Patent Application
20210276022
The present invention relates to a biosensor and more particularly to a dielectrophoresis-used microelectrode biosensor configured to further improve sensitivity and detection width of a sensor by forming, between microelectrodes, a receptor specifically reacting with a target biomaterial and by increasing a probability of specifically reacting with a target biomaterial using concentration effect based on dielectrophoresis phenomenon, and a method for detecting a bio material using the same.
Recently, bio sensors are developed on a large scale configured to detect, by an electric method, presence and absence of various biomaterials such genes and proteins, and concentrations thereof.
One of these examples is to use an IDE (Interdigitated Electrode), and the IDE is so evaluated as to properly measure concentration of biomaterials even if a region where receptors fixed to specifically react with biomaterials is substantially very broad in a zigzag shape.
Referring to
That is, an impedance component is determined by allowing an antibody to be fixed between electrodes to form an electric field, where disturbance is generated on an electric field formed between electrodes by allowing target molecules to be positioned by pushing out a bio buffer solution when the target molecules are specifically fixed to the antibody, which resultantly induces the change in impedance.
An amount of change in impedance may be determined by an amount of target biomolecules, whereby quantitative analysis of target biomolecules within a sample is made to be enabled by ascertaining the amount of change in impedance.
Meantime, in order to improve the sensitivity of the said IDE sensor, a technology is disclosed by a registered patent No.: 10-1727107 entitled as ‘microelectrode bio-sensor using dielectrophoresis’ to allow an IDE sensor to use a dielectrophoresis phenomenon.
Here, the ‘dielectrophoresis (dielectric electrophoresis)’ is a phenomenon in which a net force is exerted in an electric field by allowing a bipolarity to be induced on particles when non-polar particles exist in non-uniform AC electrical field.
The strength of dielectrophoresis force may be increased by adjusting a distance between electrodes of the IDE sensor or a size of non-uniform electrical field formed through the intensity change of applied voltage.
However, in a case when a biosensor is so formed as to use the dielectrophoresis, there is generated a possibility of incurring a damage to a bio particle when the intensity of voltage applied in order to obtain the dielectrophoresis is increased.
Furthermore, in consideration of applications in various aspects including commercialization of IDE sensor as a health care sensor for disease diagnosis, or manufacturing of the same for portability, the increase in the intensity of voltage in order to obtain a dielectrophoresis effect may create another problem.
Korea registered patent No.: 1727107 (Published on Apr. 17, 2017)
The present invention is provided to solve the aforementioned problems and it is an object of the present invention to provide a dielectrophoresis-used microelectrode bio sensor configured to further improve sensitivity and detection width of a sensor by increasing a probability of specifically reacting with a target bio material through concentration effect using the dielectrophoresis to thereby lower the intensity of voltage for use of the dielectrophoresis.
It is another object of the present invention to provide a method for detecting a bio material through a bio sensor using the dielectrophoresis.
In one general aspect of the present invention, there may be provided a dielectrophoresis-used microelectrode bio sensor comprising:
Preferably, but not necessarily, the first microelectrode and the second microelectrode may be applied with an AC (Alternating Current) for generating dielectrophoresis forces.
Preferably, but not necessarily, the micropattern may be formed in any one shape of square, rectangular and line(ar) shapes.
Preferably, but not necessarily, size of AC for generating the dielectrophoresis may be greater than 0.25V but less than 0.35V, and frequency may be 50 MHz.
In another general aspect of the present invention, there may be provided method for detecting biomaterial using a biosensor, comprising:
applying a voltage to the first microelectrode and the second microelectrode using the dielectrophoresis according to any one preceding claim of 1 to 8;
measuring an impedance between the first microelectrode and the second microelectrode; and
calculating one or more of presence or absence of target biomaterial and concentration thereof based on the measured impedance.
According to the present invention, a probability capable of specifically reacting with a target biomaterial can be increased through concentration effect using the dielectric electrophoresis, whereby sensitivity and detection width of a biosensor can be enhanced.
Because target biomaterials inside a sample are collected, the impedance signal of the biosensor can be increased, which leads to increase in sensitivity of the biosensor whereby non-specific formation can be effectively inhibited to thereby exhibit a greater effect in an environment like plasma or serum.
Particularly, a greater electric field intensity can be obtained compared with a biosensor without micropattern because of use of micropattern between electrodes, whereby target biomaterials can be more effectively concentrated. Furthermore, damage to biomolecules can be prevented by lowering the intensity of voltage for dielectric electrophoresis, and the biosensor can be easily applied to various ways including commercialization or portability of the biosensor as a health care sensor for disease diagnosis.
Exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawing so as to allow a person skilled in the art to which the present invention belongs to easily implement the present invention. However, it should be understood that the present invention is not limited to particular embodiments, but encompasses all changes, modifications, equivalents and substitutes included within the ideas and technical scopes of the present invention. In describing the present invention, detailed descriptions of well-known technologies are omitted for brevity and clarity so as not to obscure the description of the present invention with unnecessary detail.
It will be further understood that the terms “comprises” and/or “comprising,” or “includes” and/or “including” when used in this specification, specify the presence of stated features, regions, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, regions, integers, steps, operations, elements, components, and/or groups thereof.
As may be used herein across the specification, the terms “about”, “approximately” and “substantially” are used to provide a meaning approximate to a number or a number adjacent to that number, when an intrinsic manufacturing error and material allowable error are provided, and, in order to assist understanding of the present invention, may be used to prevent an unscrupulous infringer from using the disclosed contents mentioned with accurate or absolute numbers unlawfully
It will be understood that, although the terms first, second, etc. may be used herein to describe various elements, these elements should not be limited by these terms. These terms are only used to distinguish one element from another.
The biosensor (100) may include a first microelectrode (110), a second microelectrode (120), a micropattern (130), and a plurality of receptors.
As illustrated in
At this time, each first protruding electrode formed on the first microelectrode (110) and each second protruding electrode formed on the second microelectrode (120) may be structurally arranged to mutually intersect.
A reaction region (140) may be formed in a space between the first microelectrode (110) and the second microelectrode (120).
As shown in
The micropattern (130) may be formed in any one shape of a square, a rectangle and a line(ar) shape as shown in
Furthermore,
The square-shaped micropattern (131), the rectangular-shaped micropattern (132), and the line(ar)-shaped micropattern (133) may come in various sizes and shapes.
As a more detailed example, the square-shaped micropattern (131) may be formed with a length of about 2.5 μm for each side. However, the present invention is not limited thereto. For example, each side of the square-shaped micropattern (131) may be formed with a length of greater than 2.0 μm but less than 3.0 μm.
Furthermore, the rectangular-shaped micropattern (132) may be formed with a length of about 2.5 μm for one side and of about 7.5 μm for other sides. However, the present invention is not limited thereto. For example, one side of the rectangular-shaped micropattern (132) may be formed with a length of greater than 2.0 μm but less than 3.0 μm while the other sides may be formed with a length of greater than 7.0 μm but less than 8.0 μm.
The width of the line(ar)-shaped micropattern (1331) may be formed with about 2.5 μm. However, the present invention is not limited thereto. For example, the line(ar)-shaped micropattern (133) may be formed with a width greater than 2.0 μm but less than 3.0 μm.
The plurality of receptors may be fixed in a space between the first microelectrode (110) and the second microelectrode (120) to function as specifically reacting to a target biomaterial.
The receptor and the target biomaterial to which the receptor specifically reacts may be formed in a variety way. As one of the examples, the target biomaterial detected by using a biosensor (100) may be amyloid beta antibody, where the receptor may be amyloid beta antibody, aptamer, peptide and the like.
In light of impedance detection characteristic of biosensor using reaction of a target biomaterial, an impedance between the first microelectrode (110) and the second microelectrode (120) may be expressed in the following Equation 1.
Z=R+jX=R+j(XL−XC)=R−jXC=R−j(1/wC) [Equation 1]
Where, Z is an impedance, R is a resistance, X is a reactance, C is a capacitance, and w is an angular frequency. The reactance X may be divided to an inductor component XL and a capacitor component XC, where because the first microelectrode (110) and the second microelectrode (120) are directly connected electricity-wise, it can be said that the inductor component (XL) is disregarded, and only the capacitor component (XC) exists.
When a plurality of 5 (five) receptors is fixedly arranged in a space between the first microelectrode (110) and the second microelectrode (120), most of the electric fields and impedance changes are generated to a horizontal direction where the first microelectrode (110) and the second microelectrode (120) are arranged across the plurality of receptors. An amount of target biomaterials can be detected by ascertaining the changes in the said resistance and reactance.
That is, when the plurality of receptors is fixed in a space between the first microelectrode (110) and the second microelectrode (120), and when the changes in the impedance at a space faced by the first microelectrode (110) and the second microelectrode (120) are ascertained when the target biomaterial reacts to the receptor, it is possible to qualitatively analyze the target biomaterial.
Meantime, an AC may be applied to the first microelectrode (110) and the second microelectrode (120) in order to generate the dielectric electrophoretic forces.
Referring to
Here, the induced net force may be defined as DEFs (Di-electrophoresis Forces).
That is, when an AC voltage is applied to the biosensor (100), a non-uniform AC electric field is electrically formed, and non-polar particles existing within the electric field may possess the bipolarity to be affected by the DEFs to a predetermined direction.
The intensity (size) and direction of DEFs induced to each particle forming the target biomaterials may differ depending on dielectric properties including, but not limited to, voltage of electric field, frequency, conductivity (σ) of particle and medium and permittivity (ε).
Therefore, a force that a spherical particle receives by the di-electrophoresis forces may be expressed by the following Equation 2.
F
DEP=2πεmr3Re[K(ω)]∇|Ersm|2 [Equation 2]
Where, εm is permittivity of medium, r is a radius of a particle, Re[k(ω)] is a real number portion of Clausius Mossotti factor, and Ersm means a root-mean square of electric field.
At this time k(ω) may be determined by the following Equation 3 based on a relative permittivity (ε*p) of particle and relative permittivity (ε*m) of medium, and polarity of particle may be determined by the value thereof.
When k(ω) value is greater than 0, the particle may move to a direction where the electrical field gradient is great by receiving the force. Conversely, when k(ω) value is smaller than 0, the particle may move to a direction where the electric field gradient is small in response to a formed shape of electrical field. The said phenomenon may be respectively called positive and negative DEP (Di-electrophoresis).
Thus, when a voltage is applied to allow the di-electrophoresis forces to be generated by the electric field between the first microelectrode (110) and the second microelectrode (120) using the dielectrophoresis, molecules may be moved to a direction where the electrical field gradient is great, or to a direction where the electrical field gradient is small in response to the shape formed between the first microelectrode (110) and the second microelectrode (120).
Conversely,
As noted above, when a voltage is applied to the first microelectrode (110) and the second microelectrode (120) to allow the negative (−) dielectrophoresis forces to be generated in response to non-uniform electrical field shape between the first microelectrode (110) and the second microelectrode (120) and its gradient thereof, molecules may be moved and concentrated by the induced di-electrophoresis forces.
Particularly, the target biomaterials may be reacted by concentration thereof by moving the target biomaterials to where the electrical field gradient is small (between electrodes) using the negative (−) dielectrophoresis forces in response to the formed electrical field shape.
That is, quantitative analysis of target biomolecules may be realized by fixing receptors specifically binding to target biomolecules to between the first microelectrode (110) and the second microelectrode (120) in order to detect the moving biomaterials, and by ascertaining impedance change when the target biomolecules react to the receptors.
The impedance signal is increased due to collection of target biomolecules within a sample compared to a case where the dielectrophoresis forces are not acted, which leads to sensitivity enhancement of biosensor (100). Furthermore, a greater effect can be demonstrated under plasma or serum environment because an effect of effectively generating inhibition of non-specific binding formation is generated within a sample.
As shown in
The dielectrophoresis forces may be increased by adjusting a distance between electrodes inside the biosensor and the size of non-uniform electric field formed through changes in intensity of applied voltage.
Particularly, the lowering of voltage necessary for concentration of biomolecules can prevent the damage to biomolecules caused by voltage applied for di-electrophoresis effect, and integration with battery or communication elements can be further eased when the biosensor is commercialized as a disease diagnosis health care sensor in the future as well.
Now, referring to
First of all, a voltage is applied to each microelectrode of biosensor in order to generate the dielectrophoresis forces (S210). Here, the biosensor means the biosensor (100) formed with micropatterns between the first microelectrode and the second microelectrode as shown in each exemplary embodiment thus explained.
The voltage applied in the step S210 is an AC voltage for generating the di-electrophoresis forces, and may be about 0.3V, and a frequency may be about 50 MHz. However, the present invention is not limited thereto. For example, the AC voltage for generating the d dielectrophoresis forces may be greater than 0.25V but less than 0.35V.
When an AC voltage is applied at the step S210, the target biomaterials may be moved to where the electric field gradient is small (between each electrode) in response to the formed shape of electric field by the dielectrophoresis forces, and the target biomaterials may be concentrated between the electrodes.
Now, an impedance may be measured between each electrode (S220).
Then, presence or absence of target biomaterials or concentration thereof may be calculated (S230) based on the impedance values measured at step S220.
The method of calculating the presence or absence of target biomaterials or concentration thereof at the step S230 may be made in various ways.
The target biomaterials to be detected by the present invention may include various types, and particularly, may be a biomaterial with a weight of about 4.5 kDa. However, the present invention is not limited thereto. For example, a weight of target biomaterial to be detected by the present invention may be greater than 4.0 kDa but less than 5.0 kDa. Here, an example of biomaterial with a weight of about 4.5 kDa may include an amyloid beta protein.
Meantime, a position condition for collecting amyloid beta between each electrode may be determined by an applied voltage.
It should be apparent that the biosensor according to the present invention may be applied to detect a protein heavier than the said amyloid beta protein or a protein lighter than the said amyloid beta protein, which can be made possible by changing and/or adjusting a voltage condition in response to weight of protein to be detected.
As expected from
Meantime, it can be known that an electric field greater than that of the micropattern-less biosensor having an electrode width of 5 μm under the same applied voltage is formed, even if a distance between electrodes comes to be increased to 10 μm in the micropattern-existent biosensor.
Now, an experimental example related to the biosensor (100) according to the present invention will be explained. The present experiment is to ascertain a possibility of enhancing the performance of the biosensor by implementing a qualitative analysis in order to detect amyloid beta existent within a serum.
Toward this end, an optimal voltage condition has been established in order to concentrate the amyloid beta within the biosensor (100) disposed with a micropattern.
A biosensor has been manufactured using a batch process-enabled MEMS (Micro Electro Mechanical System) process, and a sensor surface activation for fixation of antibody has been proceeded. Sensitivity of biosensor has been compared through quantitative analysis of amyloid beta within 1×PBS buffer using a biosensor having various micropattern structures, and quantitative analysis of amyloid beta existent within serum has been made using optimized biosensor.
As shown in an example of
The micropattern (130) has been respectively manufactured in a square structure with each side of 2.5 μm, a rectangular structure with one side of 2.5 μm and the other side of 7.5 μm, and a linear structure with a width of 2.5 μm. The Type #1 shows a square-shaped micropatterned biosensor, the Type #2 shows a rectangular micropatterned biosensor and the Type #3 shows a linear micropatterned biosensor.
As shown in
As a result, integration becomes easy that forms a plurality of IDE sensors on one chip through a mask manufacturing in light of characteristics of MEMS process, whereby a multiple analysis is made possible that is capable of analyzing various proteins from one chip.
It has been confirmed/ascertained that antibody had been fixed through brightness difference between a portion within a circle and a portion outside of the circle. Furthermore, it has been also ascertained that the antibody had been uniformly fixed across the entire surface-treated region through the brightness at a portion outside of the circle being uniform across the surface.
In light of the fact that, in order to evaluate the performance of biosensor disposed with a micropattern, the first thing is that a surface activation condition must be applied and an effect of the dielectrophoresis forces must be confirmed, an optimal condition that may shows the dielectrophoresis effect must be established, and therefore, experiments of qualitative analysis have been performed using these prerequisites.
Toward this end, characteristics of amyloid beta with a weight of about 4.5 kDa has been ascertained and a voltage condition for generating the di-electrophoresis effect has been confirmed by way of simulation.
Therefore, the presence and/or absence of amyloid beta concentration have been confirmed using five (5) ambient voltages of 0.1, 0.2, 0.3, 0.5 and 1.0V.
The confirmation experiment has confirmed the effect by allowing the antibody to be fixed on the biosensor using the same protocol, and by applying dielectrophoresis voltage and frequency conditions corresponding to each condition while reacting amyloid beta protein of 10 pg/mL, where the same frequency of 50 MHz was applied.
As shown in
It was confirmed that the size of the optimized dielectrophoresis voltage was an about 50% reduced value over the 0.5V used in the IDE sensor (micropattern-less biosensor) having a distance of 5 μm between electrodes.
A quantitative analysis has been made on amyloid beta protein using the established dielectrophoresis voltage condition, and preparation has been made by melting, in 1×PBS, the amyloid beta sample increased in concentration by 10 times from 100 fg/mL to 100 pg/mL.
The analysis process has been made using three (3) steps including: a signal stabilization inside the buffer; reaction and application of signal for dielectrophoresis; and washing and signal stabilization.
As illustrated in
The sensitivity has been increased from 0.146±0.005 to 0.545±0.049 by the dielectrophoresis effect in the micropattern-less biosensor (IDT).
On the other hand, in the micropattern-formed biosensor and depending on micropattern structure, it has been confirmed that the sensitivity had been increased from 0.337±0.043 to 0.724±0.027 in the Type #1 (square pattern), the sensitivity had been increased from 0.281±0.083 to 0.731±0.033 in the Type #2 (rectangular pattern), and the sensitivity had been increased from 0.337±0.031 to 0.757±0.051 in the Type #3 (linear pattern).
It can be noted through these experiments that the micropattern-existent IDE sensor has demonstrated a higher sensitivity than that of micropattern-less IDE sensor due to the dielectrophoresis effect.
Each result from the present experiments has been measured under an environment inside the PBS buffer, and it has been confirmed that the structure of the most optimized biosensor is Type #3 formed with the line(ar) micropattern, with sensitivity thereof being of 0.757±0.051, detection limit being of 100 fg/mL, detection section (dynamic Range) being of 100 pg/mL at 100 fg/mL.
Amyloid beta quantitative analysis has been performed within a plasma using an amyloid beta quantitative analysis sensor based on the optimized dielectrophoresis effect, and the type of biosensor used herein was the Type #3 (sensor using linear micropattern).
In order to perform amyloid beta quantitative analysis within a plasma, a preparation has been made by melting, in plasma, the amyloid beta sample increased in concentration by 10 times from 100 fg/mL to 100 pg/mL.
The same analysis method has been used as the amyloid beta analysis process within 1×PBS as explained above. As a result of the analysis, as shown in
It has been confirmed through these experiments that, when amyloid beta within the plasma was analyzed,
It has been confirmed that the sensitivity of optimized biosensor had been 0.628±0.032, detection limit had been 100 fg/mL, and the detection section had been 100 pg/mL at 100 fg/mL.
The sensitivity of sensor during amyloid beta analysis within a plasma had been about 83% of the sensitivity confirmed while analyzed within the PBS, which is estimated as a decrease in sensitivity due to various biomaterials existent within the plasma, but the said sensitivity has shown a remarkably excellent performance over the conventional detection techniques, and therefore, may be usefully utilized for biomarker detection within an actual sample.
While the present invention has been described with reference to the particular illustrative embodiments, it is not to be restricted by the embodiments but only by the appended claims. It is to be appreciated that those skilled in the art can change or modify the embodiments without departing from the scope and spirit of the present invention.
Therefore, it should be understood that the abovementioned exemplary embodiments are simply exemplary but not restrictive. For example, each element described in a single form may be a dispersed manner, and likewise, each element described in a dispersed manner may be implemented in a combined manner.
It should be interpreted that the scope of the present invention will be described from the following claims rather than the foregoing detailed description, and all other types of changes or modifications derived from the meaning of claims, scopes and equivalent shapes are included in the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2018-0080348 | Jul 2018 | KR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2019/008461 | 7/10/2019 | WO | 00 |
