A method for improving the detection sensitivity of fluorescent microsphere biological probes

Abstract

The application discloses a method for improving the detection sensitivity of fluorescent microsphere biological probes, and uses a lateral flow test paper with different fluorescent microsphere biological probes to detect a liquid to be detected without a marker, quantitatively analyzes the fluorescence intensity at the control line of the lateral flow test paper, determines the saturated probe density of the fluorescent microsphere biological probes according to the quantitative analysis result, mixes blank probes and antibodies with different molar ratios to form mixed probes, prepares a plurality of mixed probe solutions, mixes the plurality of mixed probe solutions with a fluorescent microsphere solution after activation treatment, detects the detection limit of the plurality of fluorescent microsphere biological probes by using an experimental method, and selects the fluorescent microsphere biological probes corresponding to the minimum detection limit as the fluorescent microsphere biological probes of the liquid to be detected.

Description

Technical Field

[0001] This invention belongs to the field of point-of-care testing, and particularly relates to a method for regulating the detection sensitivity of fluorescent microsphere biological probes. Background Technology

[0002] In recent years, advancements in diagnostic and auxiliary technologies, along with improvements in disease understanding and treatment, have led to a surge in popularity for point-of-care testing (POCT) technologies. These technologies feature miniaturized instruments, simplified operation, and immediate result reporting. Optical biosensors, one such POCT technology, have played a crucial role in the transition from qualitative to quantitative medicine and have found widespread application in disease diagnosis and biological detection. Furthermore, more portable fluorescent biosensors are creating new opportunities for the development of rapid diagnostics.

[0003] Most existing fluorescent biosensors use traditional fluorescent particles to construct biological probes, but such probes have problems such as unstable fluorescence performance, which limits the improvement of detection sensitivity. However, in the early stages of many diseases, patients only have low concentrations of disease biomarkers in their bodies. In order to better meet the clinical diagnostic needs for higher detection sensitivity in different application scenarios and broaden the detection range, further optimization of fluorescent biological probes is still needed.

[0004] Europium-chelated polystyrene microspheres (EuPSMs) exhibit a longer fluorescence lifetime compared to traditional fluorescent particles and allow signal collection beyond the background fluorescence lifetime. In particular, the material's long Stokes shift ensures that the light collected by the detector is unaffected by incident light, thus giving EuPSMs superior fluorescence properties and stability, demonstrating great potential for constructing biological probes. However, while the larger particle size (250–300 nm) of EuPSMs significantly increases the number of binding sites on the microsphere surface, allowing for more biological probes to conjugate on the surface and improve target binding rates, it also reduces the conversion rate of biological signals to fluorescence signals, thereby affecting the improvement of detection sensitivity. Summary of the Invention

[0005] The purpose of this invention is to provide a method for regulating the detection sensitivity of fluorescent microsphere biological probes by introducing blank probes to eliminate non-specific binding and thus improve detection sensitivity.

[0006] This invention employs the following technical solution: a method for regulating the detection sensitivity of fluorescent microsphere biological probes, comprising the following steps:

[0007] The sideflow test strips with different fluorescent microsphere bioprobes were used to detect the test liquid without the marker. The fluorescence intensity at the control line of the sideflow test strip was quantitatively analyzed, and the saturated probe density of the fluorescent microsphere bioprobes was determined based on the quantitative analysis results. The probe density of different fluorescent microsphere bioprobes is different.

[0008] Blank probes and antibodies in different molar ratios are mixed to form mixed probes, and various mixed probe solutions are prepared.

[0009] Multiple mixed probe solutions were mixed with activated fluorescent microsphere solutions to obtain multiple fluorescent microsphere biological probes; wherein the mixed probe density in the fluorescent microsphere biological probes was equal to the saturated probe density, and the antibody probe density in the fluorescent microsphere biological probes was less than or equal to the mixed probe density.

[0010] The detection limits of various fluorescent microsphere biological probes were determined experimentally.

[0011] Based on the required detection sensitivity, select the fluorescent microsphere biological probe corresponding to the detection limit as the fluorescent microsphere biological probe for the liquid to be tested.

[0012] Furthermore, the sideflow test strips with different fluorescent microsphere bioprobes are used to detect the marker-free test liquid, and the quantitative analysis of the fluorescence intensity at the control line of the sideflow test strip specifically includes:

[0013] Adding the same volume of antibody solution but different concentrations to a predetermined volume of activated fluorescent microsphere solution yields a variety of fluorescent microsphere bioprobe solutions.

[0014] Sideflow test strips were prepared using each fluorescent microsphere bioprobe solution;

[0015] Different side-flow test strips were used to detect the test liquid without the marker, and the fluorescence intensity of the control line of different side-flow test strips was obtained;

[0016] The fluorescence intensity of different control lines on the sideflow test paper was quantitatively analyzed to obtain the saturation probe density of the fluorescent microsphere biological probe.

[0017] Furthermore, quantitative analysis of the fluorescence intensity of different control lines on the sideflow test strips was performed, including:

[0018] Select the corresponding antibody solution concentration based on the fluorescence intensity of the control line on each type of lateral flow test strip;

[0019] The saturation probe density of the fluorescent microsphere biological probe is determined based on the antibody solution concentration.

[0020] Furthermore, the experimental method is as follows:

[0021] Sideflow test strips were prepared using each type of fluorescent microsphere biological probe;

[0022] Prepare test liquids containing different concentrations of markers;

[0023] Side-flow test strips were used to test various liquids of different concentrations.

[0024] The detection limit for each fluorescent microsphere bioprobe was determined based on the fluorescence intensity of the detection line on the side-flow test strip.

[0025] Furthermore, the blank probe was BSA, and the antibody was anti-CRP Ab8#.

[0026] Furthermore, the preparation of lateral flow test strips using each type of fluorescent microsphere biological probe includes:

[0027] The anti-CRP-AB7# solution was fixed onto the surface of the NC membrane of the side-flow test paper to form a detection line;

[0028] A control line was formed by immobilizing goat anti-mouse IgG solution on the surface of the NC membrane of the lateral flow test paper.

[0029] The blank probe is BSA, and the antibody is anti-CRP Ab8#.

[0030] Furthermore, the preparation of various test liquids containing different concentrations of the marker includes:

[0031] The concentration gradients of the test liquids of various different concentrations are 0.01, 0.025, 0.05, 0.25, 0.5, 2.5, 5, 25, 75, 125, 150, 250, 350 and 500, with the concentration unit being ng / ml.

[0032] Furthermore, antibody solutions of the same volume but different concentrations were added to a predetermined volume of activated fluorescent microsphere solution to obtain various fluorescent microsphere bioprobe solutions. The antibody probe densities on the fluorescent microsphere bioprobes in the various fluorescent microsphere bioprobe solutions were 100%, 90%, 70%, 60%, 50%, 30%, and 10%, respectively.

[0033] The beneficial effects of this invention are as follows: Based on the construction of biological probes using EuPSM, this invention improves sensitivity by adjusting the density of probes on the EuPSM surface. Moreover, to avoid the negative impact of non-specific binding at blank sites on the particle surface and to ensure the uniform distribution of biological probes, the method of introducing blank probes achieves uniform and controllable probe density adjustment. At the same time, it fills blank sites, eliminates interference, makes the detection results more accurate, and improves detection sensitivity. Attached Figure Description

[0034] Figure 1This is a schematic diagram illustrating the optimization of probe density on the surface of fluorescent microspheres in an embodiment of the present invention;

[0035] Figure 2 A schematic diagram illustrating the characteristics of EuPSM fluorescent microspheres;

[0036] Figure 3 This is a graph showing the estimated experimental results of probe saturation concentration on EuPSM in an embodiment of the present invention;

[0037] Figure 4 Figure 1 shows the results of detecting standard concentration gradient CRP systems with EuPSM probes of different densities in this embodiment of the invention.

[0038] Figure 5 A schematic diagram illustrating the detection performance at different probe densities;

[0039] Figure 6 This is a standard curve diagram for CRP detection in clinical serum samples in an embodiment of the present invention. Detailed Implementation

[0040] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0041] The main challenge in improving sensitivity is amplifying the response of the fluorescence signal to the biological probe. Optimizing sensitivity by adjusting the probe density on the EuPSM surface and amplifying the fluorescence signal's response to the biological probe to achieve optimal detection performance is crucial for guiding the design of EuPSM probes to meet different application scenarios. This will also better meet the sensitivity requirements of ultrasensitive POCT platforms. Constructing an ultrasensitive probe capable of detecting low-concentration targets holds great potential and advantages in fields such as disease diagnosis, environmental monitoring, and food safety testing.

[0042] This invention discloses a method for regulating the detection sensitivity of fluorescent microsphere bioprobes, comprising the following steps: using lateral flow test strips with different fluorescent microsphere bioprobes to detect a marker-free liquid; quantitatively analyzing the fluorescence intensity at the control line of the lateral flow test strip; and determining the saturated probe density of the fluorescent microsphere bioprobes based on the quantitative analysis results; wherein the probe densities of different fluorescent microsphere bioprobes are different; mixing blank probes and antibodies in different molar ratios to form mixed probes, and preparing multiple mixed probe solutions; mixing the multiple mixed probe solutions with activated fluorescent microsphere solutions to obtain multiple fluorescent microsphere bioprobes; wherein the mixed probe density in the fluorescent microsphere bioprobes is equal to the saturated probe density, and the antibody probe density in the fluorescent microsphere bioprobes is less than or equal to the mixed probe density; experimentally detecting the detection limits of the multiple fluorescent microsphere bioprobes; and selecting the fluorescent microsphere bioprobe corresponding to the detection limit as the fluorescent microsphere bioprobe for the liquid to be detected according to the detection sensitivity requirements.

[0043] This invention improves sensitivity by controlling the density of probes on the EuPSM surface, based on the construction of biological probes using EuPSM. Furthermore, to avoid the negative impact of non-specific binding at blank sites on the particle surface and to ensure the uniform distribution of biological probes, a method of introducing blank probes is used to achieve uniform and controllable probe density adjustment. At the same time, blank sites are filled, interference is eliminated, and the detection results are more accurate, thus improving detection sensitivity.

[0044] In this embodiment of the invention, in order to obtain EuPSM with consistent morphology, controllable and stable size, the reaction time and other conditions are controlled to ensure that all EuPSM have a large particle size of 300 nm, i.e., europium chelated polystyrene microspheres (EuPSM) are prepared by suspension polymerization. Figure 2 The diagram shown illustrates the characteristics of EuPSM fluorescent microspheres. Due to their excellent fluorescence properties and stability, EuPSM shows great potential for constructing biological probes. Furthermore, the large particle size of 300 nm significantly increases the number of binding sites on the microsphere surface, allowing more biological probes to conjugate on the surface to improve the binding rate to the target, but it reduces the conversion rate from biological signal to fluorescence signal. Therefore, this invention utilizes a large particle size of 300 nm.

[0045] First, Eu(DBM)3phen was dissolved in CH2Cl2 and mixed with carboxylic acid PSM. The mixture was sonicated for 15 min to form a uniform suspension. The suspension was stirred at room temperature for 24 h, and then heated in a 50 °C water bath for 12 h to completely evaporate the organic solvent CH2Cl2. Finally, after washing with ethanol, it was dispersed in 1% w / v water to obtain EuPSM.

[0046] Next, the prepared europium chelated polystyrene microspheres (EuPSM) were washed and phase-replaced using MES buffer; then EDC and sulfonic acid NHS were added to the washed and phase-replaced EuPSM, and the mixture was stirred in a shaker at 37°C and 220 rpm for 45 min. After washing with MES buffer, the activation process was completed.

[0047] After the activated fluorescent microsphere bioprobe (EuPSM) is prepared, the above method can be implemented using the activated fluorescent microsphere bioprobe.

[0048] As a specific implementation, an equal volume of antibody solution with different concentrations is added to a predetermined volume of activated fluorescent microsphere solution to obtain multiple fluorescent microsphere bioprobe solutions. Further, an equal volume of antibody solution with different concentrations is added to a predetermined volume of activated fluorescent microsphere solution to obtain multiple fluorescent microsphere bioprobe solutions. The antibody probe densities on the fluorescent microsphere bioprobes in these multiple solutions are 100%, 90%, 70%, 60%, 50%, 30%, and 10%, respectively.

[0049] Sideflow test strips were prepared using each fluorescent microsphere bioprobe solution; different sideflow test strips were used to detect the test liquid without the marker, and the fluorescence intensity of the control lines of the different sideflow test strips was obtained; the fluorescence intensity of the control lines of the different sideflow test strips was quantitatively analyzed to obtain the saturated probe density of the fluorescent microsphere bioprobe.

[0050] More specifically, quantitative analysis of the fluorescence intensity of control lines on different lateral flow test strips includes: selecting the corresponding antibody solution concentration based on the fluorescence intensity of the control lines on each type of lateral flow test strip; and determining the saturation probe density of the fluorescent microsphere bioprobe based on the antibody solution concentration.

[0051] Specifically, the blank sample (i.e., the test liquid without the marker) was detected using the LFIA method. Different concentrations (50 μg / ml, 100 μg / ml, 200 μg / ml, 300 μg / ml, 400 μg / ml, 600 μg / ml, 800 μg / ml) of anti-CRP-8# were added to the activated EuPSM solution to form an EuPSM-probe. The blank sample was detected using LFA, and the concentration of the saturation site was determined based on the C-line fluorescence signal of the blank sample.

[0052] More specifically, different concentrations (50 μg / ml, 100 μg / ml, 200 μg / ml, 300 μg / ml, 400 μg / ml, 600 μg / ml, 800 μg / ml) of anti-CRP-8# solution (the volume ratio of CRP-8 solution to EuPSM solution was constant) were added to the activated EuPSM solution (fixed value) to form an EuPSM probe. The binding affinity of the probe was evaluated by analyzing the C-line fluorescence signal of the blank sample using an LFA. For example... Figure 3As shown, the fluorescence signal of line C increases with the increase of anti-CRP-8# concentration. When anti-CRP-8# completely occupies all sites of EuPSM particles, the fluorescence signal of line C remains unchanged or decreases. Therefore, when the concentration of the coating antibody reaches 400 μg / ml, the fluorescence intensity of line C remains basically unchanged. However, in order to make the probe in the solution supersaturate, 600 μg / ml was finally selected as the saturation concentration of the coating antibody, which is converted to a molar amount of 0.8 nmol according to the formula.

[0053] Although large-particle-size EuPSM surfaces can conjugate more probes, excessive probes can reduce the response between fluorescence signals and sample concentration, thereby reducing the detection sensitivity of the system. However, by reducing the probe density on the particle surface to a certain extent, the detection sensitivity of the system can be effectively improved.

[0054] Considering that directly reducing the probe density will lead to uneven probe modification and exposed blank sites will result in non-specific binding, this embodiment of the invention uses BSA as a blank probe. The density of the surface probe is adjusted by introducing blank probes, and the detection performance of the EuPSM probe is further optimized.

[0055] Specifically, BSA and anti-CRP Ab8# were mixed in different molar ratios to form a hybrid probe, which was then mixed with activated EuPSM, such as... Figure 1 As shown, this allows the hybrid probes to be conjugated on the EuPSM surface to form EuPSM-probes of different densities.

[0056] More specifically, the mixed probe was added to the activated EuPSM solution and stirred in a shaker at 37°C and 220 rpm for 2 hours. Then, it was centrifuged for 10 minutes, the supernatant was removed, and 500 μL of deionized water was added. This process was repeated three times to conjugate the mixed probe on the EuPSM surface, forming EuPSM-probes with densities of 100%, 90%, 70%, 60%, 50%, 30%, and 10% of the saturation density. The molar ratios for preparing the BSA & CRP-Ab8# mixed probe are shown in Table 1.

[0057] Table 1. Molar proportions of BSA & CRP-Ab8# mixed probes prepared.

[0058]

[0059] The EuPSM probes prepared in this invention can be modified according to different targets. This embodiment only uses the detection of C-reactive protein (CRP) as an example to verify and illustrate the optimization of detection sensitivity by regulating the probe density on the EuPSM surface, but the targets are not limited to CRP. For example, when detecting protein biomarkers, corresponding antibodies can be modified on the EuPSM surface, or when detecting nucleic acid biomarkers, corresponding nucleic acid fragments can be modified on the EuPSM surface, etc.

[0060] Next, a lateral flow test strip needs to be prepared. The prepared lateral flow test strip includes a substrate, a sample pad, a binding pad, an NC membrane, and an absorbent pad. The binding pad and the absorbent pad are respectively attached to the substrate on both sides of the NC membrane, and both the binding pad and the absorbent pad are in contact with the NC membrane. The sample pad is attached to the substrate on the other side of the binding pad, and the sample pad is in contact with the binding pad.

[0061] Specifically, the prepared anti-CRP-AB7# solution and goat anti-mouse IgG solution were fixed onto the NC membrane surface of the lateral flow test strip using an XYZ platform sprayer to form a detection line (T line) and a control line (C line), respectively. The prepared EuPSM probe was dissolved in a standard buffer solution and dropped onto the conjugate pad surface of the lateral flow test strip. After drying, the EuPSM probe lateral flow test strip (EuPSM-LFA) was prepared.

[0062] In other words, the anti-CRP-AB7# solution is immobilized on the NC membrane surface of the lateral flow test paper to form a detection line; the goat anti-mouse IgG solution is immobilized on the NC membrane surface of the lateral flow test paper to form a control line; the blank probe is BSA, and the antibody is anti-CRP Ab8#.

[0063] In this embodiment of the invention, the experimental method is as follows: lateral flow test strips are prepared using each type of fluorescent microsphere biological probe; multiple test liquids containing different concentrations of markers are prepared; the test liquids of different concentrations are tested using the lateral flow test strips; and the detection limit of each fluorescent microsphere biological probe is determined based on the fluorescence intensity of the detection line on the lateral flow test strip.

[0064] Specifically, the test liquids containing different concentrations of the marker are configured as follows: the concentration gradients of the test liquids are 0.01, 0.025, 0.05, 0.25, 0.5, 2.5, 5, 25, 75, 125, 150, 250, 350 and 500, and the concentration unit is ng / ml.

[0065] In other words, a CRP detection system with a standard concentration gradient was prepared, and the prepared EuPSM-LFA was used for detection. EuPSM was excited to produce fluorescence using 350nm incident light, and the fluorescence intensity results at the detection line and control line of EuPSM-LFA were photographed using a camera (such as a smartphone) under dark conditions. To avoid experimental errors, each experiment was repeated three times, and the detection limit (LOD) was finally determined based on the fluorescence result photos.

[0066] The following example, using EuPSM probes with different saturation densities to detect standard concentration gradients in CRP systems, illustrates the effectiveness of the method of this invention.

[0067] Example 1:

[0068] 1) Europium chelated polystyrene microspheres (EuPSM) were prepared by suspension polymerization. First, 150 mg of Eu(DBM)3phen was dissolved in 10 mL of CH2Cl2 and mixed with 100 mL of carboxylic acid PSM (3%, w / v, dispersed in 0.25% SDS). The mixture was added to a 200 mL Erlenmeyer flask and sonicated for 15 min to form a homogeneous suspension. The suspension was stirred at room temperature for 24 h, and then heated in a 50 °C water bath for 12 h to completely evaporate the organic solvent CH2Cl2. Finally, the mixture was washed three times with ethanol, dispersed in 1% w / v water, and stored at 4 °C to obtain EuPSM.

[0069] 2) Take 100 μL of EuPSM prepared in step 1) and wash it 4 times with MES buffer (50 mM, pH = 6.0) to complete the washing and phase replacement.

[0070] 3) Add 12.5 μL of 100 mg / ml EDC and 12.5 μL of 100 mg / ml NHS sulfonic acid to the EuPSM that has been cleaned and phase-swapped in step 2), place it in a shaker at 37°C and 220 rpm and stir for 45 min. Wash three times with MES buffer to complete the activation process.

[0071] 4) Using BSA as a blank probe, mix 12.6 μL, 0 nmol of BSA and 12.6 μL, 0.8 nmol of CRP-Ab8# to form a mixed probe. Add the mixed probe to the EuPSM solution activated in step 3), place it in a shaker at 37°C and 220 rpm and stir for 2 h. Then centrifuge for 10 min, remove the supernatant, add 500 μL of deionized water, and repeat the operation 3 times to obtain EuPSM-probe with a density of 100% saturation density. Redisperse the EuPSM-probe mixture in 200 μL of ultrapure water and store at 4°C for use.

[0072] 5) Using an XYZ platform sprayer, 3 μL of 1 mg / mL anti-CRP-AB7# solution and 1.5 μL of 1 mg / mL goat anti-mouse IgG solution were fixed in rectangular strips on the NC membrane surface of the lateral flow test strip to form a detection line (T line) and a control line (C line). 15 μL of the EuPSM-probe prepared in step 4) was diluted 10 times and sprayed evenly on the conjugate pad. The strips were dried at 37°C for more than 2 hours. Finally, the strips were cut with a programmable shear to obtain a 3.9 mm wide strip of lateral flow test strip (EuPSM-LFA).

[0073] 6) Dilute standard CRP samples with flow buffer (0.1M, pH 8.0 Hepes, 0.5% w / v NaCl, 5% v / v Tween 20, and 5% w / v BSA) to prepare CRP detection systems with concentration gradients of 0.01, 0.025, 0.05, 0.25, 0.5, 2.5, 5, 25, 75, 125, 150, 250, 350, and 500 ng / ml, and use the EuPSM-LFA prepared in step 5) for detection. During detection, add 100 μL of sample solution to the sample area. Under capillary action, the sample solution flows sequentially through the conjugate pad, the detection area, and the absorbent pad. Detection is performed after reacting at the T and C lines in the detection area for 8 minutes. The results were obtained by taking photos of the fluorescent bands generated by the T-line and C-line excited by 350nm excitation light under dark room conditions with a smartphone under exposure for 4 seconds. To avoid experimental error, each experiment was repeated three times, and the detection limit (LOD) was finally determined based on the fluorescence results photos.

[0074] Test results as follows Figure 4 As shown in (a), it can be clearly observed that as the CRP concentration increases, the fluorescence intensity of the rectangular band gradually increases at the T line and gradually decreases at the C line. When the CRP concentration is 0.5 ng / mL, there is a weak fluorescence band at the T line, while there is no fluorescence band at the T line below this concentration. Therefore, it is shown that the detection limit (LOD) of the EuPSM probe at 100% saturation density is 0.5 ng / mL.

[0075] Example 2:

[0076] Referring to steps 1), 2), 3), 5), and 6) in Example 1, step 4) is as follows:

[0077] 4) Using BSA as a blank probe, mix 12.6 μL and 0.08 nmol of BSA and 12.6 μL and 0.72 nmol of CRP-Ab8# to form a mixed probe. Add the mixed probe to the EuPSM solution activated in step 3), place it in a shaker at 37°C and 220 rpm and stir for 2 h. Then centrifuge for 10 min, remove the supernatant, add 500 μL of deionized water, and repeat the operation 3 times to obtain EuPSM-probe with a density of 100% saturation density. Redisperse the EuPSM-probe mixture in 200 μL of ultrapure water and store at 4°C for use.

[0078] Test results as follows Figure 4 As shown in (b), the fluorescence intensity variation patterns at the T and C lines are clearly consistent with those in Example 1. When the CRP concentration is 0.25 ng / mL, a weak fluorescence band exists at the T line, while no fluorescence band is observed at concentrations below this. Therefore, it is shown that the detection limit (LOD) of the EuPSM probe with a density of 90% saturation is 0.25 ng / mL, which is 2 times lower than the gradient observed in Example 1.

[0079] Example 3:

[0080] Referring to steps 1), 2), 3), 5), and 6) in Example 1, step 4) is as follows:

[0081] 4) Using BSA as a blank probe, mix 12.6 μL and 0.24 nmol of BSA and 12.6 μL and 0.56 nmol of CRP-Ab8# to form a mixed probe. Add the mixed probe to the EuPSM solution activated in step 3), place it in a shaker at 37°C and 220 rpm and stir for 2 h. Then centrifuge for 10 min, remove the supernatant, add 500 μL of deionized water, and repeat the operation 3 times to obtain EuPSM-probe with a density of 100% saturation density. Redisperse the EuPSM-probe mixture in 200 μL of ultrapure water and store at 4°C for use.

[0082] Test results as follows Figure 4 As shown in (c), it can be clearly observed that the fluorescence intensity change pattern at the T and C lines is consistent with that in Example 1. When the CRP concentration is 0.05 ng / mL, there is a weak fluorescence band at the T line, while there is no fluorescence band at concentrations below this. Therefore, it is shown that the detection limit (LOD) of the EuPSM probe with a density of 70% saturation is 0.05 ng / mL, which is 10 times lower than the two gradients that appeared in Example 1.

[0083] Example 4:

[0084] Referring to steps 1), 2), 3), 5), and 6) in Example 1, step 4) is as follows:

[0085] 4) Using BSA as a blank probe, mix 12.6 μL and 0.32 nmol of BSA and 12.6 μL and 0.48 nmol of CRP-Ab8# to form a mixed probe. Add the mixed probe to the EuPSM solution activated in step 3), place it in a shaker at 37°C and 220 rpm and stir for 2 h. Then centrifuge for 10 min, remove the supernatant, add 500 μL of deionized water, and repeat the operation 3 times to obtain EuPSM-probe with a density of 100% saturation density. Redisperse the EuPSM-probe mixture in 200 μL of ultrapure water and store at 4°C for use.

[0086] Test results as follows Figure 4 As shown in (d), it can be clearly observed that the fluorescence intensity change pattern at the T and C lines is consistent with that in Example 1. When the CRP concentration is 0.025 ng / mL, there is a weak fluorescence band at the T line, while there is no fluorescence band at the T line below this concentration. Therefore, it is shown that the detection limit (LOD) of the EuPSM probe with a density of 60% saturation is 0.025 ng / mL. Compared with Example 1, which showed 3 gradients, the LOD has decreased by 20 times, reaching the optimal value.

[0087] Therefore, to further evaluate the practical application capability of EuPSM-LFA, the concentration of CRP in clinical serum was determined using the LFA developed in this embodiment, and the detection results were compared with the standard clinical detection results (chemiluminescence immunoassay).

[0088] The specific operation process is as follows: Using clinical serum from Shanghai Chest Hospital at standard gradient concentrations as samples, the fluorescence quantification values ​​of the T-line and C-line of the standard sample system were statistically analyzed using a reading device, and a standard curve was derived (e.g., Figure 6 (As shown in the figure). Based on this curve, 11 serum samples were diluted 500-fold with flow buffer and detected using the LFA platform developed in this embodiment. The detection concentration and standard concentration were calculated according to the standard curve, and the results are shown in Table 2.

[0089] according to Figure 6 As can be seen from the clinical test results in Table 2, the EuPSM-LFA developed in this invention has no significant difference from the clinical test results and meets the requirements for clinical application.

[0090] Table 2. Detected concentrations and standard concentrations obtained from clinical testing.

[0091]

[0092] Example 5:

[0093] Referring to steps 1), 2), 3), 5), and 6) in Example 1, step 4) is as follows:

[0094] 4) Using BSA as a blank probe, mix 12.6 μL and 0.4 nmol of BSA and 12.6 μL and 0.04 nmol of CRP-Ab8# to form a mixed probe. Add the mixed probe to the EuPSM solution activated in step 3), place it in a shaker at 37°C and 220 rpm and stir for 2 h. Then centrifuge for 10 min, remove the supernatant, add 500 μL of deionized water, and repeat the operation 3 times to obtain EuPSM-probe with a density of 100% saturation density. Redisperse the EuPSM-probe mixture in 200 μL of ultrapure water and store at 4°C for use.

[0095] Test results as follows Figure 4 As shown in (e), the fluorescence intensity variation at lines T and C is consistent with that in Example 1. A weak fluorescence band is observed at line T when the CRP concentration is 0.025 ng / mL, while no fluorescence band is observed at concentrations below this. This indicates that the limit of detection (LOD) of the EuPSM probe at 50% saturation density is 0.025 ng / mL, a 20-fold reduction compared to the three gradients observed in Example 1. Although both Example 1 and Example 4 show the same LOD, with weak fluorescence bands appearing at 0.025 ng / mL, Example 4 is significantly superior in terms of the integrity and fluorescence intensity of the fluorescence bands.

[0096] Example 6:

[0097] Referring to steps 1), 2), 3), 5), and 6) in Example 1, step 4) is as follows:

[0098] 4) Using BSA as a blank probe, mix 12.6 μL and 0.56 nmol of BSA and 12.6 μL and 0.24 nmol of CRP-Ab8# to form a mixed probe. Add the mixed probe to the EuPSM solution activated in step 3), place it in a shaker at 37°C and 220 rpm and stir for 2 h. Then centrifuge for 10 min, remove the supernatant, add 500 μL of deionized water, and repeat the operation 3 times to obtain EuPSM-probe with a density of 100% saturation density. Redisperse the EuPSM-probe mixture in 200 μL of ultrapure water and store at 4°C for use.

[0099] Test results as follows Figure 4As shown in (f), it can be clearly observed from the figure that the fluorescence intensity change pattern at the T line and C line is consistent with that in Example 1. When the CRP concentration is 0.25 ng / mL, there is a weak fluorescence band at the T line, while there is no fluorescence band at the T line below this concentration. Therefore, it is shown that the detection limit (LOD) of the EuPSM probe with a density of 30% saturation density is 0.25 ng / mL. Compared with Example 1, which shows one gradient slightly earlier, the LOD is reduced by 2 times. However, compared with Example 4, which shows two gradients later, the LOD is increased by 10 times.

[0100] Example 7:

[0101] Referring to steps 1), 2), 3), 5), and 6) in Example 1, step 4) is as follows:

[0102] 4) Using BSA as a blank probe, mix 12.6 μL and 0.72 nmol of BSA and 12.6 μL and 0.08 nmol of CRP-Ab8# to form a mixed probe. Add the mixed probe to the EuPSM solution activated in step 3), place it in a shaker at 37°C and 220 rpm and stir for 2 h. Then centrifuge for 10 min, remove the supernatant, add 500 μL of deionized water, and repeat the operation 3 times to obtain EuPSM-probe with a density of 100% saturation density. Redisperse the EuPSM-probe mixture in 200 μL of ultrapure water and store at 4°C for use.

[0103] Test results as follows Figure 4 As shown in (g), it can be clearly observed from the figure that the fluorescence intensity change pattern at the T line and C line is consistent with that in Example 1. When the CRP concentration is 0.5 ng / mL, there is a weak fluorescence band at the T line, while there is no fluorescence band at the T line below this concentration. Therefore, it is shown that the detection limit (LOD) of the EuPSM probe with a density of 10% saturation density is 0.5 ng / mL, which is consistent with that in Example 1. The LOD is the same, but compared with the three gradients that appear later in Example 4, the LOD increases by 20 times.

[0104] In summary, the density of the probe on the particle surface not only affects the response of fluorescence signals to biological signals, but also influences the particle's binding ability, such as... Figure 5 As shown, when the probe density decreases, the detection sensitivity first decreases and then increases, exhibiting the best LOD result (0.025 ng / ml) at 60%, which is nearly 20 times lower than the LOD of 100% saturated probe before optimization (0.5 ng / ml). This successfully amplifies the response of the fluorescence signal to the biological probe signal and effectively improves the detection sensitivity of the fluorescent microsphere biological probe.

[0105] In summary, this invention utilizes EuPSM fluorescent microspheres to construct biological probes. By introducing blank probes, the density of EuPSM probes can be controllably adjusted from 100% to 10%. Taking C-reactive protein (CRP) as an example, the optimization of the detection sensitivity of the EuPSM probes was successfully verified based on the traditional Lateral Flow Assay (LFA) platform. Finally, the constructed high-efficiency LFA was used for clinical serum CRP detection, and the detection results were consistent with those of clinical trials, further validating its clinical applicability.

[0106] Meanwhile, the EuPSM biological probes prepared by the method of this invention are not limited to CRP. The probe type and density can be modified according to different objects being detected. It is expected to be applied to the optimization of other biological detection probes such as bacteria, viruses, and heavy metal ions, so as to realize the development of high-performance POCT platforms in the fields of disease diagnosis, environmental monitoring and food safety testing.

[0107] The method described in this invention has advantages such as simple operation, strong versatility, and no increase in cost or labor consumption. It achieves uniform and controllable probe density adjustment, amplifies the response of fluorescence signals to biological probe signals, and can better meet the sensitivity requirements of ultrasensitive POCT platforms for biological probe detection. Furthermore, by simply adjusting the type and density of probes on the EuPSM surface, the detection needs of more types of targets can be met. This is of great significance for guiding the design of EuPSM probes to meet different application scenarios, and can demonstrate great potential and advantages in fields such as disease diagnosis, environmental monitoring, and food safety testing.

Claims

1. A method for regulating the detection sensitivity of fluorescent microsphere biological probes, characterized in that, Includes the following steps: The test liquid containing no marker was detected using lateral flow test strips with different fluorescent microsphere bioprobes. The fluorescence intensity at the control line of the lateral flow test strip was quantitatively analyzed, and the saturated probe density of the fluorescent microsphere bioprobes was determined based on the quantitative analysis results. The probe density of the different fluorescent microsphere bioprobes was different. The fluorescent microspheres in the fluorescent microsphere bioprobes were EuPSM with a large particle size of 300 nm. Blank probes and antibodies in different molar ratios are mixed to form mixed probes, and various mixed probe solutions are prepared. Multiple mixed probe solutions are mixed with activated fluorescent microsphere solutions to obtain multiple fluorescent microsphere biological probes; wherein the mixed probe density in the fluorescent microsphere biological probes is equal to the saturated probe density, and the antibody probe density in the fluorescent microsphere biological probes is less than or equal to the mixed probe density. The detection limits of various fluorescent microsphere biological probes were determined experimentally. Based on the required detection sensitivity, select the fluorescent microsphere biological probe corresponding to the detection limit as the fluorescent microsphere biological probe for the liquid to be tested. The blank probe is BSA, and the antibody is anti-CRP Ab8#.

2. The method for regulating the detection sensitivity of fluorescent microsphere biological probes as described in claim 1, characterized in that, The sideflow test strips with different fluorescent microsphere bioprobes are used to detect the test liquid without the marker. Quantitative analysis of the fluorescence intensity at the control line of the sideflow test strips specifically includes: Adding the same volume of antibody solution but different concentrations to a predetermined volume of activated fluorescent microsphere solution yields a variety of fluorescent microsphere bioprobe solutions. Sideflow test strips were prepared using each fluorescent microsphere bioprobe solution; Different side-flow test strips were used to detect the test liquid without the marker, and the fluorescence intensity of the control line of different side-flow test strips was obtained; The fluorescence intensity of different control lines on the sideflow test paper was quantitatively analyzed to obtain the saturated probe density of the fluorescent microsphere bioprobe.

3. The method for regulating the detection sensitivity of fluorescent microsphere biological probes as described in claim 2, characterized in that, Quantitative analysis of fluorescence intensity at different control lines on sideflow test strips included: Select the corresponding antibody solution concentration based on the fluorescence intensity of the control line on each type of lateral flow test strip; The saturation probe density of the fluorescent microsphere biological probe is determined based on the concentration of the antibody solution.

4. The method for regulating the detection sensitivity of fluorescent microsphere biological probes as described in claim 3, characterized in that, The experimental method is as follows: Sideflow test strips were prepared using each type of fluorescent microsphere biological probe; Prepare test liquids containing different concentrations of markers; Side-flow test strips were used to test various liquids of different concentrations. The detection limit of each fluorescent microsphere bioprobe was determined based on the fluorescence intensity of the detection line on the side-flow test strip.

5. The method for regulating the detection sensitivity of fluorescent microsphere biological probes as described in claim 4, characterized in that, The preparation of lateral flow test strips using each type of fluorescent microsphere biological probe includes: The anti-CRP-AB7# solution was fixed onto the surface of the NC membrane of the side-flow test paper to form a detection line; A control line was formed by immobilizing goat anti-mouse IgG solution on the surface of the NC membrane of the lateral flow test paper. The blank probe is BSA, and the antibody is anti-CRP Ab8#.

6. The method for regulating the detection sensitivity of fluorescent microsphere biological probes as described in claim 5, characterized in that, The preparation of test liquids containing different concentrations of markers includes: The concentration gradients of the test liquids of various different concentrations are 0.01, 0.025, 0.05, 0.25, 0.5, 2.5, 5, 25, 75, 125, 150, 250, 350 and 500, with the concentration unit being ng / ml.

7. The method for regulating the detection sensitivity of fluorescent microsphere biological probes as described in claim 2, characterized in that, Adding the same volume of antibody solution with different concentrations to a predetermined volume of activated fluorescent microsphere solution yielded a variety of fluorescent microsphere bioprobe solutions. The antibody probe densities on the fluorescent microsphere bioprobes in the various fluorescent microsphere bioprobe solutions were 100%, 90%, 70%, 60%, 50%, 30%, and 10%, respectively.

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