CN113933518A - Preparation method of protein C detection test paper and handheld reading device - Google Patents
Preparation method of protein C detection test paper and handheld reading device Download PDFInfo
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- CN113933518A CN113933518A CN202111197562.6A CN202111197562A CN113933518A CN 113933518 A CN113933518 A CN 113933518A CN 202111197562 A CN202111197562 A CN 202111197562A CN 113933518 A CN113933518 A CN 113933518A
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- G—PHYSICS
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
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- G—PHYSICS
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
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Abstract
The invention discloses a preparation method of protein C test paper, namely a palm reading device, wherein an NC film is attached to a substrate of lateral flow test paper; fixing the prepared detection solution on a detection area on the NC membrane, and fixing the prepared control solution on a control area on the NC membrane; respectively attaching a combination pad and a water absorption pad on the substrate and positioned at two sides of the NC membrane, wherein the combination pad and the water absorption pad are connected with the NC membrane, and the combination pad is provided with a probe of UCNPs-CRP antibody; attaching a sample pad on the other side of the bonding pad on the substrate, wherein the sample pad is connected with the bonding pad; according to the invention, the probe of the UCNPs-CRP antibody is arranged on the bonding pad, so that the influence of background fluorescence generated by a whole blood sample on the detection performance can be avoided, and the detection zone and the control zone are arranged on the NC membrane, so that the accuracy of the detection result can be ensured.
Description
Technical Field
The invention belongs to the technical field of preparation of protein C test paper, and particularly relates to a preparation method of protein C test paper and a handheld reading device.
Background
C-reactive protein (CRP) is present in blood, can reflect bacterial and viral infections, reflects an important index of disease development degree, and has been proved to be related to the development of various diseases in recent years. Therefore, if the CRP detection can be performed quickly, sensitively, accurately, portably and at low cost, the efficiency of disease diagnosis can be effectively improved and the economic stress of patients can be reduced.
In recent years, the real-time detection technology has been widely paid attention to by researchers because of its portable detection equipment, real-time information feedback, and low equipment cost. In particular, Lateral Flow Assay (LFA), which is a paper-based sensor based on immunochromatography, has been widely used in hospitals and laboratories to detect viruses, bacteria, and some protein markers due to its advantages of high detection speed, simple operation, low cost, and visually recognizable detection results. However, most of conventional LFAs adopt gold nanoparticles as color development signals, but the conventional LFAs are low in sensitivity and weak in anti-interference capability, and if low-level CRP detection is performed in a complex whole blood environment, pretreatment links such as sample separation and enrichment must be performed, so that the detection time and the detection cost are greatly increased.
In order to improve the sensitivity, researchers have conducted a number of studies in which fluorescent particles are used as detection signals instead of colored particles, which can provide a sensitivity increase of 2 to 3 orders of magnitude. However, the conventional fluorescent particles have the problems of photobleaching, photoflickering and the like, and the detection signal is unstable. And most of the fluorescent particles are excited by ultraviolet light or visible light, and under the excitation light condition, the whole blood sample can generate serious background fluorescence interference, so that the accuracy of a detection result is influenced.
Disclosure of Invention
The invention aims to provide a preparation method of a protein C test paper and a handheld reading device, which avoid the influence of background fluorescence generated by a whole blood sample on the detection performance by adopting UCNPs (UCNPs), thereby improving the detection performance.
The invention adopts the following technical scheme: a preparation method of protein C detection test paper comprises the following steps:
attaching an NC film to a substrate of the lateral flow test paper; wherein the average pore diameter of the NC membrane is 5-16 μm;
fixing the prepared detection solution on a detection area on the NC membrane, and fixing the prepared control solution on a control area on the NC membrane; wherein the detection solution is CRP Ab7# antibody solution, and the control solution is goat anti-mouse IgG solution;
respectively attaching a combination pad and a water absorption pad on the substrate and positioned at two sides of the NC membrane, wherein the combination pad and the water absorption pad are connected with the NC membrane, and the combination pad is provided with a probe of UCNPs-CRP antibody;
and a sample pad is attached to the other side of the bonding pad on the substrate, and the sample pad is connected with the bonding pad.
Further, the preparation method of the probe comprises the following steps:
preparing rare earth doped UCNPs by adopting a thermal decomposition method;
performing surface modification on the prepared UCNPs by adopting a polymer wrapping method and using phospholipid polyethylene glycol carboxyl to obtain water-soluble UCNPs;
and (3) combining the water-soluble UCNPs and the CRP antibody by adopting a condensation reaction to prepare the probe.
Further, the detection solution was prepared by diluting the CRP Ab7# antibody with PBS buffer solution.
Further, a control solution was prepared by diluting goat anti-mouse IgG with PBS buffer solution.
Further, the control zone is adjacent to the absorbent pad and the detection zone is adjacent to the conjugate pad.
Further, the average pore diameter of the NC membrane was 8.50 μm.
Further, the detection zone is bar-shaped, circular or square.
The invention discloses another technical square plate: a palm type reading device of protein C test paper is used for detecting the protein C test paper prepared by the method and comprises a sealed shell, wherein an accommodating groove is formed in the sealed shell and is communicated with the outside through an opening of a side plate of the sealed shell;
the top of the sealed shell is provided with an observation window which faces the accommodating groove so as to conveniently observe a detection area and a contrast area of the protein C detection test paper positioned in the accommodating groove;
the sealed shell is provided with an LED lamp group and a power supply, and the irradiation direction of the LED lamp group faces the detection area and the contrast area;
the LED lamp bank is electrically connected with the power supply and is used for irradiating the detection area and the control area so as to enable the detection area or the control area to emit light and detect the protein C detection test paper according to the light.
Further, the LED lamp bank at least comprises two LED lamp beads, and the two LED lamp beads are symmetrically arranged on the two side plates of the sealing shell.
Further, two LED lamp pearls are located the both sides of observation window.
The invention has the beneficial effects that: according to the invention, the probe of the UCNPs-CRP antibody is arranged on the bonding pad, so that the influence of background fluorescence generated by a whole blood sample on the detection performance can be avoided, and the detection zone and the control zone are arranged on the NC membrane, so that the accuracy of the detection result can be ensured.
Drawings
FIG. 1 is a schematic diagram of LFA principle of UCNPs-CRP antibody in the present invention;
FIG. 2 is a SEM image and a capillary flow experiment result for NC membranes with different pore sizes in an example of the present invention;
FIG. 3 is a picture of the fluorescence results of an LFA taken using a palm-type reading device in an embodiment of the present invention;
FIG. 4 is a standard graph of a lateral flow test strip constructed from four NC membranes in accordance with an embodiment of the invention;
fig. 5 is an exploded view of a palm-type reader of the protein C detection test paper according to the embodiment of the present invention.
Wherein: 10. sealing the housing; 20. accommodating grooves; 30. a power source; 40, LED lamp beads; 50. an observation window; 60. detecting test paper; 70. and (4) a photographing device.
Detailed Description
The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.
CRP protein is an important index which is present in blood to reflect the infection of bacteria and viruses and reflect the development degree of diseases; the LFA as a paper-based sensor based on the immunochromatography technology has the advantages of high detection speed, simplicity in operation, low cost and the like, but most of conventional LFA adopts gold nanoparticles as color development signals, so that the sensitivity is low, the anti-interference capability is weak, and low-level CRP detection cannot be performed in a complex whole blood environment; compared with common color developing particles, the Upconversion fluorescent nanoparticles (UCNPs) are novel fluorescent materials with stable fluorescence performance, long fluorescence life and good biocompatibility, can provide 2-3 orders of magnitude of sensitivity improvement, can avoid background fluorescence from biological samples due to the unique light-emitting mechanism of visible fluorescence generated by near-infrared excitation, is expected to solve the problems of photobleaching, photoflickering, unstable detection signals and the like of the common color developing particles, and researches show that the flow rate of a sample on LFA can influence the combination efficiency of target objects, thereby influencing the detection performance, therefore, the invention realizes the controllable adjustment of the flow rate through the adjustment of the aperture of the NC membrane, and furthermore, the detection limit and the detection range are optimized by adjusting the flow rate, so that the developed LFA system can meet the high sensitivity requirement of low-level CRP detection, and the controllable adjustment of the LFA detection performance becomes possible.
The invention constructs a high-sensitivity fluorescence lateral flow test paper platform with adjustable detection performance, which can be used for whole blood detection based on UCNPs, and develops a corresponding handheld portable reading device to realize rapid, sensitive, accurate, portable and low-cost CRP detection.
Specifically, a preparation method of the protein C detection test paper is disclosed, as shown in FIG. 1, and comprises the following steps: attaching an NC film to a substrate of the lateral flow test paper (in the following embodiment, a PVC base plate is specifically selected as the substrate); wherein the average pore diameter of the NC membrane is 5-16 μm; fixing the prepared detection solution on a detection area on the NC membrane, and fixing the prepared control solution on a control area on the NC membrane; wherein the detection solution is CRP Ab7# antibody solution, and the control solution is goat anti-mouse IgG solution; respectively attaching a combination pad and a water absorption pad on the substrate and positioned at two sides of the NC membrane, wherein the combination pad and the water absorption pad are connected with the NC membrane, and the combination pad is provided with a probe of UCNPs-CRP antibody; and a sample pad is attached to the other side of the bonding pad on the substrate, and the sample pad is connected with the bonding pad.
According to the invention, the probe of the UCNPs-CRP antibody is arranged on the bonding pad, so that the influence of background fluorescence generated by a whole blood sample on the detection performance can be avoided, and the detection zone and the control zone are arranged on the NC membrane, so that the accuracy of the detection result can be ensured.
In one embodiment, the probe is prepared by the following method:
preparing rare earth doped UCNPs by adopting a thermal decomposition method to obtain hydrophobic UCNPs, wherein the rare earth doped UCNPs comprise hexagonal prism type beta-NaYF4: Er/Yb. Performing surface modification on the prepared hydrophobic UCNPs by adopting a polymer wrapping method and using phospholipid polyethylene glycol carboxyl (DSPE-PEG-COOH) to obtain water-soluble UCNPs; and (3) combining the water-soluble UCNPs and the CRP antibody by adopting a condensation reaction to prepare the probe. After the probe is prepared, an XYZ three-dimensional gold spraying and membrane scratching instrument is adopted, the probe is uniformly sprayed on the bonding pad (for example, 150 mu L of the probe is uniformly sprayed on the bonding pad by using a pipetting gun), and the probe is dried for later use.
As a specific embodiment, 209mg of Y (CF) was added3COO)3·6H2O、62mg Yb(CF3COO)3·6H2O、6mg Er(CF3COO)3·6H2O，136mg NaCF3COO, 10mL Oleic Acid (OA) and 10mL Octadecane (ODE) are added into a 100mL three-neck flask, the mixture is stirred for 30min at room temperature, argon is introduced, the mixture is heated to 140 ℃, low-boiling-point impurities are removed after 10min, then the temperature is raised to 320 ℃, the reaction is carried out for 1-2 h under heat preservation, finally, excessive absolute ethyl alcohol is added, the mixture is repeatedly cleaned, the OA and the ODE are washed away, and rare earth doped UCNPs are prepared4：Er/Yb。
As another specific implementation, the UCNPs surface modification specifically operates as follows: weighing 20mg of DSPE-PEG-COOH, dissolving in 3mL of chloroform, adding 2.5mL of prepared UCNPs, ultrasonically mixing for 5min, placing in a magnetic stirrer, stirring for 2h, then blowing dry the chloroform with nitrogen, adding ultrapure water for dissolving, and removing redundant DSPE-PEGCOOH through centrifugation to prepare the UCNPs with hydrophilicity and good biocompatibility.
In addition, the specific operation steps for preparing the UCNPs-CRP antibody probe are as follows: 2- (N-morpholino) ethanesulfonic acid (MES) and dimethyl sulfoxide (DMSO) MES buffer solution are selected to wash UCNPs, and the water phase on the surface is changed into an MES phase, so that the antibody coupling environment is improved; adding 5 mu L of EDC solution and sulfo-NHS solution into the obtained suspension to activate hydroxyl groups on the surface of UCNPs, and improving the success rate of antibody coupling; the antibody concentration is controlled, the density of the UCNPs surface coupling antibody is further controlled, the detection accuracy is improved, and the detection sensitivity of the lateral flow test paper is further improved. Adding 3.14 mu L of CRP Ab8# antibody into 480 mu L of MES and 120 mu L of DMSO MES buffer solution, placing the mixture in a constant temperature shaking table (37 ℃, 220rpm) for reaction for 2h, then adding 18 mu L of coating stop solution, placing the mixture in a constant temperature shaking table (37 ℃) for reaction for 30min, finally centrifuging to remove supernatant, and adding 600 mu L of probe diluent to obtain the probe of the UCNPs-CRP antibody.
In the present example, the detection solution (i.e., the detection line solution, also referred to as the T-line solution) was prepared by diluting CRP Ab7# antibody to 1mg/mL with PBS buffer solution. Control solutions (i.e., control line solutions, also referred to as C line solutions) were prepared by diluting goat anti-mouse IgG to 0.5mg/mL in PBS buffer.
Similarly, the prepared T, C line solution is fixed on the NC membrane surface of the lateral flow test paper by adopting a gold spraying and membrane scratching instrument to form a detection line and a contrast line, and the detection line and the contrast line are dried for standby. Specifically, the detection line and the control line may have different expression forms, such as a bar, a circle, a square, or the like.
In this embodiment, the control zone is adjacent to the absorbent pad and the detection zone is adjacent to the conjugate pad.
In one embodiment, after the lateral flow strip described above is prepared, a lateral flow strip having a width of 3.9mm is cut and mounted in a housing for use.
In addition, in the present invention, the flow rate of the sample solution to be tested on the lateral flow test strip is one of the important factors affecting the test result. The flow rate is too high, the sample solution to be detected and the detection line can not react sufficiently, and the detection sensitivity is reduced; the flow rate is too slow, the detection efficiency is reduced under the same condition of meeting the detection sensitivity, and the false positive phenomenon is easy to generate. Thus, the NC membrane was characterized for pore size, capillary flow and porosity before the experiment (as shown in fig. 2), and the results showed that the smaller the pore size, the smaller the porosity and the slower the flow rate of the solution to be detected. Preferably, for protein C, the detection effect is best when the average pore diameter of the NC membrane is 8.50 μm.
In the embodiment of the invention, CRP antigen solution detection systems with different known concentrations are also prepared, the prepared lateral flow test paper is used for detection, the lateral flow test paper is placed in a matched developed handheld reading device after reacting for 15min, a camera of a smart phone is used for reading and shooting a generated fluorescence result (as shown in figure 3), and a standard curve (as shown in figure 5) is analyzed and drawn.
In this embodiment, the specific operations of analyzing and plotting the standard curve are: CRP antigen solutions with different concentrations of 0.5, 5, 10, 50, 75, 100, 150 and 200mg/L are respectively dripped on the prepared lateral flow test paper, the lateral flow test paper is placed in a matched developed handheld reading device for internal reaction for 15min, a fluorescence intensity curve and the peak area of an T, C line are obtained, 3 times of parallel tests are set for each concentration, and the standard deviation of each group of data is calculated. And drawing a standard curve with CRP concentration as an abscissa and T/C value as an ordinate.
Under the optimal test condition, testing by using a CRP antigen solution, and establishing a standard curve; detecting CRP antigen solution with concentration of 50mg/L, repeatedly measuring for 10 times, calculating variation coefficient, and performing repeated analysis; detecting a blank sample without CRP, repeatedly measuring for 10 times, and calculating the lowest detection limit by using a statistical analysis method; selecting lateral flow test paper with the best comprehensive performance, detecting clinical samples with known concentration, repeatedly measuring for 3 times, substituting the obtained T/C value into a standard curve equation, calculating the detection concentration, and calculating the relative deviation of the detection concentration and the real concentration.
When the target CRP antigen exists, the UCNPs-CRP antibody probe on the detection line forms an immune complex with the target CRP antigen, the immune complex is captured by the capture probe on the detection line when flowing to the NC membrane, the fluorescence signal appears on the detection line, the excessive nano-probe is captured by the control probe on the control line, and the fluorescence signal appears on the control line. When the more CRP antigen content in the solution, the more upconverting fluorescent particle surface-modified antibody will be bound, the more immunocomplexes can be captured by the capture probe, resulting in a stronger fluorescence signal from the detection line.
When the target CRP antigen is not present, the UCNPs-CRP antibody probe on the detection line can not form immune complex, so that no fluorescence signal appears on the detection line.
The invention discloses a high-sensitivity lateral flow test paper platform and a handheld portable reading device, which have adjustable detection performance, can be used for whole blood detection and are based on up-conversion fluorescence. In view of the unique advantages of UCNPs in biological detection, the detection line is formed by modifying CRP antibodies capable of being specifically combined with CRP antigens of targets on the surfaces of the UCNPs and fixing the CRP antibodies on the NC membrane surface of the lateral flow test paper in a rectangular strip shape in combination with the characteristics of quickness, convenience, simple operation, low cost, allowance of inexperienced workers for operation analysis and the like; meanwhile, the aperture of the side flow test paper NC membrane is regulated, the detection limit and the detection range are optimized, and the detection sensitivity is improved; finally, a quick and portable handheld reading device for up-conversion of fluorescence lateral flow test paper results is creatively developed based on the LED lamp beads.
The controllable adjustment of the flow rate is realized through the aperture adjustment of a Nitrocellulose membrane (NC membrane), and the detection limit and the detection range are further optimized through the adjustment of the flow rate, so that the developed LFA system can meet the high sensitivity requirement of low-level CRP detection. Meanwhile, the method influences the detection limit and the detection range through the aperture regulation of the NC membrane, and the controllable regulation of the LFA detection performance becomes possible.
Example 1:
taking the detection of CRP protein by NC membrane with the average pore diameter of 15.52 μm as an example, the effect of the method of the invention is illustrated, and the specific experimental process comprises the following steps:
1) preparing rare earth doped UCNPs by adopting a thermal decomposition method;
2) performing surface modification on the hydrophobic UCNPs prepared in the step 1) by using phospholipid polyethylene glycol carboxyl (DSPE-PEG-COOH) by adopting a polymer wrapping method;
3) combining the water-soluble UCNPs subjected to surface modification in the step 2) with the CRP antibody by adopting a condensation reaction to prepare a probe of the UCNPs-CRP antibody;
4) uniformly spraying the probes of the UCNPs-CRP antibody prepared in the step 3) on a bonding pad by adopting an XYZ three-dimensional gold spraying and membrane scratching instrument, and drying for later use;
5) diluting CRP Ab7# antibody and goat anti-mouse IgG with PBS buffer solution respectively to obtain detection line (Test line, T line) solution and Control line (C line) solution;
6) the NC membrane (average pore diameter: 15.52 μm, mean capillary flow time: 79.95s, porosity: 66.16%) is adhered on a PVC bottom plate, T, C thread solution prepared in the step 5) is fixed on the surface of an NC membrane of lateral flow test paper in a rectangular strip shape by using a metal spraying and membrane scribing instrument to form a detection line, and the detection line is dried for standby;
7) respectively installing the combined pad prepared in the step 4), the NC membrane prepared in the step 6), the sample pad and the water absorption pad on a PVC (polyvinyl chloride) bottom plate, cutting a lateral flow test strip with the width of 3.9mm, and encasing for later use;
8) preparing CRP antigen solution detection systems with different known concentrations, detecting by using the lateral flow test paper prepared in the step 7), placing the CRP antigen solution detection systems into a matched developed handheld reading device for reaction for 15min, reading and shooting a generated fluorescence result by using a camera of a smart phone, and analyzing and drawing a standard curve.
9) The detection performance of the NC membrane is analyzed, and a standard curve y of the relation between the T/C value and the concentration is established, wherein the standard curve y is 0.02153x +0.02227(R20.99994) (as shown in fig. 4); selecting 50mg/L CRP antigen solution for detection, repeating for 10 times, and calculating the coefficient of variation to be 10.4%; the detection was repeated 10 times with blank solution without sample, and the lowest detection limit was calculated to determine the lowest detection limit to be 0.31mg/L, corresponding to a linear range of 0.31-75mg/L (as shown in Table 1).
TABLE 1 lateral flow test paper detection performance of four NC membrane constructions
Example 2:
taking the detection of CRP protein by NC membrane with the average pore diameter of 8.50 μm as an example, the effect of the method of the invention is illustrated, and the specific experimental process comprises the following steps:
reference example 1 steps 1), 2), 3), 4), 5);
6) the NC membrane (average pore diameter: 8.50 μm, mean capillary flow time: 94.98s, porosity: 53.46%) of the test solution is adhered to a PVC bottom plate, T, C line solution prepared in the step 5) is fixed on the surface of an NC membrane of lateral flow test paper in a rectangular strip shape by using a metal spraying and membrane cutting instrument to form a detection line, and the detection line is dried for standby;
7) respectively installing the combined pad prepared in the step 4), the NC membrane prepared in the step 6), the sample pad and the water absorption pad on a PVC (polyvinyl chloride) bottom plate, cutting a lateral flow test strip with the width of 3.9mm, and encasing for later use;
8) preparing CRP antigen solution detection systems with different known concentrations, detecting by using the lateral flow test paper prepared in the step 7), placing the CRP antigen solution detection systems into a matched developed handheld reading device for reaction for 15min, reading and shooting a generated fluorescence result by using a camera of a smart phone, and analyzing and drawing a standard curve.
9) The detection performance of the NC membrane is analyzed, and a standard curve y of the relation between the T/C value and the concentration is established, wherein the standard curve y is 0.0536x +0.02008(R20.99628) (as shown in fig. 4); selecting 50mg/L CRP antigen solution for detection, repeating for 10 times, and calculating the coefficient of variation to be 10.1%; detecting with blank solution without sample, repeating for 10 times, calculating lowest detection limit to determine the lowest detection limit to be 0.26mg/L, and corresponding linear range to be 0.26-150mg/L (as shown in Table 1);
10) the CRP clinical serum with known concentration obtained from Shanghai thoracic hospital is used, UCNPs lateral flow test paper made of Sartorius CN95 NC membrane and a reading device are used for detection, 10 clinical sera with different concentrations are tested for 3 times, the obtained T/C mean value is substituted into a standard curve equation y of 0.0536x +0.02008, the detection concentration is calculated, the relative deviation between the detection concentration and the clinical sample concentration is calculated, the experimental result (shown in Table 2) shows that the 10 clinical sample concentrations and the relative deviation between the detection concentration are within +/-15%, the test result accords with the registered standard of medical POCT equipment, and the lateral flow test paper made of the Sartorius CN95 NC membrane has good clinical relevance.
TABLE 2 clinical specimen test results
Example 3:
taking the detection of CRP protein by NC membrane with the average pore diameter of 6.49 μm as an example, the effect of the method of the invention is illustrated, and the specific experimental process comprises the following steps:
reference example 1 steps 1), 2), 3), 4), 5);
6) the NC membrane (average pore diameter: 6.49 μm, mean capillary flow time: 138.97s, porosity: 51.87%) on a PVC bottom plate, fixing the T, C thread solution prepared in the step 5) on the surface of an NC membrane of lateral flow test paper in a rectangular strip shape by using a metal spraying and membrane cutting instrument to form a detection line, and drying for later use;
7) respectively installing the combined pad prepared in the step 4), the NC membrane prepared in the step 6), the sample pad and the water absorption pad on a PVC (polyvinyl chloride) bottom plate, cutting a lateral flow test strip with the width of 3.9mm, and encasing for later use;
8) preparing CRP antigen solution detection systems with different known concentrations, detecting by using the lateral flow test paper prepared in the step 7), placing the CRP antigen solution detection systems into a matched developed handheld reading device for reaction for 15min, reading and shooting a generated fluorescence result by using a camera of a smart phone, and analyzing and drawing a standard curve.
9) The detection performance of the NC membrane is analyzed, and a standard curve y of the relation between the T/C value and the concentration is established, wherein the standard curve y is 0.40052x-0.39218(R20.99853) (as shown in fig. 4); selecting 50mg/L CRP antigen solution for detection, repeating for 10 times, and calculating the coefficient of variation to be 15.2%; the detection was repeated 10 times with blank solution without sample, and the lowest detection limit was calculated to determine the lowest detection limit to be 1.31mg/L, corresponding to a linear range of 1.31-100mg/L (as shown in Table 1).
Example 4:
taking the detection of CRP protein by NC membrane with the average pore diameter of 5.14 μm as an example, the effect of the method of the invention is illustrated, and the specific experimental process comprises the following steps:
reference example 1 steps 1), 2), 3), 4), 5);
6) the NC membrane (average pore diameter: 5.14 μm, mean capillary flow time: 172.58s, porosity: 50.36%) is adhered on a PVC bottom plate, T, C thread solution prepared in the step 5) is fixed on the surface of an NC membrane of lateral flow test paper in a rectangular strip shape by using a metal spraying and membrane cutting instrument to form a detection line, and the detection line is dried for standby;
7) respectively installing the combined pad prepared in the step 4), the NC membrane prepared in the step 6), the sample pad and the water absorption pad on a PVC (polyvinyl chloride) bottom plate, cutting a lateral flow test strip with the width of 3.9mm, and encasing for later use;
8) preparing CRP antigen solution detection systems with different known concentrations, detecting by using the lateral flow test paper prepared in the step 7), placing the CRP antigen solution detection systems into a matched developed handheld reading device for reaction for 15min, reading and shooting a generated fluorescence result by using a camera of a smart phone, and analyzing and drawing a standard curve.
9) The detection performance of the NC membrane is analyzed, and a standard curve y of the relation between the T/C value and the concentration is established, wherein the standard curve y is 0.14259x-0.12533(R20.99887) (as shown in fig. 4); selecting 50mg/L CRP antigen solution for detection, repeating for 10 times, and calculating the coefficient of variation to be 14.2%; the detection was repeated 10 times with blank solution without sample, and the lowest detection limit was calculated to determine the lowest detection limit to be 2.20mg/L, corresponding to a linear range of 2.20-75mg/L (as shown in Table 1).
The invention also discloses a palm type reading device of the protein C test paper, as shown in figure 5, the protein C test paper for detecting the protein C test paper prepared by the method comprises a sealing shell 10, and the sealing shell 10 can be formed by buckling an upper part and a lower part. The sealed shell 10 is internally provided with an accommodating groove 20, and the accommodating groove 20 is communicated with the outside through an opening of a side plate of the sealed shell 10 and is used for inserting the test paper 60; an observation window 50 is arranged at the top of the sealed shell 10, and the observation window 50 faces the accommodating groove 20 so as to facilitate observation of the detection area and the contrast area of the protein C detection test paper positioned in the accommodating groove 20; the sealed shell 10 is provided with an LED lamp group and a power supply 30, and the irradiation direction of the LED lamp group faces to the detection area and the contrast area; the LED lamp set is electrically connected to the power supply 30 and is used to irradiate the detection zone and the control zone to make the detection zone or the control zone emit light, and to perform detection of the protein C test paper according to the light.
In this embodiment, the LED lamp set at least includes two LED lamp beads 40, and the two LED lamp beads 40 are symmetrically installed on two side plates of the sealed housing 10. Preferably, two LED lamp beads 40 are located on both sides of the observation window 50. In addition, a photographing device 70 may be further included, and the detection accuracy may be improved by the blister device 70.
The handheld reading device is developed by matching with a protein C detection test paper, in the embodiment, a sealing shell 10 of the handheld reading device is made of plastic, printing is performed by using a laser additive manufacturing technology, the total size is 110 x 40 x 30mm, a rectangular test paper clamping groove (20 x 5mm) is positioned below the left side of the shell and plays a role in fixing, 2 LED lamp beads (980nm and 10W) are fixed on the inner side of the upper plane of the shell and are provided with 60-degree lenses and are placed in a 45-degree inclined mode, a round hole type reading window with an infrared optical filter (phi 3x 1.1mm) is arranged above a light source intersection area of the lamp beads, the optical filter is embedded in the upper plane of the shell and plays a role in filtering near infrared light emitted by the LED lamp beads, a rechargeable power supply device is arranged inside the right side of the shell, and power supply is connected with the lamp beads through a circuit. The specific operation is as follows: open switch, it is fixed to wait to examine test paper and put into the test paper draw-in groove, and LED lamp pearl arouses the test paper and produces visible fluorescence, places smart mobile phone in the device top, and it is fixed to aim at reading the window with the camera, opens the camera and shoots fluorescence testing result (or also can direct naked eye reading result, but the accuracy can be relatively poor).
The design of the light source of the device creatively uses the LED lamp beads as an excitation light source, the purpose of exciting UCNPs is achieved, due to the difficulty of experimental operation, a mode of calculating simulation and controlling a variable method is adopted, and the excitation result is optimal by comprehensively calculating the power of the LED lamp beads, the lens degree, the installation quantity, the installation angle and the distance between the LED lamp beads and test paper, the comprehensive cost, calculating the simulation result and other factors, and finally 2 LED lamp beads with 10w power, 60-degree lens degree, the distance of 17mm between the LED lamp beads and the test paper and the inclination angle of 45 degrees relative installation angle.
In the invention, in view of the unique advantages of UCNPs in biological detection, and in combination with the characteristics of rapidness, convenience, simple operation, low cost, allowance of inexperienced workers for operation and analysis and the like of LFA, an up-conversion fluorescence lateral flow test strip is developed and used for CRP detection with high sensitivity, high speed and low cost. Furthermore, the palm type reading device is creatively developed based on the LED lamp beads and used for quickly and portably reading the result of the up-conversion fluorescence lateral flow test paper, the manufacturing cost and the equipment volume of the reading device are greatly reduced, the device cost is only 380 yuan, the cost of the detection test paper is less than 10 yuan, the palm type quick detection of CRP can be realized, meanwhile, the size is smaller, the structure is more simplified, and the palm type reading device is portable and low in cost. By using the handheld reading device to obtain fluorescence values with different intensities, CRP protein detection with rapidness, sensitivity, accuracy, convenience and low cost can be realized by contrasting a standard curve graph.
The device is used for the CRP detection with high sensitivity, high speed and low cost in a whole blood sample. And the controllable adjustment of the LFA performance is realized through the regulation and control of the NC membrane, so that a new idea is provided for the future LFA performance optimization.
In conclusion, based on the unique light-emitting mechanism that UCNPs generate visible fluorescence by near-infrared excitation, the interference of background fluorescence from a biological sample can be avoided, so that the biosensor has the anti-interference capability in a clinical blood sample, the signal-to-noise ratio is obviously improved, the detection sensitivity is high, and the anti-interference capability is strong; the controllable adjustment of the flow rate is realized by regulating and controlling the pore diameter of the NC membrane, and the detection limit and the detection range are further optimized by regulating the flow rate, so that the developed LFA system can meet the high sensitivity requirement of low-level CRP detection, and the controllable adjustment of the LFA detection performance becomes possible.
The method has the advantages of high sensitivity, strong specificity, simple operation, low cost and low requirement on equipment, the detection result can be obtained only by photographing through a camera of a smart phone, meanwhile, the detection of various targets can be realized by changing the types of probes coupled with UCNPs on a detection line, the controllable adjustment of the LFA detection performance can be realized by regulating and controlling the aperture of an NC membrane, the method has obvious effect and significance in the rapid and instant detection of various targets in multiple application scenes, and the application range is very wide.
The invention takes UCNPs as fluorescence signals, has stable fluorescence performance and good biocompatibility, can shield biological background fluorescence interference, improves the signal-to-noise ratio, constructs LFA of immunochromatography, has the advantages of high detection speed, simple operation, low cost and the like, realizes the controllable adjustment of the flow rate of a sample by adjusting the aperture of an NC membrane, further optimizes the detection limit and the detection range by adjusting the flow rate, realizes the low-level CRP detection with high sensitivity and controllable adjustment of the detection performance in a complex whole blood environment, and independently develops a palm type reading device with low cost, small volume and simple operation The CRP detection kit is portable to read and realizes the rapid detection of CRP.
In summary, the detection performance-adjustable preparation method for the test paper for detecting the CRP protein in the whole blood and the handheld reading device have the advantages of stable performance, convenience in operation, low price and the like, can be used for high-sensitivity and rapid low-level CRP detection in a whole blood sample, can effectively improve the efficiency of disease diagnosis, reduce the economic pressure of a patient and realize daily 'home self-test'; the controllable adjustment of the LFA detection performance is realized through the regulation and control of the NC membrane, a new thought is provided for the future LFA performance optimization, a solution thought is also provided for food safety, environmental monitoring and more disease diagnosis problems, the rapid and instant LFA detection kit has obvious effects and significance in the field of rapid and instant detection, and the application range is very wide.
Claims (10)
1. The preparation method of the protein C detection test paper is characterized by comprising the following steps of:
attaching an NC film to a substrate of the lateral flow test paper; wherein the average pore diameter of the NC membrane is 5-16 μm;
fixing the prepared detection solution on the detection area on the NC membrane, and fixing the prepared control solution on the control area on the NC membrane; wherein the detection solution is a CRP Ab7# antibody solution, and the control solution is a goat anti-mouse IgG solution;
respectively attaching a combination pad and a water absorption pad to the two sides of the NC membrane on the substrate, wherein the combination pad and the water absorption pad are connected with the NC membrane, and the combination pad is provided with a probe of UCNPs-CRP antibody;
and a sample pad is attached to the other side of the combination pad on the substrate, and the sample pad is connected with the combination pad.
2. The method for preparing a protein C test strip according to claim 1, wherein the probe is prepared by:
preparing rare earth doped UCNPs by adopting a thermal decomposition method;
performing surface modification on the prepared UCNPs by adopting a polymer wrapping method and using phospholipid polyethylene glycol carboxyl to obtain water-soluble UCNPs;
and (3) combining the water-soluble UCNPs with the CRP antibody by adopting a condensation reaction to prepare the probe.
3. The method of preparing a protein C test strip according to claim 1 or 2, wherein the test solution is prepared by diluting CRP Ab7# antibody with PBS buffer solution.
4. The method of claim 3, wherein the control solution is prepared by diluting goat anti-mouse IgG with PBS buffer.
5. The method of claim 4, wherein the control area is adjacent to the absorbent pad and the detection area is adjacent to the conjugate pad.
6. The method of producing a protein C test strip according to claim 1, 4 or 5, wherein the average pore size of said NC membrane is 8.50 μm.
7. The method of claim 6, wherein the detection area is in the form of a strip, a circle, or a square.
8. A palm type reading device for protein C test paper, which is used for detecting protein C test paper prepared by the method of any one of claims 1 to 7, and comprises a sealed shell (10), wherein a containing groove (20) is formed in the sealed shell (10), and the containing groove (20) is communicated with the outside through an opening of a side plate of the sealed shell (10);
an observation window (50) is arranged at the top of the sealing shell (10), and the observation window (50) faces the accommodating groove (20) so as to facilitate observation of the detection area and the control area of the protein C detection test paper positioned in the accommodating groove (20);
the sealed shell (10) is provided with an LED lamp bank and a power supply (30), and the irradiation direction of the LED lamp bank faces to the detection area and the control area;
the LED lamp group is electrically connected with the power supply (30) and is used for irradiating the detection area and the control area so as to enable the detection area or the control area to emit light, and the detection of the protein C detection test paper is carried out according to the light.
9. The palm-type reading device for the protein C detection test paper as claimed in claim 8, wherein the LED lamp set comprises at least two LED lamp beads (40), and the two LED lamp beads (40) are symmetrically installed on two side plates of the sealed shell (10).
10. The palm-type reading device of protein C detection test paper of claim 8, characterized in that two of said LED lamp beads (40) are located on both sides of said observation window (50).
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