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Portable microfluidic chip for detection of Escherichia coli in produce and blood.

Wang S, Inci F, Chaunzwa TL, Ramanujam A, Vasudevan A, Subramanian S, Chi Fai Ip A, Sridharan B, Gurkan UA, Demirci U - Int J Nanomedicine (2012)

Bottom Line: The microchip showed reliable capture of E. coli in PBS with an efficiency of 71.8% ± 5% at concentrations ranging from 50 to 4,000 CFUs/mL via lipopolysaccharide binding protein.The limits of detection of the microchip for PBS, blood, milk, and spinach samples were 50, 50, 50, and 500 CFUs/mL, respectively.The presented technology can be broadly applied to other pathogens at the POC, enabling various applications including surveillance of food supply and monitoring of bacteriology in patients with burn wounds.

View Article: PubMed Central - PubMed

Affiliation: Bio-Acoustic-MEMS in Medicine Laboratory, Center for Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02139, USA.

ABSTRACT
Pathogenic agents can lead to severe clinical outcomes such as food poisoning, infection of open wounds, particularly in burn injuries and sepsis. Rapid detection of these pathogens can monitor these infections in a timely manner improving clinical outcomes. Conventional bacterial detection methods, such as agar plate culture or polymerase chain reaction, are time-consuming and dependent on complex and expensive instruments, which are not suitable for point-of-care (POC) settings. Therefore, there is an unmet need to develop a simple, rapid method for detection of pathogens such as Escherichia coli. Here, we present an immunobased microchip technology that can rapidly detect and quantify bacterial presence in various sources including physiologically relevant buffer solution (phosphate buffered saline [PBS]), blood, milk, and spinach. The microchip showed reliable capture of E. coli in PBS with an efficiency of 71.8% ± 5% at concentrations ranging from 50 to 4,000 CFUs/mL via lipopolysaccharide binding protein. The limits of detection of the microchip for PBS, blood, milk, and spinach samples were 50, 50, 50, and 500 CFUs/mL, respectively. The presented technology can be broadly applied to other pathogens at the POC, enabling various applications including surveillance of food supply and monitoring of bacteriology in patients with burn wounds.

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Comparison of the capture efficiency of E. coli by two different surface chemistries and different capturing agents. E. coli were incubated at room temperature for 1 hour. (A) Three different experimental designs (anti-LBP-LBP, anti-LBP-LBP-BSA, and LBP-BSA) were performed on NeutrAvidin based surface chemistry. Three different capture agents were immobilized via Protein G based surface chemistry. The wash flow rate was 2 μL/min. Brackets connecting individual groups indicate statistically significant difference (analysis of Variance with Tukey’s post-hoc test for multiple comparisons, n = 2–6, P < 0.05). (B) Effect of channel flow rate on capture efficiency of E. coli on chip. 75 μL of E. coli was flowed into microchannels. After sample incubation for 15 min at ambient temperature, three different wash flow rates (2, 5, and 10 μL/min) were used to optimize the capture efficiency of E. coli on chip. Statistical analysis indicated that flow rate had a significant effect on capture efficiency (nonparametric Kruskal–Wallis test), where 2 μL/min resulted in significantly greater (P < 0.05) capture efficiency compared to 10 μL/min flow rate. Brackets connecting individual groups indicate statistically significant difference. Data are presented as average ± SEM. Non-parametric upper-tailed Mann–Whitney U test for pair-wise comparisons, n = 3–8, P < 0.05.Abbreviations: BSA, bovine serum albumin; E. coli, Escherichia coli; LBP, lipopolysaccharide binding protein; SEM, standard error of the mean.
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f3-ijn-7-2591: Comparison of the capture efficiency of E. coli by two different surface chemistries and different capturing agents. E. coli were incubated at room temperature for 1 hour. (A) Three different experimental designs (anti-LBP-LBP, anti-LBP-LBP-BSA, and LBP-BSA) were performed on NeutrAvidin based surface chemistry. Three different capture agents were immobilized via Protein G based surface chemistry. The wash flow rate was 2 μL/min. Brackets connecting individual groups indicate statistically significant difference (analysis of Variance with Tukey’s post-hoc test for multiple comparisons, n = 2–6, P < 0.05). (B) Effect of channel flow rate on capture efficiency of E. coli on chip. 75 μL of E. coli was flowed into microchannels. After sample incubation for 15 min at ambient temperature, three different wash flow rates (2, 5, and 10 μL/min) were used to optimize the capture efficiency of E. coli on chip. Statistical analysis indicated that flow rate had a significant effect on capture efficiency (nonparametric Kruskal–Wallis test), where 2 μL/min resulted in significantly greater (P < 0.05) capture efficiency compared to 10 μL/min flow rate. Brackets connecting individual groups indicate statistically significant difference. Data are presented as average ± SEM. Non-parametric upper-tailed Mann–Whitney U test for pair-wise comparisons, n = 3–8, P < 0.05.Abbreviations: BSA, bovine serum albumin; E. coli, Escherichia coli; LBP, lipopolysaccharide binding protein; SEM, standard error of the mean.

Mentions: We engineered the surface chemistry using immobilized antibodies, where the performance of the chip relies on nanoscale reactions on the microchannel surface. We evaluated antibodies specific to E. coli surface proteins using commonly reported antibody immobilization methods that provide a uniform distribution of antibodies on the capture surface in microfluidic channels, ie, Protein G and NeutrAvidin based methods. To develop a rapid microchip method for E. coli detection, four different capturing agents were immobilized in microchannels via these two surface chemistry methods. As shown in Figure 2B, anti-LPS, antiflagellin, and CD14 were immobilized on the microchannel surface via Protein G and anti-LBP antibody was immobilized on the microchannel surface via NeutrAvidin. To investigate the binding of anti-LBP antibody, and to observe the effect of antibody orientation on E. coli capture. Three different experimental designs were performed. In NeutrAvidin experiments, capture efficiencies of anti-LBP-LBP, anti-LBP-LBP-BSA, and LBP-BSA were observed to be 71.8% ± 5%, 60.7% ± 2%, and 44.5% ± 5%, respectively (Figure 3A). In comparison, E. coli capture efficiencies of antiflagellin, anti-LPS and CD14 obtained by using Protein G based surface chemistry, were 46.9% ± 3%, 41.5% ± 5%, and 41.0% ± 2%, respectively (Figure 3A). The capture efficiency via anti-LBP-LBP was observed to be significantly greater (P < 0.05) than the other capture agents that were used with Protein G based surface chemistry (Figure 3A). In our prior study, Protein G and NeutrAvidin exhibited similar efficiency to immobilize capture agents on microchannel surfaces.26 Thus, the difference in capture efficiency of E. coli was mainly due to the affinity of capture agents, ie, anti-LPS, antiflagellin, CD14, and LBP. The highest capture efficiency (71.8% ± 5%) was observed in microchannels immobilized with anti-LBP in the presence of LBP. This observation is in accordance with a previous report, in which LBP was shown to bind E. coli with high affinity; anti-LBP antibody helped the protein to take favorable orientation for E. coli capture.27 BSA blocking was used to prevent nonspecific binding in microchannels in addition to preventing the binding of anti-LBP antibodies and LBP onto the succinimide group of GMBS. The use of BSA as a blocking agent did not result in a statistically significant difference in capture efficiency with both anti-LBP-LBP and anti-LBP-LBP-BSA. The LBP-BSA experiment showed that the orientation of the LBP protein is critical to capture of E. coli, which was supported by statistical analysis of experimental results as shown in Figure 3A. As an overall result, NeutrAvidin- mediated anti-LBP antibody-LBP immobilization performed on the microchannel surface to attain high capture efficiency of E. coli.


Portable microfluidic chip for detection of Escherichia coli in produce and blood.

Wang S, Inci F, Chaunzwa TL, Ramanujam A, Vasudevan A, Subramanian S, Chi Fai Ip A, Sridharan B, Gurkan UA, Demirci U - Int J Nanomedicine (2012)

Comparison of the capture efficiency of E. coli by two different surface chemistries and different capturing agents. E. coli were incubated at room temperature for 1 hour. (A) Three different experimental designs (anti-LBP-LBP, anti-LBP-LBP-BSA, and LBP-BSA) were performed on NeutrAvidin based surface chemistry. Three different capture agents were immobilized via Protein G based surface chemistry. The wash flow rate was 2 μL/min. Brackets connecting individual groups indicate statistically significant difference (analysis of Variance with Tukey’s post-hoc test for multiple comparisons, n = 2–6, P < 0.05). (B) Effect of channel flow rate on capture efficiency of E. coli on chip. 75 μL of E. coli was flowed into microchannels. After sample incubation for 15 min at ambient temperature, three different wash flow rates (2, 5, and 10 μL/min) were used to optimize the capture efficiency of E. coli on chip. Statistical analysis indicated that flow rate had a significant effect on capture efficiency (nonparametric Kruskal–Wallis test), where 2 μL/min resulted in significantly greater (P < 0.05) capture efficiency compared to 10 μL/min flow rate. Brackets connecting individual groups indicate statistically significant difference. Data are presented as average ± SEM. Non-parametric upper-tailed Mann–Whitney U test for pair-wise comparisons, n = 3–8, P < 0.05.Abbreviations: BSA, bovine serum albumin; E. coli, Escherichia coli; LBP, lipopolysaccharide binding protein; SEM, standard error of the mean.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3368510&req=5

f3-ijn-7-2591: Comparison of the capture efficiency of E. coli by two different surface chemistries and different capturing agents. E. coli were incubated at room temperature for 1 hour. (A) Three different experimental designs (anti-LBP-LBP, anti-LBP-LBP-BSA, and LBP-BSA) were performed on NeutrAvidin based surface chemistry. Three different capture agents were immobilized via Protein G based surface chemistry. The wash flow rate was 2 μL/min. Brackets connecting individual groups indicate statistically significant difference (analysis of Variance with Tukey’s post-hoc test for multiple comparisons, n = 2–6, P < 0.05). (B) Effect of channel flow rate on capture efficiency of E. coli on chip. 75 μL of E. coli was flowed into microchannels. After sample incubation for 15 min at ambient temperature, three different wash flow rates (2, 5, and 10 μL/min) were used to optimize the capture efficiency of E. coli on chip. Statistical analysis indicated that flow rate had a significant effect on capture efficiency (nonparametric Kruskal–Wallis test), where 2 μL/min resulted in significantly greater (P < 0.05) capture efficiency compared to 10 μL/min flow rate. Brackets connecting individual groups indicate statistically significant difference. Data are presented as average ± SEM. Non-parametric upper-tailed Mann–Whitney U test for pair-wise comparisons, n = 3–8, P < 0.05.Abbreviations: BSA, bovine serum albumin; E. coli, Escherichia coli; LBP, lipopolysaccharide binding protein; SEM, standard error of the mean.
Mentions: We engineered the surface chemistry using immobilized antibodies, where the performance of the chip relies on nanoscale reactions on the microchannel surface. We evaluated antibodies specific to E. coli surface proteins using commonly reported antibody immobilization methods that provide a uniform distribution of antibodies on the capture surface in microfluidic channels, ie, Protein G and NeutrAvidin based methods. To develop a rapid microchip method for E. coli detection, four different capturing agents were immobilized in microchannels via these two surface chemistry methods. As shown in Figure 2B, anti-LPS, antiflagellin, and CD14 were immobilized on the microchannel surface via Protein G and anti-LBP antibody was immobilized on the microchannel surface via NeutrAvidin. To investigate the binding of anti-LBP antibody, and to observe the effect of antibody orientation on E. coli capture. Three different experimental designs were performed. In NeutrAvidin experiments, capture efficiencies of anti-LBP-LBP, anti-LBP-LBP-BSA, and LBP-BSA were observed to be 71.8% ± 5%, 60.7% ± 2%, and 44.5% ± 5%, respectively (Figure 3A). In comparison, E. coli capture efficiencies of antiflagellin, anti-LPS and CD14 obtained by using Protein G based surface chemistry, were 46.9% ± 3%, 41.5% ± 5%, and 41.0% ± 2%, respectively (Figure 3A). The capture efficiency via anti-LBP-LBP was observed to be significantly greater (P < 0.05) than the other capture agents that were used with Protein G based surface chemistry (Figure 3A). In our prior study, Protein G and NeutrAvidin exhibited similar efficiency to immobilize capture agents on microchannel surfaces.26 Thus, the difference in capture efficiency of E. coli was mainly due to the affinity of capture agents, ie, anti-LPS, antiflagellin, CD14, and LBP. The highest capture efficiency (71.8% ± 5%) was observed in microchannels immobilized with anti-LBP in the presence of LBP. This observation is in accordance with a previous report, in which LBP was shown to bind E. coli with high affinity; anti-LBP antibody helped the protein to take favorable orientation for E. coli capture.27 BSA blocking was used to prevent nonspecific binding in microchannels in addition to preventing the binding of anti-LBP antibodies and LBP onto the succinimide group of GMBS. The use of BSA as a blocking agent did not result in a statistically significant difference in capture efficiency with both anti-LBP-LBP and anti-LBP-LBP-BSA. The LBP-BSA experiment showed that the orientation of the LBP protein is critical to capture of E. coli, which was supported by statistical analysis of experimental results as shown in Figure 3A. As an overall result, NeutrAvidin- mediated anti-LBP antibody-LBP immobilization performed on the microchannel surface to attain high capture efficiency of E. coli.

Bottom Line: The microchip showed reliable capture of E. coli in PBS with an efficiency of 71.8% ± 5% at concentrations ranging from 50 to 4,000 CFUs/mL via lipopolysaccharide binding protein.The limits of detection of the microchip for PBS, blood, milk, and spinach samples were 50, 50, 50, and 500 CFUs/mL, respectively.The presented technology can be broadly applied to other pathogens at the POC, enabling various applications including surveillance of food supply and monitoring of bacteriology in patients with burn wounds.

View Article: PubMed Central - PubMed

Affiliation: Bio-Acoustic-MEMS in Medicine Laboratory, Center for Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02139, USA.

ABSTRACT
Pathogenic agents can lead to severe clinical outcomes such as food poisoning, infection of open wounds, particularly in burn injuries and sepsis. Rapid detection of these pathogens can monitor these infections in a timely manner improving clinical outcomes. Conventional bacterial detection methods, such as agar plate culture or polymerase chain reaction, are time-consuming and dependent on complex and expensive instruments, which are not suitable for point-of-care (POC) settings. Therefore, there is an unmet need to develop a simple, rapid method for detection of pathogens such as Escherichia coli. Here, we present an immunobased microchip technology that can rapidly detect and quantify bacterial presence in various sources including physiologically relevant buffer solution (phosphate buffered saline [PBS]), blood, milk, and spinach. The microchip showed reliable capture of E. coli in PBS with an efficiency of 71.8% ± 5% at concentrations ranging from 50 to 4,000 CFUs/mL via lipopolysaccharide binding protein. The limits of detection of the microchip for PBS, blood, milk, and spinach samples were 50, 50, 50, and 500 CFUs/mL, respectively. The presented technology can be broadly applied to other pathogens at the POC, enabling various applications including surveillance of food supply and monitoring of bacteriology in patients with burn wounds.

Show MeSH
Related in: MedlinePlus