General microcell or biomacromolecule recovery method and device for SEEG implant system

Abstract

The application discloses a general microcell or biomacromolecule recovery method of SEEG implant system, which comprises the following steps: placing a temperature-adjustable and vibratable back plate and a pre-filled eluent pusher in a suitable temperature; inserting the pre-filled eluent pusher of the pre-filled eluent into the bottom of a corresponding elution tube, pushing the piston of the pre-filled eluent pusher to make the pre-filled eluent fill the elution tube and discharge the air in the tube; then, inserting an electrode or a probe into the elution tube from top to bottom, adjusting the liquid level of the eluent in the elution tube to make the electrode or the probe be totally soaked in the eluent, adjusting the temperature of the temperature-adjustable and vibratable back plate and starting vibration, rapidly eluting the cells adhered to the surface of the electrode or the probe, and then recovering the eluent containing the micro living cells or cell lysate after elution into the pre-filled eluent pusher by pulling the pre-filled eluent pusher.

Description

Technical Field

[0001] This invention relates to the medical or scientific research fields, and in particular to a method and apparatus for the recovery of trace cells or biological macromolecules in a SEEG implantation system. Background Technology

[0002] The basic principle of SEEG is to precisely implant multi-contact deep electrodes into specific areas of the brain using stereotactic neurosurgical techniques. This allows for the direct recording of electrophysiological activities in the cortex and deep structures, thereby reconstructing the spatial relationship between the epileptogenic network and functional brain regions in three dimensions. By implanting deep electrodes (approximately 0.8-1.1 mm in diameter) using stereotactic techniques, the electrical activity of deep brain structures (such as the hippocampus, insula, and cingulate gyrus) is directly recorded, compensating for the insufficient spatial resolution of scalp EEG and subdural electrode (ECoG). Its indications mainly include the following: 1) drug-resistant epilepsy; 2) epileptogenic foci that cannot be clearly identified by non-invasive examinations (such as MRI, PET, and scalp EEG); 3) suspected epileptogenic networks involving multiple lobes or deep structures; 4) the need to assess the spatial relationship between epileptogenic areas and functional areas (such as language, motor, and other brain functions). The specific operational procedures include: 1) Preoperative planning: Designing electrode pathways based on MRI / CT fusion images, avoiding blood vessels (often combined with vascular imaging such as DSA or MRV); 2) Electrode implantation: Using a robot-assisted or frame stereotactic system, firstly, a probe is used to pre-open the implantation tunnel, and then the electrode implantation operation is completed, covering the target brain region unilaterally or bilaterally; 3) Monitoring and electrical stimulation: Continuously record the EEG during the seizure and interictal periods (usually 1-2 weeks), and combine electrical stimulation to locate the functional area; 4) Radiofrequency thermocoagulation therapy: After the EEG monitoring is completed and the location of the epileptogenic focus is determined, the electrodes can be connected to a radiofrequency generator, and the epileptogenic focus can be thermally ablated using radiofrequency energy.

[0003] Traditional SEEG technology primarily involves implanting multiple electrodes at different spatial locations within the brain to collect and analyze electroencephalogram (EEG) signals. This analysis is used to determine whether brain tissue at different locations is epileptogenic, thereby determining the extent of surgical resection. Therefore, the core function of implanted SEEGs is to distinguish between epileptogenic lesions and healthy brain tissue. Currently, the differences in EEG characteristics at different electrode locations are routinely used as biomarkers to differentiate these regions. Recently, advancements in next-generation sequencing technology have enabled multi-omics sequencing using nucleic acids collected from trace amounts of cells. Researchers have simultaneously performed transcriptomic and DNA methylome analyses on trace amounts of brain tissue on the electrode surface after SEEG monitoring. This provides multi-dimensional information for epileptogenic network research and epileptogenic focus localization. Intracranial monitoring electrodes can not only obtain electrophysiological information from brain tissue but also acquire molecular diagnostic markers through multi-omics detection, significantly improving the information scale for SEEG localization of epileptogenic foci and making epileptogenic focus localization more precise.

[0004] Meanwhile, in the scientific research field, because SEEG electrodes can be implanted at any spatial location within the human brain, cells from any spatial location within the brain can remain attached to the removed electrode surface after monitoring. These living cells possess clear spatial positioning information within the brain, and the biomolecules extracted from these cells are invaluable research materials for studying the molecular mechanisms of brain science. There are existing reports of implanting intracranial monitoring electrodes into ectopic gray matter nuclei in patients with periventricular gray matter ectopicness. After SEEG monitoring, a small amount of brain tissue attached to the electrode surface was collected, cellular DNA was extracted, and whole-exome sequencing was performed, identifying important gene variations related to periventricular gray matter ectopicness. This provides important genetic information for etiological research of the disease.

[0005] The existing technology has the following main shortcomings:

[0006] 1. When recovering cells from the trace amounts of brain tissue attached to the surface of intracranial electrodes, it is difficult to wash out all the cells attached to the electrodes. Furthermore, some cells will be lost during the washing process due to centrifugation and other operations, resulting in a small total amount of cells recovered and a small total amount of nucleic acid extracted. The cost of amplifying and building libraries for trace amounts of nucleic acid is high, making it difficult to widely promote its clinical application. Therefore, there is an urgent need for new methods that can improve the washing efficiency.

[0007] 2. Multimodal detection technology should not only include nucleic acid detection, but also be able to achieve protein quantification to obtain more dimensions of molecular information. However, existing technologies can only obtain nucleic acid information through micro-volume nucleic acid amplification, but cannot obtain protein quantification information. Micro-volume proteins are difficult to amplify and detect. Therefore, there is an urgent need to improve the total protein recovery efficiency of micro-volume cells to meet the needs of protein quantification analysis.

[0008] 3. In order to improve the recovery efficiency of trace cells, it is necessary to fully immerse the intracranial electrode in a liquid environment so that the cells can be fully eluted to improve the recovery efficiency. However, if the volume of the liquid environment is too large, it will affect the yield of biomolecule extraction. It is necessary to add a cell centrifugation step to enrich the cells in order to reduce liquid dilution. However, the centrifugation step will be accompanied by a certain degree of cell loss, which will reduce the yield of biomolecules and lower the detection success rate.

[0009] 4. After the intracranial electrode is removed from the body, the live cells attached to the electrode surface will undergo molecular changes in transcriptome and proteome as the time of removal from the body increases and as temperature and environment change. Therefore, it is necessary to quickly "fix" the various molecular expression information of the cells when they are removed from the body. However, washing, enriching, centrifuging and other operations on live cells will greatly prolong the time of removal from the body, resulting in subsequent molecular detection results that cannot truly reflect the physiological functions in the body.

[0010] 5. After radiofrequency thermocoagulation treatment, the cells attached to the intracranial electrodes undergo degeneration and necrosis due to thermal damage. After electrode removal, it is impossible to effectively obtain information on the cells and biomolecules at the electrode implantation site. However, during the electrode implantation surgery, a probe is used to pre-expand the puncture channel at the electrode location. The puncture probe and the electrode have the same spatial position, and a small number of cells can be attached to their surfaces. By washing away the cells on the probe surface, local cell and biomolecule information can be obtained in advance before SEEG EEG monitoring, thus providing more molecular marker information for epileptogenic focus localization. Summary of the Invention

[0011] The purpose of this invention is to provide a universal method and device for recovering trace cells and biomolecules in a SEEG implantation system. This method is used in neurosurgical procedures, during the implantation of stereotactic electroencephalography (SEEG) intracranial electrodes, to recover the trace cells attached to the surface of the surgical probe and / or, after SEEG monitoring, the trace cells attached to the surface of the intracranial monitoring electrodes, as well as the recovery and extraction of biomolecules, thereby solving the bottleneck problems existing in the prior art.

[0012] According to one aspect of the present invention, a method for recovering trace amounts of cells or biomacromolecules from a SEEG implantation system includes:

[0013] Disposable right-hand threaded probe used during electrode implantation;

[0014] At least two removable, disposable elution tubes are mounted on the elution device body for eluting cells attached to the surfaces of at least two electrodes or probes, wherein the electrodes or probes (500) have threads (503) for increasing the amount of cells attached.

[0015] An adjustable temperature and vibrating back plate for contacting the elution tube is installed on the back of the elution device body.

[0016] Connect at least two pre-filled eluent propellers, which are located below the body of the elution device, to at least two elution tubes.

[0017] Before eluting and recovering cells attached to each electrode surface, the temperature-adjustable and vibrating backplate and the pre-filled eluent propeller involved in the elution and recovery are pre-filled with eluent and adjusted to the required operating temperature.

[0018] At the start of elution and recovery of cells attached to the surface of each electrode or probe, a pre-filled eluent pusher pre-filled with eluent is inserted into the bottom of the corresponding elution tube. By pushing the piston of the pre-filled eluent pusher, the pre-filled eluent fills the elution tube and expels the air inside the tube.

[0019] After the elution and recovery of cells attached to the surface of each electrode or probe begins, the electrode or probe is inserted into the elution tube from top to bottom. The level of the eluent in the elution tube is adjusted so that the electrode or probe is completely immersed in the eluent. The temperature of the adjustable and vibrating backplate is then adjusted and vibration is started to quickly elute the cells attached to the surface of the electrode or probe. Then, by pulling the pre-filled eluent pusher, the eluent containing trace amounts of live cells or cell lysate is recovered into the pre-filled eluent pusher.

[0020] Preferably, the eluent is prevented from becoming inactive by setting the appropriate temperature of the temperature-adjustable and vibrating back plate and the pre-filled eluent in the pre-filled eluent propeller 300 to the eluent operating temperature.

[0021] Preferably, the temperature is adjusted to the most suitable working temperature according to the intended use of different biomolecules and the different characteristics of the selected buffer solution, and the temperature is maintained at a constant temperature to reduce the degradation of biomolecules caused by temperature fluctuations.

[0022] Preferably, the temperature-adjustable and vibrating backplate includes: a micro-vibrating plate that is attached to the side of the backplate facing the washing tube to achieve horizontal vibration; and a semiconductor temperature controller that realizes heating and cooling functions.

[0023] Preferably, the difference between the inner diameter of the elution tube and the diameter of the electrode is less than 0.3 mm, so as to avoid the cells attached to the electrode surface being diluted by the elution solution to the point that cell molecules cannot be effectively extracted.

[0024] Preferably, the eluent is one of the following:

[0025] Cell lysis buffer used for RNA extraction;

[0026] Cell lysis buffer used for DNA extraction;

[0027] Cell lysis buffer used for protein extraction;

[0028] Cell lysis buffer used for lipid extraction;

[0029] Live cell buffer for live cell extraction.

[0030] Preferably, the pre-filled eluent propulsion device is connected to the eluent tube via a hose.

[0031] Preferably, the pre-filled eluent propulsion device includes: a needle that engages with the tubing; and a piston inserted into the needle.

[0032] Preferably, the general method for recovering trace cells or biomacromolecules in the SEEG implantation system further includes installing a brown observation window at the front of the elution device body to maintain the eluent in a light-proof state when extracting cellular lipid components.

[0033] Preferably, the eluent containing trace amounts of cells recovered from the two pre-filled eluent propellers is subjected to extraction of biomacromolecules and subsequent detection and identification.

[0034] According to a second aspect of the present invention, a universal micro-cell or biomacromolecule recovery device for a SEEG implantation system comprises:

[0035] At least two elution tubes installed in the middle of the elution device are used to elute cells attached to the surfaces of two electrodes or probes at at least two intracranial sites after completion of EEG monitoring, wherein the electrodes or probes (500) have threads (503) for increasing the amount of cells attached.

[0036] An adjustable temperature and vibrating back plate installed on the back of the elution device and connected to the elution tube;

[0037] An observation window installed at the front of the washing and elution unit; and

[0038] A pre-filled eluent propeller located outside the elution apparatus for connecting at least two pre-filled eluents to the at least two elution tubes;

[0039] Specifically, before eluting and recovering cells attached to the surface of each electrode or probe, the temperature-adjustable and vibrating backplate and the pre-filled eluent in the pre-filled eluent pusher are placed at a suitable temperature.

[0040] At the start of elution and recovery of cells attached to each electrode surface, a pre-filled eluent pusher pre-filled with eluent is inserted into the bottom of the corresponding elution tube, so that the pre-filled eluent pusher fills the elution tube with pre-filled eluent and expels the air in the tube.

[0041] After the elution and recovery of cells attached to each electrode surface begins, the electrode or probe is inserted into the elution tube from top to bottom. The level of the eluent in the elution tube is adjusted so that the electrode or probe is completely immersed in the eluent. The temperature of the adjustable and vibrating backplate is then adjusted to the working temperature of the eluent and vibration is started to quickly elute the cells attached to the probe surface. Then, by pulling the pre-filled eluent pusher, the eluent containing trace amounts of live cells or cell lysate is recovered into the pre-filled eluent pusher.

[0042] Compared with the prior art, the present invention has the following technical effects:

[0043] By using pre-filled elution buffer, live cells attached to the electrode or probe surface are directly lysed in a "one-step" manner, avoiding cell elution and centrifugation operations. This shortens the time window during which biomolecules may change after being removed from the body. Furthermore, the application of surfactants in the lysate reduces the probability that live cells may not be completely eluted from the electrode surface during enrichment, thereby increasing the yield of target molecules.

[0044] The pre-filled elution buffer can be freely selected according to the different needs of extracting biological macromolecules, which makes up for the shortcomings of existing technologies that can only collect nucleic acids, and realizes the comprehensive collection and detection of biological macromolecules in the brain.

[0045] Using a "micro-volume liquid elution system" results in a small liquid environment with controllable volume. The volume of elution solution can be added quantitatively according to subsequent detection requirements, reducing the degree of dilution of target molecules. Furthermore, it maintains constant temperature conditions during lysis, reducing molecular denaturation or cell apoptosis. The addition of ultrasonic oscillation improves elution efficiency, ensuring both the quality and quantity of target molecules for subsequent detection.

[0046] Meanwhile, addressing the clinical challenge of not being able to effectively obtain cellular and biomolecular information at the electrode implantation site after the electrode is removed due to the thermal damage effect of cells attached to the intracranial electrode after radiofrequency thermocoagulation treatment, the material selection for extraction has been optimized. Cells on the surface of the surgical probe during the SEEG implantation procedure are used as elution material, which allows for the acquisition of local cellular and biomolecular information in advance before SEEG EEG monitoring, thereby providing more molecular marker information for epileptogenic focus localization. Attached Figure Description

[0047] Figure 1 This is a schematic diagram of the universal micro-cell and biomacromolecule recovery method of the SEEG implantation system of the present invention;

[0048] Figure 2 , Figure 3 and Figure 4 These are, respectively, a front view, a side view, and a top view of the first embodiment of the universal micro-cell and biomacromolecule recovery device for the SEEG implantation system that implements the method of the present invention;

[0049] Figure 5 , Figure 6 and Figure 7 These are, respectively, a front view, a side view, and a top view of the second embodiment of the universal micro-cell and biomacromolecule recovery device for the SEEG implantation system that implements the method of the present invention;

[0050] Figure 8 This is a front view of the back panel of the present invention;

[0051] Figure 9 and Figure 10 This is a schematic diagram of electrode 500 inserted into elution tube 200.

[0052] Figure 11 This is a schematic diagram of the disposable thread probe of the present invention;

[0053] Figure 12This is a schematic diagram of the sealing cap 340 installed at the front end of the pre-filled eluent propulsion device 300 of the present invention;

[0054] Figure 13a yes Figure 12 Front view of the center sealing cap 340. Figure 13b yes Figure 13a A bottom view;

[0055] Figure 14 This is a schematic diagram of differentially expressed genes in electrode EZ and electrode CTR;

[0056] Figure 15 This is a KEGG functional enrichment analysis diagram of differentially expressed genes in electrode EZ and electrode CTR. Detailed Implementation

[0057] like Figures 1-8 As shown, a universal method for recovering trace amounts of cells and biomacromolecules from a SEEG implantation system according to the present invention includes:

[0058] At least two removable, disposable elution tubes 200 are mounted on the elution apparatus body 100 for eluting cells attached to the surfaces of at least two electrodes or probes.

[0059] An adjustable temperature and vibrating back plate 130 for contacting the elution tube 200 is installed on the back of the elution device body 100.

[0060] At least two pre-filled eluent propellers 300 located below the body 100 of the elution device are connected to at least two elution tubes 200.

[0061] Before eluting and recovering cells attached to the surface of each electrode or probe, the pre-filled eluent in the temperature-adjustable and vibrating backplate 130 and the pre-filled eluent propeller 300 involved in the elution and recovery is brought to a suitable temperature.

[0062] At the start of elution and recovery of cells attached to the surface of each electrode or probe, a pre-filled eluent pusher 300 pre-filled with eluent is inserted into the bottom of the corresponding elution tube 200. By pushing the piston of the pre-filled eluent pusher 300, the pre-filled eluent fills the elution tube 200 and expels the air inside the tube.

[0063] After the elution and recovery of cells attached to the surface of each electrode or probe begins, the electrode or probe 500 is inserted into the elution tube 200 from top to bottom. The level of the eluent in the elution tube 200 is adjusted so that the electrode or probe 500 is completely immersed in the eluent. The temperature of the adjustable temperature and vibrating backplate 130 is then adjusted and vibration is started to quickly elute the cells attached to the probe surface. Then, the eluent containing trace amounts of live cells or cell lysate is recovered into the pre-filled eluent pusher 300 by pulling it out.

[0064] Figures 2-4 This invention illustrates a first embodiment of a universal micro-cell and biomacromolecule recovery device for a SEEG implantation system, comprising:

[0065] At least two detachable disposable elution tubes 200 installed in the middle of the elution device are used to elute cells attached to the surfaces of two electrodes or probes 500 at at least two intracranial sites after completion of EEG monitoring. The elution tubes 200 are made of light-transmitting material.

[0066] An adjustable temperature and vibrating back plate 130 is installed on the back of the washing device and connected to the washing tube 200;

[0067] A semi-transparent brown observation window 110 installed at the front of the elution device to maintain the eluent in a light-proof state during the extraction of cellular lipid components; and

[0068] A pre-filled eluent propeller 300 located outside the elution apparatus for connecting at least two pre-filled eluents to at least two elution tubes 200;

[0069] Before eluting and recovering cells attached to each electrode surface, the temperature-adjustable and vibrating backplate 130 and the pre-filled eluent in the pre-filled eluent propeller 300 are kept at a suitable temperature.

[0070] At the start of elution and recovery of cells attached to each electrode surface, a pre-filled eluent pusher 300 pre-filled with eluent is inserted into the bottom of the corresponding elution tube 200, so that the pre-filled eluent pusher 300 fills the elution tube 200 with pre-filled eluent and expels the air inside the tube.

[0071] After the elution and recovery of cells attached to the surface of each electrode or probe begins, the electrode 500 is inserted into the elution tube 200 from top to bottom. The level of the eluent in the elution tube 200 is adjusted so that the electrode or probe 500 is completely immersed in the eluent. Then, the temperature of the adjustable temperature and vibrating back plate 130 is adjusted and vibration is started to quickly elute the cells attached to the probe surface. Then, by pulling the pre-filled eluent pusher 300, the eluent or cell lysate containing trace amounts of live cells is recovered into the pre-filled eluent pusher 300.

[0072] Specifically, by setting the appropriate temperature of the pre-filled eluent in the adjustable temperature and vibrating back plate 130 and the pre-filled eluent propeller 300 to the working temperature of the eluent, the deactivation of the eluent is prevented.

[0073] In the recycling device of the present invention, except for the elution tube 200 and the observation window 110, all other components are made of opaque material.

[0074] In this process, the temperature is adjusted to the most suitable working temperature and maintained at a constant temperature according to the purpose of extracting different biomolecules and the different characteristics of the added buffer solution, so as to reduce the degradation of biomolecules caused by temperature fluctuations.

[0075] For example, the present invention can select eluents with different components to be added according to extraction needs, including one of the following:

[0076] Cell lysis buffer for RNA extraction; the lysis buffer formulation is as follows: prepared with DEPC water, other components include 4M guanidine isothiocyanate, 25mM sodium citrate, 0.5% N-lauroyl sarcosine, 1% NP-40; 0.1M β-mercaptoethanol (added immediately before use); lysis temperature is 4°C, lysis time is 5 minutes;

[0077] Cell lysis buffer for DNA extraction; lysis buffer formulation: 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 10 mM EDTA (pH 8.0), 0.5% Triton X-100, 1 mg / mL proteinase K (added immediately before use); lysis temperature: 37°C, rapidly cooled to 4°C after 1 hour of lysis;

[0078] Cell lysis buffer for protein extraction; the lysis buffer formulation is: 50 mM Tris-HCl (pH 7.4-8.0), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA. Before use, add the following components: 1 mM PMSF (phenylmethylsulfonyl fluoride), 1× protease inhibitor cocktail (containing leucopeptide, aprotinin, etc.), 1 mM DTT (dithiothreitol); lysis temperature 4°C, lysis time 30 minutes;

[0079] Cell lysis buffer for lipid extraction; the lysis buffer formulation is: chloroform: methanol: water = 8:4:3 (volume ratio), and the additives include: 0.01% butylated hydroxytoluene, 1 mM EDTA, 10 mM Tris-HCl (pH 8.0), lysis temperature 4°C, lysis time 15-30 minutes, and operation in the dark throughout the process;

[0080] The live cell buffer used for live cell extraction can be supplemented with commercial cell culture medium depending on the target cells to be extracted, in order to maintain the maximum survival rate of the target cells. Live cells are eluted at 37°C throughout the process to maintain the physiological activity of the cells.

[0081] The adjustable temperature and vibrating backplate 130 includes: a micro-vibrating plate 131 that is attached to the side of the backplate 130 facing the eluent tube 200 to achieve horizontal vibration; and a semiconductor temperature controller 132 that achieves heating and cooling functions.

[0082] The difference between the inner diameter of the elution tube 200 and the diameter of the electrode 500 is less than 0.3 mm, so as to avoid the cells attached to the surface of the electrode 500 being diluted by the elution solution to the point that cell molecules cannot be effectively extracted.

[0083] The elution buffer is a nucleic acid lysis buffer used for RNA extraction.

[0084] The pre-filled eluent pusher 300 includes: a needle 330 connected to the elution tube 200; and a piston or needle handle 310 inserted into the needle 330. This invention delivers the eluent from the needle 330 into the elution tube 200 by pushing the piston 310, and recovers the cell-containing eluent back into the needle 330 by pulling the piston 310.

[0085] The pre-filled eluent pusher 300 has a needle 330 connected to the eluent tube 200, and a sealing ring 210 is installed between the needle 330 and the eluent tube 200.

[0086] In the first embodiment of the SEEG implantation system universal micro-cell recovery device, the pre-filled eluent propulsion device 300 is perpendicular to the eluent tube 200.

[0087] Figures 5-7 This illustration shows a second embodiment of the universal micro-cell or biomacromolecule recovery device for the SEEG implantation system of the present invention. The difference from the first embodiment is that the eluent tube 200 is connected to a pre-filled eluent propeller 300 via a hose 400. The hose 400 and the pre-filled eluent propeller 300 are L-shaped, i.e., the hose 400 is perpendicular to the eluent tube 200, while the pre-filled eluent propeller 300 is parallel to the eluent tube 200. The pre-filled eluent propeller 300 is inserted into the hose 400. A sealing ring 210 is installed between the hose 400 and the eluent tube 210.

[0088] The pre-filled eluent pusher 300 includes: a needle tube 330 that is connected to a tubing 400; and a piston or needle handle 310 inserted into the needle tube 330. This invention delivers the eluent from the needle tube 330 into the eluent tube 200 by pushing the piston 310, and recovers the cell-containing eluent back into the needle tube 330 by pulling the piston 310.

[0089] The needle 330 of the pre-filled eluent pusher 300 is inserted into the tubing 400. A sealing ring 210 is installed between the tubing 400 and the eluent tube 210.

[0090] Furthermore, this invention extracts RNA from the eluent containing trace amounts of cells recovered from the two pre-filled eluent propulsion devices 300 to obtain differentially expressed genes characterizing the two electrodes; and identifies the main signaling pathways affected by the differentially expressed genes through KEGG enrichment analysis.

[0091] The micro-cell recovery device of the present invention is applicable to stereotactic intracranial electrode implantation systems. It can recover cells attached to the surface of the puncture probe before electrode implantation, and can also be used to recover cells attached to the electrode surface after the completion of EEG monitoring following electrode implantation.

[0092] The recovery device consists of four main parts: an ultrasonic vibration temperature control backplate 130, a detachable disposable recovery pipeline 200, a pre-filled eluent pusher 300, and a disposable right-hand threaded probe.

[0093] Figure 11 A specific embodiment of the disposable right-hand threaded probe 500 of the present invention is shown. For example... Figure 11 As shown, the disposable right-hand threaded probe 500 includes an anti-slip nut 501 with longitudinal grooves; a screw 502; and a right-hand thread 503 etched on the screw 502. The screw 502 has a diameter of approximately 0.8 mm and a length of approximately 20 cm; the right-hand thread 503 has a thread width of 0.1 mm and a thread depth of 0.1 mm. The right-hand thread 503 increases the contact area between the probe and brain tissue, increasing the amount of cells attached to the surface. When the right-hand threaded probe 500 is implanted into the expansion tunnel, it enters the brain tissue with a right-hand rotation. During rotation, the thread grooves can cut through part of the brain tissue, allowing it to enter the thread. During removal, it is pulled out vertically without rotation, which can remove trace amounts of cells embedded in the thread gaps during insertion, increasing the number of cells collected.

[0094] Figure 12The pre-filled eluent injector 300 is shown in its sealed state. The pre-filled eluent injector 300 needs to be sealed and stored separately before performing micro-cell or biomacromolecule recovery operations to seal the eluent already loaded into the syringe 330. To this end, the present invention installs a sealing cap 340 at the front end of the syringe 330 to seal the eluent in the syringe. During use, the sealing cap is removed, and the syringe 330 is connected to the tubing 400 to assemble a complete working tubing. After elution, the syringe 330 is separated from the tubing 400, and the sealing cap 340 is replaced to ensure the eluent remains sealed.

[0095] Figure 13a and Figure 13b The structure of the sealing cap 340 is shown. The sealing cap 340 is a circular cap with a cap cavity 341, a cap top 342, and a cylindrical cap wall 343 for fitting onto the front end of the syringe 330.

[0096] This invention has the following features: A disposable right-hand threaded probe can obtain cell samples at the implantation site during electrode implantation surgery, while expanding the implantation tunnel (it does not require waiting for SEEG monitoring to finish; samples are obtained after electrode removal, avoiding the drawbacks of cell inactivation and apoptosis caused by thermocoagulation therapy). This provides molecular marker information and electrophysiological monitoring information simultaneously for qualitative diagnosis of epileptogenic areas. Simultaneously, the threaded structure effectively increases the number of cells carried on the probe surface, increasing both the quality and quantity of extracted samples, thereby improving the diagnostic efficacy of biomarkers. The ultrasonic oscillation temperature-controlled base has a vertical backplate made of aluminum alloy, which can be regulated by a semiconductor temperature controller or connected to an ice storage box or heater. Ice can be added to the ice storage box to maintain the backplate temperature at 4°C. The heater is a stepless constant temperature device that can freely adjust and maintain the backplate temperature between 25°C and 50°C. The backplate has multiple vertical independent grooves that can accommodate and fix multiple disposable recycling lines. Each groove is equipped with an ultrasonic oscillation device. When the device is turned on, it can cause the probe or electrode in the line to oscillate at high frequency, forming shear friction with the side wall of the line, which promotes the elution of attached cells.

[0097] The detachable disposable recycling tubing is a silicone tube that can be embedded and fixed in the groove of the temperature control base's thermostatic back plate. It is used to contain the eluent and carry the probes or electrodes to be eluted during the elution process.

[0098] The pre-filled eluent injector, similar in shape to a syringe, is for single use. It is pre-filled with eluent and individually packaged. Different types of eluent can be selected for pre-filling depending on the product to be recovered. For recovering live cells, live cell buffer can be used (the live cell buffer can be optimized by selecting neuronal culture medium, neural stem cell culture medium, or glial cell culture medium according to the type of cells to be extracted, prioritizing the survival rate of the target cells); for recovering RNA, RNA extraction cell lysis buffer can be used; for recovering DNA, DNA extraction cell lysis buffer can be used; for recovering proteins, protein extraction cell lysis buffer can be used; and for recovering lipids, lipid extraction cell lysis buffer can be used. After opening, the injector can be directly connected to the recovery tubing. Pushing the plunger fully fills the recovery tubing with pre-filled eluent and vents the air, then the probe or electrode to be recovered can be inserted.

[0099] The following examples illustrate how the SEEG electrode microcell elution and mRNA sequencing of this invention reveal potential biomarkers for epileptogenic foci.

[0100] I. Experimental Materials:

[0101] 1. Subject: A patient with drug-resistant epilepsy diagnosed with focal cortical dysplasia (FCD) underwent SEEG intracranial electrode implantation surgery. Postoperatively, long-term stereotactic video EEG monitoring was performed to determine the seizure onset zone and the unaffected zone. After monitoring, one SEEG electrode implanted in each of the two zones was removed. The electrode in the seizure onset zone was named EZ, and the electrode in the unaffected zone was named CTR. The trace cells attached to these two electrodes were used as material for RNA recovery.

[0102] 2. Intracranial implanted electrodes: The intracranial implanted electrodes used are deep intracranial electrodes (SDE-08 / 10 / 12 / 16) manufactured by Huake Precision (Beijing) Medical Equipment Co., Ltd.

[0103] 3. RNA extraction kit: Total RNA was extracted using the miRNeasy Mini Kit (Qiagen, 217004), and the lysis buffer provided in the kit was used as the elution buffer for RNA extraction.

[0104] 4. Use a 1mm diameter silicone tube as the elution tubing, fix the tubing vertically on the backplate, and connect the end of the tubing to a syringe pre-filled with 100ul of elution buffer.

[0105] II. Experimental Methods: 1. Preparation of the elution apparatus: The prototype device was pre-cooled by refrigerating it at 4°C for half an hour. During elution, the apparatus was removed from the refrigerator, and an ice pack was placed on the back plate to maintain the low temperature during the elution process. The lysis buffer from the miRNeasyMini Kit was pre-cooled at 4°C for half an hour. 100 μL of the pre-cooled buffer was injected into a syringe, and the syringe was connected to the end of the shaking tubing. The syringe plunger was slowly pushed to raise the buffer level to the top of the shaking tubing to expel air from the tubing.

[0106] 2. Elution of Electrode-Attached Cells: Immediately after the electrode is removed from the subject, insert the implanted end into the vibrating tubing. As the electrode insertion depth increases, slowly pull back the syringe plunger to lower the liquid level, ensuring it does not overflow the tubing, until the buffer completely submerges all metal contacts of the electrode. Place a high-frequency vibrator against the backplate and vibrate for 1 minute. Pull back the syringe plunger to completely re-enter the buffer solution into the syringe. Remove the syringe from the vibrating tubing and transfer all the buffer solution into a pre-cooled 1.5ml centrifuge tube for later use.

[0107] 3. MicroRNA Extraction and Quality Control: Following the instructions for use of the miRNeasy Mini Kit, the buffer was used for subsequent total RNA extraction, and the total RNA was finally eluted with 20 μL of RNase-free water. RNA quantification and quality control were performed using the Agilent 2100 microarray analysis system and the Agilent RNA 6000 Pico Kit (Agilent, 5067-1513).

[0108] 4. mRNA library construction and sequencing: After the mRNA passed quality control, subsequent library construction and sequencing were performed. For micro-volume RNA library construction, the SMART-Seq_V4 Ultra Low Input RNA Kit for Sequencing (clontech, 634891) was used. mRNA sequencing was performed using the NovaSeq X Plus platform (Illumina).

[0109] III. Experimental Results:

[0110] 1. RNA extraction quantification and quality

[0111] A total of trace RNA was extracted from two electrodes. The total amount extracted and the quality control results are shown in Table 1.

[0112] Table 1. Total RNA amount and quality extracted from electrode samples

[0113]

[0114] 2. Differentially expressed genes by RNAseq

[0115] Figure 14 Differentially expressed genes between electrode EZ and electrode CTR were displayed. Through library construction and sequencing of trace mRNA samples, and differential expression analysis, 2257 genes were significantly upregulated and 1809 genes were significantly downregulated in electrode EZ compared to electrode CTR. 58637 genes showed no significant difference in expression levels.

[0116] 3. KEGG functional enrichment analysis of differentially expressed genes:

[0117] Figure 15 KEGG functional enrichment analysis of differentially expressed genes in electrode EZ and electrode CTR is shown.

[0118] KEGG enrichment analysis of differentially expressed genes on electrode EZ and electrode CTR identified the MAPK signaling pathway as the main signaling pathway affected by the differentially expressed genes. At the same time, the differentially expressed genes were also enriched in signaling pathways related to immune activation, such as the TNF signaling pathway and NF-kappa B signaling pathway.

[0119] IV. Experimental Conclusions:

[0120] Using this prototype device, RNA can be efficiently extracted from a small number of cells attached to the electrodes, meeting the requirements of next-generation sequencing for both the total amount and quality of RNA. It can also be used to locate the epileptogenic focus area for the electrodes, providing additional molecular pathological information in addition to electrophysiological information, thus meeting the multi-dimensional data needs for epileptogenic focus localization and providing diversified technical support for scientific research and clinical diagnosis and treatment.

[0121] Although the present invention has been described in detail above, it is not limited thereto, and those skilled in the art can make various modifications based on the principles of the present invention. Therefore, all modifications made in accordance with the principles of the present invention should be understood to fall within the protection scope of the present invention.

Claims

1. A general-purpose microcell or biological macromolecule recovery method for SEEG implant systems, characterized by include: At least two removable, disposable elution tubes (200) are mounted on the elution device body (100) for eluting cells attached to the surfaces of at least two electrodes or probes (500), wherein the electrodes or probes (500) have threads (503) for increasing the amount of cells attached. An adjustable temperature and vibrating back plate (130) for contacting the elution tube (200) is installed on the back of the elution device body (100). Connect at least two pre-filled eluent propellers (300) located below the body (100) of the elution device to at least two elution tubes (200); Before eluting and recovering cells attached to each electrode surface, the pre-filled eluent in the temperature-adjustable and vibrating backplate (130) and the pre-filled eluent propeller (300) is placed at a suitable temperature; When preparing to elute and recover cells attached to each electrode surface, a pre-filled eluent pusher (300) pre-filled with eluent is inserted into the bottom of the corresponding elution tube (200). By pushing the piston of the pre-filled eluent pusher (300), the pre-filled eluent fills the elution tube (200) and the air in the tube is expelled. During the elution and recovery of cells attached to each electrode surface, the electrode or probe (500) is inserted into the elution tube (200) from top to bottom. The level of the eluent in the elution tube (200) is adjusted so that the electrode or probe (500) is completely immersed in the eluent. The temperature of the adjustable temperature and vibrating backplate (130) is then adjusted to the required operating temperature and vibration is started to rapidly elute the cells attached to the probe surface. Then, by pulling the pre-filled eluent pusher (300), the eluent or cell lysate containing trace amounts of live cells is recovered into the pre-filled eluent pusher (300).

2. The general method for recovering trace cells or biomacromolecules in the SEEG implantation system according to claim 1, wherein the temperature of the pre-filled eluent in the temperature-adjustable and vibrating backplate (130) and the pre-filled eluent pusher (300) is set to the corresponding working temperature of the eluent, thereby preventing the eluent from becoming inactive.

3. The method of claim 2, wherein the SEEG implant system is a general microcell or biological macromolecule recovery method. Depending on the intended use of the extracted biomolecules and the characteristics of the selected buffer solution, the temperature is adjusted to the most suitable operating temperature and maintained at a constant temperature to reduce the degradation of biomolecules caused by temperature fluctuations.

4. The method of claim 2, wherein the SEEG implant system is a general microcell or biological macromolecule recovery method. The temperature-adjustable and vibration-enabled backplate (130) includes: A micro-vibrating plate (131) that vibrates horizontally against the back plate (130) facing the elution tube (200); and A semiconductor thermostat (132) that enables heating and cooling functions.

5. The method for recovering trace amounts of cells or biomacromolecules from the SEEG implantation system according to claim 2, characterized in that, The difference between the inner diameter of the elution tube (200) and the diameter of the electrode (500) is less than 0.3 mm, so as to avoid the cells attached to the surface of the electrode (500) being diluted by the elution solution to the point that cell molecules cannot be effectively extracted.

6. The method of claim 2, wherein the SEEG implant system is a general microcell or biological macromolecule recovery method. Depending on the extraction requirements, select an eluent with different components, including one of the following: Cell lysis buffer for RNA extraction; the lysis buffer formulation is as follows: prepared with DEPC water, other components include 4M guanidine isothiocyanate, 25mM sodium citrate, 0.5% N-lauroyl sarcosine, 1% NP-40; 0.1M β-mercaptoethanol (added immediately before use); lysis temperature is 4°C, lysis time is 5 minutes; Cell lysis buffer for DNA extraction; lysis buffer formulation: 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 10 mM EDTA (pH 8.0), 0.5% Triton X-100, 1 mg / mL proteinase K (added immediately before use); lysis temperature: 37°C, followed by rapid cooling to 4°C after 1 hour of lysis; Cell lysis buffer for protein extraction; the lysis buffer formulation is: 50 mM Tris-HCl (pH 7.4-8.0), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA. Before use, add the following components: 1 mM PMSF (phenylmethylsulfonyl fluoride), 1× protease inhibitor cocktail (containing leucopeptide, aprotinin, etc.), 1 mM DTT (dithiothreitol); lysis temperature 4°C, lysis time 30 minutes; Cell lysis buffer for lipid extraction; the lysis buffer formulation is: chloroform: methanol: water = 8:4:3 (volume ratio), and the additives include: 0.01% butylated hydroxytoluene, 1 mM EDTA, 10 mM Tris-HCl (pH 8.0), lysis temperature 4°C, lysis time 15-30 minutes, and operation in the dark throughout the process; The live cell buffer used for live cell extraction can be supplemented with commercial cell culture medium depending on the target cells to be extracted, in order to maintain the maximum survival rate of the target cells. Live cells are eluted at 37°C throughout the process to maintain the physiological activity of the cells.

7. The method of claim 2, wherein the SEEG implant system is a general method for recovering microcells or biological macromolecules. The pre-filled eluent propulsion unit (300) is connected to the eluent tube (200) via a hose (400).

8. The method of claim 1, wherein the SEEG implant system is a general microcell or biological macromolecule recovery method. It also includes a brown observation window (110) installed at the front of the elution device body (100) to keep the eluent in a light-proof state when extracting cellular lipid components.

9. The method of claim 6, wherein the SEEG implant system is a general microcell or biological macromolecule recovery method. The eluent containing trace amounts of cells or biomacromolecules recovered from the two pre-filled eluent propulsion devices (300) is subjected to live cell or target molecule extraction and subsequent qualitative or quantitative detection.

10. A general-purpose microcellular or biological macromolecule recovery device for SEEG implant systems, characterized by include: At least two elution tubes (200) installed in the middle of the elution device are used to elute cells attached to the surfaces of two electrodes or probes (500) at at least two intracranial sites after completion of EEG monitoring, wherein the electrodes or probes (500) have threads (503) for increasing the amount of cells attached. A temperature-adjustable and vibrating back plate (130) installed on the back of the elution device and connected to the elution tubes (200); and a pre-filled elution propeller (300) located outside the elution device for connecting at least two pre-filled elution tubes (200) with elution solution. In this process, before eluting and recovering the cells attached to each electrode surface, the pre-filled eluent in the temperature-adjustable and vibrating backplate (130) and the pre-filled eluent propeller (300) is placed at a suitable temperature. At the start of elution and recovery of cells attached to each electrode surface, a pre-filled eluent pusher (300) pre-filled with eluent is inserted into the bottom of the corresponding elution tube (200) so that the pre-filled eluent pusher (300) fills the elution tube (200) with pre-filled eluent and expels air from the tube. After the elution and recovery of cells attached to the surface of each electrode begins, the electrode (500) is inserted into the elution tube (200) from top to bottom. The level of the eluent in the elution tube (200) is adjusted so that the electrode (500) is completely immersed in the eluent. The temperature of the adjustable temperature and vibrating backplate (130) is adjusted to the working temperature of the buffer solution and vibration is started to rapidly elute the cells attached to the probe surface. Then, by pulling the pre-filled eluent pusher (300), the eluent or cell lysate containing trace amounts of live cells is recovered into the pre-filled eluent pusher (300).

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