KR101113793B1 - System and Method for Detecting Thrombin Using Micro-cantilever - Google Patents

Abstract

Micro cantilever sensors; A DNA aptamer immobilized on the micro cantilever; And a micro cantilever sensor-based thrombin detection system comprising a measuring device for measuring the resonant frequency of the micro cantilever and a thrombin detection method using the system is disclosed. The thrombin detection system and method can effectively detect trace amounts of thrombin.

Micro cantilever, aptamer, resonant frequency, thrombin

Description

System and Method for Detecting Thrombin Using Micro-cantilever}

The present invention relates to a thrombin detection system and detection method using a micro cantilever.

Micro cantilever has not only achieved structural and material advances with the development of micro electromechanical systems (MEMS) and nano electromechanical systems (NEMS), but also has greatly expanded the field of application due to the rise of nano-biotechnology. Micro-cantilevers as biosensors feature high sensitivity, high selectivity and labeling-free detection and include DNA, disease marker proteins, and small molecule biomaterials. Pathogens are targeted for analysis.

Microcantilever sensors are largely divided into microbalance principle and surface stress principle in their application. The former is a dynamic mode that measures the change in resonant frequency caused by the change in mass and spring constant of the cantilever, and the latter is a static mode that measures the displacement caused by surface stress change due to the specific reaction on the microcantilever. Static mode.

In the case of a microcantilever as a biosensor, there is a view that the output signal (the change in resonant frequency or the degree of warpage) is more critical due to the change in surface stress generated when a small amount of biological material binds to the surface. That is, the surface stress is generated by the structural change generated by the force and the specific coupling between the neighboring materials while the biological material is specifically coupled, and thus the bending phenomenon or the change of the resonance frequency of the microcantilever sensor occurs.

For biosensors, sensitivity is one of the important factors along with selectivity and speed. The biosensor is composed of a receptor element that accepts a biomaterial and a transducer element that converts it into an electrical signal, and converts the recognition of the biomaterial into an electrical signal through an optical or mechanical change. do. In order to amplify the signal of the biosensor and to improve the sensitivity, the improvement of the transducer element in the electrical and optical aspects and the receptor element in the chemical and biological aspects have been simultaneously made.

An object of one embodiment of the present invention is to provide a thrombin detection system using a micro cantilever.

Another object of an embodiment of the present invention to provide a thrombin detection method using a micro cantilever.

The present invention provides a micro-cantilever sensor; A DNA aptamer immobilized on the micro cantilever; And it provides a micro cantilever sensor-based thrombin detection system comprising a measuring device for measuring the resonant frequency of the micro cantilever and a thrombin detection method using the system.

The thrombin detection system and method according to the present invention will be able to effectively detect trace amounts of thrombin.

Thrombin detection system according to an embodiment of the present invention, the micro-cantilever sensor; A DNA aptamer immobilized on the micro cantilever; And it characterized in that it comprises a measuring device for measuring the resonant frequency of the micro cantilever.

In one embodiment, the DNA aptamer may be a single stranded DNA aptamer or a DNA aptamer-gDNA aptamer duplex structure. In another embodiment, the DNA aptamer may have a structure in which an alkyl thiol group is connected to an end thereof. More specifically, the DNA aptamer may be a thiolated single strand DNA aptamer or a thiolated DNA aptamer-g-DNA duplex structure.

In one embodiment, in order to amplify the signal of the micro-cantilever biosensor to increase the sensitivity, (1) PZT (lead zirconate titanate; Pb [Zr _x Ti _{1 -x} ] O ₃ , 0 <x <1) layer It may be a resonant micro-cantilever, and (2) may be a structure in which a gold foil layer is coated on the lower surface of the micro-cantilever.

In another embodiment, the micro cantilever is a PZT-based resonant micro cantilever sensor gilded on the lower surface of the micro cantilever sensor, and the DNA aptamer immobilized on the micro cantilever is a bioreceptor on the gold foil surface of the micro cantilever. As a single stranded DNA aptamer or DNA aptamer-g-DNA duplex (DNA aptamer-g-DNA duplex) may be combined structure. Through this, it is possible to amplify the signal of the micro cantilever sensor to increase the sensitivity.

The thrombin detection system according to an embodiment of the present invention uses a dynamic mode for analyzing the resonant frequency of the cantilever. The resonant frequency analysis is performed by analyzing the change in the phase angle of the impedance when the stress change (change of free energy) of the surface generated when the thrombin is combined with the aptamer immobilized on the micro cantilever changes the natural frequency of the cantilever. In one embodiment, the DNA aptamer immobilized on the micro cantilever can cause structure switching by binding to thrombin.

In one embodiment, the detection system may further include a liquid cell into which the lower end of the micro cantilever sensor is impregnated and inserted. In addition, the system may further include a spacer for backfilling to prevent nonspecific adsorption, for example, the spacer may be ethylene glycol. Backfilling with spacers allows for specific binding to thrombin.

The present invention also provides a micro cantilever sensor-based thrombin detection method. Specifically, the detection method may include the following steps:

(a) immobilizing the DNA aptamer on the micro cantilever;

(b) measuring the resonant frequency of the micro cantilever;

(c) reacting a sample containing a biomaterial with the micro cantilever;

(d) measuring the resonant frequency of the micro cantilever after the reaction; And

(e) calculating the resonant frequency variation of the cantilever before and after the reaction and performing quantitative analysis of thrombin contained in the sample.

In one embodiment, the DNA aptamer may be a single stranded DNA aptamer or a DNA aptamer-guard-DNA duplex. In another embodiment, the DNA aptamer may have a structure in which an alkyl thiol group is connected to an end thereof. More specifically, the DNA aptamer may be a thiolated single strand DNA aptamer or a thiolated DNA aptamer-g-DNA duplex structure.

In one embodiment, the measuring method comprises the steps of: (1) preparing a PZT-based resonant micro-cantilever sensor to amplify the signal of the micro-cantilever biosensor to increase the sensitivity; (2) A gold foil is coated on the lower surface of the microcantilever sensor, and a single stranded DNA aptamer or DNA aptamer-g-DNA duplex is used as a bioreceptor on the gold foil surface. Adding to combine; And (3) reacting thrombin as the target substance. Through this, the thrombin is quantitatively analyzed by measuring the variation of the resonance frequency before and after the reaction between the biomaterial and the microcantilever sensor to be analyzed. More specifically, the micro cantilever is a PZT-based resonant microcantilever sensor coated with gold leaf on the lower side, and the step (a) is a single stranded DNA aptamer as a bioreceptor on the gold foil surface of the micro cantilever. aptamer) or DNA aptamer-g-DNA duplex may be added to bind.

In addition, after fixing the aptamer to the (a) micro cantilever, the method may further include inserting the liquid into the liquid cell so that the lower end of the micro cantilever is impregnated. After inserting the micro cantilever into the liquid cell, the method may further include a step of backfilling using a spacer, ethylene glycol.

The thrombin detection method according to an embodiment of the present invention uses a dynamic mode for analyzing the resonance frequency of the cantilever. The resonant frequency analysis is performed by analyzing the change in the phase angle of the impedance when the stress change (change of free energy) of the surface generated when the thrombin is combined with the aptamer immobilized on the micro cantilever changes the natural frequency of the cantilever. In one embodiment, the DNA aptamer immobilized on the micro cantilever can cause structure switching by binding to thrombin.

Hereinafter, the present invention will be described in detail by specific examples such as the following examples. However, the following specific examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples.

Experimental Example 1

1-1. device  Manufacture and Liquid  Cell design

PZT layer embedded micro cantilevers were fabricated by a micromatching process. The size of the micro cantilever used for thrombin analysis is 30 μm × 90 μm × 2.48 μm (width × length × thickness). A 1.7 μm thick PZT layer is included, which is thicker while improving the Q-factor in the liquid phase (see FIG. 1 (b)). The prepared micro cantilever was insulated by coating with parylene-C (parylene-C; ˜0.5 μm). The Q-element of the insulated micro-cantilever is generally 10 or less. On the other hand, in order to measure the resonant frequency of the micro cantilever in the liquid phase, the liquid cell was made of Terfon. The prepared liquid cell had a 800 μm wide channel, a 30 μl volume reaction chamber, and a glass window for laser transmission (see FIG. 2 (a)).

1-2. Resonant frequency  Measuring system

The resonant frequency of the micro cantilever was measured using an optical heterodyne laser Doppler vibrometer (LDV) (see FIG. 2 (b)). While sweeping the frequency range, the output signal (speed and displacement) of the LDV measured the resonant frequency with the microcantilever set to select the initial resonant frequency of the microcantilever at the point of maximum deflection. .

1-3. Aptamer  Immobilization and Thrombin  analysis

The micro cantilever was coated with a gold film and impregnated in 1 μM DNA aptamer, without g-DNA or DNA-g-DNA duplex solution (in TE), for 6 hours at room temperature, and with ethylene glycol spacer (5 In a solution of mM HS-C ₁₁ -EG ₃ -OH (in EtOH) at room temperature for 1.5 hours. Sequences of DNA aptamer and g-DNA were 5'-CAC TGT GGT TGG, respectively TGT GGT TGG- C ₁₂ -SH-3 'and 5'-CCA ACC ACA GTG-5' (thrombin binding sites are underlined). The aptamer-immobilized micro cantilever was inserted into the liquid cell and then 100 ng / ml α-thrombin solution (in 150 mM PBS with 3 mM MgCl ₂ ) was injected into the reaction chamber. After observing the saturation point, washing buffer (150 mM PBS with 3 mM MgCl ₂ ) was injected to remove nonspecific bound thrombin. All analyzes were monitored in situ with the LDV system in standing state rather than liquid flow.

1-4. Experiment Result Analysis

Prior to thrombin binding analysis in liquid, the aptamer-based recognition layer was allowed to be placed on the surface of the micro cantilever Au film outside the liquid cell. The micro-cantilever was sequentially treated with thiolated aptamer-g-DNA duplex (or aptamer without g-DNA) and ethylene glycol spacer, thereby producing a reliable self-assembled monolayer. (See FIG. 3).

In addition, using the aptamer-immobilized micro cantilever, the effect of structure-switching of the aptamer by thrombin binding on the resonant frequency variation of the micro cantilever was studied. First, the specificity of the aptamer receptor applies a heterogeneous protein such as Prostate Specific Antigen (PSA, 100 ng / ml) and human serum albumin (HAS, 1mg / ml). It confirmed by doing. As shown in Figure 4 (a, b), micro cantilever hardly responds to nonspecific proteins regardless of protein concentration. They commonly exhibit a resonant frequency shift of about -70 Hz. The results of specific thrombin analysis are shown in FIG. 4 (c). The resonance frequency change induced by structure-switching of aptamer molecules by thrombin binding was observed. Interestingly, the curve shape of the resonant frequencies of the aptamer-g-DNA duplex and the aptamer without g-DNA appeared to be apparently different, including the equilibrium resonant frequency. Thrombin binding was commonly saturated at ˜15 min, but there were differences in terms of dissociation of nonspecifically bound trobines. The resonant frequency of the microcantilever functionalized with the aptamer-g-DNA duplex remained unchanged (final Δf = -1800 Hz), while the resonant frequency of the microcantilever functionalized with the aptamer without g-DNA was 500 Hz (final). Δf = -750 Hz).

Duplex-to-complex g-DNA-mediated structure switching by thrombin binding is more likely to generate stresses on the surface than to simple coplex formation by thrombin binding. It seems to have a lot of influence. In addition, g-DNA appears to increase stability by thrombin binding. 4 (d) shows the importance of ethylene glycol backfilling. Without spacer backfilling, no specific thrombin binding occurred in any case.

As a result, the resonant frequency of the microcantilever functionalized with the single stranded aptamer and aptamer-g-DNA duplex due to thrombin binding in Observed in situ . From these results, in relation to g-DNA, the effect of structure switching of duplex-to-complex by thrombin binding in the nanomechanical response can be explained.

1 is a SEM image of (a) micro cantilever and (b) PZT-embedded Pt / SiNx / Si wiper cross section;

2 is a schematic diagram illustrating (a) a liquid cell, (b) a picture of a measurement system, and (c) a measurement principle.

FIG. 3 is a schematic of thrombin analysis using (a) DNA-aptamer-g-DNA duplex and (b) single stranded DNA aptamer without g-DNA; FIG.

4 shows (a) 100 ng / ml PSA, (b) 1 mg / ml HSA injection respectively, and 100 ng / ml alpha-thrombin injection and (c) spacer backfilling and (d) spacer About backfilling in This graph shows the results of observing the situ resonant frequency.

Claims (14)

Micro cantilever sensors; A DNA aptamer immobilized on the micro cantilever; A measuring device for measuring a resonance frequency of the micro cantilever; And A spacer for backfilling, The DNA aptamer comprises a 5'-GGT TGG TGT GGT TGG-3 'sequence and is a micro cantilever sensor-based thrombin detection device characterized in that the DNA aptamer-g-DNA duplex (DNA aptamer-guard-DNA duplex) structure . delete The method of claim 1, The DNA aptamer is a thiolated DNA aptamer-g-DNA duplex structure of the micro-cantilever sensor-based thrombin detection device. The method of claim 1, The micro cantilever is a microcantilever sensor-based thrombin detection device comprising a lead zirconate titanate (PZT) Pb [Zr _x Ti _1-x ] O ₃ , 0 <x <1) layer or a gold foil layer. The method of claim 1, The micro cantilever is a PZT-based resonant microcantilever sensor coated with a gold leaf on the lower side of the micro cantilever sensor, The DNA aptamer immobilized on the micro cantilever has a structure in which a DNA aptamer-g-DNA duplex is coupled to a gold foil surface of the micro cantilever as a bioreceptor. Thrombin detection system. The method according to any one of claims 1 and 3 to 5, The DNA aptamer microcantilever sensor-based thrombin detection device characterized in that structure switching occurs by binding to thrombin. The method of claim 6, The detection device, the micro-cantilever sensor-based thrombin detection apparatus further comprises a liquid cell (liquid cell) is inserted into the lower end of the micro-cantilever sensor. The method of claim 1, The backfilling spacer is a micro-cantilever sensor-based thrombin detection apparatus, characterized in that the ethylene glycol (ethylene glycol). (a) immobilizing the DNA aptamer on the micro cantilever; (b) measuring the resonant frequency of the micro cantilever; (c) reacting a sample containing a biomaterial with the micro cantilever; (d) measuring the resonant frequency of the micro cantilever after the reaction; And (e) calculating the resonant frequency variation of the cantilever before and after the reaction to perform quantitative analysis of thrombin contained in the sample, After the step (a), further comprising the step of backfilling using a spacer (spacer), The DNA aptamer comprises a 5'-GGT TGG TGT GGT TGG-3 'sequence and is a micro cantilever sensor-based thrombin detection method characterized in that the DNA aptamer-guard-DNA duplex structure . delete The method of claim 9, The micro cantilever is a resonant micro-cantilever sensor based on PZT coated with a gold leaf on the lower side, The step (a) is a micro cantilever sensor-based thrombin detection method, characterized in that the step of adding a DNA aptamer-g-DNA duplex (DNA aptamer-g-DNA duplex) as a bioreceptor to the gold foil surface of the micro-cantilever . The method of claim 9, Before the step of backfilling using the spacer, The microcantilever sensor-based thrombin detection method further comprises the step of inserting into the liquid cell so that the lower end of the cantilever is impregnated. The method of claim 9, In the step of backfilling using a spacer The spacer is a micro-cantilever sensor-based thrombin detection method characterized in that the ethylene glycol (ethylene glycol). The method according to any one of claims 9 and 11 to 13, The DNA aptamer micro-cantilever sensor-based thrombin detection method characterized in that the structure switching occurs by binding to thrombin.

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