TWI589855B - Chip for monitoring transportation behavior and method for monitoring transportation behavior with the same - Google Patents

Description

Sensing wafer and method for monitoring material transport behavior

The present invention is basically directed to a sensing wafer for monitoring the transport behavior of a substance. More specifically, the present invention relates to a sensing wafer that can simultaneously capture surface plasmon resonance (SPR) and plasmon waveguide resonance (PWR) signals while monitoring material transport behavior.

The study of transmembrane transport of membrane proteins on membranes plays an important role in understanding biological mechanisms and applications, and is also helpful in understanding the effects of drugs on functional proteins on membranes. Nowadays, in the study of channel protein operation (especially in the study of ion migration), the main tool used is patch clamp (Patch Clamp) technology, which has developed quite mature and accurate, but requires high-intensity training and experience. A wealth of operators to operate, as well as the need for relatively high equipment costs. In addition, other known techniques, such as fluorescence dyes, radioactive flux assays, and fluorescence resonance energy transfer-based voltages (fluorescence resonance energy transfer-based voltages) Measurement) There are mainly restrictions on the need to look for additional labeling, the need to chemically modify the surface of the cell membrane, or to measure only charged species. Therefore, there is still a need to construct a technology platform that provides dynamic information on the total amount of channel protein transported substances without the use of fluorescent or radioactive labels.

The present invention provides a sensing wafer for monitoring a substance transport behavior, comprising: a substrate; and a grating structure formed on the substrate. The grating structure includes: a layer having an array of holes; and a metal layer formed at a bottom of the array hole and exposed from the array hole, and the metal layer serves as a surface plasma resonance layer.

According to an embodiment of the invention, the material of the metal layer is selected from at least one of the group consisting of gold, silver, aluminum, nickel and titanium. According to another embodiment of the invention, the metal layer has a thickness ranging between 1 nm and 1000 nm. According to another embodiment of the invention, the metal layer has a thickness of 50 nm.

According to an embodiment of the invention, the material having the layer of the array of holes is selected from the group consisting of cerium oxide (SiO ₂ ), titanium oxide (TiO ₂ ), aluminum oxide (Al ₂ O ₃ ), and tetra-nitridium tetrachloride ( At least one of a group consisting of SiN ₃ ), Teflon, a polymer material, and graphene. According to a specific embodiment, the layer having the array holes is a plasma waveguide layer and has a thickness of 50 nm to 1000 nm.

According to a particular embodiment of the invention, the layer having the array of holes has a thickness of from 50 nm to 1000 nm. According to a specific embodiment, the period of the array holes is 0.01 μm to 10 μm, and the aperture is 0.01 μm to 10 μm.

In accordance with an embodiment of the present invention, the sensing wafer includes a film for transporting a monitoring substance disposed on the grating structure such that at least a portion of the array of holes form an interior space. According to a specific embodiment, the membrane is a biological membrane. Preferably, the biofilm system has at least one transport membrane protein.

In one aspect of the invention, a method of monitoring a material transport behavior is also provided, comprising using the sense wafer to detect a plasma waveguide resonance signal and/or a surface plasma resonance signal.

According to an embodiment of the invention, the method further comprises laying a film on the grating structure of the sensing wafer such that the array of holes of the grating structure form an internal space. In a preferred embodiment of the present invention, the method further comprises contacting the sensing wafer with a solution containing the substance to be monitored, and detecting changes in the plasma waveguide resonance signal and/or the surface plasma resonance signal. According to a specific embodiment, the plasma waveguide resonance signal is generated by the layer having the array holes, and the surface plasma resonance signal is generated by the metal layer.

In the sensing wafer of the present invention, the metal layer acts as a layer that produces a surface plasma resonance signal. By simultaneously having a metal layer capable of generating a surface plasma resonance signal and a grating structure capable of generating a plasma waveguide resonance, the present invention can achieve a signal for simultaneously monitoring surface plasmon resonance and plasma waveguide resonance at the same time.

The sensing wafer and method disclosed by the present invention can be measured without using a specific marking substance, complementing the field that cannot be detected by the prior art, and can be applied to a wider range of mass transfer measurement.

100,200‧‧‧Sensor wafer

102‧‧‧Light source

104‧‧‧Detector

110‧‧‧Grating structure

111‧‧‧Substrate

112‧‧‧metal layer

113‧‧‧ joint layer

114‧‧‧layer with array holes

116‧‧‧Array holes

120, 220‧ ‧ film

122‧‧‧ transport protein

124‧‧‧ substances

211‧‧‧Substrate

212‧‧‧metal layer

213‧‧‧ joint layer

214‧‧‧layer with array holes

216‧‧‧Array holes

230‧‧‧ Giant cell membrane derived vesicles

BRIEF DESCRIPTION OF THE DRAWINGS The accompanying drawings, which are incorporated in and constitute The principles of the invention are taught.

Fig. 1 shows the position of the wafer of the present invention using the array holes formed thereon as a support film. Figures 1a and 1c show that the overall solution outside the hole increases the refractive index (n) due to the increased concentration of the substance to be monitored. Figures 1b and 1d show that as the material to be monitored accumulates inside the etched pores, the refractive index of the solution inside the pore increases as the concentration increases.

Figure 2 shows the structure of a sensing wafer in accordance with one embodiment of the present invention.

Figure 3 shows that the sensing wafer of one embodiment of the present invention can include the film laid on the grating structure and its resonant correlation peaks.

Figure 4 shows a schematic of monitoring the transmembrane transport behavior of a substance using a membrane-mounted sensing wafer for real time detection.

Figure 5 is a schematic cross-sectional view showing a sensing wafer in accordance with an embodiment of the present invention.

Figure 6 shows the reflectance versus angle of incidence for different aperture and array periods when the sense wafer is in water and the hole height is fixed, in accordance with an embodiment of the present invention.

Figure 7 is a cross-sectional view of a sensing wafer and a side view of a scanning electron microscope of one embodiment of the present invention.

Figure 8 shows the method and steps for laying a cell membrane on a sensing wafer.

Figure 9 shows the performance of the unlaid sensing wafers for instant measurements.

Figure 10 shows the performance of the transmissive sensing wafer when it is being measured.

Figure 11 shows the performance of the sensed wafer when it is subjected to an immediate measurement after the addition of an inhibitor that inhibits the activity of the transport protein on the membrane.

Fig. 12 is a view showing different detection regions formed by combining surface plasma resonance and plasma waveguide resonance in a sensing wafer according to an embodiment of the present invention.

Figure 13 is a cross-sectional view showing a sensing wafer in accordance with an embodiment of the present invention.

Fig. 14 is a view showing the detection results of the sensing wafer in the case of having different array aperture periods and different size apertures in an embodiment of the present invention.

Figure 15 shows a cross-sectional view and a scanning electron microscope side view of a sensing wafer of the present invention having both SPR and PWR signals.

Figure 16 shows the performance of a sensor wafer that can simultaneously monitor SPR and PWR signals without film laying for immediate measurement.

Figure 17 shows the performance of a sensor wafer that can be simultaneously monitored by SPR and PWR signals for real-time measurement.

Figure 18 shows the performance of the sensed wafer when it is immediately measured by the addition of an inhibitor that inhibits the activity of the transport protein on the membrane.

Figure 19 is a graph showing the effect of the reflectance of the sensing wafer of the present invention for simultaneously monitoring the SPR and PWR signals in different environments on the angle of incidence.

It should be noted that the figures are not to scale and the elements of the structure or function that are substantially represented by the reference number are intended to be illustrative. It should also be noted that the figures are only intended to facilitate the various embodiments disclosed herein. The drawings do not describe the scope of all aspects of the teachings herein and are not intended to be limited.

The embodiments of the present invention are provided to exemplify the advantages and technical features of the present invention, and the present invention is not intended to limit the present invention. Any one skilled in the art can, without departing from the spirit and scope of the present invention, Various modifications and refinements are made, therefore, the scope of the present invention is defined by the scope of the appended claims.

The present invention relates to a sensing wafer for monitoring the transport behavior of a substance, as shown in FIG. 1, using a micron-sized array of pore structures formed on the sensing wafer 100 as a supported lipid bilayer membrane having a channel protein. It serves as a barrier to distinguish the space between the pore area and the bulk area. In Fig. 1a, when the bulk area outside the hole increases the refractive index due to the increase in the concentration of the substance to be monitored, and the area inside the hole remains unchanged due to the barrier, the change is mainly caused by the resonance of the plasma waveguide. The result is shown in Fig. 1c. The formant of this shift is called "plasma waveguide resonance correlation peak"; in Fig. 1b, when the glucose as the substance to be monitored accumulates inside the etched hole, the hole is made The refractive index of the internal solution increases due to the increase in concentration, and its change is sensed by surface plasma resonance. The resonance angle shift result is shown in Fig. 1d. The resonance peak of this shift is called "surface plasma resonance correlation. peak".

In one embodiment, the sensing wafer of the present invention includes a substrate 111 and a grating structure 110 formed on the substrate 111. The grating structure 110 includes a layer 114 having an array of holes and a metal layer 112, as shown in FIG. Show.

The layer 114 having array holes can induce surface plasma resonance, and the material thereof can be, but not limited to, cerium oxide (SiO ₂ ), titanium oxide (TiO ₂ ), aluminum oxide (Al ₂ O ₃ ), and tetra-nitriding. Dielectric materials such as SiN ₃ and Teflon, or polymer materials, or conductive non-metallic materials such as graphene. The layer 114 having the array holes may have a thickness of 50 nm to 1000 nm, which is a preferable condition for forming a plasma waveguide resonance.

Further, the array aperture may have a pore diameter of from 0.01 micrometer to 10 micrometers, and the array aperture may have a period of from 0.01 micrometer to 10 micrometers. As used herein, "period" means the distance between a hole and a hole in a layer having an array of holes.

The substrate material used in the present invention may be a low refractive index material such as fused silica (refractive index (n) = 1.458), which distinguishes two resonance peaks of surface plasma resonance and plasma waveguide resonance. Be more clear.

In the sensing wafer of the present invention, in addition to bonding using a bonding layer (such as a titanium layer and a chromium layer) between the grating structure and the substrate, a thermal annealing technique may also be used to increase the variation of the curve signal reaction, and further Improve signal resolution.

The method for inducing and detecting the vibration of the plasma waveguide and/or the surface plasma resonance used in the present invention may be a method known in the art. For example, the light source for inducing surface plasma resonance may be Any light source known to induce surface plasma resonance, such as light with a wavelength of 632.8 nm, or not See commonly used excitation electromagnetic waves in the light interval.

Referring to Fig. 3, the sensing wafer of the present invention may further comprise a film 120 laid on the grating structure. Therefore, the sensing wafer can be used to monitor the transport behavior of the substance 124 through the film. In a specific embodiment of the present invention, by laying a film on the sensing wafer, the array holes on the sensing wafer can be separated from the external environment to form an internal space, thereby monitoring the substance inside and outside the film. Transmission behavior. The film can be applied to the sensing wafer using any conventional means.

In transmembrane-laid sensing wafers, real time detection can be used to monitor the transmembrane transport behavior of the material. Referring to Fig. 4, since the accumulation of the substance 124 in the internal space formed by the film 120 and the holes in the layer 114 having the array of holes is detected by the surface plasma correlation peak, the angle of the incident light can be fixed. And measuring the signal reflected by the metal layer 112 over time to observe the change of the surface resonance resonance peak. It is found that the more the substance 124 accumulated in the internal space through the transport protein 122 on the membrane 120, the higher the average refractive index of the solution inside the pore, the higher the signal intensity, and stops until a new equilibrium is reached.

Please refer to FIG. 5, which shows a schematic cross-sectional view of a sensing wafer in accordance with an embodiment of the present invention. The sensing wafer 200 includes a substrate 211 and a grating structure formed on the substrate 211. The grating structure includes a layer 214 having an array of holes and a metal layer 212. In the present embodiment, the material of the substrate 211 is molten cerium oxide, the metal layer 212 as the plasma resonance layer is composed of gold and has a thickness of 50 nm, and the material of the layer 214 having the array holes is also molten oxidized. Hey. In addition, in the embodiment, the sensing The wafer further includes a titanium layer as the bonding layer 213 having a thickness of 1.5 nm.

The manufacturing method of one embodiment of the sensing wafer of the above case will be further explained below. First, a substrate is provided, and the material forming the substrate is molten cerium oxide. Prior to forming the metal layer on the substrate, it may be pre-cleaned with O ₂ plasma. Thereafter, the pre-cleaned substrate was spin-coated to apply a photomask. Next, after exposure to ultraviolet light, the unreacted photomask is removed from the substrate. Thereafter, the substrate can be etched using, for example, inductively coupled plasma. After etching, it can be cleaned using O ₂ plasma and placed in an electron beam evaporator to form a metal layer. In this embodiment, a 1.5 nm titanium metal layer is first formed on the bottom of the hole formed by the etching process and the remaining mask to form a 50 nm metal layer. Thereafter, the remaining reticle and the metal layer deposited thereon were removed with acetone, and the wafer was washed with alcohol and deionized water. Through the above manufacturing steps, a sensing wafer having a grating structure as shown in FIG. 5 can be obtained.

Referring to Figure 6, there is shown the reflectance versus incident angle for different aperture and array periods when the wafer is sensed in water and the hole height is fixed at 510 nm in accordance with an embodiment of the present invention. As shown in Fig. 6, it is found that when the aperture and the array period are less than 3000 nm, the detection result will have severe signal interference; in contrast, under the condition that the aperture and the array period are greater than 3000 nm, From the range of 75° to 90°, a clear characteristic angle can be obtained, and in the case where the width of the aperture and the array period is more than 3000 nm, 3000 nm, 3500 nm, and 4000 nm are preferable. Referring to Figure 7, there is shown a sensing crystal of a preferred embodiment of the present invention. A cross-sectional view of the sheet and a scanning electron microscope (SEM) side view in which the aperture and array period of the array holes are both about 3 microns and the depth of the holes is about 510 nm.

Furthermore, the sensing wafer of the present invention may further comprise a film laid on the grating structure using conventional methods. In one embodiment, the membrane is a cell membrane of HeLa cells, and the membrane comprises glucose transport proteins (Glut 1 and Glut 2).

It will be understood by those of ordinary skill in the art that cell membranes have a variety of transport membrane proteins (also referred to herein as transport proteins) that can be used to transport substances. This example is based solely on glucose transport proteins on cell membranes for monitoring glucose. The transmission behavior. Those of ordinary skill in the art can select a cell membrane having a corresponding transport membrane protein depending on the type of substance to be monitored.

Please refer to Fig. 8, which shows a method and a step of laying a cell membrane of HeLa cells on the sensing wafer. First, in order to prepare the HeLa cells as a giant plasma membrane-derived vesicles (GPMV) 230 solution, the HeLa cells were immersed in a 25 mM paraformaldehyde (PFA) and 2 mM dithiothreose. A phosphate buffered saline (PBS) of dithiothreitol (DTT) was allowed to stand in an environment of pH 7.4 for 3 hours to obtain a GPMV solution. The GPMV solution was contacted with the surface of the sensing wafer for 1 hour, and evaporated by ginger water (e.g., at a humidity of 50% and 25 ° C) to allow the film 220 to be laid on the sensing wafer. Thereafter, the uncoated GPMV and impurities can be washed away using GPMV buffer.

Please refer to Figures 9 and 10, which show the unlayed film and the transmembrane, respectively. The performance of the laid sensing wafers during instant measurements. As shown in Fig. 9, the time for injecting the substance to be monitored (here, glucose is taken as an example) is about 10 seconds, and after the completion of the implantation, the response signal of the uncoated film sensing wafer is balanced; in contrast, It takes about 350 seconds for the transmissive sensing chip to reach the signal balance. In addition, please refer to Fig. 11, which shows the performance of the sensed wafer when it is subjected to an instant measurement after the addition of an inhibitor which inhibits the activity of the transport protein on the membrane. It was apparent that after the addition of the inhibitor, the signal reached equilibrium after 5 seconds after the glucose injection.

In the sensing wafer according to an embodiment of the present invention, the grating structure has a structure of a material capable of generating a plasma waveguide resonance signal and a material capable of generating a surface plasma resonance signal, so that the electricity can be detected at the same time. The signal of the slurry waveguide resonance and surface plasma resonance. Please refer to Fig. 12, which shows different detection areas combining surface plasma resonance and plasma waveguide resonance. The inner space of the array hole 116 is mainly detected by a surface plasma resonance sensing region formed by a metal layer 112 (such as composed of gold), which can be used to monitor the accumulation or adsorption of a substance in a hole; and the area outside the hole is Detected by a plasma waveguide resonance sensing region formed by a layer 114 of array holes (composed of SiO ₂ ), which can be used to monitor changes in solution concentration outside the pore, formation of cell membranes on the grating structure, or adsorption of substances on the membrane. event.

Please refer to FIG. 13, which shows a schematic diagram of a sensing wafer 200 in accordance with an embodiment of the present invention. In the present embodiment, the material of the substrate 211 is molten cerium oxide, the metal layer 212 as the plasma resonance layer is composed of gold, and the thickness thereof is 50 nm, and the material of the layer 214 having the array holes is dioxide.矽, used to capture the signal of the plasma waveguide resonance. In addition, In the present embodiment, the sensing wafer further includes a titanium layer as the bonding layer 213 having a thickness of 1.5 nm.

The manufacturing method of one embodiment of the sensing wafer of the present invention will be further described below. First, a substrate is provided, the material of which is molten cerium oxide. In this embodiment, a 1.5 nm titanium metal layer is first formed on the molten ceria substrate as a bonding layer, and a 50 nm gold metal layer is formed to serve as a plasmon resonance active layer. 510 nm cerium oxide is then deposited on the metal layer. Next, in order to form a grating structure on the wafer, the photoresist is first spin-coated on the wafer, and then exposed through a photomask to form a pattern on the wafer. Then, after exposure by ultraviolet light, the unreacted light is removed from the substrate. Thereafter, the substrate can be etched using, for example, inductively coupled plasma. After etching, the O2 plasma can be used for cleaning, and the remaining photoresist is removed with acetone and isopropyl alcohol. The wafer can then be rinsed with deionized water and blown dry with nitrogen. Through the above manufacturing steps, a sensing wafer having a grating structure as shown in Fig. 13 can be obtained.

The sensing wafer in this embodiment simultaneously extracts signals of surface plasma resonance and plasma waveguide resonance. Figure 14 shows the detection results of the sensing wafer when it has apertures of different sized array apertures and apertures of different sized apertures.

Referring to Figure 14, the sensing wafer was measured in water (n = 1.333). It is found that the sensing wafer of the present embodiment has severe signal interference under the condition that the hole diameter and the period are less than 3000 nm; in contrast, under the condition that the hole diameter and the period are greater than 3000 nm, In order to obtain a clear characteristic angle, in the case where the pore diameter and the period are larger than 3000 nm, it is preferable to use 3000 nm, 3500 nm, and 4000 nm. Compared with the embodiment shown in Fig. 6, the grating structure of this embodiment has the characteristic of inducing resonance of the plasma waveguide, so that two characteristic peaks can be obtained. Referring to Figure 15, there is shown a cross-sectional view and SEM side view of a sensing wafer having simultaneously detectable SPR and PWR signals in accordance with a preferred embodiment of the present invention, wherein the aperture of the array of hole junctions is about 3.54 microns. The period is about 2.57 microns and the depth of the holes is about 510 nm.

Similarly, the sensing wafer described above that can simultaneously monitor the SPR and PWR signals can further include a film that is laid over the grating structure. In the present embodiment, HeLa cells are used as an example, but the scope of the film which can be used in the present invention is not limited thereto. The method of forming a film using the HeLa cells in the sensing wafer of the present invention is the same as the method described above, and thus will not be described in detail in the present embodiment.

Please refer to Figures 16 and 17, which show the performance of the sensing wafers that can be simultaneously monitored by SPR and PWR signals without film laying and film laying. As shown in Fig. 16, after injecting the substance to be monitored (here, glucose is taken as an example), the response signal of the sensing wafer without the film laying is balanced immediately after the completion of the injection; in contrast, the film is laid. It takes about 330 seconds for the sensing chip to reach the signal balance. Further, please refer to Fig. 18, which shows the performance of the sensed wafer when it is immediately measured after the addition of an inhibitor which inhibits the activity of the transport protein on the membrane. It can be clearly observed that after the addition of the inhibitor, the signal reaches equilibrium after 5 seconds after the glucose injection, indicating that the glucose transport protein activity on the membrane is inhibited and cannot be Glucose transport causes the concentration of glucose in the pores to rise, further affecting the change in refractive index in the pores.

Referring again to Fig. 19, there is shown the performance of the reflectance of the sensing wafer of the present invention for simultaneously monitoring the SPR and PWR signals in different environments versus the angle of incidence. A blue curve is obtained initially in the buffer. After laying the supported lipid bilayer film under the same environment, an orange curve can be obtained. Since the refractive index of the surface of the ceria has an average refractive index rise due to the formation of the film, the detection of the block is performed. The resonance-resonant peak of the plasma waveguide is offset, and the resonance angle is increased from 67.14 degrees in blue to 67.63 degrees. However, the solution environment inside the cavity is still a buffer, so the surface plasma resonance correlation peak has only a relatively small offset of 0.05 degrees. Then, the solution environment was changed to a buffer solution containing 4.6% glucose in the same membrane environment to obtain a green curve. Since the refractive index of the glucose buffer solution is higher than that of the original buffer solution, when the inside of the hole is detected by the surface plasma resonance correlation peak, the resonance angle can be found to be shifted from 77.18 degrees to 77.98 degrees. However, because the solution is replaced with a solution of all the solutions outside the pores into a glucose buffer solution, and this interval is the detection range of the resonance peak of the plasma waveguide, the resonance angle is also observed to shift from 67.63 degrees to 67.88 degrees. The situation. In summary, Fig. 19 shows the effect that the double peaks measured by the sensing wafer of the present invention do have respective resonance refractive index shifts corresponding to changes in the external refractive index.

100‧‧‧Sensor wafer

102‧‧‧Light source

104‧‧‧Detector

110‧‧‧Grating structure

111‧‧‧Substrate

112‧‧‧metal layer

114‧‧‧layer with array holes

116‧‧‧Array holes

Claims (13)

A sensing wafer for monitoring a substance transport behavior, comprising: a substrate; and a grating structure formed on the substrate, and the grating structure comprises: a layer having an array of holes, wherein the material layer having the layer of the array hole And at least one selected from the group consisting of cerium oxide, titanium nitride, aluminum oxide, tri-n-trifluoride, Teflon, and polymer materials; and a metal layer formed at the bottom of the array hole to extend to The array of holes is below the layer and is exposed from the array of holes, and the metal layer acts as a surface plasma resonance layer. The sensing wafer of claim 1, wherein the material of the metal layer is selected from the group consisting of gold, silver, aluminum, nickel, and titanium. The sensing wafer of claim 1, wherein the metal layer has a thickness ranging from 1 nm to 1000 nm. The sensing wafer of claim 1, wherein the layer having the array of holes is a plasma waveguide layer. The sensing wafer of claim 1, wherein the layer having the array holes has a thickness of 50 nm to 1000 nm. The sensing wafer of claim 1, wherein the array of holes has a period of from 0.01 μm to 10 μm. The sensing wafer of claim 1, wherein the array aperture has a pore diameter of 0.01 μm to 10 μm. The sensing wafer of claim 1, further comprising a film for transporting a monitoring substance, disposed on the grating structure such that at least a portion of the array of holes form an internal space. The sensing wafer of claim 8, wherein the film is a biological film. The sensing wafer of claim 9, wherein the biological membrane system has at least one transport membrane protein. A method of monitoring a substance transporting behavior, comprising detecting a plasma waveguide resonance signal and/or a surface plasma resonance signal using a sensing wafer according to any one of claims 1 to 10. The method of claim 11, further comprising contacting the sensing wafer with a solution containing the substance to be monitored, and detecting a change of the plasma waveguide resonance signal and/or the surface plasma resonance signal. The method of claim 11, wherein the plasma waveguide resonance signal is generated by the layer having the array of holes, and the surface plasma resonance signal is generated by the metal layer.