A biomolecule detection method and device based on water submergence effect and application thereof

Abstract

The application discloses a biomolecule detection method and device based on water plowing effect and application thereof, and belongs to the technical field of biology. The method comprises the following steps: introducing a liquid sample to be detected into a micro-nano flow channel, the inner wall surface of which is decorated with specific recognition molecules for target biomolecules, so that the liquid sample to be detected flows in the micro-nano flow channel and generates a water plowing electric signal; collecting the water plowing electric signal; and judging whether the target biomolecules are contained in the liquid sample to be detected according to the change of the water plowing electric signal. The detection process of the application does not depend on fluorescence, enzymes or other markers, and does not involve complex biochemical reaction steps. The detection signal is generated by the liquid flow itself, and an external excitation power source is not needed, so that the target biomolecules can be rapidly detected.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a biomolecular detection method, device, and application based on the water voltaic effect. Background Technology

[0002] The highly sensitive and rapid detection of biomolecules (such as proteins, nucleic acids, and pathogen antigens) is of paramount importance in disease diagnosis, pathogen screening, environmental monitoring, and life science research. To achieve this goal, immunoassay technology has become the cornerstone of this field, converting biorecognition events into readable signals through the high-affinity binding between target biomolecules and immobilized specific recognition elements (such as antibodies and aptamers).

[0003] Currently, the detection of biomolecules mainly relies on immunoassay techniques, such as enzyme-linked immunosorbent assay (ELISA), electrochemical immunoassay, and luminescent immunoassay. While these methods can detect biomolecules under laboratory conditions and have a certain level of sensitivity and specificity, they still have some limitations in practical applications. Specifically, existing detection methods typically require the introduction of fluorescent, enzyme, or electrochemical labels and rely on multi-step reaction or incubation processes, making the overall operation relatively complex. Furthermore, most detection methods require external light sources, power supplies, or dedicated detection equipment, making them highly dependent on the detection environment and instrument conditions. In addition, due to the involvement of labeling reactions, incubation, or signal amplification steps, the detection process often takes a considerable amount of time, making it difficult to meet the application requirements of rapid and on-site detection. These factors, to some extent, limit the application of biomolecule detection technologies in point-of-care testing scenarios.

[0004] Taking the early diagnosis of human immunodeficiency virus (HIV) infection as an example, its core antigen, HIV-1 p24, appears in the blood at a very early stage after infection, making it a key biomarker for shortening the detection "window period." However, although existing detection methods for p24 (such as fourth-generation ELISA) have integrated antigen detection, due to the aforementioned limitations, they are still difficult to achieve truly rapid, convenient, and low-cost widespread adoption in primary healthcare institutions and emergency screening sites.

[0005] Therefore, there is an urgent need to provide a biomolecule detection method that can obtain detection signals without the introduction of markers or external power supply, so as to achieve rapid detection of biomolecules and meet the application needs of on-site detection and instant detection. Summary of the Invention

[0006] To address the shortcomings of existing technologies and practical needs, this invention provides a biomolecule detection method, device, and application based on the water voltaic effect. This method introduces specific recognition molecules for biomolecules into a sensing structure possessing the water voltaic effect. The binding behavior of biomolecules in the sample causes changes in the electrical state within the sensing structure, which are then manifested as changes in the water voltaic electrical signal, thereby enabling the detection of biomolecules. The detection process does not rely on fluorescence, enzymes, or other markers, nor does it involve complex biochemical reaction steps. The detection signal is generated by the liquid flow itself, requiring no external excitation power source, making it suitable for rapid detection applications.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a biomolecule detection method based on the water voltaic effect. The method includes: introducing a liquid sample to be tested into a micro / nanochannel whose inner surface is modified with a specific recognition molecule targeting the target biomolecule; allowing the sample to flow within the micro / nanochannel and generating a water voltaic signal; acquiring the water voltaic signal; and determining whether the liquid sample contains the target biomolecule based on changes in the water voltaic signal. A schematic diagram of the detection method of the present invention is shown below. Figure 1 As shown.

[0009] In this invention, the water voltaic effect constitutes the fundamental physical mechanism for generating the detection signal. When liquid flows within a micro / nanochannel, a measurable potential difference is formed across the micro / nanochannel due to the interaction between the inner wall surface of the channel and ions in the solution. When a target biomolecule is present in the sample and specifically binds to the recognition molecules on the inner wall of the micro / nanochannel, this binding behavior causes changes in the channel interface charge state and local electric field distribution, thereby affecting the migration behavior of ions within the micro / nanochannel and further reflecting as changes in the water voltaic electrical signal. Through this process, a correspondence is established between the binding of the target biomolecule and the water voltaic electrical response, allowing the presence of the target biomolecule to be characterized by an electrical signal. A schematic diagram of the synergistic mechanism of the water voltaic effect and biorecognition is shown below. Figure 2 As shown.

[0010] This invention introduces the water voltaic effect as a signal generation and conversion mechanism, reducing the dependence on markers and external excitation conditions during the detection process, and simplifying the overall detection process while ensuring detection effectiveness.

[0011] In this invention, the water voltaic effect serves as the primary source of the detection signal, and its implementation is not limited to any specific structure or configuration. Any technical solution that utilizes the flow of liquid within micro / nano-scale channels, pores, or interface structures to generate potential or current signals, and uses these electrical signals for the detection of target biomolecules, is a reasonable extension of the inventive concept and falls within the scope of protection of this invention.

[0012] Preferably, the target biomolecule includes any one or a combination of at least two of proteins, nucleic acids, viral particles, or cytokines.

[0013] Preferably, the specific recognition molecule includes any one or a combination of at least two of the following: antibody, antigen-binding fragment, aptamer, or antigen-binding peptide.

[0014] In this invention, after the target biomolecule binds to its corresponding specific recognition molecule, the amplitude, phase, or waveform of the initial hydrovoltaic signal generated by the flow of the liquid sample to be tested changes.

[0015] Preferably, the method for immobilizing the specific recognition molecule on the inner wall surface of the micro / nano channel includes: immobilizing the specific recognition molecule on the inner wall surface of the micro / nano channel through physical adsorption, chemical bonding or a biotin-avidin system.

[0016] The micro / nano channels described in this invention can be varied in morphology according to actual application requirements. For example, the arrangement, number, and overall structure of the channels can be adjusted. The nanochannels can be single-channel structures, multi-channel combinations, or planar, membrane, or other micro / nano structures with equivalent functions.

[0017] As long as the above structure can generate a water-voltaic electrical response during liquid flow and modulate the response through biometric events, it should be considered as an equivalent technical solution of the present invention.

[0018] Preferably, the liquid sample to be tested includes any one of serum, plasma, whole blood, or body fluid.

[0019] Preferably, the water-volt signal includes an open-circuit voltage signal and / or a short-circuit current signal.

[0020] Preferably, the change in the hydroelectric signal includes: comparing and judging any one or at least two of the amplitude, frequency, phase, waveform or integral area of ​​the acquired hydroelectric signal with a preset threshold.

[0021] Preferably, the preset threshold is determined by testing the water voltaic signal of a negative control sample or a sample known to not contain the target biomolecule.

[0022] Preferably, the criteria for judgment include: when the difference between the water voltaic signal generated by the liquid sample to be tested and the water voltaic signal generated by the negative control sample or the sample known to not contain the target biomolecule is greater than 0.1V, it is determined that the target biomolecule exists in the liquid sample to be tested.

[0023] In this invention, the concentration of the target biomolecule can also be determined based on the correspondence between any one or at least two of the amplitude, frequency, phase, waveform, or integral area of ​​the water voltaic signal and the standard curve.

[0024] In one embodiment of the present invention, the determination criterion can be determined as follows: Under the same detection conditions, firstly, the water voltaic signal generated by a negative control sample that does not contain the target biomolecule is collected, and the statistical characteristics of this signal are used as a baseline signal; then, the water voltaic signal generated by the liquid sample to be tested is compared with the baseline signal, and when the difference between the two exceeds a preset determination threshold, it is determined that the target biomolecule exists in the liquid sample to be tested. The determination threshold is obtained based on the negative control sample under repeated testing conditions and is used to characterize the system noise and background fluctuation range, thereby achieving a reliable determination of the presence or absence of the target biomolecule.

[0025] In this invention, the acquisition and output format of the water voltaic signal can be adjusted according to specific application requirements. The water voltaic signal can be output in the form of voltage or current, or characterized in other electrical forms through signal conversion. Any electrical signal form based on the water voltaic effect and capable of reflecting the presence or changing trend of target biomolecules should be considered as an equivalent technical solution of this invention.

[0026] In a second aspect, the present invention provides an apparatus for implementing the biomolecule detection method based on the water voltaic effect described in the first aspect, the apparatus comprising: a sensing unit, a signal acquisition unit, and an analysis unit;

[0027] The sensing unit includes a substrate with micro-nano channels and micro-nano channels with inner wall surfaces modified with specific recognition molecules for target biomolecules. The sensing unit is used to perform actions including: allowing the liquid sample to flow and generating a water voltaic signal.

[0028] The signal acquisition unit is electrically connected to the sensing unit and is used to perform the following: acquiring water volt-electric signals;

[0029] The analysis unit is used to perform the following: comparing the collected water voltaic signal with a preset reference signal or threshold, and outputting a judgment result on the presence or concentration of the target biomolecule.

[0030] The above functional units can be designed as an integrated structure according to specific application requirements, or they can be combined and integrated in a modular form.

[0031] The detection process of this invention can be implemented either through an integrated detection device or through the collaborative operation of multiple functional modules. Without altering the core technical concept of this invention, the order of the steps in the detection process can be reasonably adjusted, and the integration method of each functional module can also be structurally changed according to specific implementation conditions.

[0032] This invention can be implemented either as a detection method or as a detection device for performing that method. Any device that employs essentially the same technical means as this invention and achieves the same or similar technical effects should be considered an equivalent substitute for this invention.

[0033] Preferably, the micro / nano channel is composed of hydrophilic materials, inorganic nanowire arrays, porous polymer membranes, or nanomaterials with surface charges.

[0034] Thirdly, the present invention provides a kit for detecting biomolecules, the kit comprising the apparatus described in the second aspect.

[0035] Preferably, the kit further includes any one or a combination of at least two of the following: a pretreatment buffer for the liquid sample to be tested, a negative control, or a positive control.

[0036] Fourthly, the present invention provides the application of the biomolecule detection method based on the water voltaic effect described in the first aspect, the device described in the second aspect, or the reagent kit for detecting biomolecules described in the third aspect in the preparation of products for early diagnosis or screening of viral infections.

[0037] In one embodiment of the present invention, the virus includes human immunodeficiency virus.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] (1) This invention introduces the water volt effect into the biomolecular detection process, using the water volt potential generated by the flow of liquid in micro-nano channels as the source of detection signal, which can directly generate measurable electrical signals, avoiding dependence on fluorescence, enzymes or electrochemical labels, and eliminating the need for complex reaction and incubation steps.

[0040] (2) This invention utilizes the change in the charge state of the micro-nano channel interface caused by the binding of biomolecules with their specific recognition molecules to modulate the water voltage signal, thereby realizing the direct correlation and conversion between bio-recognition events and electrical signals, which is beneficial to shorten the detection time.

[0041] (3) The present invention uses a water-voltaic sensing unit with a micro-nano flow channel structure. The influence of the interfacial electrical effect on the electrical signal during the liquid flow process is enhanced, thereby improving the modulation efficiency of the water-voltaic signal by the binding of the target biomolecule-specific recognition molecule and helping to improve the stability and repeatability of the detection signal. The detection signal comes from the water-voltaic effect generated by the liquid flow itself. The entire detection process does not require an external excitation power source, which helps to reduce system energy consumption and simplify the device structure. The detection method and device of the present invention are more suitable for application scenarios such as portable detection, on-site detection and instant detection. Attached Figure Description

[0042] Figure 1 This is a schematic diagram of the detection method of the present invention;

[0043] Figure 2 This is a schematic diagram illustrating the synergistic mechanism between the water-based effect and biometric recognition.

[0044] Figure 3 The voltage results for detecting HIV-1 p24 antigen using the method of this invention are shown in the figure.

[0045] Figure 4 The figure shows the results of the practicality test of the method of the present invention;

[0046] Figure 5 The graph shows the accuracy test results of the method of the present invention.

[0047] Figure 6 This is a diagram showing the specific detection results of the present invention. Detailed Implementation

[0048] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0049] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0050] Example 1

[0051] This embodiment uses the biomolecular detection method based on the water voltaic effect described in this invention to detect HIV-1 p24 antigen.

[0052] In this embodiment, the micro / nanochannel can be fabricated using conventional micro / nano fabrication or molding methods in the art, such as molding, etching, electrospinning, or porous membrane assembly to obtain a substrate with a micro / nano-scale channel structure. The specific recognition molecule can be immobilized on the inner wall surface of the micro / nanochannel through physical adsorption, chemical bonding, or a biotin-avidin system, enabling the channel to specifically bind to the HIV-1 p24 antigen. The acquisition of the water-voltaic signal can be achieved through a signal acquisition circuit electrically connected to both ends of the micro / nanochannel. The signal acquisition circuit includes an electrode interface and an electrical signal acquisition module for acquiring open-circuit voltage signals. The acquired water-voltaic signal can be analyzed by examining the signal amplitude, time evolution characteristics, or stable plateau level to determine the presence of the target biomolecule.

[0053] In this embodiment, pure water was used as a blank control sample, and solutions containing different concentrations of HIV-1 p24 antigen were used as test samples, covering a concentration range from low (pg / mL) to high (mg / mL). During the detection process, serum samples were introduced into the water-voltaic sensing unit, and the open-circuit voltage signal generated across the sensing unit was acquired without applying an external power supply. Experimental results showed that when the blank control sample was introduced, the measured water-voltaic voltage signal exhibited no voltage response; when HIV-1 p24 antigen was present in the sample, the generated water-voltaic voltage signal changed significantly compared to the blank sample, and this change showed a consistent trend with increasing antigen concentration. Within the tested concentration range, the peak value of the water-voltaic voltage signal gradually increased with increasing antigen concentration, forming a distinguishable electrical response. The water-voltaic signal could exhibit transient changes or form a relatively stable plateau signal over a certain time scale. The determination of the water-voltaic electrical signal was based on the transient response peak voltage generated after sample introduction. The results are as follows: Figure 3 As shown, in the initial stage when the serum sample enters the micro / nano channel and begins to flow, the water-voltaic voltage signal changes rapidly and reaches a peak. This peak voltage shows a significant difference compared to the blank control sample, and exhibits a consistent increasing trend with increasing antigen concentration, indicating the presence of HIV-1 p24 antigen in the serum sample. To verify the stability and repeatability of the detection results, water-voltaic sensors prepared in different batches were used, and the above detection process was repeatedly tested on multiple independent sensors. The water-voltaic signal change trends obtained from different batches of sensors and different devices were consistent, and no obvious abnormal responses or indistinguishable situations were observed, indicating that the detection method has good repeatability and consistency under multiple test conditions. To further verify the practicality of the method of the present invention, transient peak voltage responses were recorded under several representative antigen concentration conditions. The results are as follows: Figure 4As shown, the experimental results indicate that the transient peak voltages corresponding to different concentrations of HIV-1 p24 antigen (concentration 1: 500 pg / mL; concentration 2: 375 pg / mL; concentration 3: 250 pg / mL; concentration 4: 125 pg / mL) are distinguishable, and the peak voltages show a consistent increasing trend with increasing antigen concentration. Compared with the blank control samples, the samples containing the target antigen showed significant differences in the transient peak voltage response, thus enabling effective determination of the presence or absence of the target antigen and its relative concentration changes.

[0054] The results of the above embodiments demonstrate that the detection method based on the water voltaic effect proposed in this invention can effectively distinguish the presence or absence of target analytes under actual operating conditions, and produce stable, repeatable and discriminative electrical responses to HIV-1 p24 antigen in different concentration ranges.

[0055] Example 2

[0056] This embodiment is used to verify the accuracy of the biomolecule detection method based on the water voltaic effect described in this invention.

[0057] In this embodiment, samples known to be free of the target biomolecule HIV-1 p24 antigen (confirmed negative serum samples) were selected as the matrix samples. Known amounts of the target biomolecule HIV-1 p24 antigen were added to these matrix samples to construct test samples with different spiking levels, simulating the different concentrations of the target biomolecule in actual samples. These spiking levels covered low, medium, and high concentration ranges.

[0058] The spiked samples described in this invention were tested using the method described herein. The detection process, signal acquisition method, and judgment criteria were all performed in accordance with Example 1. By comparing the water voltaic signal responses generated by samples with different spiking levels, the consistency between the detection results and the actual spiking of the target biomolecules in the samples was evaluated.

[0059] The results are as follows Figure 5 As shown, the experimental results indicate that for samples with different spiking levels, the measured water voltaic signal response can accurately reflect the changing trend of the target biomolecule content. The detection results are in good agreement with the known spiking of the target biomolecules in the samples, and no detection results that deviate significantly from the actual situation were observed.

[0060] The above results demonstrate that the biomolecule detection method based on the water voltaic effect proposed in this invention can accurately reflect the presence and relative content changes of target biomolecules in samples with known actual conditions, verifying the accuracy of the method under actual detection conditions.

[0061] Example 3

[0062] This embodiment demonstrates the specificity of the method of the present invention.

[0063] In this embodiment, ascorbic acid, hemoglobin, oxalic acid, and bilirubin, which are non-target biomolecules that have certain similarities with the target biomolecule HIV-1 p24 antigen in terms of physicochemical properties or biological origin, were selected as endogenous control analytes. The detection method is the same as in Example 1.

[0064] During the detection process, the water voltaic signal response generated under conditions where only non-target biomolecules were present was recorded and compared with the water voltaic signal under conditions where target biomolecules were present. Simultaneously, a sample containing no target or non-target biomolecules (serum sample) was used as a blank control.

[0065] The results are as follows Figure 6 As shown, the experimental results indicate that when only non-target biomolecules are present in the sample, the collected water voltaic signal does not show significant changes compared with the blank control sample, and does not exhibit characteristic signal responses related to the binding of target biomolecules; however, when target biomolecules are present in the sample, water voltaic signal changes consistent with those in Example 1 with discriminative characteristics can be observed.

[0066] The above results demonstrate that the detection method of the present invention has good selectivity for target biomolecules, and non-target biomolecules do not elicit a water voltaic signal response similar to that of target biomolecules, thus verifying the specific detection capability of the method in complex sample environments.

[0067] In summary, the detection process of this invention does not rely on fluorescence, enzymes or other markers, nor does it involve complex biochemical reaction steps. The detection signal is generated by the liquid flow itself, without the need for an external excitation power source, enabling rapid detection of target biomolecules.

[0068] The present invention has been illustrated with the above embodiments to explain the detailed method of the present invention. However, the present invention is not limited to the detailed method described above, that is, it does not mean that the present invention must rely on the detailed method described above to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A biomolecule detection method based on the water voltaic effect, characterized in that, The method includes: introducing a liquid sample to be tested into a micro / nano channel whose inner wall surface is modified with a specific recognition molecule for the target biomolecule, allowing it to flow within the micro / nano channel and generating a water voltaic signal, acquiring the water voltaic signal, and determining whether the liquid sample to be tested contains the target biomolecule based on the changes in the water voltaic signal.

2. The biomolecule detection method according to claim 1, characterized in that, The target biomolecule includes any one or a combination of at least two of the following: proteins, nucleic acids, viral particles, or cytokines.

3. The biomolecule detection method according to claim 1, characterized in that, The specific recognition molecules include any one or a combination of at least two of the following: antibodies, antigen-binding fragments, aptamers, or antigen-binding peptides.

4. The biomolecule detection method according to claim 1, characterized in that, The method for fixing the specific recognition molecule to the inner wall surface of the micro / nano channel includes fixing the specific recognition molecule to the inner wall surface of the micro / nano channel through physical adsorption, chemical bonding or biotin-avidin system.

5. The biomolecule detection method according to claim 1, characterized in that, The liquid sample to be tested includes any one of serum, plasma, whole blood, or body fluids.

6. The biomolecule detection method according to claim 1, characterized in that, The water-volt signal includes an open-circuit voltage signal and / or a short-circuit current signal.

7. The biomolecule detection method according to claim 1, characterized in that, The changes in the hydroelectric signal include: comparing and judging any one or at least two combinations of the amplitude, frequency, phase, waveform or integral area of ​​the collected hydroelectric signal with a preset threshold. The preset threshold is determined by testing the water voltaic signal of a negative control sample or a sample known to contain no target biomolecule; The criteria for judgment include: when the difference between the water voltaic signal generated by the liquid sample to be tested and the water voltaic signal generated by the negative control sample or the sample known to not contain the target biomolecule is greater than 0.1, it is determined that the liquid sample to be tested contains the target biomolecule.

8. An apparatus for implementing the biomolecular detection method based on the water voltaic effect according to any one of claims 1-7, characterized in that, The device includes: a sensing unit, a signal acquisition unit, and an analysis unit; The sensing unit includes a substrate with micro-nano channels and micro-nano channels with inner wall surfaces modified with specific recognition molecules for target biomolecules. The sensing unit is used to perform actions including: allowing the liquid sample to flow and generating a water voltaic signal. The signal acquisition unit is electrically connected to the sensing unit and is used to perform the following: acquiring water volt-electric signals; The analysis unit is used to perform the following: comparing the collected water voltaic signal with a preset reference signal or threshold, and outputting a judgment result on the presence or concentration of the target biomolecule.

9. A reagent kit for detecting biomolecules, characterized in that, The test kit includes the apparatus of claim 8; The kit also includes any one or a combination of at least two of the following: a pretreatment buffer for the liquid sample to be tested, a negative control, or a positive control.

10. The application of the biomolecule detection method based on the water voltaic effect according to any one of claims 1-7, the device according to claim 8, or the reagent kit for detecting biomolecules according to claim 9 in the preparation of products for early diagnosis or screening of viral infections.

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