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Highly sensitive detection of Cronobacter sakazakii based on immunochromatography coupled with surface-enhanced Raman scattering

  • Siyuan Gao
    Affiliations
    Engineering Research Center of Agricultural Microbiology Technology, Ministry of Education, Heilongjiang University, Harbin 150500, China

    Key Laboratory of Molecular Biology of Heilongjiang Province, College of Life Science, Heilongjiang University, Harbin 150080, China
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  • Jinhui Wu
    Affiliations
    Engineering Research Center of Agricultural Microbiology Technology, Ministry of Education, Heilongjiang University, Harbin 150500, China

    Key Laboratory of Molecular Biology of Heilongjiang Province, College of Life Science, Heilongjiang University, Harbin 150080, China
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  • Hong Wang
    Affiliations
    Key Laboratory of Functional Inorganic Material Chemistry, Ministry of Education, Heilongjiang University, Harbin 150080, China
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  • Shengying Hu
    Affiliations
    Engineering Research Center of Agricultural Microbiology Technology, Ministry of Education, Heilongjiang University, Harbin 150500, China

    Key Laboratory of Molecular Biology of Heilongjiang Province, College of Life Science, Heilongjiang University, Harbin 150080, China
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  • Li Meng
    Correspondence
    Corresponding author
    Affiliations
    Engineering Research Center of Agricultural Microbiology Technology, Ministry of Education, Heilongjiang University, Harbin 150500, China

    Key Laboratory of Molecular Biology of Heilongjiang Province, College of Life Science, Heilongjiang University, Harbin 150080, China
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Open ArchivePublished:January 14, 2021DOI:https://doi.org/10.3168/jds.2020-18915

      ABSTRACT

      The presence of Cronobacter sakazakii must be controlled in infant powder plants, because it may cause infectious disease in infants, with high mortality. Testing for C. sakazakii in powdered infant formula should be performed before delivery, and it requires rapid and specific detection methods. In this study, we established a surface-enhanced Raman scattering (SERS) immunochromatographic test strip for the quantitative determination of C. sakazakii in powdered infant formula. Monoclonal antibodies for C. sakazakii were labeled with p-aminothiophenol-bound colloidal gold nanoparticles. Color change in the test line indicated the presence of C. sakazakii. A highly sensitive and quantitative test method was developed based on the Raman signal produced by the p-aminothiophenol bonding on gold nanoparticles. The SERS immunochromatographic test strip assay required a short analysis time (12 min) and exhibited a linearity range from 102 to 107 cfu/mL. The limit of detection was 201 cfu/mL without preculture. The SERS immunochromatographic test strip assay is a promising tool for the simple and rapid quantitative analysis of C. sakazakii and other pathogenic bacteria.

      Key words

      INTRODUCTION

      Cronobacter sakazakii, formerly Enterobacter sakazakii (
      • Monroe P.W.
      • Tift W.L.
      Bacteremia associated with Enterobacter sakazakii (yellow, pigmented Enterobacter cloacae).
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      • Bougatef A.
      • Naessens A.
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      Outbreak of necrotizing enterocolitis associated with Enterobacter sakazakii in powdered milk formula.
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      • Bidlas E.
      • Cleenwerck I.
      • Marugg J.
      • Fanning S.
      • Stephan R.
      • Joosten H.
      The taxonomy of Enterobacter sakazakii: Proposal of a new genus Cronobacter gen. nov. and descriptions of Cronobacter sakazakii comb. nov. Cronobacter sakazakii subsp sakazakii, comb. nov., Cronobacter sakazakii subsp malonaticus subsp. nov., Cronobacter turicensis sp. nov., Cronobacter muytjensii sp. nov., Cronobacter dublinensis sp. nov. and Cronobacter genomospecies I.
      ), is a pathogen that causes infectious diseases such as bacteremia, neonatal meningitis, and necrotizing enterocolitis, associated with high mortality rates in infants. Powdered infant formula is frequently contaminated with C. sakazakii (
      • Drudy D.
      • Mullane N.R.
      • Quinn T.
      • Wall P.G.
      • Fanning S.
      Enterobacter sakazakii: An emerging pathogen in powdered infant formula.
      ;
      • Baumgartner A.
      • Grand M.
      • Liniger M.
      • Iversen C.
      Detection and frequency of Cronobacter spp. (Enterobacter sakazakii) in different categories of ready-to-eat foods other than infant formula.
      ). The European Union requires that Cronobacter spp. be absent from foodstuffs (
      • European Commission
      Commission regulation (EC) No. 2073/2005 of 15 November 2005 on microbiological criteria for foodstuffs.
      ) in powdered infant formula. Therefore, a rapid and sensitive detection method is urgently needed by industries and regulatory agencies for the detection and quantification of C. sakazakii (
      • ISO (International Organization for Standardization)
      ISO 22964:2017(en): Microbiology of the Food Chain—Horizontal Method for the Detection of Cronobacter spp.
      ).
      Cronobacter sakazakii has been detected using traditional selective culture methods, biochemical reactions, and PCR technology (
      • Minami J.
      • Soejima T.
      • Yaeshima T.
      • Iwatsuki K.
      Direct real-time PCR with ethidium monoazide: A method for the rapid detection of viable Cronobacter sakazakii in powdered infant formula.
      ;
      • ISO (International Organization for Standardization)
      ISO 22964:2017(en): Microbiology of the Food Chain—Horizontal Method for the Detection of Cronobacter spp.
      ;
      • de Benito A.
      • Gnanou Besse N.
      • Desforges I.
      • Gerten B.
      • Ruiz B.
      • Tomás D.
      Validation of standard method EN ISO 22964:2017—Microbiology of the food chain—Horizontal method for the detection of Cronobacter spp.
      ;
      • Akineden O.
      • Wittwer T.
      • Geister K.
      • Plotz M.
      • Usleber E.
      Nucleic acid lateral flow immunoassay (NALFIA) with integrated DNA probe degradation for the rapid detection of Cronobacter sakazakii and Cronobacter malonaticus in powdered infant formula.
      ), but these conventional methods are laborious and time-consuming. Indeed, PCR-based methods require total DNA extraction and target DNA amplification, which may result in false positives (
      • Friedemann M.
      Enterobacter sakazakii in food and beverages (other than infant formula and milk powder).
      ), and they require several hours for detection (
      • Srikumar S.
      • Cao Y.
      • Yan Q.Q.
      • Van Hoorde K.
      • Nguyen S.
      • Cooney S.
      • Gopinath G.R.
      • Tall B.D.
      • Sivasankaran S.K.
      • Lehner A.
      • Stephan R.
      • Fanning S.
      RNA sequencing-based transcriptional overview of xerotolerance in Cronobacter sakazakii SP291.
      ;
      • Yuan Y.
      • Wu X.
      • Liu Z.
      • Ning Q.
      • Fu L.
      • Wu S.
      A signal cascade amplification strategy based on RT-PCR triggering of a G-quadruplex DNAzyme for a novel electrochemical detection of viable Cronobacter sakazakii..
      ). Real-time fluorescence PCR has been developed to quantify C. sakazakii, but it requires technical proficiency and comes with high costs (
      • Tutar E.
      • Akinci K.S.
      • Akyol I.
      Development and application of a new multiplex real-time PCR assay for simultaneous identification of Brucella melitensis, Cronobacter sakazakii and Listeria monocytogenes in raw milk and cheese.
      ).
      The immunochromatographic test strip, based on immunological recognition, has attracted increasing attention because of its high specificity and ease of use in on-site testing (
      • Aragay G.
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      Nanomaterials for sensing and destroying pesticides.
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      Advances in paper-based point-of-care diagnostics.
      ;
      • Koczula K.M.
      • Gallotta A.
      Lateral flow assays.
      ). However, the sensitivity and accuracy of the test strip need to be confirmed for pathogen detection (
      • Scharinger E.J.
      • Dietrich R.
      • Wittwer T.
      • Märtlbauer E.
      • Schauer K.
      Multiplexed lateral flow test for detection and differentiation of Cronobacter sakazakii serotypes O1 and O2.
      ;
      • Pan R.
      • Jiang Y.
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      • Wang R.
      • Zhuang K.
      • Zhao Y.
      • Wang H.
      • Ali M.A.
      • Xu H.
      • Man C.
      Gold nanoparticle-based enhanced lateral flow immunoassay for detection of Cronobacter sakazakii in powdered infant formula.
      ). In test strip assays, gold nanoparticles (AuNP) are used to label the antibody, resulting in color changes upon interaction of the antibody with antigens (
      • Bu T.
      • Jia P.
      • Liu J.H.
      • Liu Y.N.
      • Sun X.Y.
      • Zhang M.
      • Tian Y.M.
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      • Wang J.L.
      • Wang L.
      Diversely positive-charged gold nanoparticles based biosensor: A label-free and sensitive tool for foodborne pathogen detection.
      ;
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      • Zhou Y.F.
      • Huang H.
      • Chen X.R.
      • Leng Y.K.
      • Lai W.H.
      • Huang X.L.
      • Xiong Y.H.
      Engineered gold nanoparticles as multicolor labels for simultaneous multi-mycotoxin detection on the immunochromatographic test strip nanosensor.
      ). However, before color changes are observed, the antigen–antibody combination has already occurred (
      • Sajid M.
      • Kawde A.-N.
      • Daud M.
      Designs, formats and applications of lateral flow assay: A literature review.
      ); more sensitive signals of antigen–antibody combination are needed.
      In the 1970s, individual silver colloidal nanoparticles were used to obtain a 1014- to 1015-fold amplification of the Raman signal from single rhodamine 6G molecules compared to conventional detection methods. The Raman signals showed higher stability and strength on colloidal nanoparticles compared to single molecules (
      • Nie S.
      • Emory S.R.
      Probing single molecules and single nanoparticles by surface-enhanced Raman scattering.
      ). Light resonance stimulates electromagnetic molecules and enhances the signals by 101- to 103-fold. Due to resonance, the surface-enhanced Raman scattering (SERS) effects of single molecules may yield a 1014- to 1015-fold signal amplification (
      • Hwang J.
      • Lee S.
      • Choo J.
      Application of a SERS-based lateral flow immunoassay strip for the rapid and sensitive detection of staphylococcal enterotoxin B.
      ).
      Nanoscale levels of colloidal elements such as Ag and Au can form plasmonic nanoparticles and magnify the signal by resonance absorption or incident light scattering (
      • Anker J.N.
      • Hall W.P.
      • Lyandres O.
      • Shah N.C.
      • Zhao J.
      • Van Duyne R.P.
      Biosensing with plasmonic nanosensors.
      ;
      • Lombardi J.R.
      • Birke R.L.
      A unified view of surface-enhanced Raman scattering.
      ;
      • Zong C.
      • Xu M.X.
      • Xu L.J.
      • Wei T.
      • Ma X.
      • Zheng X.S.
      • Hu R.
      • Ren B.
      Surface-enhanced Raman spectroscopy for bioanalysis: Reliability and challenges.
      ). Nanoscale colloidal particles amplify Raman spectroscopy signals by combining with incident light energy (
      • Aizpurua J.
      • Hanarp P.
      • Sutherland D.S.
      • Kall M.
      • Bryant G.W.
      • Garcia de Abajo F.J.
      Optical properties of gold nanorings.
      ;
      • Zong C.
      • Xu M.X.
      • Xu L.J.
      • Wei T.
      • Ma X.
      • Zheng X.S.
      • Hu R.
      • Ren B.
      Surface-enhanced Raman spectroscopy for bioanalysis: Reliability and challenges.
      ). Previously, SERS sensitivity has been applied in immunochromatographic assays (
      • Deng D.
      • Yang H.
      • Liu C.
      • Zhao K.
      • Li J.
      • Deng A.
      Ultrasensitive detection of Sudan I in food samples by a quantitative immunochromatographic assay.
      ;
      • Li X.
      • Yang T.
      • Song Y.
      • Zhu J.
      • Wang D.
      • Li W.
      Surface-enhanced Raman spectroscopy (SERS)-based immunochromatographic assay (ICA) for the simultaneous detection of two pyrethroid pesticides.
      ). However, SERS-based strips have rarely been used to detect pathogens. Because of the large size of bacteria compared to other antigens, the preparation of colloidal particles with Raman molecular and strip detecting conditions should be carefully optimized.
      In this study, a new SERS-based immunochromatographic test strip was developed for the simple and rapid quantification of C. sakazakii. Our method relied on the use of Raman reporter gold nanoparticles instead of the traditional gold nanoparticles used in typical test strip analyses. The Raman signal of SERS nanotubes was monitored to achieve high sensitivity and quantitative assessment of C. sakazakii. This new SERS immunochromatographic test strip assay is a promising tool for the rapid and simple quantitative detection of C. sakazakii.

      MATERIALS AND METHODS

      Chemicals and Instruments

      Chloroauric acid (HAuCl4), sodium citrate (Na3-citrate), p-aminothiophenol (PATP), goat anti-rat IgG, and BSA were purchased from Sigma-Aldrich (St. Louis, MO). Anti-C. sakazakii monoclonal antibodies were prepared by the laboratory. Powdered infant formula was purchased from YeLi Dairy (Heilongjiang, China). Polyvinylpyrrolidone was purchased from Tianjin Guangfu Fine Chemical Research Institute (Tianjin, China). Absorbent pads, sample pads, and nitrocellulose membranes were purchased from Sartorius Trading Co. Ltd. (Shanghai, China). All other chemicals were analytical-grade reagents. Ultraviolet-visible absorption spectra were assayed by using a Lambda 35 UV/VIS spectrometer (PerkinElmer, Waltham, MA). Transmission electron microscopy images were captured with an HT7800 transmission electron microscope (Hitachi, Tokyo, Japan). Raman spectra were obtained using a Raman microspectrometer system (inVia Raman microscope; Renishaw, Wotton-under-Edge, UK).

      Bacterial Strains

      We used 3 strains of C. sakazakii ATCC 29544, 12868, and 29004, as well as Cronobacter muytjensii ATCC 51329, Cronobacter universalis NCTC 9529, Staphylococcus aureus ATCC 25923, Salmonella typhimurium ATCC 14028, Shigella flexneri ATCC 29931, Listeria monocytogenes ATCC 19114, and Escherichia coli ATCC 25922. All strains were cultured in Luria-Bertani broth (Aladdin China Ltd., Shanghai, China) for 24 h at 37°C.

      Preparation of Monoclonal Antibodies

      Cronobacter sakazakii ATCC 29544 was activated and subcultured in at 37°C for 24 h. The C. sakazakii cells were centrifuged at 5,000 × g for 10 min. The pellets were ultrasonically disrupted 70 times for 5 s at 350 W with 9-s intervals at 26°C (VCX 605; Sonics, Newtown, CT). The lysates were extracted with equal amounts of 90% phenol, kept at 68°C for 30 min, and dialyzed using dialysis tubing of 6,000 to 8,000 molecular weight (Biomol GmbH, Hamburg, Germany) at room temperature against distilled water for 48 h. The dialysate was concentrated with polyethylene glycol 20000 and purified to obtain crude LPS, which was used to immunize 8-wk-old BALB/c female mice with Freund's complete adjuvant emulsion, followed by 5 subsequent immunizations at 2-wk intervals (
      • Scharinger E.J.
      • Dietrich R.
      • Kleinsteuber I.
      • Märtlbauer E.
      • Schauer K.
      Simultaneous rapid detection and serotyping of Cronobacter sakazakii serotypes O1, O2, and O3 by using specific monoclonal antibodies.
      ). After the mice were killed, spleen lymphocytes were collected and fused with myeloma cells at a 5:1 ratio in polyethylene glycol 1500. Hybridoma cells with high antibody activity were selected, cloned in RPMI-1640 medium with 10% calf serum, and stored frozen in liquid nitrogen. Another group of 8-wk-old female BALB/c mice were injected intraperitoneally with 0.5 mL of paraffin. After 7 d, the hybridoma cells were resuspended in RPMI-1640 medium and intraperitoneally injected (
      • Wu X.
      • Wang W.
      • Liu L.
      • Kuang H.
      • Xu C.
      Monoclonal antibody-based cross-reactive sandwich ELISA for the detection of Salmonella spp. in milk samples.
      ;
      • Scharinger E.J.
      • Dietrich R.
      • Kleinsteuber I.
      • Märtlbauer E.
      • Schauer K.
      Simultaneous rapid detection and serotyping of Cronobacter sakazakii serotypes O1, O2, and O3 by using specific monoclonal antibodies.
      ). The ascitic fluid was then collected and the mAb were purified using the caprylic acid-ammonium sulfate method (
      • Shi Q.
      • Huang J.
      • Sun Y.
      • Yin M.
      • Hu M.
      • Hu X.
      • Zhang Z.
      • Zhang G.
      Utilization of a lateral flow colloidal gold immunoassay strip based on surface-enhanced Raman spectroscopy for ultrasensitive detection of antibiotics in milk.
      ). A mouse mAb Ig subclass detection kit (Thermo Scientific, West Palm Beach, FL) was used to determine the antibody subtype (
      • Jaradat Z.W.
      • Rashdan A.M.
      • Ababneh Q.O.
      • Jaradat S.A.
      • Bhunia A.K.
      Characterization of surface proteins of Cronobacter muytjensii using monoclonal antibodies and MALDI-TOF mass spectrometry.
      ).

      Preparation of SERS Immunoprobe

      Preparation of the SERS immunoprobe was performed as previously described, with minor modifications (Figure 1A;
      • Li M.X.
      • Yang H.
      • Li S.Q.
      • Liu C.W.
      • Zhao K.
      • Li J.G.
      • Jiang D.N.
      • Sun L.L.
      • Wang H.
      • Deng A.P.
      An ultrasensitive competitive immunochromatographic assay (ICA) based on surface-enhanced Raman scattering (SERS) for direct detection of 3-amino-5-methylmorpholino-2-oxazolidinone (AMOZ) in tissue and urine samples.
      ). Briefly, the immunoprobe (mAb-Au-PATP) was assembled by sequential addition of the Raman reporter (PATP) and the antibody to colloidal gold particles. Colloidal gold particles were prepared using the citrate reduction method (
      • Liang J.
      • Liu H.
      • Lan C.
      • Fu Q.
      • Huang C.
      • Luo Z.
      • Jiang T.
      • Tang Y.
      Silver nanoparticle enhanced Raman scattering-based lateral flow immunoassays for ultra-sensitive detection of the heavy metal chromium.
      ). Ultrapure water was heated to boiling, and HAuCl4 was added to obtain 300 mL of 0.01% aqueous solution. Next, 4.5 mL of 1% trisodium citrate solution was added to the boiling solution for 15 min to obtain colloidal gold particles, and the colloidal solution was cooled to room temperature. Then, 0.1 mol/L K2CO3 was added to the mixture to adjust the pH to 9.0 before addition of PATP. Then, 10 mL of the colloidal gold solution was mixed with 10 μL of 1 mmol/L PATP solution and stirred at 25°C for 15 min. The mixture was kept at 4°C for 1 h, and the mAb against C. sakazakii were added to 1 mL of PATP-labeled colloidal gold particle solution, followed by stirring at room temperature for 15 min and cooling at 4°C for 1 h to obtain the mAb-Au-PATP preparation. To block nonspecific binding sites, 100 μL of 5% BSA was added to the solution and kept overnight at 4°C. Finally, mAb-Au-PATP was obtained by centrifuging at 5,000 × g for 10 min, and resuspended with PBS (0.5 M, pH 7.4) to the original volume. The final mAb-Au-PATP solutions were stored at 4°C.
      Figure thumbnail gr1
      Figure 1Schematics of the surface-enhanced Raman scattering (SERS) immunochromatographic test strip assay for the detection of Cronobacter sakazakii. (A) Preparation and structure of the mAb-Au-PATP probe. (B) Assembly of the strip and SERS immunochromatographic test strip procedure for C. sakazakii detection. AuNP = gold nanoparticle; C = control; NC = nitrocellulose; PATP = p-aminothiophenol; T = test.

      Assembly of the Lateral Flow Strip

      The immunochromatographic strip was assembled using a nitrocellulose membrane, sample pad, and absorbent pad on a polyvinyl chloride plate (Figure 1B). Then, 10 μL of 1 mg/mL mAb was layered on the nitrocellulose membrane to form the test line, and 10 μL of goat anti-rat IgG was layered to form the control line. The membranes were dried at room temperature for 1 h. A glass-fiber membrane, serving as a sample pad, was soaked in PBS for 24 h (0.5 M, pH 7.4, containing 20% polyvinylpyrrolidone, 20% Tween-20). A nitrocellulose film was adhered to the polyvinyl chloride board. The sample and absorbent pad were attached to each end of the nitrocotton film, with an overlap of 1.5 mm. Then, the attached films were cut into strips 4 mm wide. The strips were sealed in glass vials and stored at 4°C. The immunochromatographic strips were directly dipped into wells containing mAb-Au-PATP and sample solutions.

      SERS Immunochromatographic Test Strip Procedure

      Detection using the SERS immunochromatographic test strips was carried out by immersing the sample pad in the mAb-Au-PATP solution containing 100 μL of the sample. The sample solution, together with the SERS immunoprobe, flowed onto the absorption pad. The reaction was considered complete when the control line turned red. The Raman signal reflected the extent of Ag-mAb-Au-PATP/antibody complex formation on the test line. The resonance was excited using a 10 mW 785 nm laser with an integration time of 10 s. The Raman spectrum between 800 and 1,900 cm−1 was analyzed. The intensity at 1,081 cm−1 of 10 different points on the test line was selected for quantitative calculation of signal intensity.

      Quantitative Calculation of Signal Intensity

      Commercial powdered infant formula free of C. sakazakii was used to generate artificially contaminated samples. First, 10 g of powdered infant formula was reconstituted with 90 mL of buffered peptone water preheated to 44°C, and C. sakazakii bacteria were added at final concentrations between 106 and 102 cfu/mL. Then, the plate counting method was used to establish the initial concentration of the C. sakazakii standard suspension. The samples were centrifuged for 10 min at 4,500 × g and 4°C, and the cell pellets were suspended in normal saline. Then, 100 μL of the suspended solution was loaded onto the SERS immunochromatographic test strip for detection.

      RESULTS AND DISCUSSION

      SERS Immunochromatographic Test Strip Method

      Purified mAb were obtained using the caprylic acid-ammonium sulfate method. The main IgG subclass was IgG1. The structure of the mAb-Au-PATP immunoprobe is shown in Figure 1A. Raman reporters (PATP) and antibodies were sequentially bound to AuNP, and the remaining sites on the nanoparticles were blocked with BSA. The principles and workflow of the SERS immunochromatographic test strip method for C. sakazakii are illustrated in Figure 1B. First, a test sample (possibly containing C. sakazakii) was introduced by immersing the 0.5 cm test strip into the sample solution, which flowed to the absorbent pad by capillarity. Then, the sample interacted with SERS immunoprobes (mAb-Au-PATP), forming an immunoprobe–C. sakazakii complex that migrated together with the sample liquid. Next, as the immunoprobes reached the test line with pre-immobilized coating antibody on the nitrocellulose membrane, an antibody–antigen–antibody sandwich complex was formed. Excessive uncombined immunoprobes were absorbed by the control line, where the immunoprobes reacted with pre-immobilized goat anti-mouse IgG. Finally, Raman signal was generated on the test line by the Raman reporter (PATP), and was positively correlated with the number of C. sakazakii cells.

      Characterization of the Immunoprobes

      To prepare the mAb-AuNP-PATP immunoprobes for detection of C. sakazakii, a colloidal gold suspension with a suitable particle diameter was prepared by chemical chloroauric acid reduction with trisodium citrate. Next, PATP were labeled with AuNP to generate the Raman signal, and the mAb were absorbed on the AuNP surface to form the mAb-AuNP-PATP complexes.
      The physicochemical properties of the AuNP were examined by transmission electron microscopy (Figure 2A), and UV-visible spectra of AuNP, Au-PATP, and mAb-Au-PATP were compared (Figure 2B). The average diameter of the AuNP was 46 nm, and the particles appeared uniform. The diameter of the AuNP is crucial for Raman signal amplification (
      • Shao W.
      • Liu X.F.
      • Min H.H.
      • Dong G.H.
      • Feng Q.Y.
      • Zuo S.L.
      Preparation, characterization, and antibacterial activity of silver nanoparticle-decorated graphene oxide nanocomposite.
      ). The AuNP diameter of 46 nm has been shown to be the most suitable for Raman signal amplification (
      • Liang J.
      • Liu H.
      • Lan C.
      • Fu Q.
      • Huang C.
      • Luo Z.
      • Jiang T.
      • Tang Y.
      Silver nanoparticle enhanced Raman scattering-based lateral flow immunoassays for ultra-sensitive detection of the heavy metal chromium.
      ). The maximum absorption wavelengths of the AuNP, Au-PATP, and mAb-Au-PATP were 534, 535, and 537 nm, respectively (Figure 2B). The shift in Au-PATP absorption demonstrated the successful combination of AuNP and PATP.
      Figure thumbnail gr2
      Figure 2Characterization of the mAb-Au-PATP immunoprobe. (A) Transmission electron microscopy image of AuNP. (B) Ultraviolet-visible spectra of AuNP, Au-PATP, and mAb-Au-PATP. AuNP = gold nanoparticle; PATP = p-aminothiophenol.

      Optimization of SERS Immunochromatographic Test Strip

      To improve the performance of the SERS immunochromatographic test strip, several key parameters were optimized, including mAb concentration and assay time, using the bacterial concentration of 106 cfu/mL. Because the concentration of anti-C. sakazakii mAb on the test line had a great influence on detection sensitivity, this parameter was optimized first. As shown in Figure 3A, at mAb concentrations exceeding 1 mg/mL, strong Raman signals were observed from positive reactions, and low background signals were detected in negative reactions (PBS). Therefore, 1 mg/mL of anti-C. sakazakii mAb was identified as the optimal concentration.
      Figure thumbnail gr3
      Figure 3Optimization of the surface-enhanced Raman scattering (SERS) immunochromatographic test strip assay. (A) Optimization of mAb concentration on test (T) line. (B) Optimization of assay time; values represent the mean of 3 replicates (n = 3). (C) Optimization of the amount of mAb-labeled Au-PATP. PATP = p-aminothiophenol. Error bars indicate SD.
      Assay time was also crucial for optimal performance of the SERS immunochromatographic test strip. As shown in Figure 3B, the intensity at 1081 cm−1 increased steadily until 12 min, and then reached a plateau. Therefore, an assay time of 12 min was adopted. The appropriate amount of anti–C. sakazakii mAb for Au-PATP labeling was also investigated, and the highest Raman signal was obtained with a concentration of 10 μg/mL (Figure 3C).

      Quantitative Detection of C. sakazakii

      To define the detection limit of the SERS immunochromatographic test strip for C. sakazakii analysis, the assay was used to test bacterial concentrations of 0 to 107 cfu/mL (Figure 4A). The color of the test line became stronger with increases in C. sakazakii concentration. The concentration limit could be visually assessed and was determined to be 105 cfu/mL.
      Figure thumbnail gr4
      Figure 4Quantitative performance of the surface-enhanced Raman scattering (SERS) immunochromatographic test strip assay. (A) Color intensity of the test (T) line as a function of Cronobacter sakazakii concentration. (B) SERS spectra of the T line. (C) Calibration curves of SERS signal as a function of C. sakazakii concentration; values represent the mean of 3 replicates (n = 3). C = control. Error bars indicate SD.
      Bacteria combined with mAb-AuNP-PATP immunoprobes and antigen–antibody complexes were captured by the coated antibodies on the test line. Raman signal intensity increased with the accumulation of PATP in the bacteria–mAb-AuNP-PATP complexes. Therefore, Raman signal intensity increased with C. sakazakii concentration (Figure 4B). A bacterial concentration of 102 cfu/mL yielded a Raman signal intensity of 1,126.87 ± 24.90 a.u., significantly higher than the blank (957.51 ± 57.28 a.u.; P = 0.04). Quantitative analysis revealed a nonlinear calibration curve (Figure 4C). The limit of detection was estimated to be 201 cfu/mL, calculated by 3 times the standard deviation in the presence of 0 cfu/mL. The strip showed positive results when the sample with C. sakazakii was enriched at 37°C for 18 h. Under these conditions, the bacterial titer reached approximately 201 cfu/mL in buffered peptone water broth according to the ISO 22964:2017 method (
      • ISO (International Organization for Standardization)
      ISO 22964:2017(en): Microbiology of the Food Chain—Horizontal Method for the Detection of Cronobacter spp.
      ). With the precultivation procedure, the SERS immunochromatographic test strip assay may be a suitable method for detecting C. sakazakii infection in industrial plants.
      The antibody-conjugated SERS nanoparticles significantly improved the limit of detection of the strip, increasing its sensitivity by approximately 2 orders of magnitude compared to that achieved by visual assessment of color changes.

      Reproducibility and Selectivity of the SERS Immunochromatographic Test Strip Method

      To assess the reproducibility of the SERS-based method, 10 independent measurements of Raman signal intensity at 1,081 cm−1 were performed at each C. sakazakii concentration from 107 to 103 cfu/mL (Figure 5A). Signal intensity was monitored in the middle of the test lines. Relative standard deviations were 3.4, 2.8, 5.2, 5.5, and 5.3% for the different bacterial concentrations, respectively, suggesting high precision. Reproducibility was similar to the technology applied to detect other hazardous substances (
      • Zong C.
      • Xu M.X.
      • Xu L.J.
      • Wei T.
      • Ma X.
      • Zheng X.S.
      • Hu R.
      • Ren B.
      Surface-enhanced Raman spectroscopy for bioanalysis: Reliability and challenges.
      ). The relative standard deviation was lower than the visible light signal of the strip (
      • Wang X.
      • Zhu C.
      • Xu X.
      • Zhou G.
      Real-time PCR with internal amplification control for the detection of Cronobacter spp. (Enterobacter sakazakii) in food samples.
      ;
      • Akineden O.
      • Wittwer T.
      • Geister K.
      • Plotz M.
      • Usleber E.
      Nucleic acid lateral flow immunoassay (NALFIA) with integrated DNA probe degradation for the rapid detection of Cronobacter sakazakii and Cronobacter malonaticus in powdered infant formula.
      ).
      Figure thumbnail gr5
      Figure 5Repeatability and specificity of the surface-enhanced Raman scattering (SERS) immunochromatographic test strip assay. (A) Repeatability: SERS intensity of p-aminothiophenol (PATP) at 1,081 cm−1 from 10 different spots along the middle parts of the test lines of the strips with Cronobacter sakazakii concentrations of 107, 106, 105, 104, and 103 cfu/mL, respectively. (B) Specificity: SERS signal intensity obtained with 6 pathogenic bacteria: Cronobacter sakazakii, Staphylococcus aureus, Salmonella typhimurium, Shigella flexneri, Listeria monocytogenes, and Escherichia coli at concentrations of 107 cfu/mL.
      The strain used to obtain the antibody was C. sakazakii ATCC29544. Because other Enterobacter spp. may occur concurrently with C. sakazakii, common foodborne pathogens were used to examine the specificity of the SERS strip. To guarantee the specificity of the strip developed, mAb were used instead of polyclonal antibodies. For high sensitivity, lipopolysaccharide was used as an adjuvant to obtain mAb. To verify the specificity of the mAb obtained, C. sakazakii ATCC 128868, C. sakazakii ATCC 29004, C. muytjensii ATCC 51329, C. universalis NCTC 9529, Staphylococcus aureus ATCC 25923, Salmonella typhimurium ATCC 14028, Shigella flexneri ATCC 29931, L. monocytogenes ATCC 19114, and E. coli ATCC 25922 were used to verify the specificity of the mAb obtained. A competitive ELISA method was used to detect the specificity of the mAb. As listed in Table 1, the 3 strains of C. sakazakii showed positive results, and the 7 strains of other bacteria showed negative results. No cross-reactivity with other pathogens was found (Table 1). These results indicated that the SERS strip developed in our study was specific to C. sakazakii. The SERS strip had high specificity for C. sakazakii and no cross-reaction to Staphylococcus aureus, Salmonella typhimurium, Shigella flexneri, L. monocytogenes, or E. coli. The SERS strip had the same specificity for the mAb used in the strip (Figure 5B). Strains of C. sakazakii gave out Raman signals, but the other bacterial strains did not. These results were consistent with those of
      • Pan R.
      • Jiang Y.
      • Sun L.
      • Wang R.
      • Zhuang K.
      • Zhao Y.
      • Wang H.
      • Ali M.A.
      • Xu H.
      • Man C.
      Gold nanoparticle-based enhanced lateral flow immunoassay for detection of Cronobacter sakazakii in powdered infant formula.
      , except that the strip tested in the present study showed no cross-reaction with Staphylococcus aureus, different from the report of
      • Pan R.
      • Jiang Y.
      • Sun L.
      • Wang R.
      • Zhuang K.
      • Zhao Y.
      • Wang H.
      • Ali M.A.
      • Xu H.
      • Man C.
      Gold nanoparticle-based enhanced lateral flow immunoassay for detection of Cronobacter sakazakii in powdered infant formula.
      . The reason for this may have been that the lipopolysaccharide adjuvant evaluated the specificity of the antibody generated (
      • Scharinger E.J.
      • Dietrich R.
      • Wittwer T.
      • Märtlbauer E.
      • Schauer K.
      Multiplexed lateral flow test for detection and differentiation of Cronobacter sakazakii serotypes O1 and O2.
      ).
      Table 1Specificity of mAb testing (by competitive ELISA) and the surface-enhanced Raman scattering (SERS) immunochromatographic test strip assay
      No.StrainResult
      + = positive; − = negative.
      mAbSERS
      1Cronobacter sakazakii ATCC 29544++
      2Cronobacter sakazakii ATCC 12868++
      3Cronobacter sakazakii ATCC 29004++
      4Cronobacter muytjensii ATCC 51329
      5Cronobacter universalis NCTC 9529
      6Staphylococcus aureus ATCC 25923
      7Salmonella typhimurium ATCC 14028
      8Shigella flexneri ATCC 29931
      9Listeria monocytogenes ATCC 19114
      10Escherichia coli ATCC 25922
      1 + = positive; − = negative.

      Application of SERS Immunochromatographic Test Strip for Detection of C. sakazakii

      To verify the accuracy of the method, the SERS immunochromatographic test strip was applied to real samples. The amount of C. sakazakii in milk powder (2 × 102 to 1.6 × 106 spiked-in bacteria) was measured using the SERS immunochromatographic test strip; the curves obtained are summarized in Figure 6. Concentrations of C. sakazakii were also determined using the plate counting method, which is currently the gold standard (Table 2). Test accuracy was calculated based on the ratio between the values obtained using the SERS immunochromatographic test strip and those obtained by plate counting. The results showed accuracy in the range of 99 to 105%, demonstrating that the SERS immunochromatographic test strip assay can be used for the accurate measurement of C. sakazakii titers in milk powder. The limit of detection was 201 cfu/mL, and the concentration limit for visual detection was 105 cfu/mL. As well, using an enrichment procedure at 37°C for 18 h, this method could detect 1 cfu/100 g, which was more sensitive than the ELISA method (
      • Song X.
      • Teng H.
      • Chen L.
      • Kim M.
      Cronobacter species in powdered infant formula and their detection methods.
      ).
      Figure thumbnail gr6
      Figure 6Results of sensitivity test for spiked sample. In spiked samples 1, 2, 3, and 4, the amounts of Cronobacter sakazakii added were 255 ± 23, 456 ± 38, 18,267 ± 907, and 1,650,000 ± 75,498 cfu/mL, respectively, using the plate counting method.
      Table 2Detection of Cronobacter sakazakii in infant milk powder using the plate counting method and the surface-enhanced Raman scattering (SERS) immunochromatographic test strip assay (mean ± SD; n = 5)
      Plate counting method (cfu/mL)SERS (cfu/mL)Accuracy (%)
      255 ± 23280 ± 24105 ± 9
      456 ± 38473 ± 108104 ± 5
      18,267 ± 90718,133 ± 80899 ± 3
      1,650,000 ± 75,4981,645,178 ± 30,970100 ± 3

      CONCLUSIONS

      The SERS immunochromatographic test strip assay proved to be highly specific for the detection of C. sakazakii; it was also highly sensitive, because of the Raman signal. In this system, the presence of bacteria could be visually assessed in 12 min based on color change in the test line. The intensity of the Raman signal produced by the mAb-Au-PATP immunoprobes on the test line was highly correlated with bacterial concentration in the samples, providing accurate quantification of the C. sakazakii titers. The range of quantitative detection was 102 to 107 cfu/mL, with a limit of detection of 201 cfu/mL and an accuracy of 99 to 104%, compared to plate counting without preculture. Based on the ISO 22964:2017 enrichment procedure, the method met the detection requirements of the European Commission. Because of its short assay time and ease of implementation, the SERS immunochromatographic test strip assay described in this article proved to be an effective tool for simple and rapid quantification of specific pathogens in industrial food.

      ACKNOWLEDGMENTS

      This work was financially supported by the Heilongjiang Province University Basic Research Operating Costs Heilongjiang University Special Project (no. KJCX201817). Siyuan Gao performed the experiments and wrote the manuscript draft; Jinhui Wu helped with the monoclonal antibody experiments; Hong Wang and Shengying Hu helped with data analysis; Li Meng designed the experiments, provided guidance, and finalized the manuscript. The authors have not stated any conflicts of interest.

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