Kit for qualitative / quantitative detection of fluoroquinolone compounds and method thereof

Abstract

The application discloses a kit and a method for qualitatively and quantitatively detecting fluoroquinolones, wherein the detection method comprises the following steps: providing a solution to be detected; contacting an immune probe with the solution to be detected, so that the immune probe is enriched with the fluoroquinolones, so as to obtain an enriched immune probe; and qualitatively and quantitatively analyzing and detecting the enriched immune probe. The method uses the immune probe for enrichment and extraction, and the fluoroquinolones can be extracted without sample pretreatment, so that the extraction method is simple and efficient.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of analytical chemistry, in particular, to a method and a kit for qualitative / quantitative detection of fluoroquinolone compounds. BACKGROUND

[0002] Veterinary antibiotic residues existing in food and environment can have adverse side effects on humans, such as allergic reactions and bacterial antibiotic resistance. Therefore, it is necessary to accurately and quickly monitor the level of antibiotic residues. Open mass spectrometry (AMS) is favored by researchers for its powerful qualitative and high-throughput analysis capabilities, and the development of label-free rapid detection platforms based on mass spectrometry is increasingly concerned.

[0003] Therefore, a method for qualitative and quantitative detection of trace residues of veterinary antibiotics in food and environment needs to be researched. SUMMARY

[0004] The present application aims to at least solve one of the technical problems existing in the prior art. To this end, one object of the present application is to provide a method for qualitative / quantitative detection of fluoroquinolone compounds, which is online, rapid, high-throughput, high-sensitivity, high-accuracy, and especially suitable for trace extraction and detection of fluoroquinolone drugs (FQs) without sample pretreatment.

[0005] According to one aspect of the present application, a method for qualitative / quantitative detection of fluoroquinolone compounds is provided. According to an embodiment of the present application, the method comprises providing a solution to be tested; contacting an immune probe with the solution to be tested for adsorption extraction treatment, so that the immune probe is enriched with the fluoroquinolone compounds, so as to obtain an enriched immune probe; and performing qualitative / quantitative analysis and detection on the enriched immune probe, wherein the immune probe comprises: a metal probe; a graphene oxide layer covering at least part of the surface of the end of the metal probe, and the surface of the graphene oxide layer is modified with streptavidin; an antibody layer covering at least part of the surface of the graphene oxide layer, the antibody layer comprising biotin-modified broad-spectrum antibodies, wherein the broad-spectrum antibodies specifically bind to fluoroquinolone compounds.

[0006] The method for qualitatively and quantitatively detecting fluoroquinolones according to the embodiment of the application uses an immunological probe for extraction, the immunological probe is coated with graphene oxide (GO) on the surface of a metal probe (TN), the GO has a large specific surface area and rich oxygen-containing functional groups such as carboxyl groups on the surface, which can provide abundant reaction sites for covalent coupling of biomolecules, a large amount of streptavidin can be coupled through the graphene oxide, and then a broad-spectrum antibody can be adsorbed on the surface of the metal probe through the coupling of streptavidin and biotin, the fixed amount of the antibody is large, and the accuracy and precision of detection are high; and the method for detecting fluoroquinolones does not need sample pretreatment for extraction, the extraction method is simple, and the efficiency is high.

[0007] In addition, the method for qualitatively and quantitatively detecting fluoroquinolones according to the above embodiment of the application can further have the following additional technical features:

[0008] According to the embodiment of the application, the preparation method of the immunological probe comprises: coating the end surface of the metal probe with an adhesive liquid and performing first contact with graphene oxide powder to obtain a probe coated with graphene oxide; performing activation treatment on carboxyl groups of the graphene oxide coated on the probe to obtain an activated probe; performing second contact of the activated probe with streptavidin to make streptavidin react with the activated graphene oxide to obtain a streptavidin-modified probe; performing protein blocking treatment on the streptavidin-modified probe to obtain a blocked probe; and performing third contact of the blocked probe with a broad-spectrum antibody modified with biotin to obtain the immunological probe for fluoroquinolones, wherein the broad-spectrum antibody specifically binds to fluoroquinolones.

[0009] According to the embodiment of the application, the thickness of the graphene oxide powder is 0.8-1.2 nanometers, and the width is 1-15 micrometers.

[0010] According to the embodiment of the application, the activation treatment is performed by using 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC).

[0011] According to the embodiment of the application, the time of the second contact is 0.5-1.5 hours.

[0012] According to the embodiment of the application, the time of the third contact is 15-45 minutes, and the temperature is 30-45 degrees Celsius.

[0013] According to the embodiment of the application, the metal probe is a tungsten needle.

[0014] According to the embodiment of the application, the length of the tungsten needle is 2-10 centimeters, and the tip is 1-10 micrometers.

[0015] According to an embodiment of the present application, the thickness of the graphene oxide layer is 1-3 microns.

[0016] According to an embodiment of the present application, the immobilized amount of the broad-spectrum antibody is 10-25 micrograms per root.

[0017] According to an embodiment of the present application, the extraction treatment is performed for 5-15 minutes, preferably 10 minutes.

[0018] According to an embodiment of the present application, the extraction treatment is performed at a pH of 5-8.

[0019] According to an embodiment of the present application, the concentration of the fluoroquinolone compound is not higher than 20 ng / mL.

[0020] According to an embodiment of the present application, the fluoroquinolone compound is at least one selected from the group consisting of norfloxacin (NOF), enoxacin (ENO), perfloxacin (PEF), ofloxacin (OFL), lomefloxacin (LOM) and sarafloxacin (SAR).

[0021] According to an embodiment of the present application, the analytical detection is an open electrospray mass spectrometry detection.

[0022] According to an embodiment of the present application, the spray desorption solvent of the open electrospray mass spectrometry detection is a 5% formic acid methanol solution.

[0023] According to an embodiment of the present application, the detection conditions of the open electrospray mass spectrometry are as follows: scanning mode: ESI + ; detection mode: multiple reaction monitoring (MRM); gas curtain pressure: 30; residence time: 100 milliseconds.

[0024] According to an embodiment of the present application, the tip of the immunoprobe is 2-10 mm, preferably 5 mm, away from the open electrospray mass spectrometry inlet.

[0025] According to another aspect of the present invention, a kit for qualitative / quantitative detection of fluoroquinolone compounds is provided. According to an embodiment of the present invention, the kit comprises: the aforementioned immunoprobe, prepared using the aforementioned method, the immunoprobe comprising: a metal probe; a graphene oxide layer covering at least a portion of the surface of the metal probe tip, the graphene oxide layer being modified with streptavidin; and an antibody layer covering at least a portion of the surface of the graphene oxide layer, the antibody layer comprising a biotin-modified broad-spectrum antibody, wherein the broad-spectrum antibody specifically binds to fluoroquinolone compounds. Thus, this kit utilizes the specific adsorption and enrichment of fluoroquinolone compounds by the immunoprobe, exhibiting high specificity in extraction and enrichment, requiring no sample pretreatment, simplifying the extraction process, and providing high detection sensitivity, making it particularly suitable for trace extraction and detection of fluoroquinolone drugs (FQs).

[0026] According to an embodiment of the present invention, the kit further comprises: a spray desorption solvent and an immune probe.

[0027] According to embodiments of the present invention, the fluoroquinolone compound is at least one selected from norfloxacin (NOF), enoxacin (ENO), pefloxacin (PEF), ofloxacin (OFL), lomefloxacin (LOM), and sarafloxacin (SAR).

[0028] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0029] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0030] Figure 1 A schematic diagram of the structure of an immune probe according to an embodiment of the present invention is shown;

[0031] Figure 2 The diagram illustrates the relationship between the number of sprays and the proportion according to an embodiment of the present invention, wherein (a) NOF; (b) ENO; (c) PEF; (d) LOM; (e) OFL; and (f) SAR.

[0032] Figure 3 This diagram illustrates the selective detection results of TN@GO-SA-biotin-Ab according to an embodiment of the present invention.

[0033] Figure 4A schematic diagram showing enrichment results under different conditions according to an embodiment of the present invention is provided, wherein (a) biotin-Ab concentration, (b) reaction time, (c) pH, and (d) FQs concentration;

[0034] Figure 5 A schematic diagram of solvent standard curves obtained by using the logarithm of different FQs concentrations as the abscissa is shown according to an embodiment of the present invention, wherein (a) NOF; (b) ENO; (c) PEF; (d) LOM; (e) OFL; (f) SAR;

[0035] Figure 6 The diagram shows the MS / MS analysis results of blank milk and undiluted milk with 50 ng / mL FQs added, according to an embodiment of the present invention, wherein (a) NOF; (b) ENO; (c) PEF; (d) LOM; (e) OFL; (f) SAR;

[0036] Figure 7 The relationship between the quantity and ratio of TN@GO-SA-biotin-Ab used according to an embodiment of the present invention is shown. Detailed Implementation

[0037] Embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0038] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0039] According to one aspect of the present invention, a method for qualitative / quantitative detection of fluoroquinolone compounds is provided. The method for qualitative / quantitative detection of fluoroquinolone compounds according to embodiments of the present invention utilizes an immunoprobe for extraction. This immunoprobe has graphene oxide (GO) coated on the surface of a metal probe. GO has a large specific surface area and abundant oxygen-containing functional groups, such as carboxyl groups on its surface, which can provide abundant reaction sites for covalent coupling of biomolecules. A large amount of streptavidin can be coupled through graphene oxide, and then a broad-spectrum antibody is adsorbed onto the surface of the metal probe through streptavidin-biotin coupling. The antibody immobilization is large, resulting in high accuracy and precision of detection. Furthermore, this detection method can extract fluoroquinolone compounds without sample pretreatment, making the extraction method simple and efficient. The detection method of the embodiments of the present invention uses open-circuit mass spectrometry, which has sensitivity comparable to HPLC-MS / MS. For example, the detection limit is 0.08-0.16 ng / mL in phosphate buffered saline and 0.21-0.32 ng / mL in diluted milk. Compared with HPLC-MS / MS, this method is simple and rapid, with a sample enrichment time of only about 10 min, a detection time of <1 min, and a high recovery rate of 81.94% to 102.08%.

[0040] The detection method of this invention utilizes an immunoassay probe for extraction, significantly reducing sample preparation (dilution only) and enrichment time (only 10 minutes). Compared to existing non-mass spectrometry analysis techniques (such as enzyme-linked immunosorbent assay, magnetic relaxation immunosensors, and fluorescence sensors), this mass spectrometry-based approach can rapidly and simultaneously determine multiple analytes without chemical or enzyme labeling, providing accurate molecular weight information and avoiding false positive results.

[0041] For ease of understanding, the method for qualitative / quantitative detection of fluoroquinolone compounds is explained herein. According to an embodiment of the present invention, the method includes:

[0042] S100 provides test solution

[0043] According to an embodiment of the present invention, a test solution is provided. It should be noted that the test solution can be the original sample or a diluted sample.

[0044] S200 Extraction Processing

[0045] According to an embodiment of the present invention, an immunoprobe is contacted with the test solution for adsorption and extraction, thereby enriching the fluoroquinolone compounds in the immunoprobe to obtain an enriched immunoprobe. Thus, extraction is performed using an immunoprobe coated with graphene oxide (GO). GO has a large specific surface area and abundant oxygen-containing functional groups, such as carboxyl groups, which can provide abundant reaction sites for covalent coupling of biomolecules. A large amount of streptavidin can be coupled through graphene oxide, and then broad-spectrum antibodies are adsorbed onto the surface of the metal probe through streptavidin-biotin coupling. This results in a large amount of antibody immobilization and high detection accuracy and precision.

[0046] refer to Figure 1 According to an embodiment of the present invention, the fluoroquinolone immune probe comprises:

[0047] Metal Probe 100:

[0048] According to an embodiment of the present invention, the metal probe 100 is a tungsten needle (TN). Therefore, the tungsten needle tip spraying technique does not require complex sample pretreatment, has good conductivity and stable spraying, and offers advantages such as high sensitivity and high analysis speed when coupled with mass spectrometry. Furthermore, the tungsten needle has high chemical stability, can be cleaned with 5% nitric acid to remove adsorbed compounds, leaves no residue after cleaning, and can be reused. It features simplicity, real-time processing, speed, and low cost.

[0049] According to an embodiment of the present invention, the tungsten needle has a length of 2-10 cm and a tip length of 1-10 micrometers. Therefore, the tungsten needle with the above specifications exhibits stable electrical conductivity, high tip ionization efficiency under high voltage, low organic solvent elution, and high sensitivity for target compound detection.

[0050] Graphene oxide layer 200:

[0051] According to an embodiment of the present invention, the graphene oxide layer 200 covers at least a portion of the surface of the end of the metal probe 100, and the surface of the graphene oxide layer is modified with streptavidin. Thus, graphene oxide (GO) has a large specific surface area and abundant oxygen-containing functional groups, such as carboxyl groups on its surface, which can provide abundant reaction sites for the covalent coupling of biomolecules. A large amount of streptavidin can be coupled through graphene oxide, thereby facilitating subsequent antibody conjugation.

[0052] Antibody layer 300:

[0053] According to an embodiment of the present invention, the antibody layer is coated on at least a portion of the surface of the graphene oxide layer, and the antibody layer comprises a biotin-modified broad-spectrum antibody, wherein the broad-spectrum antibody specifically binds to fluoroquinolone compounds. Thus, through streptavidin and biotin conjugation, the broad-spectrum antibody is adsorbed onto the surface of the metal probe, resulting in a large amount of antibody immobilization and high detection accuracy and precision.

[0054] According to an embodiment of the present invention, the thickness of the graphene oxide layer is 1-3 micrometers.

[0055] According to an embodiment of the present invention, the amount of broad-spectrum antibody immobilized is approximately 10-25 micrograms per probe, that is, 10-25 μg of broad-spectrum antibody is immobilized on the surface of one fluoroquinolone compound immunoprobe. The large amount of immobilized broad-spectrum antibody is beneficial to the accuracy and sensitivity of extraction and detection.

[0056] According to an embodiment of the present invention, the broad-spectrum antibody comprises: a heavy chain variable region (VH) containing antigen-determining regions (CDRs) 1, 2, and 3; and a light chain variable region (VL) containing antigen VL CDRs 1, 2, and 3; wherein the amino acid sequences of the VH CDRs 1, 2, and 3 are as shown in SEQ ID NO: 3, 4, and 5, respectively, and the amino acid sequences of the VL CDRs 1, 2, and 3 are as shown in SEQ ID NO: 6, 7, and 8, respectively.

[0057] According to an embodiment of the present invention, the VH comprises the amino acid sequence shown in SEQ ID NO: 1, and the VL comprises the amino acid sequence shown in SEQ ID NO: 2.

[0058] According to an embodiment of the present invention, the extraction time is 8-15 minutes, preferably 10 minutes. This allows for sufficient contact and enrichment of the antibody with the fluoroquinolone compounds in the sample, which is beneficial for improving the accuracy of detection. If the extraction time is too short, the antibody will not have sufficient contact with the fluoroquinolone compounds in the sample, resulting in incomplete enrichment and leading to a lower-than-expected result. If the extraction time is too long, the antibody enrichment will have reached a plateau, resulting in an excessively long reaction time.

[0059] According to an embodiment of the present invention, the extraction process is carried out under conditions of pH 5-8. This results in high antibody binding activity on the surface of the immunoprobe and a high proportion of adsorbed fluoroquinolone compounds.

[0060] According to an embodiment of the present invention, the concentration of the fluoroquinolone compound is not higher than 20 ng / mL. If the concentration of the fluoroquinolone compound is higher than 20 ng / mL, the antibody on the surface of the immune probe is saturated and the remaining fluoroquinolone compound cannot be enriched. To further understand the aforementioned fluoroquinolone-based immunoprobe, this invention provides a method for preparing the immunoprobe, characterized by comprising: coating the surface of a metal probe tip with an adhesive liquid and making a first contact with graphene oxide powder to obtain a graphene oxide-coated probe; activating the carboxyl groups of the graphene oxide coated on the probe to obtain an activated probe; making a second contact with streptavidin to allow streptavidin to react with the activated graphene oxide to obtain a streptavidin-modified probe; performing a protein blocking treatment on the streptavidin-modified probe to obtain a blocked probe; and making a third contact with a biotin-modified broad-spectrum antibody to obtain the fluoroquinolone-based immunoprobe, wherein the broad-spectrum antibody specifically binds to the fluoroquinolone compound.

[0061] According to the method for preparing the aforementioned fluoroquinolone compound immune probes according to embodiments of the present invention, graphene oxide is adhered to the surface of a metal probe using an adhesion liquid. Graphene oxide has a large specific surface area and abundant oxygen-containing functional groups, such as carboxyl groups on its surface, which can provide abundant reaction sites for covalent coupling of biomolecules. A large amount of streptavidin can be coupled through graphene oxide, and then a broad-spectrum antibody can be adsorbed onto the surface of the metal probe through the coupling of streptavidin and biotin. The prepared immune probe has a large amount of antibody immobilization, high detection accuracy and precision, and is simple to use.

[0062] According to an embodiment of the present invention, the graphene oxide powder has a thickness of 0.8-1.2 nanometers and a width of 1-15 micrometers.

[0063] According to embodiments of the present invention, the adhesive liquid can be used as long as it can adhere GO powder to the probe, and the type is not particularly limited. For example, it can be anti-mildew 580 glass glue.

[0064] According to an embodiment of the present invention, the activation treatment is carried out using 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC). Therefore, the activation effect is good.

[0065] According to an embodiment of the present invention, the second contact time is 0.5-1.5 hours. This facilitates the full coupling of streptavidin to GO.

[0066] According to an embodiment of the present invention, the third contact time is 15-45 minutes, and the temperature is 30-45 degrees Celsius. Thus, broad-spectrum antibodies that are beneficial for biotin modification are coupled to the surface of the graphene oxide layer via the binding of biotin and streptavidin.

[0067] S300 Analysis and Testing

[0068] According to an embodiment of the present invention, the enriched immune probe is subjected to qualitative / quantitative analysis and detection.

[0069] According to embodiments of the present invention, the fluoroquinolone compound is at least one selected from norfloxacin (NOF), enoxacin (ENO), pefloxacin (PEF), ofloxacin (OFL), lomefloxacin (LOM), and sarafloxacin (SAR).

[0070] According to an embodiment of the present invention, the analytical detection is an open-type electrospray mass spectrometry detection. Using the immunoprobe of this embodiment as the sample introduction substrate, when a high voltage is applied, an electrospray is formed at the tip of the probe, which then enters the mass spectrometer for analysis.

[0071] The inventors discovered that when the spray voltage applied to the metal probe is fixed, the distance between the tip of the metal probe and the mass spectrometer inlet has a significant impact on the mass spectrometer response; both excessively large and small distances will reduce the mass spectrometer response signal. According to an embodiment of the present invention, the tip of the immunoassay probe is 2-10 mm from the open electrospray mass spectrometer inlet, preferably 5 mm.

[0072] According to an embodiment of the present invention, the spray ionization conditions for the electrospray mass spectrometry are: spray voltage: +2000-5000V. The inventors have found that when the high-voltage power supply voltage is +2000-3600V, the signal intensity of quinolone compounds increases with the increase of voltage. When the voltage is +3600-5000V, the signal intensity of six fluoroquinolone compounds decreases with the increase of voltage. Preferably, the high-voltage power supply voltage is selected as +3600V.

[0073] According to an embodiment of the present invention, the spray desorption solvent for open-cell electrospray mass spectrometry detection is a methanol solution containing 5% formic acid, and an internal standard of 30 ng / mL can be added. -1 NOFD5 and OFLD3.

[0074] According to an embodiment of the present invention, the detection conditions for the open-type electrospray mass spectrometry are: scanning mode: ESI. + Detection method: Multiple reaction monitoring (MRM); Air curtain pressure: 30 psi; Residence time: 100 ms; This makes the detection of the target object more stable, and the accuracy and sensitivity of the detection are high.

[0075] According to embodiments of the present invention, the multiple reaction monitoring (MRM) parameters of mass spectrometry for six fluoroquinolone compounds are shown in Table 1.

[0076] Table 1. MRM parameters of 6 fluoroquinolone drugs and isotope internal standards

[0077]

[0078]

[0079] * indicates quantitative ions

[0080] According to another aspect of the present invention, a kit for qualitative / quantitative detection of fluoroquinolone compounds is provided. According to an embodiment of the present invention, the kit comprises: the aforementioned immunoprobe, wherein the metal probe is prepared using the aforementioned method, the immunoprobe comprising: a metal probe; a graphene oxide layer covering at least a portion of the surface of the tip of the metal probe, and the surface of the graphene oxide layer being modified with streptavidin; and an antibody layer covering at least a portion of the surface of the graphene oxide layer, the antibody layer comprising a biotin-modified broad-spectrum antibody, wherein the broad-spectrum antibody specifically binds to fluoroquinolone compounds. Thus, this kit utilizes the specific adsorption and enrichment of fluoroquinolone compounds by the immunoprobe, exhibiting high specificity in extraction and enrichment, requiring no sample pretreatment, simplifying the extraction process, and providing high detection sensitivity, making it particularly suitable for trace extraction and detection of fluoroquinolone drugs (FQs).

[0081] According to an embodiment of the present invention, the kit further comprises: a spray desorption solvent and an immune probe.

[0082] According to embodiments of the present invention, the fluoroquinolone compound is at least one selected from norfloxacin (NOF), enoxacin (ENO), pefloxacin (PEF), ofloxacin (OFL), lomefloxacin (LOM), and sarafloxacin (SAR).

[0083] The present invention will now be described with reference to specific embodiments. It should be noted that these embodiments are merely illustrative and should not be construed as limiting the present invention.

[0084] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products, such as those purchased from Sigma.

[0085] Example 1

[0086] The method for preparing fluoroquinolone extraction probes according to embodiments of the present invention is as follows:

[0087] 1. Materials and reagents

[0088] A conical tungsten needle with a 5 cm length and a 3 μm tip was used; Submarine Anti-Mold 580 glass glue was purchased from a supermarket; Graphene Oxide (GO) solid powder (0.8-1.2 nm, thickness × 1-15 μm width, prepared using the Staudenmaier method) was provided by Beijing Innocare Technology Co., Ltd.; Streptavidin (SA), Bovine Serum Albumin (BSA), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), and a single-component ultrasensitive TMB colorimetric kit were provided by Beijing Biosen Biotechnology Co., Ltd.; and a BCA (Bicinchoninic acid) protein analysis kit was provided by Beijing Bio-Rad Biotechnology Co., Ltd.

[0089] Solid standards for norfloxacin (NOF), enoxacin (ENO), pefloxacin (PEF), ofloxacin (OFL), lomefloxacin (LOM), sarafloxacin (SAR), norfloxacin-D5, ofloxacin-D3, sulfadiazine (SDZ), and doxycycline (DXY) were provided by Shanghai Anpu Experimental Technology Co., Ltd.

[0090] The antibodies against quinolone drugs and biotinylated quinolone antibodies (species: mouse, concentration: 2.0 mg / mL, measured by NanoDrop) were donated by China Agricultural University. Sequence information is as follows:

[0091] Light chain variable region (VL)

[0092] ENVLTQSPAIMSASPGEKVTMTC RSSTGAVTTSSYLH WYQQKSSASPRLWIY STSNLAS GVPARFSGSGSGTSYSLTISSVEAEDGATYYC ALWYGYPLIFTFGSGTKLEIK (VL: SEQ ID NO: 2, CDRs1, 2 and 3: SEQ ID NO: 6, 7 and 8)

[0093] Heavy chain variable region

[0094] QVQLKESGPGLVAPSQSLSITTCTVSDFSLN NSGVH WVRQSPGKGLEWLG RIVPANGNTNFNSAFKS RLSISKDNSKSQVFLKLNSLQAVDSAKYYCAK ALSLYDY RYSMDYWGQGTSVTVSS(VH: SEQ ID NO: 1, CDRs1, 2 and 3: SEQ ID NO: 3, 4 and 5)

[0095] Biotinylated mouse IgG was provided by Beijing Bio-Sens Biotechnology Co., Ltd.

[0096] PBS buffer (pH: 7.3-7.5) was provided by Shanghai Aladdin Biochemical Technology Co., Ltd.; milk samples were provided by a local supermarket.

[0097] 2. Preparation method of extraction probe

[0098] (a) Preparation of tungsten needle tips coated with graphene oxide

[0099] 580 glass glue was uniformly coated on the surface of the tungsten needle (TN), and then it was gently pressed into GO powder so that GO was uniformly adhered to the TN surface to prepare TN@GO; then the TN@GO was placed in an oven at 60°C to dry overnight; the uncoated GO on the surface of the graphene tungsten needle tip was washed with anhydrous ethanol, dried slightly with nitrogen, and then stored in deionized water.

[0100] (b) Preparation of tungsten needle tips coated with broad-spectrum antibody-modified graphene oxide

[0101] (1) After washing TN@GO 3 times with PBS, add EDC and react at room temperature for 15 min; add 20 mg / mL SA dissolved in PBS to the reagent tube and react at room temperature for 1 h;

[0102] (2) Remove the liquid reagent in the reagent tube after the reaction, wash with PBS 3 times to obtain streptavidin-modified graphene oxide-coated tungsten needles (graphene oxide-coated tungsten needle tips).

[0103] (3) The tungsten needle tip coated with graphene oxide was placed in 50 mg / mL BSA for 1 h to block the remaining reaction sites, and then washed 3 times with PBS.

[0104] (4) The tungsten needle tip coated with graphene oxide obtained above was placed in a 20 μg / mL biotinylated fluoroquinolone antibody solution and reacted at 37°C for 30 min to obtain a tungsten needle tip coated with graphene oxide modified with a broad-spectrum antibody.

[0105] Example 2

[0106] The extraction probe prepared in Example 1 was used to detect fluoroquinolone compounds in milk, and the method is as follows:

[0107] 1. Sample preparation

[0108] Take 100 μL of milk, dilute it 10 times with PBS, and then perform the assay. Place the extraction probe prepared in Example 1 into the prepared sample and shake at room temperature for 10 min to complete the enrichment process. Finally, remove the tungsten needle tip coated with graphene oxide modified with a broad-spectrum antibody, wash it twice quickly in deionized water, and let it air dry. Analyze the dried metal probe using TNS-MS.

[0109] 2. TNS-MS analysis

[0110] (1) The tip of the extraction probe is mounted on the three-dimensional moving platform of xyz, so that the tip of the tungsten needle tip coated with graphene oxide modified with broad-spectrum antibody is located 5 mm in front of the MS inlet.

[0111] (2) Add 3 μL of methanol (containing 5% formic acid, 30 ng / mL NOFD5 and OFLD3) to the surface of the tungsten needle tip coated with graphene oxide modified with broad-spectrum antibody.

[0112] (3) A high voltage of +3.6kV was applied to the tip of the extraction probe to generate spray ionization at the tip. The analysis was performed in positive ion mode using a Qtrap 5500 mass spectrometer (AB SCIEX, Redwood, USA) and the drug was quantitatively determined in MRM mode.

[0113] (4) The ratio of the quantitative ion peak area generated by the analyte to the quantitative ion peak area of ​​the IS was used as the signal for analysis. The sum of the signals from the first three sprays was used to determine the amount of analyte. Data acquisition and analysis were performed using Analyst 1.5.1 software (AB SCIEX, Redwood, USA), and the results are as follows: Figure 2 As shown in the figure. The results indicate that the ratio gradually decreases with the increase of the number of sprays, and after the third spray, the ratio gradually approaches the measured value of 0 ng / mL. The target signal intensity is taken as the sum of the signals from the first three sprays (accounting for more than 80%).

[0114] 3. Investigate the selectivity of TN@GO-SA-biotin-Ab

[0115] Specifically, 50 ng / mL sulfadiazine (SDZ) and doxycycline (DXY) were added as system interferants to a 5 ng / mL FQs solution to investigate the selectivity of TN@GO-SA-biotin-Ab for FQs analytes. The results are as follows: Figure 3 a. Compared with TN@GO, TN@GO-SA, and TN@GO-SA-IgG, TN@GO-SA-biotin-Ab significantly increased the signal intensity of FQs. Furthermore, in the presence of high concentrations of interfering drugs, the TN@GO-SA-biotin-Ab system significantly enhanced the enrichment of FQs. The TN@GO, TN@GO-SA, and TN@GO-SA-IgG systems relied solely on non-specific adsorption of GO. QNs could be specifically recognized by TN@GO-SA-biotin-Ab. Similarly, using actual blank milk matrix samples as a control group, the addition of 5 ng / mL of FQs to the control group and milk matrix samples was compared with the results shown below. Figure 3 b indicates that TN@GO and TN@GO-SA showed no significant difference compared to the control group, while the signal of TN@GO-SA-biotin-Ab was significantly higher than that of the control group. The experimental results demonstrate that the immune probes of this invention exhibit good specificity for fluoroquinolone compounds.

[0116] 4. Optimization of TN@GO-SA-biotin-Ab conditions

[0117] Specifically, the key parameter affecting the enrichment effect was optimized: biotin-Ab concentration ( Figure 4 a) Reaction time ( Figure 4 b) pH Figure 4 c) FQs concentration ( Figure 4 d).

[0118] 5. Determination of Standard Curve

[0119] Specifically, under optimal system conditions, the linearity of fungicides (FQs) (spray solvent: methanol containing 30 ng / mL IS) was evaluated in a PBS matrix using the prepared TN@GO-SA-biotin-Ab combined with TNS-MS. The solvent standard curve obtained by plotting the logarithms of the six FQ concentrations on the x-axis is shown below. Figure 5 As shown in the results, the six FQs exhibited good exponential linearity at concentrations ranging from 0.1 to 10 ng / mL, with an LOD range of 0.08 to 0.16 ng / mL.

[0120] 6. Evaluate the analytical values ​​of TNS@GO-SA-biotin-Ab in actual samples.

[0121] Due to matrix interference in mass spectrometry detection, the MS / MS spectra of blank milk and undiluted milk supplemented with 50 ng / mL FQs were examined. Firstly, the presence of the six FQs was confirmed by peaks obtained from the adsorption and determination of the added milk using TNS@GO-SA-biotin-Ab. The verification results are as follows. Figure 6 As shown, this demonstrates that TNS@GO-SA-biotin-Ab exhibits excellent qualitative and quantitative performance in the matrix after employing this simple undiluted strategy.

[0122] 7. Methodological validation of mass spectrometry detection

[0123] This study investigated the detection of six different concentrations of fluoroquinolone antibiotics in a blank milk matrix using an open-air electrospray ionization mass spectrometry (ESI-MS) method. Specifically, certain amounts of fluoroquinolone antibiotics (FQs) were added to the blank milk matrix to achieve final concentrations of 1, 5, and 10 ng / mL. 100 μL of the spiked milk was diluted 10-fold with PBS before analysis. TN@GO-SA-biotin-Ab was placed in the diluted sample and reacted at room temperature for 10 min to complete enrichment. Finally, the TN@GO-SA-biotin-Ab was removed, washed with deionized water, dried, and analyzed by TNS-MS.

[0124] Meanwhile, using HPLC-MS / MS analysis as a control, the above milk samples were analyzed. Specifically, the spiked milk was extracted with acetonitrile, vortexed for 10 min, and centrifuged at 10000 rpm / min for 10 min. The supernatant was collected, dried under nitrogen at 40℃, and then reconstituted with 5% acetonitrile aqueous solution before HPLC-MS / MS analysis. The HPLC-MS / MS detection conditions were as follows: Chromatographic system: Shimazu LC-30AD; Mass spectrometer: AB Sciex Qtrap 5500; Zorbaxeclipse+C18 column (2.1 mm × 150 mm, 3.5 μm) (Agilent Technologies Inc., Pala). FQs were separated by chromatography at Alto, CA, USA; flow rate: 0.40 mL / min; injection volume: 5 μL; mobile phase: acetonitrile (phase A) and 0.1% FA aqueous solution (phase B); gradient elution program was used, the elution program was: initial volume ratio of phase B was 95% at 0.1 min, decreased to 85% from 0.1 min to 3 min, decreased to 0% from 3 min to 7 min, increased to 95% within 0.1 min, and held at 95% for 2.9 min, with a total run time of 10 min; mass spectrometry was performed in MRM mode with positive ion source mode; mass spectrometry parameters of FQs (precursor ion, daughter ion, collision energy, declustering voltage) were optimized by peristaltic pump injection; the spray ionization voltage was +5500 V. Data acquisition and processing were performed using Analyst 1.5.1 software. Measurements were conducted using a matrix-matched standard curve. The RSDs ranged from 1.11% to 14.44% when the corresponding spiked concentrations were 5, 20, and 50 ng / mL, respectively. The results are shown in Table 2. Compared with traditional HPLC-MS / MS analysis, this technique exhibits better accuracy, indicating that TN@GO-SA-biotin-Ab can effectively enrich fibrillated quantifiers (FQs), and TNS-MS can effectively analyze FQs.

[0125] Table 2 Precision results of TNS-MS and HPLC-MS / MS

[0126]

[0127] 9. Reproducibility test of immune probes

[0128] The reusability of TN@GO-SA-biotin-Ab was tested using 5 ng / mL FQs. Figure 7 After repeated use of TN@GO-SA-biotin-Ab three times, it was found that the proportion dropped sharply after only one use, and then tended to stabilize. This may be because antibody inactivation reduces enrichment capacity.

[0129] In the description of this specification, references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0130] Although embodiments of the invention have been shown and described, those skilled in the art will understand that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.

Claims

1. A method for qualitative or quantitative detection of fluoroquinolone compounds, characterized in that, include: Provide the solution to be tested; The immunoprobe is contacted with the test solution for adsorption and extraction, thereby enriching the immunoprobe with the fluoroquinolone compounds to obtain the enriched immunoprobe. as well as The enriched immune probes were subjected to qualitative / quantitative analysis for detection. The immune probe includes: Metal probe; A graphene oxide layer, the graphene oxide layer covering at least a portion of the surface of the metal probe tip, and the surface of the graphene oxide layer being modified with streptavidin; An antibody layer, said antibody layer being coated on at least a portion of the surface of said graphene oxide layer, said antibody layer comprising a biotin-modified broad-spectrum antibody, wherein said broad-spectrum antibody specifically binds to fluoroquinolone compounds, wherein the HCDRs:1-3 of the heavy chain variable region of said antibody are the amino acid sequences as described in SEQ ID NO:3-5, and the LCDRs:1-3 of the light chain variable region of said antibody are the amino acid sequences as described in SEQ ID NO:6-8.

2. The method according to claim 1, characterized in that, The method for preparing the immune probe includes: The surface of the metal probe tip is coated with an adhesive liquid and then comes into initial contact with graphene oxide powder to obtain a graphene oxide-coated probe. The carboxyl groups of the graphene oxide coated on the probe are activated to obtain an activated probe. The activated metal probe is brought into a second contact with streptavidin so that streptavidin reacts with the activated graphene oxide to obtain a streptavidin-modified probe. The streptavidin-modified probe was subjected to protein blocking treatment to obtain a blocked probe; and The blocked probe is then subjected to a third contact with a biotin-modified broad-spectrum antibody to obtain the fluoroquinolone compound immune probe, wherein the broad-spectrum antibody specifically binds to the fluoroquinolone compound.

3. The method according to claim 2, characterized in that, The graphene oxide powder has a thickness of 0.8-1.2 nanometers and a width of 1-15 micrometers.

4. The method according to claim 2, characterized in that, The activation treatment was carried out using 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC).

5. The method according to claim 2, characterized in that, The second contact time is 0.5-1.5 hours.

6. The method according to claim 2, characterized in that, The third contact lasts for 15-45 minutes at a temperature of 30-45 degrees Celsius.

7. The method according to claim 2, characterized in that, The metal probe is a tungsten needle.

8. The method according to claim 7, characterized in that, The tungsten needle is 2-10 cm long and has a tip of 1-10 micrometers.

9. The method according to claim 1, characterized in that, The thickness of the graphene oxide layer is 1-3 micrometers.

10. The method according to claim 1, characterized in that, The immobilization amount of the broad-spectrum antibody is 10-25 micrograms / unit.

11. The method according to claim 1, characterized in that, The extraction process takes 5-15 minutes.

12. The method according to claim 11, characterized in that, The extraction process takes 10 minutes.

13. The method according to claim 1, characterized in that, The extraction process was carried out under conditions of pH 5-8.

14. The method according to claim 1, characterized in that, The concentration of the fluoroquinolone compound is not higher than 20 ng / mL.

15. The method according to claim 1, characterized in that, The fluoroquinolone compound is selected from at least one of norfloxacin (NOF), enoxacin (ENO), pefloxacin (PEF), ofloxacin (OFL), lomefloxacin (LOM), and sarafloxacin (SAR).

16. The method according to claim 1, characterized in that, The analysis and detection were performed using open-type electrospray mass spectrometry.

17. The method according to claim 16, characterized in that, Spray voltage for open-type electrospray mass spectrometry: +2000-5000 V. The spray desorption solvent for the open-cell electrospray mass spectrometry detection contains 5% formic acid and is an internal standard. and The methanol solution, wherein the volume of the desorption solvent is 1-10 μL.

18. The method according to claim 17, characterized in that, The spray voltage for open-type electrospray mass spectrometry detection is +3600 V; the volume of the desorption solvent is 3 μL.

19. The method according to claim 16, characterized in that, The detection conditions for the open-type electrospray mass spectrometry are as follows: Scanning method: ESI + ; Detection method: Multiple reaction monitoring (MRM); Air curtain pressure: 30 psi; Dwell time: 100 milliseconds.

20. The method according to claim 16, characterized in that, The tip of the metal probe is 2-10 mm away from the open electrospray mass spectrometer inlet.

21. The method according to claim 16, characterized in that, The tip of the metal probe is 5 mm away from the open electrospray mass spectrometer inlet.

22. A kit for qualitative / quantitative detection of fluoroquinolone compounds, characterized in that, include: The immune probe according to any one of claims 1-21.

23. The reagent kit according to claim 22, characterized in that, Further includes: The spray desorption solvent is a methanol solution containing 5% formic acid and an internal standard. and .

24. The kit according to claim 22, characterized in that, The fluoroquinolone compound is selected from at least one of norfloxacin (NOF), enoxacin (ENO), pefloxacin (PEF), ofloxacin (OFL), lomefloxacin (LOM), and sarafloxacin (SAR).

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