High Specific DNAzyme-Aptamer Sensor for Salmonella paratyphi A Using Single-Walled Nanotubes–Based Dual Fluorescence-Spectrophotometric Methods

Abstract

In this work, single-stranded DNA aptamers that are highly specific to enterotoxigenic Salmonella paratyphi A were obtained from an enriched oligonucleotide pool using Systematic Evolution of Ligands by Exponential Enrichment (SELEX) to target the flagellin protein. The selected aptamers were confirmed to have high sensitivity and specificity to the flagellin. In addition, a probe (P0) containing the DNAzyme and fluorescein isothiocyanate–labeled aptamer3 sequences was employed as a dual probe for observing fluorescence and absorbance changes. The flagellin demonstrated low detection limits of 5 ng/mL by fluorescence and 20 ng/mL by spectrophotometry. Moreover, milk samples spiked with Salmonella paratyphi A were also detected, with the low detection limits increasing to 10⁵ CFU/mL by fluorescence and 10⁶ CFU/mL by spectrophotometry. The combination of fluorescence and spectrophotometry offers a specific, rapid, and sensitive way for detecting Salmonella paratyphi A and has potential for detecting other pathogens in food.

Introduction

Salmonella paratyphi A, a pathogenic microorganism found in food-borne infections, is a major source of diarrhea, fever, nausea, gastroenteritis, food poisoning, and other symptoms in humans and has a great impact on public health.^1,2 These symptoms are actually closely related to the flagellum. The flagellum extends from the cell membrane to form a helical propeller that enables the thallus to migrate to more favorable environments. ³ The flagellum can also play an important role in other processes such as substrate adhesion and host invasion in pathogenic bacteria. ⁴ Hence, flagella antigen can be used as a surface molecular marker as it not only can be detected but also can be used to study the mechanism of interaction with the host.

S. paratyphi A are generally detected using conventional microbiological culture methods, immunology, or PCR.^5,6 However, all three methods have their shortfalls. Conventional microbiological culture methods are labor intensive, slow, and unsatisfactory for responding promptly to cases of contamination. Immunological methods are similarly time-consuming and costly, require complex sample pretreatment procedures, and produce false-positive results from cross-reaction. PCR requires multiple touch conditions and contaminates easily during nucleic acid sample extraction. An alternative method that is quick and highly sensitive and has a short detection time is needed.

Aptamers, or chemical antibodies, are ssDNA and RNA molecules obtained from the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process that can bind to a wide range of target molecules with high specificity and affinity, including proteins, metal ions, small molecules, whole cells, and even tissues.^7–11 They have been used to detect pathogenic bacteria, treat cancer, and identify biomarkers.^{12 –14} They have also been tested as a bioprobe for detecting ATP, mercury ion, proteins, and cells.^15–18 Their application in detecting pathogens, such as Staphylococcus aureus, Salmonella typhimurium, and Escherichia coli K88, has previously been reported.^19–21

Catalytic nucleic acids (DNAzymes) have attracted substantial efforts for developing labels with amplified signals. One of the most extensively studied DNAzymes has been the hemin/G-quadruplex horseradish peroxidase–mimicking DNAzyme, which catalyzes the oxidation of 2,2′-azino-bis (3-ethylBenzthiazoline-6-sulfonic acid) ABTS²⁻ to the colored product ABTS⁻ by H₂O₂. ²² Many studies have already employed DNAzymes as catalytic labels for the colorimetric detection of DNA, aptamer complexes, and metal ions.^23,24 Thus, using analyte-dependent DNAzymes as catalysts to amplify sensing events is especially attractive as a platform for designing biosensors.

Carbon nanotubes, first discovered by Iijima in 1991, have been extensively studied because of their unique chemical, electrical, and mechanical properties.^25–27 Previous experiments have found that ssDNA wrapped around single-walled nanotubes (SWNTs) by π-stacking interactions between the nucleotide bases and the SWNT sidewalls can form stable ssDNA/SWNTs complexes. ²⁸ In addition, SWNTs can act as quenchers for many dyes, which provides a new platform for detecting protein via fluorescence signals.

In this article, a sensitive and cost-effective detection method using noncovalently assembled SWNTs and probe (P0) was constructed for detecting S. paratyphi A. The P0 was composed of two regions: I and II. Region I was an fluorescein isothiocyanate (FITC)–labeled aptamer sequence that bound specifically to the target and emit fluorescence. Region II was a DNAzyme sequence that amplified absorbance signals. In the absence of the target, no detectable signals were generated as the P0 bound to the SWNTs blocked the formation of free hemin-containing active DNAzymes, which quenched the dye fluorescence. Upon adding the hemin and target, the P0 was released from the SWNTs and self-assembled into a DNAzyme unit that catalyzed the ABTS²⁻ and fluorophores away from SWNTs and increased fluorescent intensity. This dual detection method successfully detected S. paratyphi A using both fluorescence and spectrophotometry.

Materials and Methods

Materials

Carboxy1-modified SWNTs, with a carboxyl mass fraction of 2.73%, were purchased from Organic Chemicals Co., Ltd. (Chengdu, China). All DNA oligonucleotides including the FITC-labeled probe were synthesized by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd (Shanghai, China). All materials used in the experiment were dissolved in distilled water purified by a Milli-Q water purification system (electric resistivity 18 MΩ cm⁻¹). The S. paratyphi A strain was given by the Changsha Central Hospital (Changsha, China). E. coli TOP10 was obtained from the Laboratory of Plant Developmental and Molecular Biology of the Hunan Normal University (Changsha, China). Hemin was purchased from Geling NanoTech Co., Ltd (Changsha, China). ABTS²⁻ was purchased from Sigma. The pUC-T Simple Cloning Kit was obtained from Shanghai Sangon Biological Engineering Technology & Services Co., Ltd (Shanghai, China). The fluorescent emission spectra were recorded in a quartz cuvette by an LS55 luminescence spectrometer (PerkinElmer, UK) at room temperature. Both excitation and emission spectrometer slits were set at 10.0 nm. The ultraviolet (UV) spectrophotometer used for absorbance detection at 414 nm was purchased from Tianmei Techcomp (Shanghai, China). The milk used for analysis was purchased from the local market.

Methods

Separation and Purification of Flagellin

After culturing in nutrient medium (pH 7.4) at 37 °C for 18 h, S. paratyphi A were collected via centrifugation (10,286 × g) for 30 min at 4 °C and then resuspended in normal saline. Then, 1 mol/L HCl was added and the bacterial suspension continuously stirred for 30 min until the pH reached 2.0. The sediment was discarded after centrifuging (36,000 × g) for 60 min at 4 °C. NaOH 1 mol/L and then (NH₄)₂SO₄ were added to the supernatant to obtain a pH of 7.2 in the supernatant. The mixture was placed at 4 °C overnight and then the precipitation dissolved in double-distilled H₂O after centrifuging (36,000 × g) for 10 min at 4 °C. Native polyacrylamide gel electrophoresis (PAGE) was performed and the target protein purified from the gel with electroelution and lyophilized. The target protein powder was then conserved at −20 °C prior to use.

SELEX Procedure

A synthetic ssDNA library containing a 35-base central random sequence (5′-GGGAGCTCAGAATAAACGCTCAA-N₃₅-TTCGACATGAGGCCCGGATC-3′) had primer sites on both sides: 5′-GGGAGCTCAGAATAAACGCTCAA-3′ for the forward site and 5′-GATCCGGGCCTCATGTCGAA-3′ for the reverse.

The procedure for selecting the flagellin DNA aptamers was implemented as follows. The flagellin with 0.05 mol/L NaHCO₃ (pH 9.6) was coated on a 96-well enzyme-linked immunosorbent assay plate at 4 °C overnight. Three percent bovine serum albumin (BSA) was added at 37 °C for 2 h to block the wells coated with flagellin and blank wells. The ssDNA pools were heated at 95 °C for 5 min in binding buffer containing 20 mmol/L HEPES buffer (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid) at pH 7.35, 120 mmol/L NaCl, 1 mmol/L MgCl₂, 1 mmol/L CaCl₂, and 5 mmol/L KCl and then immediately placed into iced water. To decrease background binding, yeast tRNA was added to the binding buffer. For negative selection, ssDNA libraries added to BSA-blocked blank wells were maintained at 37 °C for 40 min. Uncombined ssDNA was then transferred to the wells coated with flagellin at 37 °C for 40 min. The wells were washed five times with washing buffer (binding buffer + 0.05% Tween 20) and filled with eluting buffer (20 mmol/L Tris-HCl, 4 mol/L guanidinium isothiocyanate, 1 mmol/L DTT, pH 8.3) at 80 °C for 10 min. Sodium acetate 3 mol/L (pH 5.2) and dehydrated ethanol were added and kept at −80 °C for 40 min and then centrifuged at 30,857 × g for 10 min at 4 °C. The sediment was dissolved in 20 µL double-distilled water after drying. The ssDNA was amplified by PCR (5 min at 95 °C, then 30 s at 95 °C, 30 s at 65 °C, and 30 s at 72 °C, 20 cycles, followed by 5 min at 72 °C). The asymmertric PCR (5 min at 95 °C, then 30 s at 95 °C, 30 s at 65 °C, and 30 s at 72 °C, 40 cycles, followed by 5 min at 72 °C; concentration of forward primer was 100 times greater than the down primer) was used to acquire the ssDNA as the enriched library for the next selection round. The amount of ssDNAs at each step was measured by UV-Vis spectroscopy. The aptamer binding ratio was obtained by analyzing the fluorescence of eluted ssDNA every two rounds.

After 14 rounds of aptamer enrichment, the PCR products selected from the 14th round were subcloned into pUC-T vectors using the pUC-T Simple Cloning Kit. The vectors were transformed into E. coli TOP10 cells. Thirty positive clones were then inoculated into test tubes. After shaking at 37 °C for 24 h (200 rpm/min), 1 mL of inocula was taken from each single colony and sent to sequence. Their secondary structures were performed using the Mfold program.

Determination of Dissociation Constant

The affinities of the selected aptamers against their targets were confirmed with binding assays. The equation Y = BmaxX/(K_d + X) and a calibration plot of the mean fluorescent intensities of the aptamers bound to flagellin were used to calculate the binding dissociation constant, K_d. Bmax was the maximal fluorescent intensity measured by this experiment, Y the mean fluorescent intensity, and X the concentration of the aptamer that was added.

Representation of Binding Parameters of Aptamer to Bacteria

Various amounts of S. paratyphi A cells ranging from 10⁴ to 10¹¹ CFU/mL were incubated with 400 nmol/L FAM-labeled aptamer3. Other bacteria, such as S. typhimurium, E. coli and S. aureus, were also incubated at 10¹¹ CFU/mL with 400 nmol/L FAM-labeled aptamer 3 to test for selectivity. After binding and washing, the bacterium-ssDNA complex sediment was resuspended in 400 µL of normal saline. Fluorescent intensity (measurement conditions explained in the Materials and Methods section) was used to determine the binding affinity of the aptamer3 against various bacteria. Confocal laser scanning microscopy was also used to assess the binding of S. paratyphi A and S. typhimurium cells and aptamer3.

Simultaneous Fluorescence Spectrophotometric Assay and Specificity Detection

SWNTs were dissolved in Milli-Q water and sonicated for 30 min in an ice bath before use to obtain a homogeneous black solution. For the detection assays, the working solution (50 nmol/L P0, 20 mmol/L Tris-HCl) was mixed with 0.02 mg/mL SWNTs at 37 °C for 2 h. Hemin solution (1 µmol/L) and target flagellin were then added at 37 °C and the mixture incubated for 2 h. The solution was centrifuged at 20,571 × g for 10 min, and the supernatant was collected. The fluorescent intensity was measured at 515 nm with an excitation wavelength of 480 nm and recorded. ABTS²⁻ (3 mmol/L) and H₂O₂ (5 mmol/L) were added, and the absorbance was measured again at 414 nm in 20 KH buffer (50 mmol/L HEPES-NH₄OH, pH 8.0; 20 mmol/L KCl; 200 mmol/L NaCl; 0.05% Triton X-100; 1% DMSO). S. typhimurium flagellin, BSA, and the control were chosen to verify the selectivity of this approach. The concentration of P0 was set at 50 nmol/L, S. paratyphi A flagellin at 60 ng/mL, and S. typhimurium flagellin and BSA at 120 ng/mL.

Milk Sample Analysis

Milk was chosen as the sample matrix for detecting pathogens in food. Different concentrations of bacteria were added to 1 mL of milk in centrifuge tubes. The P0/SWNTs complexes in working solution (20 mmol/L Tris-HCl) were mixed with 1 µmol/L hemin and the various milk mixtures spiked with different concentrations of bacteria at 37 °C for 2 h; the fluorescent intensities were then measured. ABTS²⁻ (3 mmol/L) and H₂O₂ (5 mmol/L) were added to the systems, and the absorbance was measured in 20 KH buffer at 414 nm. Specificity was examined using four different bacteria (10⁸ CFU/mL)—S. paratyphi A, S. typhimurium, E. coli, and S. aureus—and detected under the same conditions. All experiments were repeated in triplicates.

Results and Discussion

Supplementary Figure 1S displays the results of native PAGE (a) and sodium dodecyl sulfate (SDS) PAGE (b). One band was visible between 66.7 and 45 KD in SDS PAGE, which was consistent with previous work and indicated that pure flagellin was acquired. A linear regression analysis of BSA concentration and optical density (OD value) was performed to calculate the flagellin concentration. Supplementary Figure 2S reveals that the OD value increased with increasing BSA concentration with a linear range of 0.25 to 1.25 mg/mL (R² = 0.9926). These results indicated that the flagellin concentration could be measured provided that the OD value was confirmed.

The ssDNA concentration decreased with each subsequent selection round. After 14 selection rounds, the aptamers developed strong affinities for the flagellin. The amounts of flagellin and ssDNA pools added in each round are shown in Supplementary Table S1. The enrichment of target-specific aptamers was determined during the selection process, and increasing ratios of aptamers against the flagellin protein were observed after rounds 11 to 14 (Suppl. Fig. 3S). The binding ratio of ssDNA was higher at round 11 than round 9, whereas the percentage of bound ssDNA did not increase substantially after round 11. These results indicated that the affinity of the ssDNA pool to the flagellin protein remained nearly constant after round 11. Nonetheless, the affinity of round 14 was higher than round 11. Therefore, ssDNAs eluted from the 14 selection rounds were then amplified with nonmodified primers and cloned into pUC-T simple vectors using a pUC-T Simple Cloning Kit. After 30 white colonies were identified and chosen, 26 positive colonies with plasmids containing inserted ssDNA were successfully identified by molecular cloning and sequencing techniques using an eluted sample from the final selection round. The selection process was terminated after round 14, and the selected aptamer pool was cloned and characterized. Four independent inserts were acquired and nominated aptamers 1, 2, 3, and 4 (Suppl. Table S2). It should be noted that certain sequences appeared more than once in the four nominated aptamers. For example, the aptamer3 nucleotide sequence appeared nine times, aptamer2 five times, aptamer1 three times, and aptamer4 twice. This clearly demonstrated that the aptamers increased affinity with each round.

The nonlinear regression analyses of select aptamers are shown in Supplementary Figure S4a. Aptamers 1, 2, 3, and 4 produced the best results, with dissociation constants in the low nanomolar range compared with the initial pool. Aptamer3 yielded the lowest K_d at 27 ± 5 nmol/L, whereas the other three aptamers were slightly higher: 49 ± 6 nmol/L for aptamer1, 61 ± 7 nmol/L for aptamer2, and 65 ± 9 nmol/L for aptamer4. These results suggested that the four aptamers were good candidates for detecting flagellin. Moreover, the low K_d value of aptamer3 was notably lower than previously proposed aptamers. Thus, these oligonucleotides would be effective for detecting S. paratyphi A flagellin.

The secondary structures of aptamers 1 to 4 were predicted by DNAMAN software, and their typical stem and loop motifs are shown in Supplementary Figure S4b. Aptamers have been known to form special secondary structures composed of stems, loops, hairpin, pseudoknots, and G-quadruplexes when bound to their targets. Loops 1–1, 1–2, 2–1, 2–2, 3–1, 3–2, and 4–1 were the most likely active sites for our four aptamers, with each stem-loop structure binding to different epitopes of the protein. For aptamer1, the conserved motif formed two looped structures via a stem ring joint with the stem and loop rich in adenine-thymine and cytosine-guanine base pairs, respectively. The aptamer2 sequence was completely different, with its two stems composed completely of cytosine-guanine base pairs. As for aptamer3 and aptamer4, the configurations of loop 3–1 and loop 4–1 in the secondary structures were also observed in the insert sequences containing the primers. These results convincingly demonstrated that the stem-loop structures played an important role in the target recognition and binding of the four aptamers. In addition, it was presumed that the stems had a stabilizing function, whereas the bases in the loops were responsible for binding. The optimal aptamer (aptamer3), with its strong affinity, was selected for the following experiments.

Figure 1a shows the proportional increase of fluorescence intensity to the number of cells, especially in the range from 10⁶ to 10¹¹ CFU/mL (R² = 0.9927). The low detection limit was 10⁴ CFU/mL, which confirmed that aptamer3 bound specifically to the bacteria and served as an ideal tool for monitoring them. Moreover, the fluorescent intensities of nontargeted bacteria were much lower than that of the target ( Fig. 1b ), despite having similar cell numbers (10¹¹ CFU/mL). S. typhimurium, a bacteria within the same species and with similar serotypes to S. paratyphi A, yielded higher fluorescent intensities than that of the less related bacteria E. coli and S. aureus, suggesting that the flagellin of S. typhimurium and S. paratyphi A possessed similar binding epitopes. The fluorescent value of S. paratyphi A, however, was 2.8 times that of S. typhimurium, confirming that aptamer3 was more selective toward S. paratyphi A. Sequence alignment also found that, when compared with S. paratyphi A, the homologies of S. typhimurium and E. coli flagellin were 71.313% and 52.456%, respectively. Thus, the higher the homology, the more similar the spatial structure and higher affinity, which was consistent with the results above. Confocal laser scanning microscopy ( Fig. 1c ) further confirmed these results as FITC-specific green fluorescence appeared on the surfaces of S. paratyphi A rather than S. typhimurium cells.

Figure 1.

The binding capacity of candidate aptamer (aptamer3) against S. paratyphi A measured by fluorescence analysis. (a) Different amounts of the bacteria varied from 10⁴ to 10¹¹ CFU/mL from a to h were measured by fluorescence (the inserted figure shows that the increase in fluorescence intensity is proportional to amounts of the bacteria ranging from 10⁶ to 10¹¹CFU/mL). (b) Fluorescence intensities of different strains of bacteria (a, S. paratyphi A; b, S. typhimurium; c, E. coli; d, S. aureus). (c) Confocal imaging of a: S. paratyphi A cells and b: S. typhimurium cells by aptamers3 labeled with fluorescein isothiocyanate; left is the fluorescence image, and right is the optical image.

To develop a versatile detection system, a simultaneous fluorescence-spectrophotometric method employing SWNTs with a P0 was devised ( Fig. 2 ). The P0 included two parts—an FITC-labeled aptamer3 (I) and a DNAzyme sequence (II; Suppl. Table S3)—that wrapped around SWNTs via π-stacking interactions between the nucleotide bases and SWNT sidewalls to form a stable complex. It was first necessary to examine the ratio of SWNTs and P0 to improve the detection efficiency. Supplementary Figure 5S shows that SWNT concentration increased with decreasing P0 fluorescent intensity. Approximately 90% of FITC’s fluorescence was quenched by 0.02 mg/mL SWNTs, suggesting that 0.02 mg/mL SWNTs could effectively quench the fluorescence of 50 nM P0 via energy transfer. No detectable signals should be generated in the absence of the target because P0/SWNTs block the formation of free hemin-containing active DNAzymes, preventing the dye from being released from the SWNTs and thereby quenching dye fluorescence. When ABTS²⁻ and H₂O₂ were added, the P0 separated from the SWNTs, which increased fluorescent intensity and changed the absorbance. Supplementary Figure 3a reveals that fluorescent intensity increased with increasing flagellin and stopped when the flagellin concentration reached 160 ng/mL, indicating that all P0 were released from the SWNTs. The linear range was from 5 ng/mL to 160 ng/mL (R² = 0.9958), and the detection limit increased to 5 ng/mL. The absorbance value increased rapidly to a steady level within 140 s ( Fig. 3b ). When the absorbance values were compared with different quantities of targets, the absorbance accelerated as the quantity of targets increased, implying that the more P0 discharged from SWNTs, the more DNAzymes were formed. A linear increase in absorbance value was observed between 60 and 160 ng/mL (R² = 0.9983), and the detection limit increased to 20 ng/mL. Milk spiked with S paratyphi A cells was also detected with low detection limits of 10⁵ CFU/mL by fluorescence and 10⁶ CFU/mL by spectrophotometry ( Fig. 4a , b ). Thus, the dual fluorescence-spectrophotometric method was a highly sensitive and affinitive way for detecting S. paratyphi A with flagellin.

Figure 2.

Schematic representation of the dual fluorescence-spectrophotometric platform for the detection of S. paratyphi A flagellin using aptamer3. (a) P0 included DNAzyme sequence and fluorescein isothiocyanate–labeled aptamer sequence bound to single-walled nanotubes (SWNTs), which results in the formation of a stable P0/SWNTs complex. (b) In the absence of target, the solution shows no obvious changes; upon adding target and hemin to the solution, P0 was discharged from SWNTs, which resulted in the increase in fluorescence intensity and the absorbance changes after adding ABTS²⁻ and H₂O₂.

Figure 3.

Quantitative detection of S. paratyphi A flagellin via simultaneous fluorescence-spectrophotometric method. (a) In the presence of target and hemin, the fluorescence intensities of flagellin ranging from 5 ng/mL to 180 ng/mL (from the bottom to the top) increased gradually. (The inset displays the linear relationship ranging from 5 ng/mL to 160 ng/mL.) (b) After adding ABTS²⁻ and H₂O₂, time-dependent absorbance changes were occurred. Amounts of the flagellin varied from 20 ng/mL to 180 ng/mL (from a to i) and were measured by absorbance values. (The inset is the linear relation that corresponds to the absorbance values from 60 ng/mL to 160 ng/mL.)

Figure 4.

The detection of the sample milk spiked with target bacteria with different concentrations. (a) Fluorescence intensity detection. The milk spiked with target bacteria concentrations are varied from 10⁵ to 10¹⁰ CFU/mL from the bottom to the top. (b) Absorbance detection. The target bacteria concentrations ranged from 10⁶ to 10¹⁰ CFU/mL from the bottom to the top. (The inserted figure shows that the increases in fluorescence intensity and absorbance value are proportional to the concentration of target bacteria.)

To test for selectivity, the fluorescent intensity of target flagellin was compared with nontarget proteins and found to be greater even when the concentrations of the control groups were twice that of the experimental group ( Fig. 5a ). The absorbance value changed similarly ( Fig. 5b ). These results suggested that only the target protein could enhance the fluorescent intensity and absorbance value of the solution, which, in this case, confirmed that the aptamers could specifically recognize the flagellin of S. paratyphi A and not nontarget proteins. Pathogen detection was also assessed, and it was found that the fluorescent intensity and absorbance triggered by S. paratyphi A were greater than that of nontargets ( Fig. 6a , b ). It should be stressed that our biosensor was highly selective and specific not only for S. paratyphi A flagellin but also for the bacteria itself, making the biosensor highly effective for pathogen detection and food safety control in the future. Compared with conventional methods, the biosensor was very sensitive to the addition of small volumes of the targets, and its detection limit was lower than enzyme immunoassays but similar to multiple PCR.^29,30 As for selectivity, our biosensor and enzyme immunoassays could detect Salmonella bacteria with different serotypes but multiple PCR could not. Therefore, our new approach for Salmonella detection was simple, selective, and sensitive.

Figure 5.

Dual signals triggered by different targets. (a) Fluorescence intensity changes. (b) Absorbance values changes. a: S. paratyphi A flagellin (60 ng/mL); b: S. typhimurium flagellin (120 ng/mL); c: bovine serium albumin (120 ng/mL); d: background signal.

Figure 6.

Specific detection of the sample milk spiked with S. paratyphi A compared with other bacteria strains. (a) Variation in fluorescence intensities. (b) Variation in absorbance values. a: S. paratyphi A; b: S. typhimurium; c: E. coli; d: S. aureus. The numbers of these four bacteria are 10⁸ CFU/mL, respectively.

In conclusion, the ssDNA aptamers against S. paratyphi A flagellin were selected by employing asymmertric PCR-based SELEX. Four aptamers were authenticated with high affinities (K_d: aptamer1 = 49 ± 6 nmol/L, aptamer2 = 61 ± 7 nmol/L, aptamer3 = 27 ± 5 nmol/L, and aptamer4 = 65 ± 9 nmol/L). A simple and highly selective and sensitive fluorescence-spectrophotometric aptasensor has been developed for flagellin detection, with a low detection limit of 5 ng/mL for fluorescence and 20 ng/mL for spectrophotometry. The aptasensor, with its prominent fluorescence-spectrophotometric signal in cellular assays (low detection limits of 10⁵ CFU/mL for fluorescence and 10⁶ CFU/mL for spectrophotometry), was ideal for detecting real samples, such as S. paratyphi A. Furthermore, the simultaneous fluorescence-spectrophotometric method was more precise than current methods. Future work will focus on developing a kit for rapid detection of food-borne pathogens based on the simultaneous fluorescence-spectrophotometry.