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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 conventional culture method and the microchip based E. coli detection. (A) Conventional procedure for bacteria detection in clinical facilities. Blood sample collection. (1) Blood samples are incubated in an automated blood culture system. (2) Pathogen or bacteria grown on agar plate are subject to Gram-staining for differentiation between Gram-positive and negative strains. (3) The sample is sub-cultured into a nutrient-rich agar plate for the identification of the species and to determine the bacterial concentration. (B) POC testing approach for rapid detection. Blood sample collection (spiked with GFP-expressing E. coli BL21 stock as a model microorganism). (1) The blood sample is analyzed in microchannels functionalized with E. coli antibodies. E. coli were specifically captured by antibodies on the microchannel surface. (2) Unbound E. coli are washed away with PBS using a syringe micropump. (3) GFP-expressing E. coli are imaged/counted under a fluorescence microscope.Abbreviations:E. coli, Escherichia coli; GFP, green fluorescent protein; LBP, lipopolysaccharide binding protein; PBS, phosphate buffered saline; POC, point-of-care.
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f1-ijn-7-2591: Comparison of the conventional culture method and the microchip based E. coli detection. (A) Conventional procedure for bacteria detection in clinical facilities. Blood sample collection. (1) Blood samples are incubated in an automated blood culture system. (2) Pathogen or bacteria grown on agar plate are subject to Gram-staining for differentiation between Gram-positive and negative strains. (3) The sample is sub-cultured into a nutrient-rich agar plate for the identification of the species and to determine the bacterial concentration. (B) POC testing approach for rapid detection. Blood sample collection (spiked with GFP-expressing E. coli BL21 stock as a model microorganism). (1) The blood sample is analyzed in microchannels functionalized with E. coli antibodies. E. coli were specifically captured by antibodies on the microchannel surface. (2) Unbound E. coli are washed away with PBS using a syringe micropump. (3) GFP-expressing E. coli are imaged/counted under a fluorescence microscope.Abbreviations:E. coli, Escherichia coli; GFP, green fluorescent protein; LBP, lipopolysaccharide binding protein; PBS, phosphate buffered saline; POC, point-of-care.

Mentions: Currently, the gold standard detection method for bacteria is agar plate culture. However, this method is limited by the culturing time and volume of sample required to determine the presence of pathogens (Figure 1). Due to the challenge of obtaining enough sample volume, agar plate cultures give false negative results at rates ranging from 7.2% to 21.2%.10,11 In addition, the process is complicated by the fact that clinical samples need to go through multiple post-cultural steps for analysis, including Giemsa staining and differentiation on MacConkey plates.12 The whole process takes 48 to 72 hours.13 Although polymerase chain reaction (PCR) has high sensitivity and specificity,14 the need for a thermal cycler makes it unsuitable for point-of-care (POC) testing.15 Therein lies the niche for which microfluidic technologies are ideal; they have been employed to develop POC testing devices because of low manufacturing cost, reduced consumption of samples and reagents, and shortened assay time.15–20 However, existing microfluidic devices for bacterial detection, either based on PCR21 or enzyme-linked immunosorbent assay (ELISA), require multiple sample processing steps prior to detection.22,23 All of these methods suffer from challenges including culture time, need of high sample volumes and reagents, the requirement for preprocessing of samples, low accuracy of the pathogen detection, and high cost. Further, for the detection of rare bacteria, PCR and ELISA based methods require large initial sample volumes, and preprocessing of samples, and sample amplification. Thus, there is an unmet need to develop POC devices that can address these issues, and capture, isolate and detect bacteria from biologically complex samples such as blood and produce.


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 conventional culture method and the microchip based E. coli detection. (A) Conventional procedure for bacteria detection in clinical facilities. Blood sample collection. (1) Blood samples are incubated in an automated blood culture system. (2) Pathogen or bacteria grown on agar plate are subject to Gram-staining for differentiation between Gram-positive and negative strains. (3) The sample is sub-cultured into a nutrient-rich agar plate for the identification of the species and to determine the bacterial concentration. (B) POC testing approach for rapid detection. Blood sample collection (spiked with GFP-expressing E. coli BL21 stock as a model microorganism). (1) The blood sample is analyzed in microchannels functionalized with E. coli antibodies. E. coli were specifically captured by antibodies on the microchannel surface. (2) Unbound E. coli are washed away with PBS using a syringe micropump. (3) GFP-expressing E. coli are imaged/counted under a fluorescence microscope.Abbreviations:E. coli, Escherichia coli; GFP, green fluorescent protein; LBP, lipopolysaccharide binding protein; PBS, phosphate buffered saline; POC, point-of-care.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3368510&req=5

f1-ijn-7-2591: Comparison of the conventional culture method and the microchip based E. coli detection. (A) Conventional procedure for bacteria detection in clinical facilities. Blood sample collection. (1) Blood samples are incubated in an automated blood culture system. (2) Pathogen or bacteria grown on agar plate are subject to Gram-staining for differentiation between Gram-positive and negative strains. (3) The sample is sub-cultured into a nutrient-rich agar plate for the identification of the species and to determine the bacterial concentration. (B) POC testing approach for rapid detection. Blood sample collection (spiked with GFP-expressing E. coli BL21 stock as a model microorganism). (1) The blood sample is analyzed in microchannels functionalized with E. coli antibodies. E. coli were specifically captured by antibodies on the microchannel surface. (2) Unbound E. coli are washed away with PBS using a syringe micropump. (3) GFP-expressing E. coli are imaged/counted under a fluorescence microscope.Abbreviations:E. coli, Escherichia coli; GFP, green fluorescent protein; LBP, lipopolysaccharide binding protein; PBS, phosphate buffered saline; POC, point-of-care.
Mentions: Currently, the gold standard detection method for bacteria is agar plate culture. However, this method is limited by the culturing time and volume of sample required to determine the presence of pathogens (Figure 1). Due to the challenge of obtaining enough sample volume, agar plate cultures give false negative results at rates ranging from 7.2% to 21.2%.10,11 In addition, the process is complicated by the fact that clinical samples need to go through multiple post-cultural steps for analysis, including Giemsa staining and differentiation on MacConkey plates.12 The whole process takes 48 to 72 hours.13 Although polymerase chain reaction (PCR) has high sensitivity and specificity,14 the need for a thermal cycler makes it unsuitable for point-of-care (POC) testing.15 Therein lies the niche for which microfluidic technologies are ideal; they have been employed to develop POC testing devices because of low manufacturing cost, reduced consumption of samples and reagents, and shortened assay time.15–20 However, existing microfluidic devices for bacterial detection, either based on PCR21 or enzyme-linked immunosorbent assay (ELISA), require multiple sample processing steps prior to detection.22,23 All of these methods suffer from challenges including culture time, need of high sample volumes and reagents, the requirement for preprocessing of samples, low accuracy of the pathogen detection, and high cost. Further, for the detection of rare bacteria, PCR and ELISA based methods require large initial sample volumes, and preprocessing of samples, and sample amplification. Thus, there is an unmet need to develop POC devices that can address these issues, and capture, isolate and detect bacteria from biologically complex samples such as blood and produce.

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