Foodborne pathogens that cause numerous illnesses have become a worldwide health problem [1
]. Clostridium perfringens
is one of the most common foodborne pathogens. This predominant pathogen is a spore-forming, rod-shaped, gram-positive bacterium widely found in different environments (e.g., soils and waters) and various foods (e.g., meats and dairy products). Many countries have implemented a limit for C. perfringens
since it is associated with two kinds of foodborne diseases: diarrhea and enteritis necroticans [2
]. Thus, the development of approaches to detect C. perfringens
is urgent and important.
Up to now, several methods for C. perfringens
detection have been developed. Conventional methods including bacteria cultivation and biochemical tests are usually reliable and accurate but labor-consuming and time-costing [5
]. In contrast, some newly developed methods based on molecule detection, such as polymerase chain reaction (PCR) [6
], enzyme-linked immunosorbent assay (ELISA) [8
], and rolling circle amplification (RCA) [9
], can achieve rapid detection compared with conventional methods. Nevertheless, these methods often require complex procedures, expensive instruments and experts [10
]. Therefore, it is significant to develop a facile, cheap and effective approach to detect C. perfringens
In recent years, electrochemical DNA biosensors are widely applied to the specific detection of target DNA (tDNA) via hybridization with complementary DNA probe owing to their obvious advantages such as fast response, facilitate manipulation, low cost and high sensitivity [12
]. The immobilization of DNA probe on electrode surface is an essential issue to construct an electrochemical DNA biosensor [13
]. There are two types of DNA immobilization: covalent and non-covalent strategies [14
]. Covalent approaches can be prepared through covalent bonding interaction, for example, Au-S bond [15
], Ag-N bond [16
], and amide bond [17
]. However, there are some disadvantages in the application of labeled DNA due to the complex operation and expensive biochemical preparation. Contrarily, biocompatible nanomaterials are widely applied to immobilize DNA via non-covalent strategies, such as aromatic stacking [18
], hydrogen bonding [19
], electrostatic interaction [20
], and hydrophobic force [18
]. To efficiently immobilize DNA probe and decrease non-specific adsorption of DNA, the interaction between nanomaterials and DNA is more considered [21
Cerium oxide (CeO2
), an important rare earth material, was chosen for electrochemical DNA biosensor owing to its exclusive properties, such as nontoxicity, good biocompatibility, high stability and forceful absorption capability [23
]. Over the years, various morphologies of CeO2
have been prepared, such as nanoparticles [23
], nanorods [24
], nanocubes [25
], nanoshuttles [26
], nanoplates [27
], etc. and the morphology of nanoceria has a conclusive effect on its properties. Tan et al. reported that Pd/CeO2
nanocubes showed higher catalytic activity than octahedrons and rods [28
]. Kang et al. found that the CeO2
nanorods have a higher adsorption capacity for fluoride compared with octahedron and nanocubes [29
]. A reason that different morphologies of CeO2
expose different crystal planes is they display reaction activity. Therefore, it is interesting to investigate the DNA adsorption properties of CeO2
nanomaterials with different shapes. In addition, to enhance the sensitivity of the DNA biosensor, electrical conductivity of synthesized nanoceria should be considered. Hence, nanostructured CeO2
with controlled size, specific morphology, excellent conductivity and high surface charge is attracting much attention in the development of electrochemical DNA biosensor.
Chitosan (CHIT), a macromolecule polysaccharide comprising plentiful amino and hydroxyl groups, is obtained from the deacetylation of chitin. Its application in designing a DNA biosensor is becoming increasingly popular due to its biocompatibility, non-toxicity, good adhesion and attractive film-forming ability [30
]. If CHIT is modified with CeO2
nanorods, it is possible to obtain novel functionalized materials that simultaneously have the properties of CHIT (specific surface area and electrical conductive properties) and CeO2
(high DNA adsorption capacity) through incorporating their individual characteristics. To our best knowledge, there is no report on employing CeO2
/CHIT nanocomposite as sensing material to detect Clostridium perfringens
nanorods were synthesized via a simple hydrothermal method and used as sensing materials to detect Clostridium perfringens
DNA sequence in dairy products. The rod-like CeO2
/CHIT nanocomposite was used for immobilizing the DNA probe on the electrode surface without employing any functional groups or intermediate linker. The preparation process of the DNA biosensor is described in Scheme 1
. The surface density of single-strand DNA (ssDNA) probe on the modified electrode was investigated using methylene blue (MB) as electrochemical probe. Electrochemical characterizations of the fabricated biosensor were carried out by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Under optimum conditions, the fabricated DNA biosensor exhibited high sensitivity, wide dynamic range, excellent selectivity, and satisfactory reproducibility and stability. Thus, the rod-shaped CeO2
-based biosensor can be utilized as a potential sensing platform to detect foodborne pathogens effectively and conveniently.
2.1. Materials and Reagents
Cerium nitrate hexahydrate (Ce(NO3)3·6H2O, 99.9%), methylene blue (MB) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) were provided by Shanghai Adamas Reagent Co., Ltd. (Shanghai, China). Chitosan (90% deacetylation degree) was purchased from Sam Chemical Technology Co., Ltd. (Guangdong, China). Tris-HCl was obtained from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). EDTA was provided by Tianjin Zhiyuan Chemical Reagent Co., Ltd. (Tianjin, China). Trichloroacetic acid (CCl3COOH) and acetonitrile (CH3CN) were purchased from Acros Organics (Beijing, China). All other chemicals were purchased from Tianjin Fengchuan Chemical Reagent Technologies Co., Ltd. (Tianjin, China), and were of analytical reagent grade. Milli-Q ultra-pure water (18.25 MΩ cm) was used to prepare the aqueous solutions.
Sequence of C. perfringens
gene was obtained from the National Center for Biotechnology Information (NCBI) database (accession No. NR_121697.2) [31
]. Specificities of the oligonucleotide sequences were identified by Basic Local Alignment Search Tool (BLAST). The oligonucleotides used in this work were synthesized and purified by Tsingke Biological Technology Co., Ltd. (Kunming, China) with the following sequences:
ssDNA: GCT CCT TTG GTT GAA TGA TG
tDNA: CAT CAT TCA ACC AAA GGA GC
one base-mismatched DNA: CAG CAT TCA ACC AAA GGA GC
three base-mismatched DNA: CAG CAT TCA ACT AAC GGA GC
non-complementary DNA: GGC GAG CGT TAT CCG GAT TT
The stock solutions of 20-mer oligonucleotides sequence (100 µmol/L) were prepared with TE buffer (10 mmol/L Tris-HCl and 1 mmol/L EDTA, pH 8.0), and kept frozen. The hybridization solutions consisted of 10 mmol/L Tris-HCl, 1 mmol/L EDTA and 0.1 mol/L NaCl (pH 7.4).
2.2. Synthesis of Nanoceria
nanorods were prepared by a modified hydrothermal method [32
]. Briefly, 3.84 g NaOH was dissolved in 15 mL of ultra-pure water to form a clear solution. Then, 1 mL of 0.8 mol/L Ce(NO3
O aqueous solution was added drop wise into 15 mL NaOH aqueous solution under continuously stirring and the white precipitate was generated immediately. After constantly stirring for 0.5 h, the mixed solution was transferred into an autoclave and heated at 100 °C for 24 h. The white products after hydrothermal treatment were washed by ultra-pure water and ethanol alternatively several times, and dried at 60 °C overnight. The CeO2
nanoparticles were prepared through the same methods at 100 °C for 24 h, while CNaOH
was 0.01 mol/L.
2.3. Apparatus and Characterization
The synthesized samples were characterized by X-ray powder diffraction (XRD) performed on a Bruker D8-Advance diffractometer with Cu Kα radiation (λ = 0.154 nm) in the 2θ range from 10° to 90°. Morphologies of obtained nanoparticles and nanorods were studied by transmission electron microscope (TEM, JEM-2100, JEOL Ltd., Tokyo, Japan). The ultraviolet-visible light (UV–vis) absorption spectra were recorded using a spectrophotometer (U-2001 Hitachi, Tokyo, Japan) and the analyzed range was 200–800 nm. X-ray photoelectron spectroscopy (XPS) was recorded by a K-Alpha+ (Thermo Fisher Scientific, Waltham, MA, USA) operating with Mono Al Kα radiation. The values of zeta potential were measured using a Malvern Zeta Sizer Nano (Malvern Instruments Ltd., Worcestershire, UK).
2.4. Preparation of the CeO2/CHIT/GCE
CHIT solution (2.0 mg/mL) was prepared by dissolving chitosan (100 mg) in 50 mL aqueous solution containing 1.0% acetic acid. CeO2 nanorods (5 mg) were stirred and sonicated in the CHIT solution for 30 min at room temperature to form a highly dispersed colloidal solution.
Before each experiment, a glassy carbon electrode (GCE) was sequentially polished with 0.3 µm and 0.05 µm Al2O3 powders, respectively. Then, it was cleaned ultrasonically in ultra-pure water and ethanol for 3 min. The CeO2/CHIT composite (7 µL) was dropped onto electrode surface and dried in air. The CHIT/GCE and CeO2/GCE were carried out through similar procedure.
2.5. DNA Probe Immobilization and Hybridization
Five microliters of ssDNA probe (1.0 × 10−7 mol/L) solution was pipetted onto the CeO2/CHIT electrode surface and dried at 25 °C for 30 min. Then, the ssDNA/CeO2/CHIT/GCE was rinsed with ultra-pure water to remove non-specific adsorption of DNA. The hybridization reaction was conducted by dropping 5 µL of tDNA solution onto the surface of ssDNA/CeO2/CHIT electrode and the reaction was kept at 50 °C for 30 min. Then, the dsDNA/CeO2/CHIT/GCE was rinsed with water to prevent unhybridized tDNA. The hybridization of ssDNA probe with one base-mismatched DNA, three base-mismatched DNA and non-complementary DNA were performed through similar procedure.
2.6. Electrochemical Measurements
Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) experiments were performed by utilizing a CHI 660E Electrochemical Workstation (Shanghai Chenhua Instrument) and connected to a three-electrode system including a modified CeO2/CHIT/GCE as a working electrode, a platinum wire served as counter electrode, and a saturated calomel reference electrode, which were performed in 0.1 mol/L sodium phosphate buffer (PBS, pH 7.4) containing 2.0 mmol/L [Fe(CN)6]3−/4− and 0.1 mol/L KCl. CV measurements was scanned between −0.2 V and 0.5 V (vs. SCE) at a scan rate of 0.05 V/s. The frequencies of EIS measurements were recorded from 0.1 Hz to 10 KHz at the amplitude of 5 mV. All electrochemical experiments were conducted in triplicate.
2.7. Real Sample Assay
Clostridium perfringens (ATCC 13124) was provided by Guangdong Huankai Microbial Sci. & Tech. Co., Ltd. (Guangdong, China). The bacteria were grown in cooked meat medium for 15 h at 37 °C. The DNA extraction process was carried out using a kit manufacturer (Tiangen Biotech Co., Ltd., Beijing, China). The DNA extraction efficacy was estimated by measuring absorbance at 260 nm using an ultraviolet spectrophotometer.
The pure milk and milk powder were purchased from a local supermarket. According to the methods reported previously [33
], 1 mL of CCl3
COOH, 1 mL of CH3
CN, 7 mL (for pure milk)/9 mL (for milk powder) of ultra-pure water were added into 2 mL of pure milk or 1 g of milk powder. Then, the mixture was ultrasonically extracted for 0.5 h and centrifugated at 11,500× g
for 10 min to eliminate protein. The resultant supernatant was adjusted to pH 7.40 with PBS and then used to prepare different concentrations of C. perfringens
gene from (1 to 50 pmol/L). Before the hybridization process, DNA sequence was denatured at 95 °C for 5 min into ssDNA. Detection process was performed under the optimum conditions.