A system, method and kit for detecting biochemical oxygen demand of wastewater

Abstract

The application discloses a wastewater biochemical oxygen demand detection system, method and kit, and belongs to the technical field of environmental monitoring. + The detection system comprises at least two independent detection channels and a signal processing module, each detection channel independently comprising: an enzymatic conversion module comprising a hydrolytic enzyme system, an oxidoreductase system and a coenzyme NAD + for a specific category of organic matter; a signal transduction and amplification module comprising a CRISPR-Cas effector protein, a fluorescent reporter probe and a molecular switch; and a signal processing module for detecting the fluorescence signal intensity of each detection channel and converting the fluorescence signal into the biochemical oxygen demand contribution value of each detection channel according to a pre-stored exclusive standard curve, and accumulating to obtain the total biochemical oxygen demand. The application effectively overcomes the detection uncertainty caused by the fluctuation of microbial activity and the difference in substrate degradation rate in the traditional method, significantly shortens the detection period, and the detection period can be controlled within 30 min.

Description

Technical Field

[0001] This invention relates to the field of environmental monitoring technology, and in particular to a system, method and reagent kit for detecting biochemical oxygen demand in wastewater. Background Technology

[0002] Biochemical oxygen demand (BOD) is a core indicator for evaluating the degree of organic pollution in municipal wastewater and the efficiency of wastewater treatment. Its value reflects the total amount of organic matter in water that can be degraded by microorganisms. Currently, the standard detection method for this indicator is the five-day BOD5 method. However, this method has an excessively long detection cycle (five days) and depends on the metabolic activity of microorganisms. The test results are easily affected by fluctuations in microbial population status, inoculum size, and culture conditions, resulting in poor reproducibility. At the same time, the operation steps are cumbersome and the risk of human error is relatively high.

[0003] To shorten detection time, various rapid detection technologies have been developed, such as biosensors based on immobilized microbial membranes and respiration rate measurement methods. While these methods have improved detection efficiency to some extent, they still cannot escape dependence on live microorganisms. Inherent problems such as activity decay and poisoning inactivation of microbial membranes persist, severely limiting the long-term stability and reliability of sensors.

[0004] The organic composition of actual municipal wastewater is complex, with carbohydrates and proteins being the main contributors to BOD. During transport and treatment, these substances are gradually hydrolyzed and converted into intermediate metabolites such as small-molecule organic acids (primarily acetic acid). Existing rapid detection technologies have limitations in their consistent response to different forms of organic matter, making it difficult to comprehensively and accurately reflect the overall oxygen-consuming characteristics of organic matter in wastewater, leading to significant discrepancies between detection results and actual BOD values. For example, conventional carbohydrate detection methods can only generally determine total sugar content and cannot distinguish between soluble, easily degradable carbohydrates and recalcitrant polysaccharides such as cellulose, resulting in a mismatch with the true BOD composition. Natural organic matter such as humic acid in water bodies causes spectral absorption interference, severely affecting the accuracy of spectrophotometric detection of organic matter. Protein detection can only characterize the overall protein content and cannot identify free amino acid components individually, while amino acids are also important oxygen-consuming organic matter in wastewater. Small-molecule organic acids such as acetic acid and free amino acids cannot be directly quantified using conventional rapid detection methods. Relying on instruments such as gas chromatography for precise detection results in long detection cycles, cumbersome operations, and high costs, making them unsuitable for rapid on-site monitoring of municipal wastewater. Summary of the Invention

[0005] In view of this, the present invention provides a system, method and kit for detecting biochemical oxygen demand in wastewater. The present invention combines the broad-spectrum organic matter conversion catalyzed by multi-enzyme cascade with the signal amplification function of the CRISPR-Cas system, thereby realizing the detection of BOD without the need for living microorganisms, and can respond rapidly and comprehensively to various easily degradable organic matter.

[0006] In a first aspect, the present invention provides a system for detecting the biochemical oxygen demand (BOD) of wastewater, comprising at least two independent detection channels and a signal processing module, wherein each detection channel independently includes: The enzymatic conversion module contains an enzyme system and the coenzyme NAD that can convert corresponding types of organic matter into NADH. + ; The signal transduction and amplification module includes a CRISPR-Cas effector protein, a fluorescent reporter probe, and a molecular switch; the molecular switch is composed of a double-stranded structure formed by complementary hybridization of an NADH-specific nucleic acid aptamer and a repressor strand. The signal processing module is used to detect the fluorescence signal intensity of each detection channel, and convert the fluorescence signal into the biochemical oxygen demand contribution value of each detection channel according to the pre-stored exclusive standard curve, and accumulate them to obtain the total biochemical oxygen demand.

[0007] Preferably, the at least two independent detection channels include at least two of the following: a carbohydrate detection channel, a protein detection channel, and an acetic acid detection channel.

[0008] Furthermore, the enzymatic conversion module of the carbohydrate detection channel includes α-amylase, glucoamylase, glucose dehydrogenase, and coenzyme NAD. + ; The enzymatic conversion module of the protein detection channel includes endopeptidase, exopeptidase, L-α-amino acid transaminase, glutamate dehydrogenase, α-ketoglutarate, and coenzyme NAD. + ; The enzymatic conversion module of the acetic acid detection channel includes acetyl-CoA synthase, malate dehydrogenase, citrate synthase, and coenzyme NAD. + ATP, coenzyme A and L-malic acid.

[0009] Preferably, the wastewater biochemical oxygen demand (BOD) detection system further includes at least one additional detection channel, wherein the organic matter targeted by the additional detection channel is selected from lactic acid, alcohols, or aldehydes; the enzymatic conversion module of the additional detection channel includes corresponding types of dehydrogenases and coenzymes NAD. + .

[0010] Preferably, the NADH-specific nucleic acid aptamer is configured to undergo a conformational change upon binding to NADH, causing the inhibitory strand to dissociate and release, thereby triggering the trans-cleavage activity of the CRISPR-Cas effector protein, cleaving the fluorescent reporter probe to generate a detectable signal.

[0011] Preferably, the CRISPR-Cas effector protein is Cas12a or Cas14a.

[0012] Preferably, the fluorescent reporter probe is an oligonucleotide probe labeled with a fluorescent group and a quencher group.

[0013] Secondly, the present invention provides a method for detecting the biochemical oxygen demand (BOD) of wastewater, using the above-mentioned detection system, comprising the following steps: Divide the wastewater sample to be tested into at least two portions and add them to at least two independent detection channels; In each detection channel, the enzyme-catalyzed conversion module is used to perform an enzyme-catalyzed conversion reaction to convert the corresponding type of organic matter into NADH; then, the signal transduction and amplification module is used to perform a signal transduction and amplification reaction to generate a fluorescence signal related to the NADH concentration. The fluorescence signal intensity of each detection channel is detected by the signal processing module, and the fluorescence signal is converted into the biochemical oxygen demand contribution value of each detection channel according to the pre-stored exclusive standard curve. The total biochemical oxygen demand of the wastewater is obtained by summing them up.

[0014] Preferably, the temperature of the enzymatic conversion reaction is 20~39℃ and the time is 5~20 minutes; the temperature of the signal transduction and amplification reaction is 20~39℃ and the time is 10~25 minutes.

[0015] Thirdly, the present invention provides a reagent kit for detecting biochemical oxygen demand in wastewater, including the above-mentioned detection system.

[0016] Compared with the prior art, the present invention has achieved the following beneficial effects: This invention, by setting up at least two independent detection channels and integrating an enzymatic conversion module for specific types of organic matter and a CRISPR-Cas signal transduction and amplification module based on NADH aptamer molecular switches in each channel, can convert different types of readily biodegradable organic matter in wastewater into NADH and generate corresponding fluorescence signals. The signal processing module then converts the fluorescence signals into corresponding BOD contribution values ​​based on pre-stored standard curves for each channel, and the total BOD value is obtained by summing these values. Thus, without relying on active microorganisms, it achieves simultaneous typing and precise summation of major BOD contributors such as sugars and proteins in wastewater. This effectively overcomes the detection uncertainties caused by fluctuations in microbial activity and differences in substrate degradation rates in traditional methods, significantly shortening the detection cycle to within 30 minutes. Simultaneously, classification calibration eliminates the measurement bias caused by differences in NADH yields of different organic matter, improving the accuracy of the detection results and adaptability to different water quality compositions. Attached Figure Description

[0017] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments and descriptions of the invention are used to explain the invention and do not constitute an undue limitation thereof. Obviously, those skilled in the art can obtain other drawings based on these drawings without any inventive effort.

[0018] Figure 1 This is a fitting graph of the standard curves and correction coefficients of different types of organic compounds in Example 1 of the present invention. Detailed Implementation

[0019] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0020] In this invention, CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats, a gene editing technology derived from the bacterial immune system. In this invention, it specifically refers to the non-specific "trans-cleavage" activity of its associated Cas proteins (such as Cas12a) for signal amplification.

[0021] In this invention, NAD +It refers to oxidized nicotinamide adenine dinucleotide, which is an important coenzyme in organisms and serves as an electron acceptor for redox reactions in multi-enzyme cascades in this invention.

[0022] In this invention, NADH refers to reduced nicotinamide adenine dinucleotide (NADH), which is the form of NAD+. + The reduced form of the substance, in this invention, is produced by the oxidation of organic matter as a unified signal molecule.

[0023] This invention provides a system for detecting biochemical oxygen demand (BOD) in wastewater, comprising at least two independent detection channels and a signal processing module, wherein each detection channel independently includes: The enzymatic conversion module contains an enzyme system and the coenzyme NAD that can convert corresponding types of organic matter into NADH. + ; The signal transduction and amplification module includes a CRISPR-Cas effector protein, a fluorescent reporter probe, and a molecular switch; the molecular switch is composed of a double-stranded structure formed by complementary hybridization of an NADH-specific nucleic acid aptamer and a repressor strand. The signal processing module is used to detect the fluorescence signal intensity of each detection channel, and convert the fluorescence signal into the biochemical oxygen demand contribution value of each detection channel according to the pre-stored exclusive standard curve, and accumulate them to obtain the total biochemical oxygen demand.

[0024] In this invention, the detection system achieves simultaneous and separate detection of different types of organic matter in wastewater by setting up multiple physically independent detection channels (like different well positions in a multi-well plate). Each channel responds only to a specific type of organic matter, thus avoiding signal crosstalk between NADH from different sources. Different types of organic matter (such as carbohydrates, proteins, and acetic acid) inherently differ in the number of NADH moles produced per unit BOD. If all types of organic matter are mixed and detected in the same reaction system, the total amount of NADH produced can be measured, but the contribution ratio of NADH from each source cannot be distinguished, and directly using a mixed standard curve for conversion inevitably introduces systematic calculation bias. This invention, by producing NADH separately through separate channels, establishing dedicated standard curves for each, and converting and summing them separately, fundamentally eliminates the measurement error caused by different NADH conversion factors for different organic matter, providing a reliable foundation for subsequent accurate classification calibration and total accumulation.

[0025] In this invention, the at least two independent detection channels preferably include at least two of the following: a carbohydrate detection channel, a protein detection channel, and an acetic acid detection channel. More preferably, three channels are provided simultaneously to cover the main BOD-contributing components in municipal wastewater and domestic sewage. This invention is particularly suitable for water quality where easily biodegradable organic matter is the main component, such as municipal wastewater and domestic sewage.

[0026] The enzymatic conversion module of the carbohydrate detection channel includes α-amylase, saccharifying enzyme, glucose dehydrogenase, and coenzyme NAD. + Starch is converted to dextrin by α-amylase, dextrin is converted to glucose by saccharifying enzymes, and glucose is catalyzed to produce NADH by glucose dehydrogenase. Therefore, the BOD contribution of carbohydrates composed of α-1,4 glycosidic bonds is fully accounted for. The preferred amount of α-amylase is 100-300 U / L, more preferably 150-250 U / L, for example, 200 U / L; the preferred amount of saccharifying enzyme is 200-500 U / L, more preferably 300-400 U / L, for example, 350 U / L; the preferred amount of glucose dehydrogenase is 50-100 U / L, more preferably 70-90 U / L, for example, 80 U / L; and the coenzyme NAD... + The final concentration is preferably 0.5~3.0 mmol / L, more preferably 0.8~1.5 mmol / L, for example, it can be 1.0 mmol / L.

[0027] The enzymatic conversion module of the protein detection channel includes endopeptidase, exopeptidase, transaminase, glutamate dehydrogenase, as well as α-ketoglutarate and coenzyme NAD. + Proteins are cleaved into polypeptide fragments by endopeptidases; polypeptides are then hydrolyzed from their ends by exopeptidases, releasing free amino acids; amino acids are then converted to glutamate by L-α-amino acid transaminases, using α-ketoglutarate as the amino acceptor; glutamate is then dehydrogenated by glutamate dehydrogenases to produce α-ketoglutarate and NADH (while consuming NAD). + Therefore, the BOD contribution of the protein is fully accounted for.

[0028] In this invention, the preferred amount of endopeptidase is 50-150 U / L, more preferably 80-120 U / L, for example, 100 U / L; the preferred amount of exopeptidase is 80-200 U / L, more preferably 100-150 U / L, for example, 120 U / L; the preferred amount of L-α-amino acid transaminase is 50-150 U / L, more preferably 100-120 U / L, for example, 110 U / L; the preferred amount of glutamate dehydrogenase is 50-300 U / L, more preferably 150-250 U / L, for example, 170 U / L; α-ketoglutarate is used as a co-substrate, and its final concentration is preferably 0.5-5 mM, for example, 1 mM; coenzyme NAD... + The final concentration is preferably 0.5~3.0 mmol / L, more preferably 0.8~1.5 mmol / L, for example, it can be 1.0 mmol / L.

[0029] The enzymatic conversion module of the acetic acid detection channel includes acetyl-CoA synthase, malate dehydrogenase, citrate synthase, and coenzyme NAD. + Acetic acid, ATP, coenzyme A, and L-malate. Acetic acid, ATP, and coenzyme A (CoA) are converted to acetyl-CoA by acetyl-CoA synthase; L-malate is oxidized to oxaloacetate by malate dehydrogenase (which requires no exogenous addition), simultaneously reducing NAD. + NADH is generated; subsequently, acetyl-CoA and oxaloacetate condense under the action of citrate synthase to generate citrate and release coenzyme A. In the entire reaction pathway, the NADH generation process corresponding to acetic acid after conversion can be coupled and transferred through L-malate mediation, thereby realizing the complete conversion of the oxygen consumption equivalent of acetic acid, so that its BOD contribution value can be accurately and completely included in the detection results.

[0030] In this invention, the preferred amount of acetyl-CoA synthase is 50-300 U / L, more preferably 100-200 U / L, for example, 150 U / L; the preferred amount of malate dehydrogenase is 80-400 U / L, more preferably 150-300 U / L, for example, 200 U / L; the preferred amount of citrate synthase is 60-350 U / L, more preferably 120-250 U / L, for example, 180 U / L; and the preferred amount of coenzyme NAD is... +The final concentration of the ATP is preferably 0.5-3.0 mmol / L, more preferably 0.8-1.5 mmol / L, for example, 1.0 mmol / L; the final concentration of the ATP is preferably 0.2-2.0 mmol / L, more preferably 0.5-1.0 mmol / L, for example, 0.8 mmol / L; the final concentration of the coenzyme A (CoA) is preferably 0.1-1.5 mmol / L, more preferably 0.3-0.8 mmol / L, for example, 0.5 mmol / L; the final concentration of the L-malic acid is preferably 0.3-3.0 mmol / L, more preferably 0.5-2.0 mmol / L, for example, 1.2 mmol / L.

[0031] In addition, the detection system may include at least one additional detection channel for detecting specific small-molecule organic compounds present in the wastewater, such as lactic acid, alcohols, or aldehydes. The enzymatic conversion module of the additional detection channel contains corresponding types of dehydrogenases (such as lactate dehydrogenase, alcohol dehydrogenase, and aldehyde dehydrogenase) and the coenzyme NAD. + It may also contain other intermediate auxiliary conversion substances. The standard curve for the additional channels needs to be established in advance using the corresponding standard substances (such as sodium lactate, ethanol, formaldehyde).

[0032] In this invention, coenzyme NAD + It is an indispensable electron carrier and substrate in redox reactions. In the reaction system, its final concentration is preferably 0.5~5 mM, more preferably 1~2 mM, for example, 1 mM, to ensure that sufficient acceptor is reduced to NADH to meet the requirements of dehydrogenation reactions in each channel.

[0033] In this invention, the signal transduction and amplification module in each detection channel is used to convert the NADH concentration generated in that channel into a detectable fluorescent signal. This module includes a CRISPR-Cas effector protein, a fluorescent reporter probe, and a double-stranded molecular switch formed by complementary hybridization of an NADH-specific nucleic acid aptamer and a repressor strand.

[0034] Molecular switches are the core of signal recognition. NADH-specific nucleic acid aptamers are mixed with their perfectly complementary repressor strands at a molar ratio of 1:1 to 1:1.5 (e.g., 1:1.2), heated at 95°C for 5 minutes to denature the double strands, and then slowly cooled to room temperature for renaturation hybridization, forming a stable double-stranded complex. In the absence of NADH, this double-stranded structure is stable, and the repressor strand competitively binds to the guide RNA (crRNA) of the Cas protein, inactivating the Cas protein. When NADH generated by this channel is present, NADH specifically binds to the aptamer, inducing a conformational change in the aptamer, thereby dissociating it from the repressor strand and releasing it. The released repressor strand can activate the trans-cleavage activity of CRISPR-Cas effector proteins. The final concentration of the repressor strand in the reaction system is preferably 20–100 nM, more preferably 50–80 nM, for example, 50 nM.

[0035] The cascade signal amplification system based on NADH aptamer molecular switches and CRISPR-Cas effector proteins provided by this invention exhibits extremely low nonspecific background signal compared to traditional direct fluorescent labeling or single-enzyme amplification methods. The principle is as follows: when the molecular switch is not bound to NADH, the double-stranded structure formed by the aptamer and the repressor strand is highly stable, effectively blocking the activation site of the Cas protein and preventing accidental activation by nucleic acid fragments or other small molecules in the system. Simultaneously, the CRISPR-Cas effector protein itself does not possess trans-cleavage activity when it does not form a complete complex with the guide RNA (crRNA), further reducing background leakage. Only when the target analyte NADH is present and induces a conformational change in the aptamer, releasing the repressor strand, can the Cas protein be precisely activated and cleave the fluorescent reporter probe. Therefore, even in complex wastewater matrices with trace amounts of interfering substances, this invention maintains high sensitivity and specificity, effectively avoiding false positive results.

[0036] CRISPR-Cas effector proteins are the executors of signal amplification. Proteins with trans-cleavage activity on single-stranded DNA are preferred, such as Cas12a or Cas14a, with Cas12a being more preferred due to its mature technology and high activity. In the reaction system, the final concentration of Cas12a protein is preferably 10–50 nM, more preferably 20–30 nM, for example, 20 nM. The activated Cas protein non-specifically cleaves all single-stranded DNA probes in the system.

[0037] A fluorescent reporter probe is a carrier of signal output, consisting of a short oligonucleotide single-stranded DNA segment doubly labeled with a fluorescent group and a quencher group. When the probe is intact, fluorescence is not emitted due to quenching; upon cleavage by the activated Cas protein, the fluorescent group separates from the quencher group, generating a fluorescent signal. Because the detection channels are physically isolated, different channels can use the same labeled fluorescent reporter probe (e.g., all using FAM-BHQ), and the signals will not interfere with each other during separate detection; alternatively, probes with different emission wavelengths can be selected as needed (e.g., FAM-labeled probes for carbohydrate channels, HEX-labeled probes for protein channels, and ROX-labeled probes for acetic acid channels) to distinguish signals by wavelength during simultaneous multi-channel detection. The sequence of the fluorescent reporter probe is typically a continuous thymine (poly-T), such as 5'-FAM-TTTTTTTTTTTTTT-BHQ-3' (SEQ ID NO: 1). Here, FAM refers to Carboxyfluorescein, a commonly used green fluorescent reporter group; HEX refers to Hexachlorofluorescein; ROX refers to Rhodamine X; and BHQ refers to BlackHole Quencher. The final concentration of the fluorescent reporter probe is preferably 50~200 nM, more preferably 100~150 nM, for example, 100 nM.

[0038] The detection system of the present invention also includes a common signal processing module for detecting the fluorescence signal intensity of each detection channel, converting the fluorescence signal into the BOD contribution value of each detection channel according to a pre-stored dedicated standard curve, and accumulating them to obtain the total BOD value.

[0039] The signal processing module typically includes a fluorescence detection unit (such as a photomultiplier tube or CCD sensor) and a computing unit (such as a microprocessor). The fluorescence detection unit sequentially reads the fluorescence intensity of each detection channel and subtracts the blank control to obtain the net fluorescence value. The computing unit pre-stores the dedicated standard curve for each channel (e.g., a linear equation determined in advance using standard substances), substitutes the net fluorescence value into the corresponding equation to calculate the BOD contribution value of that channel, and then sums the contribution values ​​of all channels to output the total BOD value of the wastewater.

[0040] To ensure the efficient execution of enzymatic and CRISPR reactions in each detection channel, the detection system also includes a standardized reaction buffer. This buffer provides a suitable pH and ionic environment for the multi-enzyme catalytic reaction and the CRISPR nucleic acid reaction. The buffer is preferably a tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) buffer system with a pH of 7.0–7.6, more preferably pH 7.4. Its composition preferably includes: 40–100 mM Tris-HCl, more preferably 50 mM; 5–15 mM MgCl2, more preferably 10 mM; and 50–200 mM NaCl, more preferably 150 mM. This buffer can simultaneously meet the activity requirements of all channels.

[0041] This invention also provides a method for detecting the biochemical oxygen demand (BOD) of wastewater, using the aforementioned detection system. The method includes the following steps: Divide the wastewater sample to be tested into at least two portions and add them to at least two independent detection channels; In each detection channel, the enzyme-catalyzed conversion module is used to perform an enzyme-catalyzed conversion reaction to convert the corresponding type of organic matter into NADH; then, the signal transduction and amplification module is used to perform a signal transduction and amplification reaction to generate a fluorescence signal related to the NADH concentration. The fluorescence signal intensity of each detection channel is detected by the signal processing module, and the fluorescence signal is converted into the biochemical oxygen demand contribution value of each detection channel according to the pre-stored exclusive standard curve. The total biochemical oxygen demand of the wastewater is obtained by summing them up.

[0042] The wastewater samples to be tested in this invention also require a pretreatment step, namely, filtering the wastewater samples through a 0.22~0.45 μm filter membrane to remove suspended particulate matter. If the expected BOD value of the sample is too high, it needs to be appropriately diluted with deionized water or water of the same type as the sample matrix to bring the BOD value into the linear range of the detection method. For wastewater containing interfering substances that may inhibit enzyme activity or CRISPR system function (such as heavy metal ions, high salinity, oxidizing / reducing substances), appropriate interference control reagents can be added in the pretreatment step, such as heavy metal chelating agents (such as EDTA) and reducing substance scavengers (such as catalase).

[0043] This invention divides the pretreated wastewater sample into at least two equal portions (the number of portions being the same as the number of detection channels to be used), and adds each portion to at least two independent detection channels. In each detection channel, the corresponding enzyme-catalyzed conversion module reagent (including the corresponding enzyme system and coenzyme NAD) is added to the sample. + The enzyme is mixed with a buffer solution and then subjected to an enzymatic conversion reaction.

[0044] The preferred temperature for the enzymatic conversion reaction is 20–39°C, more preferably 30–37°C, for example, 37°C; the preferred reaction time is 5–20 minutes, more preferably 10–15 minutes, for example, 15 minutes. During this reaction time, the macromolecular organics in each channel are hydrolyzed into monomers, and the monomers and the original small organic molecules are oxidized and dehydrogenated, leading to the formation of coenzyme NAD+. + Simultaneous reduction to NADH. Because the enzyme system of each channel is specific, the NADH produced by each channel comes only from the organic category corresponding to that channel.

[0045] After the enzymatic conversion reaction is complete, without changing the container or performing intermediate processing, directly add the reagents for the signal transduction and amplification module, including CRISPR-Cas effector proteins, pre-hybridized molecular switches, and fluorescent reporter probes, to the reaction system of each detection channel. After mixing, proceed with the signal transduction and amplification reaction.

[0046] The preferred temperature for the signal transduction and amplification reaction is 20–39°C, more preferably 30–37°C, for example, 37°C; the preferred reaction time is 10–25 minutes, more preferably 15–20 minutes, for example, 15 minutes. During this stage, the NADH generated in each channel specifically binds to the molecular switch, releasing the repressor chain and activating the trans-cleavage activity of the CRISPR-Cas effector protein. The activated Cas protein cleaves the fluorescent reporter probe extensively, generating a strong fluorescent signal proportional to the NADH concentration. Since each channel is physically independent, the signals do not interfere with each other.

[0047] After the reaction, the fluorescence signal intensity of each detection channel is sequentially detected using the fluorescence detection unit of the signal processing module. The excitation and emission wavelengths are set according to the labeling group of the fluorescent reporter probe used. For example, when using FAM labeling, the excitation wavelength is preferably 480–500 nm (e.g., 488 nm), and the emission wavelength is preferably 510–530 nm (e.g., 520 nm). The fluorescence reading for each channel is subtracted from the background value of the blank control (using the same volume of buffer or deionized water instead of the sample) to obtain the net fluorescence intensity (ΔF).

[0048] To achieve quantification, a dedicated standard curve for each detection channel needs to be pre-established. The standard curves are stored in the computation unit of the signal processing module. The establishment method is as follows: Standard curve for carbohydrate detection channel: Using glucose as the standard substance, prepare a series of glucose standard solutions with known BOD concentrations (BOD concentration range preferably 0~200 mg / L, e.g., 0, 20, 50, 100, 150, 200 mg / L). Perform detection according to steps 2~4 above (using the reagents of the carbohydrate detection channel). Plot the theoretical BOD concentration as the abscissa (x) and the net fluorescence intensity as the ordinate (y), and perform linear fitting to obtain the regression equation y = k1x + b1, with a linear correlation coefficient R. 2 ≥0.98. The slope k1 is the correction coefficient for carbohydrates.

[0049] Standard curve for protein detection channel: Using L-glutamic acid as the standard substance, the regression equation y = k2x + b2 was established in the same way.

[0050] Acetic acid detection channel standard curve: Using acetic acid as the standard substance, the regression equation y = k3x + b3 is established in the same way.

[0051] Additional detection channel standard curves: These are established using corresponding standard substances based on the target analyte (lactic acid, ethanol, formaldehyde, etc.).

[0052] All standard curves should be prepared using the same matrix as the test sample (e.g., filtered supernatant of actual wastewater) to eliminate matrix effects.

[0053] For actual wastewater samples, after measuring the net fluorescence intensity ΔF1, ΔF2, ΔF3… for each channel, the calculation unit of the signal processing module automatically calls the pre-stored corresponding standard curves to calculate the BOD contribution value for each channel: BOD 糖类 = (ΔF1- b1) / k1; BOD 蛋白类 = (ΔF2 - b2) / k2; BOD 乙酸 = (ΔF3- b3) / k3; Additional channels follow the same pattern.

[0054] Then, the BOD contribution values ​​from all detection channels are summed to obtain the total BOD value of the wastewater: Total BOD = BOD 糖类 +BOD 蛋白类 + BOD 乙酸 + …。 If the sample has been diluted, the total BOD value needs to be multiplied by the dilution factor to obtain the actual BOD value of the original wastewater sample.

[0055] To facilitate use and promotion, this invention also provides a reagent kit for detecting biochemical oxygen demand (BOD) in wastewater, which includes the core components of the aforementioned detection system. Specifically, the reagent kit comprises: At least two independently packaged enzyme reaction reagent units. Each enzyme reaction reagent unit corresponds to a class of organic compounds (e.g., sugars, proteins, acetic acid, etc.) and is packaged separately. Each unit contains lyophilized enzyme powder or stock solution for that class of organic compounds, as well as coenzyme NAD. + The reagents are lyophilized powders or solutions. The concentrations of each enzyme and coenzyme are designed so that, when used according to the instructions, the final concentration in the final reaction system is the aforementioned optimal concentration. Multiple reagent units are physically isolated to avoid cross-contamination.

[0056] At least two independently packaged CRISPR assay kit units. Each CRISPR assay kit unit corresponds one-to-one with the multi-enzyme reaction kit unit. Each kit contains CRISPR-Cas effector protein lyophilized powder or stock solution, NADH-responsive molecular switch (aptamer-inhibitor double-stranded complex) solution, and fluorescent reporter probe solution. All kits are also individually packaged to ensure the stability of each component before use.

[0057] Signal Processing Module: The kit may include an integrated signal processing module (such as a small fluorescence detector), or it may only provide a storage medium (such as a USB flash drive or QR code) pre-stored with standard curve information, or a standard module for users to create their own standard curves. The standard module includes: carbohydrate standards (glucose), protein standards (L-glutamic acid), acetate standards (acetic acid), and other optional standards. Users can use these standards to calibrate and create their own standard curves.

[0058] Buffer Components: Provides a uniform reaction buffer, preferably a 10× concentrate, which should be diluted to a 1× working concentration with nuclease-free water before use. The buffer composition is the same as described above (pH 7.4 Tris-HCl, containing MgCl2 and NaCl).

[0059] Instruction manual: The manual describes in detail the sample pretreatment method, the amount of each reagent unit and the reconstitution method, the detection operation steps (including the reaction temperature and time for enzyme conversion and signal amplification), the method for establishing the standard curve, the setting of fluorescence detection parameters, and the result calculation formula.

[0060] To improve stability and portability, the enzyme components in the multi-enzyme reaction reagent unit and / or the CRISPR-Cas effector protein in the CRISPR detection reagent unit are preferably in lyophilized powder form in the reaction tubes or microplates, allowing for rapid reconstitution simply by adding buffer and sample. The kit is further preferably in the form of pre-filled eight-tube or 96-well microplates, with each well or tube pre-filled with lyophilized beads of the corresponding multi-enzyme reaction reagent and CRISPR detection reagent. Users can directly add sample and buffer without complex preparation, making it suitable for high-throughput screening or rapid on-site detection.

[0061] The technical solution of the present invention will be further described below with reference to specific embodiments. The present invention does not impose any special restrictions on the source of reagents used in the following embodiments; commercially available products well known to those skilled in the art can be used. Experimental methods not specified with specific conditions in the embodiments are generally performed under conventional conditions in the art or according to the manufacturer's recommendations.

[0062] Example 1 In this embodiment, three independent detection channels (carbohydrate detection channel, protein detection channel, and acetic acid detection channel) were constructed. Each channel had its own independently prepared reaction system and the same fluorescently labeled probes were used to establish a dedicated standard curve for each channel.

[0063] 1. General Reagents and Materials Reaction buffer: Tris-HCl buffer at pH 7.4, final concentration 50 mM Tris-HCl, 10 mM MgCl2, 150 mM NaCl.

[0064] Coenzyme NAD + Prepare a 12.5 mM stock solution using buffer solution.

[0065] Fluorescent reporter probe: Sequence 5'-FAM-TTTTTTTTTTTTTT-BHQ-3' (SEQ ID NO: 1), dissolved in RNase-free water to 2.5 μM and stored at -20°C protected from light. The same probe was used for all channels.

[0066] Molecular switch: NADH-specific nucleic acid aptamer (5'-GGTTGGTGTGGTTGG-3', SEQ ID NO: 2) and fully complementary repressor strand (5'-CCAACCACACCAACC-3', SEQ ID NO: 3) were mixed at a molar ratio of 1:1.2, heated at 95°C for 5 min, and slowly cooled to room temperature to hybridize and form a double-stranded complex. The storage concentration was 625 nM.

[0067] Cas12a protein: Commercially available Cas12a, diluted to 500 nM with buffer.

[0068] 2. Preparation of multi-enzyme systems for each detection channel The multi-enzyme system for each channel was prepared according to Table 1 (the final concentration of each enzyme was optimized in advance by single-factor experiments).

[0069] Table 1. Preparation of multi-enzyme systems for each detection channel

[0070] Note: Enzyme activity unit (U) is defined as the amount of enzyme required to catalyze the conversion of 1 μmol of substrate per minute under specific conditions. The final concentrations of all components in Table 1 refer to the concentrations in a final 25 μL reaction system.

[0071] 3. Establishment of dedicated standard curves for each channel (1) Preparation of standard solutions Using municipal wastewater treatment plant secondary effluent (background BOD < 5 mg / L) filtered through a 0.22 μm filter membrane and sterilized as the dilution matrix, the following three single-category standard series solutions were prepared: Standard series of carbohydrates: glucose, theoretical BOD concentrations of 0, 10, 20, 30, 50, 80, 100, and 150 mg / L.

[0072] Protein standard series: L-glutamic acid, theoretical BOD concentrations of 0, 10, 20, 30, 50, 80, 100, and 150 mg / L.

[0073] Acetic acid standard series: Acetic acid, theoretical BOD concentrations of 0, 10, 20, 30, 50, 80, 100, and 150 mg / L.

[0074] (2) Standard curve of carbohydrate channels In separate reaction tubes, add 16 μL of premixed carbohydrate detection solution, then add 5 μL of glucose standard solution of each concentration, and mix well. Incubate at 37°C in the dark for 15 min for enzymatic conversion. Then add 1 μL of Cas12a protein (final concentration of 20 nM), 2 μL of molecular switch (final concentration of 50 nM), and 1 μL of fluorescent reporter probe (final concentration of 100 nM) to each tube, mix well, and incubate at 37°C for another 15 min, for a total reaction volume of 25 μL. Detect using a fluorescence microplate reader (excitation wavelength 485 nm, emission wavelength 525 nm). Perform triple replicates for each concentration, and subtract the blank (5 μL matrix solution instead of standard solution) to obtain the net fluorescence intensity. Plot BOD concentration as the x-axis and net fluorescence intensity as the y-axis to obtain a linear curve for carbohydrates: y1 = 12.5x1 + 46.2(R²). 2 =0.989). For example... Figure 1 As shown.

[0075] (3) Standard curve for protein channels The procedure is the same as for “(2) Carbohydrate Channel Standard Curve”, but the protein detection channel premix and L-glutamate standard solution are used. A linear fit yields the protein standard curve: y² = 11.8x² + 42.5(R² / 2) 2 =0.988). For example... Figure 1As shown.

[0076] (4) Acetic acid channel standard curve The procedure is the same as in "(2) Standard Curve for Carbohydrate Channel", using the acetic acid detection channel premix and acetic acid standard solution. The acetic acid standard curve was obtained by linear fitting: y3 = 13.2x3 + 48.7 (R²) / 2. 2 =0.990). For example Figure 1 As shown.

[0077] 4. Testing process For any sample to be tested, take three equal portions (5 μL each) and add them to the premixed solution (16 μL) of each of the three channels. The subsequent steps are the same as the incubation and detection in "(2) Standard Curve of Carbohydrate Channel". According to the standard curve of the corresponding channel, output the BOD contribution value of each channel and sum them up.

[0078] Example 2 This embodiment uses the three independent detection channels constructed in Embodiment 1 to simultaneously detect actual municipal sewage inlet samples and evaluate their accuracy, anti-interference ability, and repeatability.

[0079] 1. Actual wastewater sample testing and accuracy verification (1) Sample processing: A water sample was collected from the inlet of a municipal sewage treatment plant and immediately filtered through a 0.45 μm filter membrane. The filtrate was then used for further processing.

[0080] (2) Channel-specific detection: Take three PCR tubes and add 16 μL of premixed carbohydrate channel buffer, 16 μL of premixed protein channel buffer, and 16 μL of premixed acetic acid channel buffer to each tube, respectively. Add 5 μL of pretreated water sample to each tube and mix well. Place all three tubes in a constant temperature metal bath and incubate at 37°C in the dark for 15 min.

[0081] Add 1 μL of Cas12a protein, 2 μL of molecular switch, and 1 μL of fluorescent reporter probe to each tube sequentially, for a total reaction volume of 25 μL. After mixing, incubate at 37°C for 15 min.

[0082] Fluorescence detection (excitation wavelength 485 nm, emission wavelength 525 nm) was performed, and the net fluorescence intensity of each tube was read.

[0083] (3) Calculation of results: Net fluorescence intensity measured: ΔF1=456.2 RFU (carbohydrate channel), ΔF2=328.3 RFU (protein channel), ΔF3=228.7 RFU (acetic acid channel).

[0084] Substitute the respective standard curves: BOD contribution from carbohydrates = (456.2 - 46.2) / 12.5 = 32.8 mg / L; Protein BOD contribution = (328.3 - 42.5) / 11.8 = 24.2 mg / L; Acetic acid BOD contribution = (228.7 – 48.7) / 13.2 = 13.6 mg / L; Total BOD = 32.8 + 24.2 + 13.6 = 70.6 mg / L.

[0085] (4) Comparison using the national standard method: The same sample was measured according to HJ 505-2009 "Determination of Five-Day Biochemical Oxygen Demand (BOD5) in Water - Dilution and Inoculation Method", and the BOD5 value was 78.3 mg / L. The relative error was within 10%.

[0086] 2. Anti-interference capability verification Adding common interfering substances to the same wastewater sample: Cu 2+ 10 mg / L sodium hypochlorite and 5 mg / L sodium hypochlorite were added. Following the steps in "1. Actual Wastewater Sample Detection and Accuracy Verification," the total BOD was calculated to be 74.2 mg / L. Compared with the result without interfering substances (70.6 mg / L), the relative error was 4.6%. This indicates that the present invention has good tolerance to common interfering substances in wastewater.

[0087] 3. Repeatability and stability verification For the same wastewater sample, the same operator performed six consecutive measurements following the steps in "1. Actual Wastewater Sample Detection and Accuracy Verification". The total BOD results were 71.9, 70.5, 73.3, 72.1, 75.8, and 74.7 mg / L, respectively. The average value was 73.05 mg / L, the standard deviation (SD) was 1.95, and the relative standard deviation (RSD) was 2.67%. The RSD of the fluorescence signal in each channel was <1%, demonstrating excellent repeatability of the method.

[0088] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A system for detecting biochemical oxygen demand (BOD) in wastewater, characterized in that, It includes at least two independent detection channels and signal processing modules, each detection channel independently containing: The enzymatic conversion module contains an enzyme system and the coenzyme NAD that can convert corresponding types of organic matter into NADH. + ; The signal transduction and amplification module includes a CRISPR-Cas effector protein, a fluorescent reporter probe, and a molecular switch; the molecular switch is composed of a double-stranded structure formed by complementary hybridization of an NADH-specific nucleic acid aptamer and a repressor strand. The signal processing module is used to detect the fluorescence signal intensity of each detection channel, and convert the fluorescence signal into the biochemical oxygen demand contribution value of each detection channel according to the pre-stored exclusive standard curve, and accumulate them to obtain the total biochemical oxygen demand. The dedicated standard curve is stored in the computing unit of the signal processing module and is pre-established in the following manner: Standard curve for carbohydrate detection channel: Using glucose as the standard substance, a series of glucose standard solutions with known BOD concentrations were prepared. The reagents from the carbohydrate detection channel were used for detection. A linear fit was performed with theoretical BOD concentration as the x-axis and net fluorescence intensity as the y-axis to obtain the regression equation y = k1x + b1, and the linear correlation coefficient R0. 2 ≥0.98, the slope k1 is the correction coefficient for carbohydrates; Standard curve for protein detection channel: Using L-glutamic acid as the standard substance, the regression equation y = k2x + b2 was established in the same way; Acetic acid detection channel standard curve: Using acetic acid as the standard substance, the regression equation y = k3x + b3 was established in the same way; Additional detection channel standard curves: established using corresponding standard substances according to the target analyte; All standard curves should use the same matrix as the test sample to prepare the standard solution in order to eliminate matrix effects; For actual wastewater samples, after measuring the net fluorescence intensity ΔF1, ΔF2, ΔF3… for each channel, the calculation unit of the signal processing module automatically calls the pre-stored corresponding standard curves to calculate the BOD contribution value for each channel: THAT 糖类 = (ΔF1- b1) / k1; BOD 蛋白类 = (ΔF2- b2) / k2; BOD 乙酸 = (ΔF3- b3) / k3; Additional channels follow the same pattern; Then, the BOD contribution values ​​from all detection channels are summed to obtain the total BOD value of the wastewater: Total BOD = BOD 糖类 + BOD 蛋白类 +BOD 乙酸 + … 2. The detection system as described in claim 1, characterized in that, The at least two independent detection channels include at least two of the following: a carbohydrate detection channel, a protein detection channel, and an acetic acid detection channel.

3. The detection system as described in claim 2, characterized in that, The enzymatic conversion module of the carbohydrate detection channel includes α-amylase, saccharifying enzyme, glucose dehydrogenase, and coenzyme NAD. + ; The enzymatic conversion module of the protein detection channel includes endopeptidase, exopeptidase, L-α-amino acid transaminase, glutamate dehydrogenase, α-ketoglutarate, and coenzyme NAD. + ; The enzymatic conversion module of the acetic acid detection channel includes acetyl-CoA synthase, malate dehydrogenase, citrate synthase, and coenzyme NAD. + ATP, coenzyme A and L-malic acid.

4. The detection system as described in claim 1, characterized in that, The wastewater biochemical oxygen demand (BOD) detection system also includes at least one additional detection channel, the additional detection channel targeting organic compounds selected from lactic acid, alcohols, or aldehydes; the enzymatic conversion module of the additional detection channel contains corresponding types of dehydrogenases and coenzyme NAD. + .

5. The detection system as described in claim 1, characterized in that, The NADH-specific nucleic acid aptamer is configured to undergo a conformational change upon binding to NADH, causing the repressor strand to dissociate and release, thereby triggering the trans-cleavage activity of the CRISPR-Cas effector protein, which cleaves the fluorescent reporter probe to generate a detectable signal.

6. The detection system as described in claim 1, characterized in that, The CRISPR-Cas effector protein is either Cas12a or Cas14a.

7. The detection system as described in claim 1, characterized in that, The fluorescent reporter probe is an oligonucleotide probe labeled with a fluorescent group and a quencher group.

8. A method for detecting biochemical oxygen demand (BOD) in wastewater, characterized in that, The detection system according to any one of claims 1 to 7 comprises the following steps: Divide the wastewater sample to be tested into at least two portions and add them to at least two independent detection channels; In each detection channel, the enzyme-catalyzed conversion module is used to perform an enzyme-catalyzed conversion reaction to convert the corresponding type of organic matter into NADH; then, the signal transduction and amplification module is used to perform a signal transduction and amplification reaction to generate a fluorescence signal related to the NADH concentration. The fluorescence signal intensity of each detection channel is detected by the signal processing module, and the fluorescence signal is converted into the biochemical oxygen demand contribution value of each detection channel according to the pre-stored exclusive standard curve. The total biochemical oxygen demand of the wastewater is obtained by summing them up. The dedicated standard curve is stored in the computing unit of the signal processing module and is pre-established in the following manner: Standard curve for carbohydrate detection channel: Using glucose as the standard substance, a series of glucose standard solutions with known BOD concentrations were prepared. The reagents from the carbohydrate detection channel were used for detection. A linear fit was performed with theoretical BOD concentration as the x-axis and net fluorescence intensity as the y-axis to obtain the regression equation y = k1x + b1, and the linear correlation coefficient R0. 2 ≥0.98, the slope k1 is the correction coefficient for carbohydrates; Standard curve for protein detection channel: Using L-glutamic acid as the standard substance, the regression equation y = k2x + b2 was established in the same way; Acetic acid detection channel standard curve: Using acetic acid as the standard substance, the regression equation y = k3x + b3 was established in the same way; Additional detection channel standard curves: established using corresponding standard substances according to the target analyte; All standard curves should use the same matrix as the test sample to prepare the standard solution in order to eliminate matrix effects; For actual wastewater samples, after measuring the net fluorescence intensity ΔF1, ΔF2, ΔF3… for each channel, the calculation unit of the signal processing module automatically calls the pre-stored corresponding standard curves to calculate the BOD contribution value for each channel: THAT 糖类 = (ΔF1- b1) / k1; BOD 蛋白类 = (ΔF2- b2) / k2; BOD 乙酸 = (ΔF3- b3) / k3; Additional channels follow the same pattern; Then, the BOD contribution values ​​from all detection channels are summed to obtain the total BOD value of the wastewater: Total BOD = BOD 糖类 + BOD 蛋白类 +BOD 乙酸 + … 9. The detection method as described in claim 8, characterized in that, The temperature of the enzymatic conversion reaction is 20~39℃ and the time is 5~20 minutes; the temperature of the signal transduction and amplification reaction is 20~39℃ and the time is 10~25 minutes.

10. A reagent kit for detecting biochemical oxygen demand (BOD) in wastewater, characterized in that, The detection system includes any one of claims 1 to 7.

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