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We demonstrated a sensitive quartz crystal microbalance DNA sensor using streptavidin-coated ferrofluid nanoparticles as “mass enhancers” for detection of E. coli O157:H7

A nanoparticle amplification based quartz crystal microbalance DNA sensor for detection of Escherichia coli O157:H7.

Biosensors and Bioelectronics, no. 7 (2006): 1178-1185

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摘要

A quartz crystal microbalance (QCM) DNA sensor, based on the nanoparticle amplification method, was developed for detection of Escherichia coli O157:H7. A thiolated single-stranded DNA (ssDNA) probe specific to E. coli O157:H7 eaeA gene was immobilized onto the QCM sensor surface through self-assembly. The hybridization was induced by exp...更多

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简介
  • Escherichia coli O157:H7, a foodborne pathogen first discovered in 1982, has been recognized as an emerging threat to the public health.
  • Coli O157:H7 often leads to hemorrhagic colitis and hemolytic uremic syndrome (HUS), both of which cause watery or bloody diarrhea, kidney failure, and even death, especially in children (Rowe et al, 1991; Buchanan and Dolye, 1997).
  • Rapid and sensitive detection methods are needed to meet the challenge for the detection of this pathogenic bacteria.
  • To achieve the desired rapidity, sensitivity, and detection limit, DNA based pathogen detection
重点内容
  • Escherichia coli O157:H7, a foodborne pathogen first discovered in 1982, has been recognized as an emerging threat to the public health
  • We demonstrated a sensitive quartz crystal microbalance (QCM) DNA sensor using streptavidin-coated ferrofluid nanoparticles as “mass enhancers” for detection of E. coli O157:H7
  • The detection limit for E. coli O157:H7 was 2.67 × 102 colony forming unit (CFU)/ml cells by detecting the PCR products without any enrichment of the culture, which was about one order lower than the lowest detection limit achieved by PCR gel-based detection method
  • Compared to the fluorescence measurements based on gel electrophoresis for PCR products, this QCM DNA sensor greatly improved the detection limit and reduced the assay time
  • It is believed that an ideal DNA piezoelectric sensor for bacteria detection would be the one that does not require the use of PCR; future studies are needed for the further improvement of the sensor’s sensitivity to minimize the need of pre-detection amplification, or evetually elimate the PCR procedure
结果
  • When two quartz crystals were modified with thiolated probe and thiolated probe + blocking BSA, respectively, and treated with 500 ␮l 1:25 PBS diluted nanoparticle stock solution, there was a significant frequency drop (∼75 Hz) for the crystal without BSA blocking, while much less frequency change (<3 Hz) was found on the BSA blocked crystal.
  • Upon the introduction of BSA solution, a drastic frequency drop was observed partially due to the significant change of properties of the liquid contacting with quartz crystal.
  • The 40 Hz frequency shifts before and after BSA blocking suggested the adsorption of BSA to the electrode
结论
  • The authors demonstrated a sensitive QCM DNA sensor using streptavidin-coated ferrofluid nanoparticles as “mass enhancers” for detection of E. coli O157:H7.
  • Coli O157:H7 was 2.67 × 102 CFU/ml cells by detecting the PCR products without any enrichment of the culture, which was about one order lower than the lowest detection limit achieved by PCR gel-based detection method.
  • Compared to the fluorescence measurements based on gel electrophoresis for PCR products, this QCM DNA sensor greatly improved the detection limit and reduced the assay time.
  • It is believed that an ideal DNA piezoelectric sensor for bacteria detection would be the one that does not require the use of PCR; future studies are needed for the further improvement of the sensor’s sensitivity to minimize the need of pre-detection amplification, or evetually elimate the PCR procedure
表格
  • Table1: Sequences of oligonucleotides used in sensor development
Download tables as Excel
基金
  • This work was supported in part by the Food Safety Consortium and the Center of Excellence for Poultry Science at the University of Arkansas
研究对象与分析
cases: 73000
Escherichia coli O157:H7, a foodborne pathogen first discovered in 1982, has been recognized as an emerging threat to the public health. It is estimated to cause 73,000 cases of illness and 61 deaths in the United States annually (CDC, 2004). Infection of E. coli O157:H7 often leads to hemorrhagic colitis and hemolytic uremic syndrome (HUS), both of which cause watery or bloody diarrhea, kidney failure, and even death, especially in children (Rowe et al, 1991; Buchanan and Dolye, 1997)

data: 3
The signal versus concentration (10−12 to 10−8 M) relation was plotted in a semi-logarithmic scale. The measurements were highly reproducible for all the concentrations (n = 3, R.S.D. < 12.5%). Linear relationships were found between the frequency shift and the log number of the positive control concentration for higher concentrations (10−10 to 10−8 M) and lower concentrations (10−12 to 10−10 M), respectively

data: 3
The sensorgram for the sensor fabrication and detection. a) Frequency shifts of the DNA sensor as a function of time for oligonucleotide controls. (b) Signals for the different concentration of positive controls. Linear relationships were found between the frequency shift vs. log (concentration) from 10−10 to 10−8 M (y1) and 10−12 to 10−10 M (y2), respectively. Error bars indicate the standard deviation (n = 3). SEM images of bare electrode (a), electrode after probe immobilization and nanoparticle treatment (b), electrode after probe immobilization, BSA blocking and nanoparticle treatment (c), and electrode after the detection of positive control (d)

data: 3
Frequency shifts of the DNA sensor as a function of time for different PCR samples. The responses of the sensor to different PCR samples. The detection limit is determined as 2.67 × 102 CFU/ml. A linear correlation between the frequency change and logarithmic number of cell concentration was found from 2.67 × 102 to 2.67 × 106 CFU/ml. Error bars indicate the standard deviation (n = 3).

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