Fluorescent probe array for distinguishing testosterone, estrone and estradiol and preparation method and application thereof

Abstract

This application provides a fluorescent probe array for distinguishing testosterone, estrone, and estradiol, its preparation method, and its application, relating to the field of fluorescence sensing technology. The fluorescent probe array of this application includes a first probe and a second probe. The first probe comprises fluorescein isothiocyanate-labeled human serum albumin f-HSA and a first fluorescent indicator MML as shown in Formula I. The second probe comprises human serum albumin HSA and a second fluorescent indicator MeOC as shown in Formula II; wherein the molar ratios of f-HSA to MML and HSA to MeOC are 1:1. The fluorescent probe array of this application, relying on a competitive substitution mechanism, generates differentiated fluorescence responses. No complex sample pretreatment is required, enabling accurate identification and quantitative detection of the three steroid hormones. The operation is simple and rapid, providing a reliable solution for clinical testing, food hormone residue screening, and doping screening.

Description

Technical Field

[0001] This application relates to the field of fluorescence sensing technology, specifically to a fluorescent probe array for distinguishing testosterone, estrone, and estradiol, its preparation method, and its application. Background Technology

[0002] Testosterone (TST), estrone (EST), and estradiol (ESD) are key endogenous steroid hormones that participate in important physiological functions such as regulating sexual development, maintaining reproductive homeostasis, and regulating metabolic balance. However, due to their significant biological activity, these hormones are often illegally added to cosmetics and health foods. Long-term exposure or abuse can easily lead to health problems such as endocrine disorders and precocious puberty, seriously endangering human health.

[0003] Among existing analytical methods, fluorescent probe arrays offer an effective approach to distinguishing structurally similar compounds. They utilize multiple cross-reactive sensing units to generate differentiated, multidimensional fluorescent response signals for the analyte, enabling precise identification of similar molecules through pattern recognition. However, currently, there is no mature fluorescent probe array capable of efficiently distinguishing highly structurally similar steroid hormones such as TST, EST, and ESD.

[0004] Therefore, developing a fluorescent probe array that can distinguish and identify TST, EST, and ESD is of great significance in regulatory efforts to combat illegal additives, clinical assessment of endocrine function, and anti-doping in sports events. Summary of the Invention

[0005] The purpose of this application is to provide a fluorescent probe array for distinguishing testosterone, estrone, and estradiol, as well as its preparation method and application.

[0006] To achieve the above objectives, the embodiments of this application propose the following technical solutions:

[0007] In a first aspect, embodiments of this application provide a fluorescent probe array for distinguishing testosterone, estrone, and estradiol, the fluorescent probe array comprising a first probe and a second probe, each independently distributed on a carrier;

[0008] The first probe includes:

[0009] Fluorescein isothiocyanate-labeled human serum albumin;

[0010] The first fluorescent indicator is a compound of formula I;

[0011]

[0012] Formula I;

[0013] The second probe includes:

[0014] Human serum albumin;

[0015] The second fluorescent indicator is a compound of formula II;

[0016]

[0017] Formula II;

[0018] The fluorescein isothiocyanate-labeled human serum albumin is coated on the surface of the first fluorescent indicator; the human serum albumin is coated on the surface of the second fluorescent indicator.

[0019] In one embodiment, the molar ratio of the fluorescein isothiocyanate-labeled human serum albumin to the first fluorescent indicator is 1:1; the molar ratio of the human serum albumin to the second fluorescent indicator is 1:1.

[0020] Secondly, embodiments of this application provide a method for preparing the fluorescent probe array described in the first aspect, the method comprising:

[0021] Prepare human serum albumin solution, fluorescein isothiocyanate-labeled human serum albumin solution, first fluorescent indicator solution, and second fluorescent indicator solution respectively;

[0022] The first probe solution is obtained by mixing the fluorescein isothiocyanate-labeled human serum albumin solution with the first fluorescent indicator solution, and the second probe solution is obtained by mixing the human serum albumin solution with the second fluorescent indicator solution.

[0023] The first probe solution and the second probe solution are independently distributed on the carrier to obtain a fluorescent probe array.

[0024] As one embodiment, the method for preparing the human serum albumin solution includes:

[0025] Human serum albumin was added to deionized water to obtain a human serum albumin solution.

[0026] As one embodiment, the method for preparing the fluorescein isothiocyanate-labeled human serum albumin solution includes:

[0027] Human serum albumin solution was mixed with fluorescein isothiocyanate solution and purified by dialysis to obtain fluorescein isothiocyanate-labeled human serum albumin solution.

[0028] As one embodiment, the method for preparing the first fluorescent indicator solution includes:

[0029] 2-(3,5,5-trimethyl-2-cyclohexene-1-ylidene)malonitrile and 4-morpholinobenzaldehyde were dissolved in ethanol, piperidine was added dropwise, and the mixture was heated, rotary evaporated, and eluted to obtain the first fluorescent indicator.

[0030] The first fluorescent indicator solution was prepared by dissolving the first fluorescent indicator in dimethyl sulfoxide.

[0031] As one embodiment, the method for preparing the second fluorescent indicator solution includes:

[0032] 1-(2-hydroxy-4-methoxyphenyl)ethyl-1-one and 4-(piperidin-1-yl)benzaldehyde were dissolved in ethanol, and aziridine was added dropwise. After desolvation under reduced pressure and purification, a second fluorescent indicator was obtained.

[0033] The second fluorescent indicator solution was prepared by dissolving the second fluorescent indicator in dimethyl sulfoxide.

[0034] In one embodiment, the first probe solution and the second probe solution are freeze-dried to obtain the first probe and the second probe.

[0035] Thirdly, embodiments of this application provide an application of the fluorescent probe array described in the first aspect in distinguishing and / or quantitatively detecting the content of testosterone, estrone, and estradiol in a sample.

[0036] In one embodiment, the sample may include food or urine.

[0037] Compared with the prior art, the embodiments of this application have at least the following beneficial effects:

[0038] Compared to existing technologies, the core innovation of the sensor array in this application lies in the ingenious construction of a binary array using two cross-reactive host-guest probes, MML@f-HSA and MeOC@HSA. By leveraging the subtle differences in the binding ability of TST, EST, and ESD probes to compete with these probes, differentiated fluorescence responses are triggered, forming a unique fluorescent fingerprint for each hormone. This method achieves high-precision differentiation and quantification of the three hormones without complex pretreatment. Furthermore, the array maintains robust differentiation performance in urine and food matrices, and its simple and rapid operation provides an efficient and practical solution for doping screening, clinical testing, and rapid on-site food safety detection.

[0039] Additional aspects and advantages of this disclosure will be set forth in part in the description which follows, and will become apparent from the description or may be learned by practice of this disclosure. Attached Figure Description

[0040] Figure 1 A schematic diagram illustrating the construction principle of a fluorescent probe array is shown.

[0041] Figure 2 The fluorescence titration spectra of two probes for different concentrations of TST, ESD, and EST are shown; where bd is the response of MeOC@HSA and eg is the response of MML@f-HSA.

[0042] Figure 3 The following diagrams are shown: (a) fluorescence ratio response histogram, (b) thermogram, (c) LDA plot, and (d) HCA plot of the fluorescent probe array in the presence of EST, ESD, and TST (250 μM).

[0043] Figure 4 The following diagrams are shown: (a) array radar image, (b) heat map, (c) LDA image, and (d) LD1 score fitted to TST, ESD, and EST hormone concentrations at different concentrations.

[0044] Figure 5 The following are shown: (a) cuvette photographs of the RGB visualization array, (b) a circular clustering heatmap, (c) an LDA plot of 10 blind sample arrays, and (d) a confusion matrix.

[0045] Figure 6 The following graphs are shown: (a) heatmap, (b) LDA plot, (c) HCA plot, and (d) LD1 score versus hormone concentration ratio fitting plot for binary mixtures of ESD / TST, EST / ESD, and EST / TST with different ratios.

[0046] Figure 7 The following diagrams show (a) a ring clustering heatmap and (b) an LDA plot of the ternary mixture.

[0047] Figure 8 The following diagrams are shown: (a) ratio histogram, (b) ring clustering heatmap, (c) LDA plot, (d) LDA plot of 50 blind sample arrays, (e) confusion matrix, (f) heatmap of EST / ESD binary mixture, (g) LDA plot, (i) LD1 score fitting plot, and (h) LDA plot of ternary mixture in whole milk matrix.

[0048] Figure 9 The following diagrams are shown: (a) LDA plot, (b) HCA plot, and (c) thermogram of EST / TST, EST / ESD, and ESD / TST binary mixtures in urine samples.

[0049] Figure 10 The following diagrams show (a) a heatmap, (b) an LDA plot, and (c) an HCA plot of the ternary mixture in a urine sample. Detailed Implementation

[0050] The technical solutions in the embodiments of this disclosure will be clearly and completely described below with reference to the accompanying drawings. Obviously, the embodiments described below are only some embodiments of this disclosure, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this disclosure without creative effort are within the scope of protection of this disclosure.

[0051] The following will describe in detail a fluorescent probe array for distinguishing testosterone, estrone, and estradiol, its preparation method, and its application.

[0052] First, let me describe the fluorescent probe array of the first aspect of this embodiment.

[0053] Fluorescent probe array.

[0054] In existing reports, research on sensor arrays for the identification and differentiation of TST, EST and ESD is still relatively limited. In 2025, Yui Sasaki et al. first reported a colorimetric sensor array based on indicator displacement analysis (IDA). This system was assembled using three azobenzene derivatives and cucurbitaureine (CB[8]), and combined with ultraviolet-visible spectroscopy and chemometrics methods to achieve qualitative and quantitative analysis of 15 steroid hormones in saliva (including TST, EST and ESD).

[0055] However, this array still has significant limitations in practical applications: 1) Complex system construction: It relies on the precise assembly of various artificially synthesized indicators and macrocyclic substrates, making the preparation steps cumbersome, and the proportions of each component must be strictly controlled, affecting the robustness and reproducibility of the method. 2) Stringent detection conditions: It must be carried out in an acidic buffer system at a specific pH (3.2) and in a DMSO / water mixed solvent. These limitations largely stem from the inherent dependence of the IDA mechanism on the specific protonation state of the indicator, as well as the solubility requirements of the host-guest complex and hydrophobic steroid analytes, thus limiting its adaptability to routine or field applications. 3) Single signal dimension: This system is a colorimetric sensing mode, relying solely on visible light color changes to provide a single response signal, resulting in limited identification accuracy and anti-interference capabilities.

[0056] In view of this, this embodiment provides a binary fluorescent probe array. The array utilizes the differences and cross-reactivity between two sensing units and three hormones to achieve simultaneous and accurate differentiation and identification of TST, EST and ESD. It also has advantages such as simple process, mild detection conditions, strong anti-interference ability and wide applicability.

[0057] Specifically, the fluorescent probe array in this embodiment includes a first probe (denoted as MML@f-HSA) and a second probe (denoted as MeOC@HSA) that are independently distributed on the carrier, and both probes are host-guest structures.

[0058] The main body of the first probe is an engineered fluorescent albumin constructed by labeling human serum albumin (HSA) with fluorescein isothiocyanate (FITC), denoted as f-HSA; the guest is a first fluorescent indicator, which is a compound shown in Formula I, denoted as MML.

[0059] Formula I.

[0060] The main component of the second probe is natural human serum albumin, and the guest component is a second fluorescent indicator, which is the compound shown in Formula II, denoted as MeOC;

[0061] Formula II.

[0062] It is understood that the fluorescent probe in this embodiment utilizes the hydrophobic cavity of the protein host (f-HSA or HSA) to coat the surface of a dye guest (MML or MeOC) with complementary photophysical properties, and the binding molar ratio of the host to the guest is controlled at 1:1. The fluorescent indicator molecule is encapsulated within the HSA-specific binding site, creating a stable and constant molecular microenvironment, which enables the probe to possess stable intrinsic fluorescence emission performance, forming characteristic fixed fluorescence emission peaks at wavelengths of 535 nm and 680 nm.

[0063] Those skilled in the art will recognize that TST, EST, and ESD are hydrophobic small molecules, and their affinity binding to the HSA protein binding site is significantly stronger than that of fluorescent indicators MeOC and MML. Based on the indicator displacement analysis (IDA) mechanism, the hormone to be tested can competitively occupy the specific binding site of HSA, displacing the fluorescent indicator molecule originally bound to the protein, causing the fluorescent indicator to change from a bound state to a free state; the molecular microenvironment changes accordingly, resulting in characteristic fluorescence response changes and forming a differentiated multidimensional signal combination.

[0064] This embodiment uses two cross-reactive host-guest probes, MML@f-HSA and MeOC@HSA, to construct a binary fluorescence sensing array. Based on the difference in competitive binding ability between TST, EST, ESD, and the two probes, quantifiable changes in dual-probe fluorescence intensity are generated, forming a fluorescence recognition fingerprint specific to each hormone. Fluorescence intensities at 535 nm and 680 nm are collected using a microplate reader or fluorescence spectrometer, and the ratiometric fluorescence signal I is calculated. 535 / I 680 This ratio detection mode can effectively avoid external interference such as excitation light fluctuations, probe concentration, and instrument detection errors, and has better anti-interference ability and detection accuracy compared with single-wavelength detection.

[0065] Based on the above, this embodiment introduces chemometric methods such as linear discriminant analysis (LDA) and hierarchical cluster analysis (HCA) to analyze the multidimensional fingerprint signal, enabling accurate differentiation and quantitative detection of three structurally similar steroid hormones. Simultaneously, the system possesses visual RGB response characteristics. Under UV excitation, indicator displacement induces inverse changes in dual-wavelength fluorescence intensity, resulting in a clearly visible difference in the overall fluorescence hue of the system. Fluorescence images are acquired using imaging equipment, and the R, G, and B characteristic parameters of the fluorescent regions are extracted, converting the visually perceptible color changes into quantified RGB signals, thus achieving intuitive and visual discrimination of the detection results.

[0066] In summary, the binary fluorescent probe array of this application utilizes the differential response and cross-reaction characteristics between the two sensing units and the three hormones to make the ratio fluorescence signal and RGB color signal corresponding to each hormone show significant distinction, which can efficiently achieve specific identification and accurate differentiation of three structurally similar steroid hormones, TST, EST, and ESD.

[0067] Next, the method for preparing the fluorescent probe array of the second aspect of this embodiment will be described.

[0068] Preparation method.

[0069] The method for preparing the fluorescent probe array in this embodiment includes:

[0070] (1) Prepare fluorescein isothiocyanate-labeled human serum albumin solution (f-HSA), human serum albumin solution (HSA), first fluorescent indicator solution (MML), and second fluorescent indicator solution (MeOC) respectively.

[0071] (2) Mix the fluorescein isothiocyanate-labeled human serum albumin solution with the first fluorescent indicator solution to obtain the first probe solution, and mix the human serum albumin solution with the second fluorescent indicator solution to obtain the second probe solution;

[0072] (3) The first probe solution and the second probe solution are independently distributed on the carrier to obtain a fluorescent probe array.

[0073] Specifically, the preparation of the HSA solution in step (1) includes: dissolving HSA powder in deionized water to prepare an HSA solution.

[0074] The preparation of f-HSA solution involves: slowly adding FITC solution dropwise to HSA solution through a constant-pressure dropping funnel; after sufficient reaction, purifying by dialysis to obtain f-HSA solution. For long-term storage, the solution can be freeze-dried to obtain f-HSA solid powder.

[0075] The preparation of MML includes: dissolving 2-(3,5,5-trimethyl-2-cyclohexene-1-yl)malononitrile and 4-morpholinobenzaldehyde in ethanol, adding piperidine dropwise, and heating to mix; after the reaction, solid MML is obtained by rotary evaporation and elution; then MML is dissolved in dimethyl sulfoxide (DMSO) to obtain MML solution.

[0076] The preparation of MeOC includes: dissolving 1-(2-hydroxy-4-methoxyphenyl)ethyl-1-one and 4-(piperidin-1-yl)benzaldehyde in ethanol, and adding azacyclobutane dropwise to react completely; after the reaction, the solid MeOC is obtained by desolvation under reduced pressure and purification by silica gel column chromatography; then the MeOC is dissolved in DMSO to obtain a MeOC solution.

[0077] In step (2), the above f-HSA solution is mixed with MML solution and HSA solution with MeOC solution at a molar ratio of 1:1. After vortexing and standing, MML@f-HSA solution and MeOC@HSA solution are obtained respectively.

[0078] To facilitate long-term storage and standardized use of the sensing material, the MML@f-HSA and MeOC@HSA solutions prepared above were freeze-dried to obtain solid probe powder, which was then stored away from light. Before subsequent detection, an appropriate amount of probe powder was taken and reconstituted with a conventional buffer solution.

[0079] For example, the buffer solution is selected from PBS buffer or HEPES buffer.

[0080] It is understood that the HSA molecule in this embodiment has a unique hydrophobic cavity structure. The fluorescent dye molecule mainly embeds into the binding site of HSA through weak interactions such as hydrophobic interaction, van der Waals force, and hydrogen bond, so as to achieve stable physical encapsulation of the fluorescent dye without involving covalent chemical reaction.

[0081] Step (3) Adjust the MML@f-HSA and MeOC@HSA probe solutions obtained in step (2) to a suitable working concentration, such as 10 μM; according to the preset spatial arrangement, add (distribute) the two probe working solutions onto the carrier to construct a fluorescent probe array that is physically independent and parallel to each other.

[0082] For example, the carrier is selected from a multi-well plate (such as a 96-well plate) or a cuvette.

[0083] After the sample to be tested is added to the fluorescent probe array, the two probes in the array will interact specifically with the sample, producing differentiated colorimetric or fluorescent responses. After the reaction is fully completed, the fluorescence signal changes of each well are detected by an ELISA reader or a fluorescence spectrometer, and the corresponding fluorescence data are collected and recorded to provide data support for the qualitative identification and quantitative analysis of the target analytes (TST, EST, ESD).

[0084] In summary, the preparation method provided in this application is simple and utilizes the hydrophobic cavity of albumin to non-covalently physical encapsulate the fluorescent dye, resulting in high binding stability and mild assembly conditions. The constructed dual-probe array has independent spatial arrangement and strong anti-interference ability, enabling efficient and accurate detection of three types of steroid hormones: TST, EST, and ESD. It has good practical application value and promotion prospects.

[0085] Next, the application of the fluorescent probe array in the third aspect of this embodiment will be explained.

[0086] application.

[0087] Based on the first aspect of the discussion, the fluorescent probe array provided in this embodiment constructs a binary sensing system with MML@f-HSA and MeOC@HSA as the main and guest probes, which have cross-reactivity. Relying on the competitive binding differences between TST, EST, ESD and the two probes, it can not only efficiently achieve accurate differentiation and identification of three structurally similar steroid hormones, but also has the advantages of simple process, mild detection conditions, strong anti-interference ability and wide applicability.

[0088] Specifically, the probe in this embodiment can achieve rapid identification and quantitative detection of three types of hormones in urine, and is applicable to the screening and identification of hormone residues in complex matrices such as food.

[0089] Understandably, this probe array possesses dual-wavelength fluorescence emission characteristics. Relying on the indicator substitution competitive binding mechanism, the affinity differences between the three target hormones and the binding sites of HSA proteins can trigger characteristic differential responses in the fluorescence intensity of the probe system, forming a unique identification fingerprint for each hormone. It has the dual advantages of fluorescence ratio quantification and RGB visualization naked-eye recognition.

[0090] For example, this fluorescent probe array can be widely used in the field of rapid on-site screening of hormone residues in food processing, agricultural product traceability and other fields. Its ratiometric signal output mode can effectively offset the background interference caused by components such as proteins, lipids and natural pigments in the food matrix, improve detection sensitivity and accuracy, and meet the strict limit requirements for the residues of the three types of steroid hormones in food testing.

[0091] Secondly, in the field of biosample testing, urine, as the main carrier of steroid hormone metabolism and excretion in the body, plays a crucial role in the accurate screening of these hormones for physiological and biochemical analysis and clinical sample identification. The fluorescent probe array in this embodiment is adaptable to the complex matrix environment of urine, effectively avoiding interference from endogenous impurities such as urea and uric acid. Utilizing the cross-response characteristics of dual probes combined with chemometric analysis methods, it achieves efficient differentiation of structurally similar hormones. Simultaneously, the ratio fluorescence detection mode can mitigate detection errors caused by environmental pH, temperature, and instrument fluctuations, providing technical support for the accurate detection of steroid hormones.

[0092] In summary, the fluorescent probe array in this embodiment generates differentiated fluorescence responses based on the host-guest recognition and competitive substitution mechanism. It enables accurate identification and quantitative detection of three types of steroid hormones without the need for complex sample pretreatment. The operation is simple and quick, providing a reliable solution for clinical testing, food hormone residue screening, and doping screening.

[0093] The present application will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustration / explanation only and are not intended to limit the scope of the present application.

[0094] Unless otherwise specified, all materials, reagents and instruments used in the following embodiments are commercially available.

[0095] Example 1:

[0096] (1) Preparation of the first probe MML@f-HSA.

[0097] Synthesis of S1 and MML.

[0098] The synthetic route for MML is shown below:

[0099]

[0100] Specifically, it includes the following steps:

[0101] 2-(3,5,5-trimethyl-2-cyclohexene-1-yl)malononitrile (500 mg, 2.7 mmol) and 4-morpholinobenzaldehyde (1.55 g, 8.1 mmol) were dissolved in 15 mL of ethanol, and then 2-5 drops of piperidine were added. The mixture was heated under reflux at 80°C for 12 h. After the reaction was completed, the organic solvent was removed by rotary evaporation. The resulting red solid was separated by silica gel column chromatography using dichloromethane / petroleum ether (DCM / PE) as the eluent to give 435 mg / mL, with a yield of 45%.

[0102] The 1H NMR characterization data of MML are as follows:

[0103] 1H NMR (600 MHz, d6-DMSO)δ: 7.58 (d, J = 9.1 Hz, 2H), 7.22 (d, J =7.9 Hz, 2H), 6.96 (d, J = 9.1 Hz, 2H), 6.79 (s, 2H), 3.73 (t, J = 4.9 Hz,4H) , 3.24 (t, J = 5.0 Hz, 4H) , 2.59 (s, 2H) , 2.53 (s, 2H) , 1.01 (s, 6H) .

[0104] Preparation of S2 and f-HSA.

[0105] Weigh 133 mg of human serum albumin powder, dissolve it completely in 2 mL of deionized water, and place it in a 20 mL reaction flask for later use; at the same time, dissolve 1.9 mg of FITC solid in 10 mL of deionized water to prepare a 0.5 mM FITC standard solution.

[0106] Under continuous stirring, the FITC solution was slowly and dropwise added to the reaction flask containing HSA using a constant pressure dropping funnel, and the reaction was maintained for 24 hours. After the reaction was completed, in order to fully remove unreacted free FITC molecules, the mixture was transferred to a dialysis bag with a molecular weight cutoff of 3000 Da and dialyzed continuously in deionized water for 24 hours. The solution in the dialysis bag was collected and transferred to a 20 mL volumetric flask and diluted to volume to obtain a 0.1 mM f-HSA standard stock solution.

[0107] Preparation of S3 and MML@f-HSA.

[0108] Weigh 18.0 mg of MML and dissolve it completely in 5 mL of dimethyl sulfoxide (DMSO) to prepare a 10 mM MML standard stock solution for later use. Dilute the f-HSA stock solution to a final concentration of 10 μM using PBS buffer (1 mM, pH 7.4) as the solvent. Add an equimolar concentration (10 μM) of MML stock solution to the above f-HSA solution, let stand for 1 min, shake well and continue to incubate for 3 min to allow the host and guest molecules to fully self-assemble and form a stable MML@f-HSA complex solution.

[0109] (2) Preparation of the second probe MeOC@HSA.

[0110] Synthesis of S1 and MeOC.

[0111] The synthetic route for MeOC is shown below:

[0112]

[0113] Specifically, it includes the following steps:

[0114] 0.0033 mol of 1-(2-hydroxy-4-methoxyphenyl)ethyl-1-one and 0.004 mol of 4-(piperidin-1-yl)benzaldehyde were added to 50 mL of ethanol, and 5 drops of aziridine were added to the reaction mixture. The mixture was stirred at room temperature for 24 h, and the solvent was removed under reduced pressure. The crude product was purified by silica gel column chromatography, with dichloromethane as the eluent. The gradient elution ratio was adjusted from pure dichloromethane to dichloromethane / petroleum ether = 20:1, and the yield was 53%.

[0115] The 1H NMR characterization data for MeOC are as follows:

[0116] 1 H NMR (600 MHz, CDCl3) δ: 13.84 (s, 1H), 7.88(d, J = 15.2 Hz, 1H), 7.86–7.81(m, 1H), 7.58–7.52(m, 2H), 7.35(d, J = 15.2 Hz, 1H), 6.60–6.53(m,2H), 6.50–6.44(m, 2H), 3.85(s, 3H), 3.40–3.33(m, 4H), 2.09–1.99(m, 4H).

[0117] Preparation of S2 and MeOC@HSA.

[0118] Accurately weigh 16.3 mg of MeOC and dissolve it completely in 3 mL of DMSO to prepare a 10 mM MeOC stock solution. Simultaneously, weigh 66.5 mg of HAS powder and dissolve it completely in 1 mL of deionized water to prepare a 1 mM HSA stock solution. Then, precisely pipette 2 μL of the MeOC stock solution and 20 μL of the HSA stock solution into 2 mL of PBS buffer. Vortex the mixture vigorously at 2000 rpm for 3 min, then allow it to stand for 30 s to ensure that the MeOC is uniformly and stably encapsulated in the HSA.

[0119] (3) Lyophilization and reconstitution of probes

[0120] To obtain sensing materials that are easy to store long-term and standardized, the prepared and stable MML@f-HSA and MeOC@HSA solutions were freeze-dried separately to obtain solid powder probes, which were then stored in the dark. Before each array test, an appropriate amount of probe powder was taken and reconstituted with PBS buffer.

[0121] (4) Construction of fluorescent probe array.

[0122] The reconstituted MML@f-HSA and MeOC@HSA probe solutions were adjusted to a working concentration of 10 μM. The two probe working solutions were then added to 96-well plates according to the pre-defined spatial arrangement to construct fluorescent probe arrays.

[0123] Specifically, the spatial arrangement of the array follows a multi-parallel and multi-concentration gradient design: five parallel experiments are independently set up for each probe, and each experiment covers six hormone concentration gradients (i.e., five effective concentration gradients and one blank control). By rationally dividing the microwell matrix of the 96-well plate, the physical independence of different probes and samples of different concentrations is ensured, providing a reliable guarantee for the accuracy of subsequent high-throughput signal acquisition and the reproducibility of data.

[0124] Figure 1 This is a schematic diagram illustrating the construction principle of the fluorescent probe array. The left side shows two supramolecular probes assembled from MeOC and HSA, and MML and f-HSA, respectively. By utilizing their differentiated fluorescence cross-responses to the three analytes TST, ESD, and EST, a pattern recognition algorithm is used to map different target analytes into unique feature fingerprints, thereby achieving the differentiation and recognition of the three types of steroid hormones.

[0125] Example 2:

[0126] In the embodiments of this application, the TST, ESD, and EST hormone stock solutions were all prepared using dimethyl sulfoxide (DMSO) as solvent, with a uniform concentration of 10 mM, and were sealed and stored at -4°C for later use.

[0127] The detection was performed using a HORIBA Duette fluorescence spectrometer. The excitation wavelength of the sensor arrays (MeOC@HSA and MML@f-HSA) was uniformly set to 450 nm, and the excitation and emission slit widths were both set to 3 nm. Before each test, 2 mL of the corresponding complex probe solution was taken, vortexed at 2000 rpm for 2 min, and allowed to stand for 2 min to stabilize the sensing system. Then, different volumes of hormone stock solution were added dropwise to the above 2 mL probe solution to prepare a gradient titration system with a concentration of 0-250 μM. After thorough shaking and short-term standing, the fluorescence emission spectra from 500-800 nm were scanned and acquired to complete the characteristic signal extraction.

[0128] The results of the fluorescence titration are as follows Figure 2 As shown. Among them, Figure 2The bd section shows the fluorescence titration spectra of MeOC@HSA under increasing TST, ESD, and EST concentrations. As the concentrations of the three analytes increased from 0 to 250 μM, the fluorescence characteristic peak of MeOC@HSA at 535 nm was quenched to varying degrees, with the quenching degree being b > c > d. At the same time, new fluorescence emission was excited in the long wavelength region of 680 nm.

[0129] Figure 2 The middle eg section shows the fluorescence titration spectrum of MML@f-HSA under increasing concentrations of TST, ESD, and EST. The fluorescence peak of MML@f-HSA at 535 nm shows an increasing trend, and the response sensitivity of MML@f-HSA to TST is significantly better than that of the two estrogens, ESD and EST. In the long wavelength region of 680 nm, the fluorescence peaks corresponding to TST and ESD are quenched, while EST shows an enhanced fluorescence peak.

[0130] To further evaluate the detection performance of the array, this embodiment extracts the fluorescence ratio signal of the dual sensing units to construct a two-dimensional feature vector.

[0131] Figure 3 The following diagrams are shown: (a) fluorescence ratio response histogram, (b) thermogram, (c) LDA plot, and (d) HCA plot of the fluorescent probe array in the presence of EST, ESD, and TST (250 μM).

[0132] Specifically, Figure 3 Part a demonstrates that all three hormones can induce a characteristic ratio response in the dual-channel system (I). 535 / I 680 Among them, EST has the highest signal-to-weight ratio at MeOC@HSA, and TST has the highest signal-to-weight ratio at MML@f-HSA. Figure 3 Section b shows the specific fluorescent fingerprints of the three analytes, demonstrating the array's excellent cross-recognition capability.

[0133] Figure 3 Part c shows the LDA analysis results. The algorithm transforms the training matrix (2 channels × 3 hormones × 5 parallels) into standardized scores. The ellipse in the figure represents the 95% confidence interval. The variance contribution rates of LD1 and LD2 are 98.7% and 1.3%, respectively. The scatter plot shows clearly separated data clusters. After cross-validation, the qualitative classification accuracy of each analyte reached 100%, indicating that the array can effectively distinguish the three hormones. Figure 3 Part d shows the HCA clustering results. Parallel samples of the same hormone clustered closely together, and the Euclidean distance between samples of different hormones differed significantly, further confirming that all steroid samples were accurately clustered and classified without any cross-classification errors.

[0134] In summary, the fluorescent probe array constructed in this embodiment utilizes the synergistic effect of dual sensing units, enabling the array to accurately capture subtle differences in the target hormone's ability to replace different dyes, thereby achieving efficient differentiation of the three hormones.

[0135] Example 3:

[0136] Standard solutions of TST, ESD, and EST with concentration gradients of 40 μM, 80 μM, 120 μM, 180 μM, and 250 μM were prepared and added to the fluorescent probe array. After scanning and collecting fluorescence signals by a fluorescence spectrometer, radar charts, thermal images, LDA analysis, and concentration fitting were performed.

[0137] Figure 4 The following diagrams are presented: (a) array radar image, (b) heat map, (c) LDA plot, and (d) LD1 score and TST, ESD, and EST concentration fitting plot, to further evaluate the quantitative analysis capability of the array.

[0138] Specifically, Figure 4 Part b visually confirms that the array can effectively distinguish different concentrations of steroid hormones. As the hormone concentration increases, the heatmap color contributed by the MeOC@HSA unit shows a systematic positive correlation evolution; while the color change of the MML@f-HSA unit is relatively mild, but shows differentiated correlation characteristics. Figure 4 The LDA analysis results shown in section c indicate that EST and TST samples with different concentrations can be completely clustered and separated, while ESD shows slight cluster overlap in the 120-250 μM concentration range. Figure 4 The middle d part uses the LD1 score as the response signal and fits it with the analyte concentration to successfully construct quantitative standard curves for three single hormones, confirming that this array can reliably quantify TST, ESD, and EST.

[0139] Example 4:

[0140] The fluorescence spectrum is converted into discrete digital color components, and images of the detection solution are captured via a smartphone. The coordinate values ​​of the red (R), green (G), and blue (B) color channels are then extracted. The original dual-probe system is expanded into a six-channel sensor array (2 probes × 3 colors).

[0141] Figure 5 The following are shown: (a) cuvette photographs of the RGB visualization array, (b) a circular clustering heatmap, (c) an LDA plot of 10 blind sample arrays, and (d) a confusion matrix.

[0142] Specifically, Figure 5The fluorescence images in part a visually present the characteristic color differences of the six-channel RGB visualization array and the response of different steroid hormones. Each hormone can be accurately represented by a unique six-element RGB vector. Figure 5 The ring-shaped clustering heatmap in section b further confirms that TST, ESD, and EST exhibit highly recognizable exclusive fluorescent fingerprint patterns, providing a clear signal basis for cross-identification. Figure 5 The blind LDA test results shown in section c indicate that all 10 random unknown samples were accurately classified into the corresponding steroid signal clusters, with no cross-classification errors, and the qualitative classification accuracy reached 100%. Figure 5 The confusion matrix results in part d further validated this conclusion, confirming that the array does not make any misclassifications in the classification prediction of unknown samples, demonstrating its broad application prospects for rapid visual detection on portable devices such as smartphones.

[0143] Example 5:

[0144] In clinical endocrinology, the precise quantification of specific sex hormone ratios (such as TST / ESD, EST / ESD, TST / EST) can reflect the dynamic metabolic interactions in the body, which is crucial for the diagnosis, treatment, and monitoring of complex endocrine diseases. Based on this, this embodiment delves into the ability of this dual-channel array to distinguish target mixtures.

[0145] Under constant total concentration (200 μM), solutions of EST / ESD, EST / TST, ESD / TST binary mixtures, and EST / ESD / TST ternary mixtures were prepared in different proportions. Fluorescent probe arrays were added to each solution, fluorescence signals were collected, and ratio responses were calculated. Chemometric analysis was then performed. The specific experimental steps are as follows:

[0146] Preparation of binary mixture systems: Using ESD, EST, and TST as subjects, three binary mixture systems, EST / ESD, EST / TST, and ESD / TST, were constructed by combining them in pairs. By precisely adjusting the molar ratio of the two hormones, evolving it within a gradient range from 2:8, 6:4... up to 8:2, a series of binary mixtures covering different concentration ratios were obtained.

[0147] Preparation of ternary mixture systems: Under the same total concentration limit, the relative molar ratios of the three components EST, ESD and TST are synergistically adjusted (2:2:6, 7:1:2, etc.) to prepare ternary mixture solutions with different concentration distributions.

[0148] Figure 6The following figures are shown: (a) heatmap, (b) LDA plot, (c) HCA plot, and (d) LD1 score versus hormone concentration ratio fitting plot for binary mixtures of different ratios of ESD / TST, EST / ESD, and EST / TST. Under the condition of a constant total concentration of 200 μM, the original ratio signal heatmaps of the three binary mixtures at five ratio gradients are shown below. Figure 6 As shown in part a; Figure 6 The LDA results in part b show that samples with different ratios form clearly separated data clusters; Figure 6 In Part c, HCA analysis achieved 100% correct sample clustering with no misclassifications; by fitting LD1 to the hormone ratio, a quantitative standard curve for the binary hormone ratio was successfully established, and the results are shown in [Figure number missing]. Figure 6 Part d in the middle.

[0149] Figure 7 The results show (a) a ring clustering heatmap and (b) an LDA plot of the ternary mixture. The test results further demonstrate that this array can finely distinguish EST / ESD / TST ternary mixtures with different proportions, fully showcasing its potential as an innovative diagnostic tool. It is expected to be applied to the subtype identification of complex endocrine diseases such as polycystic ovary syndrome and hormone-sensitive tumors, as well as the health supervision of illegal additives in cosmetics and health products.

[0150] Example 6:

[0151] This embodiment was tested and verified in whole milk and urine samples from healthy individuals to simulate real-world scenarios involving illegal food additives and endocrine fluctuations.

[0152] 6.1 Detection of Milk Matrix Samples

[0153] In the experiment, hormones (TST, EST, or ESD) or their mixtures were added to untreated whole milk samples at a final concentration of 120 μM. The spiked milk was then pretreated according to a standardized procedure of methanol precipitation, centrifugation, filtration, nitrogen purging, and PBS reconstitution. The reconstituted milk solution without any added hormones served as a blank control group; the spiked samples with added hormones were randomly numbered 1-50 as the unknown blind sample test group. Fluorescence spectroscopy was performed: different volumes (0-32 μL) of the reconstituted extract were added to 2 mL of 10 μM probe working solution in quartz cuvettes and thoroughly mixed. Throughout the blind sample screening process, the spectral data acquisition and subsequent chemometric analysis of all samples were conducted under completely consistent experimental conditions, strictly following the aforementioned established protocol.

[0154] Figure 8The following diagrams are shown: (a) ratio histogram, (b) ring clustering heatmap, (c) LDA plot, (d) LDA plot of 50 blind sample arrays, (e) confusion matrix, (f) heatmap of EST / ESD binary mixture, (g) LDA plot, (i) LD1 score fitting plot, and (h) LDA plot of ternary mixture in whole milk matrix.

[0155] In a whole milk matrix, the dual-channel sensing unit still maintains a differentiated ratio response to the three target hormones, forming a specific fingerprint pattern. Figure 8 Parts a and b); LDA can completely cluster and separate the three hormone samples, with 100% accuracy in multi-concentration cross-validation. In a blind test of 50 milk matrix samples, only 1 case was misjudged, achieving an overall prediction accuracy of 98%. Figure 8 (Parts d and e). Simultaneously, the array can achieve precise classification and proportional quantification of binary and ternary mixed hormones in milk matrix. Figure 8 The middle part (f-i) can be used for qualitative and quantitative screening of illegal hormone addition in food matrices.

[0156] 6.2 Detection of urine matrix samples

[0157] This experiment further evaluated the array's detection performance in urine samples from healthy volunteers. The urine samples required no pretreatment and were used for testing after a five-fold dilution. Spectral acquisition and data processing were performed in accordance with the previously described methods. Experimental results showed that the sensor array exhibited excellent anti-interference performance in a urine environment.

[0158] Figure 9 The following diagrams are shown: (a) LDA plot, (b) HCA plot, and (c) thermogram of EST / TST, EST / ESD, and ESD / TST binary mixtures in urine samples. Figure 10 The following diagrams show (a) a heatmap, (b) an LDA plot, and (c) an HCA plot of the ternary mixture in a urine sample.

[0159] Despite the complex composition of urine, the LDA model successfully achieved 100% accurate classification of the three target steroid hormones. The high resolution exhibited by this array in physiological fluids further confirms that the subject-object recognition mechanism based on HSA is primarily driven by thermodynamic affinity, rather than simple electrostatic adsorption, and therefore can effectively resist interference from high ionic strength in urine.

[0160] In summary, the fluorescent probe array provided in this embodiment generates differentiated fluorescence responses based on a competitive substitution mechanism. It enables accurate identification and quantitative detection of three types of steroid hormones without the need for complex sample pretreatment. The operation is simple and quick, providing a reliable solution for clinical testing, food hormone residue screening, and doping screening.

[0161] The technical solutions provided by the embodiments of this application have been described in detail above. Specific examples have been used in this document to illustrate the principles and implementation methods of the embodiments of this application. The descriptions of the embodiments above are only for helping to understand the principles of the embodiments of this application. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the embodiments of this application. Therefore, the content of this specification should not be construed as a limitation of this application.

Claims

1. A fluorescent probe array for distinguishing testosterone, estrone, and estradiol, characterized in that, The fluorescent probe array includes a first probe and a second probe, each independently distributed on the carrier; The first probe includes: Fluorescein isothiocyanate-labeled human serum albumin; The first fluorescent indicator is a compound of formula I; Formula I; The second probe includes: Human serum albumin; The second fluorescent indicator is a compound represented by Formula II; Formula II; The fluorescein isothiocyanate-labeled human serum albumin is coated on the surface of the first fluorescent indicator; the human serum albumin is coated on the surface of the second fluorescent indicator.

2. The fluorescent probe array according to claim 1, characterized in that, The molar ratio of the fluorescein isothiocyanate-labeled human serum albumin to the first fluorescent indicator is 1:1; the molar ratio of the human serum albumin to the second fluorescent indicator is 1:

1.

3. A method for preparing a fluorescent probe array for distinguishing testosterone, estrone, and estradiol as described in claim 1, characterized in that, The preparation method includes: Prepare human serum albumin solution, fluorescein isothiocyanate-labeled human serum albumin solution, first fluorescent indicator solution, and second fluorescent indicator solution respectively; The first probe solution is obtained by mixing the fluorescein isothiocyanate-labeled human serum albumin solution with the first fluorescent indicator solution, and the second probe solution is obtained by mixing the human serum albumin solution with the second fluorescent indicator solution. The first probe solution and the second probe solution are independently distributed on the carrier to obtain a fluorescent probe array.

4. The preparation method according to claim 3, characterized in that, The method for preparing the human serum albumin solution includes: Human serum albumin was added to deionized water to obtain a human serum albumin solution.

5. The preparation method according to claim 3, characterized in that, The method for preparing the fluorescein isothiocyanate-labeled human serum albumin solution includes: Human serum albumin solution was mixed with fluorescein isothiocyanate solution and purified by dialysis to obtain fluorescein isothiocyanate-labeled human serum albumin solution.

6. The preparation method according to claim 3, characterized in that, The method for preparing the first fluorescent indicator solution includes: 2-(3,5,5-trimethyl-2-cyclohexene-1-ylidene)malonitrile and 4-morpholinobenzaldehyde were dissolved in ethanol, piperidine was added dropwise, and the mixture was heated, rotary evaporated, and eluted to obtain the first fluorescent indicator. The first fluorescent indicator solution was prepared by dissolving the first fluorescent indicator in dimethyl sulfoxide.

7. The preparation method according to claim 3, characterized in that, The method for preparing the second fluorescent indicator solution includes: 1-(2-hydroxy-4-methoxyphenyl)ethyl-1-one and 4-(piperidin-1-yl)benzaldehyde were dissolved in ethanol, and aziridine was added dropwise. After desolvation under reduced pressure and purification, the second fluorescent indicator was obtained. The second fluorescent indicator solution was prepared by dissolving the second fluorescent indicator in dimethyl sulfoxide.

8. The preparation method according to claim 3, characterized in that, The first probe solution and the second probe solution are freeze-dried to obtain the first probe and the second probe.

9. The use of the fluorescent probe array according to any one of claims 1-2 in distinguishing and / or quantitatively detecting the content of testosterone, estrone and estradiol in a sample.

10. The application according to claim 9, characterized in that, The samples may include food or urine.

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