Two birds, one stone: long-wavelength carbon dots enables ratiometric detection of ciprofloxacin and Co 2+ for smartphone, logic gate, and imaging applications

Two birds, one stone: long-wavelength carbon dots enables ratiometric detection of ciprofloxacin and Co 2+ for smartphone, logic gate, and imaging applications | 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 Two birds, one stone: long-wavelength carbon dots enables ratiometric detection of ciprofloxacin and Co 2+ for smartphone, logic gate, and imaging applications Yanan Yan, Hongping Zhang, Youhong Tang, Shengmei Song, Shaomin Shuang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7544936/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Nov, 2025 Read the published version in Microchimica Acta → Version 1 posted 9 You are reading this latest preprint version Abstract Environmental pollution poses a significant threat to human health and sustainable development, highlighting the need for rapid and sensitive contaminant detection. Here, we present a long-wavelength ratiometric fluorescent carbon dots to detect ciprofloxacin (CIP) and cobalt ion (Co 2+ ). The carbon dots (D-CDs) exhibiting outstanding dual emission at 445 nm and 662 nm are successfully synthesized through a one-pot hydrothermal approach, with methylene blue serving as the sole precursor. Interestingly, the blue fluorescence at 445 nm is significantly enhanced since the effect of formation of hydrogen bonds and charge transfer between D-CDs and CIP, while the 662 nm emission remains unchanged, yielding a ratiometric fluorescence response (F 445nm /F 662nm ) across a range of 0.048 − 3.58 nM, with a detection limit of 16.7 pM. Additionally, the fluorescence of CDs/CIP can be efficaciously ratiometric restored by right of a particular reaction of Co 2+ with CIP, achieving ratio fluorescence quantitative assay of Co 2+ (LOD = 14.7 nM). Density functional theory (DFT) calculations has been used to illustrate the potential interaction mechanisms, which shows strong agreement with the experimental results. Notably, a smartphone-integrated colorimetric test strip enables on-site monitoring of CIP and Co 2+ , expanding environmental applications, which has been demonstrated by effectively detection of CIP in milk and river water. Further exploration has been conducted by developing a logic gate sensor which harnesses the activated cascade effect to serve as an intelligent probe for monitoring trace levels of CIP and Co 2+ . Furthermore, D-CDs are applied in cellular imaging, demonstrating their strong potential in sensing, bioimaging, and environmental analysis. Carbon dots Ciprofloxacin Co2+ Ratiometric Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction The progress of society induces the emergence of increasing environmental issues [ 1 ] . The arbitrary discharge of industrial wastewater and the use of a large amount of antibiotics have led serious environmental pollution problems, among them heavy metal ions and antibiotic pollution cannot be underestimated [ 2 , 3 ] . Therefore, the measuring of heavy metal ions and antibiotic residues are of great significance for environment and human health. Carbon dots (CDs), a class of photoluminescent nanomaterials with illustrious optical properties [ 4 ] , low toxicity [ 5 ] and favorable biocompatibility [ 6 ] possess an extensive range of potential applications, including optical sensing, biological imaging, light emitting diode (LED) and so forth[7, 8]. Especially in the detection of environmental pollutants, CDs are good candidates as sensors [ 9 – 11 ] . However, most of the reported CDs for the detection of environmental pollutant respond via a single fluorescence signal, which is easily subjected to strong influence from multiple factors, for instance probe concentration, fluctuation of excitation source, background absorption [ 12 ] . Ratiometric fluorescence sensing can overcome these matters through simultaneously gauging the ratio of two well-tackled emission peaks to enhance detection capability [ 13 ] . Therefore, development of ratiometric fluorescence sensors have profound significance toward the analysis of environmental pollutants. In addition, analysis is often limited to semi quantitative detection in laboratory conditions, which severely restricts the applications in several fields, especially in resources-limited areas, poverty countries, and clinical diagnosis [ 14 ] . The problem may be solved by developing fluorescent colorimetric strategies and visual fluorescent test papers. Paper sensors have been widely used in the many fields due to their advantages of low cost, portability, and versatility, especially for environmental pollutants detection [ 15 ] . In recent years, with the continuous upgrade of smartphones equipped with HD cameras, smartphone has received wide attention as a good analysis device in point-of-care testing (POCT) sensors [ 16 ] . Therefore, how to establish an integrated intelligent detection platform to realize sensitive, accurate and convenient monitoring of environmental pollutants by combining CDs with smartphone assistance combined with test strips in an uncomplicated way is of great significance. Ciprofloxacin (CIP) has the strongest antibacterial activity among the third-generation fluoroquinolone antibiotic, so it is broadly applied in the treatment of human and animal infectious diseases [ 17 ] . With the widespread application of CIP, it inevitably leads to a large amount of residues in animal bodies, which may exist in edible substances, such as milk or eggs [ 18 ] . Furthermore, CIP cannot be completely absorbed and will enter the environment in its original form with excreta through human metabolism. Even at low concentrations, it may trigger tremendous harm to the ecological environment and mankind health, bringing about the emergence of various diseases. Global concerns have been induced by the increasing existence of CIP residues and most countries have established maximum residue limits (MRLs) for CIP. Furthermore, China’s Ministry of Agriculture (CMA) has similarly issued corresponding regulations to regulate the usage of CIP [ 19 ] . Hence, efficacious monitoring of CIP residues has turned into an essential matter. The traditional detection approaches of CIP mainly comprise high-performance liquid chromatography (HPLC), enzyme linked immunoassay (ELI), capillary electrophoresis (CE), electrochemical assay. However, these traditional approaches are restricted in realistic applications. Fluorimetry is deemed to be a credible analytical tactic owing to uncomplicated of operation, technical simplicity and expansive adaptability. To this end, CDs-based ratiometric probe has been developed for CIP sensing. Gui et al. [ 20 ] first introduced a novel ratiometric fluorescence (FL) probe for the detection of CIP based on CDs/SiDs–BPMA hybrids. Subsequently, Lu et al. [ 21 ] raised a ratiometric tactics for the determination of CIP based on CDs/riboflavin system. Gao et al. [ 22 ] designed a platform between CDs and 2, 3-diaminophenazine for ratiometric monitoring of CIP. However, there are still some limitations with the reported ratiometric fluorescence CDs for detecting CIP, such as time-consuming and cumbersome procedures. Up to now, to the best of our knowledge, one-pot synthesis of ratiometric fluorescence CDs for rapid measure of CIP have not been reported. In addition, its on-site, rapid, and sensitive measurements also pose challenges. Therefore, it is urgently necessary to design and fabricate a portable device for tracing CIP accurately and conveniently on the spot or special sites. Cobalt ions (Co 2+ ) is a trace element that acts an essential role in the human body. However, excessive intake of Co 2+ wreaks a woeful threat to human health, such as low blood pressure, diarrhea and even death by reason of myocardial infarction [ 23 ] . The World Health Organization (WHO) has regulate the utmost permitted content of Co 2+ in drinking water less than 40 µg/L [ 24 ] . Therefore, it is in great demand to construct an approach for detecting Co 2+ . Until now, numerous techniques for the detection of Co 2+ have been exploited, including atomic absorption spectrometry (AAS), liquid chromatography (LC), plasma-atomic emission spectrometry (ICP-OES) and fluorescence method. Since the exceptional selectivity, low detection limit, and unsophisticated operation of fluorescent nanoprobes, it has mesmerized immense focus of researchers [ 25 ] . Due to the outstanding properties of CDs, some CDs ratiometric fluorescent nanoprobes have been discovered to monitor Co 2+ . Dong et al. [ 26 ] designed a dual-emission CDs to ratiometric recognition of Co 2+ . Nevertheless, CDs-dependent Co 2+ ratiometric FL sensors have been rarely built. Consequently, it remains a challenge to get ahold of a ratiometric CDs for the detection of Co 2+ . In this work, we design a sensing platform based on multifunctional D-CDs for the ratiometric rapid measuring of CIP and Co 2+ firstly (Scheme 1 ).The obtained D-CDs, manifesting dual emission of blue emission at 445 nm and weak red fluorescence at 662 nm under 306 nm excitation, are synthesized via the one-step hydrothermal route through adopting methylene blue as the only precursors. Interestingly, the fluorescence at 445 nm increases and remains at 662 nm with the injection of CIP, which appears ratiometric natures (F 445nm /F 662nm ). In addition, the fluorescence of CDs/CIP can be efficaciously reinstated by replacing CIP with Co 2+ . More importantly, the colorimetric test strips are exploited and a sensing platform combining with smartphone is established to achieve rapid, sensitive and accurate visual monitoring of CIP and Co 2+ . Meanwhile, we explore the potential use of the synthesized D-CDs for constructing a logic gate sensor capable of detecting CIP and Co 2+ based on the fluorescence cascaded switching mechanism. Furthermore, the D-CDs is triumphantly utilized to detect CIP and Co 2+ intracellular imaging. 2. Experimental 2.1 Material The materials required for the experiment were provided in the Support Information. 2.2 Preparation of D-CDs. The D-CDs were prepared through a one-pot solvothermal tactics. 0.0156 g of methylene blue was dissolved with 25 mL DDI water and heated at 200°C for 6 h. Next, the obtained product was centrifuged (8000 rpm, 20 min) and dialyzed (MWCO 800, 12 h). Ultimately, the D-CDs powders were stored for subsequent use. 2.3 Detection of CIP and Co 2+ To begin with, 20 µL of D-CDs liquid (4.5 mg/mL) was added into 2.0 mL of DDI water. To assess the specificity of the D-CDs, various antibiotics were injected into the solution at the concentration of 0.01 M. Subsequently, different volumes of Co 2+ (0.1 M) was added into the CDs/CIP (36.9 µM) system. The anti-interference tests were implemented through introducing other antibiotics (10 mM) instead of CIP (1 mM) in mixed solution. 2.4 Cellular imaging The Hela cells were hatched with the D-CDs (4.5 mg/mL) for 30 min. Afterwards, being washed with PBS buffer (pH = 7.4) to get rid of redundant D-CDs. The pretreated cells were then incubated with CIP (7.5 µL, 1 mM) firstly. The cells loaded with CDs/CIP were further treated with Co 2+ (70 µL, 0.1 M) for imaging. 2.5 Monitoring of CIP and Co 2+ in real samples Milk was bought from native shop in Taiyuan, China. The pretreated procedure refers to the literature [ 27 ] . Real water samples (Lingde Lake water from Shanxi University, China) were filtered through filtered membrane (0.22 µm) and centrifuged (4000 rpm, 15 min) for subsequent use. Finally, the prepared samples were used for the detection of CIP and Co 2+ according to conventional procedures. The water samples were gauged by the proposed ratiometric fluorescence and smartphone assisted. 2.6 Smartphone application for the detection of CIP and Co 2+ . Different volumes of CIP (0.01 M) were introduced into the D-CDs solution (0.045 mg/mL), and then the fluorescent color photos were captured by a smartphone under a 365 nm UV lamp. The color parameters (RGB values) were derived through a smartphone application (Color Identification APP) [ 28 ] . Subsequently, different volumes of Co 2+ (0.1 M) was added into the CDs/CIP (208.1 µM) system. The fluorescence images were captured for further analysis. For paper-based sensing, the filter paper was cut into approximately 1 cm 2 as the test strips firstly. Afterward, the test strips were dipped in the D-CDs solution (0.045 mg/mL) and the test papers loaded with D-CDs were dipped in solution of different concentrations of CIP and dried spontaneously. The color variation of the test strips was taken by the smartphone. The color information (RGB values) was derived through a smartphone application (Color Identification APP). Subsequently, different concentrations of Co 2+ was dropped onto the prepared filter paper strips. After spontaneous drying of the test paper, the fluorescence images were captured for further analysis. 3. Results and Discussion 3.1 Characterizations The morphology of the D-CDs were examined by transmission electron microscopy (TEM) and atomic force microscope (AFM) [ 29 ] . The D-CDs presents a globular morphology, with a mean value of 2.28 ± 0.22 nm (Fig. 1 (a) and Fig. 1 (b)). The lattice spacing of D-CDs is 0.22 nm in accord with the (100) planes of graphite carbon, manifesting that D-CDs have highly crystalline carbon structures (inserted in Fig. 1 (a)). Furthermore, AFM is applied to confirm the morphology (Fig. 1 (c) and Fig. 1 (d)), and D-CDs emerges granule heights of roughly 2.55 nm, which corresponding to the result of TEM. X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared (FTIR) were performed to reveal the composition of the D-CDs [ 30 ] . We characterized the surface functional groups of D-CDs by FTIR and XPS. The full XPS spectrum of D-CDs exhibit six main peaks at 529 eV (O 1s), 397.2 eV (N 1s), 283.1 eV (C 1s), 226.4 eV (Cl 2p) and 162.8 eV (S 2p) respectively (Fig. 2 (a)). The C 1s spectrum (Fig. 2 (b)) contains three parts at 284.6 eV (C − C/C = C), 285.9 eV (C − O/C − S) and 287.0 eV (C = O). The N 1s band displays two constituent peaks of C-N and N-H, which situated at 398.6 eV and 400.9 eV, individually (Fig. 2 (c)). The O 1s spectrum can be divided into two sections at 531.4 and 532.5 eV, which corresponding to C − OH and C = O (Fig. 2 (d)). The spectrum of S 2p shows three peaks of 164.0 eV, 165.2 eV, and 168.3 eV, corresponding to C-S (S 2p 3/2 ), C-S (S 2p 1/2 ), and S = O, respectively (Fig. 2 (e)). The band at 3436.7 cm − 1 presents the existence of O-H. Peaks at about 2953 cm − 1 indicated the presence of C-H. It is evident that the peaks at 1602 cm − 1 (C = O), 1324 cm − 1 (C-O) and 1142 cm − 1 (S = O) can be observed. Importantly, peak at 909 cm − 1 was due to the N-H (Fig. 2 (f)). Above results indicate that the D-CDs is plentiful in hydroxyl and carboxylic groups. The results of FTIR are unanimous with the confirmation of XPS. The UV–vis absorption spectrum of D-CDs portrayed two peaks centered at 261 nm and 280 nm, which attribute to π-π* transition of the C = C bond and the n-π* transition of the C = O/C-O bond. In 500 nm -700 nm, a wide absorption band is at 600 nm, which corresponds to the MB itself (Fig. 3 (a)). The D-CDs exhibit two well-resolved fluorescence peaks at 455 and 662 nm at the optimal excitation wavelength. The emission peak of D-CDs slightly alters when the excitation wavelength ranging from 230 nm to 320 nm (Fig. 3 (b)). The λ ex -dependent fluorescence behavior may be attributed to the molecular state. Furthermore, the fluorescence quantum yield of the D-CDs was found to be 16.8% using Rhodamine B as a standard (Figure S1 and Table S1 ). The fluorescent stability of D-CDs was investigated. The fluorescence intensity of D-CDs persists steady upon persistent illumination for 1 h, indicating that the D-CDs has remarkable photobleaching resistance (Figure S2). Simultaneously, the emission intensity remains stable in the high ionic strength (2.0 M) (Figure S3) and wider pH rang (5–13) (Figure S4), which is beneficial for elaborate environment and living systems. 3.2 Ratiometric detection of CIP By introducing a variety of metal ions, the selectivity performance of D-CDs was investigated. The common metal ion almost exhibits no effects (Figure S5). As shown in Fig. 4 (a), the ratio value (F 445nm /F 662nm ) of D-CDs adding antibiotics at a concentration of 0.01M were collected and recorded. Among these materials, the ratio of F 445nm /F 662nm is considerably increased along with the addition of CIP, while other antibiotics did not show the equivalent phenomenon, indicating the terrific selectivity of D-CDs for CIP. CIP belongs to the fluoroquinolones (FQs) antibacterial drug. The influence of other FQs residues on the fluorescent behavior of D-CDs is evaluated as depicted in Figure S6. The fluorescence of D-CDs does not significant changes after adding other FQs. When CIP was injected into D-CDs including interferences, the PL intensity of D-CDs could still be dramatically increased (Figure S7), demonstrating that the D-CDs can be applied to detect CIP with good anti-interference. Figure 4 (b) presents the ratio value of D-CDs reaches equilibrium with CIP in only 10 s, indicating that as-acquired D-CDs have the potential for rapid monitoring of CIP. With increasing the concentration of CIP, the red fluorescence at 662 nm remained following with a ratio growth at 445 nm (Fig. 4 (c)). In addition, the value of F 445nm /F 662nm shows exceptional linear relationship with the CIP concentration in the range of 0.047 nM-3.59 nM, the fitting curve equation is F 445nm /F 662nm = 0.8040 + 1.0783C CIP , R 2 = 0.9989. Simultaneously, corresponding LOD for CIP was gauged to be 16.8 pM (S/N = 3) (Fig. 4 (d)). As described in Table S2, the ratiometric fluorescence CDs applied to detect CIP currently have drawbacks such as long detection time or high detection limits. Above results displayed that D-CDs can act as a nanosensor for rapid measure of CIP by a ratio sensing model, with a fast response and very low LOD value of 16.8 pM, which is excellent to existing data. 3.3 Ratiometric detection of Co 2+ . A holistic exploration on the influences of diverse metal ions on the fluorescence of the CDs/CIP was executed (Fig. 5 (a)). The CDs/CIP system proves particular identification to Co 2+ beyond other competitive ions. Meanwhile, the time for the reaction between CDs/CIP and Co 2+ was conducted (Fig. 5 (b)), indicating CDs/CIP system can rapid monitor of CIP (15 s). Upon gradually increasing Co 2+ concentrations (0 − 369.95 µM), the PL intensity at 445 nm recuperates (Fig. 5 (c)). Meanwhile, the value of F 445nm/ F 662nm exhibits a good linear correlation (F 445nm /F 662nm = 2.0762-0.0090C Co 2+ , R 2 = 0.9996) by varying Co 2+ in the range of 45.65 − 89.39 nM, and the LOD is computed to be 14.68 nM, which is below the WHO regulations (Fig. 5 (d)). 3.4 Sensing mechanisms FTIR, zeta potential, UV-vis were operated to explore the mechanism. Both FTIR and XPS information manifest the existence of abundant carboxylic and hydroxyl groups on the surface of the D-CDs. CIP as well contains numerous groups, embodying C-F, C-O, and C-N. Taking inspiration from previous literature, we speculate that the excellent response of D-CDs to CIP may be attributed to hydrogen bonding between CIP and oxygen-containing functional groups (-COOH or -OH) on the surface of D-CDs. To corroborate this hypothesis, the FTIR of the D-CDs system before and after the addition of CIP were comprehensively measured. In Figure S8, there are two blue shifts. To start with, contrasted to the C-OH vibrations (3431 cm − 1 ) of D-CDs (red line), there is a blue shift of 103 cm − 1 of the CD/CIP (black line). Next, the vibrations of N-H situated at around 2943 cm − 1 in CDs/CIP, there is as well a blue movement of 90 cm − 1 contrasted to the CIP (3033 cm − 1 ). These hint the formation of hydrogen bonding between CIP and D-CDs. The zeta potential as well supplied the unanimous results (Figure S9). The initial value of the testing system is -6.63 mV, which is due to ionization of negative groups (-COOH and -OH) adhered to the surface of D-CDs. When CIP is added, the potential of the system rapidly increases to -1.52 mV. This indicates that the CIP in the system consumed some negative functional groups and successfully combined with them, causing potential reversal. On the other hand, charge transfer also plays an important role. It can be also related to the electron accepting nature of the -SO 3 H groups, and the electron donating character of the target CIP. The conjugation system of CIP can be connected to D-CDs by charge transfer. Subsequently, further research was conducted on the UV spectra before and after the interaction between D-CDs and CIP (Figure S10). Along with the addition of CIP, the UV absorption peak had undergone a significant red shift, which due to the combined effect of hydrogen bonding and charge transfer, which expands the conjugated system. The synergy effect of the hydrogen bond and charge transfer [ 32 ] greatly enhances the fluorescence intensity of D-CDs (Scheme 2 ). Fluorescence enhancement only occurred in the system containing CIP. The possible reason of high selectivity is described as follows: FQs is a fluorinated 4-quinolone ring containing carboxylic acid, fluorine and a piperazine ring substitution at position 3, 7 and 8. Strong hydrogen bond occurs between the carboxyl of D-CDs and amine of the piperazine ring preferentially. As shown in Table S3, the differences between all molecular structures are proposed. For most FQs, the amine of the piperazine ring is substituted by methyl, which results in the inability to form a strong hydrogen bond. It is negative for charge transfer and further hinders the production of sensitization. In addition, the benzene ring in the quinolone ring is replaced by a pyridine ring and the cyclohexyl group on the quinolone is replaced by a methyl group. This causes the conjugation system and electron donating character of ENO and NOR to be weakened. Thus, the system enables the specific determination of CIP. Hybrid conjugated structures are designed to simulate D-CDs, and the adsorption energies of various FQs on D-CDs are assessed through density functional theory (DFT) calculations, as depicted in Table S4. The results from these calculations are in good agreement with the experimental findings. It has been reported that the carboxylic acid groups in CIP can be combined with metal ions, such as Ca 2+ , Cd 2+ , Cu 2+ , Pb 2+ , Al 3+ and Co 2+ , especially with Co 2+ . In this work, the addition of Co 2+ destroyed the synergy effect of the hydrogen bond and charge transfer, forming the Co 2+ -CIP complex. In the system for detecting Co 2+ , the pH value of the solution is neutral, and some ions, such as Al 3+ and Cu 2+ , undergo hydrolysis. Furthermore, relative to metal ions such as Ca 2⁺ , Cd 2⁺ , and Pb 2⁺ , Co 2+ —with its smaller ionic radius, reduced steric hindrance, and higher charge density—more readily forms robust interactions with CIP, thereby effectively promoting the reaction. After the introduction of Co 2+ into CDs/CIP system, the blue emission of CDs/CIP at 445 nm is sensibly reinstated. According to the literature, we conclude that the coordination ability between Co 2+ and CIP is stronger than the hydrogen bonding force between CIP and D-CDs. As observed, the zeta potential of CDs/CIP/Co 2+ aqueous solution exhibits a remarkable enhancement in comparison with the CDs/CIP system and it further proves that upon the addition of Co 2+ , the electron donating groups of CDs/CIP may react with Co 2+ to change the system charge (Figure S10).The existence of Co-N and Co-O bonds in the FTIR further confirms the coordination between Co 2+ and CIP (Figure S11). Thus, the gained D-CDs were of great potential for effectively ratiometric sensing of CIP and Co 2+ . 3.5 Practical applicability To comprehend the accuracy and applicability of the proposed ratiometric sensor, spiking and recovery tests were conducted applying the lake water and milk as real sample. The CIP and Co 2+ recoveries were acquired in the range of 97.0% -104.8%, 96% − 99.9%, individually (Table S5 and Table S6), with the relative standard deviations (RSD) of 1.7–4.5% for the spiked samples, which manifested the prepared method can be successfully utilized to real samples determination. 3.6 Smartphone application for the detection of CIP and Co 2+ Recently, the portable detection of analytes is still limited by the complex operation and the cumbersome of laboratory instruments. Therefore, it is necessary to establish a simple quantitative analysis equipment that can be carried manually [ 33 ] . We design a convenient paper-based CD sensor to quickly detect the samples with the assistance of the smartphone platform. As shown in Fig. 6 (a), when the concentration of CIP increases from 0 to 208 µM, the fluorescent probe exhibits a continuous color change from orange to blue under 365 nm. Conversely, the fluorescence intensity is gradually enhanced from blue to tangerine when the incremental concentrations of Co 2+ are mixed to the CDs/CIP system. At the same time, the paper-based sensor shows the same phenomenon in the presence of analytes as depicted in Fig. 6 (b). However, it is s difficult to directly determine the actual content of the measured object because the subtle color changes are not easily observed by the naked eye. To address this problem, a smartphone sensor platform is designed to analyze the color parameters. And the high-resolution camera of smartphone and third-party software are applied as tools for analyzing results. Furthermore, the RGB values are acquired through the color recognizer for further statistical analysis. The calculation displays a linear relationship between B/(R + G) and CIP and Co 2+ , respectively. Photographs of paper strips and solutions containing CIP and Co 2+ are collected, and the calibration equation is established Fig. 6 (c) and Fig. 6 (d). There is an excellent linear relationship between the B/(R + G) value and the CIP at a range of 0–208 µM and could be expressed as B/(R + G) = 0.0034C CIP + 0.39 (R 2 = 0.9966). Then, the value of B/(R + G) decrease with increasing concentration of Co 2+ (0.5–22.4 mM) and an excellent liner relationship could be expressed as B/(R + G) = -0.025C Co2+ + 1.2 (R 2 = 0.9914), and the color of the paper strip gradually changed from blue to tangerine. In short, the results indicate that the smartphone-assisted fluorescence sensing platform have a fast speed detection, convenient reading and intelligent. The above findings indicate that D-CDs possess the potential for cascade detection of CIP and Co 2+ . The solution of D-CDs can emit orange fluorescence when exposed to a 365 nm UV lamp. When adding 208 µM of CIP, the fluorescence of D-CDs solution present blue. However, when 22.4 mM of Co 2+ are introduced into the CDs/CIP mixture, the corresponding mixture display a tangerine fluorescence under 365 nm UV lamp. A fluorescence logic system with a dual-input logic operation function is designed based on the cascade reaction of D-CDs to CIP and Co 2+ (Figure. 7(a)). In this logic system, the “NAND” gate is assigned to D-CDs, the “OR” gate is assigned to CDs/CIP., and CIP and Co 2+ are used as chemical inputs (referred to as “Input 1” and “Input 2”). The presence and absence of them corresponds to the Boolean logic functions of “1” and “0”, respectively. The reduction and enhancement of fluorescence at 445 nm were defined as “0” and “1” respectively. The truth table (Fig. 7 (b)) presents the parameters and results of the fluorescence cascade logic operation based on D-CDs. The output signal combined with the fluorescence color make the logic gate applicable for detection of CIP and Co 2+ , thus providing a new perspective for the application of D-CDs in digital encryption and other aspects. 3.7 Biocompatibility experiment The toxicity of D-CDs was assessed by the MTT tests [34] . The viability of HeLa cells was more than 80% after incubating with D-CDs (0–600 µg/mL) (Figure S12). The result manifested minor toxicity of the D-CDs. There initially displayed a weak blue fluorescence in channel 1 accompanied by weak red fluorescence in channel 2 (Fig. 8 (a)). Upon the introduction of CIP, the red fluorescence intensity maintained, and the blue fluorescence intensity brighten (Fig. 8 (b)). After continuing to add Co 2+ , blue fluorescence emission was reinstated, and the red fluorescence was still no altered (Fig. 8 (c)). These results reveal the practical use of D-CDs for CIP and Co 2+ in the living system. 4. Conclusion Here, we cover some new observations on the luminescence of carbon dots (CDs) that are passivated with methylene blue and explored them, for the first time, as speedy and detection of CIP and Co 2+ . Specifically, CIP is found to enhance the fluorescence of D-CDs due to the strong adsorption energy, while Co 2+ can disrupt the acting force between D-CDs and CIP, leading to the quenching of fluorescence at 445 nm. The as prepared nanosensor display a sensitive, linear dependence, and rapid response to CIP and Co 2+ . Moreover, a logic gate sensor is constructed using the cascade switching fluorescence of D-CDs, allowing for trace detection of both CIP and Co 2+ . In addition, the fluorescence test paper based on D-CDs is fabricated for portable and disposable detection. The synthesized D-CDs are successfully utilized for detecting CIP and Co 2+ in real samples. The construction of this portable fluorescent sensing device not only achieves the cascade detection of CIP and Co 2+ , but also holds the potential to improve the detection efficiency and reduce the cost, shows great practical significance. We anticipate that this investigation can supply a cornerstone and motivate novel thoughts for precise analysis in the fields of environmental protection. Declarations Funding Declaration This work was financially supported by the National Natural Science Foundation of China (No. 22274090). Y Yan also thanks the financial support from Shanxi University to support her Flinders University, Australia visiting as a PhD student. Competing Interest The author(s) declares that they have no competing interests. Author Contribution The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. These authors contributed equally. 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10:03:03","extension":"png","order_by":39,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":25688,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage17.png","url":"https://assets-eu.researchsquare.com/files/rs-7544936/v1/a6d4cb75589c150cc236f4b2.png"},{"id":91842946,"identity":"9953b108-6e4d-4c00-9498-a27bd3b9836f","added_by":"auto","created_at":"2025-09-22 10:03:03","extension":"xml","order_by":40,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":106268,"visible":true,"origin":"","legend":"","description":"","filename":"a996119d718a4117bfd8f1d64a3cec241structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7544936/v1/02ee21496f85dc7a7179343c.xml"},{"id":91842934,"identity":"d7953b87-a088-411c-bd6a-dbb43d9932e8","added_by":"auto","created_at":"2025-09-22 10:03:03","extension":"html","order_by":41,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":113440,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7544936/v1/acd863db265498a82f8c631e.html"},{"id":91842898,"identity":"924c56a3-51cd-4804-971e-06bc4350fc48","added_by":"auto","created_at":"2025-09-22 10:03:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":220871,"visible":true,"origin":"","legend":"\u003cp\u003e(a) TEM image and HRTEM image (top right inset); (b) the dimensions distribution of prepared D-CDs; (c) AFM image of D-CDs; (d) AFM 3D image of D-CDs.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7544936/v1/0e08cb51adc0f91f9d649a56.png"},{"id":91842897,"identity":"875a3381-2cf9-489d-a63c-a6e110f2db24","added_by":"auto","created_at":"2025-09-22 10:03:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":129843,"visible":true,"origin":"","legend":"\u003cp\u003e(a) XPS full-survey and high-resolution of (b) C 1s; (c) N 1s; (d) O 1s; (e) S 2p; (f) FTIR spectrum for the D-CDs.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7544936/v1/3fd50c43a510dba34ba72591.png"},{"id":91842900,"identity":"112f5897-7fe1-4d06-9cf0-63791b725166","added_by":"auto","created_at":"2025-09-22 10:03:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":117529,"visible":true,"origin":"","legend":"\u003cp\u003e(a) UV−vis absorption and PL spectra of D-CDs; (b) excitation-emission matrix for D-CDs.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7544936/v1/a468eca1fec1208558ac8075.png"},{"id":91842954,"identity":"6c35798e-9af7-4d2c-8773-a219b1868683","added_by":"auto","created_at":"2025-09-22 10:03:20","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":157630,"visible":true,"origin":"","legend":"\u003cp\u003e(a) The ratio value F\u003csub\u003e445nm\u003c/sub\u003e/F\u003csub\u003e662nm\u003c/sub\u003e of D-CDs (0.45 mg/mL) in the presence of distinct antibiotic (0.01 M); (b) the influence time on the FL of D-CDs when 7.5 μL (1 mM) CIP was added; (c) fluorescence spectra of D-CDs with increasing CIP (0.0-3.58 nM); (d) relevance of F\u003csub\u003e445nm\u003c/sub\u003e/F\u003csub\u003e662nm\u003c/sub\u003e and CIP (0.047-3.58 nM).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7544936/v1/95e9499aeb173ed7976dddb9.png"},{"id":91842902,"identity":"778679ea-ab35-43e1-bd2d-5bc7a85d025d","added_by":"auto","created_at":"2025-09-22 10:03:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":148126,"visible":true,"origin":"","legend":"\u003cp\u003e(a) The ratio value F\u003csub\u003e445nm\u003c/sub\u003e/F\u003csub\u003e662nm\u003c/sub\u003e of CDs/CIP in the presence of various metal ions (0.1 M); (b) effect of time on the F\u003csub\u003e445nm\u003c/sub\u003e/F\u003csub\u003e662nm\u003c/sub\u003e of CDs/CIP vs. Co\u003csup\u003e2+\u003c/sup\u003e (77.8 μL, 0.1 M); (c) fluorescence spectra of CDs/CIP with gradually increased Co\u003csup\u003e2+\u003c/sup\u003e; (d) relevance of F\u003csub\u003e445nm\u003c/sub\u003e/F\u003csub\u003e662nm\u003c/sub\u003e and Co\u003csup\u003e2+\u003c/sup\u003e (0-369.95 μM). Inset: the linear fitting region of (d).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7544936/v1/d87f2ca7b578835ce04fa613.png"},{"id":91845359,"identity":"46dd686d-3ea6-4889-8e1a-595fa2bfc168","added_by":"auto","created_at":"2025-09-22 10:11:02","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":171463,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Photographs of D-CDs-impregnated paper strips in CIP and Co2+ containing different concentrations taken under a 365 nm UV lamp; (b) change of color in Lab modes of D-CDs paper strips; linear relationship between B/(R+G) value with (c) CIP and (d) Co2+ concentrations, respectively.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7544936/v1/4f9850c90166dcae872eff7a.png"},{"id":91845366,"identity":"61a69d0d-b665-4cf2-8412-60601d98a5ab","added_by":"auto","created_at":"2025-09-22 10:11:02","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":72156,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Logic gate scheme; (b) the corresponding truth table based on the D-CDs.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7544936/v1/698ef779c2824a3a48935ae3.png"},{"id":91846325,"identity":"762a3d98-190e-4b22-a2dc-8b4663917f2b","added_by":"auto","created_at":"2025-09-22 10:19:02","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":241958,"visible":true,"origin":"","legend":"\u003cp\u003e(a) HeLa cells were hatched only with D-CDs (0.45 mg/mL); (b) fluorescence image after further incubation with 7.5 μL of CIP (1 mM) for 10 min; (c) 70 μL of Co\u003csup\u003e2+\u003c/sup\u003e (0.1 M) was brought into system.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-7544936/v1/d3f02103792b0e3170cf6170.png"},{"id":97178426,"identity":"8d8d81e1-6b16-4866-bcc5-22f09adc493a","added_by":"auto","created_at":"2025-12-01 16:09:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1904051,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7544936/v1/e1c23b2e-c3d5-432d-8b9e-38642c3f539f.pdf"},{"id":91842901,"identity":"6081a011-0a7f-4825-affa-4b21445696fe","added_by":"auto","created_at":"2025-09-22 10:03:02","extension":"doc","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1463256,"visible":true,"origin":"","legend":"","description":"","filename":"SupportInformation.doc","url":"https://assets-eu.researchsquare.com/files/rs-7544936/v1/40543966dc4d295823e90852.doc"},{"id":91842959,"identity":"8bcb4358-306d-400a-b0c4-c8230ce9d959","added_by":"auto","created_at":"2025-09-22 10:03:34","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":836420,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstracts.docx","url":"https://assets-eu.researchsquare.com/files/rs-7544936/v1/3f444d9a9a2cc4baa94a8e99.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Two birds, one stone: long-wavelength carbon dots enables ratiometric detection of ciprofloxacin and Co 2+ for smartphone, logic gate, and imaging applications","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe progress of society induces the emergence of increasing environmental issues\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. The arbitrary discharge of industrial wastewater and the use of a large amount of antibiotics have led serious environmental pollution problems, among them heavy metal ions and antibiotic pollution cannot be underestimated\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Therefore, the measuring of heavy metal ions and antibiotic residues are of great significance for environment and human health.\u003c/p\u003e\u003cp\u003eCarbon dots (CDs), a class of photoluminescent nanomaterials with illustrious optical properties\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e, low toxicity\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e and favorable biocompatibility\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e possess an extensive range of potential applications, including optical sensing, biological imaging, light emitting diode (LED) and so forth[7, 8]. Especially in the detection of environmental pollutants, CDs are good candidates as sensors\u003csup\u003e[\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. However, most of the reported CDs for the detection of environmental pollutant respond via a single fluorescence signal, which is easily subjected to strong influence from multiple factors, for instance probe concentration, fluctuation of excitation source, background absorption\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. Ratiometric fluorescence sensing can overcome these matters through simultaneously gauging the ratio of two well-tackled emission peaks to enhance detection capability\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Therefore, development of ratiometric fluorescence sensors have profound significance toward the analysis of environmental pollutants. In addition, analysis is often limited to semi quantitative detection in laboratory conditions, which severely restricts the applications in several fields, especially in resources-limited areas, poverty countries, and clinical diagnosis\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. The problem may be solved by developing fluorescent colorimetric strategies and visual fluorescent test papers. Paper sensors have been widely used in the many fields due to their advantages of low cost, portability, and versatility, especially for environmental pollutants detection\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. In recent years, with the continuous upgrade of smartphones equipped with HD cameras, smartphone has received wide attention as a good analysis device in point-of-care testing (POCT) sensors\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Therefore, how to establish an integrated intelligent detection platform to realize sensitive, accurate and convenient monitoring of environmental pollutants by combining CDs with smartphone assistance combined with test strips in an uncomplicated way is of great significance.\u003c/p\u003e\u003cp\u003eCiprofloxacin (CIP) has the strongest antibacterial activity among the third-generation fluoroquinolone antibiotic, so it is broadly applied in the treatment of human and animal infectious diseases\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. With the widespread application of CIP, it inevitably leads to a large amount of residues in animal bodies, which may exist in edible substances, such as milk or eggs\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. Furthermore, CIP cannot be completely absorbed and will enter the environment in its original form with excreta through human metabolism. Even at low concentrations, it may trigger tremendous harm to the ecological environment and mankind health, bringing about the emergence of various diseases. Global concerns have been induced by the increasing existence of CIP residues and most countries have established maximum residue limits (MRLs) for CIP. Furthermore, China\u0026rsquo;s Ministry of Agriculture (CMA) has similarly issued corresponding regulations to regulate the usage of CIP\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Hence, efficacious monitoring of CIP residues has turned into an essential matter. The traditional detection approaches of CIP mainly comprise high-performance liquid chromatography (HPLC), enzyme linked immunoassay (ELI), capillary electrophoresis (CE), electrochemical assay. However, these traditional approaches are restricted in realistic applications. Fluorimetry is deemed to be a credible analytical tactic owing to uncomplicated of operation, technical simplicity and expansive adaptability. To this end, CDs-based ratiometric probe has been developed for CIP sensing. Gui et al.\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e first introduced a novel ratiometric fluorescence (FL) probe for the detection of CIP based on CDs/SiDs\u0026ndash;BPMA hybrids. Subsequently, Lu et al.\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e raised a ratiometric tactics for the determination of CIP based on CDs/riboflavin system. Gao et al.\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e designed a platform between CDs and 2, 3-diaminophenazine for ratiometric monitoring of CIP. However, there are still some limitations with the reported ratiometric fluorescence CDs for detecting CIP, such as time-consuming and cumbersome procedures. Up to now, to the best of our knowledge, one-pot synthesis of ratiometric fluorescence CDs for rapid measure of CIP have not been reported. In addition, its on-site, rapid, and sensitive measurements also pose challenges. Therefore, it is urgently necessary to design and fabricate a portable device for tracing CIP accurately and conveniently on the spot or special sites.\u003c/p\u003e\u003cp\u003eCobalt ions (Co\u003csup\u003e2+\u003c/sup\u003e) is a trace element that acts an essential role in the human body. However, excessive intake of Co\u003csup\u003e2+\u003c/sup\u003e wreaks a woeful threat to human health, such as low blood pressure, diarrhea and even death by reason of myocardial infarction\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. The World Health Organization (WHO) has regulate the utmost permitted content of Co\u003csup\u003e2+\u003c/sup\u003e in drinking water less than 40 \u0026micro;g/L\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Therefore, it is in great demand to construct an approach for detecting Co\u003csup\u003e2+\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eUntil now, numerous techniques for the detection of Co\u003csup\u003e2+\u003c/sup\u003e have been exploited, including atomic absorption spectrometry (AAS), liquid chromatography (LC), plasma-atomic emission spectrometry (ICP-OES) and fluorescence method. Since the exceptional selectivity, low detection limit, and unsophisticated operation of fluorescent nanoprobes, it has mesmerized immense focus of researchers\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. Due to the outstanding properties of CDs, some CDs ratiometric fluorescent nanoprobes have been discovered to monitor Co\u003csup\u003e2+\u003c/sup\u003e. Dong et al.\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e designed a dual-emission CDs to ratiometric recognition of Co\u003csup\u003e2+\u003c/sup\u003e. Nevertheless, CDs-dependent Co\u003csup\u003e2+\u003c/sup\u003e ratiometric FL sensors have been rarely built. Consequently, it remains a challenge to get ahold of a ratiometric CDs for the detection of Co\u003csup\u003e2+\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn this work, we design a sensing platform based on multifunctional D-CDs for the ratiometric rapid measuring of CIP and Co\u003csup\u003e2+\u003c/sup\u003e firstly (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).The obtained D-CDs, manifesting dual emission of blue emission at 445 nm and weak red fluorescence at 662 nm under 306 nm excitation, are synthesized via the one-step hydrothermal route through adopting methylene blue as the only precursors. Interestingly, the fluorescence at 445 nm increases and remains at 662 nm with the injection of CIP, which appears ratiometric natures (F\u003csub\u003e445nm\u003c/sub\u003e/F\u003csub\u003e662nm\u003c/sub\u003e). In addition, the fluorescence of CDs/CIP can be efficaciously reinstated by replacing CIP with Co\u003csup\u003e2+\u003c/sup\u003e. More importantly, the colorimetric test strips are exploited and a sensing platform combining with smartphone is established to achieve rapid, sensitive and accurate visual monitoring of CIP and Co\u003csup\u003e2+\u003c/sup\u003e. Meanwhile, we explore the potential use of the synthesized D-CDs for constructing a logic gate sensor capable of detecting CIP and Co\u003csup\u003e2+\u003c/sup\u003e based on the fluorescence cascaded switching mechanism. Furthermore, the D-CDs is triumphantly utilized to detect CIP and Co\u003csup\u003e2+\u003c/sup\u003e intracellular imaging.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"2. Experimental","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Material\u003c/h2\u003e\u003cp\u003eThe materials required for the experiment were provided in the Support Information.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Preparation of D-CDs.\u003c/h2\u003e\u003cp\u003eThe D-CDs were prepared through a one-pot solvothermal tactics. 0.0156 g of methylene blue was dissolved with 25 mL DDI water and heated at 200\u0026deg;C for 6 h. Next, the obtained product was centrifuged (8000 rpm, 20 min) and dialyzed (MWCO 800, 12 h). Ultimately, the D-CDs powders were stored for subsequent use.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Detection of CIP and Co\u003csup\u003e2+\u003c/sup\u003e\u003c/h2\u003e\u003cp\u003eTo begin with, 20 \u0026micro;L of D-CDs liquid (4.5 mg/mL) was added into 2.0 mL of DDI water. To assess the specificity of the D-CDs, various antibiotics were injected into the solution at the concentration of 0.01 M. Subsequently, different volumes of Co\u003csup\u003e2+\u003c/sup\u003e (0.1 M) was added into the CDs/CIP (36.9 \u0026micro;M) system. The anti-interference tests were implemented through introducing other antibiotics (10 mM) instead of CIP (1 mM) in mixed solution.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Cellular imaging\u003c/h2\u003e\u003cp\u003eThe Hela cells were hatched with the D-CDs (4.5 mg/mL) for 30 min. Afterwards, being washed with PBS buffer (pH\u0026thinsp;=\u0026thinsp;7.4) to get rid of redundant D-CDs. The pretreated cells were then incubated with CIP (7.5 \u0026micro;L, 1 mM) firstly. The cells loaded with CDs/CIP were further treated with Co\u003csup\u003e2+\u003c/sup\u003e (70 \u0026micro;L, 0.1 M) for imaging.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Monitoring of CIP and Co\u003csup\u003e2+\u003c/sup\u003e in real samples\u003c/h2\u003e\u003cp\u003eMilk was bought from native shop in Taiyuan, China. The pretreated procedure refers to the literature\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. Real water samples (Lingde Lake water from Shanxi University, China) were filtered through filtered membrane (0.22 \u0026micro;m) and centrifuged (4000 rpm, 15 min) for subsequent use. Finally, the prepared samples were used for the detection of CIP and Co\u003csup\u003e2+\u003c/sup\u003e according to conventional procedures. The water samples were gauged by the proposed ratiometric fluorescence and smartphone assisted.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003e2.6 Smartphone application for the detection of CIP and Co\u003c/b\u003e\u003csup\u003e\u003cb\u003e2+\u003c/b\u003e\u003c/sup\u003e.\u003c/h2\u003e\u003cp\u003eDifferent volumes of CIP (0.01 M) were introduced into the D-CDs solution (0.045 mg/mL), and then the fluorescent color photos were captured by a smartphone under a 365 nm UV lamp. The color parameters (RGB values) were derived through a smartphone application (Color Identification APP)\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. Subsequently, different volumes of Co\u003csup\u003e2+\u003c/sup\u003e (0.1 M) was added into the CDs/CIP (208.1 \u0026micro;M) system. The fluorescence images were captured for further analysis.\u003c/p\u003e\u003cp\u003eFor paper-based sensing, the filter paper was cut into approximately 1 cm\u003csup\u003e2\u003c/sup\u003e as the test strips firstly. Afterward, the test strips were dipped in the D-CDs solution (0.045 mg/mL) and the test papers loaded with D-CDs were dipped in solution of different concentrations of CIP and dried spontaneously. The color variation of the test strips was taken by the smartphone. The color information (RGB values) was derived through a smartphone application (Color Identification APP). Subsequently, different concentrations of Co\u003csup\u003e2+\u003c/sup\u003e was dropped onto the prepared filter paper strips. After spontaneous drying of the test paper, the fluorescence images were captured for further analysis.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Characterizations\u003c/h2\u003e\u003cp\u003eThe morphology of the D-CDs were examined by transmission electron microscopy (TEM) and atomic force microscope (AFM)\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. The D-CDs presents a globular morphology, with a mean value of 2.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(a) and Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(b)). The lattice spacing of D-CDs is 0.22 nm in accord with the (100) planes of graphite carbon, manifesting that D-CDs have highly crystalline carbon structures (inserted in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(a)). Furthermore, AFM is applied to confirm the morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(c) and Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(d)), and D-CDs emerges granule heights of roughly 2.55 nm, which corresponding to the result of TEM.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eX-ray photoelectron spectroscopy (XPS) and Fourier transform infrared (FTIR) were performed to reveal the composition of the D-CDs\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. We characterized the surface functional groups of D-CDs by FTIR and XPS. The full XPS spectrum of D-CDs exhibit six main peaks at 529 eV (O 1s), 397.2 eV (N 1s), 283.1 eV (C 1s), 226.4 eV (Cl 2p) and 162.8 eV (S 2p) respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(a)). The C 1s spectrum (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(b)) contains three parts at 284.6 eV (C\u0026thinsp;\u0026minus;\u0026thinsp;C/C\u0026thinsp;=\u0026thinsp;C), 285.9 eV (C\u0026thinsp;\u0026minus;\u0026thinsp;O/C\u0026thinsp;\u0026minus;\u0026thinsp;S) and 287.0 eV (C\u0026thinsp;=\u0026thinsp;O). The N 1s band displays two constituent peaks of C-N and N-H, which situated at 398.6 eV and 400.9 eV, individually (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(c)). The O 1s spectrum can be divided into two sections at 531.4 and 532.5 eV, which corresponding to C\u0026thinsp;\u0026minus;\u0026thinsp;OH and C\u0026thinsp;=\u0026thinsp;O (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(d)). The spectrum of S 2p shows three peaks of 164.0 eV, 165.2 eV, and 168.3 eV, corresponding to C-S (S 2p\u003csub\u003e3/2\u003c/sub\u003e), C-S (S 2p\u003csub\u003e1/2\u003c/sub\u003e), and S\u0026thinsp;=\u0026thinsp;O, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(e)). The band at 3436.7 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e presents the existence of O-H. Peaks at about 2953 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e indicated the presence of C-H. It is evident that the peaks at 1602 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e(C\u0026thinsp;=\u0026thinsp;O), 1324 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e(C-O) and 1142 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (S\u0026thinsp;=\u0026thinsp;O) can be observed. Importantly, peak at 909 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was due to the N-H (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(f)). Above results indicate that the D-CDs is plentiful in hydroxyl and carboxylic groups. The results of FTIR are unanimous with the confirmation of XPS.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe UV\u0026ndash;vis absorption spectrum of D-CDs portrayed two peaks centered at 261 nm and 280 nm, which attribute to π-π* transition of the C\u0026thinsp;=\u0026thinsp;C bond and the n-π* transition of the C\u0026thinsp;=\u0026thinsp;O/C-O bond. In 500 nm -700 nm, a wide absorption band is at 600 nm, which corresponds to the MB itself (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(a)). The D-CDs exhibit two well-resolved fluorescence peaks at 455 and 662 nm at the optimal excitation wavelength. The emission peak of D-CDs slightly alters when the excitation wavelength ranging from 230 nm to 320 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(b)). The λ\u003csub\u003eex\u003c/sub\u003e-dependent fluorescence behavior may be attributed to the molecular state. Furthermore, the fluorescence quantum yield of the D-CDs was found to be 16.8% using Rhodamine B as a standard (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe fluorescent stability of D-CDs was investigated. The fluorescence intensity of D-CDs persists steady upon persistent illumination for 1 h, indicating that the D-CDs has remarkable photobleaching resistance (Figure S2). Simultaneously, the emission intensity remains stable in the high ionic strength (2.0 M) (Figure S3) and wider pH rang (5\u0026ndash;13) (Figure S4), which is beneficial for elaborate environment and living systems.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Ratiometric detection of CIP\u003c/h2\u003e\u003cp\u003eBy introducing a variety of metal ions, the selectivity performance of D-CDs was investigated. The common metal ion almost exhibits no effects (Figure S5). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(a), the ratio value (F\u003csub\u003e445nm\u003c/sub\u003e/F\u003csub\u003e662nm\u003c/sub\u003e) of D-CDs adding antibiotics at a concentration of 0.01M were collected and recorded. Among these materials, the ratio of F\u003csub\u003e445nm\u003c/sub\u003e/F\u003csub\u003e662nm\u003c/sub\u003e is considerably increased along with the addition of CIP, while other antibiotics did not show the equivalent phenomenon, indicating the terrific selectivity of D-CDs for CIP. CIP belongs to the fluoroquinolones (FQs) antibacterial drug. The influence of other FQs residues on the fluorescent behavior of D-CDs is evaluated as depicted in Figure S6. The fluorescence of D-CDs does not significant changes after adding other FQs. When CIP was injected into D-CDs including interferences, the PL intensity of D-CDs could still be dramatically increased (Figure S7), demonstrating that the D-CDs can be applied to detect CIP with good anti-interference. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(b) presents the ratio value of D-CDs reaches equilibrium with CIP in only 10 s, indicating that as-acquired D-CDs have the potential for rapid monitoring of CIP. With increasing the concentration of CIP, the red fluorescence at 662 nm remained following with a ratio growth at 445 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(c)). In addition, the value of F\u003csub\u003e445nm\u003c/sub\u003e/F\u003csub\u003e662nm\u003c/sub\u003e shows exceptional linear relationship with the CIP concentration in the range of 0.047 nM-3.59 nM, the fitting curve equation is F\u003csub\u003e445nm\u003c/sub\u003e/F\u003csub\u003e662nm\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.8040\u0026thinsp;+\u0026thinsp;1.0783C\u003csub\u003eCIP\u003c/sub\u003e, R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9989. Simultaneously, corresponding LOD for CIP was gauged to be 16.8 pM (S/N\u0026thinsp;=\u0026thinsp;3) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(d)). As described in Table S2, the ratiometric fluorescence CDs applied to detect CIP currently have drawbacks such as long detection time or high detection limits. Above results displayed that D-CDs can act as a nanosensor for rapid measure of CIP by a ratio sensing model, with a fast response and very low LOD value of 16.8 pM, which is excellent to existing data.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Ratiometric detection of Co\u003csup\u003e2+\u003c/sup\u003e.\u003c/h2\u003e\u003cp\u003eA holistic exploration on the influences of diverse metal ions on the fluorescence of the CDs/CIP was executed (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e(a)). The CDs/CIP system proves particular identification to Co\u003csup\u003e2+\u003c/sup\u003e beyond other competitive ions. Meanwhile, the time for the reaction between CDs/CIP and Co\u003csup\u003e2+\u003c/sup\u003e was conducted (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e(b)), indicating CDs/CIP system can rapid monitor of CIP (15 s). Upon gradually increasing Co\u003csup\u003e2+\u003c/sup\u003e concentrations (0\u0026thinsp;\u0026minus;\u0026thinsp;369.95 \u0026micro;M), the PL intensity at 445 nm recuperates (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e(c)). Meanwhile, the value of F\u003csub\u003e445nm/\u003c/sub\u003eF\u003csub\u003e662nm\u003c/sub\u003e exhibits a good linear correlation (F\u003csub\u003e445nm\u003c/sub\u003e/F\u003csub\u003e662nm\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;2.0762-0.0090C\u003csub\u003eCo\u003c/sub\u003e\u003csup\u003e2+\u003c/sup\u003e, R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9996) by varying Co\u003csup\u003e2+\u003c/sup\u003e in the range of 45.65\u0026thinsp;\u0026minus;\u0026thinsp;89.39 nM, and the LOD is computed to be 14.68 nM, which is below the WHO regulations (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e(d)).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Sensing mechanisms\u003c/h2\u003e\u003cp\u003eFTIR, zeta potential, UV-vis were operated to explore the mechanism. Both FTIR and XPS information manifest the existence of abundant carboxylic and hydroxyl groups on the surface of the D-CDs. CIP as well contains numerous groups, embodying C-F, C-O, and C-N. Taking inspiration from previous literature, we speculate that the excellent response of D-CDs to CIP may be attributed to hydrogen bonding between CIP and oxygen-containing functional groups (-COOH or -OH) on the surface of D-CDs. To corroborate this hypothesis, the FTIR of the D-CDs system before and after the addition of CIP were comprehensively measured. In Figure S8, there are two blue shifts. To start with, contrasted to the C-OH vibrations (3431 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) of D-CDs (red line), there is a blue shift of 103 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of the CD/CIP (black line). Next, the vibrations of N-H situated at around 2943 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in CDs/CIP, there is as well a blue movement of 90 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e contrasted to the CIP (3033 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). These hint the formation of hydrogen bonding between CIP and D-CDs. The zeta potential as well supplied the unanimous results (Figure S9). The initial value of the testing system is -6.63 mV, which is due to ionization of negative groups (-COOH and -OH) adhered to the surface of D-CDs. When CIP is added, the potential of the system rapidly increases to -1.52 mV. This indicates that the CIP in the system consumed some negative functional groups and successfully combined with them, causing potential reversal. On the other hand, charge transfer also plays an important role. It can be also related to the electron accepting nature of the -SO\u003csub\u003e3\u003c/sub\u003eH groups, and the electron donating character of the target CIP. The conjugation system of CIP can be connected to D-CDs by charge transfer. Subsequently, further research was conducted on the UV spectra before and after the interaction between D-CDs and CIP (Figure S10). Along with the addition of CIP, the UV absorption peak had undergone a significant red shift, which due to the combined effect of hydrogen bonding and charge transfer, which expands the conjugated system. The synergy effect of the hydrogen bond and charge transfer\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e greatly enhances the fluorescence intensity of D-CDs (Scheme \u003cspan refid=\"Sch2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFluorescence enhancement only occurred in the system containing CIP. The possible reason of high selectivity is described as follows: FQs is a fluorinated 4-quinolone ring containing carboxylic acid, fluorine and a piperazine ring substitution at position 3, 7 and 8. Strong hydrogen bond occurs between the carboxyl of D-CDs and amine of the piperazine ring preferentially. As shown in Table S3, the differences between all molecular structures are proposed. For most FQs, the amine of the piperazine ring is substituted by methyl, which results in the inability to form a strong hydrogen bond. It is negative for charge transfer and further hinders the production of sensitization. In addition, the benzene ring in the quinolone ring is replaced by a pyridine ring and the cyclohexyl group on the quinolone is replaced by a methyl group. This causes the conjugation system and electron donating character of ENO and NOR to be weakened. Thus, the system enables the specific determination of CIP. Hybrid conjugated structures are designed to simulate D-CDs, and the adsorption energies of various FQs on D-CDs are assessed through density functional theory (DFT) calculations, as depicted in Table S4. The results from these calculations are in good agreement with the experimental findings.\u003c/p\u003e\u003cp\u003eIt has been reported that the carboxylic acid groups in CIP can be combined with metal ions, such as Ca\u003csup\u003e2+\u003c/sup\u003e, Cd\u003csup\u003e2+\u003c/sup\u003e, Cu\u003csup\u003e2+\u003c/sup\u003e, Pb\u003csup\u003e2+\u003c/sup\u003e, Al\u003csup\u003e3+\u003c/sup\u003e and Co\u003csup\u003e2+\u003c/sup\u003e, especially with Co\u003csup\u003e2+\u003c/sup\u003e. In this work, the addition of Co\u003csup\u003e2+\u003c/sup\u003e destroyed the synergy effect of the hydrogen bond and charge transfer, forming the Co\u003csup\u003e2+\u003c/sup\u003e-CIP complex. In the system for detecting Co\u003csup\u003e2+\u003c/sup\u003e, the pH value of the solution is neutral, and some ions, such as Al\u003csup\u003e3+\u003c/sup\u003e and Cu\u003csup\u003e2+\u003c/sup\u003e, undergo hydrolysis. Furthermore, relative to metal ions such as Ca\u003csup\u003e2⁺\u003c/sup\u003e, Cd\u003csup\u003e2⁺\u003c/sup\u003e, and Pb\u003csup\u003e2⁺\u003c/sup\u003e, Co\u003csup\u003e2+\u003c/sup\u003e\u0026mdash;with its smaller ionic radius, reduced steric hindrance, and higher charge density\u0026mdash;more readily forms robust interactions with CIP, thereby effectively promoting the reaction. After the introduction of Co\u003csup\u003e2+\u003c/sup\u003e into CDs/CIP system, the blue emission of CDs/CIP at 445 nm is sensibly reinstated. According to the literature, we conclude that the coordination ability between Co\u003csup\u003e2+\u003c/sup\u003e and CIP is stronger than the hydrogen bonding force between CIP and D-CDs. As observed, the zeta potential of CDs/CIP/Co\u003csup\u003e2+\u003c/sup\u003e aqueous solution exhibits a remarkable enhancement in comparison with the CDs/CIP system and it further proves that upon the addition of Co\u003csup\u003e2+\u003c/sup\u003e, the electron donating groups of CDs/CIP may react with Co\u003csup\u003e2+\u003c/sup\u003e to change the system charge (Figure S10).The existence of Co-N and Co-O bonds in the FTIR further confirms the coordination between Co\u003csup\u003e2+\u003c/sup\u003e and CIP (Figure S11). Thus, the gained D-CDs were of great potential for effectively ratiometric sensing of CIP and Co\u003csup\u003e2+\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Practical applicability\u003c/h2\u003e\u003cp\u003eTo comprehend the accuracy and applicability of the proposed ratiometric sensor, spiking and recovery tests were conducted applying the lake water and milk as real sample. The CIP and Co\u003csup\u003e2+\u003c/sup\u003e recoveries were acquired in the range of 97.0% -104.8%, 96% \u0026minus;\u0026thinsp;99.9%, individually (Table S5 and Table S6), with the relative standard deviations (RSD) of 1.7\u0026ndash;4.5% for the spiked samples, which manifested the prepared method can be successfully utilized to real samples determination.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.6 Smartphone application for the detection of CIP and Co\u003csup\u003e2+\u003c/sup\u003e\u003c/h2\u003e\u003cp\u003eRecently, the portable detection of analytes is still limited by the complex operation and the cumbersome of laboratory instruments. Therefore, it is necessary to establish a simple quantitative analysis equipment that can be carried manually\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. We design a convenient paper-based CD sensor to quickly detect the samples with the assistance of the smartphone platform. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003e(a), when the concentration of CIP increases from 0 to 208 \u0026micro;M, the fluorescent probe exhibits a continuous color change from orange to blue under 365 nm. Conversely, the fluorescence intensity is gradually enhanced from blue to tangerine when the incremental concentrations of Co\u003csup\u003e2+\u003c/sup\u003e are mixed to the CDs/CIP system. At the same time, the paper-based sensor shows the same phenomenon in the presence of analytes as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003e(b). However, it is s difficult to directly determine the actual content of the measured object because the subtle color changes are not easily observed by the naked eye. To address this problem, a smartphone sensor platform is designed to analyze the color parameters. And the high-resolution camera of smartphone and third-party software are applied as tools for analyzing results. Furthermore, the RGB values are acquired through the color recognizer for further statistical analysis. The calculation displays a linear relationship between B/(R\u0026thinsp;+\u0026thinsp;G) and CIP and Co\u003csup\u003e2+\u003c/sup\u003e, respectively. Photographs of paper strips and solutions containing CIP and Co\u003csup\u003e2+\u003c/sup\u003e are collected, and the calibration equation is established Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003e(c) and Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003e(d). There is an excellent linear relationship between the B/(R\u0026thinsp;+\u0026thinsp;G) value and the CIP at a range of 0\u0026ndash;208 \u0026micro;M and could be expressed as B/(R\u0026thinsp;+\u0026thinsp;G)\u0026thinsp;=\u0026thinsp;0.0034C\u003csub\u003eCIP\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;0.39 (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9966). Then, the value of B/(R\u0026thinsp;+\u0026thinsp;G) decrease with increasing concentration of Co\u003csup\u003e2+\u003c/sup\u003e (0.5\u0026ndash;22.4 mM) and an excellent liner relationship could be expressed as B/(R\u0026thinsp;+\u0026thinsp;G) = -0.025C\u003csub\u003eCo2+\u003c/sub\u003e + 1.2 (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9914), and the color of the paper strip gradually changed from blue to tangerine. In short, the results indicate that the smartphone-assisted fluorescence sensing platform have a fast speed detection, convenient reading and intelligent.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe above findings indicate that D-CDs possess the potential for cascade detection of CIP and Co\u003csup\u003e2+\u003c/sup\u003e. The solution of D-CDs can emit orange fluorescence when exposed to a 365 nm UV lamp. When adding 208 \u0026micro;M of CIP, the fluorescence of D-CDs solution present blue. However, when 22.4 mM of Co\u003csup\u003e2+\u003c/sup\u003e are introduced into the CDs/CIP mixture, the corresponding mixture display a tangerine fluorescence under 365 nm UV lamp. A fluorescence logic system with a dual-input logic operation function is designed based on the cascade reaction of D-CDs to CIP and Co\u003csup\u003e2+\u003c/sup\u003e (Figure. 7(a)). In this logic system, the \u0026ldquo;NAND\u0026rdquo; gate is assigned to D-CDs, the \u0026ldquo;OR\u0026rdquo; gate is assigned to CDs/CIP., and CIP and Co\u003csup\u003e2+\u003c/sup\u003e are used as chemical inputs (referred to as \u0026ldquo;Input 1\u0026rdquo; and \u0026ldquo;Input 2\u0026rdquo;). The presence and absence of them corresponds to the Boolean logic functions of \u0026ldquo;1\u0026rdquo; and \u0026ldquo;0\u0026rdquo;, respectively. The reduction and enhancement of fluorescence at 445 nm were defined as \u0026ldquo;0\u0026rdquo; and \u0026ldquo;1\u0026rdquo; respectively. The truth table (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003e(b)) presents the parameters and results of the fluorescence cascade logic operation based on D-CDs. The output signal combined with the fluorescence color make the logic gate applicable for detection of CIP and Co\u003csup\u003e2+\u003c/sup\u003e, thus providing a new perspective for the application of D-CDs in digital encryption and other aspects.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.7 Biocompatibility experiment\u003c/h2\u003e\u003cp\u003eThe toxicity of D-CDs was assessed by the MTT tests\u003csup\u003e[34]\u003c/sup\u003e. The viability of HeLa cells was more than 80% after incubating with D-CDs (0\u0026ndash;600 \u0026micro;g/mL) (Figure S12). The result manifested minor toxicity of the D-CDs.\u003c/p\u003e\u003cp\u003eThere initially displayed a weak blue fluorescence in channel 1 accompanied by weak red fluorescence in channel 2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003e(a)). Upon the introduction of CIP, the red fluorescence intensity maintained, and the blue fluorescence intensity brighten (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003e(b)). After continuing to add Co\u003csup\u003e2+\u003c/sup\u003e, blue fluorescence emission was reinstated, and the red fluorescence was still no altered (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003e(c)). These results reveal the practical use of D-CDs for CIP and Co\u003csup\u003e2+\u003c/sup\u003e in the living system.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eHere, we cover some new observations on the luminescence of carbon dots (CDs) that are passivated with methylene blue and explored them, for the first time, as speedy and detection of CIP and Co\u003csup\u003e2+\u003c/sup\u003e. Specifically, CIP is found to enhance the fluorescence of D-CDs due to the strong adsorption energy, while Co\u003csup\u003e2+\u003c/sup\u003e can disrupt the acting force between D-CDs and CIP, leading to the quenching of fluorescence at 445 nm. The as prepared nanosensor display a sensitive, linear dependence, and rapid response to CIP and Co\u003csup\u003e2+\u003c/sup\u003e. Moreover, a logic gate sensor is constructed using the cascade switching fluorescence of D-CDs, allowing for trace detection of both CIP and Co\u003csup\u003e2+\u003c/sup\u003e. In addition, the fluorescence test paper based on D-CDs is fabricated for portable and disposable detection. The synthesized D-CDs are successfully utilized for detecting CIP and Co\u003csup\u003e2+\u003c/sup\u003e in real samples. The construction of this portable fluorescent sensing device not only achieves the cascade detection of CIP and Co\u003csup\u003e2+\u003c/sup\u003e, but also holds the potential to improve the detection efficiency and reduce the cost, shows great practical significance. We anticipate that this investigation can supply a cornerstone and motivate novel thoughts for precise analysis in the fields of environmental protection.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was financially supported by the National Natural Science Foundation of China (No. 22274090). Y Yan also thanks the financial support from Shanxi University to support her Flinders University, Australia visiting as a PhD student.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author(s) declares that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. These authors contributed equally.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLi Y, Jiang XX, Xie JX, Lv Y kai (2022) Recent advances in the application and mechanism of carbon dots/metal-organic frameworks hybrids in photocatalysis and the detection of environmental pollutants. 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J Hazard Mater 408:124422. https://doi.org/10.1016/j.jhazmat.2020.124422\u003c/li\u003e\n\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":"Carbon dots, Ciprofloxacin, Co2+, Ratiometric","lastPublishedDoi":"10.21203/rs.3.rs-7544936/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7544936/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEnvironmental pollution poses a significant threat to human health and sustainable development, highlighting the need for rapid and sensitive contaminant detection. Here, we present a long-wavelength ratiometric fluorescent carbon dots to detect ciprofloxacin (CIP) and cobalt ion (Co\u003csup\u003e2+\u003c/sup\u003e). The carbon dots (D-CDs) exhibiting outstanding dual emission at 445 nm and 662 nm are successfully synthesized through a one-pot hydrothermal approach, with methylene blue serving as the sole precursor. Interestingly, the blue fluorescence at 445 nm is significantly enhanced since the effect of formation of hydrogen bonds and charge transfer between D-CDs and CIP, while the 662 nm emission remains unchanged, yielding a ratiometric fluorescence response (F\u003csub\u003e445nm\u003c/sub\u003e/F\u003csub\u003e662nm\u003c/sub\u003e) across a range of 0.048\u0026thinsp;\u0026minus;\u0026thinsp;3.58 nM, with a detection limit of 16.7 pM. Additionally, the fluorescence of CDs/CIP can be efficaciously ratiometric restored by right of a particular reaction of Co\u003csup\u003e2+\u003c/sup\u003e with CIP, achieving ratio fluorescence quantitative assay of Co\u003csup\u003e2+\u003c/sup\u003e (LOD\u0026thinsp;=\u0026thinsp;14.7 nM). Density functional theory (DFT) calculations has been used to illustrate the potential interaction mechanisms, which shows strong agreement with the experimental results. Notably, a smartphone-integrated colorimetric test strip enables on-site monitoring of CIP and Co\u003csup\u003e2+\u003c/sup\u003e, expanding environmental applications, which has been demonstrated by effectively detection of CIP in milk and river water. Further exploration has been conducted by developing a logic gate sensor which harnesses the activated cascade effect to serve as an intelligent probe for monitoring trace levels of CIP and Co\u003csup\u003e2+\u003c/sup\u003e. Furthermore, D-CDs are applied in cellular imaging, demonstrating their strong potential in sensing, bioimaging, and environmental analysis.\u003c/p\u003e","manuscriptTitle":"Two birds, one stone: long-wavelength carbon dots enables ratiometric detection of ciprofloxacin and Co 2+ for smartphone, logic gate, and imaging applications","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-22 10:02:57","doi":"10.21203/rs.3.rs-7544936/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-08T05:14:43+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-08T04:48:59+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-24T02:51:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"37381309599712529675047902654819092237","date":"2025-09-23T02:33:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"224152675600751473505774942258562810911","date":"2025-09-19T09:20:03+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-11T21:48:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-10T08:31:41+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-10T06:23:34+00:00","index":"","fulltext":""},{"type":"submitted","content":"Microchimica Acta","date":"2025-09-05T13:50:04+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":"0fac97be-053b-4e3b-82d3-a206eac0b4d1","owner":[],"postedDate":"September 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-01T16:02:31+00:00","versionOfRecord":{"articleIdentity":"rs-7544936","link":"https://doi.org/10.1007/s00604-025-07715-8","journal":{"identity":"microchimica-acta","isVorOnly":false,"title":"Microchimica Acta"},"publishedOn":"2025-11-25 15:57:57","publishedOnDateReadable":"November 25th, 2025"},"versionCreatedAt":"2025-09-22 10:02:57","video":"","vorDoi":"10.1007/s00604-025-07715-8","vorDoiUrl":"https://doi.org/10.1007/s00604-025-07715-8","workflowStages":[]},"version":"v1","identity":"rs-7544936","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7544936","identity":"rs-7544936","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}