Overview
Our project is to develop a portable rapid device for simultaneously detecting four common
strains of
food-borne bacterial pathogens in food samples.
It is more sensitive and rapid than current professionalized approaches for detecting bacteria,
requiring no laboratory equipment and special expertise.
The target pathogens of our device involve pathogenic E. coli (EC), V. parahaemolyticus (VP), S.
aureus (SA), and S. enteritidis (SE). The device uses four
corresponding specific receptor binding proteins (RBPs) attached to nanomagnetic beads (MB) to
specifically isolate the targetpathogens from food samples by magnetic force and utilizes a
paper-based sensor to quickly, cheaply and simultaneously identify these bacteria.
The four RBPs are tail fiber protein (TFP), gp15, cell binding domain (CBD) and tail spike
protein
(TSP) which can specifically bind to the four bacteria, respectively. As the project is designed
for
operating in crude and simple conditions short of professional personnel,
the feedback of detection of our project should be visualized and investigated easily. Thus, we
prepared a reaction system with the optimal reaction concentrations
of a lysing agent and four corresponding chromogenic substrates--chlorophenol
red-β-D-galactopyranoside (CPRG), X-β-glu, 4-nitrophenyl β-D-glucopyanoside (pNPG), and magenta
caprylate (MC)--on a paper-based carrier.
Aspects to be proved
As for whether this design can accomplish the expected effect, it is required to be proved in two
aspects: the effect of RBP-MBs on isolating target bacteria from samples and the efficiency of
chromogenic substrates for recognizing target bacteria strains.
(1)For the first aspect, we divided samples of each bacteria strain into two groups: Test group
was
processed by the four RBP-MBs for bacteria isolation, and control group was used for comparison.
The
separation rate of target bacteria by RBP-MBs was reckoned.
(2)To prove the second aspect, we used two phases in the laboratory. In phase one, we tested the
specificity of each chromogenic substrate for the corresponding bacterium. In phase two, we
analyzed
the visual effect of these chromogenic substrates for different
concentrations of bacterial solutions and investigated the lowest concentration limits of our
device
by modelling the color’s changes of each substrate mathematically.
Laboratory experiments
For laboratory work, we used bacteria of VP, SA, SE and EC to test the performance of our
product.
For safety concerns, we chose K12 MG1655 as our test EC strain. We succeeded to ascertain that
our
RBP-MBs are able to separate their corresponding target bacteria from solution.
We also confirmed that the chromogenic substrates in our paper-based sensors could give specific
visual signals for target bacteria and were concentration dependent. The detailed experiment
results
are as follows.
Experiment 1
3.1.1 Materials
The bacteria samples and their PCR products were all stored in the Biology Engineering College
of
South China University of Technology. Nanomagnetic beads (0.2µm-diameter carboxyl magnetic
beads)
were products of Sangon Biotech (China). Receptor binding proteins (RBPs) were conjugated
with nanomagnetic beads which are activated by EDC and Sulfo-NHS via stable amide bond and
formed
complexes named RBP-MBs. RBP-MBs can be separated from the solution by magnetic separation
device,
and used for downstream purification and separation tests.
3.1.2 Sample preparation
Bacteria solutions of SA (1×109 CFU/mL), SE (1×109 CFU/mL), EC (1×109 CFU/mL) and VP (1×108
CFU/mL)
were prepared.
The following samples were prepared.
(1)(Control group 1) SA, SE, EC and VP solutions were diluted to 1×103 CFU/mL by adding PBS
buffer
(total volume:1000μL) respectively and were labeled as Control group SA, Control group SE,
Control
group EC and Control group VP.
(2)(Control group 2) The four bacteria solutions and LB broth were added in one sample tube
labeled
Control group MIX, and all bacteria strains were diluted to 1×105 CFU/mL (total volume:1000μL).
(3)(Test Group 1) SA, SE, EC and VP solutions were diluted to 1×105 CFU/mL by adding LB broth
(total
volume:1000μL) respectively and were labeled as Test group SA, Test group SE, Test group EC and
Test
group VP.
(4)(Test Group 2) The four bacteria solutions and LB broth were added in one sample tube labeled
Test group MIX, and all bacteria strains were diluted to 1×105 CFU/mL (total volume:1000μL).
3.1.3 Experiments
For each RBP-MB, control and test groups of the related bacteria strain and strain mix were used.
For example, for MBs-CDB to separate SA, Control sample SA, Test sample SA, Control sample Mix
and Test sample Mix were used. The following experiments were conducted.
(1)(Control group) Samples were centrifuged at 1000 rpm for a minute, and the supernatant was
discarded. Bacteria were resuspended with 990μL PBS, and smeared on the TSA solid medium, and
culture overnight at 4 ℃.
(2)(Test Group) Solid RBP-MBs and 5 mL coupling buffer were added to each sample EP tube and
reacted at 37℃ for 30min. The tube was placed in the rotary instrument for 90min, and placed in
a
magnetic frame for 60s. The liquid in the tube was discarded.
(3)(Test Group) 5mL of sealing solution was added and mixed. The tube was placed in the rotary
instrument for 30min and then placed on the magnetic frame for 60s. Then, the liquid transfer
gun was used to remove the upper clear liquid.
(4)(Test Group) 3mL sealing liquid was added into the tube and mixed well. The tube was fixed on
the magnetic frame for 60s, and the upper clear liquid was discarded.
(5)(Test Group) 3mL preservation solution was added into the tube and mixed well. The tube was
fixed on the magnetic frame for 60s, and the upper clear solution was discarded.
(6)(Test Group) Step (5) was repeated twice.
(7)(Test Group) 1mL preservation solution was added into the tube, and mixed well. This tube
containing the separated bacteria solution was stored in 4℃ refrigerator.
(8)(Test Group) The separated bacterial solution was smeared onto the TSA solid medium at 4℃
overnight.
(9)The number of colonies of each bacterial strain was compared between the test group and its
corresponding control group after cultivation overnight. Separation rate was equal to the ratio
between the number of colonies in the test group and that in the control group.
3.1.3Results
The separation rates of the samples showed that MBs-TFP and MBs-gp15 had high efficiencies for
isolating target bacteria EC and VP, respectively (Table 1). MBs-CBD was not ideal but met the
basic
demand for separating SA. MBs-TSP was able to separate SE,
although the separation rate was low.
Table 1:Separation rates (capture efficiency) of RBP-MBs for
target bacteria
| Protein |
MBs-TFP |
MBs-gp15 |
| Strains
| EC |
Mix(SA,SE,VP,EC) |
VP |
(SA,SE,VP,EC) |
| Input |
2.30×10⁵ |
10⁵ |
1.60×10⁵ |
1.60×10⁵ |
| Capture |
2.20×10⁵ |
2.04×10⁵ |
1.56×10⁵ |
1.55×10⁵ |
| Capture efficiency |
95.65% |
88.70% |
97.50% |
96.88% |
| RSD |
9.45% |
8.45% |
6.86% |
8.53% |
| Protein |
MBs-TFP |
MBs-gp15 |
| Strains
| EC |
Mix(SA,SE,VP,EC) |
VP |
(SA,SE,VP,EC) |
| Input |
0.85×10⁵ |
2.97×10⁵ |
2.97×10⁵ |
2.97×10⁵ |
| Capture |
0.52×10⁵ |
0.50×10⁵ |
0.33×10⁵ |
0.30×10⁵ |
| Capture efficiency |
61.17% |
58.82% |
11.11% |
10.10% |
| RSD |
5.67% |
8.43% |
7.65% |
9.41% |
Experiment2
3.2.1 Materials
The bacteria samples and their PCR products were all stored in the Biology Engineering College
of
SCUT. Chromogenic substrates were all
from the storage of Biology Engineering College of South China Univeristy of Technology. The
solvent
of bacteria solutions was PBS buffer.
3.2.2 Sample preparation
Bacteria solutions of SA (9.5μl 1×10⁸CFU/mL), SE (9.5μl 1×10⁸CFU/mL), EC (9.5μl 1×10⁸CFU/mL) and
VP
(9.5μl 1×10⁸CFU/mL) were prepared.
Bacteria solution of each strain was divided into four groups to test the specificity of the
four
chromogenic substrates, respectively (Table 2).
Table 2:Theoretical color changes of each group
| Group |
substrate |
Original color |
Target Bacteria |
Color after reaction |
| Group 1 |
CPRG |
Yellow |
EC |
Purple |
| Group 2 |
pNPG |
Colorless |
SA |
Yellow |
| Group 3 |
X-β-glu |
Colorless |
VP |
Blue |
| Group 4 |
magenta caprylate |
Colorless |
SE |
Purple |
For Group 1, Group 2 and Group 4 of each strain, 20μl bacteria solution, 0.2μl colistin and 0.5μl
chromogenic substrate were mixed in one well of a 96-well plate.
For the Group 3, 20μl bacteria solution, 0.2μl polymiyxin and 0.5μl chromogenic substrate were
mixed
in one well of a 96-well plate.
3.2.3Experiments
The mixtures were preserved at 37℃ for 2h, and then the color of the mixtures was checked.
3.2.4Result and revision
The original color of each substrate,
its target strain and the expected result of color change after
encountering the target strain are listed in Table 2.
Our experiments showed that all the bacteria samples reacted with the corresponding chromogenic
substrates according to our expectation (Figure1), and showed ideal color change.
CPRG, X-β-glu, and PNPG showed high specificity in distinguishing target bacteria from other
strains, but magenta caprylate showed low specificity(Figure1).
So, we further tested the specificity of the paper-based sensors in combination with RBP-MBs, as
would be used in our product. We used the same samples with concentration of 10⁷CFU/mL and same
preparation.
The result showed that all four sensors had high specificity for target bacteria, including the
magenta caprylate sensor.
Figure1 Experiment results of specificity of chromogenic substrates toward target bacteria
Figure 2 Experiment results of bacteria detection using RBP-MBs combined with paper-based
sensors
Experiment3
3.3.1Materials
The bacteria samples and their PCR products were stored in the Biology Engineering College of
South
China University of Technology. Paper-based sensors were prepared by mixing with 0.5μ of
chromogenic
substrates
with the concentration 10mg/mL and 0.2μL lysis in the wells of a 96-well plate. The plate was
put
into a 37℃ incubator to evaporate the water for 30min. Four paper-based sensors were prepared in
the plate: Sensor 1 with CPRG,
Sensor 2 with PNPG, Sensor 3 with X-β-glu, and Sensor 4 with magenta caprylate.
3.3.2Sample preparation
Bacteria solution samples with different concentrations were prepared (Table 3).
PBS buffer was used for dilution.
Table 3 Chromogenic substrates, bacteria and bacteria concentrations for each sensor
| Paper-based sensor |
Chromogenic substrate in the sensor |
Bacteria |
Concentration (CFU/mL) |
| Sensor 1 |
CPRG |
EC |
2.5×10⁴, 5×10⁴, 7.5×10⁴, 2.5×10⁵, 5×10⁵, 7.5×10⁵, 2.5×10⁶, 5×10⁶, 7.5×10⁶ |
| Sensor 2 |
PNPG |
SA |
10⁴, 10⁵, 10⁶, 10⁷, 10⁸ |
| Sensor 3 |
X-β-glu |
VP |
10⁴, 10⁵, 10⁶, 10⁷, 10⁸ |
| Sensor 4 |
magenta caprylate |
SE |
10³, 5×10³, 10⁴, 5×10⁴, 10⁵, 5×10⁵, 10⁶, 5×10⁶, 10⁷ |
3.3.3Experiments
(1)Paper-based sensors were prepared in a 96-well plate as described in “3.3.1 Material”.
Sensors with the same chromogenic substrate were arranged in the wells of the same row of the plate.
(2)Bacteria samples 20.7μL of each strain were added into the wells containing the corresponding
sensor as shown in Table 3. And the 96-well plate was incubated at 37℃ for 2h.
(3)The color change of the sensors was observed by naked eye. Images were taken by a screen
capture App, Snipaste, from Windows system or a Wechat screen capture tool and
analyzed using mathematic method and model for the RGB (red, green, blue) color values.
3.3.4 Results and revision
Our experiments showed that the degree of color change of the sensors for each strain was all
positively related to the concentrations of bacterial solution. By naked eye, the color change
of Sensor 1was apparent at all concentrations.
The color change of Sensor 3 began to be distinguishable at the concentration 10⁶ CFU/mL.
The color change of Sensor 4 began to be distinguishable at the concentration 10⁷ CFU/mL.
By analyzing the RGB color values of the screen capture images of the sensors, we found that all
four sensors showed a strong correlation between the blue value and the bacterial concentration.
We drew the graph of Blue 1 value against log10(concentration of bacteria).
Using the blue value, the lower limit in the detectable concentration for all bacteria
could reach around 10⁶ CFU/mL except for EC whose color changes were quite apparent
from the concentration of 7.5×10⁵ CFU/mL. Results are shown in Figure 1 to 4.
Figure 2 Results of Sensor 2 for VP
Figure 3 Results of Sensor 3 for SA
Figure 4 Results of Sensor 4 for SE
Conclusion
According to the results of laboratory work, our product can isolate efficiently
of target bacteria
from food samples and show consistent and sensitive visual signals for target bacteria with
different concentrations.
In general, this technology has been proved technically accessible.
