Based on Aptamer-carbon quantum dots and silver nanoparticles FRET sensor for sensitive detection of E. coli

Based on Aptamer-carbon quantum dots and silver nanoparticles FRET sensor for sensitive detection of E. coli | 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 Based on Aptamer-carbon quantum dots and silver nanoparticles FRET sensor for sensitive detection of E. coli Xiaolian Bai, Lu Ga, Jun Ai This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2984200/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract In this work, carbon quantum dots were prepared from grapefruit peel as carbon source by microwave heating method. The prepared carbon quantum dots CQDs were analyzed by fluorescence spectroscopy, TEM analysis, XPS analysis, etc., as well as their optical properties were also investigated. The prepared CQDs have high green fluorescence with excitation wavelength of 320 nm and maximum emission wavelength of 415 nm. the average particle size is about 7.4 nm, uniform dispersion and good stability. And a FRET (fluorescence resonance energy transfer) based fluorescence method was constructed by combining carbon quantum dots with silver nanoparticles (Ag NPs) for the rapid detection of E. coli. Its fluorescence transduction is based on the spectral overlap between the donor (CQDs) emission and the acceptor (nanoparticles) absorbance. The fluorescence of the aptamer-attached CQDs is burst in the presence of and silver nanoparticles. Upon addition of the specific E. coli solution, an aptamer-target complex is formed and the preferential interaction of the aptamer with the specific bacteria leads to the release of CQDs and Ag NPs. After incubation time, the bacterial cells are centrifuged, leading to the precipitation of E. coli aptamer couples and Ag NPs, resulting in the recovery of CQDs fluorescence. This method allows specific detection of E. coli in a wide range of pathogenic bacteria. The final results showed that the linear range of the sensor was 2×10 3 ~ 2×10 8 CFU·mL − 1 and the detection limit for E. coli was as low as 77 CFU·mL − 1 . CQDs Escherichia coli biosensor FRET Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Carbon quantum dots (CQDs) are one of the fluorescent nanoparticles that have been widely used in optical biosensors in recent years. This is due to the advantages of CQDs such as good biocompatibility, low toxicity, ease of synthesis, good stability in aqueous solution, and small particle size.CQDs have many hydroxyl and carboxyl groups on their surface and bind to other biomolecules through these functional groups [ 1 ] . Chemical and natural substances as precursors are prepared by different synthetic methods [ 2 ] such as laser ablation, electrical discharge, chemical oxidation, microwave irradiation and hydrothermal methods.The fine optical properties of CQDs lead to a wide range of applications of these particles in different fields such as bioimaging, sensing, drug and gene delivery [ 3 ] .One of the best methods for the synthesis of CQDs is microwave heating using natural sources [ 4 ] . Microwave heating method is a simple, cost-effective and time-saving method. In this study, white grapefruit peel was used as a carbon source for green synthesis of CQDs. Grapefruit peel is a by-product of grapefruit processing and is rich in soluble and insoluble carbohydrates. In addition, it has many functional components such as vitamins, minerals and flavonoids. Therefore, it is considered as a wonderful carbon precursor. These rich compositions allow the synthesis of quanta with good physical and chemical properties (e.g. excellent fluorescence, water solubility and biocompatibility). Fluorescence methods for E. coli have been widely studied for their high sensitivity and simplicity [ 5 ] . Biosensor technology is widely used for pathogenic bacteria detection. The use of antibodies in biosensors has declined due to high cost, complexity of conjugation methods and surface modification methods, their long selection process and low stability. However, aptamers are single-stranded oligonucleotides of DNA or RNA that can bind to their targets with high affinity and specificity. Biosensor technology for bacterial detection has also been developed. The folding of aptamers with intramolecular interactions leads to secondary and tertiary conformations with high specificity for their targets. Aptamers have good stability in different organic solvents, temperatures and pH (4–9), have the flexibility to be used as biosensors, are easy to synthesize by the SELEX process and can be chemically modified and coupled [ 6 ] . Other advantages of aptamers compared to antibodies are as follows: aptamers are highly stable at high temperatures and in long-term storage. Antibodies are very expensive to produce, whereas aptamers are easily synthesized by chemical reactions. Aptamers are less immunogenic because nucleic acids are not recognized as foreign substances by the human immune system. Antibodies, on the other hand, have significant immunogenicity. Aptamers show high affinity for substances that cannot be recognized by antibodies, such as ions or small molecules. Based on these advantages, aptamers are considered as an alternative to antibodies in many biological applications [ 7 , 8 ] . Hu et al. designed an aptamer sensor system for the detection of E. coli based on carbon quantum dots and magnetic nanoparticles to prepare fluorescent probes for specific detection of E. coli using aptamer-attached fluorescent probes. In this work, a fluorescence-adapted sensor based on FRET detection was designed for E. coli detection (Fig. 1 ). FRET is a technique that utilizes the principle of fluorescence resonance energy transfer between the acceptor and donor fluorophores. In the FRET process, carbon quantum dots can be employed as a donor for the FRET system due to their unique fluorescence bursting properties. On the other hand, silver nanoparticles can be used as effective acceptors for FRET systems due to their high extinction coefficient and wide absorption band [ 9 ] . CQDs (donors) emit green fluorescence under UV light and their fluorescence intensity decreases when specific oligonucleotide aptamers surround them. In the presence of metals, the efficiency of fluorescence resonance energy transfer (FRET) can be improved [ 10 ] . Silver nanoparticles (Ag NPs) (receptors) burst the fluorescence of CQDs-attendees during FRET [ 11 ] . In the presence of E. coli, the preferential interaction of the aptamer with specific bacteria leads to the release of CQDs and Ag NPs. centrifugation of bacterial cells after a period of incubation leads to the precipitation of E. coli-aptamer coupling and Ag NPs, which results in the re-emission of CQDs fluorescence in the supernatant before measurement. After the addition of the control bacterial solution, no interaction with the aptamer occurs; therefore, the fluorescence intensity of the supernatant cannot be recovered should remain unchanged. The strong burst of CQDs fluorescence during FRET leads to higher detection sensitivity. Ag NPs have attracted a lot of attention in FRET methods because of their excellent surface plasmon resonance absorption [ 12 ] . However, the use of CQDs-Ag NPs in FRET-based sensors has been rarely reported [ 13 ] . To the best of our knowledge, FRET between CQDs and Ag NPs has not been applied to the detection of E. coli. The aim of this study was to develop a novel, simple, rapid, non-toxic method with maximum possible ultra-sensitive detection of E. coli. Most CQDs are used for bioimaging of bacterial cells [ 14 , 15 ] . There are few studies on the detection of specific bacteria as targets by CQDs. No studies have been reported on the use of green CQDs and silver nanoparticles for the detection of E. coli during FRET. The sensor has a high sensitivity in detecting E. coli in a short period of time. 2. Experimental section 2.1. Materials and reagents AgNO 3 were obtained from Kaima Reagent Co., Ltd. (Tianjin, China). Glutathione purchased from Biotoped. NaBH 4 were obtained from Fuchen Reagent Co., Ltd. (Tianjin, China). Polyethylene Pyrrole K-30(PVP)were obtained from Chemical Reagent of China National Pharmaceutical Group. o-Phenylenediamine were obtained from Tianjin Chemical Reagent Co., Ltd (Tianjin, China). o-Phenylenediamine were obtained from Tianjin Chemical Reagent Co., Ltd (Tianjin, China). Ethanolamine and Ethylene glycol were obtained from Tianjin Beilian Fine Chemicals Development Co., Ltd (Tianjin, China). All the solutions were prepared with pure water and stored at 4◦ C before use. All chemical reagents were of analytical grade and used without further purification. The DNA used in the experiment was purchased from Shanghai Sangong Biotechnology Co., Ltd. The sequence of E. coli specific aptamer was 5 '- FAM-CCG GAC GCT TAT GCC TTG CCA TCT ACA GAG CAG GTG TGA CGG-3'. Bacterial sample E Coli (BL21) is provided by the School of Biology, where bacterial colonies are diluted with PBS. 2.2. Apparatus Transmission electron microscope (TEM) images were obtained with a JEOL-2100F TEM (Japan) operating voltage of 200 kV. All fluorescence (FL) spectra were recorded on a Hitachi F-4600 fluorescence spectrophotometer.The ultraviolet–visible (UV–vis) absorption spectra and fluorescence spectrum were recorded using a Hitachi U-2900 (Hitachi, Japan) UV–vis spectrophotometer.X-ray photoelectron spectroscopy (XPS) data were collected by ESCALab 250Xi. 2.3 CQDs的合成 Carbon quantum dots were synthesized by microwave heating. First, fresh white grapefruit peels were washed three times with ethanol and deionized water and dried at room temperature. The dried grapefruit peel was ground into a powder. The powder (1 g) was mixed with 10 mL of ethylene glycol and then heated in a household microwave oven over medium heat for 3 min. The solution turned dark brown, indicating the formation of CQDs. The brown solution was then purified by centrifugation at 15,000 rpm with dialysis membranes (molecular weight cutoff = 1000) to remove insoluble material and small molecules. The prepared CQDs were stored at 4°C for further studies. Figure 1 shows a schematic diagram of the preparation of CQDs from grapefruit peel. 2.4 Aptamer attachment to CQDs The E. coli aptamer was annealed and heated in a water bath at 95°C for 5 min, and slowly cooled down to room temperature at room temperature. The annealed DNA was diluted by PBS (0.01 M, pH = 7.40) and set aside. Then, 200 µL of CQDs were added to 20 µL of aptamer solution and shaken for 30 min at 37°C in the dark. To block the free carboxyl groups on the surface of CQDs, we added ethanolamine (10 mM) and shaken the solution for 2 hr. Finally, we measured the fluorescence intensity of the solution. 2.5 Preparation of silver nanoparticles The preparation was carried out by the original method of laboratory sisters. Take 3 mLAgNO3 solution (1M) and slowly add it to 3 mLPVP solution (1M), mix the solution thoroughly, then add 1.5 mL o-phenylenediamine solution (0.1M) and stir for 2 h at room temperature to obtain the primary product, then centrifuge at 15000 rpm, and wash the final product with ethanolamine in the ratio of 1:1 for three times to obtain PVP-Ag NPs. 2.6 FRET-based detection of E. coli First, in order to achieve the desired results, we have to optimize some parameters to optimize the volume of aptamer-CQDs hybridized with Ag NPs. The FRET process occurs when the excited state donor transfers energy to the ground state acceptor through physicochemical interactions. Fluorescence transduction is based on the spectral overlap between the emission of the donor and the absorbance of the acceptor [127].FRET between CQDs-Ag NPs aptamers is constructed by the following steps: 80 µL of different concentrations of Ag NPs are added to 400 µL of CQDs-aptamer solution and shaken for 15 min.The fluorescence intensity of this mixture is measured and then, the above solution is added with different concentrations of E. coli were added to the above solution and shaken at room temperature for 2 h. The fluorescence of the supernatant was centrifuged and detected. The selectivity of the sensor was tested with other strains such as Salmonella, Salmonella and Carbonic Anhydrase. A concentration of 2 × 108 CFU-mL-1 of each bacterium was diluted with PBS and evaluated separately using this method. 2.7 Temperature sensing of CQDs After the temperature of the water bath had risen to a certain temperature and stabilized, one of the centrifuge tubes was heated in the water bath to make the reaction complete, and the fluorescence intensity was measured. The fluorescence intensity of CQDs at different temperatures was recorded. 3 Results and discussion 3.1 Principle of FRET sensor A fluorescence resonance energy transfer (FRET) biosensor based on green synthetic carbon quantum dots (CQDs) and silver nanoparticles (Ag NPs) (excellent surface plasmon resonance absorption ability) was designed to detect E. coli, and the fluorescence transduction was based on the spectral overlap between the donor (CQDs) emission and the acceptor (nanoparticles) absorbance. The excitation and emission wavelengths of CQDs are shown in Fig. 2 (a), where the prepared CQDs have an excitation peak at 320 nm and an emission peak at 415 nm. The inset in Fig. 3 (a) demonstrates that the bright green fluorescence of CQDs can be observed under UV lamp irradiation at 365 nm. In addition, the emission wavelength of CQDs at 400 nm overlaps with the absorbance of Ag NPs. This indicates that Ag NPs can successfully achieve the burst of carbon quantum dot fluorescence by mechanisms such as fluorescence resonance energy transfer (FRET) or internal filtering effect (IFE). It can be seen that the fluorescence intensity of CQDs-aptamer-Ag NPs was burst and then the fluorescence recovered with the increase of E. coli in the system (Fig. 3 b). The fluorescence intensity also decreases when the aptamer is added to the CQD solution, and the decrease increases when Ag NPs are added, which finally succeeds in bursting the CQDs fluorescence, leading to the FRET process. Due to the surface of CQDs with amine groups, adsorption is easily achieved by electrostatic interaction with the surface of Ag NPs, and this interaction leads to the bursting of the fluorescence signal emitted by CQDs [11,16]. However, the preferential binding of specific aptamers through H-bonds upon addition of bacteria [17] leads to the release of CQDs and Ag NPs, and after a period of incubation, the bacterial cells are centrifuged, leading to the precipitation of E. coli aptamer couples and Ag NPs, at which point CQDs fluorescence is re-emitted. The fluorescence intensity of CQDs in the supernatant was measured. These aptamers can specifically bind to specific bacterial surface compounds, which can serve as recognition elements for new aptamer systems. As the principle belongs, the black line in (Fig. 2 b) is the CQDs alone, and the fluorescence is burst when the aptamer is attached and Ag NPs are added, as in the red line in (Fig. 2 b). The FRET process was confirmed to proceed efficiently, and the fluorescence was restored after the final addition of E. coli broth and incubation for a period of time, as shown in the blue line in (Fig. 3 b). 3.2 Optimization of experimental conditions In order to better observe the FRET results, the volumes of CQDs, Ag NPs and aptamers were optimized during the experiments. The effect of different material dosage on the fluorescence intensity was recorded. The premise of the optimization is to ensure the fluorescence burst effect of CQDs as much as possible, on the other hand, the volume of CQDs we prepared is sufficient, but the volume of aptamer is very small (248 µL for one tube) so the optimization was carried out by reading the literature as much as possible within the previous experiments to avoid waste. Through the optimization experiment, we can see from Fig. 2 (c) that the Apt: CQDs: Ag NPs = 1:15:1 burst intensity is the largest when CQDs is the variable, but we can also see that the fluorescence intensity burst of Apt: CQDs: Ag NPs = 1:10:1 is about 150, which has burst 90% of the original CQDs, so the Apt: CQDs: Ag NPs is chosen when CQDs is the variable. Therefore, the ratio of Apt: CQDs: Ag NPs = 1: 10: 1 was chosen for the variable CQDs. As above, the fluorescence intensity of both Apt: CQDs: Ag NPs = 1: 10: 2 and Apt: CQDs: Ag NPs = 1: 10: 3 was below 100 when Ag NPs was the variable, so the ratio of Apt: CQDs: Ag NPs = 1: 10: 2 was chosen for the sake of drug consumption, and the subsequent experiments were conducted with this ratio. In Fig. 2 (d), we can also see the relative fluorescence difference of each volume ratio compared with the fluorescence intensity of the original CQDs, and we can observe that the fluorescence intensity difference is about 1000 for Apt: CQDs: Ag NPs = 1: 10: 2, which again confirms that we can choose the ratio of Apt: CQDs: Ag NPs = 1: 10: 2 for the subsequent experiments. 3.3 Characterization of carbon quantum dots The CQDs were comprehensively characterized using transmission electron microscopy images (TEM), X-ray diffraction (XRD), Fourier infrared spectroscopy (FT-IR), and X-ray photoelectron spectroscopy (XPS). 3.3.1Transmission electron microscopy (TEM) The carbon quantum dots prepared by microwave heating method using grapefruit peel as carbon source, we cannot distinguish the morphology and size of the prepared substances by naked eyes only, and transmission electron microscopy has been widely used to characterize the size and morphology of CQDs. The synthesized carbon quantum dots solution was precisely dropped onto the copper grid of the carbon support film with a pipette gun and dried naturally at room temperature for about one hour when the solvent was water, and then observed under the transmission electron microscope. The prepared CQDs samples showed good dispersion and proper dispersion under transmission electron microscopy (TEM) with spherical shape and relatively small particle size, the diameter distribution ranged from 5–9 nm with an average particle size of 7.4 nm, accordingly, no obvious deposition of large particles was observed in these samples(Fig. 3 ). 3.3.2 XRD analysis and FT-IR analysis of CQDs In this work, the crystallinity of CQDs was investigated in depth using X-ray diffraction analysis (XRD) technique, and the relevant experimental results are presented in Fig. 3 (c). The synthesized CQDs exhibit highly disordered carbon atoms, which is well reflected by the diffraction peaks detected by XRD located around 2θ = 22° [ 18 ] . The functional groups on the surface of CQDs were investigated by FT-IR spectroscopy. As shown in Fig. 3 (d), the FT-IR absorption bands of CQDs were 3416, 3140, 2350, 1630, 1380, and 1082 cm-1. The peaks at 3416 cm-1 and 3140 cm-1 can be attributed to primary amino (N - H) or hydroxyl group (O - H) vibrations. In addition, the spectral peaks at 2350 cm-1 and 1630 cm-1 are associated with the vibrations of the carboxyl group C = O or C = C [ 19 ] . The spectral peaks at 1380 cm-1 and 1082 cm-1 can confirm that CQDs have amide bonds [20], corresponding to the C - N and C - O stretching vibrations. The FT-IR test results showed that CQDs are rich in functional groups on the surface, and functional groups such as carboxyl and amino groups are particularly prominent. The presence of these functional groups can not only indicate the water solubility of CQDs, but also provide the necessary support for their good biocompatibility. The elemental composition and surface groups of CQDs can be studied and analyzed by applying X-ray photoelectron spectroscopy (XPS) techniques. Specifically, Fig. 4 of this paper presents XPS full spectrum scans of the synthesized CQDs and high-resolution spectra for the elements O1s, C1s and N1s. In the full spectrum range as shown in (Fig. 4 a), three main peaks can be found, and the peaks correspond to energies of 284.6 eV, 401.2 eV and 532.3 eV, which correspond to C1s, N1s and O1s, respectively, so it can be inferred that the CQDs mainly contain carbon, nitrogen, oxygen and hydrogen elements. The percentages of carbon, nitrogen and oxygen atoms are about 75.96%, 9.36% and 14.68%. Three typical peaks of 284.7 eV (C - C/C = C), 286.5 eV (C - O/C - N) and 288.6 eV (C = O/C = N) were present in the high-resolution C1s spectrum (Fig. 4 b). In addition, the XPS analysis results are in agreement with the FT-IR analysis. After the detection of the high-resolution O1spectrum peaks, the corresponding three characteristic peaks were found to be located at 531.5 eV, 532 eV, and 533.4 eV, representing the C - O, C = O, and C - O - C groups, respectively (Fig. 4 c). Meanwhile, Fig. 4 d presents the N1s spectrum of CQDs, where the main peak at 399.7 eV represents C - N. These results suggest the presence of more oxygen-containing, carbon-containing groups on the synthesized quantum surface. In addition, the oxygen element may be the main dopant element that plays an important role in the change of fluorescence properties [ 21 , 22 ] . 3.5 Stability analysis of CQDs For the synthesized carbon quantum dots, the stability of the nanoparticles under different environments was then investigated. In Fig. 5 (a), it can be clearly seen that the fluorescence intensity of the carbon quantum dots decreases linearly from 25 to 65°C, and between 75 and 95°C, the fluorescence intensity also decreases, but the decrease is less than 10 negligible and basically remains stable. It proves that the nanoparticles remain stable between 75–95℃. In Fig. 5 (b), we can observe the above conclusion more clearly. By comparing the fluorescence intensity, we can know that it has the highest fluorescence intensity at room temperature. Therefore, in our subsequent experiments, there is no need for heating and other steps, and it can be performed at room temperature. Figure 5 (c) shows that the fluorescence change of CQDs is less than 150, or almost no change, after 5 months of preparation at 4℃, which indicates that the CQDs are very stable. If the amount of subsequent experiments is large, we can prepare a large amount at one time and use it in the subsequent experiments for several months without further preparation. The uncertainty of repeated preparation of CQDs is well avoided. These experimental results also demonstrate that it can maintain its stability under more complex conditions. 3.6 Optical properties of CQDs The fluorescence stability of CQDs was investigated for their photobleaching properties, as shown in Fig. 6 .a. The prepared CQDs exhibited insignificant photobleaching after continuous irradiation with a 365 nm UV lamp for 1 h. A finer decrease in fluorescence intensity of less than 200 can be observed in (Fig. 6 b). The results indicate that the fluorescence intensity maintained its stability during continuous irradiation with the UV lamp. Figure 6 (b) shows the emission spectra of CQDs at different excitation wavelengths from 290 nm to 380 nm, and it can be seen that the emission intensity is highest at 330 nm excitation. A redshift in the emission spectrum can be clearly observed in the fluorescence emission spectra as the excitation wavelength gradually increases. As observed in Fig. 6 (c), in the ultraviolet-visible (UV-vis) absorption spectrum, higher absorbance in the UV-visible spectrum can be clearly detected in the region of 200 ~ 400 nm. Specifically, 2 unique absorption peaks were exhibited in the UV-vis absorption region. They appear at 220 nm and 283 nm, respectively. The leap energy band at 220 nm corresponds to the π-π leap of C = C, while the absorption at 283 nm represents the n-π leap of the C = O bond [ 23 ] .The optical absorption peaks of CQDs show a gradual decrease in intensity with increasing dilution and a slight blue shift, which may be due to surface or edge defects of CQDs. 3.7 Sensibility analysis for Escherichia coli The effect of different concentrations of E. coli in the set-up ligand sensor on the fluorescence intensity was investigated under optimized conditions. The extent to which the fluorescence was restored after the reaction of this CQDs with different concentrations of E. coli was further experimented. Figure 7 (a) shows the fluorescence intensity of the CQDs after the addition of different concentrations of E. coli. It can be observed that the fluorescence intensity of CQDs increases with increasing concentration of E. coli. E. coli solutions in the concentration range of 2 × 10 2 ~ 2 × 10 8 CFU-mL − 1 were examined. The linear relationship between the fluorescence values of the CQDs and the concentration of E. coli solution at concentrations between 2×10 3 ~ 2×10 8 CFU-mL-1 was extremely strong, as shown in Fig. 7 (b), with a correlation coefficient of 0.9961 and a detection limit of 77 CFU-mL-1. The sensitivity of the system was investigated, ensuring a fairly wide dynamic range and The sensitivity of the system was investigated, ensuring a wide dynamic range and a low detection limit. A simple method based on the FRET process between aptamer-CQDs and Ag NPs was designed. CQDs were prepared from natural sources and are cost-effective compared to commercially available QD nanoparticles. Compared to other techniques, the fluorescent biosensor technology further confirms the reliability of the method while improving the detection sensitivity, and thus is widely used in practical applications. 3.8 Specificity of the assay for Escherichia coli To investigate the specificity of the assay in the detection of E. coli, the method was applied to the detection of other bacteria under optimal conditions. such as carbonic anhydrase bacteria, Salmonella typhimurium, Salinophilus and Bacillus, and LB cultures. As shown in Fig. 8 , the fluorescence intensity was enhanced in the presence of E. coli in the mixture. In the absence of the addition of E. coli, the fluorescence intensity of other bacterial solutions remained significantly unchanged even when added. The experimental results proved that the fluorescence intensity enhancement of non-target bacteria and culture solution was much lower than that of E. coli. When other non-specific bacteria were added, there was no interaction between the bacteria and the aptamer; therefore, the FRET process could not be disrupted, and the fluorescence of the supernatant remained unchanged. The results clearly indicate that the sensor is less responsive to other bacteria; therefore, the biosensor has good specificity. 4. Conclusion This work presents a new strategy for the detection of E. coli based on green synthetic carbon quantum dots (CQDs) and silver nanoparticles (Ag NPs) designed as fluorescence resonance energy transfer (FRET) biosensors. CQDs materials were successfully synthesized by microwave reaction technique using white grapefruit peel as a carbon source. The materials exhibited excellent green luminescence properties. The fluorescence of CQDs was burst in the presence of aptamers and Ag NPs. After the addition of specific bacteria, an aptamer-target complex is formed and the emission of free CQDs is restored. The linear range of the aptamer sensor for E. coli concentration was 2 × 10 3 ~ 2 × 10 8 CFU-mL-1 and the detection limit for E. coli was as low as 77 CFU-mL-1. These results indicate that the analytical performance of the system can be used as an effective sensor for E. coli detection. This response is caused by the specific binding of the aptamer to E. coli, which is much higher than the response to other non-specific bacteria, indicating the high specificity of the bioassay. Because of its simplicity and effectiveness, the FRET fluorescent biosensor holds promise for a wide range of applications in other bacterial and biomolecular assays. Declarations Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant No.21864020，82160652), the Natural Science Foundation of Inner Mongolia (Grant No. and 2019MS02014, 2018MS02012), ‘‘Young Science and Technology Talents Program’’ (Leading Person) in Inner Mongolia Autonomous Region Colleges and Universities (Grant No. NJYT-19-A04), the Fundamental Research Funds for the Inner Mongolia Normal University, China, (Grant No. 2022JBZD013). Availability of data and materials All data and materials in this study are included in the published article and its additional file. Ethics approval and consent to participate Not applicable. Consent for publication All authors agree to publish. Competing interests The authors declare that there is no conflict of interest. Author details 1 College of Chemistry and Environmental Science, College of Geographi- cal Science, Inner Mongolia Key Laboratory of Environmental Chemistry, Inner Mongolia Normal University, 81 Zhaowudalu, Hohhot 010022, China. 2 College of Pharmacy, Inner Mongolia Medical University, Jinchuankaifaqu, Hohhot 010110, China. References Arkan E, Barati A, Rahmanpanah M, et al. Green synthesis of carbon dots derived from walnut oil and an investigation of their cytotoxic and apoptogenic activities toward cancer cells[J]. Advanced pharmaceutical bulletin, 2018, 8(1): 149–155. Sun X, Lei Y. Fluorescent carbon dots and their sensing applications[J]. TrAC Trends in Analytical Chemistry, 2017, 89: 163-180. Sachdev A, Gopinath P. Green synthesis of multifunctional carbon dots from coriander leaves and their potential application as antioxidants, sensors and bioimaging agents[J]. Analyst, 2015, 140(12): 4260-4269. Motaghi H, Mehrgardi M A, Bouvet P. 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One-pot green synthesis of oxygen-rich nitrogen-doped graphene quantum dots and their potential application in pH-sensitive photoluminescence and detection of mercury (II) ions[J]. Talanta, 2015, 142: 131-139. Ding H, Li X H, Chen X B, et al. Surface states of carbon dots and their influences on luminescence. Journal of Applied Physics, 2020, 127(23): 231101-21. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2984200","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":204471828,"identity":"6d120d1a-8f8d-4597-9ffc-f067e521fc3e","order_by":0,"name":"Xiaolian Bai","email":"","orcid":"","institution":"Inner Mongolia Normal University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiaolian","middleName":"","lastName":"Bai","suffix":""},{"id":204471829,"identity":"7ffc8912-2a49-4b20-97e3-0ace7d229fde","order_by":1,"name":"Lu Ga","email":"","orcid":"","institution":"Inner Mongolia Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lu","middleName":"","lastName":"Ga","suffix":""},{"id":204471830,"identity":"05ca1cb4-7fdd-49f2-b88f-55ebf7e6aeaf","order_by":2,"name":"Jun Ai","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuklEQVRIiWNgGAWjYBACNjDJI8HDz8x8+AEpWmxkJNvZ0gxIsSzNxuA8j4IEUWr5xA4//PBD5jCP8WEeBgOGGptowg6TTjOW7OE5zGN2mPfAA4ZjabkNhLUkmDHwgLXwJRgwNhwmRkv6N8Y/QC3GzTwGEkRqyTFj5uFJ4zFgJkFLsbQMjw2PxGFgICcQ4xf52ekbP77tkbDn7z98+MGHGhvCWsCAsQfKSCBKORj8IF7pKBgFo2AUjEAAAMyaNN5cEHPPAAAAAElFTkSuQmCC","orcid":"","institution":"Inner Mongolia Normal University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Ai","suffix":""}],"badges":[],"createdAt":"2023-05-26 07:14:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2984200/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2984200/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":37677962,"identity":"33b809c5-8713-474b-b96a-63c6c0abc6f4","added_by":"auto","created_at":"2023-05-30 15:54:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":215100,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram of using FRET technology to detect Escherichia coli.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-2984200/v1/1706487671f5e4bf21418c84.png"},{"id":37679503,"identity":"4f8ff1f0-54ab-4c07-8ee8-1467b1bca7c0","added_by":"auto","created_at":"2023-05-30 16:02:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":172866,"visible":true,"origin":"","legend":"\u003cp\u003e(a). Fluorescence spectrogram of CQDs overlaid with the absorption spectrum of Ag NPs. (Inset visible light (light yellow) UV lamp (green) (b). Fluorescence spectra of CODs, Apt-CQDs+AgNPs and Apt-CQDs+AgNPs+E.coli. (c). Fluorescence spectra of CQDs/Ag NPs/aptamers with different volume ratios.; (d)Histogram of different volume ratios of CQDs/Ag NPs/Aptamer fluorescence difference\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-2984200/v1/b223f31c7a6a7ccf036b03f5.png"},{"id":37680338,"identity":"b865be12-51ec-444d-8975-e28e41517181","added_by":"auto","created_at":"2023-05-30 16:10:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":190457,"visible":true,"origin":"","legend":"\u003cp\u003e(a).TEM plots of CQDs; (b)CQDs particle size distribution map; (c). XRD analysis of CQDs.; (d)FT-IR analysis of CQDs\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-2984200/v1/7bbc86032742c7da04f927c9.png"},{"id":37677964,"identity":"c4357f95-4399-4b9f-9014-a9ac79545988","added_by":"auto","created_at":"2023-05-30 15:54:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":104877,"visible":true,"origin":"","legend":"\u003cp\u003e(a) XPS full range survey spectrum and high-resolution (b) C1s, (c) N1s , and (d) O1s spectra of as-prepared CQDs.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-2984200/v1/b637c76e1611f37df1a833a6.png"},{"id":37677961,"identity":"fcad59ff-1719-4f0c-8303-60b870403690","added_by":"auto","created_at":"2023-05-30 15:54:11","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":85529,"visible":true,"origin":"","legend":"\u003cp\u003e(a).Fluorescence spectrum of CQDs at 25 - 95 ℃; (b). Fluorescent line graph of CQDs at 25 - 95 ℃; (c). Fluorescence spectra of CQDs after 1h and 5 months of preparation.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-2984200/v1/5fbf6ed8f74fd5fd5ba8912b.png"},{"id":37677968,"identity":"e32b3a80-59f1-41df-8ddc-159e94c8d533","added_by":"auto","created_at":"2023-05-30 15:54:11","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":155444,"visible":true,"origin":"","legend":"\u003cp\u003e(a) CQDs photobleaching fluorescence spectrum. (b) CQDs photobleaching fluorescence scatter plot. (c) Fluorescence emission spectra of CQDs at different excitation wavelengths.(d)UV-Vis absorption spectra of CQDs.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-2984200/v1/73f5593adcc49dce2ab09d70.png"},{"id":37677966,"identity":"b8ad2620-64bb-49e8-b49b-cdf303a18ac4","added_by":"auto","created_at":"2023-05-30 15:54:11","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":87726,"visible":true,"origin":"","legend":"\u003cp\u003e(a). Fluorescence spectra of CQDs after the addition of different concentrations of E.coli; (b). Linear relationship between the fluorescence intensity of CQDs and the concentration of E. coli.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-2984200/v1/5366d3ee7cbefd9081c4799b.png"},{"id":37679501,"identity":"a0f90d01-84df-4b46-ae67-cc5c785ea167","added_by":"auto","created_at":"2023-05-30 16:02:11","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":58002,"visible":true,"origin":"","legend":"\u003cp\u003eSelectivity of FRET biosensors.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-2984200/v1/e31b18f13f81946ef2943921.png"},{"id":37788503,"identity":"765c5726-1a2a-42ab-ab76-8eecde375f06","added_by":"auto","created_at":"2023-05-31 17:59:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1346509,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2984200/v1/bf041c60-0573-45e8-87de-bd4c46d2fdfa.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Based on Aptamer-carbon quantum dots and silver nanoparticles FRET sensor for sensitive detection of E. coli","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCarbon quantum dots (CQDs) are one of the fluorescent nanoparticles that have been widely used in optical biosensors in recent years. This is due to the advantages of CQDs such as good biocompatibility, low toxicity, ease of synthesis, good stability in aqueous solution, and small particle size.CQDs have many hydroxyl and carboxyl groups on their surface and bind to other biomolecules through these functional groups \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Chemical and natural substances as precursors are prepared by different synthetic methods\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e such as laser ablation, electrical discharge, chemical oxidation, microwave irradiation and hydrothermal methods.The fine optical properties of CQDs lead to a wide range of applications of these particles in different fields such as bioimaging, sensing, drug and gene delivery\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e.One of the best methods for the synthesis of CQDs is microwave heating using natural sources\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Microwave heating method is a simple, cost-effective and time-saving method. In this study, white grapefruit peel was used as a carbon source for green synthesis of CQDs. Grapefruit peel is a by-product of grapefruit processing and is rich in soluble and insoluble carbohydrates. In addition, it has many functional components such as vitamins, minerals and flavonoids. Therefore, it is considered as a wonderful carbon precursor. These rich compositions allow the synthesis of quanta with good physical and chemical properties (e.g. excellent fluorescence, water solubility and biocompatibility). Fluorescence methods for E. coli have been widely studied for their high sensitivity and simplicity\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Biosensor technology is widely used for pathogenic bacteria detection. The use of antibodies in biosensors has declined due to high cost, complexity of conjugation methods and surface modification methods, their long selection process and low stability. However, aptamers are single-stranded oligonucleotides of DNA or RNA that can bind to their targets with high affinity and specificity. Biosensor technology for bacterial detection has also been developed. The folding of aptamers with intramolecular interactions leads to secondary and tertiary conformations with high specificity for their targets. Aptamers have good stability in different organic solvents, temperatures and pH (4\u0026ndash;9), have the flexibility to be used as biosensors, are easy to synthesize by the SELEX process and can be chemically modified and coupled \u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Other advantages of aptamers compared to antibodies are as follows: aptamers are highly stable at high temperatures and in long-term storage. Antibodies are very expensive to produce, whereas aptamers are easily synthesized by chemical reactions. Aptamers are less immunogenic because nucleic acids are not recognized as foreign substances by the human immune system. Antibodies, on the other hand, have significant immunogenicity. Aptamers show high affinity for substances that cannot be recognized by antibodies, such as ions or small molecules. Based on these advantages, aptamers are considered as an alternative to antibodies in many biological applications \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Hu et al. designed an aptamer sensor system for the detection of E. coli based on carbon quantum dots and magnetic nanoparticles to prepare fluorescent probes for specific detection of E. coli using aptamer-attached fluorescent probes.\u003c/p\u003e \u003cp\u003eIn this work, a fluorescence-adapted sensor based on FRET detection was designed for E. coli detection (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). FRET is a technique that utilizes the principle of fluorescence resonance energy transfer between the acceptor and donor fluorophores. In the FRET process, carbon quantum dots can be employed as a donor for the FRET system due to their unique fluorescence bursting properties. On the other hand, silver nanoparticles can be used as effective acceptors for FRET systems due to their high extinction coefficient and wide absorption band \u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. CQDs (donors) emit green fluorescence under UV light and their fluorescence intensity decreases when specific oligonucleotide aptamers surround them. In the presence of metals, the efficiency of fluorescence resonance energy transfer (FRET) can be improved \u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. Silver nanoparticles (Ag NPs) (receptors) burst the fluorescence of CQDs-attendees during FRET \u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. In the presence of E. coli, the preferential interaction of the aptamer with specific bacteria leads to the release of CQDs and Ag NPs. centrifugation of bacterial cells after a period of incubation leads to the precipitation of E. coli-aptamer coupling and Ag NPs, which results in the re-emission of CQDs fluorescence in the supernatant before measurement. After the addition of the control bacterial solution, no interaction with the aptamer occurs; therefore, the fluorescence intensity of the supernatant cannot be recovered should remain unchanged. The strong burst of CQDs fluorescence during FRET leads to higher detection sensitivity. Ag NPs have attracted a lot of attention in FRET methods because of their excellent surface plasmon resonance absorption\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. However, the use of CQDs-Ag NPs in FRET-based sensors has been rarely reported \u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. To the best of our knowledge, FRET between CQDs and Ag NPs has not been applied to the detection of E. coli. The aim of this study was to develop a novel, simple, rapid, non-toxic method with maximum possible ultra-sensitive detection of E. coli. Most CQDs are used for bioimaging of bacterial cells\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. There are few studies on the detection of specific bacteria as targets by CQDs. No studies have been reported on the use of green CQDs and silver nanoparticles for the detection of E. coli during FRET. The sensor has a high sensitivity in detecting E. coli in a short period of time.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"2. Experimental section","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Materials and reagents\u003c/h2\u003e \u003cp\u003eAgNO\u003csub\u003e3\u003c/sub\u003e were obtained from Kaima Reagent Co., Ltd. (Tianjin, China). Glutathione purchased from Biotoped. NaBH\u003csub\u003e4\u003c/sub\u003e were obtained from Fuchen Reagent Co., Ltd. (Tianjin, China). Polyethylene Pyrrole K-30(PVP)were obtained from Chemical Reagent of China National Pharmaceutical Group. o-Phenylenediamine were obtained from Tianjin Chemical Reagent Co., Ltd (Tianjin, China). o-Phenylenediamine were obtained from Tianjin Chemical Reagent Co., Ltd (Tianjin, China). Ethanolamine and Ethylene glycol were obtained from Tianjin Beilian Fine Chemicals Development Co., Ltd (Tianjin, China). All the solutions were prepared with pure water and stored at 4◦ C before use. All chemical reagents were of analytical grade and used without further purification. The DNA used in the experiment was purchased from Shanghai Sangong Biotechnology Co., Ltd. The sequence of E. coli specific aptamer was 5 '- FAM-CCG GAC GCT TAT GCC TTG CCA TCT ACA GAG CAG GTG TGA CGG-3'. Bacterial sample E Coli (BL21) is provided by the School of Biology, where bacterial colonies are diluted with PBS.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Apparatus\u003c/h2\u003e \u003cp\u003eTransmission electron microscope (TEM) images were obtained with a JEOL-2100F TEM (Japan) operating voltage of 200 kV. All fluorescence (FL) spectra were recorded on a Hitachi F-4600 fluorescence spectrophotometer.The ultraviolet\u0026ndash;visible (UV\u0026ndash;vis) absorption spectra and fluorescence spectrum were recorded using a Hitachi U-2900 (Hitachi, Japan) UV\u0026ndash;vis spectrophotometer.X-ray photoelectron spectroscopy (XPS) data were collected by ESCALab 250Xi.\u003c/p\u003e \u003cp\u003e2.3 CQDs的合成\u003c/p\u003e \u003cp\u003eCarbon quantum dots were synthesized by microwave heating. First, fresh white grapefruit peels were washed three times with ethanol and deionized water and dried at room temperature. The dried grapefruit peel was ground into a powder. The powder (1 g) was mixed with 10 mL of ethylene glycol and then heated in a household microwave oven over medium heat for 3 min. The solution turned dark brown, indicating the formation of CQDs. The brown solution was then purified by centrifugation at 15,000 rpm with dialysis membranes (molecular weight cutoff\u0026thinsp;=\u0026thinsp;1000) to remove insoluble material and small molecules. The prepared CQDs were stored at 4\u0026deg;C for further studies. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows a schematic diagram of the preparation of CQDs from grapefruit peel.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Aptamer attachment to CQDs\u003c/h2\u003e \u003cp\u003eThe E. coli aptamer was annealed and heated in a water bath at 95\u0026deg;C for 5 min, and slowly cooled down to room temperature at room temperature. The annealed DNA was diluted by PBS (0.01 M, pH\u0026thinsp;=\u0026thinsp;7.40) and set aside. Then, 200 \u0026micro;L of CQDs were added to 20 \u0026micro;L of aptamer solution and shaken for 30 min at 37\u0026deg;C in the dark. To block the free carboxyl groups on the surface of CQDs, we added ethanolamine (10 mM) and shaken the solution for 2 hr. Finally, we measured the fluorescence intensity of the solution.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Preparation of silver nanoparticles\u003c/h2\u003e \u003cp\u003eThe preparation was carried out by the original method of laboratory sisters. Take 3 mLAgNO3 solution (1M) and slowly add it to 3 mLPVP solution (1M), mix the solution thoroughly, then add 1.5 mL o-phenylenediamine solution (0.1M) and stir for 2 h at room temperature to obtain the primary product, then centrifuge at 15000 rpm, and wash the final product with ethanolamine in the ratio of 1:1 for three times to obtain PVP-Ag NPs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.6 FRET-based detection of E. coli\u003c/h2\u003e \u003cp\u003eFirst, in order to achieve the desired results, we have to optimize some parameters to optimize the volume of aptamer-CQDs hybridized with Ag NPs. The FRET process occurs when the excited state donor transfers energy to the ground state acceptor through physicochemical interactions. Fluorescence transduction is based on the spectral overlap between the emission of the donor and the absorbance of the acceptor [127].FRET between CQDs-Ag NPs aptamers is constructed by the following steps: 80 \u0026micro;L of different concentrations of Ag NPs are added to 400 \u0026micro;L of CQDs-aptamer solution and shaken for 15 min.The fluorescence intensity of this mixture is measured and then, the above solution is added with different concentrations of E. coli were added to the above solution and shaken at room temperature for 2 h. The fluorescence of the supernatant was centrifuged and detected. The selectivity of the sensor was tested with other strains such as Salmonella, Salmonella and Carbonic Anhydrase. A concentration of 2 \u0026times; 108 CFU-mL-1 of each bacterium was diluted with PBS and evaluated separately using this method.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.7 Temperature sensing of CQDs\u003c/h2\u003e \u003cp\u003eAfter the temperature of the water bath had risen to a certain temperature and stabilized, one of the centrifuge tubes was heated in the water bath to make the reaction complete, and the fluorescence intensity was measured. The fluorescence intensity of CQDs at different temperatures was recorded.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3 Results and discussion","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Principle of FRET sensor\u003c/h2\u003e \u003cp\u003eA fluorescence resonance energy transfer (FRET) biosensor based on green synthetic carbon quantum dots (CQDs) and silver nanoparticles (Ag NPs) (excellent surface plasmon resonance absorption ability) was designed to detect E. coli, and the fluorescence transduction was based on the spectral overlap between the donor (CQDs) emission and the acceptor (nanoparticles) absorbance. The excitation and emission wavelengths of CQDs are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(a), where the prepared CQDs have an excitation peak at 320 nm and an emission peak at 415 nm. The inset in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(a) demonstrates that the bright green fluorescence of CQDs can be observed under UV lamp irradiation at 365 nm. In addition, the emission wavelength of CQDs at 400 nm overlaps with the absorbance of Ag NPs. This indicates that Ag NPs can successfully achieve the burst of carbon quantum dot fluorescence by mechanisms such as fluorescence resonance energy transfer (FRET) or internal filtering effect (IFE). It can be seen that the fluorescence intensity of CQDs-aptamer-Ag NPs was burst and then the fluorescence recovered with the increase of E. coli in the system (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). The fluorescence intensity also decreases when the aptamer is added to the CQD solution, and the decrease increases when Ag NPs are added, which finally succeeds in bursting the CQDs fluorescence, leading to the FRET process. Due to the surface of CQDs with amine groups, adsorption is easily achieved by electrostatic interaction with the surface of Ag NPs, and this interaction leads to the bursting of the fluorescence signal emitted by CQDs [11,16]. However, the preferential binding of specific aptamers through H-bonds upon addition of bacteria [17] leads to the release of CQDs and Ag NPs, and after a period of incubation, the bacterial cells are centrifuged, leading to the precipitation of E. coli aptamer couples and Ag NPs, at which point CQDs fluorescence is re-emitted. The fluorescence intensity of CQDs in the supernatant was measured. These aptamers can specifically bind to specific bacterial surface compounds, which can serve as recognition elements for new aptamer systems. As the principle belongs, the black line in (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb) is the CQDs alone, and the fluorescence is burst when the aptamer is attached and Ag NPs are added, as in the red line in (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). The FRET process was confirmed to proceed efficiently, and the fluorescence was restored after the final addition of E. coli broth and incubation for a period of time, as shown in the blue line in (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Optimization of experimental conditions\u003c/h2\u003e \u003cp\u003eIn order to better observe the FRET results, the volumes of CQDs, Ag NPs and aptamers were optimized during the experiments. The effect of different material dosage on the fluorescence intensity was recorded. The premise of the optimization is to ensure the fluorescence burst effect of CQDs as much as possible, on the other hand, the volume of CQDs we prepared is sufficient, but the volume of aptamer is very small (248 µL for one tube) so the optimization was carried out by reading the literature as much as possible within the previous experiments to avoid waste. Through the optimization experiment, we can see from Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (c) that the Apt: CQDs: Ag NPs = 1:15:1 burst intensity is the largest when CQDs is the variable, but we can also see that the fluorescence intensity burst of Apt: CQDs: Ag NPs = 1:10:1 is about 150, which has burst 90% of the original CQDs, so the Apt: CQDs: Ag NPs is chosen when CQDs is the variable. Therefore, the ratio of Apt: CQDs: Ag NPs = 1: 10: 1 was chosen for the variable CQDs. As above, the fluorescence intensity of both Apt: CQDs: Ag NPs = 1: 10: 2 and Apt: CQDs: Ag NPs = 1: 10: 3 was below 100 when Ag NPs was the variable, so the ratio of Apt: CQDs: Ag NPs = 1: 10: 2 was chosen for the sake of drug consumption, and the subsequent experiments were conducted with this ratio. In Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (d), we can also see the relative fluorescence difference of each volume ratio compared with the fluorescence intensity of the original CQDs, and we can observe that the fluorescence intensity difference is about 1000 for Apt: CQDs: Ag NPs = 1: 10: 2, which again confirms that we can choose the ratio of Apt: CQDs: Ag NPs = 1: 10: 2 for the subsequent experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Characterization of carbon quantum dots\u003c/h2\u003e \u003cp\u003eThe CQDs were comprehensively characterized using transmission electron microscopy images (TEM), X-ray diffraction (XRD), Fourier infrared spectroscopy (FT-IR), and X-ray photoelectron spectroscopy (XPS).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.3.1Transmission electron microscopy (TEM)\u003c/h2\u003e \u003cp\u003eThe carbon quantum dots prepared by microwave heating method using grapefruit peel as carbon source, we cannot distinguish the morphology and size of the prepared substances by naked eyes only, and transmission electron microscopy has been widely used to characterize the size and morphology of CQDs. The synthesized carbon quantum dots solution was precisely dropped onto the copper grid of the carbon support film with a pipette gun and dried naturally at room temperature for about one hour when the solvent was water, and then observed under the transmission electron microscope. The prepared CQDs samples showed good dispersion and proper dispersion under transmission electron microscopy (TEM) with spherical shape and relatively small particle size, the diameter distribution ranged from 5–9 nm with an average particle size of 7.4 nm, accordingly, no obvious deposition of large particles was observed in these samples(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.3.2 XRD analysis and FT-IR analysis of CQDs\u003c/h2\u003e \u003cp\u003eIn this work, the crystallinity of CQDs was investigated in depth using X-ray diffraction analysis (XRD) technique, and the relevant experimental results are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(c). The synthesized CQDs exhibit highly disordered carbon atoms, which is well reflected by the diffraction peaks detected by XRD located around 2θ = 22° \u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. The functional groups on the surface of CQDs were investigated by FT-IR spectroscopy. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(d), the FT-IR absorption bands of CQDs were 3416, 3140, 2350, 1630, 1380, and 1082 cm-1. The peaks at 3416 cm-1 and 3140 cm-1 can be attributed to primary amino (N - H) or hydroxyl group (O - H) vibrations. In addition, the spectral peaks at 2350 cm-1 and 1630 cm-1 are associated with the vibrations of the carboxyl group C = O or C = C \u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. The spectral peaks at 1380 cm-1 and 1082 cm-1 can confirm that CQDs have amide bonds [20], corresponding to the C - N and C - O stretching vibrations. The FT-IR test results showed that CQDs are rich in functional groups on the surface, and functional groups such as carboxyl and amino groups are particularly prominent. The presence of these functional groups can not only indicate the water solubility of CQDs, but also provide the necessary support for their good biocompatibility.\u003c/p\u003e \u003cp\u003eThe elemental composition and surface groups of CQDs can be studied and analyzed by applying X-ray photoelectron spectroscopy (XPS) techniques. Specifically, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e of this paper presents XPS full spectrum scans of the synthesized CQDs and high-resolution spectra for the elements O1s, C1s and N1s. In the full spectrum range as shown in (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea), three main peaks can be found, and the peaks correspond to energies of 284.6 eV, 401.2 eV and 532.3 eV, which correspond to C1s, N1s and O1s, respectively, so it can be inferred that the CQDs mainly contain carbon, nitrogen, oxygen and hydrogen elements. The percentages of carbon, nitrogen and oxygen atoms are about 75.96%, 9.36% and 14.68%. Three typical peaks of 284.7 eV (C - C/C = C), 286.5 eV (C - O/C - N) and 288.6 eV (C = O/C = N) were present in the high-resolution C1s spectrum (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). In addition, the XPS analysis results are in agreement with the FT-IR analysis. After the detection of the high-resolution O1spectrum peaks, the corresponding three characteristic peaks were found to be located at 531.5 eV, 532 eV, and 533.4 eV, representing the C - O, C = O, and C - O - C groups, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Meanwhile, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed presents the N1s spectrum of CQDs, where the main peak at 399.7 eV represents C - N. These results suggest the presence of more oxygen-containing, carbon-containing groups on the synthesized quantum surface. In addition, the oxygen element may be the main dopant element that plays an important role in the change of fluorescence properties \u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Stability analysis of CQDs\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFor the synthesized carbon quantum dots, the stability of the nanoparticles under different environments was then investigated. In Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(a), it can be clearly seen that the fluorescence intensity of the carbon quantum dots decreases linearly from 25 to 65°C, and between 75 and 95°C, the fluorescence intensity also decreases, but the decrease is less than 10 negligible and basically remains stable. It proves that the nanoparticles remain stable between 75–95℃. In Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(b), we can observe the above conclusion more clearly. By comparing the fluorescence intensity, we can know that it has the highest fluorescence intensity at room temperature. Therefore, in our subsequent experiments, there is no need for heating and other steps, and it can be performed at room temperature. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e (c) shows that the fluorescence change of CQDs is less than 150, or almost no change, after 5 months of preparation at 4℃, which indicates that the CQDs are very stable. If the amount of subsequent experiments is large, we can prepare a large amount at one time and use it in the subsequent experiments for several months without further preparation. The uncertainty of repeated preparation of CQDs is well avoided. These experimental results also demonstrate that it can maintain its stability under more complex conditions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Optical properties of CQDs\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe fluorescence stability of CQDs was investigated for their photobleaching properties, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e.a. The prepared CQDs exhibited insignificant photobleaching after continuous irradiation with a 365 nm UV lamp for 1 h. A finer decrease in fluorescence intensity of less than 200 can be observed in (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). The results indicate that the fluorescence intensity maintained its stability during continuous irradiation with the UV lamp. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e (b) shows the emission spectra of CQDs at different excitation wavelengths from 290 nm to 380 nm, and it can be seen that the emission intensity is highest at 330 nm excitation. A redshift in the emission spectrum can be clearly observed in the fluorescence emission spectra as the excitation wavelength gradually increases.\u003c/p\u003e \u003cp\u003eAs observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e(c), in the ultraviolet-visible (UV-vis) absorption spectrum, higher absorbance in the UV-visible spectrum can be clearly detected in the region of 200 ~ 400 nm. Specifically, 2 unique absorption peaks were exhibited in the UV-vis absorption region. They appear at 220 nm and 283 nm, respectively. The leap energy band at 220 nm corresponds to the π-π leap of C = C, while the absorption at 283 nm represents the n-π leap of the C = O bond \u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e.The optical absorption peaks of CQDs show a gradual decrease in intensity with increasing dilution and a slight blue shift, which may be due to surface or edge defects of CQDs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Sensibility analysis for Escherichia coli\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe effect of different concentrations of E. coli in the set-up ligand sensor on the fluorescence intensity was investigated under optimized conditions. The extent to which the fluorescence was restored after the reaction of this CQDs with different concentrations of E. coli was further experimented. Figure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e(a) shows the fluorescence intensity of the CQDs after the addition of different concentrations of E. coli. It can be observed that the fluorescence intensity of CQDs increases with increasing concentration of E. coli. E. coli solutions in the concentration range of 2 × 10\u003csup\u003e2\u003c/sup\u003e ~ 2 × 10\u003csup\u003e8\u003c/sup\u003e CFU-mL\u003csup\u003e− 1\u003c/sup\u003e were examined. The linear relationship between the fluorescence values of the CQDs and the concentration of E. coli solution at concentrations between 2×10\u003csup\u003e3\u003c/sup\u003e ~ 2×10\u003csup\u003e8\u003c/sup\u003e CFU-mL-1 was extremely strong, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e (b), with a correlation coefficient of 0.9961 and a detection limit of 77 CFU-mL-1. The sensitivity of the system was investigated, ensuring a fairly wide dynamic range and The sensitivity of the system was investigated, ensuring a wide dynamic range and a low detection limit. A simple method based on the FRET process between aptamer-CQDs and Ag NPs was designed. CQDs were prepared from natural sources and are cost-effective compared to commercially available QD nanoparticles. Compared to other techniques, the fluorescent biosensor technology further confirms the reliability of the method while improving the detection sensitivity, and thus is widely used in practical applications.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.8 Specificity of the assay for Escherichia coli\u003c/h2\u003e \u003cp\u003eTo investigate the specificity of the assay in the detection of E. coli, the method was applied to the detection of other bacteria under optimal conditions. such as carbonic anhydrase bacteria, Salmonella typhimurium, Salinophilus and Bacillus, and LB cultures. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e, the fluorescence intensity was enhanced in the presence of E. coli in the mixture. In the absence of the addition of E. coli, the fluorescence intensity of other bacterial solutions remained significantly unchanged even when added. The experimental results proved that the fluorescence intensity enhancement of non-target bacteria and culture solution was much lower than that of E. coli. When other non-specific bacteria were added, there was no interaction between the bacteria and the aptamer; therefore, the FRET process could not be disrupted, and the fluorescence of the supernatant remained unchanged. The results clearly indicate that the sensor is less responsive to other bacteria; therefore, the biosensor has good specificity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThis work presents a new strategy for the detection of E. coli based on green synthetic carbon quantum dots (CQDs) and silver nanoparticles (Ag NPs) designed as fluorescence resonance energy transfer (FRET) biosensors. CQDs materials were successfully synthesized by microwave reaction technique using white grapefruit peel as a carbon source. The materials exhibited excellent green luminescence properties. The fluorescence of CQDs was burst in the presence of aptamers and Ag NPs. After the addition of specific bacteria, an aptamer-target complex is formed and the emission of free CQDs is restored. The linear range of the aptamer sensor for E. coli concentration was 2 × 10\u003csup\u003e3\u003c/sup\u003e ~ 2 × 10\u003csup\u003e8\u003c/sup\u003e CFU-mL-1 and the detection limit for E. coli was as low as 77 CFU-mL-1. These results indicate that the analytical performance of the system can be used as an effective sensor for E. coli detection. This response is caused by the specific binding of the aptamer to E. coli, which is much higher than the response to other non-specific bacteria, indicating the high specificity of the bioassay. Because of its simplicity and effectiveness, the FRET fluorescent biosensor holds promise for a wide range of applications in other bacterial and biomolecular assays.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (Grant No.21864020，82160652), the Natural Science Foundation of Inner Mongolia (Grant No. and 2019MS02014, 2018MS02012), \u0026lsquo;\u0026lsquo;Young Science and Technology Talents Program\u0026rsquo;\u0026rsquo; (Leading Person) in Inner Mongolia Autonomous Region Colleges and Universities (Grant No. NJYT-19-A04), the Fundamental Research Funds for the Inner Mongolia Normal University, China, (Grant No. 2022JBZD013). \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data and materials in this study are included in the published article and its additional file.\u003c/p\u003e\n\n\u003cp\u003eEthics approval and consent to participate \u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors agree to publish.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there is no conflict of interest.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e1 College of Chemistry and Environmental Science, College of Geographi-\u003c/p\u003e\n\u003cp\u003ecal Science, Inner Mongolia Key Laboratory of Environmental Chemistry,\u003c/p\u003e\n\u003cp\u003eInner Mongolia Normal University, 81 Zhaowudalu, Hohhot 010022, China.\u003c/p\u003e\n\u003cp\u003e2 College of Pharmacy, Inner Mongolia Medical University, Jinchuankaifaqu,\u003c/p\u003e\n\u003cp\u003eHohhot 010110, China.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eArkan E, Barati A, Rahmanpanah M, et al. 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Green fluorescent carbon quantum dots as a label-free probe for rapid and sensitive detection of hematin[J]. Spectrochimica Acta Part A Molecular \u0026amp; Biomolecular Spectroscopy, 2019, 212:167-172.\u003c/li\u003e\n\u003cli\u003eLi Y, Zhao Y, Cheng H, et al. Nitrogen-doped graphene quantum dots with oxygen-rich functional groups[J]. Journal of the American Chemical Society, 2011, 134(1): 15-18.\u003c/li\u003e\n\u003cli\u003eShi B, Zhang L, Lan C, et al. One-pot green synthesis of oxygen-rich nitrogen-doped graphene quantum dots and their potential application in pH-sensitive photoluminescence and detection of mercury (II) ions[J]. Talanta, 2015, 142: 131-139.\u003c/li\u003e\n\u003cli\u003eDing H, Li X H, Chen X B, et al. Surface states of carbon dots and their influences on luminescence. Journal of Applied Physics, 2020, 127(23): 231101-21.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"CQDs, Escherichia coli, biosensor, FRET","lastPublishedDoi":"10.21203/rs.3.rs-2984200/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2984200/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn this work, carbon quantum dots were prepared from grapefruit peel as carbon source by microwave heating method. The prepared carbon quantum dots CQDs were analyzed by fluorescence spectroscopy, TEM analysis, XPS analysis, etc., as well as their optical properties were also investigated. The prepared CQDs have high green fluorescence with excitation wavelength of 320 nm and maximum emission wavelength of 415 nm. the average particle size is about 7.4 nm, uniform dispersion and good stability. And a FRET (fluorescence resonance energy transfer) based fluorescence method was constructed by combining carbon quantum dots with silver nanoparticles (Ag NPs) for the rapid detection of E. coli. Its fluorescence transduction is based on the spectral overlap between the donor (CQDs) emission and the acceptor (nanoparticles) absorbance. The fluorescence of the aptamer-attached CQDs is burst in the presence of and silver nanoparticles. Upon addition of the specific E. coli solution, an aptamer-target complex is formed and the preferential interaction of the aptamer with the specific bacteria leads to the release of CQDs and Ag NPs. After incubation time, the bacterial cells are centrifuged, leading to the precipitation of E. coli aptamer couples and Ag NPs, resulting in the recovery of CQDs fluorescence. This method allows specific detection of E. coli in a wide range of pathogenic bacteria. The final results showed that the linear range of the sensor was 2\u0026times;10\u003csup\u003e3\u003c/sup\u003e ~ 2\u0026times;10\u003csup\u003e8\u003c/sup\u003e CFU\u0026middot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and the detection limit for E. coli was as low as 77 CFU\u0026middot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e","manuscriptTitle":"Based on Aptamer-carbon quantum dots and silver nanoparticles FRET sensor for sensitive detection of E. coli","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-05-30 15:54:07","doi":"10.21203/rs.3.rs-2984200/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"77c62587-5c1a-4adb-b8a1-3df9615d7395","owner":[],"postedDate":"May 30th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-05-31T17:59:29+00:00","versionOfRecord":[],"versionCreatedAt":"2023-05-30 15:54:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2984200","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2984200","identity":"rs-2984200","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}