In this study, we developed a new detection strategy for Bacillus cereus (BC) and Vibrio parahaemolyticus (VP) based on the colorimetric detection of smartphones and bacterial binding proteins. This assay enabled the detection of BC and VP at low concentrations without pre-enrichment. Bacterial levels could be determined by the naked eye or quantified by smartphone image analysis, meeting the criteria for point-of-care testing (POCT). Our key experimental findings were as follows:
1. Construction of plasmids pET28a-His-autolysinCBD and pET28a-His-MS18
2. Expression and purification of autolysinCBD and MS18
3. Detection system optimization
4. Colorimetric detection with bacteria-binding proteins
5. Mobile app development
We designed plasmids encoding the BC autolysin cell wall binding domain (CBD) and VP phage tail fiber protein (MS18), i.e., pET28a-His-autolysinCBD and pET28a-His-MS18. To construct these plasmids, we first amplified the autolysinCBD and MS18 fragments from the synthetic plasmids and obtained the pET28a vector by enzymatic digestion reaction (Figure 1.1 A-B). Then, we homologous recombined the target fragment (autolysinCBD or MS18) with the corresponding pET28a vector. The constructed plasmids were transformed into E. coli and plated on LB agar containing kanamycin. The plates were incubated at 37°C overnight (Figure 1.1 C-D).
Figure 1.1 Construction of plasmids pET28a-His-MS18 & pET28a-His-autolysinCBD.
(A) Double enzyme digestion result of pET28a.
(B) Amplification result of MS18 (495 bp) and autolysinCBD (402 bp).
(C-D) Transformation results of recombinant plasmids.
We selected the transformants grown on solid plates for colony PCR, and the results showed that both pET28a-His-autolysinCBD and pET28a-His-MS18 showed the correct bands, which initially indicated that our constructs were successful (Figure 1.2).
Figure 1.2 Colony PCR results of transformants.
To further confirm that the plasmid was constructed successfully, we sent the transformants to the company (Aznta) for sequencing. As shown in Figure 1.3, we did obtain the correct recombinant plasmid.
Figure 1.3 Sequencing results of transformants.
2. Expression and purification of the target protein
Two plasmids were transformed into E. coli BL21(DE3) for protein production. Cultures were grown to OD600 0.6-0.8 and induced with IPTG for 24 h. Cells were lysed by sonication and His-tagged proteins were purified by Ni-affinity chromatography. SDS-PAGE analysis demonstrated successful expression and purification of autolysinCBD (15.3 KDa) and MS18 (17.84 KDa) in Figure 2.
Figure 2 SDS-PAGE result of autolysinCBD and MS18.
3. Detection system optimization
To improve the detection and to obtain optimal reaction conditions, we explored the two most important experimental conditions in the autolysinCBD and MS18 detection system, i.e., the catalytic reaction time and the TMB substrate concentration. We first diluted the bacterial suspension to 106 CFU/mL and used different concentrations of TMB 0.01-0.05% (w/v) as the colorimetric substrate. The concentration of bacteria was determined at different times (0.5-2 h).
The results showed that for MS18, the optimal assay conditions were the optimal conditions for the 0.04% TMB substrate concentration for 30 min; for autolysinCBD, the optimal assay conditions were at 0.01% TMB for 1 h (Figure 3).
Figure 3 Optimal reaction conditions for detection systems.
4. Colorimetric detection with bacteria-binding proteins
For testing the ability of the system to detect Bacillus cereus (BC) or Vibrio parahaemolyticus (VP). We diluted overnight bacterial cultures of BC or VP to 102-107 CFU/mL using PBS, and then 1 mL of each concentration was taken for the assay. The two bacterial solutions were reacted under previously determined optimal conditions (Figure 3).
We performed curve fitting of the detection results, and the experimental results are shown in Figure 4. For BC bacteria, the fitted curve of the detection was y = 0.1922x - 0.1994 (R² = 0.9423), and for VP bacteria, the fitted curve of the detection was y = 0. 1797x - 0.1067 (R² = 0.9775). The detection values of the two bacteria showed a favorable linear relationship with the concentration of the bacteria. However, it was also seen that when the concentration of the bacterium reached 107 CFU/mL, the detection value decreased instead, which suggested that the optimal detection interval of the bacteria concentration shall be between 102-106 CFU/mL when using this detection system.
Figure 4 Testing results of different concentrations of bacteria.
5. Mobile App Development
In order to facilitate our bacteria detection method, we also worked on developing a mobile app in which people can use their phone camera to scan the color of the test sample to obtain the value of the bacteria concentration directly. The app installation package (not fully finished) is available here for showcase only.
Figure 5 Screenshots of the mobile app from left to right: Homepage; Select Bacteria Type; Test Scanning Results (including RGB values and the detected bacteria concentration)
In conclusion, we have successfully developed and optimized a novel smartphone-based detection strategy for the detection of major foodborne pathogens BC and VP.
Currently, we have produced a prototype of the mobile app with the curve of bacteria concentration and OD450 only, and in the future, we will further investigate the limit of the detection the quantity relationship between RGB values and the bacteria concentration of the test sample by obtaining more experimental data to upgrade our model so as to develop the mobile app, besides evaluating the detection sensitivity for refinement.