Electrochemical Biosensing Interface Based on Carbon Dots-Fe3O4 Nanomaterial for the Determination of Escherichia coli O157:H7

Escherichia coli (E. coli) O157:H7 can cause many food safety incidents, which seriously affect human health and economic development. Therefore, the sensitive, accurate, and rapid determination of E. coli O157:H7 is of great significance for preventing the outbreak and spread of foodborne diseases. In this study, a carbon dots-Fe3O4 nanomaterial (CDs-Fe3O4)-based sensitive electrochemical biosensor for E. coli O157:H7 detection was developed. The CDs have good electrical conductivity, and the surface of carbon dots contains abundant carboxyl groups, which can be used to immobilize probe DNA. Meanwhile, the CDs can be used as a reducing agent to prepare CDs-Fe3O4 nanomaterial. The Fe3O4 nanomaterial can improve the performance of the electrochemical biosensor; it also can realize the recovery of CDs-Fe3O4 due to its magnetism. As expected, the electrochemical biosensor has excellent specificity of E. coli O157:H7 among other bacteria. The electrochemical biosensor also exhibited good performance for detecting E. coli O157:H7 with the detection range of 10–108 CFU/ml, and the detection limit of this electrochemical biosensor was 6.88 CFU/ml (3S/N). Furthermore, this electrochemical biosensor was successfully used for monitoring E. coli O157:H7 in milk and water samples, indicating that this electrochemical biosensor has good application prospect. More importantly, this research can provide a new idea for the detection of other bacteria and viruses.


INTRODUCTION
In recent years, food poisoning caused by foodborne pathogens has frequently occurred worldwide, which is the most prominent public health problem in the world (Hou et al., 2020;Duan et al., 2021). As one of the most hazardous foodborne pathogens, Escherichia coli (E. coli) O157:H7 can cause some diseases such as hemorrhagic colitis and hemolytic uremic syndrome. What is more, low-dose E. coli O157:H7 can make humans sick, and in severe cases, it can even cause kidney failure and death. Currently, more than two million acute foodborne diseases worldwide are attributed to E. coli O157:H7 each year (Li et al., 2015a;Zhou et al., 2020). Therefore, it is very important to monitor the E. coli O157:H7 with high sensitivity. At present, some bioanalytical methods have been applied for E. coli O157: H7 detection, including enzyme-linked immunosorbent assay (ELISA) (Guo et al., 2016;Zhao et al., 2020), lateral flow immunochromatographic detection technology (Xie et al., 2014;Ye et al., 2020), polymerase chain reaction (PCR) (Dong et al., 2015), and so on. These alternative assays are the most rapidly used tests because of their good specificity and stability. Nevertheless, they are still faced with some limitations in detecting foodborne pathogens, including low sensitivity, complicated pretreatment, and high environmental impact. Therefore, there is an urgent need to establish a sensitive, simple, and highly specific method for detecting E. coli O157: H7. At present, many different electrochemical sensors have been used in food safety due to their some advantages, such as high sensitivity, economy, and accuracy (Li et al., 2015b;Li et al., 2016;Dong et al., 2020). Therefore, electrochemical biosensors for the detection of E. coli O157:H7 have received extensive attention.
In this study, a CDs-Fe 3 O 4 nanomaterial-based electrochemical biosensor is constructed for detecting E. coli O157:H7, which is highly sensitive and reliable ( Figure 1). The CDs have good electrical conductivity and large specific surface areas to improve the sensitivity of the sensor. The CDs also can be used as the reducing agent to synthesize the CDs-Fe 3 O 4 nanomaterial. The Fe 3 O 4 can improve the catalytic performance of the electrochemical biosensor, and it also can realize the recovery of CDs-Fe 3 O 4 due to its magnetism. This electrochemical biosensor can achieve a good linear and detection limit for monitoring E. coli O157:H7. In addition, the biosensor has been also successfully applied to measure the E. coli O157:H7 in milk, indicating that the electrochemical biosensor has a broad application prospect. What is more, this study can provide a new idea for the preparation of electrochemical biosensors for other bacteria and virus detection, such as Staphylococcus aureus (S. aureus), Listeria, SARS-CoV-2, Hepatitis B virus, etc.

Synthesis of carbon dots Fe 3 O 4 nanomaterial
The preparation of CDs-Fe 3 O 4 nanocomposite was based on the method of instrument preparation of our group (Kuang et al., 2020): First, 3 g of citric acid and 1 g of glucose were added in 5 ml H 2 O and reacted under microwave with a radiation power of 900 W for 6 min to synthesize CDs. Second, the above solution was cooled and added 30 ml H 2 O, centrifuged them at 12,000 rpm for 15 min and took the supernatant, then dialyzed the supernatant for 24 h and then freeze dried. Third, the abovementioned CD solid was dissolved in 25 ml of the aqueous solution, then 1 g of sodium acetate and 1 g of ferric chloride was added into 15 ml of CD solution, the mixed solution was sonicated for 8 h, then placed in an autoclave, and reacted at 210°C for 24 h; after cooling, they were centrifuged at 12,000 rpm for 10 min to acquire CDs-Fe 3 O 4 nanomaterial.

Preparation of electrodes
First, 5.0 μl of CDs-Fe 3 O 4 nanomaterial was slowly dropped on the glassy surface of the carbon electrode (GCE) to prepare a CDs-Fe 3 O 4 /GCE working electrode. Second, the CDs-Fe 3 O 4 / GCE electrode was immersed in 1 ml of 0.1 mM PBS, 8.00 mM EDC, and 8.00 mM NHS mixed solution for 3 h to activate the electrode, then it was rinsed with TE buffer solution. Third, the above electrode was immersed in 1 ml of 0.1 μM probe DNA to react for 24 h, and it was rinsed with TE buffer solution to prepare the DNA/CDs-Fe 3 O 4 /GCE probe electrode. Finally, the prepared electrode was stored at 4°C.

Preparation of the biosensor
The prepared DNA/CDs-Fe 3 O 4 /GCE was immersed into different concentrations (C) of E. coli O157:H7 solutions and reacted for 40 min at 42°C in a water bath. After the reaction, TE buffer solution was used to wash off non-specifically adsorbed Frontiers in Chemistry | www.frontiersin.org November 2021 | Volume 9 | Article 769648 E. coli on the surface to obtain the E. coli O157:H7-DNA/CDs-Fe 3 O 4 /GCE. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) have characterized the layer-by-layer assembly of each modified electrode: the [Fe (CN) 6 ] 3-/4has been applied as the electrolyte. For the test parameters of CV, the potential scanning range was −0.2-0.8 V, and the scan rate(5) was 0.1 V/s. DPV was used to record the electrochemical signal changes before and after the identification of the electrode and different substances, and the potential interval was −0.2-0.8 V, the amplitude was 0.05 V, the pulse width was 0.05 s.

Characterization of nanomaterial
Transmission electron microscopy (TEM) has characterized the morphologies of CDs and CDs-Fe 3 O 4 nanomaterial. As shown in Figure 2A, the particle size distribution of the prepared CDs is relatively uniform, mainly at 3-4 nm. Figure 2B shows the highresolution TEM (HRTEM) image of CDs. It shows that CDs have a good crystal lattice, and the lattice spacing is 0.210 nm, which is a typical (002) carbon crystal plane. As shown in Figure 2C, the particle size of the CDs-Fe 3 O 4 nanomaterial was in the range of 15-40 nm. The HRTEM characterization of CDs-Fe 3 O 4 ( Figure 2D) shows that the nanomaterial has two lattice spacings, 0.345 and 0.210 nm, respectively. The corresponding crystal planes are (220) of the Fe 3 O 4 and (002) of the carbon. The above morphology characterization can indicate that CDs-Fe 3 O 4 nanomaterial has been successfully prepared.

Cyclic voltammetry behavior of electrodes
It is well known that [Fe(CN) 6 ] 3−/4− is often used as a supporting electrolyte for electrochemical biosensors (Fan et al., 2018;Li et al., 2021;Nano et al., 2021). The working electrodes were electrochemically characterized by CVs in 1.0 mM [Fe(CN) 6 ] 3−/4− and 0.1 M KCl solution ( Figure 3A). On the surface of GCE, the  Frontiers in Chemistry | www.frontiersin.org November 2021 | Volume 9 | Article 769648 redox peak current (I) has good reversibility, and the oxidation peak potential (I pa ) and reduction peak potential (I pc ) are −6.

Effects of scan rate
The effect of different v on the electrochemical response of DNA/ CDs-Fe 3 O 4 /GCE in [Fe(CN) 6 ] 3−/4− has also been studied. Figure 4A shows that in the process of increasing v, the I also keeps increasing.
As shown in Figure 4B, the I pa and I pc show an excellent linear relationship with ] 1/2 , and the linear regression equations are I pa 3.598 + 80.265 v 1/2 and I pa −2.551-85.187 v 1/2 , respectively. The results show that the electrochemical behavior of [Fe(CN) 6 ] 3−/4− on the surface of DNA/CDs-Fe 3 O 4 /GCE is mainly affected by the diffusion-controlled process (Alam and Deen, 2020).

Effect of interaction time
The interaction time between the probe electrode and the target substance is one of the key factors affecting the performance of the biosensors. Therefore, the interaction time of E. coli O157:H7 with DNA/CDs-Fe 3 O 4 /GCE was studied. When the C of E. coli O157:H7 was 10 CFU/Ml, the optimum hybridization time was determined to be 40 min (Supplementary Figure S1). However, when the C of E. coli O157:H7 was 10 8 CFU/Ml, the optimum hybridization time was determined to be 35 min (Supplementary Figure S2). Therefore, in this electrochemical biosensor, the best reaction time between E. coli O157:H7 and DNA/CDs-Fe 3 O 4 / GCE is 40 min.

Specificity of the biosensor
The specificity of the electrochemical biosensor is a necessary factor to ensure the accuracy of the biosensor. Therefore, the interferences of different bacteria with 10 5 CFU/ml on the DNA/ CDs-Fe 3 O 4 /GCE through DPV have been investigated, such as S. aureus, Salmonella, Staphylococcus lactis (S. lactis), and Listeria. Figure 5A shows that DNA/CDs-Fe 3 O 4 /GCE has almost no effect on other bacteria. However, the DNA/CDs-Fe 3 O 4 /GCE has a very strong response to E. coli O157:H7, and the I pa drops by 53.42%. As shown in Figure 5B, other interfering substances have almost no effect on the determination of E. coli O157:H7. The above results show that the electrochemical biosensor based on DNA/CDs-Fe 3 O 4 /GCE for detecting E. coli O157:H7 has very excellent specificity and is expected to realize the determination of E. coli O157:H7 in real samples.

Sensitivity of the biosensor
As is known, DPV has been extensively applied in the construction of electrochemical sensors because it has good detection sensitivity. This study explores the sensitivity and   Figure 6A shows that when E. coli O157:H7 interacts with DNA/CDs-Fe 3 O 4 /GCE, I pa decreases as the C of E. coli O157: H7 increases. The main possible reason is E. coli O157:H7 has poor electrical conductivity, which affects the electrical conductivity of the working electrode's interface, leading to a weakening of the electrochemical signal. When the C of E. coli O157:H7 is in the range of 10-107 CFU/ml, the lgC and I pa show a good linear relationship ( Figure 6B): I pa 1.5422 lgC-14.387. At the same time, the detection limit (LOD) can reach 6.88 CFU/ ml. Compared with other electrochemical biosensors for monitoring E. coli O157:H7 (Table 1), this electrochemical biosensor based on DNA/CDs-Fe 3 O 4 /GCE has very good sensitivity.

Repeatability and stability of the biosensor
To investigate the reproducibility of this biosensor, six DNA/ CDs-Fe 3 O 4 /GCE working electrodes were prepared, and they have been applied to detect 10 5 CFU/ml of E. coli O157:H7 respectively. Supplementary Figure S3 shows that the relative standard deviation (RSD) was 2.6%, which indicates the electrochemical biosensor based on DNA/ CDs-Fe 3 O 4 /GCE for E. coli O157:H7 detection has good reproducibility. The stability of the electrochemical biosensor has been also studied; the DNA/CDs-Fe 3 O 4 /GCE was placed in a dryer at 4°C, and the above working electrode was applied to measure 10 5 CFU/ml of E. coli O157:H7 every 3 days. As shown in Supplementary Figure S4 after 30 days, the I pa was only dropped by 2.7%, which shows that the electrochemical biosensor based on DNA/CDs-Fe 3 O 4 /GCE has good stability.

Analysis of real samples
Since milk and water are often contaminated by E. coli O157:H7, it is very necessary to efficiently determine E. coli O157:H7 in milk and water. In this study, to verify the practical applicability of the biosensor, the electrochemical biosensor is used to determine the E. coli O157:H7 in milk and water.   Frontiers in Chemistry | www.frontiersin.org November 2021 | Volume 9 | Article 769648 shows that 10 3 , 10 4 , and 10 5 CFU/ml of E. coli O157:H7 were added to the samples, and the recovery rates are between 95.2% and 102.0%, At the same time, the results were consistent with ELISA results, indicating that the method is feasible for monitoring E. coli O157:H7 in milk and water samples.

CONCLUSIONS
In conclusion, a sensitive electrochemical DNA biosensor has been prepared for monitoring E. coli O157:H7 by CDs-Fe 3 O 4 nanomaterial. Under the best conditions, when the C of E. coli O157:H7 is in the range of 10-10 8 CFU/ml, the lgC has a good linear relationship with the I pa , and the LOD reaches 6.88 CFU/ml. At the same time, the electrochemical DNA biosensor has been successfully used to determine E. coli O157:H7 in milk and water samples. Therefore, this research provides an effective biosensor manufacturing strategy, which is expected to provide a reference for the preparation of electrochemical DNA biosensors for other bacteria or viruses detection.  9.96 × 10 3 99.6 9.53 × 10 3 10 5 1.02 × 10 5 102.0 9.91 × 10 4 Water 10 3 9.93 × 10 2 99.3 9.86 × 10 2 10 4 9.89 × 10 3 98.9 9.81 × 10 3 10 5 9.79 × 10 4 97.9 9.69 × 10 4