Colorimetric-Fluorescent-Magnetic Nanosphere-Based Multimodal

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Colorimetric-Fluorescent-Magnetic Nanosphere-Based Multimodal Assay Platform for Salmonella Detection Jiao Hu, Yong-Zhong Jiang, Man Tang, Ling-Ling Wu, Hai-yan Xie, Zhi-Ling Zhang, and Dai-Wen Pang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05154 • Publication Date (Web): 05 Dec 2018 Downloaded from http://pubs.acs.org on December 6, 2018

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Analytical Chemistry

Colorimetric-Fluorescent-Magnetic Nanosphere-Based Multimodal Assay Platform for Salmonella Detection Jiao Hu,†,‡ Yong-Zhong Jiang,† Man Tang,† Ling-Ling Wu,† Hai-yan Xie,†,§ Zhi-Ling Zhang,† Dai-Wen Pang*,† †

Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, State Key Laboratory of Virology, The Institute for Advanced Studies, and Wuhan Institute of Biotechnology, Wuhan University, Wuhan 430072, People’s Republic of China

‡ Wuhan

academy of agricultural sciences, Wuhan 430072, People's Republic of China

§ School

of Life Science and Technology, Beijing Institute of Technology, Beijing, 100081, P. R. China

*Address correspondence to [email protected]; phone 0086-27-68756759; fax 0086-27-68754067

ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract: Rapid and sensitive foodborne pathogen detection assay, which can be applied in multiple fields, is essential to timely diagnosis. Herein, we proposed a multi-signal readout lateral flow immunoassay for Salmonella typhimurium (S. typhi) detection. The assay employs colorimetricfluorescent-magnetic nanospheres (CFMNs) as labels, which possess multi-functional: target separation and enrichment, multi-signal readout and two formats of quantitation. The assay for S. typhi detection involves magnetic separation and chromatography. First, the S. typhi were separated and enriched from matrix by antibody labelled CFMNs, and then the S. typhi-containing suspension is added onto the sample pad to flow up the test strip. The introduction of magnetic separation enhances anti-interference ability and 10-fold sensitivity, making the assay possible for practical application. The assay has realized naked eye detection of 1.88×104 CFU/mL S. typhi, and 3.75×103 CFU/mL S. typhi can be detected with a magnetic assay reader, which is 2–4 orders of magnitude lower than other label-based LFIAs, with a quantitation range of 1.88×104 to 1.88×107 CFU/mL by measuring the fluorescence intensity and magnetic signal. Moreover, the successful detection of S. typhi in complex matrix (tap water, milk, fetal bovine serum and whole blood) indicated its potential application in real samples. Introduction Infectious diseases caused by foodborne pathogenic bacteria continue to present a major threat to public health.1,2 Centers for Disease Control and Prevention (CDC) estimated that approximately 9.4 million episodes of foodborne illness occurred annually in the United States of America.3,4 In 2013, the frequent outbreak of foodborne diseases have resulted in a total of 19,056 infections, 4,200 hospitalizations, and 80 deaths in America.5 The occurrence of foodborne diseases are even highly frequent in many developing countries due to the poor medical conditions. Currently, the commonly used method for bacteria detection is the conventional culture-based method, however, it suffers from 3–5 days for obtaining results.6 Although enzyme-linked immunosorbent assay (ELISA)7,8 and polymerase chain reaction (PCR)9,10 have been developed as replacements for the culture method, they are limited to tedious procedures and poor efficiency, and are not as yet user-friendly for average users who have limited training in chemical or biological laboratories. They are also limited to intensive infrastructures.11,12 In recent years, numerous approaches have been developed for pathogen detection, such as lateral flow immunoassay (LFIA),13,14 optical biosensor,15,16 surface enhanced Raman scattering biosensor,17,18 electrochemical biosensors.19,20 However, among these methods, only LFIA can meet the requirements of simple, rapid and point-of-care (POC) detection simultaneously. LFIA as the most prominent rapid POC diagnostic assay exhibits advantages in portability, speed, cost-effectiveness, and ease-of-use.21–23 These unique characteristics have spurned intense interests in the use of LFIA for rapid POC detection of foodborne pathogen.


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In addition, the greatest advantage of the colorimetric-based LFIA is that the result can be determined with naked eyes without the requirement for any specialist facilities and healthcare professionals. Moreover, breakthroughs in LFIA have been realized through the use of functional nanoparticles, such as dyed beads, up-conversion phosphors and quantum dots.24–26 These fluorescent materials employed as labels have improved sensitivity and made quantitative detection a reality.27,28 Compared to Au NPs used in colorimetric LFIA, which is yes-or-no diagnosis, quantitative detection often provides more valuable information to physicians to help them make better therapeutic measures to fit the patients. However, the fluorescent materials require an extra excitation light source to have them emit fluorescence, which might impede their application in POC diagnosis, especially under poor medical conditions. As another alternative label for quantitative detection, magnetic nanospheres can also provide a higher sensitivity.29– 31

Moreover, magnetic nanospheres can be used to separate and enrich targets under an external magnetic

field,32–34 expanding the application of LFIA in the detection of targets within complex biological matrix. The other merit of magnetic nanospheres is that they offer the possibility for trace target detection via the measurement of magnetic signal compared to optical density.35,36 Additionally, the intraband transition of magnetic nanoparticles results in its absorption spectrum covering almost the whole visible range. As a result, the magnetic nanospheres present a dark brown color. Hence, besides magnetic signal, magnetic nanospheres can also provide colorimetric signal which can be seen with naked eyes.37 However, both fluorescent materials and magnetic nanospheres can only provide one or two signals, and could not meet the requirements under different medical conditions simultaneously. Herein, we fabricated colorimetric-fluorescent-magnetic nanospheres (CFMNs) as the label of LFIA. They were fabricated by assembling Fe3O4 and quantum dots (QDs) on the surface of polymer nanospheres in turns. Owing to the high extinction coefficients of Fe3O4 and QDs within the visible range, which is rather comparable to that of Au NPs, the CFMNs can provide colorimetric signal. Simultaneously, the CFMNs can provide fluorescence signal and magnetic signal that are attributable to QDs and Fe3O4. As labels, CMFNs possess multi-functional: target separation and enrichment, multisignal readout and two formats of quantitation. Taking advantages of the CFMNs, a sensitive multi-signal readout LFIA for foodborne pathogen Salmonella typhimurium (S. typhi) has been developed for both naked eye readout and quantitative detection based on fluorescence signal as well as magnetic signal. The multi-signal readout enables the assay applied flexibly at different areas with different conditions to meet the detection requirements of real samples. As depicted in Scheme 1, the S. typhi can be separated and enriched from matrix by using antibody-conjugated CFMNs (ICFMNs). Then the S. typhi-containing suspension is added onto the sample pad. By the assay, S. typhi can be visualized with naked eyes and quantitatively detected by measuring fluorescence signal and magnetic signal.



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Scheme 1. Schematic diagram for multi-signal readout detection of S. typhi by CFMNs-based lateral flow immunoassay.

EXPERIMENTAL SECTION Reagents and Instruments. Mouse monoclonal antibody to S. typhimurium (S. typhi) was purchased from Abcam. Hydrophobic CdSe/ZnS quantum dots (QDs) were purchased from JiaYuan Quantum Dots Co., Ltd. (Wuhan). Hoechst 33342 was from Invitrogen Corp. Goat anti-mouse IgG antibody, bovine serum albumin (BSA), FITC labeled goat anti-mouse IgG (FITC-IgG), branched poly(ethylene imine) (PEI, MW 25 kDa and MW 750 kDa), tetraethylorthosilicate (TEOS), (3-aminopropyl)triethoxysilane (APTES), polyvinylpyrrolidone (K-30), N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), and N-Hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich. Fetal bovine serum (FBS) was obtained from Gibco. Human peripheral blood plasma samples were supplied by Hubei Cancer Hospital. Ultrapure water (18.2 MΩ•cm) was obtained with a Millipore Milli-Q system and used for the preparation of all solutions. Nitrocellulose membrane, sample pad, absorbent pad, and plastic adhesive card were supplied by JieYi BioTech Co., Ltd. (Shanghai) and used as provided. The transmission electron microscopy (TEM) images were acquired on a FEI Tecnai G2 20 TWIN electron microscope. Magnetic hysteresis loops were measured with a vibrating sample magnetometer (Lake Shore 7410 VSM). Fluorescence emission spectra were collected on a Fluorolog-3 fluorescence spectrometer (HORIBA JOBIN YVON). Fluorescence images were obtained with an inverted fluorescence microscope (Nikon TIU) which was equipped with a CCD camera (Nikon DS-Ri1). The magnetic signal of


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the strip was recorded with a magnetic assay reader (MagnaBioSciences, CA, USA). The fluorescence signal of the strip was measured with a fiber optic spectrometer. Fabrication of Colorimetric-Fluorescent-Magnetic Nanospheres (CFMNs). CFMNs were fabricated according to layer-by-layer (LBL) assembly method previously reported by our group.38,39 Briefly, PStAAm-COOH nanosphers were reacted with branched poly(ethylene imine) and Fe3O4 or QDs repeatedly, and an outer layer of silica was introduced to increase the stability of the CFMNs. Through further modification with succinic anhydride, the CFMNs were equipped with carboxyl groups for further coupling with antibody. Detection of S. typhi with the Multi-Modal Readout LFIA. S. typhi and other bacteria were diluted in the solution of 1% BSA−1% Tween-20−0.9% NaCl to obtain the bacterial samples. First, 1 mL of bacteria sample was incubated with ICFMNs and then washed twice. The complex was resuspended in 100 μL of PBS (0.01 M, pH = 7.4, containing 1% BSA and 1% Tween-20). Then, the solution was added onto the sample pad, allowing all liquid to be absorbed and migrate along the strip. A few minutes later, the qualitative results were obtained through observing the brown color band by the naked eyes or the fluorescence signal with a portable ultraviolet lamp. The quantitative detection was realized by two means: measuring the magnetic signal by a magnetic assay reader, and the fluorescence intensity by a fiber optic spectrometer. Detection of S. typhi in the Spiked Samples. S. typhi were spiked to tap water, milk, fetal bovine serum, and whole blood to obtain the complex samples. The tap water, milk and fetal serum sample volume was 1 mL. The volume of whole blood sample was 200 μL. Thus, all the spiked samples which contained 107 CFU S. typhi were incubated with ICFMNs. After washing, separation and chromatography as described above, signals were detected with a magnetic assay reader or a fiber optic spectrometer. Control experiments were done as described here expect no S. typhi was added.

Results and Discussion Fabrication of Colorimetric-Fluorescent-Magnetic Nanospheres (CFMNs). Colorimetric-fluorescentmagnetic nanospheres (CFMNs) were prepared according to the layer-by-layer assembly method previously reported by our group.38,39 To get a rapid magnetic response and strong fluorescence intensity, the layers of Fe3O4 and QDs that assembled on the surface of PSt-AAm-COOH nanospheres were investigated. As we know, the more layers of Fe3O4, the stronger colorimetric signal and higher magnetic signal; the more layers of QDs, the



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stronger fluorescence signal. Due to the high absorbance index of Fe3O4, the fluorescence signal of QDs will be absorbed. Additionally, the more layers of nanoparticles were assembled, the larger size of CMFNs. Hence, the layers of Fe3O4 and QDs must be adjusted. As shown in Figure 1, the magnetic saturation value of three layers of Fe3O4 is 24.8 emu/g, ensured it can be captured quickly by a commercial magnetic scaffold (Figure S1D). The fluorescence intensity after assembling different layers of QDs was also investigated. With the increase of QDs, the fluorescence intensity increased (Figure 1B). As a label of LFIA, the size should be lower than 400 nm to make it flow quickly. The size of PSt-AAmCOOH was about 270 nm, and it increased about 15 nm with one layer of Fe3O4 or QDs assembled. Hence, three layers of Fe3O4 and QDs were chosen to fabricate the CFMNs, which process strong magnetic signal and fluorescence signal and appropriate size (Figure S1). Furthermore, the colorimetric intensity of the CFMNs increased linearly with increasing nanosphere concentration (Figure 1C), which can be used for qualitative and semi-quantitative detection with naked eye.

Figure 1. (A) Magnetic hysteresis loop of the nanospheres with different layers of Fe3O4. (B) Fluorescence intensity of the nanospheres with different layers of QDs. (C) Proportional relationship of absorbance at 600 nm versus CFMNs concentration.

The Proof-of-Concept Assay for the Detection of S. typhi. The proof-of-concept of the assay for S. typhi detection is introduced. The fluorescence microscopy and TEM results demonstrated that the S. typhi could be efficiently captured by the ICFMNs. Figure 2A shows fluorescence microscopic image of S. typhi/ICFMNs complexes. The bacteria were stained by a nuclear dye Hoechst 33342. With a long pass ultraviolet filter, the S. typhi exhibited blue fluorescence and ICFMNs showed red fluorescence. It can be seen that the blue and red fluorescence signals were overlapped. The TEM image (Figure 2B) showed that many ICFMNs bound to the S. typhi, further demonstrated the successful association between ICFMNs and S. typhi. After chromatography, as expected, the S. typhi/ICFMNs complexes were captured on the test line by the antibody to S. typhi, and the presence of S. typhi can be identified through a brown color change or the appearance of a red fluorescence band on the test line. Representative digital images of LFIA strips of negative and positive samples were shown in Figure 2C, D. At the same time, the S. typhi/ICFMNs were also used as magnetic detection for the quantitative detection of S. typhi. For the positive sample, with a


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magnetic strip reader, magnetic signal on the test line and control line can be detected simultaneously (Figure 2F). In contrast, no magnetic signal can be detected on the test line in the absence of S. typhi (Figure 2E). Consequently, the presence of S. typhi could be identified by the naked eyes. Additionally, quantitative analysis of S. typhi was achieved by monitoring the magnetic signal of the S. typhi/ICFMNs.

Figure 2. Fluorescence microscopic image (A) and TEM image (B) of S. typhi captured by ICFMNs, respectively. Representative photographic images of the lateral flow test in the absence (negative, C, E) and presence (positive, D, F) of S. typhi, respectively.

Analytical Performance of CFMNs Based LFIA for S. typhi Detection. To evaluate the sensitivity and dynamic range of the CFMNs-based LFIA for S. typhi detection, under optimized detection conditions (Figure S3), the analytical performance of the CFMNs-based LFIA was examined with different concentrations of the S. typhi ranging from 0 to 1.88×107 CFU/mL. Figure 3A showed the typical response of the assay for S. typhi with different concentrations of 0, 3.75×103, 1.88×104, 1.88×105, 3.75×105, 1.88×106 and 1.88×107 CFU/mL, respectively. As shown in Figure 3A, only brown color on control line was observed in the absence of S. typhi. In contrast, two lines were observed in the presence of S. typhi.



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As the S. typhi concentration increased, more sandwich complexes were formed, and the brown color intensity became darker. The brown band could be visualized at concentrations as low as 1.88×104 CFU/mL. By recording the magnetic signal of the test line, as shown in Figure 3B, well-defined peaks were obtained and the peak intensity increased along with the increasing of S. typhi concentration. While no obvious magnetic signal for the control sample could be detected. When the concentration is 3.75×103 CFU/mL, the magnetic peak (Figure 3B, inset image, red line) is obvious compared with the control one (Figure 3B, inset image, black line), indicated the higher sensitivity of magnetic signal than colorimetric signal. The assay also exhibited a good linear range at the concentration 1.88×104–1.88×107 CFU/mL with a detection limit of 3.5×103 CFU/mL (Figure 3C). The assay can also realize qualitative and quantitative detection of S. typhi by measuring fluorescence signal. With a portable ultraviolet lamp, as low as 3.75×103 CFU/mL S. typhi can be observed, and the quantitation range is also 1.88×104 to1.88×107 CFU/mL (Figure S4). The results also demonstrated that magnetic signal and fluorescence signal have advantage in sensitivity compared with colorimetric signal.

Figure 3. (A) Bright field photographs of the test strips for 0, 3.75×103, 1.88×104, 1.88×105, 3.75×105, 1.88×106, 1.88×107 CFU/mL S. typhi in buffer (from left to right). (B) CFMNs-based LFIA response for 0, 3.75×103, 1.88×104, 1.88×105, 3.75×105, 1.88×106 and 1.88×107 CFU/mL S. typhi (curves a–g), respectively. (C) CFMNs-based LFIA linear response for detection by measuring the magnetic signal; concentration range is 1.88×104–1.88×107 CFU/mL in buffer.

Compared with some other assays for Salmonella detection, the assay developed in this work showed better performance (Table 1).40–45 The sensitivity was increased by 2–4 orders of magnitude. Recently, a two-step LFIA, which consists of magnetic separation and chromatography was reported,44 the assay developed here also shows superiority in sensitivity and rapidity. It is also more sensitive than some commercial test strips for Salmonella detection (105 to 107 CFU/mL). The higher sensitivity is attributed to higher signal-to-noise of fluorescence and magnetic signal. In addition, the magnetic separation also realized the enrichment of the target. Thirdly, large quantity of QDs and Fe3O4 were coated on one nanosphere, which can provide higher signal than one QD or Fe3O4.


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Table 1. Summary of Analytical Performance of Bacterium Detection by Different Methods method

target

label

signal

detection limit (CFU/mL)

assay time

linearity range

ref

SPRa)

Salmonella

Au NPs

single

7400

80 min

103–106

40

PADsb)

S. typhi

β-galactosidase

single

102

75 min

qualitative

41

thermal sensor

S. typhi

magnetic nanomaterials

single

300

1.5 h

300–103

42

optical biosensor

S. typhi

fluorescent nanospheres

single

10

>1 h

105–107

15

LFIA c)

Bacillus anthracis spores

magnetic nanobeads

double

6×104 spores/g milk powder

20 min

103–105

13

LFIA

Salmonella enteritidis

Gold nanoparticles

single

107

qualitative

43

Salmonella choleraesuis

Fluorescent Microspheres

single

1.5×105

>70 min

7.6×104–7.6×106

44

LFIA

S. typhi.

UP-coversion phosphor

single

104

20 min

104–107

45

LFIA

S. typhi

CFMNs

triple

3.5×103

35 min

1.88×104–1.88×107

this work

Two-step LFIAd)

a)

SRP: Surface Raman scattering. b) PADs: Paper-based analytical devices. c) LFIA: lateral flow immunoassay. d) Two-step LFIA: consists

of magnetic separation and chromatography

Reliability and Specificity of the Assay. After demonstrating that the CFMNs-based LFIA has better performance than some other labels based LFIA, the specificity of this strategy was evaluated. First, the specificity of the interactions between ICFMNs and S. typhi were tested (Figure S5). The results demonstrated that the S. typhi could be captured by ICFMNs specifically. Then the feasibility of ICFMNs-based LFIA for practice application was also investigated. The S. aureus and E. coli were employed as negative controls to investigate the specificity and selectivity of the assay. As shown in Figure 4, the samples of S. aureus and E. coli showed similar phenomenon as that of the blank, in which no obvious magnetic signal was detected. In contrast, 1.88×105 CFU/mL S. typhi as well as those S. typhi in the presence of 1.88×107 CFU/mL S. aureus or E. coli showed strong magnetic signal, indicating that these interference bacteria did not interfere with the detection of S. typhi. All these results suggested the strong specificity and selectivity of the CFMNs-based LFIA and validated the feasibility of the assay for practical application.

Figure 4. Histogram of magnetic value for the specificity and anti-interference ability test.



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Application to Complex Samples. Encouraged by these initial experiments, we next applied the assay into the detection of S. typhi in the spiked complex samples (tap water, milk, fetal bovine serum, and whole blood). The results were shown in Figure 5. All positive samples detected the magnetic signal, while no signal of ICFMNs was found in the negative samples. The corresponding magnetic signal was slightly lower than that of the S. typhi in buffer sample (1%BSA−1% Tween-20−0.9% NaCl solution), which was due to the complexity of the matrixes. The result demonstrated that the proposed assay is thus promising to detect S. typhi in real, complex samples.

Figure 5. Detection results of the spiked samples of S. typhi.

Conclusions We presented a simple, sensitive and selective LFIA for S. typhi detection based on a multi-signal readout nanospheres (CFMNs) as the reporter. With the unique magnetic property, the assay enables capture and enrichment of S. typhi, and led to higher sensitivity and anti-interference ability of the assay. In addition, the CFMNs can simultaneously provide three different signals: colorimetric signal, fluorescence signal and magnetic signals, which can be applied flexibly at areas with different conditions, especially for the resource-poor area. Compared with some other reported assays for Salmonella, especially the commercial LFIA (105 to 107 CFU/mL), the CFMNs-based LFIA has increased the sensitivity by 2–4 orders of magnitude, and realized two formats quantitative detection of S. typhi, making the assay promising for sensitive detection of bacteria. Moreover, this method is easy to operate, time-saving, and its success in detecting S. typhi in complex matrix validates its potential use in real samples. The merits of the novel reporter, multi-signal readout, and two formats of quantitation, high sensitivity, and strong anti-interference ability may substantially broaden the applications of lateral flow assays for early detection of foodborne pathogens, and hold great potential in clinical diagnosis, food safety, environmental monitoring and so forth.


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ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Additional text, one scheme, and five figures describing fabrication and characterization of colorimetric-fluorescent-magnetic nanospheres (CFMNs), fabrication and verification of ICFMNs, optimization of detection conditions, analytical performance of CFMNs based LFIA for S. typhi detection. Reliability and specificity analysis of the assay (PDF). AUTHOR INFORMATION Corresponding Authors *(D. W. Pang) E-mail: [email protected]; phone 0086-27-68756759; fax: 0086-27-68754067. ORCID Dai-Wen Pang: 0000-0002-7017-5725 Zhi-Ling Zhang: 0000-0001-7807-2264 Hai-yan Xie: 0000-0002-6330-7929 Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This work was supported by the National Science and Technology Major Project of China (2018ZX10301405), the National Natural Science Foundation of China (21535005, 21775111 and 21804103), the 111 Project (No. 111-2-10), and Collaborative Innovation Center for Chemistry and Molecular Medicine. REFERENCES (1) Taubes, G. The Bacteria Fight Back. Science 2008, 321, 356–361. (2) Havelaar, A. H.; Kirk, M. D.; Torgerson, P. R.; Gibb, H. J.; Hald, T.; Lake, R. J.; Praet, N.; Bellinger, D. C.; de Silva, N. R.; Gargouri, N.; et al. World Health Organization Global Estimates and Regional Comparisons of the Burden of Foodborne Disease in 2010. PLoS Med. 2015, 12, e1001923. (3) Scallan, E.; Hoekstra, R. M.; Angulo, F. J.; Tauxe, R. V.; Widdowson, M. A.; Roy, S. L.; Jones, J. L.; Griffin, P. M. Foodborne Illness Acquired in the United States-Major Pathogens. Emerg. Infect. Dis. 2011, 17, 7–15. (4) Lipkin, W. I. The Changing Face of Pathogen Discovery and Surveillance. Nat. Rev. Microbiol. 2013, 11, 133–141. (5) Crim, S. M.; Iwamoto, M.; Huang, J. Y.; Griffin, P. M.; Gilliss, D.; Cronquist, A. B.; Cartter, M.; Tobin-D'Angelo, M. Blythe, D; Smith, K. S.; et al. Incidence and Trends of Infection with Pathogens Transmitted Commonly Through Food - Foodborne Diseases Active Surveillance Network, 10 US Sites, 2006-2013. MMWR-Morb. Mortal. Wkly. Rep. 2014, 63, 328–332.



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Colorimetric-Fluorescent-Magnetic Nanosphere-Based Multimodal

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