Rapid and Sensitive Detection of Salmonella Typhimurium on Plastic Food

Processing Plates by Using Wireless Biosensors

Yuzhe Liua, Shin Horikawaa, Songtao Dua, Yating Chaia, Jiajia Hua,b, Fengen Wanga,c,
and Bryan A. China

a. Materials Research & Education Center, Auburn University, Auburn, Alabama 36830, USA
b. Material Science and Engineering, Changzhou University, Changzhou, Jiangsu, P.R. China
c. Institute of Quality Standard and Testing Technology for Agro-Products, Shandong Academy of
Agricultural Sciences, Jinan, Shandong, P.R. China

Abstract

This paper presents an investigation into rapid, sensitive detection of Salmonella

Typhimurium on plastic food processing plates by using wireless magnetoelastic (ME)
biosensors. In this research, a planar spiral coil was microfabricated and employed as
a surface-scanning detector for real-time, in-situ measurement of the biosensors
without sample preparation and/or enrichment in the testing process. The ME
biosensor consists of an ME resonator, made from metallic glass (Metglas alloy
2826MB), as the signal transducer and E2 phage as the bio-molecular recognition
element that is engineered to selectively bind with Salmonella Typhimurium. Both
measurement sensors (multiple E2 phage-coated biosensors) and control sensors
(multiple biosensors without phage) were prepared and distributed on a food grade
plastic plate contaminated with Salmonella Typhimurium with known volumes and
concentrations. The resonant frequency changes of the measurement sensors due to
specific binding of Salmonella Typhimurium were found to be statistically different
from those of the control sensors.

Introduction

Bacteria are widely found in nature and in our living environment, such as in food,
water, soil and the intestinal tracts of humans and animals. Every year in the United
States over 48 million Americans become ill due to foodborne illness. More than 60%
of pathogen outbreak reports in the previous five years are associated with poultry
and meat products.1 An ideal pathogen detection system requires not only quick,
sensitive and specific detection, but also should be portable for use in the field.

The methods for pathogen detection that the FDA uses right now, such as
polymerase chain reaction (PCR), can offer very high sensitivity.2 However, there are
obviously disadvantages such as: time-consuming sample preparation, laboratory
testing only, and enrichment and culturing is needed. So, a low-cost, portable, label-
free, in-field analysis and real-time detection system is needed urgently to insure the
safety of our food.
 In the recent two decades, ME sensors have attracted significant interest of
researchers because of their excellent and wireless performance. They have been
used to measure a wide range of parameters of the environment including pressure3,
humidity4, pH5, temperature4,5 and the viscosity of liquids6. In previous reported
research by our group, ME sensors have been used as biosensors because they show
promise for in-situ, real-time detection of pathogenic bacteria on food surfaces. The
freestanding, phage-based ME biosensor is composed of an ME resonator platform
coated with a bio-molecular recognition element that binds specifically with a target
pathogen.7 Once the biosensor comes into contact with the target pathogen, the
pathogen binds with the biosensor causing the mass of the biosensor to increase
resulting in a decrease of its resonant frequency.8 An equation (equation 1) was given
which can be used to calculate the mass change based on the change of resonant
frequency. In the past decade, an electro-magnetic solenoid was used to generate the
oscillating magnetic field and measure the biosensors resonant frequency.9 To make
the testing even faster, a planar spiral coil10 which can give a much larger signal
amplitude at resonance was used to replace the solenoid coil measurement method.

$
" = ' $ [1]
$%
(
where " is mass-loaded resonant frequency, $ is the fundamental resonant
frequency, is the original mass of resonator, is the loaded mass or mass
change of the resonator.

In this paper, we demonstrate this revolutionary alternate using phage-coated ME

biosensors to capture, concentrate, isolate and identify Salmonella on plastic food
surfaces to improve the safety of food production and processing (Fig. 1). At first,
phage-coated magnetoelastic sensors are distributed randomly onto the plastic food
processing surface to capture multiple target pathogens as shown in Fig. 1a. A magnet
can be used to concentrate the sensors, thereby concentrating and isolating the target
pathogens. Finally, our wireless, remote technology was employed for the
identification of the isolated pathogens.
 a

Fig. 1. Methodology used for the capture, concentration, isolation, and identification
of pathogens. a. capture; b. concentration & isolation; c. identification

Experimental details

Coil detector fabrication

Our planar coil detector was made using micro-electronic fabrication methods on
a transparent glass wafer with an electro-deposition method. The pattern of
fabrication mask was designed using AutoCAD 2016. The performance of the planar
coil, which was determined by the coils self-resistance, self-inductance and self-
capacitance, was controlled by the geometry of the coil. After the fabrication was
done, a thin layer of SU-8 photoresist was used to package the device, making the
whole device resistant to physical damage, oxidization and contamination.

Sensor fabrication

ME resonant biosensors were fabricated by METGLAS 2826MB alloy (Honeywell

International Inc.). An automated dicing saw (DAD 3220, Disco Corp, Tokyo, Japan)
was employed to dice the metallic glass ribbon into 1mm*0.2mm*0.028mm platforms.
The platforms were cleaned with methanol and acetone and then annealed at 220
in vacuum (103 Torr) for 2 h to remove residual stress generated by the dicing process.
Cr and Au were deposited onto the platform surfaces using sputtering so that Au layer
can provide corrosion resistance and a ready surface for bio-probe immobilization. A
well fabricated coil detector with two ME sensors is shown in Fig. 2a.

E2 phage immobilization and bovine serum albumin (BSA) surface blocking

Dr. James M. Barbaree's lab in the Department of Biological Sciences at Auburn

University prepared and provided the filamentous E2 phage for this research. The E2
phage which is specific for Salmonella Typhimurium was developed by Dr. Valery
Petrenko at Auburn University. The concentration of E2 phage solution was 5*1012
vir/ml in a tris-buffered saline (TBS) solution. The ME sensors were immersed in the
phage solution for 2 hours for physical loading. Then, the biosensors were washed
with deionized water twice to remove unbound phage and salt originating from the
TBS. To compensate for environmental effects and non- specific binding, control
biosensors were prepared following the same steps but without E2 phage
immobilization. Both measurement (with phage) and control (without phage)
biosensors were blocked with BSA to prevent non-specific binding. The biosensors
were immersed in a 1 mg/ml BSA solution for 40 min and washed with deionized water
twice.

Bacterial contamination on plastic food preparing surfaces

Ultra-high-molecular-weight polyethylene (UHMW-PE) (food-grade) was used to

simulate the plastic food preparation plate in this work. Salmonella enterica-serovar
Typhimurium culture in a concentration of 5*108 CFU/ml was provided by Dr. James
M. Barbaree's laboratory. In this work, 5*108 CFU/ml Salmonella Typhimurium
solution was diluted to lower concentrations (started from 5*107 CFU/ml) with
deionized water. The same volume (30) of pathogen solutions were pipetted and
inoculated onto the UHMW-PE surface, forming a contamination area, circular in
shape with a diameter of about 3-4 mm. Equation 2 was used to determine the
corresponding surface Salmonella Typhimurium concentrations.
/0 2
= [2]
34 5
where is the surface concentration of Salmonella, 6 is the concentration of
Salmonella in the solution, V is the volume of the solution which dropped onto the
surface, R is the radius of the drop on the surface. So, an average level of the
concentrations was calculated as 1.5*106 CFU/mm2 and 1.5*105 CFU/mm2 separately.

Two sensors Real-time, in-situ detection process

After 1h dry in a fuming hood, two ME biosensors, in which one is a measurement

sensor loaded with phage and the other is a control sensor all blocked by BSA, were
placed on contaminated regions and the sample with biosensors was placed in a two
planar coils measurement system, shown in Fig. 2b. The AC signal was generated by
and then feed back to a Network Analyzer.

a b

Figure 2. a. A well fabricated coil detector with two ME sensors placed on it; b. the
two coil measurement system.

Results & discussion

A diced UHMW-PE board (food grade), which was used to simulate a real food
preparing plate, is shown in Fig. 3a. In Fig. 3b & 3c, the results of optical microscope
under 5X and 50X magnification show us the roughness of surface of this UHMW-PE
board. As the requirement of the food preparing process, the surface roughness of
the plate is relatively high. This high roughness keeps the food from slipping on the
preparation board, however, this kind of surface also gives bacteria a very good
environment to hide and multiple in numbers. Fig. 3d is a microscope picture of the
ring of bacteria on the UHMW-PE board with a concentration of the bacteria
solution at 5*108 CFU/ml. The contaminated region can be clearly found by the
microscope so that we can make sure both the measurement sensor and the control
sensor were placed on the contaminated region.

a b

c d

Fig. 3. a. UHMW-PE board (food grade); b. testing surface of UHMW-PE under 5X

magnification; c. 50X; d. UHMW-PE board spiked with 1.5*108CFU/mm2 Salmonella.

A typical measurement result is shown in Fig. 4. Thanks for a new designed

measurement supporting software, the testing speed can be very fast with
measurements completed in less than 5 seconds for measurement and control
sensors. The resonant frequency changes of the measurement and control sensors
over time at 1.5*106 CFU/mm2 is shown in Fig. 4a. The drop of resonant frequency
happens at about 60s in the measurement process and saturates at about 120s. So,
the total measurement time for the measurement sensor to capture and respond to
the captured Salmonella takes about 60s. During the entire measurement process, the
control sensor shifted 200Hz range which is in the acceptable noise. In Fig. 4b, an
original curve of the measurement sensor and a final curve is shown. A significant shift
of the resonant frequency peak (9K Hz) which indicates a large number of Salmonella
bacteria has been caught by the measurement sensor can be easily noticed. The same
measurement and data treatment method was also used on the test of plastic board
sample contaminated with 1.5*105 CFU/mm2 Salmonella. The results, shown as Fig. 4c
& 4d, indicates that the measurement process takes about 160s to saturate while the
shift between the original resonance peak and final resonance peak is about 6kHz.
 2000 0

0 1000
Measurement sensor
Control sensor
2000
2000
Delta-Frequency(kHz)

Delta-Frequency(Hz)
Measurement sensor
Control sensor 3000
4000

4000

6000

8000 a 5000

6000
c
10000 7000
20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300
Seconds(S) Seconds(S)
1.0010 1.004

1.0005 Origin curve Original curve

Final curve 1.003 Final curve

1.0000
1.002

0.9995
1.001
Intensity

Intensity
0.9990

1.000
0.9985

b d
0.999
0.9980

0.9975 0.998

0.9970 0.997
6 6 6 6
2.1010 2.15 2.20 2.25 2.3010 2.1010 2.15 2.20 2.25 2.3010
Frequency(Hz) Frequency(Hz)

Fig. 4. a. Resonant frequency change of measurement and control sensors over time
at 1.5*106 CFU/mm2; b. Initial and final curves of the measurement sensor; c.
Resonant frequency change of measurement and control sensors over time at
1.5*105CFU/mm2; d. Initial and final curves of the measurement sensor.

From the results above, we can see that our two sensors enable real-time, in-situ
detection of Salmonella on plastic preparation boards. This technology has detection
times of minutes with measured resonance peak changes of 9KHz versus a background
noise of 200Hz. Since the specificity of our measurement is based on the type of phage
that was loaded on the sensors, with further modification of the design of phage which
can catch the pathogens in need especially, we can detect not only Salmonella, but
also other kinds of bacteria, virus, or even multiple kinds of pathogens mixture.

Conclusion

In this research, both measurement sensors, which is loaded with E2 phage, and
control sensors, which is covered by BSA blocking without phage, were prepared. A
food grade UHMW-PE plate which was contaminated with Salmonella Typhimurium
at concentrations of 1.5*106 CFU/mm2 and 1.5*105 CFU/mm2 were measured
separately. Significant resonant frequency changes of the measurement sensors of
about 9kHz and 6kHz due to specific binding of Salmonella Typhimurium were
recorded. Time to detection of Salmonella Typhimurium was less than 120 seconds in
each case.

Acknowledgments
 This work was supported by the United States Department of Agriculture (USDA),
National Institute of Food and Agriculture (NIFA) and the Specialty Crop Research
Initiative (SCRI).

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