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Sensors and Materials, Vol. 35, No. 10 (2023) 4761–4768
MYU Tokyo
S & M 3428

Development of Organic–Inorganic Nanostructure Labels
for Food-poisoning Bacteria
Shogo Nakamura,1 Akihiro Nakao,1 Satohiro Itagaki,1 Kyohei Matsui,1
Shigeki Nishii,1 Xueling Shan,2 Yojiro Yamamoto,1Yasuhiro Sadanaga,1 Zhidong Chen,2 and Hiroshi Shiigi1*
1Department of Applied Chemistry, Osaka Metropolitan University,

Sakai 599-8570, Japan

2Jiangsu Key Laboratory of Advanced Catalytic Materials and Technology, Changzhou University,

Changzhou 213164, China(Received April 17, 2023; accepted September 22, 2023)

Keywords: Staphylococcus aureus, copper nanostructures, electrochemical labels, organic–inorganic
hybrids, food-poisoning bacteria

In this paper, we describe a simple strategy to detect food-poisoning bacteria using thespectroscopic and electrochemical properties of organic–inorganic nanostructures composed ofelectrically conductive polyaniline-coated copper nanoparticles. Antibody-introduced
nanostructures specifically bind to cells via antigens and function as nanometer-sized labels.
Attempting the quantification of Staphylococcus aureus using nanostructure labels, we foundthat the absorbance of the free label in the supernatant decreased as the number of bacterial cells
increased, indicating that the labeled cells were obtained as precipitates. Furthermore, the peakcurrent (Ip) based on the number of labels bound to cells on the electrode increased with thenumber of bacterial cells. Thus, the introduction of antibodies into nanostructures has made it
possible to electrochemically and spectroscopically quantify the causative bacteria of foodpoisoning.

1.

IntroductionMore than 200 diseases are known to be caused by the foodborne transmission of various
pathogens, including bacteria, viruses, and parasites.(1) The prevention of diseases associatedwith foodborne pathogens remains a major public health challenge, but the risk of foodborne
diseases, particularly those caused by Salmonella, Escherichia coli, and Staphylococcus aureus,
has increased significantly.(2–4) To reduce those risks, bacteriological examinations have become
important. Most bacteriological tests are performed by colony counting with culture on agar
plates. This method is time-consuming, requiring 2–3 days for initial results and up to a week ormore for the confirmation of specific pathogenic organisms.(5,6) In recent years, rapid tests using
polymerase chain reaction (PCR), enzyme-linked immunosorbent (ELISA), and*Corresponding author: e-mail: shii@omu.ac.jp

https://doi.org/10.18494/SAM4432

ISSN 0914-4935 © MYU K.K.
https://myukk.org/

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Sensors and Materials, Vol. 35, No. 10 (2023)

immunochromatography have been extensively studied. The PCR method is the best-known andestablished nucleic acid amplification method.(7,8) In this method, double-stranded DNA isdenatured into single strands, specific primers are annealed to these DNA strands, and primers
complementary to the single-stranded DNA are extended by a thermostable DNA polymerase.
These steps are repeated, doubling the target sequence to the initial number in each cycle.(9,10)
ELISA is based on the antibody-sandwich technique, in which a food pathogen antigen
specifically bound to a support-immobilized antibody is combined with an enzymaticallyconjugated secondary antibody. This method is useful because it is accurate, sensitive, andspecific.(11–14) However, these two methods have problems in that they are complicated tooperate and difficult to introduce into the field owing to the need for large-scale and expensive
equipment. On the other hand, in immunochromatography, the lateral flow test strip
immunoassay using gold nanoparticle labels is known as a simple and inexpensive method. As
the sample fluid flows laterally across the test strip, the antibody-immobilized gold nanoparticles
interact with target analytes within the fluid and subsequently aggregate at the test line owing to
specific interactions between the antigen and the antibody. At this time, the test line is coloredby the localized surface plasmons of the gold nanoparticles. This method is rapid and simple, but
the quantification is difficult and the detection limit is high (>105 cells/mL).(15) We previously
reported that in a mixed solution of aniline and metal ions, the polymerization and nanoparticle
formation reactions proceed simultaneously in the same reaction field, forming nanometer-sizedorganic–inorganic hybrid particles.(16,17) Furthermore, we clarified that these organic–inorganic
hybrid particles have optical and electrochemical characteristics.(18–23) In this study, we utilized
this method to form copper nanostructures. As the nanostructures exhibit characteristic
electrochemical properties based on copper nanoparticles, we used them as electrochemicallabels to detect S. aureus. The binding properties of the nanostructures to bacterial cells were
evaluated spectroscopically and electrochemically.

2.

Materials and Methods

2.1

Bacterial culture and sample preparation

S. aureus was purchased from the National Institute of Technology and Evaluation Biological
Resource Center (NBRC, Japan). Bacterial cultures and experiments were performed in abiosafety level 2 laboratory and were developed and managed in accordance with the appropriate
safety regulations (WHO Laboratory Biosafety Manual). The bacterial strain was cultured in anagar growth medium (E-MC35, Eiken Chemical Co., Japan) for 18 h. A single colony was thenselected and placed in a liquid growth medium and incubated for 18 h. The suspension cultures
were then centrifuged for 5 min, and the precipitate was resuspended in sterilized ultrapure
water by shaking for 1 min.
2.2

Preparation of copper nanostructures

The ultrapure water (>18 MΩ) used in this experiment was sterilized using UV light. In
addition, reagent-grade chemicals such as copper(II)sulfate pentahydrate, aniline, and ethanol

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were used for the preparation of copper nanostructures, all of which were purchased from
Fujifilm Wako Chemicals, Co. (Japan). An aqueous copper sulfate solution and a 0.10 M aniline
ethanol solution were added to the ultrapure water, stirred at 298 K for 30 min, and thencentrifuged. The supernatant was disposed to remove unreacted substances and the precipitate
was dispersed in ultrapure water. The obtained dispersion was ultrafiltrated to obtain copper
nanostructures. The size distribution of the nanostructures in ultrapure water was measured
with a zeta-potential and particle size analyzer (ELSZ-2Plus, Otsuka Electronics, Japan).
2.3

Immobilization of antibodies on copper nanostructures

The goat anti-S. aureus antibody (Kirkegaard & Perry Laboratories, Inc., U.S.) was
immobilized on copper nanostructures using N-hydroxy-succinimide sodium salt (NHS) and1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) as a cross-linking agent.
EDC and NHS were added to an anti-S. aureus antibody and stirred for 30 min. Phosphatebuffered saline (PBS, pH 7.0) was added to the mixed solution and centrifuged to obtain the
EDS/NHS-modified antibody. Copper nanostructure dispersion was added to the EDC/NHSmodified antibody and stirred for 2 h.
2.4

Optical characterization of copper nanostructures

Copper nanostructures were observed using a transmission electron microscope (TEM, JEM2000FXII, JEOL, Japan) at an acceleration voltage of 200 kV. Bacterial cells labeled with copper
nanostructures were observed using a field emission scanning electron microscope (FE-SEM,
SU8010, Hitachi High-Tech, Japan) at an acceleration voltage of 5 kV.
2.5

Pretreatment of carbon chip electrodes

In this experiment, a carbon chip electrode was used as shown in Fig. 1. Carbon ink was used
to screen-print the working, reference, and counter electrodes onto the resin substrate. The Ag/

Fig. 1.

(Color online) Illustration of carbon chip electrode.

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AgCl ink was applied to the reference electrode and allowed to dry for 5 min. After that, theresist portion of the carbon chip electrode was masked with a Teflon tape, and plasma wasirradiated at 10 mA for 1 min.
2.6

Electrochemical and absorbance measurements

The prepared S. aureus (106 –109 cells mL−1) and anti-S. aureus antibody-conjugated copper
nanostructures were mixed and stirred for 15 min. Then, each mixed dispersion was centrifuged
at 6,100 rpm (3,500 ×g) and 278 K for 30 min. The spectrum of the obtained supernatant wasmeasured with an ultraviolet-visible spectrophotometer (V-750, JASCO, Japan). The obtained
precipitate was dispersed in ultrapure water and then again centrifuged under the same
conditions as above (hereinafter called “washing operation”). The obtained supernatant was also
subjected to spectral measurement. The precipitate thus washed was dispersed in ultrapure
water. Three microliters of the dispersion was dropped onto the working electrode on the carbon
chip electrode and vacuum-dried at 298 K for 1 h. Fifty microliters of PBS was dropped on the
carbon chip electrode, and cyclic voltammetry (CV) and differential pulse voltammetry (DPV)
were carried out by potential scanning from −0.2 to +0.2 V using a potentio-galvanostat
ECstat-302 (EC Frontier Inc., Japan) under the following conditions: initial potential, −0.2 V;
initial scan direction, positive. CV was performed by repeating three cycles of potential scans.

3.

Results and Discussion

Copper nanostructures were obtained as a bluish dispersion consisting of spheres with amean diameter of 146.4 ± 41.3 nm. In the TEM image, a structure in which numerous small
copper nanoparticles are tightly encapsulated inside the polymer was observed with distinctly
different contrasts [Fig. 2(a)]. In the SEM image, it was confirmed that the copper nanostructures
bound to the bacterial cells [Fig. 2(b)]. This indicates that the antibodies introduced into thenanostructure bound to the cell surface through specific binding with the antigen.

(a)

(b)

Fig. 2. (a) TEM image of copper nanostructure. The scale bar is 100 nm. (b) SEM image of copper-nanostructurelabeled cell. The scale bar is 500 nm.

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In CV and DPV, current responses derived from two-step oxidation reactions of Cu0 to Cu(I)and Cu(I) to Cu(II) were observed (Fig. 3).(24) This indicates that the nanostructures not only
function as electrochemical labels but also hold the copper nanoparticles in a stable form. The
decrease in current response with increasing number of cycles was attributed to the oxidation of
Cu0 in the nanostructures and Cu2+ diffused into the electrolyte [Fig. 3(a)]. The current response
at +0.08 V based on the oxidation reactions of Cu(I) into Cu(II) was observed prominently in
DPV [Fig. 3(b)].
The presence of polyaniline with a low degree of polymerization made it possible to introduce
antibodies into the nanostructures via an amide coupling reaction.(16,21) It was easily predictedthat antibody-introduced nanostructures would specifically bind to cells via the antigen andfunction as labels. Specific binding proceeded spontaneously in the mixture of labels and target
bacterial cells. Centrifuging the mixture of S. aureus (1.0 × 109 cells mL−1) and labels yielded a
bluish supernatant [Fig. 4(A-a)]. This indicated that there were free labels that were unbound tothe cells. On the other hand, the supernatant after the washing operation became colorless andtransparent [Fig. 4(A-b)]. At this time, the resulting bluish precipitate originating from thecopper nanostructures was obtained, confirming the binding of the label to the target bacterial
cells. To investigate the nonspecific adsorption of nanostructures to bacterial cells, absorbanceand electrochemical measurements were performed using copper nanostructures without
antibody modification. A slight absorption peak at 620 nm derived from the copper nanostructure
was observed in the supernatant after the washing operation. This is due to the exfoliation ofcopper nanostructures nonspecifically adsorbed to bacterial cells. Nonspecifically adsorbed
copper nanostructures were removed by washing, but it was presumed that some of them werestill adsorbed. However, the precipitates did not exhibit the current response attributed to thecopper nanostructures. It was suggested that nonspecific adsorption was completely inhibited or
did not affect electrochemical measurements owing to sensitivity.
A significant absorption peak was observed at 620 nm in the absorption spectrum of themixed dispersion of bacterial cells and labels. The peak intensity did not change even when thenumber of bacterial cells was increased. Therefore, centrifugation was performed to separate(a)Fig. 3.

(b)(Color online) (a) CV and (b) DPV results of copper nanostructures in PBS (pH 7.0).

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(A)

(a)

(b)

(a)

(b)

(B)

(c)
Fig. 4. (Color online) (A) Photos and (B) absorbance spectra of the mixture of bacterial cells and labels (a) beforeand (b) after the washing operation, and (c) plot of ΔI at 620 nm before the washing operation. ΔI was obtained bysubtracting the peak intensity of the supernatant at the number of each bacteria (I) from that without bacteria(I0 = 0.24).

unbound free labels and nonspecifically adsorbed labels from labeled bacterial cells. A
prominent absorption peak was also obtained at 620 nm in the absorption spectrum in the
supernatant, and the peak intensity decreased as the number of bacterial cells increased [Fig.
4(B-a)]. On the other hand, in the absorption spectrum of the supernatant after the washingSensors and Materials, Vol. 35, No. 10 (2023)(a)Fig. 5.

4767(b)(Color online) (a) DPV results of precipitate and (b) plot of peak current (Ip) at +0.08 V (n = 3).

operation, the absorption peak at 620 nm disappeared [Fig. 4(B-b)]. This change in absorbance
was based on free and nonspecifically adsorbed labels obtained by removing labeled bacterial
cells from the mixed dispersion [Fig. 4(B-a)]. Therefore, ΔI increased with the number ofbacterial cells owing to the decrease in the number of labels not bound to the bacterial cells [R2 =0.9997, Fig. 4(B-c)]. In this way, we succeeded in the spectroscopic quantification of target
bacteria by focusing on labels that did not specifically bind.
In the DPV of labeled bacterial cells obtained as precipitates, the current response resulting
from the oxidation of copper nanostructures was obtained at +0.08 V [Fig. 5(a)]. The peakcurrent (Ip), based on the number of labels bound to S. aureus on the electrode, increased with
the number of bacterial cells [R2 = 0.9850, Fig. 5(b)]. On the basis of this result, theelectrochemical quantification of specifically bound labels was also successful.

4.

Conclusions

We have developed copper nanostructures with characteristic spectroscopic andelectrochemical properties and clarified that they function as labels by introducing antibodies
into these structures. Complementary results were obtained by evaluating supernatants andprecipitates from mixtures of labels and target bacterial cells. Therefore, it was clarified that theuse of such labels enables the detection of target bacterial cells by spectroscopic andelectrochemical techniques. However, the detection limits of both techniques were notsatisfactory. In the future, it will be necessary to investigate the increase in the number of copper
nanoparticles in the nanostructures. Since this method can respond to various target bacterial
cells by introducing antibodies into nanostructures, it is useful for measuring various bacteriathat cause not only food poisoning but also infectious diseases.

Acknowledgments
We gratefully acknowledge the financial support provided by a JST START Grant (Number
JPMJST1916). We also acknowledge the financial support from the Japan Society for the4768

Sensors and Materials, Vol. 35, No. 10 (2023)

Promotion of Science Grant-in-Aid for Scientific Research (A) (KAKENHI 21H04963) and
Grant-in-Aid for Challenging Exploratory Research (22K18442).

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