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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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Related in: MedlinePlus

Correlation of E. coli quantification by microchip and LB plating. This experiment was performed to establish the correlation between bacteria cell counts obtained by colony count from LB agar plates and cell count after capture on a microfluidic device (A, C, E and G). Bland–Altman analysis between the microchip count and E. coli stock concentrations did not display an evidence for a systematic bias for chip counts. (A) 75 μL of varying concentrations (up to 500 CFUs/mL) of E. coli spiked in PBS was injected into microchannels functionalized with anti-LBP antibody. For comparison, 75 μL of each concentration of E. coli was plated out on ampicillin containing LB agar plates and incubated overnight. The number of E. coli colonies was counted the next day and compared to the E. coli counted on chip. The detection limit of microchip was found as 50 CFUs/mL. Data are presented as average ± SEM (n = 3) (r = 0.960, P = 0.009). (B) The mean bias for E. coli spiked in PBS was −70 CFUs/mL sample in microchip counts compared to E. coli stock concentrations. (C) Varying concentrations (up to 400 CFUs/mL) of E. coli spiked in blood were injected into microchannels functionalized with anti-LBP antibody and the detection limit of microchip was found as 50 CFUs/mL (r = 0.989, P = 0.011). (D) The mean bias was −165 CFUs/mL of blood in microchip counts compared to E. coli stock concentrations. (E) Varying concentrations (up to 400 CFUs/mL) of E. coli spiked in milk were injected into microchannels functionalized with anti-LBP antibody and the detection limit of microchip was found as 50 CFUs/mL (r = 0.962, P = 0.038). (F) The mean bias was −163 CFUs/mL of milk in microchip counts compared to E. coli stock concentrations. (G) Varying concentrations (up to 4,000 CFUs/mL) of E. coli spiked in spinach were injected into microchannels functionalized with anti-LBP antibody and the detection limit of microchip was found as 500 CFUs/mL (r = 0.977, P = 0.023). (H) The mean bias was −1869 CFUs/mL of spinach sample in microchip counts compared to E. coli stock concentrations. (“r” indicates Pearson product-moment correlation coefficient, “P” indicates the statistical significance of correlation).Abbreviations: CFU, colony forming unit; E. coli, Escherichia coli; LB, Luria–Bertani; LBP, lipopolysaccharide binding protein; SD, standard deviation; SEM, standard error of the mean.
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f5-ijn-7-2591: Correlation of E. coli quantification by microchip and LB plating. This experiment was performed to establish the correlation between bacteria cell counts obtained by colony count from LB agar plates and cell count after capture on a microfluidic device (A, C, E and G). Bland–Altman analysis between the microchip count and E. coli stock concentrations did not display an evidence for a systematic bias for chip counts. (A) 75 μL of varying concentrations (up to 500 CFUs/mL) of E. coli spiked in PBS was injected into microchannels functionalized with anti-LBP antibody. For comparison, 75 μL of each concentration of E. coli was plated out on ampicillin containing LB agar plates and incubated overnight. The number of E. coli colonies was counted the next day and compared to the E. coli counted on chip. The detection limit of microchip was found as 50 CFUs/mL. Data are presented as average ± SEM (n = 3) (r = 0.960, P = 0.009). (B) The mean bias for E. coli spiked in PBS was −70 CFUs/mL sample in microchip counts compared to E. coli stock concentrations. (C) Varying concentrations (up to 400 CFUs/mL) of E. coli spiked in blood were injected into microchannels functionalized with anti-LBP antibody and the detection limit of microchip was found as 50 CFUs/mL (r = 0.989, P = 0.011). (D) The mean bias was −165 CFUs/mL of blood in microchip counts compared to E. coli stock concentrations. (E) Varying concentrations (up to 400 CFUs/mL) of E. coli spiked in milk were injected into microchannels functionalized with anti-LBP antibody and the detection limit of microchip was found as 50 CFUs/mL (r = 0.962, P = 0.038). (F) The mean bias was −163 CFUs/mL of milk in microchip counts compared to E. coli stock concentrations. (G) Varying concentrations (up to 4,000 CFUs/mL) of E. coli spiked in spinach were injected into microchannels functionalized with anti-LBP antibody and the detection limit of microchip was found as 500 CFUs/mL (r = 0.977, P = 0.023). (H) The mean bias was −1869 CFUs/mL of spinach sample in microchip counts compared to E. coli stock concentrations. (“r” indicates Pearson product-moment correlation coefficient, “P” indicates the statistical significance of correlation).Abbreviations: CFU, colony forming unit; E. coli, Escherichia coli; LB, Luria–Bertani; LBP, lipopolysaccharide binding protein; SD, standard deviation; SEM, standard error of the mean.

Mentions: To determine the microchip’s limit of detection for E. coli capture, we used LB agar plate culture as the gold standard for E. coli detection. We correlated agar plate results for a series of known concentrations of E. coli with the microchip capture results. E. coli concentration and microchip-based E. coli detection showed a positive correlation for E. coli concentrations ranging from 50 to 4,000 CFUs/mL (Figure 5). The linearity of the correlation indicates that microchip-based E. coli detection can be used as an alternative to agar plate culture.


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)

Correlation of E. coli quantification by microchip and LB plating. This experiment was performed to establish the correlation between bacteria cell counts obtained by colony count from LB agar plates and cell count after capture on a microfluidic device (A, C, E and G). Bland–Altman analysis between the microchip count and E. coli stock concentrations did not display an evidence for a systematic bias for chip counts. (A) 75 μL of varying concentrations (up to 500 CFUs/mL) of E. coli spiked in PBS was injected into microchannels functionalized with anti-LBP antibody. For comparison, 75 μL of each concentration of E. coli was plated out on ampicillin containing LB agar plates and incubated overnight. The number of E. coli colonies was counted the next day and compared to the E. coli counted on chip. The detection limit of microchip was found as 50 CFUs/mL. Data are presented as average ± SEM (n = 3) (r = 0.960, P = 0.009). (B) The mean bias for E. coli spiked in PBS was −70 CFUs/mL sample in microchip counts compared to E. coli stock concentrations. (C) Varying concentrations (up to 400 CFUs/mL) of E. coli spiked in blood were injected into microchannels functionalized with anti-LBP antibody and the detection limit of microchip was found as 50 CFUs/mL (r = 0.989, P = 0.011). (D) The mean bias was −165 CFUs/mL of blood in microchip counts compared to E. coli stock concentrations. (E) Varying concentrations (up to 400 CFUs/mL) of E. coli spiked in milk were injected into microchannels functionalized with anti-LBP antibody and the detection limit of microchip was found as 50 CFUs/mL (r = 0.962, P = 0.038). (F) The mean bias was −163 CFUs/mL of milk in microchip counts compared to E. coli stock concentrations. (G) Varying concentrations (up to 4,000 CFUs/mL) of E. coli spiked in spinach were injected into microchannels functionalized with anti-LBP antibody and the detection limit of microchip was found as 500 CFUs/mL (r = 0.977, P = 0.023). (H) The mean bias was −1869 CFUs/mL of spinach sample in microchip counts compared to E. coli stock concentrations. (“r” indicates Pearson product-moment correlation coefficient, “P” indicates the statistical significance of correlation).Abbreviations: CFU, colony forming unit; E. coli, Escherichia coli; LB, Luria–Bertani; LBP, lipopolysaccharide binding protein; SD, standard deviation; SEM, standard error of the mean.
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Related In: Results  -  Collection

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f5-ijn-7-2591: Correlation of E. coli quantification by microchip and LB plating. This experiment was performed to establish the correlation between bacteria cell counts obtained by colony count from LB agar plates and cell count after capture on a microfluidic device (A, C, E and G). Bland–Altman analysis between the microchip count and E. coli stock concentrations did not display an evidence for a systematic bias for chip counts. (A) 75 μL of varying concentrations (up to 500 CFUs/mL) of E. coli spiked in PBS was injected into microchannels functionalized with anti-LBP antibody. For comparison, 75 μL of each concentration of E. coli was plated out on ampicillin containing LB agar plates and incubated overnight. The number of E. coli colonies was counted the next day and compared to the E. coli counted on chip. The detection limit of microchip was found as 50 CFUs/mL. Data are presented as average ± SEM (n = 3) (r = 0.960, P = 0.009). (B) The mean bias for E. coli spiked in PBS was −70 CFUs/mL sample in microchip counts compared to E. coli stock concentrations. (C) Varying concentrations (up to 400 CFUs/mL) of E. coli spiked in blood were injected into microchannels functionalized with anti-LBP antibody and the detection limit of microchip was found as 50 CFUs/mL (r = 0.989, P = 0.011). (D) The mean bias was −165 CFUs/mL of blood in microchip counts compared to E. coli stock concentrations. (E) Varying concentrations (up to 400 CFUs/mL) of E. coli spiked in milk were injected into microchannels functionalized with anti-LBP antibody and the detection limit of microchip was found as 50 CFUs/mL (r = 0.962, P = 0.038). (F) The mean bias was −163 CFUs/mL of milk in microchip counts compared to E. coli stock concentrations. (G) Varying concentrations (up to 4,000 CFUs/mL) of E. coli spiked in spinach were injected into microchannels functionalized with anti-LBP antibody and the detection limit of microchip was found as 500 CFUs/mL (r = 0.977, P = 0.023). (H) The mean bias was −1869 CFUs/mL of spinach sample in microchip counts compared to E. coli stock concentrations. (“r” indicates Pearson product-moment correlation coefficient, “P” indicates the statistical significance of correlation).Abbreviations: CFU, colony forming unit; E. coli, Escherichia coli; LB, Luria–Bertani; LBP, lipopolysaccharide binding protein; SD, standard deviation; SEM, standard error of the mean.
Mentions: To determine the microchip’s limit of detection for E. coli capture, we used LB agar plate culture as the gold standard for E. coli detection. We correlated agar plate results for a series of known concentrations of E. coli with the microchip capture results. E. coli concentration and microchip-based E. coli detection showed a positive correlation for E. coli concentrations ranging from 50 to 4,000 CFUs/mL (Figure 5). The linearity of the correlation indicates that microchip-based E. coli detection can be used as an alternative to agar plate culture.

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