Smartphone-based homogeneous fluorescent immunoassay method and application thereof

Abstract

The application discloses a kind of homogeneous fluorescence immunoassay method based on smart phone and application thereof, method includes the following steps: S1, with 1,1,2,2-tetrakis (4-carboxyl phenyl) ethylene and zirconium tetrachloride preparation Zr-MAF fluorescent material;S2, preparation CNP nanometer carbon ball;S3, the Zr-MAF fluorescent material obtained in S1 is prepared non-activation label monoclonal antibody as donor probe;S4, the CNP nanometer carbon ball obtained in S2 is prepared label monoclonal antibody as receptor probe;S5, configuration measured material standard serum solution, the donor probe prepared in S3 and the receptor probe prepared in S4 are added to the sample unit of homogeneous fluorescence immunoassay device based on smart phone, after adding measured material standard serum solution, and read light intensity using the analysis unit of homogeneous fluorescence immunoassay device based on smart phone then fitting standard curve;S6, the same method as S5 is used to read the light intensity of test sample after measured sample, and the corresponding concentration value is converted using standard curve.The method provided by the application is low in cost, fast and convenient.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological immune detection analysis, and more particularly to a homogeneous fluorescence immunoassay method based on a smart phone and application. BACKGROUND

[0002] The fluorescence immunoassay technology has the advantages of strong specificity, high sensitivity, good practicability, etc., and has been widely used in the fields of chemistry, biology, medicine, health, agriculture and environmental protection. The fluorescence immunoassay technology is mainly a quantitative analysis method for determining the content of the measured substance by using the instrument to detect the fluorescence intensity of the system under the condition that the concentration of the measured substance in the system and the fluorescence intensity are linearly related.

[0003] The traditional fluorescence immunoassay technology is relatively complex to operate, requires professional technical operation, is not conducive to popularization, and the instrument is large in size and high in manufacturing cost, which is difficult to apply to on-site detection. For example, Chinese patent application 202310153361.9 provides a carcinoembryonic antigen fluorescence immunoassay kit, in which a carcinoembryonic antigen monoclonal antibody and a chicken IgY antibody are used as coating antibodies on the detection line and the quality control line respectively, and a carcinoembryonic antigen antibody and a goat anti-chicken IgY are mixed as a fluorescent microsphere labeled antibody. The fluorescence immunoassay method is used to determine the fluorescence signal value, which is converted into the CEA concentration in the sample by a dry-type fluorescence immunoassay instrument. The operation process is complex and requires special instruments with high cost, which is not suitable for use in mobile medical points and primary health institutions.

[0004] The smart phone, as a convenient mobile device, brings new development opportunities for portable detection technology. With the continuous improvement of the multifunctional features of smart phones, the wide use of application programs and the improvement of data processing capabilities, they can serve as image acquisition tools and digital processing devices, and play an important role. Usually, they capture the light signal in the reaction process through the light sensor or analyze the color characteristics of the photographed measured substance, and then use special picture processing software or application programs installed on the smart phone to realize the quantification of the signal, and finally obtain the data results. Therefore, how to combine the smart phone with the traditional detection technology to provide a fluorescence immunoassay method with low cost, simple operation and fast processing speed is an important research topic in the field of immune detection methods. SUMMARY

[0005] Therefore, it is necessary to provide a homogeneous fluorescence immunoassay method based on a smart phone and application in view of the above technical problems.

[0006] In order to solve the above technical problems, the present application provides a homogeneous fluorescence immunoassay method based on a smart phone and application, which adopts the following technical solutions:

[0007] The first aspect of the application provides a homogeneous fluorescent immunoassay method based on a smart phone, which comprises the following steps:

[0008] S1, preparing Zr-MAF fluorescent material from 1,1,2,2-tetrakis(4-carboxyphenyl) ethylene and zirconium tetrachloride;

[0009] S2, preparing CNP nanocarbon balls;

[0010] S3, preparing the Zr-MAF fluorescent material obtained in S1 into an unactivated labeled monoclonal antibody as a donor probe;

[0011] S4, preparing the CNP nanocarbon balls obtained in S2 into a labeled monoclonal antibody as an acceptor probe;

[0012] S5, configuring a standard serum solution of a to-be-tested substance, adding the donor probe prepared in S3 and the acceptor probe prepared in S4 into a sample inlet unit of a homogeneous fluorescent immunoassay device based on a smart phone, reading the light intensity of the test sample after adding the standard serum solution of the to-be-tested substance, and then fitting a standard curve by using an analysis unit of the homogeneous fluorescent immunoassay device based on the smart phone;

[0013] S6, reading the light intensity of the test sample in the same way as in S5, and then converting the light intensity into a corresponding concentration value by using the standard curve.

[0014] Further, the Zr-MAF fluorescent material is prepared from 1,1,2,2-tetrakis(4-carboxyphenyl) ethylene and zirconium tetrachloride in a mass ratio of 3-4:1-2, and the CNP nanocarbon balls are prepared from Triton X-100 and nanocarbon black in a mass ratio of 5-10:1.

[0015] Further, the method for preparing the Zr-MAF fluorescent material is as follows: 1,1,2,2-tetrakis(4-carboxyphenyl) ethylene and zirconium tetrachloride in a mass ratio of 4:1 are ultrasonically dissolved in a dimethylformamide solution, acetic acid is added, and then the Zr-MAF fluorescent material is generated by heating reaction.

[0016] Further, the method for preparing the CNP nanocarbon balls in step S2 is as follows: Triton X-100 and nanocarbon black in a mass ratio of 5:1 are added to a borate buffer solution, thoroughly stirred, and then ultrasonically treated to obtain the CNP nanocarbon balls.

[0017] Further, the method for preparing the Zr-MAF fluorescent material is as follows: 100 mg of 1,1,2,2-tetrakis(4-carboxyphenyl) ethylene and 25 mg of zirconium tetrachloride are ultrasonically dissolved in 6 mL of a dimethylformamide solution, 3 mL of glacial acetic acid is added, 95℃ constant temperature stirring is performed, heating reaction is performed for 30 min, then 100 μL of deionized water is added, stirring and heating reaction are performed for 2 h.

[0018] Further, the non-activated labeled monoclonal antibody in step S3 is composed of Zr-MAF fluorescent material and monoclonal antibody mAb1, and the CNP labeled monoclonal antibody in step S4 is monoclonal antibody mAb2.

[0019] Further, the smartphone-based homogeneous fluorescence immunoassay detection device comprises an analysis unit and a sample injection unit, the analysis unit comprises a control chamber, a through slot, a smartphone and a substrate from top to bottom, and the sample injection unit slides into the analysis unit from the through slot; the control chamber comprises an LED light source and a light guide channel from top to bottom, and the through slot is provided with an optical filter at a position corresponding to the light guide channel, and the light source emitted by the LED light source is received by the smartphone after sequentially passing through the light guide channel, the sample injection unit and the optical filter.

[0020] Further, the LED light source is 365nm excitation light, and the optical filter is a narrow-band optical filter allowing wavelength 470nm light to pass through.

[0021] Further, the sample injection unit comprises an ELISA microwell and a bearing base loaded with the ELISA microwell, and the bearing base is provided with a light transmission hole at the bottom.

[0022] The second aspect of the application provides the use of any of the above methods in detecting carcinoembryonic antigen CEA.

[0023] Compared with the prior art, the application has the following beneficial effects:

[0024] The application provides a smartphone-based homogeneous fluorescence immunoassay detection method and application thereof, wherein a metal cation zirconium is combined with 1,1,2,2-tetrakis(4-carboxyphenyl) ethylene to prepare Zr-MAF fluorescent material through a simple method, and the Zr-MAF fluorescent material has stable properties; the Zr-MAF fluorescent material is combined with monoclonal antibody mAb1 to form Zr-MAF-mAb1; nanometer carbon black and Triton X-100 are used to obtain nanometer carbon spheres with strong stability and easy to label through a simple one-step ultrasonic stirring method; the nanometer carbon spheres are combined with monoclonal antibody mAb2 to form CNP-mAb2; and Zr-MAF-mAb1, CEA and CNP-mAb2 are combined to form Zr-MAF-mAb1-CEA-CNP-mAb2; due to the formation of the immune complex, the distance between CNP and Zr-MAF is shortened, and fluorescence quenching is caused. The method does not need pre-coating, washing, large instruments, professionals and technical personnel, and can directly complete detection through a one-step method, and has the advantages of high precision and sensitivity, convenient and fast operation, low cost and on-site instantaneity.

[0025] The application provides a homogenous fluorescence immunoassay method based on a smart phone, which combines the smart phone with traditional detection technology, fully combines the advantages of the smart phone, such as portability, low cost, simple operation and fast processing speed, with the principle and applicability of the traditional detection method, and provides a broad development platform for the research of a rapid, efficient and accurate detection method, and is very suitable for mobile medical points and primary health institutions. BRIEF DESCRIPTION OF DRAWINGS

[0026] In order to more clearly illustrate the scheme in the application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced as follows. Obviously, the drawings in the following description are some embodiments of the application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of the drawings.

[0027] Figure 1 A decomposition schematic view of the handheld homogenous fluorescence immunoassay device based on the smart phone proposed for the embodiment 1 is shown in the figure.

[0028] Figure 2 A side view of the handheld homogenous fluorescence immunoassay device based on the smart phone proposed for the embodiment 1 is shown in the figure.

[0029] Figure 3 A structure schematic view of the sample inlet unit of the handheld homogenous fluorescence immunoassay device based on the smart phone proposed for the embodiment 1 is shown in the figure.

[0030] Figure 4 A test flowchart of the homogenous fluorescence immunoassay device based on the smart phone proposed for the embodiment 1 is shown in the figure.

[0031] Figure 5 A transmission electron microscope view of the Zr-MAF fluorescent material and the CNP nanocarbon ball proposed for the embodiment 2 is shown in the figure.

[0032] Figure 6 A reaction principle of the homogenous fluorescence immunoassay device based on the smart phone proposed for the embodiment 2 for detecting CEA is shown in the figure.

[0033] Figure 7 A standard curve graph of the homogenous fluorescence immunoassay device based on the smart phone proposed for the embodiment 2 for detecting CEA is shown in the figure.

[0034] The mark in the figure is explained as follows.

[0035] Radiator 1, LED light source 2, ELISA microwell 3, bearing base 4, switch 5, smart phone 6, light sensor 7, base plate 8, through slot 9, optical filter 10, variable resistor 11, rechargeable lithium battery 12, top cover 13, light transmission hole 14, positioning line 15. DETAILED DESCRIPTION

[0036] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0037] In the description of the invention, it should be understood that the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", "clockwise", "counterclockwise", "axial", "radial", "circumferential" and the like to indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as limiting the invention.

[0038] In order to enable those skilled in the art to better understand the solution of the present application, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the accompanying drawings.

[0039] Example 1 A homogeneous fluorescence immunoassay device based on a smartphone

[0040] like Figures 1-3 As shown, this embodiment provides a smartphone-based homogeneous fluorescence immunoassay device, which includes an analysis unit and a sample injection unit. The analysis unit comprises, from top to bottom, a top cover 13, a control chamber, a through slot 9, a base plate 8, and a smartphone 6. The sample injection unit slides into the analysis unit through the through slot 9, and the base plate 8 is used to place the smartphone 6 for reading data.

[0041] Furthermore, the control room includes a heat sink 1, an LED light source 2, and a light guide channel from top to bottom, and the light emitted by the LED light source 2 enters the sample injection unit through the light guide channel.

[0042] Furthermore, a filter 10 is provided at a position corresponding to the light guide channel in the through slot 9. The light generated by the LED light source 2 passes through the light guide channel, the sample injection unit, and the filter 10, and is finally received by the smartphone 6. Specifically, a light sensor 7 is provided on the top of the smartphone 6. During detection, the LED light source 2, the sample injection unit, the filter 10, and the light sensor 7 are on the same center line.

[0043] Furthermore, the through slot 9 is also provided with an indicator scale for assisting the positioning of the injection unit.

[0044] Furthermore, a rechargeable lithium battery 12 and a variable resistor 11 are provided in the control room. The rechargeable lithium battery 12, the radiator 1, the LED light source 2, the variable resistor 11, the switch 5 provided outside the control room and the wires form a complete control circuit.

[0045] Furthermore, the sample injection unit includes an ELISA micropore 3 and a supporting base 4 for loading the ELISA micropore 3. The bottom of the supporting base 4 has a light-transmitting hole 14 that allows light to pass through and a positioning line 15 for fixing the position. After the immunoassay is completed, the ELISA micropore 3 is placed in the supporting base 4 and slides into the through groove 9 under the control chamber, and the positioning line 15 and the indicator scale of the through groove 9 correspond. During the test, the LED light source 2, the light guide channel, the ELISA micropore 3, the light-transmitting hole 14, the filter 10, and the light sensor 7 are on the same light path.

[0046] Furthermore, the LED light source 2 is 365nm excitation light; and the filter 10 is a narrow-band filter that allows light with a wavelength of 470nm to pass through.

[0047] Furthermore, the light sensor 7 is a photosensitive element built into the smart phone 6 .

[0048] Furthermore, the smartphone 6 is provided with a light intensity detection application, which can convert the light signal captured by the light sensor 7 into a digital signal. After the light sensor 7 receives the light intensity transmitted through the light hole 14, the light intensity detection application reads the value.

[0049] Specifically, in this embodiment, the resistance range of the variable resistor 11 is 0 to 10 KΩ.

[0050] When using, such as Figure 1 As shown, the light sensor 7 is located next to the front-facing camera on the top of the smartphone. The light intensity detection application includes a detection interface and a user help interface. After the immunoassay is completed, the ELISA microwell 3 and the supporting base 4 are placed into the through-slot 9. Simultaneously, the smartphone 6 is inserted into the base plate 8. Using the scale, the light sensor 7 is positioned directly below the light-transmitting hole 14. Then, the light intensity detection application is launched to read the reading.

[0051] like Figure 4The test process of the smartphone-based homogeneous fluorescence immunoassay device is shown. Monoclonal antibody mAb1 is first coupled to the surface of self-made Zr-MAF material with fluorescence emission function without activation and forms monoclonal antibody Zr-MAF-mAb1 (donor probe), and monoclonal antibody mAb2 is coupled to the surface of self-made CNP nanocarbon sphere and forms monoclonal antibody CNP-mAb2 (acceptor probe). The donor and acceptor probes are first added to the ELISA microwell 3. When the sample containing the target is added to the ELISA microwell 3, an immune reaction occurs, and an antibody-target analyte-antibody complex is formed, which causes fluorescence to be quenched through resonance energy transfer. The ELISA microwell 3 is loaded into the bearing base 4, and after the LED light source 2 of the detection device is turned on, the higher the concentration of the target analyte, the higher the concentration of the immune complex formed, the stronger the fluorescence quenching effect, and the smaller the light intensity value of the ELISA microwell 3. The light intensity is inversely proportional to the concentration of the target sample to be tested

[0052] Example 2: A smartphone-based homogeneous fluorescence immunoassay method

[0053] This example applies the smartphone-based homogeneous fluorescence immunoassay device proposed in Example 1 to the detection of carcinoembryonic antigen A (CEA), as shown in Figure 4 The specific steps include the following:

[0054] 1. Preparation of Zr-MAF fluorescent material and CNP nanocarbon sphere

[0055] 1.1 Weigh 100 mg of ligand 1,1,2,2-tetrakis(4-carboxyphenyl)ethylene (TCPE) and 25 mg of zirconium tetrachloride, and ultrasonically dissolve them in 6 mL of dimethylformamide (DMF) solution. After adding 3 mL of glacial acetic acid, heat and stir at 95°C for 30 min, then add 100 μL of deionized water and stir and heat for 2 h. Then, the prepared Zr-MAF fluorescent material is naturally cooled to room temperature, washed and resuspended with ethanol, and stored at 4°C for standby.

[0056] 1.2 Add 0.1 g of nanocarbon black (purchased from Degussa company, model number: SB4) to 100 mL of 0.02 M borate buffer (BB) and stir thoroughly. Then, add 0.5 mL of Triton X-100 (purchased from Beijing Solaybao Technology Co., Ltd.) to the carbon solution and stir at room temperature for 30 min. Then, ultrasonically treat at 300 W and 4°C for 10 s, stop for 5 s, and repeat for 80 cycles. Centrifuge at 4°C and 5000 rpm for 10 min, and take the supernatant as the CNP nanocarbon sphere solution.

[0057] The morphology of the Zr-MAF fluorescent material and the CNP nanocarbon sphere material is shown in Figure 5 .

[0058] 2. Preparation of non-activated labeled monoclonal antibody Zr-MAF-mAb1 and CNP-mAb2

[0059] 2.1 Zr-MAF material stored in ethanol was diluted 50 times with deionized water, 40 μg of monoclonal antibody mAb1 was added to 1 mL of diluted Zr-MAF material, mixed at room temperature for 2 h in the dark, 100 μL of 10% bovine serum albumin was added for blocking for 30 min, centrifuged at 8000 rpm for 10 min at 4°C, the supernatant was discarded, and the precipitate was resuspended with 300 μL of PBST buffer to obtain the Zr-MAF-mAb1 complex, which was stored at 4°C for later use.

[0060] 2.2 The CNP nanocarbon ball solution was washed twice with 0.02M BB buffer, 40 μg of monoclonal antibody mAb2 was added to 1 mL of washed CNP and mixed by rotation for 2 h, 100 μL of 10% bovine serum albumin was added for blocking for 30 min, centrifuged at 10000 rpm for 10 min at 4°C, the supernatant was discarded, and the precipitate was resuspended with 500 μL of 0.02M BB buffer containing 2% trehalose and 0.5% BSA to obtain the CNP-mAb2 complex, which was stored at 4°C for later use.

[0061] 3. Fitting of light intensity standard curve

[0062] (1) 100 μL of a series (0.02-2.56 ng / mL) of CEA serum standards was added to ELISA microwells 3 with Zr-MAF-mAb1 and CNP-mAb2, incubated at 37°C for 20 min, and the fluorescence intensity was directly detected. The reaction principle is shown in Figure 6 .

[0063] (2) The standard solution loaded in ELISA microwells 3 was tested using a smartphone-based homogeneous fluorescence immunoassay detection device. The serial concentrations of CEA standard serum were 0.02, 0.04, 0.08, 0.16, 0.32, 0.64, 1.28, and 2.56 ng / mL, and the average light intensity values at different sample concentrations were 142.67, 101.67, 81.33, 54.33, 34.67, 21, 8.67, and 2.33 LUX, respectively. With the increase of CEA concentration, the brightness of fluorescence light intensity gradually weakened, and the measured light intensity value gradually decreased. Taking log2 concentration as the abscissa and light intensity as the ordinate, the fitting equation Y = 12.14X 2 -34X + 24.5 (R 2 = 0.9983) was obtained, and the fitting standard curve is shown in Figure 7 .

[0064] Sample testing: after the operation of the test sample according to step 3(1), the light intensity value is read by the smartphone-based homogeneous fluorescence immunoassay detection device, and then the CEA concentration of the sample is obtained according to the fitting standard curve of step 3(2).

[0065] Obviously, the above-described embodiments are only some of the embodiments of the present application, rather than all the embodiments, and the preferred embodiments of the present application are given in the drawings, but do not limit the patent scope of the present application. The present application can be implemented in many different forms, and conversely, the purpose of providing these embodiments is to make the disclosure of the present application more thorough and comprehensive. Although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions recorded in the foregoing specific embodiments, or equivalently replace some of the technical features. Any equivalent structure made by using the content of the specification and drawings, directly or indirectly applied to other related technical fields, is also within the patent protection scope of the present application.

Claims

1. A homogeneous fluorescence immunoassay method based on a smartphone, characterized in that: The following steps are involved: S1. Preparing a Zr-MAF fluorescent material using 1,1,2,2-tetrakis(4-carboxyphenyl)ethylene and zirconium tetrachloride. The method for preparing the Zr-MAF fluorescent material is as follows: ultrasonically dissolving 1,1,2,2-tetrakis(4-carboxyphenyl)ethylene and zirconium tetrachloride in a mass ratio of 4:1 in a dimethylformamide solution, adding glacial acetic acid, and heating the solution to react to generate the Zr-MAF fluorescent material. S2, preparing CNP nanocarbon spheres; the method for preparing CNP nanocarbon spheres is to add Triton X-100 and nanocarbon black in a mass ratio of 5:1 to borate buffer, stir thoroughly, and then ultrasonically treat; S3, using the Zr-MAF fluorescent material obtained in S1 to prepare a non-activated labeled monoclonal antibody as a donor probe; S4, using the CNP nanocarbon spheres obtained in S2 to prepare labeled monoclonal antibodies as receptor probes; S5. Prepare a standard serum solution of the substance to be tested, add the donor probe prepared in S3 and the acceptor probe prepared in S4 to the injection unit of the smartphone-based homogeneous fluorescent immunoassay device, add the standard serum solution of the substance to be tested, and use the analysis unit of the smartphone-based homogeneous fluorescent immunoassay device to read the light intensity and then fit the standard curve; S6. Read the light intensity of the sample to be tested in the same manner as in S5 and convert it into the corresponding concentration value using the standard curve.

2. The handheld homogeneous fluorescent immunoassay method based on a smart phone according to claim 1, characterized in that: The method for preparing Zr-MAF fluorescent material is as follows: 100 mg of 1,1,2,2-tetrakis(4-carboxyphenyl)ethylene and 25 mg of zirconium tetrachloride are ultrasonically dissolved in 6 mL of dimethylformamide solution, 3 mL of glacial acetic acid is added, and the mixture is stirred and heated at a constant temperature of 95°C for 30 minutes, and then 100 μL of deionized water is added and stirred and heated for 2 hours.

3. The handheld homogeneous fluorescent immunoassay method based on a smart phone according to claim 1, characterized in that: The non-activated labeled monoclonal antibody in step S3 is composed of Zr-MAF fluorescent material and monoclonal antibody mAb1, and the CNP-labeled monoclonal antibody in step S4 is monoclonal antibody mAb2.

4. The homogeneous fluorescence immunoassay method based on a smartphone according to claim 1, characterized in that: The smartphone-based homogeneous fluorescence immunoassay device comprises an analysis unit and a sample injection unit. The analysis unit comprises, from top to bottom, a control chamber, a through slot (9), a smartphone (6), and a base plate (8). The sample injection unit slides into the analysis unit from the through slot (9). The control chamber comprises, from top to bottom, an LED light source (2) and a light guide channel. A filter (10) is provided at a position corresponding to the light guide channel in the through slot (9). Light emitted by the LED light source (2) passes through the light guide channel, the sample injection unit, and the filter (10) in sequence and is then received by the smartphone (6).

5. The homogeneous fluorescent immunoassay method based on a smartphone according to claim 4, characterized in that: The LED light source (2) is 365nm excitation light; the filter (10) is a narrow-band filter that allows light with a wavelength of 470nm to pass through.

6. The homogeneous fluorescence immunoassay method based on a smartphone according to claim 4, characterized in that: The sample injection unit comprises an ELISA microwell (3) and a supporting base (4) for loading the ELISA microwell (3), and a light-transmitting hole (14) is provided at the bottom of the supporting base (4).

7. Use of any one of the methods of claims 1 to 6 for in vitro detection of carcinoembryonic antigen (CEA) for non-disease diagnosis purposes.

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