One-step monoclonal antibody electrochemical detection biosensor and preparation method and application thereof

Abstract

The application discloses a one-step monoclonal antibody drug electrochemical detection biosensor and a preparation method and application thereof. The application designs and screens an affinity specific DNA aptamer hairpin probe and uses the same as a specific capture unit, then uses a gold-sulfur bond to fix the DNA aptamer hairpin probe on a screen-printed electrode surface, meanwhile, uses a self-assembly interaction between a metal and a mercapto group to fix a blocking agent introduced on the screen-printed electrode surface, and blocks the excess electrode surface reaction sites, so that the one-step monoclonal antibody drug electrochemical detection biosensor is obtained. The one-step monoclonal antibody drug electrochemical detection biosensor has the advantages of simple operation, high sensitivity, less non-specific adsorption, high capture efficiency, stable physical and chemical properties, strong specificity and good selectivity, and can be used for capturing antibody drugs in complex biological samples.

Description

Technical Field

[0001] This invention belongs to the field of biomedical sensing and detection, specifically relating to a one-step electrochemical detection biosensor for monoclonal antibody drugs, its preparation method, and its application. Background Technology

[0002] Specific antibodies play an important role in clinical diagnosis and disease treatment. For example, some anti-double-stranded DNA (dsDNA) antibodies are valuable biomarkers for diagnosing autoimmune diseases [1]. For example, viruses often produce some specific antibodies in the early stages of infection, which are usually difficult to detect [2,3]. In addition to these disease-specific antibodies, the effectiveness of appropriate doses of therapeutic antibodies in treating cancer patients has been increasingly recognized in the past decade [4]. Therefore, it can be said that whether it is antibody biomarkers or therapeutic drug antibodies, real-time dynamic monitoring of their accurate concentration in vivo is of great practical significance and research value for effective diagnosis and personalized treatment of diseases. However, the current detection strategy dominated by ELISA often has problems such as cumbersome operation process, high price, and easy loss of reagent activity when detecting real blood samples. Therefore, the development of simple, rapid and effective antibody detection strategies is an urgent need to achieve accurate and personalized diagnosis and treatment.

[0003] Aptamers are single-stranded oligonucleotides that are systematically screened for ligands by exponential enrichment (SELEX) and can bind to targets with high specificity [5]. Nucleic acid aptamers are essentially functional nucleic acids with target-specific binding. Therefore, aptamers have many advantages over antibodies, such as low cost, programmable replication, high physicochemical stability, and ease of modification [6]. In addition, aptamers often undergo significant conformational changes after binding to the target, which provides great flexibility for designing novel biosensors with high sensitivity and high selectivity [7]. After nearly 30 years of development, the effectiveness of aptamers in clinical diagnosis, targeted therapy, and other fields has been increasingly recognized and focused on [8]. Among them, affinity biosensing strategies based on aptamers have been of great interest in the detection of different targets such as ions, small molecules, proteins, viruses, or cells.

[0004] Hairpin DNA is a secondary DNA structure in which two regions of the same strand are complementary in nucleotide sequence, and the base pairs form a double helix with each other, eventually forming an unpaired loop [9]. The chemical properties of nucleic acids make them easy to synthesize and modify. Therefore, a large number of studies have reported the modification of hairpin DNA structures into DNA structures with high bioavailability, regulatory capacity and multifunctionality

[10] . Hairpin DNA probes have high hybridization specificity and unique selectivity for target molecules such as DNA, small molecules and proteins

[11] . Kevin et al. pioneered the development of electrochemical switch biosensors based on hairpin DNA and successfully applied them to the direct and rapid detection of antibodies in whole blood

[12] . Due to the advantages of hairpin DNA-based electrochemical switch biosensors such as simplicity and speed, a large number of related studies have been reported in recent years. However, most of these reported strategies are used for nucleic acid detection based on Watson-Crick base pairing and are rarely used for the detection of other targets, especially some large protein molecules. This is mainly because some challenges have been encountered in designing and developing target protein affinity hairpin DNA, so the examples of using hairpin DNA-based electrochemical switch biosensors to detect proteins are relatively limited.

[0005] Based on the above background, the technical solution of this invention is to modify a specific aptamer chimeric hairpin DNA probe on a screen-printed electrode matrix. This not only combines the flexibility of DNA structure but also retains the advantage of high affinity between the aptamer and its target, which is a promising solution to the above problems.

[0006] Therefore, a simple, single-step electrochemical switch biosensor strategy was constructed for the first time to detect the therapeutic antibody bevacizumab. Compared with reported switch-based detection strategies, this Ach probe-based electrochemical switch biosensor does not require additional labeled antigens or other affinity ligands. Therefore, the Ach probe-based electrochemical switch biosensor is simpler in both operation and structural design. Furthermore, this aptamer-chimeric probe-based electrochemical switch biosensor retains the flexibility of hairpin DNA, which is of great significance for the rapid and efficient detection of therapeutic antibody drugs in plasma samples.

[0007] References:

[0008] [1]Bragazzi N L,Watad A,Damiani G,et al.Role of anti-DNAauto-antibodies as biomarkers of response to treatment in systemic lupuserythematosus patients:hypes and hopes.Insights and implications from acomprehensive review of the literature[J].Expert review of moleculardiagnostics,2019,19(11):969-978.

[0009] [2]Ita S,Hill AK,Lam E C,et al.High-Resolution Sequencing of ViralPopulations During Early SIV Infection Reveals Evolutionary Strategies forRapid Escape from Emerging Env-Specific Antibody Responses[J].Journal ofVirology,2018,DOI:10.1128 / JVI.01574-17.

[0010] [3]Tsai C,Robinson P V,de Jesus Cortez F,et al.Antibody detection byagglutination–PCR(ADAP)enables early diagnosis of HIV infection by oral fluidanalysis[J].Proceedings of the National Academy of Sciences,2018,115(6):1250-1255.

[0011] [4]Weiner L M,Surana R,Wang S.Monoclonal antibodies:versatileplatforms for cancer immunotherapy[J].Nature Reviews Immunology,2010,10(5):317-327.

[0012] [5]Tuerk C,Gold L.Systematic evolution of ligands by exponentialenrichment:RNAligands to bacteriophage T4 DNA polymerase[J].science,1990,249(4968):505-510.

[0013] [6]Chen K,Zhou J,Shao Z,et al.Aptamers as versatile molecular toolsfor antibody production monitoring and quality control[J].Journal of theAmerican Chemical Society,2020,142(28):12079-12086.

[0014] [7]Song S,Wang L,Li J,et al.Aptamer-based biosensors[J].TrAC Trendsin Analytical Chemistry,2008,27(2):108-117.

[0015] [8]Li J,Mo L,Lu C H,et al.Functional nucleic acid-based hydrogels forbioanalytical and biomedical applications[J].Chemical Society Reviews,2016,45(5):1410-1431.

[0016] [9]Bonanni A,Pumera M.Graphene platform for hairpin-DNA-basedimpedimetric genosensing[J].ACS nano,2011,5(3):2356-2361.

[0017]

[10] Zheng J, Yang R, Shi M, et al. Rationally designed molecular beacons for bioanalytical and biomedical applications [J]. Chemical Society reviews, 2015, 44(10): 3036-3055.

[0018]

[11] Riccelli PV, Merante F, Leung KT, et al.Hybridization of single-stranded DNA targets to immobilized complementary DNA probes: comparison of hairpin versus linear capture probes[J]. Nucleic acids research, 2001, 29(4):996-1004.

[0019]

[12] Vallée-Bélisle A, Ricci F, Uzawa T, et al. Bioelectrochemical switches for the quantitative detection of antibodies directly in whole blood [J]. Journal of the American Chemical Society, 2012, 134(37): 15197-15200. Summary of the Invention

[0020] To overcome the shortcomings and disadvantages of the prior art, the primary objective of this invention is to provide a method for preparing affinity-specific DNA aptamer hairpin probes. The affinity-specific DNA aptamer hairpin probes prepared by this method can be applied to one-step monoclonal antibody drug electrochemical detection biosensors, and its sensing and capture unit is specifically customized to obtain a development strategy and preparation method with higher affinity.

[0021] The second objective of this invention is to provide an affinity-specific DNA aptamer hairpin probe prepared by the above-described preparation method. This DNA aptamer hairpin probe has a secondary DNA structure in which two regions of the same strand are complementary in nucleotide sequence, and the base pairs form a double helix with each other, finally forming an unpaired loop.

[0022] The third objective of this invention is to provide a method for preparing a one-step electrochemical detection biosensor for monoclonal antibody drugs. This method includes a method for preparing an affinity-specific DNA aptamer hairpin probe. This method utilizes gold-sulfur bonding to bind a hairpin DNA probe (affinity-specific DNA aptamer hairpin probe) with a thiol-modified end to a pretreated screen-printed gold electrode (SPCE). A blocking agent is then used to self-assemble a monolayer on the screen-printed gold electrode to block excess active sites, ultimately obtaining a functionalized bioligand-modified biosensor material, i.e., a one-step electrochemical detection biosensor for monoclonal antibody drugs.

[0023] The fourth objective of this invention is to provide a one-step electrochemical detection biosensor for monoclonal antibody drugs prepared by the above-described preparation method. The sensing and capture unit of this sensor can be specifically customized to obtain a strategy with higher affinity. This sensor has the advantages of simple strategy, easy operation, high sensitivity, low non-specific adsorption, high capture efficiency, stable physicochemical properties, strong specificity, and good selectivity. It can be used for the capture of antibody drugs in complex biological samples.

[0024] The fifth objective of this invention is to provide an application of a one-step electrochemical detection biosensor for monoclonal antibody drugs, which can be used to detect clinical monoclonal antibody drugs.

[0025] The primary objective of this invention is achieved through the following technical solution:

[0026] A method for preparing an affinity-specific DNA aptamer hairpin probe includes the following steps: Optimizing the partial binding conditions of a linear DNA aptamer with bevacizumab by incubation to obtain a linear DNA aptamer-bevacizumab; then, diluting the linear DNA aptamer-bevacizumab at different folds and detecting its fluorescence value using a microthermophoresis apparatus (MST); further optimizing the binding conditions by setting different reaction temperatures and concentrations of the linear DNA aptamer and bevacizumab to find the optimal reaction temperature and concentration for binding between the linear DNA aptamer and bevacizumab; finally, designing complementary affinity base pairs with varying numbers and sequences at both ends of the linear DNA aptamer to form an affinity-specific DNA aptamer hairpin probe.

[0027] Preferably, the concentration of the linear DNA aptamer is 100 nM to 200 nM, the concentration of the bevacizumab is 0.001 to 1000 nM, the temperature is 22 to 25°C, and the incubation time is 20 to 30 min.

[0028] The second objective of this invention is achieved through the following technical solution:

[0029] An affinity-specific DNA aptamer hairpin probe was prepared by the above-described method.

[0030] Preferably, the structure of the affinity-specific DNA aptamer hairpin probe is as follows: Figure 10 As shown.

[0031] The hairpin structure of the affinity-specific DNA aptamer hairpin probe has a redox signal tag at one end and a conventional capping sequence at the other end.

[0032] Preferably, the affinity-specific DNA aptamer hairpin probe has a secondary DNA structure, in which two regions of the same strand are complementary in nucleotide sequence, and the base pairs form a double helix with each other, finally forming an unpaired loop.

[0033] Preferably, the redox signal probe is one of a fluorescent signal probe, methylene blue, ferrocene, toluidine blue, or porphyrin iron.

[0034] Preferably, the conventional end-capping sequence is a multi-carbon chain thiol sequence.

[0035] The third objective of this invention is achieved through the following technical solution:

[0036] A one-step method for preparing a biosensor for electrochemical detection of monoclonal antibody drugs includes the following preparation steps:

[0037] (1) Design and screen affinity-specific DNA aptamer hairpin probes;

[0038] (2) Pretreatment of screen printing electrode: The screen printing electrode in sulfuric acid solution is subjected to electrochemical pretreatment to obtain the pretreated screen printing electrode.

[0039] (3) Reduction treatment of affinity-specific DNA aptamer hairpin probe: The affinity-specific DNA aptamer hairpin probe powder prepared in step (1) is prepared into a solution of 1 μM to 10 μM. The disulfide bonds of the affinity-specific DNA aptamer hairpin probe are reduced by tris(2-carboxyethyl)phosphine (TCEP) reducing agent to obtain the reduced affinity-specific DNA aptamer hairpin probe.

[0040] (4) Construction of a one-step monoclonal antibody drug electrochemical detection biosensor: The affinity-specific DNA aptamer hairpin probe reduced in step (3) is incubated with the pretreated screen-printed electrode surface in step (2) to fix the DNA aptamer hairpin probe electrode surface. Then, the active sites on the electrode surface are blocked with a blocking agent solution. Finally, PBS solution is added to the electrode surface to preserve the electrode, thus obtaining the final functionalized one-step monoclonal antibody drug electrochemical detection biosensor modified with biological ligands.

[0041] Preferably, in step (1), in order to ensure the activity of the nucleic acid aptamer in the affinity-specific DNA aptamer hairpin probe, it is necessary to precisely control the number of bases in the neck of the affinity-specific DNA aptamer hairpin probe; wherein the bevacizumab aptamer Ach sequence is embedded in the affinity-specific DNA aptamer hairpin probe, and a fluorescent group is labeled at the 3' end of each affinity-specific DNA aptamer hairpin probe for the output of the micro thermophoresis (MST) experimental signal.

[0042] Preferably, the concentration of the sulfuric acid solution in step (2) is 0.5M to 2M.

[0043] Preferably, in step (2), the electrochemical test mixed solution of the screen-printed electrode pretreatment contains [Fe(CN)6]. 3- / 4- The solution concentration was 1 mM to 5 mM, and the potassium chloride solution concentration was 0.1 M to 0.2 M. Cyclic voltammetry parameters were set as follows: initial potential 0.5 V to 1 V, high potential 0.5 V to 1 V, low potential -0.2 V to 0.2 V, with other parameters set to instrument defaults. Electrochemical impedance spectroscopy parameters were set as follows: high frequency (High Freq) 1 to 2 × 10⁻⁶. 5 Hz, LowFreq is 0.1~1Hz, Amplitude is 0.01~0.1V, and the other parameters are the instrument's default settings.

[0044] Preferably, the screen-printed electrode in step (2) is a screen-printed gold electrode.

[0045] Preferably, the blocking agent in step (4) is one of 2-mercaptoethanol (MCH), polyadenylate sequence, bovine serum albumin (BSA), and poly(ethylene glycol) self-assembled monolayer (PEG-SAMSs), and the concentration of the blocking agent is 1 mM to 2 mM.

[0046] The fourth objective of this invention is achieved through the following technical solution:

[0047] A one-step electrochemical detection biosensor for monoclonal antibody drugs is prepared by the above-described method.

[0048] The fifth objective of this invention is achieved through the following technical solution:

[0049] An application of a one-step electrochemical detection biosensor for monoclonal antibody drugs, wherein the target analyte is dissolved in PBS and healthy human plasma, and the target analyte is detected by square wave voltammetry using the one-step electrochemical detection biosensor for monoclonal antibody drugs.

[0050] Preferably, the target substance is one of trastuzumab, rituximab, infliximab, cetuximab, BSA, human immunoglobulin, or bevacizumab.

[0051] Preferably, when the square wave voltammetry is used to detect the target object, the frequency is 25-50Hz, the pulse width is 0.005-0.045V, and the step potential is 0.002-0.004V.

[0052] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0053] (1) The one-step monoclonal antibody drug electrochemical detection biosensor obtained in this invention has the advantages of simple strategy, easy operation, high sensitivity, low non-specific adsorption, high capture efficiency, stable physicochemical properties, strong specificity and good selectivity. It can be used to capture antibody drugs in complex biological samples.

[0054] (2) The technical solution of this invention application is developed through a one-step detection strategy: using screen-printed electrodes as the substrate material, micro-thermophoresis (MST) technology is used to design and screen the thiol hairpin sequence of the affinity-specific DNA aptamer hairpin probe to select a structure with suitable affinity. Then, the affinity-specific DNA aptamer hairpin probe binds to the target antibody with specific affinity. At the same time, the binding will cause the end of the affinity-specific DNA aptamer hairpin probe carrying the signal molecule to be opened. The signal molecule moves away from the electrode surface, resulting in a signal difference before and after binding. Therefore, the instantaneous electrical signal difference can be used to show the detection result of the target analyte. There is no need to go through other signal amplification strategies or operations to amplify the signal, nor is it necessary to label the antigen or other affinity ligands. The detection result can be shown through one step.

[0055] (3) The technical solution of this invention uses micro-thermophoresis (MST) to screen affinity-specific DNA aptamer hairpin probes and applies them to the construction of a one-step sensor. They are modified and fixed on a screen-printed electrode matrix. The affinity-specific DNA aptamer hairpin probes can be used to achieve precise and comprehensive capture of the target by directly recognizing the target, thereby improving the accuracy of capture.

[0056] (4) The technical solution of the present invention introduces an appropriate blocking agent after modifying the ligand on the screen-printed electrode, so as to effectively block the excess reaction sites and reduce the non-specific adsorption caused by the electrode material.

[0057] (5) The technical solution of this invention uses screen-printed electrodes as the matrix. Compared with traditional gold disk electrodes or glassy carbon electrodes, screen-printed electrodes have advantages such as simple operation, simple steps, rich reaction sites, large specific surface area, good stability, and strong portability. Therefore, they can significantly improve the bonding density of ligands to increase the capture capacity of target substances.

[0058] (6) The one-step monoclonal antibody drug electrochemical detection biosensor prepared by the present invention can be applied to the capture of target antibody drugs in serum samples, which is conducive to industrialization and provides a potential platform for in vivo analysis of antibody drugs. Attached Figure Description

[0059] Figure 1 This is a schematic diagram of the affinity-specific DNA aptamer hairpin probes (H5-H10) in Example 1;

[0060] Figure 2 This is a curve fitting diagram of the affinity screening experiment of affinity-specific DNA aptamer hairpin probes (H5-H10) in Example 1;

[0061] Figure 3 To determine and optimize the initial concentration of affinity-specific DNA aptamer hairpin probes (H5-H10) in Example 1, the concentration of affinity-specific DNA aptamer hairpin probes (H5-H10) was first optimized so that its fluorescence intensity was between 200 and 1500, in order to meet the detection limit of the instrument.

[0062] Figure 4 This is a temperature diagram illustrating the optimization of the binding of linear DNA aptamers to bevacizumab using the MST binding assay in Example 1.

[0063] Figure 5 This is a diagram illustrating the time required to optimize the binding of linear DNA aptamers to bevacizumab using the MST binding assay in Example 1.

[0064] Figure 6 The following are characterization diagrams and feasibility verification diagrams of the one-step monoclonal antibody drug electrochemical detection biosensor obtained in Example 2, including cyclic voltammetry, electrochemical impedance spectroscopy and square wave voltammetry.

[0065] Figure 7 This is a graph showing the specificity and selectivity analysis of the one-step monoclonal antibody drug electrochemical detection biosensor obtained in Example 4;

[0066] Figure 8 The graph shows the linearity and sensitivity evaluation of the one-step monoclonal antibody drug electrochemical detection biosensor obtained in Example 5.

[0067] Figure 9 This is a performance evaluation graph showing the reusability, stability, and reproducibility of the one-step monoclonal antibody drug electrochemical detection biosensor obtained in Example 6.

[0068] Figure 10 This is a structural diagram of an affinity-specific DNA aptamer hairpin probe. Detailed Implementation

[0069] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0070] Example 1: Preparation of affinity-specific DNA aptamer hairpin probes

[0071] As a core component of a one-step electrochemical sensor, the affinity-specific DNA aptamer hairpin probe first requires sequence design and screening using the MST affinity assay. In this experiment, the concentration of the linear DNA aptamer and its binding temperature and time with bevacizumab were optimized. The optimization process was characterized using MST affinity binding curves. According to the instrument instructions, the fluorescence value of the linear DNA aptamer should ideally be between 200 and 1500 counts (the instrument's detection limit). Before determining the binding affinity, a binding check experiment was performed to determine whether there is affinity between the receptor (bevacizumab) and the ligand (linear DNA aptamer).

[0072] Because the loop in the affinity-specific DNA aptamer hairpin probe is composed of a bevacizumab-specific linear DNA aptamer, the optimal reaction temperature for the linear DNA aptamer hairpin probe and bevacizumab can be used for subsequent binding of the DNA aptamer hairpin probe to bevacizumab. In this experiment, the concentration of bevacizumab was 10 μM, and the concentration of the affinity-specific DNA aptamer hairpin probe was 100 nM. The experimental results optimized by MST are as follows: Figure 4 As shown in the experimental results, it was found that as the temperature increased from 22℃ to 35℃, the MST curve of the linear DNA aptamer-bevacizumab complex increased, while the MST curve of the linear DNA aptamer alone gradually decreased. The relative fluorescence values ​​of the two also gradually decreased, indicating that the binding gradually weakened. It can be observed that the binding of the linear DNA aptamer-bevacizumab gradually weakens with increasing temperature, and this trend of decreasing binding force due to temperature changes is very significant. The experiment found that the optimal experimental results were achieved between 22 and 25℃. Therefore, 22–25℃ can also be considered the optimal screening temperature for the affinity-specific DNA aptamer hairpin probes prepared later. Through this experiment, the reaction conditions for affinity-specific DNA aptamers were optimized, which are the suitable reaction temperatures for the affinity-specific DNA aptamer hairpin probes prepared later.

[0073] To save reaction time in subsequent screening experiments and obtain accurate and effective reaction times, the reaction time between the linear DNA aptamer and bevacizumab was further optimized, serving as a reference for subsequent reaction times. We set reaction times of 15 min, 30 min, 45 min, and 60 min. The optimized reaction time experimental results are shown below. Figure 5As shown, we found that the effect was slightly worse with a reaction time of only 15 minutes. After a reaction time of more than 30 minutes, the reaction fitting curves could basically overlap completely. The experiment found that the best reaction effect was achieved between 20 and 30 degrees Celsius, and the optimal incubation time was 20 to 30 minutes.

[0074] Similarly, to further determine and optimize the initial concentration of affinity-specific DNA aptamer hairpin probes, we first optimized the concentration before conducting the screening experiment for affinity-specific DNA aptamer hairpin probes.

[0075] The concentration of linear DNA aptamers (100 nM–500 nM) should be such that their fluorescence intensity is between 200 and 1500 ppm, in order to meet the instrument's detection limit. Figure 3 As shown, tests revealed...

[0076] The optimal concentration of linear DNA aptamers is between 100 nM and 200 nM.

[0077] After obtaining the optimal reaction conditions, complementary sequences of varying numbers and orders of affinity base pairs are designed and added to the extended strands at both ends of the specific linear DNA aptamer according to the base affinity rules. These base pair sequences consist of A / C / G / T, i.e., the neck structure in the hairpin probe shown in the diagram. This constitutes the affinity-specific DNA aptamer hairpin probe. This section only describes the hairpin probe structure design strategy. This experiment mainly involves designing different base sequences for the extended strands, and then adjusting these different base sequences. Based on the base affinity, we select an optimal base as the main base structure. Affinity hairpin probes are screened by adjusting the number of bases. The double-strand extension technique is relatively common and can usually be performed by a bioengineering company. For specific structures, please refer to [link to relevant documentation]. Figure 1 (H5-H10).

[0078] Example 2: Preparation of a one-step electrochemical biosensor for monoclonal antibody drug detection

[0079] The affinity-specific DNA aptamer hairpin probe (H8) obtained in Example 1 was applied to the fabrication of a specific biosensor. Characterization of the electrode fabrication process is crucial for clarifying the effectiveness of each step. In this experiment, we primarily used cyclic voltammetry and electrochemical impedance spectroscopy to characterize the electrode fabrication process. Cyclic voltammetry was used to characterize each step of the electrode process.

[0080] The specific steps for the pretreatment of the screen-printed gold electrode are as follows: Using the electrochemical cyclic voltammetry method, the standardized screen-printed gold electrode is cleaned after setting relevant parameters with a 0.5M sulfuric acid solution. This process is repeated multiple times until a stable voltammetric curve appears. Afterward, the electrode is rinsed thoroughly with PBS solution and pure water multiple times. The electrode pretreatment process is then complete. The electrochemical testing solution environment for the screen-printed gold electrode pretreatment is 5mM [Fe(CN)6]. 3- / 4- The solution contained 0.1M potassium chloride solution; the cyclic voltammetry parameters were set as follows: initial potential 0.5V, high potential 0.5V, low potential -0.2V, and other parameters were the instrument default settings. The specific preparation steps of the one-step electrochemical biosensor for monoclonal antibody detection are as follows:

[0081] Using a pretreated screen-printed gold electrode as the substrate material, the thiol hairpin sequence of the affinity-specific DNA aptamer hairpin probe selected in Example 1 was immobilized on the surface of the screen-printed gold electrode through gold-sulfur bonding. The affinity-specific DNA aptamer hairpin probe then binds to the target antibody through its specific affinity. A self-assembled monolayer of the blocking agent MCH is then used to modify the electrode to block excess active sites, ultimately yielding a functionalized, one-step electrochemical biosensor for monoclonal antibody detection. The blocking agent includes, but is not limited to, 2-mercaptoethanol (MCH), polyadenylate sequences, bovine serum albumin (BSA), and poly(ethylene glycol) self-assembled monolayers. This method utilizes reported blocking agents such as PEG-SAMSs. When affinity-specific DNA aptamer hairpin probes bind to screen-printed gold electrodes, the end of the hairpin probe carrying the signal molecule is opened. The electrode surface is first characterized using cyclic voltammetry and electrochemical impedance spectroscopy, followed by detection using square wave voltammetry (SWV). Because the signal molecule is far from the electrode surface, the signal difference before and after binding allows for the immediate presentation of the target analyte detection result using the instantaneous electrical signal difference. No additional signal amplification strategies or operations are required to amplify the signal, nor is additional labeling of antigens or other affinity ligands necessary. Detection can be completed in a single step. For details, please refer to [link to relevant documentation]. Figure 6

[0082] For a series of designed H5-H10 hairpin probes, a series of affinity experiments were conducted under the same experimental conditions, and a set of affinity data for comparison was obtained. (See attached data.) Figure 2 .

[0083] Example 3

[0084] The one-step monoclonal antibody detection electrochemical biosensor prepared in Example 2 was tested by square wave voltammetry.

[0085] (1) Optimization of parameters and frequency in square wave voltammetry

[0086] In square wave voltammetry, parameter settings are crucial. This experiment optimized several key parameters, primarily frequency, step potential (Incr E), and amplitude (pulse height). First, the frequency of the square wave voltammetry technique was optimized by setting different frequencies and comparing the detection results. It was found that as the frequency increases, the current signal also strengthens, and the background noise of the current response increases accordingly. However, the background noise is particularly low at 25Hz and 50Hz. Considering both background noise and current intensity, a more suitable frequency was selected as the optimized value for the next stage of the experiment.

[0087] (2) Optimization of square wave voltammetry parameters-amplitude

[0088] After optimizing the frequency, our next step was to optimize the amplitude, also known as the pulse width. We defined it as ΔEp, which determines the potential range and potential resolution involved in each cycle. Based on the previous optimization, the remaining parameters used the instrument's default values. In this experiment, the amplitude at which the current intensity was maximum was selected as the final optimized value. Besides frequency and amplitude, the step potential is also a crucial parameter in square wave voltammetry. It determines the potential rise in each step and plays a dominant role in the total detection time and sensitivity. Therefore, we optimized the step potential in the next step and selected the optimal step potential as the optimized value for the next stage of the experiment.

[0089] Example 4

[0090] The specificity and selectivity of the one-step monoclonal antibody drug electrochemical detection biosensor obtained in Example 2 were analyzed.

[0091] (1) Biosensor specificity assessment:

[0092] Sensor specificity emphasizes the sensor's ability to recognize a target analyte in complex environments containing structural analogues or other related substances. The specificity of this sensor was evaluated using rituximab, trastuzumab, infliximab antibody (10 μM), BSA (10 μM), human immunoglobulin, bevacizumab (10 nM), and mixtures thereof dissolved in PBS. Figure 7 As shown in Figure A, the current signals generated by proteins such as rituximab, infliximab, human immunoglobulin, and BSA are almost equivalent to those of the blank sample and the sensor's background value. However, when bevacizumab and other protein mixtures are added to the system, the sensor produces a current drop of almost the same intensity. This indicates that the sensor has good specificity for bevacizumab. Figure 7Using a histogram of current difference intensity, B more intuitively reveals that the signals generated by non-target proteins such as rituximab, infliximab, human immunoglobulin, and BSA are almost equivalent to the current signals generated by the blank sample, while the current signals generated by the target substance bevacizumab and the mixture are much stronger than those of other proteins. This more intuitively illustrates that the sensor has a high specificity for bevacizumab.

[0093] (2) Selectivity evaluation of biosensors

[0094] Selectivity is defined as the ability of a biosensor to measure and distinguish the presence of a target analyte in a blank biological matrix sample. For example... Figure 7 As shown in Figure C, the current difference between PBS and blank plasma is almost zero, while the current intensity generated by bevacizumab in PBS is relatively high, indicating that the sensor has good selectivity for bevacizumab in PBS. To further evaluate the selectivity of the sensor for bevacizumab in real plasma, we tested the signal generated by bevacizumab in plasma. Similarly, the current intensity generated by the sensor for bevacizumab in plasma is almost equivalent to the signal generated by bevacizumab in PBS, with only a 6.02% difference. These results further demonstrate that the sensor still has strong selectivity for bevacizumab in the complex plasma matrix, almost completely overcoming the interference in the plasma matrix.

[0095] Example 5

[0096] Linearity and sensitivity analysis of the one-step monoclonal antibody detection electrochemical biosensor obtained in Example 2.

[0097] The linearity and sensitivity of a sensor determine its applicability in practical applications. Considering that the developed sensor is a signal-decreasing type sensor, the initial signal is very high. Therefore, as... Figure 8 As shown in Figure A, the addition of a very low concentration (0.00001 nM) of bevacizumab (dissolved in blank plasma) did not result in a significant decrease in the electrochemical signal. Further, the concentration of the target analyte was gradually increased, and exponentially ( Figure 8 B) The linear range and sensitivity of the sensor were evaluated by concentration increase. It was found that the sensor exhibited good linearity within the concentration range of 0.001–1000 nM. Figure 8 C).

[0098] Example 6

[0099] The performance of the one-step monoclonal antibody detection electrochemical biosensor obtained in Example 2 was evaluated and analyzed, including its reusability, stability, and reproducibility.

[0100] In addition to the specificity, selectivity, linearity, and sensitivity evaluated above, the reusability, stability, and reproducibility of the sensor are also key parameters for evaluating its performance. First, the sensor interface capturing bevacizumab was washed with 6M guanidine hydrochloride at room temperature. After rinsing, PBS was added and the mixture was allowed to stand for 10 minutes before the electrical signal of the sensor was tested again. Figure 9 As shown in Figure A, the results demonstrate that the initial signal of the regenerated sensor can reach over 96%, which is within an acceptable relative standard deviation, indicating that the sensor has good regeneration performance. Next, the long-term stability of the sensor was evaluated. Figure 9 (B) Overall, the sensor exhibited good stability over 8 days. Subsequently, the reproducibility of the sensor fabrication process was evaluated. Five electrodes were fabricated, and three processes involving these five electrodes were characterized. We found that the electrode exhibited good inter-electrode stability in all three processes during fabrication, indicating that the sensor fabrication process has good reproducibility.

[0101] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing an affinity-specific DNA aptamer hairpin probe, characterized in that, The preparation steps include the following: The binding conditions of linear DNA aptamers and bevacizumab were optimized by incubation to obtain linear DNA aptamer-bevacizumab. Then, the linear DNA aptamer-bevacizumab was diluted at different folds and its fluorescence value was detected by MST. The binding conditions were further optimized by setting different reaction temperatures and concentrations of linear DNA aptamers and bevacizumab to find the optimal reaction temperature and concentration for the binding of linear DNA aptamers and bevacizumab. Finally, the linear DNA aptamer was designed with complementary affinity base pairs of different numbers and sequences at both ends to form an affinity-specific DNA aptamer hairpin probe. The structure of the affinity-specific DNA aptamer hairpin probe is shown below: ； The hairpin structure of the affinity-specific DNA aptamer hairpin probe has a redox signal tag at one end and a conventional capping sequence at the other end, wherein the Ach sequence is 5'-AAAAAGCGGTTGGTGGTAGTTACGTTCGCTTTTT-3'.

2. The method for preparing the affinity-specific DNA aptamer hairpin probe according to claim 1, characterized in that, The concentration of the linear DNA aptamer is 100 nM to 200 nM, the concentration of the bevacizumab is 0.001 to 1000 nM, the temperature is 22 to 25°C, and the incubation time is 20 to 30 min.

3. An affinity-specific DNA aptamer hairpin probe, characterized in that, It is prepared by the preparation method of any one of claims 1 or 2.

4. A method for preparing a one-step electrochemical detection biosensor for monoclonal antibody drugs, characterized in that, The preparation steps include the following: (1) Design and screen the affinity-specific DNA aptamer hairpin probe as described in claim 3; (2) Pretreatment of screen printing electrode: The screen printing electrode in sulfuric acid solution is subjected to electrochemical pretreatment to obtain the pretreated screen printing electrode; (3) Reduction treatment of affinity-specific DNA aptamer hairpin probe: The affinity-specific DNA aptamer hairpin probe powder prepared in step (1) is prepared into a 1μM~10μM solution, and the disulfide bonds of the affinity-specific DNA aptamer hairpin probe are reduced by tris(2-carboxyethyl)phosphine reducing agent to obtain the reduced affinity-specific DNA aptamer hairpin probe. (4) Construction of a one-step monoclonal antibody drug electrochemical detection biosensor: The affinity-specific DNA aptamer hairpin probe reduced in step (3) is incubated with the pretreated screen-printed electrode surface in step (2) to fix the DNA aptamer hairpin probe electrode surface. Then, the active sites on the electrode surface are blocked with a blocking agent solution. Finally, PBS solution is added to the electrode surface to preserve the electrode, and the functionalized one-step monoclonal antibody drug electrochemical detection biosensor modified with biological ligands is finally prepared.

5. The method for preparing a one-step electrochemical detection biosensor for monoclonal antibody drugs according to claim 4, characterized in that, In step (4), the blocking agent is one of 2-mercaptoethanol, polyadenylate sequence, bovine serum albumin, or polyethylene glycol self-assembled monolayer, and the concentration of the blocking agent is 1 mM to 2 mM.

6. A one-step electrochemical detection biosensor for monoclonal antibody drugs, characterized in that, It is prepared by the preparation method of any one of claims 4 or 5.

7. An application of the one-step electrochemical detection biosensor for monoclonal antibody drugs according to claim 6, characterized in that, The application involves dissolving the target substance in PBS and healthy human plasma, and then using a one-step monoclonal antibody drug electrochemical detection biosensor to detect the target substance via square wave voltammetry; the target substance is bevacizumab.

8. The application of the one-step monoclonal antibody drug electrochemical detection biosensor according to claim 7, characterized in that, When the square wave voltammetry is used to detect the target object, the frequency is 25~50Hz, the pulse width is 0.005~0.045V, and the step potential is 0.002~0.004V.

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