Colorimetric Detection System for Salmonella typhimurium Based on Peroxidase-Like Activity of Magnetic Nanoparticles with DNA Aptamers

Recently, much attention has been devoted to food-related health issues. In particular, food-poisoning bacteria are becoming a serious threat to human health. So far, techniques used to detect these bacteria are time-consuming and laborious. To overcome these challenges, a biosensor with a simple platform was developed to detect Salmonella typhimurium. The colorimetric strategy is attractive because it enables simple and rapid sensing with the naked eyes. We used magnetic nanoparticles (MNPs), specific aptamers, and a colorimetric substrate, 3,3,5,5-tetramethylbenzidine (TMB) in the presence of H 2 O 2 . Because MNPs display enzyme-like activities, they can undergo color changes with the help of a colorimetric substrate. In this system, MNPs were first incubated with aptamers that specifically interact with the Salmonella species, reducing the peroxidase activity of the MNPs via DNA-mediated shielding of catalytic activity. After the addition of Salmonella cells to the solution, specific aptamers on the MNPs interact with the Salmonella, consequently enhancing the peroxidase activity of the MNPs. Considering their low cost, easy separation, and stable activity, MNPs could be applied to various detection systems.


Introduction
Food-poisoning bacteria have been a serious threat to human health for the past years.Salmonella especially is one of the major pathogens causing intestinal infection.They can spread from poultry, eggs, and vegetables to humans or livestock during handling and distribution [1].The consumption of food contaminated with bacteria can lead to immune deficiencies and other symptoms such as fever, diarrhea, and even death within 12 to 72 hours.Lately, great attention about well-being food becomes a cause for increasing infection to human bodies, leading to medical costs and mass recall in the food industry.According to the Center for Disease Control and Prevention (CDC), infection by Salmonella has been growing every year, and value is estimated to be about 19,000 hospitalizations and 380 deaths [2,3].Thus, the whole world has demanded a fast and simple detection method before people consume the contaminated food.
Common procedures for the detection of food-poisoning bacteria involve culture methods and polymerase chain reaction (PCR) followed by gel electrophoretic analysis [4,5].However, PCR-based methods require tedious experimental procedures and long analysis times, which cannot prevent distribution among food markets.Recently, colorimetric methods for the detection of bacteria, employing noble metal nanoparticles such as gold and TiO 2 , have become popular [6][7][8][9].For example, the aggregation of gold nanoparticles (AuNPs) results in a shift in the absorption spectrum and a color change from red to purple.Furthermore, nanomaterials conjugated with antibodies have a unique possibility in detecting bacteria or other molecules.Nevertheless, these methods have several limitations that depend on experimental conditions (e.g., salt concentration, pH, and temperature) and are subjected to complicated steps during the preparation and conjugation of antibodies on the nanoparticles [10].In addition, fluorescence-based assays based on natural enzymes like horseradish peroxidase have been studied.However, enzymes as biological catalysts can be easily digested and denatured [11].
To overcome such challenges, aptamers and magnetic nanoparticles (MNPs) have been provided a substitute choice to meet the requirements.Aptamers are oligonucleotides by SELEX (systematic evolution of ligands by exponential enrichment).Created aptamers can specifically bind to target molecule forming a unique structure similar with antibodies.Furthermore, owing to inexpensive and stable features in various experimental conditions, aptamers are useful in biotechnology [12,13].In this study, aptamers that interact with outer membrane protein on the surface of S. typhimurium were used [14,15].As another well-known materials, MNPs exhibit catalytic stability, ease to separation, and chemical inertness.In this system, MNPs promote the oxidation of 3,3  ,5,5 tetramethylbenzidine (TMB) as a colorimetric substrate by peroxidase-like activity within several minutes in the presence of H 2 O 2 [16,17] 18].Because of the high oxidizing ability of radical, it mediates oxidation of TMB.This reaction produces a blue-colored product, which enables colorimetric detection with naked eyes.Herein, the purpose of colorimetric method using MNPs and DNA aptamers is the prior detection of contaminated food before its distribution among market places.Furthermore, the development of methods for a fast and stable detection of bacteria is of great significance in avoiding and controlling Salmonella pathogens.(hydrogen peroxide) was obtained from Junsei (Tokyo, Japan) and ethanol was purchased from Samchun Chemical (Pyeongtaek, Korea).DNA aptamers with or without fluorophores (oligomer: 5  -GAGGAAAGTCTA-TAGCAGAGGAGATGTGTGAACCGAGTAA-3  ) were synthesized by Macrogen (Seoul, Korea) with MOPC purification method [14,15].Luria-Bertani (LB) broth and agar media were obtained from LPS solution (Daejeon, Korea).All experiments were conducted with ultrapure deionized (DI) water using a Milli-Q water purifier from Merck Millipore (Billerica, MA).

Synthesis of Fe 3 O 4 Magnetic Nanoparticles (MNPs).
MNPs were synthesized by a simple and convenient method using precipitation and ultrasonication.FeCl 2+ (0.25 M) and FeCl 3+ (0.25 M) (Fe 3+ /Fe 2+ = 2) were added to 50 mL of ultrapure DI water.Then, a 1 M NaOH solution was added dropwise until the pH reached 10.0.The color of the ferrous and ferric solution changed from bright brown to black.The solution was sonicated at a frequency of 40 kHz and an ultrasonic power of 100 W at 80 ∘ C.After the reaction for 35 min, the resulting black MNPs were collected by neodymium magnetic separation and washed with water and ethanol several times.The final suspension was dried overnight in a vacuum oven at 60 ∘ C.

Characterizations.
Field-emission transmission electron microscopy (FE-TEM, 200 kV) (JEM-2100F, Jeol, Japan) and X-ray diffractometry (XRD, NEW D8-Advance, Bruker-AXS, Madison, WI) were used to analyze the morphology and structural features of the synthesized MNPs.The potential of the MNPs was obtained by measuring the zeta potential (ELSZ-1000, Otsuka, Japan) to determine the capture efficiency.A multimode microplate reader (Synergy H1, BioTek, Winooski, VT) was used at all experimental steps.

MNP-Based Colorimetric Assay with TMB.
TMB is used as a typical chromogenic substrate and can act as a donor of hydrogen for reduction of H 2 O 2 by peroxidase.In common with enzyme, the resulting change of colors could verify the catalytic activity of the MNPs.To prepare well-dispersed solutions, MNPs in ultrapure DI water (1 mg/mL) were placed in the sonicator bath for 30 min.The white TMB powder (0.5 M) was dissolved in DMSO, and the solution was diluted in ultrapure water.MNPs were diluted in 0.1 M acetate buffer (pH 4.0) and incubated with TMB and H 2 O 2 for 10 min at 42 ∘ C.After the reaction, the mixture was immediately separated using an external magnet for 30 s.The supernatant was used to obtain the absorbance intensity at 650 nm.

Microorganism and Culture Conditions.
To validate the colorimetric detection system, we used S. typhimurium ATCC 14028.Bacteria were grown in LB broth at 37 ∘ C with gentle shaking at 200 rpm.Furthermore, we carried out visible plate counting using agar plates after incubation for 24 h at 37 ∘ C. Finally, we determined that the number of S. typhimurium ATCC 14028 was 3.75 × 10 8 CFU/mL at an optical density (OD) of 1.0.In the experiment, the cultured bacteria (1 mL) were centrifuged at 13,000 rpm for 1 min at an OD of 1.0, and the pellet was then diluted in ultrapure DI water before the experimental step.

Preparation and Characterization of MNPs.
MNPs were prepared by the coprecipitation method with sonication.After introducing sonochemistry, the peroxidase-like activities of Fe 3 O 4 MNPs increased and resulted in small spherical particles that were below 20 nm in diameter (Figures 1(a) and 1(b)).The decrease in particle size was necessary to improve the peroxidase-like activities of MNPs [19].To further identify characteristic features, MNPs were examined using XRD (Figure 1(c)).As a result, all peaks corresponded to Fe 3 O 4 (JCPDF card number 00-024-0081).

Effect of Conditions on Catalytic Properties of MNPs.
Before starting the experiment, we assessed the catalytic activity of each component in the system.Where MNPs were present with TMB and H 2 O 2 , a colorimetric response was observed with a high absorption peak at 650 nm compared with the other samples (red solid line in Figure 3).On the other hand, significantly reduced or no signals were generated when H 2 O 2 (blue solid line), TMB (blue dashed line), or MNPs (black dashed line) were excluded.These results indicated that the catalytic activity of MNPs was induced when TMB and H 2 O 2 were mixed together.Importantly, no colorimetric signal was observed in the DNA@TMB@H 2 O 2 sample (red dash line), showing that the DNA aptamers did not contribute to the oxidation of TMB.
The absorption of DNA aptamers on the surface of the MNPs contributed to colorimetric assay, and this phenomenon led to the inhibition of the peroxidase-mimicking activity.We first evaluated the charge of the MNP surface using zeta potential analysis in order to explore the absorption property of MNPs with or without DNA aptamers.Thus, we confirmed the change in charge on the surface of the MNPs after introducing the DNA aptamers.The potential of pristine MNPs was positive (+16.89),but it turned into a negative value (−27.18) with the presence of DNA aptamers, as shown in Figure 4(a).This result showed that the negatively charged DNA aptamers immediately reacted with the positively charged surface of the MNPs via electrostatic interactions.To further validate the interaction, the amount of DNA aptamers adsorbed onto the MNPs was calculated by measuring the relative fluorescence units (RFU).However, a decline in MNP concentration would decrease the capture efficiency.This means that the shielding effect of MNPs exhibited differences in ability at various concentrations as a result of steric hindrance and competitive reaction.Additionally, we determined the optimal conditions of this detection system, as shown in Figure 6.After performing buffer tests using Tris-HCl, HEPES, PBS, and acetate buffer in various pH conditions (data not shown), we found that acetate buffer at pH 4.0 was a suitable buffer solution for this system (Figure 6(a)).The significant signal from the oxidation of TMB was correlated with the concentration of MNPs.A concentration that is either too high or too low Wavelength (nm) Figure 3: UV-vis absorption spectra (a) and bar graph at 650 nm (b).The effect of each reagent was tested in the colorimetric system.MNPs@TMB@H 2 O 2 : red solid line, MNPs@TMB: blue solid line, MNPs@DNA@H 2 O 2 : red dashed line, MNPs@H 2 O 2 : blue dashed line, and TMB@H 2 O 2 : black dash line.The insets in (b) represent images of the well plates for the different colors in the five cases.would lead to a weak intensity in the UV-vis spectrum.We confirmed that the best MNP concentration was 400 g/mL (Figure 6   MNP antibody PCR 10 1 -10 7 CFU/mL 10 3 CFU/mL [5] line) displayed a significant reduction in color response compared with the control (red solid line) owing to the shielding effect.To demonstrate the capability of the colorimetric system, Salmonella (7.5 × 10 5 CFU/mL) was added to the MNPs@DNA aptamers solution.Immediately, DNA aptamers were detached from the surface of the MNPs because of their strong affinity to Salmonella, which led to the reexposure of the MNP surface to TMB.Finally, the intensity of absorbance and color response were regained in comparison with a sample containing DNA aptamers only.Table 1 summarizes the detection of Salmonella using various methods.It is presented that our proposed method has no outstanding performance compared to other spectrophotometric or fluorometry methods in the lower detection limit.However, most of the assays introduce antibodies and other nanomaterials.Detection method based on the peroxidaselike activity of MNP and DNA aptamer does not exist.Thus, this study is very meaningful as a proof-of-concept (POC) experiment for the colorimetric detection of pathogen.Much remains to be done with enhancement of sensitivity at further improvement.

Conclusion
In summary, a simple and rapid colorimetric system based on MNPs and DNA aptamers was developed for the detection of S. typhimurium, which relies on increasing signal from the peroxidase-like activities of MNPs.The developed colorimetric system required a short assay time of only 10 min, and results could be verified with the naked eyes.Furthermore, MNPs and DNA aptamers did not require surface modification.The method was cost-effective and simple, unlike biosensors based on antibodies or fluorophores.After optimization, the system was able to visibly detect bacteria up to 7.5 × 10 5 CFU/mL in buffer solution.Compared with antibody-immobilized MNPs that have been reported, the sensitivity of this colorimetric system was efficient.The advantage of the new system reveals its great potential application as a point-of-care testing sensor.Thus, we are still investigating the development of MNPs to enhance the detection limit and to extend the cross-reactivity to other bacteria.
Using the Peroxidase-Like Activities of MNPs.The basic principle of colorimetric detection is shown in Figure2.In the procedure, the MNPs

Figure 1 :
Figure 1: TEM images and XRD data of MNPs.Scale bars are 50 nm (a) and 20 nm (b).XRD patterns (c) indicate that the synthesized MNPs were Fe 3 O 4 based on JCPDF data.

H 2 O 2 Figure 2 :
Figure 2: Schematic illustration of the MNP-based colorimetric detection using label-free DNA aptamers and TMB.

Figure 4 :
Figure 4: Absorption of DNA aptamers onto the MNPs confirmed by zeta potential (a) and relative fluorescence units (RFU) using FAMaptamers (b).
(b)).In order to determine the effect of TMB on the generation blue color, g/mL of MNPs has reacted with various concentrations of TMB (Figure 6(c)) and H 2 O 2 (Figure 6(d)).The concentrations of TMB and H 2 O 2 chosen in this study were 40 M and 35 mM, respectively.Finally, the solution was incubated for 10 min (Figure 6(e)).

3. 4 .Figure 5 :
Figure 5: UV-vis absorption spectra (a) and bar graph at 650 nm (b) obtained from solutions containing different concentrations of MNPs with DNA aptamers.The insets in (b) represent images of the well plates for the different cases, with or without DNA aptamers.

Figure 6 :
Figure 6: Optimization of parameters for the colorimetric system.Acetate buffer at pH 4.0 was optimal for TMB oxidation (a).MNP concentration of 400 g/mL (b), TMB concentration of 40 M (c), H 2 O 2 concentration of 35 mM (d), and incubation time of 10 min (e) yielded optimal effectiveness.

Figure 7 :
Figure 7: UV-vis absorption spectra (a) and bar graph at 650 nm (b).The insets in (b) represent images of the well plates for the colorimetric detection of Salmonella sp.
. Nonetheless, mechanism of enzymelike activity is not known exactly.Once H 2 O 2 molecules are adsorbed onto the surface of MNPs, Fe 2+ and Fe 3+ in the MNPs catalyze the disassembly of H 2 O 2 to radicals like • OH and O 2 −• /HO 2 • [

Table 1 :
Comparison of different methods for detection of Salmonella.