CN105241872B - Detect the method and agents useful for same of half lactadherin -1 in blood - Google Patents

Abstract

The present invention provides a kind of method and agents useful for same of detection sample to be tested half lactadherin 1 especially in blood, the method for the present invention includes：Using the gold nanorods of Lactose-modified as probe, it is made to contact mixing with sample to be tested, 20~37 DEG C of maintenance system temperature stands 10 minutes to 2 hours, half lactadherin 1 is detected by detecting the color of mixed system.The present invention is with the gold nanorods (Lac GNRs) of Lactose-modified for probe, using ultraviolet-visible near infrared spectrometer as detection means, efficient and special detection is carried out to gelactin 1 in molecular level and cellular level, and the galectin 1 in blood of human body is accurately detected, make a kind of means of cancer metastasis early monitoring.

Description

Method for detecting galectin-1 in blood and reagent used

technical field

The invention relates to a method for detecting galectin-1 in blood and a reagent used therefor.

Background technique

Cancer is a disease that seriously threatens human life and health around the world. According to statistics, more than 7 million people die of cancer in the world every year (Ahmedin, J., Rebecca S., Elizabeth W., Hao, Y.P., Xu, J.Q., Taylor , M., Michael, J.T. Cancer Statistics, CA: Cancer J. Clin. 2008, 58, 71-96). The incidence of cancer in my country is on a linear upward trend, and a large number of patients are already in the middle and late stages when they seek treatment, basically losing the possibility of cure. An important reason why patients with advanced cancer are difficult to cure is that cancer is prone to metastasis. The primary tumor can be removed surgically or treated with radiation, but cancer that has spread is often difficult to treat without damaging normal tissue. Tumor metastasis is accompanied by poor prognosis and low survival rate. If effective detection methods can be developed for early monitoring, it will help timely detection and early intervention of metastatic events, which will help prolong the life of patients and improve their quality of life. (Liao Zijun, Lei Guangyan, Zhang Guanjun, Liu Wenchao, Li Hongsheng, Yu Guozheng, Tumor Metastasis, Shaanxi Science and Technology Press, 2007, p322-p331).

Tumor metastasis is a complex cascading process. In this complex process, tumor cells often need to express a large number of specific proteins (such as integrins, cadherins, immunoglobulins, matrix metalloproteinases, plasmin activating factors, etc.) , vascular growth factors, etc.) to achieve the functions of degrading extracellular matrix, invading adjacent cell layers, inducing cancer cell movement, evading immune system attack, promoting new angiogenesis and metastatic tumor cell proliferation (Zeng Yixin, Lv Youyong, Zhu Minghua, Chen Guoqiang, Gong Jianping, Guo Zhuming, Oncology (Third Edition), People's Health Publishing House, 2012, p241-p252). Some secreted proteins may enter the circulatory system and be detected in the blood, and become effective biomarkers of tumor metastasis. The detection of these biomarkers can help realize the early diagnosis of tumor metastasis and become an important means of tumor metastasis prediction (Xue , H., Li, B.J., Zhang, J., Wu, M., Huang, Q., Wu, Q., Sheng, H., Wu, D., Hu, J., Lai, M., Identification of Serum Biomarkers for Colorectal Cancer Metastasis Using a Differential Secretome Approach. J. Proteome. Res., 2010, 9, 545-555).

Galectin-1 (galectin-1) is an important serum marker, which plays an important role in the proliferation, invasion, metastasis, angiogenesis and immune escape of tumor cells (Liu, F.T., Rabinovich, G.A. Galectinsas Modulators of Tumor Progression. Nature Reviews Cancer. 2005, 5, 29-41). A large number of studies have shown that galectin-1 is overexpressed in a variety of tumors (lung cancer, liver cancer, gastric cancer, cervical cancer, breast cancer, glioma, melanoma, etc.), and its expression level is significantly higher than that of normal people. Gao (Kuo, P.L., Hung, J.Y., Huang, S.K., Chou, S.H., Cheng, D.E., Jong, Y.J., Hung, C.H., Yang, C.J., Tsai, Y.M., Hsu, Y.L., Huang, M.S., Lung Cancer-Derived Galectin-1Mediates Dendritic Cell Anergythrough Inhibitor of DNA Binding 3/IL-10Signaling Pathway.J.Immunol.2011,186,1521-1530; Chen,J.,Tang,d.,Wang,S.,Li,Q.G.,Zhang, J.R.,Li,P.,Lu,Q.,Niu,G.,Gao,J.,Ye,N.Y.,Wang,D.R.,High Expressions of Galectin-1 and VEGF are Associated with Poor Prognosis in Gastric Cancer Patients,Tumor Biol.2014 , 35, 2513-2519). At the same time, as an overexpressed substance, galectin-1 will be secreted into tumor stroma and human blood to play its role, so galectin-1 can be used as a serum marker of tumor metastasis. Detection of galectin-1 in blood may become a new method for early monitoring of tumor metastasis. However, although the blood galectin-1 concentration of cancer patients is higher than that of normal people, its absolute content is still very low (ng/ml level), and there is still a lack of effective and convenient detection methods for early detection and intervention treatment of cancer metastasis Not much help.

Contents of the invention

An object of the present invention is to provide a method for effectively and conveniently detecting galectin-1 in blood to improve detection sensitivity in view of the lack of effective detection of galectin-1 in blood in the prior art And specificity, to help early detection and intervention treatment of cancer metastasis.

Another object of the present invention is to provide a detection reagent specially designed for effective and convenient detection of galectin-1 in blood, which is applied to detect galectin-1 in blood and has high sensitivity and specificity.

In order to achieve the above object, on the one hand, the present invention provides a method for detecting galectin-1 (galectin-1) in a sample to be tested, the method mainly comprising lactose-modified gold nanorods (Lac-GNRs) As a probe, an ultraviolet-visible-near-infrared spectrophotometer is used as a detection method to efficiently and specifically detect gelactin-1.

Specifically, the present invention provides a method for detecting galectin-1 in a test sample, the method comprising:

Using lactose-modified gold nanorods as probes, make them contact and mix with the sample to be tested, keep the system temperature at 20-37°C for 10 minutes to 2 hours, and detect galectin-1 by detecting the color of the mixed system.

The method of the present invention can accurately detect galectin-1 at the molecular level or cellular level or directly in human blood. Since galectin-1 is closely related to tumor metastasis, the method of the present invention may become a cancer metastasis A novel means of early detection.

According to a specific embodiment of the present invention, in the method for detecting galectin-1 (galectin-1) in a test sample of the present invention, the test sample can be an aqueous solution of galectin-1, a cancer cell culture fluid or It is the blood or serum of the individual to be tested, and the method of the present invention has high detection sensitivity and specificity.

According to a specific embodiment of the present invention, when the method for detecting galectin-1 (galectin-1) in a test sample of the present invention is specifically implemented, the test sample (such as an aqueous solution of galectin-1, cancer cells, etc. culture medium or human serum, etc.) into the lactose-modified gold nanorods (Lac-GNRs) aqueous solution, shake well and let it stand for 10 minutes to 2 hours, and use the ultraviolet-visible-near-infrared spectrophotometer (UV-Vis-Near-infrared spectrophotometer (UV-Vis- NIS spectrophotometer) optically detects the system, and judges whether the substance to be tested contains galectin-1 through the change of the absorption curve, or can also detect the galectin-1 in the sample to be tested by visually observing the color change of the system.

According to a specific embodiment of the present invention, in the method for detecting galectin-1 in a test sample of the present invention, the concentration of lactose-modified gold nanorods in the aqueous solution of lactose-modified gold nanorods is 10 nM to 100 nM.

According to a specific embodiment of the present invention, in the method for detecting galectin-1 in a test sample of the present invention, the volume ratio of the test sample to the lactose-modified gold nanorod aqueous solution is 1:1-100.

The method for detecting galectin-1 in the test sample of the present invention has high sensitivity, and the concentration of Galectin-1 in the test sample can be detected as low as 1 pM.

On the other hand, the present invention also designs a detection reagent specially designed for effective and convenient detection of galectin-1 in blood, which mainly includes lactose-modified gold nanorods (Lac-GNRs). According to a specific embodiment of the present invention, the lactose-modified gold nanorods are complexes formed by linking sulfhydryl-containing lactose to gold nanorods through gold-sulfur bonds.

Gold nanorods are a very important tool in the detection of trace substances due to their unique surface plasmon resonance effect. The surface plasmon resonance effect enables it to absorb incident light at its resonant frequency, resulting in a loss of light energy at this frequency, which is easily detected by an optical detector. This surface plasmon resonance is extremely sensitive to the surface environment of gold, and microscopic changes on the gold surface, including surface modification, agglomeration or deagglomeration caused by external factors, can cause obvious changes in optical absorption (Cao, J., Sun, T. ., Grattan, K.T.V. Gold Nanorod-Based Localized Surface PlasmonResonance Biosensors: A Review, Sensors and Actuators B, 2014, 195, 332–351). At the same time, gold nanorods have special spatial anisotropy, and their rod-like shape makes them have surface plasmon resonance peaks in both horizontal and vertical directions, making them particularly sensitive to trace substances (Huang, H.W.Qu, C.T., Liu, X.Y.,Huang,S.W.,Xu,Z,J.,Zhu,Y.J.,Chu,P.K.,Amplification of Localized Surface PlasmonResonance Signals by a Gold Nanorod Assembly and Ultra-Sensitive Detection ofMercury,Chem.Commun.,2011,47,6897- 6899). It is because of these properties that gold nanorods may become a tool for galectin-1 detection.

However, in order to achieve efficient and specific detection of galectin-1, in addition to using gold nanorods as the base material, it is also necessary to load functional substances that can specifically recognize galectin-1. In the present invention, lactose, a small chemical molecule capable of specifically binding to galectin-1, is selected as a recognition substance to functionalize the gold nanorods. Lactose is loaded on gold nanorods, lactose is used to realize the recognition function, and gold nanorods are used to realize the optical response function, which can realize the efficient and specific detection of galectin-1, and can avoid the traditional substances that play a recognition role, such as the ability to interact with Antibodies or small molecular substances that specifically bind to the analyte have defects such as high cost, complicated operation, variability, and easy self-agglomeration of gold nanorods.

According to a specific embodiment of the present invention, the lactose-modified gold nanorods (Lac-GNRs) of the present invention are prepared according to the following method:

The gold nanorod and the lactose containing sulfhydryl group are mixed, and left standing at room temperature for 8-15 hours to prepare a biological probe for detecting the tumor marker galectin-1. The biological probe is a complex formed by linking the sulfhydryl-containing lactose with the gold nanorod through a gold-sulfur bond.

According to a specific embodiment of the present invention, when the lactose-modified gold nanorods (Lac-GNRs) of the present invention are prepared, the length of the gold nanorods is about 60-80 nm and the width is about 20-25 nm. Preferably, the surface of the gold nanorods is coated with hexadecyltrimethylammonium bromide molecules, which can be prepared by seed induction in an aqueous solution: first reduce chloroauric acid with sodium borohydride to prepare 1 ~2nm gold seeds, and then gold nano-seeds were added to chloroauric acid (HAuCl ₄ ), silver nitrate (AgNO ₃ ), dilute hydrochloric acid (HCl), ascorbic acid, lauryl dimethyl ammonium bromide (CTAB) In the mixed aqueous solution, gold nanorods with a width of about 20-25nm and a length of about 60-80nm, whose surface is covered by CTAB, are produced. The peak position of the transverse plasmon resonance absorption peak of the gold nanorod prepared in the present invention is about 510 nm, and the peak position of the longitudinal plasmon resonance absorption peak is 700-800 nm.

According to a specific embodiment of the present invention, when the lactose-modified gold nanorods (Lac-GNRs) of the present invention are prepared, the thiol-containing lactose has the structural formula It can be glycosylated, mercaptolated and deacetylated using peracetyl lactose (peracetyllactose can be obtained commercially or prepared by yourself with reference to the existing technology, usually obtained by the reaction of acetic anhydride, sodium acetate and lactose under heating conditions) It is prepared by introducing a sulfhydryl-containing linking arm at the terminal carbon atom of lactose through reaction steps such as chemical reaction. Specifically, the thiol-containing lactose can be prepared as follows:

Whole acetyl lactose (compound 1, structural formula ) and chlorotriethylene glycol (compound 2, structural formula ) under the effect of catalyst, stirred and reacted at room temperature for 36～72 hours, after purification, the obtained structural formula is The compound 3; the molar ratio of the compound 1 and the compound 2 is 1:1.2～2;

(b) The compound 3 and potassium thioacetate were stirred and reacted at room temperature for 3-5 hours, and after purification, the obtained structural formula was The compound 4; the molar ratio of the compound 3 and the potassium thioacetate is 1:1.2～2;

(c) The compound 4 is dissolved in methanol, then sodium metal is added, stirred overnight at room temperature, after purification, the lactose containing mercapto group is obtained (compound 5, structural formula ).

According to a specific embodiment of the present invention, when the lactose-modified gold nanorods (Lac-GNRs) of the present invention are prepared, the molar ratio of the sulfhydryl-containing lactose to the gold nanorods is preferably 1×10 ⁴ :1- 3×10 ⁴ : 1. The prepared lactose-modified gold nanorods are coated with hexadecyltrimethylammonium bromide bilayers on the surface of the gold nanorods, and the thiol-containing lactose is not coated with ten Part or all of the hexaalkyltrimethylammonium bromide bilayer is connected to the gold nanorods through gold-sulfur bonds to form a complex.

According to a specific embodiment of the present invention, the method of the present invention can utilize the lactose-modified gold nanorods (Lac-GNRs) to detect galectin-1 at the molecular level, cell level or blood level.

In a specific embodiment of the present invention, the present invention evaluates the detection of galectin-1 at the molecular level, and finds that Lac-GNRs can detect the presence of galectin-1 very sensitively, and the detection limit can reach 1pM. And the response value varies with the concentration of galectin-1. The combination of Galectin-1 and lactose can induce the agglomeration of gold nanorods, thereby changing the light absorption in the ultraviolet-visible-near-infrared region (UV-Vis-NIS). When 1nM galectin-1 was added to the system, the absorption peak at 770nm decreased sharply, and a new absorption peak appeared at 1100nm, indicating that the gold nanorods had been seriously agglomerated, and the color of the Lac-GNR solution changed from orange to When the concentration of galectin-1 decreased to 100pM, the absorption peak at 770nm also decreased significantly, and a new peak appeared at 900nm, indicating that the agglomeration of gold nanorods was also more obvious. The concentration of galectin-1 can be reduced to 1pM, and the peak at 770nm is still significantly reduced at this time, indicating that Lac-GNRs are particularly sensitive to galectin-1, and can detect extremely low concentrations of galectin-1.

In addition to responding to galectin-1, Lac-GNRs respond very poorly to other proteins that cannot recognize lactose (such as bovine serum albumin BSA). For example, adding 1 nm of galectin-1 to the system can induce a significant change in the absorption peak, and even adding BSA at a much larger concentration, or adding the same concentration of galectin-3 (galectin-3) with a structure similar to galectin-1 -3), none of them can significantly change the absorption of gold nanorods, indicating that the specificity of Lac-GNRs is very good.

In another specific embodiment of the present invention, the present invention studies the detection of galectin-1 at the cellular level. Since galectin-1 is a secreted protein, it will be secreted into the extratumoral matrix to play a role. The cancer cells are cultured by the method of cell culture, and the culture fluid thereof is taken for detection, and the existence of galectin-1 should be detected. In the present invention, HUH-7 (human liver cancer cells), MCF-7 (human breast cancer cells), JEG-3 (human choriocarcinoma cells), HeLa (human cervical cancer cells), Hep-G2 (human liver cancer cells) have been cultured in total. ), C6 (mouse glioma cells) and other 6 kinds of tumor cells, and their cell culture fluids were detected, and it was found that the culture fluids of these 6 kinds of tumor cells could cause a significant decrease in the Lac-GNRs light absorption curve, It shows that Lac-GNRs can accurately detect galectin-1.

In another specific embodiment of the present invention, the present invention studies the detection of galectin-1 at human blood level. Since galectin-1 is expressed in large quantities in the metastatic stage of cancer and is secreted into the blood to play a role, the concentration of galectin-1 in the blood of patients with cancer metastasis is significantly higher than that of normal people. The present invention collects the blood of 5 cases of normal people and the blood of 15 cases of cancer patients who have been confirmed as metastases, and detects them, and finds that Lac-GNRs can significantly distinguish the blood of normal people and cancer patients: the blood of normal people only It can cause a slight red shift (shift to the right) of the Lac-GNRs absorption curve, but the blood of cancer patients can not only cause a large red shift of the Lac-GNRs absorption curve, but also cause a significant decrease in the absorption curve. The results indicate that Lac-GNRs can accurately detect galectin-1 in blood, and may become a clinically available method for monitoring cancer metastasis.

In addition to measuring the light absorption in the UV-Vis-NIS region, the test results can also be judged directly with the naked eye: the Lac-GNR solution added with normal human serum has the same color as before adding serum; Serum Lac-GNR solution, its color is obviously lightened. This distinct change in color greatly enhances the visibility and convenience of inspection.

In summary, the method for detecting galectin-1 in the sample to be tested of the present invention can detect the tumor prognosis marker galectin-1 in human blood in vitro, and lactose-modified gold nanorods (Lac-GNRs ) as a probe, using UV-visible-near-infrared spectrophotometer as a detection method, efficiently and specifically detect gelactin-1 at the molecular and cellular levels, and accurately detect galectin-1 in human blood , after using the method of the present invention to detect galectin-1 in the sample to be tested from a human body to be tested, the method of the present invention can be used to detect galectin-1 in the same sample from a healthy individual at the same time Detection, compare the test results of the sample to be tested with the test results of samples from healthy individuals, and judge the health of the individual to be tested according to the difference in light absorption curve or color difference between the two, making it a tool for early monitoring of cancer metastasis a means. The main beneficial technical effects of the present invention include:

The present invention successfully uses the small sugar molecule lactose to stably modify the gold nanorods, and constructs a new molecular probe that can be used to detect the tumor prognosis marker galectin-1; using the molecular probe to The tumor prognostic marker galectin-1 in the blood has been accurately detected, which can not only realize the efficient and specific detection of galectin-1, but also distinguish the blood of normal people and patients with cancer metastasis, which may have real clinical value , and become a new method for early monitoring of tumor metastasis. In addition, since Lac-GNRs avoids the use of biological macromolecules such as enzymes and antibodies, the detection cost is greatly reduced and the operation steps are simplified. Since the detection method is a commonly used optical detection tool—ultraviolet-visible light-near-infrared spectrophotometer, the technology of the present invention also has the advantages of simple operation, convenience and easy operation.

Description of drawings

Figure 1 is a schematic diagram of the synthesis of seed-induced gold nanorods in Example 1 of the present invention.

FIG. 2A is an absorption curve diagram of gold nanorods prepared in Example 1 of the present invention.

FIG. 2B is a microstructure diagram of gold nanorods prepared in Example 1 of the present invention.

Fig. 3 is a synthetic route diagram of the lactose ligand in Example 1 of the present invention.

Fig. 4 is a schematic diagram of lactose modification on the surface of gold nanorods in Example 1 of the present invention.

Fig. 5A is the spectrogram (XPS) of element composition changes of gold nanorods before and after lactose modification in Example 1 of the present invention.

FIG. 5B is a histogram comparing the change of surface charge (zeta potential) of gold nanorods before and after lactose modification in Example 1 of the present invention.

FIG. 5C is a comparison diagram of light absorption changes in the ultraviolet-visible-near-infrared region of gold nanorods before and after lactose modification in Example 1 of the present invention.

FIG. 5D is a microstructure diagram of gold nanorods modified by lactose in Example 1 of the present invention.

Fig. 6A is a graph showing the relationship between the detection absorption curve of Lac-GNRs and galectin-1 in Example 2 of the present invention as a function of concentration.

6B is a photo of the aggregation degree of Lac-GNRs when 1 nM galectin-1 is added in the detection of galectin-1 by using Lac-GNRs in Example 2 of the present invention.

Fig. 6C is a graph showing the color change of the solution in the detection of galectin-1 by using Lac-GNRs in Example 2 of the present invention.

Fig. 7 is a comparison chart of the specificity detected by using Lac-GNRs to detect galectin-1 in Example 2 of the present invention.

Fig. 8 is the detection result of galectin-1 in tumor cell culture medium by using Lac-GNRs in Example 3 of the present invention.

Fig. 9 is the detection result of galectin-1 in human blood by using Lac-GNRs in Example 4 of the present invention.

Fig. 10 is the result of solution color change when Lac-GNRs are used to detect galectin-1 in human blood in Example 4 of the present invention.

Detailed ways

In order to understand the essence of the present invention more clearly, the present invention will be further described in detail below through specific embodiments and accompanying drawings, but the present invention is not limited thereby. The experimental methods without specific conditions indicated in the following examples are usually carried out according to the conventional operations in the field or according to the conditions suggested by the manufacturer.

Example 1, Preparation of lactose-modified gold nanorod detection probes

In this example, the seed-induced gold nanorod synthesis method is used to prepare gold nanorods with good dispersion performance, single particle size, suitable long-short axis ratio, and stable existence; synthesize suitable lactose ligands; Lactose ligands were loaded onto the surface of gold nanorods to achieve lactosylation on the surface of gold nanorods, and a lactose-modified gold nanorod detection probe was prepared and its structure was confirmed.

1. Preparation of gold nanorods

Gold rods were prepared by seed-induced synthesis of gold nanorods in aqueous solution. First use sodium borohydride to reduce chloroauric acid to prepare 1-2nm gold seeds, then add gold nano-seeds to chloroauric acid (HAuCl ₄ ), silver nitrate (AgNO ₃ ), dilute hydrochloric acid (HCl), ascorbic acid, dodecanoic acid In a mixed aqueous solution of alkyldimethylammonium bromide (CTAB), gold nanorods with a width of about 20 to 25 nm and a length of about 60 to 80 nm, whose surface is covered with CTAB, are produced. The specific experimental operation is as follows (can be combined and shown in Figure 1):

Dissolve 0.5 mM HAuCl ₄ in 0.2 M CTAB solution, stir vigorously, add 600 microliters of 10 mM cold NaHB ₄ solution, and stir at 37° C. for 2 hours to obtain a gold nanometer seed solution;

Then add 0.2M CTAB, 0.1M AgNO ₃ , 1.2M HCl and 10mM ascorbic acid to the 5mM HAuCl ₄ solution. When the solution turns from dark red to colorless, add 50 microliters of the above-prepared gold nano-seed solution. After incubation for 12 hours, centrifuge at 10,000 rpm for 10 minutes, remove the supernatant, and redisperse the residue with ultrapure water to obtain an aqueous solution of gold nanorods (CTAB-GNRs) whose surface is covered by CTAB.

The optical absorption spectrum shows that the prepared gold nanorods have weak and strong absorptions at 510nm and 770nm respectively (corresponding to the short-axis absorption and long-axis absorption of gold nanorods, respectively, as shown in Figure 2A). Transmission electron microscopy (TEM) shows that the width of the gold nanorods is about 20nm-25nm, and the length is about 60nm-80nm (as shown in Figure 2B).

2. Synthesis of lactose ligand

Starting from peracetyllactose, through the three-step reaction of glycosylation, sulfhydrylation and deacetylation, a sulfhydryl-containing linking arm was introduced at the terminal carbon atom of lactose to prepare a lactose complex capable of interacting with gold nanorods. body (the synthetic route is shown in Figure 3). The specific experimental process is as follows:

(1) Preparation of 1-β-(chlorotriethylene glycol)-2,3,6,2',3',4',6'-heptaacetyllactose (compound 3):

Add sodium acetate to acetic anhydride, heat to 130°C, slowly add lactose to obtain a reaction solution, the molar ratio of acetic anhydride, sodium acetate and lactose is 12:12:1, then lower the temperature to 110°C and continue the reaction for 5 hours, The reaction solution was poured into ice water and left for 12 hours, the solid was precipitated, filtered, and the obtained filter cake was recrystallized with ethanol to obtain peracetyl lactose, whose structural formula was as follows:

Peracetyl lactose (compound 1, 3.5 g) and chlorotriethylene glycol ( Compound 2, 1.3 g) was dissolved in dichloromethane (CH ₂ Cl ₂ , 5 mL), and 500 mg Molecular sieve, stirred for 20 minutes, added boron trifluoride diethyl ether (BF ₃ -Et ₂ O, 7.8 ml), stirred at room temperature for 72 hours. After the reaction, remove boron trifluoride ether by filtration, dilute the filtrate with dichloromethane, wash the diluted solution successively with saturated sodium bicarbonate solution and saturated saline solution, dry over anhydrous sodium sulfate, then remove sodium sulfate by filtration, and use the filtrate to Rotary evaporator was evaporated to dryness, and the remaining residue was separated by chromatographic column to obtain compound 3. The structural formula of compound 3 is as follows:

The NMR data of compound 3 are as follows: ¹ HNMR (400MHz, CDCl ₃ , 25°C): δ=1.97(s,3H),2.05(m,12H),2.12(s,3H)2.16(s,3H),3.6- 3.8(m,12H),3.8-3.95(m,2H),4.05-4.15(m,4H),4.47-4.58(m,2H),4.56(d,2H,J=7.8),4.89(t,1H ), 4.95(dd, 1H, J=10.5, 3.3Hz, CH), 5.1(dd, 1H, J=10.5, 7.8), 5.2(t, 1H, J=9.3Hz) 5.34(d, 1H, J= 3.3Hz, CH).

(2) Preparation of 1-β-(thioacetyltriethylene glycol)-2,3,6,2',3',4',6'-heptaacetyllactose (compound 4):

Compound 3 (770 mg) was dissolved in N,N-dimethylformamide (DMF, 3 mL), potassium thioacetylate (KSAc, 550 mg) was added, and stirred at room temperature for 4 hours. After the reaction stopped, add ethyl acetate to the reaction solution, wash with water, saturated sodium bicarbonate solution and saturated saline solution successively (2 times each), dry over anhydrous sodium sulfate, then filter to remove sodium sulfate, and use a rotary evaporator for the filtrate Evaporation to dryness afforded compound 4. The structural formula of compound 4 is as follows:

The NMR data of compound 4 are as follows: ¹ HNMR (400MHz, CDCl ₃ , 25°C): δ=1.855(s,3H),1.935(s,3H),1.941(m,6H),1.955(s,3H),2.014 (s,3H),2.046(s,3H),2.23(s,3H),2.92(tr,J＝6.3Hz,2H),3.6-3.8(m,12H),3.85(m,2H),4.01( m,2H),4.38(dd,1H,J=1.8Hz,12.6Hz),4.42(d,1H,J=7.8Hz),4.49(d,1H,J=7.8Hz),4.78(dd,1H, J＝7.8Hz, 9.3Hz), 4.86(dd, 1H, J＝10.5, 3.3Hz), 4.9(dd, 1H, J＝10.5, 7.8Hz), 5.1(t, 1H, JH＝9.3Hz), 5.24 (d, 1H, J=3.6Hz). ¹³ C NMR (400MHz, CDCl ₃ ): 19.920, 20.049, 20.049, 20.079, 20.130, 20.22, 20.27, 28.212, 29.982, 60.298, 61.526, 66.131, 68.4397, 668.58 .

(3) Preparation of 1-β-(mercaptotriethylene glycol) lactose (compound 5):

Compound 4 (600 mg) was dissolved in 2 ml of methanol, an appropriate amount of sodium metal was added to the solution, the weight of sodium metal was 5% of the weight of compound 4, and stirred overnight at room temperature. After the reaction is finished, an appropriate amount of acidic cationic resin is added to adjust the pH of the solution to 7. After filtration, the filtrate was evaporated to dryness with a rotary evaporator, the residue was dissolved in an appropriate amount of water, and freeze-dried to obtain compound 5. The structural formula of compound 5 is as follows:

The NMR data of compound 5 are as follows: ¹ HNMR (400MHz, MeOD, 25°C): δ=2.8(t, 2H, J=6.6Hz), 3.35(2H, s), 3.5-3.8(20H, m), 3.9( d, 2H, J=3.3Hz), 4.1(2H, m), 4.45(d, 1H, J=7.8Hz)), 4.51(1H, d, J=7.8Hz). ¹³ C NMR (400MHz, MeOD, 25°C): δ=102.54, 101.65, 77.82, 74.88, 73.89, 72.29, 70.41, 69.25, 68.27, 60.6, 59.61, 48.51, 42.79.

3. Lactose modification of gold nanorods

The ligand exchange reaction was carried out with the aqueous solution of the lactose ligand and the gold nanorod solution, and the lactose ligand was loaded on the surface of the gold nanorod through Au-S coordination (as shown in Figure 4). (XPS), surface charge (Zeta potential), ultraviolet-visible-near-infrared light absorption and microstructure (TEM) were used to characterize the modification results. The specific experimental operation is as follows:

Add 100 microliters of an aqueous solution of mercaptolactose ligand (compound 5) to 5 ml of the above-prepared gold nanorod solution, the molar ratio of thiol-containing lactose to gold nanorods is 1×10 ⁴ : 1, at room temperature Leave it to stand for 15 hours without stirring, stop the reaction, and prepare a bioprobe (expressed as Lac-GNRs) for detecting the tumor marker galectin-1. A composite formed by linking gold nanorods. The reaction product obtained in this experiment is the Lac-GNR aqueous solution.

As shown in Figure 5A-Figure 5D, the XPS pattern analysis indicated that the carbon element (C) content of lactose-modified gold nanorods (Lac-GNRs) was lower than that of gold nanorods (CTAB-GNRs) before modification, while the oxygen element ( O) content increased (Figure 5A), indicating that part of CTAB on the surface of gold nanorods was replaced by lactose (the extra oxygen came from lactose); Zeta test showed that the surface charge of Lac-GNRs was lower than that of CTAB-GNRs (Figure 5B), It is further verified that part of CTAB is replaced by lactose (CTAB is positively charged, while lactose is uncharged); the light absorption in the ultraviolet-visible-near-infrared region shows that the transverse absorption peak of gold nanorods does not change after lactose modification, while the longitudinal absorption front changes. A small red shift occurred (Fig. 5C); Fig. 5D shows the microstructure of Lac-GNRs.

The above results indicated that the gold nanorods were successfully modified by lactose, and the lactose-modified gold nanorod detection probes were successfully constructed.

Example 2, the detection of galectin-1 by molecular level gold nanorods (Lac-GNRs)

This example evaluates the detection of galectin-1 at the molecular level.

The method used to detect galectin-1: In operation, add 100 microliters of the substance to be tested (aqueous solution of galectin-1) to 1 milliliter of the Lac-GNR aqueous solution prepared in Example 1, shake well and let stand at room temperature for one Hours, use a UV-Vis-NIS spectrophotometer (UV-Vis-NIS spectrophotometer) to optically detect the system, measure the light absorption from 250nm to 1100nm, and judge whether the test substance contains galectin-1 through the change of the absorption curve, It can even be judged by the naked eye through the change in color.

The detection results of this example show that Lac-GNRs can detect the presence of galectin-1 extremely sensitively, and the detection limit can reach 1pM, and the response value changes with the concentration of galectin-1 ( FIG. 6A ). The combination of Galectin-1 and lactose can induce the agglomeration of gold nanorods, thereby changing the light absorption in the ultraviolet-visible-near-infrared region (UV-Vis-NIS). When 1nM galectin-1 was added to the system, the absorption peak at 770nm decreased sharply, and a new absorption peak appeared at 1100nm, indicating that the gold nanorods had been seriously aggregated (Figure 6B). At this time, the Lac-GNR solution The color of galectin-1 changed from orange to purple (Figure 6C); and when the concentration of galectin-1 was reduced to 100pM, the absorption peak at 770nm also decreased significantly, and a new peak appeared at 900nm, indicating that gold nanorods The reunion is also more obvious. The concentration of Galectin-1 can be reduced to 1pM (Figure 6A), and the peak at 770nm is still significantly reduced at this time, indicating that Lac-GNRs are particularly sensitive to the response of Galectin-1, and can achieve extremely low concentration of Galectin-1 detection.

In addition to responding to galectin-1, Lac-GNRs respond very poorly to other proteins that cannot recognize lactose (such as bovine serum albumin BSA). For example, adding 1 nM galectin-1 to the Lac-GNR aqueous solution prepared in Example 1 can induce a significant change in the absorption peak, and even if a much larger concentration of BSA is added, or the same concentration of Similar galectin-3 (galectin-3), none can significantly change the absorption of gold nanorods (as shown in Figure 7), indicating that the specificity of Lac-GNRs is very good.

Example 3. Detection of galectin-1 by Lac-GNRs at the cellular level

In this example, the detection of galectin-1 is studied at the cellular level, because galectin-1 is a secreted protein, which will be secreted into the extratumor matrix to play a role. The cancer cells are cultured by the method of cell culture, and the culture fluid thereof is taken for detection, and the existence of galectin-1 should be detected.

In this example, HUH-7 (human liver cancer cells), MCF-7 (human breast cancer cells), JEG-3 (human choriocarcinoma cells), HeLa (human cervical cancer cells), Hep-G2 (human liver cancer cells) were cultured in total. Cells), C6 (mouse glioma cells) and other six kinds of tumor cells, and their cell culture fluids were detected. The culture conditions of the cells are as follows: the cells are planted in H-DMEM medium containing 10% fetal bovine serum (high-glucose Dulbecco's minimum essential medium, the cell concentration is about 2x 10 ⁶ ), at 37°C Incubate in medium for 24 hours, then remove the culture medium, add the same amount of new culture medium, incubate again for 48 hours, take out the culture medium for measurement.

The method used to detect galectin-1: In operation, 100 microliters of the substance to be tested (the cancer cell culture medium of galectin-1) was added to 1 milliliter of the Lac-GNR aqueous solution prepared in Example 1, and after shaking well, room temperature Let stand for one hour, use UV-Vis-NIS spectrophotometer (UV-Vis-NIS spectrophotometer) to optically detect the system, and judge whether the substance to be tested contains galectin-1 through the change of absorption curve

As a result of the test, it was found that the culture fluids of the six tumor cells used in this example could all cause a significant decrease in the light absorption curve of Lac-GNRs ( FIG. 8 ), indicating that Lac-GNRs can accurately detect galectin-1 therein.

Example 4. Detection of galectin-1 by Lac-GNRs in blood level

This example studies the detection of galectin-1 at the level of human blood. Since galectin-1 will be expressed in large quantities in the metastatic stage of cancer and secreted into the blood to play a role, the galectin-1 in the blood of patients with cancer metastasis 1 concentration was significantly higher than normal.

The method used to detect galectin-1: in operation, add 100 microliters of the substance to be tested (human serum of galectin-1) to 1 milliliter of the Lac-GNR aqueous solution prepared in Example 1, shake well and let stand at room temperature For one hour, use a UV-Vis-NIS spectrophotometer (UV-Vis-NIS spectrophotometer) to optically detect the system, measure the light absorption from 250nm to 1100nm, and judge whether the test substance contains galectin-1 through the change of the absorption curve , and can even be judged by the naked eye through the change in color.

In this example, the blood of 5 normal people and 15 cancer patients who have been confirmed as metastases were collected and tested, and it was found that Lac-GNRs can significantly distinguish the blood of normal people and cancer patients: the blood of normal people It can only cause a slight red shift of the Lac-GNRs absorption curve (see Figure 9, moving to the right), but the blood of cancer patients can not only cause a large red shift of the Lac-GNRs absorption curve, but also cause a significant decrease in the absorption curve. The results indicate that Lac-GNRs can accurately detect galectin-1 in blood, and may become a clinically available method for monitoring cancer metastasis.

In addition to measuring the light absorption in the UV-Vis-NIS region, the test results can also be judged directly with the naked eye: the Lac-GNR solution added with normal human serum has the same color as before adding serum (as shown in Figure 10 Shown in row 1); while the color of Lac-GNR solution added to cancer patient serum is obviously lightened (shown in row 2-4 in FIG. 10 ). This distinct change in color greatly enhances the visibility and convenience of inspection.

Claims (9)

1. A method for detecting galectin-1 in a sample to be tested, the method comprising: Use lactose-modified gold nanorods as probes, make them contact and mix with the sample to be tested, maintain the system temperature at 20-37°C and let it stand for 10 minutes to 2 hours, and detect galectin-1 by detecting the color of the mixed system; Wherein, the gold nanorods have a length of 60-80 nm and a width of 20-25 nm; Wherein, in the lactose-modified gold nanorod aqueous solution, the concentration of the lactose-modified gold nanorod is 10nM to 100nM; the detection limit of galectin-1 in the sample to be tested is 1pM. 2. The method according to claim 1, wherein the sample to be tested is an aqueous solution of galectin-1, cancer cell culture fluid, or blood or serum of an individual to be tested. 3. The method of claim 1, comprising: Add the sample to be tested into the lactose-modified gold nanorod aqueous solution, shake it well and let it stand for 10 minutes to 2 hours, and use the ultraviolet-visible-near-infrared spectrophotometer to optically detect the system or observe the color change of the system with the naked eye. Detect galectin-1 in the sample to be tested. 4. The method according to claim 1 or 3, wherein the volume ratio of the sample to be tested to the aqueous solution of lactose-modified gold nanorods is 1:1-100. 5. The method according to claim 1, wherein the lactose-modified gold nanorods are complexes formed by linking sulfhydryl-containing lactose to gold nanorods through gold-sulfur bonds. 6. The method according to claim 1, wherein the lactose-modified gold nanorods are prepared according to the following method: The gold nanorod and the lactose containing sulfhydryl group are mixed, and left standing at room temperature for 8-15 hours to prepare a biological probe for detecting the tumor marker galectin-1. 7. The method according to claim 6, wherein the gold nanorods are gold nanorods with a length of 60 to 80 nm, a width of 20 to 25 nm, and a surface covered by CTAB; Prepared by introducing a mercapto-containing linking arm at the carbon atom. 8. The method according to claim 6, wherein the molar ratio of the thiol-containing lactose to the gold nanorods is 1×10 ⁴ :1˜3×10 ⁴ :1. 9. The method according to claim 1, wherein galectin-1 is detected at molecular level, cellular level or blood level.