enteritidis (red arrows), respectively. (I)

471

Photographs of S. enteritidis bacterial colonies after separately incubation with 1.5

472

mg/mL of MoS2 and Graphene.

473

Figure 2. Optimization results of varied influencing factors. (A) and (B), results of

474

the ICT with different concentrations and volumes of MoS2 and Graphene. (C), ICT

475

results for different incubation times of 2D nanosheets and S. enteritidis. (D), results

476

of the ICT with different pH values of solution.

477

Figure 3. Assay performances of the MoS2-ICT (left) and Graphene-ICT (right)

478

biosensors for S. enteritidis detection. (A) and (B), results for sensitivity test. (C) and

479

(D), quantitative results of the 2D nanosheets based ICT biosensor analyzed by Image

480

J. (E) and (F), calibration curves of S. enteritidis detection separately using the

22



481

MoS2-ICT and Graphene-ICT (inset image: the variation rule in the whole detection

482

range).

483

Figure 4. Specificity evaluation of the 2D nanosheets based ICT to 9 types of isolated

484

strains (∼108 CFU/mL). Nos. 1–11 correspond to S. enteritidis, S. london, S.

485

typhimurium, S. paratyphi B, S. aureus, S. hadar, C. albicans, E. coli, C. coli, L.

486

monocytogenes and control group, respectively.

487

Figure 5. Detection results of S. enteritidis in drinking water and watermelon juice

488

samples separately by the MoS2-ICT (A, B) and Graphene-ICT (C, D) biosensors.

23


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489

Table 1 Comparison of sensitivity for Salmonella detection by ICTs based on

490

different signals in pure bacterial culture Label GMBNsb-Abc Collidal gold-Ab GNPsd growth-Ab HRPe-Ab GNPs growth-DNA MoS2 Graphene

491

a

492

b

493 494 495

Target pathogen

LODa (CFU/mL)

Reference

S. choleraesuis S. typhimurium S. enteritidis S. typhimurium S. typhimurium S. enteritidis S. enteritidis

105 105 104

29 30 18 31 32 This work This work

9.2  103 104 103 104

LOD, limit of detection. GMBNs, gold magnetic bifunctional nanobeads. c Ab, antibody. d GNPs, gold nanoparticles. e HRP, horseradish peroxidase.

24



Page 26 of 33

496

Table 2 Recovery efficiencies of S. enteritidis spiked in drinking water and

497

watermelon juice samples detected by the MoS2-ICT (A) and Graphene-ICT (B) Sample

Spiked (Log CFU/mL)

Found (Log CFU/mL) A

B

Recovery (%) A

B

RSD (%, n = 3) A

B

drinking

4

4.06

4.04

101.48

101.01

2.5

3.3

water

6

5.70

5.32

94.98

88.75

3.7

4.2

8

7.77

8.11

97.15

90.10

4.8

1.8

watermelon

4

3.64

4.02

91.08

100.55

2.4

2.9

juice

6

5.13

5.92

85.5

98.59

5.6

3.0

8

7.47

7.72

93.39

96.47

3.2

5.8

25


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498


Scheme 1

26



499

Figure 1

27


Page 28 of 33

Page 29 of 33

500


Figure 2

28



501

Figure 3

29


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502


Figure 4

30



503

Figure 5

31


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504


For TOC only

505

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