Integrated circuit impedance sensing system for tumor detection

Abstract

The invention discloses an integrated circuit impedance sensing system for tumor detection, relates to the technical field of tumor detection, and solves the problems that tumor detection needs a long time, is not suitable for rapid detection in an operation, is low in detection efficiency and automation degree, and is easy to cause detection errors caused by manual operation errors. An integrated circuit impedance sensing system for tumor detection comprises a semiconductor chip, a signal generator, an analog-to-digital converter, a field programmable gate array and an embedded silicon fan-out packaging structure, and a cell-sized microelectrode array is integrated on the surface of the semiconductor chip and used for capturing and adhering single cells. Through cooperation of the field programmable gate array and the transimpedance amplifier, when the semiconductor chip is detected, reliable detection and real-time processing of an ultra-weak electric signal are realized, so that the efficiency and the automation degree of the system are improved, and detection errors caused by manual misoperation are avoided.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of tumor detection, and particularly to an integrated circuit impedance sensing system for tumor detection. BACKGROUND

[0002] The incidence and mortality of malignant tumors are rising, and early diagnosis screening and treatment are conducive to improving the prognosis and quality of life of patients. At present, the early diagnosis screening methods of tumors mainly rely on tissue biopsy and imaging methods such as X-ray, ultrasound, computed tomography, magnetic resonance imaging, endoscopy, etc. Their application range is limited, and some examinations are invasive operations, which lead to poor patient tolerance. In addition, most of the above examination methods are complex in operation, require high professional requirements for detection personnel, have low accuracy and high cost, and are not conducive to wide promotion. Studies have shown that folate receptor-alpha (FR-α) is overexpressed on the surface of most tumor cells, while it is in a low expression state on the surface of normal cells. Therefore, FR-α is an important biomarker and has important application value in the rapid screening of epithelial tumor cells. Folic acid molecules and their derivatives have specific binding with FR-α and are widely used in tumor detection and imaging research.

[0003] The existing tumor detection requires a long time, and the efficiency and automation degree of detection are low, and detection errors caused by human operation errors are easy to occur; therefore, the existing needs are not met, and a kind of integrated circuit impedance sensing system for tumor detection is proposed. SUMMARY

[0004] The present application aims to provide an integrated circuit impedance sensing system for tumor detection to solve the problems of long time required for tumor detection, low efficiency and automation degree of detection, and detection errors caused by human operation errors in the background art.

[0005] To achieve the above-mentioned purpose, the present application provides the following technical scheme: an integrated circuit impedance sensing system for tumor detection, comprising a semiconductor chip, a signal generator, an analog-to-digital converter, a field programmable gate array and an embedded silicon fan-out package structure, the surface of the semiconductor chip is integrated with a microelectrode array of cell size, which is used for capturing and adhering single cells, and an integrated circuit is embedded below the microelectrode array, which is used for real-time amplification and transmission of impedance signals.

[0006] The signal generator is used to provide a sinusoidal excitation signal with a frequency range of 1 kHz-10 MHz to the semiconductor chip, so as to activate the target electrode channel in the microelectrode array;

[0007] The analog-to-digital converter is connected with the output end of the semiconductor chip, and is used for converting the analog impedance signal into a digital signal, the resolution of the analog-to-digital converter is not less than 16 bits, and the sampling rate is not less than 1 MS / s.

[0008] The field programmable gate array is in communication connection with the analog-to-digital converter and the signal generator, and is used for controlling the activation timing of the electrode channel, storing impedance data, and performing spectrum analysis and classification processing on the data.

[0009] The embedded silicon fan-out packaging structure adopts a polymer material to batch package the semiconductor chip, and realizes the patterning of the surface electrodes of the semiconductor chip and high-throughput manufacturing.

[0010] Preferably, the microelectrode array is composed of 22x16 electrodes, the diameter of a single electrode in the microelectrode array ranges from 10 to 50 μm, and the electrode spacing ranges from 20 to 100 μm.

[0011] Preferably, the integrated circuit includes a transimpedance amplifier, the transimpedance amplifier is responsible for processing signals from a row of electrodes, the transimpedance gain of the transimpedance amplifier can be programmed digitally, and ranges from 87 to 142 dBΩ, so as to adapt to different cell types and excitation amplitudes, the transimpedance amplifier is biased by a constant transconductance bias circuit to stabilize the transimpedance gain within a target temperature range, thereby ensuring the operation stability under environmental changes.

[0012] Preferably, the signal generator can programmably adjust the voltage amplitude of the output sine wave, and the range is 10 mVpp-1Vpp, the signal generator has a multi-channel switching function, and supports selective activation of target electrodes in the microelectrode array in row and column addressing modes.

[0013] Preferably, the embedded silicon fan-out packaging structure uses poly-p-xylylene C as a filling material, and the height difference between the embedded chip and the silicon substrate is less than 1 μm.

[0014] Preferably, the surface electrodes of the semiconductor chip form a metal layer through photolithography and metal sputtering process, the metal layer material is composed of 95% aluminum and 5% copper, and the thickness is 925 nm, platinum is deposited on the surface of the metal layer, and the excess metal is removed by ion beam etching, so as to create a patterned electrode without damaging the integrated circuit below the surface electrode.

[0015] Preferably, the microfluidic channel module is integrated with the semiconductor chip, and is used for automatically delivering the tumor biopsy sample to the surface of the microelectrode array.

[0016] An integrated circuit impedance sensing system for tumor detection, comprising the following steps:

[0017] S1: dissociate the tumor tissue sample into a single cell suspension and resuspend it in fresh culture medium, then transport it to the surface of the semiconductor chip through the microfluidic channel;

[0018] S2: output a sinusoidal excitation by the field programmable gate array to activate the corresponding electrode channel to measure the impedance of the single cell;

[0019] S3: continuously monitor the impedance between the electrodes through the external circuit and send it to the field programmable gate array for further processing;

[0020] S4: analyze and process the data by the field programmable gate array, and evaluate the tumor risk level according to the impedance measurement results.

[0021] Compared with the prior art, the beneficial effects of the present application are:

[0022] 1、The field programmable gate array and the transimpedance amplifier cooperate to enable the semiconductor chip to reliably detect and real-time process ultra-weak electrical signals during detection, thereby improving the efficiency and automation level of the system and avoiding detection errors caused by human operation errors.

[0023] 2、The tumor biopsy sample is separated into single cells and loaded onto the semiconductor chip for impedance measurement, the integrated circuit located directly below the surface electrode for electrical cell-substrate impedance sensing maximizes the signal-to-noise ratio, thereby distinguishing cancer cells from non-cancer cells at the single cell level, the proportion of cancer cells in the biopsy sample can indicate the risk level of tumor benignity or malignancy, and the entire process from sample collection to turnover can be completed within 20 minutes, so that the system can be adapted for intraoperative rapid detection. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 It is a single cell impedance sensing platform schematic diagram of the IC-ECIS system of the present application;

[0025] Figure 2 It is a schematic diagram of the patterned electrode capturing single cells of the IC-ECIS system of the present application;

[0026] Figure 3 It is a graph of impedance size of human gastric cancer cell line (SGC-7901) and its corresponding gastric epithelial cell line (GES-1), B lymphocytes, T lymphocytes and mesenchymal stem cells (MSC) verified by the single cell classification method based on IC-ECIS of the present application;

[0027] Figure 4 It is a schematic diagram of the single cell classifier based on impedance size of the present application;

[0028] Figure 5Impedance differentiation chart of mixed dyeing cell samples (GES-1 and SGC-7901) of the application;

[0029] Figure 6 Impedance-based cancer risk assessment standard chart of the application;

[0030] Figure 7 Single cell quantity chart separated from tumors of different sizes of the application;

[0031] Figure 8 Gain simulation result schematic diagram of semiconductor chips at different temperatures of the application;

[0032] Figure 9 Gain simulation result of semiconductor chips in a frequency range of the application;

[0033] Figure 10 Chip image schematic diagram bonded with gold wire of the application. DETAILED DESCRIPTION

[0034] The technical solutions in the embodiments of the application will be apparently and completely described below with reference to the drawings in the embodiments of the application. Obviously, the described embodiments are only part of the embodiments of the application, rather than all the embodiments of the application.

[0035] Please refer to Figures 1 to 7 An embodiment provided by the application is an integrated circuit impedance sensing system for tumor detection, which comprises a semiconductor chip, a signal generator, an analog-to-digital converter, a field programmable gate array and an embedded silicon fan-out package structure. The surface of the semiconductor chip is integrated with a microelectrode array of cell size, which is used for capturing and adhering single cells. The microelectrode array is embedded with an integrated circuit below, which is used for amplifying and transmitting impedance signals in real time.

[0036] The signal generator is used to provide a sine wave excitation signal with a frequency range of 1 kHz-10 MHz to the semiconductor chip, so as to activate the target electrode channel in the microelectrode array.

[0037] The analog-to-digital converter is connected with the output end of the semiconductor chip, and is used to convert the analog impedance signal into a digital signal. The resolution of the analog-to-digital converter is not less than 16 bits, and the sampling rate is not less than 1 MS / s.

[0038] The field programmable gate array is in communication connection with the analog-to-digital converter and the signal generator, and is used to control the activation timing of the electrode channel, store impedance data, and perform frequency spectrum analysis and classification processing on the data.

[0039] The embedded silicon fan-out package structure adopts a polymer material to batch package the semiconductor chip, so as to realize the patterning of the surface electrodes of the semiconductor chip and high-throughput manufacturing.

[0040] The microelectrode array is composed of 22x16 electrodes, the diameter of a single electrode in the microelectrode array ranges from 10 to 50 microns, and the electrode spacing ranges from 20 to 100 microns.

[0041] The integrated circuit includes a transimpedance amplifier responsible for processing signals from a row of electrodes, the transimpedance gain of the transimpedance amplifier can be programmed by numbers, ranging from 87 to 142 dBΩ, to adapt to different cell types and excitation amplitudes, the transimpedance amplifier is biased by a constant transconductance bias circuit to stabilize the transimpedance gain within the target temperature range, thereby ensuring operation stability under environmental changes.

[0042] The voltage amplitude of the sine wave output by the signal generator can be programmed to adjust, ranging from 10 mVpp to 1 Vpp, and the signal generator has a multi-channel switching function, supporting selective activation of target electrodes in the microelectrode array in row and column addressing mode.

[0043] The embedded silicon fan-out package structure uses poly-p-xylylene C as the filling material, and the height difference between the embedded chip and the silicon substrate is less than 1 microns.

[0044] The surface electrode of the semiconductor chip forms a metal layer through photolithography and metal sputtering process, the metal layer material is composed of 95% aluminum and 5% copper, the thickness is 925 nm, the surface of the metal layer is deposited with platinum, and the excess metal is removed by ion beam etching, to create a patterned electrode without damaging the integrated circuit below the surface electrode.

[0045] It also includes a microfluidic channel module integrated with the semiconductor chip, for automatically delivering tumor biopsy samples to the surface of the microelectrode array.

[0046] An integrated circuit impedance sensing system for tumor detection, comprising the following steps:

[0047] S1: dissociate the tumor tissue sample into a single cell suspension and resuspend it in fresh culture medium, then deliver it to the surface of the semiconductor chip through the microfluidic channel;

[0048] S2: output a sinusoidal excitation by the signal generator controlled by the field programmable gate array, activate the corresponding electrode channel to measure the impedance of the single cell;

[0049] S3: continuously monitor the impedance between the electrodes through an external circuit and send it to the field programmable gate array for further processing;

[0050] S4: analyze and process the data by the field programmable gate array, and evaluate the tumor risk level according to the impedance measurement results.

[0051] The integrated circuit-based battery substrate impedance sensing (IC-ECIS) system for distinguishing commercialized cancer cells and non-cancer cells:

[0052] To evaluate the performance of the IC-ECIS system in distinguishing cancer cells and non-cancer cells at the single-cell level, a commercialized human gastric cancer cell line (SGC-7901) was chosen as the model cancer cell line, and a gastric epithelial cell line (GES-1) was chosen as the corresponding non-cancer cell line for the study. In addition, non-cancer cells, including immune cells (B and T lymphocytes) that can exist in tumor tissues and mesenchymal stem cells (MSCs) that are sometimes used for cancer treatment, were also studied to evaluate their accuracy. The impedance of a single cell between two adjacent electrodes was measured and compared.

[0053] As shown in Figure 3 , cancer cells exhibit significantly higher impedance compared to all four non-cancerous cells.

[0054] The higher impedance of cancer cells (SGC-7901) compared to non-cancerous tissue cells (GES-1) is due to the unique physical properties and metabolic activity of cancer cells.

[0055] When cells transform into a cancerous state, their transmembrane potential, surface charge, ion concentration, and other properties change significantly.

[0056] Cancer cells tend to release a large amount of lactate ions, which can pass through the cytoplasmic membrane, causing a large accumulation of negative charges on the cell membrane surface.

[0057] When an electric field is applied, these accumulated negative charges generate an opposite current, thereby increasing the impedance.

[0058] In addition, cancer cells express more proteins related to adhesion, migration, and invasion. When cells adhere to the electrode surface through these proteins, charge transfer becomes more difficult because they must pass through a layer of non-conductive extracellular adhesion proteins, resulting in increased resistance to charge transfer.

[0059] Both of these mechanisms result in an increase in the impedance amplitude of cancer cells relative to non-cancerous cells or precancerous cells. Immune cells (B and T cells) are reluctant to adhere to the electrodes, resulting in lower impedance. Among non-cancerous cells, T cells have the lowest impedance amplitude, which may be attributed to their smaller size. The impedance of MSCs is significantly lower than that of cancer cells but slightly higher than that of immune cells. This is due to the complex differentiation state of MSCs, leading to the overexpression of certain proteins.

[0060] To establish a reliable standard for distinguishing individual cancer cells from non-cancer cells, the impedance range was carefully studied. Considering that MSCs rarely appear in biopsy samples, only normal tissue cells and immune cells were analyzed.

[0061] like Figure 4 As shown, the impedance values ​​of non-cancerous stomach cells ranged from 7 to 24 MΩ, while those of gastric cancer cells ranged from 21 to 35 MΩ. Cells with impedance values ​​between 21 and 24 MΩ could not be identified. These data were excluded from the analysis. However, cells with impedance values ​​above the upper limit of non-cancerous cells (24 MΩ) were most likely cancerous, while cells with impedance values ​​below the lower limit of cancer cells (21 MΩ) were most likely normal.

[0062] To evaluate the accuracy of cell classification based on IC-ECIS, SGC-7901 and GES-1 cells were labeled with red and green fluorescence, respectively, for biochemical cell identification. The labeled cells were then classified as cancer cells or non-cancerous cells by IC-ECIS according to the above criteria.

[0063] Impedance differentiation diagram Figure 5 As shown, the samples in the second and fourth quadrants were accurately classified, where GES-1 cells were correctly identified as non-cancerous cells and SGC-7901 cells were identified as cancer cells, while the samples in the first and third quadrants were incorrectly identified.

[0064] Clinical tumor samples, even if they are malignant, contain both cancerous and non-cancerous cells. The percentage of cancerous cells varies depending on the stage of tumor development. Here, the cancer risk level is categorized as low risk, intermediate risk, or high risk based on the percentile of cancer cells in the tumor biopsy.

[0065] As attached Figure 6 As shown, single-cell impedance measurements were performed using IC-ECIS on tumor cells extracted from the tumor and non-cancerous cells extracted from areas outside the tumor. A threshold was set as the upper limit of the impedance of all cells in the non-tumor sample. The risk level was determined based on the proportion of tumor cells exceeding the threshold, which are most likely to be cancerous. When the proportion is below 20%, the risk of the detected tumor being cancerous is low, as this may be due to errors in the impedance sensing system. When more than 40% of cells have abnormal impedance, the risk is considered high. When the proportion is between 20% and 40%, the risk is considered moderate, and in this case, the physician needs to consider clinical symptoms for a more accurate diagnosis.

[0066] Tumor processing time is crucial for guiding surgical decision-making. Sample turnaround time on the IC-ECIS platform is limited by the ability to dissociate tumors into single cells. To validate the minimum time required to obtain sufficient primary tumor cells for IC-ECIS, three tumor samples were collected from the same patient and their dissociation rates were systematically evaluated. Sample sizes ranged from 0.13 cm to 0.78 cm to 3.11 cm.

[0067] like Figure 7As shown, while larger tumor samples yielded more cells in the same amount of time due to their greater surface area exposed to the tumor digestion buffer, they required longer to complete the dissociation process. Remarkably, even the smallest samples yielded over 3,000 single cells in 15 minutes, a sufficient number for subsequent on-chip assays. The entire assay process, including impedance measurement, can be completed in 20 minutes. Compared to the 1-hour turnaround time for frozen pathology, the 20-minute tumor assay time based on IC-ECIS allows physicians to easily determine whether to expand the dissection area during surgery.

[0068] Semiconductor chip manufacturing process:

[0069] The integrated circuits were designed and simulated using Cadence software (Cadence Design Systems, Inc., USA) and subsequently fabricated using a 0.35 μm commercial standard complementary metal oxide semiconductor (CMOS) process from ams-OSRAM AG (Premstaetten, Austria).

[0070] The internal structure of the semiconductor chip consists of semiconductor elements, an insulating layer, vias, and four metal layers. The peripheral circuit was designed using Altium Designer (Altium, Australia) and simulated using Multisim (National Instruments, USA). The chip backside was ground using a WG-1211S thinning machine (Hyopto-tech, China).

[0071] like Figure 8 and Figure 9 As shown in Figure 1, the transimpedance gain and temperature effects of the transimpedance amplifier are simulated. The transimpedance gain variation within the system temperature range of 20 to 40°C is less than 0.07 dBΩ and can therefore be ignored.

[0072] The transimpedance gain of the transimpedance amplifier is digitally programmable between 87 and 142 dBΩ to accommodate different cell types and excitation amplitudes.

[0073] like Figure 10 As shown, to achieve physical integration, the semiconductor chip uses 14 gold wires, where each gold wire has a diameter of 1 mil and there are 7 gold wires on each side to connect the semiconductor chip to the printed circuit board, and then the contact pads are sealed with black epoxy to prevent external interference.

[0074] Clinical trial process: Tumors and surrounding tissues are obtained from tumor resection surgery or biopsy, and clinical specimens are obtained from the resected tissue immediately after surgery.

[0075] The tissue was then transferred to a 15 ml centrifuge tube, to which 5 ml of preservation solution (DMEM / F12, containing 1 % penicillin / streptomycin, 1 % Glutamax and 10 mM HEPES) was added.

[0076] The specimen was removed and in a biological safety cabinet the specimen was minced into 0.1 cm 3 pieces and placed in a 10 cm Petri dish. The tissue pieces were incubated in EBSS (with the addition of 125 U / ml collagenase II and 0.1 mg / ml DNase I) at 37°C, after which the degree of digestion was observed and the mixture was stirred once every 5 minutes with a pipette to accelerate the digestion process, until no large pieces remained.

[0077] After the digestion was complete, the digestion was stopped by the addition of cold DMEM / F12 and the mixture was filtered through a 40 μιη nylon cell strainer (Solarbio) to obtain a single cell suspension. The filtered cell suspension was transferred to a 50 ml tube and centrifuged at 300 x g for 5 minutes. The resulting pellet was suspended in erythrocyte lysis buffer (Invitrogen TM ) for 2 minutes, after which the lysis process was stopped by the addition of sufficient EBSS.

[0078] Detection procedure: cells were isolated and resuspended in fresh medium according to standard protocols. Then 2-5 μΐ^of the cell suspension was transferred to the IC chip using a microfluidic channel. After 5 minutes of settling, cells naturally adhered to the surface electrodes under the influence of gravity, and impedance measurements were performed when a single cell fell between two electrodes, and the cell position was verified under a microscope. A signal generator applied a sinusoidal excitation to the semiconductor chip, which activated the corresponding electrode channel. An external circuit continuously monitored the impedance between the electrodes and sent it to a field-programmable gate array for further processing. After each detection, the chip was thoroughly cleaned using isopropanol, then rinsed with deionized water and dried with nitrogen.

[0079] It will be obvious to a person skilled in the art that the application is not limited to the details of the above-described exemplary embodiments but can be implemented in other embodiments without departing from the scope of the application. The embodiments are to be considered in all respects as being illustrative and not restrictive, the scope of the application being indicated by the appended claims rather than by the description above, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. No reference signs in the claims should be considered as limiting the scope of the claims with respect to the features they indicate.

Claims

1. An integrated circuit impedance sensing system for tumor detection, comprising a semiconductor chip, a signal generator, an analog-to-digital converter, a field programmable gate array, and an embedded silicon fan-out packaging structure, characterized in that: The surface of the semiconductor chip is integrated with a cell-sized microelectrode array for capturing and adhering single cells, and an integrated circuit is embedded under the microelectrode array for real-time amplification and transmission of impedance signals; The signal generator is used to provide a sinusoidal wave excitation signal with a frequency range of 1kHz to 10MHz to the semiconductor chip to activate the target electrode channel in the microelectrode array; The analog-to-digital converter is connected to the output terminal of the semiconductor chip and is used to convert the analog impedance signal into a digital signal; The field programmable gate array is in communication with the analog-to-digital converter and the signal generator, and is used to control the activation timing of the electrode channels, store impedance data, and perform spectrum analysis and classification processing on the data; The embedded silicon fan-out packaging structure uses polymer materials to batch package semiconductor chips.

2. The integrated circuit impedance sensing system for tumor detection according to claim 1, characterized in that: The resolution of the analog-to-digital converter is not less than 16 bits, and the sampling rate is not less than 1MS / s. The microelectrode array consists of 22×16 electrodes. The diameter of a single electrode in the microelectrode array ranges from 10 to 50 μm, and the electrode spacing is 20 to 100 μm.

3. The integrated circuit impedance sensing system for tumor detection according to claim 2, characterized in that: The integrated circuit includes a transimpedance amplifier, which is responsible for processing signals from a row of electrodes. The transimpedance gain of the transimpedance amplifier can be digitally programmed in a range of 87 to 142 dBΩ to accommodate different cell types and excitation amplitudes. The transimpedance amplifier is biased using a constant transconductance bias circuit to stabilize the transimpedance gain within a target temperature range, thereby ensuring operational stability under environmental changes.

4. The integrated circuit impedance sensing system for tumor detection according to claim 3, characterized in that: The voltage amplitude of the sine wave output by the signal generator is programmable and adjustable within a range of 10mVpp to 1Vpp. The signal generator has a multi-channel switching function and supports selective activation of target electrodes in a microelectrode array according to row and column addressing modes.

5. The integrated circuit impedance sensing system for tumor detection according to claim 4, characterized in that: The embedded silicon fan-out packaging structure uses parylene C as a filling material, and the height difference between the embedded chip and the silicon substrate is less than 1 μm.

6. The integrated circuit impedance sensing system for tumor detection according to claim 5, characterized in that: The surface electrode of the semiconductor chip forms a metal layer through photolithography and metal sputtering processes. The metal layer material is composed of 95% aluminum and 5% copper, and its thickness is 925nm. Platinum is deposited on the surface of the metal layer, and excess metal is removed by ion beam etching to create a patterned electrode without damaging the integrated circuit under the surface electrode.

7. The integrated circuit impedance sensing system for tumor detection according to claim 6, characterized in that: The invention also includes a microfluidic channel module, which is integrated with the semiconductor chip and is used to automatically transport the tumor biopsy sample to the surface of the microelectrode array.

8. A tumor detection method using an integrated circuit impedance sensing system for tumor detection according to claim 7, characterized in that: The following steps are involved: S1: Tumor tissue samples are dissociated into single-cell suspensions, resuspended in fresh culture medium, and then delivered to the surface of a semiconductor chip through a microfluidic channel; S2: Controlling the signal generator through the field programmable gate array to output sinusoidal excitation and activate the corresponding electrode channel to perform impedance measurement on the single cell; S3: The impedance between the electrodes is continuously monitored by an external circuit and sent to a field programmable gate array for further processing; S4: The data is analyzed and processed by a field programmable gate array to assess the tumor risk level based on the impedance measurement results.

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