Membrane anchoring fluorescent probe as well as preparation method and application thereof

Abstract

The invention relates to the technical field of preparation and application of fluorescent probes, in particular to a membrane anchoring fluorescent probe as well as a preparation method and application thereof. The membrane anchoring fluorescent probe provided by the invention can specifically image cell membranes of living cells, can quantitatively detect the pH values of different types of cell membranes, successfully distinguish non-alcoholic fatty liver tissues and detect non-alcoholic fatty liver tissues with different lesion degrees. Membrane-anchored fluorescent probe with structural formula of # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent probe preparation and application, and in particular to a membrane-anchored fluorescent probe and a preparation method and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] In recent years, with the continuous development of biochemistry, people have been studying more and more deeply on the active species of living cells, cell signal transduction and cell apoptosis. The cell membrane plays a very important role in the life activities of the body. First, it provides a relatively stable internal environment for the life activities of the cell. Secondly, it provides recognition sites for the cell and completes the transmembrane conduction of substances inside and outside the cell. Thirdly, it provides binding sites for a variety of enzymes, so that the enzymatic reaction can proceed efficiently and orderly. The cell membrane is composed of a phospholipid bilayer and membrane proteins. Fluctuations in the pH value of the cell membrane can lead to changes in the structure of the phospholipid bilayer and changes in the permeability of the cell membrane. Disturbances in the pH value of the cell membrane can seriously affect receptors such as ion channels and transport proteins on the cell membrane, trigger oxidative stress and cell apoptosis, affect normal lipid and energy metabolism processes, and lead to further imbalance of the pH value of the cell membrane. In addition, fluctuations in the pH value of the cell membrane affect enzyme activity, membrane potential and cytoskeleton stability. Therefore, it is particularly important to expand and study new cell membrane pH sensors.

[0004] Non-alcoholic fatty liver disease has become the most common chronic liver disease, accounting for about 25% of the total population. If timely and effective intervention is not carried out, non-alcoholic fatty liver disease is likely to develop into cirrhosis or even liver cancer. Generally speaking, non-alcoholic fatty liver disease is a curable disease that can be cured as long as it is diagnosed and treated in time, so early diagnosis of non-alcoholic fatty liver disease is of great significance.

[0005] In recent years, fluorescence imaging technology based on fluorescent probes has been widely used in cell imaging and biological detection due to its advantages such as high sensitivity, easy operation, and real-time in situ detection. Summary of the invention

[0006] In order to overcome the above problems, the present invention provides a membrane-anchored fluorescent probe and its preparation method and application. The membrane-anchored fluorescent probe provided by the present invention can specifically image the cell membrane of living cells and can quantitatively detect the pH of different types of cell membranes and successfully distinguish non-alcoholic fatty liver tissue and detect non-alcoholic fatty liver tissue with different degrees of pathology.

[0007] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0008] The first aspect of the present invention provides a membrane-anchored fluorescent probe, the structural formula of which is shown in formula (I):

[0009]

[0010] The second aspect of the present invention provides a method for preparing the above-mentioned membrane-anchored fluorescent probe, comprising:

[0011] Dissolving compound 1 in a solvent, adding compound 2, and synthesizing a membrane-anchored fluorescent probe represented by formula (I) under the catalysis of a catalyst;

[0012] The structural formula of compound 1 is shown in formula (II):

[0013]

[0014] The structural formula of compound 2 is shown in formula (III):

[0015]

[0016] In one or more embodiments, the solvent includes anhydrous methanol.

[0017] In one or more embodiments, the molar ratio of compound 1 to compound 2 is 0.8 to 1.2:1, preferably 1:1.

[0018] In one or more embodiments, the concentration of Compound 1 in the solvent is 27-40 mM, preferably 34 mM.

[0019] In one or more embodiments, the catalyst includes piperidine.

[0020] In one or more embodiments, the reaction temperature is 90-100° C., preferably 95° C.; the reaction time is 40-60 h, preferably 48 h.

[0021] The third aspect of the present invention provides the use of the membrane-anchored fluorescent probe described in the first aspect and / or the membrane-anchored fluorescent probe prepared by the preparation method described in the second aspect in cell membrane localization fluorescence imaging.

[0022] In one or more embodiments, the cells include: MIHA, RAW, A549, HeLa and SiHa cells.

[0023] A fourth aspect of the present invention provides the use of the membrane-anchored fluorescent probe described in the first aspect and / or the membrane-anchored fluorescent probe prepared by the preparation method described in the second aspect in detecting the pH value of a cell membrane.

[0024] In one or more embodiments, the cells include: MIHA, RAW, A549, HeLa and SiHa cells.

[0025] The fifth aspect of the present invention provides use of the membrane-anchored fluorescent probe described in the first aspect and / or the membrane-anchored fluorescent probe prepared by the preparation method described in the second aspect in preparing a diagnostic product for non-alcoholic fatty liver disease.

[0026] In one or more embodiments, the diagnostic product includes a detection reagent or a detection kit.

[0027] A sixth aspect of the present invention provides a diagnostic product for non-alcoholic fatty liver disease, comprising the membrane-anchored fluorescent probe described in the first aspect and / or the membrane-anchored fluorescent probe prepared by the preparation method described in the second aspect.

[0028] In one or more embodiments, the diagnostic product includes a detection reagent or a detection kit.

[0029] The beneficial effects of the present invention are:

[0030] The present invention designs and synthesizes a membrane-anchored fluorescent probe, which can be used for fluorescent imaging of cell membranes. Thanks to the lipophilic interaction between the long alkyl chain of the probe and the phospholipids of the cell membrane and the electrostatic interaction between the positively charged pyridinium salt of the probe and the negatively charged phosphate of the cell membrane, the probe can specifically target the cell membrane under these two forces. At the same time, the probe exhibits excellent pH sensitivity and reversibility in both in vitro and cell imaging experiments. 1 H NMR spectroscopy and Gaussian simulation confirmed its pH response mechanism. The highest occupied molecular orbital (HOMO) of the membrane-anchored fluorescent probe was mainly concentrated in the methoxy and naphthalene ring fluorophore regions before protonation, while the lowest unoccupied molecular orbital (LUMO) was significantly distributed on the pyridine ring. The energy gap between HOMO and LUMO was calculated to be 2.37 eV. This separation distribution of frontier orbitals indicates the existence of intramolecular charge transfer (ICT) transition in the membrane-anchored fluorescent probe. After protonation, the HOMO energy is reduced, the distribution is transferred to the alkyl chain, and the methoxyl electron-donating ability is weakened, blocking the ICT process and causing the fluorescence intensity to decrease. Furthermore, the membrane-anchored fluorescent probe can sensitively detect pH changes through the ICT mechanism. It can not only detect the pH of different types of cell membranes and analyze the changes in cell membrane pH during different physiological activities, but also detect different degrees of non-alcoholic fatty liver by imaging the clarity of the liver cell membrane boundary, and can also visualize their pH heterogeneity, thereby realizing the detection of early non-alcoholic fatty liver. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0032] Figure 1 The preparation route for membrane-anchored fluorescent probes;

[0033] Figure 2 Absorption and fluorescence spectra of the probe Mem-pH (10 μM) vary with pH value (a, b), (c) fluorescence intensity of Mem-pH varies with pH value, (d) pH reversibility of Mem-pH (10 μM) between pH 3.0 and 12.0;

[0034] Figure 3 is the photostability of the probe Mem-pH;

[0035] Figure 4 HOMO-LUMO energy level (a) and electrostatic potential (b) of Mem-pH before and after protonation;

[0036] Figure 5 MTT results of HeLa (a), A549 (b), SiHa (c), RAW (d), MIHA (e) and RAW (f) after incubation in different incubation concentrations of Mem-pH for 24 hours; the black bars in (a) to (f) represent the control group, and the red bars represent the Mem-pH treatment group;

[0037] Figure 6 Fluorescence images of GUVs with different pH values ​​stained with 10 mM Mem-pH (a-c); d) Average fluorescence intensity obtained in the circular areas of a, b, and c, Ex = 405 nm, Em = 550-620 nm, scale bar = 20 μm;

[0038] Figure 7 Fluorescence, DIC and merged images of HeLa cells stained with Mem-pH (2uM, 10 minutes) and Dil (0.2uM, 10 minutes); Merged 1 is the merged image of Mem-pH and DIC; Merged 2 is the merged image of Mem-pH and Dil channels; Mem-pH: Ex = 405nm, Em = 550-620nm; Dil: Ex = 532nm, Em = 550-580nm; Scale bar = 20μm;

[0039] Figure 8A in the figure is the fluorescence and merged image of SiHa and HeLa cells stained by Mem-pH (2 μM, 20 minutes), Lyso-Red (0.5 μM, 10 minutes) and MTDR (0.5 μM, 10 minutes), Mem-pH value: Ex = 405 nm, Em = 550-620 nm; Lyso-Red: Ex = 552 nm, Em = 570-610 nm; MTDR: Ex = 635 nm, Em = 650-680 nm; Scale bar = 20 μm; B is the fluorescence distribution along the direction of the white arrow in the merged image of the stained SiHa cells; C is the fluorescence distribution along the direction of the white arrow in the merged image of the stained HeLa cells;

[0040] Fig. 9 Fluorescence, DIC, merged and quantified pseudo-color images of HeLa cells stained by Mem-pH (2 μM, 20 min) at different pH values, Ex = 405 nm, Em = 550-620 nm, scale bar = 20 μm;

[0041] Fig.10 Fluorescence, DIC, merged and quantified pseudo-color images of different cell types stained by Mem-pH (2 μM, 20 min), Ex = 405 nm, Em = 550-620 nm, scale bar = 20 μm;

[0042] Fig.11 Fluorescence, DIC and merged images of HeLa cells stained with Mem-pH (2 μM, 20 min) under normal conditions and after incubation with LPS for different time periods, Ex = 405 nm, Em = 550-620 nm, scale bar = 20 μm;

[0043] Fig.12 Fluorescence, DIC and merged images of HeLa cells stained by Mem-pH (2 μM, 20 min) after incubation with H2O2 for different time periods under normal conditions, Ex = 405 nm, Em = 550-620 nm, scale bar = 20 μm;

[0044] Fig.13 Fluorescent, merged and quantified pseudo-color images of normal and NAFLD tissues stained by Mem-pH staining (2 μM, 20 minutes), Ex=405 nm, Em=550-620 nm, scale bar=20 μm. DETAILED DESCRIPTION

[0045] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0046] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0047] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0048] The cells used in the following examples were cultured in a 37°C 5% CO2 incubator. HeLa, SiHa, MIHA and A549 cells were cultured in f12k medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin, and RAW cells were cultured in RPMI1640 medium containing 10% FBS and 1% penicillin / streptomycin.

[0049] Example 1

[0050] Figure 1 For the preparation route of membrane-anchored fluorescent probes, refer to Figure 1 , synthesized membrane-anchored fluorescent probe (hereafter referred to as Mem-pH).

[0051] Preparation of Mem-pH:

[0052] Compound 1 (0.400 g, 0.845 mmol) and anhydrous methanol (25 mL) were added to a three-necked flask and heated at 55°C until completely dissolved, followed by compound 2 (0.198 g, 0.845 mmol) and piperidine (6 drops). The temperature was raised and refluxed at 95°C for 48 h until a reddish-brown solid precipitated. After the reaction was completed, the system was cooled to room temperature and filtered to obtain the membrane-anchored fluorescent probe Mem-pH. 1H NMR (DMSO-d6., 300MHz): δ (ppm): 8.83 (d, J = 8Hz, 2H), 8.13 (d, J = 8Hz, 2H), 8.04 (d, J = 16Hz, 2H), 7.79 (s, 3H), 7.47 (m, 1H),7.28(d,1H),7.09(s,1H),4.37(d,J=16Hz,2H),3.78(s,2H),1.79(s,2H),1.13(m,30H),0.72(d,J=12Hz,3H).13C NMR (101MHz, DMSO-d6): δ (ppm): 158.55, 152.92, 144.08, 141.11, 135.36, 130.47, 130.11, 129.68, 128.17, 127.58, 124.14, 123.54, 1 22.32,119.33,106.30,59.58,55.31,31.19,30.38,28.94,28.89,28.77,28.65,28.60,28.26,25.29,21.99,13.85.HRMS(m / z):calcd for C 36 H 52 INO:641.30; found:514.41(MI) + .

[0053] Example 2

[0054] In order to verify whether the probe Mem-pH can sense pH, a pH titration experiment was performed. The absorption and fluorescence spectra of Mem-pH are shown in Figure 2 As shown. Figure 2 As shown in a-b, the maximum absorption and emission peaks of the probe Mem-pH are approximately 390 nm and 580 nm, respectively. In addition, the fluorescence intensity of Mem-pH gradually increases with the increase of pH value, indicating that the ICT process of the probe is restored in an alkaline environment. In addition, the linear fluorescence response of the probe Mem-pH to pH is demonstrated by fitting the curve to the fluorescence intensity at different pH values ​​( Figure 2 c). More importantly, after several acid-base cycles, the fluorescence intensity of Mem-pH remained almost unchanged, demonstrating that Mem-pH has an excellent reversible pH response. ( Figure 2 d) In addition, the photostability of Mem-pH was studied, e.g. Figure 3 As shown in the figure, even after 20 minutes of continuous irradiation with a xenon lamp, the fluorescence intensity of Mem-pH remained stable, which is conducive to long-term imaging of cells by Mem-pH. These experimental results show that Mem-pH can respond to different pH values ​​and can fit the linear relationship well, which provides a basis for detecting and calculating pH changes in cell membranes.

[0055] Furthermore, to understand the relationship between the optical properties and electronic transitions, the energy and electrostatic potential of Mem-pH were calculated using density functional theory (DFT). Figure 4 a shows that the highest occupied molecular orbital (HOMO) of Mem-pH is mainly concentrated in the methoxy and naphthalene ring fluorophore regions before protonation, while the lowest unoccupied molecular orbital (LUMO) is significantly distributed on the pyridine ring, and the energy gap between HOMO and LUMO is calculated to be 2.37 eV. This separation distribution of frontier orbitals indicates the existence of ICT transition in Mem-pH. After protonation, the HOMO energy decreases, the distribution is transferred to the alkyl chain, the methoxy electron donor is weakened, the ICT process is blocked and the fluorescence intensity is reduced. Figure 4 b further shows that the potential energy of the protonated methoxy molecules increases, which inhibits the electron donor ability and ICT process of the methoxy group and ultimately leads to a decrease in its fluorescence intensity.

[0056] Example 3

[0057] The cytotoxicity of the probe is an important parameter before cell staining and imaging, so 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent was used to quickly and easily identify the cytotoxicity of Mem-pH. Figure 5 As shown in Figure 3, after incubation with different concentrations of Mem-pH (2, 4, 6, 8, and 10 μM) for 24 h, the cell viability of HeLa, A549, SiHa, RAW, and MIHA was still above 85%. Therefore, the cytotoxicity of Mem-pH at short time and low dose is negligible.

[0058] Example 4

[0059] Giant unilamellar vesicles (GUVs) are a model membrane that is often used to mimic cell membranes to analyze the physical properties of probes. Here, the ability of the probe Mem-pH to detect membrane pH using GUVs with different pH values ​​was first tested. Figure 6 As shown in Figure 2, the probe Mem-pH can clearly image GUVs, and its fluorescence intensity gradually increases with the increase of pH value. Figure 6 As shown in Figure 3, as the pH value of GUVs increased from 4.00 to 9.18, the fluorescence intensity of Mem-pH increased by nearly 10 times. Therefore, the probe Mem-pH can visualize GUVs composed of phospholipids and give different fluorescence intensity signals according to different pH values.

[0060] Then, live cells were stained with the probe Mem-pH to verify whether it could be targeted to the cell membrane. Figure 7As shown, the Mem-pH fluorescence from differential interference microscopy (DIC) and fluorescence merged images completely outlines the cell contours. In addition, the fluorescence of the probe Mem-pH can be completely covered by the commercial cell membrane probe Dil, and the colocalization coefficient reaches 0.89. In addition, Mem-pH was co-stained with the commercially available lysosomal probe Lyso-Red and mitochondrial probe MTDR to further confirm that it can stain the cell membrane. Figure 8 As shown, the fluorescence of the probe Mem-pH is distributed around Lyso-Red and MTDR and does not overlap with them at all. Therefore, the probe Mem-pH can specifically stain the cell membrane.

[0061] Example 5

[0062] In order to prove that Mem-pH can detect the pH value of cell membrane, HeLa cells were selected and stained with Mem-pH in different acid-base environments. Fig. 9 As shown in Figure 2, as the pH value increases from 4.00 to 9.18, the cell membrane fluorescence emitted by Mem-pH gradually increases. This may be because the ICT process of Mem-pH is inhibited by the protonation of the methoxyl group in an acidic environment and is restored in an alkaline environment. Figure 2 c) The linear relationship between pH and the corresponding fluorescence intensity is calculated. As the pH increases, the pseudo color gradually changes from purple to yellow, indicating a gradual increase in fluorescence intensity. These data clearly show that Mem-pH is able to image and monitor cell membrane pH using fluorescence intensity.

[0063] Example 6

[0064] Mem-pH probes can be used to distinguish normal cells from cancer cells and study cell physiological activities:

[0065] According to relevant reports, the cell membrane will become acidic after canceration, so the probe Mem-pH can be used to distinguish and detect normal cells and cancer cells. For this purpose, five cell types, including MIHA, RAW, A549, HeLa and SiHa cells, were selected for Mem-pH staining. Fig.10 As shown in the figure, the probe Mem-pH completely analyzed all cells, which shows that Mem-pH has the ability to stain the cell membranes of normal cells and cancer cells. However, since the pH value of cancer cell membranes (pH = 6.2-6.8) is not much different from that of normal cell membranes (pH = 7.2-7.4), the fluorescence intensity of Mem-pH is different from that of normal cell membranes. Fig.10 The fluorescence channel does not change much. Figure 2The equation in c calculates the pH values ​​of different cell membranes. By comparing the pH value pseudo-color bands, the probe Mem-pH mainly exhibits yellow fluorescence in normal cell membranes and purple fluorescence in cancer cell membranes. This indicates that the pH value of cancer cell membranes is lower than that of normal cell membranes. Therefore, the probe Mem-pH successfully distinguishes and detects the pH values ​​of cancer cell membranes and normal cell membranes through fluorescence intensity calculation.

[0066] In the physiological process of cellular inflammation, the pH value of the cell membrane is affected by the increase of cellular metabolic activity. For this reason, HeLa cells were selected to construct an inflammation model by treating them with lipopolysaccharide (LPS) to study this cellular behavior. Fig.11 As shown in the figure, with the increase of LPS treatment time, the Mem-pH fluorescence intensity of the cell membrane continued to decrease. This should be because inflammation leads to dysfunction of ion channels, transporters and proton pumps on the cell membrane, which in turn leads to the outflow of metabolites such as hydrogen ions, organic acids and bicarbonate, thereby reducing the pH value of the cell membrane. In addition, cell apoptosis is also an important physiological activity that plays a key role in processes such as cell development, tissue repair and immune response. Therefore, HeLa cells were then treated with H2O2 to trigger the apoptosis process. As Fig.12 As shown in the figure, as the H2O2 treatment time increases, the cell membrane begins to swell and deform, and the fluorescence intensity of Mem-pH continues to decrease. Therefore, during apoptosis, the pH value of the cell membrane also decreases due to the dysfunction of blood permeability and cell membrane imbalance. In summary, the probe Mem-pH can not only detect the pH value of normal cells and cancer cells, but also analyze the changes in cell membrane pH during different physiological activities.

[0067] Example 6

[0068] Non-alcoholic fatty liver disease is a typical hepatic fatty degeneration disease that can be cured through early diagnosis and timely treatment. Metabolites produced during lipid deacidification will reduce the pH value, so liver tissue acidification is also an important parameter reflecting the degree of non-alcoholic fatty liver disease, so it is very meaningful to detect pH changes in fatty liver. Considering the pH response characteristics of Mem-pH, it is used to detect pH changes in non-alcoholic fatty liver disease. Fig.13As shown, the probe Mem-pH can visualize the outline of liver tissue, and the outline gradually becomes blurred as fatty liver worsens. In addition, compared with normal tissue, the fluorescence of Mem-pH in non-alcoholic fatty liver tissue is weaker and gradually decreases as fatty liver worsens. According to the pH pseudo-color test paper, the probe Mem-pH shows yellow fluorescence at the clear and regular boundaries of the hepatocyte membrane, and blue fluorescence at the blurred boundaries. These data indicate that the deterioration of non-alcoholic fatty liver increases the acidification of liver tissue and disrupts the boundaries of tissue cells. Therefore, the probe Mem-pH can not only detect different degrees of non-alcoholic fatty liver by imaging the clarity of the hepatocyte membrane boundaries, but also visualize their pH heterogeneity. Therefore, the probe Mem-pH can not only distinguish non-alcoholic fatty liver tissue, but also detect early fatty liver lesions, providing hope for the timely diagnosis of non-alcoholic fatty liver.

[0069] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A membrane-anchored fluorescent probe, characterized in that: Its structural formula is shown in formula (I):

2. The method for preparing a membrane-anchored fluorescent probe according to claim 1, characterized in that: include: Dissolving compound 1 in a solvent, adding compound 2, and synthesizing a membrane-anchored fluorescent probe represented by formula (I) under the catalysis of a catalyst; The structural formula of compound 1 is shown in formula (II): The structural formula of compound 2 is shown in formula (III):

3. The method according to claim 2, characterized in that The solvent includes anhydrous methanol; Or, the molar ratio of compound 1 to compound 2 is 0.8 to 1.2:1, preferably 1:1; Alternatively, the concentration of compound 1 in the solvent is 27-40 mM, preferably 34 mM. or, the catalyst comprises piperidine; Alternatively, the reaction temperature is 90-100° C., preferably 95° C.; the reaction time is 40-60 h, preferably 48 h.

4. Use of the membrane-anchored fluorescent probe according to claim 1 and / or the membrane-anchored fluorescent probe prepared by the preparation method according to any one of claims 2 to 3 in cell membrane localization fluorescence imaging.

5. The use according to claim 4, characterized in that Cells include: MIHA, RAW, A549, HeLa and SiHa cells.

6. Use of the membrane-anchored fluorescent probe according to claim 1 and / or the membrane-anchored fluorescent probe prepared by the preparation method according to any one of claims 2 to 3 in detecting the pH value of cell membrane.

7. The use according to claim 6, characterized in that Cells include: MIHA, RAW, A549, HeLa and SiHa cells.

8. Use of the membrane-anchored fluorescent probe according to claim 1 and / or the membrane-anchored fluorescent probe prepared by the preparation method according to any one of claims 2 to 3 in the preparation of a diagnostic product for non-alcoholic fatty liver disease.

9. A diagnostic product for non-alcoholic fatty liver disease, characterized in that: It comprises the membrane-anchored fluorescent probe according to claim 1 and / or the membrane-anchored fluorescent probe prepared by the preparation method according to any one of claims 2 to 4.

10. The diagnostic product according to claim 9, characterized in that The diagnostic products include detection reagents and detection kits.

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