with a charge-coupled device (CCD) camera. Five microliters of the solution containing the amplicon-labelled microbeads was placed on the microelectrode and covered with a glass cover slip. An AC voltage of 5 VPP (peak to peak) amplitude at 100 kHz was applied to the microelectrode to generate the DEP force.
4.2.5 DEPIM of DNA-labeled microbeads
DEPIM for electrical measurement of DEP-trapped DNA-labeled microbeads was performed with an experiment set up used in chapter 3 shown as figure 3.7. Fifty microliters of a solution containing the DNA labeled microbeads was placed on the microelectrode for 1 min before DEP and impedance measurements for the DNA-labeled beads to precipitate onto the microelectrode surface by gravity. This provided experimental consistency by ensuring that most beads were within the influence of the DEP field. An AC voltage of 2 VPP and 100 kHz was applied to the microelectrode to generate DEP forces and perform DEPIM.
4.2.6 Selective detection of target bacteria
In environmental or clinical samples, comparing to the background bacterial, the amount of target bacterial is small. Therefore, in order to study the selectivity of our method, serially diluted E. coli solutions mixed with yeast solutions that have considerably high concentration have been used. The yeast and E. coli were cultured the concentration of bacterial solutions were confirmed by colony count as described in 4.2.1.
The cultured E. coli solution was serially diluted in liquid LB. Then, 2.5 µl of each diluted E. coli solution was mixed with 2.5 µl of cultured yeast solution and used immediately.
The DNA extraction and PCR procedures were similarly performed for pure E. coli suspension, as described in Section 4.2. The amplicons were confirmed by standard agarose gel electrophoresis and the concentration of amplicons was confirmed using the Qubit® 3.0 Fluorometer. The amplicons were labeled on microbeads and used for electrical detection, as described in 4.2.3.
shown in figure 4.2, the concentration of amplicons depended on bacterial concentration.
This is because the higher bacterial concentration would result in a higher concentration of extracted DNA. Since theoretically PCR amplified DNA exponentially, the amount of amplified DNA is related to the extracted DNA. Therefore, as shown in figure 4.2, the concentration of amplicons increased in a semi-logarithmic way with bacterial concentration, when the bacterial concentration was above 2.4×104 CFU/ml. It should be noticed that DNA extracted from bacteria cannot be amplified efficiently when bacterial concentration was below 2.4×104 CFU/ml.
4.3.2 Microbeads DEP behavior observation
Figure 4.3. shows the DEP behavior of DNA-labeled microbeads. Microbeads labeled with amplicons from E. coli at a concentration of 2.4×103 CFU/ml, were repelled from the electrode gaps, which were the high electric field regions, under the n-DEP force.
In contrast, microbeads labeled with amplicons from E. coli at a concentration of 2.4×104 CFU/ml, were trapped between the electrode gaps under the action of p-DEP.
4.3.3 Detection for pure E. coli solution.
Figure 4.4. shows the DEPIM results obtained with microbeads labeled by amplicons from serially diluted E. coli. When E. coli concentrations were above 2.4×104 CFU/ml, the electrode conductance increased rapidly after AC voltage was applied.
In contrast, for E. coli concentrations below 2.4×104 CFU/ml, electrode conductance decreased after AC voltage was applied. This is because the concentration of amplicons is not amplified efficiently when the bacterial concentration is below 2.4×104 CFU/ml, as shown in figure. 4.2. Therefore, there would not be enough DNA to attached onto the microbeads for alter the microbeads DEP from negative to positive. Hence, the microbeads would be repelled from the high electric field region under n-DEP force, as shown in figure 4.3 (a), and result in the decrease of electrode conductance.
The relationship between bacterial concentration and the initial rate of increase in microelectrode conductance (tangent slope of conductance increase at time t=0) in figure.
4.4., is depicted in figure. 4.5. For bacteria at concentrations above 2.4×104 CFU/ml, the rate of conductance increase grew in a semi-logarithmic way with bacterial concentration.
This because the concentration of amplicons, which would be attached to the microbeads, increased in a semi-logarithmic way with bacterial concentration, when the bacterial
concentration was above 2.4×104 CFU/ml. Therefore, this electrical detection method has potential for quantitative detection of bacterial at concentrations above 2.4×104 CFU/ml.
For bacteria at concentrations below 2.4×104 CFU/ml, the rate of conductance increase was nearly zero, this is mainly due to the un-efficiently amplification of DNA extracted from bacteria as shown in figure 4.2. Therefore, it can be considered that the sensitivity of this method is strongly depended on the sensitivity of PCR. The sensitivity of PCR varies for different targets and has been widely studied. The factor affects the sensitivity of PCR can be mainly considered to be the selected primer and the conditions of the reaction. For instance, comparing to the primer designed for the 16S rRNA, which is used in this study, the primer designed for fgbE is much more sensitive. The primer used in PCR that targeting the fgbE gene for E. coli can result in the detection of E. coli below 2.4×104 CFU/ml. Therefore, it is possible to increase the sensitivity of this method by select the primer depend on the purpose. Furthermore, the PCR amplification is mainly related to the bacterial number exist in the solution used for DNA extraction and PCR. Therefore, the sensitivity can also be easily improved by applying pre-concentration of bacteria, such as immunomagnetic separation or centrifugation90.
4.3.4 Selective detection of E. coli from E. coli and yeast mixture
Figure 4.6. shows the gel electrophoresis results of the amplification from extracted DNA, which are from mixtures of yeast solution and serially diluted E. coli. The gel electrophoresis results showed that the concentration of amplicons increased with the E.
coli concentration. Furthermore, DNA extracted from yeast was not amplified by PCR with primer pA and pH.
Figure 4.7 shows DEPIM results obtained with microbeads labeled by amplicons of mixtures from yeast and serially diluted E. coli. For solutions containing only yeast (concentration of 6.5×107 CFU/ml), the electrode conductance decreased after AC voltage was applied. When the mixture contained E. coli at concentrations above 105 CFU/ml, the conductance between the electrodes increased rapidly after the AC voltage was applied.
The selectivity of our method is mainly based on the PCR. For the detection of E.
coli, the primers pA and pH were used in this research, which are not target the DNA of yeast. As shown in figure 4.5, it is confirmed that the DNA extracted from yeast is not
amplified. Therefore, for yeast solution, there will be no amplicons to label on the microbeads to alter the DEP from negative to positive and result in the increase of the microelectrode conductance. As a result, the conductance of the microelectrode decreases because the microbeads were repelled from the electrode gap for yeast solution due to the n-DEP force as shown in figure 4.7.
The relationship between E. coli and yeast mixture and the rate of conductance increase at time t=0 obtained from figure 4.7 is depicted in figure 4.8. As shown in figure 4.8, the rate of the conductance increase grew in a semi-logarithmic way when the concentration of mixed E. coli was above 105 CFU/ml. Therefore, this method has potential for quantitative detection of target bacterial at concentration above 105 CFU/ml, when there is other bacterial exist. The selectivity can be achieved by using specific primers designed for selected bacterial. For instance, the selective detection of E. coli O157:H7 and O55:H7 can be achieved by apply the primers design for the eaeA gene50.
The traditional methods used for bacterial detection is based on culturing, which is far more time consuming than PCR based methods. For instance, Arthur et al compared the detection time of E. coli in ground beef for culture based methods and PCR based methods94. It showed that the PCR based methods (7.5-12 h) were at least 9 hours shorter than the culture based methods (21-48 h). One major component in the detection time of PCR based methods is the confirmation of the amplified DNA. The gel electrophoresis, which takes 1 to 2 hours, is generally used for the confirmation of the amplified DNA.
Our novel DNA detection method based on p-DEP of microbeads can reduce the detection time of amplified DNA to 15 min. Furthermore, it is much easier to operate, since it only need to mix the amplicons with the microbeads and used for electric detection. Therefore, this method can lead to a more rapid and simple bacterial detection.
Furthermore, since this method uses magnetic microbeads, it has the potential for automatic detection in the future.
It should be pointed out that the amplified DNA is attached to the microbeads through biotin–streptavidin interaction. Because the biotin–streptavidin interaction can be reversibly broken in water at elevated temperatures95. Therefore, by collecting the DNA labeled microbeads after the detection, the labeled DNA can be used for further application, such as DNA sequencing for identification of bacterial96.