Direct Detection of Bacteria on Fresh Produce

Shin Horikawa

, Yating Chai

, Howard C. Wikle

, James M. Barbaree

and Bryan A. Chin

Materials Research and Education Center, Auburn University, Auburn, Alabama 36849, U.S.A.

Department of Biological Sciences, Auburn University, Auburn, Alabama 36849, U.S.A.

Keywords:

Direct Detection, Bacteria, Fresh Produce, Magnetoelastic Biosensor, Phage, Surface-Scanning Detector.

Abstract:

This paper presents a revolutionary method of bacterial detection that directly detects and quantiﬁes the pres-

ence of speciﬁc bacteria on the surfaces of fresh produce without sample preparation (water rinse, soak, stom-

aching) and/or enrichment. The speed of detection is from 2 to 10 minutes with a limit of detection in a

range of 10

to 10

cfu/mm

. The speciﬁcity of detection is 2 in 10

background bacteria. This technology

was awarded a $20,000 prize in the ﬁrst United States Food and Drug Administration (FDA) Food Safety

Challenge. The method combines wireless magnetoelastic (ME) biosensors and a surface-scanning detector

for rapid determination of bacterial contamination. Tests were conducted on tomatoes and grapes spiked with

different concentrations of Salmonella Typhimurium. The resonant frequency changes of the biosensors were

found to be dependent on the surface concentration of Salmonella. Detection limits were found to be affected

by the surface roughness of the food. A 90-second video of a test for Salmonella on tomato can be viewed at

http://eng.auburn.edu/food-safety. The method presented in this paper is envisioned for use at ports of entry

for the swift screening of foods.

1 INTRODUCTION

The past decades have been marked by a global in-

crease in the outbreaks of food poisoning and asso-

ciated illnesses. These public health problems are

caused by the accidental supply and consumption of

contaminated food, largely due to improper safety

knowledge, perspectives, and practices of food pro-

ducers as well as insufﬁcient consumer awareness.

Although substantial progress on food safety reg-

ulations has been made worldwide, up to 30% of

the population even in industrialized countries suffer

from foodborne illnesses each year (WHO, 2007). In

the United States, approximately 48 million cases of

foodborne illnesses are estimated to occur annually,

resulting in 128,000 hospitalizations, 3,000 deaths,

and $51.0 to $77.7 billion economic losses (Scallan

et al., 2011a; Scallan et al., 2011b; Scharff, 2012).

Salmonella is one of the most pervasive food prob-

lems today. In the past years in the United States,

$1.1 billion was lost, and over 3,000 individuals were

conﬁrmed sick due to Salmonella food contamination

(2008 tomato, 2009 peanut butter, and 2010 egg out-

breaks). Since foodborne illnesses spread so easily,

rapid, on-site detection can play an important role

in preventing the spread of contamination. However,

current detection methods of Salmonella can take up

to several days, causing delays in contacting con-

sumers, removing food items from the marketplace,

and preventing producers from selling products while

the commodity remains stored in warehouses. In this

work, we present a revolutionary bacterial detection

method that allows for testing of contamination to be

completed in only a matter of minutes without sample

preparation (water rinse, soaking, stomaching, con-

centration, etc.) and/or enrichments in the testing pro-

cess. The method combines phage-coated magnetoe-

lastic (ME) biosensors and a surface-scanning detec-

tor, which can be used on site at ports of entry, food

processing facilities and in agriculture ﬁelds for food

inspection and outbreak investigation.

2 MATERIAL AND METHODS

2.1 Fabrication of ME Biosensor

Platforms

Strip-shaped ME biosensor platforms (length × width

× thickness: 1 mm × 0.2 mm × 30 µm) were fab-

ricated by dicing a commercially available Metglas

2826MB ribbon (Metglas, Inc.). The diced biosensor

platforms were, then, coated successively with thin

Horikawa, S., Chai, Y., Wikle, H., Barbaree, J. and Chin, B.

Direct Detection of Bacteria on Fresh Produce.

DOI: 10.5220/0005670100480053

In Proceedings of the 9th International Joint Conference on Biomedical Engineering Systems and Technologies (BIOSTEC 2016) - Volume 1: BIODEVICES, pages 48-53

ISBN: 978-989-758-170-0

layers of chromium (90 nm in thickness) and gold

(150 nm in thickness) by electron-beam induced de-

position. The chromium layer acts as an adhesive in-

terlayer. The gold layer provides corrosion resistance

as well as a ready surface for the immobilization of

a phage probe. Before and after the metal deposi-

tion, annealing was performed in vacuum at 220

◦

for 2 hrs to relieve residual internal stresses and cor-

rect any surface defects from the fabrication processes

(Chai et al., 2012; Chai et al., 2013c; Chai et al.,

2013b; Chai et al., 2013a; Li et al., 2010; Horikawa

et al., 2011; Horikawa et al., 2014b; Horikawa et al.,

2014a).

2.2 Preparation of E2 Phage and

Salmonella Typhimurium

E2 phage was derived from a landscape f8/8 phage

library (Sorokulova et al., 2005) and used as the

biomolecular recognition element for the ME biosen-

sor. E2 phage possesses 10- to 1,000-fold greater

binding afﬁnity for S. Typhimurium over other bacte-

ria. The speciﬁcity of detection has been reported to

be 2 in 10

background bacteria (Lakshmanan, 2008).

Suspensions of E2 phage (1 × 10

virions/ml

in TBS) and S. Typhimurium cells (ATCC 13311,

5 × 10

cfu/ml in sterile distilled water) were pre-

pared and provided by Dr. James Barbaree’s group

at Auburn University, Auburn, Alabama, U.S.A. The

concentrated Salmonella suspension was diluted with

sterile distilled water as desired prior to use.

2.3 Phage Immobilization and Surface

Blocking

The fabricated biosensor platforms were individually

immersed in 330 µL of the E2 phage suspension in a

polypropylene PCR tube. The tubes were, then, ro-

tated with a Barnstead LabQuake tube rotator (Fisher

Scientiﬁc, Inc.) at 8 rpm for 1 hr. In this way, the

phage was allowed to uniformly attach to the biosen-

sor platforms via physical adsorption. Finally, a wa-

ter rinse was performed to remove loosely attached

phages and TBS buffer residues from the platform

surfaces.

In order to reduce non-speciﬁc binding of S. Ty-

phimurium on biosensor surfaces, surface blocking

was performed. The phage-coated biosensors, or

what we call ”measurement sensors,” were individ-

ually immersed in a 330-µl solution of SuperBlock

Blocking Buffer (Thermo Fisher Scientiﬁc, Inc.) in a

PCR tube. After 2 hrs of tube rotation at 8 rpm, the

biosensors were collected from the solution and thor-

oughly rinsed with sterile distilled water to be ready

for use. ”Control sensors,” which are not coated with

E2 phage but only surface-blocked with the blocking

buffer, were also prepared and used for determination

of the limits of detection.

∆f ≈ −

∆m

(1− ν)

, (1)

2.4 Principle of Detection

The ME biosensor used in this work is made of

Metglas 2826MB, a magnetostrictive alloy (Li et al.,

2012). Hence, the biosensors can be placed into me-

chanical resonance when subjected to an externally

applied time-varying magnetic ﬁeld at the right fre-

quency. In this study, the fundamental resonant fre-

quency of longitudinal vibration, f, is of interest, and

thus, an external magnetic ﬁeld was applied in the di-

rection parallel to the length of the biosensor to excite

such a mode of vibration. For a freestanding, strip-

shaped biosensor, f can be expressed by (Liang et al.,

2007)

f =

ρ(1− ν)

, (2)

where L, E, ρ, and ν denote the length, modulus of

elasticity, density, and Poisson’s ratio of the biosen-

sor, respectively. When this biosensor comes into

contact with S. Typhimurium, E2 phage that is coated

on the biosensor binds speciﬁcally with the bacteria,

thereby increasing the total mass of the biosensor by

∆m. This change in mass causes a corresponding

decrease in the biosensor’s resonant frequency. The

mass-induced resonant frequency change, ∆ f, can be

approximated by (Grimes et al., 2011)

where W and T represent the width and thickness

of the biosensor, respectively. By recording ∆ f as a

function of time, real-time monitoring of the presence

of Salmonella on food surfaces can be performed.

Unlike a measurement sensor (with E2 phage), a con-

trol sensor (without E2 phage) does not bind speciﬁ-

cally with S. Typhimurium, which gives a background

resonant frequency shift in the testing environment.

2.5 Construction of the

Surface-Scanning Detector

The surface-scanning detector is a microelectroni-

cally fabricated planar coil that serves as: (1) a driving

coil to magnetically excite the longitudinal vibration

of the biosensor and (2) a pick-up coil to read the res-

onant frequency of the biosensor. The detector was

fabricated using standard microelectronic fabrication

techniques. As shown in Fig. 1, the coil turns, leads,

Direct Detection of Bacteria on Fresh Produce

Network analyzer

Humidifier

Environmental chamber

Three-axis translation stage

Humidity sensor

Bias magetic field

Detector

Sensors

Pair of magnetic plates

Figure 2: Measurement setup for the direct detection of S. Typhimurium on food.

Glass (500 m)

Cu turns (8 m)

Ti (10 nm)

SU-8 (5 m)

SU-8 (10 m)

Cu lead and pad (200 nm)

Section AA'

(schematic, not to scale)

A'A

1 mm

Contact pads

Coil turns

Figure 1: Design of the surface-scanning detector.

and contact pads are made of copper, and they reside

on a glass substrate. To promote good adhesion of the

coil to the substrate, titanium was used as the inter-

layer. In addition, SU-8 3005 (MicroChem Corp.), an

epoxy-based photoresist, was used as the insulating

ﬁller and topmost layer.

2.6 Spiking of Fresh Produce with S.

Typhimurium

Fresh tomatoes and grapes were purchased from a lo-

cal grocery store (Kroger) and used as-received. 20-µl

drops of S. Typhimurium with various concentrations

(5 × 10

to 5 × 10

cfu/ml) were spot-inoculated on

the surfaces of the foods. Salmonella were, then, al-

lowed to dry in air for 2 hrs. Finally, the area of

the Salmonella-spiked spots were measured, which

allows conversion of cfu/ml into cfu/mm

(i.e., sur-

face density of S. Typhimurium).

2.7 Measurement Setup and

Experimental Procedure

The measurement setup for the direct Salmonella de-

tection on food consists of a surface-scanning detec-

tor, a 3-axis translation stage, a network analyzer (Ro-

hde & Schwarz ZNC3), a humidiﬁer, a pair of mag-

netic plates, and an environmental chamber as shown

in Fig. 2. ME biosensors were ﬁrst placed on a

Salmonella-spiked spot on food. The food was, then,

placed between the magnetic plates in the environ-

mental chamber. At this time, the biosensors on the

food can spontaneously align parallel to the direction

of the external magnetic ﬁeld, which is perpendicular

to the magnetic plates. The 3-axis translation stage

was next used to position the biosensors under the

surface-scanning detector. The detector is connected

to the ZNC3 network analyzer, operated in the S

reﬂection mode. An incident AC power is applied

across the detector to magnetically excite the longi-

BIODEVICES 2016 - 9th International Conference on Biomedical Electronics and Devices

Time (seconds)

Resonant frequency shift, Δf (kHz)

0 50 100 150 200 250 300

-0.5

-1

-1.5

-2

-3

-2.5

-3.5

-3

-4

Measurement sensor

Control sensor

Measurement sensor

Control sensor

Figure 3: Resonant frequency shifts of biosensors on a

tomato that is spiked with 5.6 × 10

cfu/mm

S. Ty-

phimurium (open squares: control sensor and open circles:

measurement sensor). Scanning electron micrographs con-

ﬁrm the resonant frequency shift results. Salmonella cells

are shown as the black spots in the micrographs (scale bars:

50 µm).

tudinal vibration of the biosensor, and the resultant

reﬂected power is compared to the incident power

over a selected range of frequencies. In this man-

ner, frequency-dependant reﬂection coefﬁcient, S

can be determined for the circuit. At the resonant fre-

quency of the biosensor, the largest change in the re-

ﬂected power of the circuit occurs, which is visible

as a downward peak in the network analyzer output.

To enhance the magnitude of the resonance peak, a

proper bias magnetic ﬁeld was applied to the biosen-

sor by adjusting the distance between the magnetic

plates. The measurement was conducted at 23

◦

and 85% relative humidity. Data were collected every

10 seconds (power: 0 dBm, bandwidth: 1 kHz, fre-

quency span: 100 kHz, number of date points: 2001,

averaging: 10 times, and smoothing aperture: 1 %).

3 RESULTS AND DISCUSSION

3.1 Direct Detection of S. Typhimurium

on Food

Resonant frequency shifts of ME biosensors placed

on the Salmonella-spiked foods were recorded every

10 seconds using the measurement setup described

in Subsection 2.7. Typical test results for a tomato

2 3 4

2 3

99.7%

96.8%

95.2%

66.2%

95.9%

95.3%

50%65.7%

Figure 4: Dose-response results for (a) tomatoes and (b)

grapes. The numbers shown above the bars are the con-

ﬁdence levels of difference between the measurement and

control sensors at each Salmonella concentration.

spiked with 5.6 × 10

cfu/mm

Salmonella are shown

in Fig. 3. The resonant frequency shift of a con-

trol sensor (open squares) was found to be negligible

during the measurement. By contrast, a much larger

resonant frequency shift was observed for a measure-

ment sensor (open circles), due to the occurrence of

the phage-based speciﬁc binding of the bacteria on

the sensor. Depending on the surfaces of foods and

concentration of S. Typhimurium, the rates of reso-

nant frequencyshifts and detection speeds were found

to vary. In this particular example shown in Fig.

3, S. Typhimurium (5.6 × 10

cfu/mm

) was de-

tected within 2 minutes. Finally, scanning electron

microscopy was used to conﬁrm the frequency mea-

surement results. A large cell count was found on

the measurement sensor as anticipated, indicating that

speciﬁc Salmonella binding had occurred.

In order to determine the limits of detection

Direct Detection of Bacteria on Fresh Produce

Spinach leaf

atermelon

Figure 5: Surfaces of various fresh produce (scale bars: 100 µm).

(LODs), the biosensors were tested at different

Salmonella concentrations on the food surfaces for 10

minutes. The dose-response results for tomatoes and

grapes are shown in Fig. 4. As anticipated, the reso-

nant frequency shifts of measurement sensors (black

bars) were found to be larger than those of control

sensors (red bars) at high Salmonella concentrations,

which is due to the speciﬁc binding of the bacteria

through E2 phage. By contrast, comparable sensor re-

sponses were observed at low Salmonella concentra-

tions, indicating that the limits of detection had been

reached. A one-tailed, unpaired Student’s t-test was

conducted to analyze the degree of dissimilarity be-

tween the measurement and control sensors. The con-

ﬁdence levels of difference were calculated and pre-

sented above the bars at each Salmonella concentra-

tion in Fig. 4. With a conﬁdence level of difference

higher than 95% (p < 0.05), the LODs were deter-

mined to be lower than: (a) 5.6 × 10

cfu/mm

for

tomatoes and (b) 2.7 × 10

cfu/mm

for grapes. The

method presented in this paper is a direct bacterial

detection method without sample preparation (con-

centration, puriﬁcation, washing, etc.) and/or enrich-

ments in the testing process. The speed of detection

was found to be from 2 to 10 minutes, suited for rapid,

on-site pre-screening of food products.

4 FUTURE WORK

The current LODs can be improved by using ME

biosensors with smaller sizes. As can be seen in

Eq. 1, the mass-induced resonant frequency change,

∆f, is inversely proportional to L

WT, where L,

W, and T are the length, width, and thickness of

the biosensor, respectively. Smaller biosensors are,

hence, more mass-sensitive and capable of detecting

smaller amounts of pathogens that may be present on

food surfaces. The LOD is also largely dependent on

the surface topography of foods. As shown in Fig.

5, the surface pattern and roughness vary from one

food to another, which necessitates the use of appro-

priate sizes of biosensors for improved pathogen de-

tection. The authors have reported previously an ini-

tial model to calculate the LOD and probability of de-

tection as a function of the size and quantity of biosen-

sors (Horikawa, 2013). The model will be improved

BIODEVICES 2016 - 9th International Conference on Biomedical Electronics and Devices

and used to demonstrate a proof-in-concept in the fu-

ture.

5 CONCLUSIONS

A unique, revolutionary method of bacterial detection

on the surfaces of foods was presented. By combining

phage-coated ME biosensors and a surface-scanning

detector, S. Typhimurium was detected in a range of 2

to 10 minutes without sample preparation and/or en-

richment in the testing process. The method presented

in this paper can be used for rapid, on-site screening

of food products. The pathogen-positive foods deter-

mined by this method can then be sent to lab for con-

ﬁrmation and identiﬁcation tests.

ACKNOWLEDGEMENT

This work was supported by the Auburn University

Detection and Food Safety Center and a grant from

the U.S. Department of Agriculture (USDA-2011-

51181-30642A).

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