Electrochemical Detection of Escherichia coli from Aqueous Samples Using Engineered Phages

Article abstract of DOI:10.1021/acs.analchem.6b03752

In this study, an enzyme-based electrochemical method was developed for the detection of Escherichia coli (E. coli) using the T7 bacteriophages engineered with lacZ operon encoding for beta-galactosidase (β-gal). The T7_lacZ phages can infect E. coli, and have the ability to trigger the overexpression of β-gal during the infection of E. coli. The use of the engineered phages resulted in a more sensitive detection of E. coli by (1) overexpression of β-gal in E. coli during the specific infection and (2) release of the endogenous intracellular β-gal from E. coli following infection. The endogenous and phage-induced β-gal was detected using the electrochemical method with 4-aminophenyl-β-galactopyranoside (PAPG) as a substrate. The β-gal catalyzed PAPG to an electroactive species p-aminophenol (PAP) which could be monitored on an electrode. The electrochemical signal was proportional to the concentration of E. coli in the original sample. We demonstrated the application of our strategy in aqueous samples (drinking water, apple juice, and skim milk). Using this method, we were able to detect E. coli at the concentration of approximately 10⁵ CFU/mL in these aqueous samples in 3 h and 10² CFU/mL after 7 h. This strategy has the potential to be extended to detect different bacteria using specific bacteriophages engineered with gene encoding for appropriate enzymes.

Full text of DOI:10.1021/acs.analchem.6b03752

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Article
Electrochemical detection of Escherichia coli
from aqueous samples using engineered phages
Danhui Wang, Juhong Chen, and Sam R. Nugen
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03752 • Publication Date (Web): 11 Jan 2017
Downloaded from http://pubs.acs.org on January 11, 2017
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Analytical Chemistry
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Electrochemical detection of Escherichia coli from aqueous samples
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using engineered phages
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†,‡
†,‡
,†,‡
Danhui Wang, Juhong Chen, Sam R. Nugen*
†
Department of Food Science, University of Massachusetts, Amherst, MA 01003, United States
Department of Food Science, Cornell University, Ithaca, NY 14853, United States
‡
ABSTRACT: In this study, an enzymeꢀbased electrochemical method was developed for the detection of Escherichia coli (E. coli)
using the T7 bacteriophages engineered with lacZ operon encoding for betaꢀgalactosidase (βꢀgal). The T7lacZ phages can infect E.
coli, and have the ability to trigger the overexpression of βꢀgal during the infection of E. coli. The use of the engineered phages
resulted in a more sensitive detection of E. coli by: (1) overexpression of βꢀgal in E. coli during the specific infection; (2) release of
the endogenous intracellular βꢀgal from E. coli following infection. The endogenous and phageꢀinduced βꢀgal was detected using
the electrochemical method with 4ꢀaminophenylꢀβꢀgalactopyranoside (PAPG) as a substrate. The βꢀgal catalyzed PAPG to an elecꢀ
troactive species pꢀaminophenol (PAP) which could be monitored on an electrode. The electrochemical signal was proportional to
the concentration of E. coli in the original sample. We demonstrated the application of our strategy in aqueous samples (drinking
5
water, apple juice, and skim milk). Using this method, we were able to detect E. coli at the concentration of approximately 10
CFU/mL in these aqueous samples in 3 hours, and 10 CFU/mL after 7 hours. This strategy has the potential to be extended to deꢀ
tect different bacteria using specific bacteriophages engineered with gene encoding for appropriate enzymes.
2
Food safety remains an important issue for public health
protection. Foodborne illnesses resulting from the bacterial
pathogens pose an increasing threat to the human health
been widely used as a reporter enzyme to determine the conꢀ
centration of E. coli in food and aqueous samples. There are
many colorimetric, electrochemical, and fluorescent substrates
that can be catalyzed by βꢀgal which allows for various methꢀ
1
worldwide. The ability to more rapidly and sensitively detect
11,16,17
bacteria in food and water samples is critical to ensure the
food safety and minimize the risk of human exposure to potenꢀ
ods to measure the activity of this enzyme.
Here, an electrochemical method was used to specifically
measure the activity of βꢀgal in order to estimate the E. coli
contamination. Electrochemical methods measure the free
electron generation of an electroactive species when it is oxiꢀ
dized and reduced on the electrode. The electroactive comꢀ
pound is usually produced via the catalysis from an enzyme.
Electrochemical methods offer a promising alternative to conꢀ
ventional analytical methods, providing a convenient detection
by obtaining instantly quantitative signals with minimal
equipment required. Moreover, electrochemical detection has
some advantages over the other rapid methods, for example it
is not affected by the turbidity of sample solution which can
be problematic in colorimetric assays for the sample such as
2,3
tial hazzards. Escherichia. coli (E. coli) has been among the
most studied bacteria, and significant research efforts have
been placed on improving the ability to detect these bacteria in
food and water supplies. The United States Food and Drug
Administration (FDA) has suggested to use coliforms or “geꢀ
neric” E. coli as indicators for the bacterial contamination
4,5
level in fresh produce. Additionally, the Environmental Proꢀ
tection Agency (EPA) identified E. coli as an indicator of the
6
fecal contamination. The most commonly used methods to
determine the presence of E. coli involve culturing and plate
counting using selective medium. Although these methods are
accurate and reliable, they require long incubation periods
3,7
18ꢀ20
which are timeꢀconsuming and laborious. Nucleicꢀbased
method such as PCR is an alternative for the rapid identificaꢀ
tion and quantification of bacteria. Unfortunately, it requires
significant equipment and sample preparation, and also cannot
juice or milk.
The detection of these samples could benefit
from the development of an electrochemical method. The lowꢀ
cost nature of electrochemical detection can best be exempliꢀ
fied by the common blood glucose meter which is available
for under $10.
8
,9
distinguish viable and nonviable cells. Therefore, significant
efforts are continuously being invested in the development of
new strategies to improve the detection of bacteria with better
sensitivity, specificity, and less time. One alternative used for
rapid and sensitive bacterial detection is based on the measꢀ
urement of the activity of a specific enzyme which reflects the
However, the concentration of endogenous enzyme βꢀgal in
E. coli may not be at high enough for a rapid detection methꢀ
11,17
od.
In order to solve this problem, engineered phages were
used in this strategy to overexpress βꢀgal in order to achieve a
more sensitive detection. Bacteriophages (phages) are viruses
which specifically recognize, attach to, and infect target bacteꢀ
Phages replicate using the molecular machinery of the
host bacteria. At the final stages of the infection, lytic phages
can induce the disruption of the host cells, releasing of the
10ꢀ12
contamination of the target bacteria.
21,22
In E. coli, the activity of intracellular enzyme βꢀ
ria.
galactosidase (βꢀgal) is able to reflect the concentration of
13ꢀ15
indicator strain E. coli.
Betaꢀgal is a wellꢀknown bacteriaꢀ
enclosed enzyme of E. coli, encoded by lacZ operon. It has
1
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intracellular components and replicated cells to the surroundꢀ ing environment. Genetic engineering techniques allow
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Figure 1. The scheme representation of electrochemical detection of E. coli using engineered phage. (a) The designed construct of
genome of T7lacZ phage. (b) Specific capture and infection of E. coli by T7lacZ phage resulted in the release and overexpression of
enzyme βꢀgal. PAPG was catalyzed by βꢀgal into an electroactive species PAP that can be quantified by electrochemical device.
the insertion of genes encoding for marker enzyme such as
alkaline phosphatase (ALP), green fluorescent protein (GFP)
and luciferase (luc) into phage genome, and these enzymes
can be expressed in the host cells during the phage life cycle
lowꢀcost and sensitive quantification of E. coli with minimal
equipment.
EXPERIMENTAL SESSION
2
4ꢀ26
and measured by various methods.
This expression is unꢀ
Chemical, materials and instrument. 4ꢀaminophenylꢀβꢀ
galactopyranoside (PAPG) was purchased from Sigmaꢀ
Aldrich (St. Loius, MO). All other analytical grade chemicals
were purchased from Fisher Scientific (Fair Lawn, NJ). The
MilliꢀQ water with 18 MΩ/cm resistivity (EMD Millipore,
Billerica, MA) was used for the preparation of all solution.
der a stronger promoter resulting in increased transcription.
Therefore, in addition to intracellular βꢀgal produced by E.
coli itself, the bacteriophages engineered with lacZ operon
encoding for βꢀgal are able to induce the expression of this
enzyme in E. coli during the infection, resulting the high proꢀ
duction of βꢀgal which can facilitate to achieve a more sensiꢀ
tive detection. Because phages can only replicate in a live
bacterial cell, phageꢀbased detection is able to distinguish viaꢀ
The Potentiostat/Galvanostat used in the electrochemical
detection was from PalmSens (Utrecht, Netherlands). The
Dropꢀcell connector which connected the potentiostat and
electrode and thinꢀfilm single platinum electrodes were obꢀ
tained from MicruX Technologies (Austurias, Spain). Platiꢀ
num is a commonly used standard material for the electrode in
the electrochemical detection and has been widely used in
many studies. It has high corrosion resistance and high conꢀ
2
7,28
ble and nonꢀviable host cells.
This reduces the possibility
of falseꢀpositive results caused by nonꢀviable bacteria which
may have already succumb to mitigation steps such as antibiꢀ
otics, chlorine, cleaners, or other antimicrobials.
In this study, we proposed a new strategy to rapidly and
specifically detect E. coli in aqueous samples based on elecꢀ
trochemical quantification of βꢀgal overexpressed by engiꢀ
neered phages. This is an attempt to make a phageꢀbased deꢀ
tection assay field deployable. The lacZ operon encoding for
βꢀgal was inserted into the genome of T7 phages to form engiꢀ
neered bacteriophages. The engineered phages were first used
to specifically infect the target E. coli. Following the infection
cycle, βꢀgal, both endogenous and phageꢀenabled, was reꢀ
leased into the sample solution. Then the enzyme βꢀgal hydroꢀ
lyzed the electrochemical substrate to generate an electroacꢀ
tive product which was quantified by the subsequent electroꢀ
chemical detection (Fig. 1). The use of engineered T7 phages
enables this strategy detect live E. coli cells and overexpress
reporter enzyme into solution without the need of adding lysoꢀ
zyme to lyse bacteria cells. Additionally, the engineered phagꢀ
es coupled with an electrochemical method offers a simple,
29
ductivity.
Bacterial culture samples. E. coli BL21 (ATCC 25922)
was grown overnight (18ꢀ20 hours) in 50 mL of LuriaꢀBertani
broth (LB broth, 10 g tryptone, 5.0 g yeast extract, 10.0 g soꢀ
dium chloride in 1 L distilled water, pH 7.2) under 200 rpm at
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7℃. The overnight culture was then centrifuged at 7,000x g
for 2 minutes, washed two times with phosphate buffered saꢀ
line (PBS buffer, 137.0 mM NaCl, 2.7 mM KCl, 10.0 mM
Na HPO , 2.0 mM KH PO , pH 7.2) and resuspended in 1x
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PBS buffer. The concentration of E. coli BL21 were deterꢀ
mined by enumeration after plating on the LB agar plates and
incubation for overnight. And E. coli BL21 was serially dilutꢀ
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ed into the desired concentration (10 , 10 , 10 , 10 , 10 , and
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0 CFU/mL) in LB broth for the subsequent experiments.
Construction, propagation and purification of engi-
neered phage. The method to construct engineered phages
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Analytical Chemistry
for overexpression of βꢀgal in E. coli was based on the strateꢀ
gy previously described for T7ALP phage and T7TEV phages.
0.05 V; t pulse: 0.05 s; Scan rate: 0.05 V/s. The electroactive
compound PAP was produced by catalyze of βꢀgal from the
electroꢀinactive substrate PAPG. DPV allowed a relatively
short analysis of the sample solution. The current was measꢀ
ured prior to and after the applied potential and the change in
30,31
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Briefly, the lacZ construct was synthesized in a pUC57 plasꢀ
mid containing lacZ operon from GenScript (Piscataway, NJ).
The lacZ construct was then amplified using a Phusion PCR
kit (Ipswich, MA). Next, all PCR products were purified and
digested by PCR Purification Kit (Qiagen, Valencia, CA) and
EcoRI and HindIII, respectively. After T4 DNA ligase
32,33
current was plotted against the applied potential.
The reꢀ
sponse was determined to be the peak current of the DPV
curve which was proportional to the concentration of PAP
generated on the electrode. Therefore, the peak current was
recorded as the signal of the electrochemical detection.
(Promega, Madison, WI) facilitated the insertion of the conꢀ
struct into the T7Select415 (EMD Millipore, Billerica, MA)
genome vector arms, T7lacZ phage was formed by packing the
construct using the T7Select packing kit (Fig. 1(a)). In order to
prove the effectiveness of the T7lacZ phages on the overexpresꢀ
sion of βꢀgal, a control DNA which contains Sꢀtag was also
packaged using T7Select kit to create T7control. After T7lacZ and
T7control were propagated and plated, the individual colonies
were confirmed with the appropriate size insert by the
T7Select up and down primers using Phusion PCR kit.
Detection in the real aqueous samples. Drinking water
was obtained from the drinking water fountain at University of
Massachusetts, Amherst. Apple juice and skim milk were purꢀ
chased in a local supermarket. The safety of drinking water is
a critical health issue, and is especially a problem for the peoꢀ
ple in developing countries. In many areas drinking water is
obtained from private sources which can become contaminatꢀ
ed with bacteria. Many of these areas also suffer from a lack
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The engineered phages, T7lacZ and T7control, were amplified
and purified prior to the use in the detection assay. An aliquot
of 100 ꢀL E. coli BL21 overnight culture was inoculated into
a 150 mL of sterile flask with 35 mL LB broth and then incuꢀ
bated at 37℃ with 200 rpm until the OD600 of the culture
reached 0.6. Then engineered phage stock (15 µL) was inocuꢀ
lated in the culture and incubated for 2 hours with the same
conditions to allow infection and replication. The phage lysate
was then centrifuged at 8,000x g for 10 min, followed by filꢀ
tration of the supernatant through a 0.22 ꢀm sterile filter
of water safety monitoring. Foods such as juice and raw milk
are often associated with the foodborne outbreaks. Apple juice
and milk which are not compatible with colorimetric detection
methods can be measured by electrochemical methods. The
pH of drinking water is 7.01, the pH of skim milk is 6.70 and
the pH of apple juice is 3.61. The apple juice was adjusted to
approximately pH 7.2 prior to the detection in order to mainꢀ
tain an optimal condition for phage infection and enzymatic
reaction. E. coli BL21 was inoculated in the drinking water,
apple juice, and skim milk, respectively, to final concentration
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(Corning Life Science, Corning, NY). The lysates were then
of 10 , 10 , 10 , 10 , 10 , and 10 CFU/mL. Next, a 200 ꢀL
aliquot of five times concentrated LB broth supplemented with
PAPG with final concentration of 15 mM and T7lacZ phages
ultracentrifuged at 35,000x g for 2 hours. The phage pellets
were finally resuspended in 4 mL of 1x PBS buffer at 4 ℃.
The phage titer (PFU/mL) was determined by plaque assay
using a double agar overlay. Briefly, the phages (100 ꢀL) were
added into the melt top LB agar (3ꢀ4 mL) containing the overꢀ
night E. coli culture (200 ꢀL) and then placed on solid LB
agar, followed by the counting of plaques after an incubation
of 3ꢀ4 hours at 37℃.
4
with final concentration of 10 PFU/mL was added into 1mL
of each sample (drinking water, apple juice, and skim milk) in
a 2 mL microcentrifuge tube (Eppendorf, Hauppauge, NY,
USA). An aliquot of samples without E. coli were used as a
negative control. The microcentrifuge tubes were incubated
under 150 rpm agitation for 3 hours and 7 hours at 37℃, reꢀ
spectively. Then the samples were analyzed using the electroꢀ
chemical assay following the steps described in the previous
section.
Electrochemical detection of E. coli using engineered
2
phage. Aliquots (1 mL) of E. coli with concentration of 10 ,
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0 , 10 , 10 , 10 , and 10 CFU/mL in LB broth were infected
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Statistical analysis. SAS software was used to analyze daꢀ
ta with ANOVA (SAS Institute, Inc., Cary, NC). The mean
values among different groups were determined significantly
different (P<0.05) with Tukey’s test. The data presented repreꢀ
sent a mean of a minimum of three independent samples and
error bars represent the standard deviation of the replicates.
with 100 ꢀL of engineered phages (10 PFU/mL). The subꢀ
strate PAPG (100 ꢀL, 15 mM) was also added into the sample
prior to the incubation, allowing for the enzyme reaction at the
same time with phage infection. The simultaneous phage inꢀ
fection and enzymatic reaction shortened the assay time. Next,
the samples were incubated in 37℃ for various times (2, 3, 4,
5
, 6 and 7 hours) at 150 rpm to allow for the phage infection
RESULTS AND DISCUSSION
of E. coli and overexpression of βꢀgal. An aliquot of 1 mL LB
broth without E. coli was also incubated with substrate PAPG
and engineered phage under the same conditions above as a
negative control. All the samples after incubation were used
for the following electrochemical detection.
Optimization of condition for enzyme reaction. There
are various factors (e.g. temperature, pH, and concentration of
substrate) that may affect the phage infection and enzyme
reaction. It is therefore necessary to optimize the reaction conꢀ
dition in order to increase the sensitivity of our proposed apꢀ
proach. The effect of subtract (PAPG) concentration as well as
the effect of incubation temperature (37℃ and 23℃) were
investigated. The pH range of this method was determined by
activity of phages and βꢀgal. In general, phages are usually
stable at a large pH range (pH 4.0ꢀ10.0). Phages show the
The electrochemical detection system consisted of a Palmꢀ
sens Potentiostat/Galvanostat, Dropꢀcell connector and a Miꢀ
cruX thinꢀfilm single platinum. Following the incubation
steps, 20 ꢀL of sample solution was deposited on the platinum
electrode, covering all the three electrodes. So the surface area
of the working electrode for each electrochemical detection
was consistent. Differential pulse voltammetry (DPV), a
commonly used model to measure the electric signal, was used
to perform the electrochemical detection for all the measureꢀ
ments with the following conditions: Time for equilibration:
35,36
optimum for physical stability and infection at pH 6.0ꢀ8.0.
Betaꢀgal is a neutral enzyme which has the optimal activity at
pH 6.7 to 7.2 and is stable from pH 6.0 to 9.0. The product
PAP is also more stable at a neutral pH and it is oxidized and
37,38
reduced on the electrode at pH 7.5.
Therefore, this method
3
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s; Potential range: ꢀ0.1 V – 0.4 V; E step: 0.01 V; E pulse:
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can be applied in a relatively large pH range and is optimal at
neutral pH. The pH of the enzyme reaction was then selected
at pH 7.2. The concentration of phage used here was based on
parison of incubation temperature at room temperature (23℃)
and 37℃. Peak current obtained for detecting 10 CFU/mL of
E. coli at 23℃ (red bar) and 37℃ (blue bar), respectively after
3 or 5 hours of incubation.
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our previous studies on the phage amplification matrix.
There is a dynamic interaction between the bacteria growth
and phage replication. The total enzyme finally expressed are
related to the total number of cells infected and the initial
phage concentration. The more phages used for the infection
there are more gene encoding for βꢀgal, resulting in more proꢀ
duction of βꢀgal. On the other hand, high initial phage concenꢀ
trations cause the fast lysis of E. coli cells and the uninfected
cells do not have time for doubling to provide more cells
available for infection. Therefore, based on the previous reꢀ
Incubation temperatures of 37℃ and room temperature
(23℃) were also compared to investigate their effect on detecꢀ
tion limits. Although the optimal temperature for phage repliꢀ
cation and growth of E. coli is 37℃, room temperature is more
convenient for assays in lowꢀresource settings. Aliquots conꢀ
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of 15 mM PAPG were used to detect 10 CFU/mL E. coli after
3 hours or 5 hours of incubation at 37℃ and room temperature
(23℃), respectively. Fig. 2(b) shows the peak current recorded
after 3 hours and 5 hours of incubation in the presence of
PAPG and T7lacZ phage at either 37℃ or 23℃. It can be obꢀ
served that the signal obtained after incubation at 37℃ was
significantly higher than that at 23℃ for incubation time of
both 3 hours and 5 hours as expected. As incubation time inꢀ
creased from 3 hours to 5 hours, the signal of reaction at 23℃
did not show a significant increase while the peak current obꢀ
tained for reaction at 37℃ increased steadily. Therefore, in
order to obtain a high signal, it was necessary to incubate the
sample at 37℃.
4
search a concentration of 10 PFU/mL engineered phages were
used for this study based on the previous research.
Then the concentration of substrate PAPG was investigated
to determine the optimal enzymatic activity. An aliquot of 100
4
ꢀ
L engineered phage (10 PFU/mL) and 100 ꢀL PAPG with
varying concentration (0, 5, 10, 12, 15, 18, and 20 mM) in
PBS buffer were used for the phage infection and enzyme
6
reaction, respectively, to detect 10 CFU/mL of E. coli after
incubation for 4 hours at 37℃. The DPV peak current model
was used to compare the signal obtained for samples with
increasing concentration of PAPG. Fig. 2(a) shows that the
concentration of PAPG significantly affected the peak current
obtained from the DPV curve. The results demonstrate that the
peak current increased gradually with the increase of concenꢀ
tration of substrate PAPG and leveled off at approximately 15
mM, after which the peak current increased only slightly.
Therefore, 15 mM was used for the concentration of substrate
PAPG for the subsequent experiments in order to allow the
optimal conversion of PAPG to PAP.
Comparison of engineered phages (T7lacZ phages),
control phages (T7control phages) and no phage for E. coli
detection. One of the functions of engineered phages was to
release endogenous and phageꢀenabled βꢀgal into the sample
solution during the phage infection. T7 phage is a lytic phage
which can lyse the host cell at the end of the infection cycle.
This results in a release of the intracellular proteins such as βꢀ
gal. Additionally, the T7lacZ phages we used here were engiꢀ
neered with a lacZ operon for βꢀgal, so the enzyme βꢀgal
would be expressed during the phage infection. To prove the
effectiveness of engineered phages, we conducted an investiꢀ
gation to compare the signal obtained using the engineered
phages, control phages (no operon for βꢀgal), and without
phage. Aliquots containing 100 ꢀL of engineered phages, conꢀ
6
trol phages, respectively were used to infect 10 CFU/mL of E.
coli. The same concentration of E. coli was also incubated
with substrate PAPG in the absence of phage. Following the
incubation for 3, 4 or 5 hours at 37℃, an electrochemical deꢀ
tection was performed and the peak current from each DPV
curve was recorded as the signal.
6
Figure 3. The signal obtained for detection of 10 CFU/mL E.
coli without phage (pink bar), with control phages (red bar)
and with engineered phages (dark red bar), respectively, after
incubation of 3, 4 or 5 hours at 37℃.
Figure 2. Optimization of condition of enzyme reaction. (a)
Dependence of the current on varying concentration of subꢀ6
strate PAPG (0, 5, 10, 12, 15, 18, and 20 mM) detecting 10
CFU/mL E. coli after incubation of 4 hours at 37℃. (b) Comꢀ
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Analytical Chemistry
The results were shown in Fig. 3, demonstrating that with
and 100 ꢀL of 15 mM PAPG were used to detect the samples.
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the same incubation conditions, the signal obtained using enꢀ
gineered phages was significantly higher than that using conꢀ
trol phages or no phage. These data suggest that the concentraꢀ
tion of βꢀgal generated using T7lacZ phages was significantly
higher than the other two groups. The enzyme βꢀgal is an inꢀ
tracellular enzyme which can be produced by E. coli. The
results also demonstrated that when using engineered phages,
more βꢀgal was expressed in E. coli during the infection in
addition to the intracellular βꢀgal produced by E. coli, and
released into the sample solution, resulting in the signal ampliꢀ
fication. The T7control phage without lacZ gene did not faciliꢀ
tate the expression of βꢀgal but did lysed the E. coli cells alꢀ
lowing endogenous enzymes to react. In absence of phages,
the intracellular enzyme βꢀgal was not able to be released from
E. coli cells. The free βꢀgal in the sample solution, mostly
from the natural lysis of the bacterial cells and the diffusion of
The concentration of each bacteria strain inoculated into the
6
sample was approximately 10 CFU/mL. After incubation for
3 hours at 37℃, the signal was obtained from the peak current
of the DPV curve. The results shown in Fig. 4 demonstrated
that only samples containing E. coli resulted in a significant
signal. The electrochemical signal obtained for other bacteria
had no significant difference with the control (no bacteria).
The mixture of all the four strains gave the similar signal as
the sample inoculated with only E. coli. These results suggest
that engineered phages are specifically target to E. coli. Thereꢀ
fore, bacteriophages could be used both for their specificity as
well as their ability to express reporter enzymes during infecꢀ
tion.
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1,40,41
PAPG across the cell membrane was limited.
The negaꢀ
tive control sample consisting of E. coli (no phage) also reꢀ
sulted in a high signal after 5 hours. This is most likely due to
E. coli cells which were able to produce higher concentrations
of βꢀgal with time increased and raised the enzyme to a high
concentration during the incubation. Conversely, the E. coli
infected with the control phage were lysed and could not proꢀ
duce additional enzyme. Overall, these results demonstrated
that the engineered phage was more effective for obtaining a
higher signal in order to achieve a lower limit of detection
.
6
Figure 4. The signal obtained for detecting 10 CFU/mL E.
coli, S. enterica, S. aureus, P. aeruginosa and a mixture of all
the strains, respectively, after incubation of 3 hours at 37℃. A
sample without any bacteria was used as the negative control.
Figure 5. Results of electrochemical detection of E. coli. (a)
Differential pulse voltammetry (DPV) curve for increasing
concentration of E. coli after 5 hours of incubation at 37℃.
The inset is the amplification of the plot of control and low
Specificity of engineered phages. Another use of engiꢀ
neered phages in this strategy was to specifically target E. coli
using the natural specificity of phages towards its host. A bacꢀ
teriophage is known to specifically infect a subset of bacteria
strains. The specificity of phages is based on the nature and
structure of receptors on the surface of bacterial cells. The
receptors which are recognized by the tail fibers of bacterioꢀ
phages are dependent on the different taxonomic groups. T7
phages attach the tail fibers specifically to the lipopolysacchaꢀ
2
3
concentrations (10 , 10 CFU/mL). (b) Dependence of peak
current obtained from DPV curve on varying concentration of
E. coli for different incubation time.
Electrochemical detection of E. coli after varying in-
cubation time. After determining the condition of enzyme
reaction and proving the effectiveness and specificity of the
engineered phages, the detection of E. coli with varying conꢀ
4
2
rides (LPS) on the outer membrane of E. coli. An investigaꢀ
tion on the specificity of this strategy was conducted by using
four different single strains including E. coli BL 21, Salmonel-
la enterica (S. enterica), Staphylococcus aureus (S. aureus),
and Pseudomonas aeruginosa (P. aeruginosa), as well as a
mixture of all these four different bacteria strains. A sample
without any bacteria was used as a negative control. Engiꢀ
2
3
4
5
6
7
centrations (0, 10 , 10 , 10 , 10 , 10 and 10 CFU/mL) using
electrochemical method was performed. The detection of E.
coli was based on the measurement of activity of βꢀgal generꢀ
ated during the infection by T7lacZ phages. The simultaneous
addition of engineered phages and the substrate PAPG alꢀ
lowed simultaneous phage replication and enzyme reactions.
The expressed βꢀgal converted the substrate PAPG into an
4
neered phages (100 ꢀL) with concentration of 10 PFU/mL
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Page 6 of 16
electroactive product PAP which was reflected by the electroꢀ Antibody preparation is laborious and expensive. Conversely,
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chemical signal. The current was measured immediately beꢀ
fore each potential change by DPV model, and the current
difference is plotted as a function of potential. The peak potenꢀ
tial is proportional to the concentration of the electroactive
product. The DPV curve was obtained for the detection of
each concentration of E. coli BL21. Fig. 5(a) shows one of the
electrochemical results ꢀ the DPV curves obtained when deꢀ
6
phages can easily grow in the lab which is costꢀeffective. In
addition, compared with methods using phages without gene
17
encoding for reporter enzyme, our results have demonstrated
that engineered phages not only have high specificity, but also
facilitate the overexpression of reporter enzymes and lyse the
bacterial cells, resulting in a higher signal. Furthermore, since
βꢀgal is originally intracellular enzyme of E. coli, it is relativeꢀ
ly easy to trigger the overexpression of βꢀgal in E. coli. Thus,
our strategy has shown the improvement for the current elecꢀ
trochemical detection of E. coli.
2
3
4
5
tected E. coli with concentration of 0, 10 , 10 , 10 , 10 , 10
7
and 10 CFU/mL after incubation for 5 hours. We can observe
from the results that the negative control had a background
signal because the substrate has a formal potential. It is necesꢀ
sary to maintain the background signal at a relatively low levꢀ
0
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el. PAPG has been shown to be an ideal and commonly used
43,44
substrate of βꢀgal for electrochemical detection
and the
product PAP is reversibly oxidized on the electrode at a mildly
positive potential. Therefore, there is no need for additional
reagent to form the redox cycling. Additionally, PAP causes
very low level of electrode fouling, allowing the reuse of the
11
electrode. The results also demonstrate that the signal inꢀ
creased with the increasing concentration of E. coli. The workꢀ
ing concentration range of detection after 5 hours is between
3
6
1
0 and 10 CFU/mL of E. coli and the method is able to deꢀ
3
tect approximately 5×10 CFU/mL E. coli. The detection was
also performed for each concentration of E. coli after varying
incubation times (2, 3, 4, 5, 6 and 7 hours) and the peak curꢀ
rent obtained from each DPV curve dependent on concentraꢀ
tion of E. coli for different incubation time were shown in Fig.
5
(b). The DPV result for each incubation time was respectiveꢀ
ly shown in Fig. S1(a)ꢀ(f). A limit of detection was obtained
for each incubation time and the limit of detection decreased
with the time increasing. After 7 hours, we were able to detect
2
the sample with the lowest concentration (10 CFU/mL). As
the incubation time was increased, the signal for each concenꢀ
tration of E. coli was also increased due to the utilization of
substrate. A longer incubation time allowed a more sufficient
phage infection and allowed for additional bacterial doubling.
Electrochemical detection in real aqueous samples. The
electrochemical detection was then conducted in the real
aqueous samples including drinking water, apple juice, and
skim milk. We incorporated engineered phages into an elecꢀ
trochemical method, and thereby enable the detection of E.
coli from aqueous samples by measuring the activity of βꢀgal
generated. Fig. 6 shows the signal obtained for these three
samples inoculated with varying concentration of E. coli after
Figure 6. Peak current obtained for varying concentration of
E. coli after (a) 3 hours and (b) 7 hours of incubation in drinkꢀ
ing water (pink bars), apple juice (red bars) and skim milk
(
dark red bars), respectively. Bars with different letters (a, b, c,
d or e) are significantly different (P<0.05).
When compared with other
bacteriophages, the engineered T7 phages and electroꢀ
chemical methods also demonstrated benefits. For example,
methods
using
3
hours and 7 hours. After 3 hours of incubation, the limit of
26,47
5
detection was approximately 10 CFU/mL for all the three
2
samples. The limit of detection was able to reach 10 CFU/mL
47
the study of Derda used filterꢀbased colorimetric detection of
E. coli coupled with phages amplification. The bacteriophages
used in their study was a nonlytic M13 phage. While we used
T7 phage is a lytic phage, thereby eliminating the need for the
addition of lysozyme, and the engineered T7 phages can faciliꢀ
tate the overexpression of enzyme directly. When detecting in
turbid or colored samples, they required a pretreatment step
after 7 hours of incubation. There were no significant differꢀ
ences among the signal obtained for the three aqueous samples
(drinking water, apple juice and skim milk). This demonstrates
the feasibility of this enzymeꢀbased electrochemical assay
with engineered phages in beverages. Bacteriophages are not
only able to specific capture target bacteria, but also can be
engineered to overexpress enzyme that produce electrical,
visual and fluorescent signal.
(filtration) for the sample to obtain a relatively clean sample
for the detection. Electrochemical methods are able to detect
such samples directly, thus simplify the detection procedure.
Additionally, electrochemical methods offer the advantage of
instant quantification of bacteria with minimal equipment. In
their research, filtration was also used as a preꢀconcentration
step. If our detection is coupled with preꢀconcentration, we
have the potential to reach a lower limit of detection with less
time. Therefore, the use of engineered phages coupled with
There have been a variety of strategies developed for rapid
and sensitive bacterial detection. Our presented research
showed advantages based on the obtained results over the othꢀ
er reported electrochemical methods. First, compared with the
19,45,46
electrochemical methods commonly using antibodies,
bacteriophages have the ability to distinguish the live cells and
have a large range of stability in nonꢀbiological matrices with
36
the same capture efficiency for bacteria with antibodies.
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Analytical Chemistry
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electrochemical detection made this method sensitive, easy
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(
CONCLUSION
There is increasing need for the rapid quantification of bacꢀ
teria in the food and water sample in order to ensure the health
of the public. Here, an alternative strategy based on electroꢀ
chemical methods using engineered bacteriophages was deꢀ
veloped to detect E. coli in aqueous samples. Following the
infection of E. coli with the engineered phages, overexpressed
β ꢀgal was released into sample solution that allows for the
electrochemical detection. This approach was able to detect
(
(
0
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2
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5
6
7
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9
0
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0
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9
0
5
2
1
0 CFU/mL E. coli after 3 hours and 10 CFU/mL E. coli
after 7 hours from aqueous samples (drinking water, apple
juice, and skim milk). The standards and regulations for moniꢀ
toring bacterial contamination is very stringent. EPA requires
a public water supply to maintain less than one CFU of coliꢀ
form bacteria in 100 mL of water. To achieve this low limit of
detection, the EPAꢀapproved analytical method using memꢀ
brane filtration for preꢀconcentration needs incubation of the
plate for 24 hours at 35 ℃, followed by the fluorescent measꢀ
4
8
urement. Compared with this method, our approach is able to
2
detect 10 CFU/mL of E. coli in 7 hours without preꢀ
concentration. When coupled with preꢀconcentration (such as
filtration or immunomagnetic beads) and preꢀenrichment steps,
we have the potential to detect one CFU E. coli per 100 mL
sample while still requiring much less time than the EPAꢀ
approved method. Moreover, our presented strategy is more
easyꢀofꢀuse due to the simplicity of the equipment.
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and 3) lyse the host cell, allowing the release of reporter enꢀ
zymes. The sensitivity of this assay benefits from signal amꢀ
plification through the overexpression of the marker enzyme
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(
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(
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Differential pulse voltammetry (DPV) curve for the detection of
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(
(
AUTHOR INFORMATION
Corresponding Author
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*
(“S.R.N.”). Phone: +1ꢀ607ꢀ255ꢀ9185. Email:
snugen@cornell.edu
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
The authors would like to acknowledge USDA NIFA (USDA
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Analytical Chemistry
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Analytical Chemistry
Page 10 of 16
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Figure 1. The scheme representation of electrochemical detection of E. coli using engineered phage. (a) The
designed construct of genome of T7lacZ phage. (b) Specific capture and infection of E. coli by T7lacZ phage
resulted in the release and overexpresꢀsion of enzyme βꢀgal. PAPG was catalyzed by βꢀgal into an
electroactive species PAP that can be quantified by electrochemical device.
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Page 11 of 16
Analytical Chemistry
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Figure 2. Optimization of condition of enzyme reaction. (a) Dependence of the current on varying
concentration of sub-strate PAPG (0, 5, 10, 12, 15, 18, and 20 mM) detecting 106 CFU/mL E. coli after
incubation of 4 hours at 37℃. (b) Comparison of incubation temperature at room temperature (23℃) and
3
7℃. Peak current obtained for detecting 106 CFU/mL of E. coli at 23℃ (red bar) and 37℃ (blue bar),
respectively after 3 or 5 hours of incubation.
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Analytical Chemistry
Page 12 of 16
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Figure 3. The signal obtained for detection of 106 CFU/mL E. coli without phage (pink bar), with control
phages (red bar) and with engineered phages (dark red bar), respectively, after incubation of 3, 4 or 5
hours at 37℃.
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Page 13 of 16
Analytical Chemistry
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Figure 4. The signal obtained for detecting 106 CFU/mL E. coli, S. enterica, S. aureus, P. aeruginosa and a
mixture of all the strains, respectively, after incubation of 3 hours at 37℃. A sample without any bacteria
was used as the negative control.
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Analytical Chemistry
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Figure 5. Results of electrochemical detection of E. coli. (a) Differential pulse voltammetry (DPV) curve for
increasing concentration of E. coli after 5 hours of incubation at 37℃. The inset is the amplification of the
plot of control and low concentrations (102, 103 CFU/mL) (b) Dependence of peak current obtained from
DPV curve on varying concentration of E. coli for different incubation time.
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Analytical Chemistry
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Figure 6. Peak current obtained for varying concentration of E. coli after (a) 3 hours and (b) 7 hours of
incubation in drinking water (pink bars), apple juice (red bars) and skim milk (dark red bars), respectively.
Bars with different letters (a, b, c, d or e) are significantly different (P<0.05).
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Analytical Chemistry
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