3.4 Detection Step
QCM-based biosensor represents a new technology for the rapid detection, ease of
use, low cost, online monitoring, shorter analysis time to measure pathogens, toxins,
pesticides, and any analyte that can be recognized by aptamers, antibodies, or
complementary DNA strand. Nevertheless it is worth to highlight that the
dimensions of the analyte to recognize play a key role in the detection step because
in case of large molecules no strategy is needed and a direct frequency shift will be
shown. On contrary, when the molecules to detect are of medium or small size, it is
indispensable to use different strategies to ballast them and to improve the limit of
detection (LOD).
Starting from the detection of large molecules, Escherichia coli O157:H7 (E. coli
O157:H7) is one of the most studied water contaminants because it is a dangerous
pathogen. It causes serious illnesses such as bloody diarrhea, bloody feces, anemia,
and kidney failure. Hence, an establishment of rapid and sensitive methods for
E. coli O157:H7 detection is strongly needed to control this pathogenic bacterium
in water supplies or food. Traditional methods for testing of E. coli O157:H7 include
plating and culturing, enumeration methods, and biochemical testing. Although the
detection limits for these methods are very low (about a few colony-forming units
(CFU)/ml), the testing time is time-consuming (from 1 day to 1 week). Besides,
some new techniques for rapid detection of this bacteria have been developed
including immunoassays, polymerase chain reaction (PCR) [8], DNA microarrays,
and immunomagnetic separations.
It has been shown that sensitivity and selectivity of these methods are good and
detection time for these methods is from about 2 h to 24 h. However, these methods
have a disadvantage in that they are expensive or complicated due to the use of
laboratories equipped with specific instruments and chromospheres. Therefore, they
are not suitable for rapid test of E. coli O157:H7 bacteria. Nurliyana et al. [53]
developed a QCM-based biosensor anchoring the antibodies onto gold surface with
SAMs method. The LOD they were able to reach was 10
2
–10
3 CFU/mL of E. coli
O157:H7 observing frequency shifts of about 15 Hz and 34 Hz, respectively. This
result allows QCM system to be used for qualitative and quantitative analysis of cell
concentration in solution. Fulgione et al. [54] reached an LOD of 10
0 CFU/mL in the
detection of Salmonella typhimurium in chicken meat with a relatively simple
protocol which requires a pre-enrichment step lasting only 4 h at 37
C.
The reliability of the proposed immunosensor has been demonstrated through the
validation of the experimental results with ISO standardized culture method which
takes up to 10 days to provide a reliable response. In order to further improve the LOD
of bacteria, some authors developed a quartz crystal microbalance immunosensor for
detection of E. coli O157:H7 by self-assembling of protein A and affinity-purified
anti-E. coli O157:H7 antibodies on the gold electrode of an AT-cut piezoelectric
quartz crystal. To enhance the sensitivity of the QCM immunosensor, nanoparticle–
antibody conjugates, which were prepared using streptavidin-conjugated
nanoparticles (145 nm diameter) and biotinylated anti-E. coli antibodies, were used
336
B. Della Ventura et al.
QCM-based biosensor represents a new technology for the rapid detection, ease of
use, low cost, online monitoring, shorter analysis time to measure pathogens, toxins,
pesticides, and any analyte that can be recognized by aptamers, antibodies, or
complementary DNA strand. Nevertheless it is worth to highlight that the
dimensions of the analyte to recognize play a key role in the detection step because
in case of large molecules no strategy is needed and a direct frequency shift will be
shown. On contrary, when the molecules to detect are of medium or small size, it is
indispensable to use different strategies to ballast them and to improve the limit of
detection (LOD).
Starting from the detection of large molecules, Escherichia coli O157:H7 (E. coli
O157:H7) is one of the most studied water contaminants because it is a dangerous
pathogen. It causes serious illnesses such as bloody diarrhea, bloody feces, anemia,
and kidney failure. Hence, an establishment of rapid and sensitive methods for
E. coli O157:H7 detection is strongly needed to control this pathogenic bacterium
in water supplies or food. Traditional methods for testing of E. coli O157:H7 include
plating and culturing, enumeration methods, and biochemical testing. Although the
detection limits for these methods are very low (about a few colony-forming units
(CFU)/ml), the testing time is time-consuming (from 1 day to 1 week). Besides,
some new techniques for rapid detection of this bacteria have been developed
including immunoassays, polymerase chain reaction (PCR) [8], DNA microarrays,
and immunomagnetic separations.
It has been shown that sensitivity and selectivity of these methods are good and
detection time for these methods is from about 2 h to 24 h. However, these methods
have a disadvantage in that they are expensive or complicated due to the use of
laboratories equipped with specific instruments and chromospheres. Therefore, they
are not suitable for rapid test of E. coli O157:H7 bacteria. Nurliyana et al. [53]
developed a QCM-based biosensor anchoring the antibodies onto gold surface with
SAMs method. The LOD they were able to reach was 10
2
–10
3 CFU/mL of E. coli
O157:H7 observing frequency shifts of about 15 Hz and 34 Hz, respectively. This
result allows QCM system to be used for qualitative and quantitative analysis of cell
concentration in solution. Fulgione et al. [54] reached an LOD of 10
0 CFU/mL in the
detection of Salmonella typhimurium in chicken meat with a relatively simple
protocol which requires a pre-enrichment step lasting only 4 h at 37
C.
The reliability of the proposed immunosensor has been demonstrated through the
validation of the experimental results with ISO standardized culture method which
takes up to 10 days to provide a reliable response. In order to further improve the LOD
of bacteria, some authors developed a quartz crystal microbalance immunosensor for
detection of E. coli O157:H7 by self-assembling of protein A and affinity-purified
anti-E. coli O157:H7 antibodies on the gold electrode of an AT-cut piezoelectric
quartz crystal. To enhance the sensitivity of the QCM immunosensor, nanoparticle–
antibody conjugates, which were prepared using streptavidin-conjugated
nanoparticles (145 nm diameter) and biotinylated anti-E. coli antibodies, were used
336
B. Della Ventura et al.