A Novel Steady-state Chemiluminescent Aptasensor using Rolling Circle Amplification Products DNA Hydrogel for Rapid and Ultra-sensitive Detection of Salmonella Typhimurium in Chicken

A Novel Steady-state Chemiluminescent Aptasensor using Rolling Circle Amplification Products DNA Hydrogel for Rapid and Ultra-sensitive Detection of Salmonella Typhimurium in Chicken | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A Novel Steady-state Chemiluminescent Aptasensor using Rolling Circle Amplification Products DNA Hydrogel for Rapid and Ultra-sensitive Detection of Salmonella Typhimurium in Chicken Liling Hao, Jifan Huang, Mengqiu Li, Yihang Yang, Jiaqi Han, Hui Cao, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8569290/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Apr, 2026 Read the published version in Microchimica Acta → Version 1 posted 9 You are reading this latest preprint version Abstract Salmonella typhimurium ( S.typhimurium ) is one of the common foodborne pathogens, posing a serious global food safety risks. It is thus important to develop a rapid and sensitive detection method for S.typhimurium . In this study, a steady-state chemiluminescent DNA hydrogel aptasensor was constructed for ultrasensitive detection of S.typhimurium in chicken. The sensor exploited the dense network of rolling circle amplification products (RCPs) DNA hydrogel to restrict luminol diffusion, thereby generating a stable chemiluminescent signal. A dual rolling circle amplification (DRCA) strategy was employed to accelerate hydrogel formation within 33 min, which increased RCP yield and strengthened intermolecular interactions. In the presence of S.typhimurium , the aptamers specifically binds to the bacteria, leading to the dissociation of the aptamer–primer complex and the subsequent release of primers that initiate two rolling circle amplification (RCA) reactions on partially complementary circular templates. When mixing the two parallel RCA products, the long ssDNA strands instantaneously assembled into hydrogels through complementary base pairing and physical entanglement, subsequently incorporating luminol for quantitative analysis. The chemiluminescence intensity exhibited a concentration-dependent enhancement with S.typhimurium , displaying a well-defined linear response from 10 to 7.6 × 10⁸ CFU/mL under optimal conditions, with a limit of detection of 8 CFU/mL. The assay also exhibited reliable specificity and stability in chicken samples, supporting its potential application in food safety monitoring. Aptamer Rolling circle amplification DNA hydrogel Steady-state Chemiluminescent Salmonella typhimurium Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Foodborne pathogens, particularly Salmonella typhimurium , are widely recognized as a major threat to food safety[ 1 , 2 ]. Even at low infectious doses, S.typhimurium is capable of causing gastrointestinal disorders, including vomiting, fever, and intestinal inflammation, and even death in severe cases[ 3 , 4 ]. Poultry products, particularly inadequately cooked meat and raw eggs, are recognized as major sources of S.typhimurium contamination and transmission[ 5 ]. Surveillance studies have revealed that approximately 22.2% and 18% of retail chicken samples in Egypt and America were contaminated with S.typhimurium [ 6 ]. It is therefore necessary to develop rapid, sensitive, and reliable analytical techniques for the detection of S.typhimurium in poultry-derived foods. Traditional culture-based methods, such as plate colony counting, are reliable but inherently time-consuming[ 7 ]. PCR-based assays have become widely adopted because of their high sensitivity and shorter detection time. However, the high cost of PCR instruments and the requirement for trained personnel restrict their use in field or on-site testing[ 8 ]. These limitations highlight the need for rapid, straightforward, and cost-effective alternatives for the detection of S.typhimurium . Aptasensors have attracted increasing attention in recent years, particularly when combined with functional nanomaterials[ 9 – 11 ]. Aptamers are short single-stranded oligonucleotides generated through systematic evolution of ligands by exponential enrichment (SELEX) that can bind specifically to their targets through hydrogen bonding, electrostatic forces, π–π stacking, and other noncovalent interactions[ 12 , 13 ]. The integration of aptamers with fluorescent or chemiluminescent nanomaterials combines the strong target affinity and structural versatility of aptamers with the superior signal transduction efficiency of nanomaterials[ 14 , 15 ]. Such hybrid aptasensors provide a robust platform for the rapid, sensitive, and facile detection of pathogenic bacteria. Chemiluminescent aptasensors have been increasingly used for pathogenic bacteria detection owing to their high sensitivity and no need of external excitation source[ 16 ]. However, conventional substrates such as luminol produce flash luminescence with about 5–10 s, which compromises detection accuracy and limits rapid applications[ 17 ]. Dang et al.[ 18 ] reported a steady-state chemiluminescence systems using iron porphyrin metal–organic frameworks based peroxidase mimic with natural glucose oxidase (GOx), in this system, hydrogen peroxide continuously generated by GOx and oxidized luminol, yielding an intensive and persistent chemiluminescence. Yet, the activity of GOx is easily influenced by temperature and pH, which adversely affect sensing in food matrices. Our group improved this system by incorporating p-iodophenol (PIP) into cobalt ion–enhanced gold nanoparticles, in this system, PIP is oxidized by hydrogen peroxide and yield more stable iodophenoxy radical[ 19 ]. These stabilized radicals suppress rapid self-coupling and modulate the oxidation kinetics of luminol, thereby enabling a sustained and steady-state chemiluminescence emission. Nonetheless, PIP raises environmental concerns, as even low concentrations can adversely affect aquatic organism[ 20 ]. The dense structure of DNA hydrogel provides an alternative of steady-state chemiluminescence through delaying the diffusion of chemiluminescence reagents[ 21 ]. Rolling circle amplification products (RCPs) DNA hydrogel is formed by DNA self-assembly and can be easily coupled with recognition elements such as aptamers, which is more conducive to sensing and signal amplification[ 22 – 24 ]. However, the gelation time of RCPs DNA hydrogels usually takes 24 to 72 h, limiting its application in food safety rapid detection[ 25 ]. Wildan Hanif et al.[ 26 ] constructed multi-primer rolling circle amplification (MCA) strategy to shorten the gelation time to 4 h by increasing the yield of RCPs. To further accelerate the gelation process, it was hypothesized that enhancing both the yield and intermolecular interactions of the RCPs could promote a more rapid formation of the DNA hydrogel. Herein, in this study, we proposed a novel strategy using dual rolling circle amplification (DRCA) to accelerate the gelation of RCP-based DNA hydrogels. Based on this, a RCPs DNA hydrogel steady-state chemiluminescence detection system was constructed for rapid and highly sensitive detection of S.typhimurium . Two partially complementary template strands were designed to synergistically promote hydrogel assembly by both increasing the concentration of RCPs and strengthening intermolecular interactions. In the presence of S.typhimurium , the aptamer binded to the target, disrupting the double-stranded structure and releasing primers. The released primers hybridized with circular templates 1 and circular templates 2 to initiate parallel RCA reactions, generating long ssDNA1 and ssDNA2. Upon mixing, the two products instantaneously assembled into DNA hydrogel through base pairing and physical entanglement within 3 min. The resulting dense and porous network of DNA hydrogel delayed the diffusion of chemiluminescence reagents, thereby producing steady-state chemiluminescence, which made the system more favorable for rapid and reliable detection of pathogenic bacteria. 2. Experimental 2.1 Materials and instruments All HPLC-purified oligonucleotides were synthesized from Generay Biotech Co, Ltd. (Shanghai, China), and the sequence information was shown in Supplementary Table S1 . S.typhimurium (ATCC 14028), Listeria monocytogenes (ATCC 19115), Staphylococcus aureus (ATCC 29213), Escherichia coli (ATCC 25922), Escherichia coli (ATCC 25922), Bacillus cereus (ATCC 10876) were purchased from Shanghai Preservation Microbiology Center. T4 DNA ligase (400 U/mL) and Phi 29 DNA polymerase (1000 U/mL) were purchased from New England Biolabs, Inc. (Ipswich, MA). Exonuclease I (Exo I), Exonuclease III (Exo III) was purchased from Takara Biomedical Technology (Beijing) Co., Ltd. dNTP were purchased from Sangon Biotechnology (Shanghai, China). DNA Marker (10–150 bp), GelRed, SYBR Gold were obtained from Thermo Fisher Scientific Co., USA. The chemiluminescent image of the DNA hydrogel was taken with a Huawei phone under a dark box ultraviolet analyzer (Shanghai Baoshan Optoelectronic Instrument Factory). Scanning electron microscopy (SEM) was performed using a Hitachi Regulus 8100 scanning electron microscope. Polyacrylamide gel electrophoresis (PAGE) was performed using the electrophoresis instrument of Mini protein Tetra System. 2.2 Cultivation and quantitation of bacteria All the bacteria strains were cultured in Luria-Bertani (LB) broth at 37°C for 24 h. Bacterial were harvested by centrifugation at 4500 rpm for 10 min at 4°C. After washing with phosphate-buffered saline (PBS, pH 7.4, 137 mM NaCl, 2.7 mM KCl, 8 mM Na 2 HPO 4 , 2 mM KH 2 PO 4 ), the bacteria were redispersed in PBS. The concentration of bacterial was quantified using the standard plate counting method. 2.3 Prepration of aptamer-primer hybridized product and Circ-DNA The aptamer (10 µM) was mixed with primer (10 µM). After being heated at 95°C for 10 min, the mixture was cooled to room temperature to obtain hybridized product. Then 14 µL Exo I was added and incubated at 37°C for 3 h to obtain pure hybridized product by digesting the free aptamer and primer. The aptamer-primer hybridized product were investigated by using 15% native PAGE. The gel was run at 180 V for 45 min in TBE buffer (45 mM Tris-borate, 2 mM EDTA), then scanned using a electrophoresis instrument. RCA primer (20 µM) was mixed with template 1/2 (20 µM) for annealing. The annealing parameters was listed in Supplementary Table S2. Then 7.5 µL T4 DNA ligase was added and incubated at 37°C for 2 h. After ligation, the reaction mixture was heated to 65°C and incubated for 10 min to inactivate T4 DNA ligase. In order to purify the obtained circular template (Circ-DNA), 10 µL Exo I and 5 µL Exo III were added and incubated at 37°C and 80°C for 1 h and 20 min, respectively. The purified Circ-DNA was placed at 4°C for later use. The purified Circ-DNA was investigated by using 15% native PAGE. The gel was run at 180 V for 45 min in TBE buffer, then scanned using a electrophoresis instrument. 2.4 Determination of chemiluminescence kinetics The chemiluminescence kinetic curves of luminol, luminol incorporated RCA hydrogel and luminol incorporated DRCA hydrogel were studied respectively. RCA hydrogel was prepared by mixing long ssDNA1 and long ssDNA1. DRCA hydrogel was prepared by mixing long ssDNA1 and long ssDNA2, the DNA hydrogel was then transferred to luminol solution (0.1 M) and incubated at 600 rpm for 30 min, after washing with water, chemiluminescence buffer (1.9 mmol/L Na 2 HPO 4 •2H 2 O, 8.1 mmol/L NaH 2 PO 4 •2H 2 O, 0.2 mmol/L NaOH), CoCl 2 (100 µmol/L) and H 2 O 2 (0.5 mol/L) were added. Photographs were taken every 5 min under dark condition. The RGB value of chemiluminescence images was analyzed by Image J software. 2.5 Quantitative analysis of S.typhimurium A series of S.typhimurium standard solutions (0–10⁸ CFU/mL) were prepared and incubated with aptamers at 37°C with shaking at 600 rpm for 45 min. Subsequently, the samples were passed through a 0.22 µm membrane filter to remove both bacteria and the bacteria-aptamer complexes. The filtrate obtained previously were mixed with circ-DNA1/circ-DNA2, dNTPs, phi29 DNA polymerase and incubated at 37°C with shaking at 600 rpm for 45 min to generate long ssDNA1 and ssDNA2. The resulting long ssDNA1 and ssDNA2 were subsequently mixed, vortexed for 20 s, and centrifuged at 4500 rpm for 3 min to form the DNA hydrogel. The DNA hydrogel was then transferred into luminol solution, washed, added chemiluminescence buffer and analyzed the chemiluminescence intensity following the procedure of 2.5. 2.6 Preparation of chicken samples and determination of S.typhimurium Chicken samples were purchased from a local market. 25 g chicken was added to PBS buffer for homogenization and sterilization. After centrifugation at 1000 rpm for 5 min, the precipitate was discarded and the supernatant was collected. Then, 300 µL supernatant was mixed with 600 µL bacteria to obtain the actual sample. Finally, the chemiluminescence detection was performed according to the method described above. 3. Results and discussion 3.1 Principle of the steady-state chemiluminescent DNA hydrogel aptasensor The detection process was shown in Fig. 1 . Initially, the RCA primer was designed to be partially complementary pairing with the S.typhimurium aptamer, allowing the two oligonucleotides to pre-hybridized and formed duplex complex. In the presence of S.typhimurium , the aptamer tended to bind to the targets, thus leading to the dissociation of aptamer–primer duplex. The released primers hybridized with Circ-DNA1 and Circ-DNA2, thereby initiating two parallel RCA reactions that generated long single-stranded DNA products, ssDNA1 and ssDNA2. Owing to the pre-designed partial complementarity between Circ-DNA1 and Circ-DNA2, the RCA products ssDNA1 and ssDNA2 instantaneously self-assembled to form DRCA hydrogel through synergistic complementary pairing and physical chain entanglement. Luminol was then added to form chemiluminescent DNA hydrogel. The densely cross-linked and porous structure of the DNA hydrogel effectively retarded the diffusion of reactants, resulting the transition from flash-type chemiluminescence into steady-state chemiluminescence. The higher concentration of S. typhimurium resulting the larger amount of DNA hydrogel produced, led to more luminol entrapment and consequently stronger chemiluminescent intensity. The chemiluminescence images were recorded by a smartphone, and the corresponding RGB values were analyzed by Image J software to determine the concentration of S.typhimurium . 3.2 Characterization of the process and products of RCA The successful circularization of DNA templates was verified by 15% native PAGE. As shown in Fig. 2 a, compared with the corresponding linear template (lane 3, lane 6), the products treated with T4 DNA ligase (lane 4, lane 7) exhibited a markedly slower mobility, indicating the successful synthesis of circular DNA. In addition, faint bands corresponding to unreacted primers were observed in the low-molecular-weight region of lane 4 and 7. Therefore, EXO I and EXO III were employed to remove residual linear DNA and byproducts. After enzymatic purification, the free single-stranded bands in lane 5 and 8 disappeared, confirming the high-molecular-weight byproduct bands were substantially reduced. This result indicates successful preparation of the highly purified cyclized DNA products. Circ-DNA1 and Circ-DNA2 were designed to be partially complementary, enabling the RCA-generated long ssDNA1 and long ssDNA2 to hybridize and form cross-linking sites, thereby promoting the formation of the hydrogel network. After 30 min of RCA reaction, three experimental groups were prepared under identical conditions of mixing, vortexing, and centrifugation: (A) ssDNA1 (incorporated with GelRed) with ssDNA1, (B) ssDNA1 with ssDNA2 (incorporated with SYBR Gold), and (C) ssDNA2 with ssDNA2. As shown in Fig. 2 b, the DNA hydrogel in group B formed more rapidly and exhibited a larger volume within 3 min, with a total gelation of 33 min, demonstrating that the DRCA strategy effectively accelerates DNA hydrogel formation through complementary pairing. The microstructural of the DRCA hydrogel were further characterized by scanning electron microscopy (SEM). As shown in Fig. 2 c, the hydrogel displayed a distinct nanoflower-like morphology, whereas Fig. 2 d revealed a highly porous network structure. The nanoflower morphology was ascribed to the self-assembly of ssDNA with magnesium pyrophosphate (Mg₂PPi) crystals, which were formed via the reaction between pyrophosphate ions generated during RCA and Mg²⁺ in the buffer. The porous, sponge-like structure provided a highly liquid-loading capacity and allowed the free diffusion of small molecules. This structural feature was advantageous for delaying the diffusion of chemiluminescent reagents, thereby extending the chemiluminescence emission time and contributing to the steady-state luminescent behavior. 3.3 Chemiluminescence kinetics study of DRCA DNA hydrogel As illustrated in Fig. 3 , the chemiluminescence intensity of the luminol system (blue curve) rapidly reached its maximum within a short period and then decayed sharply, exhibiting a typical flash-type chemiluminescence. In contrast, the luminol incorporated DNA hydrogel (black and red curve) displayed a markedly different kinetic behavior. The chemiluminescence intensity gradually increased within the first 500 s, remained relatively stable for a prolonged period, and subsequently declined slowly, demonstrating a characteristic steady-state chemiluminescence. This transformation in chemiluminescence kinetics can be attributed to the slow diffusion effect of the DNA hydrogel. To further elucidate the structural influence of DNA hydrogels on steady-state chemiluminescence, RCA hydrogels (ssDNA1) and DRCA hydrogels (the mixture of ssDNA1 and ssDNA2) incorporated with luminol were prepared, and the chemiluminescence kinetic profiles were recorded. As shown in Fig. 3 , compared with RCA hydrogels, the luminol incorporated DRCA hydrogel exhibited longer steady-state chemiluminescence time and more stable chemiluminescence intensity. This distinction may be attributed to the structural differences between the two hydrogels. The DRCA hydrogel, formed through both complementary base pairing and physical entanglement, possessed a more compact and cross-linked three-dimensional network. While the RCA DNA hydrogel relied solely on physical entanglement, resulting in a looser and less ordered structure. The looser structure was less effective in restricting the diffusion of chemiluminescent reactants, leading to a rapider consumption of reactive species and a shorter emission lifetime. 3.4 Optimization of experimental conditions To improve detection sensitivity, the concentrations of Co 2+ , H 2 O 2 , and luminol in the chemiluminescence system were optimized. As the catalyst in the chemiluminescence reaction, the concentration of Co 2+ must be systematically optimized. As shown in Fig. 4 a, the chemiluminescence intensity increased with the Co 2+ concentration, reaching a maximum at 100 µmol/L. A further increase led to signal declination, likely due to the formation of cobalt hydroxide precipitates, which reduced the effective concentration of free Co 2+ available for catalysis. Therefore, 100 µmol/L was selected as the optimal Co 2+ concentration. As the critical oxidant in the chemiluminescence reaction, the concentration of H₂O must be systematically optimized. As shown in Fig. 4 b, the chemiluminescence intensity initially increased and then decreased with increasing H 2 O 2 concentration, reaching its peak at 0.5 mol/L. Excessive H 2 O 2 tended to decompose and generate bubbles, thereby interfering with the measurement process. As the chemiluminescent substrate in the chemiluminescence reaction, the concentration of luminol must be systematically optimized. As shown in Fig. 4 c, the signal intensity rose with increasing luminol concentration and reached a plateau beyond 100 mmol/L, may suggesting that the hydrogel’s adsorption sites for luminol had become saturated. 3.5 Sensitivity and specificity analysis The sensitivity of the proposed aptasensor was subsequently studied under the optimal conditions. As shown in Fig. 5 a, the RGB value of chemiluminescence image increased progressively with the concentration of S.typhimurium . A good linear relationship was observed between the RGB value and the logarithm of S. typhimurium concentration in the range of 10 − 7.6 × 10⁸ CFU/mL, following the regression equation: Y = 20.92 + 2.420 × log X (R² = 0.9927). The limit of detection (LOD) was 8 CFU/mL calculated by 3σ/K, demonstrating the high sensitivity of the developed sensor. Table 1 summarized representative detection methods and LOD for S. typhimurium reported in recent years. The detection limit and detection time achieved in this study were lower than those reported in most previous works, highlighting the superior analytical performance and potential practical utility of the proposed chemiluminescent sensing strategy. Table 1 Reported methods of S.typhimurium detection in recent years Methods Detection Strategy LOD (CFU/mL) Detection Time (min) Electrochemiluminescence[ 27 ] β-CD host-guest recognition 10 240 Colorimetry[ 28 ] HCR 7 205 Colorimetry[ 29 ] AuNPs accumulation/dispersion 23 150 Fluorometry[ 30 ] LAMP CRISPR/Cas12a 1 120 Fluorometry[ 31 ] RCA CRISPR/Cas12a 193 120 This work RCPs DNA hydrogel 8 123 The specificity of the sensing system was assessed by comparing with other common foodborne pathogens, including Bacillus cereus , Listeria monocytogenes , Staphylococcus aureus , and Escherichia col . As illustrated in Fig. 5 b, only S. typhimurium induced a distinct chemiluminescence response, whereas the signals from the other four pathogens were negligible. These results indicate that the sensor exhibits excellent specificity toward S. typhimurium detection. 3.6 Application in real samples To validate the practical application of the steady-state chemiluminescent DNA hydrogel aptasensor for detecting S. typhimurium , we simulated a real-life complex environment by introducing the bacterium into chicken samples. The results were compared with those obtained by the standard plate counting method. As presented in Table 2 , no significant difference was observed between the two methods, and the recovery rates ranged from 83.9% to 111.4%, confirming the accuracy and applicability of the developed assay for real sample analysis. Table 2 Detection results of the S.typhimurium in chicken samples* Measured by the developed method (CFU/mL) Measured by the plate counting method (CFU/mL) Recovery ratio of the developed method (%) 1 (1.70 ± 0.24)×10 2a (1.80 ± 0.10)×102 a 96.7 2 (4.51 ± 0.44)×10 2a (4.80 ± 0.50)×10 2a 93.4 3 (3.25 ± 0.33)×10 3a (3.62 ± 0.31)×10 3a 89.8 *The same letter indicates that there is no significant difference between the two test results (p > 0.05, n = 3). 4. Conclusion In summary, a steady-state chemiluminescent aptasensor based on a DRCA DNA hydrogel was successfully established for the ultrasensitive detection of S.typhimurium . Compared with conventional flash-type chemiluminescence systems, this aptasensor utilizes the densely cross-linked and porous structure of the DNA hydrogel to retard the diffusion of luminol reactants, thereby maintaining a steady and long-lasting luminescent signal for up to 40 min. The DRCA strategy markedly improved both the yield and intermolecular association of RCPs, thereby shortening the gelation time to 33 min and substantially enhancing the detection sensitivity. The resulting DNA hydrogel produces a steady-state chemiluminescent signal that can be visualized by a smartphone and quantitatively analyzed by Image J software, with a detection limit as low as 8 CFU/mL and the detection time of 123 min. The assay exhibited excellent sensitivity and specificity in chicken samples, demonstrating its reliability in real food matrices. Overall, the proposed steady-state chemiluminescent DNA hydrogel aptasensor provides a promising and practical approach for rapid, sensitive, and on-site monitoring of foodborne pathogens in complex food environments. Declarations Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Funding This work was supported by The National Natural Science Foundation of China (32202173, 32572696) and Shanghai Rising-Star Program (23QC1401300). Author Contribution Liling Hao: Writing – review & editing, Supervision, Project administration, Methodology, Funding acquisition, Conceptualization. Jifan Huang: Writing – original draft, Validation, Methodology, Investigation, Formal analysis, Data curation. Mengqiu Li: Resources, Investigation, Formal analysis. Yihang Yang: Formal analysis, Data curation. Jiaqi Han: Formal analysis, Data curation. Hui Cao: Formal analysis, Data curation. Tai Ye: Formal analysis, Data curation. Xiuxiu Wu: Formal analysis, Data curation. Huajie Gu: Formal analysis, Data curation. Yuzheng Li: Formal analysis, Data curation. Fei Xu: Writing – review & editing, Resources, Investigation, Funding acquisition. 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Supplementary Files GraphicalAbstract.tiff Supplementarydata.docx Cite Share Download PDF Status: Published Journal Publication published 17 Apr, 2026 Read the published version in Microchimica Acta → Version 1 posted Editorial decision: Revision requested 08 Feb, 2026 Reviews received at journal 07 Feb, 2026 Reviewers agreed at journal 30 Jan, 2026 Reviews received at journal 30 Jan, 2026 Reviewers agreed at journal 21 Jan, 2026 Reviewers invited by journal 21 Jan, 2026 Editor assigned by journal 15 Jan, 2026 Submission checks completed at journal 15 Jan, 2026 First submitted to journal 10 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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1","display":"","copyAsset":false,"role":"figure","size":111878,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic illustration of the developed steady-state chemiluminescent DNA hydrogel aptasensor for the detection of \u003cem\u003eS.typhimurium\u003c/em\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8569290/v1/e5128d0bf4600e66ae125cad.png"},{"id":100984653,"identity":"fe2d5acc-4071-41ba-973d-d78914d3a5e6","added_by":"auto","created_at":"2026-01-23 12:56:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":449591,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Native PAGE image, lane 1: DNA marker (10−150 bp); lane 2: primer; lane 3: linear template 1; lane 4: Circ-DNA 1; lane 5: purified Circ-DNA1; lane 6: linear template 2; lane 7: Circ-DNA2. (b) Fluorescence images of the DNA hydrogel formation process. (c、d) SEM image of DRCA DNA hydrogel under different proportions\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8569290/v1/842ed04dd37f6dd256f2fa41.png"},{"id":100984662,"identity":"80e3c020-f7d2-438f-9651-59a5a21505b1","added_by":"auto","created_at":"2026-01-23 12:56:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":40183,"visible":true,"origin":"","legend":"\u003cp\u003eChemiluminescence kinetics curves of luminol, luminol incorporated RCA hydrogel and luminol incorporated DRCA hydrogel\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8569290/v1/026bd3d3f22a483316b58bc4.png"},{"id":100984675,"identity":"89fe5b98-d6a6-4b3a-bd97-2b685bfec3fd","added_by":"auto","created_at":"2026-01-23 12:56:13","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":24994,"visible":true,"origin":"","legend":"\u003cp\u003eInfluence of the concentration of (a) Co\u003csup\u003e2 +\u003c/sup\u003e, (b) H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and (c) luminol\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8569290/v1/7daf1d8a9c4e004a5e9ced2d.png"},{"id":100984658,"identity":"a32ce63a-aa96-4e19-bb38-d0d53ebb271f","added_by":"auto","created_at":"2026-01-23 12:56:08","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":105910,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Standard curve of the RGB value of chemiluminescence image versus logarithm of \u003cem\u003eS.typhimurium\u003c/em\u003e concentration. (b) Specificity investigation of the DNA hydrogel aptasensor for \u003cem\u003eS.typhimurium\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8569290/v1/8e39ac2c50744e445345566b.png"},{"id":107351866,"identity":"b16e6661-6c58-4593-88b9-5df96c12fe04","added_by":"auto","created_at":"2026-04-20 16:12:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1038861,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8569290/v1/48038e84-2be0-40c9-b5e9-7a65f6058424.pdf"},{"id":100984743,"identity":"f5a27465-24c1-4779-b2b2-0bb6e44a2c87","added_by":"auto","created_at":"2026-01-23 12:56:25","extension":"tiff","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":96017,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.tiff","url":"https://assets-eu.researchsquare.com/files/rs-8569290/v1/7bd54e419bf199415c8493e9.tiff"},{"id":100984671,"identity":"441a6cb9-f2cb-43f2-aa69-c6c5b5a550f1","added_by":"auto","created_at":"2026-01-23 12:56:12","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":415487,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarydata.docx","url":"https://assets-eu.researchsquare.com/files/rs-8569290/v1/df8bf9e1808a7fa6d1631a22.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"A Novel Steady-state Chemiluminescent Aptasensor using Rolling Circle Amplification Products DNA Hydrogel for Rapid and Ultra-sensitive Detection of Salmonella Typhimurium in Chicken","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eFoodborne pathogens, particularly \u003cem\u003eSalmonella typhimurium\u003c/em\u003e, are widely recognized as a major threat to food safety[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Even at low infectious doses, \u003cem\u003eS.typhimurium\u003c/em\u003e is capable of causing gastrointestinal disorders, including vomiting, fever, and intestinal inflammation, and even death in severe cases[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Poultry products, particularly inadequately cooked meat and raw eggs, are recognized as major sources of \u003cem\u003eS.typhimurium\u003c/em\u003e contamination and transmission[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Surveillance studies have revealed that approximately 22.2% and 18% of retail chicken samples in Egypt and America were contaminated with \u003cem\u003eS.typhimurium\u003c/em\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. It is therefore necessary to develop rapid, sensitive, and reliable analytical techniques for the detection of \u003cem\u003eS.typhimurium\u003c/em\u003e in poultry-derived foods.\u003c/p\u003e \u003cp\u003eTraditional culture-based methods, such as plate colony counting, are reliable but inherently time-consuming[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. PCR-based assays have become widely adopted because of their high sensitivity and shorter detection time. However, the high cost of PCR instruments and the requirement for trained personnel restrict their use in field or on-site testing[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. These limitations highlight the need for rapid, straightforward, and cost-effective alternatives for the detection of \u003cem\u003eS.typhimurium\u003c/em\u003e. Aptasensors have attracted increasing attention in recent years, particularly when combined with functional nanomaterials[\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Aptamers are short single-stranded oligonucleotides generated through systematic evolution of ligands by exponential enrichment (SELEX) that can bind specifically to their targets through hydrogen bonding, electrostatic forces, π\u0026ndash;π stacking, and other noncovalent interactions[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The integration of aptamers with fluorescent or chemiluminescent nanomaterials combines the strong target affinity and structural versatility of aptamers with the superior signal transduction efficiency of nanomaterials[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Such hybrid aptasensors provide a robust platform for the rapid, sensitive, and facile detection of pathogenic bacteria.\u003c/p\u003e \u003cp\u003eChemiluminescent aptasensors have been increasingly used for pathogenic bacteria detection owing to their high sensitivity and no need of external excitation source[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. However, conventional substrates such as luminol produce flash luminescence with about 5\u0026ndash;10 s, which compromises detection accuracy and limits rapid applications[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Dang et al.[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] reported a steady-state chemiluminescence systems using iron porphyrin metal\u0026ndash;organic frameworks based peroxidase mimic with natural glucose oxidase (GOx), in this system, hydrogen peroxide continuously generated by GOx and oxidized luminol, yielding an intensive and persistent chemiluminescence. Yet, the activity of GOx is easily influenced by temperature and pH, which adversely affect sensing in food matrices. Our group improved this system by incorporating p-iodophenol (PIP) into cobalt ion\u0026ndash;enhanced gold nanoparticles, in this system, PIP is oxidized by hydrogen peroxide and yield more stable iodophenoxy radical[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. These stabilized radicals suppress rapid self-coupling and modulate the oxidation kinetics of luminol, thereby enabling a sustained and steady-state chemiluminescence emission. Nonetheless, PIP raises environmental concerns, as even low concentrations can adversely affect aquatic organism[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe dense structure of DNA hydrogel provides an alternative of steady-state chemiluminescence through delaying the diffusion of chemiluminescence reagents[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Rolling circle amplification products (RCPs) DNA hydrogel is formed by DNA self-assembly and can be easily coupled with recognition elements such as aptamers, which is more conducive to sensing and signal amplification[\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. However, the gelation time of RCPs DNA hydrogels usually takes 24 to 72 h, limiting its application in food safety rapid detection[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Wildan Hanif et al.[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] constructed multi-primer rolling circle amplification (MCA) strategy to shorten the gelation time to 4 h by increasing the yield of RCPs. To further accelerate the gelation process, it was hypothesized that enhancing both the yield and intermolecular interactions of the RCPs could promote a more rapid formation of the DNA hydrogel.\u003c/p\u003e \u003cp\u003eHerein, in this study, we proposed a novel strategy using dual rolling circle amplification (DRCA) to accelerate the gelation of RCP-based DNA hydrogels. Based on this, a RCPs DNA hydrogel steady-state chemiluminescence detection system was constructed for rapid and highly sensitive detection of \u003cem\u003eS.typhimurium\u003c/em\u003e. Two partially complementary template strands were designed to synergistically promote hydrogel assembly by both increasing the concentration of RCPs and strengthening intermolecular interactions. In the presence of \u003cem\u003eS.typhimurium\u003c/em\u003e, the aptamer binded to the target, disrupting the double-stranded structure and releasing primers. The released primers hybridized with circular templates 1 and circular templates 2 to initiate parallel RCA reactions, generating long ssDNA1 and ssDNA2. Upon mixing, the two products instantaneously assembled into DNA hydrogel through base pairing and physical entanglement within 3 min. The resulting dense and porous network of DNA hydrogel delayed the diffusion of chemiluminescence reagents, thereby producing steady-state chemiluminescence, which made the system more favorable for rapid and reliable detection of pathogenic bacteria.\u003c/p\u003e"},{"header":"2. Experimental","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials and instruments\u003c/h2\u003e \u003cp\u003eAll HPLC-purified oligonucleotides were synthesized from Generay Biotech Co, Ltd. (Shanghai, China), and the sequence information was shown in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. \u003cem\u003eS.typhimurium\u003c/em\u003e (ATCC 14028), \u003cem\u003eListeria monocytogenes\u003c/em\u003e (ATCC 19115), \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (ATCC 29213), \u003cem\u003eEscherichia coli\u003c/em\u003e (ATCC 25922), \u003cem\u003eEscherichia coli\u003c/em\u003e (ATCC 25922), \u003cem\u003eBacillus cereus\u003c/em\u003e (ATCC 10876) were purchased from Shanghai Preservation Microbiology Center. T4 DNA ligase (400 U/mL) and Phi 29 DNA polymerase (1000 U/mL) were purchased from New England Biolabs, Inc. (Ipswich, MA). Exonuclease I (Exo I), Exonuclease III (Exo III) was purchased from Takara Biomedical Technology (Beijing) Co., Ltd. dNTP were purchased from Sangon Biotechnology (Shanghai, China). DNA Marker (10\u0026ndash;150 bp), GelRed, SYBR Gold were obtained from Thermo Fisher Scientific Co., USA.\u003c/p\u003e \u003cp\u003eThe chemiluminescent image of the DNA hydrogel was taken with a Huawei phone under a dark box ultraviolet analyzer (Shanghai Baoshan Optoelectronic Instrument Factory). Scanning electron microscopy (SEM) was performed using a Hitachi Regulus 8100 scanning electron microscope. Polyacrylamide gel electrophoresis (PAGE) was performed using the electrophoresis instrument of Mini protein Tetra System.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Cultivation and quantitation of bacteria\u003c/h2\u003e \u003cp\u003eAll the bacteria strains were cultured in Luria-Bertani (LB) broth at 37\u0026deg;C for 24 h. Bacterial were harvested by centrifugation at 4500 rpm for 10 min at 4\u0026deg;C. After washing with phosphate-buffered saline (PBS, pH 7.4, 137 mM NaCl, 2.7 mM KCl, 8 mM Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 2 mM KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e), the bacteria were redispersed in PBS. The concentration of bacterial was quantified using the standard plate counting method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Prepration of aptamer-primer hybridized product and Circ-DNA\u003c/h2\u003e \u003cp\u003eThe aptamer (10 \u0026micro;M) was mixed with primer (10 \u0026micro;M). After being heated at 95\u0026deg;C for 10 min, the mixture was cooled to room temperature to obtain hybridized product. Then 14 \u0026micro;L Exo I was added and incubated at 37\u0026deg;C for 3 h to obtain pure hybridized product by digesting the free aptamer and primer. The aptamer-primer hybridized product were investigated by using 15% native PAGE. The gel was run at 180 V for 45 min in TBE buffer (45 mM Tris-borate, 2 mM EDTA), then scanned using a electrophoresis instrument. RCA primer (20 \u0026micro;M) was mixed with template 1/2 (20 \u0026micro;M) for annealing. The annealing parameters was listed in Supplementary Table S2. Then 7.5 \u0026micro;L T4 DNA ligase was added and incubated at 37\u0026deg;C for 2 h. After ligation, the reaction mixture was heated to 65\u0026deg;C and incubated for 10 min to inactivate T4 DNA ligase. In order to purify the obtained circular template (Circ-DNA), 10 \u0026micro;L Exo I and 5 \u0026micro;L Exo III were added and incubated at 37\u0026deg;C and 80\u0026deg;C for 1 h and 20 min, respectively. The purified Circ-DNA was placed at 4\u0026deg;C for later use. The purified Circ-DNA was investigated by using 15% native PAGE. The gel was run at 180 V for 45 min in TBE buffer, then scanned using a electrophoresis instrument.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Determination of chemiluminescence kinetics\u003c/h2\u003e \u003cp\u003eThe chemiluminescence kinetic curves of luminol, luminol incorporated RCA hydrogel and luminol incorporated DRCA hydrogel were studied respectively. RCA hydrogel was prepared by mixing long ssDNA1 and long ssDNA1. DRCA hydrogel was prepared by mixing long ssDNA1 and long ssDNA2, the DNA hydrogel was then transferred to luminol solution (0.1 M) and incubated at 600 rpm for 30 min, after washing with water, chemiluminescence buffer (1.9 mmol/L Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e\u0026bull;2H\u003csub\u003e2\u003c/sub\u003eO, 8.1 mmol/L NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e\u0026bull;2H\u003csub\u003e2\u003c/sub\u003eO, 0.2 mmol/L NaOH), CoCl\u003csub\u003e2\u003c/sub\u003e (100 \u0026micro;mol/L) and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (0.5 mol/L) were added. Photographs were taken every 5 min under dark condition. The RGB value of chemiluminescence images was analyzed by Image J software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Quantitative analysis of \u003cem\u003eS.typhimurium\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eA series of \u003cem\u003eS.typhimurium\u003c/em\u003e standard solutions (0\u0026ndash;10⁸ CFU/mL) were prepared and incubated with aptamers at 37\u0026deg;C with shaking at 600 rpm for 45 min. Subsequently, the samples were passed through a 0.22 \u0026micro;m membrane filter to remove both bacteria and the bacteria-aptamer complexes. The filtrate obtained previously were mixed with circ-DNA1/circ-DNA2, dNTPs, phi29 DNA polymerase and incubated at 37\u0026deg;C with shaking at 600 rpm for 45 min to generate long ssDNA1 and ssDNA2. The resulting long ssDNA1 and ssDNA2 were subsequently mixed, vortexed for 20 s, and centrifuged at 4500 rpm for 3 min to form the DNA hydrogel. The DNA hydrogel was then transferred into luminol solution, washed, added chemiluminescence buffer and analyzed the chemiluminescence intensity following the procedure of 2.5.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Preparation of chicken samples and determination of \u003cem\u003eS.typhimurium\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eChicken samples were purchased from a local market. 25 g chicken was added to PBS buffer for homogenization and sterilization. After centrifugation at 1000 rpm for 5 min, the precipitate was discarded and the supernatant was collected. Then, 300 \u0026micro;L supernatant was mixed with 600 \u0026micro;L bacteria to obtain the actual sample. Finally, the chemiluminescence detection was performed according to the method described above.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Principle of the steady-state chemiluminescent DNA hydrogel aptasensor\u003c/h2\u003e \u003cp\u003eThe detection process was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Initially, the RCA primer was designed to be partially complementary pairing with the \u003cem\u003eS.typhimurium\u003c/em\u003e aptamer, allowing the two oligonucleotides to pre-hybridized and formed duplex complex. In the presence of \u003cem\u003eS.typhimurium\u003c/em\u003e, the aptamer tended to bind to the targets, thus leading to the dissociation of aptamer\u0026ndash;primer duplex. The released primers hybridized with Circ-DNA1 and Circ-DNA2, thereby initiating two parallel RCA reactions that generated long single-stranded DNA products, ssDNA1 and ssDNA2. Owing to the pre-designed partial complementarity between Circ-DNA1 and Circ-DNA2, the RCA products ssDNA1 and ssDNA2 instantaneously self-assembled to form DRCA hydrogel through synergistic complementary pairing and physical chain entanglement. Luminol was then added to form chemiluminescent DNA hydrogel. The densely cross-linked and porous structure of the DNA hydrogel effectively retarded the diffusion of reactants, resulting the transition from flash-type chemiluminescence into steady-state chemiluminescence. The higher concentration of \u003cem\u003eS. typhimurium\u003c/em\u003e resulting the larger amount of DNA hydrogel produced, led to more luminol entrapment and consequently stronger chemiluminescent intensity. The chemiluminescence images were recorded by a smartphone, and the corresponding RGB values were analyzed by Image J software to determine the concentration of \u003cem\u003eS.typhimurium\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Characterization of the process and products of RCA\u003c/h2\u003e \u003cp\u003eThe successful circularization of DNA templates was verified by 15% native PAGE. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, compared with the corresponding linear template (lane 3, lane 6), the products treated with T4 DNA ligase (lane 4, lane 7) exhibited a markedly slower mobility, indicating the successful synthesis of circular DNA. In addition, faint bands corresponding to unreacted primers were observed in the low-molecular-weight region of lane 4 and 7. Therefore, EXO I and EXO III were employed to remove residual linear DNA and byproducts. After enzymatic purification, the free single-stranded bands in lane 5 and 8 disappeared, confirming the high-molecular-weight byproduct bands were substantially reduced. This result indicates successful preparation of the highly purified cyclized DNA products.\u003c/p\u003e \u003cp\u003eCirc-DNA1 and Circ-DNA2 were designed to be partially complementary, enabling the RCA-generated long ssDNA1 and long ssDNA2 to hybridize and form cross-linking sites, thereby promoting the formation of the hydrogel network. After 30 min of RCA reaction, three experimental groups were prepared under identical conditions of mixing, vortexing, and centrifugation: (A) ssDNA1 (incorporated with GelRed) with ssDNA1, (B) ssDNA1 with ssDNA2 (incorporated with SYBR Gold), and (C) ssDNA2 with ssDNA2. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, the DNA hydrogel in group B formed more rapidly and exhibited a larger volume within 3 min, with a total gelation of 33 min, demonstrating that the DRCA strategy effectively accelerates DNA hydrogel formation through complementary pairing.\u003c/p\u003e \u003cp\u003eThe microstructural of the DRCA hydrogel were further characterized by scanning electron microscopy (SEM). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, the hydrogel displayed a distinct nanoflower-like morphology, whereas Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed revealed a highly porous network structure. The nanoflower morphology was ascribed to the self-assembly of ssDNA with magnesium pyrophosphate (Mg₂PPi) crystals, which were formed via the reaction between pyrophosphate ions generated during RCA and Mg\u0026sup2;⁺ in the buffer. The porous, sponge-like structure provided a highly liquid-loading capacity and allowed the free diffusion of small molecules. This structural feature was advantageous for delaying the diffusion of chemiluminescent reagents, thereby extending the chemiluminescence emission time and contributing to the steady-state luminescent behavior.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Chemiluminescence kinetics study of DRCA DNA hydrogel\u003c/h2\u003e \u003cp\u003eAs illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, the chemiluminescence intensity of the luminol system (blue curve) rapidly reached its maximum within a short period and then decayed sharply, exhibiting a typical flash-type chemiluminescence. In contrast, the luminol incorporated DNA hydrogel (black and red curve) displayed a markedly different kinetic behavior. The chemiluminescence intensity gradually increased within the first 500 s, remained relatively stable for a prolonged period, and subsequently declined slowly, demonstrating a characteristic steady-state chemiluminescence. This transformation in chemiluminescence kinetics can be attributed to the slow diffusion effect of the DNA hydrogel.\u003c/p\u003e \u003cp\u003eTo further elucidate the structural influence of DNA hydrogels on steady-state chemiluminescence, RCA hydrogels (ssDNA1) and DRCA hydrogels (the mixture of ssDNA1 and ssDNA2) incorporated with luminol were prepared, and the chemiluminescence kinetic profiles were recorded. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, compared with RCA hydrogels, the luminol incorporated DRCA hydrogel exhibited longer steady-state chemiluminescence time and more stable chemiluminescence intensity. This distinction may be attributed to the structural differences between the two hydrogels. The DRCA hydrogel, formed through both complementary base pairing and physical entanglement, possessed a more compact and cross-linked three-dimensional network. While the RCA DNA hydrogel relied solely on physical entanglement, resulting in a looser and less ordered structure. The looser structure was less effective in restricting the diffusion of chemiluminescent reactants, leading to a rapider consumption of reactive species and a shorter emission lifetime.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Optimization of experimental conditions\u003c/h2\u003e \u003cp\u003eTo improve detection sensitivity, the concentrations of Co\u003csup\u003e2+\u003c/sup\u003e, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and luminol in the chemiluminescence system were optimized. As the catalyst in the chemiluminescence reaction, the concentration of Co\u003csup\u003e2+\u003c/sup\u003e must be systematically optimized. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, the chemiluminescence intensity increased with the Co\u003csup\u003e2+\u003c/sup\u003e concentration, reaching a maximum at 100 \u0026micro;mol/L. A further increase led to signal declination, likely due to the formation of cobalt hydroxide precipitates, which reduced the effective concentration of free Co\u003csup\u003e2+\u003c/sup\u003e available for catalysis. Therefore, 100 \u0026micro;mol/L was selected as the optimal Co\u003csup\u003e2+\u003c/sup\u003e concentration.\u003c/p\u003e \u003cp\u003eAs the critical oxidant in the chemiluminescence reaction, the concentration of H₂O must be systematically optimized. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, the chemiluminescence intensity initially increased and then decreased with increasing H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentration, reaching its peak at 0.5 mol/L. Excessive H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e tended to decompose and generate bubbles, thereby interfering with the measurement process.\u003c/p\u003e \u003cp\u003eAs the chemiluminescent substrate in the chemiluminescence reaction, the concentration of luminol must be systematically optimized. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, the signal intensity rose with increasing luminol concentration and reached a plateau beyond 100 mmol/L, may suggesting that the hydrogel\u0026rsquo;s adsorption sites for luminol had become saturated.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Sensitivity and specificity analysis\u003c/h2\u003e \u003cp\u003eThe sensitivity of the proposed aptasensor was subsequently studied under the optimal conditions. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, the RGB value of chemiluminescence image increased progressively with the concentration of \u003cem\u003eS.typhimurium\u003c/em\u003e. A good linear relationship was observed between the RGB value and the logarithm of \u003cem\u003eS. typhimurium\u003c/em\u003e concentration in the range of 10\u0026thinsp;\u0026minus;\u0026thinsp;7.6 \u0026times; 10⁸ CFU/mL, following the regression equation: Y\u0026thinsp;=\u0026thinsp;20.92\u0026thinsp;+\u0026thinsp;2.420 \u0026times; log X (R\u0026sup2; = 0.9927). The limit of detection (LOD) was 8 CFU/mL calculated by 3σ/K, demonstrating the high sensitivity of the developed sensor. Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e summarized representative detection methods and LOD for \u003cem\u003eS. typhimurium\u003c/em\u003e reported in recent years. The detection limit and detection time achieved in this study were lower than those reported in most previous works, highlighting the superior analytical performance and potential practical utility of the proposed chemiluminescent sensing strategy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eReported methods of \u003cem\u003eS.typhimurium\u003c/em\u003e detection in recent years\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMethods\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDetection Strategy\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLOD (CFU/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDetection Time\u003c/p\u003e \u003cp\u003e(min)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eElectrochemiluminescence[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eβ-CD host-guest recognition\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e240\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eColorimetry[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHCR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e205\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eColorimetry[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAuNPs accumulation/dispersion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFluorometry[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLAMP\u003c/p\u003e \u003cp\u003eCRISPR/Cas12a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e120\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFluorometry[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRCA\u003c/p\u003e \u003cp\u003eCRISPR/Cas12a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e193\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e120\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThis work\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRCPs DNA hydrogel\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e123\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe specificity of the sensing system was assessed by comparing with other common foodborne pathogens, including \u003cem\u003eBacillus cereus\u003c/em\u003e, \u003cem\u003eListeria monocytogenes\u003c/em\u003e, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, and \u003cem\u003eEscherichia col\u003c/em\u003e. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, only \u003cem\u003eS. typhimurium\u003c/em\u003e induced a distinct chemiluminescence response, whereas the signals from the other four pathogens were negligible. These results indicate that the sensor exhibits excellent specificity toward \u003cem\u003eS. typhimurium\u003c/em\u003e detection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Application in real samples\u003c/h2\u003e \u003cp\u003eTo validate the practical application of the steady-state chemiluminescent DNA hydrogel aptasensor for detecting \u003cem\u003eS. typhimurium\u003c/em\u003e, we simulated a real-life complex environment by introducing the bacterium into chicken samples. The results were compared with those obtained by the standard plate counting method. As presented in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, no significant difference was observed between the two methods, and the recovery rates ranged from 83.9% to 111.4%, confirming the accuracy and applicability of the developed assay for real sample analysis.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDetection results of the \u003cem\u003eS.typhimurium\u003c/em\u003e in chicken samples*\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMeasured by the developed method (CFU/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMeasured by the plate counting method (CFU/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRecovery ratio of the developed method (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(1.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24)\u0026times;10\u003csup\u003e2a\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(1.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10)\u0026times;102\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e96.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(4.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44)\u0026times;10\u003csup\u003e2a\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(4.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50)\u0026times;10\u003csup\u003e2a\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e93.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(3.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33)\u0026times;10\u003csup\u003e3a\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(3.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31)\u0026times;10\u003csup\u003e3a\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e89.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e*The same letter indicates that there is no significant difference between the two test results (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05, n\u0026thinsp;=\u0026thinsp;3).\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn summary, a steady-state chemiluminescent aptasensor based on a DRCA DNA hydrogel was successfully established for the ultrasensitive detection of \u003cem\u003eS.typhimurium\u003c/em\u003e. Compared with conventional flash-type chemiluminescence systems, this aptasensor utilizes the densely cross-linked and porous structure of the DNA hydrogel to retard the diffusion of luminol reactants, thereby maintaining a steady and long-lasting luminescent signal for up to 40 min. The DRCA strategy markedly improved both the yield and intermolecular association of RCPs, thereby shortening the gelation time to 33 min and substantially enhancing the detection sensitivity. The resulting DNA hydrogel produces a steady-state chemiluminescent signal that can be visualized by a smartphone and quantitatively analyzed by Image J software, with a detection limit as low as 8 CFU/mL and the detection time of 123 min. The assay exhibited excellent sensitivity and specificity in chicken samples, demonstrating its reliability in real food matrices. Overall, the proposed steady-state chemiluminescent DNA hydrogel aptasensor provides a promising and practical approach for rapid, sensitive, and on-site monitoring of foodborne pathogens in complex food environments.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eDeclaration of competing interest\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by The National Natural Science Foundation of China (32202173, 32572696) and Shanghai Rising-Star Program (23QC1401300).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eLiling Hao: Writing \u0026ndash; review \u0026amp; editing, Supervision, Project administration, Methodology, Funding acquisition, Conceptualization. Jifan Huang: Writing \u0026ndash; original draft, Validation, Methodology, Investigation, Formal analysis, Data curation. Mengqiu Li: Resources, Investigation, Formal analysis. Yihang Yang: Formal analysis, Data curation. Jiaqi Han: Formal analysis, Data curation. Hui Cao: Formal analysis, Data curation. Tai Ye: Formal analysis, Data curation. Xiuxiu Wu: Formal analysis, Data curation. Huajie Gu: Formal analysis, Data curation. Yuzheng Li: Formal analysis, Data curation. Fei Xu: Writing \u0026ndash; review \u0026amp; editing, Resources, Investigation, Funding acquisition.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThis work was financially supported by The National Natural Science Foundation of China (32202173, 32572696) and Shanghai Rising-Star Program (23QC1401300).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZhan K, Chen L, Li S, Yu Q, Zhao Z, Li J, Xing Y, Ren H, Wang N, Zhang G (2024) A novel metal\u0026ndash;organic framework based electrochemical immunosensor for the rapid detection of Salmonella typhimurium detection in milk. 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J Agric Food Chem 36:13518\u0026ndash;13526. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acs.jafc.3c03799\u003c/span\u003e\u003cspan address=\"10.1021/acs.jafc.3c03799\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"microchimica-acta","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"miac","sideBox":"Learn more about [Microchimica Acta](https://link.springer.com/journal/604)","snPcode":"604","submissionUrl":"https://submission.springernature.com/new-submission/604/3","title":"Microchimica Acta","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Aptamer, Rolling circle amplification, DNA hydrogel, Steady-state Chemiluminescent, Salmonella typhimurium","lastPublishedDoi":"10.21203/rs.3.rs-8569290/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8569290/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eSalmonella typhimurium\u003c/em\u003e (\u003cem\u003eS.typhimurium\u003c/em\u003e) is one of the common foodborne pathogens, posing a serious global food safety risks. It is thus important to develop a rapid and sensitive detection method for \u003cem\u003eS.typhimurium\u003c/em\u003e. In this study, a steady-state chemiluminescent DNA hydrogel aptasensor was constructed for ultrasensitive detection of \u003cem\u003eS.typhimurium\u003c/em\u003e in chicken. The sensor exploited the dense network of rolling circle amplification products (RCPs) DNA hydrogel to restrict luminol diffusion, thereby generating a stable chemiluminescent signal. A dual rolling circle amplification (DRCA) strategy was employed to accelerate hydrogel formation within 33 min, which increased RCP yield and strengthened intermolecular interactions. In the presence of \u003cem\u003eS.typhimurium\u003c/em\u003e, the aptamers specifically binds to the bacteria, leading to the dissociation of the aptamer\u0026ndash;primer complex and the subsequent release of primers that initiate two rolling circle amplification (RCA) reactions on partially complementary circular templates. When mixing the two parallel RCA products, the long ssDNA strands instantaneously assembled into hydrogels through complementary base pairing and physical entanglement, subsequently incorporating luminol for quantitative analysis. The chemiluminescence intensity exhibited a concentration-dependent enhancement with \u003cem\u003eS.typhimurium\u003c/em\u003e, displaying a well-defined linear response from 10 to 7.6 \u0026times; 10⁸ CFU/mL under optimal conditions, with a limit of detection of 8 CFU/mL. The assay also exhibited reliable specificity and stability in chicken samples, supporting its potential application in food safety monitoring.\u003c/p\u003e","manuscriptTitle":"A Novel Steady-state Chemiluminescent Aptasensor using Rolling Circle Amplification Products DNA Hydrogel for Rapid and Ultra-sensitive Detection of Salmonella Typhimurium in Chicken","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-23 12:54:31","doi":"10.21203/rs.3.rs-8569290/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-08T14:47:55+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-07T14:25:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"87373594393383391990005548085052206110","date":"2026-01-30T13:55:04+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-30T10:04:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"186828892071710953630215138618759119699","date":"2026-01-21T13:08:05+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-21T11:51:48+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-15T23:33:37+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-15T23:33:27+00:00","index":"","fulltext":""},{"type":"submitted","content":"Microchimica Acta","date":"2026-01-10T15:22:21+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"microchimica-acta","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"miac","sideBox":"Learn more about [Microchimica Acta](https://link.springer.com/journal/604)","snPcode":"604","submissionUrl":"https://submission.springernature.com/new-submission/604/3","title":"Microchimica Acta","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"88573e63-5250-4ac2-855c-2f4e2a85bd81","owner":[],"postedDate":"January 23rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-20T16:08:24+00:00","versionOfRecord":{"articleIdentity":"rs-8569290","link":"https://doi.org/10.1007/s00604-026-07998-5","journal":{"identity":"microchimica-acta","isVorOnly":false,"title":"Microchimica Acta"},"publishedOn":"2026-04-17 15:58:47","publishedOnDateReadable":"April 17th, 2026"},"versionCreatedAt":"2026-01-23 12:54:31","video":"","vorDoi":"10.1007/s00604-026-07998-5","vorDoiUrl":"https://doi.org/10.1007/s00604-026-07998-5","workflowStages":[]},"version":"v1","identity":"rs-8569290","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8569290","identity":"rs-8569290","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}