healthmedicinet

Reagents and materials

Two kinds of nanoparticles were synthesized: MNPs and AuNPs. Aniline, iron (III) oxide
nanopowder, ammonium persulfate, methanol, and diethyl ether (Sigma-Aldrich, St. Louis,
MO) were used for the synthesis of the MNPs. Gold (III) chloride trihydrate (Aldrich,
MO) and dextrin (Fluka, MO) were used for the synthesis of AuNPs under alkaline conditions
22].

MNPs were functionalized with monoclonal anti-E. coli O157:H7 antibody obtained from Meridian Life Science, Inc (Saco, ME). AuNPs were
conjugated with polyclonal anti- E. coli O157:H7 antibody from Meridian Life Science, Inc (Saco, ME). Protein A from Staphylococcus aureus (Sigma-Aldrich, St. Louis, MO) was used as the linkage agent for AuNP and antibody
conjugation.

Triton-X100, phosphate buffered saline (PBS), casein, bovine serum albumin (BSA) and
sodium phosphate (dibasic and monobasic) were obtained from Sigma-Aldrich (St. Louis,
MO). PBS buffer (0.01 M and 0.1 M, pH 7.4), 0.01 M PBS buffer with 0.05 % (w/v) Triton-X100,
phosphate buffer (0.1 M sodium phosphate, pH 7.4), 0.01 M PBS buffer with 0.01 % casein,
0.01 M PBS buffer with 0.1 % (w/v) BSA were prepared with deionized water from Millipore
Direct-Q system. PBS buffer and phosphate buffer were used in preparing nanoparticle-Ab
conjugates and in washing. PBS buffer with casein or BSA was used to block nanoparticle
surface against nonspecific binding. PBS buffer with Triton-X100 was used for washing
off unbound or nonspecifically bound reactants after capture.

Bacterial culture

E. coli O157:H7 Sakai strain was obtained from the Nano-Biosensors Lab collection at Michigan
State University. The colonies from frozen (stored at –70 °C) culture were grown on
trypticase soy agar (BD Biosciences, MD) plates. A single colony was isolated and
inoculated in tryptic soy broth (BD Biosciences, MD) and grown overnight at 37 °C.
One milliliter of the liquid culture was transferred to another tube of tryptic soy
broth and incubated overnight at 37 °C. One milliliter of this liquid culture was
transferred to a new tube of broth and incubated at 37 °C for 6 h before each experiment.
The serial dilutions of bacterial culture were prepared using 0.1 % (w/v) peptone
water (Fluka-Biochemika, Switzerland) before each experiment. Viable cells were enumerated
by microbial plating on Sorbitol MacConkey agar (SMAC, BD Biosciences, MD).

Apparatus

Electrochemical measurement was performed with a potentiostat/galvanostat (263A, Princeton
Applied Research, MA) with a software operating system (PowerSuite, Princeton Applied
Research, MA) on a computer connected to the potentiostat. The measurement was performed
by introducing each sample onto a SPCE chip (Gwent Inc. England). The SPCE chip consisted
of a working carbon electrode and a counter and reference silver/silver chloride electrode.
One hundred microliters of each sample were introduced to the electrode area on the
SPCE chip.

Synthesis of nanoparticles

PANI coated MNPs were synthesized according to our method 6]. Fifty milliliters of 1 M hydrochloric acid, 10 ml of water and 0.4 ml of aniline
monomer were mixed in a flask, and then 0.65 g of iron (III) oxide nanopowder were
added to the solution to maintain a final ?-Fe
₂
O
₃
: aniline weight ratio of 1: 0.6. The mixture was put in a beaker filled with ice
and sonicated for 1 h. The solution was stirred while it was still on ice. During
the stirring, ammonium persulfate (1 g of ammonium persulfate in 20 ml deionized water)
was added to the solution slowly for 30 min. The solution was stirred for another
1.5 h. After the reaction, the solution was filtered using 2.5 ?m filter paper and
washed with 20 % methanol. Hydrochloric acid (1 M) was used to wash until the filtrate
became clear, followed by washing with 10 ml of 20 % methanol. The filtrate was filtered
again using a 1.2 ?m filter paper, and 10 ml of 20 % methanol solution was added to
the filter. The hydrochloric acid and methanol wash was repeated. The nanoparticles
on the filter paper were left under a fume hood to dry for 24 h at room temperature
and stored in a vacuum desiccator after drying.

AuNPs were synthesized under alkaline conditions following the approach published
by Anderson et al 22]. Briefly, 20 ml of dextrin stock solution (25 g/l) and 20 ml of sterile water were
mixed in a 50 ml sterile orange cap tube (disposable). Five milliliters of HAuCl
₄
stock solution (8 g/ml) were then added, and the pH of the solution was adjusted to
9 with sterile 10 % (w/v) Na
₂
CO
₃
solution. The final volume was brought to 50 ml with pH 9 water. The reaction was
carried out by incubating the solution in a sterile flask in the dark at 50 °C with
continuous shaking (100 rpm) for 6 h. A red solution was obtained at the end of the
reaction.

Functionalization of nanoparticles

MNPs were functionalized with a monoclonal anti-E. coli O157:H7 antibody 6]. MNPs (2.5 mg) were suspended in 150 ?l of 0.1 M phosphate buffer, and sonicated
for 15 min. Monoclonal anti-E. coli O157:H7 antibody (2.5 mg/ml, 100 ?l) was added to the suspension, and hybridized
on tube rotator for 5 min. Twenty five microliters of PBS buffer (0.1 M) were added.
Then the conjugation was carried on for 55 min on the tube rotator. The MNPs were
separated from the solution by magnetic separation, and blocked by adding 250 ?l of
0.1 M tris buffer with 0.01 % casein and incubated for 5 min. This step was repeated
three times, and the suspension was put on tube rotator for 1 h to hybridize. Finally,
the MNPs were magnetically separated and resuspended in 2.5 ml of 0.1 M phosphate
buffer. The MNP-Ab conjugate was stored at 4 °C before use.

AuNPs were conjugated with a polyclonal anti-E. coli O157:H7 antibody through protein A linkage. Two hundred microliters of 1:2 diluted
suspension of AuNPs in water were put into a 2 ml microcentrifuge tube and sonicated
for 10 min. Then the suspension was centrifuged for 6 min at 13,000 rpm. The supernatant
was removed after centrifugation. To modify the surface of the AuNPs, protein A (0.25 mg/ml)
in 200 ?l of 0.01 M PBS buffer was used to resuspend the AuNPs. The conjugation was
conducted by rotating the mixture for 1 h. The modified AuNPs were separated from
the suspension by centrifugation for 6 min at 13,000 rpm. The nanoparticles were washed
by adding 200 ?l of 0.01 M PBS buffer and centrifuged. After removing the supernatant,
100 ?l of 1 mg/ml antibody and 100 ?l of 0.01 M PBS buffer were added to the tube
and mixed for 60 min by rotating. After separating the AuNP-antibody (AuNP-Ab) conjugates from
the supernatant, 200 ?l of PBS buffer with 0.1 % (w/v) BSA were added to the tube.
The mixture was rotated for 30 min. Finally, the AuNP-Ab conjugates were separated
from the suspension by centrifugation, and the final suspension of the conjugates
in 200 ?l PBS buffer with BSA was stored at 4 °C.

Detection of target pathogenic bacteria

Detection of the target pathogen is presented in Fig. 1. Blank control for the tests was peptone water in the same volume as the sample.
Firstly, 400 ?l of 0.01 M PBS buffer, 50 ?l of cell dilution (or peptone water for
the blank) and 50 ?l of MNP-Ab conjugates were combined in a 2 ml sterile tube. After
15 min hybridization, PBS buffer (55 ?l, 0.01 M) with 0.1 % BSA was added to the mixture
as a blocking agent. Then, the MNP-E. coli complexes were magnetically separated from the solution and resuspended in 450 ?l
of 0.01 M PBS buffer. Secondly, 50 ?l of the AuNP-Ab conjugates were introduced to
the system, followed by 15 min hybridization. After washing the complexes once with
0.01 M PBS buffer, the complexes were resuspended in 500 ?l of PBS buffer with 0.05 %
Triton-X100, and let stand for 3 min. Finally, the complexes were magnetically separated
from the buffer and resuspended in 500 ?l of 0.01 M PBS buffer. One hundred microliters
of the suspension were plated on SMAC for cell counting. The rest of the complexes
were magnetically separated from the supernatant (400 ?l).

Electrochemical measurement

The target bacteria were detected by measuring the electrochemical signal of AuNPs.
Each sample from the last section (complexes magnetically separated from supernatant)
in 100 ?l 1 M hydrochloric acid was introduced to the SPCE chip. An oxidation potential
of 1.4 V vs. Ag/AgCl was applied to the working electrode. After oxidation, a differential
pulse voltammetric (DPV) measurement was performed. The scan was from 1.5 V to ?1.5 V.
The potential and currents were recorded. All measurements were performed at room
temperature. Each sample was measured three times. At least three samples of each
concentration of bacteria were tested.