CN107748147B - White luminous up-conversion nano-particles and test strip based on same and capable of simultaneously realizing detection of multi-component tumor markers - Google Patents
White luminous up-conversion nano-particles and test strip based on same and capable of simultaneously realizing detection of multi-component tumor markers Download PDFInfo
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Abstract
The invention discloses a white luminous up-conversion nanoparticle and a test strip based on the same and capable of simultaneously realizing multi-component tumor marker detection, wherein the nanoparticle has a three-layer core-shell structure and emits white light under the excitation of near infrared light; after the antibody of multiple tumor markers to be detected is marked, the antibody is fixed in a marker compound binding region of the test strip, so that the simultaneous detection of the multiple tumor markers can be realized. The invention realizes the sensitive detection of the single up-conversion nano-particle on various tumor markers, and has the advantages of simple operation, good specificity and high sensitivity of qualitative, quantitative and real-time detection.
Description
Technical Field
The invention belongs to the technical field of biomedical diagnosis, and particularly relates to an up-conversion test paper detection method of a multi-component tumor marker.
Background
Malignant tumor, commonly referred to as cancer, is a cancer in which partial gene expression is uncontrolled, and malignant proliferation of cells destroys normal vital activities of the body, resulting in rapid growth of the cancer, leading to emaciation, fever and severe organ damage of the human body, and ultimately life threatening. Currently, cancer has become the first killer to endanger human life. Therefore, early detection and clinical timely monitoring of cancer have become important issues in modern biomedicine. With the maturation of nanotechnology, the platform for combining nanotechnology and cancer markers for tumor marker detection has become a hot spot. The colloidal gold test paper is the earliest immunochromatographic test paper widely applied, has a simple and quick structure, does not need any instrument or equipment, and can distinguish results by naked eyes. However, compared with the up-conversion test paper, the colloidal gold test paper has poor sensitivity, is difficult to realize quantitative measurement, and has poor stability. Rare earth doped up-conversion luminescent nano materials (UCNPs) are nano materials which can absorb near infrared light and emit short-wavelength near ultraviolet visible light, and have the advantages of weak fluorescence background, no biological interference, strong tissue penetration capability, high sensitivity and the like. Therefore, the combination of UCNPs and immunochromatographic test paper can bring about breakthrough change, and the unique up-conversion luminescence phenomenon of the anti-Stokes displacement of the UCNPs can eliminate the self-luminescence interference phenomenon of a biological sample when the UCNPs are used for marking bioactive molecules, thereby improving the signal-to-noise ratio, enhancing the sensitivity and stability and realizing the high-sensitivity quantitative detection of cancer markers.
The up-conversion test paper detection method can be used for serological examination and helps doctors to perform auxiliary diagnosis, which has important values on curative diagnosis and follow-up visits, and the test paper detection method has obvious advantages in the aspects of operation, reaction speed and price. The tumor marker detected may be an enzyme, a hormone, a glycoprotein, an embryonic antigen or a tumor metabolite. Currently, there are:
1. alpha-fetoprotein
Alpha-fetoprotein (AFP) can provide an important basis for early diagnosis of liver cancer. The AFP in the body of the patients with liver cancer metastasis such as endoderm tumor, malignant teratoma, gastric cancer, etc. is increased.
2. Carcinoembryonic antigen
Carcinoembryonic antigen (CEA) can be found in elevated levels in gastrointestinal tumors, lung cancer, breast cancer, urinary tumors, and the like. The CEA content in the chest, ascites, secretion and digestive juice of cancer patients is increased, and the more advanced the cancer is, the higher the CEA content is, the higher the positive rate is.
However, when the existing upconversion test strip is used for simultaneously detecting multiple tumor markers, different upconversion nanoparticles are needed to be adopted for different T lines, the manufacturing process is complex, and the detection efficiency is seriously affected.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides the up-conversion nano-particles with white luminescence and the test strip based on the up-conversion nano-particles and capable of simultaneously detecting the multi-component tumor markers, aiming at simultaneously detecting various tumor markers by using one up-conversion nano-material and improving the detection efficiency.
In order to realize the purpose of the invention, the invention adopts the following technical scheme:
the invention firstly discloses a white luminous up-conversion nano particle which is characterized in that: the up-conversion nano particles are used in test paperOn the strip, the detection of the multi-component tumor marker is realized simultaneously; the 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.
The up-conversion nano particles are prepared by adopting a seed crystal method, and the method comprises the following specific steps:
a. synthesis of NaGdF doped with Yb, Tm and Er4Nanoparticles as inner core
Weighing Oleic Acid (OA)10mL, Octadecene (ODE)10mL, Gd (OAc)30.0841g、Yb(OAc)3Adding 0.0858g, 25 mu L of 0.1mol/LTm aqueous solution and 0.4200g of solid 2 mu L, NaF of 0.2mol/L Er aqueous solution into a three-neck flask A, magnetically stirring, heating to 110-120 ℃, keeping for 10min, and then vacuumizing to remove water and oxygen; removing all and then introducing N2Heating to 300 ℃ and reacting for 1.5 h;
b. wrapping Eu-doped NaGdF outside the inner core4First shell
Weighing 4mL Oleic Acid (OA), 4mL Octadecene (ODE), and Gd (OAc)30.0568g、Eu(OAc)30.0098g, adding into a three-neck flask B, magnetically stirring, heating to 110-120 ℃, keeping for 10min, and then vacuumizing to remove water and oxygen; removing all and then introducing N2Heating to 220 ℃, injecting the mixture into the three-neck flask A at the speed of 1mL/min by using a needle tube after the reaction in the step a is finished, and reacting for 0.5-1h at the temperature of 300 ℃;
c. wrapping NaYF outside the first shell layer4Second shell
Weighing Oleic Acid (OA)4mL, Octadecene (ODE)4mL, Y (OAc)30.0532g, adding into a three-neck flask C, magnetically stirring, heating to 110-120 ℃, keeping for 10min, and then vacuumizing to remove water and oxygen; removing all and then introducing N2And reacted in step bAfter the reaction is finished, injecting the mixture into the three-neck flask A by using a needle tube at the speed of 1mL/min, and reacting for 0.5-1h at 300 ℃; cooling to room temperature after the reaction is finished, putting the reaction liquid in the three-neck flask A into a centrifugal tube, and centrifugally separating to obtain the obtained nano particles;
d. stripping oleic acid from the surface of the nanoparticles obtained in step c
C, taking the nano particles obtained in the step c, adding a mixed solution of ethanol with the pH value of 1 and concentrated hydrochloric acid into the nano particles, performing ultrasonic dispersion uniformly, then performing vibration and ultrasonic treatment for 30min, centrifuging the mixture, removing supernatant, adding a mixed solution of ethanol with the pH value of 4 and concentrated hydrochloric acid into the nano particles, performing ultrasonic dispersion uniformly, then performing vibration and ultrasonic treatment for 40-60min, performing centrifugal separation again, and washing the obtained nano particles with water to obtain water-soluble white luminous up-conversion nano particles;
and dispersing the upconversion nanoparticles in a 0.01M PBS buffer solution with the pH value of 6.5-7.4 for storage.
The method for simultaneously realizing the detection of the multi-component tumor marker by the white luminous up-conversion nano particles is characterized in that: the up-conversion nanoparticles are used for emitting light in blue, green and red wave bands, so that the combination of one wave band and any two wave bands and the detection of three wave bands corresponding to one tumor marker can realize the simultaneous detection of 7 tumor markers; the upconversion nanoparticles are used as a tracer marker of the immunochromatography test strip, and white luminescence of the upconversion nanoparticles or Fluorescence Resonance Energy Transfer (FRET) action between a fluorescent dye and the upconversion nanoparticles is utilized to quench light of one or two corresponding wave bands of the upconversion nanoparticles, so that the upconversion nanoparticles show the remaining luminescence color. And judging the tumor marker in the sample to be detected according to the color change of the nano particles on the detection area of the test strip, thereby realizing the qualitative detection of the tumor marker type in the sample to be detected.
The invention also discloses a test strip for simultaneously realizing the detection of the multi-component tumor marker, which comprises a sample adding area, a labeled compound binding area, a detection area, a quality control area and a handheld area, and is characterized in that: the white luminescent up-conversion nanoparticles are fixed on the labeling compound binding region, and antibodies of various tumor markers to be detected are labeled on the white particles; 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; and 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 detection 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.
Specifically, during detection, a sample to be detected is dripped into the sample addition area, if antigens corresponding to the antibodies of the multiple tumor markers modified on the upconversion nanoparticles in the labeling compound binding area exist in the sample to be detected, the antigens are combined with the corresponding antibodies marked on the upconversion nanoparticles to form new combinations, the new combinations enter the detection area, are combined with the tumor marker antibodies corresponding to the upper surface of the T line of the detection area, and are fixed on the corresponding T line of the detection area; under the excitation of 980nm laser, the fluorescent dye on each T line and the up-conversion nanoparticles can quench the light of the corresponding wave band of the up-conversion nanoparticles through the action of Fluorescence Resonance Energy Transfer (FRET), so that the up-conversion nanoparticles show the rest luminescent color, and each T line also shows different colors to emit light;
meanwhile, during detection, a sample to be detected is dripped into the sample adding area, the upconversion nanoparticles fixed on the labeling compound binding area flow forwards along with the detection sample, part of the upconversion nanoparticles is fixed on the detection area T line, the rest part of the upconversion nanoparticles flows into the quality detection area along with the detection sample, and the antibody of the tumor marker to be detected, which is labeled on the upconversion nanoparticles, is combined with the IgG antibody coated on the quality detection area C line and is fixed on the quality detection area C line; the IgG antibody coated on the C line is not marked by dye, and the particles on the C line emit light in all wave bands under the excitation of 980nm laser, namely white light.
Further, dripping a series of standard antigen solutions with known concentration on the sample adding region for detection; after the detection is finished, the fluorescence intensity of the corresponding T line and the C line of the quality detection area in the detection area is obtained through 980nm laser excitation, and a standard curve of the ratio of the fluorescence intensity of the T line and 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 in the sample to be detected is realized.
The manufacturing method of the test strip comprises the following steps:
(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.
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.
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 6.5-7.4, 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 of 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 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).
The method for modifying the fluorescent dye on the antibody of the tumor marker comprises the following steps: 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 ℃.
Preferably, 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.
The invention has the beneficial effects that:
1. the invention realizes the rapid detection and screening of the multi-component tumor marker by using the up-conversion nano particles, and has high accuracy and specificity.
2. The test strip takes UCNPs as biomarker antibodies, can directly observe results under the excitation of near infrared light, eliminates background interference due to the unique optical characteristics of the UCNPs, and greatly improves the sensitivity; and UCNPs in the test paper are harmless to testers and the environment, and the safety is good.
3. The sample detected by the test paper does not need much pretreatment, quantitative measurement can be directly realized by virtue of the up-conversion luminescence sensor, the operation is simple and rapid, and the on-site operation can be realized.
Drawings
FIG. 1 is a fluorescence emission spectrum of the upconverting nanoparticle obtained in example 1;
FIG. 2 is a schematic diagram of the structure of an upconversion test strip;
FIG. 3 is a schematic diagram showing a reaction in which an immune reaction is negative;
FIG. 4 is a diagram showing a reaction in which an immune reaction is positive.
Detailed Description
The technical solution of the present invention will be explained in detail below with reference to examples and drawings.
Example 1 detection of CEA, AFP tumor markers
In this embodiment, CEA and AFP are taken as tumor markers to be detected, and the details are as follows.
1. Preparation of UCNPs-antibodies
1.1, preparing white luminous up-conversion nano particles with a three-layer core-shell structure by adopting a seed crystal method, wherein the up-conversion nano particles are NaGdF doped with Yb, Tm and Er4The nano particles are taken as an inner core, and Eu-doped NaGdF is wrapped outside the inner core4A first shell layer, and the outside of the first shell layer is wrapped withNaYF4A second shell layer which forms Yb, Tm, Er and Eu co-doped NaGdF4:Yb/Tm/Er@NaGdF4:Eu@NaYF4A core-shell structure; the preparation method comprises the following specific steps:
a. synthesis of NaGdF doped with Yb, Tm and Er4Nanoparticles as inner core
Weighing Oleic Acid (OA)10mL, Octadecene (ODE)10mL, Gd (OAc)30.0841g、Yb(OAc)3Adding 0.0858g, 25 mu L of 0.1mol/L Tm aqueous solution and 0.4200g of solid 2 mu L, NaF of 0.2mol/L Er aqueous solution into a three-neck flask A, magnetically stirring, heating to 110-120 ℃, keeping for 10min, and then vacuumizing to remove water and oxygen; removing all and then introducing N2Heating to 300 ℃ and reacting for 1.5 h;
b. wrapping Eu-doped NaGdF outside inner core4First shell
Weighing 4mL Oleic Acid (OA), 4mL Octadecene (ODE), and Gd (OAc)30.0568g、Eu(OAc)30.0098g, adding into a three-neck flask B, magnetically stirring, heating to 110-120 ℃, keeping for 10min, and then vacuumizing to remove water and oxygen; removing all and then introducing N2Heating to 220 ℃, injecting the mixture into a three-neck flask A at the speed of 1mL/min by using a needle tube after the reaction in the step a is finished, and reacting for 1h at the temperature of 300 ℃;
c. wrapping NaYF outside the first shell layer4Second shell
Weighing Oleic Acid (OA)4mL, Octadecene (ODE)4mL, Y (OAc)30.0532g, adding into a three-neck flask C, magnetically stirring, heating to 110-120 ℃, keeping for 10min, and then vacuumizing to remove water and oxygen; removing all and then introducing N2After the reaction in the step b is finished, injecting the mixture into the three-neck flask A by using a needle tube at the speed of 1mL/min, and reacting for 1h at 300 ℃; cooling to room temperature after the reaction is finished, putting the reaction liquid in the three-neck flask A into a centrifugal tube, and centrifugally separating to obtain the obtained nano particles;
d. stripping oleic acid from the surface of the nanoparticles obtained in step c
C, taking the nano particles obtained in the step c, adding a mixed solution of ethanol with the pH value of 1 and concentrated hydrochloric acid (7.5mL of ethanol and 62.5 mu L of concentrated hydrochloric acid), carrying out ultrasonic dispersion uniformly, then carrying out vibration while carrying out ultrasonic treatment for 30min, then centrifuging, removing supernate, adding a mixed solution of ethanol with the pH value of 4 and concentrated hydrochloric acid (7.5mL of ethanol and 7.5mL of concentrated hydrochloric acid), carrying out ultrasonic dispersion uniformly, carrying out vibration while carrying out ultrasonic treatment for 40min, then carrying out centrifugal separation again, and washing the obtained nano particles with water to obtain water-soluble white luminous up-conversion nano particles; the upconverting nanoparticles were dispersed in 0.01MPBS buffer solution at pH 6.5 at a concentration of 5mg/mL and stored until use. FIG. 1 shows the fluorescence emission spectrum of the upconversion nanoparticle obtained in this example.
1.2 tumor marker antibody labeled on surface of upconversion nanoparticle by electrostatic adsorption and the like
Taking 1mL of the upconversion nanoparticles dispersed in 0.01M PBS buffer solution with pH of 6.5 obtained in the step 1.1, adding 10 μ L of 2mg/mL CEA monoclonal antibody and 8 μ L of 2.5mg/mL AFP monoclonal antibody, carrying out shaking table reaction at normal temperature for 2h, then carrying out centrifugal separation at 4 ℃ to obtain white luminescent upconversion nanoparticles labeling the antibodies of the multiple tumor markers to be detected, dispersing the white luminescent upconversion nanoparticles in 0.01M, pH-7.0 PBS buffer solution containing 2% BAS by mass, and storing the white luminescent upconversion nanoparticles at 4 ℃ to obtain a solution labeling the antibodies of the multiple tumor markers to be detected.
1.3 fluorescent dye modified on antibody of tumor marker
Diluting 50 μ L of 1.9mg/mL CEA antibody with 0.1M, pH ═ 9.0 carbonate buffer solution to 500 μ L, adding 3 μ L of 1mg/mL fluorescent dye solution of isothiocyanato Rhodamine B (RBITC), and reacting for 4h in a shaker; the reaction solution was dialyzed overnight against 0.01M, pH ═ 7.4 PBS buffer solution to remove unbound RBITC, i.e., the CEA monoclonal antibody was modified with the fluorescent dye RBITC, and stored at 4 ℃.
Diluting 30 mu L of 3.3mg/mLAFP antibody to 500 mu L with carbonate buffer solution of 0.1M, pH ═ 9.0, adding 3 mu L of 1mg/mL fluorescent dye solution 4-chloro-7-nitro-2, 1, 3-benzoxadiazole (NBD-Cl) for fluorescence, and reacting for 4h by a shaking table; the reaction solution was dialyzed overnight against 0.01M, pH ═ 7.4 PBS buffer solution to remove unbound NBD-Cl, i.e., fluorescent dye NBD-Cl modified with AFP antibody, and stored at 4 ℃.
2. Preparation of test paper strip
As shown in fig. 2, the test strip for simultaneously detecting a multi-component tumor marker in this embodiment includes a sample application region, a labeled complex binding region, a detection region, a quality control region, and a handheld region. The white luminous up-conversion nanoparticles are fixed in the labeling compound binding region, and antibodies of two tumor markers to be detected are labeled on the up-conversion nanoparticles; two detection T lines are arranged in different areas of the detection area, T1 is coated with CEA antibody, T2 is coated with AFP antibody, and the antibodies of the two tumor markers are modified by fluorescent dyes with different colors; the quality control area is provided with a quality control C line, and the quality control C line is coated with a goat anti-mouse IgG antibody.
Specifically, the test strip is prepared by the following method:
(1) sample pad handling
Soaking the sample pad in the sample pad treatment solution for 5min by using a cellulose membrane as a sample pad material, and drying at 37 ℃ to finish the treatment of the sample pad; the sample pad treatment solution was a PBS buffer solution of 0.01M, pH ═ 8.0 containing 0.5% BAS and 1% Tween-20 by mass concentration;
(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 5min, then drying at 37 ℃, then placing the bonding pad in the solution of the white luminous up-conversion nanoparticles for marking the antibodies of the multiple tumor markers to be detected, obtained in the step 1.2, soaking for 5min, and then drying at 37 ℃ to finish the treatment of the bonding pad; the conjugate pad treatment solution was a PBS buffer solution containing 1% BAS, 2% Tween-20, and 5% sucrose at 0.01M, pH ═ 7.4 by mass.
(3) Treatment of nitrocellulose membranes
The nitrocellulose membrane was divided into two parts:
marking off CEA antibodies and AFP antibodies of two tumor markers to be detected in different areas of the first part according to the parameters of 1 mu L/cm, and respectively forming a detection T1 line and a detection T2 line coated with the antibodies of the corresponding tumor markers, wherein the CEA antibodies are modified with an isothiocyanatodorhodamine B (RBITC) fluorescent dye, and the AFP antibodies are modified with a 4-chloro-7-nitro-2, 1, 3-benzoxadiazole (NBD-Cl) fluorescent dye;
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.
3. Detection process of up-conversion test strip
During detection, a test sample to be detected is dripped in a sample adding area, if tumor markers AFP and CEA exist in the test sample to be detected, the test sample reacts with UCNPs marked with tumor markers AFP and CEA antibodies to be detected on the surface of a marking compound binding area, the tumor markers AFP and CEA are combined with the AFP and CEA antibodies marked on the UCNPs and enter a detection area, the tumor markers AFP and CEA fixed on the UCNPs respectively react with the CEA fixed on a detection area T1 line for modifying RBITC and the AFP antibody fixed on a T2 line for modifying NBD-Cl, part of the tumor markers AFP and CEA are fixed on the detection area T line, the rest part of the tumor markers AFP and CEA fixed on the UCNPs flow into a quality detection area along with the test sample to be detected, and are combined with the goat anti-mouse IgG antibodies fixed on the quality control area C line and fixed on the quality control.
And after the detection is finished, observing the color change of the detection area and the quality detection area by 980nm laser excitation. At this time, under the irradiation of 980nm excitation light, Fluorescence Resonance Energy Transfer (FRET) exists between the RBITC on the T1 line and the upconversion nanoparticles, so that the green light emission peak of the upconversion nanoparticles is weakened or even disappears, and the upconversion nanoparticles fixed on the T1 line show purple luminescence. Fluorescence Resonance Energy Transfer (FRET) exists between NBD-Cl and the upconversion nanoparticles on the T2 line, so that an emission peak in a blue light region of the upconversion nanoparticles is weakened or even disappears, and the upconversion nanoparticles fixed on the T2 line show yellow luminescence. The emission peak of the upconversion nanoparticles fixed on the quality control region detection line C is unchanged, and the upconversion nanoparticles display white light under the irradiation of 980nm exciting light. FIG. 3 is a diagram showing a reaction in which an immune response is negative, and FIG. 4 is a diagram showing a reaction in which an immune response is positive.
In addition, CEA antigen (10-200ng/ml) and AFP antigen (100-1000ng/ml) with different concentration gradients are prepared, sample standard solutions of the CEA antigen and the AFP antigen with different concentrations are dripped on the sample pad, three identical test strips are prepared at each concentration, after the test strips are kept still for five minutes, the test strips are scanned by an up-conversion luminescence sensor, and a standard curve is established by comparing fluorescence intensities before and after reaction and comparing fluorescence intensities of a T line and a C line, so that quantitative measurement can be realized.
The above description is only exemplary of the present invention and should not be taken as limiting the invention, and any modifications, equivalents, improvements and the like that are made within the spirit and principle of the present invention should be included in the scope of the present invention.
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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