A new patch clamp amplifier system based on single chip microcomputer

introduction

Patch clamp is an important tool for detecting cell membrane ion channel current. The patch clamp amplifier is relatively large and expensive, usually between tens of thousands and hundreds of thousands. More importantly, since the analog acquisition system is directly connected to the PC, the interference caused by the PC is very large.

In order to solve the above problems, we studied a new type of patch clamp amplifier. This system is divided into two parts: the upper computer and the lower computer. The lower computer is an acquisition system with a single-chip microcomputer as the control core. It can work independently to complete the acquisition, amplification, capacitance and resistance compensation of microcurrent signals, waveform display and data storage. In addition, the lower computer can also communicate with the upper computer. The communication is realized by infrared transmission. The serial port drives the infrared transmitter to realize the communication between the upper computer and the lower computer. The upper computer mainly completes the processing and analysis of the signals transmitted by the lower computer.

System Structure

In order to realize signal acquisition, display and transmission, the system has the following basic functions:

·Ion channel current collection and amplification

Clamp voltage generator

Resistor and capacitor compensation

Analog to digital signal conversion

Human-machine interface

System and PC communication

To achieve the above functional requirements, the system is mainly divided into six main modules: micro-current collection and amplification, clamping voltage generator, resistance and capacitance compensation circuit, ADμC841 control core, liquid crystal display module LCM3202401 and key control, and communication between the system and the PC. Figure 1 shows the functional block diagram of the system.

Figure 1 Functional block diagram of the patch clamp amplifier system

As shown in Figure 1, the ion channel current signal obtained through the electrode is collected and amplified by micro-current, and the resistance and capacitance are compensated at the same time before entering the A/D conversion part of the microcontroller to digitize the analog signal. The collected signal is also sent to the LCD for display. In addition, the storage and transmission of the collected signal can also be realized. The key module can realize the control of multiple operating functions in a friendly and convenient way.

System hardware design

Control module - single chip microcomputer system ADmC841

ADmsC841 is a single-chip microcomputer recently launched by ADI. It integrates the core of 8052 microprocessor and provides a large storage space. In addition, the chip also integrates many peripheral components. Among them, the precise and high-speed 8-channel 12-bit analog-to-digital conversion (its conversion rate can reach up to 420Ksps) can easily realize the interface with the front-stage sensor; UART, SPI, I2C communication interface, time interval counter, watchdog timer and power monitor, etc. These modules can easily realize communication with other single-chip microcomputers or PCs (level conversion circuit is required at this time), and can also effectively ensure the normal operation of the single-chip microcomputer power supply and the normal operation of the program.

Clamp voltage generator

There are two methods for monitoring the current of cell membrane ion channels: voltage clamping and current clamping. We use the voltage clamping method, that is, a clamping voltage is connected to the in-phase input of the IV converter to clamp the cell membrane potential at a fixed voltage value. The amplitude of this voltage is in the range of tens to hundreds of mV, and the pulse time is 10~50ms. Figure 2 shows the circuit of the clamping voltage generator. The circuit uses 555 to form a multi-vibrator to achieve the generation of square waves. The amplitude of the square wave signal directly generated by 555 is close to the power supply voltage, and the clamping voltage used should be a signal with a voltage amplitude of about hundreds of millivolts, so the amplitude of the signal generated by 555 should be adjusted. The square wave signal generated by 555 passes through resistor R3 and voltage regulator D1, and a stable 2.4V voltage is output at both ends of D1. Then, a potentiometer R4 is connected at both ends of this voltage, and the voltage is taken from its sliding end as the clamping voltage. In this way, the clamping voltage can be flexibly adjusted to obtain the required amplitude. The period of the generated square wave can be changed between 14ms and 154ms by adjusting potentiometer R2.

Figure 2 Clamp voltage generation circuit [page]

Micro-current collection, amplification and resistance-capacitance compensation

The most important part of the patch clamp amplifier is the current collection, IV conversion and amplification, and various compensation circuits. Since the current signal is measured, the current must be converted into voltage first. Since the cell membrane ion channel current is very weak, only a few pA, the performance requirements of the amplifier used in the current-voltage conversion part are relatively high, requiring it to have a very high input impedance and a very low bias current. To meet the above requirements, the author selected ADI's high-precision, low-power, rail-to-rail amplifier AD8627. It has an extremely low bias current, only 1pA at most; it can be powered by a single power supply of 5~26V or ±2.5 to ±13V; the maximum offset voltage is 500mV. The specific circuit is shown in Figure 3.

Figure 3 Current-voltage conversion circuit

When using a patch clamp amplifier to record the cell membrane ion channel current, there are stray electrode capacitance Cp, cell membrane capacitance Cm and series resistance Rs between the electrode input and the cell membrane at the electrode input; if a step voltage is applied to the clamp voltage Vc, it will inevitably cause transient charging currents of Cp and Cm and voltage drops on Rs. The charging current passes through the resistor Rf, resulting in dynamic errors in the output voltage, and may saturate the amplifier, so that it cannot work normally. To correct these errors, corresponding compensation measures must be adopted. Figure 4 shows the circuit diagram of the resistor-capacitor compensation circuit. The amplifiers in this circuit all use ADI's OP4177. OP4177 integrates four operational amplifiers and uses a 5V power supply. It can be powered by the same power supply as other parts of the circuit. Its offset voltage is 60mV, bias current is 2nA, and noise is very low, which can well meet the design requirements.

Figure 4 RC compensation circuit

The electrode potential Vp is the sum of the series resistance compensation signal V1 and the corrected control voltage 10 Vc, which is realized by a one-tenth attenuation circuit composed of two resistors. The voltage output by A6 enters the follower after passing through a potentiometer, and then realizes the fast capacitance compensation through a 1pF capacitor. The potentiometer can realize compensation adjustment, making the circuit flexible and convenient. The slow capacitance compensation signal is obtained by Vc passing through the state variable loop composed of A3, A4 and A5. The error voltage V2 generated by the predicted injection current on Rs is also obtained by the state variable loop and added to the control voltage Vc through A2. Due to the positive feedback, A2 passes through the state variable loop to generate an overshoot voltage Vc corresponding to Vc, thereby generating an overcharging effect. At the same time, the compensation circuit of the slow capacitor also realizes the prediction of the series resistance error. The voltage output from the current monitoring output terminal passes through A1 and then is synchronously adjusted by the prediction circuit to realize the compensation of the series resistance. The fast capacitor and slow capacitor compensation circuits are both shown in Figure 4, and are connected to the electrode input terminal through their respective current injection capacitors.

LCD display module

This system uses the graphic LCD LCM3202401 from Beijing Qingyun Company, which has a dot matrix of 320240 and uses SED1335 as the controller, which can realize two display modes: graphic and text. The LCD module is directly controlled by ADmC841.

Button module and menu interface

In the system, three buttons are provided, corresponding to the relevant menus on the LCD screen. Each level of the menu provides users with simple prompts for easy use, so you only need to press a button (there are three buttons A, B, and C) under the prompt of the menu to complete the required operation.

This system uses independent buttons, which directly use I/O lines to form a single button circuit. Each button occupies an I/O line, and its working state will not affect the working state of other I/O lines. The control lines are controlled by p1.2, p1.3 and p1.4 respectively, and the button input is valid at high level. Since each button is connected to the LCD menu during use, the current design of the system is that one button corresponds to one function.

System software design

The system software is mainly used to complete the acquisition and storage of analog signals by the single chip microcomputer, the playback of original data, the communication between the system and the PC, and the control of the LCD and buttons to achieve human-computer interaction for easy operation. The system software design adopts a modular structure, which is mainly divided into measurement module, printing module and wireless transmission module. The system adopts a Chinese menu-friendly user interface for easy operation. After powering on, the system is initialized first, and then the main menu is displayed. After the main menu is displayed, the function menu is displayed after a delay of 5 seconds. The function menu consists of three parts: playback of original data, real-time sampling display and infrared transmission.

Conclusion

The circuit designed in this paper is suitable for the collection of micro-current signals. It overcomes the shortcomings of the existing diaphragm embedded system, such as large size and high price, to a certain extent. It also weakens the interference caused by the direct connection between the measurement and the PC by using wireless communication.