Appl Biochem Biotechnol DOI 10.1007/s12010-014-0904-4
Specific Detection of Vibrio Parahaemolyticus by Fluorescence Quenching Immunoassay Based on Quantum Dots Ling Wang & Junxian Zhang & Haili Bai & Xuan Li & Pintian Lv & Ailing Guo
Received: 19 September 2013 / Accepted: 3 April 2014 # Springer Science+Business Media New York 2014
Abstract In this study, anti-Vibrio parahaemolyticus polyclonal and monoclonal antibodies were prepared through intradermal injection immune and lymphocyte hybridoma technique respectively. CdTe quantum dots (QDs) were synthesized at pH 9.3, 98 °C for 1 h with stabilizer of 2.7:1. The fluorescence intensity was 586.499, and the yield was 62.43 %. QD probes were successfully prepared under the optimized conditions of pH 7.4, 37 °C for 1 h, 250 μL of 50 mg/mL EDC·HCl, 150 μL of 4 mg/mL NHS, buffer system of Na2HPO4-citric acid, and 8 μL of 2.48 mg/mL polyclonal antibodies. As gold nanoparticles could quench fluorescence of quantum dots, the concentration of V. parahaemolyticus could be detected through measuring the reduction of fluorescence intensity in immune sandwich reaction composed of quantum dot probe, gold-labeled antibody, and the sample. For pure culture, fluorescence intensity of the system was proportional with logarithm concentration of antigen, and the correlation coefficient was 99.764 %. The fluorescence quenching immunoassay based on quantum dots is established for the first time to detect Vibrio parahaemolyticus. This method may be used as rapid testing procedure due to its high simplicity and sensitivity. Keywords CdTe QD probes . Gold-labeled antibodies . Fluorescence quenching immunoassay . Vibrio parahaemolyticus
Introduction As a halophilic Gram-negative bacterium, Vibrio parahaemolyticus which frequently causes food-borne human gastroenteritis is widely distributed in coastal and estuarine environments throughout the world [1]. With the rapidly increasing global incidence of V. parahaemolyticus infection in recent years, consumption of raw or undercooked seafood proves to be the major cause [2]. Traditional detection methods are based on bacterium culture, including enrichment Ling Wang and Junxian Zhang contributed equally to this paper.
L. Wang : J. Zhang : H. Bai : X. Li : P. Lv : A. Guo (*) Key Laboratory of Environment Correlative Dietology of Ministry of Education, College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, China e-mail: [email protected]
Appl Biochem Biotechnol
in liquid media and isolation from selective culture medium [3]. Whereas, it takes 7 days to get the conclusion and high appearance of false negative results is the limitation for its current application [4]. Therefore, rapid and accurate detection methods of V. parahaemolyticus are urgently needed in prevention of food poisoning and food safety. Current techniques of detecting bacteria include immunological methods, molecular means, and equipment tests. Molecular means such as dot blotting, PCR are relatively high-cost, meanwhile professional skills are required [5]. Equipment detections are rapid but have not been used broadly. Immunological methods with simplicity, cheapness, and sensitivity and rapidness properties are ideal for detecting pathogens [6]. Semiconductor quantum dots (QDs) are nanometer-sized crystals with unique photochemical and photophysical properties which have aroused great interest as a new class of fluorescent labels in past decades in the fields of biology, medicine, sensing, and environmental analysis and so on [7]. Compared to traditional organic dyes, QDs have their own unique nature of a wide range of excitation wavelength, narrow emission wavelength, size-adjustment, long fluorescence lifetime, highly photochemical stability, high photoluminescence, and narrow and symmetrical emission peak [8]. The method of QDs coupled with biological molecules has a very broad application in biology, medicine, sensing, and environment analysis [9, 10]. QDs will be the most promising biological fluorescent probe. Researchers have devoted a lot of effort to studying fluorescent ways that involve the use of nanometer materials, especially QDs combined with AuNPs [11]. AuNPs have caused great attention for their high extinction coefficient and broad absorption spectrum in a visible light. AuNPs are unique quenchers for organic dyes and QDs through electron-transfer or energy-transfer procedure [12]. Up to now, fluorescent sensors based on QDs and AuNPs still have extensive potential in detection of small molecules or protein [13]. The most prevalent explanation of the quenching phenomenon between AuNPs and QDs focus on two theories, which are fluorescence resonance energy transfer (FRET) and inner filter effect (IFE). The FRET process involves intermolecular connection of QDs and AuNPs and a particular distance or geometry to enable interaction between them. Usually, the FRETrelated sensors are based on complicated chemical reactions with regards to modification of AuNPs or QDs surface. The IFE refers to complementary overlap of absorption band of the absorber (quenchers, such as AuNPs) and excitation or emission bands of the fluorophore (such as QDs) [10]. Therefore, facilely tuned absorption or emission spectrum can be used as IFE-based fluorescent assay [14]. In this research, monoclonal antibody modified by AuNPs and polyclonal antibody modified by QDs were prepared, and these two decorated materials were further conjugated with V. parahaemolyticus or any other antigens to form an immune sandwich reaction. Based on the FRET process of QDs/AuNPs, the fluorescence of CdTe QDs is very weak in the presence of V. parahaemolyticus or other antigens. Compared to other bacterium, the fluorescence was very sensitive to V. parahaemolyticus. It demonstrated that the result of our work based on FRET to determine V. parahaemolyticus is a specific, rapid, and sensitive approach.
Materials and Methods Apparatus, Materials, and Reagents Fluorescence spectrophotometer (RF-5301p) and UV-vis spectrophotometer (UV-1077) were from Shimadzu, Japan. 5415-R frozen high-speed centrifuge were bought from Eppendorf. Other instruments were bought from China. Chemical reagents were purchased from Sigma
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Company. V. parahaemolyticus ATCC17802 was purchased from Pharmaceutical Biological Product Detection Station in China. SP2/0 mouse myeloma cells were stored in our laboratory. Preparation of Polyclonal Antibody Liquid culture of V. parahaemolyticus was boiled for 2 h. Then, the inactive bacterium was assembled after centrifugation at 4,000 r/min for 5 min. The bacterial suspension was adjusted to 1010 cfu/L with normal saline, and consequently antigen was gained and stored at −20 °C. Japanese large-eared rabbits were immunized by subcutaneous injection of 1 mL bacterial suspension (1×107 cfu/L) once 2 weeks. Blood was collected via the ear-rim vein at the 10th day after each immunization, then put at 37 °C for 1 h, and finally stored at 4 °C for a night. The stored blood was centrifuged at 10,000 r/min, 4 °C for 5 min to obtain serum later for detection of polyclonal antibody with ELISA method. Preparation of Monoclonal Antibody Female BAL b/C mice 6–8 weeks old were immunized with 0.5 mL (107 cfu/L) antigen by abdominal subcutaneous injection once 2 weeks. The mice were docked and blood was collected at 1 week after each immunization later for detecting mice serum antibody titers via ELISA method. BAL b/C mice immuned were used for preparing immune splenocyte, and SP2/0 mice were used to prepare myeloma cell. The prepared splenocyte (1×108 cfu/L) and myeloma cell (2×107 cfu/L) were complexed with the ratio of 5:1 for cell fusion, and then the positive hybridoma cells were screened. The hybridoma cells were cloned and then for scale-up culture to screen monoclonal antibody further. Bacteria including V. parahaemolyticus, V. alginolyticus, V. metschnikovii, V. cholerae, and V. vulnificus were used as antigens. They were cross-reacted with various monoclonal antibodies and verified the specificity of monoclonal antibody. Preparation of CdTe QDs and CdTe QDs/Polyclonal Antibody Fluorescence Probes In a typical experiment, water-soluble CdTe QDs coated with thioglycolic acid (TGA) were prepared in a 10 mL airtight three-necked bottle with 50.0 mg NaHB4 and 31.9 mg tellurium (Te) powder at 40 °C, until the solution color turned to transparent purple. Then, 99.0 mL ultrapure water was injected to the three-necked bottle, and at the same time, CdCl2 was added to the bottle with N2 protection. After heated for 30 min at 95 °C, TGA (the molar ratio of CdCl2 and TGA was 3:1) was added to the solution under the condition of pH 10 and heating reflux at 95 °C. CdTe QDs prepared (0.5 mL) were added into 50 mg/mL EDC·HCl mixed with 4 mg/mL N-hydroxysuccinimide (NHS), vibrated for 10∼20 min, and then centrifuged for 5 min at 12,000 r/min. Precipitate collected was suspended in 5 mL buffer solution after twice centrifugation and finally stored at 4 °C. Preparation of Gold-Labeled Antibody Distilled water (99.0 mL) and HAuCl4 (1 %, 1 mL) were boiled in a 250-mL triangular flask with turbine. Then, 1 % Na3C6H5O7 (1.5 mL) was added into the system. The color turned from black to red after boiling for 15 min. When the temperature of the solution cooled down to ambient temperature, AuNPs began to form. Monoclonal antibody (0.0545 mg) was added into 30 mL aforesaid colloidal gold solution at pH 8.5 with stirring for 15 min. It was mixed
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for 15 min again after 1/100 volume 2 % PEG 20000 was added. Gold-labeled antibody was purified through the two-step centrifugation: the pellet was discarded after the first centrifugation at 3,000 r/min for 30 min. The second centrifugation was at10,000 r/min for 30 min. Then, the precipitate was dissolved in 1.5 mL water and stored at 4 °C. Detection of Vibrio Parahaemolyticus by Fluorescence Quenching Immunoassay One hundred microliters of antigen solutions of different concentrations were added into 80 μL Au-labeled antibody mixed with 5 mL CdTe-antibody fluorescent probe solution. The various concentrations are 0, 1×105, 1×106, 1×107, 1×108, 1×109, and 1×1010 cfu/L. Fluorescence intensity was then measured. Detection parameters were as follows. The scanning scope was 450–720 nm, excitation wavelength was 370 nm, EX was 5.0 nm, and EM was 5.0 nm.
Results and Discussion Preparation of CdTe QD Probes The fluorescence intensity of the system significantly increased at the same position of absorbance peak after antibody was added (Fig. 1a), demonstrating that coupling with antibody did not affect its intrinsic fluorescence property. The enhanced fluorescence intensity of CdTe QDs modified by antibody was most probably because the coverage of antibody could overcome interference outside, meanwhile passivate the surface of QDs, and resist surface defection effectively [15]. Figure 1b shows the image of SDS-PAGE, with separating gel concentration of 15 % and stacking gel of 4 %. There were two clear bands both in lanes 1 3, while there was no band in lane 4. Referring to the marker in lane 2, the mobility speed of bands in lane 1 was faster than that in lane 3 (Fig. 1b A). It might be due to the fact that QDs had a large amount of negative charge, thus, electric charge of CdTe-antibody was much higher than that of unlabeled antibody, leading to changes of electrophoresis mobility. The positions of bands in Fig. 1b B were the same as the ones in Fig. 1b A with bright bands only in lane 1. Since unlabeled antibodies did not emit fluorescence, Fig. 1 confirmed that QD probes were successfully prepared. The Influence of Coupling Agent (EDC·HCl and NHS) on the Fluorescence Intensity of CdTe QD Probes Two kinds of coupling agents (EDC·HCl and NHS) were used to activate QDs. When optimizing EDC·HCl, 150 μL of NHS (4 mg/mL) and different volumes (0, 100, 150, 200, 250, 300 μL) of EDC·HCl (5 mg/mL) were mixed; and when optimizing NHS, 250 μL of EDC·HCl (5 mg/mL) and different volumes (0, 60, 90, 120, 150, 180 μL) of NHS (4 mg/mL) were mixed. Figure 2 shows the effect of EDC·HCl and NHS on fluorescence intensity of CdTe QDs-antibody probes. With increase of EDC·HCl and NHS, fluorescence intensity firstly climbed up and then declined, and when the volume of EDC·HCl was 250 μL and NHS was 150 μL, it reached the maximum. Meanwhile, as the amount of EDC·HCl and NHS increased, active QDs increased, resulting in more conjugation and stronger fluorescence. However, when the coupling agent reached saturation, excessive EDC·HCl and NHS could quench the fluorescence instead. EDC·HCl could activate carboxyl groups of mercaptoacetic acid coating on CdTe QDs to form midbody O-acyl urea, and the reaction was reversible and easily hydrolyzed. NHS could replace the EDC part of midbody O-acyl urea and prevent the
Appl Biochem Biotechnol
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Fig. 1 The speciality of CdTe QDs-antibodies. a Fluorescence spectra of QDs in presence and absence of antibodies. b Light image and transmission image of SDS-PAGE for CdTe-antibodies (A light image of SDS-PAGE; B transmission image of SDS-PAGE. Lanes 1–4, CdTe QDs-antibodies, marker, antibodies, CdTe)
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reversible reaction to increase the using ratio of EDC·HCl. Then, NHS reacted with primary amine of the antibody for covalent conjugation. Optimization of Coupling Conditions of QDs-Antibody To optimize coupling conditions of QDs-antibody, influences of pH value, type of buffer solution, temperature, and reaction time were investigated (Fig. 3). Fluorescence intensity of QD probes were detected at different pH values (pH 5, 6, 7, 7.4, 8, 9, 10) and in various buffer solutions (Na2HPO4-citric acid, BBS, Tris-HCl, barbital sodium-HCl, PBS) at 0.01 mol/L, pH 7.4. As showed in Fig. 3a, at pH 7.4, fluorescence intensity of QDs-antibody was the maximal. If pH value was higher or lower than 7.4, coupling could be obstructed and accordingly fluorescence was quenched. When pH stayed the same with that of cell sap, it was just the optimum condition for coupling. There was no obvious distinction in different buffer solutions, but Na2HPO4-citric acid buffer solution possessed the highest fluorescence intensity (Fig. 3b). The change of fluorescence intensity at varied temperature and reaction time was displayed in Fig. 3c. At 25 °C, fluorescence intensity reached the maximum after 2 h, which shifted to 1 h at 37 °C. The chemical reaction rate was related to temperature. Because with higher temperature,
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more reagents were active, leading to more effective crash times in the system. At 37 °C, bioactivity of antibody was superior, benefiting to the coupling process of QDs and antibodies. Therefore, the optimal parameters were 37 °C, 1 h. Preparation of Gold-Labeled Antibody Colloidal gold solution had strong absorption peak at 450 nm in Fig. 4, due to surface plasma resonance absorption [16]. UV-visible absorption peak was red-shifted and lower after coupling with antibody. According to previous report, interaction between colloidal gold and absorbing mediums or host materials could cause red-shift of absorption peak [17], which could preliminarily determine whether antibody interacted with colloidal gold. Another reason for that was the increase of pH value of colloidal gold solution after being labeled, which could neutralize the negative charge on AuNP’s surface. Intercoagulation further increased the grain diameter of AuNPs, resulting in red-shift of absorption peak. Establishment of Fluorescence Quenching Immunoassay One hundred microliters of antigen solutions of varied concentration were added into 80 μL Au-labeled antibody and 5 mL CdTe-antibody fluorescent probe. Influence of antigen concentration on fluorescence intensity was shown in Fig. 5. With increased antigen concentration, fluorescence intensity decreased gradually. FRET could account for fluorescence quenching. FRET was a nonradiative process whereby an excited state donor (QDs-antibody) transferred energy to a proximal ground state acceptor (gold-labeled antibody) through long-range dipole–dipole interactions. The rate of energy transfer was highly dependent on many factors, such as extent of spectral overlap, relative orientation of the transition dipoles, and most importantly, distance between the donor and the acceptor molecules. The absorption band of gold-antibody nanoparticles coincided partially with the emission band of QDs-antibody. After antibodies were labeled, V. parahaemolyticus and quantum dot probes were mixed, and consequently, immune sandwich reaction occurred, creating shorter distance between quantum dots and colloidal gold. That is, the molecule distance between QDs and gold nanoparticles was very near. In turn, the excited state energy of quantum 1.0
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Appl Biochem Biotechnol
dots transferred to colloidal gold, resulting in fluorescence quenching of quantum dots. Equation was established on the basis of the connection between fluorescence intensity of the system and logarithm concentration of antigens for detecting V. parahaemolyticus. Fluorescence intensity of the system was proportional with logarithm concentration of antigen. The correlation coefficient was 99.764 %, and the detection limit of V. parahaemolyticus was 5.03×104 cfu/L (Fig. 5 inserted). Specificity of the Fluorescence Quenching Immunoassay Two groups of bacterial species were selected to inspect the specificity of fluorescence quenching immunoassay. They were inter-genus and cross-genus of Vibrio. In Fig. 6, it was obvious that
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lg(CAg) Fig. 5 The effect of concentration of antigen on fluorescence intensity of the system. a Fluorescence spectra of the system. The antigen concentration from top to bottom 0, 1×105, 1×106, 1×107, 1×108, 1×109, and 1×1010 cfu/L. The dashed lines were fluorescence spectra of GNPs-mAb. b The linear spectra between fluorescence intensity and logarithm concentration of antigens at 575 nm. Experiments were performed in triplicate, and the error bars represent the standard deviation of the average
Appl Biochem Biotechnol
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Bacteria Fig. 6 Results of the fluorescence quenching immunoassay. a Inter-generic cross specificity (1, unadded bacteria; 2, Vibrio parahaemolyticus; 3, Shigella boydii; 4, Proteus mirabilis; 5, Listeria monocytogenes; 6, Staphylococcus aureus; 7, Escherichia coli; 8, Salmonella paratyphosa; 9, Bacillus subtilis; 10, Enteroaerogen; 11, Pseudomonas aeruginosa). b Cross-genus specificity (1, unadded bacteria; 2, V. parahaemolyticus; 3, V. vulnificus; 4, V. cholera; 5, V. metschnikovii; 6, V. alginolyticus). F, Fluorescence intensity of the system; F*, Fluorescence intensity of CdTe QDs. Reactions were performed in triplicate, and the error bars represent the standard deviation of the average
with the same concentration of non-Vibrio bacteria or non-Vibrio parahaemolyticus strains added, fluorescence intensity of the system hardly changed, and fluorescence quenching did not occur. Only when V. parahaemolyticus was added, fluorescence quenching would arise in the system, explaining why the fluorescence quenching immunoassay could detect V. parahaemolyticus of inter-genus and cross-genus with high specificity. Figure 6 shows that there was no change of relative fluorescence intensity after different bacteria were added, except for V. parahaemolyticus. The addition of V. parahaemolyticus largely decreased fluorescence intensity. Apparent quenching of fluorescence caused by V. parahaemolyticus could be used to specifically detect V. parahaemolyticus.
Conclusion Heat-inactive V. parahaemolyticus thallus was used as immune antigen for preparation of multiantibodies and monoantibodies. Water-soluble CdTe QDs coated with thioglycolic acid were labeled on multiantibodies of V. parahaemolyticus as fluorescent probe. Colloidal gold
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nanoparticles were coupled with monoantibodies of V. parahaemolyticus as gold-labeled antibody. Due to electric dipole interaction between molecules, the donor energy transferred to the receptor, and consequently, the donor fluorescence intensity was decreased. Therefore, CdTe quantum dots and colloidal gold acted as energy donor and receptor, respectively. Goldlabeled antibody could quench the fluorescent intensity of QDs in light of FRET theory through immune sandwich reaction. Then, V. parahaemolyticus was detected through that immune method. The equation was established on the basis of connection between fluorescence intensity of the system and logarithm concentration of antigen to detect V. parahaemolyticus. Compared to other immunological test techniques, this method was superior in specificity, sensitivity, and accuracy. Acknowledgments The research was supported by the Fundamental Research Funds for the Central Universities (NO.2013PY104).
References 1. Su, C. P., Jane, W. N., & Wong, H. C. (2013). International Journal of Food Microbiology, 160, 360–366. 2. Deepanjali, A., Kumar, H. S., & Karunasagar, I. (2005). Applied and Environmental Microbiology, 71, 3575–3580. 3. Wang, L., Shi, L., Su, J., Ye, Y., & Zhong, Q. (2013). Gene, 515, 421–425. 4. Guo, A., Sheng, H. L., Zhang, M., Wu, R. W., & Xie, J. (2012). Journal of Food Quality, 35, 366–371. 5. Di Pinto, A., Terio, V., Di Pinto, P., Colao, V., & Tantillo, G. (2012). Letters in Applied Microbiology, 54, 494–498. 6. Kawatsu, K., Ishibashi, M., & Tsukamoto, T. (2006). Journal of Clinical Microbiology, 44, 1821–1827. 7. Bailey, R. E., Smith, A. M., & Nie, S. (2004). Low-dimensional Systems and Nanostructures, 25, 1–12. 8. Huang, D., Niu, C., Wang, X., Lv, X., & Zeng, G. (2013). Analytical Chemistry, 85, 1164–1170. 9. Liu, Y., Liu, C. Y., & Zhang, Z. Y. (2012). Applied Surface Science, 263, 481–485. 10. Negi, D. P. S., & Chanu, T. I. (2008). Nanotechnology, 19, 465503. 11. Zhang, M., Cao, X., Li, H., Guan, F., Guo, J., Shen, F., et al. (2012). Food Chemistry, 135, 1894–1900. 12. Kamat, P. V., Barazzouk, S., & Hotchandani, S. (2002). Angewandte Chemie-International Edition, 41, 2764. 13. Mandal, G., Bardhan, M., & Ganguly, T. (2011). Journal of Physical Chemistry C, 115, 20840–20848. 14. Shang, L., & Dong, S. (2009). Analytical Chemistry, 81, 1465–1470. 15. Hui, Y. (2008). Master thesis, Jilin University, Changchun, China. 16. DiScipio, R. G. (1996). Analytical Biochemistry, 236, 168–170. 17. Li, G. A. (2006). Journal of Shaanxi Normal University (Natural Science Edition), 34, 32–35.
Specific Detection of Vibrio Parahaemolyticus by Fluorescence Quenching Immunoassay Based on Quantum Dots Ling Wang & Junxian Zhang & Haili Bai & Xuan Li & Pintian Lv & Ailing Guo
Received: 19 September 2013 / Accepted: 3 April 2014 # Springer Science+Business Media New York 2014
Abstract In this study, anti-Vibrio parahaemolyticus polyclonal and monoclonal antibodies were prepared through intradermal injection immune and lymphocyte hybridoma technique respectively. CdTe quantum dots (QDs) were synthesized at pH 9.3, 98 °C for 1 h with stabilizer of 2.7:1. The fluorescence intensity was 586.499, and the yield was 62.43 %. QD probes were successfully prepared under the optimized conditions of pH 7.4, 37 °C for 1 h, 250 μL of 50 mg/mL EDC·HCl, 150 μL of 4 mg/mL NHS, buffer system of Na2HPO4-citric acid, and 8 μL of 2.48 mg/mL polyclonal antibodies. As gold nanoparticles could quench fluorescence of quantum dots, the concentration of V. parahaemolyticus could be detected through measuring the reduction of fluorescence intensity in immune sandwich reaction composed of quantum dot probe, gold-labeled antibody, and the sample. For pure culture, fluorescence intensity of the system was proportional with logarithm concentration of antigen, and the correlation coefficient was 99.764 %. The fluorescence quenching immunoassay based on quantum dots is established for the first time to detect Vibrio parahaemolyticus. This method may be used as rapid testing procedure due to its high simplicity and sensitivity. Keywords CdTe QD probes . Gold-labeled antibodies . Fluorescence quenching immunoassay . Vibrio parahaemolyticus
Introduction As a halophilic Gram-negative bacterium, Vibrio parahaemolyticus which frequently causes food-borne human gastroenteritis is widely distributed in coastal and estuarine environments throughout the world [1]. With the rapidly increasing global incidence of V. parahaemolyticus infection in recent years, consumption of raw or undercooked seafood proves to be the major cause [2]. Traditional detection methods are based on bacterium culture, including enrichment Ling Wang and Junxian Zhang contributed equally to this paper.
L. Wang : J. Zhang : H. Bai : X. Li : P. Lv : A. Guo (*) Key Laboratory of Environment Correlative Dietology of Ministry of Education, College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, China e-mail: [email protected]
Appl Biochem Biotechnol
in liquid media and isolation from selective culture medium [3]. Whereas, it takes 7 days to get the conclusion and high appearance of false negative results is the limitation for its current application [4]. Therefore, rapid and accurate detection methods of V. parahaemolyticus are urgently needed in prevention of food poisoning and food safety. Current techniques of detecting bacteria include immunological methods, molecular means, and equipment tests. Molecular means such as dot blotting, PCR are relatively high-cost, meanwhile professional skills are required [5]. Equipment detections are rapid but have not been used broadly. Immunological methods with simplicity, cheapness, and sensitivity and rapidness properties are ideal for detecting pathogens [6]. Semiconductor quantum dots (QDs) are nanometer-sized crystals with unique photochemical and photophysical properties which have aroused great interest as a new class of fluorescent labels in past decades in the fields of biology, medicine, sensing, and environmental analysis and so on [7]. Compared to traditional organic dyes, QDs have their own unique nature of a wide range of excitation wavelength, narrow emission wavelength, size-adjustment, long fluorescence lifetime, highly photochemical stability, high photoluminescence, and narrow and symmetrical emission peak [8]. The method of QDs coupled with biological molecules has a very broad application in biology, medicine, sensing, and environment analysis [9, 10]. QDs will be the most promising biological fluorescent probe. Researchers have devoted a lot of effort to studying fluorescent ways that involve the use of nanometer materials, especially QDs combined with AuNPs [11]. AuNPs have caused great attention for their high extinction coefficient and broad absorption spectrum in a visible light. AuNPs are unique quenchers for organic dyes and QDs through electron-transfer or energy-transfer procedure [12]. Up to now, fluorescent sensors based on QDs and AuNPs still have extensive potential in detection of small molecules or protein [13]. The most prevalent explanation of the quenching phenomenon between AuNPs and QDs focus on two theories, which are fluorescence resonance energy transfer (FRET) and inner filter effect (IFE). The FRET process involves intermolecular connection of QDs and AuNPs and a particular distance or geometry to enable interaction between them. Usually, the FRETrelated sensors are based on complicated chemical reactions with regards to modification of AuNPs or QDs surface. The IFE refers to complementary overlap of absorption band of the absorber (quenchers, such as AuNPs) and excitation or emission bands of the fluorophore (such as QDs) [10]. Therefore, facilely tuned absorption or emission spectrum can be used as IFE-based fluorescent assay [14]. In this research, monoclonal antibody modified by AuNPs and polyclonal antibody modified by QDs were prepared, and these two decorated materials were further conjugated with V. parahaemolyticus or any other antigens to form an immune sandwich reaction. Based on the FRET process of QDs/AuNPs, the fluorescence of CdTe QDs is very weak in the presence of V. parahaemolyticus or other antigens. Compared to other bacterium, the fluorescence was very sensitive to V. parahaemolyticus. It demonstrated that the result of our work based on FRET to determine V. parahaemolyticus is a specific, rapid, and sensitive approach.
Materials and Methods Apparatus, Materials, and Reagents Fluorescence spectrophotometer (RF-5301p) and UV-vis spectrophotometer (UV-1077) were from Shimadzu, Japan. 5415-R frozen high-speed centrifuge were bought from Eppendorf. Other instruments were bought from China. Chemical reagents were purchased from Sigma
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Company. V. parahaemolyticus ATCC17802 was purchased from Pharmaceutical Biological Product Detection Station in China. SP2/0 mouse myeloma cells were stored in our laboratory. Preparation of Polyclonal Antibody Liquid culture of V. parahaemolyticus was boiled for 2 h. Then, the inactive bacterium was assembled after centrifugation at 4,000 r/min for 5 min. The bacterial suspension was adjusted to 1010 cfu/L with normal saline, and consequently antigen was gained and stored at −20 °C. Japanese large-eared rabbits were immunized by subcutaneous injection of 1 mL bacterial suspension (1×107 cfu/L) once 2 weeks. Blood was collected via the ear-rim vein at the 10th day after each immunization, then put at 37 °C for 1 h, and finally stored at 4 °C for a night. The stored blood was centrifuged at 10,000 r/min, 4 °C for 5 min to obtain serum later for detection of polyclonal antibody with ELISA method. Preparation of Monoclonal Antibody Female BAL b/C mice 6–8 weeks old were immunized with 0.5 mL (107 cfu/L) antigen by abdominal subcutaneous injection once 2 weeks. The mice were docked and blood was collected at 1 week after each immunization later for detecting mice serum antibody titers via ELISA method. BAL b/C mice immuned were used for preparing immune splenocyte, and SP2/0 mice were used to prepare myeloma cell. The prepared splenocyte (1×108 cfu/L) and myeloma cell (2×107 cfu/L) were complexed with the ratio of 5:1 for cell fusion, and then the positive hybridoma cells were screened. The hybridoma cells were cloned and then for scale-up culture to screen monoclonal antibody further. Bacteria including V. parahaemolyticus, V. alginolyticus, V. metschnikovii, V. cholerae, and V. vulnificus were used as antigens. They were cross-reacted with various monoclonal antibodies and verified the specificity of monoclonal antibody. Preparation of CdTe QDs and CdTe QDs/Polyclonal Antibody Fluorescence Probes In a typical experiment, water-soluble CdTe QDs coated with thioglycolic acid (TGA) were prepared in a 10 mL airtight three-necked bottle with 50.0 mg NaHB4 and 31.9 mg tellurium (Te) powder at 40 °C, until the solution color turned to transparent purple. Then, 99.0 mL ultrapure water was injected to the three-necked bottle, and at the same time, CdCl2 was added to the bottle with N2 protection. After heated for 30 min at 95 °C, TGA (the molar ratio of CdCl2 and TGA was 3:1) was added to the solution under the condition of pH 10 and heating reflux at 95 °C. CdTe QDs prepared (0.5 mL) were added into 50 mg/mL EDC·HCl mixed with 4 mg/mL N-hydroxysuccinimide (NHS), vibrated for 10∼20 min, and then centrifuged for 5 min at 12,000 r/min. Precipitate collected was suspended in 5 mL buffer solution after twice centrifugation and finally stored at 4 °C. Preparation of Gold-Labeled Antibody Distilled water (99.0 mL) and HAuCl4 (1 %, 1 mL) were boiled in a 250-mL triangular flask with turbine. Then, 1 % Na3C6H5O7 (1.5 mL) was added into the system. The color turned from black to red after boiling for 15 min. When the temperature of the solution cooled down to ambient temperature, AuNPs began to form. Monoclonal antibody (0.0545 mg) was added into 30 mL aforesaid colloidal gold solution at pH 8.5 with stirring for 15 min. It was mixed
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for 15 min again after 1/100 volume 2 % PEG 20000 was added. Gold-labeled antibody was purified through the two-step centrifugation: the pellet was discarded after the first centrifugation at 3,000 r/min for 30 min. The second centrifugation was at10,000 r/min for 30 min. Then, the precipitate was dissolved in 1.5 mL water and stored at 4 °C. Detection of Vibrio Parahaemolyticus by Fluorescence Quenching Immunoassay One hundred microliters of antigen solutions of different concentrations were added into 80 μL Au-labeled antibody mixed with 5 mL CdTe-antibody fluorescent probe solution. The various concentrations are 0, 1×105, 1×106, 1×107, 1×108, 1×109, and 1×1010 cfu/L. Fluorescence intensity was then measured. Detection parameters were as follows. The scanning scope was 450–720 nm, excitation wavelength was 370 nm, EX was 5.0 nm, and EM was 5.0 nm.
Results and Discussion Preparation of CdTe QD Probes The fluorescence intensity of the system significantly increased at the same position of absorbance peak after antibody was added (Fig. 1a), demonstrating that coupling with antibody did not affect its intrinsic fluorescence property. The enhanced fluorescence intensity of CdTe QDs modified by antibody was most probably because the coverage of antibody could overcome interference outside, meanwhile passivate the surface of QDs, and resist surface defection effectively [15]. Figure 1b shows the image of SDS-PAGE, with separating gel concentration of 15 % and stacking gel of 4 %. There were two clear bands both in lanes 1 3, while there was no band in lane 4. Referring to the marker in lane 2, the mobility speed of bands in lane 1 was faster than that in lane 3 (Fig. 1b A). It might be due to the fact that QDs had a large amount of negative charge, thus, electric charge of CdTe-antibody was much higher than that of unlabeled antibody, leading to changes of electrophoresis mobility. The positions of bands in Fig. 1b B were the same as the ones in Fig. 1b A with bright bands only in lane 1. Since unlabeled antibodies did not emit fluorescence, Fig. 1 confirmed that QD probes were successfully prepared. The Influence of Coupling Agent (EDC·HCl and NHS) on the Fluorescence Intensity of CdTe QD Probes Two kinds of coupling agents (EDC·HCl and NHS) were used to activate QDs. When optimizing EDC·HCl, 150 μL of NHS (4 mg/mL) and different volumes (0, 100, 150, 200, 250, 300 μL) of EDC·HCl (5 mg/mL) were mixed; and when optimizing NHS, 250 μL of EDC·HCl (5 mg/mL) and different volumes (0, 60, 90, 120, 150, 180 μL) of NHS (4 mg/mL) were mixed. Figure 2 shows the effect of EDC·HCl and NHS on fluorescence intensity of CdTe QDs-antibody probes. With increase of EDC·HCl and NHS, fluorescence intensity firstly climbed up and then declined, and when the volume of EDC·HCl was 250 μL and NHS was 150 μL, it reached the maximum. Meanwhile, as the amount of EDC·HCl and NHS increased, active QDs increased, resulting in more conjugation and stronger fluorescence. However, when the coupling agent reached saturation, excessive EDC·HCl and NHS could quench the fluorescence instead. EDC·HCl could activate carboxyl groups of mercaptoacetic acid coating on CdTe QDs to form midbody O-acyl urea, and the reaction was reversible and easily hydrolyzed. NHS could replace the EDC part of midbody O-acyl urea and prevent the
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reversible reaction to increase the using ratio of EDC·HCl. Then, NHS reacted with primary amine of the antibody for covalent conjugation. Optimization of Coupling Conditions of QDs-Antibody To optimize coupling conditions of QDs-antibody, influences of pH value, type of buffer solution, temperature, and reaction time were investigated (Fig. 3). Fluorescence intensity of QD probes were detected at different pH values (pH 5, 6, 7, 7.4, 8, 9, 10) and in various buffer solutions (Na2HPO4-citric acid, BBS, Tris-HCl, barbital sodium-HCl, PBS) at 0.01 mol/L, pH 7.4. As showed in Fig. 3a, at pH 7.4, fluorescence intensity of QDs-antibody was the maximal. If pH value was higher or lower than 7.4, coupling could be obstructed and accordingly fluorescence was quenched. When pH stayed the same with that of cell sap, it was just the optimum condition for coupling. There was no obvious distinction in different buffer solutions, but Na2HPO4-citric acid buffer solution possessed the highest fluorescence intensity (Fig. 3b). The change of fluorescence intensity at varied temperature and reaction time was displayed in Fig. 3c. At 25 °C, fluorescence intensity reached the maximum after 2 h, which shifted to 1 h at 37 °C. The chemical reaction rate was related to temperature. Because with higher temperature,
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more reagents were active, leading to more effective crash times in the system. At 37 °C, bioactivity of antibody was superior, benefiting to the coupling process of QDs and antibodies. Therefore, the optimal parameters were 37 °C, 1 h. Preparation of Gold-Labeled Antibody Colloidal gold solution had strong absorption peak at 450 nm in Fig. 4, due to surface plasma resonance absorption [16]. UV-visible absorption peak was red-shifted and lower after coupling with antibody. According to previous report, interaction between colloidal gold and absorbing mediums or host materials could cause red-shift of absorption peak [17], which could preliminarily determine whether antibody interacted with colloidal gold. Another reason for that was the increase of pH value of colloidal gold solution after being labeled, which could neutralize the negative charge on AuNP’s surface. Intercoagulation further increased the grain diameter of AuNPs, resulting in red-shift of absorption peak. Establishment of Fluorescence Quenching Immunoassay One hundred microliters of antigen solutions of varied concentration were added into 80 μL Au-labeled antibody and 5 mL CdTe-antibody fluorescent probe. Influence of antigen concentration on fluorescence intensity was shown in Fig. 5. With increased antigen concentration, fluorescence intensity decreased gradually. FRET could account for fluorescence quenching. FRET was a nonradiative process whereby an excited state donor (QDs-antibody) transferred energy to a proximal ground state acceptor (gold-labeled antibody) through long-range dipole–dipole interactions. The rate of energy transfer was highly dependent on many factors, such as extent of spectral overlap, relative orientation of the transition dipoles, and most importantly, distance between the donor and the acceptor molecules. The absorption band of gold-antibody nanoparticles coincided partially with the emission band of QDs-antibody. After antibodies were labeled, V. parahaemolyticus and quantum dot probes were mixed, and consequently, immune sandwich reaction occurred, creating shorter distance between quantum dots and colloidal gold. That is, the molecule distance between QDs and gold nanoparticles was very near. In turn, the excited state energy of quantum 1.0
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dots transferred to colloidal gold, resulting in fluorescence quenching of quantum dots. Equation was established on the basis of the connection between fluorescence intensity of the system and logarithm concentration of antigens for detecting V. parahaemolyticus. Fluorescence intensity of the system was proportional with logarithm concentration of antigen. The correlation coefficient was 99.764 %, and the detection limit of V. parahaemolyticus was 5.03×104 cfu/L (Fig. 5 inserted). Specificity of the Fluorescence Quenching Immunoassay Two groups of bacterial species were selected to inspect the specificity of fluorescence quenching immunoassay. They were inter-genus and cross-genus of Vibrio. In Fig. 6, it was obvious that
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Bacteria Fig. 6 Results of the fluorescence quenching immunoassay. a Inter-generic cross specificity (1, unadded bacteria; 2, Vibrio parahaemolyticus; 3, Shigella boydii; 4, Proteus mirabilis; 5, Listeria monocytogenes; 6, Staphylococcus aureus; 7, Escherichia coli; 8, Salmonella paratyphosa; 9, Bacillus subtilis; 10, Enteroaerogen; 11, Pseudomonas aeruginosa). b Cross-genus specificity (1, unadded bacteria; 2, V. parahaemolyticus; 3, V. vulnificus; 4, V. cholera; 5, V. metschnikovii; 6, V. alginolyticus). F, Fluorescence intensity of the system; F*, Fluorescence intensity of CdTe QDs. Reactions were performed in triplicate, and the error bars represent the standard deviation of the average
with the same concentration of non-Vibrio bacteria or non-Vibrio parahaemolyticus strains added, fluorescence intensity of the system hardly changed, and fluorescence quenching did not occur. Only when V. parahaemolyticus was added, fluorescence quenching would arise in the system, explaining why the fluorescence quenching immunoassay could detect V. parahaemolyticus of inter-genus and cross-genus with high specificity. Figure 6 shows that there was no change of relative fluorescence intensity after different bacteria were added, except for V. parahaemolyticus. The addition of V. parahaemolyticus largely decreased fluorescence intensity. Apparent quenching of fluorescence caused by V. parahaemolyticus could be used to specifically detect V. parahaemolyticus.
Conclusion Heat-inactive V. parahaemolyticus thallus was used as immune antigen for preparation of multiantibodies and monoantibodies. Water-soluble CdTe QDs coated with thioglycolic acid were labeled on multiantibodies of V. parahaemolyticus as fluorescent probe. Colloidal gold
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nanoparticles were coupled with monoantibodies of V. parahaemolyticus as gold-labeled antibody. Due to electric dipole interaction between molecules, the donor energy transferred to the receptor, and consequently, the donor fluorescence intensity was decreased. Therefore, CdTe quantum dots and colloidal gold acted as energy donor and receptor, respectively. Goldlabeled antibody could quench the fluorescent intensity of QDs in light of FRET theory through immune sandwich reaction. Then, V. parahaemolyticus was detected through that immune method. The equation was established on the basis of connection between fluorescence intensity of the system and logarithm concentration of antigen to detect V. parahaemolyticus. Compared to other immunological test techniques, this method was superior in specificity, sensitivity, and accuracy. Acknowledgments The research was supported by the Fundamental Research Funds for the Central Universities (NO.2013PY104).
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