Quinolone antibiotic bimodal sensing detection method and device
By constructing photoelectric active films and specific nucleic acid aptamers on conductive substrates, and combining a dual-modal sensing method with fluorescence and photoelectrochemical signal output, the complexity and field applicability of quinolone antibiotic detection in existing technologies have been solved, achieving highly sensitive, interference-resistant, and portable detection results.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-26
- Publication Date
- 2026-03-24
AI Technical Summary
Existing methods for detecting quinolone antibiotics are expensive, complex to operate, require professional personnel, have long analysis cycles, and are susceptible to interference from complex matrices, making it difficult to meet the needs of rapid on-site screening. Furthermore, there is a lack of portable dual-modal detection devices.
A dual-modal sensing method based on nucleic acid aptamers is adopted, which combines fluorescence and photoelectrochemical signal output. The sensor is constructed on a conductive substrate using photoelectroactive materials such as TiO2, g-C3N4, and CdS and specific nucleic acid aptamers. The sensor is verified by the dual-signal ratio method or logic gate criterion, and combined with the anti-fouling layer to suppress non-specific adsorption, so as to achieve high specificity and anti-interference detection.
It achieves highly sensitive, interference-resistant, and portable detection of quinolone antibiotics, reducing the risk of false positives and false negatives, providing rapid on-site detection capabilities, and is low in cost, making it suitable for complex water bodies.
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Figure CN121720993A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of environmental analysis and detection technology, specifically relating to a sensing method based on the mechanism of nucleic acid aptamer-specific recognition of quinolone antibiotics, combined with fluorescence and photoelectrochemical dual signal output modes, and a corresponding detection device. Background Technology
[0002] Quinolone antibiotics (QNs) are widely used in human medicine and animal husbandry due to their broad-spectrum antibacterial activity. However, improper discharge of these antibiotics leads to their frequent detection in aquatic environments, posing a significant source of antibiotic resistance (AMR) spread and ecological risks. While existing detection methods (such as high-performance liquid chromatography-mass spectrometry and enzyme-linked immunosorbent assay) are sensitive and accurate, they generally suffer from drawbacks such as expensive equipment, complex operation, the need for specialized personnel, and long analysis cycles, making it difficult to meet the needs of rapid on-site screening.
[0003] In recent years, aptamer-based biosensing technologies have attracted widespread attention due to their high specificity, designability, and stability. Aptamers are single-stranded DNA or RNA molecules obtained through SELEX screening, capable of recognizing targets with high affinity and selectivity. Previous studies have utilized aptamers to construct fluorescent or photoelectrochemical biosensors for QNs detection; however, single-modal signals are susceptible to interference from complex matrices (such as humic acid, metal ions, and particulate matter in wastewater), leading to false positives or false negatives and affecting detection reliability. Dual-modal sensing strategies, by cross-validating the same target through two independent signaling pathways, can significantly improve the reliability and anti-interference capability of detection results. However, there are currently no reports of combining aptamer recognition with fluorescence-photoelectrochemical dual-modality for on-site QNs detection, and integrated, portable dual-modal detection devices are also lacking. Summary of the Invention
[0004] In view of the aforementioned existing problems, the present invention is proposed.
[0005] Therefore, this invention provides a dual-modal detection method for quinolone antibiotics that is highly specific, highly sensitive, and has strong anti-interference ability, and is applicable to complex water bodies. It also develops a portable, easy-to-operate, and field-use detection device to solve the technical bottleneck of existing technologies that are difficult to balance sensitivity, reliability, and field applicability.
[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution:
[0007] In a first aspect, the present invention provides a dual-modal sensing detection method for quinolone antibiotics, the method comprising the following core steps:
[0008] A photoelectric active film is constructed on the surface of a conductive substrate. The photoelectric active material is selected from titanium dioxide (TiO2), graphitic carbon nitride (g-C3N4), cadmium sulfide (CdS) quantum dots, metal-organic frameworks (MOFs), or their binary / ternary composites. A stable photoelectric conversion layer is formed by spin coating, electrodeposition, or self-assembly. Subsequently, quinolone antibiotic-specific nucleic acid aptamers (single-stranded DNA or RNA sequences) are immobilized on the surface of the photoelectric active layer using covalent coupling or physical adsorption methods to form a recognition probe array. The aptamer sequences are specifically optimized for one or more targets of ciprofloxacin, norfloxacin, and ofloxacin. A fluorescent signal probe is further introduced. The probe is a fluorescein-labeled complementary strand, a molecular beacon structure, an aggregation-induced emission (AIE) material, or a fluorescence quenching pair. Its fluorescence output state is dynamically regulated by conformational changes of the aptamer.
[0009] When quinolone antibiotics are present in the water sample, the aptamer specifically binds to the target and triggers a conformational change. This change simultaneously leads to two quantifiable events: (1) the fluorescence intensity of the fluorescent signal probe is significantly quenched due to energy transfer or steric hindrance; (2) the aptamer-target complex hinders charge transport at the photoelectric active interface, causing a decrease in photocurrent signal. By establishing a two-parameter response model of fluorescence signal change (ΔF) and photocurrent change (ΔI) to quinolone antibiotic concentration, and using the two-signal ratio method or logic gate criterion (AND / OR logic) for cross-validation, errors caused by environmental temperature fluctuations, pH changes, interference from dissolved organic matrix, and long-term sensor drift are eliminated, thus achieving accurate quantitative detection of trace quinolone antibiotics in water samples.
[0010] Preferably, the electrode surface is further modified with an anti-nonspecific adsorption coating. The coating is composed of polyethylene glycol (PEG) segments, zwitterionic polymers (such as sulfobetaine methacrylate) or bovine serum albumin (BSA) forming a two-dimensional or three-dimensional antifouling network, which effectively inhibits the nonspecific adsorption of humic acid, protein and suspended particles in complex water bodies at the sensing interface and improves the detection signal-to-noise ratio.
[0011] A second aspect of the present invention provides a dual-modal detection device for implementing the above-described method, the device being a modular integrated design, comprising:
[0012] The photoelectric detection module includes a UV-LED light source with a wavelength of 365 nm and a power of 30-80 mW, used to excite photoactive materials to generate electron-hole pairs; a potentiostat chip (supporting a three-electrode system) drives the sensing chip to complete the photo-assisted electrochemical reaction; and an electrochemical signal acquisition and amplification circuit realizes the precise extraction of picoampere-level photocurrent signals.
[0013] Fluorescence detection module: Includes a blue LED excitation source with a wavelength of 450-480 nm and a power of 5-20 mW, a bandpass filter with a center wavelength of 520 nm and a bandwidth of 20-40 nm, and a silicon-based photodiode array with a response wavelength range of 400-700 nm, which, together with the signal amplification circuit, realizes the quantitative detection of fluorescence intensity.
[0014] Disposable sensor chip slot: It adopts a spring contact design to form a stable electrical contact with the working electrode, counter electrode and reference electrode of the pre-modified adapter sensing electrode, and supports plug-and-play quick replacement.
[0015] Microprocessor control unit: Controls the photoelectric detection module via I²C bus and the fluorescence detection module via SPI bus, realizing synchronous acquisition timing control of dual-channel signals, analog-to-digital conversion, digital filtering, concentration inversion algorithm execution, and dual-modal signal consistency scoring (setting thresholds to automatically trigger repeated detection or alarm).
[0016] Power management module: integrates lithium battery charge and discharge management and multiple regulated outputs to provide low-noise power supply for microprocessors, light sources and signal processing circuits.
[0017] Human-computer interaction module: including LCD display and function buttons, which displays the dual-modal signal curve, concentration detection results and quality control parameters (such as correlation coefficient R² and signal-to-noise ratio SNR) in real time.
[0018] Wireless communication module: Connects to the microprocessor via UART interface, supports Bluetooth 4.0 or WiFi data transmission protocols, and enables remote reporting and analysis of detection data to smart terminals or cloud servers.
[0019] Preferably, the disposable sensor chip is an integrated screen-printed electrode, the structure of which, from top to bottom, consists of: a PET flexible substrate layer; a carbon working electrode, an Ag / AgCl reference electrode, and a carbon counter electrode prepared by screen printing; and a photoactive material layer, an aptamer recognition layer, and an anti-fouling layer sequentially modified on the surface of the working electrode, with the total thickness controlled within the range of 200-500 μm, achieving mass production at low cost and single-use, avoiding cross-contamination.
[0020] Compared with existing technologies, the advantages of this invention are as follows: This invention significantly reduces the risk of false positives / false negatives by using dual-modal signals for mutual verification; the aptamers have nanomolar affinity for QNs, can distinguish structural analogs, and can achieve cross-identification of multiple QNs; the disposable sensor chip using screen printing technology is low-cost, avoids cross-contamination, and ensures detection accuracy; the built-in consistency scoring algorithm can quantitatively evaluate the reliability of detection results; and the modules are connected through a standardized bus interface, facilitating maintenance and upgrades. Attached Figure Description
[0021] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0022] Figure 1 Schematic diagram of dual-modal sensing principle
[0023] Figure 2 Overall structural diagram of portable dual-modal detection device
[0024] Includes: device housing (1), sensor chip slot (2), LCD display (3), function buttons (4), USB charging interface (5), and power switch (6).
[0025] Figure 3 Internal module connection diagram of the device
[0026] It includes: a microprocessor control unit (10), a photoelectric detection module (11), a fluorescence detection module (12), a sensor chip interface (13), a power management module (14), and a wireless communication module (15).
[0027] The photoelectric detection module (11) includes a UV-LED light source (11a), an electrochemical signal acquisition circuit (11b), and a potentiostat chip (11c).
[0028] The fluorescence detection module (12) includes an excitation light source (12a), a bandpass filter (12b), a photodiode array (12c), and a signal amplification circuit (12d).
[0029] Figure 4 : Detection process flowchart Detailed Implementation
[0030] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the protection scope of the present invention.
[0031] Example 1: Detection of Ciprofloxacin (CIP)
[0032] 1. Sensing interface construction: (1) Take the screen-printed ITO electrode, ultrasonically clean it with ethanol and deionized water for 5 min each, and blow it dry with nitrogen; (2) Take 5 μL of g-C3N4 nanosheet dispersion (2 mg / mL, solvent is DMF) and drop it onto the surface of the working electrode, dry it at 110℃ for 30 min to form a uniform photoactive layer; (3) Immerse the electrode in MES buffer (pH 6.0) containing EDC (50 mM) and NHS (50 mM) for 30 min to activate it; (4) Add 5′-amino modified CIP aptamer solution (sequence: 5′-NH2-CCGGGTTGGTGTGGTTGG-3′, concentration 1 μM), and incubate at 37℃ for 2 h; (5) Rinse three times with PBS buffer to remove unbound aptamers; (6) Add 1% BSA solution to block for 30 min, and then treat with 0.1 mM PEG-SH solution for 1 minute. h, forming an anti-fouling coating; (7) after rinsing with PBS, store at 4°C for later use.
[0033] 2. Dual-modal detection: (1) Take a domestic sewage sample and filter it through a 0.22 μm filter membrane to remove suspended particles; (2) Take 10 μL of the filtered sample and add 40 μL of PBS buffer (pH 7.4) containing 100 nM FAM-labeled complementary chain (sequence: 5′-FAM-CCAACCACACCAACCCGG-3′) and mix well; (3) Inject the mixture into the reaction chamber of the sensor chip and incubate at 37℃ for 10 min; (4) Insert the sensor chip into the portable detection device; (5) Fluorescence detection: The device automatically turns on the 470 nm blue LED excitation (power 10 mW), and after passing through a 520 nm bandpass filter, the photodiode collects the fluorescence intensity, with an integration time of 1 s; (6) Photoelectrochemical detection: UV-LED (365 nm, power 50 mW) irradiates the electrode surface, and a constant potential of +0.2 V (vs. Ag / AgCl) is applied to record the steady-state photocurrent value.
[0034] 3. Detection performance: (1) In the CIP concentration range of 0-100 ng / L, the fluorescence signal change value ΔF = F0 - F has a good linear relationship with the CIP concentration: ΔF = 12.5×[CIP] + 3.2 (R² = 0.996); (2) The photocurrent change value ΔI = I0 - I also has a linear relationship with the CIP concentration: ΔI = 0.85×[CIP] + 0.12 (R² = 0.993); (3) Detection limit (LOD, S / N=3): 0.3 ng / L for fluorescence channel and 0.5 ng / L for photoelectrochemical channel; (4) Relative standard deviation (RSD, n=6): 3.2% for fluorescence channel and 4.1% for photoelectrochemical channel.
[0035] 4. Actual sample verification: Water samples were collected from the effluent of a domestic wastewater treatment plant. The CIP concentration was measured to be 8.2 ng / L, and the HPLC-MS / MS verification result was 8.5 ng / L, with a relative error of 3.5%; the dual-modal signal consistency score was 96.2%.
[0036] Example 2: Norfloxacin (NOR) Detection
[0037] 1. Sensing interface construction: The same substrate preparation and photoelectric active layer modification methods as in Example 1 were used, but the aptamer was replaced with a NOR-specific aptamer (sequence: 5′-NH2-TGGGGGTTGAGGCTAAGCCGA-3′, concentration 1 μM), and the other steps were the same.
[0038] 2. Dual-modal detection: The operation steps are the same as in Example 1, except that the fluorescent complementary strand sequence is replaced with 5′-FAM-TCGGCTTAGCCTCAACCCCA-3′.
[0039] 3. Detection performance: (1) Good linearity in the NOR concentration range of 0-150 ng / L: ΔF = 10.8×[NOR] +2.8 (R² = 0.994), ΔI = 0.72×[NOR] + 0.09 (R² = 0.991); (2) Detection limit: 0.5 ng / L for fluorescence channel and 0.8 ng / L for photoelectrochemical channel; (3) Relative standard deviation (RSD, n=6): 3.8% for fluorescence channel and 4.5% for photoelectrochemical channel.
[0040] 4. Actual sample verification: Wastewater samples were collected from a farm. The NOR concentration detected by this device was 25.6 ng / L, and the HPLC-MS / MS verification result was 26.8 ng / L, with a relative error of 4.5%; the consistency score of the dual-modal signal was 94.8%.
[0041] Example 3: Detection of ofloxacin (OFL)
[0042] 1. Sensing interface construction: The same substrate preparation method as in Example 1 was used, except that the photoactive material was replaced with a CdS quantum dot / g-C3N4 composite material (mass ratio 1:5), and the aptamer was an OFL-specific aptamer (sequence: 5′-NH2-GGAAGTGGTGGGGAGGATA-3′, concentration 1 μM). The remaining steps were the same.
[0043] 2. Detection performance: (1) Good linearity in the range of OFL concentration from 0 to 120 ng / L: ΔF = 11.2×[OFL] +3.0 (R² = 0.995), ΔI = 0.92×[OFL] + 0.15 (R² = 0.992); (2) Detection limit: 0.4 ng / L for fluorescence channel and 0.6 ng / L for photoelectrochemical channel.
[0044] 3. Actual sample verification: Wastewater samples were collected from a hospital. The OFL concentration detected by this device was 42.3 ng / L, and the HPLC-MS / MS verification result was 44.1 ng / L, with a relative error of 4.1%; the consistency score of the dual-modal signal was 95.5%.
[0045] Example 4: Anti-interference performance test
[0046] 1. Test objective: To verify the detection reliability of the dual-modal sensor under interference from complex aquatic matrix.
[0047] 2. Interference Preparation: The following common interfering substances were added to a standard solution containing 50 ng / L CIP: humic acid: 10 mg / L; Fe³⁺: 1 mg / L; Cu²⁺: 0.5 mg / L; Pb²⁺: 0.1 mg / L; tetracycline (a structurally similar antibiotic): 100 ng / L; amoxicillin (a different class of antibiotic): 100 ng / L; Na⁺, K⁺, Ca²⁺, Mg²⁺ (common ions): 10 mg / L each.
[0048] 3. Test Results
[0049] Interference concentration Fluorescence signal deviation Photocurrent signal deviation Bias after dual-modal correction Blank control - 0% 0% 0% humic acid 10 mg / L -8.5% -3.2% -1.8% Fe³⁺ 1 mg / L -5.2% -6.8% -2.1% Cu²⁺ 0.5 mg / L -4.1% -5.5% -1.5% Pb²⁺ 0.1 mg / L -2.3% -3.1% -0.8% tetracycline 100 ng / L +3.5% +2.8% +1.2% Amoxicillin 100 ng / L +1.2% +0.8% +0.5% Mixed ions 10 mg / L each -2.8% -2.1% -0.9%
[0050] 4. Conclusions: (1) Single-mode detection has a signal deviation of 5%-10% in complex matrices; (2) By correcting the dual-mode signal ratio (ΔF / ΔI internal standard method), the detection deviation can be reduced to less than 2%; (3) The sensor has good selectivity for structural analogs (tetracycline) and other antibiotics (amoxicillin), with a cross-reactivity rate of <5%.
[0051] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.
Claims
1. A dual-modal sensing detection method for quinolone antibiotics, characterized in that: The method includes immobilizing quinolone antibiotic-specific nucleic acid aptamers on the surface of an electrode modified with photoactive materials and introducing fluorescent signal probes. When quinolone antibiotics are present in the water sample to be tested, the aptamers specifically bind to them and undergo conformational changes, simultaneously causing fluorescence signal quenching and photocurrent signal decrease. Quantitative detection of quinolone antibiotics is achieved through the combined output of dual-modal signals.
2. The dual-modal sensing and detection method as described in claim 1, characterized in that: The quinolone antibiotics include one or more of ciprofloxacin, norfloxacin, and ofloxacin; the nucleic acid aptamer is a single-stranded DNA or RNA sequence capable of specifically recognizing the quinolone antibiotics.
3. The dual-modal sensing and detection method as described in claim 1, characterized in that: The photoelectric active material is selected from titanium dioxide (TiO2), graphitic carbon nitride (g-C3N4), cadmium sulfide (CdS) quantum dots, metal-organic frameworks (MOFs) or their composites; the electrode is conductive glass, gold electrode or screen-printed electrode.
4. The dual-modal sensing and detection method as described in claim 1, characterized in that: The fluorescent signal probes include fluorescein-labeled complementary strands, molecular beacons, aggregation-induced emission (AIE) materials, or fluorescence quenching pairs, and their fluorescence output state is regulated by aptamer conformational changes.
5. The dual-modal sensing and detection method as described in claim 1, characterized in that: The electrode surface is also modified with an antifouling coating, which is composed of polyethylene glycol (PEG), zwitterionic polymers or bovine serum albumin (BSA), and is used to suppress the interference of non-specific adsorption in complex water bodies on the detection signal.
6. The dual-modal sensing and detection method as described in claim 1, characterized in that: The quantitative detection includes establishing the response relationship between changes in fluorescence signal and changes in photocurrent to the concentration of quinolone antibiotics, and performing cross-validation through dual-signal ratio or logic gate criteria to eliminate errors caused by environmental fluctuations, matrix interference, or sensor drift.
7. A dual-modal detection device for implementing the method according to any one of claims 1-6, characterized in that, include: The photoelectric detection module includes a UV-LED light source, a potentiostat chip, and an electrochemical signal acquisition and amplification circuit. The fluorescence detection module includes a blue LED excitation source, a bandpass filter, a photodiode array, and a signal amplification circuit. The disposable sensor chip slot is equipped with spring contacts for forming electrical contact with the working electrode, counter electrode, and reference electrode of the pre-modified adapter's sensing electrode; The microprocessor control unit is electrically connected to the photoelectric detection module via an I²C bus and to the fluorescence detection module via an SPI bus. It is used to synchronously control the timing of signal acquisition, process dual-channel signal data, and calculate the target concentration and the consistency score of the dual-modal signal. The power management module is electrically connected to the microprocessor control unit, the photoelectric detection module, and the fluorescence detection module, and is used to provide regulated power supply. The human-computer interaction module includes an LCD display and function buttons for displaying test results; The wireless communication module is connected to the microprocessor control unit via a UART interface and supports Bluetooth or WiFi data transmission for data interaction with external devices.
8. The dual-modal detection device as described in claim 7, characterized in that: The UV-LED light source has a wavelength of 365 nm and a power of 30-80 mW, and is used to excite photoactive materials to generate photocurrent; the blue LED excitation light source has a wavelength of 450-480 nm and a power of 5-20 mW, and is used to excite fluorescent probes to generate fluorescence signals; the bandpass filter has a center wavelength of 520 nm and a bandwidth of 20-40 nm; the photodiode array is a silicon-based photodiode with a response wavelength range of 400-700 nm.
9. The dual-modal detection device as described in claim 7, characterized in that: The disposable sensing chip is an integrated screen-printed electrode, comprising: PET flexible substrate; A carbon working electrode, an Ag / AgCl reference electrode, and a carbon counter electrode are screen-printed on the substrate layer; A photoactive material layer, an aptamer recognition layer, and an anti-fouling layer are sequentially applied to the surface of the working electrode. The PDMS microreactor covering the electrode area is equipped with an inlet and an exhaust port; The chip has a standardized interface that matches the position of the spring contacts in the device slot.
10. The dual-modal detection device as described in claim 7, characterized in that: The microprocessor control unit incorporates a dual-modal signal processing algorithm, including: The signal preprocessing module is used to filter and perform baseline correction on fluorescence and photocurrent signals; The concentration calculation module calculates the fluorescence channel concentration value C_F and the photoelectrochemical channel concentration value C_PEC based on the pre-stored standard curves. The consistency scoring module calculates the consistency score of the dual-mode signal according to the formula S = [1 - C_F - C_PEC / ((C_F + C_PEC) / 2)] × 100%; The result determination module outputs the average concentration value when the consistency score S≥90%, and prompts for retesting or sample abnormality when S<90%.