A beta-lactoglobulin fluorescent probe, a preparation method and application thereof

Abstract

The application discloses a beta-lactoglobulin fluorescent probe, a preparation method and application thereof, and the structural formula of the probe is (C 30 H 25 N4I), the probe is combined with beta-lactoglobulin through non-covalent interaction, triggers significant enhancement of a fluorescence signal, and realizes low-background fluorescence detection of the beta-lactoglobulin. The probe has low cytotoxicity, has excellent mitochondrion targeting capability, and provides a powerful tool for visualization of the beta-lactoglobulin in living cells. After being combined with the beta-lactoglobulin, the sample produces obvious color change under ultraviolet light irradiation, and the change can be used for evaluating the content of active beta-lactoglobulin in the sample, and further realizing rapid detection of milk protein level in beverages. The beta-lactoglobulin fluorescent probe has wide application prospects in the fields of biological imaging, milk protein detection and beverage quality control.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probe technology, specifically relating to a method for preparing and using a β-lactoglobulin fluorescent probe. Background Technology

[0002] Milk, as a comprehensive source of nutrition, plays an important role in daily life, providing the human body with a range of key metabolic products, including protein, carbohydrates, fats, vitamins, and minerals. Among these diverse nutrients, high-quality milk protein, especially β-lactoglobulin, has attracted much attention due to its crucial role in maintaining physiological processes such as tissue repair and muscle growth. β-lactoglobulin, a key member of the lipoprotein family, is the most abundant protein in ruminant whey, possessing significant nutritional value and various physiological activities, such as enhancing mineral absorption, regulating gut microbiota, and modulating angiotensin-converting enzyme activity. However, β-lactoglobulin is also a significant allergen, frequently triggering adverse reactions in consumers with milk allergies, particularly infants and young children.

[0003] The market currently offers a wide variety of dairy products designed to meet consumers' protein nutritional needs. Unfortunately, some counterfeit or misleading products also exist, which may lack sufficient protein or have inaccurate protein content labeling, posing a serious threat to consumer health and trust. While traditional methods for verifying protein content in dairy products, such as the Kjeldahl method, high-performance liquid chromatography, spectrophotometry, electrophoresis, and enzyme-linked immunosorbent assay (ELISA), are accurate, they are complex, costly, and have limited accessibility, posing a significant challenge for ordinary consumers.

[0004] In recent years, fluorescent probe technology has gradually emerged as a real-time, rapid, and highly sensitive detection method in the field of substance detection. In food testing, this technology enables rapid on-site analysis and testing of various analytes, such as natural antioxidants and hypochlorous acid. However, despite the great potential of fluorescent probe technology in the detection field, the development and utilization of fluorescent probes specifically for milk proteins, particularly β-lactoglobulin, remains relatively limited. Developing highly sensitive and selective fluorescent probes specifically for the detection of β-lactoglobulin in complex food matrices remains a challenge that urgently needs to be addressed. Given the significant advantages that fluorescent probe technology may bring to the dairy industry, the development of fluorescent probes specifically for milk proteins is particularly important to meet the market demand for accurate and convenient detection. Summary of the Invention

[0005] Purpose of the invention: This invention addresses the problems existing in the prior art by providing a β-lactoglobulin fluorescent probe, its preparation method, and its application, which enables efficient, accurate, and convenient detection of β-lactoglobulin and meets the needs of fields such as bioimaging, milk protein detection, and beverage quality control.

[0006] Technical Solution: To achieve the above-mentioned objectives, the present invention adopts the following technical solution: a β-lactoglobulin fluorescent probe, having the following structure:

[0007] .

[0008] The present invention also provides a method for preparing the above-mentioned β-lactoglobulin fluorescent probe, comprising the following steps:

[0009] S1, 2-(1-(4-(pyridin-4-yl)phenyl)ethylene)malonitrile, 6-(dimethylamino)-2-naphthal, and piperidine were dissolved in dichloromethane and refluxed for 4-8 h. After concentration under reduced pressure, the crude product was purified to obtain 2-(3-(6-(dimethylamino)naphthylacetamide-2-yl)1-(4-(pyridin-4-yl)phenyl)allyl)malonitrile.

[0010] S2, dissolve 2-(3-(6-(dimethylamino)naphthylacetamide-2-yl)1-(4-(pyridine-4-yl)phenyl)allyl)malonium obtained in step S1 and iodomethane in acetonitrile and reflux for 20-36 h. After concentration under reduced pressure, add a small amount of acetonitrile to dissolve, then precipitate with diethyl ether, filter and wash to obtain the β-lactoglobulin fluorescent probe.

[0011] Furthermore, the crude product obtained in step S1 is purified by silica gel column chromatography using a mixture of dichloromethane, ethyl acetate, and petroleum ether in a volume ratio of 1:1:17.

[0012] The present invention also provides the application of the above-mentioned β-lactoglobulin fluorescent probe in in vitro β-lactoglobulin detection or β-lactoglobulin-mediated cell imaging for non-therapeutic or diagnostic purposes, as well as in milk protein detection and beverage quality control.

[0013] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0014] (1) The β-lactoglobulin fluorescent probe provided by the present invention is characterized by not emitting fluorescence itself, but exhibiting selective fluorescence enhancement response to specific proteins in the presence of various proteins including β-lactoglobulin. It shows a high sensitivity response to β-lactoglobulin, while only producing a weak response to α-lactalbumin, casein, serum albumin, etc., and has strong β-lactoglobulin response specificity.

[0015] (2) The fluorescent probe of the present invention has a strong β-lactoglobulin binding ability and good anti-interference performance, and can accurately and specifically identify and bind β-lactoglobulin in complex samples.

[0016] (3) The probe exhibits low cytotoxicity, can easily penetrate cell membranes, and demonstrates excellent mitochondrial targeting properties. This unique property provides a powerful technical means for visualizing β-lactoglobulin in living cells, greatly expanding its potential in biomedical research and clinical applications.

[0017] (4) When bound to β-lactoglobulin, samples rich in β-lactoglobulin will exhibit a significant color change under ultraviolet light irradiation. This color change is closely related to the content of active β-lactoglobulin in the sample, and therefore can be used to quickly and easily assess the content of β-lactoglobulin in the sample, thereby enabling rapid detection of milk protein levels in beverages. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is the structural formula of the β-lactoglobulin fluorescent probe described in this invention.

[0020] Figure 2 The above is the 1H NMR spectrum of the fluorescent probe of this invention, with d6-DMSO as the solvent.

[0021] Figure 3 The image shows the carbon NMR spectrum of the fluorescent probe of this invention, with d6-DMSO as the solvent.

[0022] Figure 4 This is a comparison of the fluorescence intensity changes of the fluorescent probe in different proteins (λex = 490 nm).

[0023] Figure 5 A comparison of fluorescence intensity changes of the fluorescent probe in different concentrations of β-lactoglobulin (λex = 490nm).

[0024] Figure 6 The fluorescence quenching pattern of the fluorescent probe on β-lactoglobulin (λex = 280 nm).

[0025] Figure 7 The effects of various common commercial drugs on the responsiveness of the fluorescent probe β-lactoglobulin.

[0026] Figure 8 This is a diagram of the HepG2 cytotoxicity assay using a fluorescent probe.

[0027] Figure 9 Fluorescence imaging of HepG2 cells after co-incubation with fluorescent probes and mitochondrial dye MitoTracker Green.

[0028] Figure 10 The images show the results of a probe-based polyamide thin-film sensor being immersed in various liquid and solid beverages rich in and without β-lactoglobulin, and then air-dried, under 365 nm ultraviolet light. Detailed Implementation

[0029] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. After reading this invention, any modifications of the invention in various equivalent forms by those skilled in the art will fall within the scope defined by the appended claims. To enable those skilled in the art to better understand the technical solutions in the embodiments of the present invention, and to make the above-mentioned objectives, features, and advantages of the present invention more apparent, the specific embodiments of the present invention will be further described below.

[0030] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0031] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0032] This invention provides a β-lactoglobulin fluorescent probe, the structural formula of which is shown below:

[0033]

[0034] The method for preparing the β-lactoglobulin fluorescent probe according to embodiments of the present invention specifically includes the following steps:

[0035] Step 1: Dissolve 2-(1-(4-(pyridin-4-yl)phenyl)ethylene)malononitrile, 6-(dimethylamino)-2-naphthaldehyde, and piperidine in dichloromethane and heat under reflux. After cooling, separate by column chromatography to obtain 2-(3-(6-(dimethylamino)naphthylacetamide-2-yl)1-(4-(pyridin-4-yl)phenyl)allyl)malononitrile.

[0036] Step 2: Dissolve 2-(3-(6-(dimethylamino)naphthylacetamide-2-yl)1-(4-(pyridin-4-yl)phenyl)allyl)malonium and iodomethane in acetonitrile, stir and heat under reflux, cool and filter, and wash with anhydrous diethyl ether to obtain the β-lactoglobulin fluorescent probe. The preparation route is shown below:

[0037]

[0038] The present invention will be further illustrated by the following examples.

[0039] Example 1: Preparation of β-lactoglobulin fluorescent probe

[0040] Step 1: 0.40 g of 1.65 mmol of 2-(1-(4-(pyridin-4-yl)phenyl)ethylene)malonitrile, 0.30 g of 1.5 mmol of 6-(dimethylamino)-2-naphthaldehyde, and 22 drops of piperidine were dissolved in 20 mL of dichloromethane and refluxed for 6 h. After concentration under reduced pressure, the crude product was purified on a silica gel column (dichloromethane / ethyl acetate / petroleum ether, 1 / 1 / 17, v / v / v) to give 2-(3-(6-(dimethylamino)naphthylacetamide-2-yl)1-(4-(pyridin-4-yl)phenyl)allyl)malonitrile.

[0041] Step 2: Dissolve 0.23 g of 2-(3-(6-(dimethylamino)naphthylacetamide-2-yl)1-(4-(pyridin-4-yl)phenyl)allyl)malonidonitrile 0.53 mmol and 0.75 g of iodomethane 5.3 mmol in 20 mL of acetonitrile and reflux for 24 h. After concentration under reduced pressure, dissolve in a small amount of acetonitrile, precipitate with diethyl ether, filter and wash to obtain the β-lactoglobulin fluorescent probe. 1 H NMR (400MHz, DMSO-d6) δ (ppm): 9.11 (t, J =4.4 Hz, 2H), 8.66–8.58 (m, 2H), 8.37–8.32(m, 2H), 8.23–8.11 (m, 2H), 7.97–7.81 (m, 3H), 7.77–7.55 (m, 3H), 7.24–7.12(m, 1H), 7.05-6.95 (m, 1H), 4.39 (d,J = 2.8 Hz, 3H), 3.71 (s, 3H), 3.07 (s, 3H). 13 C NMR (101 MHz, DMSO-d6) δ (ppm): 170.2, 169.9, 153.5, 150.6,146.3,137.2, 137.2, 136.2, 136.0, 133.8, 132.8, 130.7, 129.0, 127.6, 127.4, 125.6,124.9, 124.9, 124.3, 121.4, 116.8, 114.8, 113.9, 113.2, 105.7, 83.5, 79.1,56.8, 47.8, 47.8.

[0042] Please refer to the following: Figure 2 and Figure 3 , Figure 2 The above is the 1H NMR spectrum of the β-lactoglobulin fluorescent probe of the present invention, with d6-DMSO as the solvent; Figure 3 The image shows the carbon NMR spectrum of the β-lactoglobulin fluorescent probe of this invention, with d6-DMSO as the solvent.

[0043] Example 2: Selective Response Assay of Fluorescent Probe Proteins

[0044] Stock solution preparation: Dissolve the probe in DMSO to prepare a 2 mM stock solution. Dissolve α-lactalbumin, β-lactoglobulin, γ-globulin, bovine serum albumin, trypsin, lysozyme, α-chymotrypsin, and β-lactoglobulin in 0.1 M PBS (pH 7.4) to prepare an 80 μM stock solution. Dissolve casein in 0.2 mol / L sodium hydroxide to prepare a 10 g / L stock solution.

[0045] In volumetric flasks, α-lactalbumin, β-lactoglobulin, γ-globulin, bovine serum albumin, trypsin, lysozyme, α-chymotrypsin, β-lactoglobulin, and casein were first added to a final concentration of 5 μM, followed by a probe to a final concentration of 5 μM. The mixture was then brought to volume using 0.1 M PBS buffer. The solution was allowed to stand for at least 30 minutes. The fluorescence emission spectrum of the sample was measured using an F-7000 fluorescence spectrophotometer at an excitation wavelength of 490 nm.

[0046] like Figure 4As shown, this invention provides a probe with selective responsiveness to β-lactoglobulin. The probe itself has no intrinsic fluorescence, but its fluorescence is significantly enhanced upon the addition of β-lactoglobulin. Under the same experimental conditions, when α-lactalbumin, casein, and bovine serum albumin were added to the probe, fluorescence enhancement was also observed, but the degree of enhancement was relatively weak. Notably, even when the amount of casein added was much higher than that of β-lactoglobulin, its fluorescence enhancement effect on the probe remained weak. Furthermore, when other proteins such as γ-globulin, trypsin, lysozyme, and α-chymotrypsin were added to the probe, the fluorescence of the probe remained unchanged, further demonstrating the high selectivity of the probe for β-lactoglobulin. Considering practical application environments, taking milk as an example, the content of β-lactoglobulin is approximately 3 times that of α-lactalbumin and approximately 10 times that of bovine serum albumin. Therefore, in practical applications, the response of the probe to α-lactalbumin and bovine serum albumin is almost negligible. In summary, the probe of this invention can be considered to have a high selective responsiveness to β-lactoglobulin in practical applications.

[0047] Example 3: Sensitivity test of fluorescent probe β-lactoglobulin

[0048] In a volumetric flask, first add β-lactoglobulin to a final concentration of 0-5 μM, then add the probe to a final concentration of 5 μM, and bring the mixture to volume using 0.1 M PBS buffer (pH = 7.4). Let the solution stand for at least 30 minutes. Using an F-7000 fluorescence spectrophotometer, measure the fluorescence emission spectrum of the sample at an excitation wavelength of 490 nm.

[0049] like Figure 5 As shown, the probe fluorescence significantly increased with increasing β-lactoglobulin concentration. From... Figure 5 The inset shows that the change in fluorescence intensity (ΔF) at the probe emission wavelength of 640 nm exhibits a good linear correlation (R0) with the concentration of β-lactoglobulin (0-5 μM) within a certain range. 2 =0.9983). Based on the signal-to-noise ratio of S / N = 3, the detection limit of the probe for β-lactoglobulin was calculated to be 0.046 μM. The probe exhibits extremely high sensitivity in detecting β-lactoglobulin.

[0050] Example 4: Fluorescence quenching assay of β-lactoglobulin by fluorescent probe

[0051] In a volumetric flask, first add β-lactoglobulin to a final concentration of 5 μM, then add the probe to a final concentration of 0-7 μM, and bring the mixture to volume using 0.1 M PBS buffer (pH = 7.4). Let the solution stand for at least 30 minutes. Using an F-7000 fluorescence spectrophotometer, measure the fluorescence emission spectrum of the sample at an excitation wavelength of 280 nm.

[0052] like Figure 6 As shown, the intrinsic fluorescence of β-lactoglobulin was significantly quenched with increasing probe concentration. From... Figure 6 The inset shows that the fluorescence intensity of β-lactoglobulin at an emission wavelength of 337 nm exhibits a good linear correlation (R0) with the probe concentration (0-7 μM) within a certain range. 2 = 0.9941). Based on the classic Stern-Volmer equation, the association constant between the probe and β-lactoglobulin was calculated to be 4.47 × 10⁻⁶. 5 M -1 The probe has a strong binding ability to β-lactoglobulin.

[0053] Example 5: Effect of common commercial drugs on the responsiveness of the fluorescent probe β-lactoglobulin

[0054] Common commercial drug stock solutions preparation: Ibuprofen, phenylbutazone, ibrutinib, anastrozole, erlotinib, regorafenib, capecitabine, moxifloxacin hydrochloride, repaglinide, simvastatin, and telaprevir are dissolved in anhydrous ethanol to prepare a stock solution with a concentration of 1 mM.

[0055] In volumetric flasks, first add β-lactoglobulin to a final concentration of 5 μM and a probe to a final concentration of 5 μM. Then add various commercially available drugs (ibuprofen, phenylbutazone, ibrutinib, anastrozole, erlotinib, regorafenib, capecitabine, moxifloxacin hydrochloride, repaglinide, simvastatin, and telaprevir) to a final concentration of 5 μM each. Dilute the mixture to volume using 0.1 M PBS buffer (pH 7.4). Let the solution stand for at least 30 minutes. Measure the fluorescence emission spectrum of the sample using an F-7000 fluorescence spectrophotometer at an excitation wavelength of 490 nm.

[0056] like Figure 7As shown, the fluorescence response of the probe to β-lactoglobulin in this invention exhibits a certain degree of enhancement in the presence of specific drugs. Specifically, drugs such as phenylbutazone, ibrutinib, anastrozole, and erlotinib have a slight enhancing effect on the probe's β-lactoglobulin response. However, it is noteworthy that many other drugs, including ibuprofen, regorafenib, capecitabine, moxifloxacin hydrochloride, repaglinide, simvastatin, and telaprevir, did not significantly affect the probe's β-lactoglobulin fluorescence response. This result fully demonstrates that the probe of this invention has strong anti-interference ability and can maintain a precise fluorescence response to β-lactoglobulin against complex backgrounds. Therefore, the probe of this invention is suitable for various complex environments and can accurately and reliably detect β-lactoglobulin, showing broad application prospects.

[0057] Example 7: Cytotoxicity test

[0058] The inoculation density in the 96-well plate was 1×10⁻⁶. 4 HepG2 liver cancer cells were collected per well. Different concentrations of probes were added to the corresponding wells, and the samples were incubated at 37 °C in a 5% CO2 incubator for 24 h. The cytotoxicity of the samples was then determined using the MTT assay.

[0059] like Figure 8 As shown, HepG2 cells maintained extremely high cell viability levels as the probe concentration gradually increased. This result clearly demonstrates that the probe has very low cytotoxicity and has no significant adverse effects on cell growth and survival, even at relatively high concentrations.

[0060] Example 8: Live Cell Imaging Assay

[0061] HepG2 cells were cultured in confocal culture dishes and initially incubated with 200 nM Mito-tracker Green for 40 minutes. The cells were then divided into two groups. One group had its original culture medium replaced with fresh medium; the other group had its original culture medium replaced with fresh medium containing β-lactoglobulin (5 μM). Both groups were then incubated for another 30 minutes. After incubation, 5 μM of the probe was added to both groups of cells, and incubation continued for another 30 minutes. The culture medium was removed, and the cells were washed three times with PBS buffer. Fluorescence imaging of the treated HepG2 cells was observed using an inverted fluorescence microscope.

[0062] like Figure 9As shown, when HepG2 cells were stained with the probe at a concentration of 5 μM, a weak fluorescence signal was observed in the red channel. This phenomenon can be attributed to the internalization of exogenous bovine serum albumin by HepG2 cells during culture in a medium containing 10% fetal bovine serum, leading to a slight enhancement of the probe's fluorescence. Notably, the fluorescence signal of this probe closely overlapped with that of Mito-Tracker Green, with a Pearson's colocalization coefficient (Pr) as high as 0.90. This result fully demonstrates the probe's excellent mitochondrial targeting ability, enabling it to accurately locate this important organelle.

[0063] The probe's fluorescence intensity was significantly enhanced upon the introduction of β-lactoglobulin. This change clearly demonstrates the probe's high sensitivity to β-lactoglobulin levels, enabling it to respond accurately and rapidly to changes in intracellular β-lactoglobulin. β-lactoglobulin accumulates primarily in mitochondria after internalization, which aligns with the probe's mitochondrial targeting capability. In the presence of β-lactoglobulin, the probe's fluorescence signal overlapped more closely with the Mito-Tracker Green fluorescence signal (Pr = 0.92). These properties further confirm the reliability and accuracy of the probe as a mitochondrial targeting and β-lactoglobulin detection tool, providing a powerful tool for real-time monitoring of dynamic changes in β-lactoglobulin at the cellular level.

[0064] Example 9: An experiment to assess milk protein levels in beverages using a β-lactoglobulin detection sensor developed based on polyamide films.

[0065] The probe was dissolved in methanol to obtain a 0.5 mM probe stock solution. Simultaneously, the polyamide membrane was cut into 10 mm discs using a punch. The discs were then immersed in the probe stock solution for 10 minutes and allowed to air dry. The β-lactoglobulin detection sensor was obtained through the natural evaporation of methanol.

[0066] Twelve commercially available liquid beverages (including seven types of milk, one type of soy milk with added lactoglobulin powder, one type of milk tea, one type of carbonated beverage, and two types of unsweetened tea beverages) and seven types of solid beverages (including four types of milk powder, two types of whey protein powder, and one type of instant coffee) were selected. The β-lactoglobulin detection sensor was immersed in the liquid beverage or a solid beverage solution prepared at 167 mg / ml for 5 minutes, then removed and air-dried naturally, and its imaging was observed under a 365 nm lamp.

[0067] like Figure 10As shown, the β-lactoglobulin detection sensor exhibits significant fluorescence signals when in contact with samples rich in milk protein; however, almost no fluorescence signals are generated in samples lacking milk protein (such as sample 5 carbonated beverage, sample 7 unsweetened tea beverage, sample 8 unsweetened tea beverage, and sample 19 instant coffee) or samples with low milk protein content (such as sample 6 milk tea). This characteristic indicates that the probe can serve as a rapid and effective detection tool for assessing milk protein levels in beverages.

[0068] In summary, this invention discloses a novel β-lactoglobulin fluorescent probe and its applications. This probe is synthesized using simple chemical raw materials and exhibits high selectivity and sensitivity for β-lactoglobulin. Through non-covalent interactions, it specifically binds to β-lactoglobulin, significantly enhancing the fluorescence signal and achieving efficient detection under low background fluorescence. This probe not only possesses low cytotoxicity but also demonstrates excellent mitochondrial targeting capability, providing a powerful tool for visualizing β-lactoglobulin in living cells. Upon binding to β-lactoglobulin, the sample exhibits a significant color change under UV light irradiation; this characteristic can be used to rapidly assess the content of active β-lactoglobulin in a sample, thereby enabling accurate detection of milk protein levels in beverages. Therefore, the β-lactoglobulin fluorescent probe of this invention shows broad application prospects in fields such as bioimaging, milk protein detection, and beverage quality control.

[0069] The above description is only a partial embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A β-lactoglobulin fluorescent probe having the following structure: 。 2. A method for preparing the β-lactoglobulin fluorescent probe according to claim 1, characterized in that... Includes the following steps: S1, compound 1 6-(dimethylamino)-2-naphthaldehyde and piperidine were dissolved in dichloromethane and refluxed for 4-8 hours. After concentration under reduced pressure, the crude product was purified to obtain compound 2. , S2, Compound 2 obtained in step S1 and iodomethane were dissolved in acetonitrile and refluxed for 20-36 h. After concentration under reduced pressure, a small amount of acetonitrile was added to dissolve the compound, and then precipitated with diethyl ether. After filtration and washing, the β-lactoglobulin fluorescent probe was obtained.

3. The preparation method according to claim 2, characterized in that: The crude product obtained in step S1 was purified by silica gel column chromatography using dichloromethane, ethyl acetate and petroleum ether in a volume ratio of 1:1:

17.

4. The use of the β-lactoglobulin fluorescent probe of claim 1 in in vitro β-lactoglobulin detection or β-lactoglobulin-mediated cell imaging for non-therapeutic or diagnostic purposes.

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