Biotin-labeled small molecule substance as well as preparation method and application thereof

Abstract

The invention relates to a biotin-labeled small molecule substance as well as a preparation method and application thereof. Biotin is used for labeling the small molecule substance through a long-chain molecule; the molecular weight of the long-chain molecules is not less than 1000Da, and the molecular weight of the small-molecular substances is less than 1000Da. The biotin-labeled small molecular substance disclosed by the invention is applied to light-activated chemiluminescence immunoassay to detect substances with small molecular weight, and has higher signal value and discrimination.

Description

Technical Field

[0001] The present invention belongs to the technical field of biochemistry, and particularly relates to a biotin-labeled small molecule substance, a preparation method thereof and an application thereof. Background Art

[0002] Common enzyme-linked immunosorbent assay (ELISA) experimental methods include the competitive method, the capture method, the indirect method, and the sandwich method. The competitive method can be used to measure antigens or antibodies; taking the measurement of antigens as an example, the antigen to be detected and the enzyme-labeled antigen compete for binding to the solid-phase antibody. Therefore, the amount of enzyme-labeled antigen bound to the solid phase is inversely proportional to the amount of the antigen to be detected; this method is generally used to detect small molecule substances with fewer epitopes.

[0003] Light Initiated Chemiluminescent Assay is a homogeneous immunoassay technique and one of the common methods in chemiluminescence analysis technology. It can be used to study the interactions between biomolecules and is mainly used for disease detection clinically. This technology integrates research in related fields such as polymer particle technology, organic synthesis, protein chemistry, and clinical detection. It generates the transfer of ionic oxygen energy and emits a light signal by the combination of photosensitive particles and luminescent particles within a certain range, thereby detecting the sample to be measured. Among them, the photosensitive particles are filled with photosensitive compounds inside, and the luminescent particles are filled with luminescent compounds and lanthanide elements. Under the excitation of red laser (600 - 700 nm), the photosensitive particles release high-energy singlet oxygen ions (4 μS), and the propagation distance is about 200 nm. When the distance between the photosensitive particles and the luminescent particles is close enough, the singlet oxygen ions released by the photosensitive particles can reach the luminescent particles and emit light at a high energy level of 520 - 620 nm through a series of chemical reactions, which is detected by the instrument. In the Light Initiated Chemiluminescent Immunoassay reaction system, the concentration of the particles is very low, the collision probability is small, and the background signal is weak. Only after the photosensitive particles and the luminescent particles are combined through an immune reaction, will obvious light be emitted. Therefore, the detection sensitivity is very high. Compared with the traditional enzyme-linked immunoassay method, it has the characteristics of homogeneity, high sensitivity, simple operation and easy automation. Therefore, its application prospect is very broad.

[0004] The detection by the method of photochemiluminescence immunoassay is based on specific pairing binding to capture target molecules and make them emit signals for detection. When this method is used to detect small molecule substances, due to the small molecular weight of the small molecule substances themselves, the molecular weight remains small after biotinylation by common methods, resulting in low detection signal values and discrimination. Moreover, the difference in molecular weight between the two substances before and after labeling is small, making it difficult to perform conventional dialysis purification, and it is difficult to obtain a relatively pure biotinylated substance. When preparing the reagent for detection, more mixtures containing the biotinylated substance need to be added, introducing more impurities, which easily interfere with normal detection, resulting in low detection signal values and discrimination. Therefore, there is an urgent need to develop a competitor that can be applied to photochemiluminescence immunoassay based on the principle of competitive method to detect substances with small molecular weights and obtain high signal values and discrimination. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the present invention provides a biotinylated small molecule substance, its preparation method and application. Applying the biotinylated small molecule substance of the present invention to detect substances with small molecular weights in photochemiluminescence immunoassay has high signal values and discrimination.

[0006] To this end, the first aspect of the present invention provides a biotinylated small molecule substance, wherein the biotin labels the small molecule substance through a long-chain molecule; the molecular weight of the long-chain molecule is not less than 1000 Da, and the molecular weight of the small molecule substance is less than 1000 Da.

[0007] In some embodiments of the present invention, the molecular weight of the biotinylated small molecule substance is 1500 Da to 20000 Da, preferably 2000 Da to 16000 Da, more preferably 3000 Da to 11000 Da, and further preferably 4000 Da to 6000 Da. For example, the molecular weight of the biotinylated small molecule substance can be 4000 Da, 4063 Da, 4500 Da, 5000 Da, 5500 Da, 6000 Da, etc.

[0008] In some embodiments of the present invention, the molecular weight of the long-chain molecule is 1000 Da to 19000 Da, preferably 1500 Da to 15000 Da, more preferably 2000 Da to 10000 Da, and further preferably 3000 Da to 5000 Da.

[0009] In some embodiments of the present invention, the long-chain molecule is a hydrophilic long-chain polymer molecule.

[0010] In some embodiments of the present invention, the long-chain molecule is selected from polyethylene glycol or dextran.

[0011] In some embodiments of the present invention, the polyethylene glycol is selected from at least one of PEG25 to PEG200; preferably, selected from at least one of PEG30 to PEG180; more preferably, selected from at least one of PEG40 to PEG150; still more preferably, selected from at least one of PEG70 to PEG120.

[0012] In some embodiments of the present invention, the dextran [C6H 10 O5]n is selected from at least one of dextrans with n = 7 to 55; preferably, selected from at least one of dextrans with n = 10 to 50; more preferably, selected from at least one of dextrans with n = 16 to 40; still more preferably, selected from at least one of dextrans with n = 20 to 30.

[0013] In some embodiments of the present invention, the molecular weight of the small molecule substance is less than 800 Da, more preferably less than 600 Da.

[0014] In some embodiments of the present invention, the small molecule substance can specifically pair and bind with a specific pairing binding member; preferably, the specific pairing binding member can also specifically pair and bind with a target molecule to be detected; more preferably, when the target molecule to be detected specifically pairs and binds with the specific pairing binding member, it has a stronger binding force than the biotin-labeled small molecule substance.

[0015] The second aspect of the present invention provides a method for preparing a biotin-labeled small molecule substance as described in the first aspect, comprising the following steps:

[0016] (1) Activate the small molecule substance with DSS to obtain small molecule substance-LC-NHS;

[0017] (2) Mix and react the small molecule substance-LC-NHS obtained in step (1) with biotin-long chain molecule-NHS to obtain a mixture containing the biotin-labeled small molecule substance;

[0018] (3) Use a desalting column to desalt and purify the mixture containing the biotin-labeled small molecule substance obtained in step (2).

[0019] In some embodiments of the present invention, in step (2), the amount of substance of the small molecule substance-LC-NHS is greater than or equal to the amount of substance of the biotin-long chain molecule-NHS.

[0020] In some embodiments of the present invention, in step (2), desalting and purification are carried out multiple times, such as 2 to 5 times.

[0021] In some embodiments of the present invention, the retention efficiency of the desalting column for substances with a molecular weight less than 1000 Da is ≥95%.

[0022] In some embodiments of the present invention, the desalting column is a Zeba desalting column.

[0023] In some embodiments of the present invention, in step (3), desalting and purification are carried out using a 0.1 M NaHCO3 buffer solution with a pH of 8.5 for elution.

[0024] The third aspect of the present invention provides an application of a biotin-labeled small molecule substance as described in the first aspect or a biotin-labeled small molecule substance prepared by the preparation method as described in the second aspect in determining whether a sample for luminescent oxygen channeling immunoassay contains a target molecule to be detected or in determining the concentration of the target molecule to be detected in a sample for luminescent oxygen channeling immunoassay.

[0025] In some embodiments of the present invention, both the target molecule to be detected and the small molecule substance can specifically pair and bind with the specific pairing binding member, and the specific pairing binding force between the target molecule to be detected and the specific pairing binding member is stronger.

[0026] In some embodiments of the present invention, the molecular weight of the target molecule to be detected is less than 1000 Da.

[0027] In some embodiments of the present invention, the target molecule to be detected is a hapten.

[0028] In some embodiments of the present invention, the target molecule to be detected is selected from hormones.

[0029] In some embodiments of the present invention, the target molecule to be detected is selected from any one of free tetraiodothyronine (FT4, molecular weight 776.93), tetraiodothyronine (T4, molecular weight 776.93), triiodothyronine (T3, molecular weight 650.97), free triiodothyronine (FT3, molecular weight 650.97), progesterone (PROG, molecular weight 314.46), testosterone (TESTO, molecular weight 288.42), estradiol (E2, molecular weight 274.39), etc.

[0030] In some embodiments of the present invention, the target molecule to be detected is free tetraiodothyronine, the small molecule substance is free triiodothyronine, and the specific pairing binding member is a tetraiodothyronine antibody.

[0031] In some embodiments of the present invention, the target molecule to be detected is thyroxine, the small molecule substance is triiodothyronine, and the specific pairing binding member is a thyroxine antibody.

[0032] In some embodiments of the present invention, the target molecule to be detected is free triiodothyronine, the small molecule substance is free 3,5-diiodo-L-thyronine, and the specific pairing binding member is a triiodothyronine antibody.

[0033] In some embodiments of the present invention, the target molecule to be detected is triiodothyronine, the small molecule substance is 3,5-diiodo-L-thyronine, and the specific pairing binding member is a triiodothyronine antibody.

[0034] The beneficial effects of the present invention are as follows:

[0035] The present invention provides a small molecule substance labeled with biotin through a long-chain molecule. The labeled substance has a relatively large molecular weight. When the small molecule substance undergoes specific pairing binding with the specific pairing binding member, the formed specific pairing binding complex also has a relatively large molecular weight, which can effectively overcome the defects of low detection signal value and low discrimination degree caused by the small molecular weight of the specific pairing binding complex in the prior art. Secondly, the long-chain molecule between biotin and the small molecule substance increases the distance between the small molecule substance and biotin, which can effectively reduce the steric hindrance of the specific pairing binding between the specific pairing binding member and the small molecule substance, thereby increasing the success rate of the specific pairing binding between the specific pairing binding member and the small molecule substance, increasing the detected signal value and the discrimination degree between the sample containing the target molecule to be detected and the sample not containing the target molecule to be detected; ultimately improving the sensitivity and accuracy of the detection.

[0036] The long-chain molecule of the present invention is selected from hydrophilic long-chain polymer molecules. First, the hydrophilicity can meet the requirements of biotin labeling small molecule substances in the aqueous phase; secondly, the molecular weight of the long-chain polymer molecule is relatively easy to control, and long-chain polymer molecules with different molecular weights can be selected according to the detection needs to prepare biotin-labeled small molecule substances. The hydrophilic long-chain polymer molecule is preferably selected from polyethylene glycol or dextran. The methods for obtaining polyethylene glycol and dextran with different molecular weights are simple and the cost is low. Different molecular weights of polyethylene glycol and dextran can be flexibly selected according to the molecular weight of the selected small molecule substance to form a biotin-labeled small molecule substance with a suitable molecular weight, so as to ensure high accuracy and sensitivity in the luminescence immunoassay of the specific pairing binding complex.

[0037] In the present invention, the molecular weight of the long-chain molecule is defined as not less than 1000 Da, and the molecular weight of the small molecule substance is less than 1000 Da; the molecular weight of the small molecule substance labeled with biotin through the long-chain molecule is quite different from that of free biotin and unlabeled small molecule substances. When preparing the target label of the present invention, it is beneficial to separate free biotin and unlabeled small molecule substances from the prepared mixture through a simple desalting and purification step, so as to obtain a target label with high purity and reduce the interference of free biotin and unlabeled small molecule substances on the detection result. The retention efficiency of a conventional desalting column for salts and small molecules (with a molecular weight less than 1000 Da) is ≥95%, and substances with a molecular weight slightly greater than 1000 Da are also easily retained in the desalting column; the molecular weight of the small molecule substance labeled with biotin in the present invention is 1500 Da to 20000 Da, which is much larger than 1000 Da, and it is not easily retained in the desalting column when using the desalting column for desalting and purification. Therefore, the desalting and purification yield of the small molecule substance labeled with biotin is also relatively high.

[0038] In addition, the inventors found that within a certain range, appropriately increasing the molecular weight of the long-chain molecule to prepare the small molecule substance labeled with biotin can increase the molecular weight difference between the small molecule substance labeled with biotin and the unlabeled small molecule substance, so that it is easier to remove the unlabeled small molecule substance from the mixture obtained after the reaction and obtain a small molecule substance labeled with biotin with higher purity. However, when the molecular weight of the long-chain molecule reaches a certain value, further increasing the molecular weight of the long-chain molecule, the increase in the purity of the small molecule substance labeled with biotin is not obvious. Similarly, within a certain range, the farther the molecular weight of the small molecule substance labeled with biotin is from 1000 Da, the less the small molecule substance labeled with biotin is retained in the desalting column during desalting and purification using a conventional desalting column, and the higher the yield of the small molecule substance labeled with biotin. However, when the molecular weight of the small molecule substance labeled with biotin reaches a certain value, further increasing the molecular weight of the small molecule substance labeled with biotin, the increase in the yield of the small molecule substance labeled with biotin is not obvious. Moreover, when the molecular weight of the small molecule substance labeled with biotin is too high, the immune complex formed during the detection process (such as: luminescent particle - specific pairing binding member - small molecule substance - long-chain small molecule - biotin - avidin - photosensitive particle) will have too large a distance between the luminescent particle and the photosensitive particle due to the too long chain, which will lead to a decrease in the signal transmission efficiency between the two or even cause the luminescent particle not to receive the singlet oxygen ion released by the photosensitive particle and thus unable to emit light, resulting in a long detection time or an error in the detection result; controlling the molecular weight of the small molecule substance labeled with biotin within 20000 Da can ensure the detection efficiency and the accuracy of the detection result.

[0039] The biotin-labeled small molecule of the present invention is used as a competitor and applied to the detection of small molecular weight substances in a luminescent oxygen channeling immunoassay (LOCI) based on the principle of competitive method, which has a high signal value and discrimination; it can be used to detect small molecular weight substances such as hormones, and solve the problems of low detection signal value and low discrimination existing in the prior art when detecting small molecular weight hormones, and has good application prospects. Detailed implementation manners

[0040] To make the present invention easy to understand, the present invention will be described in detail below. However, before describing the present invention in detail, it should be understood that the present invention is not limited to the specific implementation manners described. It should also be understood that the terms used herein are only for describing specific implementation manners and do not represent restrictive.

[0041] When a numerical range is provided, it should be understood that each intermediate value between the upper and lower limits of the range and any other specified or intermediate value in the specified range is covered within the present invention. The upper and lower limits of these smaller ranges can be independently included in the smaller ranges and are also covered within the present invention, subject to any explicit exclusions in the specified range. When the specified range includes one or both of the limits, ranges excluding either or both of the included limits are also included in the present invention.

[0042] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention, the preferred methods and materials are now described.

[0043] In the present invention, "the biotin labels the small molecule through a long-chain molecule" means that biotin is connected to the small molecule through a long-chain molecule, and can be abbreviated as "biotin-long-chain molecule-small molecule or small molecule-long-chain molecule-biotin".

[0044] In the present invention, the molecule of "biotin (abbreviated as Biotin in English)" has two ring structures, namely an imidazolone ring and a thiophene ring, and the imidazolone ring is the main part for binding to streptavidin. Activated biotin can be conjugated to almost all known biological macromolecules under the mediation of a protein cross-linking agent, including proteins, nucleic acids, polysaccharides, and lipids, etc. The "avidin" molecule is composed of 4 identical peptide chains, and each peptide chain can bind one biotin. Therefore, each antigen or antibody can be conjugated with multiple biotin molecules at the same time, thereby producing a "tentacle effect" to improve the analysis sensitivity.

[0045] In the present invention, "polyethylene glycol" (abbreviated as "PEG" in English), with the chemical formula HO(CH2CH2O) n H, is non-irritating, slightly bitter in taste, has good water solubility, and has good compatibility with many organic components.

[0046] In the present invention, "dextran" (with the chemical formula [C6H 10 O5]n) refers to a homopolysaccharide composed of glucose as the monosaccharide. Dextran has a relatively high molecular weight, and the glucose units are connected by glycosidic bonds, mainly composed of D-glucopyranose connected by α,1→6 bonds, and the branching points are connected by 1→2, 1→3, and 1→4.

[0047] In the present invention, "DSS" is dithiobis(succinimidyl propionate), with the molecular formula C 16 H 20 N2O8. DSS is an uncleavable membrane-permeable crosslinking agent, with an amine-reactive N-hydroxysuccinimide (NHS) ester at each end of an 8-carbon atom spacer arm. The NHS ester reacts with primary amines under the condition of pH = 7 - 9 to form stable amide bonds, while releasing the N-hydroxysuccinimide leaving group. Proteins (including antibodies) usually have several primary amines on the side chains of lysine (K) residues and at the N-terminus of each polypeptide, and these primary amines can be used as targets for NHS ester crosslinking reagents. DSS is first dissolved in an organic solvent (such as DMF or DMSO), and then added to the aqueous crosslinking reaction.

[0048] In the present invention, "NHS" is N-hydroxysuccinimide, with the chemical formula C4H5NO3, a white crystalline substance, soluble in water, and easily soluble in acetone, alcohol, and ethyl acetate; it can be used for bioconjugation, crosslinking, labeling, and immobilization.

[0049] In the present invention, "DMSO" is dimethyl sulfoxide, with the chemical formula C2H6OS, a sulfur-containing organic compound, a colorless, odorless, transparent liquid at room temperature, and a hygroscopic flammable liquid; DMSO has the characteristics of high polarity, high boiling point, good thermal stability, aprotic, and miscible with water, and can dissolve in most organic substances such as ethanol, propanol, benzene, and chloroform, and is known as the "universal solvent".

[0050] In the present invention, "DIPEA" is N,N-diisopropylethylamine, with the chemical formula C8H 19 N, a colorless or light yellow transparent liquid, used in organic synthesis.

[0051] In the present invention, the luminescent particles can react with reactive oxygen species to produce detectable chemiluminescence signals. The photosensitive particles can generate reactive oxygen species in the excited state.

[0052] Example

[0053] To make the present invention more easily understood, the present invention will be further described in detail below in conjunction with examples. These examples are for illustrative purposes only and are not limited to the application scope of the present invention. The raw materials or components used in the present invention can be obtained through commercial channels or conventional methods without special instructions.

[0054] Example 1: Preparation of Biotinylated Small Molecule Substance (T2-PEG-Biotin)

[0055] 1.1 Experimental Raw Materials and Equipment

[0056] The raw materials and equipment used in the preparation process are shown in Table 1 and Table 2 respectively.

[0057] Table 1: Raw Materials

[0058] 3,5-Diiodo-L-thyroxine (T2) DSS DMSO DIPEA Biotin-PEG-NHS (588 Da) Biotin-PEG-NHS (1000 Da) Biotin-PEG-NHS (3400 Da) Biotin-PEG-NHS (10000 Da) Biotin-PEG-NHS (20000 Da) <![CDATA[0.1M NaHCO3 (pH = 8.5)]]> Zeta desalting column

[0059] Table 2: Equipment

[0060] LCD digital control rotary mixer Freezing centrifuge

[0061] 1.2 Preparation Process

[0062] (1) Weigh 8 μmol of T2 and dissolve it in 600 μL of DMSO. Take a centrifuge tube and transfer the prepared solution to the centrifuge tube.

[0063] (2) Weigh 8 μmol of DSS and dissolve it in 114.25 μL of DMSO, and then add the prepared DSS solution to the centrifuge tube in step (1).

[0064] (3) Add 174 μL of DMSO and 100 μL of DIPEA to the centrifuge tube obtained in step (2) in sequence. After vortex mixing, place it on a rotary mixer and react overnight at room temperature and 60 rpm to obtain 8 μmol / mL of T2-LC-NHS.

[0065] (4) Take 5 centrifuge tubes and add 0.4 μmol of Biotin-PEG-NHS (588 Da), Biotin-PEG-NHS (1000 Da), Biotin-PEG-NHS (3400 Da), Biotin-PEG-NHS (10000 Da), and Biotin-PEG-NHS (20000 Da) respectively. Then add 0.4 μmol of the T2-LC-NHS obtained in step (3) to each tube, and supplement 0.1 M NaHCO3 buffer to make the concentration of T2-LC-NHS 0.4 μmol / mL. React at room temperature with 60 rpm for 3 - 6 h to obtain a mixed solution containing T2-PEG-Biotin.

[0066] (5) Use a Zeba desalting column to desalt and purify the mixed solution containing T2-PEG-Biotin obtained in step (4), and thus obtain purifications of T2-PEG-Biotin (1250 Da), T2-PEG-Biotin (1663 Da), T2-PEG-Biotin (4063 Da), T2-PEG-Biotin (10663 Da), and T2-PEG-Biotin (20663 Da).

[0067] Among them, the desalting and purification process in step (5) includes the following steps:

[0068] First centrifugation: Take the desalting column, remove the tail of the desalting column, and loosen the lid of the desalting column. Place it in a suitable centrifuge tube and weigh it on an electronic balance for balance. Centrifuge at 2 - 8 °C (set at 4 °C), 1000 g, for 5 min.

[0069] Second centrifugation: Replace with a new centrifuge tube, add 0.1 M NaHCO3 (pH = 8.5) buffer to the desalting column, and weigh it on an electronic balance for balance. Centrifuge at 2 - 8 °C (set at 4 °C), 1000 g, for 5 min.

[0070] Third centrifugation: Replace with a new centrifuge tube, add 0.1 M NaHCO3 (pH = 8.5) buffer to the desalting column, and weigh it on an electronic balance for balance. Centrifuge at 2 - 8 °C (set at 4 °C), 1000 g, for 5 min.

[0071] Fourth centrifugation: Replace with a new centrifuge tube, add the mixed solution containing T2-PEG-Biotin obtained in step (4) to the desalting column, and weigh it on an electronic balance for balance. Centrifuge at 2 - 8 °C (set at 4 °C), 1000 g, for 5 min. Collect the liquid in the centrifuge tube, which is the purified reagent.

[0072] Example 2: Preparation of biotin-labeled small molecule substance (T3-dextran-Biotin)

[0073] 2.1 Experimental raw materials and equipment

[0074] The raw materials and equipment used in the preparation process are shown in Table 3 and Table 4 respectively.

[0075] Table 3: Raw materials

[0076] Triiodothyronine (T3) DSS DMSO DIPEA Biotin-dextran-NHS (3000 Da) Biotin-dextran-NHS (10000 Da) Zeta desalting column <![CDATA[0.1M NaHCO3 (pH = 8.5)]]>

[0077] Table 4: Equipment

[0078] LCD digital control rotary mixer Freezing centrifuge

[0079] 2.2 Preparation process

[0080] (1) Weigh 7 μmol of T3 and dissolve it in 600 μL of DMSO. Take a centrifuge tube and transfer the prepared solution into the centrifuge tube.

[0081] (2) Weigh 7 μmol of DSS and dissolve it in 114.25 μL of DMSO, and then add the prepared DSS solution into the centrifuge tube in step (1).

[0082] (3) Add 174 μL of DMSO and 100 μL of DIPEA into the centrifuge tube obtained in step (2) in sequence. After vortex mixing, place it on a rotary mixer and react overnight at room temperature and 60 rpm to obtain T3-LC-NHS with a concentration of 7 μmol / mL.

[0083] (4) Take 2 centrifuge tubes, add 0.4 μmol of Biotin-dextran-NHS (3000 Da) and Biotin-dextran-NHS (10000 Da) respectively, and then add 0.45 μmol of T3-LC-NHS obtained in step (3) to each tube. Add 0.1 M NaHCO3 buffer to make the concentration of T3-LC-NHS 0.45 μmol / mL, and react at room temperature and 60 rpm for 3 - 6 h to obtain a mixed solution containing T3-dextran-Biotin.

[0084] (5) Use a Zeba desalting column to desalt and purify the mixed solution containing T3-dextran-Biotin obtained in step (4), and two pure substances containing T3-dextran-Biotin are obtained.

[0085] Among them, the desalting and purification process in step (5) includes the following steps:

[0086] First centrifugation: Take the desalting column, remove the tail of the desalting column, and loosen the lid of the desalting column. Place it in a suitable centrifuge tube and weigh it on an electronic balance for balancing. Centrifuge at 2 - 8 °C (set at 4 °C), 1000 g, for 5 min.

[0087] Second centrifugation: Replace with a new centrifuge tube, add 0.1M NaHCO3 (pH = 8.5) buffer solution to the desalting column, and weigh and balance with an electronic balance. Centrifuge at 2 - 8°C (set at 4°C), 1000g, for 5 minutes.

[0088] Third centrifugation: Replace with a new centrifuge tube, add 0.1M NaHCO3 (pH = 8.5) buffer solution to the desalting column, and weigh and balance with an electronic balance. Centrifuge at 2 - 8°C (set at 4°C), 1000g, for 5 minutes.

[0089] Fourth centrifugation: Replace with a new centrifuge tube, add the mixed solution containing T3 - dextran - Biotin obtained in step (4) to the desalting column, and weigh and balance with an electronic balance. Centrifuge at 2 - 8°C (set at 4°C), 1000g, for 5 minutes. Collect the liquid in the centrifuge tube, which is the purified reagent.

[0090] Application Example 1: The process and results of using the biotin - labeled small molecule (T2 - PEG - Biotin) prepared in Application Example 1 as a competitor to detect free triiodothyronine (FT3) on a photo - excited chemiluminescence immunoassay platform are as follows:

[0091] (1) Dilute the T3 - coated luminescent microparticles with HEPES buffer to 30 μg / mL as Reagent 1; dilute the purified product containing T2 - PEG - Biotin obtained after desalting and purification in step (5) of Example 1 with Tris buffer to a concentration of 1 μg / mL as Reagent 2; dilute the streptavidin - coated photosensitive microparticles with HEPES buffer to 30 μg / mL as Reagent 3.

[0092] (2) Take 25 μL each of Reagent 1, Reagent 2, and the sample to be detected, mix well, and incubate at 37°C for 15 minutes.

[0093] (3) Add 175 μL of Reagent 3 to the mixed solution obtained in step (2), and incubate at 37°C for 10 minutes.

[0094] (4) Perform photo - excited chemiluminescence immunoassay on the mixed solution obtained in step (3).

[0095] Six samples with FT3 concentrations of 0, 1.53, 4.00, 7.96, 13.73, 52.14 pg / mL were detected, and the signal values and signal value inhibition rates obtained from the photo - excited chemiluminescence immunoassay are shown in Table 5.

[0096] Table 5 Detection signal values and inhibition rate data of samples containing FT3

[0097]

[0098] In Table 5:

[0099] 1. Inhibition rate = current signal value / zero - point signal value, where the zero - point signal value is the signal value of the sample with a concentration of 0 pg / mL. The inhibition rate can illustrate the sensitivity of the detected mixture to the change in FT3 concentration. A low inhibition rate indicates high sensitivity.

[0100] 2. Overall discrimination degree = multiple between the signal value of the lowest concentration and the signal value of the highest concentration.

[0101] It can be known from the results in Table 5:

[0102] When the molecular weight of T2 - PEG - Biotin is 1250 Da, the overall sample signal value is relatively low, and the overall discrimination degree of the detected mixture is 17.7;

[0103] When the molecular weight of T2 - PEG - Biotin increases from 1250 Da to 1663 Da, based on the data when the molecular weight of T2 - PEG - Biotin is 1250 Da, the average signal value of samples with different FT3 concentrations increases by 12 times, the inhibition rate at the highest concentration increases by 2 times, and the overall discrimination degree of the detected mixture is 36.2;

[0104] When the molecular weight of T2 - PEG - Biotin increases from 1663 Da to 4063 Da, based on the data when the molecular weight of T2 - PEG - Biotin is 1250 Da, the average signal value of samples with different FT3 concentrations increases by 24 times, the inhibition rate at the highest concentration increases by 14 times, and the overall discrimination degree of the detected mixture is 243.2;

[0105] When the molecular weight of T2 - PEG - Biotin increases from 4063 Da to 10663 Da, based on the data when the molecular weight of T2 - PEG - Biotin is 1250 Da, the average signal value of samples with different FT3 concentrations increases by 24 times, the inhibition rate at the highest concentration increases by 2 times, and the overall discrimination degree of the detected mixture is 39.6;

[0106] When the molecular weight of T2 - PEG - Biotin increases from 10663 Da to 20663 Da, based on the data when the molecular weight of T2 - PEG - Biotin is 1250 Da, the average signal value of samples with different FT3 concentrations increases by 4 times, the inhibition rate at the highest concentration is the same, and the overall discrimination degree of the detected mixture is 15.0.

[0107] Application Example 2: The process and results of using the biotin - labeled small molecule substance (T3 - dextran - Biotin) prepared in Application Example 2 as a competitor to detect free tetraiodothyronine (FT4) on a photo - initiated chemiluminescence immunoassay platform are as follows:

[0108] (1) Dilute the T4-coated luminescent microparticles with HEPES buffer to 30 μg / mL as Reagent 1; dilute the purified product containing T3-dextran-Biotin obtained by desalting and purification in step (5) of Example 2 with Tris buffer to a concentration of 1 μg / mL as Reagent 2; dilute the streptavidin-coated photosensitive microparticles with HEPES buffer to 30 μg / mL as Reagent 3.

[0109] (2) Take 25 μL each of Reagent 1, Reagent 2, and the sample to be detected, mix well, and incubate at 37 °C for 15 min.

[0110] (3) Add 175 μL of Reagent 3 to the mixture obtained in step (2), and incubate at 37 °C for 10 min.

[0111] (4) Perform photoactivated chemiluminescence immunoassay on the mixture obtained in step (3).

[0112] Six samples with concentrations of 0, 4.55, 7.46, 15.45, 27.9, and 85.55 pg / mL of FT4 were detected, and the signal values and signal value inhibition rates obtained from the photoactivated chemiluminescence immunoassay are shown in Table 6.

[0113] Table 6 Detection signal values and inhibition rate data of samples containing FT4

[0114]

[0115] From the comprehensive application of the results of Example 1 and Application Example 2, it can be seen that the biotin-labeled small molecule substances of the present invention have universality. Using PEG or dextran as the long-chain molecule and connecting biotin to the small molecule through the long-chain molecule to form biotin-long-chain molecule-small molecule substances for the detection of substances with relatively small molecular weights can obtain relatively high signal values and discrimination degrees.

[0116] It should be noted that the above-described embodiments are only used to explain the present invention and do not constitute any limitation to the present invention. The present invention has been described by referring to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory words rather than limiting words. Modifications can be made to the present invention within the scope of the claims of the present invention as provided, and the present invention can be revised without departing from the scope and spirit of the present invention. Although the present invention described therein relates to specific methods, materials, and embodiments, it does not mean that the present invention is limited to the specific examples disclosed therein. On the contrary, the present invention can be extended to all other methods and applications with the same functions.

Claims

1. A biotin-labeled small molecule substance, characterized in that, The biotin labels the small molecule through a long-chain molecule; the molecular weight of the long-chain molecule is not less than 1000 Da, and the molecular weight of the small molecule is less than 1000 Da.

2. The biotin-labeled small molecule substance according to claim 1, wherein The molecular weight of the biotin-labeled small molecule is 1500 Da to 20000 Da, preferably 2000 Da to 16000 Da, more preferably 3000 Da to 11000 Da, and further preferably 4000 Da to 6000 Da; and / or The molecular weight of the long-chain molecule is 1000 Da to 19000 Da, preferably 1500 Da to 15000 Da, more preferably 2000 Da to 10000 Da, and further preferably 3000 Da to 5000 Da; and / or The molecular weight of the small molecule is less than 800 Da, and more preferably less than 600 Da.

3. The biotin-labeled small molecule substance according to claim 1 or 2, characterized in that, The long-chain molecule is a hydrophilic long-chain polymer molecule.

4. The biotin-labeled small molecule substance according to any one of claims 1-3, characterized in that The long-chain molecule is selected from polyethylene glycol or dextran.

5. The biotin-labeled small molecule substance according to claim 4, characterized in that, The polyethylene glycol is selected from at least one of PEG25 to PEG200; more preferably, selected from at least one of PEG30 to PEG180; further preferably, selected from at least one of PEG40 to PEG150; still more preferably, selected from at least one of PEG70 to PEG120; and / or The dextran [C6H 10 O5]n is selected from at least one of dextrans with n = 7 to 55; more preferably, selected from at least one of dextrans with n = 10 to 50; further preferably, selected from at least one of dextrans with n = 16 to 40; still more preferably, selected from at least one of dextrans with n = 20 to 30.

6. The biotin-labeled small molecule substance according to any one of claims 1-5, characterized in that, The small molecule can specifically pair and bind with a specific pairing binding member; preferably, the specific pairing binding member can also specifically pair and bind with a target molecule to be detected; more preferably, when the target molecule to be detected specifically pairs and binds with the specific pairing binding member, it has a stronger binding force than the biotin-labeled small molecule.

7. A method for preparing a biotin-labeled small molecule substance according to any one of claims 1-6, characterized in that, The preparation method includes the following steps: (1) Activate the small molecule with DSS to obtain small molecule-LC-NHS; (2) Mix and react the small molecule-LC-NHS obtained in step (1) with biotin-long-chain molecule-NHS to obtain a mixture containing the biotin-labeled small molecule; (3) Use a desalting column to desalt and purify the mixture containing the biotin-labeled small molecule obtained in step (2).

8. The preparation method according to claim 7, wherein In step (2), the amount of substance of the small molecule-LC-NHS is greater than or equal to the amount of substance of the biotin-long-chain molecule-NHS.

9. Use of a biotin-labeled small molecule as described in any one of claims 1-6 or a biotin-labeled small molecule prepared by the preparation method as described in any one of claims 7-8 in determining whether a target molecule to be detected is contained in a sample for photochemiluminescence immunoassay or in determining the concentration of the target molecule to be detected in a sample for photochemiluminescence immunoassay; preferably, both the target molecule to be detected and the small molecule can specifically pair and bind with a specific pairing binding member, and the specific pairing binding force between the target molecule to be detected and the specific pairing binding member is stronger.

10. The application according to claim 9, wherein The molecular weight of the target molecule to be detected is less than 1000 Da; preferably, the target molecule to be detected is a hapten; more preferably, the target molecule to be detected is selected from hormones; further preferably, the target molecule to be detected is selected from any one of free tetraiodothyronine, tetraiodothyronine, triiodothyronine, free triiodothyronine, progesterone, testosterone, and estradiol.

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