TWI825704B - Micropore microfluidic chip system and use thereof for measuring electrical properties of bacteria - Google Patents

Abstract

The present invention provides a micropore microfluidic chip system and use thereof for continuous dynamic measuring electrical properties of individual bacterium. The micropore microfluidic chip system comprises: a body having a tunnel electrode, an upper flow channel and a lower flow channel, and the body forms a micropore structure, which is the upper flow channel and the lower flow channel, wherein the tunnel electrode is formed in the lower flow channel. The present invention further provides a micropore microfluidic chip system comprising a recognition layer with imprinted polymer. The micropore microfluidic chip system of the present invention can measure the real-time electrical properties of a single bacterium.

Description

Micropore microfluidic chip system and its use for measuring bacterial electrical properties

The present invention relates to a micron pore microfluidic chip system and its use for measuring the electrical properties of bacteria. In particular, it relates to a micron pore microfluidic chip system with a tunneling electrode perpendicular to the micron pore axis and its use for instantaneous measurement of single bacteria. Electrical uses.

Bacteria are one of the main groups of living things, and they are also the most numerous group of living things. The skin, intestines, mouth and other parts of the human body are home to a large number of bacteria. Although most bacteria are harmless, some bacteria are pathogens. When humans are invaded by such bacteria and cause disease, it will cost a huge amount of money. It takes manpower, material resources and time to identify the type of bacteria. Different bacteria will cause different symptoms in infected people. Taking Escherichia coli ( E. coli ) as an example, the length of the bacteria lasts about 2 to 3 years. m, width is about 0.8 to 1 m. Escherichia coli usually lives in the intestines of humans and animals, and most of them are harmless. In fact, they are also important beneficial bacteria for healthy human intestines. However, some Escherichia coli are pathogenic, which means that they may cause host disease. Diarrhea or other symptoms, and the route of entry into individuals may be contaminated water, food, or through contact with animals or humans.

Diseases caused by microorganisms (especially bacteria and viruses) are becoming more and more serious around the world. Approximately 600 million people fall ill after eating contaminated food, causing 420,000 deaths worldwide each year. The bacterial detection market is expected to reach $13.98 billion by 2022, growing at a CAGR of 7.8% from 2017 to 2022. Bacterial contamination of food includes E. coli, salmonella, staphylococcus, listeria, etc. According to the CDC, approximately 265,000 people are infected each year with Shiga toxin-producing E. coli, 1.35 million with salmonella, 119,000 with Staphylococcus aureus, and 1,600 with listeria.

Microbial infections have always been a major threat to public health and human life, and timely detection of pathogenic bacteria and inhibition of bacterial growth are of great significance for preventing infections and improving survival rates. The current examination is based on biochemical and molecular biology methods, such as observing colony types, fluorescent PCR sequencing, and antibiotic testing. It takes about 3 to 5 days to get the results, which is time-consuming and laborious. If the identification time is too long, Organisms will be attacked by bacteria that multiply in large numbers, resulting in increased mortality. Technologies based on immunology and nucleic acids require a large amount of sample preparation and are not suitable for miniaturization of on-site testing. As the culture of the most commonly used microorganisms, the culture results play an important role in the treatment of infectious diseases. However, in the presence of a large number of contaminants, if the storage time is too long, the contaminating bacteria will overproduce and the culture results will be distorted. , masking the real pathogenic bacteria. Although there have been studies on the electrical properties of bacteria, they can only detect the electrical properties of a group of bacteria in the culture medium, and cannot accurately detect and subsequently analyze the electrical properties of a single or a few bacteria.

In summary, it is necessary to develop a method that can quickly and accurately measure the electrical properties of single bacteria in real time to improve the efficiency of detection and analysis of microbial infections, especially for miniaturization in on-site detection. sex.

Therefore, one object of the present invention is to provide a micron pore microfluidic chip system, which includes: a body with a tunneling electrode, an upper flow channel and a lower flow channel, and the body forms a micron pore structure with a dielectric structure. A micron hole between the upper flow channel and the lower flow channel, wherein the tunneling electrode is disposed perpendicular to the axis of the micron hole; preferably, the tunneling electrode can be formed in the lower flow channel, and The lower flow channel is between the positive electrode and the negative electrode.

According to a preferred embodiment of the present invention, the micropore microfluidic chip system of the present invention may further include a shift electrode. The shift electrode has at least one positive electrode and at least one negative electrode, and the positive electrode and the negative electrode are respectively arranged in parallel. on both sides of the micron hole axis.

According to a preferred embodiment of the present invention, the tunneling electrode includes at least one positive electrode and at least one negative electrode, and the lower flow channel is between the positive electrode and the negative electrode, wherein the positive electrode and the negative electrode can be respectively disposed perpendicular to the Both sides of the micron hole axis.

According to a preferred embodiment of the present invention, the micropore structure has a thin film layer and a supporting substrate, wherein the thin film layer forms the micron pores, and the supporting substrate can be formed on the upper flow channel.

According to a preferred embodiment of the present invention, the front end of the upper flow channel has an injection port, and the micron microfluidic chip system of the present invention can further include a molecular imprinting polymer recognition layer disposed on the upper flow channel. Between the injection port and the micron pore structure, it is composed of a polymer film imprinted with one molecule of a substance to be tested.

According to a preferred embodiment of the present invention, the molecular imprinting polymer film is formed on a microelectrode or a piezoelectric film, and the thickness of the piezoelectric film can be 100 nm to 1 m.

Another object of the present invention is to provide a method for preparing a molecular imprinting polymer recognition layer as described above, which includes: (i) mixing the test substance and a polymer monomer into a solution; (ii) mixing the The solution is in contact with a piezoelectric film and a voltage is applied; and (iii) the polymer monomer is electropolymerized on the piezoelectric film using the object to be measured as a template; wherein the voltage can be 1.8 to 2.5 volt.

Another object of the present invention is to provide a method for detecting the electrical properties of a analyte using the micropore microfluidic chip system as described above, including: (a) adding a sample containing the analyte to the injection inlet; (b) causing the analyte in the sample to flow from the upper flow channel to the lower flow channel through the micron pore; and (c) measuring a passage of the analyte in the sample through the micron pore. Tunnel current signal.

According to a preferred embodiment of the present invention, before step (b), the analyte in the sample is first captured by the molecular imprinting polymer recognition layer.

According to a preferred embodiment of the present invention, the analyte can be a bacterium, a cell, or the like.

According to a preferred embodiment of the present invention, step (c) further includes: using a tunneling signal equivalent circuit model to obtain a first electrical impedance value from the tunneling current signal.

According to a preferred embodiment of the present invention, the method for detecting the electrical properties of an object to be tested further includes a step (d): measuring a displacement signal of the object to be tested passing through the micron hole in the sample.

According to a preferred embodiment of the present invention, step (d) reconstructs the apparent three-dimensional geometry of the single or multiple objects under test based on the displacement signal.

According to a preferred embodiment of the present invention, step (d) further includes: using a shift event equivalent circuit model to obtain a second electrical impedance value from the shift signal.

The micron pore microfluidic chip system of the present invention integrates the micron pore microfluidic channel of the tunnel electrode, which enables real-time and continuous dynamic detection of the electrical signals of individual passing bacteria, and further cooperates with the equivalent circuit model to measure the electrical properties of the bacteria. The impedance value can be used to monitor the differences in individual properties of bacteria, such as drug resistance, surface attachment molecules, three-dimensional structure, etc., through electrical changes, and can be used to identify different bacteria, such as Escherichia coli or Bacillus subtilis; at the same time, the micropore microfluidic of the present invention The chip system can further include a molecular imprinting polymer recognition layer to specifically capture the bacteria to be tested and increase the linear range of detecting bacteria in the sample.

In order to enable those familiar with the art to understand the purpose, features and effects of the present invention, the present invention is described in detail below with reference to the following specific embodiments and the accompanying drawings.

The embodiments of the present invention will be further described below with reference to the drawings. The examples listed below are used to illustrate the features and applications of the present invention, but not to limit the scope of the present invention. Anyone familiar with this art will not deviate from the Some modifications and modifications may be made within the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be determined by the appended patent application scope.

The numerical values used in this article are approximate, and all experimental data are expressed within the range of 20%, preferably within the range of 10%, and optimally within the range of 5%.

In the technical field to which the present invention belongs, micropores can be divided into biological micropores and solid-state micropores; among them, biological micropores are composed of proteins and lipids existing in nature, and are the micropores that were proposed earlier and are more complete. , different from biological micropores, solid micropores are completely artificially processed on solid materials.

The micropore microfluidic chip system of the present invention includes a tunneling current electrode (hereinafter referred to as a tunneling electrode) perpendicular to the axis of the solid micron pore (hereinafter referred to as the micron pore), and can be equipped with a tunneling current electrode parallel to the axis of the micron pore. The displacement electrode can simultaneously measure the displacement electrical signal and the tunneling electrical signal of the bacteria, thereby accurately and instantly converting the electrical parameters of each bacteria; further, in the micron pore microfluidic chip system of the present invention It can further include a Molecularly Imprinted Polymers recognition layer located in the front-end flow channel of the micron pores, which uses electropolymerization to quickly and accurately imprint specific bacteria on the polymer film, which can increase the number of targets. Bacterial selectivity, and can also significantly increase the linear range of bacterial detection.

In a preferred embodiment of the present invention, the operating steps of the micropore microfluidic chip system of the present invention can be as follows: first, thoroughly mix the sample to be tested with phosphate buffered saline (PBS), take it out and place it in the The inlet of the invented micron pore microfluidic chip system allows the analyte (such as bacteria or cells) in the sample to pass through the micron pore, and measures the displacement signal and tunneling current; and further, The object to be measured in the sample to be measured can be first captured by the molecular imprinting polymer recognition layer, and then the captured object to be measured can be passed through the micron hole, and the displacement signal and tunneling current can be measured. The preparation method and efficacy test results of the micropore microfluidic chip system of the present invention will be described in detail below. Example 1 Preparation of a micron microfluidic chip system with micron holes of tunneling electrodes according to the present invention

In one embodiment of the present invention, the micropores and the tunneling electrodes perpendicular to the axis of the micropore microfluidic chip system of the present invention will be described. More specifically, the micron holes of the present invention will be described in detail. The arrangement of the micron pore structure in the well microfluidic chip system, the perforation process of the micron pores, and the arrangement of the tunneling electrode perpendicular to the axis of the micron pores.

The micron pore microfluidic chip system of the present invention includes a tunneling electrode perpendicular to the axis of the micron pore. When the bacteria to be tested pass through the micron pore, the ion flow will be temporarily blocked, causing the current to decrease, and tunneling will be added. type electrode, which can provide better tunneling current for spatial resolution. Tunneling electrodes use a concept similar to the quantum tunneling effect to construct lateral electrodes perpendicular to the axis of the micron pores. The quantum tunneling effect used in electronic components can be understood as when two conductors that are very close together (about tens of nanometers) are applied with a certain bias voltage, electrons can pass through the middle dielectric layer and end up between the two electrodes. A stable current is formed between them. In the micron pores of the present invention constructed by utilizing this phenomenon, when a bias voltage is applied to the lateral electrodes, a tunneling current will be generated. At this time, if the bacteria to be tested pass through the micron pores due to electrophoresis, the passage of blocked electrons will be temporarily blocked. causing the tunneling current to decrease. Unlike general resistive pulse sensors that use ions as transfer carriers, in the present invention, the transfer carriers of the tunneling current are electrons. Since the moving speed of electrons is much faster than that of ions, it provides better sensitivity and spatial resolution. Spend.

1 and 2A to C are schematic structural diagrams of the micropore microfluidic chip system of the present invention. The micropore microfluidic chip system of the present invention includes: a body 1 with a tunneling electrode C1, C2, and an upper layer. The flow channel 11 and the lower flow channel 12, and the body 1 forms a micron pore structure 13 with a micron hole 133 between the upper flow channel and the lower flow channel, and may further include a shift electrode A1 , A2, wherein the tunneling electrodes C1 and C2 can be disposed around the micron hole 133 and formed in the upper flow channel 11 or the lower flow channel 12 .

More specifically, the front end of the upper flow channel 11 has an injection port for adding a sample to be tested, and the end thereof has an upper chamber, and the front end of the lower flow channel 12 has a lower chamber; wherein the upper flow The channel 11 and the lower flow channel 12 can be connected to each other only through the micropores in the micropore structure 13, and the upper chamber and the lower chamber are directly connected to the micropore structure 13 and connected through the micropores. .

In addition, the tunneling electrode 14 may have at least one positive electrode C1 and at least one negative electrode C2, and the lower flow channel 12 may be between the positive electrode C1 and the negative electrode C2, so that the positive electrode C1 and the negative electrode C2 are respectively disposed perpendicular to on both sides of the micron hole axis. The displacement electrode may have at least one positive electrode A1 and at least one negative electrode A2, and the positive electrode A1 and the negative electrode A2 are respectively disposed on both sides parallel to the axis of the micron hole. More specifically, the positive electrode A1 and the negative electrode A2 may They are respectively provided at the injection inlet and outlet of the integral microfluidic channel.

Alternatively, in a preferred embodiment of the present invention, the micron pores and the tunneling electrode perpendicular to the axis of the micron pores can be packaged into an integrated sticker flow channel to form the micron pore microfluidic chip system of the present invention. More specifically, in the process of using the micropore microfluidic chip system of the present invention, the micropores must be immersed in the solution throughout the entire process. Therefore, in order to make the components closely adhere to each other and prevent solution leakage, polymethyl chloride is preferably used. Poly (methyl methacrylate) (PMMA) stickers are used for encapsulation; more specifically, with the micron pore structure 133 as the center, the encapsulated micron pore structure 133 is divided into two layers of electrolyte chambers: an upper chamber and a lower chamber. chamber to be integrated into a microfluidic system including an upper flow channel and a lower flow channel to prepare the micron pore microfluidic chip system of the present invention, in which the micron pore is the only connection between both sides of the upper chamber and the lower chamber. at.

Refer to FIG. 3 , which is a schematic diagram of the micron microfluidic chip system of the present invention integrated into the sticker flow channel 20 . A total of ten layers of stickers can be used in the sticker flow channel 20 and can be sequentially pasted on the lower substrate 20a. The lower substrate 20a can be made of a polymer material with better hydrophilicity instead of glass. For example, a ring can be used. Cyclic olefin copolymer (COC), which makes it easier for the solution to enter the flow channel and less likely to generate bubbles in each chamber. The first layer of stickers 21a and the second layer of stickers 21b can be lower flow channels, and the thickness can be 48~72 m, preferably 60 m, which includes the solution filling and extraction ports and the circular chamber directly below the micron holes. Its diameter can be 2.4~3.6 mm, preferably 3 mm. The third layer of sticker 22 is the electrode interface, and the material can be ethylene phthalate (PET) and the thickness can be 120~180 m, preferably 150 m, the third layer of sticker 22 contains a silver electrode above, which is the main bridge connecting the tunneling electrode with the outside. The fourth layer of stickers 23a, the fifth layer of stickers 23b, and the sixth layer of stickers 23c are increased layers, and the total thickness can be 288~432 m, preferably 360 m, in order to allow the eighth layer of stickers 24b and the ninth layer of stickers 25 to be attached flatly, while the seventh layer of stickers 24a and the eighth layer of stickers 24b are the upper and lower sealing layers, and the thicknesses can be 80~120 respectively. m, and 48~72 m, and preferably 100 respectively m, 60 m, the purpose is to ensure that in the micron hole structure 13, except for the micron holes, the rest are isolated from the upper and lower fluids, and to fix the micron hole structure 13 in the sticker flow channel 20. The ninth layer of sticker 25 is the spacer layer, and the thickness can be 120~180 m, preferably 150 m is the lower wall of the upper flow channel, which can prevent fluid from entering the gap between the eighth layer 24b and the sixth layer 23b. The tenth layer of sticker 26 is the upper flow channel, and the thickness can be 48~72 m, preferably 60 mIt includes an upper fluid injection port and an upper chamber. Finally, it is covered with an upper substrate 20b, which is made of the same material as the lower substrate 20a. It is mainly to facilitate the passage of liquid and is less likely to generate bubbles.

In the measurement structure of the micropore microfluidic chip system of the present invention, buffer solutions (such as PBS solutions or physiological saline solutions) and solutions containing bacterial samples can be added to the upper flow channel through the inlet, and By applying a bias voltage to encourage bacteria to flow through the micron pores into the lower flow channel, the displacement events and tunneling signal changes caused by the passage of bacteria are measured and recorded.

More specifically, referring to Figure 2A, the micropore structure 13 of the micropore microfluidic chip system of the present invention mainly consists of the following structure: a support substrate 131, a thin film layer 132 disposed on the support substrate 131, A micron hole 133 on the thin film layer 132, and preferably the tunnel electrodes C1 and C2 can be disposed on the thin film layer 132, and the upper chamber of the upper flow channel 11 and the lower flow channel 12 The lower chambers can be directly connected to the micropore structure 13 and communicate with each other through the micropores 133 . For example, the support substrate 131 can be formed to connect the upper chamber of the upper flow channel 11 .

In a preferred embodiment of the present invention, as shown in FIGS. 2B and 2C , the micropore structure 13 is a top view (viewed from the upper chamber to the lower chamber) and a bottom view (viewed from the lower chamber to the upper chamber) respectively. Viewed from the direction of the room), the support substrate 131 can be a silicon substrate or a plastic sheet with a thickness of 150~600 nm, and the thickness is preferably 200 nm. It mainly provides support for the overall structure, and the appearance can be 10 10 mm ² ~50 For ^a 50 mm square sheet, the center connected to the upper chamber can be an inverted pyramid-shaped square hole, with the widest part about 5 5 m ² ~300 300 m ² extends to the other side where the narrowest point is 1 1 m ² ~100 100 m ² , and the width is preferably 75 75 m ² , preferably 30 in the narrow part 30 m ² , so that the thin film layer 132 is in a suspended state. The thin film layer 132 is preferably made of silicon nitride (Si ₃ N ₄ ) and has a size of between 3 and 10 m micron holes 133 are formed on the film layer 132, preferably 5 m, to avoid the pore diameter being too small to cause clogging when the bacteria to be tested passes through, and to avoid the pore diameter being too large to be able to distinguish the signal, and the thickness of the film layer 132 can be 5~200 nm, preferably 30 nm, and the thickness of the film layer 132 The tunneling electrodes C1 and C2 can use gold (Au) or silver (Ag) as the conductive material. The thickness of the tunneling electrodes C1 and C2 can be 10 nm, and the distance between the tunneling electrodes C1 and C2 can be this micron. Hole 133 is the center, and the minimum diameter of hole 133 can be up to 100 micrometers. m, the smaller the distance, the more sensitive it is.

In embodiments of the present invention, the process of forming the micron holes on the thin film layer may be an electron beam using a tunneling electron microscopy (TEM) or a focused ion beam microscope system. FIB) ion beam. Since tunneling electron microscopy has stricter requirements for samples, compared to focused ion beam microscopy, the sample requirements are more relaxed and only need to be less than 25 mm in diameter. Therefore, it is better to use focused ion beam microscopy to prepare the sample. Micron holes in the microfluidic system of the present invention.

More specifically, in a preferred embodiment of the present invention, a focused ion beam electron beam scanning microscope system (FEI Helios Nanolab 600i System) is used to prepare the micron holes in the microfluidic system of the present invention. In order to obtain higher resolution, the system's maximum accelerating voltage of 30 kV and minimum current of 1.1 pA were used, and in order to produce a diameter of 5 m circular micron holes, set the relevant parameters of the FIB patterning process, including substrate settings, acceleration voltage, probe current, circular size and focus depth as listed in Table 1, to produce the product shown in Figure 4 Micron pores. Table 1 Substrate settings accelerating voltage Probe current Pattern size focus depth silicon nitride 30kV 1.1 pA 5 m round 10nm Example 2 The micropore microfluidic chip system of the present invention is used to measure the real-time electrical properties of bacteria

In one embodiment of the present invention, it will be described that the micrometer hole microfluidic chip system of the invention is used to analyze the equivalent circuit model of the measured displacement event and tunneling current, so as to obtain a test signal passing through the micrometer hole. The three-dimensional geometric parameters of the bacteria and the impedance of a single bacteria are further used to reconstruct the appearance of the bacteria to be tested and observe their real-time electrical properties.

In the measurement structure of the micropore microfluidic chip system of the present invention, the ionic solution (such as PBS solution or physiological saline solution) and the sample solution containing the bacteria to be tested are added to the upper flow channel through the injection port, And by applying a bias voltage to promote the bacteria to flow through the micron pores into the lower flow channel, the displacement events and tunneling signal changes caused by the passage of the bacteria to be measured are measured and recorded. In order to analyze these two signal, the present invention further proposes a shift event equivalent circuit model and a tunneling signal equivalent circuit model respectively.

The experimental configuration of measuring displacement current and tunneling current using the micron hole microfluidic chip system of the present invention is shown in Figure 5. The two can be measured using different waveform signal inputs, such as constant voltage, 1 Hz ~ 1 MHz sine wave, square wave, or triangle wave, etc., in which the lower flow channel and lower chamber are filled with ionic solution (such as PBS solution or physiological saline solution), and the upper flow channel and upper chamber are filled with The sample solution containing the bacteria to be tested is measured using two sets of electrodes in order to drive the bacteria through the micron pores. The first set is the displacement electrodes A1 and A2 parallel to the axis of the micron pores. The positive electrode is connected to the upper chamber just above the micron pores. chamber, the negative electrode is connected to the lower chamber below the micron hole and output to the patch clamp amplifier probe I, which mainly provides bias voltage to promote bacteria to pass through the micron hole and the measurement shift event when the bacteria to be measured pass through the micron hole. The second set of electrodes are tunneling electrodes C1 and C2 perpendicular to the axis of the micron pore. They are mainly used to measure the tunneling current when the bacteria to be tested pass through the micron pore, and output them to the patch clamp amplifier probe II. The relevant experimental conditions are shown in Table 2. Table 2 Instrument settings Feedback resistor (MultiClamp 700B) 50 MΩ Low pass filter (MultiClamp 700B) 10kHz Sampling rate (NI-USB 6361) 200kHz Patch Clamp Amplifier Probe I (A1 & A2) Shift current Patch Clamp Amplifier Probe II (C1 & C2) tunneling current Sample filling upper chamber Bacteria to be tested in 0.1xPBS lower chamber 0.1xPBS

The equivalent circuit model of the shift event is shown in Figure 6A. When the ionic solution fills the flow channel, the entire fluid can be regarded as a conductor. In this model, the resistance of the upper chamber (R _up ), the resistance of the lower chamber It is composed of resistance (R _down ), resistance (R _micropore ) and capacitance (C _micropore ) of the microporous silicon substrate. In the derivation of the formula, in order to simplify the formula, s is used to replace the imaginary part j2 in the impedance. f, the overall impedance R _total is shown in Equation 2-1. Since the DC voltage is applied in the experiment and the frequency is zero, after simplification, as shown in Equation 2-3, the equivalent circuit can be regarded as three resistors in series. Among them, the resistance can be expressed as Equation 2-4, R is the equivalent resistance of the fluid section, is the resistivity (that is, the reciprocal of the solution conductivity), L is the length of the fluid, and A is the cross-sectional area of the fluid. When bacteria pass through a micron pore, they will occupy part of the cross-sectional area of the hole, as shown in Figure 6B. Assuming that the diameter of the micron pore is D1 and the diameter of bacteria B is D2, the equivalent cross-sectional area through which the ion flow can pass becomes A', which The impedance is R' _micropore in Equation 2-5, and the difference between the two impedances is the impedance change caused by bacteria. R _micropore , the degree of this impedance change is proportional to the diameter of the bacteria. The passage of bacteria with larger diameters will cause greater impedance changes. [Formula 2-1] [Formula 2-2] [Formula 2-3] [Formula 2-4] [Formula 2-5] [Formula 2-6]

The tunneling signal equivalent circuit model is shown in Figure 7A, in which the bacterial cell is assumed to be a single-shell particle. The bacteria suspended in the solution between the two electrodes provide components in the equivalent circuit, including the resistance and capacitance of the cell wall B1 (R _wall , C _wall ) and the resistance (R _cyt ) of B2 inside the cell (i.e., the cytoplasm), and are connected in parallel with the resistance and capacitance (R _sol , C _sol ) in the solution. The overall total impedance Z _total is shown in Equation 2-7, In the experiment, a DC voltage is applied when measuring the tunneling current. Therefore, when the frequency is zero, the above formula can be simplified to Equation 2-10, which means that under the condition of DC bias, this circuit model can be regarded as each part of the The resistors are connected in series and parallel (Equation 2-12), and the simplified equivalent circuit is shown in Figure 7B. [Formula 2-7] [Formula 2-8] [Formula 2-9] [Formula 2-10] [Formula 2-11] [Formula 2-12]

A bias voltage of -100 mV is applied to both ends of the electrodes A1 and A2 parallel to the axis of the micron pore to drive the bacteria through the micron pore. When the bacteria pass through the micron pore, it will cause both a displacement event and a change in the tunneling current. In this paper In one embodiment of the invention, Escherichia coli is used as the bacterium to be tested, and the corresponding diagram between the translocation event and the tunneling current is shown in Figure 8. In addition to providing the translocation event, the corresponding diagram also provides two important information, such as As shown in Figure 9A and 9B, respectively, the measured waveform relative current changes Ib and relative time changes Tb, and the magnitude of the increase in tunneling current I _t and the length of time it affects T _t , the increase in tunneling current is mainly due to the fact that when bacteria pass through the tunneling electrode, the equivalent resistance between the two electrodes is reduced, thereby increasing the current.

Based on the tunneling current and translocation events of bacteria, three modes of movement can currently be statistically calculated. The first mode of movement is shown in Figure 10A, in which a single bacterium passes through a micron pore and accurately passes through the middle of the micron pore. This kind of movement This method can measure the translocation event and tunneling current of a single bacterium. The second movement method is shown in Figure 10B, where the bacteria enter the micron pore along the periphery of the micron pore. This movement method will cause the signal to be slow at first. changes and remains at the peak position for a period of time, and then quickly returns to the current level before the bacteria passed through; the third mode of movement is shown in Figure 10C, where multiple bacteria continuously pass through the micron pores, and their tunneling current and shift events will have multiple peaks.

The distribution diagrams of tunneling current events and translocation events of E. coli are shown in Figures 11 and 12. The tunneling current rise range is 3.4 to 21.2 nA, the translocation event size is 1.2 to 5.3 nA, and the impact time length is 0.1 to 19.7 ms.

Through bacterial translocation events, the average morphological change of bacterial cross-section can be inferred, so the ups and downs of the bacterial surface can be simply depicted. Based on the ups and downs of the signal in Figure 13, drawing software is used to simulate the large intestine as shown in Figure 13 Bacillus appearance. When a position with a larger bacterial diameter passes through a micron pore (b in the bacterial reconstruction model in Figure 13), a larger area occupied by bacteria in the micron pore results in a smaller area for ion flow to pass through, resulting in a decrease in current. The amplitude is larger (b of the measured current in Figure 13). Using displacement events to reconstruct the appearance of bacteria mainly uses the size of the displacement event to reconstruct the diameter of the bacteria, which affects the length of time to convert the length of the bacteria. Therefore, through the displacement signal of E. coli through the micron pores, the three-dimensional geometric reconstruction of the appearance of single and multiple bacteria can be performed.

Regarding the equivalent circuit model of the shift event, the equivalent circuit model of the micron pore electronic signal is used to convert the distribution of the shift event into the impedance change caused by E. coli passing through the micron pore. The calculation method is shown in Figure 14 shows that the voltage applied to both sides of the micron pore is 100 mV, the current size when the bacteria does not pass through the micron pore is I ₀ , at this time the equivalent impedance of the micron pore is R _micorpore , and the current size when the bacteria passes through is I, at this time The equivalent impedance of a micron pore is R' _micropore . The subtraction between the two is the impedance change caused by bacteria passing through the micron pore. R _micropore (Formula 4-1). The resulting distribution is shown in Figure 15. The impedance changes caused by E. coli fall between 12.5 and 57.6 k . [Formula 4-1]

Regarding the tunneling signal equivalent circuit model, the distribution diagram of tunneling current events is used to calculate the equivalent impedance of a single E. coli. The calculation method is shown in Figure 16. The current when the bacteria does not pass through the micron pore is I ₀ . The resistance at this time is the equivalent impedance R _sol of the solution between the tunneling electrodes. When the bacteria pass through the micron pore, the current is I. The equivalent impedance Z _total at this time is the equivalent impedance of the bacteria R. The equivalent impedance of _bacteria and the solution R _sol is connected in parallel, and its derived relationship is shown in Equation 4-4. The distribution of calculation results is shown in Figure 17. The equivalent impedance value of a single E. coli particle ranges from 4.7 to 28.6 M , with an average of 14.1 M . Therefore, by using the tunneling current signal of E. coli through the micron pore, and using the signal equivalent circuit model of the present invention, the impedance of a single E. coli can be calculated, and the more precise electrical changes in the electrode orientation of each cross-section of the bacteria can be understood. And used to monitor the differences in individual properties of bacteria. [Formula 4-2] [Formula 4-3] [Formula 4-4]

The present invention uses a solid-state micron hole integrated with a tunneling electrode to detect electrical signals of bacteria, especially the electrical signals of a single bacteria, including qualitative measurement of voltage and current, shift events, and exploration of tunneling current, and further Establish an equivalent circuit model to simulate the impedance response during displacement and the role of bacteria in the tunneling current. This can be used to calculate the impedance of the bacteria to be measured and analyze the structure and electrical characteristics of the bacteria. Therefore, it can be used to identify the type of bacteria. . Example 3 Molecular imprinting polymer recognition layer of the micron pore microfluidic chip system of the present invention

In one embodiment of the present invention, the front end of the micropore microfluidic chip system additionally has a molecular imprinting polymer recognition layer (hereinafter referred to as the recognition layer); more specifically, see Figure 18, which shows the present invention including the recognition layer. A schematic structural diagram of a micron pore microfluidic chip system. The micron pore microfluidic wafer system of the present invention includes: an upper flow channel 11, a lower flow channel 12, and a micron pore structure 13, and the identification layer 14 can be disposed on the Between the injection port of the upper flow channel 11 and the upper chamber, the molecular imprinting polymer uses appropriate functional monomers and bacteria to be detected as templates, and electropolymerization is used on the piezoelectric polymer sensor. The bacterial molecule imprinting polymer is made on the piezoelectric film to serve as the identification layer in the micron pore microfluidic chip system of the present invention; alternatively, in the micron pore microfluidic chip system of the present invention, the molecular imprinting The polymer identification layer can perform imprinting on two or more target bacteria to capture more than two target bacteria at the same time; in addition, the micron pore microfluidic chip system of the present invention can further include a waste liquid area 15 to discharge unused liquid. The waste liquid captured by the identification layer 14.

In an embodiment of the present invention, the recognition layer of the micropore microfluidic chip system is a molecular imprinting high-precision material prepared by using Escherichia coli as a template and o-phenylenediamine as a monomer. Molecule, in which E. coli transformed with fluorescent genes is used to facilitate the observation of the results, and each process stage of the molecular imprinting polymer of the present invention is verified by fluorescence photography and scanning electron microscope, and Its effectiveness in capturing target bacteria.

The preparation method of the molecular imprinting polymer recognition layer of the present invention is as follows: in the embodiment of the present invention, modified electrodes (such as modified with polyvinylidene fluoride (PVDF)) are used, and other materials can also be substituted according to needs. , first prepare the monomer for rubbing (such as o-phenylenediamine) in a buffer solution (such as acetate buffer solution), and add E. coli at a concentration of 10 ⁷ CFU/mL as a template, which can replace Escherichia coli peripheral polysaccharide was used as a template, and then the prepared solution was dispersed in a droplet size of 2.5 to 4.0 L is dropped on the electrode to completely cover a single electrode, and then a voltage of 1.8 to 2.5 volts is applied for electropolymerization for 10 to 30 minutes, so that the molecular imprinting polymer recognition layer is polymerized on the piezoelectric film, in which the size of the microelectrode negative electrode greater than 25 m (to avoid being burned during electropolymerization), the positive electrode is less than 2 cm, and the thickness of the piezoelectric film is 100 nm to 1 m.

After E. coli and the o-phenylenediamine monomer used for rubbing are polymerized on the electrode surface, place the electrode in a crystallizing dish filled with methanol solution, shake and clean it for 30 minutes with a horizontal oscillator rotating at 35 rpm. The template E. coli was removed, and the result is shown in Figure 19A. It can be seen that the bright spots on the electrode have obviously disappeared after the template was removed, so it is verified that the methanol solution can be used to remove the template; alternatively, after using In the micron pore microfluidic chip system of the present invention, sodium dodecyl sulfate (SDS) solution can be used to wash away the identified substances such as E. coli, so that the identified substances can continue to pass through the micron pores to the lower flow channel. And the waste liquid that has not been captured can be removed through the waste liquid area. Then, Escherichia coli with fluorescent transgenic genes was used to verify the ability of the molecular imprinting polymer recognition layer of the present invention to capture target bacteria. The results are shown in Figure 19B, in which it can be seen that the brightness of the fluorescence is significantly greater than that of Figure 19A shows the fluorescence brightness after removing the template. This result shows that the molecular imprinting polymer of the present invention has the ability to grasp the target, and therefore can be used to detect the target in the micron pore microfluidic chip system of the present invention. identification layer.

In addition, in order to verify the specificity of the molecular imprinting polymer recognition layer of the present invention, after removing the template E. coli with methanol, the molecular imprinting polymer recognition layer of the present invention was used to capture Bacillus subtilis with fluorescent transgenic genes. ( Bacillus subtilis ), the results are shown in Figure 19C, in which it can be seen that the brightness of the fluorescence is significantly lower than the results of capturing E. coli, indicating that if E. coli is used as the template for the molecular imprinting polymer of the present invention, it will The grabbing ability of other bacteria other than E. coli is far less than that of E. coli. Therefore, the molecular imprinting polymer recognition layer of the present invention has considerable specificity.

The molecular imprinting polymer recognition layer of the present invention has high selectivity and sensitivity for target bacteria, can greatly increase the linear range of bacterial detection, and also has high chemical/mechanical stability, reusability, low limit of detection (LOD), It has the advantages of easy preparation, low cost, miniaturization, automation, etc., and the bacterial molecular imprinting polymer technology produced by electropolymerization in the present invention not only has the advantages of fast and simple process, but more importantly, it can be accurate and uniform. A porous film is created on the electrode to reduce material loss.

To sum up, the micropore microfluidic chip system of the present invention integrates the micropore microfluidic channel of the tunnel electrode, so that it can real-time and continuous dynamic detection of the electrical signals of individual single passing bacteria, and further cooperates with the calibrated circuit model to The electrical impedance value of the bacteria is measured, so that the electrical changes can be used to monitor the differences in individual properties of the bacteria, such as drug resistance, surface attachment molecules, three-dimensional structure, etc., and can be used to identify different bacteria, such as Escherichia coli or Bacillus subtilis; at the same time, the present invention The micropore microfluidic chip system can further include a molecular imprinting polymer recognition layer to specifically capture the bacteria to be tested and increase the linear range of the detection of bacteria in the sample.

1: Ontology 11: Upper flow channel 12: Lower flow channel 13:Micropore architecture 131: Support base material 132:Thin film layer 133: Micron pores 14:Identification layer 15:Waste liquid area 20: Sticker runner 20a: Lower substrate 20b: Upper substrate 21a: First layer of stickers 21b: Second layer of stickers 22:The third layer of stickers 23a: The fourth layer of stickers 23b: The fifth layer of stickers 23c:Sixth layer of stickers 24a:Seventh layer of stickers 24b: The eighth layer of stickers 25: Ninth layer sticker 26:Tenth layer of stickers A1, A2: shift electrode B: Bacteria B1: cell wall B2: Inside the cell (cytoplasm) C1, C2: Tunneling electrode D1: Micron hole diameter D2: Bacterial diameter

FIG. 1 shows a schematic structural diagram of a micropore microfluidic chip system according to an embodiment of the present invention. FIG. 2A shows a cross-sectional view of the micropore structure with tunneling electrodes in the micropore microfluidic chip system of the present invention. 2B shows a top view of the micropore structure with tunneling electrodes in the micropore microfluidic chip system of the present invention. 2C shows a bottom view of the micropore structure with tunneling electrodes in the micropore microfluidic chip system of the present invention. Figure 3 shows a schematic diagram of the micron hole microfluidic chip system of the present invention integrated into the sticker flow channel. Figure 4 shows the preparation of micron holes of the microfluidic chip of the present invention using a focused ion beam electron beam scanning microscope system. Figure 5 shows the experimental configuration for measuring displacement current and tunneling current in the micron hole microfluidic chip system of the present invention. FIG. 6A shows the equivalent circuit model of the displacement event in the micron pore microfluidic chip system of the present invention. FIG. 6B shows a schematic diagram of the micropore blocking principle in the micropore microfluidic chip system of the present invention. FIG. 7A shows the equivalent circuit model of the tunneling signal in the micron hole microfluidic chip system of the present invention. FIG. 7B shows a simplified equivalent circuit model of the tunneling signal in the micron hole microfluidic chip system of the present invention. Figure 8 shows the translocation events and tunneling current of E. coli using the micropore microfluidic chip system of the present invention. Figure 9A shows the actual situation of the translocation event of E. coli in Figure 7. FIG. 9B shows the actual tunneling current of E. coli in FIG. 7 . Figure 10A shows the displacement events and tunneling current of a single bacterium accurately passing through the middle of the micron pore measured using the micron pore microfluidic chip system of the present invention. Figure 10B shows the translocation events and tunneling current of a single bacterium entering the micron pore along the periphery of the micron pore measured using the micron pore microfluidic chip system of the present invention. Figure 10C shows the translocation events and tunneling current of multiple bacteria passing through the micropores measured using the micropore microfluidic chip system of the present invention. Figure 11 shows the tunneling current event distribution diagram of E. coli. Figure 12 shows the distribution of translocation events for E. coli. Figure 13 shows the three-dimensional geometric reconstruction of the appearance of bacteria using the translocation events obtained by the micropore microfluidic chip system of the present invention. Figure 14 shows a schematic diagram showing impedance changes caused by bacteria passing through micron pores deduced from translocation events. Figure 15 shows a distribution diagram of micropore impedance changes for bacterial translocation events. Figure 16 shows a schematic diagram of estimating the impedance of a single bacterium from the tunneling current. Figure 17 shows the distribution diagram of the equivalent impedance of a single bacterium. Figure 18 shows a schematic structural diagram of a micron micropore microfluidic chip system according to another embodiment of the present invention. Figure 19A shows a photo under a fluorescence microscope of a piezoelectric film in which the template E. coli is removed from the molecularly imprinted polymer using a methanol solution. Figure 19B shows a photo of E. coli captured using a molecular imprinting polymer recognition layer under a fluorescence microscope. Figure 19C shows a photo of Bacillus subtilis captured under a fluorescence microscope using a molecular imprinting polymer recognition layer.

without.

1: Ontology 11: Upper flow channel 12: Lower flow channel 13:Micropore architecture A1, A2: shift electrode C1, C2: Tunneling electrode

Claims (11)

A micron pore microfluidic chip system includes: a body with a tunneling electrode, an upper flow channel and a lower flow channel, and the body forms a micron pore structure with a structure between the upper flow channel and the lower flow channel There is a micron hole between them, wherein the tunneling electrode is arranged perpendicular to the axis of the micron hole, the front end of the upper flow channel has an injection port; and a molecular imprinting polymer recognition layer is arranged on the upper flow channel between the injection port and the micron pore structure, and is composed of a polymer film imprinted with one molecule of a test object. The micropore microfluidic chip system according to claim 1, further comprising a shift electrode, the shift electrode has at least one positive electrode and at least one negative electrode, and the positive electrode and the negative electrode are respectively arranged parallel to the axis of the micrometer hole. both sides. The micropore microfluidic chip system as claimed in claim 1 or 2, wherein the tunneling electrode includes at least one positive electrode and at least one negative electrode, and the positive electrode and the negative electrode can be respectively disposed on both sides perpendicular to the axis of the micropore. . The micropore microfluidic chip system of claim 1 or 2, wherein the micropore structure has a thin film layer and a supporting substrate, wherein the thin film layer forms the micropore. The micropore microfluidic chip system according to claim 1 or 2, wherein the molecular imprinting polymer film is formed on a microelectrode or a piezoelectric film. The micropore microfluidic chip system as claimed in claim 1 or 2, wherein the analyte is a bacterium or a cell. A method for detecting the electrical properties of an analyte using the micropore microfluidic chip system as described in any one of claims 1 to 6, including: (a) adding a sample containing the analyte to the injection inlet; (b) causing the analyte in the sample to flow from the upper flow channel to the lower flow channel through the micron pore; (c) measuring a tunnel of the analyte in the sample through the micron pore current signal; and (d) measuring a displacement current signal of the analyte in the sample passing through the micron pore; wherein before step (b), the analyte in the sample is first imprinted with the molecule by the polymer Identification layer capture. The method of claim 7, wherein the analyte is a bacterium or a cell. The method of claim 7, wherein step (c) further includes: using a tunneling signal equivalent circuit model to obtain a first electrical impedance value from the tunneling current signal. The method of claim 7, wherein step (d) reconstructs the apparent three-dimensional geometry of a single or multiple objects under test based on the displacement signal. The method of claim 7, wherein step (d) further includes: applying the shift signal to a shift event equivalent circuit model to obtain a second electrical impedance value.