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870 MOLECULAR MEDICINE REPORTS 17: 870-876, 2018

Application of F0F1‑ATPase immuno‑biosensors
for detecting Escherichia coli O157:H7

Department of Biotechnology, Beijing Centre for Physical and Chemical Analysis, Beijing 100094;
Department of Clinical Laboratory Medicine, Chinese People's Liberation Army General Hospital &
Medical School of Chinese People's Liberation Army, Beijing 100853; 3Department of Laboratory Medicine,
School of Pharmacy and Medical Technology, Putian University, Putian, Fujian 351100, P.R. China

Received August 30, 2016; Accepted May 18, 2017

DOI: 10.3892/mmr.2017.7996

Abstract. Escherichia coli (E. coli) O157:H7 is an important contaminated food. It can cause abdominal pain, hemorrhagic
food‑borne pathogen with a low infective threshold and high fever or bloody stools, and can induce secondary haemolytic
resistance to treatment. There are currently a number of detection uraemic syndrome in infants, preschool children or weak,
methods available, however, the majority are time‑consuming, elderly individuals. In addition, due to its strong drug resis-
complex and expensive, thus it is hard for these methods to be tance, it is very hard to eliminate O157:H7 from contaminated
applied in routine detection. Therefore, there is urgent require- food sources. O157:H7 contamination has now become an
ment to develop more sensitive, rapid and specific detective international food security concern (1). The American Centers
techniques. In the present study, an immuno‑biosensor based for Disease Control has revealed that E. coli O157:H7 is one
on the interference of load to the F0F1‑ATPase rotation, indi- of the major pathogenic bacteria causing food‑borne diseases;
cated by the fluorescence fluctuation, was constructed to detect thus, poses a serious threat to public health. Furthermore, this
O157:H7. The results demonstrated a good linear relationship strain has been detected in pork, beef and mutton in China (2).
(R2=0.9818) between antigen concentration (range, 102  cfu to There are several detection methods currently used for
104  cfu) and the fluorescence intensity. The detection signals pathogenic bacteria, including culture‑based, enzyme‑linked
of the samples containing 102 cfu/well and 104 cfu/well E. coli immunosorbent assay (ELISA) and polymerase chain reac-
O157:H7 were significantly stronger than the signal produced tion (PCR)‑based methods (3‑5). However, these methods are
by the control sample (P<0.01). Due to its higher sensibility and usually time‑consuming, expensive and insensitive, which
simplicity when compared with the current methods applied, the makes them unsuitable for the detection of this pathogen.
results of the present study indicate a promising future for the Therefore, it is necessary to develop more efficient detection
application of this technique in detecting food source pathogens. apparatus.
F0F1‑ATPase, located in the mitochondria and/or the chlo-
Introduction roplast thylakoid of eukaryotic organisms and the bacterial
plasma membrane, catalyzes the synthesis of ATP using the
Escherichia coli (E. coli) O157:H7 is a type of pathogenic transmembrane proton gradient. In E. coli, the soluble F1 and
bacterium that infects humans and livestock primarily through transmembrane F0 parts are comprised of the α3β3γδε and
ab2cn subunits, respectively. These two parts are connected
by the stalks of γε in the centre and b2 δ on the outside. When
the downhill proton passes through F0, the c and γε subunits
are rotated leading to conformational changes in F1, which
Correspondence to: Dr Yong‑Zhi Lun, Department of Laboratory
Medicine, School of Pharmacy and Medical Technology, Putian
promotes the formation of ATP from ADP and inorganic
University, 450 Dongzhen West Street, Chengxiang, Putian, phosphate, and vice versa. As a result, F0F1‑ATPase forms a
Fujian 351100, P.R. China molecule size motor, which can transform the electric chem-
E‑mail: ical potential energy into chemical energy. If this process is
disturbed by other factors, the rate of ATP synthesis and proton
Dr Hui‑Juan Wu, Department of Biotechnology, Beijing Center
for Physical and Chemical Analysis, 27 North Xisanhuan Road,
flux maybe altered; this phenomenon maybe reflected by pH
Haidian, Beijing 100094, P.R. China sensitive substances (6‑7). F1300 is a pH sensitive fluorescent
E‑mail: probe that can be used as an indicator of changes in the pH of
F0F1‑ATPase. During ATP synthesis, protons are pumped out
Key words: F0F1‑ATPase, immuno‑biosensors, Escherichia coli of the chromatophore and this transfer of protons is detected
O157:H7, detection by F1300 (4‑5,8). This concept was used to construct the
immuno‑rotary biosensor (IRB) for detecting specific targets,
which achieved great success (4‑5,9‑10). Although this type

Materials and methods Bacterial strains. Waltham. The capture system complexes and chromatophores tion. detection of much larger antigen such as a single bacterium has not been reported.000 x g for 30 min at 4˚C and resuspended in buffer (20 mM Tris‑HCl. completely. The chromatophore provides H+ for ATP‑synthesis. The cell‑free Abcam. 1 mM DTT. 5 mM MgCl 2 and 5 mM KCl). strain no. Manassas. Cambridge. tion. b. Varioskan Flash spectral scanning multimode reader (excita- odology to estimate the specificity of O157:H7. Subsequently. 24 h. Schematic view of F1300 chromatophores. Surplus bacteria were in activated by heating to 80˚C for 1 h. then free biotin was were centrifuged at 12. A total of 100 µl was transferred to a panel for further cultivation and each dilution gradient sample was tripled. USA) and Escherichia coli (E.1 mM PMSF and 2 mM MgCl2. coli O157:H7 871 of biosensor has been used to detect a virus (3. A total of tion was set up that contained equal amounts of β ‑subunit 1‑2 µl F1300 (1 mg/ml) was added to 600 µl chromatophores antibody‑biotin complex (200 µl. WEI et al: FOF1-ATPase IMMUNO-BIOSENSORS DETECT E. Salmonella (strain no. antibody [Homemade. subunit a. The supernatant was discarded and the precipitate was resuspended with sterile normal saline (NS) to the original volume. 5 mM MgCl2 and 5 mM KCl. 50 mM).0) followed by ultrasonication for 10 min in an ice bath. To create the capture system complex. The chromatophores for 15‑30 min at room temperature.000 x g for 1 h at 4˚C to of 10 mM biotin was added to 500 µl of 3 mg/ml β‑subunit collect membrane vesicles. Merck KGaA. volume following the third centrifugation.6‑7). CMCC‑44101. 538 nm. a. ‘Signal into signal did not decrease further. antibody‑biotin complex. ATCC35150) was obtained from the Guangdong Microbial Culture Collection Center (Guangzhou. complex. subunit b. the free F1300 was removed components’ is a chromatophore with the pH sensitive fluores. chromatophores was assayed using the enzyme coupling method with pyruvate kinase and lactate dehydrogenase. In addition. porate the probe into the inner part of the chromatophores. VA. 1. to incor.1 mM tricine. subunit c. China) and was incubated in nutrient broth medium at 37˚C for 24 h. 12. China) were also subjected to the same then the relative fluorescence signal was detected using the procedure as O157:H7. The arrow indicates the repeated twice to remove medium complex components and F0F1‑ATPase carrier.5) and Collection. Thermo Fisher Scientific. A ATPase β ‑subunit 100 mM NaCl.. 50 mM) and O157:H7 prior to ultrasonication for 3 min in an ice bath. as previously described (10)]. F1300.000 x g for 30 min at 4˚C. E. described previously (4). When the relative fluorescence Preparation of ‘signal into components’. AB81131. the precipitate was resuspended with sterile NS to the original The chromatophore and the F0F1‑ATPase are the organic combination. The cells were harvested by centrifugation at 4. 485 nm. (Fig. 0. The antibody for 15‑30 min at room temperature. then 10 ml was centrifuged for 30 min at 4. then free chromatophores were stored in 50% glycerol at ‑80˚C. MA. pH 6. The purification process was This produced the capture system complex: the β ‑subunit repeated three times to remove free F1300. This process was Figure 1. The biotin was removed via dialysis to produce the β ‑subunit chromatophore structure is presented in Fig.000 x g at 4˚C for 30 min to remove removed via dialysis to produce the O157:H7 antibody‑biotin glycerol. The aim of the present study was to investigate the potential use of this method for the detection of E. 200 µl 55 mM streptavidin (Sigma‑Aldrich.0.000 x g and 4˚C. The bacterial suspension was then diluted to 10‑4. a reac- 0. they were tested with the same meth. 2 µl of 10 mM biotin was added to 500 µl of 3 mg/ml O157:H7 antibody Labeling of chromatophores with F1300. biotin pH 8. The ATP hydrolysis activity of the labeled cent probe. USA). antibody‑biotin complex (200 µl.000 x g for 30 min at an antibody targeted against O157:H7 (cat. as Preparation of chromatophores. American Type Culture Collection. UK) were used for the following: 2 µl supernatant was centrifuged at 40. (Sigma‑Aldrich. Cn. Darmstadt. Germany) and The suspension was centrifuged at 10. Merck KGaA) The free F1300 fraction was purified by centrifugation at was added and incubated for 15‑30 min at room temperature. Inc. coli O157:H7. The bacterial clone in the incubated sample was counted 24 h later.1 mM tricine. termed chromatophores. The unit of enzyme activity was roseumwa0073 (ATCC27502) were purchased from the defined by hydrolyzing 1 µmol ATP per minute with 1 mg American Type Culture Collection and incubated at 60˚C for chromatophores (Fig. Thermomicrobium. then they were resuspended with buffer (pH 6. Beijing. 4˚C to remove unbroken cells and cell fractions. 3). and aliquots of antibody‑biotin‑Streptavidin‑biotin‑O157:H7 antibody the supernatant were collected to assess the level of purifica. coli. The precipitate was resuspended in tricine‑NaOH buffer labeled with F1300 (F1300‑ch) were then mixed to a 4:1 . Constructing the O157:H7 detector. no. China Medical Culture (0. 10‑5 and 10‑6 in bacteria‑free PBS. emission. ATCC14028. 2). coli O157:H7 (strain no.

10% glycerol. F1300‑ch‑ β ‑subunit antibody‑biotin‑Strepta. biotin. Group B. USA). Group 4. Statistical analysis was performed using SPSS 10 sion was adjusted to 5x103. dilution. a. O157:H7 antibody. The bacterial concentration for each Group C.05 was considered to 102 cfu bacteria/well. 6. pH 8. 2.. F1300‑ch. subunit c. Figure 4. Statistical analysis. 3. Group 3.0) was added to each well for further phores were successfully labeled with the fluorescent pH incubation at 45˚C for 15 min. 485 nm. To each well. Table II). Group Band Group C. P<0. Group A. a. The was detected using the Varioskan Flash spectral scanning fluorescence intensity of the control chromatophores was the multimode reader (excitation. subunit a. subunit b. This produced the 5. 2 mmol/ADP. Schematic view of the biosensor constructed based on F0F1‑ATPase. 5. F1300‑ch control. group and pathogenic bacteria are presented in Table I. were subjected to the same protocols in order to determine the F1300‑ch‑β ‑subunit antibody‑biotin‑Streptavidin complex. Inc. Data are presented as the mean ± standard Fluorescence assay. however. phores and F1300 was much higher than the control. ATPase. biosensor used in the present study (Fig. ATCC14028 Salmonella and CMCC44101 E. 538 nm. β ‑subunit antibody. 103 cfu bacteria/well. indicate a statistically significant difference. 8. The inner chromato- 5 mmol/l MgCl2. Group 2. Cn. 2018 Figure 2. The relative fluorescence signal indicator F1300 as a unidirectional label (Fig. streptavidin. 50 µl biosensor and 20 µl of the bacterial suspension were added. subunit a. The concentration of the bacterial suspen. Inc. 5 mmol/l NaH2PO4 and Construction of the immuno‑biosensor. b. Cn. Three different subunit c. based on the fluorescence of F1300‑ch Specificity.872 MOLECULAR MEDICINE REPORTS 17: 870-876. reactions were set up. four assessed by linear regression and a Dunnett's T3 post hoc test groups were set up: Group 1. respectively. In order to software (SPSS. and were incubated at 37˚C for 1 h. detection carrier. Schematic view of the process applied to establish the capture system complex. subunit b. Chicago. 5x104 and 5x105 cfu/ml. . Following incubation Results for 30 min at 37˚C. 1. emission. IL. 103 and 104 cfu bacteria/well. deviation. 70 µl ATP synthesis buffer (50 mmol/l tricine. 104 cfu bacteria/well.). The fluorescence intensity of the mixture of chromato- Thermo Fisher Scientific. was used for multiple comparisons. 4). lowest. chromatophore complex. vidin‑biotin‑O157:H7 antibody complex. control (sterile NS). b. specificity to O157:H7. coli with different loads: Group A. 7. The correlation was generate 102. to demonstrate that the construction of the immuno‑biosensor was successful. Figure 3. Schematic view of the F1300‑labelled inner chromatophores (green dots).

01). 7. producing high fluorescence intensity.052 for Salmonella. The results revealed that free F1300 was completely removed. Time of detection. it revealed that the greater the load on F0F1‑ATPase. 0. preparation of biosensors. In addition. coli O157:H7 873 Table I. coli groups was 0.01). 6. The time required for each step was 2. coli centrifugation groups. a part of F1300 entered the chromatophore with ultrasonication.5 h and the total time for testing samples was <16 h. coli O157:H7 0 27 133 265 1. 1.4 µmol/mg/min. Therefore.75. Specificity for O157:H7.035‑0.8 times higher than that of the Group Relative fluorescence value control. thus 4.043 and 0. The fluorescence value gradually decreased with the increasing concentration of O157:H7 (Fig. WEI et al: FOF1-ATPase IMMUNO-BIOSENSORS DETECT E. cfu. by combining it with the immune magnetic separa- tion technique.519 chromatophores. Control.25 h. the F1300‑labeled chromatophores were used in the present study. Table III). fluorescence labelled and the steps: bacterium solution treatment.48 antibody‑biotin‑Streptavidin‑biotin‑substrate antibody) was successfully established. The fluorescence intensity did not change following the fourth round of centrifugation. Following three centrifugation F0F1‑ATPase has the following two characteristics: i) it procedures. coli centrifugation groups. Escherichia coli. coli.075. the enzyme activity was 106. centrifugation steps. In addition.275 E. the lower the relative fluorescence. 3rd centrifugation 1. This was performed to verify that the inner chromato- 0. Though this phores were labeled with F1300 successfully.063‑0. the lower the enzyme activity and thus. The relative fluorescence value of the CMCC44101 E. Following statistical analysis. Bacterial concentration (cfu/well) ‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑ Strain of pathogenic bacteria Control Group 2 Group 3 Group 4 Group 5 E. the limit of detection was 100 cfu. while the fluorescent probe F1300 labeled the inner Chromatophores 0. These results demonstrated that this biosensor has specificity for O157:H7 (Fig. the results revealed that there were significant differences between the control and 104  cfu group (P<0. method requires separation and enrichment of target bacteria when testing samples. and between the 102  and 10 4  cfu groups (P<0. 4 E.12 Fig. 9.325 E. Table IV). the remaining Discussion free F1300 stayed out of the chromatophore.49. the time required for sample pretreatment was it decreased with ultrasonication and the four subsequent 8.5 h in total. which is consistent with the positive threshold value for O157:H7.750 Salmonella ATCC14028 0 26 128 255 1.49 This indicated that the capture system complex (β ‑subunit 4th centrifugation 1.69 As presented in Fig. the fluorescence intensity of 2nd centrifugation 2. respectively. The synthetic activity of F1300‑chisshown in Fluorescence labeling 18. chromatophores to produce ATP and it can also hydrolyze .9818). Figure 5. the chromatophore the mix was centrifuged 4 times to produce the 4 E. it was 2. while group C was the lowest.82 group A was the highest.035‑0. Activity of F1300‑ch in the control. The results of O157:H7 detection using this method identified a strong positive gradient between 102‑104 cfu (R2=0. Table II. contained sterile normal saline and 0 cfu/well O157:H7. Sensitivity for O157:H7. 8.5 and 0. Initially. This method is short and includes only four phores. colony forming units. Relative fluorescence value of the F1300‑labeled inner chromato. 1st centrifugation 6. The curve of 101‑103  cfu identified a good separation. the fluorescence intensity decreased to 1. Following the incorporation of F1300 into loading and testing. however. coli CMCC44101 0 35 175 350 1. This can use the H+ gradient between the inside and outside of indicated that the free F1300 were removed by centrifugation. Bacterial concentration in assay wells to estimate the specificity of the O157:H7 biosensor. Results of F1300 labelling in the inner chromatophores.

When the O157:H7 loads into the chromatophore.03x10‑4a 102 104 0. the specific underlying mechanism will be studied further in the future. coli and salmonella groups. At present. Liu et al (8) directly observed the mechanically driven proton influx or efflux in F0 coupled with rotation of F1 at a single molecular level. the F0F1‑ATPase nano‑biosensor is labeled by pH sensitive F1300.00327a Group 2 2. Concentration group (cfu/well) P‑value Group comparison P‑value 0 102 0. E. which can be used as a fast detection technique (7. 102 vs. coli. F0F1‑ATPase activity is regulated by external links on b subunits with different molecular weights.15x10‑5b Group 3 2. The present study used biosensors to detect O157:H7. Group B. coli. as well as the feasibility of this method using standard strains. Synthetic activity of the F1300‑labelled chromatophores link on to the β subunits successively (7). E. vs. 2018 Table III. Group 1 0. In another way. Results of the comparison between the O157:H7.63584 Group 3 1. Results of the comparison between the concentration Table IV. F1300‑ch.36 Group 2 2.15).60539 Group 5 9.82x10‑7a O157:H7 vs. vs.00x10‑4b Group 4 1. It is inhibited when anti‑b subunit antibodies. streptavidin and H9 antibodies Figure 6. bP<0.16x10‑6b ATP by reverse transporting H+.10). the present study investigated its specificity.9. the sophisticated microbial method. F1300‑ch. In addition. bP<0. the detection of O157:H7 was constructed successfully.11‑13). aP<0. more sensitive and easier to operate.19239 O157:H7 vs.01. coli 0 103 ‑0.01.00015b Group 4 1. this is similar to the compe- tition method in ELISA.89x10‑7a 103 104 0.874 MOLECULAR MEDICINE REPORTS 17: 870-876. a inside the chromatophores. ii) F0F1‑ATPase rotation speed and the loads on its subunits are positively correlated. The holoenzyme (F1300‑ch).69 0 104 0. Salmonella Comparisons were performed using Dunnett's T3 test. When compared with other novel detection methods. Table V presents a comparison of the results between testing methods of pathogenic microorganisms include the the current different methods. Group C.92x10‑5b protons are pumped to the outside from the inside of chro- matophores. The F1300‑ch combined with the capture system achieves the molecular motor nano‑biosensor. This strating that this method is rapid. the stronger the concentration. Effects of the biosensor based on F0F1‑ATPase for O157:H7. The results demonstrated that it has a good specificity to E. including Chloramphenicol.81x10‑5a 102 103 0. which leads to a change in proton concentration P<0.01. a fluo- rospectrophotometric probe.06347 Group1 0. Based on its two enzymology characteristics. sensitive. colony forming units. 0 vs. 104 cfu. activity was inhibited as it linked to more external substances. F1300‑ch‑β ‑subunit antibody‑biotin‑Streptavidin‑biotin‑O157:H7 anti- this method is faster. therefore the alteration in relative fluorescence intensity should be gener- ated by those non‑O157:H7‑loaded chromatophores in each detection well. groups. The microbial method . 104.7. salmonella.01. demon- Figure 7. Clenbuterol and Deoxynivalenol (4‑5. simple and has a experiment was performed to verify that the F0F1‑ATPase‑based biosensor for low cost. PCR and ELISA. Group 5 4. the lower the changing biosensors and so the smaller the change value (14. body complex. Listeria monocytogenes. to produce the functional unit. H9 virus. Group A. F1300‑ch‑β‑subunit antibody‑biotin‑Streptavidin complex. The application of F0F1‑ATPase immuno‑biosensors for the detection of O157:H7 has not been reported previously. the chromatophore cannot move completely and there are no alterations in protons between the internal and external chromatophore. E. During ATP synthesis.

coli and Salmonella. 2013YQ140405). comparing O157:H7. Specificity for O157:H7 in Groups 1 to 5. colony forming units. Chinese Academy of Sciences. R2=0. Due to the complexity of the sample and the variety of bacteria in different samples. Group 1. the application of biosensors in pathogenic bacteria detection is rarely reported. the Beijing Academy of Science and Technology of China (grant no. Sensitivity for O157:H7 in the 0 (control). A comparison of the current methods used for detection. 350 and 255 cfu/well for O157:H7. however. capil- lary zone electrophoresis and other technologies are also being studied by the researchers. respectively. The present study performed preliminary research on the feasibility of applying F0F1‑ATPase immuno‑biosensors Figure 8. Group 5. Group 3. cfu.009163. however. 2010D002022000009) and the National Key Foundation of pathogenic microorganisms. and increasing bacteria and bacteria solution treatment. 1. In addition. it requires skilled operation and a detection limit of 106  cfu/l. coli and made to this paper. WEI et al: FOF1-ATPase IMMUNO-BIOSENSORS DETECT E.01 vs. coli. Surface plasmon resonance. Group 4. its reagents are for Exploring Scientific Instrument (grant no. respectively. The present study was supported by the Salmonella. polymerase chain reaction. Surface plasmon resonance. To promote its application. **P<0. O157:H7.325. high specificity immune magnetic beads could be used to enrich the target bacterial.275 cfu/well for O157:H7. 265. capillary zone electrophoresis. coli and Salmonella. 0 cfu/well (control). Method Time Detection limit Specificity Cost F0F1‑ATPase immuno‑biosensor 13 h 1‑5 cfu/25 g 95% Very low Microbial method 5‑7 d 1 cfu/25 g 100% Low PCR >48 h 10 ‑7x103 cfu/ml 2 99% (DNA easily polluted) High ELISA 36‑48 h 106 cfu/ml 95% Low Reverse transcription PCR 48 h 1‑5 cfu/25 g 99% (DNA easily polluted) High SPR biosensor >24 h 2x106 cfu/ml 95% High CEZ >24 h 105‑106 cfu/ml 95% Low PCR. Ministry of Science and Technology of the People's Republic of China (grant no. E. as well as 128 cfu/well for O157:H7. therefore. improper controls. The ELISA method is relatively time‑efficient. as this method is extremely sensitive. **P<0. biosensors. which will aid rapid clinical diagnosis. Group 2. E. the exogenous DNA. 35 and 26 cfu/well for O157:H7. CEZ. 2008IM021600). Escherichia coli. is time‑consuming and involves complicated processes. primer design and the target selection of sequence will all affect the results. sample pretreatment. as they are quicker than the microbial method and simpler than PCR. the experimental condi- tions. China) for the revisions and modifications tively. 103 and 104 cfu/well for O157:H7 detection. China) for his technical assistance. respectively. The authors would like to thank Professor Jia‑Chang Yue Salmonella and E. . novel fluorescent material could be chosen as fluorescent probe to improve the sensitivity and accuracy of this method (21‑25).750 and 1. expensive and the procedure is complex. SPR.9787. will require extensive research in practical sample testing.133. E. IG200905N). 102. 1. E. coli and Salmonella. control (sterile normal saline). Acknowledgements Figure 9. respec. which can minimize the interference of other bacte- rial (26‑28). 175 and Beijing. however. coli. P=0. Furthermore. The detection techniques based on groups.01 vs. coli O157:H7 875 Table V. E. Professor Yan‑Qun Li (South of China Normal University. they require expensive instruments and the low detection limit is 105‑106 cfu/ml (16‑23). (Institute of Biophysics. the application of a biosensor for the detection of pathogens is rarely reported. PCR the Beijing Municipal Party Organization of China (grant is the most mature method in the national testing methods no. In addition. Guangdong. F0F1‑ATPase can rapidly detect the disease markers in patient serum or feces. 27.

Naugle AL. Gao LJ. Wang F. Cui S. WL and Liu QJ: Application of nano biosensortech. Tang F and Wei Q: Green and orange 21. Yue J. 2006. Appl Environ Microbiol 70:  5. Int para‑digm. Zhengtao D. Wu HJ. 203‑206. 20. Rasooli I. Food Sci 31: 167‑170. 2009. Cooper CL. Clark LC Jr and Lyons C: Electrode systems for continuous 28. Nou X.876 MOLECULAR MEDICINE REPORTS 17: 870-876. detecting clenbuterol. Food Science 28: 446‑450. Wu HJ. 2006. Li F. Luo ZY and Jiang PD: tion of Staphylococcus aureus by a combination of Mechanically driven proton conduction in single delta‑free monoclonal antibody‑coated latex and capillary electrophoresis. 2010 (In Chinese). progress of four pathogenic bacterias. Yue J. Zhang Y. Cui YB . Science 281: 2016‑2018. 122‑127. 195‑199. Treadway JA. Xu G. Arthur TM. Zordan MD. Yue JC. Zhang W. Malo AT. Meng J. Paoli GC and Brewster JD:  7. 2009. Nanduri  V. for dual simultaneous and independent detection of viruses. Karplus M and Gao YQ: Biomolecular motors: The F1‑ATPase O157:H7 by immunomagnetic separation and real‑time PCR. Wei L. Electrophoresis 27: 1784‑1789. Larson JP. J Clin Microbiol 43: 6086‑6090. Ge N. monocytogenes using cadmium telluride quantum dots as a proton flux sensor and phage‑displayed antibody. J Food Microbiol 99: 47‑57. Curr Opin Struct Biol 14: 250‑259. Kang ZJ and Zhao L: The preliminary 527‑534. Science 297: 237‑240. 23. Kalchayanand N and Koohmaraie M: Improvement Chinese). inspection service regulatory testing program for Escherichia coli 17. 1998. . 2005. 2002. Capaldi RA and Aggeler R: Mechanism of the F(1)F(0)‑type detection by the PickPen magnetic particle separation device. 22. Deng Z. 2002. Zhang Y. 25. Biochem Biophys Res using sol‑gel derived waveguides. Wu X. Zhongguo Weishengtai Xue ramification amplification assay for detection of Escherichia coli Zazhi 22: 743‑745. Wang P. Shiyong Yufang imaging cytometry device. et al: The pollution and detection research fecal samples. Cytometry A 75: 155‑162. Li BM. Zhou H. Biosens Bioelectron 23: 248‑252. Chapman PA. Wu HJ. 2004. Appl Environ Microbiol 63: 2549‑2553. a biological rotary motor. Wei L. 2010 (In Chinese). Aslan MH and Oral AY: DNA biosen-  3. Liu H. 2010 (In Chinese). Park K and Leary JF: Detection of pathogenic  2. applying to detect H9 avian influenza virus. Wu HJ. Tu SI. Yue J and Ha Y: A Peale F and Bruchez MP: Immunofluorescent labeling of cancer novel biosensor regulated by the rotator of F0F1‑ATPase to detect marker Her2 and other cellular targets with semiconductor deoxynivalenolrapidly. Rogelj S and Kieft TL: Rapid detection of Escherichia coli 12. Iranian Journal  1. Buyukaksoy A. coli O157:H7 by a hybrid microfluidic SPR and molecular food‑borne pathogens in chilled broilers. Trends Biochem J Food Prot 69: 2870‑2874. Bhunia AK. Biochem Biophys Res Commun  423: quantum dots. Siddons CA and Harkin M: Use of monitoring in cardiovascular surgery. Li W. J Food Prot 68: 462‑468. F0F1‑ATPase. 13. Bosilevac JM. Wei L. Mousavi SL. Wu J and Zhang DY: Use of genes based on immunobiosensor. Oda M. Nat Biotechnol 21: 41‑46. 2005. Fu Z. Jiachang Y. Li BM. 2007 (In Guerini MN. Holt KG. 2011 (In Chinese). Gao P. Levine P and Eckel R: Food safety and of Clinical Infectious Diseases 4: 97‑103. Liu X. 1962. study of a rapid detecting technology for Listeria monocyto. Nazarian S and Amani J: Simultaneous detection of Escherichia coli O157:H7. Acharya G. 15. Food Sci 32: 302‑306. Kotov NA and Giersig  M: Spontaneous organiza- CdTe quantum dots as effective pH‑sensitive fluorescent probes tion of single CdTe nanoparticles into luminescent nanowires. 2004. Shi X. 2006. 2003. Liu J. Zhao C. 27. 2012. South Biomed Eng Conf 24: Commun 342: 1319‑1322. Zhongguo Shipin Weisheng Zazhi 22: 498‑501. Tang  Z. 24. 2007 . separation systems for detecting Escherichia coli O157 in bovine 14. Lu HT. Liu X L. Grafton MM. 18.  9. Zhao Y. Zhang Y. O157:H7 and other E. et al: Application of immuno‑rotary tive nonisotopic detection. O157:H7 in raw ground beef. Zhang XL and Kang ZJ: An immunobiosensor for rapid detection of Staphylococcus aureusenes. Brichta‑Harhay DM. Lun YZ. Yue JC . Haley KN. Fangqiong T and Qun W: Using SPR biosensor for the detection of L. Reece LM. Anal Biochem 364: 2007. coli O157:H7 detection in drinking water resources as biosensor to detect single virus. J Phys Chem B 111: 12024‑12031. 19. Unno H and Tanji Y: Rapid detection nology in chloramphenicol detection. Yuan K and Tian J: Rapid detec-  8. 1997. Chan WC and Nie S: Quantum dot bioconjugates for ultrasensi- 10. ATP syn‑thase. Jiang P and Zhang Z: F0F1‑ATPase sors for E. Morita M. of immunomagnetic separation for Escherichia coli O157:H7 11. Zhang XA . biosensor based on FoF1‑ATPase in Chromatophores for 26. Lun YZ. protein‑labeled PP01 bacteriophage. 2018 References 16. Sci 27: 154‑160.  6. Bahşi ZB. 2005. Yixue 17: 1314‑1315. Aronson AI. Ann N Y Acad Sci 102: commercial enzyme immunoassays and immunomagnetic 29‑45. 2009. Yun Z. Biochem Biophys Res Commun 347: 752‑757. Wang HH and Wu RM: Survey on contamination status of E. 2010 of Escherichia coli O157: H7 by using green fluorescent (In Chinese). 2006. 2007. toxigenic Vibrio cholerae and Salmonella typhimurium by multiplex PCR.  4.