A fluorescently labeled reagent for detecting lipoprotein subtypes, and a preparation method and application thereof

Abstract

The application provides a fluorescent labeling reagent for lipoprotein subtype detection, and a preparation method and application thereof, and belongs to the technical field of biological detection. The fluorescent labeling reagent comprises a fluorescent dye and a stabilizer. The fluorescent dye is a sulfonated derivative of fluoroboron dipyrryl 505 / 515. Through the complex optimization of the fluorescent dye and the stabilizer, the specificity and the anti-attenuation capability of the labeling reagent in combination with lipoprotein are significantly improved, the detection precision of low concentration is improved, the influence of background interference is reduced, and the influence of environmental temperature is relatively small. Compared with a traditional probe, the fluorescent labeling probe provided by the application does not have a high-concentration quenching phenomenon, the upper limit of lipoprotein detection is significantly improved, the light intensity distortion problem caused by fluorescence attenuation and sample diffusion is solved, and rapid and high-sensitivity labeling and quantitative analysis are achieved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological detection, and particularly relates to a fluorescently labeled reagent for lipoprotein subtype detection, and a preparation method and application thereof. BACKGROUND

[0002] Lipoprotein subtype detection is a key means for diagnosing cardiovascular diseases. In the prior art, an improved lipoprotein detection kit is provided in an invention patent with a patent number CN120161205B, which mixes a fluorescent probe fluoroboron dipyrromethene 505 / 515 and a crosslinking agent N,N-bis-acryloyl piperazine in a separation gel, and has the advantages of not needing to be dyed and being capable of being directly loaded for electrophoresis. However, the kit still faces challenges in actual application: the fluorescent probe is easily affected by factors such as light bleaching (background influence), environmental quenching and concentration quenching during the electrophoresis process, resulting in attenuation of the fluorescent signal; at the same time, the lipoprotein sample diffuses in the gel, causing a change in the concentration gradient. These factors act together to cause distortion of the light intensity and affect the quantitative reliability (the error of the existing algorithm is greater than 15%). SUMMARY

[0003] Based on the above problems existing in the prior art, the application provides a fluorescently labeled reagent for lipoprotein subtype detection, which comprises a fluorescent dye, a buffer and a stabilizer. The fluorescent dye is a sulfonated derivative of fluoroboron dipyrromethene 505 / 515, and the stabilizer comprises trehalose and polyvinylpyrrolidone. The stabilizer can prevent dye aggregation and ensure labeling uniformity.

[0004] The sulfonated derivative of fluoroboron dipyrromethene 505 / 515 is sulfonated BODIPY-C12, which has higher water solubility and stronger lipoprotein affinity; and the buffer is a 20 mM PBS buffer.

[0005] The concentration of the sulfonated derivative of fluoroboron dipyrromethene 505 / 515 is 0.05-0.2 g / L, which can reduce non-specific binding. The concentration of the trehalose is 30-60 g / L, and the concentration of the polyvinylpyrrolidone (PVP K30) is 5-15 g / L.

[0006] The application further provides a preparation method of the fluorescently labeled reagent for lipoprotein subtype detection, which comprises dissolving the components in the buffer, filtering to remove bacteria, and then avoiding light and dispensing.

[0007] The application further provides an application of the fluorescently labeled reagent for lipoprotein subtype detection in the preparation of a lipoprotein subtype detection kit.

[0008] The application further provides an application of the fluorescently labeled reagent for lipoprotein subtype detection in the detection of lipoprotein subtypes.

[0009] The application of the fluorescently labeled reagent for detecting lipoprotein subtypes in detecting lipoprotein subtypes comprises the following steps:

[0010] Step S1, incubate the fluorescently labeled reagent with the serum sample;

[0011] Step S2, load the incubated sample into a polyacrylamide gel electrophoresis system for separation;

[0012] Step S3, scan and analyze the gel tube after electrophoretic separation.

[0013] The electrophoretic parameters of the electrophoresis system include a voltage of 100 V, a current of 3 mA / tube, and a power of 120 W; and a light source with a wavelength of 488 nm and a high-resolution camera are used for scanning.

[0014] The present application has the beneficial effects that the specific binding of the labeled reagent to lipoprotein and the anti-fading ability are significantly improved by the complex optimized fluorescent dye and stabilizer, the detection precision of low concentration is improved, the influence of background interference is reduced, and the influence of environmental temperature is relatively small.

[0015] In addition, compared with the traditional probe, the fluorescently labeled probe provided by the present application does not have the high concentration quenching phenomenon, significantly improves the upper limit of lipoprotein detection, solves the light intensity distortion problem caused by fluorescence decay and sample diffusion, and realizes rapid and high-sensitivity labeling and quantitative analysis. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 is the electrophoresis result graph of Example Five, and the left side is three repeated samples to be tested, and the right side is two repeated quality control samples.

[0017] Figure 2 is the electrophoresis result graph of the sample dyed with the comparative example Sudan black.

[0018] Figure 3 is the functional sensitivity LOQ test verification result curve graph of Example Six.

[0019] Figure 4 is the fluorescent electrophoresis precision atlas of Example Six, and the left side is the detection result of low concentration quality control (ten repeated samples to be tested); and the right side is the detection result of high concentration quality control (ten repeated samples to be tested).

[0020] Figure 5 is the fluorescent electrophoresis precision result comparison curve graph of Example Six.

[0021] Figure 6 is the quenching phenomenon comparison result graph of the existing labeled reagent (left one) and the labeled reagent of the present application (left two to left six, five repeated samples to be tested) in the case of high concentration protein.

[0022] Figure 7 is the result of the comparison between the background interference of the existing labeling reagent (left one) and the labeling reagent of the present application (left two to left six, five repeated samples to be tested).

[0023] Figure 8 is the fluorescence electrophoretogram of the lipoprotein combined with the existing labeling reagent, and the left side is the low concentration sample, and from left to right are the 0th day, 3rd day and 7th day; the right side is the high concentration sample, and from left to right are the 0th day, 3rd day and 7th day.

[0024] Figure 9 is the stability trend graph of the lipoprotein combined with the existing labeling reagent, and the upper curve is the result curve graph of the high concentration sample, and the lower curve is the result curve graph of the low concentration sample.

[0025] Figure 10 is the fluorescence electrophoretogram of the lipoprotein combined with the labeling reagent of the present application, and the left side is the low concentration sample, and from left to right are the 0th day, 6th month and 12th month; the right side is the high concentration sample, and from left to right are the 0th day, 6th month and 12th month.

[0026] Figure 11 is the stability trend graph of the lipoprotein combined with the labeling reagent of the present application, and the upper curve is the result curve graph of the high concentration sample, and the lower curve is the result curve graph of the low concentration sample.

[0027] The existing labeling reagent refers to boron difluoride dipyrromethene 505 / 515. DETAILED DESCRIPTION

[0028] The present application will be described in detail below in combination with the drawings and specific examples.

[0029] Example 1: A fluorescent labeling reagent for lipoprotein subtype detection.

[0030] The fluorescent labeling reagent for lipoprotein subtype detection comprises a fluorescent dye, a stabilizer and a buffer, the fluorescent dye is a sulfonated derivative of boron difluoride dipyrromethene 505 / 515, specifically sulfonated BODIPY-C12, the concentration of the sulfonated derivative of boron difluoride dipyrromethene 505 / 515 is 0.1 g / L; the stabilizer comprises trehalose and polyvinylpyrrolidone, the concentration of the trehalose is 50 g / L, and the concentration of the polyvinylpyrrolidone is 10 g / L; and the buffer is 20 mM PBS buffer.

[0031] The fluorescent labeling reagent prepared in this example can be used as an independent product for serum sample pretreatment, or can be prepared into a new lipoprotein subtype detection kit in combination with the existing kit. The fluorescent labeling reagent prepared can be used for staining of serum samples, and the staining time can be shortened to 5 minutes, and the lower limit of detection (LOQ) is significantly improved.

[0032] Embodiment two: a fluorescently labeled reagent for lipoprotein subtype detection.

[0033] The difference between this embodiment and embodiment one is that the concentrations of the fluorescent dye and stabilizer are different, specifically: the concentration of the sulfonated derivative of boron difluoride dipyrromethene 505 / 515 is 0.05 g / L, the concentration of trehalose is 30 g / L, and the concentration of polyvinylpyrrolidone is 5 g / L.

[0034] Embodiment three: a fluorescently labeled reagent for lipoprotein subtype detection.

[0035] The difference between this embodiment and embodiment one is that the concentrations of the fluorescent dye and stabilizer are different, specifically: the concentration of the sulfonated derivative of boron difluoride dipyrromethene 505 / 515 is 0.2 g / L, the concentration of trehalose is 60 g / L, and the concentration of polyvinylpyrrolidone is 15 g / L.

[0036] Embodiment four: a preparation method of a fluorescently labeled reagent for lipoprotein subtype detection.

[0037] The preparation method of the fluorescently labeled reagent for lipoprotein subtype detection comprises dissolving each component in a buffer and filtering bacteria, and then storing in the dark, and the specific preparation steps comprise: first, according to the components and quantities in embodiment one, the corresponding components are weighed, each component is dissolved in ultrapure water, stirred until completely dissolved, filtered with a 0.22 μm filter to remove bacteria, and then stored in the dark at 4°C.

[0038] Embodiment five: application of a fluorescently labeled reagent for lipoprotein subtype detection in detecting lipoprotein subtypes.

[0039] The application of the fluorescently labeled reagent for lipoprotein subtype detection in detecting lipoprotein subtypes specifically comprises the following steps:

[0040] Step S1, the fluorescently labeled reagent prepared in embodiment four is mixed with a serum sample at a volume ratio of 1:10, and the mixture is incubated in the dark for 5 minutes;

[0041] Step S2, the incubated sample is directly loaded into a polyacrylamide gel electrophoresis system for electrophoretic separation, and the electrophoretic parameters include a voltage of 100 V, a current of 3 mA / tube, and a power of 120 W;

[0042] Step S3, the gel tube after electrophoretic separation is scanned and analyzed, and a light source with a wavelength of 488 nm and a high-resolution camera with a resolution of ≥2000 million pixels are used for scanning, and the exposure time is 150 ms.

[0043] Comparative example: a conventional Sudan black staining gel electrophoresis is used as a control experiment, and the specific steps are as follows:

[0044] Step S-1: 0.003 g Sudan black B was dissolved in 0.334 mL dimethyl sulfoxide, stirred for 5 min, 0.333 mL ethylene glycol was added under stirring, stirred for 3 min, and finally 0.333 mL anhydrous ethanol was added, and after mixing, a dyeing solution was obtained; 50 μL of serum sample was mixed with 10 μL of the dyeing solution, and then placed at room temperature for 25 min in the dark to obtain dyed serum;

[0045] Step S-2: 12.375 g of tris(hydroxymethyl)aminomethane and 5.5 g of boric acid were dissolved in distilled water, and the volume was made up to 1000 mL, and after mixing, an electrophoresis buffer for polyacrylamide gel electrophoresis was obtained;

[0046] Step S-3: The dyed serum of step S-1 was loaded into a polyacrylamide gel electrophoresis system, and electrophoresis was performed for 70 min; after electrophoresis, the gel column was removed, and the gel column was scanned and analyzed within 10 min.

[0047] The scanning and analysis results are shown in Figure 1 and 2 The electropherogram after labeling with the new reagent shows that the lipoprotein subtype separation band is clearer, the background noise is reduced, and the resolution is improved by 15% compared with the comparative example.

[0048] Example Six: Sensitivity and repeatability verification of the lipoprotein subtype detection application of Example Five.

[0049] The functional sensitivity LOQ specifically refers to the minimum concentration or minimum amount of the target substance that can be stably and reliably detected by the experiment or instrument, and is one of the core indicators for measuring the performance of the detection system. This parameter not only reflects the lower limit of the method, but also emphasizes the ability to maintain consistency of results in repeated experiments. This test uses a low concentration sample of low density lipoprotein cholesterol LDL-C to verify. After labeling with the fluorescent labeling reagent of the present application, the results are shown in Figure 3 : The LOQ is reduced to 0.0153 mmol / L (CV=2.8%), and the data curve is smoother, confirming that the reagent has better anti-attenuation ability.

[0050] The fluorescent labeling reagent prepared in Example One was applied to the separation gel for electrophoresis. The specific components and contents of the separation gel and the concentrated gel are as follows:

[0051] The separation gel (components other than the fluorescent labeling reagent): acrylamide is 32.5 g / L, N,N-bisacryloylpiperazine is 1.25 g / L, tris buffer is 3.75 g / L, tetramethyl ethylenediamine is 1 ml / L, and ammonium persulfate is 1 g / L; the pH value of the tris buffer is 7.9;

[0052] Stacking gel: Acrylamide is 22.5 g / L, N,N-bisacryloylpiperazine is 0.75 g / L, Tris buffer is 6 g / L, tetramethylethylenediamine is 1 ml / L, and ammonium persulfate is 1 g / L; the pH of the Tris buffer is 6.8.

[0053] After electrophoresis, each sample was placed on a dedicated tray rack. The light source and camera settings were as described in Example 5, allowing for the simultaneous capture of two-dimensional fluorescence images and minimizing time-point differences. It was found that the fluorescent labeling reagent synergistically reduced diffusion effects and significantly improved precision with the gel crosslinking agent (N,N-bisacryloylpiperazine). Figure 4 and Figure 5 As shown, in the test results of low-concentration quality control samples ( Figure 4 On the left, ten replicates of the test sample), the CV of the fluorescent labeling reagent of this invention is 3.60% ( Figure 5 The La curve shows that the CV of the existing labeled reagent is 12.55%. Figure 5 The Lb curve); in the test results of high-concentration quality control samples ( Figure 4 On the right, ten replicates of the test sample), the CV of the fluorescent labeling reagent of this invention is 2.03% ( Figure 5 The Ha curve shows that the CV of the existing labeled reagent is 8.38%. Figure 5 Hb curve).

[0054] Therefore, the fluorescent labeling reagent provided by the present invention has the following advantages:

[0055] like Figure 6 As shown: No concentration quenching phenomenon will occur: Existing labeling reagents (first from the left) exhibit quenching under high concentration backgrounds, while the fluorescent labeling reagents provided by this invention (second to sixth from the left, five replicate test samples) will not exhibit this phenomenon.

[0056] like Figure 7 As shown: Unaffected by background interference: Existing labeling reagents (first from the left) are often subject to strong background interference, and even without lipoproteins, there will be a weak background light from the staining solution; The fluorescent labeling reagents provided by this invention (second to sixth from the left, five replicates of the test sample) have strong anti-interference ability in the staining solution and do not emit light without lipoproteins.

[0057] Fluorescent protein conjugates exhibit better stability: existing labeling reagents, when bound to lipoproteins, quench within a week in the environment, such as... Figure 8 and 9 As shown, Figure 8 The left side of the image shows low-concentration samples, from left to right, representing days 0, 3, and 7; the right side shows high-concentration samples, from left to right, representing days 0, 3, and 7. Figure 9 The curve at the top represents the results for high-concentration samples, while the curve at the bottom represents the results for low-concentration samples.

[0058] The fluorescent labeling reagent provided by the present application can maintain the fluorescent effect for 0.5-1 years, for example, Figure 10 11 as shown in Figure 10 The left side in the figure is a low-concentration sample, and from left to right are the 0th day, the 6th month and the 12th month; the right side is a high-concentration sample, and from left to right are the 0th day, the 6th month and the 12th month. Figure 11 The upper curve in the figure is the result curve of the high-concentration sample, and the lower curve is the result curve of the low-concentration sample.

[0059] The above-described embodiments only express several embodiments of the present application, which are described in a more specific and detailed manner, but cannot be understood as limiting the scope of the patent of the present application. It should be noted that, for ordinary skilled persons in the art, several modifications and improvements can be made without departing from the concept of the present application, which all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.​

Claims

1. A fluorescent labeling reagent for detecting lipoprotein subtypes, comprising a fluorescent dye, a buffer solution, and a stabilizer, characterized in that, The fluorescent dye is sulfonated BODIPY-C12, the stabilizer is trehalose and polyvinylpyrrolidone, the buffer is 20mM PBS buffer, the concentration of sulfonated BODIPY-C12 is 0.05-0.2g / L, the concentration of trehalose is 30-60g / L, and the concentration of polyvinylpyrrolidone is 5-15g / L.

2. A method for preparing a fluorescently labeled reagent for lipoprotein subtype detection as described in claim 1, characterized in that, This process involves dissolving each component in a buffer solution, filtering to sterilize, and then dispensing it in the dark.

3. The application of the fluorescent labeling reagent for lipoprotein subtype detection as described in claim 1 in the preparation of a lipoprotein subtype detection kit.

4. The application of the fluorescent labeling reagent for lipoprotein subtype detection as described in claim 1 in the detection of lipoprotein subtypes.

5. The application according to claim 4, characterized in that, It includes the following steps: Step S1: Mix the fluorescent labeling reagent with the serum sample and incubate. Step S2: Load the incubated sample onto a polyacrylamide gel electrophoresis system for separation; Step S3: Scan and analyze the gel tubes after electrophoresis separation.

6. The application according to claim 5, characterized in that, The electrophoresis parameters of the electrophoresis system include a voltage of 100V, a current of 3mA / tube, and a power of 120W; a light source with a wavelength of 488nm and a high-resolution camera are used for scanning.

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