KR20060089613A - Sensing switch and detecting method using the same - Google Patents

Abstract

The present invention relates to a detection device for biomolecules or chemicals, more specifically, a substrate; A support placed on the substrate; Sensing plates extending in parallel and spaced apart from the substrate connected from the side of the support; A receptor coupling region located on an upper surface of the sensing plate end; Electric or magnetic field generating means for inducing deflection of the sensing plate when the receptor bound to the receptor binding region selectively binds to a ligand that is electrically or magnetically active; And a pair of switching electrodes spaced apart from each other by a predetermined distance, which may be connected when the sensing plate contacts the substrate by bending of the sensing plate, and a sensing method using the same. According to the present invention, there is no need to fluorescently label the target material, and there is no need to separately signal the fluorescence or electrical detection signal in the analyzer, and the signal can be directly transmitted or not by the current flow by switching on or off. It has the advantage of being readable.

Description

Sensing switch and detecting method using the same

1 is a front view of a sensing switch (cantilever) according to an embodiment of the present invention.

2 is an exploded perspective view of a sensing switch (cantilever) according to another embodiment of the present invention.

Figure 3a is a front view of the sensing switch (yangbobo) according to another embodiment of the present invention, Figure 3b is a view showing that the switch is activated by the sensing, Figure 3c is a sensing according to another embodiment of the present invention A perspective view of a switch (bottle beam).

4A is a conceptual diagram illustrating an on-state of the sensing switch of FIG. 3, and FIG. 4B is a conceptual diagram illustrating an off-state of the sensing switch of FIG. 3.

5A conceptually illustrates a conventional DNA chip detection system, and FIG. 5B conceptually illustrates a DNA chip detection system according to the present invention.

6A conceptually illustrates a differential sensing system in a conventional manner, and FIG. 6B illustrates a differential sensing system according to the present invention.

FIG. 7 conceptually illustrates an example of the differential sensing result of FIG. 6.

FIG. 8A illustrates an 'and' logic circuit of a sensing circuit according to an embodiment of the present invention, FIG. 8B illustrates an 'or' logic circuit, and FIG. 8C illustrates an 'and (and) ) 'And' or (or) 'logic circuits.

9A and 9B illustrate scales for calculating the operating torque of the sensing switch of FIG. 3.

10 is a schematic diagram of an apparatus used for the simulation of a sensing switch in one embodiment of the invention.

11 shows a simulation result of the sensing switch according to the present invention.

FIG. 12 is a graph showing the force acting between the magnetic beads and the distance between the magnetic generating means in the sensing switch of FIG. 2.

FIG. 13A illustrates a scale of a sensing plate used for simulation of the sensing switch of FIG. 2.

13B and 13C illustrate simulation results performed by using the sensing plate of FIG. 13A.

FIG. 14 conceptually illustrates a manufacturing process of a sensing switch according to the present invention of FIG. 2.

15A and 15B are enlarged SEM photographs of a sensing plate of a sensing switch manufactured through the above process.

FIG. 16 is a graph illustrating a result of effectively detecting a receptor by the sensing switch of FIG. 15.

The present invention relates to a device for detecting biomolecules or chemicals, and more particularly, by using an electrostatic force between electrodes of the same or opposite polarity of the ligand or a magnetic force between the magnetic beads and the magnetic field generating means coupled to the ligand. The present invention relates to a sensing switch that physically moves itself as a switch and a detection method using the same.

Effective detection of biomolecules and chemicals is required in various fields. Biochips are a typical field for detecting biomolecules. A biochip is a chip in which a high density of biomolecules receptors such as DNA and protein to be analyzed are attached onto a substrate, and the gene expression patterns, gene defects, protein distributions, and reaction patterns in the sample can be analyzed. . Biochips may be classified into microarray chips attached to a solid substrate and lab-on-a-chips attached to microchannels according to the attachment form of the receptor. In such biochips, a system capable of detecting whether a target material is bound to a receptor immobilized on a substrate is required to determine whether a target material capable of binding to a receptor exists in a sample.

Most DNA chips for genetic analysis now label fluorescent dyes on sample DNA, react with a probe on the chip, and detect condensed phosphors on the chip surface using a confocal microscope or CCD camera. Method is used (see US Pat. No. 6,141,096). However, since such an optical detection method is difficult to miniaturize and the digitized output cannot be seen, much research is being conducted on the development of a new detection method capable of producing an electrical signal.

Many research institutes, including Clinical Micro Sensors, are investigating a method of electrochemically detecting DNA hybridization using metal compounds that are easy to oxidize / reduce (see US Patent No. 6096273, 6090933). When DNA hybridizes, other compounds, including metals that are easily oxidized / reduced, form a complex together and are detected electrochemically (Anal. Chem., Vol. 70, pp. 4670-4677). , 1998, J. Am. Chem. Soc., Vol. 119, pp. 9861-9870, 1997, Analytica Chimica Acta, Vol. 286, pp. 219-224, 1994., Bioconjugate Chem., Vol. 8, pp 906-913, 1997.) However, electrochemical methods also have the disadvantage of requiring separate labeling.

In addition to the above methods, there are optical detection methods and detection methods using piezoelectric materials. The optical detection method measures the movement of the sensor from the change in reflectance of the irradiated light. The detection method using the piezoelectric material is to manufacture the sensor with multiple layers of various piezoelectric materials, and to measure the movement of the sensor from the change of the resistance of the sensor, which is difficult to design and manufacture of the sensor (US Patent No. 5,807,758). Reference).

The conventional fluorescence detection method, electrochemical detection method, optical detection method and detection method using piezoelectric materials are separate from signal processing of signals obtained from the measurement equipment as well as measurement equipment for measuring the sensor separately from the sensor itself. Because of the demand for analytical devices, the entire system is huge, requires expensive equipment and requires skilled technicians at each stage, but it takes a lot of time to make final decisions and connection noise between the devices. ) Has the disadvantage that occurs.

Accordingly, the present inventors have diligently researched to overcome the problems of the prior arts. As a result, the present inventors have used the electrostatic force between electrodes having the same or opposite polarity as the ligand or the magnetic force between magnetic beads and magnetic field generating means coupled with the ligand The sensor itself is physically moved to act as a switch, confirming that a miniaturized sensing switch that does not require any further signal processing is completed, and the present invention has been completed.

Accordingly, a main object of the present invention is to provide a new sensing switch capable of simultaneously performing mechanical sensing and electrical switching without the need for separate signal processing after conventional sensing.

Another object of the present invention is to provide a sensing circuit including the plurality of sensing switches in a logical circuit relationship.

Another object of the present invention is to provide a method for detecting the binding of a ligand using the sensing switch.

In order to achieve the object of the present invention, the present invention is a substrate; A support placed on the substrate; Sensing plates extending in parallel and spaced apart from the substrate connected from the side of the support; A receptor coupling region located on an upper surface of the sensing plate end; Electric or magnetic field generating means for inducing deflection of the sensing plate when the receptor bound to the receptor binding region selectively binds to a ligand that is electrically or magnetically active; And a pair of switching electrodes spaced apart from each other by a bending of the sensing plate, which may be connected when the sensing plate contacts the substrate.

One embodiment of the sensing switch according to the present invention is a substrate; A support placed on the substrate; Sensing plates extending in parallel and spaced apart from the substrate connected from the side of the support; A receptor coupling region located on an upper surface of the sensing plate end; A push-out electrode of the same polarity as the ligand located above the sensing plate, a pull-in electrode of opposite polarity to the ligand located below the sensing plate, and the repulsive force and attraction of the evacuation electrode and the attracting electrode By providing a sensing switch comprising a pair of switching electrodes spaced apart from each other that can be connected when the sensing plate is in contact with the substrate.

Another embodiment of the sensing switch according to the present invention is a substrate; A support placed on the substrate; Sensing plates extending in parallel and spaced apart from the substrate connected from the side of the support; A receptor coupling region located on an upper surface of the sensing plate end; Magnetic beads that selectively bind to a receptor bound to the receptor binding region through a ligand; Magnetic field generating means located below the sensing plate; And a pair of switching electrodes spaced apart from each other by a generated magnetic field, which may be connected when the sensing plate contacts the substrate.

In the present invention, the sensing plate may be a cantilever or may be a cantilever (seesaw shape), it is preferably characterized in that the cantilever. In the case of a two-palm beam, the sensing plate extends in parallel and spaced apart from the substrate to both sides from the center of the connecting beam connecting the two supports placed on the substrate, two receptor coupling on the upper surface of both arms of the sensing plate It is characterized by the presence of an area. Both arms of the sensing plate move upward and downward like a seesaw by rotation of the connecting beam to open and close a pair of switching electrodes spaced apart from each other on the substrate.

In the present invention, the support serves to support the floating sensing plate or the connecting beam which is placed on the substrate and spaced apart from the substrate at a predetermined distance, and also serves as a pivot (pivot) that the connecting beam can rotate.

In the present invention, the sensing plate is preferably made of a material that can be easily bent by the repulsive force and attraction force of the reject electrode and the attracting electrode, and when bent down to open or close the switching electrode on the substrate in whole or at least The portion in contact with the switching electrode should be an electrical conductor.

In the present invention, the connecting beam is preferably narrower or thinner than the sensing plate so that it can be easily bent by the repulsive force and attractive force of the reject electrode and the attracting electrode. The connecting beam may be formed integrally or separately from the sensing plate.

In the present invention, the ligand may be any biomolecule or chemical which can be charged, and preferably a nucleic acid, protein, peptide, antibody, antigen, polymer, liquid or gaseous chemical that selectively binds to the receptor. Can be.

In the present invention, the receptor binding region may be directly bound to the ligand, but is preferably characterized in that the receptor capable of binding the ligand is bound. In the latter case, the receptor may be any substance capable of binding to a ligand, but may preferably be DNA, RNA, peptide nucleic acid (PNA), locked nucleic acid (LNA), protein, peptide, and chemicals. In the present specification, binding is to be understood to include specific binding, such as hybridization of nucleic acid and antigen-antibody (Ag-Ab) interaction.

In the present invention, the receptor binding region may be made of any material to which biomolecules or chemicals can be bonded, but is preferably made of a material selected from the group consisting of glass, metal, plastic, and silicon. In addition, the receptor binding region may be a carboxyl group (-COOH), a thiol group (-SH), a hydroxyl group (-OH), a silane group, an amine group or the like so that a receptor or a ligand can be bound using a surface modification method of a conventional biochip. It may be surface modified to have an epoxy group.

The magnetic beads may be attached with a ligand that selectively binds to a receptor, or a secondary receptor that selectively binds to a ligand that selectively binds to the receptor.

In the present invention, the receptor coupling region may be immobilized in only one of two regions, but preferably, two different types of receptors are immobilized in the two receptor coupling regions. In the latter case, one of the two types of receptors acts as a reference receptor for the other, allowing differential detection as well as effectively eliminating background signals.

In order to achieve another object of the present invention, the present invention is a plurality of sensing switches of the present invention are connected in series and / or parallel to constitute a logic circuit 'and' and / or 'or' It provides a sensing circuit.

In the present invention, the logic circuit of 'and (and)' current flows only when two or more sensing switches are connected in series so that all the switches are in an on state, and logic of 'or' In a circuit, two or more sensing switches are connected in parallel so that current flows when at least one of them is on. In the present invention, a plurality of these logic circuits 'and' and / or 'or' may be connected in combination to form various logic circuits.

In the present invention, the sensing and analysis of a plurality of ligands at the same time by looking at the output signal output through the logic circuit of and (and) and / or (or) sensing Provide a circuit. In the circuit of the present invention, one or more input lines and one or more output lines may exist, and current may be measured from the output lines after applying current to the input lines to simultaneously detect and analyze a plurality of receptors. In addition, the present invention provides a method for the detection of different receptors for each sensing switch constituting a circuit by turning on / off the current to the output line without the need for signal processing after signal detection. Effectively analyze the presence or absence of binding.

In order to achieve another object of the present invention, the present invention comprises the steps of introducing a ligand to the receptor binding region of the sensing switch according to the invention using an electric field; Subjecting the ligand to a receptor fixed to the receptor binding region; Applying a voltage of the same polarity as the ligand to the reject electrode and a voltage of the opposite polarity to the ligand to the attracting electrode; Sensing plates are moved up and down by repulsion and attraction between the ligand, the reject electrode and the attracting electrode to open and close a pair of switching electrodes on the substrate; And it provides a ligand binding detection method comprising the step of measuring the flow of current between the switching electrode.

In addition, to achieve another object of the present invention, the present invention includes the steps of attaching a magnetic bead to a ligand that selectively binds to the receptor, or a secondary receptor that selectively binds to the ligand; Introducing a ligand or secondary receptor to which the magnetic beads are attached to a receptor fixed to a receptor binding region of a sensing switch according to the present invention using a magnetic field; Attaching magnetic beads to the receptor binding region by a selective binding reaction between the ligand and the receptor; Removing the ligand or secondary receptor of magnetic bead attachment that did not participate in the binding reaction; Generating a magnetic field from a magnetic field generating means located below the sensing plate; Moving the sensing plate up and down by the magnetic field to open and close a pair of switching electrodes on the substrate; And it provides a ligand binding detection method comprising the step of measuring the flow of current between the switching electrode.

In the present invention, the receptor binding region of the sensing switch may be immobilized in only one of two regions, but preferably, different types of receptors are immobilized in two receptor binding regions to discriminate between different ligands. (differential) provides a method for detecting ligand binding whether characterized in that the binding. In the latter case, the two receptor binding regions act like a seesaw, causing the sensing plate ends to which more of the two receptors bind move downward. In this case, one of the two types of receptors may act as a reference receptor for the other to effectively remove the background signal.

According to the present invention, unlike the signal processing after measuring the resistance, impedance, and current in a conventional electrical detection method with a separate measuring device, and simply read the presence or absence of current flow of the sensing switch, for example, the on / off of the lamp You can easily detect whether the coupling.

Hereinafter, with reference to the accompanying drawings will be described the present invention in more detail.

1 is a front view of a sensing switch (cantilever) according to an embodiment of the present invention. The sensing switch shown in the figure is a sensing plate 4 extending in parallel and spaced apart from the substrate 1, the support 2 placed on the substrate 1, and the substrate 1 connected from the side of the support 2. Receptor binding region (5) located on the upper surface of the sensing plate (4), push-out electrode (6) of the same polarity as the ligand located on the sensing plate (4), of the sensing plate (4) A pull-in electrode 7 of opposite polarity to the ligand located below, and the sensing plate 4 is applied to the substrate 1 by the repulsive force and attractive force of the reject electrode 6 and the attracting electrode 7. Provided is a sensing switch comprising a pair of switching electrodes 8 spaced apart from each other that can be connected when touched.

In FIG. 1, a receptor capable of binding a ligand may be immobilized in the receptor binding region 5. When a ligand is directly or indirectly bound to the receptor binding region 5, the repulsive force acts on the ligand and the reject electrode 6 with the same polarity and the attraction force of the ligand and the attractant electrode 7 act on the opposite polarity. To bend the sensing plate 4 downward. At this time, the end of the sensing plate 4 touches the pair of switching electrodes 8 on the substrate to allow current to flow through.

In FIG. 1, the pair of switching electrodes 8 is represented by a single line for convenience, but is composed of a pair of switching electrodes that do not pass current at a predetermined interval from each other before the sensing plates contact each other.

2 is an exploded perspective view of a sensing switch (cantilever) according to another embodiment of the present invention. 2, a sensing switch according to another embodiment of the present invention is a substrate (1); A support (2) on the substrate (1); Sensing plates (4) extending in parallel and spaced apart from the substrate (1) connected from the side of the support (2); A receptor coupling region 5 located on an upper surface of the sensing plate 4 end; Magnetic beads (10) that selectively bind to the receptor (12) bound to the receptor binding region (5) via a ligand (11); Magnetic field generating means (13) located below the sensing plate (4); And a pair of switching electrodes 9 and 9 'spaced apart from each other by which the sensing plate 4 can be connected when the sensing plate 4 touches the substrate by the generated magnetic field.

In FIG. 2, a receptor 12 is immobilized in the receptor coupling region 5. In addition, the magnetic beads 10 are coupled to the ligand 11, and through the selective binding between the ligand 11 and the receptor 12, the beads are bound to the receptor located on the upper surface of the sensing plate (4) end Engage with region (5). The magnetic bead 10 coupled to the receptor engagement region 5 is moved in the direction of the substrate by the magnetic field generated by the magnetic field generating means 13, whereby the sensing plate 4 is also bent.

The magnetic beads can be used mainly in the form of suspensions, for example, available from Dynal AS. The magnetic beads include tonic, paramagnetic and superparamagnetic beads. The magnetic beads can be produced, for example, by the method disclosed in EP 0106873.

3A is a front view of a sensing switch (bottle beam) according to another embodiment of the present invention, and FIG. 3B is a view showing that the switch is operated by sensing. The sensing switch of the figure is located in the center of the substrate 1, the two supports (2, 2 ') placed on the substrate, the connection beam (3) connecting the support (2, 2'), the connection beam (3) in the center A sensing plate 4 positioned parallel to the substrate 1 and spaced apart from the substrate 1 in parallel, two receptor coupling regions 5 and 5 ′ positioned on upper surfaces of both arms of the sensing plate 4, and the sensing plate 4. A push-out electrode 6 of the same polarity as the ligand located above, and a pull-in electrode 7,7 'of the opposite polarity to the ligand located below the sensing plate, and the rejection. And a pair of switching electrodes (not shown) spaced apart from each other, which may be connected when the sensing plate 4 contacts the substrate due to the repulsive force and attraction of the electrode 6 and the attracting electrodes 7, 7 '. .

In FIG. 3A, different receptors such as oligonucleotides are immobilized in the receptor binding regions 5 and 5 ', and more receptor DNA regions 5' have more target DNA molecules than other receptor binding regions 5 '. Combined to give more negative charge, the reject electrode 6 on the sensing plate has an anode of the same polarity as the DNA, and the attracting electrodes 7, 7 ′ under the sensing plate at all times. Is a cathode of opposite polarity to DNA. The attracting electrodes 7 and 7 'may be separated from each other but may be integral. In FIG. 3B, the receptor coupling region 5 draws more negative charge so that the effective electrostatic forces between the negative reject electrode 6 and the positive attracting electrode 7, i.e. It is moved down like a seesaw by the attraction.

Figure 3c is a perspective view of a sensing switch (yangpalbo) according to another embodiment of the present invention. For convenience, the two receptor coupling regions located on the upper surface of both arms of the sensing plate 4, the rejection and attracting electrodes located above and below the sensing plate, and the switching electrodes on the substrate are omitted.

4A is a conceptual diagram illustrating an on-state of the sensing switch of FIG. 3, and FIG. 4B is a conceptual diagram illustrating an off-state of the sensing switch of FIG. 3.

In FIG. 4A, the switch is turned on to allow current to flow between the switching electrodes on the substrate. In FIG. 3B, the switch is turned off to prevent current from flowing between the switching electrodes on the substrate. Therefore, the sensing switch of the present invention performs a role of sensing and switching at the same time so that the sensing of the charge amount change during the binding reaction with the ligand (switching-on) immediately. As described above, the present invention is a sensing switch having an on-state and an off-state, so that a logic circuit such as a normal electronic circuit can be made.

The sensing switch of the present invention solves the inconvenience of processing the acquired signal after sensing by performing mechanical sensing and electrical switching at the same time. In addition, by requiring minimal circuitry and minimal power, it can be smaller than any sensing and signal processing method ever encountered (Miniaturization). In addition, it has minimal circuit nose (flicker, white noise) and can minimize noise by reducing connection noise between sensor and post processor (Noise minimization).

5A conceptually illustrates a conventional DNA chip detection system, and FIG. 5B conceptually illustrates a DNA chip detection system according to the present invention. In the conventional method of FIG. 5A, since the spots A, B, and C on the DNA chip are measured by a scanner and then signal processed by an analysis device, the target patient may be determined to be a MODY patient since the spots A, B, and C exhibit more than one intensity. However, in the method of the present invention of FIG. 5B, three sensing switches on one DNA chip perform both sensing and analysis, and the final output line is turned on. It can be easily identified as a patient.

6A conceptually illustrates a differential sensing system in a conventional manner, and FIG. 6B illustrates a differential sensing system according to the present invention. Differential sensing is a sensing method that reduces S / N ratio by reducing common noise factor / back ground noise, and all existing methods are another reference spot / sensor (identical) identical to the actual analysis sensor as shown in FIG. The results from both sensors were analyzed using an analytical tool / electrical circuit, requiring at least two identical spot / sensors and differential amplification / post analysis of the signals obtained. In contrast, in the differential sensing of the present invention, as shown in FIG. 6B, one sensing switch performs all functions performed by the conventional devices, thereby miniaturizing, minimizing power consumption, and minimizing power consumption. High S / N ratio noise and remarkably high sensitivity can be obtained.

FIG. 7 conceptually illustrates an example of the differential sensing result of FIG. 6. FIG. 7A is a conventional method, which is measured by a scanner for differential sensing of spots A, B, and C of a DNA chip, and after the intensity of the background spot B is subtracted from the intensity of each spot by signal processing. 7b is a method of the present invention, the sensing switch compares the sensing amount of both arms of the sensing plate like a seesaw, and enables differential sensing of spots A and C. Will be removed automatically.

FIG. 8A illustrates an 'and' logic circuit of a sensing circuit according to an embodiment of the present invention, and FIG. 8B illustrates an 'or' logic circuit. In the 'and' logic circuit of FIG. 8A, when sensing switches 1 and 2 are both on (eg, infected with two HP2 and HP3), the output of the circuit is also on. In the 'or' logic circuit of 8b, when sensing switches 1 or 2 are turned on (eg, when HP2 or HP3 is infected), the output of the circuit also turns on.

FIG. 8C illustrates a composite circuit in which a plurality of “and” and “or” logic circuits exist in a sensing circuit according to an embodiment of the present invention, and thus, a single composite circuit may be formed. By measuring on (1) or off (0) it is possible to accurately diagnose the infection of HP2 and / or HP3. Diagnosis results from the output signal of FIG. 8C are summarized in Table 1 below.

TABLE 1

Highly sensitive sensing logic circuit according to the present invention will be a breakthrough design of LOC that can replace the existing device sensor (DNA chip) + Scanner / controller + analysis tool (PC).

Looking at the working principle of the sensing switch of the present invention, the four torques (Torque = Forces X distance) acting on the sensing plate is as follows.

Electrostatic or magnetic torque (Me): Induces the movement of the sensing plate and depends on the placement / geometric of the electrode or magnetic generating means, the electric or magnetic field, the charge or magnetic beads of the receptor.

Inertia torque (I): proportional to angular acceleration and dependent on the rotational inertia (I) of the rotating connecting beam.

Damping torque (D): proportional to the rotational speed and depends on the damping factor (F) (medium's viscosity, sensor dimension).

Mechanical restoring torque (Mm): proportional to the tilting angle, depending on the material and geometry / design of the sensor.

The relationship between the four torques is as follows.

[Equation 1]

9A and 9B illustrate scales for calculating the operating torque of a sensing switch in accordance with another embodiment of the present invention. The receptor coupling area is 80 × 30 um ² , the material of the sensor is doped polysilicon, E (Young's modulus) = 150 Gpa, ν (poisson ratio) = 0.22, ε _b (relative dielectric constant of buffer) = 50, Voltage = 10V. Therefore, in steady state, the following equation can be concluded.

[Equation 1]

[Equation 2]

Ramen

[Equation 3]

As such, design parameters / requirements can be determined through analytical solutions, and above all, operation can be determined. The dynamical response, such as the response speed of the sensor, must be solved for the full equation, but it is not important at the time of feasibility.

The mechanical torque is calculated as follows (see B.R. Hopkins, design analysis of shafts and beams, 2nd edition):

[Equation 4]

[Equation 5]

[Equation 6]

You can see the result below.

K (Stiffness coeff. Of the beam) = 2.25 × 10 ^-24 m ⁴ ,

G (elastic modulus of the beam in shear) = 61.47 × 10 ⁹ N / m ²

Restoring torque = 9.22 × 10 ^-9 Nm

Electrostatic torque is given by

Electrostatic torque = electrostatic force × distance

[Equation 7]

Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

<Example 1>

Torque was calculated under the following conditions using the sensing switch of the present invention shown in FIG. 9 (A. Peterson, the effect of surface probe density on DNA hybridization, Nucleic Acid Research 2001, 29, 24,5163- 5168). 80% non-specific binding to mismatched probe (single mismatch probe, hypothetically), Immobilized 25mer probe DNA density = 2 × 10 ¹² molecules / cm ² , 100% hybridization with perfect matched DNA (25mer), Probe: 5'-SH- C6-AGATCAGTGCGTCTGTACTAGCACA-3 '

The steady state analysis results are as follows.

Total effective charge (charge difference) q = reactive surface area × molecule / area × # of electrons / molecule × charge / electron = 6 × 10 ^-11 C

Electric Field = 10V / (50 × 10um) = 2 × 10 ⁵ N / C

Fe = 1.2 × 10 ^-5 N

Therefore, as shown below, the electrostatic torque is smaller than the mechanical restoring torque so that the sensor does not move.

Electrostatic Torque = Fe × 40 um = 4.8 × 10 ^-10 Nm

<Restoring torque = 9.22 × 10 ^-9 Nm

If some of the designs / experiments are modified in the permanent embodiment, different results are obtained as follows.

First, if the voltage is increased to 200V, the result is as follows.

Electrostatic Torque = 9.6 × 10 ^-9 Nm> 9.22 × 10 ^-9 Nm (Restoring torque)

Electrodes with an oxide insulating layer interfere with hydrolysis. Theoretically, the voltage can increase to dielectric breakdown voltage (Oxide> 10 MV / cm, liquid ~ 1MV / cm) (~ 1000V / 10 um in our case). The voltage increase causes the electrostatic torque to be greater than the mechanical restorative torque, causing the switch to move.

Secondly, if RCA is used to increase the effective charges, it becomes 100 times rolling multiplication (few minutes), and the electrostatic torque is greater than the mechanical recovery torque as shown below.

Electrostatic Torque = 48 × 10 ^-9 Nm> 9.22 × 10 ^-9 Nm (Restoring torque)

Third, modifying beam designs and materials yields lower young's modulus and longer beam length. This allows the switch to move by lowering the mechanical recovery torque compared to the electrostatic torque.

<Example 2>

In order to find out whether the sensing switch according to the present invention of FIG. 10 can act as a real switch, a simulation experiment was performed using nucleic acid as a biomolecule (A. Peterson, the effect of surface probe density on DNA hybridization, Nucleic Acid Research). 2001, 29, 24,5163-5168). ANSYS 8.0 (ANSYS, Inc., USA) was used as the simulation tool, and the simulation conditions were as follows. Element: shell 93 (suited for large deflection), Beam material: Gold, Young's modulus = 75 GPa (silicon ~ 169 GPa), Poisson's ratio = 0.42, Density = 1.932 × 10 ⁴ kg / m ³ (silicon ~ 2.33 × 10 ³ kg / m ³ ), Beam thickness = 1um, Relative permitivity = 80 (experimental buffer solution), Gap between two electrodes = 100 um, Applied potential = 10 V, Immobilized 25mer probe DNA density = 2 × 10 ¹² molecules / cm ² , 100% hybridization with perfect matched DNA (25mer), Probe: 5 'SH-C6-AGATCAGTGCGTCTGTACTAGCACA 3'. Depending on the number of beams (arms of the plate), different designs were applied as shown in Table 2 below. The simulation results are shown in FIGS. 11a (three beams) and 11b (six beams) (Etch holes: 5um × 5um, Distance between holes: 5um). As shown in the simulation results, it is shown that it can be bent sufficiently under a given condition and can act as a switch by connecting two electrodes.

TABLE 2

<Example 3>

In the apparatus shown in Fig. 2, it was examined whether the force less than the coupling force between the receptor and the ligand and enough force to move the switch occurred between the magnetic beads and the magnetic field generating means.

BioMag ^® BM551 was used as magnetic beads. The magnetic beads have a streptavidin immobilized on the surface as a ligand. In addition, a neodymium magnet was used as the magnetic generating device. The properties of the magnetic beads and neodymium magnets are as follows: Br = 1.22 Tesla, μ ₀ = 1.26 × 10 ^-6 H / m, magnetic mass susceptibility = 2.54 × 10 ^-3 m ³ / kg, density = 1.70 × 10 ³ kg / m ³ , magnetic susceptibility = 4.31, bead diameter = 1.50 × 10 ⁻⁶ m, V = 1.77 × 10 ⁻¹⁸ m ³ , L_magnet = 0.003 m.

Using the above values and Equation 8, the force according to the distance between the magnetic beads and the magnetic generating means was calculated.

[Equation 8]

The calculation result is shown in FIG. As shown in FIG. 12, for example, it had a value of 90 pN / μm ² at a distance of 1 mm.

On the other hand, the mutual binding force of streptavidin and biotin is known to be 260 ± 20 pN (Proc. IEEE 85 (4), 672-680, 1997).

From the above results, in the sensing switch according to the present invention, a force (90 pN) sufficient to move the switch while not disturbing the coupling is weaker than the coupling force between the receptor-ligand (260 ± 20 pN) and generates magnetic beads and magnetic fields. It can be seen that it occurs between means.

<Example 4>

In order to find out whether the sensing switch according to the present invention of FIG. 2 can serve as a real switch, a simulation experiment was applied by applying the method of Example 2. FIG. 13A illustrates a scale of a sensing plate used for simulation of the sensing switch of FIG. 2. The simulation conditions were as follows: the sensing plate thickness was 3 μm and the material was silicon (single crystal, Young's modulus = 169 GPa, Density = 2330 kg / m ³ ).

The pressure was 0.1 × 90 N / m ² = 9 Pa as a result of calculation under the above conditions. Based on the pressure, the distance between the sensing plate and the switching electrode to which the sensing plate is sufficiently curved to connect the pair of switching electrodes was calculated. It is assumed that magnetic beads only bind to 10% of the sensing plate cross section. Figure 13b shows the simulation result.

From the above results, it can be seen that the distance between the sensing plate and the switching electrode should be 180 μm or less so that the sensing plate can be connected to the switching electrode when the magnetic field is applied.

In addition, the sensing plate was bent downward due to the weight of the sensing plate itself and the magnetic beads, so that it was connected to the switching electrode even when no magnetic field was applied. It is assumed that magnetic beads bind to the entirety of the sensing plate cross section, ie 100%. Figure 13c shows the simulation results.

From the above results, it can be seen that when the distance between the sensing plate and the switching electrode is 1.6 μm or more, the sensing plate is not connected to the switching electrode when the magnetic field is not applied.

That is, it can be seen that the distance between the sensing plate and the switching electrode for the sensing plate to act as a switch is 1.6 to 180 μm.

Example 5

The sensing switch according to the present invention of FIG. 2 was actually manufactured by applying a semiconductor process technology.

FIG. 14 conceptually illustrates a manufacturing process of a sensing switch according to the present invention of FIG. 2.

Referring to FIG. 14, in order to manufacture a sensing plate, an oxide layer 22 is first formed on an SOI wafer 21 and the SOI wafer 23 is again placed thereon. A PR 24 is coated on the surface of the SOI wafer 21 and the SOI wafer 21 is etched in a conventional manner using mask 1 (step (a)). Next, PR 25 is coated on the surface of the opposite SOI wafer 23 and the SOI wafer 23 is etched in a conventional manner using mask 2 (step (b)). Thereafter, the oxide layer 22 exposed in the direction of the SOI wafer 23 is etched to manufacture a sensing plate (step (c)).

Next, the poly-Si 27 is coated and the oxide layer 26 is etched using mask 3 (step (d)), and a pair of switching electrodes 29 and contact pads connected thereto are used using mask 4 ( contact pad) (step (e)).

The substrate and the sensing switch manufactured above are combined (step (f)). The sensing switch shown in step (f) of FIG. 14 corresponds to that of FIG. 2, and its identification number is shown in accordance with FIG.

15A and 15B are enlarged SEM photographs of a sensing plate of a sensing switch manufactured through the above process.

<Example 6>

It was confirmed whether the sensing switch manufactured in Example 5 actually performs the detection function.

BioMag ^® BM551 was used as magnetic beads. The magnetic beads have a streptavidin immobilized on the surface as a ligand. A neodymium magnet was used as the magnetic generating device. 1V DC voltage was applied to the pair of switching electrodes.

The experiment was performed by dividing the biotin into the case where the biotin was bound to the receptor binding region located on the upper end of the sensing plate.

The result is shown in FIG. 16, it can be seen that when biotin is bound to the receptor binding region of the sensing plate, current flows when the magnetic field is operated, but no current flows when the biotin is not bound. Therefore, it was confirmed that the sensing switch according to the present invention can effectively perform the detection of the receptor.

As described above, according to the present invention, there is no need to fluorescently label target substances such as biomolecules and chemicals, and there is no need to separately signal the fluorescence or electrical detection signals in the analyzer, and The advantage is that the signal can be read directly with or without current flow due to the off. That is, the sensing switch of the present invention solves the inconvenience of processing the acquired signal after sensing by performing mechanical sensing and electrical switching at the same time. In addition, by requiring minimal circuitry and minimal power, it can be smaller than any sensing and signal processing method ever encountered (Miniaturization). In addition, it has minimal circuit nose (flicker, white noise) and can minimize noise by reducing connection noise between sensor and post processor (Noise minimization).

Claims (13)

Board; A support placed on the substrate; Sensing plates extending in parallel and spaced apart from the substrate connected from the side of the support; A receptor coupling region located on an upper surface of the sensing plate end; Electric or magnetic field generating means for inducing deflection of the sensing plate when the receptor bound to the receptor binding region selectively binds to a ligand that is electrically or magnetically active; And a pair of switching electrodes spaced apart from each other by a bending of the sensing plate, which may be connected when the sensing plate contacts the substrate. Board; A support placed on the substrate; Sensing plates extending in parallel and spaced apart from the substrate connected from the side of the support; A receptor coupling region located on an upper surface of the sensing plate end; A push-out electrode of the same polarity as the ligand located above the sensing plate, a pull-in electrode of opposite polarity to the ligand located below the sensing plate, and the repulsive force and attraction of the evacuation electrode and the attracting electrode And a pair of switching electrodes spaced apart from each other by which a sensing plate may be connected when the sensing plate contacts the substrate. Board; A support placed on the substrate; Sensing plates extending in parallel and spaced apart from the substrate connected from the side of the support; A receptor coupling region located on an upper surface of the sensing plate end; Magnetic beads that selectively bind to a receptor bound to the receptor binding region through a ligand; Magnetic field generating means located below the sensing plate; And a pair of switching electrodes spaced apart from each other by a generated magnetic field, which may be connected when the sensing plate contacts the substrate. The method according to any one of claims 1 to 3, Wherein said ligand is a nucleic acid, protein, peptide, antibody, antigen, polymer, liquid or gaseous chemical agent that selectively binds to a receptor. The method of claim 3, wherein The magnetic bead is attached to the magnetic bead selectively binds to the receptor, or a second receptor that selectively binds to the ligand selectively binding to the sensing switch. The method according to any one of claims 1 to 3, The sensing plate extends in parallel and spaced apart from the substrate to both sides from the center of the connecting beam connecting the two supports placed on the substrate, there are two receptor coupling regions on the upper surface of both arms of the sensing plate Sensing switch characterized by. The method of claim 6, And a different type of receptor is immobilized in the two receptor coupling regions. A sensing circuit in which a plurality of sensing switches of any one of claims 1 to 7 are connected in series and / or in parallel to form a logic circuit of 'and' and / or 'or'. The method of claim 8, And a detection circuit for simultaneously detecting and analyzing a plurality of ligands by viewing an output signal output through the logic circuits of 'and' and / or 'or'. Introducing a ligand into the receptor binding region of the sensing switch of claim 2; Subjecting the ligand to a receptor fixed to the receptor binding region; Applying a voltage of the same polarity as the ligand to the reject electrode and a voltage of the opposite polarity to the ligand to the attracting electrode; Sensing plates are moved up and down by repulsion and attraction between the ligand, the reject electrode and the attracting electrode to open and close a pair of switching electrodes on the substrate; And Ligand binding detection method comprising the step of measuring the flow of current between the switching electrode. The method of claim 10, And a different type of receptor is immobilized in two receptor binding regions of the sensing switch to detect whether there is a differential binding between different ligands. Attaching a magnetic bead to a ligand that selectively binds to the receptor, or to a secondary receptor that selectively binds to the ligand; Introducing a ligand or secondary receptor to which the magnetic beads are attached to a receptor fixed to the receptor binding region of the sensing switch of claim 3 or 6; Attaching magnetic beads to the receptor binding region by a selective binding reaction between the ligand and the receptor; Removing the ligand or secondary receptor of magnetic bead attachment that did not participate in the binding reaction; Generating a magnetic field from a magnetic field generating means located below the sensing plate; Moving the sensing plate up and down by the magnetic field to open and close a pair of switching electrodes on the substrate; And Ligand binding detection method comprising the step of measuring the flow of current between the switching electrode. The method of claim 12, And a different type of receptor is immobilized in two receptor binding regions of the sensing switch to detect whether there is a differential binding between different ligands.

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