A method of accelerating analyte detection

Abstract

The present application belongs to the field of biological detection technology, and particularly relates to a method for accelerating analyte detection. In the biosensor for real-time monitoring of analyte, the response time of low-concentration analyte is greatly shortened by introducing non-analyte, and the analyte concentration is calculated in combination with the initial binding rate, so that the analysis time is greatly saved and the detection efficiency is improved. The present application lays a foundation for the further development of fast, high-sensitivity and label-free biosensors and POCT.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, and specifically relates to a method for accelerating the detection of analytes. Background Technology

[0002] Currently, significant progress has been made in the research and development of precise biomolecular detection technologies both domestically and internationally, particularly in molecular detection, sensor technology, artificial intelligence, and big data analysis, demonstrating strong growth momentum. Human health indicator detection technologies are developing towards ultra-sensitivity, high throughput, and multifunctional integration. Key technologies include: 1. Simoa (Single-Molecular Array) technology, which uses high-affinity antibodies or nucleic acid probes to bind target molecules and further enhances molecular capture efficiency through magnetic beads or other solid-phase carriers; the smaller reaction volume and efficient capture mechanism significantly improve the detection probability of target molecules. 2. Real-time quantitative PCR (qPCR), a powerful tool for detecting and quantifying biomolecules. By monitoring fluorescence signals in real time during the PCR reaction, the amplification of DNA or RNA can be tracked in each amplification cycle, thereby achieving precise quantification of nucleic acids. 3. Electrochemical sensing technology, which quantitatively analyzes target substances by detecting the current, potential, or resistance signals generated by the electrochemical reactions occurring at the interface between biomolecules and sensors. 4. Optical sensing technology, which detects signal changes based on the interaction between light and biomolecules; the main principles include absorbance, fluorescence, light scattering, light interference, and surface plasmon resonance (SPR).

[0003] Optical biosensors enable real-time, convenient, and cost-effective monitoring of biomolecules. Plasma biosensors, as an optical sensor, do not require relatively expensive proprietary instruments and possess high sensitivity and multiplexing potential. Plasma resonance biosensors, due to their ability to perform label-free testing of analytes, are increasingly being used in medical diagnostics, food safety, and environmental monitoring. The main working principle of plasma resonance biosensors lies in the sensitive response of surface plasmon resonance (SPR) between a noble metal layer and the surrounding medium to changes in the refractive index (RI) of the surrounding medium under the excitation of an external light source. 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 characteristic makes plasmonic resonance biosensors an indispensable tool for real-time and label-free analysis. Surface refractive index sensors, with their high sensitivity and versatility, have demonstrated enormous application potential in multiple fields.

[0004] Despite remarkable progress in improving the limits of detection (LOD) of biosensors, the requirements for detection metrics in actual clinical testing far exceed the LOD levels currently achieved in biosensor strategy research. From a practical perspective, a sensor is clinically useful only if it can accurately quantify the analyte within an acceptable timeframe (e.g., a few minutes). However, especially at ultra-low analyte concentrations, the detection time is often unacceptable due to significant delays caused by mass transport limitations in delivering biomolecules from the stock solution to the sensor capture volume.

[0005] To reduce response time, various 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. Furthermore, chaotic mixing in sample flow using three-dimensional serpentine microchannels has been shown to increase analyte binding by up to 2 times. Additionally, based on electrodynamic effects (electrophoresis, two-dimensional electrophoresis, and electrothermal plasma effects), target molecules can be more efficiently transported into plasma sensing structures by applying an external electric field and / or generating a temperature gradient. However, these strategies either require more complex microfluidic systems (such as bilayer microchannels or structured microchannels) or additional electrical and heating elements, which hinders the miniaturization of sensing systems. Summary of the Invention

[0006] To achieve rapid, label-free analysis of analytes, this invention provides a method for rapidly analyzing the concentration of analytes in complex systems within a real-time refractive index sensing system. This method accelerates the binding of analytes to receptors on the sensor surface and combines this with initial binding rate analysis (IRA) to quickly analyze the concentration of analytes.

[0007] The first objective of this invention is to provide a method for accelerating the detection speed of analytes.

[0008] The second aspect of this invention aims to provide an application of the method of the first aspect of this invention.

[0009] To achieve the above-mentioned objectives of this invention, the technical solution adopted by this invention is as follows:

[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 detection system is used to detect the sample to be tested, and the analyte concentration is calculated using the initial binding rate analysis.

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

[0013] When the sample to be tested is a simple system, an additional non-analyte is introduced before detection.

[0014] When the sample to be tested is a complex system, the sample is tested 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 are known. Examples include detection methods such as nasal swabs, throat swabs, and sampling brushes, where tissue samples are taken and placed in a detection buffer.

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

[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 standards in a laboratory or clinical setting, wherein the simple system consists of the analyte to be detected and a buffer solution.

[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 present 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, and tissue fluid.

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

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

[0025] Label-free analyte detection refers to the detection of analyte concentration by directly binding the analyte to receptor molecules on the sensor surface, causing a change in the sensor's refractive index. Here, the label refers to all other linking ligands, groups, molecules, etc., besides the analyte molecule itself. This includes not only traditional fluorescent, radioactive, or enzyme-labeled molecules, but also other macromolecules, metal particles, etc.

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

[0027] In some embodiments of the present invention, the sample to be tested does not contain artificially modified groups.

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

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

[0030] In some embodiments of the present invention, the nonanalyte is a protein.

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

[0032] In some embodiments of the present invention, the nonanalyte solution includes a buffer solution.

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

[0034] In some embodiments of the present invention, the ratio of the concentration of the non-analyte to the mass concentration of the analyte in the non-analyte solution is 1 to 1 × 10⁻⁶. 7 Preferably, it is 1 to 1 × 10⁻⁶. 5 More preferably, it is 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 binding initial reaction rate includes: obtaining a linear concentration fitting relationship using the initial binding rate over a period of time during 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 a constant coefficient, C is the analyte concentration, and B is a constant intercept.

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

[0039] In some embodiments of the present invention, the initial stage of the reaction is a stage in which the receptor-analyte binding reaction is dominant, occurring within 10 to 100 seconds of the start of the response. More preferably, it is 10 to 80 seconds. More preferably, it is 10 to 60 seconds.

[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 maintain a constant volume concentration, the reaction between the immobilized acceptor and the analyte in 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] In the formula, C is the concentration of the analyte in the solution, and R max Let R(t) be the maximum sensor response, and k be the sensor response at time t. a and k d These are the association rate and the dissociation rate, respectively.

[0044] In a first aspect of the invention, the fluid detection system with microchannels refers to a detection system (device) that requires the liquid to be tested to flow in the microchannel in real time, such as a microfluidic device (a system based on microchannels to process or manipulate tiny fluids), a biochip (based on protein-protein interactions), a biosensor (based on optical and / or electrical methods to identify enzymes or antibodies), an electrochemical sensor (based on changes in current generated by an electrochemical probe), spectrophotometry (based on detection of optical absorbance), or a combination of the above systems (technologies).

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

[0046] In some embodiments of the present invention, the detection method is a label-free detection method, where no external detection molecules, such as primers, nucleic acid aptamers, antibodies, etc., are introduced into the detection system, or the components of the detection system do not require artificial modification, such as modification with isotopes, biotin, fluorescent groups, etc. The method for improving the detection speed 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 analytes include at least one of nucleic acids, proteins, lipids, and carbohydrates.

[0048] The analyte detection includes real-time detection.

[0049] The term "point-of-care testing" refers to technologies or methods that can rapidly obtain test results after sample collection. Such testing is typically completed within a short time and aims to provide immediate feedback for timely decision-making and intervention. It is widely used in fields such as medicine, environmental monitoring, and food safety.

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

[0051] The analyte detection is not for diagnostic or therapeutic purposes.

[0052] The beneficial effects of this invention are:

[0053] This invention significantly shortens the response time of low-concentration analytes in a biosensor for real-time analyte monitoring by introducing non-analytes, and calculates analyte concentrations based on initial binding rates, greatly saving analysis time and improving detection efficiency. It lays the foundation for point-of-care testing (POCT) and pharmacokinetic analysis (POCT) by further developing rapid, highly sensitive, and label-free biosensors. Attached Figure Description

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

[0055] Figure 1 A flowchart for methods to accelerate the analysis and detection of analytes.

[0056] Figure 2 The response curves are for the pure antigen and the introduction of 4 times the amount of mixed protein.

[0057] Figure 3 The diagram illustrates the loss effect caused by the adsorption of the analyte on the inner wall of the tube in a microfluidic system.

[0058] Figure 4 The response curves are for the pure antigen and the introduction of a high concentration of contaminating proteins.

[0059] Figure 5 A schematic diagram of a method for accelerating the analysis of the initial binding rate in complex systems.

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

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

[0062] Figure 8 This is a comparison of the detection results of the method of the present invention with the detection results of commercial equipment.

[0063] Figure 9 The detection results of different AFP concentrations in Comparative Example 1 of this invention. Detailed Implementation

[0064] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0065] Figure 1 To accelerate the flowchart of analytical methods for analytes, for complex systems such as blood, serum, urine, and saliva, the surface refractive index sensor typically exhibits a real-time response based on the specific binding of the receptor and analyte. Due to the complex bodily fluid environment, non-specific consumption of the analyte in the tubing is negligible, accelerating binding kinetics and significantly shortening the response time. However, if the sample composition is relatively simple, containing only the analyte and buffer solution, non-specific consumption of the analyte occurs in the tubing system as the sample passes through, leading to a slow binding response. Furthermore, using Initial Reaction Binding Analysis (IRA) to calculate the analyte concentration can further improve the analyte detection rate by 228 times compared to no processing. Specific data are shown in the following examples.

[0066] Example 1: Effect of introducing 4-fold heterozygotes on the detection response

[0067] 1. Sample preparation:

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

[0069] Prepare a 10 ng / mL AFP antigen solution using PBS buffer.

[0070] Prepare a 100 μg / mL Anti-AFP solution using PBS buffer.

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

[0072] The UGNA sensor (disclosed in patent 2024113072459) was used to detect mixed antigen solution (40 ng / mL) and pure AFP antigen solution (10 ng / mL), and the detection data were analyzed. Specifically, the sensor chip was placed on the test stage, the syringe pump flow rate was set to 1 mL / h, phosphate-buffered saline (PBS) was injected into the microfluidic channel, the structural regions of the sensor chip were identified by microscope, the system was then switched to dark field mode, and reflectance spectra were continuously acquired. The reflectance spectrum was monitored in real time, and the entire liquid permeation process was monitored in real time.

[0073] 2. Sensor channel surface finishing:

[0074] 1) PBS washing: 15 min;

[0075] 2) Inject a 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. Wash with PBS for 15 minutes.

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

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

[0078] 3. Testing 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 min.

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

[0081] 4. Experimental Results:

[0082] Experimental results are as follows Figure 2 As shown, the mixed antigen reached adsorption equilibrium at 2160 s, while the pure AFP antigen solution reached adsorption equilibrium at 9910 s. For AFP only, the response (Δλ) after adsorption equilibrium was consistent for both solutions, proving that the sensor can accurately detect 10 ng / mL AFP antigen in both solutions. However, the response of the mixed antigen was significantly faster than that of the pure antigen. This is because the mixed antigen contains a total antigen concentration of 40 ng / mL, which is four times that of the pure AFP antigen. The microfluidic channel's adsorption of proteins in the analytical solution is non-specific, so the consumption of the analyte antigen in the mixed antigen solution is less than that in the pure antigen, resulting in an earlier response time with virtually no delay.

[0083] The relevant principles are as follows Figure 3 As shown, this illustrates the loss effect caused by analyte adsorption on the inner wall of the tube in a microfluidic system. I and II represent two cases with different antigen binding efficiencies on the sensor surface: (I) flow of a solution containing only the target antigen, and (II) antigen solution containing additional contaminating proteins. Figure 4 In the red curve (response curve for 10 ng / mL AFP), the response only begins after 4000 seconds. This delay is likely due to non-specific binding of antigen molecules to the inner wall of the injection tube. This leads 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, such as... Figure 3 As shown in Case I. To avoid this response delay at low concentrations, this embodiment adds a high concentration of impurity proteins to the antigen solution to block non-specific binding to the inner wall of the injection tube, as shown in Case II. This effectively prevents non-specific 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 The time response curves for two antigen detection methods are shown, with the blue and red curves representing scenarios I and II, respectively. When a low concentration of analyte is injected into the sensor channel, the pure analyte is non-specifically adsorbed by the tube wall in the early stages, requiring a relatively long time to accumulate to saturation. Therefore, the actual number of analyte molecules reaching the detection site in the initial detection phase is far less than the detection concentration, resulting in a delayed response. However, with the addition of a large amount of contaminating protein, the non-specific adsorption of analyte in the initial tube almost ceases to consume it, leading to a rapid response. Furthermore, the combination of contaminating protein addition and the initial rate analysis (IRA) method further shortens the detection time (t). I ≥t II ).

[0085] Example 2: The effect of high concentrations of contaminating proteins on the detection response

[0086] 1. Sample preparation:

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

[0088] Prepare a 1 ng / mL AFP antigen solution (1 ng / mL AFP-PBS solution) using PBS buffer.

[0089] Prepare a 100 μg / mL Anti-AFP solution using PBS buffer.

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

[0091] The UGNA sensor (disclosed in patent 2024113072459) was used to detect 1 ng / mL AFP-BSA solution and 1 ng / mL AFP-PBS solution, and the detection data were analyzed. Specifically, the sensor chip was placed on the test stage, the syringe pump flow rate was set to 1 mL / h, phosphate buffered saline (PBS) was injected into the microfluidic channel, the structural regions of the sensor chip were identified by microscope, the system was then switched to dark field mode, and reflectance spectra were continuously acquired. The reflectance spectrum was monitored in real time, and the entire liquid permeation process was monitored in real time.

[0092] 2. Sensor channel surface finishing:

[0093] 1) PBS washing: 15 min;

[0094] 2) Inject a 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. Wash with PBS for 15 minutes.

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

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

[0097] 3. Testing 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] 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 solution injection (within seconds), while in PBS, the response only began after approximately 5500 s. With increasing AFP antigen concentration, the amount of antigen adsorbed corresponding to the response moved slowly over time, consistent with the expected trend of increased adsorption on the sensor surface due to increased antigen concentration. However, waiting for the reaction to reach equilibrium (approximately 15-20 minutes), then rinsing off excess sample from the channel and calculating the shift, is both time-consuming and inefficient, unsuitable for developing POCT strategies. By combining IRA with the former, analyzing the slope of the response starting 40–50 s, this embodiment achieved a faster concentration analysis than the latter equilibrium state (~11400 s), thereby improving detection and analysis time by 228 times.

[0102] Clinically relevant biosensor samples contain not only analytes but also a large number of background molecules. During incubation of the sample with the receptor surface, background proteins compete with the analyte for receptor binding sites. The binding of analytes to background proteins depends on the receptor specificity and the concentration of all molecules; this model is known as the Langmuir competition model. All non-analyte molecules are collectively referred to as background molecules, and the concentration fraction of the analyte, c, is used. a Concentration fraction c of background molecules b It has the following relationship:

[0103] c a +c b =1

[0104] In this case, all molecules other than the analyte are collectively referred to as background molecules. Therefore, the surface coverage of the competitively adsorbed analyte a and background molecule b to the acceptor molecule in the solution are respectively:

[0105]

[0106]

[0107] Where K a and K b Let θ be the adsorption constant. a and θ b These represent the surface fractions covered by adsorbed molecules a and b, respectively. When C a <<C b θ bThis can be ignored. It is directly reflected in the resonance response, so the introduction of a large number of non-analytes such as extraneous proteins does not affect the test results.

[0108] Example 3: Introducing a high concentration of contaminating proteins and using IRA analysis to plot a standard curve.

[0109] 1. Sample preparation:

[0110] Prepare BSA-PBS buffer solutions of 60 g / L, 70 g / L and 80 g / L using PBS buffer, and set up AFP antigen gradients (0, 1, 10, 100, 1000 ng / mL). Prepare AFP antigen solutions of corresponding concentration gradients using the above three BSA-PBS buffer solutions.

[0111] Prepare a 1 ng / mL AFP antigen solution using PBS buffer.

[0112] Prepare a 100 μg / mL Anti-AFP solution using PBS buffer.

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

[0114] The antigen solution was detected and the data analyzed using a UGNA sensor (disclosed in patent 2024113072459). Specifically, the sensor chip was placed on the test stage, 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 regions of the sensor chip were identified using a microscope. The system then switched to dark-field mode and began to continuously acquire reflectance spectra, monitor the reflectance spectra in real time, and monitor the entire liquid flow process in real time.

[0115] 2. Sensor channel surface finishing:

[0116] 1) PBS washing: 15 min;

[0117] 2) Inject a 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. Wash with PBS for 15 minutes.

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

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

[0120] 3. Testing Process:

[0121] Inject the following solutions into different channels: 0 ng / mL AFP (60 g / L BSA-PBS), 1 ng / mL AFP (60 g / L BSA-PBS), 10 ng / mL AFP (60 g / L BSA-PBS), 100 ng / mL AFP (60 g / L BSA-PBS), 1000 ng / mL AFP (60 g / L BSA-PBS), 0 ng / mL AFP (70 g / L BSA-PBS), 1 ng / mL AFP (70 g / L BSA-PBS), 10 ng / mL AFP (70 g / L BSA-PBS), 100 ng / mL AFP (70 g / L BSA-PBS), 1000 ng / mL AFP (70 g / L BSA-PBS), 0 ng / mL AFP (80 g / L BSA-PBS), 1 ... The following solutions were tested: 10 ng / mL 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).

[0122] 4. Experimental Results:

[0123] Test results as follows Figure 6 , 7 As shown, the results represent the reading wavelength response and light intensity response, respectively. Figure 6 In this study, the AFP concentration response in 60 g / L, 70 g / L, and 80 g / L BSA-PBS buffers was tested three times to obtain the CI / t relationship, and the fitted linear relationship was as follows: Figure 7 In this study, 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 as follows: Furthermore, it was found that the concentration of BSA-PBS buffer had no effect on the relationship between C and ΔI / t, thus the influence of differences in serum composition among different individuals on the test results could be ignored.

[0124] The fitted curve, serving as the standard curve for the initial slope of clinical testing, needs to account for individual differences. Therefore, when different individuals have varying total protein concentrations, it is necessary to determine whether to introduce a compensation factor for correction.

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

[0126] Example 4: Detection of Complex Systems

[0127] 1. Sample preparation:

[0128] Prepare a 100 μg / mL Anti-AFP solution using PBS buffer.

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

[0130] In addition, serum samples were collected from seven patients for analysis.

[0131] The antigen solution was detected and the data analyzed using a UGNA sensor (disclosed in patent 2024113072459). Specifically, the sensor chip was placed on the test stage, 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 regions of the sensor chip were identified using a microscope. The system then switched to dark-field mode and began to continuously acquire reflectance spectra, monitor the reflectance spectra in real time, and monitor the entire liquid flow process in real time.

[0132] 2. Sensor channel surface finishing:

[0133] 1) PBS washing: 15 min;

[0134] 2) Inject a 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. Wash with PBS for 15 minutes.

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

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

[0137] 3. Testing Process:

[0138] The test substance (the aforementioned human serum sample) was injected into different channels respectively.

[0139] 4. Experimental Results:

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

[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) testing shows that the antigen to be tested does not need to be modified and can obtain a response at a concentration of 1 ng / mL (the reference cutoff value for clinical 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 the solvent.

[0146] Sample testing:

[0147] The test spot was aligned with the sensing area in the microchannel. The syringe pump continuously injected liquid at a rate of 1 mL / h, and the detector continuously recorded the reflectivity curve of the sensor surface.

[0148] 1) PBS washing: 15 min;

[0149] 2) Inject (40mM EDC and 10mM 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 for 50 min to fix, then wash with PBS for 15 min.

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

[0152] 5) Inject the analyte into different channels at concentrations of 1, 10, 100, 200, 400, 600, 800, and 1000 ng / mL of AFP solution.

[0153] 2. Experimental Results

[0154] The results are as follows Figure 9 As shown.

[0155] It can be seen that at concentrations of 100-1000 ng / mL, the analyte reaches equilibrium in approximately 1000-2000 s (within 30 min). However, at concentrations of 1 and 10 ng / mL, the time to initiate a response and reach equilibrium is very long, even exceeding 12000 s (2 h). The reasons for this phenomenon include: 1. The influence of fluid mass exchange and reaction kinetics; low-concentration reactants have slower reaction rates. 2. Non-specific consumption of fluid proteins by microfluidic channels, leading to the initial consumption of the low-concentration antigen fluid itself, thus causing a response lag.

Claims

1. A method for improving the detection speed of analytes for non-disease diagnosis and treatment purposes in the field of analyte detection; The analyte was AFP; The method describes a simple system as the test sample, where more than 90% of the substances in the test sample are known. The method includes the following steps: The detection system is used to detect the sample to be tested. Non-analytes are introduced into the sample to be tested, and the analytes are detected quantitatively or qualitatively using the initial binding rate. The analyte is a substance with a particle size of 0.1 to 1000 nanometers; The non-analyte is BSA protein; The testing method is a label-free testing method; The detection system includes a fluid detection system with microchannels; After the introduction of the non-analyte, the ratio of the concentration of the non-analyte to the mass concentration of the analyte is 10. 2 ~10 4 .

2. The application according to claim 1, characterized in that: The analyte detection includes real-time detection.

3. The application according to claim 1, characterized in that: The initial binding rate includes: obtaining a linear concentration fitting relationship using the initial binding rate over a period of time during the initial stage of the reaction, as shown in the following formula: lg( R / t) = A × lgC - B; in R represents the response change, t is time, A is a constant coefficient, C is the analyte concentration, and B is a constant intercept. The R includes at least one of intensity change I, wavelength change λ, and angle change θ; The initial stage of the reaction is dominated by the binding reaction between the receptor and the analyte, which occurs within 10 to 100 seconds after the response begins.

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