CN107748147B - A white light-emitting up-conversion nanoparticle and a test strip for simultaneous multi-component tumor marker detection based thereon - Google Patents

Abstract

The invention discloses a white light-emitting up-conversion nanoparticle and a test strip based on the same for realizing multi-component tumor marker detection. The nanoparticle has a three-layer core-shell structure and emits white light under the excitation of near-infrared light; After it is labeled with the antibodies of multiple tumor markers to be detected, it is fixed on the labeled complex binding area of the test strip, so that the simultaneous detection of multiple tumor markers can be realized. The invention realizes the sensitive detection of multiple tumor markers by a single up-conversion nanoparticle, and has simple operation, good specificity, and high qualitative and quantitative real-time detection sensitivity.

Description

A white-emitting upconversion nanoparticle and its simultaneous multicomponent realization Test strips for tumor marker detection

technical field

The invention belongs to the technical field of biomedical diagnosis, in particular to an up-conversion test strip detection method for multi-component tumor markers.

Background technique

Malignant tumors are often referred to as cancers, which are uncontrolled gene expression in part of the body, and malignant proliferation of cells destroying normal life activities of the body. The rapid growth rate will cause weight loss, fever and serious organ damage in the human body, and eventually crisis. life. At present, cancer has become the number one killer that endangers people's lives. Therefore, the early detection of cancer and the timely clinical monitoring have become a major subject of modern biomedicine. With the maturity of nanotechnology, the combination platform of nanotechnology and cancer markers for the detection of tumor markers has become a hot spot. Colloidal gold test strips are the earliest widely used immunochromatographic test strips. They are simple in structure, fast, do not require any equipment, and can identify the results with the naked eye. However, compared with up-conversion test strips, colloidal gold test strips have poor sensitivity, are difficult to achieve quantitative measurement, and are slightly less stable. Rare earth-doped upconversion luminescent nanomaterials (UCNPs) are a class of nanomaterials that can absorb near-infrared light and emit short-wavelength near-ultraviolet visible light. advantage. Therefore, combining UCNPs with immunochromatographic test strips can bring breakthrough changes. The unique up-conversion luminescence phenomenon of UCNPs anti-Stokes shift can make it possible to exclude the self-luminescence interference phenomenon of biological samples when labeling biologically active molecules. Improve signal-to-noise ratio, enhance sensitivity and stability, and achieve high-sensitivity quantitative detection of cancer markers.

The up-conversion test strip detection method can be used in serological examinations to help doctors in auxiliary diagnosis, which is of great value for efficacy diagnosis and follow-up, and the test strip detection method has obvious advantages in terms of operation, response speed, and price. The tumor markers detected can be enzymes, hormones, glycoproteins, embryonic antigens or tumor metabolites. The most common ones are:

1. Alpha-fetoprotein

Alpha-fetoprotein (AFP) can provide an important basis for the early diagnosis of liver cancer. Endodermal tumor, malignant teratoma, gastric cancer and other patients with liver cancer metastasis increased AFP in vivo.

2. Carcinoembryonic antigen

Carcinoembryonic antigen (CEA) can be increased in gastrointestinal tumors, lung cancer, breast cancer, and urinary tract tumors. The content of CEA in the chest, ascites, secretions, and digestive juice of cancer patients increased, and the more advanced the cancer, the higher the CEA, and the higher the positive rate.

However, when the existing upconversion test strips detect multiple tumor markers at the same time, different T lines need to use different upconversion nanoparticles, the manufacturing process is complicated, and the detection efficiency is seriously affected.

SUMMARY OF THE INVENTION

In order to overcome the deficiencies of the above-mentioned prior art, the present invention provides a white light-emitting up-conversion nanoparticle and a test strip for simultaneous detection of multi-component tumor markers based thereon, aiming to utilize an up-conversion nanoparticle based on the same. The conversion of nanomaterials can simultaneously detect multiple tumor markers and improve the detection efficiency.

To achieve the purpose of the invention, the present invention adopts the following technical solutions:

The invention firstly discloses a white light-emitting up-conversion nanoparticle, which is characterized in that: the up-conversion nano-particle is used on a test strip to realize the detection of multi-component tumor markers at the same time; The particle has a three-layer core-shell structure, with NaGdF ₄ nanoparticles doped with Yb, Tm and Er as the core, and a first shell layer of Eu-doped NaGdF ₄ is wrapped outside the core, and the first shell is The second shell layer of NaYF ₄ is wrapped outside the layer to form a Yb, Tm, Er and Eu co-doped NaGdF ₄ :Yb/Tm/Er@NaGdF ₄ :Eu@NaYF ₄ core-shell structure; the upconversion nanoparticles Under the excitation of near-infrared light, it emits three primary colors of blue, green and red, and it generally displays white light emission.

The above-mentioned up-conversion nanoparticles are prepared by the seed crystal method, and the specific steps are as follows:

a, Synthesis of _NaGdF nanoparticles doped with Yb, Tm and Er as the core

Weigh oleic acid (OA) 10mL, octadecene (ODE) 10mL, Gd(OAc) ₃ 0.0841g, Yb(OAc) ₃ 0.0858g, 0.1mol/LTm aqueous solution 25μL, 0.2mol/L Er aqueous solution 2μL, NaF solid 0.4200g was added to the three-necked flask _A , the temperature was raised to 110℃～120℃ with magnetic stirring, kept for 10min, and then the water and oxygen were removed by vacuum;

b. The first shell layer of Eu-doped NaGdF ₄ is wrapped around the inner core

Weigh 4 mL of oleic acid (OA), 4 mL of octadecene (ODE), 0.0568 g of Gd(OAc) ₃ , and 0.0098 g of Eu(OAc) ₃ , add them to the three-necked flask B, and heat up to 110 ℃ ~ 120 ℃ with magnetic stirring, Hold for 10min, and then vacuum to remove water and oxygen; after removal, pass N ₂ , heat up to 220 ° C, and after the reaction in step a, inject it into the three-necked flask A at a speed of 1 mL/min with a syringe, 300 ℃ reaction 0.5-1h;

c, the second shell layer of NaYF ₄ is wrapped outside the first shell layer

Weigh 4 mL of oleic acid (OA), 4 mL of octadecene (ODE), and 0.0532 g of Y(OAc) ₃ , add them to a three-necked flask C, and heat up to 110 ℃ ~ 120 ℃ with magnetic stirring, hold for 10 min, and then remove water by vacuuming and oxygen; after removing it, pass N ₂ , and after the reaction in step b, inject it into the three-necked flask A at a speed of 1 mL/min with a syringe, and react at 300 ° C for 0.5-1 h; after the reaction is completed, it is lowered to room temperature , the reaction solution in the three-necked flask A is placed in a centrifuge tube, and the obtained nanoparticles are centrifuged;

d, peeling off the oleic acid on the surface of the nanoparticles obtained in step c

Take the nanoparticles obtained in step c, add a mixture of ethanol and concentrated hydrochloric acid with pH=1 to them, and ultrasonically disperse them evenly, then ultrasonically oscillate for 30 min, then remove the supernatant after centrifugation, and add pH to the nanoparticles. = 4 mixed solution of ethanol and concentrated hydrochloric acid, after ultrasonically dispersing uniformly, ultrasonically oscillating for 40-60min, and then centrifuging again, the obtained nanoparticles are water-soluble white luminescent upconversion nanoparticles after washing with water;

The upconversion nanoparticles were dispersed in 0.01M PBS buffer solution with pH=6.5-7.4 and stored for later use.

The method for simultaneously realizing multi-component tumor marker detection with the white light-emitting up-conversion nanoparticles is characterized in that: using the blue, green and red light emission of the up-conversion nanoparticles to make one band, any combination of two bands, and the detection of one tumor marker in each of the three bands, a total of 7 tumor markers can be detected simultaneously; the up-conversion nanoparticles are used as immunochromatographic test strips The tracer marker, using the white light emission of the up-conversion nanoparticles, or using the fluorescence resonance energy transfer (FRET) between the fluorescent dyes and the up-conversion nanoparticles, the up-conversion nanoparticles correspond to one or two wavelengths of light burst extinction, causing the upconverting nanoparticles to exhibit the remaining luminescent color. According to the color change of the nanoparticles on the detection area of the test strip, the tumor markers present in the sample to be detected are judged, and the qualitative detection of the types of tumor markers contained in the sample to be detected is realized.

The invention also discloses a test strip that simultaneously realizes the detection of multi-component tumor markers, including a sample adding area, a labeling complex binding area, a detection area, a quality control area and a hand-held area, which is characterized in that: in the marker The above-mentioned white light-emitting up-conversion nanoparticles are immobilized in the complex binding area, and the white light particles are labeled with antibodies of various tumor markers to be detected; different regions of the detection area are provided with the tumor markers to be detected. There are several detection T lines with the same type and number of antibodies, each detection T line is respectively coated with an antibody of a tumor marker to be detected, and the antibody of each tumor marker is modified with fluorescent dyes of different colors; The quality control area is provided with a quality control C line, and the quality control C line is coated with goat anti-mouse IgG antibody.

When in use, drop the sample to be detected in the sample application area, if the sample to be detected contains antibodies corresponding to multiple tumor markers modified on the up-converted nanoparticles in the binding area of the labeled complex. The antigen is bound to the corresponding antibody labeled on the up-conversion nanoparticle, and enters the detection area together and is partially fixed on the corresponding T line in the detection area, and the rest flows into the quality inspection area with the sample to be detected; after the detection is completed, pass the 980nm laser excitation, observe the color change of T line and C line to judge the tumor markers in the sample to be tested, and realize the qualitative detection of the types of tumor markers contained in the sample to be tested.

Specifically, during detection, drop the sample to be detected in the sample application area, if the sample to be detected has multiple tumor markers modified on the upconversion nanoparticles in the binding area of the labeled complex in the sample to be detected The antigen corresponding to the antibody, then the antigen is combined with the corresponding antibody labeled on the up-conversion nanoparticle to form a new conjugate and enter the detection area. Under the excitation of 980nm laser, the fluorescent dye on each T line and the upconversion nanoparticles will quench the light in the corresponding band of the upconversion nanoparticles through fluorescence resonance energy transfer (FRET), resulting in the The converted nanoparticles showed the remaining luminescent colors, and each T line also showed different colors of luminescence;

At the same time, during detection, the sample to be detected is dropped into the sample adding area, and the upconversion nanoparticles immobilized on the binding area of the labeled complex flow forward together with the detection sample, and part of it is immobilized on the T line in the detection area. The remaining part flows into the quality inspection area with the sample to be detected, and the antibody of the tumor marker to be detected labeled on the up-converted nanoparticles binds to the IgG antibody coated on the C line of the quality inspection area, and is fixed on the C line of the quality inspection area. Top; the IgG antibody coated on the C line is not labeled with the dye, and the particles on the C line show all wavelengths of luminescence, that is, white light, when excited by a 980 nm laser.

Further, a series of standard antigen solutions of known concentration were added dropwise to the sample addition area for detection; after the detection was completed, the fluorescence intensity of the corresponding T line in the detection area and the C line in the quality inspection area were obtained by excitation with a 980 nm laser, and the T line was established. and the standard curve of the ratio of fluorescence intensity of C-line and antigen concentration, so as to realize the quantitative detection of the concentration of tumor markers contained in the sample to be detected.

The production method of the above test strips is as follows:

(1) Handling of sample pads

Using cellulose film as the material of the sample pad, soak the sample pad in the sample pad treatment solution for no more than 30 minutes, and then dry it at 37°C to complete the processing of the sample pad;

(2) Handling of bonding pads

Using glass cellulose membrane as the binding pad material, first soak the binding pad in the binding pad treatment solution for no more than 30 minutes, then dry it at 37°C, and then put the binding pad into the antibody labeled with various tumor markers to be detected. After soaking in the solution of white luminescent upconversion nanoparticles for no more than 30 minutes, drying at 37°C to complete the treatment of the bonding pad;

(3) Treatment of nitrocellulose membrane

The nitrocellulose membrane is divided into two parts; the antibodies of each tumor marker to be detected are drawn in different areas of the first part with a parameter of 1 μL/cm to form several detection T cells coated with an antibody of a tumor marker. The antibody of each tumor marker is modified with fluorescent dyes of different colors; the goat anti-mouse IgG antibody is drawn in the second part with a parameter of 2 μL/cm to form the quality control line C; drying is completed, the nitrocellulose is completed treatment of membranes;

(4) Assembly of test strips

From the front end to the back end of the adhesive plastic base plate, the sample pads after bonding treatment form the sample application area, the bonding pads after bonding treatment form the labeling complex bonding area, the nitrocellulose membrane after bonding treatment forms the detection area and The quality control area and the adhesive absorbent paper form a hand-held area, wherein the detection area is located between the labeled complex binding area and the quality control area.

Wherein: the sample pad treatment solution described in step (1) is a 0.01M PBS buffer solution with a mass concentration of 0.05-10% BAS and 0.1%-2% Tween-20, pH=7.0-8.0; The binding pad treatment solution is a 0.01M PBS buffer solution with a mass concentration of 0.05-10% BAS, 0.1%-3% Tween-20 and 1%-10% sucrose, pH=7.0-8.0.

The method for obtaining the solution of white luminescent up-conversion nanoparticles labeled with antibodies of various tumor markers to be detected in step (2) is: dispersing the up-conversion nanoparticles in 0.01M PBS buffer with pH=6.5-7.4 In the solution, according to the ratio of the mass of the up-converted nanoparticles to the mass of the antibody of each tumor marker to be detected is 100-300:1, then add the antibody of various tumor markers to be detected into the PBS buffer solution , shake reaction at room temperature for 2 to 4 hours, and then centrifuge at 4 °C to obtain white luminescent up-conversion nanoparticles labeled with antibodies to various tumor markers to be detected, and then dispersed in 0.01 of 0.01 of BAS containing 0.5 to 5% BAS by mass. M, pH=7.0-8.0 PBS buffer solution, and store at 4°C, that is, the preparation of the solution of white luminescent upconversion nanoparticles labeled with antibodies to various tumor markers to be detected in step (2) is completed.

The method of modifying the fluorescent dye on the antibody of the tumor marker is as follows: the antibody is diluted in a carbonate buffer solution of 0.1M, pH=8.0-9.0, and then according to the mass ratio of the antibody to the dye is 10～100:1, add The fluorescent dye solution was shaken for 2-4 hours; the obtained reaction solution was dialyzed with 0.01M PBS buffer solution with pH=7.0-8.0 overnight to remove the unbound dye, that is, the antibody of the tumor marker was modified with fluorescence Dye, stored at 4°C.

Preferably, the length of the sample pad, the binding pad, the nitrocellulose membrane and the absorbent paper is 8-17 mm, and the contact areas of each part are overlapped with each other by 1-3 mm during assembly; the length of the adhesive plastic bottom plate is 60 mm.

The beneficial effects of the present invention are embodied in:

1. The present invention realizes the rapid detection and screening of multi-component tumor markers with an up-conversion nanoparticle with high accuracy and specificity.

2. The test strip of the present invention uses UCNPs as biomarkers to label antibodies, and can directly observe the results under near-infrared light excitation. Due to the unique optical properties of UCNPs, background interference is eliminated, and the sensitivity is greatly improved; The detector and the environment are harmless, and the safety is good.

3. The sample detected by the test strip of the present invention does not need much pretreatment, and can directly realize quantitative measurement with the help of the up-conversion luminescence sensor, and the operation is simple and fast, and can be operated on site.

Description of drawings

Fig. 1 is the fluorescence emission spectrogram of the up-conversion nanoparticles obtained in Example 1;

Fig. 2 is the structural representation of up-conversion detection test strip;

Fig. 3 is the reaction schematic diagram that the immune reaction is negative;

Figure 4 is a schematic diagram of the reaction when the immune reaction is positive.

Detailed ways

The technical solutions of the present invention will be described in detail below through embodiments and accompanying drawings.

Example 1. Detection of CEA and AFP tumor markers

In this embodiment, CEA and AFP are used as tumor markers to be detected as examples, and the details are as follows.

1. Preparation of UCNPs-antibodies

1.1. The white luminescent upconversion nanoparticles with a three-layer core-shell structure were prepared by the seed method. The NaGdF ₄ nanoparticles doped with Yb, Tm and Er were used as the core, and the Eu-doped nanoparticles were wrapped outside the core. The first shell of NaGdF ₄ , and the second shell of NaYF ₄ is wrapped around the first shell to form a Yb, Tm, Er and Eu co-doped NaGdF ₄ : Yb/Tm/Er@NaGdF ₄ :Eu@NaYF ₄ core-shell structure; the specific preparation steps are as follows:

a, Synthesis of _NaGdF nanoparticles doped with Yb, Tm and Er as the core

Weigh oleic acid (OA) 10mL, octadecene (ODE) 10mL, Gd(OAc) ₃ 0.0841g, Yb(OAc) ₃ 0.0858g, 0.1mol/L Tm aqueous solution 25μL, 0.2mol/L Er aqueous solution 2μL, NaF The solid 0.4200g was added to the three-necked flask _A , and the temperature was raised to 110℃～120℃ with magnetic stirring, kept for 10min, and then the water and oxygen were removed by vacuum;

b. The first shell of Eu-doped NaGdF ₄ is wrapped around the inner core

Weigh 4 mL of oleic acid (OA), 4 mL of octadecene (ODE), 0.0568 g of Gd(OAc) ₃ , and 0.0098 g of Eu(OAc) ₃ , add them to the three-necked flask B, and heat up to 110 ℃ ~ 120 ℃ with magnetic stirring, Keep it for 10min, then vacuum to remove water and oxygen; after removal, pass N ₂ , heat up to 220°C, and after the reaction in step a, inject it into the three-necked flask A at a rate of 1mL/min with a syringe, at 300°C Reaction 1h;

c. The second shell of NaYF ₄ is wrapped outside the first shell

Weigh 4 mL of oleic acid (OA), 4 mL of octadecene (ODE), and 0.0532 g of Y(OAc) ₃ , add them to a three-necked flask C, and heat up to 110 ℃ ~ 120 ℃ with magnetic stirring, hold for 10 min, and then remove water by vacuuming and oxygen; after removing it, pass N ₂ , and after the reaction in step b, inject it into the three-necked flask A at a speed of 1 mL/min with a syringe, and react at 300 ° C for 1 h; after the reaction is completed, it is lowered to room temperature, and the The reaction solution in the three-necked flask A is placed in a centrifuge tube, and the obtained nanoparticles are separated by centrifugation;

d, peeling off the oleic acid on the surface of the nanoparticles obtained in step c

Take the nanoparticles obtained in step c, add a mixture of pH=1 ethanol and concentrated hydrochloric acid (7.5 mL of ethanol, 62.5 μL of concentrated hydrochloric acid), and ultrasonically disperse them evenly, then ultrasonically oscillate for 30 min, and then remove the supernatant after centrifugation. liquid, and then add a mixture of ethanol and concentrated hydrochloric acid (7.5 mL of ethanol, 7.5 mL of concentrated hydrochloric acid) with pH=4 to the obtained nanoparticles, after ultrasonically dispersed uniformly, ultrasonically oscillate for 40 min, and then centrifuge again. After the particles are washed with water, they are water-soluble white luminescent upconversion nanoparticles; according to the concentration of 5 mg/mL, the upconversion nanoparticles are dispersed in a 0.01M PBS buffer solution with pH=6.5 and stored for later use. FIG. 1 is a fluorescence emission spectrum diagram of the up-conversion nanoparticles obtained in this example.

1.2. Antibodies labeled with tumor markers on the surface of upconversion nanoparticles by electrostatic adsorption

Take 1 mL of the upconversion nanoparticles dispersed in 0.01M PBS buffer solution with pH=6.5 obtained in step 1.1, add 10 μL 2 mg/ml CEA monoclonal antibody and 8 μL 2.5 mg/mL AFP monoclonal antibody to it, shake at room temperature bed reaction for 2 h, and then centrifuged at 4°C to obtain white luminescent up-conversion nanoparticles labeled with antibodies to various tumor markers to be detected, and then dispersed in 0.01M, pH=7.0 PBS buffer containing 2% BAS by mass The solution is stored at 4° C. to obtain the preparation of a solution of white luminescent up-conversion nanoparticles labeled with antibodies to multiple tumor markers to be detected.

1.3. Fluorescent dyes modified on antibodies of tumor markers

Dilute 50 μL of 1.9 mg/mL CEA antibody to 500 μL with 0.1 M, pH=9.0 carbonate buffer solution, add 3 μL of 1 mg/mL fluorescent dye solution isothiocyanorhodamine B (RBITC), and react on a shaking table for 4 h; the resulting reaction The solution was dialyzed with PBS buffer solution of 0.01M, pH=7.4 overnight to remove unbound RBITC, that is, the CEA monoclonal antibody was modified with fluorescent dye RBITC and stored at 4°C.

Take 30 μL of 3.3 mg/mL LAFP antibody and dilute it to 500 μL with 0.1 M, pH=9.0 carbonate buffer solution, add 3 μL of 1 mg/mL fluorescent dye solution 4-chloro-7-nitro-2,1,3-benzoxa Oxadiazole (NBD-Cl) fluorescence, shaken for 4h; the resulting reaction solution was dialyzed with 0.01M, pH=7.4 PBS buffer solution overnight to remove unbound NBD-Cl, that is, the AFP antibody was modified with fluorescence The dye NBD-Cl was stored at 4°C.

2. Preparation of test strips

As shown in FIG. 2 , this embodiment simultaneously realizes a test strip for multi-component tumor marker detection, including a sample application area, a labeled complex binding area, a detection area, a quality control area, and a handheld area. The white luminescent upconversion nanoparticles are immobilized in the binding area of the labeling complex, and the antibodies of the two tumor markers to be detected are labeled on the upconversion nanoparticles; there are two detection T lines in different areas of the detection area, T1 Coated with CEA antibody, T2 is coated with AFP antibody, and the antibodies of the two tumor markers are modified with fluorescent dyes of different colors; there is a quality control C line in the quality control area, and the quality control C line is coated with Goat anti-mouse IgG antibody.

Specifically, the test strips are made as follows:

(1) Handling of sample pads

Using cellulose film as the material of the sample pad, soak the sample pad in the sample pad treatment solution for 5 minutes, and then dry it at 37 °C to complete the treatment of the sample pad; the sample pad treatment solution contains 0.5% BAS and 1% Tween-20 0.01M, pH=8.0 PBS buffer solution;

(2) Handling of bonding pads

Using glass cellulose membrane as the binding pad material, first soak the binding pad in the binding pad treatment solution for 5 minutes, then dry it at 37°C, and then put the binding pad into the antibody labeled with various tumor markers to be detected obtained in step 1.2. After soaking in the solution of white luminescent upconversion nanoparticles for 5 min, drying at 37°C to complete the treatment of the binding pad; the binding pad treatment solution is 0.01M containing 1% BAS, 2% Tween-20 and 5% sucrose by mass. , pH=7.4 PBS buffer solution.

(3) Treatment of nitrocellulose membrane

Divide the nitrocellulose membrane into two parts:

The antibodies of the two tumor markers to be detected, CEA antibodies and AFP antibodies, are drawn in different areas of the first part with a parameter of 1 μL/cm, to form the detection T1 line and the detection T2 line of the antibody coated with the corresponding tumor marker, respectively. , among which, the CEA antibody is modified with isothiocyanorhodamine B (RBITC) fluorescent dye, and the AFP antibody is modified with 4-chloro-7-nitro-2,1,3-benzoxaoxadiazole (NBD -Cl) fluorescent dye;

The goat anti-mouse IgG antibody was drawn in the second part with a parameter of 2 μL/cm to form a quality control C line; drying, the treatment of the nitrocellulose membrane was completed;

(4) Assembly of test strips

From the front end to the back end of the adhesive plastic base plate, the sample pads after bonding treatment form the sample application area, the bonding pads after bonding treatment form the labeling complex bonding area, the nitrocellulose membrane after bonding treatment forms the detection area and The quality control area and the adhesive absorbent paper form a hand-held area, wherein the detection area is located between the labeled complex binding area and the quality control area.

3. Detection process of up-conversion test strips

During detection, drop the sample to be detected in the sample application area. If there are tumor markers AFP and CEA in the sample to be detected, the surface of the binding area of the labeled complex will be labeled with the UCNPs of the tumor marker AFP and CEA antibody to be detected. In response, the tumor markers AFP and CEA bind to the AFP and CEA antibodies labeled on UCNPs and enter the detection area. The tumor markers AFP and CEA immobilized on UCNPs are respectively bound to the CEA and T2 lines of modified RBITC immobilized on the T1 line of the detection area. The AFP antibody immobilized on the modified NBD-Cl reacted, and part of it was immobilized on the T line in the detection area, and the rest flowed into the quality inspection area with the sample to be detected, and the tumor markers AFP and CEA immobilized on the UCNPs were detected with the quality control area. The goat anti-mouse IgG antibody immobilized on line C is bound and immobilized on line C of detection line.

After the detection is completed, the 980nm laser excitation is used to observe the color change of the detection area and the quality inspection area. At this time, under the irradiation of 980 nm excitation light, there is fluorescence resonance energy transfer (FRET) between the RBITC on the T1 line and the upconversion nanoparticles, resulting in the weakening or even disappearance of the green light emission peak of the upconversion nanoparticles. The particles exhibited purple luminescence. Fluorescence resonance energy transfer (FRET) exists between the NBD-Cl above the T2 line and the upconversion nanoparticles, resulting in a weakening or even disappearance of the emission peak in the blue light region of the upconversion nanoparticles, and the upconversion nanoparticles immobilized on the T2 line exhibit yellow luminescence. The upconversion nanoparticles fixed on the detection line C in the quality control area showed no change in the emission peak, and the upconversion nanoparticles showed white light under the irradiation of 980 nm excitation light. FIG. 3 is a schematic diagram of a reaction in which the immune response is negative, and FIG. 4 is a schematic diagram of a reaction in which the immune response is positive.

In addition, by configuring different concentration gradients of CEA antigen (10-200ng/ml) and AFP antigen (100-1000ng/ml), the sample standard solution containing different concentrations of CEA antigen and AFP antigen was added dropwise on the sample pad. Make three test strips with the same concentration, and after standing for five minutes, scan the test strip with an upconversion luminescence sensor, and establish a standard curve by comparing the fluorescence intensity before and after the reaction, and the fluorescence intensity of T line and C line. Quantitative measurement is achieved.

The above descriptions are only exemplary embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the present invention. within the scope of protection.

Claims (7)

1. The utility model provides a realize test paper strip that multicomponent tumour marker detected simultaneously, includes application of sample district, mark complex binding area, detection zone, quality control district and handheld area, its characterized in that: fixing white luminous up-conversion nanoparticles on the labeling compound binding region, and labeling antibodies of multiple tumor markers to be detected on the up-conversion nanoparticles; a plurality of detection T lines with the same number and type as the tumor marker antibodies to be detected are arranged in different areas of the detection area, each detection T line is coated with the tumor marker antibody to be detected, and the tumor marker antibodies are modified by fluorescent dyes with different colors; a quality control C line is arranged in the quality control area, and a goat anti-mouse IgG antibody is coated on the quality control C line;

when the kit is used, a sample to be detected is dripped into the sample addition area, if the sample to be detected has antigens corresponding to the antibodies of the multiple tumor markers modified on the upconversion nanoparticles on the labeling compound binding area, the antigens are combined with the corresponding antibodies marked on the upconversion nanoparticles, enter the detection area together and are partially fixed on the corresponding T line of the detection area, and the rest of the antigens flow into the quality control area along with the sample to be detected; after the detection is finished, the color change of the T line and the C line is observed through 980nm laser excitation so as to judge the tumor marker in the sample to be detected and realize the qualitative detection of the tumor marker type in the sample to be detected;

the white luminous up-conversion nano-particles have a three-layer core-shell structure and are NaGdF doped with Yb, Tm and Er₄The nano particles are inner cores, and Eu-doped NaGdF is wrapped outside the inner cores₄A first shell layer, and the outside of the first shell layer is wrapped with NaYF₄A second shell layer which forms Yb, Tm, Er and Eu co-doped NaGdF₄:Yb/Tm/Er@NaGdF₄:Eu@NaYF₄A core-shell structure; the up-conversion nano particles emit blue, green and red tricolor light under the excitation of near infrared light, and the whole display shows white light to emit light; by utilizing the luminescence of blue, green and red wave bands of the up-conversion nano particles, the detection of one wave band, the combination of any two wave bands and the three wave bands respectively corresponding to one tumor marker can realize the simultaneous detection of 7 tumor markers.

2. The test strip of claim 1, wherein: dropping a series of standard antigen solutions with known concentration in the sample adding region for detection; after the detection is finished, the fluorescence intensity of the corresponding T line of the detection area and the fluorescence intensity of the C line of the quality control area are obtained through 980nm laser excitation, and a standard curve of the ratio of the fluorescence intensity of the T line to the fluorescence intensity of the C line to the antigen concentration is established, so that the quantitative detection of the concentration of the tumor marker contained in the sample to be detected is realized.

3. A method for manufacturing the test strip of claim 1 or 2, wherein:

(1) sample pad handling

Taking a cellulose membrane as a sample pad material, soaking the sample pad in a sample pad treatment solution for no more than 30min, and drying at 37 ℃ to finish the treatment of the sample pad;

(2) treatment of bond pads

Taking a glass cellulose membrane as a bonding pad material, firstly soaking the bonding pad in a bonding pad treatment solution for no more than 30min, then drying at 37 ℃, then placing the bonding pad into a solution of white luminous up-conversion nanoparticles for marking antibodies of various tumor markers to be detected, soaking for no more than 30min, and then drying at 37 ℃ to finish the treatment of the bonding pad;

(3) treatment of nitrocellulose membranes

Dividing the nitrocellulose membrane into two parts; marking the antibodies of each tumor marker to be detected in different areas of the first part by parameters of 1 mu L/cm to form a plurality of detection T lines coated with the antibodies of one tumor marker, wherein the antibodies of each tumor marker are modified by fluorescent dyes with different colors; drawing lines on the second part of the goat anti-mouse IgG antibody according to the parameters of 2 mu L/cm to form a quality control C line; drying to finish the treatment of the nitrocellulose membrane;

(4) assembly of test strips

The sample pad after the sequential bonding treatment from the front end to the rear end of the viscous plastic bottom plate forms a sample adding area, the bonding pad after the bonding treatment forms a labeling compound bonding area, the nitrocellulose membrane after the bonding treatment forms a detection area and a quality control area, and the adhesive absorbent paper forms a handheld area, wherein the detection area is positioned between the labeling compound bonding area and the quality control area.

4. The method of manufacturing according to claim 3, wherein:

the sample pad treatment solution in the step (1) is a PBS buffer solution containing 0.01M, pH-7.0-8.0 mass concentration of BAS and 0.1-2% Tween-20;

the bonding pad treatment solution in the step (2) is PBS buffer solution containing 0.05-10% of BAS, 0.1-3% of Tween-20 and 1-10% of cane sugar in mass concentration, wherein 0.01M, pH is 7.0-8.0.

5. The method of manufacturing according to claim 3, wherein: the method for obtaining the solution of the white-light-emitting up-conversion nanoparticles for labeling the antibodies of the multiple tumor markers to be detected in the step (2) comprises the following steps: dispersing the up-conversion nanoparticles in 0.01M PBS buffer solution with the pH value of 6.5-8.0, then adding the antibodies of the multiple tumor markers to be detected into the PBS buffer solution according to the ratio of the mass of the up-conversion nanoparticles to the mass of the antibodies of the multiple tumor markers to be detected being 100-300: 1, carrying out shaking table reaction for 2-4h at normal temperature, then carrying out centrifugal separation at 4 ℃ to obtain the white-light-emitting up-conversion nanoparticles for marking the antibodies of the multiple tumor markers to be detected, dispersing the white-light-emitting up-conversion nanoparticles in the PBS buffer solution with 0.01M, pH of BAS with the mass concentration of 0.5-5% of 7.0-8.0, and storing the white-light-emitting up-conversion nanoparticles at 4 ℃, thus completing the preparation of the solution of the white-light-emitting up-conversion nanoparticles for marking the antibodies of the multiple tumor markers to be detected in the step (2).

6. The method of claim 3, wherein the fluorescent dye is modified with an antibody against a tumor marker by: diluting an antibody in a carbonate buffer solution with the mass ratio of 0.1M, pH-8.0-9.0, adding a fluorescent dye solution according to the mass ratio of the antibody to the dye of 10-100: 1, and reacting for 2-4h in a shaking table; the reaction solution was dialyzed overnight against 0.01M, pH ═ 7.0 to 8.0 PBS buffer solution to remove unbound dye, i.e., the antibody to the tumor marker was modified with a fluorescent dye, and stored at 4 ℃.

7. The method of manufacturing according to claim 3, wherein: the lengths of the sample pad, the combination pad, the nitrocellulose membrane and the absorbent paper are 8-17 mm, and contact areas of all parts are mutually overlapped for 1-3 mm during assembly; the length of the viscous plastic bottom plate is 60 mm.

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