KR20180102363A - Metal ion detection method using dielectrophoretic technique - Google Patents

Abstract

The present invention relates to a method for detecting metal ions using a novel spectroscopic technique and for simply and accurately mass-measuring a wide range of metal ion concentration. More specifically, the present invention utilizes a polynucleotide-coated substrate and microspheres to increase mutual binding force between metal ion-mediated polynucleotides as the metal ion concentration increases. Therefore, the present invention can measure presence of the metal ions in a simple manner at a wide concentration range with high sensitivity by measuring unbinding force by DEP force and quantifying the same by a statistical analysis method, and mass measurement at a given time.

Description

[0001] The present invention relates to a metal ion detection method using a dielectrophoretic technique,

The present invention relates to a metal ion detection method and a metal ion concentration measurement method using a dielectrophoretic technique.

The heavy metals generated by the rapid development of industry are the factors that cause environmental pollution such as air, water quality and soil, and they are accumulated not without decomposition in vivo as well as toxicity itself, and there is a great risk of causing various diseases to humans. Accumulation of heavy metals in the human body can cause damage to the central nervous system paralysis and speech disorders, can cause osteoporosis, gastrointestinal disorders, kidney failure, miscarriage, low birth weight and infant growth disorders. Therefore, it is essential to develop metal ion detection and concentration measurement method that can easily and accurately measure the degree of heavy metal contamination.

Typical methods for quantifying trace metals include gas chromatography (GC), inductively coupled plasma spectroscopy (ICP), atomic absorption spectroscopy (AAS), and atomic emission spectroscopy (AES) Method or an electrochemical method.

However, the above-described atomic absorption spectrophotometry requires a long time for measurement, and the measurement apparatuses such as stationary apparatuses are very expensive, have a high maintenance cost, are complicated in the apparatus control method, and have a high analysis cost. Especially, the measuring instrument using spectroscopy is very expensive because the main parts of the instrument including the light source are required to have an additional maintenance cost in parts replacement and after-sales service, And accuracy. On the other hand, the electrochemical measurement method is widely used as a quick and economical analysis method compared to the spectroscopic method. However, in the case of the mercury electrode method, which is one of the electrochemical measurement methods, when the mercury source solution is used as the conduction medium, the mercury electrode is disconnected and stable and continuous measurement is impossible. (Metal Thin Film Electrode), there is a concern that secondary pollution occurs because the mercury undecaneous solution is used as a conduction medium, and the reproducibility and reliability of the analysis are somewhat low. In the case of SWV (square wave stripping voltammetry), which is most commonly used as a typical heavy metal analysis method, it is advantageous to detect heavy metal ions at a low concentration with higher sensitivity than spectroscopic methods or other electrochemical methods. However, Since it is necessary to perform a pretreatment process to concentrate the surface of the sensor, measurement time of several minutes to several tens of minutes is required. Further, there is a problem that when the sensor is used for a long period of time, the surface of the sensor is contaminated by heavy metal concentration, resulting in a decrease in measurement sensitivity.

Therefore, it is necessary to overcome the above problems and to detect a heavy metal in a stable high sensitivity even in a long term use, and to develop a new metal ion detection and concentration measurement method capable of detecting metal ions and measuring the concentration in a short time in a few seconds.

Silver has been used as an antibacterial reagent for the last 6,000 years. The ancient Egyptians, Greeks and Romans used silver bowls to keep the solution fresh and silver pads for wound healing. Recent advances in nanotechnology have led to the production of silver nanomaterials with enhanced antimicrobial activity, while generating numerous research reports on silver-based bactericidal products.

However, there has been a concern that when the silver ions emitted from the improved silver products are exposed to various ecosystems, there may arise a risk of environmental and health safety hazards thereof. Many studies have shown that Ag ⁺ can have a negative impact on humans as well as on wildlife. Also, the released Ag ⁺ forms an Ag ⁺ - polynucleotide complex, which may cause DNA damage. Therefore, it is important to develop a method that can detect the presence of Ag ⁺ in the environment and measure the formation of the Ag ⁺ - polynucleotide complex.

Previous studies have made it possible to measure Ag ⁺ using atomic absorption spectrometry and fluorescent probes based on the binding specificity between metal ions and organic molecules. However, the above approaches are complicated preparation process, including the pre-treatment process for measuring the Ag ^+, and, it is not precise enough to measure low concentrations of Ag ^+. As a possible alternative to this approach, there is a method of measuring using cytosine (C) -containing polynucleotides, which may include fluorescence-based assays (10 nM to 1 [mu] M) Cytosine was detected using electrochemical sensors (10 nM to 10 μM), resonant cantilevers (1 nM to 10 μM), and Kelvin probe force microscopy (100 pM to 100 nM) Ag ⁺ can be measured with high selectivity and specificity for abundant DNA (cytosine-rich DNA).

However, even if the measurement techniques provide high selectivity than conventional methods in the Ag ⁺ is detected, the hydrogen (H ⁺⁾ - a polynucleotide-complex with Ag ^+-related important to polynucleotide complexing coordination (coordinate) in ( & Lt ^; / RTI ^> Ag ⁺ -polynucleotides) conjugate. Therefore, it is possible to measure the Ag ⁺ , Ag ⁺ , H ^+, and intermolecular interactions produced by the polynucleotides as well as to measure various concentrations of Ag ⁺ using a simple, The development of a method is required.

The rupture force of a binding interaction is a usable candidate that can meet this need with a single probe. Nevertheless, the quantitative approach has not been reported so far. In the case of DEPFS using a recently developed internal microfluidic device, several hundreds of microspheres, either chemically or biologically functionalized under certain conditions, are probed. , It is possible to measure a number of weak binding interactions and numerous internal molecular interactions such as specific ligand-receptor binding, nonspecific interaction, and DNA-DNA interaction under various pH conditions. Thus, contrary to other force spectroscopic approaches, DEPFS can be used to obtain statistically reliable data of non-intermolecular interactions, and this data can be used for simple, It can be used for statistical investigation as a possible way.

Thus, we use the DEPFS approach to detect metals (e.g., Ag ⁺ ) with high sensitivity over a wide range of concentrations and to detect intermolecular interactions between metal poly-C DNA complexes (e.g., Ag ⁺ / H ⁺ Based on force detection, a new method has been developed to quantitatively measure the interactions between metals, hydrogen ions and poly-C DNA (eg, Ag ⁺ , H ⁺ and poly-C DNA). In addition, the rupture force (F _U ) on DNA complexes ranging from 100 pM to 100 μM in distilled water and drinking water samples was measured by this method and Ag ⁺ / H ⁺ poly-C DNA was detected by force spectroscopy. The hydrogen interaction, coordinate interaction cooperativity of Ag ⁺ - poly-C DNAs and H ⁺ -poly-C DNAs in the complex was measured and used for statistical evaluation to investigate the mechanism of Ag ⁺ interaction in DNA polynucleotides .

Therefore, the present inventors have found that, by using DEPFS, the detection of the metal ion present in the solution and the detection of the metal ion The present inventors have completed the present invention by determining that the concentration can be measured quantitatively and the mechanism of metal ion interaction in the DNA polynucleotide can be measured.

Korean Patent No. 1,599,606 Korean Patent No. 1,380,716

Measurement of the Interaction Between Hemicytosinium Duplexed Using Dielectrophoretic Tweezers (2015, 6th Asian Particle Technology Symposium 15-18th Sept. / Seungyeop Choi, Min Hyung Kim, Direct measurement of the interaction force between hemicytosinium duplexes using dielectrophoretic force spectroscopy (2015 Korea Society of Biomedical Engineering Fall Conference) (12-14th_Nov.) Detection of Silver Ions Using Dielectrophoretic Tweezers-Based Force Spectroscopy (2016 Analytical Chemistry (20th_July) / Seungyeop Choi, Sang Woo Lee et al.

The present invention provides new DEP force-based force spectrometry capable of simple and precise measurement of the presence and concentration of metal ions in solution and mass spectrometry analysis.

In order to achieve the above object,

(a) functionalizing at least one surface of a substrate on which an electrode is formed, with a polynucleotide;

(b) placing the substrate of step (a) and beads functionalized with polynucleotides into a chamber;

(c) placing a test solution containing or not containing a metal in a chamber and applying a voltage to the substrate;

(d) measuring motion of the beads to obtain video signal information;

(e) obtaining a gray scale value from the video signal information obtained in step (d);

(f) detecting a moment of rupture using the gray scale value obtained in step (e); And

(g) measuring the unbinding voltage at the rupture point and obtaining a rupture force (F _U ) through a conversion process.

In addition,

(a) functionalizing at least one surface of a substrate on which an electrode is formed, with a polynucleotide;

(b) placing the substrate of step (a) and beads functionalized with polynucleotides into a chamber;

(c) placing a test solution containing or not containing a metal in a chamber and applying a voltage to the substrate;

(d) measuring motion of the beads to obtain video signal information;

(e) obtaining a gray scale value from the video signal information obtained in step (d);

(f) detecting a moment of rupture using the gray scale value obtained in step (e);

(g) measuring an unbinding voltage at a rupture point and obtaining an unbinding force (F _U ) through a conversion process; And

(h) Provides a method of measuring the intermolecular bonding force using the Poisson statistics and the tear force value obtained in the step (g).

In addition,

A chamber containing polynucleotide-functionalized substrates and polynucleotide-functionalized beads;

An electric field generator positioned below the chamber, the electric field generator comprising an electrode unit and applying an electric voltage to the electrode unit to form an electric field;

A camera positioned above or at one side of the chamber to image the inside of the chamber;

A measuring unit measuring a gray scale value from a video signal received from the camera and detecting a moment of rupture using a gray scale value; And

Provides including a analysis to obtain; (unbinding force F _U), metal ions, the measuring device measures the voltage (unbinding voltage) of the rupture point measured by the measurement section and after the transformation process rupture strength.

The present invention provides a method for accurately and simply detecting metal ions, measuring metal ion concentrations, and interactions between metal ions and polynucleotides using DEP-based force spectroscopy and quantifying them through statistical analysis.

FIG. 1 shows the form of Ag ⁺ -poly-C-DNA bond between microspheres immobilized with poly-C DNA and a DEP chip in a solution containing Ag ⁺ . When the DEP force is increased, the microspheres are floated As a result, the bond between the poly-C DNA bound to Ag ⁺ was destroyed, and the unbinding force at that moment was measured to summarize the result of increasing the rupture force as the concentration of Ag ⁺ will be.
Figure 2 shows a schematic diagram of a measuring system. (Not to scale) depicting DEPFS, which measures the interrelationship between parallel poly-C DNA, mediated by Ag ⁺ . With the combination of hydrogen bonding and coordination bonding, the inherent forces in the H ⁺ / Ag ⁺ - poly-C complex are measured by the upward movement of the microspheres by the DEP force.
Fig. 3 schematically shows the experiment configuration. The measuring method is composed of three steps. (A) The first step is to apply a negative DEP force (48 Vp-p, 1 MHz) to hold the microspheres with poly-C DNA immobilized in the center of the electrode. (B) The next step is to induce a C-Ag ⁺ -C bond between the microsphere probe and the silicon dioxide (SiO ₂ ) surface. (C) The bursting force is then measured using a vertical DEP force (122 Vp-p, 1 MHz). (DF) represents a visual image obtained from the (AC) step in a DEP chip with interdigitated electrodes containing 100 pM (100 μm scale bar) Ag ⁺ concentration. The plot shows a single microsphere movement (10 μm scale bar) with each applied voltage.
Fig. 4 shows the surface immobilization and characteristics of poly-C DNA. (A) Microspheres with poly-C DNA covalent bonds and EDC / NHS on the SiO ₂ surface are diagrammatically illustrated.
5 shows a fluorescence image of a DEP chip surface (A) labeled with 6-FAM (excitation / emission wavelength is 495 nm / 520 nm) and a microsphere (B) ((A) scale bar 100 μm, (B) scale bar 200 μm)
6 (A) shows an SEM image of a microsphere having immobilized poly-C DNA, and (B) shows a clearly visible image of a D-box region with poly-C DNA. The illustration shows the AFM image of the carboxylated polystyrene microspheres ((A) scale bar 10 μm (B) scale bar 100 nm).
Figure 7 shows the morphological height distribution obtained from the AFM image. The illustration shows the AFM image of the carboxylated DEP chip surface (left) and the surface of the poly-C DNA immobilized DEP chip (right). Each size is 5 × 5 μm ² .
Figure 8 shows the results of quantifying the intermolecular forces between poly-C DNA and Ag ⁺ , H ⁺ having different lengths (L) in the 6-base to 36-base ranges. (A, B) Unbinding for (F _U ) represents the histogram and Gaussian fits in the presence / absence of Ag ⁺ at each L value. (C) Mean rupture force (<F _U >) and standard deviation of A and B, and the illustration showed unzipping of DNA molecules. (D) The shift force (S _F ) represents the difference in the <F _U > value obtained in the presence or absence of Ag ⁺ , which is obtained by normalizing the S _F values of the poly-C ₆ DNA .
Figure 9 shows the quantitative characterization of Ag ⁺ detection capability using DEPFS. (A) Average rupture force (<F _U >) and standard deviation from the Ag ⁺ assay using DEPFS on a semilogarithm scale. Student's t test (two-tailed) was used for statistical analysis (* P <0.0001). The dotted line corresponds to the following log function. _{<F U> = 3.65 × 10} -9 × log [Ag +] + 48.91 × 10 -9. (B) the ion selectivity test and (C) the actual application of DEPFS to the Ag ⁺ sensor (** P <0.0001).
Figure 10 shows the statistical analysis of the interaction between Ag ⁺ and poly-C DNA. (A) [Ag ^+] compared to Ag ⁺ on a semi-log scale - diagrammatically the interrelationship of the poly -C composite in Ag ⁺ and H ⁺ - poly -C complex mutual single rupture strength (Single rupture force) of the working and Ag ⁺ . (B) [Ag ⁺ ] (pentagonal, black) Contrast _indicates that the semilogarithmic plot of normalized F _Usingle (ie, F _Usingle *) matches the Hill formula (dotted line, green). F _Usingle * is obtained from the following formula. F _Usinle * = F _Usingle - MIN [F _Usingle ]) / (MAX [F _Usingle ] - MIN [F _Usingle ]). The illustration shows the operation chart of Part B.
Figure 11 shows a method for determining the unbinding force between C-Ag ⁺ -C / CH ⁺ C complexes using grayscale analysis. More specifically, the grayscale variation method is used to determine the unbinding voltage. Ⅰ) Noise level determined by more than 2 times the standard deviation (2σ) from the mean value (μ) in the normal Gaussian distribution. II) an unbinding voltage that appears as a continuous grayscale value beyond the noise level. The illustration showing the brightness (gray scale value) of the internal microsphere region increases with a function of the applied voltage. The bar represents the gray scale level (from 0 to 255).
12 shows a method of determining the bursting force F _U reflecting the unbinding voltage. I) is a diagram for explaining the calculation of the DEP force. The theoretical model of DEP force consists of three parametres (ε _p *; complex particle permittivity, ε _m *, complex medium permittivity, r, microsphere radius, burst voltage unbinding voltage, and finite element simulation (COMSOL Multiphysics 3.5a), which are unrolled from the electric field profile (E) Ⅱ) from the DEP force and applied voltage function to the unbinding event A histogram graph is shown.
Figure 13 shows a finite-element simulation of a DEP chip. (A) shows a schematic diagram of an interdigiated electrode for dielectrophoretic tweezers. (B) shows a side view of the DEP chip structure. (C) shows the surface gradient (gray scale level) of the electric field and the direction of the dielectrophoretic force (purple arrow) on the multiple linear electrodes implemented on the constraint element simulation.
14 shows a method of immobilizing poly-C DNA on the DEP chip surface and polystyrene microspheres. (I to III) show the process of immobilizing poly-C DNA on both surfaces (DEP chip, polystyrene microsphere). The EDC / NHS activates the carboxylic acid to form amides and the poly-C DNA contains stable amide bonds on each surface.
15 is a figure for demonstrating immobilization of poly-C DNA on polystyrene microsphere surface. SEM images and surface roughness represent microspheres (DNA) with carboxylated microspheres (bare) and immobilized poly-C DNA. The scale bar is 100 nm. The floor image represents a 3D image obtained using ImageJ software consisting of gray scale values (0 to 255) through an area spanning 1,280 x 960 pixels.
16 shows the roughness of the immobilized poly-C DNA on the DEP chip surface. AFM is an image of the root-mean-squared height (Rq) of the carboxyl functional group and the immobilized SiO ₂ surface of the poly-C DNA. The size of the image is 5 × 5 μm ² . The poly-C DNA immobilized SiO ₂ Surface roughness is increased by residual molecules such as unreacted carboxyl functional groups and activated reagents (EDC, NHS).
17 shows the results of repeated testing of the unbinding voltage (Vp-p) after the chip cleaning process in DEPFS.
Fig. 18 shows a schematic sequence diagram of a pair-fold state and a stretched state in poly-C DNA. (A) At the 3 'end of poly-C DNA, a unzipping model of poly-C DNA is immobilized on each surface (microsphere and DEP chip). (B) where the 3 'and 5' ends of each poly-C DNA are immobilized on different surfaces (microspheres and DEP chips).
Figure 19 shows the relationship between the intensity of CH ⁺ C bond and the medium conductivity at different Ag ⁺ concentrations (from 100 pM to 1 pM). To examine the change in DEP force that reflects the concentration of Ag ⁺ , we observed the average bursting power between the carboxylated microspheres and the carboxylated SiO ₂ surface in distilled water. The value of < _FU > was not influenced by medium conductivity when Ag ⁺ was lower than 100 μM. However, <F _U > increased dramatically when Ag ⁺ was 1 mM. Accordingly, the present inventors can select a measurement range of Ag ⁺ ions from 100 pM to 100 μM.
FIG. 20 shows Gaussian fits of different Ag ⁺ concentrations from the unbinding force (Fu) histogram. It can be seen that the histogram moves to the right as F _U and standard deviation increase.
Fig. 21 shows an evaluation value of a measurement limit (or limit of measurement; LOD) of the DEPFS system of the present invention. (A) Rupture force ( _FU ) histogram and Gaussian fit when Ag ⁺ is absent. (B) Mean breaking force (<F _U >) and standard deviation obtained from the DEPFS Ag ⁺ test with a semi-logarithm scale. According to Ag ⁺ is in the non-existent state <F _U> standard deviation of the blank (blank) (standard deviation, σ ) LOD (μ blank + 3 σ blank) that is three times the definition of, Ag ⁺ under the system of the present invention The LOD for the measurement is defined as ~ 300 pM.
Figure 22 shows medium conductivity on a 100 nM metal ion solution. The medium conductivity in various metal ion solvents was compared with that obtained from distilled water (3.1 μS / cm), and the <F _U > value was not affected by medium conductivity until 100 μM. Therefore, it can be seen that <F _U > is not affected by medium conductivity in 100 nM metal ion solution.
Figure 23 shows the results of CH ⁺ -C interactions between free poly-C DNA in distilled water using circular dichroism (CD). Each blue line represents 266 nm and 288 nm. At 266 nm and 288 nm, each peak represents the signal of the hydrogenated base (C ⁺ ) and the formation signal of CH ⁺ C in the DNA interbase. This means that in the same way as the previous study, a bond is formed between hydrogenated cytosine and cytosine in distilled water. In this approach, however, it is difficult to quantify the specific amount of hydrogenated cytosine in the medium.
Figure 24 shows the CH ⁺ C bond strength at different poly-C DNA concentration states on a DEP chip. (A) Rupture average force (<F _U >) obtained from a poly-C DNA concentration function immobilized on a DEP chip surface. < _FU > indicates a linear relationship with DNA molarity. (B) To determine the hydrogen interaction between the poly-C DNA coated microspheres and the surface of the poly-C DNA coated chip, the single burst force (F _Usingle ) is calculated. Poisson statistics Variation vs. theoretical model . < _FU > Graph was used. To compute F _Usingle , the DNA molecular densities were regulated by different <F _U > measurements. The red line represents a linear fit (128.1 × 10-12 × [<F _U >]) and the slope of the linear fit represents F _Usingle between poly-C / poly-C.
Figure 25 shows the interaction characteristics (the co-operative nature among the bulky molecules) between all poly-C DNAs interacting between the probe and the chip surface (Figure 9A) , it shows a graph of performing a normalization procedure <F _U> in order to compare. At this time, it was confirmed whether or not it conformed to the Hill formula applied to investigate the cooperative nature of the bond between Ag ⁺ and poly-C DNA. As a result, R-square showed a high reliability of 0.95.
Figure 26 shows the difference in cooperativity properties between a bulky molecule and a single molecule. The average unbinding force (F _{Usingle *;} black pentagonal and green dashed lines) and average rupture force (<F _U ^* >; red hexagons and red dashed lines) vs Ag ⁺ .
27 shows the result of measurement of the hydrogen bonding force according to the concentration of poly-cytosine DNA distributed on the chip surface.

Hereinafter, the present invention will be described in more detail.

The present invention

(a) functionalizing at least one surface of a substrate on which an electrode is formed, with a polynucleotide;

(b) placing the substrate of step (a) and beads functionalized with polynucleotides into a chamber;

(c) placing a test solution containing or not containing a metal in a chamber and applying a voltage to the substrate;

(d) measuring motion of the beads to obtain video signal information;

(e) obtaining a gray scale value from the video signal information obtained in step (d);

(f) detecting a moment of rupture using the gray scale value obtained in step (e); And

(g) measuring the unbinding voltage at the rupture point and obtaining a rupture force (F _U ) through a conversion process.

Although the type of the substrate of the step (a) is not particularly limited, it is appropriate to use a microfluidic chip, and the microfluidic chip may further include an insulating substrate, Although not particularly limited, the silicon dioxide (SiO _2, silica) may be a deposited silicon. On the other hand, more than one surface of the substrate can be functionalized with a polynucleotide, and more suitably, a single surface is functionalized with a polynucleotide. Meanwhile, the chamber of step (b) may be made of polydimethylsiloxane (PDMS).

On the other hand, in the above metal ion detection method, the kind of the metal is not particularly limited, but the metal is preferably selected from the group consisting of Ag, Hg, Li, Na, Fe, , And more preferably, it is preferably used for detecting silver ions and mercury ions.

When the present invention is used for ion detection in the test solution, it is appropriate to use poly-cytosine DNA as the polynucleotide. The poly-cytosine DNA is preferably composed of 15 to 32 cytosine bases, suitably 20 to 28, more preferably 22 to 26. Most preferably, it is 24.

When the present invention is used for mercury ion detection in a test solution, it is appropriate to use poly-cytosine thymine DNA as the polynucleotide. The poly-cytosine thymine DNA is 5 '- (CT) _n -3', wherein n is an integer of 3 to 9, more preferably 5'-CTCTCTCTCTC-3 '.

Meanwhile, the step of detecting the rupture moment in the step (f) may be performed by matching the change of the levitation displacement of the beads and the intensity of the gray scale value generated by applying the voltage, and if fluctuation occurs beyond the reference value And the moment is defined as the moment of rupture (see FIG. 11). And the reference value measures the fluctuation of the gray scale value according to the Gaussian distribution of [Equation 1], where the fluctuation level is represented by [mu]

[Formula 1]

In this formula,

x is a gray scale value,

mu is the average of the gray scale values,

and? is the standard deviation of the gray scale value.

On the other hand, the conversion process of the step (g) is performed using the following equation (2)

[Formula 2]

분수 계산값의 실수부만 취한 값을 의미하며, ε_p ^*은 복합체 소분자 유전율(complex particle permittivity)이며, ε_m ^*은 복합체 배지 유전율(complex medium permittivity)이고, r은 마이크로스피어 지름(microsphere radius)이며, E _rms 는 유한요소 시뮬레이션(finite element simulation, COMSOL Multiphysics 3.5a)으로부터 얻은 전기장 프로파일(electric field profile)인 것을 특징으로 하며, 상기 (g)단계의 평균 파열힘(<Fu>)값과 금속이온 농도가 비례하는 것을 이용하여, 금속이온의 존재 및 농도를 측정한다.(F _U ), where ε _m is the permittivity of the medium, and Re is the permittivity of the medium

Where ε _p ^* is the complex particle permittivity, ε _m ^* is the complex medium permittivity, r is the microsphere radius, And E _rms is an electric field profile obtained from finite element simulation (COMSOL Multiphysics 3.5a). The average rupture force (Fu) value of step (g) The presence and concentration of metal ions are measured using the fact that the ion concentration is proportional.

In addition,

(a) functionalizing at least one surface of a substrate on which an electrode is formed, with a polynucleotide;

(b) placing the substrate of step (a) and beads functionalized with polynucleotides into a chamber;

(c) placing a test solution containing or not containing a metal in a chamber and applying a voltage to the substrate;

(d) measuring motion of the beads to obtain video signal information;

(e) obtaining a gray scale value from the video signal information obtained in step (d);

(f) detecting a moment of rupture using the gray scale value obtained in step (e);

(g) measuring an unbinding voltage at a burst point and obtaining a unbinding force (Fu) through a conversion process; And

(h) Provides a method of measuring the intermolecular bonding force using the Poisson statistics and the tear force value obtained in the step (g).

In addition,

A chamber containing polynucleotide-functionalized substrates and polynucleotide-functionalized beads;

An electric field generator positioned below the chamber, the electric field generator comprising an electrode unit and applying an electric voltage to the electrode unit to form an electric field;

A camera positioned above or at one side of the chamber to image the inside of the chamber;

A measuring unit measuring a gray scale value from a video signal received from the camera and detecting a moment of rupture using a gray scale value; And

And an analyzer for measuring an unbinding voltage at a rupture point measured by the measuring unit and obtaining a rupture force (Fu (unbinding force) through a conversion process).

In the following Examples and Experimental Examples, the principles and interactions of DEFFS for measuring microfluidic chips, nucleotides, functionalization of nucleotides on chips and microspheres, and chemical interactions between metal ions and polynucleotide complexes , Poisson statistical analysis, and single bond strength analysis by statistical method, and the method of measuring bond cooperativity between metal and polynucleotide using Hill formula.

As a result, the present invention provides a new approach for measuring metal ions in a test substance, and a method for detecting metal ions and a method for measuring metal ion concentration using the dielectrophoretic technique of the present invention is characterized by a binding force between a polynucleotide and a specific metal ion, By using the statistical analysis method developed by the inventors of the present invention, the presence and concentration of metal ions in the test solution can be measured simply and accurately, with high sensitivity, have.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. However, the scope of the present invention is not limited thereto.

< Example  1> Microfluidic chip ( microfludic  chip manufacturing

An interdigitated electrode array pattern (40 μm wide and 10 μm apart) was fabricated on an oxidized silicon substrate (i-Nexus, Seongnam, Republic of Korea) using photolithography. Specifically, a thick chromium electrode array pattern of 1000 angstroms thick was deposited on a wafer using a thermal deposition technique and a standard lift-off process, and then the metal electrode was subjected to a thick PECVD (plasma-enhanced chemical vapor deposited silicon dioxide. A plan view of the structure is shown in FIGS. 3 D to F, and a schematic cross-sectional view of the chip is shown in FIG.

< Example  2> Oligo Nucleotide  Produce

Poly-C DNA was commercially obtained (Cosmogentech, Seoul, Rupublic of Korea). The poly-C DNA has the following sequence: 24-base poly-C DNA (5'-CCC CCCCCC CCC CCC CCC CCC CCC-3 '- (CH ₂ ) ₆ -NH ₂ and 6-FAM-5 '-CCC CCC CCC CCC CCC CCC CCC-3' - (CH ₂ ) ₆ -NH ₂ ); 12-base poly-C DNA (5'-CCC CCC CCCCCC-3 '- (CH ₂ ) ₆ -NH ₂ ); 6-base poly -C DNA (5'-CCC CCC- 3 '- (CH 2) 6 -NH 2). All oligonucleotides were synthesized at high pressure liquid chromatographic purity and used to functionalize DEP chip surfaces and microspheres.

< Example  3> Poly Microfluidic device using DNA Microfluidic  Device Functionalization

The surface of the microfluidic device prepared in Example 1 was functionalized as follows: 3-triethoxysilylpropyl succinic anhydride (TESPSA) was treated to form a carboxyl-terminated oxide layer The poly-C DNA was immobilized on the surface of the device. A silicon dioxide chip was first transferred to a solution composed of H ₂ SO ₄ -H ₂ O ₂ (1: 2) to produce a hydroxyl functionalized substrate (SiO ₂ -OH) . The hydroxyl functionalized substrate was then immersed in an organic solvent (toluene, 99.8%) containing 100 mM TEPSA (Gelest, Morrisville, PA) overnight (SiO ₂ -COOH). The carboxylated substrate was washed with three different solvents (toluene, N, N-dimethylformamide (DMF, Sigma-Aldrich, St. Louis, MO; 99.9% HPLC grade and distilled water) The carboxylated substrate was incubated with 65 mM N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride (EDC; Sigma-Aldrich, 99%) and 108 mM N-hydroxysuccinimide (Gibco, Gaithersburg, MD) solution containing 0.01 M PBS (Sigma-Aldrich, 98%) for 1 hour at pH 7.2 and then forming amide bond after each addition of the 200, 400 and 800 nM poly -C DNA solution into the solution in order to kept overnight. (SiO ₂ - poly -C DNA) -C poly after fixing the DNA, the DEP chip substrate excess PBS Rinsed with buffer solution and distilled water and dried with nitrogen gas. All the above samples were prepared at 22 캜.

< Example  4> Immobilized Poly Having -C DNA Microsphere  making

To prepare the microspheres immobilized with poly-C DNA, the carboxylated polystyrene microspheres 3 mg / mL (Kisker, Steinfurt, Germany; 15 μm) were mixed with 6.25 mg 99% EDC and 6.25 mg 98% NHS In PBS (Gibco; 0.01 M, pH 7.2) for 1 hour. Next, the reaction mixture was reacted with 1 μM poly-C DNA overnight to prepare poly-C DNA-immobilized microspheres, vortexed and washed five times in PBS with a centrifuge. The microspheres were diluted with distilled water (Gibco, Gaithersburg, MD) or drinking water (Jeju Samdasoo, JPDC, and Republic of Korea) and centrifuged before use. The preparation of all the samples was carried out at 22 ° C.

< Example  5> Fluorescence Microscopy, Scanning Electron Microscopy, SEM ) And Atomic Force Microscopy (AFM) functionalized chips and microsphere characterization methods

Poly-C DNA-immobilized microspheres and DEP chips were washed with PBS and distilled water prior to analysis. The surface of the microspheres and the plasma enhanced chemical vapor deposition (PECVD) oxide were examined with a fluorescence microscope (BX60, Olympus, Tokyo, Japan) using a WB filter. FE SEM (Field Emmission SEM) (JSM-7001F, JEOL, Tokyo, Japan) was used to observe the morphology and composition of the polystyrene microsphere surface with poly-C DNA and carboxyl functional groups.

Poly-C DNA-Immobilized Microspheres and Carboxylated Polystyrene Microspheres are nonconductive materials, so the samples were pretreated with platinum ion deposition to protect them from electron damage. The difference between the top views of the SEM images was analyzed using Image J (Fig. 15). AFM measurements were performed in air using multimode V (Veeco, Santa Barbara, Calif.) And images obtained from them were further analyzed using (5 × 5 μm ² ) Nanoscope software (Bruker). In addition, the AFM height image of a DEP chip substrate with or without poly-C DNA was quantified against the surface roughness defined as the root mean square (root mean square) value.

Specifically, R _q is a root mean square. Specifically, the height of the surface is individually measured using AFM, the average value of the measured height is obtained, the deviation between the average value and the measured value is measured, and the deviation is squared And the average value of the values divided by the total number of measurements.

n is the total number of measured samples (= height)

y _i is the i ^th sample measurement value - the average value of the sample measurement value.

< Example  6> C- Ag ⁺ -C / C-H ⁺ Method of measuring unbinding force of -C composite

The poly-C DNA was placed in contact with a 6 × 6 × 1.2 mm ³ polydimethylsiloxane (PDMS) chamber with a 3 mm diameter hole at the midpoint of the functionalized DEP chip surface. Specifically, a microsphere mixture (poly-C DNA: metal ion = 9: 1; 10 μL) having immobilized poly-C DNA with metal ions was placed in the PDMS chamber. All metal ion solutions were used in the form of metal nitrate (AgNO ₃ , LiNO ₃ , NaNO ₃ , Hg (NO ₃ ) ₂ , Zn (NO ₃ ) ₂ , and Fe (NO ₃ ) ₃ ; Sigma-Aldrich). Voltage was applied to electrodes of the microfluidic chip using a WMA 300 amplifier (Falco Systems, Amsterdam, The Netherlands) connected to a 33250 function generator (Agilent Technologies, Santa Clara, Calif.) And an oscilloscope (WaveRunner 6050, LeCroy , New York, NY). The movement of the bead was then recorded using a top-view charge coupled camera (Motionscope M3, Redlake, San Diego, Calif.). The intermolecular interaction tear force (F _U ) was quantified by using a combination of the gray scale variation method and the DEP force map obtained from finite element simulation (FES). In summary, in order to determine the point of rupture of the microspheres (probe) from the chip substrate surface, we used the grayscale value of the interior area of each microsphere of the planar visual image as the function value of the applied voltage.

The more detailed DEPFS procedure for measuring the unbinding force is as follows. To determine the rupture force between silver ions and oligonucleotides using the DEPFS system, it is important to determine the rupture displacement of the microsphere that is the subject of the levitation force by DEP.

The grayscale variation method predicts the microsphere lifting moment by matching the change in levitation displacement and intensity of the gray scale value. As the applied voltage in the chip electrode increases, that is, as the DEP forces affecting the microspheres increase, levitation displacement of the beads occurs. The low-speed image analysis of the top image shows that the intensity of the gray scale value of the inner area in each microsphere increases as the DEP force is levitated. Through these methods, it is possible to obtain the rupture variations of the microspheres immobilized with biomolecules chemophysically interacting with other biomolecules immobilized on a chip substrate. In previous studies, the fluctuation of the gray scale value of the internal microsphere region was measured for 60 seconds, and the variation follows a Gaussian distribution as follows:

x denotes a gray scale value, μ denotes an average of the gray scale values, and σ denotes the standard deviation of the gray scale values. I in Fig. 11 shows a typical example of a microsphere with a Gaussian distribution tendency at μ = 109.15, σ = 0.45. This variation of microspheres is affected by thermal fluctuations of particles according to thermodynamic theory and is caused by intermolecular interactions with some molecules on the chip surface (ie non-covalent bonding). In conclusion, the present inventors have defined a fluctuation level as a boundary, expressed as μ ± 2σ (μ, σ is a Gaussian distribution parameter) where the gray scale level of the microsphere does not exceed the fluctuated region . If the degree of fluctuation of the microspheres is changed by an external force, the force can be defined as a rupture force in a specific experimental condition (II in FIG. 11).

As the microspheres move upwards from the chip surface, each microsphere is out of focus (i.e., the chip surface ground) to match the gray scale value of the microspheres in the top view (Figures 11A-11) And began to become cloudy. Therefore, the present inventors can find the burst point (moving point) by comparing the gray scale value as the applied voltage increases while exceeding the variation level. This rupture occurred simultaneously in all the beads in the chip, and we were able to track up to 400 beads in the CCD camera system with three independent measurements.

The inventors measured each applied voltage that caused the rupture in each microsphere and converted it to a DEP unbinding force (Figure 12). In the conversion process, the inventors used a custombuilt Matlab encoded with measurements of some parameters (eg, ε _p ^* , ε _m ^* , r and E ) obtained while performing FEA (finite element analysis) with COMSOL Multiphysics. Each of the above processes has been developed by the present inventors, and a more detailed process will be described below. In order to obtain a definite silver ion concentration, ~400 different F _U values showing the normalization distribution were obtained (Fig. 12, II). By fitting to the Gaussian curve, the average unbinding force (F _U ) was obtained.

DEP force is presented as follows.

Where n is the force order, K _n is the n ^th -order Clausis-Mossotti factor, φ is the electrostatic potential of the external electric field, ε _p : particle dielectric constant, ε _m : medium permittivity, and r: particle radius. A finite element program (COMSOL), which generates an electric field profile on a 40 ㎛ wide IDT electrode structure separated by 10 μm when an AC signal of 1 V _{peak -to-peak and} 1 MHz is applied to the electrode, Matlab (R12, Mathworks) was developed for measuring total DEP force using multiphysics and the above formula, and the dielectrophoretic force was calculated using a function of the applied voltage using Matlab (Fig. 13). Since the quality of the FEM analysis may be affected by the resolution of finite element convergence, such as the quality of the mesh, the present inventors have previously found that the quality of the mesh quality ) And the sampling ratio as the function of the FEM analysis. Based on this technique, a vertical negative DEP force profile was evaluated at different voltages.

< Example  7> Calibration of DNA chip

Even when producing DEP chips in the same MEMS fabrication process, chips often exhibit performance differences. Therefore, the present inventors checked the quality of the DEP chip before using the DEP chip. The chip performance quality check result is shown in Fig. In this case, a well-cleaned chip with similar performance was selected as the result of measuring the bursting voltage of the carboxylated microspheres. The unbinding voltage was 1.002 Vp-p, which was 46.51 pN in the van der Waals interaction range, DEP (Cf., gravity of microsphere (~ 0.1 pN)).

< Experimental Example  1> Ag ⁺ / H ⁺ Poly -C DNA complexes Of DEPFS  principle

The goal of the present inventors is to provide C-Ag ⁺ -C / CH ⁺ C forces as a function of concentration of Ag ⁺ using a negative DEP (nDEP) force on a microsphere (probe) containing functionalized poly- (Figs. 2 and 3). Ag ⁺ / H ⁺ poly for measuring the mutual inter-molecular DNA of -C complex action, the inventors were functionalized hundreds of polystyrene microspheres with DEP chip substrate (SiO ₂₎ in the poly -C DNA (Figs. 2 and 14 ). The microspheres interact with the poly-C DNA functionalized chip surface in the presence or absence of Ag ⁺ . As a result, intermolecular interaction occurs between the two surfaces (i.e., the microspheres and the chip substrate). In addition, the present inventors used an unzipping mode as shown in FIG. 18 because it immobilizes poly-C DNA on two different surfaces (i.e., microspheres and DEP chips) using a 3 'amine group. Was found to be more common than shear mode.

As the DEP force increases, the interaction is broken by the upward movement of the microspheres with immobilized poly-C DNA from the chip substrate. This makes it possible to directly measure intermolecular forces (ruptures) between poly ⁺ C DNA mediated by Ag ⁺ and H ⁺ . In particular, the microspheres are randomly dispersed on a silicon dioxide film on an interdigitated (IDT) electrode in a microfluidic chamber. When an AC voltage _{(48 V p -p, 1 MHz} ) applied to the IDP (Interdigitated) electrode, and the microspheres are arrayed becomes (FIG. 3B), then interacts with the Ag ⁺ along the center of the electrode by the power nDEP , C-Ag ⁺ -C and CH ⁺ C complex (FIG. 3C). Further, the higher voltage is applied to the _{IDP (~122 V p -p, 1} MHz) electrode can break the complex when the microspheres to be perpendicular to the support in the center electrode. The movement and movement of the microspheres was assessed using an optical microscope image and the brightness of the microspheres was determined by using a grayscale method to determine the defocusing depth to be quantified to determine the rupture force of the composite ) (Fig. 3D-F). &Lt; tb &gt;&lt; TABLE &gt;

< Experimental Example  2> Immobilized Poly - DEP chip substrate with DNA Microsphere  Identify features

In order to obtain poly-C DNA-immobilized microspheres and DNA-coated chips as disclosed in Examples 3 and 4, amino-labeled poly-C DNA was immobilized on a carboxyl group Functionalized polystyrene microspheres and also a silicon dioxide substrate surface of a DEP chip functionalized with a carboxyl group were immobilized through peptide bonds (FIGS. 4 and 14). Fabrication of the microsphere and DEP chip substrate was confirmed by fluorescent microscopy (Fig. 5A, B). Meanwhile, fluorescence-labeled oligonucleotides were used to confirm whether the functionalization protocol proceeded well. Also, poly-C DNA labeled with fluorescence (6-FAM) was used to confirm functionalization of oligonucleotides on the DEP chip surface as well as probe beads (Fig. 14).

However, pure oligonucleotide sequences were used instead of fluorescence-labeled oligonucleotides in performing silver ion detection using DEPFS. This is to avoid experimental errors induced by dye molecules (ie, 6-FAM) in force measurements. Further experiments (SEM, AFM) showed the distribution of fixed poly-C DNA without fluorescent dye. Microspheres and DNA-coated chips with immobilized poly-C DNA were also examined using SEM and AFM, respectively (FIGS. 6 and 7). Since the AFM analysis of the microsphere surface changes due to the curvature and wobbling (i.e., rocking motion) of the microspheres, it is possible to use not only the shape of the microspheres to which the poly- The poly-C DNA surface was observed where the poly-C DNA was observed in numerous small grains. The results are consistent with previous reported results that the tendency of granularity increases when poly-C DNA is present. In contrast, it was confirmed that the control sample (carboxylated polystyrene microsphere) exhibits a relatively flat surface (Fig. 6B, Fig. 15).

In addition, AFM was used to detect precise topographic changes of the chip surface where biomolecules (poly-C DNA) were functionalized. In order to quantify the change of the topographic outline, the surface roughness histogram was compared before and after the poly-C DNA immobilization step. Because the oligonucleotide (size) is larger than the carboxyl functional group, the height distribution of the surface on which the DNA is immobilized is higher. The height difference on this histogram shows a large shift from left to right in equivalent AFM image conditions (FIG. 7). In general, the surface roughness is higher when DNA-immobilized than when no DNA is present, and the histogram of the topographic height shows a wider width (Fig. 16). Through these various microscopic observation analyzes, the present inventors confirmed the production method. However, this analysis does not guarantee the stability of the microspheres with immobilized poly-C DNA in the system. In addition, tensile forces or intermolecular interactions between DNAs are dependent not only on DNA length but also on coorinate interactions caused by the interaction between temperature or pH, hydrogen and Ag ⁺ , H ⁺ and poly-C DNA Dependence on the same microenvironment is well known. Therefore, the present inventors have established an optimized method as shown in <Experimental Example 3>.

< Experimental Example  3> Ag ⁺ / H ⁺ Poly -C for interaction between DNA complexes Microsphere  optimization

It is well known that hydrogen bonding occurs between cytosine and hemiprotonated cytosine (CH ⁺ C) under weak acidic conditions (pH 5). In addition, when Ag ⁺ is present in aqueous solution, the CH ⁺ -C hydrogen bond can be replaced by a coordination bond of Ag ⁺ via cross-linking between the respective nitrogen atoms of the cytosine (i.e., C-Ag ⁺ -C being). For this reason, in order to more accurately measure the interaction between Ag ⁺ / H ⁺ poly-C DNA complexes, it is necessary to make an optimal microsphere immobilized with poly-C DNA, and thus to optimize the length (L) It is necessary.

Specifically, for optimization of the microspheres with immobilized poly-C DNA, we used DEPFS to functionalize with different lengths (L) of poly-C DNA in the presence or absence of Ag ⁺ in distilled water (pH 5.4) the F _U was measured between the chip surface and the microspheres. As a result, a larger F _U was obtained as L increased. As L increases, the number of cytosine bases capable of forming CH ⁺ -C increases between the microspheres and the poly-C DNA on each surface of the chip in the absence of Ag ⁺ , and thus the binding of CH ⁺ (Fig. 8A, B). In the presence of Ag ⁺ , the F _U value is larger than when Ag ⁺ is absent, because the coordination interaction is higher than the hydrogen interaction. This indicates that poly -C ₆ DNA as a result, lead to a very small value F _U. This is probably due to the thermodynamic safety of DNA hybridization in the case of short double-stranded DNA at room temperature, and this instability can be stabilized by coordination formation due to the introduction of Ag ⁺ (Fig. 8B).

The measured Fu values are summarized in FIG. 8C as the average unbinding force (< F _U &gt;) based on the Gaussian pit can be summarized as F _U. <F _U> The difference between (S _F) appeared most strongly at ₂₄ C (Fig. 8D) when the non-existence when the Ag ⁺ present. Thus, it was confirmed that 24-base poly-C DNA can detect 10 μM Ag ⁺ . Therefore, the present inventors performed the following experiments with poly-C ₂₄ DNA.

< Experimental Example  4> Ag ⁺ / H ⁺ Poly -C Interaction between DNA complexes

To measure interactions between Ag ⁺ / H ⁺ poly-C DNA complexes mediated by hydrogen (CH ⁺ C) and coordination (C-Ag ⁺ -C) interactions using DEPFS, having a -C DNA was introduced into the Ag ⁺ into the microfluidic chip between the microspheres and the chip surface. Prior to the interaction measurements, the conductivity of the medium containing various Ag ⁺ concentrations (100 pM - 1 mM) was measured, since the medium conductivity is an important parameter in determining the dielectrophoretic force. On the other hand, except for the highest value (1 mM), the measured Ag ⁺ concentration was not significantly influenced by the medium conductivity by the conductivity value, the dielectrophoretic force value (F _MU ) (FIG. 19). Specifically, the effect of Ag ⁺ concentration on the medium conductivity was verified by measuring <F _U > at different Ag ⁺ concentrations using carboxylated microspheres and negatively charged surfaces. In conclusion, the interaction of Ag ⁺ / H ⁺ poly-C of DNA complexes was measured in the range of Ag ⁺ 100 pM to 100 μM using microspheres and chips immobilized on the surface of poly-C DNA, Is shown in Fig. In addition, it was confirmed that the mutual <F _U> is Ag ⁺ concentration increases from 12.51 ± 1.33 to 35.75 ± 3.20 nN nN of action (Fig. 20).

< Experimental Example  5> DEPFS  When using Ag ⁺ The detection capability of

In order to analyze the detection power of Ag ⁺ using DEPFS, standard deviation and mean bursting force (< F _U &gt;) were expressed using F _U data (FIG. 9A). As shown in FIG. 9A, it was confirmed that as the Ag ⁺ concentration increases on the semi-logarithm scale, < F _U > increases proportionally. More than 400 microspheres with poly-C DNA immobilized on multiple DEP chips were used for this analysis and this assay demonstrated high reliability (P <0.0001) and very short detection time (<3 min) . The limit of detection of the concentration is up to ~ 300 pM (Figure 21), which is a tripping voltammetry at chemically modified electrode (~ 2 pM) and inductively coupled plasma- mass spectrometry (~ 6 pM).

In addition, ^{Zn 2 +, Li +, Na} +, Hg 2 + and was used a variety of metal ions (100 nM) containing Fe ^{3 +} evaluate the ion selectivity (Fig. 9B). To quantify the measurement capability of DEPES of the present invention, the polyester was calculated -C DNA with Ag ⁺ or other cations (X) of two selected equilibrium coefficient is defined as the ratio constant (Selectivity coefficient) between any one of (ξ) . (I.e.,? = K _{DNA, X} / K _DNA , Ag ⁺ ),

In order to calculate the selection factor ( xi ), the inventors have defined the equilibrium constant (K) in the DEPFS system of the present invention as follows.

Where the equilibrium constant (K _{DNA, X} ) is defined as the ratio of the force between the poly-C-DNA and the complex form (poly-C DNA, X with metal ions, X) F _DNA And F _{DNA- X} . The kind of metal ion ^{(i.e., Ag +, Li +, Na} +, Fe 3 +, Hg 2 +, Zn 2 + , etc.) in the less metal ions (X) between the same present in the bare beads and DEP chip surface at a concentration of The combined force averages are considered to be equivalent. Therefore, the above formula can be simplified as K _{DNA, X} ? F _{DNA- X} / F _DNA in the same F _DNA (~ 11.22 pN) in the system of the present invention (FIG. Thus, K _DNAX is directly proportional to F _{DNA- X} , and the selectivity between two different metal ions is defined as the ratio of two equilibrium constants ( K _{DNA, X1} and K _{DNA, X2} ).

For example, based on the definition as mentioned above, the selection factor between silver ions and lithium ions can be measured to be approximately 0.6 (~ 14 pN / 23 pN) . Thus, it has been confirmed that Ag ⁺ is at least 25% more selective than other cations on the DEPFS system of the present invention.

X1 Ag ⁺ X2 Ag ⁺ Li ⁺ Na ⁺ Hg ⁺ Zn ^{2 +} Fe ^{3 +} ξ 1,000 0.637 0.516 0.557 0.741 0.673

Therefore, it was confirmed that Ag ⁺ shows excellent selectivity on the DEPFS system of the present invention by at least 25% as compared with other cations. This proved that Ag ⁺ capture has good selectivity when compared to other metal ions.

< Experimental Example  6> Within drinking water Ag ⁺ Concentration measurement Check whether

Using the method of the present invention, it was confirmed whether Ag ⁺ concentration could be measured in general drinking water (Jeju Samdasoo, JPDC, Korea), and specifically, various concentrations of AgNO ₃ (1-100 μm) in drinking water were analyzed. As shown in FIG. 9C, the <F _U > value in drinking water was about 20 nN lower than in distilled water. Nonetheless, the linear increase of <F _U > in drinking water according to [Ag ⁺ ] indicates that DEPFS measures the Ag ⁺ in the environmental water based on the sensitive requirement (46 μM) of the standard drinking water standard set forth in the US Environmental Protection Convention Which is suitable for practical applications. In addition, also the linearity of <F _U> of 9A is Ag ^+, interaction between H ⁺ and poly -C this Ag ⁺ / H ⁺ poly -C DNA complexes hydrogen (CH ⁺ -C) and coordinate (C-Ag ⁺ C) interactions between the two systems.

< Experimental Example 7> Poisson  Using statistical analysis Ag ⁺ / H ⁺ Poly -C bond-rupture force measurements in the formation of DNA complexes

We used the same < _FU > measurement data and used Poisson statistics to determine the respective bond-rupture force (ie, poly-Si) of the Ag ⁺ / H ⁺ C DNA rupture force) was calculated.

The method for calculating each combination using the Poisson statistical method is as follows.

For Poisson statistical analysis, each bond is an independent, discrete random variable, and the rupture force between the probe and the surface is defined as the sum of each individual bond (Williams, JM; Han, T .; Beebe, TP Langmuir 1996, 12, 1291-1295 / Williams, DH, Stephens, E .; Zhou, M. Journal of Molecular Biology 2003, 329, 389-399). This assumption is reasonable when considering previous studies. These statistical methods are used to calculate each combination using mean power and standard deviation derived from statistically reliable measurement data. According to Poisson statistical analysis, the distribution is the ratio of observing a random sample of n discrete events (ie, the n event for the rupture of each resulting bond), and the unbinding force And the Poisson distribution of the mean (m) and variance (σ ² ).

m = nF,

σ = nF ^{2 ,}

Within the fixed contact area, F and n individually represent the respective coupling forces and the number of combinations. The individual coupling forces can be expressed as:

F = σ ² / m .

In the present invention, each of the Ag ⁺ -poly-C DNA complexes is analyzed using the mean power and standard deviation derived from the measurement of more than 400 microsphere probes simultaneously under the same environment to obtain a high statistical reliability (P < 0.0001) The results are shown in Fig. In addition, the measurement data of the poly-C DNA complex in the state where Ag ⁺ is absent and the average force and standard deviation calculated using each bond-rupture are shown in Fig.

From the statistical point of view, it can be inferred that the loading force of the contact area is equivalent to all possible internal molecules, since the contact area between the microsphere and the electrode surface has a symmetrical geometry at the contact area midpoint. Even though the molecular long loaded forces of the contact zone vary, the average loaded forces are maintained at constant loading values.

< Experimental Example  8> Single bond strength analysis by statistical method

The inventors also measured the average force presented by each combined value and analyzed the single binding force using a statistical method from the mean power. Single bonds were analyzed by the Poisson method, although there were various mixed constituents of hydrogen and chelate bonds between individual molecules. The analyzed single bond was expressed as the average bond probability of the poly-C DNA complex mediated by the metal ion from all possible binding components. Reflecting the Poisson statistical model, a single tear force means a single molecule (ie, a pair of DNA). As shown in FIG. 10A, the respective bond-breaking force, F _Usingle , increased from 139 to 296 pN as the Ag ⁺ concentration increased. This S-shaped curve is consistent with previous studies using DNA-Ag ⁺ interactions (C-Ag ⁺ -C), and the results are not present when each internal intermolecular bond is composed of the same kind.

In addition, in the presence of heme hydrogenated cytosine (CH ⁺ C) hydrogen bond under weak acid conditions (pH 5), the respective bond-tear strength of the poly-C-DNA complex in the absence of Ag ⁺ Even at various concentrations of the complex, at 128 pN (Fig. 24). Therefore, as shown in Fig. 10A F _Usingle C-DNA complexes were affected by the degree of heterogeneity of Ag ⁺ , CH ⁺ -C and C-Ag ⁺ -C duplexes on the Ag ⁺ , poly-C DNA complexes. Under weak acidic conditions, Stranded duplex with other poly-C DNA through hydrogen bonding.

Meanwhile, the base stacking interaction is another factor that may affect the bond-rupture force depending on the degree of heterogeneity. However, according to recent AFM force spectroscopy studies, the strength of single- 2 pN, it was found that the bonding-rupture force had a slight influence on the rupture force. From the results of the above study and the present inventors, it was found that the bond-rupture force measured in the above experiment was higher than that of the base stacking Is a more common phenomenon.

Specifically, a single poly -C in mutual engagement side between the DNA, the low Ag ⁺ concentration range (0.1, 1 and 10 nM) in the poly-C -C DNA Ag ⁺ -C bonds are hardly generated Ag ⁺ ions But did not actively interact with poly-C DNA. On the other hand, in the high Ag ⁺ concentration range (eg, 1, 10, and 100 μM), it was confirmed that C-Ag ⁺ -C was formed in the Ag ⁺ / H ⁺ -poly-C-DNA complex. For example, the F _Usingle value at 1 nM Ag ⁺ was about 139 pN, which is very similar to the rupture force of each hydrogen bond (~128 pN). On the other hand, F _Usingle at 100 μM Ag ⁺ is about 2.1 times greater than the value obtained at low Ag ⁺ concentration. Also, in the high Ag ⁺ concentration range, the F _Usingle value was found to saturate.

< Experimental Example  9> Using the Hill equation Ag ⁺ Wow Poly -C DNA between Combined  Binding cooperativity ) Calculation

To further analyze the results of the calculations, we use the Hill equation, which is used to investigate more cooperativity than two different binding components, to determine the binding cooperativity between Ag ⁺ and poly-C DNA Respectively.

As a result, it was found that the relative normalization value obtained after the measurement calculation agrees with the Hill formula (FIG. 10B), and the Hill formula was applied to the normalized F _Usingle (that is, F _Usingle ^* ) (FIG. According to existing theories of co-ordination between biomolecules, the binding relevance is specified by the separation constant (K _d ) and the Hill co-operative coefficient (n) in the Hill formula (R ² = 0.99). The K _d and n values were 87.6 nM and 0.962, respectively, and the C-Ag ⁺ -C interaction had higher specificity than the nonspecific metal-ligand correlation found in other previous studies. In addition, the Hill co-operation coefficient (n) was close to 1, indicating that even if a C-Ag ⁺ -C bond is formed in the poly-C DNA, another C-Ag ⁺ (No cooperativity). The results are consistent with previous studies in which isoelectric titration calorimetry was used to analyze the binding of Ag ⁺ to the C: C base pair, especially at a molar ratio of 1: 1.

On the other hand, the S curve (FIG. 10A) shows a mixed ratio of hydrogen bonds (CH ⁺ C) and coordinate bonds (C-Ag ⁺ C) during the interaction between Ag ⁺ , H ⁺ And the effect of the In other words, at low Ag ⁺ concentration, the formation of hydrogen bonds (ie, CH ⁺ C formation) predominates and interactions between Ag ⁺ and poly-C DNA rarely occur, but as the Ag ⁺ concentration increases, _{^{i.e., K d 1 / n = 46.1}} nM) since the presence Ag ⁺ - was found to change to a stronger binding type that is managed by the linked coordinate bond (Ag + -mediated coordinate bonds). Although poly -C DNA and Ag ⁺ binding characteristics without the basis of the cooperativity (no cooperativity), this type of change in the (CH ⁺ C → -C-Ag ⁺ -C) Ag ⁺ concentration in the acid, as well as the measurement medium (medium) And the total amount of hydrogen bonds according to the degree of hydrogen bonding. On the other hand, the interaction characteristics (the co-operative nature among the bulky molecules) between all poly-C DNAs interacting with the probe surface and the chip surface with Ag ⁺ it was confirmed that there is a characteristic difference (<F _U>, the linear shape vs Figure 10B _Usingle F, S-shape of Fig. 9A). Thus, in order to confirm the co-operative properties between bulky molecules, we confirmed that it conforms to the Hill equation, which is used for observing the combined co-operative properties between Ag ⁺ and poly-C DNA. 10B, R-square was 0.95, indicating a relatively high reliability. However, the single and mean _nonbonding forces of the Ag ⁺ -poly-C composites have different cooperative constants (F _Usingle ^* , 0.962; <F _U ^* >, 0.367). This is similar to previous studies in which the interaction between a specific particle (Ag ^{+ in} this study) and a single particle (corresponding to a poly-C DNA in this study) and the interaction characteristics between bulky molecules are different. Therefore, it was confirmed that binding cooperativity decreases with decreasing binding force.

< Experimental Example  10> Poly Using CT DNA and DEP force, Hg ⁺  Identification of ion detectability

In order to confirm whether the specific binding force between nucleobase and mercury ion (Hg ²⁺ ) can be measured in DEPFS (Dielectrophoretic-tweezers based force spectroscopy) system, the specific coordination between thymine nucleobase and mercury ion CTCTCTCTCTT-3 'as a polynucleotide and dividing the poly-CT DNA into a microsphere and a DEP chip using a binding (T-Hg ^{2 +} -T) characteristic in a weakly acidic solution (distilled water) The specific binding force between the nucleobase and the mercury ion was confirmed. More specifically, each unbinding voltage was observed when Hg ^{2 +} ion concentration was 0 M and 1 μM using 10 μm Spherotech beads as the beads. In the same manner as in Example 6, the DEP rupture force (DEP rupture force) was measured (Fig. 27).

As a result, as shown in Fig. 27, it was confirmed that the binding force between the poly-CT DNA was reduced by about 50% as compared with the poly-C DNA of the same nucleating base 12. It was confirmed that the bonding force to be combined CH ⁺ -C between poly -CT DNA decreases by about 50% as a result of a decrease of 50% cytosine cytosine than the number used in the previous studies on the use form, Hg ^{+ 2} is As a result, it was found that the binding force was increased with the increase of the thymine nucleus forming selective coordination bond with Hg ^{2 +} ion. As a result, the detection of binding force with Ag ⁺ ions as well as the detection of binding force with Hg ^{2 +} ions and the feasibility of implementing the corresponding analytical model were confirmed using poly-CT DNA.

In conclusion, the present inventors have found that by using DEP-based force spectroscopy, a simple, robust, and mass spectrometric method, Ag ⁺ and H ⁺ are detected by intermolecular interaction as well as Ag ⁺ detection and high sensitivity over a wide concentration range A new approach to statistical analysis of interactions between poly-C DNA duplexes has been demonstrated. Using this approach, the rupture force (F _U ) of binding interactions between hydrogen and coordination bonds in the Ag ⁺ - poly-C complex was quantified and the optimal length of the poly-C DNA to detect distilled water and Ag ⁺ . We have also demonstrated the interaction of Ag ⁺ / H ⁺ -poly-C DNA complexes composed of CH ⁺ C and C-Ag ⁺ C duplexes using equivalent _FU measurement data, The binding cohesiveness of Ag ⁺ and poly-C DNA was measured by Poisson's statistical method. The present inventors does not exist, the Ag ⁺ poly -C DNA interactions CH ⁺ -C had to us to be dependent, on the other hand the Ag ⁺ and poly CH ⁺ -C formed as the Ag ⁺ concentration increases -C DNA C ^& lt ^{; /} RTI &gt; The present inventors quantified the weak chelation of the polynucleotide and Ag ^{+ in} the disposable microfluidic chip to demonstrate the usefulness and validity of DEPFS as a unique force spectroscopy technique.

Claims (28)

(a) functionalizing at least one surface of a substrate on which an electrode is formed, with a polynucleotide;
(b) placing the substrate of step (a) and beads functionalized with polynucleotides into a chamber;
(c) placing a test solution containing or not containing a metal in a chamber and applying a voltage to the substrate;
(d) measuring motion of the beads to obtain video signal information;
(e) obtaining a gray scale value from the video signal information obtained in step (d);
(f) detecting a moment of rupture using the gray scale value obtained in step (e); And
(g) measuring the unbinding voltage at the rupture point, and obtaining a rupture force (F _U ) through a conversion process.
The method according to claim 1,
Wherein the substrate of step (a) is a microfluidic chip.
3. The method of claim 2,
Wherein the microfluidic chip further comprises an insulating substrate.
The method of claim 3,
The insulating substrate is a metal ion detecting method, characterized in that the silicon dioxide (SiO _2, silica) of the deposited silicon.
The method according to claim 1,
Wherein the chamber of step (b) is made of polydimethylsiloxane (PDMS).
The method according to claim 1,
Wherein the metal is at least one selected from the group consisting of silver (Ag), mercury (Hg), lithium (Li), sodium (Na), iron (Fe) .
The method according to claim 1, wherein the metal ion is silver ion, and the polynucleotide is poly-cytosine DNA.
8. The method of claim 7,
Wherein the poly-cytosine DNA comprises 15 to 32 cytosine bases.
The method according to claim 1,
Wherein the metal ion is a mercury ion and the polynucleotide is poly-CytosineThymine DNA.
The method of claim 9, wherein the poly-cytosine thymine DNA is 5 '- (CT) _n -3', wherein n is an integer from 3 to 9.
10. The method of claim 9, wherein the poly-cytosine thymine DNA is 5'-CTCTCTCTCTCT-3 '.
The method according to claim 1,
The step (f) matches the change of the levitation displacement of the beads caused by application of the voltage and the change of the intensity of the gray scale value, and defines the instant when the fluctuation occurs beyond the reference value as the moment of rupture Wherein the metal ions are detected by a detector.
13. The method of claim 12, wherein the reference value is.
Measuring the fluctuation of the gray scale value according to the Gaussian distribution of [Equation 1] below,
The fluctuation level is represented by μ ± 2σ,
[Formula 1]

In this formula,
x is a gray scale value,
mu is the average of the gray scale values,
and? is the standard deviation of the gray scale value.
The method according to claim 1,
The conversion process of step (g) is performed using the following formula 2,
[Formula 2]

Through the conversion process, F _u (unbinding force) is obtained,
In this formula,
ε _m is the permittivity of the medium,
Re is the unbinding force,
epsilon _p ^* is the complex complex permittivity,
epsilon _m ^* is the complex medium permittivity,
r is the microsphere radius,
Wherein E _rms is an electric field profile obtained from finite element simulation (COMSOL Multiphysics 3.5a).
The method according to claim 1,
Wherein the average rupture force (< F _U ) value of step (g) is proportional to the metal ion concentration.
(a) functionalizing at least one surface of a substrate on which an electrode is formed, with a polynucleotide;
(b) placing the substrate of step (a) and beads functionalized with polynucleotides into a chamber;
(c) placing a test solution containing or not containing a metal in a chamber and applying a voltage to the substrate;
(d) measuring motion of the beads to obtain video signal information;
(e) obtaining a gray scale value from the video signal information obtained in step (d);
(f) detecting a moment of rupture using the gray scale value obtained in step (e);
(g) measuring an unbinding voltage at a rupture point and obtaining an unbinding force (F _U ) through a conversion process; And
(h) Measuring the intermolecular binding force using Poisson statistics and the tear force value obtained in step (g).
17. The method of claim 16, wherein the Poisson statistical analysis method of step (h) is performed using Equation (2) below,
[Formula 2]
m = nF, σ = ² nF
F = ? ² / m
Wherein F is the intermolecular bonding force,
m is the average of the bursting forces F _U ,
and σ ² is a variance.
A chamber containing polynucleotide-functionalized substrates and polynucleotide-functionalized beads;
An electric field generator positioned below the chamber, the electric field generator comprising an electrode unit and applying an electric voltage to the electrode unit to form an electric field;
A camera positioned above or at one side of the chamber to image the inside of the chamber;
A measuring unit measuring a gray scale value from a video signal received from the camera and detecting a moment of rupture using a gray scale value; And
Measuring the voltage (unbinding voltage) of the rupture point measured by the measurement section and after the transformation process rupture strength; that includes an analysis to obtain the (F _U unbinding force), metal ion measuring device.
The method of claim 18, wherein the metal is at least one selected from the group consisting of silver (Ag), mercury (Hg), lithium (Li), sodium (Na), iron (Fe) Gt; metal ion detection method.
The method of detecting a metal ion according to claim 1, wherein the metal ion is silver ion, and the polynucleotide is poly-cytosine DNA.
21. The method of claim 20,
Wherein the poly-cytosine DNA is composed of 20 to 32 cytosine bases.
22. The method of claim 21,
Wherein the metal ion is a mercury ion, and the polynucleotide is poly-cytosine thymine DNA.
The method of claim 22, wherein the poly-cytosine thymine DNA is 5 '- (CT) _n -3', wherein n is an integer from 3 to 9.
24. The method of claim 23, wherein the poly-cytosine thymine DNA is 5'-CTCTCTCTCTCT-3 '.
19. The apparatus of claim 18, wherein the measuring unit is configured to match a change in levitation displacement of a bead caused by application of a voltage and a change in intensity of a gray scale value, define a moment when a fluctuation occurs beyond a reference value as a burst moment Characterized in that the metal ion measuring device is a metal ion measuring device.
26. The method of claim 25,
Measuring the fluctuation of the gray scale value according to the Gaussian distribution of [Equation 1] below,
The fluctuation level is represented by μ ± 2σ,
[Formula 1]

In this formula,
x is a gray scale value,
mu is the average of the gray scale values,
and? is the standard deviation of the gray scale value.
19. The method of claim 18, wherein the conversion process is performed using Equation (2) below,
[Formula 2]

Through the conversion process, F _u (unbinding force) is obtained,
In this formula,
竜_m Is the medium permittivity of the medium _,
Re is the unbinding force,
epsilon _p ^* is the complex complex permittivity,
epsilon _m ^* is the complex medium permittivity,
r is the microsphere radius,
Wherein E is an electric field profile obtained from a finite element simulation (COMSOL Multiphysics 3.5a).
19. The method of claim 18,
Wherein the analysis unit detects the metal ion by using the ratio of the average rupture force (< F _U &gt;) to the metal ion concentration.

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