Method for accelerating analyte detection

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to a method for accelerating analyte detection. In the biosensor for monitoring the analyte in real time, the response time of the low-concentration analyte is greatly shortened by introducing the non-analyte, and the concentration of the analyte is calculated in combination with the initial binding rate, so that the analysis time is greatly saved, and the detection efficiency is improved. And a foundation is laid for further development of rapid, high-sensitivity and label-free biosensors and POCT (point-of-care testing).

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and in particular relates to a method for accelerating analyte detection. Background Art

[0002] At present, significant progress has been made in the research and development of precise detection technology of biomolecules at home and abroad, especially in molecular detection, sensor technology, artificial intelligence and big data analysis, showing a strong growth trend. Human health index detection technology is developing towards ultra-sensitive, high-throughput and multifunctional integration. The main technologies include: 1. Single molecule array (Simoa) technology, which uses high-affinity antibodies or nucleic acid probes to bind target molecules, and further enhances the molecular capture efficiency through magnetic beads or other solid phase carriers; the smaller reaction volume and efficient capture mechanism greatly increase the detection probability of target molecules. 2. Real-time fluorescence quantitative PCR, which is a powerful tool for detecting and quantifying biomolecules. By real-time monitoring of the fluorescence signal during the PCR reaction, the amplification of DNA or RNA can be tracked in each amplification cycle, thereby achieving accurate quantification of nucleic acids. 3. Electrochemical sensing technology, which quantitatively analyzes the target substance by detecting the current, potential or resistance signal generated by the electrochemical reaction at the interface between biomolecules and sensors. 4. Optical sensing technology, which detects signal changes based on the interaction between light and biomolecules, and the main principles include absorbance, fluorescence, light scattering, light interference and surface plasmon resonance (SPR).

[0003] Among them, optical biosensors can realize real-time, convenient and economical monitoring of biomolecules. Plasmon biosensors, as optical sensors, do not require relatively expensive proprietary instruments and have high sensitivity and multiplexing potential. Plasmon resonance biosensors are increasingly used in medical diagnosis, food safety and environmental monitoring because they can perform label-free testing of detected objects. The main working principle of plasmon resonance biosensors is that under the excitation of external light sources, the surface plasmon resonance (SPR) between the noble metal layer and the medium responds sensitively to changes in the refractive index (RI) of the surrounding medium. By measuring changes in these resonance conditions (e.g., angle, wavelength, intensity or phase), molecular interactions occurring on the sensor surface can be directly monitored in real time. This unique feature makes plasmon resonance biosensors an indispensable tool for real-time and label-free analysis. Surface refractive index sensors have shown great application potential in many fields due to their high sensitivity and versatility.

[0004] Despite remarkable progress in improving the limit of detection (LOD) of biosensors, actual clinical testing requires detection indicators that are far higher than the LOD of current biosensing strategy research levels. From a practical point of view, sensors are clinically useful only if accurate quantification of analytes can be completed within an acceptable time frame (e.g., a few minutes). However, especially at ultra-low analyte concentrations, due to the limitation of mass transport when delivering biomolecules from the stock solution to the sensor capture volume, there is a serious corresponding delay, and the detection time is usually within an unacceptable range.

[0005] In order to reduce the response time, a variety of strategies have been proposed. For example, for common binding kinetics and analytes, flow nanopore arrays can provide approximately 10 times the response time compared to existing flow sensors. In addition, chaotic mixing in three-dimensional serpentine microchannels during sample flow has also been shown to increase analyte binding rates by 2 times. In addition, based on electrokinetic effects (electrophoresis, two-dimensional electrophoresis, and electrothermal plasma effects), target molecules can be more efficiently transported to the plasma sensing structure by applying an electric field and / or generating a temperature gradient. However, these strategies either require more complex microfluidic systems (such as double-layer microchannels or structured microchannels) or require additional electrical and heating elements, which is not conducive to the miniaturization of the sensing system. Summary of the invention

[0006] In order to realize label-free rapid analysis of analytes, the present invention provides a method for rapidly analyzing the concentration of a detection object in a complex system in a real-time monitoring refractive index sensing system, by accelerating the binding of the analyte to the sensor surface receptor and combining the initial binding rate analysis (IRA) method to rapidly analyze the concentration of the detection object.

[0007] The first aspect of the present invention aims to provide a method for accelerating the detection of analytes.

[0008] The purpose of the second aspect of the present invention is to provide an application of the method of the first aspect of the present invention.

[0009] In order to achieve the above-mentioned purpose of the present invention, the technical solution adopted by the present invention is:

[0010] A first aspect of the present invention provides a method for accelerating the detection speed of an analyte, comprising the following steps:

[0011] The sample to be tested is detected using the detection system, and the analyte concentration is calculated using initial binding rate analysis.

[0012] In some embodiments of the present invention, there is a step of determining the complexity of the sample to be tested before the test;

[0013] When the sample to be tested is a simple system, the test is performed after additional introduction of non-analytes;

[0014] When the sample to be tested is a complex system, the sample to be tested is detected using a detection system, and the analyte concentration is calculated using initial binding rate analysis.

[0015] The simple system refers to a detection system in which more than 90% of the substances in the sample to be tested are known, for example, a detection method in which a tissue is sampled by a nasal swab, a throat swab, a sampling brush, etc. and then placed in a detection buffer.

[0016] In some embodiments of the present invention, the simple system also includes a solution system containing 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of known substances.

[0017] In some embodiments of the present invention, the detection method described in the first aspect of the present invention is used for the detection of a standard in a laboratory or clinical environment, and the simple system consists of an analyte to be detected and a buffer.

[0018] In some embodiments of the present invention, the analyte is a substance with a particle size of 0.1 to 1000 nanometers.

[0019] In some embodiments of the present invention, the analyte includes at least one of protein, nucleic acid, lipid, and carbohydrate.

[0020] In some embodiments of the invention, the analyte is a protein.

[0021] In some embodiments of the present invention, the protein includes at least one of alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), prostate-specific antigen (PSA), and carbohydrate antigen CA19-9.

[0022] The complex system refers to unprocessed biological samples directly derived from the body, such as plasma, serum, tissue fluid and other samples.

[0023] In some embodiments of the present invention, the complex system includes an analyte to be detected, a buffer and a non-analyte.

[0024] In some embodiments of the present invention, the detection method is a label-free detection method.

[0025] Analyte label-free detection refers to the detection of analyte concentration by directly binding the analyte to the receptor molecules on the sensor surface, causing the refractive index of the sensor surface to change. The markers here are all other connected ligands, groups, molecules, etc. except the analyte molecules themselves, that is, in addition to the traditional fluorescent, radioactive or enzyme labeled molecules, they also include other macromolecules, metal particles, etc.

[0026] In some embodiments of the present invention, the detection system comprises a fluid detection system having a real-time detection microchannel.

[0027] In some embodiments of the present invention, the sample to be detected does not have any artificial modification groups.

[0028] In some embodiments of the present invention, the artificial modification group includes at least one of biotin, a fluorescent group, an isotope, and a label protein.

[0029] In some embodiments of the present invention, the non-analyte is at least one of a protein, a fat, or a hormone that non-specifically binds to the detection system.

[0030] In some embodiments of the invention, the non-analyte is a protein.

[0031] In some embodiments of the invention, the protein comprises BSA.

[0032] In some embodiments of the invention, the non-analyte solution comprises a buffer.

[0033] In some embodiments of the present invention, the buffer comprises at least one of PBS, TBS, HBSS, HEPE, and DMEM.

[0034] In some embodiments of the present invention, the mass concentration ratio of the non-analyte to the analyte in the non-analyte solution is 1 to 1×10 7 ; Preferably, 1 to 1×10 5 ; More preferably, 10 to 10 5 ; More preferably 10 2 ~10 4 .

[0035] In some embodiments of the present invention, the detection method of the detection system includes combining the initial reaction rate.

[0036] In some embodiments of the present invention, the initial reaction rate of the binding comprises: using the initial binding rate to obtain a linear concentration fitting relationship within a period of time in the initial stage of the reaction: as shown in the following formula: lg(ΔR / t)=A×lgC-B;

[0037] Where ΔR represents the response change, t is time, A is the constant coefficient, C is the analyte concentration, and B is the constant intercept.

[0038] In some embodiments of the present invention, the R includes at least one of an intensity change I, a wavelength change λ, and an angle change θ.

[0039] In some embodiments of the present invention, the initial stage of the reaction is the stage where the binding reaction between the receptor and the analyte is dominant, and the initial stage of the response is 10 to 100 s. More preferably, it is 10 to 80 s. More preferably, it is 10 to 60 s.

[0040] In some embodiments of the present invention, the unit of ΔR is an arbitrary unit (AU), including but not limited to changes in light intensity, wavelength, resonance angle, current intensity, fluorescence intensity, refractive index, absorbance, etc.

[0041] When the analyte solution is continuously replenished to keep its volume concentration constant, the reaction between the immobilized receptor and the analyte in the solution can be considered to follow pseudo-first-order kinetics. In this case, the sensor response rate dR / dt can be written as:

[0042]

[0043] Where C is the concentration of the analyte in the solution, R max is the maximum sensor response, R(t) is the sensor response at time t, k a and k d are the association rate and dissociation rate, respectively.

[0044] In the first aspect of the present invention, the fluid detection system with microchannels refers to a detection system (device) that requires the liquid to be tested to flow in real time in the microchannel, such as a microfluidic device (a system based on microchannel processing or manipulation of tiny fluids), a biochip (based on the interaction between proteins), a biosensor (based on optical and / or electrical methods to identify enzymes or antibodies), an electrochemical sensor (based on the current changes generated by electrochemical probes), spectrophotometric technology (based on optical absorbance detection), or a combination of the above systems (techniques).

[0045] In some embodiments of the present invention, the non-analyte solution includes a miscellaneous protein solution, or an irrelevant protein solution, such as bovine serum albumin (BSA). Those skilled in the art can expect that other non-target analyte proteins such as collagen, tag proteins (Flag, HA, GST, Myc, etc.), whey protein, etc. can also achieve similar effects of the present invention.

[0046] In some embodiments of the present invention, the detection method is label-free detection, and no external detection molecules, such as primers, nucleic acid aptamers, antibodies, etc., need to be introduced into the detection system, or the components of the detection system do not need to be artificially modified, such as isotopes, biotin, fluorescent groups, etc. The method for increasing the detection speed of the detection object of the first aspect of the present invention is used for non-disease diagnosis and treatment purposes. The second aspect of the present invention provides the application of the method of the first aspect of the present invention in the field of analyte detection.

[0047] The analyte includes at least one of nucleic acid, protein, lipid and carbohydrate.

[0048] The analyte detection includes point-of-care testing.

[0049] Point-of-care testing refers to a technology or method that can quickly obtain test results after sample collection. This type of testing is usually completed in a short period of time and aims to provide immediate feedback to facilitate timely decision-making and intervention. It is widely used in the fields of medical care, environmental monitoring and food safety.

[0050] In some embodiments of the present invention, the instant detection refers to the detection completed within 30 minutes; preferably, within 20 minutes; further preferably, within 10 minutes; further preferably, within 5 minutes.

[0051] The analyte detection is for non-diagnostic and non-therapeutic purposes.

[0052] The beneficial effects of the present invention are:

[0053] The present invention greatly shortens the response time of low-concentration analytes by introducing non-analytes in the real-time monitoring biosensor of analytes, and calculates the analyte concentration in combination with the initial binding rate, thereby greatly saving analysis time and improving detection efficiency. This lays a foundation for the further development of rapid, highly sensitive, label-free biosensors and POCT. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0055] Figure 1 Flowchart of the method for accelerating the analysis of the detected substance.

[0056] Figure 2 This is the response curve of pure antigen and after the introduction of 4 times of impurity protein.

[0057] Figure 3 Schematic diagram showing the loss effect caused by adsorption of analytes on the inner wall of the tube in a microfluidic system.

[0058] Figure 4 These are the response curves for pure antigen and after the introduction of high concentrations of foreign proteins.

[0059] Figure 5 Schematic diagram of the initial binding rate analysis method for accelerated binding of complex system responses.

[0060] Figure 6 AFP concentration in different BSA concentrations and initial binding rate Linear relationship diagram

[0061] Figure 7 AFP concentration in different BSA concentrations and initial binding rate Linear relationship diagram

[0062] Figure 8 The figure is a comparison between the test results of the method of the present invention and the test results of commercial equipment.

[0063] Fig. 9 The detection results of different AFP concentrations in Comparative Example 1 of the present invention. DETAILED DESCRIPTION

[0064] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0065] Figure 1 Flow chart of the method for accelerating the analysis of detection objects, usually for complex systems such as blood, serum, urine, saliva, etc., the surface refractive index sensor surface will produce real-time response with the specific binding of receptor-analyte. Due to the complex body fluid environment, the non-specific consumption of analyte in the pipeline can be basically ignored, the binding kinetics are accelerated, and the response time is greatly shortened. However, if the test sample composition is relatively single, in the case of not having other components except the analyte to be detected and the buffer, the analyte will have non-specific consumption in the pipeline when the sample passes through the pipeline system, resulting in the problem of slow binding response. Further, the use of initial reaction binding rate analysis (IRA) to calculate the analyte concentration can further improve the rate of analyte detection compared to not doing treatment, and the analysis rate is improved by 228 times. Specific data are shown in the following examples.

[0066] Example 1 Effect of introducing 4 times of impurity protein on detection response

[0067] 1. Sample preparation:

[0068] A low concentration mixed antigen solution (40 ng / mL) was prepared using PBS buffer, and the total antigen concentration was 40 ng / mL, of which the concentrations of AFP, PSA, CEA and CA19-9 antigens were all 10 ng / mL.

[0069] A 10 ng / mL AFP antigen solution was prepared using PBS buffer.

[0070] Use PBS buffer to prepare 100 μg / mL Anti-AFP solution.

[0071] Prepare a 10 g / L BSA solution using PBS buffer.

[0072] The mixed antigen solution (40 ng / mL) and the pure AFP antigen solution (10 ng / mL) were detected using the UGNA sensor (disclosed in patent 2024113072459), and the detection data were analyzed. Specifically, the sensor chip was placed on the test bench, the injection pump flow rate was set to 1 mL / h, and phosphate buffered saline (PBS) was injected into the microfluidic channel. The structural area of ​​the sensor chip was identified by a microscope, and the system was then switched to dark field mode and began to continuously collect the reflectance spectrum, monitor the reflectance spectrum in real time, and monitor the entire liquid flow process in real time.

[0073] 2. Sensor channel surface modification:

[0074] 1) PBS washing: 15 min;

[0075] 2) Inject MES solution containing a final concentration of 40 mM EDC and 10 mM NHS for 15 minutes to activate the carboxyl groups on the sensor surface, and then wash with PBS for 15 minutes.

[0076] 3) Inject 100 μg / mL Anti-AFP and fix for 50 min, then wash with PBS for 15 min.

[0077] 4) 10 g / L BSA was injected for 40 min to block the empty sites on the sensor surface that were not bound to Anti-AFP to ensure the specificity of the sensor, and then washed with PBS for 15 min.

[0078] 3. Test process:

[0079] 1) Channel 1: Inject pure AFP antigen solution (10 ng / mL) and test until the spectrum no longer changes (reaction equilibrium), then wash with PBS for 15 minutes.

[0080] 2) Channel 2: Inject the test object mixed with the antigen solution (40 ng / mL) and test until the spectrum no longer changes (reaction equilibrium).

[0081] 4. Experimental results:

[0082] The experimental results are as follows Figure 2 As shown, the mixed antigen reaches adsorption equilibrium at 2160s, while the pure AFP antigen solution reaches adsorption equilibrium at 9910s. For AFP only, the responses (Δλ) of the two solutions after adsorption equilibrium are consistent, proving that the sensor can accurately detect 10ng / mL AFP antigen in both solutions. However, the response of the mixed antigen is significantly faster than that of the pure antigen. This is because the concentration of the total antigen in the mixed antigen is 40ng / mL, which is 4 times that of the pure AFP antigen, and the microfluidic channel is non-specific for the adsorption of the analysis liquid protein, so the consumption of the antigen to be tested in the mixed antigen solution is less than that of the pure antigen, so the response time is also relatively early, and there is basically no delay.

[0083] Related principles such as Figure 3 As shown in the figure, it shows the loss effect caused by the adsorption of analytes on the inner wall of the tube in the microfluidic system. I and II represent two situations with different antigen binding efficiencies on the sensor surface: (I) the solution containing only the target antigen flows, and (II) the antigen solution containing additional impurities. Figure 4 In the figure, the red curve (response curve of 10 ng / mL AFP) starts to respond after 4000 seconds. This delay may be caused by the non-specific binding of antigen molecules to the inner wall of the injection tube. This will lead to unexpected antigen loss, resulting in only a small number of antigen molecules being able to enter the microchannel and bind to the sensor surface. Figure 3 As shown in case I in the figure. In order to avoid such response delay at low concentration, in this embodiment, a high concentration of foreign protein is added to the antigen solution to block the nonspecific binding of the inner wall of the injection pipeline, as shown in case II. This can effectively prevent the nonspecific adsorption of the antigen, thereby significantly enhancing the binding efficiency of the antigen on the sensor surface.

[0084] like Figure 4 As shown, the two curves correspond to Figure 3 Time response curves of two antigen tests, where the blue curve and the red curve represent cases I and II, respectively. When a low concentration of analyte is injected into the sensor channel, since the pure analyte is non-specifically adsorbed by the tube wall in the early stage, it takes a long time to accumulate to saturation. The analyte molecules that actually reach the analysis site in the early stage of detection are far less than the detection concentration, so the response starts late. After adding a large amount of impurities, the consumption of analytes by non-specific adsorption in the initial pipeline almost no longer occurs, so the response is rapid. In addition, the combination of the addition of impurities and the initial rate analysis (IRA) method further shortens the detection time (t I ≥t II ).

[0085] Example 2 Effect of introducing high concentration of impurity protein on detection response

[0086] 1. Sample preparation:

[0087] PBS buffer was used to prepare a 70 g / L BSA solution, and then the 70 g / L BSA solution was used as a solvent to prepare a 1 ng / mL AFP antigen solution (1 ng / mL AFP-BSA solution).

[0088] A 1 ng / mL AFP antigen solution (1 ng / mL AFP-PBS solution) was prepared using PBS buffer.

[0089] Use PBS buffer to prepare 100 μg / mL Anti-AFP solution.

[0090] Prepare a 10 g / L BSA solution using PBS buffer.

[0091] The UGNA sensor (disclosed in patent 2024113072459) was used to detect 1ng / mL AFP-BSA solution and 1ng / mL AFP-PBS solution, and the detection data was analyzed. Specifically, the sensor chip was placed on the test bench, the injection pump flow rate was set to 1mL / h, and the phosphate buffer (PBS) was injected into the microfluidic channel. The structural area of ​​the sensor chip was identified by a microscope. The system then switched to dark field mode and began to continuously collect the reflectance spectrum, monitor the reflectance spectrum in real time, and monitor the entire liquid flow process in real time.

[0092] 2. Sensor channel surface modification:

[0093] 1) PBS washing: 15 min;

[0094] 2) Inject MES solution containing a final concentration of 40 mM EDC and 10 mM NHS for 15 minutes to activate the carboxyl groups on the sensor surface, and then wash with PBS for 15 minutes.

[0095] 3) Inject 100 μg / mL Anti-AFP and fix for 50 min, then wash with PBS for 15 min.

[0096] 4) 10 g / L BSA was injected for 40 min to block the empty sites on the sensor surface that were not bound to Anti-AFP to ensure the specificity of the sensor, and then washed with PBS for 15 min.

[0097] 3. Test process:

[0098] 1) Channel 1: Inject 1 ng / mL AFP-BSA solution and test until the spectrum no longer changes (reaction equilibrium).

[0099] 2) Channel 2: Inject 1 ng / mL AFP-PBS solution and test until the spectrum no longer changes (reaction equilibrium).

[0100] 4. Experimental results

[0101] The experimental results are as follows Figure 6 As shown, the response curves of 1 ng / mL AFP antigen in PBS and 70 g / L BSA buffer were further compared. In BSA buffer, the response was observed immediately after the solution was injected (within a few seconds), while in PBS, the response began only after about 5500 s. As the concentration of AFP antigen increased, the amount of antigen adsorption corresponding to the response moved slowly over time, which is consistent with the expected trend that the increase in antigen concentration leads to increased adsorption on the sensor surface. However, waiting for the reaction to reach equilibrium (approximately 15-20 minutes) and then calculating the displacement after flushing excess sample from the channel is time-consuming and inefficient, and is not suitable for formulating POCT strategies. By combining IRA with the former and analyzing the slope of the response starting 40 to 50 s, this embodiment achieves a faster concentration analysis than the latter equilibrium state (~11400 s), thereby increasing the detection and analysis time by 228 times.

[0102] Clinically relevant biosensing samples contain not only analytes but also a large number of background molecules. When the sample is incubated with the receptor surface, the background proteins compete with the analyte for the receptor binding sites. The binding of the analyte to the background proteins depends on the specificity of the receptor and the concentration of all molecules. This model is called the Langmuir competition model. All non-analyte molecules are collectively referred to as background molecules, and the concentration fraction of the analyte c a The concentration fraction c of the background molecule b Has the following relationship:

[0103] c a +c b =1

[0104] In this case, the molecules other than the analyte are collectively referred to as background molecules, so the surface coverage of the competitively adsorbed analyte a and background molecules b with the receptor molecules in the solution are:

[0105]

[0106]

[0107] Where K a and K b is the adsorption constant, θ a and θ b are the surface fractions covered by adsorbed molecules a and b, respectively. a <<C b ,θ bThis is directly reflected in the resonance response, so the introduction of a large amount of non-analytes such as impurities does not affect the test results.

[0108] Example 3: Introducing high concentration of impurity proteins and drawing standard curves in combination with IRA analysis

[0109] 1. Sample preparation:

[0110] Use PBS buffer to prepare three concentrations of BSA-PBS buffer: 60 g / L, 70 g / L and 80 g / L, and set an AFP antigen gradient (0, 1, 10, 100, 1000 ng / mL). Use the above three BSA-PBS buffers to prepare AFP antigen solutions with corresponding concentration gradients.

[0111] Use PBS buffer to prepare a 1 ng / mL AFP antigen solution.

[0112] Use PBS buffer to prepare 100 μg / mL Anti-AFP solution.

[0113] Prepare a 10 g / L BSA solution using PBS buffer.

[0114] The antigen solution was detected using the UGNA sensor (disclosed in patent 2024113072459) and the data was analyzed. Specifically, the sensor chip was placed on the test bench, and phosphate buffered saline (PBS) was injected into the microfluidic channel at a flow rate of 1 mL / h using a syringe pump. The structural area of ​​the sensor chip was identified by a microscope, and the system was then switched to dark field mode and began to continuously collect the reflection spectrum, monitor the reflectance spectrum in real time, and monitor the entire liquid flow process in real time.

[0115] 2. Sensor channel surface modification:

[0116] 1) PBS washing: 15 min;

[0117] 2) Inject MES solution containing a final concentration of 40 mM EDC and 10 mM NHS for 15 minutes to activate the carboxyl groups on the sensor surface, and then wash with PBS for 15 minutes.

[0118] 3) Inject 100 μg / mL Anti-AFP and fix for 50 min, then wash with PBS for 15 min.

[0119] 4) 10 g / L BSA was injected for 40 min to block the empty sites on the sensor surface that were not bound to Anti-AFP to ensure the specificity of the sensor, and then washed with PBS for 15 min.

[0120] 3. Test process:

[0121] Different channels were injected with: 0 ng / mL AFP (60 g / L BSA-PBS) solution, 1 ng / mL AFP (60 g / L BSA-PBS) solution, 10 ng / mL AFP (60 g / L BSA-PBS) solution, 100 ng / mL AFP (60 g / L BSA-PBS) solution, 1000 ng / mL AFP (60 g / L BSA-PBS) solution, 0 ng / mL AFP (70 g / L BSA-PBS) solution, 1 ng / mL AFP (70 g / L BSA-PBS) solution, 10 ng / mL AFP (70 g / L BSA-PBS) solution, 1000 ng / mL AFP (70 g / L BSA-PBS) solution, 0 ng / mL AFP (80 g / L BSA-PBS) solution, 1 ng / mL AFP (80 g / L BSA-PBS) solution. BSA-PBS) solution, 10 ng / mL AFP (80 g / L BSA-PBS) solution, 100 ng / mL AFP (80 g / L BSA-PBS) solution, and 1000 ng / mL AFP (80 g / L BSA-PBS) solution were tested.

[0122] 4. Experimental results:

[0123] Test results such as Figure 6 , 7 The following are the results of reading wavelength response and light intensity response respectively. Figure 6 In the experiment, the AFP concentration response in 60g / L, 70g / L and 80g / L BSA-PBS buffer was tested three times to obtain the CI / t relationship, and the fitted linear relationship was: Figure 7 In the experiment, the AFP concentration response in 60 g / L, 70 g / L and 80 g / L BSA buffer was tested three times to obtain the C-λ / t relationship, and the fitted linear relationship was: It was also found that the concentration of BSA-PBS buffer had no effect on the relationship between C and ΔI / t, so the impact of differences in serum composition among different individuals on the test results could also be ignored.

[0124] The fitting curve is used as the standard curve for the initial slope of clinical testing, and individual differences need to be taken into account. Therefore, it is necessary to confirm whether to introduce compensation factor correction when the total protein concentration of different individuals is different.

[0125] The above experimental results prove that the fitting curves under different BSA concentrations (simulating different serum total protein concentrations under physiological environment) are basically consistent, indicating that the influence of the difference in total protein concentration between different individuals can be eliminated.

[0126] Example 4 Complex system detection

[0127] 1. Sample preparation:

[0128] Use PBS buffer to prepare 100 μg / mL Anti-AFP solution.

[0129] Prepare a 10 g / L BSA solution using PBS buffer.

[0130] In addition, sera from 7 patients were collected for analysis.

[0131] The antigen solution was detected using the UGNA sensor (disclosed in patent 2024113072459) and the data was analyzed. Specifically, the sensor chip was placed on the test bench, and phosphate buffered saline (PBS) was injected into the microfluidic channel at a flow rate of 1 mL / h using a syringe pump. The structural area of ​​the sensor chip was identified by a microscope, and the system was then switched to dark field mode and began to continuously collect the reflection spectrum, monitor the reflectance spectrum in real time, and monitor the entire liquid flow process in real time.

[0132] 2. Sensor channel surface modification:

[0133] 1) PBS washing: 15 min;

[0134] 2) Inject MES solution containing a final concentration of 40 mM EDC and 10 mM NHS for 15 minutes to activate the carboxyl groups on the sensor surface, and then wash with PBS for 15 minutes.

[0135] 3) Inject 100 μg / mL Anti-AFP and fix for 50 min, then wash with PBS for 15 min.

[0136] 4) 10 g / L BSA was injected for 40 min to block the empty sites on the sensor surface that were not bound to Anti-AFP to ensure the specificity of the sensor, and then washed with PBS for 15 min.

[0137] 3. Test process:

[0138] Different channels are injected with test objects (human serum samples collected above)

[0139] 4. Experimental results:

[0140] AFP concentration analysis was performed and the results were compared with the hospital's commercial equipment (Roche Cobas e801). The specific comparison results are as follows Figure 8As shown, whether by extracting the wavelength shift (Δλ) signal or the intensity signal (ΔI) signal, it is consistent with the detection results of the commercial device Roche Cobas e801 (the specific working principle is to use ruthenium (Ru) markers to form a sandwich complex with the antigen to be tested and the corresponding antibody, the complex is combined with magnetic beads, and fixed on the electrode through electromagnetic action, and the light signal is excited under the electric field. The analyte concentration is calculated based on the luminescence intensity, and the detection process takes 18 minutes). It can distinguish the positive and negative of AFP, and the AFP level detection value in positive patients is also close to the result of Roche equipment.

[0141] Comparative Example 1

[0142] 1. Experimental methods

[0143] The response curve of the UGNA biosensor (disclosed in patent 2024113072459) for AFP antigen (PBS buffer environment) test. The antigen to be tested does not need to be modified and a response can be obtained at a concentration of 1 ng / mL (the clinical reference critical value for liver cancer is 7 ng / mL).

[0144] Sample configuration:

[0145] AFP solutions with concentrations of 1, 10, 100, 200, 400, 600, 800 and 1000 ng / mL were prepared using PBS as solvent.

[0146] Sample testing:

[0147] The test light spot is aimed at the sensing area in the microchannel. The syringe pump keeps pumping liquid at a rate of 1 mL / h, and the detector keeps recording the reflectivity curve of the sensor surface.

[0148] 1) PBS washing: 15 min;

[0149] 2) Inject (40 mM EDC and 10 mM NHS) / MES solution for 15 minutes to activate the carboxyl terminal groups. Wash with PBS for 15 minutes;

[0150] 3) Inject 100 μg / mL Anti-AFP and fix for 50 min, then wash with PBS for 15 min.

[0151] 4) 10 g / L BSA was injected for 40 min to block the empty sites on the sensor surface that were not bound to Anti-AFP to ensure the specificity of the sensor, and then washed with PBS for 15 min.

[0152] 5) The test substances were injected into different channels respectively: 1, 10, 100, 200, 400, 600, 800 and 1000 ng / mL AFP solution).

[0153] 2. Experimental results

[0154] The results are as follows Fig. 9 shown.

[0155] It can be seen that at a concentration of 100-1000 ng / mL, the time it takes for the analyte to reach equilibrium is about 1000-2000 s (within 30 min), but at 1 and 10 ng / ml, the time it takes to start responding and reach equilibrium is very long, even exceeding 12000 s (2 h). The reasons for this phenomenon include: 1. The influence of fluid material exchange and reaction kinetics, the reaction rate of low-concentration reactants is relatively slow. 2. The non-specific consumption of fluid proteins by the microfluidic channel causes the initial consumption of the concentration of the low-concentration antigen fluid itself, resulting in a delayed response.

Claims

1. A method for improving the detection speed of a detection object, characterized in that: The following steps are involved: The sample to be tested is detected using a detection system, and the analyte is quantitatively or qualitatively detected using the initial binding rate.

2. The method according to claim 1, characterized in that: The method also includes the step of analyzing the complexity of the components of the sample to be tested before testing; When the sample to be tested is a simple system, the test is performed after additional introduction of non-analytes; When the sample to be tested is a complex system, the sample to be tested is detected using a detection system, and the analyte concentration is calculated using an initial binding rate analysis; Preferably, the non-analyte is at least one of a protein, a fat, or a hormone that non-specifically binds to the detection system.

3. The method according to claim 2, characterized in that: The detection method is a label-free detection method.

4. The method according to claim 3, characterized in that: The detection system includes a fluid detection system having a microchannel.

5. The method according to claim 4, characterized in that: The analyte is a substance with a particle size of 0.1 to 1000 nanometers; Preferably, the analyte comprises at least one of protein, nucleic acid, lipid, and carbohydrate; Preferably, the non-analyte comprises at least one of protein, nucleic acid, lipid, and carbohydrate; Preferably, the non-analyte comprises a protein; Preferably, the protein comprises BSA.

6. The method according to claim 2, characterized in that: The non-analyte includes a buffer.

7. The method according to claim 6, characterized in that: After the non-analyte is introduced, the mass concentration ratio of the non-analyte to the analyte is 1 to 1×10 7 .

8. The method according to claim 1, characterized in that: The initial binding reaction rate includes: using the initial binding rate to obtain a linear concentration fitting relationship within a period of time in the initial stage of the reaction, as shown in the following formula: lg(ΔR / t)=A×lgC-B; Where ΔR represents the response change, t is the time, A is the constant coefficient, C is the analyte concentration, and B is the constant intercept; Preferably, the R includes at least one of intensity change I, wavelength change λ, and angle change θ; Preferably, the initial stage of the reaction is a stage where the binding reaction between the receptor and the analyte is dominant, which is 10 to 100 seconds from the beginning of the response.

9. Application of the method according to any one of claims 1 to 8 in the field of analyte detection; The analyte includes at least one of nucleic acid, protein, lipid and carbohydrate.

10. The use according to claim 9, characterized in that: The analyte detection includes point-of-care testing.

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