- Email: [email protected]
Dual FITC lateral flow immunoassay for sensitive detection of Escherichia coli O157:H7 in food samples
Author’s Accepted Manuscript Dual FITC lateral flow immunoassay for sensitive detection of Escherichia coli O157:H7 in food samples Chunmei Song, Jinxin Liu, Jianwu Li, Qing Liu www.elsevier.com/locate/bios
PII: DOI: Reference:
S0956-5663(16)30488-2 http://dx.doi.org/10.1016/j.bios.2016.05.057 BIOS8747
To appear in: Biosensors and Bioelectronic Received date: 29 March 2016 Revised date: 13 May 2016 Accepted date: 19 May 2016 Cite this article as: Chunmei Song, Jinxin Liu, Jianwu Li and Qing Liu, Dual FITC lateral flow immunoassay for sensitive detection of Escherichia coli O157:H7 in food samples, Biosensors and Bioelectronic, http://dx.doi.org/10.1016/j.bios.2016.05.057 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Dual FITC lateral flow immunoassay for sensitive detection of Escherichia coli O157:H7 in food samples ﹡
Chunmei Song, Jinxin Liu, Jianwu Li, Qing Liu
School of Medical Instrument and Food Engineering, University of Shanghai for Science and Technology, Shanghai, 200093, China *
To
whom
correspondence
should
be
addressed:
Tel:
86-021-
65710369.
E-mail:
[email protected] (Qing Liu)；
Abstract A pattern of signal amplification lateral flow immunoassay (LFIA) for pathogen detection, which used fluorescein isothiocyanate (FITC) labeled antigen and antibody for dual FITC-LFIA was developed. Escherichia coli O157:H7 (E.coli O157:H7) was selected as the model analyte. In the signal amplification LFIA method, FITC was mixed with sample culture medium, with the presence of E.coli O157:H7 in the samples, the bacteria could emit a yellow-green fluorescence after incubation, creating a fluorescent antigen probe. This antigen probe was added to LFIA, which
already
contained
E.coli
O157:H7
monoclonal
antibodies-FITC
(McAb-E.coli
O157:H7-FITC) dispersed in the conjugate pad. Another E.coli O157:H7 McAb was the test line, and goat anti-mouse IgG antibody was the control line in nitrocellulose (NC) membrane. The visual limit of detection (LOD) of the strip for qualitative detection was 105 CFU/mL while the LOD for semi-quantitative detection could down to 104 CFU/mL by using scanning reader. Signal amplification LFIA was perfectly applied to the detection of food samples with E.coli O157:H7. The LOD was substantially improved to 1 CFU/mL of the original bacterial content after 1
pre-incubation of the bread, milk and jelly samples in broth for 10, 8 and 8 h respectively. The results of this method was more sensitive by 10-fold than the conventional colloidal gold (CG) based strips and comparable to the traditional ELISA. This simple, low-cost and easy to be popularized method served as a significant step towards the development of monitoring food-borne pathogens in food-safety testing. Keywords: FITC; Signal amplification; Dual lateral flow immunoassay; Escherichia coli O157:H7
1. Introduction Food contaminated by food-borne pathogens causes diseases, affects individuals, and even kills these affected individuals. As such, rapid and sensitive detection methods should be developed to screen pathogens in food (Sharma and Mutharasan 2013). One of current detection methods is lateral flow immunoassay, an efficient technique because of several advantages, including rapidity, simplicity, stability, portability, and sensitivity (Mak et al. 2015; Posthuma-Trumpie et al. 2009). The most widely used format of strip sensor is the employment of CG as reporters for colorimetric detection (Chunmei et al. 2011). However, CG-based strip shows serious limitations when high sensitivity is needed (Xie et al. 2014). Many strategies were applied to enhance the sensitivity of lateral flow immunoassay, such as innovating new labels application (Huang et al. 2016), designing new formats of lateral flow immunoassay (Chen and Yang 2015; Song et al. 2016; Zhao et al. 2016), combining with other methods (Chen et al. 2014; Cui et al. 2013), and developing signal-amplification systems (Chen et al. 2015; Zhu et al. 2014). New labels such as colloidal carbon (Noguera et al. 2011), colloidal selenium (Wang et al. 2014), quantum dots (Bruno 2014), up-converting phosphors nanoparticles (Hua et al. 2
2015), lanthanide fluorescent nanoparticles (Zhang et al. 2014) were successfully applied to enhance the sensitivity of LFIA to detect food-borne pathogens. Note that all these series methods are based on the combination of labels and antibodies, and heavily relied on the quality of the used labels and the coupling effect. Usually, raw materials for these new labels, especially the rare-earth elements based labels, are expensive than the conventional colloidal gold labels. And it is also generally accepted that it is of difficulty to prepare the labels with a stable and excellent performance (Huang et al. 2016). FITC, a fluorochrome dye that absorbs ultraviolet or blue light causing molecules to become excited and emit a visible yellow-green light, is widely used to attach a fluorescent label to proteins via the amine group in various techniques to visualize biological cells and detect the presence of bacterial pathogens (Laguerre et al. 2015; Ma et al. 2013). Herein, we report, for the first time, the development of a novel dual FITC LFIA applied the target bacteria as a novel fluorescent probe after incubated with FITC and the FITC-McAb as the second fluorescent probe. The CG-based strip method using the same two McAbs for E.coli O157:H7 detection has already been reported by our group with the LOD at the level of 4 CFU/mL (Song et al. 2016). In this study, the dual FITC LFIA was successfully prepared to detect E.coli O157:H7 with a better sensitivity than CG-based strip. An economical, easy, rapid, and sensitive detection strategy was innovated. The specificity and stability of the LFIA were also evaluated and rapid E.coli O157:H7 detection in real samples was also demonstrated. 2. Materials and methods 2.1 Bacterial strains and Materials The McAb against E. coli O157:H7 (D3, E7) was produced in our lab. Standard strains used for testing the cross-reactivity of the strip are listed in Table 1. All the microorganisms were purchased from Shanghai Prajna biology technique Co.Ltd (Shanghai, China). The NC membrane, 3
sample pad, conjugate pad and absorbent paper were purchased from Millipore. FITC ≥90 % (HPLC) was purchased from Sigma (St. Louis, MO, USA). All solvents and other chemicals were analytical reagent grade. All the solutions used in this study were prepared with ultrapure water (>18MΩ).
Table 1 Specificity of the strip to the strains used in the study NO
Species/Strains
Detected by PCR
ICS(108)
0
FITC containing culture broth
–
–
1
Escherichia coli O157:H7
ATCC43889
+
+
2
Escherichia coli O157:H7
NCTC12900
+
+
3
Escherichia coli O157:H7
ATCC43895
+
+
4
Escherichia coli O91
ATCC:HG15
+
–
5
Escherichia coli O97
ATCC18683
+
–
6
Escherichia coli O100
ATCC18684
+
–
7
Escherichia coli O149
ATCC18685
+
–
8
Escherichia coli
ATCC25922
+
–
9
Escherichia coli
ATCC8739
+
–
10
Escherichia coli
CMCC(B)44102
+
–
11
Staphylococcus aureus
ATCC25923
+
–
12
Staphylococcus aureus
ATCC29213
+
–
13
Staphylococcus aureus
ATCC27660
+
–
14
Staphylococcus aureus
CICC21493
+
–
15
Shigella flexneri
+
–
16
Shigella boydii
CMCC51346
+
–
17
Shigella boydii
ATCC9207
+
–
18
Shigella sonnei
ATCC25931
+
–
19
Yersinia enterocolitica
CMCC52207
+
–
20
Yersinia enterocolitica
ATCC23715
+
–
21
Enterobacter sakazakii
ATCC29004
+
–
ATCC12022
4
22
Enterobacter sakazakii
ATCC29554
+
–
23
Listeria monocytogenes
ATCC19114
+
–
24
Salmonella typhiumukriunm
ATCC14028
+
–
25
Salmonella typhiumukriunm
CICC22956
+
–
26
Vibrio Parahemolyticus
+
–
ATCC17802
2.2 Conjugate of antibodies to FITC Purified McAb-E. coli O157:H7 at about 1 mg/mL antibody concentration was dialyzed extensively into bicarbonate/carbonate buffer at pH 9.0-9.5. A fresh stock solution of FITC in dimethylsulfoxide (DMSO) was added dropwise with stirring to the antibody solution until a final concentration of about 100 μg FITC per mg McAb was achieved. After the reagents were mixed, the reaction tube was covered with foil or kept in the dark while incubating for 2 hours at room temperature. At the end of 2 hours, the reaction mixture was passed through a desalting column to separate the conjugated antibody from the unconjugated FITC. The labeled conjugate was stored at 4 ℃ before use. The fluorescein to protein (F/P) molar ratio can be estimated by measuring the absorbance at 495 nm and 280 nm. The F/P ratio should be between 5 and 10 (Lyerla and Hierholzer 1975). From the absorbance readings (A280 nm and A495 nm) of the conjugate, calculate the F/P ratio of the conjugate according to the equations: Molar F/P = 2.77×A495 nm/[A280 nm -(0.35× A495 nm )]. 2.3 Fabrication of sandwich LFIA Sandwich LFIA is composed of a sample application pad, conjugation pad, NC membrane, absorption pad, and a backing card as shown in Fig.1B. Both the sample pad (15 mm×30 cm) and conjugation pad (7 mm×30 cm) were made from glass fiber. The conjugation pad was prepared by dispensing a desired volume of McAb-E. coli O157:H7(D3)-FITC conjugates onto the glass fiber pad by an XYZ Biostrip Dispenser, followed by drying at room temperature before stored at 4 ℃. 5
The NC membrane (2×30 cm) was spotted with the optimal capture McAb(E7) and goat anti-mouse IgG antibody applied in the test line and control line. After drying for 1h at 40 ℃, the membrane was sealed, and stored under dry conditions. The sample pad, conjugation pad, blotted membrane, and absorption pad were assembled on the plastic backing support board (4×30 cm) sequentially with a 1-2 mm overlap. The assembly was cut into 2.8 mm wide strips using a CM 4000 Cutter (Bio-Dot) and then sealed in a plastic bag with desiccant gel and stored at 4 ℃.
Fig.1 Schematic diagram of the principle for the detection of E. coli O157:H7 and the typically results of the dual FITC LFIA. 2.4 Bacteria culture and sample pre-treatment FITC was dissolved in DMSO. Freshly prepared FITC solution was added to the modified E. coli broth and mixed evenly. E. coli O157:H7 colonies were cultured in modified E. coli broth at 37 ℃ for 12 h before use. Total viable counts were determined by plate count. Food sample (25 g or 25 mL) was added to the prepared broth (225 mL) and incubated at 37 ℃ for 12 h before detection. 6
2.5 Analytical procedure The detection of E. coli O157:H7 was carried out by applying 80 μL sample solution to the bottom of the strip. FITC containing culture broth was used as negative control. 5 min later, the results of test line were visualized in a black box equipped with an ultraviolet light source and a filter of 525 nm (Semrock, USA) and recorded with a digital camera (Fig. 1D) or the intensity of fluorescence on the test line was determined by the ESE-Quant Lateral Flow Reader (Qiagen, Germany) for semi-quantitative analysis (Fig. 1E). 2.6 Simulated Samples detection and comparison study of ELISA and LFIA methods. The E. coli O157:H7 free bread, milk and jelly samples purchased from the local food markets were verified by cultural and PCR methods. Three samples were added to the modified E. coli broth containing FITC. E. coli O157:H7 inoculums were spiked to sample-broth at dilution of 1 CFU/mL and incubated at 37 °C. Aliquots of 1mL from each bottle at 0, 2, 4, 6, 8, 10, 12 h incubation periods were collected and immediately heat killed at 121 °C for 15 min before storage at 4 °C. The spiked samples and nonspiked samples were both detected in triplicate. Double McAb sandwich ELISA: McAb-E. coli O157:H7-E7 was diluted using carbonate buffer (pH 9.6) and immobilized on a 96-well microtiter plate for 2 h at 37 ℃. After washing three times and blocking with 3 % nonfat dried milk, standard bacterium solution or sample solutions were added to the microtiterplate for 30 min at 37 ℃. The second antibody was HRP labeled McAb-E. coli O157:H7-D3, using the substrate 3, 3′, 5, 5′-Tetramethylbenzidine (TMB). 1 M H2SO4 was added to stop the enzyme reaction. Absorbance was measured using a microplate reader at OD450 nm. FITC containing culture broth was used as a negative control. The standard curve was obtained by plotting the OD450 nm values from the standard bacterium solution against 7
its logarithm concentrations. The visual limit of detection (LOD) of the sandwich ELISA was defined as the minimum bacterial concentration producing the OD450
nm
values≥2.1×OD450
nm
values of negative control. All the collected samples were tested with strip and sandwich ELISA.
3. Results and discussion 3.1. Principle and results judgments The principle of the designed LFIA as shown in Fig.1 was based on the antigen-antibody reaction to form a sandwich format (McAb-FITC-Ag-FITC-McAb) when sample migrated to the end of the strip. When E. coli O157:H7 is present in the sample, it will emit a yellow-green fluorescence upon excitation after enrichment (Fig. S1). As sample solution migrated to the end of the strip, the fluorescent bacteria will bind to McAb-FITC probe, and then combine with capture antibodies on the test line, thereby forming a visible yellow-green line on the test zone. When FITC containing culture broth was applied onto the sample pad, the McAb-FITC probes in the conjugate pad were dissolved and migrated freely to the NC membrane and captured by control line, no yellow-green line was observed in the test line zone (Fig. 1C). Regardless of the presence of the target bacterial in the detection solution, goat anti-mouse IgG antibody in the control line will combine with McAb-FITC probes ensuring the validity of the detection. To measure the sensitivity of the developed method, the visual LOD of the strip was defined as the minimum target concentration producing the fluorescence at the test line significantly different from the test line of a negative control strip run without target bacteria. And the LOD for quantitative detection by the scanning reader was defined as the concentration of E. coli O157:H7 in the solution that could induce 10 % increase of the fluorescence intensity compared with that induced by solution without E. coli O157:H7. 8
3.2. Synthesis of McAb-FITC probes UV-Vis absorption spectrum of the McAb-FITC probe was shown in Fig. 2. McAb-FITC exhibit strong absorption at 498 nm and 280 nm, due to conjugated FITC and McAb respectively. The mole ratio of F/P was 8.6. The results were requisite for preparation of the McAb-FITC probe and the test strip.
Fig 2. UV-vis absorption spectra of the McAb-FITC probe 3.3. Optimization of the strip determination The culture enrichment of samples prior to detection is inevitable because the contamination level often falls below the detection limit. Since the detection efficiency depends mainly on the growth rate of bacteria, we first tested the impact of DMSO concentration on E. coli O157:H7 growth. A suitable DMSO concentration for E. coli O157:H7 growth was found to be less than 2 % (Table S1). Tolerance of E. coli O157:H7 to FITC was also evaluated, and the results show that high levels of FITC repressed bacterial growth, 0.4 mg/mL FITC enhanced the growth of E.coli O157:H7 (Fig. S2). In developing the strip method, many factors should be optimized to increase sensitivity. First, 9
the concentration of the FITC solution for sample incubation could affect the brightness of E. coli O157:H7, thus the final fluorescence of the test line. Prepared FITC solution was diluted to different concentrations for sample incubation. The fluorescence results of 0.4 mg/mL and 1.2 mg/mL dilution were compared and a 0.4 mg/mL of FITC solution was found to be optimal for detection (Fig. 3). For test line and control line, the reagent amount of capture antibodies (McAb-E. coli O157:H7-E7) and goat anti-mouse IgG antibody which were coated on each strip of the NC membrane were 1, 0.5 mg/mL respectively. The optimum solute composition for the sample pad buffer solution was found to 0.01 M TBS buffer (pH 7.8), containing 0.5 % PVP, 1 % sucrose, 0.5 % Tw-20, and 1 %BSA. 3.4. Sensitivity and stability of the detection Based on the above optimized detection conditions, E. coli O157:H7 at 0-108 CFU/mL cultured with different concentrations of FITC were analyzed with the prepared strip. Each sample was examined in triplicate and typical results are shown in Fig. 3. McAb-FITC probe could bound to the bacterial surface and formed a distinct yellow-green fluorescent ring (Fig. S3). We firstly introduced McAb-FITC probe to LFIA and detect unlabeled E. coli O157:H7. The visible LOD of the detection was 106 CFU/mL (Fig. 3A). We also detected the fluorescent E. coli O157:H7 by test strip without conjugation pad spotted with McAb-FITC, the visible LOD of the detection was also 106 CFU/mL(Fig.3D). From the results of Fig. 3B and C, we found that with an increase in target E. coli O157:H7 in the detection solution, the fluorescence intensity on the test line increased. Furthermore, the intensity of the yellow-green fluorescence on the test line at 105 CFU/mL of E. coli O157:H7 was significantly different from that of the negative detection 10
solution without E. coli O157:H7. Herein, 105 CFU/mL could be treated as the visual LOD of the dual FITC LFIA. Moreover, we found that 1.2 mg/mL FITC can’t effectively improve the detection sensitivity but increase the background. These detection results were 10-fold improved compare to the above single FITC based strip method and CG-based strip (Fig. S4). A quantitative calibration curve of the detection was constructed using the scanned Peak-ROD values of the test lines at different concentrations. The relationship between the target concentration and corresponding Peak-ROD is shown in Fig. 3E. The Peak-Area of test line was increased with increasing bacteria concentration. At the early period of the concentration increase, the increase of Peak-ROD was not very obvious. Till the concentration of 104 CFU/mL, the Peak-ROD increased dramatically. From the calibration curve in Fig.4, the LOD of the quantitative detection was calculated as 105 CFU/mL. Anyway, we could find a good linear relationship from 103 to107 CFU/mL (inset figure in Fig. 4). This LOD of the dual FITC LFIA was comparable to that of the ELISA method.
B
C
D
E
2500
2250 2000 1750 1500 1250 1000 750 500 250 0
2250
2000
Peak Aera (mm×mv)
A
1750 1500
1250
8
1000
7
6
5
4
3
750 500 250
0 8
7
6
5
4
3
2
1
lg[E. coli O157:H7 concentration(CFU/mL)]
Fig.3 Sensitivity results of the dual FITC LFIA (CFU/mL) and the detection curve of E. coli O157:H7 by the scanning reader.
The stability of the dual FITC LFIA was examined by using the same batch of strips at 105 CFU/mL of E. coli O157:H7 cultures after storage for 1 day, 1, 5, 10, 15 and 20 weeks at 4 ℃. The performance of the dual FITC LFIA after 20 week storage was exactly the same as that stored 11
0
for only 1 day. The dual FITC LFIA after 20 weeks storage at 4 ℃ were undoubtedly still usable. The further confirmation research of the shelf life is under way in our group. 3.5. Specificity confirmation To establish the specificity of the test strip, 3 strains of E. coli O157:H7 and 23 of other food-borne strains at 108 CFU/mL were applied for the label-free strip test. Only the E. coli O157:H7 strains displayed positive signals, whereas the other strains didn’t show any signals (Fig.4). We conclude that the dual FITC LFIA have no cross-reactivity with other likely food-borne pathogens.
Fig.4 Specificity results of the dual FITC LFIA (0-26 correspond to standard strains listed in Table 1). 3.6. Sample analysis In order to ensure the applicability and accuracy of the dual FITC LFIA, simulated bread, milk and jelly samples spiked with E. coli O157:H7 were detected in different incubation periods. As shown in Fig. 5A, bread, milk and jelly samples showed positive results after incubation for 10, 8 and 8 h, and the detection sensitivities increased to 1 CFU/mL. Test results of three kinds of nonspiked samples incubated for 12 h were all negative.
12
Absorbance OD450nm
4 E. coli O157:H7+FITC
3
E. coli O157:H7 2
1
0
2
(A)
3
4 5 6 7 Lg[dilution (CFU/mL)]
(B)
Fig.5 (A) Detection of E. coli O157:H7 in simulated food samples by the dual FITC LFIA after pre-incubation in different hours. (B) Standard curve for labeled and unlabeled E. coli O157:H7 using sandwich ELISA. 3.6 Comparative studies between test strips and ELISA The sensitivity of the sandwich ELISA was determined with the labeled and unlabeled E. coli O157:H7 standard solution for comparing with the performance of the dual FITC LFIA. In the ELISA test, the OD450 nm values were increased as the bacterium concentration increased. The standard curve was shown in Fig. 5B. The visual LOD of sandwich ELISA was 105 CFU/mL. This result demonstrated that FITC does not affect the antigen epitope of E. coli O157:H7. All detection results of simulated samples from dual FITC LFIA were confirmed by ELISA test, and the results from the two analysis methods showed good correspondence (Table S2).
4. Conclusions A novel signal amplification LFIA was developed for the detection of E. coli O157:H7. The dual FITC conjugates McAb-FITC-E. coli O157:H7-FITC-McAb resulted in higher sensitivity than traditional LFIA methods. In this study, qualitative and quantitative detection of E. coli O157:H7 were successfully realized with LODs of 105 and 104 CFU/mL by naked-eye observation 13
8
and scanning reader, respectively. All detection results meet the Chinese standards as well as the requirements of other countries. The detection sensitivity of E. coli O157:H7 improved to 1 CFU/mL after pre-incubation of the bacteria in bread, milk and jelly samples for 10, 8 and 8 h respectively. More importantly, this method could be extended to the detection of other pathogens by simply replacing the antibody. Work in our laboratory on optimization of the quantitative detections and further signal enhancement for E. coli O157:H7 detection and general applications are ongoing.
Acknowledgment This research is supported by the National Natural Science Foundation of China (No. 31371776), Capability Construction Program of Science and technology commission of Shanghai (No.13430502400), Science and technology innovation plan of Shanghai：Yangtze River Delta joint research (15395810900), China Postdoctoral Science Foundation (No.2015M581637) and the Cultivation Fund for National Project of University of Shanghai for Science and Technology (16HJPY-QN09).
Supplementary data Supplementary data can be found in the online version.
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Chen, X., Gan, M., Xu, H., Chen, F., Ming, X., Xu, H., Wei, H., Xu, F., Liu, C., 2014. Food Control 46, 225-232. Chunmei, S., Qingtang, L., Aimin, Z., Jifei, Y., Yubao, Z., Qingmei, L., Xiaofei, H., Ruiguang, D., Justin, C., Liang, T., 2011. J. Agric. Food Chem. 59(17), 9319-9326. Cui, X., Xiong, Q.R., Xiong, Y.H., Shan, S., Lai, W.H., 2013. Chin. J. Anal. Chem. 41(12), 1812-1816. Hua, F., Zhang, P., Zhang, F., Zhao, Y., Li, C., Sun, C., Wang, X., Yang, R., Wang, C., Yu, A., 2015. Sci. Rep. 5, 17178. Huang, X., Aguilar, Z.P., Xu, H., Lai, W., Xiong, Y., 2016. A review. Biosens. Bioelectron. 75, 166-180. Laguerre, A., Hukezalie, K., Winckler, P., Katranji, F., Chanteloup, G., Pirrotta, M., Perrier-Cornet, J.-M., Wong, J.M., Monchaud, D., 2015. J. Am. Chem. Soc. 137(26), 8521-8525. Lyerla, H.C., Hierholzer, J.C., 1975. J. Clin. microbiol. 1(5), 451-461. Ma, J., Wang, H., Wang, Y., Zhang, S., 2013. Mol. immunol. 53(4), 355-362. Mak, W.C., Beni, V., Turner, A.P.F., 2016. Trac-Trend. Anal. Chem. 79, 297-305. Noguera, P., Posthuma-Trumpie, G., Van Tuil, M., Van der Wal, F., De Boer, A., Moers, A., Van Amerongen, A., 2011. Anal. Bioanal. Chem. 399(2), 831-838. Posthuma-Trumpie, G.A., Korf, J., van Amerongen, A., 2009. Anal. Bioanal. Chem. 393(2), 569-582. Sharma, H., Mutharasan, R., 2013. Sens. Actuators, B 183(20), 535-549. Song, C., Liu, C., Wu, S., Li, H., Guo, H., Yang, B., Qiu, S., Li, J., Liu, L., Zeng, H., 2016. Food Control 59, 345-351. 15
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Highlights
FITC was used to make the E. coli O157:H7 to fluoresce. Target E. coli O157:H7 as fluorescent reporters. FITC-antibody was firstly introduced to lateral flow immunoassay. Dual FITC LFIA for E. coli O157:H7 detection was more sensitive by 10-fold than GC-based strip. Successful applications in actual bread, milk, jelly samples with sensitivity of 1CFU/mL
16
PII: DOI: Reference:
S0956-5663(16)30488-2 http://dx.doi.org/10.1016/j.bios.2016.05.057 BIOS8747
To appear in: Biosensors and Bioelectronic Received date: 29 March 2016 Revised date: 13 May 2016 Accepted date: 19 May 2016 Cite this article as: Chunmei Song, Jinxin Liu, Jianwu Li and Qing Liu, Dual FITC lateral flow immunoassay for sensitive detection of Escherichia coli O157:H7 in food samples, Biosensors and Bioelectronic, http://dx.doi.org/10.1016/j.bios.2016.05.057 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Dual FITC lateral flow immunoassay for sensitive detection of Escherichia coli O157:H7 in food samples ﹡
Chunmei Song, Jinxin Liu, Jianwu Li, Qing Liu
School of Medical Instrument and Food Engineering, University of Shanghai for Science and Technology, Shanghai, 200093, China *
To
whom
correspondence
should
be
addressed:
Tel:
86-021-
65710369.
E-mail:
[email protected] (Qing Liu)；
Abstract A pattern of signal amplification lateral flow immunoassay (LFIA) for pathogen detection, which used fluorescein isothiocyanate (FITC) labeled antigen and antibody for dual FITC-LFIA was developed. Escherichia coli O157:H7 (E.coli O157:H7) was selected as the model analyte. In the signal amplification LFIA method, FITC was mixed with sample culture medium, with the presence of E.coli O157:H7 in the samples, the bacteria could emit a yellow-green fluorescence after incubation, creating a fluorescent antigen probe. This antigen probe was added to LFIA, which
already
contained
E.coli
O157:H7
monoclonal
antibodies-FITC
(McAb-E.coli
O157:H7-FITC) dispersed in the conjugate pad. Another E.coli O157:H7 McAb was the test line, and goat anti-mouse IgG antibody was the control line in nitrocellulose (NC) membrane. The visual limit of detection (LOD) of the strip for qualitative detection was 105 CFU/mL while the LOD for semi-quantitative detection could down to 104 CFU/mL by using scanning reader. Signal amplification LFIA was perfectly applied to the detection of food samples with E.coli O157:H7. The LOD was substantially improved to 1 CFU/mL of the original bacterial content after 1
pre-incubation of the bread, milk and jelly samples in broth for 10, 8 and 8 h respectively. The results of this method was more sensitive by 10-fold than the conventional colloidal gold (CG) based strips and comparable to the traditional ELISA. This simple, low-cost and easy to be popularized method served as a significant step towards the development of monitoring food-borne pathogens in food-safety testing. Keywords: FITC; Signal amplification; Dual lateral flow immunoassay; Escherichia coli O157:H7
1. Introduction Food contaminated by food-borne pathogens causes diseases, affects individuals, and even kills these affected individuals. As such, rapid and sensitive detection methods should be developed to screen pathogens in food (Sharma and Mutharasan 2013). One of current detection methods is lateral flow immunoassay, an efficient technique because of several advantages, including rapidity, simplicity, stability, portability, and sensitivity (Mak et al. 2015; Posthuma-Trumpie et al. 2009). The most widely used format of strip sensor is the employment of CG as reporters for colorimetric detection (Chunmei et al. 2011). However, CG-based strip shows serious limitations when high sensitivity is needed (Xie et al. 2014). Many strategies were applied to enhance the sensitivity of lateral flow immunoassay, such as innovating new labels application (Huang et al. 2016), designing new formats of lateral flow immunoassay (Chen and Yang 2015; Song et al. 2016; Zhao et al. 2016), combining with other methods (Chen et al. 2014; Cui et al. 2013), and developing signal-amplification systems (Chen et al. 2015; Zhu et al. 2014). New labels such as colloidal carbon (Noguera et al. 2011), colloidal selenium (Wang et al. 2014), quantum dots (Bruno 2014), up-converting phosphors nanoparticles (Hua et al. 2
2015), lanthanide fluorescent nanoparticles (Zhang et al. 2014) were successfully applied to enhance the sensitivity of LFIA to detect food-borne pathogens. Note that all these series methods are based on the combination of labels and antibodies, and heavily relied on the quality of the used labels and the coupling effect. Usually, raw materials for these new labels, especially the rare-earth elements based labels, are expensive than the conventional colloidal gold labels. And it is also generally accepted that it is of difficulty to prepare the labels with a stable and excellent performance (Huang et al. 2016). FITC, a fluorochrome dye that absorbs ultraviolet or blue light causing molecules to become excited and emit a visible yellow-green light, is widely used to attach a fluorescent label to proteins via the amine group in various techniques to visualize biological cells and detect the presence of bacterial pathogens (Laguerre et al. 2015; Ma et al. 2013). Herein, we report, for the first time, the development of a novel dual FITC LFIA applied the target bacteria as a novel fluorescent probe after incubated with FITC and the FITC-McAb as the second fluorescent probe. The CG-based strip method using the same two McAbs for E.coli O157:H7 detection has already been reported by our group with the LOD at the level of 4 CFU/mL (Song et al. 2016). In this study, the dual FITC LFIA was successfully prepared to detect E.coli O157:H7 with a better sensitivity than CG-based strip. An economical, easy, rapid, and sensitive detection strategy was innovated. The specificity and stability of the LFIA were also evaluated and rapid E.coli O157:H7 detection in real samples was also demonstrated. 2. Materials and methods 2.1 Bacterial strains and Materials The McAb against E. coli O157:H7 (D3, E7) was produced in our lab. Standard strains used for testing the cross-reactivity of the strip are listed in Table 1. All the microorganisms were purchased from Shanghai Prajna biology technique Co.Ltd (Shanghai, China). The NC membrane, 3
sample pad, conjugate pad and absorbent paper were purchased from Millipore. FITC ≥90 % (HPLC) was purchased from Sigma (St. Louis, MO, USA). All solvents and other chemicals were analytical reagent grade. All the solutions used in this study were prepared with ultrapure water (>18MΩ).
Table 1 Specificity of the strip to the strains used in the study NO
Species/Strains
Detected by PCR
ICS(108)
0
FITC containing culture broth
–
–
1
Escherichia coli O157:H7
ATCC43889
+
+
2
Escherichia coli O157:H7
NCTC12900
+
+
3
Escherichia coli O157:H7
ATCC43895
+
+
4
Escherichia coli O91
ATCC:HG15
+
–
5
Escherichia coli O97
ATCC18683
+
–
6
Escherichia coli O100
ATCC18684
+
–
7
Escherichia coli O149
ATCC18685
+
–
8
Escherichia coli
ATCC25922
+
–
9
Escherichia coli
ATCC8739
+
–
10
Escherichia coli
CMCC(B)44102
+
–
11
Staphylococcus aureus
ATCC25923
+
–
12
Staphylococcus aureus
ATCC29213
+
–
13
Staphylococcus aureus
ATCC27660
+
–
14
Staphylococcus aureus
CICC21493
+
–
15
Shigella flexneri
+
–
16
Shigella boydii
CMCC51346
+
–
17
Shigella boydii
ATCC9207
+
–
18
Shigella sonnei
ATCC25931
+
–
19
Yersinia enterocolitica
CMCC52207
+
–
20
Yersinia enterocolitica
ATCC23715
+
–
21
Enterobacter sakazakii
ATCC29004
+
–
ATCC12022
4
22
Enterobacter sakazakii
ATCC29554
+
–
23
Listeria monocytogenes
ATCC19114
+
–
24
Salmonella typhiumukriunm
ATCC14028
+
–
25
Salmonella typhiumukriunm
CICC22956
+
–
26
Vibrio Parahemolyticus
+
–
ATCC17802
2.2 Conjugate of antibodies to FITC Purified McAb-E. coli O157:H7 at about 1 mg/mL antibody concentration was dialyzed extensively into bicarbonate/carbonate buffer at pH 9.0-9.5. A fresh stock solution of FITC in dimethylsulfoxide (DMSO) was added dropwise with stirring to the antibody solution until a final concentration of about 100 μg FITC per mg McAb was achieved. After the reagents were mixed, the reaction tube was covered with foil or kept in the dark while incubating for 2 hours at room temperature. At the end of 2 hours, the reaction mixture was passed through a desalting column to separate the conjugated antibody from the unconjugated FITC. The labeled conjugate was stored at 4 ℃ before use. The fluorescein to protein (F/P) molar ratio can be estimated by measuring the absorbance at 495 nm and 280 nm. The F/P ratio should be between 5 and 10 (Lyerla and Hierholzer 1975). From the absorbance readings (A280 nm and A495 nm) of the conjugate, calculate the F/P ratio of the conjugate according to the equations: Molar F/P = 2.77×A495 nm/[A280 nm -(0.35× A495 nm )]. 2.3 Fabrication of sandwich LFIA Sandwich LFIA is composed of a sample application pad, conjugation pad, NC membrane, absorption pad, and a backing card as shown in Fig.1B. Both the sample pad (15 mm×30 cm) and conjugation pad (7 mm×30 cm) were made from glass fiber. The conjugation pad was prepared by dispensing a desired volume of McAb-E. coli O157:H7(D3)-FITC conjugates onto the glass fiber pad by an XYZ Biostrip Dispenser, followed by drying at room temperature before stored at 4 ℃. 5
The NC membrane (2×30 cm) was spotted with the optimal capture McAb(E7) and goat anti-mouse IgG antibody applied in the test line and control line. After drying for 1h at 40 ℃, the membrane was sealed, and stored under dry conditions. The sample pad, conjugation pad, blotted membrane, and absorption pad were assembled on the plastic backing support board (4×30 cm) sequentially with a 1-2 mm overlap. The assembly was cut into 2.8 mm wide strips using a CM 4000 Cutter (Bio-Dot) and then sealed in a plastic bag with desiccant gel and stored at 4 ℃.
Fig.1 Schematic diagram of the principle for the detection of E. coli O157:H7 and the typically results of the dual FITC LFIA. 2.4 Bacteria culture and sample pre-treatment FITC was dissolved in DMSO. Freshly prepared FITC solution was added to the modified E. coli broth and mixed evenly. E. coli O157:H7 colonies were cultured in modified E. coli broth at 37 ℃ for 12 h before use. Total viable counts were determined by plate count. Food sample (25 g or 25 mL) was added to the prepared broth (225 mL) and incubated at 37 ℃ for 12 h before detection. 6
2.5 Analytical procedure The detection of E. coli O157:H7 was carried out by applying 80 μL sample solution to the bottom of the strip. FITC containing culture broth was used as negative control. 5 min later, the results of test line were visualized in a black box equipped with an ultraviolet light source and a filter of 525 nm (Semrock, USA) and recorded with a digital camera (Fig. 1D) or the intensity of fluorescence on the test line was determined by the ESE-Quant Lateral Flow Reader (Qiagen, Germany) for semi-quantitative analysis (Fig. 1E). 2.6 Simulated Samples detection and comparison study of ELISA and LFIA methods. The E. coli O157:H7 free bread, milk and jelly samples purchased from the local food markets were verified by cultural and PCR methods. Three samples were added to the modified E. coli broth containing FITC. E. coli O157:H7 inoculums were spiked to sample-broth at dilution of 1 CFU/mL and incubated at 37 °C. Aliquots of 1mL from each bottle at 0, 2, 4, 6, 8, 10, 12 h incubation periods were collected and immediately heat killed at 121 °C for 15 min before storage at 4 °C. The spiked samples and nonspiked samples were both detected in triplicate. Double McAb sandwich ELISA: McAb-E. coli O157:H7-E7 was diluted using carbonate buffer (pH 9.6) and immobilized on a 96-well microtiter plate for 2 h at 37 ℃. After washing three times and blocking with 3 % nonfat dried milk, standard bacterium solution or sample solutions were added to the microtiterplate for 30 min at 37 ℃. The second antibody was HRP labeled McAb-E. coli O157:H7-D3, using the substrate 3, 3′, 5, 5′-Tetramethylbenzidine (TMB). 1 M H2SO4 was added to stop the enzyme reaction. Absorbance was measured using a microplate reader at OD450 nm. FITC containing culture broth was used as a negative control. The standard curve was obtained by plotting the OD450 nm values from the standard bacterium solution against 7
its logarithm concentrations. The visual limit of detection (LOD) of the sandwich ELISA was defined as the minimum bacterial concentration producing the OD450
nm
values≥2.1×OD450
nm
values of negative control. All the collected samples were tested with strip and sandwich ELISA.
3. Results and discussion 3.1. Principle and results judgments The principle of the designed LFIA as shown in Fig.1 was based on the antigen-antibody reaction to form a sandwich format (McAb-FITC-Ag-FITC-McAb) when sample migrated to the end of the strip. When E. coli O157:H7 is present in the sample, it will emit a yellow-green fluorescence upon excitation after enrichment (Fig. S1). As sample solution migrated to the end of the strip, the fluorescent bacteria will bind to McAb-FITC probe, and then combine with capture antibodies on the test line, thereby forming a visible yellow-green line on the test zone. When FITC containing culture broth was applied onto the sample pad, the McAb-FITC probes in the conjugate pad were dissolved and migrated freely to the NC membrane and captured by control line, no yellow-green line was observed in the test line zone (Fig. 1C). Regardless of the presence of the target bacterial in the detection solution, goat anti-mouse IgG antibody in the control line will combine with McAb-FITC probes ensuring the validity of the detection. To measure the sensitivity of the developed method, the visual LOD of the strip was defined as the minimum target concentration producing the fluorescence at the test line significantly different from the test line of a negative control strip run without target bacteria. And the LOD for quantitative detection by the scanning reader was defined as the concentration of E. coli O157:H7 in the solution that could induce 10 % increase of the fluorescence intensity compared with that induced by solution without E. coli O157:H7. 8
3.2. Synthesis of McAb-FITC probes UV-Vis absorption spectrum of the McAb-FITC probe was shown in Fig. 2. McAb-FITC exhibit strong absorption at 498 nm and 280 nm, due to conjugated FITC and McAb respectively. The mole ratio of F/P was 8.6. The results were requisite for preparation of the McAb-FITC probe and the test strip.
Fig 2. UV-vis absorption spectra of the McAb-FITC probe 3.3. Optimization of the strip determination The culture enrichment of samples prior to detection is inevitable because the contamination level often falls below the detection limit. Since the detection efficiency depends mainly on the growth rate of bacteria, we first tested the impact of DMSO concentration on E. coli O157:H7 growth. A suitable DMSO concentration for E. coli O157:H7 growth was found to be less than 2 % (Table S1). Tolerance of E. coli O157:H7 to FITC was also evaluated, and the results show that high levels of FITC repressed bacterial growth, 0.4 mg/mL FITC enhanced the growth of E.coli O157:H7 (Fig. S2). In developing the strip method, many factors should be optimized to increase sensitivity. First, 9
the concentration of the FITC solution for sample incubation could affect the brightness of E. coli O157:H7, thus the final fluorescence of the test line. Prepared FITC solution was diluted to different concentrations for sample incubation. The fluorescence results of 0.4 mg/mL and 1.2 mg/mL dilution were compared and a 0.4 mg/mL of FITC solution was found to be optimal for detection (Fig. 3). For test line and control line, the reagent amount of capture antibodies (McAb-E. coli O157:H7-E7) and goat anti-mouse IgG antibody which were coated on each strip of the NC membrane were 1, 0.5 mg/mL respectively. The optimum solute composition for the sample pad buffer solution was found to 0.01 M TBS buffer (pH 7.8), containing 0.5 % PVP, 1 % sucrose, 0.5 % Tw-20, and 1 %BSA. 3.4. Sensitivity and stability of the detection Based on the above optimized detection conditions, E. coli O157:H7 at 0-108 CFU/mL cultured with different concentrations of FITC were analyzed with the prepared strip. Each sample was examined in triplicate and typical results are shown in Fig. 3. McAb-FITC probe could bound to the bacterial surface and formed a distinct yellow-green fluorescent ring (Fig. S3). We firstly introduced McAb-FITC probe to LFIA and detect unlabeled E. coli O157:H7. The visible LOD of the detection was 106 CFU/mL (Fig. 3A). We also detected the fluorescent E. coli O157:H7 by test strip without conjugation pad spotted with McAb-FITC, the visible LOD of the detection was also 106 CFU/mL(Fig.3D). From the results of Fig. 3B and C, we found that with an increase in target E. coli O157:H7 in the detection solution, the fluorescence intensity on the test line increased. Furthermore, the intensity of the yellow-green fluorescence on the test line at 105 CFU/mL of E. coli O157:H7 was significantly different from that of the negative detection 10
solution without E. coli O157:H7. Herein, 105 CFU/mL could be treated as the visual LOD of the dual FITC LFIA. Moreover, we found that 1.2 mg/mL FITC can’t effectively improve the detection sensitivity but increase the background. These detection results were 10-fold improved compare to the above single FITC based strip method and CG-based strip (Fig. S4). A quantitative calibration curve of the detection was constructed using the scanned Peak-ROD values of the test lines at different concentrations. The relationship between the target concentration and corresponding Peak-ROD is shown in Fig. 3E. The Peak-Area of test line was increased with increasing bacteria concentration. At the early period of the concentration increase, the increase of Peak-ROD was not very obvious. Till the concentration of 104 CFU/mL, the Peak-ROD increased dramatically. From the calibration curve in Fig.4, the LOD of the quantitative detection was calculated as 105 CFU/mL. Anyway, we could find a good linear relationship from 103 to107 CFU/mL (inset figure in Fig. 4). This LOD of the dual FITC LFIA was comparable to that of the ELISA method.
B
C
D
E
2500
2250 2000 1750 1500 1250 1000 750 500 250 0
2250
2000
Peak Aera (mm×mv)
A
1750 1500
1250
8
1000
7
6
5
4
3
750 500 250
0 8
7
6
5
4
3
2
1
lg[E. coli O157:H7 concentration(CFU/mL)]
Fig.3 Sensitivity results of the dual FITC LFIA (CFU/mL) and the detection curve of E. coli O157:H7 by the scanning reader.
The stability of the dual FITC LFIA was examined by using the same batch of strips at 105 CFU/mL of E. coli O157:H7 cultures after storage for 1 day, 1, 5, 10, 15 and 20 weeks at 4 ℃. The performance of the dual FITC LFIA after 20 week storage was exactly the same as that stored 11
0
for only 1 day. The dual FITC LFIA after 20 weeks storage at 4 ℃ were undoubtedly still usable. The further confirmation research of the shelf life is under way in our group. 3.5. Specificity confirmation To establish the specificity of the test strip, 3 strains of E. coli O157:H7 and 23 of other food-borne strains at 108 CFU/mL were applied for the label-free strip test. Only the E. coli O157:H7 strains displayed positive signals, whereas the other strains didn’t show any signals (Fig.4). We conclude that the dual FITC LFIA have no cross-reactivity with other likely food-borne pathogens.
Fig.4 Specificity results of the dual FITC LFIA (0-26 correspond to standard strains listed in Table 1). 3.6. Sample analysis In order to ensure the applicability and accuracy of the dual FITC LFIA, simulated bread, milk and jelly samples spiked with E. coli O157:H7 were detected in different incubation periods. As shown in Fig. 5A, bread, milk and jelly samples showed positive results after incubation for 10, 8 and 8 h, and the detection sensitivities increased to 1 CFU/mL. Test results of three kinds of nonspiked samples incubated for 12 h were all negative.
12
Absorbance OD450nm
4 E. coli O157:H7+FITC
3
E. coli O157:H7 2
1
0
2
(A)
3
4 5 6 7 Lg[dilution (CFU/mL)]
(B)
Fig.5 (A) Detection of E. coli O157:H7 in simulated food samples by the dual FITC LFIA after pre-incubation in different hours. (B) Standard curve for labeled and unlabeled E. coli O157:H7 using sandwich ELISA. 3.6 Comparative studies between test strips and ELISA The sensitivity of the sandwich ELISA was determined with the labeled and unlabeled E. coli O157:H7 standard solution for comparing with the performance of the dual FITC LFIA. In the ELISA test, the OD450 nm values were increased as the bacterium concentration increased. The standard curve was shown in Fig. 5B. The visual LOD of sandwich ELISA was 105 CFU/mL. This result demonstrated that FITC does not affect the antigen epitope of E. coli O157:H7. All detection results of simulated samples from dual FITC LFIA were confirmed by ELISA test, and the results from the two analysis methods showed good correspondence (Table S2).
4. Conclusions A novel signal amplification LFIA was developed for the detection of E. coli O157:H7. The dual FITC conjugates McAb-FITC-E. coli O157:H7-FITC-McAb resulted in higher sensitivity than traditional LFIA methods. In this study, qualitative and quantitative detection of E. coli O157:H7 were successfully realized with LODs of 105 and 104 CFU/mL by naked-eye observation 13
8
and scanning reader, respectively. All detection results meet the Chinese standards as well as the requirements of other countries. The detection sensitivity of E. coli O157:H7 improved to 1 CFU/mL after pre-incubation of the bacteria in bread, milk and jelly samples for 10, 8 and 8 h respectively. More importantly, this method could be extended to the detection of other pathogens by simply replacing the antibody. Work in our laboratory on optimization of the quantitative detections and further signal enhancement for E. coli O157:H7 detection and general applications are ongoing.
Acknowledgment This research is supported by the National Natural Science Foundation of China (No. 31371776), Capability Construction Program of Science and technology commission of Shanghai (No.13430502400), Science and technology innovation plan of Shanghai：Yangtze River Delta joint research (15395810900), China Postdoctoral Science Foundation (No.2015M581637) and the Cultivation Fund for National Project of University of Shanghai for Science and Technology (16HJPY-QN09).
Supplementary data Supplementary data can be found in the online version.
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Highlights
FITC was used to make the E. coli O157:H7 to fluoresce. Target E. coli O157:H7 as fluorescent reporters. FITC-antibody was firstly introduced to lateral flow immunoassay. Dual FITC LFIA for E. coli O157:H7 detection was more sensitive by 10-fold than GC-based strip. Successful applications in actual bread, milk, jelly samples with sensitivity of 1CFU/mL
16