Integrated light source biocell culture sensing device

Abstract

This invention discloses an integrated light source biological cell culture sensing device. The device comprises a Micro-LED array and a biosensor array, which are bonded together. A DGHFET device and the Micro-LED device form a field-effect biosensor, with the DGHFET device located within the light spot coverage area of ​​the Micro-LED device within the same field-effect biosensor. This integrated light source biological cell culture sensing device successfully integrates efficient photostimulation, high-sensitivity biosensing, and high-reliability anti-interference capabilities onto a single platform, providing a powerful, reliable, and flexible integrated solution for optogenetic research. This invention has wide applications in the semiconductor technology field.

Description

Technical Field

[0001] This invention relates to the field of semiconductor technology, and in particular to a biological cell culture sensing device with integrated light source. Background Technology

[0002] Optogenetics research subjects are mainly divided into two categories: one is live biological models, which are suitable for studying neural signal activity in the natural activity state of organisms; the other is ex vivo biological tissue samples, which can provide large-scale, highly controllable experimental data and effectively supplement the shortcomings of live experiments.

[0003] Currently, commonly used techniques for detecting neural cells mainly include microelectrode electrophysiological recording, fluorescence imaging, and immunohistochemical analysis. Microelectrode arrays (MEAs) achieve high-density integration using micro- and nano-fabrication techniques, allowing for the simultaneous recording of the electrical activity of multiple neurons. However, their invasive operation can easily cause cell damage, and the problem of decreased signal-to-noise ratio (SNR) during electrode miniaturization has not been completely resolved. While fluorescence imaging technology offers subcellular spatial resolution, it relies on exogenous fluorescent labeling, which may interfere with normal cellular metabolism, and is limited by photobleaching effects and the autofluorescence of biological samples. Immunohistochemical techniques require tissue fixation, thus preventing dynamic monitoring in vivo. Summary of the Invention

[0004] To address the technical challenges of dynamic monitoring of in vivo states in current optogenetic research, the present invention aims to provide a biological cell culture sensing device with an integrated light source.

[0005] On one hand, embodiments of the present invention include a biological cell culture sensing device with an integrated light source, the biological cell culture sensing device with an integrated light source comprising: Micro-LED array; the Micro-LED array includes multiple Micro-LED devices; A biosensor array; the biosensor array includes multiple DGHFET devices; the DGHFET devices are used to detect extracellular action potential signals of biological cells in cell culture solution; The Micro-LED array is bonded to the biosensor array, and any one of the DGHFET devices and a corresponding Micro-LED device form a field-effect biosensor. The DGHFET device is located within the light spot coverage area of ​​the Micro-LED device in the same field-effect biosensor when it emits light.

[0006] Furthermore, the DGHFET device includes: AlGaN / GaN heterojunction; the AlGaN / GaN heterojunction includes a GaN buffer layer, an AlN insertion layer and an AlGaN barrier layer stacked sequentially from bottom to top; a two-dimensional electron gas is formed at the interface between the GaN buffer layer and the AlN insertion layer; An electrolyte gate is provided on the upper part of the AlGaN / GaN heterojunction; DGHFET source lead; one end of the DGHFET source lead forms an ohmic contact with the AlGaN / GaN heterojunction; DGHFET drain lead; one end of the DGHFET drain lead forms an ohmic contact with the AlGaN / GaN heterojunction; DGHFET Schottky gate lead; one end of the DGHFET Schottky gate lead forms a Schottky contact with the AlGaN / GaN heterojunction; A third sensing passivation layer; the third sensing passivation layer covers the AlGaN / GaN heterojunction.

[0007] Furthermore, the integrated light source biological cell culture sensing device also includes: Multiple DGHFET source pads; the DGHFET source pads are connected to the other end of the DGHFET source leads; Multiple DGHFET drain pads; the DGHFET drain pads are connected to the other end of the DGHFET drain leads; Multiple DGHFET Schottky gate pads; the DGHFET Schottky gate pads are connected to the other end of the DGHFET Schottky gate leads; Furthermore, the Micro-LED device includes: The following components are stacked sequentially from bottom to top: DBR mirror, sapphire substrate, n-GaN layer, active layer, p-GaN layer, ITO layer, first passivation layer, metal electrode and lead wire, and second passivation layer. A first passivation layer; the first passivation layer is located on the n-GaN layer and surrounds the active layer, the p-GaN layer and the ITO layer; Cathode electrode; the cathode electrode is disposed on the first passivation layer; Anode electrode; the anode electrode is disposed on the first passivation layer.

[0008] Furthermore, the integrated light source biological cell culture sensing device also includes: A transimpedance amplifier circuit; the input terminal of the transimpedance amplifier circuit is connected to the output terminal of the field-effect biosensor; A low-pass filter; the input terminal of the low-pass filter is connected to the output terminal of the transimpedance amplifier circuit; The transimpedance amplifier circuit includes a first operational amplifier, a first variable resistor, and a second variable resistor. The inverting input terminal of the first operational amplifier serves as the input terminal of the transimpedance amplifier circuit, and the output terminal of the first operational amplifier serves as the output terminal of the transimpedance amplifier circuit. The output terminal of the first operational amplifier is connected to the inverting input terminal of the first operational amplifier through the first variable resistor, and the non-inverting input terminal of the first operational amplifier is connected to a bias voltage source through the second variable resistor. The low-pass filter includes a second operational amplifier, a first resistor, a second resistor, a third resistor, and a capacitor. The non-inverting input of the second operational amplifier is connected in series with the first resistor as the input of the low-pass filter. The output of the second operational amplifier is the output of the low-pass filter. The non-inverting input of the second operational amplifier is grounded through the capacitor. The second resistor and the third resistor are connected in series and grounded to form a voltage divider circuit. The output of the second operational amplifier is connected to the inverting input of the second operational amplifier through the voltage divider circuit.

[0009] On the other hand, embodiments of the present invention also include a detection method for a biological cell culture sensing device with an integrated light source, the detection method of the biological cell culture sensing device with an integrated light source comprising the following steps: The integrated light source biological cell culture sensing device was placed in PBS solution for cell culture. The Micro-LED devices in the Micro-LED array are activated to provide light stimulation to the cells; The DGHFET device in the biosensor array is invoked to detect electrical signals in cells stimulated by light.

[0010] On the other hand, embodiments of the present invention also include a method for preparing a biological cell culture sensing device with an integrated light source, the method comprising the following steps: Fabricate the Micro-LED array; Fabricate the biosensor array; By bonding the Micro-LED array to the biosensor array, a biological cell culture sensing device with integrated light source is obtained.

[0011] The beneficial effects of the embodiments of the present invention are as follows: The integrated light source biological cell culture sensing device in the embodiments integrates a Micro-LED array and a biosensor array, thereby successfully integrating efficient light stimulation function, high-sensitivity biosensing function, and high-reliability anti-interference capability into a single platform. Specifically, since the Micro-LED array is integrated as a light source, precise light stimulation can be applied to the cells on the biosensor array when using the biosensor array for in vitro cell culture and detection. This enables the biosensor array to detect the biological neural signals of cells under light stimulation, realizing dynamic monitoring of the cell in vivo state in optogenetic research, and providing a powerful, reliable, and flexible integrated solution for optogenetic research. Attached Figure Description

[0012] Figure 1 This is a schematic diagram of the integrated light source cell culture device in the example; Figure 2 This is a three-dimensional view of the epitaxial wafer used to fabricate the Micro-LED array in the embodiment; Figure 3 This is a perspective view of the Micro-LED array fabricated in this embodiment after isolating the mesa devices. Figure 4 This is a three-dimensional view of the fabricated ITO during the fabrication of the Micro-LED array in this embodiment; Figure 5 This is a three-dimensional view of the fabrication of the Micro-LED array after the preparation of the first passivation layer in the embodiment. Figure 6 This is a three-dimensional view of the fabrication of the P / N ohmic contacts during the fabrication of the Micro-LED array in the embodiment; Figure 7 This is a three-dimensional view of the fabrication of the metal leads, the second insulating passivation layer, and the DBR reflector during the fabrication of the Micro-LED array in this embodiment. Figure 8 This is a three-dimensional view of the epitaxial wafer used to fabricate the biosensor array in the embodiment; Figure 9 This is a three-dimensional view of the biosensor array fabricated in the embodiment after isolating the mesa devices. Figure 10 This is a three-dimensional view of the biosensor array fabricated after the preparation of the third sensing passivation layer in the embodiment. Figure 11 This is a three-dimensional view of the biosensor array fabricated after the ohmic contact was prepared during the fabrication process in this embodiment. Figure 12 This is a three-dimensional view of the fabrication of the Schottky contacts during the fabrication of the biosensor array in this embodiment; Figure 13This is a three-dimensional view of the fabrication of the biosensor array in the embodiment after the preparation of metal leads, pads, and the fourth passivation layer. Figure 14 This is a schematic diagram of the bonding between the Micro-LED array and the biosensor array in the embodiment; Figure 15 This is a schematic diagram showing the connection relationship between the field-effect biosensor, the transimpedance amplifier circuit, and the low-pass filter in the embodiment. Figure 16 This is a circuit diagram of the transimpedance amplifier circuit and the low-pass filter in the embodiment. Detailed Implementation

[0013] Terminology Explanation: Cellular neural electrical signals mainly include two types: one is the extracellular action potential, which characterizes neuronal firing, and the other is the local field potential (LFP), which reflects the synchronous activity of the neural network. FET: an abbreviation for Field Effect Transistor, which is a voltage-controlled semiconductor device that utilizes the field effect. It can be further subdivided into several types. DGHFET: an abbreviation for Double Gate Heterojunction FET, which stands for Double Gate High Electron Mobility Heterojunction Field Effect Transistor. It is a type of transistor with a control gate (CG) and an electrolyte gate (EG). Due to the control gate (CG), the electrical performance of DGHFET can be adjusted to the optimal level, while the electrolyte gate (EG) is used to sense weak signals. Micro-LED: Miniature light-emitting diode, with excellent luminous efficiency; Optogenetics is a biotechnology that uses genetic engineering and optical techniques to manipulate specific cells with millisecond-level precision by introducing photosensitive ion channels. It has revolutionized research in fields such as neuroscience, enabling us to analyze the causal relationships of complex living systems, especially the brain, with unprecedented accuracy.

[0014] I. Technical Problems and Solutions Current neurophysiological techniques still face challenges in detecting electrical signals from single cells and populations of neurons, including insufficient signal-to-noise ratio, slow acquisition speed, and poor stability.

[0015] In contrast, field-effect transistors (FETs) employ an active detection mechanism, modulating the potential at the interface between the gate-sensitive membrane and the electrolyte by varying neural potentials, thereby controlling the channel conductance and converting it into a measurable current signal output. This mechanism offers significant advantages in signal stability, signal-to-noise ratio, and acquisition rate; its overall performance primarily depends on the transconductance, noise characteristics, and interface properties of the FET device. Therefore, current technological trends focus on utilizing novel semiconductor materials to construct FET structures with superior electrical properties, enabling more accurate, rapid, and stable neural signal acquisition.

[0016] Currently, signal acquisition elements used in neuroelectrophysiology include traditional microelectrodes based on metal or glass materials and various field-effect transistor (FET) structures. Traditional microelectrode arrays (MEAs) are based on passive detection principles, acquiring neural electrical signals through electrochemical impedance spectroscopy. Their signal quality is highly dependent on the electrode-electrolyte interface impedance: too low an impedance results in insufficient signal capture, while too high an impedance easily introduces significant noise. Furthermore, when FET devices detect ion fluctuations in solution, an additional reference electrode is often introduced to control potential changes, which can damage cultured organisms. Additionally, this type of technology suffers from poor long-term stability.

[0017] In optogenetic physiology research, optical stimulation can be used to regulate nerve cell activity. However, common light sources are relatively large, which can lead to non-specific expansion of the stimulation area. This not only makes it difficult to precisely control the excitation of target cell populations but also interferes with the accuracy of signal acquisition, thus hindering the further development of this technology. On the other hand, traditional microelectrodes need to be directly inserted into biological tissues for in vivo measurements. The implantation process can easily cause tissue trauma and immune rejection. In long-term use, factors such as biological contamination and decreased interfacial stability can also lead to signal quality degradation and reduced detection reliability.

[0018] To address the aforementioned issues, a possible solution is to integrate a Micro-LED array as a cell optical stimulation signal source onto a biosensor array equipped with DGHFET devices. This would enable targeted photostimulation of specific cells, achieving high spatial precision point-to-point stimulation without affecting surrounding cells. It should be noted that the FET output signal is in current form, making its subsequent processing more complex than that of a voltage signal. This complexity can be addressed by designing dedicated signal processing circuits and algorithms for analysis and utilization.

[0019] The aforementioned biosensor array and Micro-LED array constitute an integrated light source biological cell culture sensing device. This integrated light source biological cell culture sensing device can achieve high signal-to-noise ratio, rapid, and stable neural signal recording by inducing cell responses through light stimulation while maintaining cell viability. Furthermore, without significantly increasing process complexity, it integrates the simultaneous detection of multiple parameters in the culture environment, including pH, specific biomolecule concentrations, and neural electrical signals. This provides an advanced integrated solution for optogenetic research, neuroscience exploration, and high-throughput drug screening.

[0020] II. Micro-LED Arrays and Their Fabrication Methods In this embodiment, the following can be used: Figure 1 The integrated light source cell culture device shown. The Micro-LED array within it can be fabricated through the following steps: P1. Obtain the Micro-LED epitaxial wafer; such as... Figure 2 As shown, the Micro-LED epitaxial wafer comprises, from bottom to top: 1. sapphire substrate, 2. n-GaN layer, 3. active layer and 4. p-GaN layer; P2. For example Figure 3 As shown, in Figure 2 Based on this, etching was performed in a gaseous environment of Cl2 and BCl3 until the 2.n-GaN layer was reached, thereby etching out the Mesa mesa layer of the InGaN / GaN Micro-LED. P3. For example Figure 4 As shown, in Figure 3 Based on this, in a high vacuum argon environment, a 5.ITO layer (indium tin oxide transparent conductive layer) is deposited on the Mesa mesa of the InGaN / GaN Micro-LED and the corresponding pattern is etched. Then, through rapid annealing, a good ohmic contact is formed between the 5.ITO layer and the 4.p-GaN layer. P4. For example Figure 5 As shown, in Figure 4 Based on this, the active layer is activated at high temperature, and the first passivation layer is prepared by photolithography and plasma-enhanced chemical vapor deposition. P5. For example Figure 6 As shown, in Figure 5 Based on this, wet etching is used to obtain the anode and cathode electrode windows on the first passivation layer. Then, using Ti / Au (50 / 150nm) material, metal thin films are prepared in a high vacuum environment through photolithography and electron beam evaporation processes. The metal thin films are then patterned using a lift-off process to prepare the anode and cathode electrodes of the InGaN / GaN Micro-LED. P6. For example Figure 7 As shown, in Figure 6 Based on this, after preparing the anode (cathode) leads and corresponding pads using Cr / Al / Ti / Pt / Ti / Pt / Au / Ti (3 / 100 / 50 / 30 / 50 / 100 / 650 / 30nm), a second passivation layer (16.) was prepared in a high vacuum and 350℃ high temperature environment with SiH4 and N2O gas environment by photolithography and plasma-enhanced chemical vapor deposition. The second passivation layer (16.) was then patterned using a dry etching process, and a DBR mirror (17.) was deposited on the back side of the substrate.

[0021] pass Figures 2-7 The steps P1-P6 shown can be used to fabricate multiple Micro-LED devices on a sapphire substrate, which together form a Micro-LED array.

[0022] In this embodiment, the working principle of the Micro-LED device is as follows: by applying a positive voltage to the anode pad and grounding the cathode pad, the current will flow into the p-GaN region through the anode electrode (anode contact) and the wire, flow into the n-GaN through the active layer, and the current will flow out from the cathode pad, thereby driving the Micro-LED device to emit light.

[0023] III. Biosensor Arrays and Their Fabrication Methods In this embodiment, the biosensor array can be prepared through the following steps: S1. Obtain the DGHFET epitaxial wafer; such as Figure 8 As shown, the DGHFET epitaxial wafer comprises, from bottom to top: 1. sapphire substrate, 31. GaN buffer layer, 32. AlN insertion layer and 33. AlGaN barrier layer; S2. For example Figure 9 As shown, in Figure 8 Based on this, the AlGaN / GaN DGHFET mesa is etched through photolithography and dry etching processes in a gas environment of Cl2 and CF4 to the 31 GaN buffer layer, thereby forming multiple sensing mesa mesa. S3. For example Figure 10 As shown, in Figure 9 Based on this, a silicon dioxide isolation layer was prepared by placing it in a gas environment of SiH4 and N2O, high vacuum and 350℃ high temperature environment through photolithography and plasma-enhanced chemical vapor deposition process, thereby obtaining the third sensing passivation layer; S4. For example Figure 11 As shown, in Figure 10Based on this, a dry etching process is first used to slowly etch the outline of the patterned insulating layer with CF4 gas to ensure its accuracy, thereby opening a Mesa window slightly smaller than the sensing Mesa mesa surface. Then, Ti / Al / Ni / Au (150 / 800 / 200 / 600nm) metal thin films are deposited in a high vacuum environment through photolithography and electron beam evaporation. The Ti / Al / Ni / Au metal thin films are then patterned using a lift-off process and subjected to rapid thermal annealing to prepare a 37-ohm contact. S5. For example Figure 12 As shown, in Figure 11 Based on the 37. Ohmic contact, AlGaN / GaN DGHFET 38. Schottky control gate is fabricated using Ti / Au (20 / 60nm); S6. For example Figure 13 As shown, in Figure 12 Based on this, after fabricating ohmic contacts and Schottky gate leads and corresponding pads using Cr / Al / Ti / Pt / Ti / Pt / Au / Ti (3 / 100 / 50 / 30 / 50 / 100 / 650 / 30nm), a fourth passivation layer (39.) is fabricated, and the AlGaN / GaN DGHFET 41. sensing gate region, metal pad window, and grounding area window are formed. The electrolyte gate fabricated in step S6 is the surface of the gate region, which should have been made of solid gate material. PBS solution is then introduced to the surface to culture cells. Biological cells (such as nerve cells) cultured in the PBS solution can attach to the 33.AlGaN barrier layer of the DGHFET device. Therefore, the PBS solution located in the electrolyte gate and the nerve cells therein serve as the gate of the DGHFET device.

[0024] pass Figures 8-13 The steps S1-S6 shown can be used to fabricate multiple DGHFET devices on a sapphire substrate, which together form a biosensor array.

[0025] Figure 14 This is a three-dimensional image of the biosensor array after each layer has been peeled off. From bottom to top, they are: 1. Sapphire substrate, sensing Mesa mesa (including 31. GaN buffer layer, 32. AlN insertion layer and 33. AlGaN barrier layer), 35. Third sensing passivation layer, 37. Ohmic contact, lead wire, pad and ground layer and 39. Fourth sensing passivation layer.

[0026] In this embodiment, the working principle of the DGHFET device is as follows: the source of the AlGaN / GaN DGHFET shares a common lead and pad, while the drain is connected by a separate lead. The current flows into the AlGaN / GaN DGHFET through the drain lead and the drain ohmic contact electrode, passes through the two-dimensional electron gas structure between the AlN insertion layer and the GaN buffer layer, and then flows out from the source ohmic contact and the source lead of the AlGaN / GaN DGHFET.

[0027] In AlGaN / GaN DGHFET devices, the control gate (CG) and the sensing electrolyte gate (EG) play crucial roles. Because the control gate is attached by a passivation layer and does not directly contact the buffer solution such as PBS, the output voltage V can be obtained from the control gate. GS The sensitivity of the device can be unilaterally adjusted to maximize the transconductance. Furthermore, under the control of the gate, no additional reference electrode is needed to affect the solution potential, thus ensuring cell viability. (Transconductance value) The calculation formula is as follows:

[0028] When an organism receives stimulation, neurons cultured in the AlGaN / GaN DGHFET electrolyte gate generate action potentials (neural signals). The extracellular potentials (neural signals) of these neurons rapidly and sensitively regulate the two-dimensional electron gas structure between the AlN insertion layer and the GaN buffer layer, causing changes in the conductivity of the two-dimensional electron gas. This, in turn, causes changes in the source-drain current of the AlGaN / GaN DGHFET. In other words, changes in the source-drain current of the AlGaN / GaN DGHFET reflect changes in the neural signals of the biological neurons.

[0029]

[0030] This refers to the source and drain current of the DGHFET device. The electron mobility of a two-dimensional electron gas. The capacitance of the two-dimensional electron gas to the surface of the AlGaN barrier layer. The width of the AlGaN / GaN DGHFET. The channel length of the AlGaN / GaN DGHFET. This is the extracellular potential (nerve signal). The threshold voltage of the AlGaN / GaN DGHFET. This represents the source-drain voltage of the DGHFET device.

[0031] DGHFET devices enable active detection of neural signals: In neural sensing applications, the gate region (electrolyte gate, also known as the solution gate) of the AlGaN / GaNDGHFET device is in close contact with the neuron. When the neuron is stimulated to generate an action potential, it mainly manifests as Na+. + Inflow (depolarization) and K + Outflow (repolarization) is usually accompanied by Ca² + Inflow (such as during synaptic transmission) causes these ions to flow across the membrane, instantly altering the local potential of the electrolyte solution in the gap between the neuron and the DGHFET gate region. This change in solution potential is equivalent to the effective gate voltage (V) applied to the electrolyte gate of the DGHFET. G The gate voltage change highly sensitively modulates the conductivity of the channel 2DEG, which in turn affects the source-drain current (Id) of the DGHFET device. DS ) undergoes corresponding changes. Detection I DS Changes in these parameters can enable highly sensitive and rapid detection of neuronal action potentials.

[0032] In a preferred embodiment, the biosensor array integrates 48 AlGaN / GaN DGHFET devices for acquiring electrical signals from single or multiple nerve cells and local field potentials of nerve cells in solution; it also includes four monitoring units, divided into two groups, to monitor the pH value and specific biomolecule concentrations of the cell culture environment in real time. This architecture achieves multi-parameter, high signal-to-noise ratio, and rapid and stable synchronous detection of neural signals on a single chip.

[0033] IV. Integrated Light Source Biological Cell Culture Sensing Equipment and Its Preparation Method In this embodiment, when performing steps P1-P6 to prepare the Micro-LED array and steps S1-S6 to prepare the biosensor array, the same number of Micro-LED devices on the Micro-LED array and DGHFET devices on the biosensor array can be fabricated. Sapphire substrates of the same size can be used to fabricate Micro-LED devices and DGHFET devices at corresponding positions on their respective sapphire substrates. This ensures that when the sapphire substrates of the Micro-LED array and the biosensor array project to overlap, the Micro-LED devices and DGHFET devices can overlap one-to-one. Therefore, after the Micro-LED array and the biosensor array are bonded, each DGHFET device is located within the light spot coverage area of ​​the corresponding Micro-LED device when it emits light.

[0034] In this embodiment, after the Micro-LED array and the biosensor array are bonded, each sensor pair is located within the light spot coverage area when the corresponding Micro-LED device emits light.

[0035] like Figure 14 As shown, the Micro-LED array obtained in steps P1-P6 is bonded to the sensing array device obtained in steps S1-S6, thereby obtaining a biological cell culture sensing device with integrated light source. Therefore, the biological cell culture sensing device with integrated light source includes multiple field-effect biosensors.

[0036] Specifically, a three-dimensional diagram of the field-effect biosensor is shown below. Figure 13 As shown. (Refer to...) Figure 13 After the Micro-LED array is bonded to the 46-sensor array device, each DGHFET device and its corresponding Micro-LED device form a field-effect biosensor.

[0037] Figure 14 The working principle of the field-effect biosensor shown is as follows: The DGHFET device in the field-effect biosensor uses a light-transmitting sapphire substrate, and the AlGaN / GaN structure in the DGHFET device is also a light-transmitting structure. Therefore, when the Micro-LED device below emits light, the light can reach the 41. electrolyte gate in the DGHFET device, thereby enabling the Micro-LED device to excite the cells in the 41. electrolyte gate as a light source. The cells are photosensitive and the membrane potential changes, causing a weak electrical signal to be generated between the source and drain of the DGHFET device, which is then output as the signal generated by the entire field-effect biosensor.

[0038] When the integrated light source biological cell culture sensing device is working, nerve cells are cultured in biological buffered saline (PBS) to simulate the in vivo environment. Nerve cells attach to the AlGaN barrier layer of the DGHFET device in some field-effect biosensors along with the PBS, forming an electrolyte gate that acts as a gate. Power is then applied to the Micro-LED device in the field-effect biosensor with the attached nerve cells, causing it to emit light. This stimulates the nerve cells attached to the DGHFET device in the same field-effect biosensor. Since the nerve cells act as the electrolyte gate, the potential generated by the nerve cell activity is tuned to the optimal operating point by the control gate CG of the DGHFET. As the electrolyte gate EG senses the signal, fluctuating current signals are generated at the source and drain of the DGHFET, enabling the DGHFET device to detect the biological neural signals after the nerve cells are stimulated by light.

[0039] V. Transimpedance Amplifier Circuit and Low-Pass Filter In this embodiment, as Figure 15As shown, each field-effect biosensor can be equipped with a transimpedance amplifier circuit and a low-pass filter. The input of the transimpedance amplifier circuit is connected to the output of the field-effect biosensor, and the input of the low-pass filter is connected to the output of the transimpedance amplifier circuit. Figure 15 The field-effect biosensor in the text can be a single DGHFET device (in which case the output signal of the field-effect biosensor is the source-drain signal of the DGHFET device).

[0040] In this embodiment, the structures of the transimpedance amplifier circuit and the low-pass filter are as follows: Figure 16 As shown. (Refer to...) Figure 16 The transimpedance amplifier circuit includes a first operational amplifier, a first variable resistor, and a second variable resistor. The inverting input terminal of the first operational amplifier serves as the input terminal of the transimpedance amplifier circuit, and the output terminal of the first operational amplifier serves as the output terminal of the transimpedance amplifier circuit. The output terminal of the first operational amplifier is connected to the inverting input terminal of the first operational amplifier through the first variable resistor, and the non-inverting input terminal of the first operational amplifier is connected to a bias voltage source through the second variable resistor.

[0041] Reference Figure 16 The low-pass filter includes a second operational amplifier, a first resistor, a second resistor, a third resistor, and a capacitor. The non-inverting input of the second operational amplifier is connected in series with the first resistor as the input of the low-pass filter, and the output of the second operational amplifier is the output of the low-pass filter. The non-inverting input of the second operational amplifier is grounded through the capacitor. The second and third resistors are connected in series and grounded to form a voltage divider circuit. The output of the second operational amplifier is connected to the inverting input of the second operational amplifier through the voltage divider circuit.

[0042] In this embodiment, the working principle of the transimpedance amplifier circuit and the low-pass filter is as follows: the feedback current of the transimpedance amplifier circuit can be calculated using the following formula:

[0043] in, i F For feedback current, u o R represents the output voltage, while R is the impedance matching provided by the two variable resistors in the transimpedance amplifier circuit. The field-effect sensor output is at the negative terminal of the operational amplifier, while the positive terminal requires a certain bias voltage. This ensures that the amplified voltage is then input to the low-pass filter, where filtering yields a more complete signal. The cutoff frequency of the low-pass filter is:

[0044] Where f is the cutoff frequency. Here, C is the resistance value of the first resistor, and C is the capacitance value of the capacitor. The first resistor and the capacitor together form a low-pass filter circuit. Compared with passive filters, active filters have the advantages of small size, high efficiency, strong load-carrying capacity, good frequency characteristics, and more stable output signals. The current signal I output by the field effect sensor DS_t After passing through the transimpedance amplifier circuit, it is converted into a voltage signal V of appropriate magnitude. DS_t After passing through a low-pass filter, unwanted high-frequency components or noise in the detected biological signal are suppressed. After passing through a transimpedance amplifier circuit and a low-pass filter circuit, the current baseline of about several hundred microamps directly output by the field-effect biosensor is converted into a voltage baseline of about 0V, and some high-frequency noise is filtered out to obtain a relatively clean detection curve. This method plays a crucial role in subsequent data processing on this baseline.

[0045] By setting up a transimpedance amplifier circuit and a low-pass filter, the current signal output by the field-effect biosensor can be converted into a high-quality voltage signal that is suitable for the current analog-to-digital converter module to receive, which facilitates subsequent signal processing.

[0046] The integrated light source biological cell culture sensing device in this embodiment can be further improved in the following ways: 1. By thinning and grinding the sapphire substrate in the DGHFET device to a thickness of tens of micrometers, refraction and scattering losses during light propagation to the sensing site can be significantly reduced. This allows for effective stimulation of nerve cell responses at lower light source power, eliminating the need for an external light source and avoiding interference with the cell culture environment. Based on the point-to-point light excitation capability of Micro-LEDs, precise spatial control can be achieved, making it suitable for targeted stimulation of cultured neurons. This design provides a flexible and reliable experimental platform for optogenetic research, supporting the construction of various control experiments and meeting the requirements of complex optogenetic operations. 2. A dual-gate FET is employed. First, we precisely control the electrical characteristics of the FET (such as threshold voltage and transconductance) through the control gate, setting the device's operating point in the region of the current-voltage curve most sensitive to charge changes. Building upon this, we further optimize the geometry and spatial position of the top gate (i.e., the electrolyte gate) to ensure that weak signals (such as charges generated by the binding of small amounts of biomolecules) can be most effectively captured and coupled into the device channel. This synergistic strategy of "globally optimizing the operating point through the control gate and locally capturing signals through the electrolyte gate" ultimately maximizes the sensor's sensitivity to weak signals. 3. Four large metal thin films are set in the p-GaN layer of the Micro-LED device, the GaN buffer layer of the biosensor array device and its grounded metal lead layer, and a copper layer is set inside the PCB, thus forming a three-layer Faraday cage structure. This multi-layer shielding system can effectively suppress optical and electrical noise and provide the necessary electromagnetic environment guarantee for high signal-to-noise ratio biosensing. 4. In field-effect biosensor devices, to avoid potential leakage current from the sidewalls of the Mesa mesa, an atomic layer deposition (ALD) technique can be used to grow a SiO2 passivation layer on the etched Mesa structure surface. This passivation layer effectively isolates the mesa sidewalls from direct contact with the electrolyte, thereby significantly suppressing leakage current. Compared to other device structures, this design can control leakage current to a low level, or even essentially eliminate the impact of leakage current on device performance. 5. During the fabrication of the field-effect biosensor device, a Mesa structure with a two-dimensional electron gas is simultaneously constructed during the Mesa mesa fabrication stage. An integrated resistor with a fixed resistance value is then formed by metal evaporation to replace the external transimpedance amplifier circuit. This integrated resistor can directly convert the flowing current signal into a voltage signal, thereby integrating the transimpedance amplification function within the sensor device.

[0047] In summary, the integrated light source biological cell culture sensing device in this embodiment has the following advantages: 1. A highly efficient, precise, and low-interference optogenetic stimulation platform High luminous efficiency and low power consumption: By thinning the sapphire substrate, light loss is significantly reduced, which allows nerve cells to be effectively stimulated with only a lower light source power, reducing system energy consumption and potential photothermal damage to cells.

[0048] Integration and interference-free: Using integrated Micro-LED as the light source eliminates the need for bulky external light sources, avoiding interference with the cell culture environment and making it more suitable for in vivo or long-term experiments.

[0049] Precise spatial control: By utilizing the point-to-point excitation capability of Micro-LEDs, high-precision targeted stimulation of neuronal populations is achieved, providing a powerful tool for studying the function of neural circuits.

[0050] Flexibility and reliability: This design constitutes a flexible and reliable experimental platform, which facilitates the construction of various control experiments and meets the requirements of complex optogenetic operations.

[0051] 2. Excellent noise immunity and electromagnetic shielding capabilities Effective noise discharge: Grounding lines are installed at the four corners of the sensing area to provide a low-impedance discharge path for environmental noise and static electricity in the sensing area, preventing noise accumulation from interfering with the signal.

[0052] Multiple electromagnetic shielding: An innovative three-layer Faraday cage shielding structure (composed of a GaN layer, a large metal thin film, and a PCB copper plating layer) is constructed, forming a multi-layer protection system that can effectively isolate external electromagnetic interference and internal crosstalk, providing a high signal-to-noise ratio electromagnetic environment guarantee for the acquisition of weak bioelectric signals.

[0053] 3. High-fidelity signal acquisition and conversion system Avoid signal attenuation: The current signal output mode avoids the signal attenuation problem caused by voltage division due to lead resistance in traditional voltage measurement, thus better maintaining the integrity of the original biological signal.

[0054] Effective signal conversion: Through the transimpedance amplification and filtering circuit, the amplified current signal is accurately converted into a voltage signal that is easy to acquire and analyze, solving the problem of the difficulty in directly processing current signals.

[0055] 4. Collaborative optimization strategy: A dual-gate FET is adopted, and a collaborative strategy of "global optimization of the operating point of the control gate and local capture of the signal by the electrolyte gate" is used.

[0056] Maximizing sensitivity: The control gate precisely adjusts the device to its optimal operating point; the optimized electrolyte gate structure ensures efficient capture of weak bioelectrical signals. This design maximizes the sensor's sensitivity to detect weak signals.

[0057] In summary, the core advantage of this design lies in its comprehensive application of advanced technologies in optics, electronics, materials, and processes. It successfully integrates efficient photostimulation, highly sensitive biosensing, and highly reliable anti-interference capabilities onto a single platform, providing a powerful, reliable, and flexible integrated solution for optogenetics research.

[0058] It should be noted that, unless otherwise specified, when a feature is referred to as "fixed" or "connected" to another feature, it can be directly fixed or connected to the other feature, or indirectly fixed or connected to the other feature. Furthermore, the descriptions of "upper," "lower," "left," and "right" used in this disclosure are only relative to the relative positional relationships of the components of this disclosure in the accompanying drawings. The singular forms "a," "an," and "the" used in this disclosure are also intended to include the plural forms, unless the context clearly indicates otherwise. Moreover, unless otherwise defined, all technical and scientific terms used in this embodiment have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used in this embodiment specification is only for describing specific embodiments and is not intended to limit the embodiments of the invention. The term "and / or" as used in this embodiment includes any combination of one or more of the associated listed items.

[0059] It should be understood that although the terms first, second, third, etc., may be used to describe various elements in this disclosure, these elements should not be limited to these terms. These terms are only used to distinguish elements of the same type from each other. For example, a first element may also be referred to as a second element without departing from the scope of this disclosure, and similarly, a second element may also be referred to as a first element. The use of any and all instances or exemplary language (“e.g.,” “such as,” etc.) provided in this embodiment is intended only to better illustrate embodiments of the invention and, unless otherwise required, does not impose a limitation on the scope of embodiments of the invention.

[0060] It should be recognized that embodiments of the present invention can be implemented or carried out by computer hardware, a combination of hardware and software, or by computer instructions stored in a non-transitory computer-readable storage medium. The method can be implemented using standard programming techniques—including a non-transitory computer-readable storage medium configured with a computer program, wherein such a storage medium causes the computer to operate in a specific and predefined manner—according to the methods and drawings described in the specific embodiments. Each program can be implemented in a high-level procedural or object-oriented programming language to communicate with the computer system. However, if desired, the program can be implemented in assembly or machine language. In any case, the language can be a compiled or interpreted language. Furthermore, for this purpose, the program can run on a programmed application-specific integrated circuit (ASIC).

[0061] Furthermore, the procedures described in this embodiment can be performed in any suitable order unless otherwise indicated by this embodiment or otherwise obviously contradict the context. The procedures (or variations and / or combinations thereof) described in this embodiment can be executed under the control of one or more computer systems configured with executable instructions, and can be implemented by hardware or a combination thereof as code (e.g., executable instructions, one or more computer programs, or one or more applications) that commonly executes on one or more processors. A computer program includes a plurality of instructions executable by one or more processors.

[0062] Furthermore, the method can be implemented in any suitable type of computing platform, including but not limited to personal computers, minicomputers, mainframes, workstations, networked or distributed computing environments, standalone or integrated computer platforms, or in communication with charged particle tools or other imaging devices, etc. Aspects of embodiments of the invention can be implemented as machine-readable code stored on a non-transitory storage medium or device, whether removable or integrated into a computing platform, such as a hard disk, optical read and / or write storage medium, RAM, ROM, etc., such that it is readable by a programmable computer, and when the storage medium or device is read by the computer, it can be used to configure and operate the computer to perform the processes described herein. Furthermore, the machine-readable code, or portions thereof, can be transmitted via wired or wireless networks. The invention of this embodiment includes these and other different types of non-transitory computer-readable storage media when such media comprises instructions or programs that implement the steps above in conjunction with a microprocessor or other data processor. Embodiments of the invention also include the computer itself when programmed according to the methods and techniques of embodiments of the invention.

[0063] A computer program can be applied to input data to perform the functions of this embodiment, thereby transforming the input data to generate output data stored in non-volatile memory. The output information can also be applied to one or more output devices, such as a display. In a preferred embodiment of the invention, the transformed data represents physical and tangible objects, including a specific visual depiction of physical and tangible objects generated on the display.

[0064] The above are merely preferred embodiments of the present invention. The embodiments of the present invention are not limited to the above-described implementations. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the embodiments of the present invention, as long as they achieve the same technical effects, should be included within the scope of protection of the embodiments of the present invention. Within the scope of protection of the embodiments of the present invention, the technical solutions and / or implementation methods can have various modifications and variations.

Claims

1. A biological cell culture sensing device with integrated light source, characterized in that, The integrated light source biological cell culture sensing device includes: Micro-LED array; the Micro-LED array includes multiple Micro-LED devices; A biosensor array; the biosensor array includes multiple DGHFET devices; the DGHFET devices are used to detect extracellular action potential signals of biological cells in cell culture solution; The Micro-LED array is bonded to the biosensor array, and any one of the DGHFET devices and a corresponding Micro-LED device form a field-effect biosensor. The DGHFET device is located within the light spot coverage area of ​​the Micro-LED device in the same field-effect biosensor when it emits light.

2. The biological cell culture sensing device with integrated light source according to claim 1, characterized in that: The biosensor array also includes multiple DGHFET devices; the DGHFET devices are used to detect the extracellular action potential signals of biological cells in cell culture solution. The sensing unit is located within the light spot coverage area of ​​the Micro-LED device in the same field-effect biosensor when it emits light.

3. The biological cell culture sensing device with integrated light source according to claim 2, characterized in that, The DGHFET device includes: AlGaN / GaN heterojunction; the AlGaN / GaN heterojunction includes a GaN buffer layer, an AlN insertion layer and an AlGaN barrier layer stacked sequentially from bottom to top; a two-dimensional electron gas is formed at the interface between the GaN buffer layer and the AlN insertion layer; The AlGaN / GaN heterojunction is provided with a Schottky metal gate and an electrolyte gate on its upper part; DGHFET source lead; one end of the DGHFET source lead forms an ohmic contact with the AlGaN / GaN heterojunction; DGHFET drain lead; one end of the DGHFET drain lead forms an ohmic contact with the AlGaN / GaN heterojunction; DGHFET Schottky gate lead; one end of the DGHFET Schottky gate lead forms a Schottky contact with the AlGaN / GaN heterojunction; The first sensing passivation layer covers the AlGaN / GaN heterojunction, the DGHFET source lead, and the DGHFET drain lead.

4. The biological cell culture sensing device with integrated light source according to claim 3, characterized in that, The integrated light source biological cell culture sensing device also includes: Multiple DGHFET source pads; the DGHFET source pads are connected to the other end of the DGHFET source leads; Multiple DGHFET drain pads; the DGHFET drain pads are connected to the other end of the DGHFET drain leads; Multiple DGHFET Schottky gate pads; the DGHFET Schottky gate pads are connected to the other end of the DGHFET Schottky gate leads.

5. The biological cell culture sensing device with integrated light source according to claim 1, characterized in that, The Micro-LED device includes: The following components are stacked sequentially from bottom to top: DBR mirror, sapphire substrate, n-GaN layer, active layer, p-GaN layer, ITO layer, first passivation layer, metal electrode and lead wire, and second passivation layer. A first passivation layer; the first passivation layer is located on the n-GaN layer and surrounds the active layer, the p-GaN layer and the ITO layer; Cathode electrode; the cathode electrode is disposed on the first passivation layer; Anode electrode; the anode electrode is disposed on the first passivation layer.

6. The biological cell culture sensing device with integrated light source according to any one of claims 1-5, characterized in that, The integrated light source biological cell culture sensing device also includes: A transimpedance amplifier circuit; the input terminal of the transimpedance amplifier circuit is connected to the output terminal of the field-effect biosensor; A low-pass filter; the input terminal of the low-pass filter is connected to the output terminal of the transimpedance amplifier circuit.

7. The biological cell culture sensing device with integrated light source according to claim 6, characterized in that: The transimpedance amplifier circuit includes a first operational amplifier, a first variable resistor, and a second variable resistor. The inverting input terminal of the first operational amplifier serves as the input terminal of the transimpedance amplifier circuit, and the output terminal of the first operational amplifier serves as the output terminal of the transimpedance amplifier circuit. The output terminal of the first operational amplifier is connected to the inverting input terminal of the first operational amplifier through the first variable resistor, and the non-inverting input terminal of the first operational amplifier is connected to a bias voltage source through the second variable resistor. The low-pass filter includes a second operational amplifier, a first resistor, a second resistor, a third resistor, and a capacitor. The non-inverting input of the second operational amplifier is connected in series with the first resistor as the input of the low-pass filter. The output of the second operational amplifier is the output of the low-pass filter. The non-inverting input of the second operational amplifier is grounded through the capacitor. The second resistor and the third resistor are connected in series and grounded to form a voltage divider circuit. The output of the second operational amplifier is connected to the inverting input of the second operational amplifier through the voltage divider circuit.

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