Rapid detection reagent in chemiluminescence mode and preparation method thereof

Abstract

The invention relates to the technical field of chemiluminiscence detection, in particular to a rapid detection reagent in a chemiluminiscence mode and a preparation method of the rapid detection reagent, and the rapid detection reagent is characterized in that a core component of the rapid detection reagent is a nitroreductase-responsive flash type end product 5 as shown in a formula I, and the concentration is 0.1 mu M; the auxiliary reagent comprises reduced nicotinamide adenosine dinucleotide (NADH) and a Tris-HCl buffer solution (pH is 7.4), and the concentration of the reduced nicotinamide adenosine dinucleotide is 5 mu M. The reagent is used for detecting nitroreductase (NTR), a high-intensity chemiluminescence signal can be generated within 1 minute after the reagent acts with the NTR at 37 DEG C, the signal duration is larger than or equal to 40 minutes, and the NTR detection limit reaches 0.951 ng / mL. The problems that an existing flash type probe is complex in synthesis process, short in signal duration and insufficient in low-concentration NTR detection capacity are solved.

Description

Technical Field

[0001] This invention relates to the field of chemiluminescence detection technology, specifically to a rapid detection reagent using a chemiluminescence mode and its preparation method. Background Technology

[0002] Pathogen detection is crucial in public health, clinical medicine, and food safety. Pathogens such as *Escherichia coli* and *Staphylococcus aureus* are core factors causing human and animal diseases, and rapid and accurate detection technologies directly impact disease control effectiveness. Current mainstream detection technologies have significant limitations: traditional bacterial culture methods, while highly specific, are time-consuming, taking days to weeks and delaying emergency treatment; molecular biology techniques (such as PCR), while shortening the detection cycle, are hampered by expensive equipment, complex operation, inability to distinguish between live and dead bacteria, and susceptibility to sample inhibitors leading to false negatives, hindering widespread application. Chemiluminescence detection technology, with its advantages of low background noise, no need for an excitation light source, and simple equipment, has become an emerging solution, but it still generally faces bottlenecks such as response delay (>1 minute) and low signal-to-noise ratio in complex environments, making it difficult to meet the clinical needs for real-time pathogen detection and in vivo imaging.

[0003] Of particular note is the fact that flash chemiluminescence technology based on pathogen-specific enzymes (such as nitroreductase NTR) can theoretically achieve rapid detection by triggering the substrate to release a transient, intense light signal. However, existing flash probes suffer from fundamental drawbacks such as complex synthesis routes, low yields, and short signal durations (typically <10 minutes), making it impossible to capture effective signals in in vivo imaging and even more difficult to achieve accurate detection of low-concentration infections (such as NTR <1 ng / mL).

[0004] These technological shortcomings severely restrict the application of flash chemiluminescence in the point-of-care diagnosis of pathogens. Therefore, it is urgent to develop a rapid detection reagent with a chemiluminescence mode and its preparation method to solve the above problems. Summary of the Invention

[0005] The purpose of this invention is to provide a rapid detection reagent in chemiluminescence mode and its preparation method, which has the advantages of fast response speed, high detection sensitivity and excellent in vivo imaging effect, and effectively solves the problems of complex synthesis process, short signal duration and insufficient detection capability of low concentration NTR of existing flash probes.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a rapid detection reagent in chemiluminescence mode, comprising: a core component being the final product 5 shown in Formula I; auxiliary reagents including reduced nicotinamide adenosine dinucleotide and buffer solution; the reagent is used for the detection of nitroreductase.

[0007] Preferably, the concentration of the final product 5 is 0.1 μM.

[0008] When the above technical solution is adopted, the final product Formula 5I achieves high sensitivity detection of nitroreductase at a concentration of 0.1 μM and has the ability to trigger high-intensity chemiluminescence signals in low-concentration environments.

[0009] Preferably, the concentration of the reduced nicotinamide adenosine dinucleotide is 5 μM.

[0010] When the above technical solution is adopted, the reduced nicotinamide adenosine dinucleotide achieves efficient catalysis of the final product 5-form I luminescence reaction at a concentration of 5 μM, which has the advantages of optimizing reaction kinetics and reducing background interference.

[0011] Preferably, the buffer solution is a Tris-HCl buffer solution with a pH of 7.4.

[0012] When the above technical solution is adopted, the Tris-HCl buffer solution achieves accurate simulation of the physiological environment under pH 7.4 conditions and has compatibility to maintain the stable progress of the chemiluminescence reaction.

[0013] Preferably, the auxiliary reagent generates a chemiluminescent signal within 1 minute after reacting with nitroreductase at 37°C.

[0014] When the above technical solution is adopted, the auxiliary reagent achieves an immediate response to nitroreductase at 37°C, which meets the timeliness requirements for rapid diagnosis of pathogens.

[0015] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing a rapid detection reagent in a chemiluminescence mode, comprising the following steps: Step 1: Synthesis of intermediate compound 1: Phospholipid acetal II was added to dry tetrahydrofuran, stirred at -78°C under argon protection, and then diisopropylaminolithium was added dropwise. Cyclobutanone was added dropwise, and the reaction was carried out at elevated temperature. The mixture was purified to obtain intermediate compound 1 III. Step 2: Synthesis of intermediate compound 2: Intermediate compound 1 (Formula III) was dissolved in tetrahydrofuran, and tetrabutylammonium fluoride aqueous solution was added. After the reaction, the mixture was purified to obtain intermediate compound 2 (Formula IV). Step 3: Synthesis of intermediate compound 3: Intermediate compound 2 (Formula IV) was dissolved in N,N-dimethylformamide, anhydrous potassium carbonate and 4-nitrobenzyl bromide were added, and after reaction and purification, intermediate compound 3 (Formula V) was obtained; Step 4: Synthesis of intermediate compound 4: Intermediate compound 3 (Formula V) was dissolved in dichloromethane, methylene blue was added, and the mixture was irradiated with yellow light for 5 hours with oxygen to obtain intermediate compound 4 (Formula VI). Step 5: Synthesis of final product 5: Dissolve intermediate compound 4 (Formula VI) in a methanol / water mixed solvent, add lithium hydroxide, adjust the pH to precipitate, and dry to obtain final product 5 (Formula I).

[0016] Preferably, the molar ratio of phospholipid acetal II to lithium diisopropylamino in step one is 1:1.5.

[0017] When the above technical solution is adopted, phospholipid acetal II and diisopropylaminolithium are subjected to a highly efficient cyclization reaction at a molar ratio of 1:1.5, which has the synthetic efficiency to increase the yield of intermediate compound I, III to 82%.

[0018] Preferably, the molar ratio of the intermediate compound Formula III to tetrabutylammonium fluoride in step two is 1:1.2.

[0019] When the above technical solution is adopted, the intermediate compound 1 (Formula III) and tetrabutylammonium fluoride achieve a highly selective deprotection reaction at a molar ratio of 1:1.2, and the process stability is achieved to obtain intermediate compound 2 (Formula IV) in 88% yield.

[0020] Preferably, in step three, the molar ratio of intermediate compound IV of formula 2 to anhydrous potassium carbonate is 1:1.5, and the molar ratio of intermediate compound IV of formula 2 to 4-nitrobenzyl bromide is 1:1.2.

[0021] When using the above technical solution, intermediate compound 2 (Formula IV) and anhydrous potassium carbonate and 4-nitrobenzyl bromide achieve a precise condensation reaction under dual parameter control, and have the repeatability to obtain high-purity intermediate compound 3 (Formula V) with a yield of 78%.

[0022] Preferably, the molar ratio of intermediate compound V3 to methylene blue in step four is 1:0.1.

[0023] When the above technical solution is adopted, the intermediate compound 3 V and methylene blue achieve a highly efficient photo-oxidation reaction at a molar ratio of 1:0.1, which has the energy-saving advantage of obtaining the intermediate compound 4 VI with a yield of 67% within 5 hours.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention achieves a high-intensity chemiluminescent response to nitroreductase NTR within 1 minute at 37°C by setting the final product 5 shown in Formula I at a concentration of 0.1 μM in combination with 5 μM of reduced nicotinamide adenosine dinucleotide (NADH) and a Tris-HCl buffer at pH 7.4. Figure 2 As shown, this solves the problem that the response time of existing flash probes exceeds 1 minute, meeting the needs of real-time detection of pathogens.

[0025] This invention achieves highly sensitive detection of nitroreductase NTR by optimizing the reaction system of final product 5 shown in Formula I, with a detection limit of 0.951 ng / mL and a linear relationship within the concentration range of 3-55 ng / mL. Figure 3As shown, this breakthrough overcomes the bottleneck of existing technologies' insufficient ability to detect low-concentration nitroreductase NTR, and can accurately indicate trace pathogen infections.

[0026] This invention was verified through in vivo experiments. A mixed solution containing the final product 5 shown in Formula I, nitroreductase NTR at a concentration of 300 ng / mL, and NADH at a concentration of 50 μM was subcutaneously injected into the back of a mouse. Significant luminescence signals were observed after one minute of exposure. Figure 4 As shown; oral administration of the final product 5 of Formula I to mice pre-orally administered E. coli bacterial solution produced dose-dependent luminescent signals, such as... Figure 5 As shown, it demonstrates that it has excellent imaging capabilities against Escherichia coli expressing nitroreductase NTR in complex biological environments, solving the problem of low signal-to-noise ratio in in vivo imaging of existing technologies.

[0027] This invention significantly improves synthesis efficiency by optimizing key ratios in the preparation method: the molar ratio of phospholipid acetal (Formula II) to lithium diisopropylaminodimethylamine (LDA) is 1:1.5; the molar ratio of intermediate compound 1 (Formula III) to tetrabutylammonium fluoride is 1:1.2; the molar ratio of intermediate compound 2 (Formula IV) to anhydrous potassium carbonate and to 4-nitrobenzyl bromide is 1:1.2; and the molar ratio of intermediate compound 3 (Formula V) to methylene blue is 1:0.1. The yield of final product 5 reaches 62%, and the yield of intermediates reaches 67% to 88%, solving the problems of complex and low yield in existing flash probe synthesis processes.

[0028] This invention achieves a chemiluminescence duration of ≥40 minutes through the signal persistence mechanism of the final product 5 shown in Formula I, such as... Figure 2 As shown in the attenuation curve, it far exceeds the limitation of existing technologies where the signal duration is less than 10 minutes, providing a sufficient signal acquisition window for live imaging. Attached Figure Description

[0029] Figure 1 This is a synthetic route diagram for the final product 5 of the present invention; Figure 2 Chemiluminescence kinetics curves of the end product 5 of this invention for NTR and NADH; Figure 3 The photon emission integral diagram of the final product of this invention for different concentrations of NTR is shown. Figure 4 This is an in vivo imaging image of mice after subcutaneous injection of the final products 5, NTR, and NADH according to the present invention. Figure 5 This is an in vivo imaging image of mice treated with end product 5 after being administered Escherichia coli via gavage according to the present invention. Detailed Implementation

[0030] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] Example 1

[0032] To achieve rapid and highly sensitive detection of pathogens, such as Figures 2 to 5 As shown, one embodiment of the present invention is a rapid detection reagent in a chemiluminescence mode, the core component of which is the final product 5 shown in Formula I, namely a flash-type chemiluminescent molecule responsive to nitroreductase, with a concentration of 0.1 μM. Auxiliary reagents include: Nitroreductase (NTR): The concentration range is 3-55 ng / mL. It is derived from pathogens that express NTR, such as Escherichia coli. The crude enzyme solution can be extracted by ultrasonic disruption of pathogens (300W power, 3 seconds on, 5 seconds off, 10 minutes in total) followed by centrifugation (12000 rpm, 4℃, 10 minutes). Reduced nicotinamide adenosine dinucleotide (NADH): at a concentration of 5 μM, it participates in the NTR-catalyzed reduction reaction as an electron donor and must be prepared and used immediately and stored away from light. Tris-HCl buffer: pH 7.4, containing 100mM sodium chloride, used to simulate the ionic strength of the physiological environment, maintain NTR activity and the stability of end product 5; The reagent reacts with NTR in a 37°C constant temperature water bath to generate a high-intensity chemiluminescent signal within 1 minute, and its kinetic characteristics are as follows: Figure 2 As shown, the relative emission unit (RLU) at 1 minute is more than 50 times that of the blank group, and the signal peak value is approximately 6 × 10⁻¹ at 10 minutes. RLU, signal duration ≥40 min; detection limit for NTR is 0.951 ng / mL, linear relationship as follows: Figure 3 As shown, the linear equation is Y=3.54604E6×X-1.89652E7, R²=0.9909, which can accurately quantify the expression level of NTR in pathogens.

[0033] This reagent enables in vivo imaging: Subcutaneous injection of 100 μL of a mixed solution [containing 51 μM of the final product shown in Formula I, 300 ng / mL of NTR, and 50 μM of NADH] into the back of anesthetized SCID / BALB-C mice revealed a significant luminescent signal with an intensity of 1.5 × 10⁻⁶ ng / mL when exposed to an IVIS imaging system for 1 minute. Photons / s / cm² / sr, control group without NTR or NADH showed no significant signal, such as Figure 4 As shown; Administer by gavage 51 μM of the final product shown in Formula I, 1 mL, to a pre-gavage Escherichia coli culture (OD200). 600 Mice treated with 2.5 μL of bacterial solution (diluted a thousandfold with physiological saline) were given luminescent signals via gavage in groups of 1 mL, 2 mL, and 4 mL. The signal intensity increased with increasing bacterial solution volume, with the 4 mL group showing a signal 2.3 times that of the 1 mL group. The control group, which did not receive any E. coli via gavage, showed no signal. Figure 5 This demonstrates its ability to specifically image Escherichia coli expressing NTR in complex biological environments.

[0034] Example 2

[0035] To achieve controllable preparation of the final product 5 shown in Equation I, such as Figure 1 As shown in this embodiment, a method for preparing a rapid detection reagent using a chemiluminescence mode is provided, specifically including the following steps: Synthesis of intermediate compound 1 (Formula III): 492 mg (1.0 mmol) of phospholipid acetal (Formula II), with a purity ≥98%, was added to 50 mL of dry tetrahydrofuran (removed by sodium wire reflux and used immediately after distillation). The mixture was stirred for 15 min in a dry ice-acetone bath at -78 °C under argon protection with a purity of 99.999%. 2.0 mL (1.5 mmol) of diisopropylaminolithium (0.7 mL) of tetrahydrofuran was slowly added dropwise over a period of 5 min. The reaction was maintained at -78 °C for 20 min, resulting in a pale yellow clear liquid. Subsequently, 100 μL (95 mg) of cyclobutanone (1.5 mmol) was added dropwise. The mixture was purified by vacuum distillation. After stirring for another 30 min, the low-temperature bath was removed, and the reaction system was allowed to naturally warm to room temperature (25 °C) and stirred for another 30 min. TLC monitoring on a silica gel plate (GF254) with a developing solvent of petroleum ether and ethyl acetate in a 3:1 volume ratio showed that the starting spot (Rf=0.8) completely disappeared, and a new spot (Rf=0.5) formed. The reaction was quenched by adding 5 mL of saturated ammonium chloride aqueous solution, followed by extraction with ethyl acetate (3 × 20 mL). The combined organic phases were dried over 5 g of anhydrous sodium sulfate for 2 h, filtered, and concentrated under reduced pressure at 40 °C and -0.09 MPa to obtain a pale yellow, oily crude product. The crude product was purified by silica gel column chromatography on 200-300 mesh silica gel (30 cm long, 2 cm inner diameter) using a 5:1 volume ratio of petroleum ether and ethyl acetate as eluent. The fraction with Rf=0.5 was collected and concentrated to give intermediate compound III (Formula 1), a white solid with a yield of 82% and a melting point of 56-58 °C.

[0036] Synthesis of intermediate compound II (Formula IV): 436 mg (1.0 mmol) of intermediate compound III (Formula III) was dissolved in 30 mL of tetrahydrofuran and stirred at 25 °C until completely dissolved. 1.2 mL (1.2 mmol) of 1.0 M tetrabutylammonium fluoride aqueous solution was added, and stirring continued at room temperature for 15 min. TLC monitoring with a 2:1 volume ratio of petroleum ether and ethyl acetate as the developing solvent showed that the starting point Rf=0.6 completely disappeared, and a new point Rf=0.3 was formed. The mixture was diluted with 20 mL of ethyl acetate, washed with 1 × 15 mL of 1.0 M hydrochloric acid solution to remove excess fluoride ions, and then washed with 2 × 15 mL of saturated brine until neutral pH=7. The organic phase was dried over 5 g of anhydrous sodium sulfate for 2 h, filtered, and concentrated under reduced pressure to obtain a colorless oily crude product. The crude product was purified by silica gel column chromatography on 200-300 mesh silica gel, using a mixture of petroleum ether and ethyl acetate in a 4:1 volume ratio as eluent. The fraction with Rf = 0.3 was collected and concentrated to give intermediate compound 2 (formula IV), a colorless liquid with a yield of 88% and a refractive index n. 20 D =1.4520.

[0037] Synthesis of intermediate compound 3, formula V: 322 mg (1.0 mmol) of intermediate compound 2, formula IV was dissolved in 8 mL of N,N-dimethylformamide (dried by a 4 Å molecular sieve). 195 mg (1.5 mmol) of anhydrous potassium carbonate was added, and the mixture was dried at 120 °C for 4 h. After stirring at room temperature for 10 min, the system formed a white suspension. 250 mg (1.2 mmol) of 4-nitrobenzyl bromide was added, and the mixture was reacted overnight at room temperature for 12 h. TLC monitoring with a 3:1 volume ratio of petroleum ether and ethyl acetate as the developing solvent showed that the starting spot at Rf=0.4 completely disappeared, and a new spot at Rf=0.6 was formed. The reaction solution was diluted with 30 mL of water, extracted with ethyl acetate (3 × 20 mL), and the organic phases were combined. The mixture was dried over 5 g of anhydrous sodium sulfate for 2 h, filtered, and concentrated under reduced pressure at 50 °C and -0.09 MPa to obtain a pale yellow oily crude product. The crude product was purified by silica gel column chromatography on 200-300 mesh silica gel, with petroleum ether and ethyl acetate mixed in a volume ratio of 3:1 as eluent. The fraction with Rf=0.6 was collected and concentrated to give intermediate compound 3, formula V, as a pale yellow solid with a yield of 78% and a melting point of 72-74℃.

[0038] Synthesis of intermediate compound 4 (Formula VI): 457 mg (1.0 mmol) of intermediate compound 3 (Formula V) was dissolved in 20 mL of dichloromethane (removed by reflux with calcium hydride and used immediately after distillation). 32 mg (0.1 mmol) of methylene blue was added. Oxygen was bubbled through the solution at a flow rate of 10 mL / min to maintain continuous bubbling. Simultaneously, the solution was irradiated for 5 h with a 5 W yellow LED lamp at a wavelength of 570-590 nm, 10 cm above the liquid surface. TLC monitoring showed that the starting material spot (Rf=0.7) completely disappeared, and a new spot (Rf=0.4) was formed. The reaction solution was diluted with 20 mL of water and extracted with ethyl acetate (3 × 20 mL). The combined organic phases were dried over 5 g of anhydrous sodium sulfate for 2 h, filtered, and concentrated under reduced pressure to obtain a yellow oily crude product. The crude product was purified by silica gel column chromatography on 200-300 mesh silica gel, with petroleum ether and ethyl acetate mixed in a volume ratio of 2:1 as eluent. The fraction with Rf=0.4 was collected and concentrated to give intermediate compound 4, formula VI, as a yellow solid with a yield of 67% and a melting point of 98-100℃.

[0039] Synthesis of final product Formula 5I: 489 mg (1.0 mmol) of intermediate compound Formula 4VI was dissolved in 20 mL of a 1:1 mixture of methanol and water. 200 mg (5 mmol) of lithium hydroxide was added, and the mixture was stirred overnight at room temperature for 12 h. TLC monitoring with a 10:1 mixture of dichloromethane and methanol as the developing solvent showed that the starting point Rf=0.5 completely disappeared, and a new point Rf=0.2 was formed. The pH of the system was adjusted to 2-3 with 1.0 M hydrochloric acid, and after standing for 30 min, a pale yellow precipitate formed. The precipitate was analyzed using a Buchner funnel. The sample was filtered through a 5 cm filter, and the precipitate was washed with 3 × 5 mL of ice water. It was then vacuum dried at 40 °C and -0.09 MPa for 2 h to obtain the final product 5 shown in Formula I, which is a pale yellow solid with a yield of 62% and a melting point of 168-170 °C.

[0040] Its key parameters are: The molar ratio of phospholipid acetal to lithium diisopropylamino, as shown in Formula II, is 1:1.5. The molar ratio of intermediate compound I (formula III) to tetrabutylammonium fluoride is 1:1.2. The molar ratio of intermediate compound IV (formula 2) to anhydrous potassium carbonate is 1:1.5, and the molar ratio of intermediate compound IV (formula 2) to 4-nitrobenzyl bromide is 1:1.2. The molar ratio of intermediate compound V (formula 3) to methylene blue is 1:0.1. When using this invention, the preparation of all reagents should be completed first. The flash-type final product 5 of the nitroreductase response shown in Formula I is dissolved in Tris-HCl buffer (pH 7.4) to prepare a 0.1 μM stock solution, ensuring it is fully dissolved in the buffer to guarantee the homogeneity of subsequent reactions. Simultaneously, a 5 μM solution of reduced nicotinamide adenosine dinucleotide (NADH) is prepared using the same Tris-HCl buffer (pH 7.4). NADH, as a key coenzyme in the reaction, must be prepared fresh to avoid oxidative degradation. For the nitroreductase (NTR) to be detected, if it originates from pathogens expressing NTR (such as Escherichia coli), the pathogens should be disrupted by ultrasonic treatment (300W, 3s on, 5s off, 10min total), followed by centrifugation (12000rpm, 4℃, 10min) to extract the crude enzyme solution. This solution is then diluted with the aforementioned buffer to a series of concentrations of 3–55 ng / mL to cover the possible detection range.

[0041] After preparing the reagents, the reaction system can be constructed. Place 90 μL of Tris-HCl buffer (pH 7.4) in a detection tube as the base system for the reaction. Add 5 μL of 0.1 μM of the flash-type final product 5 solution of nitroreductase (Formula I) and 5 μL of 5 μM NADH solution sequentially. Gently mix to ensure thorough contact of all components. Then, preheat the mixture in a 37°C water bath for 5 minutes. This step aims to bring the system temperature to the optimal temperature for the enzymatic reaction, ensuring that nitroreductase can exert its catalytic effect under optimal conditions.

[0042] After the reaction system is preheated, signal detection can be performed. Add 10 μL of nitroreductase (NTR) solution of different concentrations to the preheated system. The reaction starts instantly upon addition, and the detection tube must be immediately placed in a chemiluminescence detector (such as an IVIS system) to quickly begin signal recording. Recording includes the initial luminescence signal generated within 1 minute, which reflects the rapid response of the reaction, as well as signal changes over the subsequent 40 minutes. Continuous monitoring allows for understanding the kinetic characteristics and duration of the signal, providing complete data support for accurately determining the expression level of NTR in pathogens. The entire process must be strictly performed according to the operating sequence to ensure the accuracy and repeatability of the detection results.

[0043] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered illustrative and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the scope of the invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0044] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A rapid detection reagent using a chemiluminescence mode, characterized in that: The core component is the final product 5 shown in Formula I; the auxiliary reagents include reduced nicotinamide adenosine dinucleotide and buffer solution; the reagents are used for the detection of nitroreductase.

2. The rapid detection reagent using a chemiluminescence mode according to claim 1, characterized in that, The concentration of the final product 5 is 0.1 μM.

3. The rapid detection reagent based on a chemiluminescence mode according to claim 1, characterized in that, The concentration of the reduced nicotinamide adenosine dinucleotide was 5 μM.

4. The rapid detection reagent using a chemiluminescence mode according to claim 1, characterized in that, The buffer solution is a Tris-HCl buffer with a pH of 7.

4.

5. The rapid detection reagent using a chemiluminescence mode according to claim 1, characterized in that, The auxiliary reagent generates a chemiluminescent signal within 1 minute after reacting with nitroreductase at 37°C.

6. A method for preparing a rapid detection reagent using a chemiluminescence mode, characterized in that: The preparation method is used to prepare the reagent according to any one of claims 1-5, and includes the following steps: Step 1: Synthesis of intermediate compound 1: Phospholipid acetal II was added to dry tetrahydrofuran, stirred at -78°C under argon protection, and then diisopropylaminolithium was added dropwise. Cyclobutanone was added dropwise, and the reaction was carried out at elevated temperature. The mixture was purified to obtain intermediate compound 1 III. Step 2: Synthesis of intermediate compound 2: Intermediate compound 1 (Formula III) was dissolved in tetrahydrofuran, and tetrabutylammonium fluoride aqueous solution was added. After the reaction, the mixture was purified to obtain intermediate compound 2 (Formula IV). Step 3: Synthesis of intermediate compound 3: Intermediate compound 2 (Formula IV) was dissolved in N,N-dimethylformamide, anhydrous potassium carbonate and 4-nitrobenzyl bromide were added, and after reaction and purification, intermediate compound 3 (Formula V) was obtained; Step 4: Synthesis of intermediate compound 4: Intermediate compound 3 (Formula V) was dissolved in dichloromethane, methylene blue was added, and the mixture was irradiated with yellow light for 5 hours with oxygen to obtain intermediate compound 4 (Formula VI). Step 5: Synthesis of final product 5: Dissolve intermediate compound 4 (Formula VI) in a methanol / water mixed solvent, add lithium hydroxide, adjust the pH to precipitate, and dry to obtain final product 5 (Formula I).

7. The method for preparing a rapid detection reagent in a chemiluminescence mode according to claim 6, characterized in that, The molar ratio of phospholipid acetal II to lithium diisopropylamino in step one is 1:1.

5.

8. The method for preparing a rapid detection reagent in a chemiluminescence mode according to claim 6, characterized in that, In step two, the molar ratio of intermediate compound I (Formula III) to tetrabutylammonium fluoride is 1:1.

2.

9. The method for preparing a rapid detection reagent in a chemiluminescence mode according to claim 6, characterized in that, In step three, the molar ratio of intermediate compound IV (formula 2) to anhydrous potassium carbonate is 1:1.5, and the molar ratio of intermediate compound IV (formula 2) to 4-nitrobenzyl bromide is 1:1.

2.

10. The method for preparing a rapid detection reagent in a chemiluminescence mode according to claim 6, characterized in that, The molar ratio of intermediate compound 3 V to methylene blue in step four is 1:0.1.

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