Method for chemically cracking biological particles based on low-voltage direct current and application

Abstract

The invention discloses a biological particle chemical cracking method based on low-voltage direct current and application, the cracking method adopts an electrode to realize efficient cracking of biological particles under a working voltage of less than or equal to 4.5 V. The cracking method has the core technical advantages that: 1) low-voltage cracking can effectively avoid a Joule heating effect and ensure the integrity of nucleic acid; electrochemical damage to nucleic acid under high voltage is avoided; (2) contents released by the biological particles can be directly used for nucleic acid amplification without centrifugal purification, so that complicated procedures such as centrifugation and purification required by traditional nucleic acid extraction are omitted; and 3) compared with a commercial lysis solution, the nucleic acid extraction efficiency of electric lysis is higher than that of the commercial lysis solution by one order of magnitude. The technology is matched with a miniaturized detection system, the whole process can be completed within 25 minutes, the detection sensitivity reaches 101 CFU / mL, and compared with a traditional method, the method has the advantages that the operation time is remarkably shortened, the cost is effectively reduced, and the method is particularly suitable for microfluidic automatic nucleic acid detection.

Description

Technical Field

[0001] This invention relates to the field of biochemistry, specifically to a method and application of low-voltage direct current electrochemical lysis of biological particles. Background Technology

[0002] Airborne transmission of microorganisms is a significant route of respiratory infection. Targeted detection encompasses a variety of biological particles, including microorganisms with intact cellular structures (such as bacteria, fungi, mycoplasma, and chlamydia) and animal and plant cells, as well as non-cellular microorganisms (such as viruses). Viruses (such as influenza viruses and coronaviruses) and drug-resistant bacteria (such as Klebsiella pneumoniae, methicillin-resistant Staphylococcus aureus, Acinetobacter baumannii, and Staphylococcus aureus) can spread via aerosols in healthcare facilities, community environments, and the food supply chain, leading to high morbidity and mortality rates. World Health Organization data shows that over one million people die globally each year from hospital-acquired infections, highlighting the urgency of rapid microbial detection technologies. Traditional detection methods, such as culture and microscopy, while capable of identifying pathogens to some extent, are time-consuming, taking 24-72 hours, making them unsuitable for on-site testing. While molecular diagnostic technologies, such as polymerase chain reaction (PCR) and recombinase polymerase amplification (RPA), have significantly improved detection speed as alternatives to traditional detection methods, their effectiveness is limited by the pretreatment process, especially the efficiency of microbial lysis.

[0003] Various cell lysis methods, including chemical, mechanical, electrochemical, thermal, optical, and electrochemical approaches, are used to extract genetic material from cells. Each method has its own advantages and disadvantages. For example, chemical lysis, while simple to operate, suffers from problems such as long mixing and reaction times, and interference from chemical reagents in amplification. Thermal lysis damages intracellular proteins, severely impacting the accuracy of detection results. Electroporation is a method that induces cell rupture by creating tiny pores in the cell membrane. This technique can be successfully demonstrated at the single-cell level and is particularly useful for laboratory equipment that needs to process large numbers of samples. However, electroporation has several limitations, including reliance on high-intensity electric fields (0.3–2.0 kV / cm), the need for a high-voltage power supply system, and the generation of Joule heating and bubble / electrochemical reactions in the working solution, which may lead to nucleic acid fragmentation or degradation. In contrast, electrochemical lysis requires no additional lysis reagents or complex equipment and is easily implemented on lab-on-a-chip devices. Electrochemical lysis is a technique based on electrochemical principles to disrupt the cell wall, effectively avoiding nucleic acid damage caused by Joule heating. Therefore, electrochemical lysis may be more convenient and effective.

[0004] Electrochemical lysis technology has developed rapidly in the field of microbial detection in recent years, but its practical application still faces significant challenges. Among current technical solutions, "Integrated microfluidic cell culture and lysis on a chip" discloses an electrochemical cell lysis device that cultures and lyses four different cell lines (HeLa, Jurkat, CHO-K1, and MCF-7) on a chip under 2.5V for 5 minutes. "High surface area cobalt aluminum layered double hydroxide printed electrodes for flexible supercapacitor and on-chip electrochemical bacterial lysing" discloses a high surface area cobalt aluminum layered double hydroxide (Co-Al LDH) for electrochemical bacterial lysis chips. This chip lyses Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris) and Gram-positive bacteria (Enterococcus faecalis, Staphylococcus aureus) within 2 minutes at 4.5V (SPCE) and 3.5V (Co-Al LDH / SPCE). Although these DC voltages are relatively low, their operation is complex and requires high-level condition control. Prolonged exposure to the applied potential after membrane rupture can cause irreversible damage to genetic material. It leads to high fragmentation of nucleic acids, making them unsuitable for nucleic acid amplification assays (NAAT). Summary of the Invention

[0005] This invention provides a low-voltage DC electrochemical lysis method for biological particles and its application, aiming to solve the technical problems of existing electrochemical lysis technologies, such as reliance on high voltage leading to sample damage, limited target microbial types, and inability to achieve automated nucleic acid detection. This method achieves efficient lysis of biological particles using electrodes at a working voltage of ≤4.5V. Its core technological advantages are: 1) Low-voltage lysis (0.5~4.5V) effectively avoids the Joule heating effect, ensuring nucleic acid integrity and avoiding electrochemical damage to nucleic acids at high voltage; 2) The contents released from the biological particles can be directly used for nucleic acid amplification without centrifugation and purification, eliminating the cumbersome centrifugation and purification processes required in traditional nucleic acid extraction; 3) The nucleic acid extraction efficiency of electrolysis is an order of magnitude higher than that of commercially available lysis buffers. This technology, coupled with a miniaturized detection system, can complete the entire process within 25 minutes, achieving a detection sensitivity of 10. 1 Compared with traditional methods, this method significantly shortens the operation time and effectively reduces costs, making it particularly suitable for microfluidic automated nucleic acid detection and providing a breakthrough solution for establishing novel rapid microbial detection technologies.

[0006] The technical solution provided by this invention is as follows:

[0007] A method for pyrolyzing biological particles based on low-voltage DC electrochemical methods, wherein the method uses electrodes to achieve efficient pyrolysis of biological particles at a working voltage of ≤4.5V;

[0008] The operating voltage is a DC voltage, including square wave, triangle wave, sine wave and mixed mode.

[0009] Furthermore, the bioparticles include:

[0010] (a) Microorganisms and plant and animal cells with cellular structures;

[0011] (b) Noncellular microorganisms;

[0012] Furthermore, the electrode is a structure or array of structures with a positive-to-negative electrode spacing in the range of 20 to 200 μm.

[0013] Furthermore, the electrode is one of the following: an interdigitated electrode, a planar capacitor electrode, or a MEMS-fabricated electrode array, or a combination of multiple such electrodes.

[0014] Furthermore, the electrode material includes gold, platinum, silver, carbon, titanium, titanium nitride, and indium oxide.

[0015] Furthermore, the positive and negative spacing between the electrodes is 100 μm;

[0016] The operating voltage is a DC voltage of 2V.

[0017] The present invention also provides an application of the low-voltage DC electrochemical pyrolysis method for biological particles as described above in the detection of substances contained in biological particles.

[0018] Furthermore, the contents released from the bioparticles after efficient lysis can be directly used for detection in nucleic acid amplification or non-amplification detection systems without the need for centrifugation purification, filtration or chemical neutralization steps;

[0019] The contents released by the biological particles include nucleic acids, proteins, organelles and their fragments.

[0020] Furthermore, the detection system includes electrochemical or time-resolved fluorescence, Raman spectroscopy, single-molecule fluorescence, optical detection methods, plasmon resonance, mass spectrometry, and nanopore detection;

[0021] The nucleic acid amplification methods include asymmetric recombinase polymerase amplification, nucleic acid sequence-dependent amplification, loop-mediated isothermal amplification, asymmetric polymer chain reaction or its derivatives, and the non-amplification methods include second- or third-generation sequencing, direct nanopore analysis, and CRISPR-Cas systems.

[0022] The present invention also provides an application of the low-voltage DC electrochemical lysis of biological particles method described above in a microfluidic chip.

[0023] Compared with the prior art, the beneficial effects of the present invention are:

[0024] 1. This invention can rapidly lyse biological particles such as bacteria, viruses, and fungi, and the contents of the biological particles can be directly detected by nucleic acid amplification or non-amplification methods through the detection system;

[0025] 2. The voltages used in this invention are all low DC voltages (≤4.5V), which improves safety and portability;

[0026] 3. This invention can lyse biological particles such as bacteria, viruses, and fungi individually, or lyse a mixture of them;

[0027] 4. The voltage used in this invention ensures the integrity of nucleic acids and avoids electrochemical damage to nucleic acids under high voltage;

[0028] 5. This invention reduces the reliance on specialized equipment and complex operations in traditional microbial detection. When paired with portable analytical instruments, it can build a miniaturized detection system, which is particularly suitable for microfluidic automated nucleic acid detection. Attached Figure Description

[0029] Figure 1 This is a flowchart of the rapid detection of Staphylococcus aureus using DC low-voltage electrochemical lysis according to the present invention;

[0030] Figure 2 This is a plate image of the lysate of Staphylococcus aureus under an electrode spacing of 100 μm in an embodiment of the present invention;

[0031] Figure 3 Here are PI staining images of Staphylococcus aureus in this embodiment of the invention: (a) without voltage applied; (b) with voltage applied;

[0032] Figure 4 The following are the results of real-time PCR of Staphylococcus aureus under different conditions in the embodiments of the present invention: (a) without voltage applied; (b) with voltage applied; (c) commercial lysis buffer; (d) comparison of results without voltage applied / with voltage applied / commercial lysis buffer.

[0033] Figure 5This is a gel electrophoresis image of Staphylococcus aureus lysed at different sodium hydroxide concentrations in an embodiment of the present invention. Detailed Implementation

[0034] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings. The described embodiments are one embodiment of the present invention, but not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available, and techniques not described in detail are performed according to standard methods well known to those skilled in the art. The reagents and instruments mentioned in this application are commercially available or otherwise publicly available. Other suitable tools or materials can be substituted. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0036] like Figure 1 As shown, this invention provides a low-voltage DC electrochemical pyrolysis method for biological particles. This method utilizes electrodes operating at a voltage ≤4.5V to achieve efficient pyrolysis of biological particles. The voltage is DC, but can be square wave, triangular wave, sine wave, or a mixed mode. Furthermore, various waveforms can be used within a voltage range of 0.5–4.5V to create a varying electric field intensity without switching the positive and negative electrodes.

[0037] Bioparticles may include:

[0038] (a) Microorganisms and plant and animal cells with cellular structures, such as bacteria, fungi, mycoplasma, chlamydia, etc.

[0039] (b) Non-cellular microorganisms, such as viruses.

[0040] When performing efficient lysis using the method provided in this embodiment, the applied low DC voltage range is generally 0.5 to 4.5V. This voltage range can effectively avoid the Joule heating effect, ensure the integrity of nucleic acids, and avoid electrochemical damage to nucleic acids under high voltage.

[0041] In practical applications, electrodes can be structures or arrays of structures with a positive-to-negative electrode spacing ranging from 20 to 200 μm. Electrodes can be one of the following: interdigitated electrodes, planar capacitor electrodes, MEMS-fabricated electrode arrays, or a combination of multiple types. Electrode materials include gold, platinum, silver, carbon, titanium, titanium nitride, and indium oxide.

[0042] The contents released from the efficiently lysed biological particles of this invention can be directly used in detection systems employing either nucleic acid amplification or non-amplification methods, without the need for centrifugation purification, filtration, or chemical neutralization. Nucleic acid amplification methods include asymmetric recombinase polymerase amplification (RPA), sequence-dependent amplification (NASBA), loop-mediated isothermal amplification (LAMP), asymmetric polymerase chain reaction (PCR), or derivatives thereof. Non-amplification methods include second- or third-generation sequencing, direct nanopore analysis, and CRISPR-Cas systems. Detection systems include electrochemical or time-resolved fluorescence, Raman spectroscopy, single-molecule fluorescence, optical detection methods, plasmon resonance, mass spectrometry, and nanopore detection.

[0043] Staphylococcus aureus is a common and highly pathogenic bacterium, and infections caused by it often bring many serious problems to human health.

[0044] This embodiment provides a method for rapid detection of Staphylococcus aureus based on low-voltage DC electrolysis. This method employs the aforementioned method based on low-voltage DC electrochemical lysis of biological particles, specifically including:

[0045] 1. DNA sequence design

[0046] The sequences of the molecular probes are shown in the table below.

[0047]

[0048] 2. Configure the real-time PCR reaction system

[0049] The quantitative real-time PCR reaction system consisted of: 10 μL of 2x SGExcel GoldStar TaqMan Mixture, 1 μL of upstream primer (10 μM), 1 μL of downstream primer (10 μM), 1 μL of DNA template, and enzyme-free water to a final volume of 20 μL. The reaction program included: 95°C pre-denaturation for 10 min; 95°C denaturation for 15 s, 40 cycles; and annealing / extension at 60°C for 1 min.

[0050] 3. Optimize electrode spacing / voltage / time for electrochemical pyrolysis.

[0051] In this invention, a low DC voltage is applied to the interdigitated electrodes, and the electrode locally generates the pyrolysis agent OH. - These hydroxide ions can disrupt cell membranes, thereby causing lysis of Staphylococcus aureus. During lysis, hydrogen ions (H+) are also generated at the electrode. +The lysate reacts with excess hydroxide ions to form a neutral pH lysis solution, eliminating the need for subsequent washing. Additionally, alkaline lysis was performed on Staphylococcus aureus using sodium hydroxide solutions of varying concentrations. Tris-HCl solution was immediately added for neutralization, followed by gel electrophoresis verification. See details below. Figure 5 .

[0052] In the electrochemical lysis study of Staphylococcus aureus, a series of experiments were designed and conducted to evaluate the effects of different voltages, durations, and electrode spacings on bacterial growth (bacterial suspension concentration was 10). 4 (CFU / mL). Common gold interdigitated electrodes with different electrode spacings were purchased from the market. The electrolysis effect was observed using the plate coating method. (See...) Figure 2 Simultaneously, 1 μL of electrolyte was used for quantitative real-time PCR. Combined with plate plot analysis, the experiment concluded that Staphylococcus aureus could be successfully lysed and its nucleic acid effectively extracted when the electrode spacing was 20–200 μm, the applied voltage was 0.5–2.5 V DC, and the lysis time was 1–3 min. In this example, the electrode spacing was 100 μm, the applied voltage was 2 V, and the duration was 1 minute.

[0053] To further verify that electrochemical lysis can effectively extract nucleic acids, Staphylococcus aureus was stained with propidium iodide (PI) before and after electrochemical lysis. High-resolution fluorescence microscopy images obtained through PI staining (e.g.) Figure 3 A clear red fluorescent signal could be observed. PI could only penetrate cells with damaged cell membranes and stain the internal nucleic acids red, clearly indicating cell lysis. To evaluate the effectiveness of nucleic acid extraction, a comprehensive analysis was performed using quantitative real-time PCR and NanoDrop to determine nucleic acid concentration. After multiple experiments and comprehensive analysis (n≥3), the optimal experimental conditions were finally determined: an applied voltage of 2V for 1 min. Under these conditions, quantitative real-time PCR was performed on different bacterial culture gradients: untreated, electrochemically lysed, and treated with commercial lysis buffer. The comparative results showed that... Figure 4 This demonstrates that the method employed in this invention has an sensitivity an order of magnitude higher than that of commercially available lysis solutions. The DC low-voltage electrochemical lysis method in this invention exhibits particularly significant advantages. Furthermore, this technology, coupled with a miniaturized detection system, can complete the entire process within 25 minutes, achieving a detection sensitivity of 10. 1 CFU / mL.

[0054] The low-voltage DC electrochemical lysis method for biological particles provided by this invention can also be applied in microfluidic chips, enabling on-site lysis of biological particles in channels with a width of less than 300 μm or chambers with a height of 300 μm or less. The contents released from the biological particles can be directly used for nucleic acid amplification without centrifugation purification, eliminating the cumbersome centrifugation and purification processes required for traditional nucleic acid extraction.

[0055] In summary, this invention provides a low-voltage DC electrochemical lysis method for biological particles. This method achieves efficient lysis of biological particles using electrodes at a working voltage of ≤4.5V. Its core technological advantages are: 1) Low-voltage lysis (0.5–4.5V) effectively avoids the Joule heating effect, ensuring nucleic acid integrity and preventing electrochemical damage to nucleic acids at high voltages; 2) The contents released from the biological particles can be directly used for nucleic acid amplification without centrifugation and purification, eliminating the cumbersome centrifugation and purification processes required in traditional nucleic acid extraction; 3) The nucleic acid extraction efficiency of electrolysis is an order of magnitude higher than that of commercially available lysis buffers. This technology, coupled with a miniaturized detection system, can complete the entire process within 25 minutes, achieving a detection sensitivity of 10. 1 Compared with traditional methods, this method significantly shortens the operation time and effectively reduces costs, making it particularly suitable for microfluidic automated nucleic acid detection and providing a breakthrough solution for establishing novel rapid microbial detection technologies.

[0056] The above description is merely the preferred embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions within the technical scope disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A method for pyrolyzing biological particles based on low-voltage direct current electrochemical lysis, characterized in that, The method employs electrodes to achieve efficient pyrolysis of biological particles at a working voltage of ≤4.5V; The operating voltage is a DC voltage, including square wave, triangle wave, sine wave and mixed mode.

2. The method for pyrolyzing biological particles based on low-voltage DC electrochemical methods according to claim 1, characterized in that, The bioparticles include: (a) Microorganisms and plant and animal cells with cellular structures; (b) Noncellular microorganisms; 3. The method for pyrolyzing biological particles based on low-voltage DC electrochemical methods according to claim 1, characterized in that: The electrodes are structures or arrays of structures with a positive-to-negative electrode spacing in the range of 20 to 200 μm.

4. The method for pyrolyzing biological particles based on low-voltage DC electrochemical methods according to claim 3, characterized in that: The electrode is one of the following: interdigitated electrode, planar capacitor electrode, MEMS-fabricated electrode array, or a combination of multiple such electrodes.

5. The method for pyrolyzing biological particles based on low-voltage DC electrochemical methods according to claim 3 or 4, characterized in that: The electrode materials include gold, platinum, silver, carbon, titanium, titanium nitride, and indium oxide.

6. The method for pyrolyzing biological particles based on low-voltage DC electrochemical methods according to claim 4, characterized in that: The positive and negative spacing between the electrodes is 100 μm; The operating voltage is a DC voltage of 2V.

7. The application of the low-voltage DC electrochemical pyrolysis method for biological particles as described in any one of claims 1-6 in the detection of substances contained in biological particles.

8. The application according to claim 7, characterized in that: The contents released from the bioparticles after efficient lysis can be directly used for detection in nucleic acid amplification or non-amplification detection systems without the need for centrifugation purification, filtration or chemical neutralization steps. The contents released by the biological particles include nucleic acids, proteins, organelles and their fragments.

9. The application according to claim 8, characterized in that: The detection system includes electrochemical or time-resolved fluorescence, Raman spectroscopy, single-molecule fluorescence, optical detection methods, plasma resonance, mass spectrometry, and nanopore detection. The nucleic acid amplification methods include asymmetric recombinase polymerase amplification, nucleic acid sequence-dependent amplification, loop-mediated isothermal amplification, asymmetric polymer chain reaction or its derivatives, and the non-amplification methods include second- or third-generation sequencing, direct nanopore analysis, and CRISPR-Cas systems.

10. The application of the low-voltage DC electrochemical lysis method for biological particles as described in any one of claims 1-6 in a microfluidic chip.

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