Method for rapidly evaluating sperm motility and quality in multiple dimensions

Abstract

The invention discloses a method for rapidly constructing a fluorescent array sensor and application of the fluorescent array sensor, and the method comprises the following steps: through four-component Ugi chemical reaction, synthesizing substituted amide fluorescent small molecules by using a carboxyl chemical substance, an amino chemical substance, a ketone or aldehyde chemical substance and an isonitrile compound which are equal in molar weight to construct an element library; and screening out a plurality of elements with the maximum contribution through a machine learning algorithm to quickly and efficiently construct the array sensor. The fluorescent array sensor disclosed by the invention can simultaneously screen healthy sperm samples and sperm samples of different types of diseases, the to-be-screened sperm sample can be cryopreserved for a long time and does not need to be used after being taken, the sperm sample of a to-be-diagnosed person can be collected at home, and the embarrassment of taking the sperm sample from a hospital toilet is avoided. Meanwhile, the array sensor has the advantages of high sensitivity, short detection time, low cost, good repeatability, high accuracy, no need of professional technicians and the like, and semen quality problems caused by different factors can be quickly identified within one minute.

Description

Technical Field

[0001] The present invention relates to rapid detection of biological diseases, and in particular to a technology for rapidly constructing a fluorescence array sensor based on a combinatorial chemical strategy and used for rapidly detecting human sperm motility and quality. Background Art

[0003] At present, male infertility is clinically divided into two categories: sexual dysfunction and normal sexual function. The latter can be further divided into azoospermia, oligospermia, asthenozoospermia, asthenozoospermia, etc. according to the results of semen analysis. Male fertility is evaluated clinically by observing sperm quality or sperm motility. The common clinical indicators currently include sperm morphology, survival rate, motility, acrosome membrane integrity and sperm mitochondrial activity. The gold standard for sperm quality assessment adopted by the World Health Organization (WHO) mainly relies on microscopes and counting chambers. These methods can evaluate multiple parameters, including semen volume, sperm count, motility, morphology, survival rate and DNA integrity. In the past few years, new clinical assessment methods for sperm quality have been introduced, such as modern computer-assisted sperm analysis (CASA) and quantitative phase microscopy (QPM). Although these methods are practical and widely used, they still have some shortcomings. First, the standardized operation stipulates that microscopic sample analysis must be completed within 1 hour after semen collection to prevent dehydration or temperature changes from affecting semen quality. This regulation creates time urgency and requires that the examinee must collect samples in the hospital. Secondly, the test requires the use of advanced instruments and the training of experienced doctors, which increases the cost of the test. At the same time, the evaluation of sperm is also very subjective, and the test procedure is complicated, which cannot achieve high-throughput testing. Therefore, there is an urgent need for a simple, convenient, and highly standardized routine semen test method to make up for the shortcomings of traditional semen testing.

[0004] Array sensors are developed and designed through the mammalian taste system. Through its unique parallel detection mechanism and the cross-response of nonspecific interactions, it can better distinguish similar substances and complex samples. The composition of semen is complex, and it is very difficult to design corresponding components for it. Therefore, a strategy for quickly generating a component library is needed. Through machine learning algorithms, efficient components are screened from the library to achieve the detection of sperm samples. In order to solve this problem, combinatorial chemistry methods, especially Ugi reactions, provide a strategy for quickly constructing a variety of sensor units. Ugi reactions can achieve four-component condensation through a one-pot method under mild conditions. It has the characteristics of simple operation, high yield, good reaction selectivity, and high atom economy. The sensor unit constructed by the Ugi reaction can quickly generate a compound library with diverse structures. This method not only improves the sensitivity and reliability of detection, but also greatly reduces the workload of synthesis. Summary of the invention

[0005] Purpose of the invention: In view of the shortcomings of existing sperm quality detection technology, a technology based on fluorescent array sensors is provided to quickly identify healthy and diseased sperm samples. This technology can quickly detect multiple sperm samples at the same time, requiring a small number of samples, short detection time, and low cost. Another purpose of this patent is to provide a new array sensor unit construction strategy to achieve rapid synthesis and detection of multi-channel sensor elements.

[0006] Technical solution: A fluorescent array sensor for rapid detection of semen, comprising a plurality of fluorescent compounds as shown in the following general formula:

[0007]

[0008] in:

[0009] F is a fluorescent group, which can be independently selected from: pyrene, anthracene, coumarin, fluorescein, boron fluoride dye (BODIPY), naphthalimide, NBD, rhodamine, cyanine dye (Cyanine);

[0010] The R1 and R2 groups together constitute: substituted or unsubstituted C1-15 straight or branched alkyl and cycloalkyl, 5-6 membered saturated heterocycle or 5-6 membered aromatic heterocycle; the substitution is -N(CH3)2, -N(CH2CH2CH3)2, -NHCH3, halogen, nitrile, nitro, hydroxyl, carboxyl, ester substitution; the 5-6 membered saturated heterocycle or 5-6 membered aromatic heterocycle are heterocycles containing one or two atoms of nitrogen and oxygen;

[0011] The R3 and R4 groups can be independently selected from: hydrogen, hydroxyl-substituted or unsubstituted C1-10 straight or branched alkyl, phenol, nitrogen-containing or oxygen-containing 5-6 membered saturated heterocycle or nitrogen-containing or oxygen-containing 5-6 membered aromatic heterocycle;

[0012] or R3 and R4 groups together constitute: substituted or unsubstituted 5-6 membered cycloalkyl, substituted or unsubstituted benzene ring, substituted or unsubstituted 5-6 membered saturated heterocyclic ring or substituted or unsubstituted 5-6 membered aromatic heterocyclic ring; the substitution is C 1-6 Alkyl, halogen, nitrile, nitro, hydroxyl, carboxyl, ester substitution;

[0013] The R5 groups can be independently selected from: hydrogen, C1-10 straight or branched alkyl;

[0014] n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms.

[0015] Preferably, the fluorescent array sensor for rapid detection of semen comprises 10-20 fluorescent compounds as shown in the general formula. More preferably, the fluorescent array sensor for rapid detection of semen comprises 14 fluorescent compounds as shown in the general formula.

[0016] The fluorescent array sensor for rapid detection of semen,

[0017] The R3 and R4 groups can be independently selected from: hydrogen, hydroxy substituted or unsubstituted C1-10 straight or branched alkyl, phenol, pyridyl, furanyl;

[0018] The R5 groups can be independently selected from: hydrogen, C1-8 straight or branched alkyl;

[0019] n is 2 to 10 carbon atoms.

[0020] The fluorescent array sensor for rapid detection of semen, R1 and R2 groups together constitute:

[0021]

[0022] In the fluorescent array sensor for rapid detection of semen, the R3 and R4 groups are independently selected from:

[0023]

[0024] Or R3 and R4 groups together constitute:

[0025]

[0026] In the fluorescent array sensor for rapid detection of semen, the R5 group is:

[0027]

[0028] In the fluorescent array sensor for rapid detection of semen, the fluorescent compound is selected from the following:

[0029]

[0030]

[0031] A method for preparing the fluorescent array sensor comprises adopting Ugi chemical reaction, dissolving carboxyl chemicals, amino chemicals, ketone or aldehyde chemicals and isonitrile compounds in methanol, mixing the four components to react, obtaining a fluorescent compound shown in the general formula, and combining the synthesized multiple fluorescent compounds and diluting or not diluting with a buffer solution to obtain the fluorescent array sensor;

[0032]

[0033] R1, R2, R3, R4, R5, n, and F are as defined above.

[0034] An in vitro diagnostic reagent comprises the fluorescent array sensor.

[0035] A method for rapidly detecting human sperm motility and quality using the fluorescent array sensor comprises adding a diluted sperm sample to the fluorescent array sensor, shaking the sensor, reading the fluorescence signal change with an enzyme marker, and processing the fluorescence data using linear discriminant analysis in statistical analysis software.

[0036] The application of the fluorescent array sensor or the in vitro diagnostic reagent in rapid detection of sperm quality.

[0037] Furthermore, through the four-component Ugi chemical reaction, equimolar amounts of carboxyl chemicals, amino chemicals, ketone or aldehyde chemicals and isonitrile compounds are dissolved in methanol, and the four components are mixed and reacted at room temperature to obtain products. Combinatorial chemistry technology is used to prepare a library of amide fluorescent small molecule compounds with different fluorophores and different side chain substitutions for the construction of fluorescent array sensors. The general formula of the compound structure is as follows:

[0038]

[0039] in:

[0040] The F fluorescent groups can be independently selected from: pyrene, anthracene, coumarin, fluorescein, boron fluoride dye (BODIPY), naphthalimide, benzoxadiazole (NBD), rhodamine, cyanine dye (Cyanine);

[0041] The R1 and R2 groups can be independently selected from: optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heterocyclic group, optionally substituted heterochain hydrocarbon group, optionally substituted alkoxy, alkylsulfonyl, halogen, cyano, hydroxyl, nitro, alkyl, and haloalkyl.

[0042]

[0043] The R3 and R4 groups can be independently selected from: optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heterocyclic group, optionally substituted heterochain hydrocarbon group, optionally substituted alkoxy, alkylsulfonyl, hydrogen, halogen, cyano, hydroxyl, nitro, alkyl, and haloalkyl.

[0044] The R5 groups can be independently selected from: optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted heterochain alkyl, optionally substituted alkoxy, alkylsulfonyl, halogen, cyano, hydroxyl, nitro, alkyl, and haloalkyl.

[0045] In the "optionally substituted" group, the substituent is selected from halogen, hydroxyl, nitro, alkyl, hydroxyalkyl, cycloalkyl, alkoxy, sulfonyl, heterocyclic, and aromatic; the alkyl contained in the "alkyl", "haloalkyl", "alkylsulfonyl", and "alkoxy" is a C1-C20 straight or branched alkyl; the "cycloalkyl" is a C3-C6 cycloalkyl, and optionally, the cycloalkyl includes cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl; the "aryl" is a 5-10 membered aromatic group; optionally, the aromatic group includes phenyl , naphthyl; the "heteroaryl" is a 5-10 membered heteroaryl containing 1-3 heteroatoms selected from N, O and S; the heterocyclic structure in the "heterocyclic group" is a saturated or unsaturated, 3-6 membered monocyclic or polycyclic non-aromatic ring structure containing 1-3 heteroatoms selected from N, O and S on the ring; the heterochain hydrocarbon structure in the "heterochain hydrocarbon group" is a C1-C20 saturated or unsaturated, straight or branched heterochain structure containing 1-3 heteroatoms selected from N, O and S on the chain; the "halogen" includes fluorine, chlorine, bromine and iodine. Preferably, the R1 and R2 groups can be independently selected from: C1-C14 straight or branched alkyl, substituted or unsubstituted C3-C6.

[0046] cycloalkyl, substituted or unsubstituted aryl or heteroaryl, substituted or unsubstituted C3-C6 heterocyclyl, straight or branched C1-C10 heterochain hydrocarbon, branched or branched C1-C10 haloalkyl; R3 and R4 groups can be independently selected from: C1-C12 straight or branched alkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted aryl or heteroaryl, substituted or unsubstituted C5 or C6 heterocyclyl; R5 groups can be independently selected from: C1-C8 straight or branched alkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C3-C6 heterocyclyl, substituted or unsubstituted aryl; R4 groups can be independently selected from: monosubstituted or Polysubstituted hydrogen, halogen, trifluoromethyl, trifluoromethoxy, nitro, methoxy, amino, hydroxyl, carboxyl, anhydride, ester, amide, substituted or unsubstituted C1-C10 straight or branched alkyl and cycloalkyl, substituted or unsubstituted amino or urea, substituted or unsubstituted straight or branched alkoxy; n is 0-10; the "heteroaryl" is a C5 or C6 heteroaryl containing one heteroatom selected from N, O, and S; the "halogen" is fluorine, chlorine, and bromine; the "aryl" is a C5 or C6 monocyclic or bicyclic aromatic ring structure; the heterochain hydrocarbon structure in the "heterochain hydrocarbon group" is a C1-C6 saturated heterochain structure containing one heteroatom selected from N, O, and S on the chain.

[0047] n is 1 to 10 carbon atoms.

[0048] More preferably:

[0049] The R1 and R2 groups can be 3-methylpentyl, 2-methylpentyl, 4-methylpentyl, propyl, butyl, decyl, 2,2-dimethyl-1,3-propanediol, 1-methoxypropane, 1,1,1-trifluoropropane, butanol, 2-propoxy-1-ethanol, (S)-butane-1,2-diol, tributylamine, methyl (propyl) sulfanyl, N,N-dimethyl-1-propylamine, 4-methyltetrahydro-2H-pyranyl, methylcyclopropane 1-propyl-1H-indolyl, methyl-2-thiophene, 2-ethyl-1-methyl-2-thiophene, 2-ethyl ...

[0050] The R3 and R4 groups can be pentyl, dodecyl, octanal, undecyl, methylcyclohexyl, 2-methylpentyl, 3-methylbutan-1-ol, propional, heptal, 4-methylpyridyl, decanal, 5-hydroxypentanal, 4-methyltetrahydro-2H-pyranyl, 1.4-dimethylpiperidinyl, propylphenyl, 2-methylfuranyl, p-cresol, 3.5-dimethoxybenzaldehyde, 4-methoxybenzaldehyde, 4-chlorobenzaldehyde, 4-bromobenzaldehyde, 4-nitrobenzaldehyde, 2-naphthaldehyde, benzofuran-5-carboxaldehyde, benzo[b]thiophene-2-carboxaldehyde, and 4-(pyrrolidin-1-yl)benzaldehyde.

[0051] The R5 group can be pentyl, methylcyclopropane, 2-methylpentyl, isobutyl, neopentyl, 4-propylmorpholinyl, 2,2,4,4-tetramethylpentyl, 1-methoxybutane, isopropylcyclopropane, methylcyclohexane, 4-propylmorpholinyl, ethylphenyl, 1-(ethylsulfinyl)-4-methylphenyl

[0052] More preferably, the R1 and R2 groups may be:

[0053]

[0054] More preferably, the R3 and R4 groups may be:

[0055]

[0056] More preferably, the R3 group may be:

[0057]

[0058] More specifically, the compounds included in the present invention are as follows:

[0059] 2-(N-(pentan-3-yl)-3-(pyren-1-yl)propionamido)-N-(2,4,4-trimethylpentan-2-yl)hexanamide, N-(3-(dimethylamino)propyl)-N-(2-oxo-1-(pyridin-4-yl)-2-((2,4,4-trimethylpentan-2-yl)amino)ethyl)-3-(pyren-1-yl)propionamide, 2-(N-(4-(dibutylamino)butyl)-3-(pyren-1-yl)propylamino)-2-methyl-N-(2,4,4-trimethylpentan-2-yl)pentanamide, 4-(N-isopentyl-3-(pyran-1-yl)propylamino)-N-(2,4,4-trimethylpentan-2-yl)tetrahydro- 2H-pyran-4-carboxamide, N-(1-(4-hydroxyphenyl)-2-oxo-2-((2,4,4-trimethylpentan-2-yl)ethyl)-N-(3-hydroxypropyl)-3-(pyran-1-yl)propionamide, 4-(N-(3-(dimethylamino)propyl)-3-(pyren-1-yl)propionamido)-1-methyl-N-(2,4,4-trimethylpentan-2-yl)piperidine-4-carboxamide, N-(3-(dimethylamino)propyl)-3-(pyren-1-yl)propionamide, 2-methyl-2-(3-(pyren-1-yl)-N-(pyridin-2-ylmethyl)propionamido)-N-(2,4,4-trimethylpentan-2-yl)pentanamide, 4-(N-(1,3-dihydroxy-2-methylpropan-2-yl)-3-(pyren-1-yl)propylamino)-1-methyl-N-(2,4,4-trimethylpentan-2-yl)piperidine-4-carboxamide, 4-hydroxy-2-(N-isopentyl-3-(pyran-1-yl)propionamido)-2-methyl-N-(2,4,4-trimethylpentan-2-yl)butanamide, N-(4-butyloctyl)-N-(1-(4-hydroxyphenyl)-2-oxo-2-((2,4,4-trimethylpentan-2-yl)amino)ethyl)-3-(pyren-1-yl)propanamide, 2-(N-(1,3-dihydroxy-2-methylpropan-2-yl)-3-(pyran-1-yl) propanamide)-4-hydroxy-2-methyl-N-(2,4,4-trimethylpentan-2-yl)butanamide, N-(1-(4-hydroxyphenyl)-2-oxo-2-((2,4,4-trimethylpentan-2-yl)ethyl)-N-(3-hydroxypropyl)-3-(pyran-1-yl)propanamide, 2-(N-(1,3-dihydroxy-2-methylpropan-2-yl)-3-(pyrene-1-yl)propanamido)-2-methyl-N-(2,4,4-trimethylpentan-2-yl)pentanamide, 2-(furan-2-yl)-2-(N-(2-morpholinylethyl)-3-(pyrene-1-yl)propanamido)-N-(2,4,4-trimethylpentan-2-yl)propanamide.

[0060] Table 1 14 preferred fluorescent compounds

[0061]

[0062]

[0063] The preparation method of the fluorescence array sensor as described above is: the synthesized compounds are sorted from high to low according to the fluorescence intensity, the sperm samples are tested in sequence (Table 1), and the sensor elements are screened by linear discriminant analysis (LDA) to construct a sensor array.

[0064] As described above, the preparation method of the multi-channel fluorescence array sensor based on combinatorial chemistry mainly comprises the following steps: the pyrene-substituted fluorescent compound synthesized by combinatorial chemistry is diluted with 0.01M PBS (pH=7.4) buffer to a final concentration of 10 μM, 200 μL of each element is added to a 96-well plate to obtain a fluorescence array sensor, and shaken for 30 seconds. Then, the fluorescence intensity of the obtained system is detected under 340nm wavelength excitation, and the fluorescence intensity data is collected.

[0065] The present invention also provides the use of the fluorescent array sensor constructed by the above-mentioned combinatorial chemistry in the detection of different types of sperm diseases. Further, the different types of sperm diseases mainly include teratozoospermia (Terato), oligoasthenozoospermia (Oligo / Astheno / Terato), asthenozoospermia (Astheno / Terato) and asthenozoospermia (Astheno).

[0066] The present invention also provides a method for detecting semen using a fluorescence array sensor constructed based on the above-mentioned combinatorial chemical library: in a 96-well plate, sperm samples of healthy people collected from a hospital and samples of four different types of diseases affecting sperm quality (Terato, Oligo / Astheno / Terato, Astheno / Terato, Astheno) are diluted 100 times with 0.01M PBS (pH=7.4) buffer, 10 μL of the sperm sample is added to a 96-well plate containing 200 μL of a detection element, and then the fluorescence intensity of the obtained system is detected under excitation at a wavelength of 340 nm, and the fluorescence intensity data is read and collected by an enzyme marker.

[0067] Furthermore, the fluorescence data were processed using multiple algorithms such as linear discriminant analysis to obtain two-dimensional array fingerprints of healthy and four different types of diseases, and compared with the test results of the hospital. The obtained two-dimensional array fingerprints were used as a model to detect unknown samples.

[0068] This application synthesizes a series of pyrene fluorescent sensor element libraries with different structures based on the Ugi reaction, screens identification elements with better reactions based on the signal changes between sperm samples and sensor elements, and constructs a fluorescent sensor array for the detection of sperm samples. The fluorescence data is processed using the LDA algorithm to obtain a better recognition model, which can achieve rapid differentiation and detection of different types of sperm samples. At the same time, the constructed model can predict unknown samples and achieve rapid identification of unknown sperm samples.

[0069] Beneficial effects: The present application screens and constructs a fluorescent array sensor through a synthetic fluorescent compound library. The preparation method of the fluorescent array sensor obtained by screening is simple, and different types of clinical patient samples can be detected at the same time. A small amount of sperm samples is required, the detection time is short, the cost is low, and different types of real clinical samples can be well distinguished without the need for professional technicians. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 It is a standard LDA graph of the fluorescence response pattern obtained by the array sensor of the present invention for healthy and four disease sperm samples (95% confidence level);

[0071] Figure 2 It is a differentiation chart obtained by using existing clinical technology to directly record sperm parameters with a microscope and computer;

[0072] Figure 3 It is the distinguishing identification diagram and accuracy rate between the two diseases of the present invention;

[0073] Figure 4 is the H NMR spectrum of P2 prepared in Example 1;

[0074] Figure 5 is the mass spectrum of P2 prepared in Example 1;

[0075] Figure 6 is the H NMR spectrum of P14 prepared in Example 1;

[0076] Figure 7 It is the mass spectrum of P14 prepared in Example 1. DETAILED DESCRIPTION

[0077] The present invention is described below by means of specific embodiments, but the present invention is not limited thereto.

[0078] Example 1 Synthesis of a pyrene-substituted compound library

[0079] The Ugi reaction combinatorial chemistry used in the following examples was prepared according to the synthetic route shown below by the following steps:

[0080]

[0081] Compounds B and C (amine, aldehyde / ketone) were dissolved in methanol (concentration of 0.4 mol / L) respectively. They were then mixed and stirred at reflux for 3 hours. Compounds A and D were added in sequence, and the mixture was stirred at the same temperature for 30 minutes. The reaction was monitored by TLC. After the reaction was completed, the mixture was cooled to room temperature and distilled under reduced pressure. The residue was purified by silica gel column chromatography (DCM / MeOH=200:1 to 15:1, v / v) to obtain compounds P1-P14 as clear oil or white powder.

[0082] 1) 2-(N-(pentan-3-yl)-4-(pyrene-1-yl)butyramido)-N-(2,4,4-trimethylpentan-2-yl)hexanamide (P1)

[0083] 1 H NMR (400MHz, CDCl3) δ (ppm): 8.38 (d, J=9.2Hz, 1H, ArH), 8.18 (d, J=3.2Hz, 1H, ArH), 8.16 (d, J=3.2Hz, 1H, ArH), 8.13 (d, J=2.8Hz, 1H, A rH), 8.10 (d, J=1.6Hz, 1H, ArH), 8.05-8.02 (m, 2H, ArH), 8.00 (t, J=7.6Hz, 1H, ArH), 7.87 (d, J=7.6Hz, 1H, ArH), 3.49-3.38 (m, 2H, ArCH2 ), 3.38-3.31 (m, 1H, NCH), 2.51-2.43 (m, 2H, CONH2), 2.28-2.17 (m, 2H, ArCH2CH2), 1.91-1.45 (m, 8H, 4CH2), 1.43 (s, 3H, CH3), 1.37 (s, 3 H, CH3), 1.35-1.26 (m, 4H, 2CH2), 1.02 (s, 9H, 3CH3), 0.90 (t, J=6.8Hz, 3H, CH3), 0.84 (t, J=7.2Hz, 3H, CH3), 0.81 (t, J=5.2Hz, 3H, CH3).

[0084] 2) N-(3-(dimethylamino)propyl)-N-(2-oxo-1-(pyridin-4-yl)-2-((2,4,4-trimethylpentan-2-yl)amino)ethyl)-4-(pyrene-1-yl)butanamide (P2)

[0085] 1H NMR (300MHz, CDCl3) δ (ppm): 8.59 (d, J=5.7Hz, 2H, ArH), 8.34 (d, J=9.3Hz, 1H, ArH), 8.19 (s, 1H, ArH), 8.17 (s, 1H, ArH), 8.13 (s, 1H, ArH ), 8.10 (d, J=1.9Hz, 1H, ArH), 8.04 (s, 2H, ArH), 8.02 (d, J=7.2Hz, 1H, ArH), 7.96 (t, J=5.5Hz, 1H, ArH), 7.88 (d, J=7.8Hz, 1H, ArH), 7.30 ( s, 1H, ArH), 6.32 (s, 1H, NH), 5.58 (s, 1H, COCH), 3.45 (t, J=7.4Hz, 2H, CH2), 3.35-3.23 (m, 2H, CH2), 2.54 (t, J=7.2Hz, 2H, CH2), 2.34-2.3 3 (m, 2H, CH2), 2.08-1.91 (m, 8H, 2CH3, CH2), 1.70 (d, J=2.6Hz, 2H, CH2), 1.43 (s, 6H, 2CH3), 1.33-1.24 (m, 2H, CH2), 0.93 (s, 9H, 3CH3). For details, see Figure 4 .

[0086] LC-MS (ESI): m / z [M+H] + Calcd for C 40 H 51 N4O2: 619.40, Found: 619.23. Figure 5 .

[0087] 2-(N-(3-(dibutylamino)propyl)-4-(pyrene-1-yl)butylamino)-2-methyl-N-(2,4,4-trimethylpentan-2-yl)pentanamide (P3)

[0088] 1H NMR (300MHz, CDCl3) δ (ppm): 8.38 (d, J=9.3Hz, 1H, ArH), 8.21-8.10 (m, 4H, ArH), 8.04 (t, J=2.4Hz, 2H, ArH), 7.99 (d, J=7.6Hz, 1H, ArH), 7.89 (d, J=7.8Hz, 1H, ArH), 5.69 (s, 1H, CONH), 3.44 (t, J=7.5Hz, 2H, CH2), 3.33-3 .11(m,2H,CH2),2.55-2.36(m,2H,CH2),2.32-2.06(m,8H,4CH2),1.81-1.47(m,6H,3CH2),1.46(s,6H,2CH3 ), 1.44-1.13 (m, 13H, CH3, 5CH2), 0.99 (s, 9H, 3CH3), 0.96 (t, J=6.9Hz, 3H, CH3), 0.89 (t, J=7.1Hz, 6H, 2CH3).

[0089] LC-MS (ESI): m / z [M+H] + Calcd for C 45 H 68 N302: 682.53, Found: 682.50.

[0090] 4-(N-isopentyl-4-(pyrene-1-yl)butyramido)-N-(2,4,4-trimethylpentan-2-yl)tetrahydro-2H-pyran-4-carboxamide (P4)

[0091] 1H NMR (300MHz, CDCl3) δ (ppm): 8.33 (d, J=9.3Hz, 1H, ArH), 8.18 (s, 1H, ArH), 8.15 (d, J=1.7Hz, 1H, ArH), 8.12 (s, 1H, ArH), 8.09 (s, 1H, ArH ), 8.03 (s, 2H, ArH), 7.99 (t, J=7.8Hz, 1H, ArH), 7.85 (d, J=7.8Hz, 1H, ArH), 6.94 (s, 1H, CONH), 3.80-3.75 (m, 4H, 2CH2), 3.43 (t, J=7.2H z, 2H, CH2), 3.10 (t, J = 8.7Hz, 2H, CH2), 2.53-2.45 (m, 2H, CH2), 2.38 (t, J = 7.2Hz, 2H, CH2), 2.29-2.15 (m, 2H, CH2), 2.13-2.01 (m, 1H, CH ), 2.00-1.85 (m, 2H, CH2), 1.63 (s, 2H, CH2), 1.38 (s, 6H, 2CH3), 1.28-1.21 (m, 2H, CH2), 0.94 (s, 9H, 3CH3), 0.65 (d, J=6.3Hz, 6H, 2CH3).

[0092] N-(1-(4-Hydroxyphenyl)-2-oxo-2-((2,4,4-trimethylpentan-2-yl)amino)ethyl)-N-(3-hydroxypropyl)-4-(pyrene-1-yl)butanamide (P5)

[0093] LC-MS (ESI): m / z [M+H] + Calcd for C 39 H 47 N2O4: 607.36, Found: 607.20.

[0094] 4-(N-(3-(dimethylamino)propyl)-4-(pyrene-1-yl)butylamino)-1-methyl-N-(2,4,4-trimethylpentan-2-yl)piperidine-4-carboxamide (P6)

[0095] 1H NMR (300MHz, CDCl3) δ (ppm): 8.36 (d, J=9.3Hz, 1H, ArH), 8.19 (d, J=3.3Hz, 1H, ArH), 8.17 (d, J=2.4Hz, 1H, ArH), 8.14 (d, J=1.7Hz, 1 H, ArH), 8.11 (s, 1H, ArH), 8.04 (s, 2H, ArH), 8.00 (t, J=7.5Hz, 1H, ArH), 7.87 (d, J=7.8Hz, 1H, ArH), 6.83 (s, 1H, CONH), 3.44 (t, J=7. 4Hz, 2H, CH2), 3.24 (t, J=7.5Hz, 2H, CH2), 2.60-2.42 (m, 8H, 4CH2), 2.38-2.32 (m, 2H, CH2), 2.30 (s, 3H, CH3), 2.22 (t, J=7.5Hz, 2H, C H2), 2.01 (s, 6H, 2CH3), 1.98 (t, J=6.3Hz, 2H, CH2), 1.64 (s, 2H, CH2), 1.62-1.55 (m, 2H, CH2), 1.39 (s, 6H, 2CH3), 0.96 (s, 9H, 3CH3).

[0096] LC-MS (ESI): m / z [M+H] + Calcd for C 40 H 57 N4O2: 625.45, Found: 625.33.

[0097] 2-Methyl-2-(4-(pyrene-1-yl)-N-(pyridin-2-ylmethyl)butyramido)-N-(2,4,4-trimethylpentan-2-yl)pentanamide (P7)

[0098] 1H NMR (300MHz, CDCl3) δ (ppm): 8.37 (s, 1H, ArH), 8.29 (d, J = 5.3Hz, 1H, ArH), 8.25 (d, J = 9.3Hz, 1H, ArH), 8.17 (s, 1H, ArH), 8.14 ( s, 1H, ArH), 8.06 (s, 1H, ArH), 8.03 (s, 1H, ArH), 8.01 (d, J=1.2Hz, 2H, ArH), 7.98 (t, J=3.3Hz, 1H, ArH), 7.71 (d, J=7.8Hz, 1H, Ar H), 7.26 (s, 1H, NH), 6.94-6.71 (m, 2H, ArH), 4.49 (s, 2H, CH2), 3.44-3.15 (m, 2H, CH2), 2.24-1.93 (m, 6H, 3CH2), 1.63-1.51 (m, 2 H, CH2), 1.47 (s, 3H, CH3), 1.40 (s, 3H, CH3), 1.29 (s, 3H, CH3), 1.25 (s, 2H, CH2), 0.93 (s, 9H, 3CH3), 0.88 (t, J=7.5Hz, 3H, CH3).

[0099] LC-MS (ESI): m / z [M+H] + Calcd for C 40 H 50 N3O2: 604.39, Found: 604.29.

[0100] 4-Hydroxy-2-(N-isopentyl-4-(pyran-1-yl)butanamido)-2-methyl-N-(2,4,4-trimethylpentan-2-yl)butanamide (P9)

[0101] 1H NMR (300MHz, CDCl3) δ (ppm): 8.38 (d, J=9.3Hz, 1H, ArH), 8.20 (d, J=2.1Hz, 1H, ArH), 8.17 (d, J=2.3Hz, 1H, ArH), 8.13 (d, J =3.1Hz, 1H, ArH), 8.11 (d, J = 2.6Hz, 1H, ArH), 8.05 (s, 2H, ArH), 8.01 (t, J = 7.6Hz, 1H, ArH), 7.89 (d, J = 7.8Hz, 1H, ArH), 5. 58 (s, 1H, CONH), 3.45 (t, J = 7.3Hz, 2H, CH2), 3.25-3.12 (m, 2H, CH2), 2.38 (t, J = 7.1Hz, 2H, CH2), 2.22 (p, J = 7.3Hz, 2H, CH2 ), 1.75-1.67 (m, 1H, CH), 1.62 (s, 6H, 2CH3), 1.48-1.42 (m, 11H, CH3, 4CH2), 0.97 (s, 9H, 3CH3), 0.71 (d, J=6.2Hz, 6H, CH3).

[0102] 2-(N-(3-Hydroxypropyl)-4-(pyran-1-yl)butyramido)-2-methyl-N-(2,4,4-trimethylpentan-2-yl)pentanamide (P10)

[0103] 1 H NMR (300MHz, CDCl3) δ (ppm): 8.34 (d, J=9.3Hz, 1H, ArH), 8.16 (d, J=3.8Hz, 1H, ArH), 8.14 (d, J=3.5Hz, 1H, ArH), 8.10 ( s, 1H, ArH), 8.08 (d, J=1.9Hz, 1H, ArH), 8.01 (s, 2H, ArH), 7.98 (t, J=7.6Hz, 1H, ArH), 7.86 (d, J=7.8Hz, 1H, ArH), 5.75 ( s, 1H, CONH), 3.50 (t, J=5.6Hz, 2H, CH2), 3.39 (t, J=7.6Hz, 2H, CH2), 2.46 (t, J=7.2Hz, 2H, CH2), 2.26-1.98 (m, 4H, 2CH 2), 1.87-1.52 (m, 6H, 3CH2), 1.43 (s, 9H, 3CH3), 1.23-1.03 (m, 2H, CH2), 0.98 (s, 9H, 3CH3), 0.91 (t, J=7.3Hz, 3H, CH3).

[0104] LC-MS(ESI): m / z[M+H]+Calcd for C 37 H51 N2O3: 571.39, Found: 571.25.

[0105] N-(3-(Dibutylamino)propyl)-N-(2-oxo-1-(pyridin-4-yl)-2-((2,4,4-trimethylpentan-2-yl)amino)ethyl)-4-(pyrene-1-yl)butanamide (P11)

[0106] 1 H NMR (400MHz, CDCl3) δ (ppm): 8.60-8.55 (m, 2H, ArH), 8.33 (d, J=9.3Hz, 1H, ArH), 8.19-8.14 (m, 2H, ArH), 8.12 (d, J=2.3Hz, 1H, ArH), 8.10 (d, J =3.8Hz, 1H, ArH), 8.02 (s, 2H, ArH), 7.99 (t, J = 7.6Hz, 1H, ArH), 7.87 (d, J = 7.8Hz, 1H, ArH), 7.31-7.27 (m, 2H, ArH), 6.29 (s, 1H, CONH), 5.54 (s , 1H, CH), 3.44 (t, J = 7.6Hz, 2H, CH2), 3.32-3.25 (m, 2H, CH2), 2.57-2.53 (m, 2H, CH2), 2.31-2.08 (m, 8H, 4CH2), 1.68 (d, J = 7.2Hz, 2H, CH2), 1.4 2 (s, 6H, 2CH3), 1.38-1.31 (m, 2H, CH2), 1.27-1.22 (m, 4H, 2CH2), 1.17 (p, J=7.0Hz, 4H, 2CH2), 0.92 (s, 9H, 3CH3), 0.85 (t, J=7.0Hz, 6H, 2CH3).

[0107] LC-MS(ESI): m / z[M+H]+Calcd for C 46 H 63 N4O2: 703.50, Found: 703.35.

[0108] N-(1-(Furan-2-yl)-2-oxo-2-((2,4,4-trimethylpentan-2-yl)amino)ethyl)-N-(2-morpholinylethyl)-4-(pyrene-1-yl)butanamide (P14)

[0109] 1H NMR (300MHz, CDCl3) δ (ppm): 8.33 (d, J=9.3Hz, 1H, ArH), 8.17 (d, J=1.4Hz, 1H, ArH), 8.14 (d, J=1.1Hz, 1H, ArH), 8.11 (d, J=0.8H z, 1H, ArH), 8.09 (s, 1H, ArH), 8.02 (s, 2H, ArH), 7.99 (t, J=8.1Hz, 1H, ArH), 7.87 (d, J=7.8Hz, 1H, ArH), 7.40 (dd, J=1.9, 0.8Hz, 1 H, ArH), 6.66 (d, J=3.3Hz, 1H, ArH), 6.38 (dd, J=3.3, 1.9Hz, 1H, ArH), 6.03 (s, 1H, CH), 5.94 (s, 1H, CONH), 3.51-3.18 (m, 8H, 4CH 2), 2.52-2.22 (m, 4H, 2CH2), 2.01-1.79 (m, 6H, 3CH2), 1.78-1.56 (m, 2H, CH2), 1.38 (d, J=2.3Hz, 6H, 2CH3), 0.92 (s, 9H, 3CH3). For details, see Figure 6 .

[0110] LC-MS(ESI): m / z[M+H]+Calcd for C 40 H 50 N3O4: 636.38, Found: 636.25. Figure 7 .

[0111] Example 2 Detection of human sperm motility and quality based on fluorescence array sensor

[0112] The pyrene-substituted fluorescent compound synthesized by combinatorial chemistry was diluted with 0.01M PBS (pH=7.4) buffer to a final concentration of 10 μM, and a small amount of sodium dodecyl sulfate (SDS) solution was used to increase the solubility of the compound. 200 μL of each was added to a 96-well plate to obtain a fluorescent array sensor.

[0113] The method for distinguishing different types of sperm diseases is as follows: in a 96-well plate, sperm samples of healthy people and 4 different types of sperm samples (Terato, Oligo / Astheno / Terato, Astheno / Terato, Astheno) are diluted 100 times with 0.01 M PBS (pH=7.4) buffer, 10 μL of the sample is added to a 96-well plate containing 200 μL of the detection element, shaken for 30 seconds, and the fluorescence signal change is read by an enzyme reader. The fluorescence data is processed using linear discriminant analysis in the statistical analysis software to obtain a two-dimensional array fingerprint spectrum for distinguishing healthy and different types of diseases (seeFigure 1 ) to achieve the same test results as the hospital (see Figure 2 ) are consistent.

[0114] The AUCROC for distinguishing healthy people from patients, Asth from NonAsth, Olig from NonOlig, Terato from NonTerato was high, at 94.2%, 90.5%, 97.8%, and 95.8%, respectively (see Figure 3 ).

[0115] Table 1 Data matrix of fluorescence response of fluorescence array sensor to different sperm quality (training matrix of fluorescence response patterns from P1-P7 (378nm, 396nm) arrays)

[0116]

[0117]

[0118]

[0119] Table 2 Data matrix of fluorescence response of fluorescence array sensor to different sperm quality (training matrix of fluorescence response patterns from P8-P14 (378nm, 396nm) arrays)

[0120]

[0121]

[0122]

[0123]

[0124] Note: Terato, Oligo / Astheno / Terato, Astheno / Terato and Astheno

[0125] LDA was used to detect and identify unknown semen samples. All unknown samples were assigned to the corresponding groups defined by the training matrix according to the shortest Mahalanobis distance. According to the validation, only 5 of the 30 unknown semen samples were misclassified, with an accuracy of 83.3% (Table 3).

[0126] Table 3 LDA results of fluorescence sensor array fluorescence response data matrix for different sperm quality

[0127] Nr. Factor1 Factor2 Factor3 Factor4 Group ldentification verification 1 Blind 2.380859 -0.01287 2.67445 0.39879141 1 Astheno Astheno 2 Blind -0.59434 -1.62779 -1.99521 -0.0754741 5 Terato Terato 3 Blind 1.151986 -1.91601 2.320039 -0.3199173 1 Astheno AsthenoTerato 4 Blind -2.02338 -1.7631 0.795802 0.08517273 2 AsthenoTerato AsthenoTerato 5 Blind 3.085632 2.039599 0.491127 -0.9275794 3 Normal Normal 6 Blind 2.04237 0.53715 1.839304 1.47165732 1 Astheno Astheno 7 Blind 2.587475 1.635426 2.256266 0.46190845 1 Astheno Astheno 8 Blind -0.52135 0.485781 1.041771 0.23372595 4 OAT OAT 9 Blind -2.70809 1.310438 0.47322 1.59592181 4 OAT OAT 10 Blind 0.127417 -1.16911 2.279383 2.16659589 1 Astheno Astheno 11 Blind -0.21321 -2.10337 1.061616 0.80974634 2 AsthenoTerato Terato 12 Blind 0.496087 -1.88695 -0.34491 0.01813458 5 Terato Terato 13 Blind 0.722367 0.673787 -0.70853 -0.5203968 3 Normal Normal 14 Blind 2.377251 0.391995 -2.39329 -0.8626949 3 Normal Normal 15 Blind -0.13642 0.438943 0.868278 0.11289536 4 OAT AsthenoTerato 16 Blind -0.68307 -0.80273 0.900705 -0.4933319 2 AsthenoTerato AsthenoTerato 17 Blind -2.97703 1.587973 0.015267 -1.4030026 4 OAT OAT 18 Blind -2.79254 2.011023 0.807415 0.51309313 4 OAT OAT 19 Blind 1.583368 0.373417 1.956071 -0.2523563 1 Astheno Astheno 20 Blind -0.5282 -1.44828 -1.3039 1.48464152 5 Terato Terato 21 Blind 1.866704 -0.12512 -1.15083 -0.2622042 3 Normal Normal 22 Blind 2.027448 -1.27503 -0.85215 1.02626198 5 Terato Terato 23 Blind -1.69147 -2.23228 2.829768 -2.8890853 2 AsthenoTerato AsthenoTerato 24 Blind -0.72392 1.392602 0.912125 1.29273056 4 OAT OAT 25 Blind 1.628474 0.442854 1.252276 0.30238968 1 Astheno Astheno 26 Blind 0.43395 -0.15419 -1.48279 0.31877672 5 Terato Normal 27 Blind -1.18095 -1.39621 -1.64672 -2.238134 2 AsthenoTerato AsthenoTerato 28 Blind -1.62026 2.013011 1.835944 -2.0439547 4 OAT OAT 29 Blind 1.994669 0.341635 -0.10639 2.38447824 1 Astheno Normal 30 Blind 0.912909 -0.68386 -0.74815 -0.0869715 5 Terato Terato

[0128] This application constructs a fluorescent sensor array library based on combinatorial chemistry, and screens out 14 preferred compounds for the detection of clinical sperm diseases. The training set is analyzed and processed using SYSTAT 13.0 software through a linear discriminant analysis algorithm. The fluorescent array sensor has an identification accuracy of 83.3% for 30 unknown samples.

[0129] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

[0130] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A fluorescence array sensor for rapid detection of semen, characterized in that: Contains a variety of fluorescent compounds as shown in the following general formula: in: F is a fluorescent group, which can be independently selected from: pyrene, anthracene, coumarin, fluorescein, boron fluoride dye (BODIPY), naphthalimide, NBD, rhodamine, cyanine dye (Cyanine); The R1 and R2 groups together constitute: substituted or unsubstituted C1-15 straight or branched alkyl and cycloalkyl, 5-6 membered saturated heterocycle or 5-6 membered aromatic heterocycle; the substitution is -N(CH3)2, -N(CH2CH2CH3)2, -NHCH3, halogen, nitrile, nitro, hydroxyl, carboxyl, ester substitution; the 5-6 membered saturated heterocycle or 5-6 membered aromatic heterocycle are heterocycles containing one or two atoms of nitrogen and oxygen; The R3 and R4 groups can be independently selected from: hydrogen, hydroxyl-substituted or unsubstituted C1-10 straight or branched alkyl, phenol, nitrogen-containing or oxygen-containing 5-6 membered saturated heterocycle or nitrogen-containing or oxygen-containing 5-6 membered aromatic heterocycle; or R3 and R4 groups together constitute: substituted or unsubstituted 5-6 membered cycloalkyl, substituted or unsubstituted benzene ring, substituted or unsubstituted 5-6 membered saturated heterocyclic ring or substituted or unsubstituted 5-6 membered aromatic heterocyclic ring; the substitution is C 1-6 Alkyl, halogen, nitrile, nitro, hydroxyl, carboxyl, ester substitution; The R5 groups can be independently selected from: hydrogen, C1-10 straight or branched alkyl; n is 1 to 10 carbon atoms.

2. The fluorescent array sensor for rapid detection of semen according to claim 1, characterized in that: The R3 and R4 groups can be independently selected from: hydrogen, hydroxy substituted or unsubstituted C1-10 straight or branched alkyl, phenol, pyridyl, furanyl; The R5 groups can be independently selected from: hydrogen, C1-8 straight or branched alkyl; n is 2 to 10 carbon atoms.

3. The fluorescent array sensor for rapid detection of semen according to claim 1, characterized in that: The R1 and R2 groups together form:

4. The fluorescent array sensor for rapid detection of semen according to claim 1, characterized in that: The R3 and R4 groups are each independently selected from: Or R3 and R4 groups together constitute:

5. The fluorescent array sensor for rapid detection of semen according to claim 1, characterized in that: The R5 group is:

6. The fluorescent array sensor for rapid detection of semen according to claim 1, characterized in that: The fluorescent compound is selected from the following:

7. A method for preparing the fluorescent array sensor according to claim 1, characterized in that: Using Ugi chemical reaction, carboxyl chemicals, amino chemicals, ketone or aldehyde chemicals and isonitrile compounds are dissolved in methanol, and the four components are mixed and reacted to obtain the fluorescent compound shown in the general formula of claim 1, and the fluorescent array sensor is obtained by combining the synthesized multiple fluorescent compounds and diluting or not diluting with a buffer solution; R1, R2, R3, R4, R5, n, and F are as defined in claim 1.

8. An in vitro diagnostic reagent, characterized in that: The invention comprises the fluorescence array sensor as claimed in claim 1.

9. A method for rapidly detecting human sperm motility and quality using the fluorescent array sensor according to claim 1, characterized in that: Add the diluted sperm sample to the fluorescent array sensor, shake it, read the change of fluorescence signal with an enzyme marker, and process the fluorescence data using linear discriminant analysis in statistical analysis software.

10. Use of the fluorescent array sensor according to claim 1 or the in vitro diagnostic reagent according to claim 8 in rapid detection of sperm quality.

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