KR101776134B1 - Apparatus and method for label-free sensing by electrokinetic streaming potential in microfluidic-chip based pathogen detection - Google Patents

Apparatus and method for label-free sensing by electrokinetic streaming potential in microfluidic-chip based pathogen detection Download PDF

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KR101776134B1
KR101776134B1 KR1020150168199A KR20150168199A KR101776134B1 KR 101776134 B1 KR101776134 B1 KR 101776134B1 KR 1020150168199 A KR1020150168199 A KR 1020150168199A KR 20150168199 A KR20150168199 A KR 20150168199A KR 101776134 B1 KR101776134 B1 KR 101776134B1
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전명석
정소현
우덕하
전영민
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한국과학기술연구원
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Abstract

A novel label-free sensing method and apparatus for detecting pathogens from a change in the interface electrokinetic flow potential based on a microfluidic chip. The sensing method and apparatus are characterized in that a pathological sample liquid is flowed through a microchannel filled with one or more glass beads to which an antimicrobial peptide is immobilized and the pathogen is bound to the glass beads through the interaction with the antimicrobial peptide over time The sensing method and apparatus are based on the principle that the chargeability of beads is changed and then the flow potential is changed. The sensing method and apparatus shortens the total analysis time in detecting pathogens such as bacteria and viruses, secures detection stability, Field diagnostic capabilities can be enhanced.

Figure 112015116469018-pat00019

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method and apparatus for sensing non-labeling by microelectrophoretic chip-based pathogen detection,

Embodiments are directed to techniques for detecting pathogens based on microfluidic chips and more particularly to methods for detecting pathogens from changes in the interfacial electrical flow potential in microchannels, And a label-free sensing method and apparatus.

The detection of pathogens is very important for public health such as clinical diagnosis, disease control, food safety, and water quality monitoring. Antibodies have been widely used for the detection of pathogens, but many animals require sacrifice to obtain antibodies and are limited in terms of stability and cost. Antimicrobial peptides (AMPs) are an example of an alternative to these antibodies. The source of antimicrobial peptides to pathogens is in electrostatic and hydrophilic-hydrophobic interactions. In other words, the antimicrobial peptide is charged with a positive charge as a whole, and pathogens are electrostatically attracted between the cells because the cell surface is charged with a negative charge, and the hydrophilic head group of the antimicrobial peptide and the hydrophobic group And are bound by interaction between cell membranes.

The antimicrobial peptides can be classified into various types depending on their terminal groups. See, for example, NV Kulagina, ME Lassman, FS Ligler, CR Taitt, "Antimicrobial peptides for detection of bacteria in biosensor assays ", Anal. Chem. 2005) and Mannoor, S. Zhang, AJ Link, MC McAlpine, "Electrical detection of pathogenic bacteria via immobilized antimicrobial peptides", Proc Natl Acad Sci USA, 107, 19207, 2010) We have reported that magainin I has a high binding affinity with bacteria.

On the other hand, the sensing of pathogens in microfluidic chip-based pathogen detection is a key technology, as shown in Table 1, there are optical, electrochemical, vibration, and mechanical methods. They have advantages over conventional methods such as Enzyme-Linked ImmunoSorbent Assay (ELISA) and Polymerase Chain Reaction (PCR) in terms of analysis time, sensitivity, and cost. In the conventional electrochemical method, there is a detection method using an impedance spectrometer, which is described in DP Poenar, C. Iliescu, J. Boulaire, H. Yu, "Label-free virus identification and characterization using electrochemical impedance spectroscopy" , Electrophoresis, 35, 433, 2014). Although this method can achieve rapid detection and high sensitivity, it is troublesome to construct a somewhat complicated electrode and circuit.

Sensing  Way Related Technology Total analysis time responsiveness cost existing ELISA, PCR Slow lowness High cost Optical Fluorescence, visible-ultraviolet spectrophotometer middle middle Low cost Electrochemical Impedance spectrometer speed height Intermediate cost vibration Quartz crystal middle height Low cost Mechanical Cantilever middle height Intermediate cost

N.V. Kulagina, M.E. Lassman, F.S. Leagues, C.R. Taitt, "Antimicrobial peptides for detection of bacteria in biosensor assays ", Anal. Chem., 77, 6504, 2005 M.S. Mannoor, S. Zhang, A.J. Link, M.C. McAlpine, "Electrical detection of pathogenic bacteria via immobilized antimicrobial peptides ", Proc. Natl. Acad. Sci. U.S.A., 107, 19207, 2010 D.P. Poenar, C. Iliescu, J. Boulaire, H. Yu, "Label-free virus identification and characterization using electrochemical impedance spectroscopy", Electrophoresis, 35, 433, 2014

According to one aspect of the present invention, there is provided a novel label-free sensing method and apparatus capable of replacing the conventional method of staining pathogens with fluorescent dyes and observing microchannels by fluorescence microscopy to detect pathogens .

A label-free sensing method according to an embodiment includes the steps of injecting one or more glass beads with an antimicrobial peptide immobilized in a chamber in a channel; Introducing a sample solution containing pathogens into the channel, thereby binding the glass beads in the chamber with pathogens; And measuring a change in the flowing potential in the channel to sense pathogens attached to the glass beads.

In one embodiment, measuring the change in flow potential within the channel includes measuring a change in flow potential per unit pressure difference over time.

In one embodiment, the flow potential is generated by the inner wall of the channel and the electrical double layer formed on the surface of the glass bead filled in the chamber in contact with the sample solution, and the inner wall of the channel is negatively charged.

In one embodiment, the step of injecting the glass beads comprises preventing the glass beads from escaping from the chamber using a plurality of weirs located within the channel.

In one embodiment, the step of combining the glass beads with a pathogen comprises introducing the sample solution into the channel using a pressure differential by a pump.

An unlabeled sensing device according to an embodiment includes a chamber, and includes a channel through which a sample solution containing pathogens flows; One or more glass beads filled into the chamber and having an antimicrobial peptide immobilized thereon for binding to pathogens; And an electrode configured to measure a change in flow potential in the channel to sense a pathogen bound to the glass bead.

In one embodiment, the electrode includes a pair of electrodes located at opposite ends of the chamber spaced a distance from the chamber.

In one embodiment, the pair of electrodes is configured to measure a change in flow potential per unit pressure difference over time.

In one embodiment, the flow potential is generated by the inner wall of the channel and the electrical double layer formed on the surface of the glass bead filled in the chamber in contact with the sample solution, and the inner wall of the channel is negatively charged.

An unlabeled sensing device according to an embodiment further comprises a plurality of banks disposed in the channel to prevent the glass beads from escaping from the chamber.

An unlabeled sensing apparatus according to an embodiment includes an inlet for introducing the sample liquid using a pressure difference by a pump; And an outlet for discharging the sample liquid after pathogens are bound to the glass beads, excluding pathogens.

In one embodiment, the width of the channel is greater than the height of the channel.

In one embodiment, the pressure difference is determined based on the cross-sectional area of the channel and the flow rate of the sample liquid.

In one embodiment, the change in flow potential corresponds to a change in chargeability of the chamber filled with the glass beads due to binding of the glass beads with pathogens over time.

According to one aspect of the present invention, a method for detecting microbial pathogens by staining conventional pathogens with fluorescent dyes and observing microchannels using a fluorescence microscope is known as a method for detecting pathogens from changes in the interfacial electrokinetic flow potential in microchannels A novel label-free sensing method and apparatus can be provided. According to the non-labeled sensing method, it is possible to shorten the total analysis time, ensure the stability of analysis, and eliminate the need for fluorescence microscope equipment since the process of staining pathogens and the process of analyzing fluorescence images are not necessary. Can be enhanced, and ultimately, rapidity, selectivity and accuracy in detecting pathogens can be improved.

FIGS. 1 to 3 are side views illustrating the formation of an electric double layer and generation of interfacial electrokinetic flow potential according to various charged states of both walls in a channel. FIG.
Fig. 4 is a graph showing the change of the flow potential according to the pressure difference for each case shown in Figs. 1 to 3. Fig.
5A is a schematic diagram of a microfluidic chip of a label-free sensing device according to an embodiment.
5B is a schematic plan view of portion A shown in FIG. 5A.
Figure 5c is a schematic side view of portion A shown in Figure 5a.
6A and 6B are conceptual diagrams showing a process in which a glass bead to which an antimicrobial peptide is immobilized is bound to E. coli.
6C is an electron micrograph showing a form in which glass beads and E. coli are combined.
FIGS. 7A and 7B are conceptual diagrams illustrating charge variation in the chamber formed in the channel due to the coupling process of FIGS. 6A and 6B and the variation of the flow potential.
8 is a schematic diagram of an unlabeled sensing device in accordance with one embodiment.
FIG. 9 is a graph showing a change in flow rate per unit pressure difference obtained over time for three types of E. coli by the non-labeling sensing method according to one embodiment.
FIGS. 10A to 10D are images obtained by observing the concentration of E. coli at a concentration of 10 5 / mL with the passage of time using a conventional fluorescence microscope.
FIG. 11 is a graph showing changes in cumulative fluorescence intensity per one bead with time elapsed from the image shown in FIG. 10 by the conventional method. FIG.

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.

According to one aspect of the present invention, a sample solution containing a pathogen is flowed through a channel filled with at least one bead with an antimicrobial peptide immobilized thereon, and the interaction between the pathogen and the antimicrobial peptide Pathogen binding patterns can be used to sense pathogens from changes over time in the interfacial electrokinetic flow potential. Pathogenic bacteria such as bacteria or viruses that can be bound to glass beads by interaction with antimicrobial peptides are all pathogenic bacteria that can be detected by the non-labeling sensing method and apparatus, and pathogenic and non- pathogenic Escherichia coli (E. coli).

Generally, when a fluid flows in a channel having a surface charged therein, a counter-ion opposite to the charge sign of the channel wall surface is distributed near the channel wall surface to form an electric double layer do. The counter ions in the electric double layer generate a streaming current by a pressure difference flow and cause a flow of electric current between the upstream and the downstream of the channel due to a potential difference due to a difference in distribution of common ions and counter ions. potential. In addition, when the counter ions continue to accumulate downstream, counter ions move in the opposite direction of the fluid flow, leading to a conduction current. In the steady state, the sum of the flow current and the conduction current is zero, and the current in the channel is preserved.

The flow potential ΔE generated by the electrokinetic effect due to the flow of the electrolyte solution having a constant ion concentration (ie, ionization intensity) in a linear channel having a constant cross-section is given by Helmholtz-Smallruchowski Helmholtz-Smoluchowski, HS).

Figure 112015116469018-pat00001

In Equation (1),? P is a pressure difference across the channel. The pressure difference is a value that can be determined from the cross-sectional area of the channel and the flow rate of the sample liquid. Also, ε o is a dielectric constant or vacuum permittivity in vacuum of 8.854 × 10 -12 Coul 2 / J · m, ε r is the relative permittivity of the electrolyte solution,

Figure 112015116469018-pat00002
Is the zeta potential of the channel wall, λ is the electric conductivity of the electrolyte solution, and η is the viscosity of the electrolyte solution.

The flow potential is one of the interfacial electrokinetic phenomena and is a mechanism opposite to electroosmosis and has been used as a method to determine the unknown zeta potential, which is the surface potential of a charged object. As examples, Szymczyk et al. (A. Szymczyk, B. Aoubiza, P. Fievet, J. Pagetti, "Electrokinetic phenomena in homogeneous cylindrical pores", J. Colloid Interface Sci. 216, 285, 1999) , It can be seen that the measurement of the flow potential effectively contributes to the charging characteristic of the porous material to the pore or the surface. In addition, U.S. Patent No. 6,727,099 discloses information on the deposition of colloidal particles on the surface of a porous membrane by measurement of flow potential over time.

The physical meaning of Equation (1) is that when the electrolyte solution is flowed in the charged channel with the pressure difference? P, a potential difference by? E is generated across the channel. And connecting any external load can get the current and voltage acting on this external resistor.

Methods and apparatus for label-free sensing according to embodiments are configured to sense pathogens attached to an antimicrobial peptide using the principles described above. The non-labeling sensing method and apparatus are combined with a micro-electromechanical system (MEMS) and a microfabrication technique to form a lab-on-a-chip (microfluidic chip) having a desired channel width -on-a-chip technology, or a micro total analysis system (μTAS).

FIGS. 1 to 3 are side views showing the formation of an electric double layer and the generation of interfacial electrodynamic flow potential according to various charged states of both walls in a channel.

1 shows a state in which both of the wall surfaces 110 and 120 defining a channel are negatively charged. The inside of the channel is divided into an electric double layer region in which ions opposite to the charges of the solid wall surfaces 110 and 120 predominate and a bulk region 130 in which the positive ions and the negative ions are present in the same number and are electrically neutral. The electrical double layer thickness is usually denoted by κ -1 . In Fig. 1, an electric double layer having a predominantly larger number of positive ions is formed in the vicinity of both wall surfaces 110, 120. When a pressure difference is applied between both ends of the channel and a solution flows through the channel, the cations constituting the electric double layer accumulate on the downstream side, and an electric field due to a charge gradient is formed between both ends of the channel. In Fig. 1, the electric field due to the charge gradient is indicated by the - and + sign, and this electric field causes a flow potential E to be generated.

2 illustrates a state in which one wall 110 is negatively charged while the other wall 120 is not charged. In this case, since the electric double layer is not formed on the other wall surface 120, the amount of positive ions accumulated downstream in accordance with the pressure difference applied to the channel is reduced as compared with that in FIG. 1, .

3A shows a state in which one wall 110 is negatively charged while a part of the other wall 120 is positively charged. In this case, since the electric double layer is formed in the positively charged region of the other wall surface 120 with anion predominated, these anions cancel out the cations accumulated in the downstream due to the pressure difference applied to the channel, Is lower than in the case of FIG.

In addition, as shown in FIG. 3B, when one wall 110 is negatively charged while the other wall 120 is positively charged, the anions present in the electric double layer are negatively charged in the opposite electric double layer, So that it is completely canceled at the downstream, so that no flow potential is generated.

4 is a graph showing the change of the flow electric potential E in accordance with the pressure difference P, and the four kinds of graphs 401-404 shown in Fig. 4 are the graphs of the wall surfaces shown in Figs. 1, 2, 3A, And represents the flow potential according to the charged state. Figure 4 also shows a qualitative change in the flow potential at any pressure difference. In the Poisson-Boltzmann (PB) equation dominating the static field, if the surface potential ψ s of the charged wall surface is lower than 25.7 mV, the number of charges per unit area of the wall surface (ie, ions) The surface charge density σ s is expressed by the following relationship.

Figure 112015116469018-pat00003

Here, κ is the inverse of the electrical double layer thickness (that is, the electric double layer thickness κ - 1), and, k is the Boltzmann constant, T is absolute temperature, e is the unit charge with one dog ion. Actually, the surface potential? S Zeta potential

Figure 112015116469018-pat00004
The following equation (3) can be obtained from the equations (1) and (2).

Figure 112015116469018-pat00005

That is, the flow potential of the channel is a charge density function of the channel wall surface. Specifically, the value obtained by dividing the flow potential ΔE of the channel by the pressure difference ΔP is proportional to the surface charge density σ s of the channel wall surface. Therefore, in the non-labeling sensing method and apparatus according to the embodiments, the surface charge density of the channel wall surface is measured by filling the channel with the immobilized anti-microbial peptide immobilized on the channel and using the change of the flow potential with time, The degree of binding of pathogens to glass beads can be sensed.

FIG. 5A is a schematic view of a microfluidic chip 1 of an unlabeled sensing device according to an embodiment, and FIGS. 5B and 5C are a schematic plan view and a side view of a portion A shown in FIG. 5A, respectively.

Referring to Figures 5A-5C, an unlabeled sensing device may include a channel 10 and one or more glass beads 100 filled in the channel 10 and coupled with an antimicrobial peptide. One or more antimicrobial peptides may be immobilized on each glass bead 100. In the channel 10, a chamber 140, which is a space for filling the glass beads 100, may be formed. A plurality of (e.g., two) weirs 150 may be formed in the channel 10 such that the glass beads 100 are not separated from the chamber 140. For example, the dam 150 may be located at either end of the chamber 140, respectively.

The unlabeled sensing device includes an inlet 20 for introducing a sample solution containing pathogens and a sample inlet for discharging the sample solution after pathogens in the sample solution are bound to the antibacterial peptide of the glass beads 100 And may further include an outlet 30. The inflow section 20 and the outflow section 30 may be connected to the inflow tubing 25 and the outflow tubing 35 to respectively receive and discharge the sample solution.

In one embodiment, the unlabeled sensing device may be configured using a lower substrate 300 and an upper substrate 200 facing each other. The lower substrate 300 and the upper substrate 200 can be made of a plastic material such as polydimethylsiloxane (PDMS) that is easy to process and inexpensive, a silicon or glass-based material that is relatively difficult to process, ≪ / RTI >

The microfluidic chip according to the present invention is a microfluidic chip formed by forming a channel on a PDMS channel substrate using a MEMS process so as to prevent a charged bead from flowing back and forth between the chamber and a position where the glass beads are filled, And a PDMS channel substrate is formed through a circular knife. The diameter of the inlet and outlet is 1/16 inch, allowing 1/16 inch tubing to be installed.

In one embodiment, the length L 1 of the chamber 140 can be suitably determined so that several hundred glass beads 100 can be filled into the chamber 140, for example, about 600 to 1000 micrometers (μm) But is not limited thereto. In addition, the length L2 of the dam 150 is about 300 micrometers (m).

In one embodiment, the height H 1 of the dam 150 is about 1/4 to 1/3 of the diameter of the glass bead 100 so that the glass bead 100 is not separated from the chamber 140. The height H 2 of the channel 10 excluding the weir 150 in the region where the weir 150 is formed is slightly larger than the maximum diameter of the glass bead 100 so that the glass bead 100 can be filled with the channel do. On the other hand, the height of the channel (i.e., H 1 + H 2 ) in the region where the bank 150 is not formed is designed so that the distance between the glass bead 100 and the upper substrate 200 does not exceed 15 micrometers So that pathogens having a length of about several micrometers (μm) can be easily bonded to the glass beads 100.

In one embodiment, the channel 10 is a microchannel with micrometer-sized dimensions. Also, in one embodiment, the width W of the channel 10 is greater than the height of the channel 10 (i.e., H 1 + H 2 ). For example, the width W of the channel 10 may be at least four times the height of the channel 10 and may be less than 500 micrometers (μm).

In one embodiment, the lower substrate 300 on which the channel 10 is formed is made of PDMS and has a thickness of 1.5 to 2.5 millimeters (mm). In one embodiment, the upper substrate 200 covering the lower substrate 300 is made of glass and has a thickness of 1.0 to 1.5 millimeters (mm).

The channel 10 and the chambers 140 and dams 150 included therein may be formed on the lower substrate 300 using a MEMS process and the upper substrate 200 may be penetrated So that the inflow portion 20 and the outflow portion 30 can be formed. The inflow portion 20 and the outflow portion 30 may be formed by punching out the upper substrate 200 with a circular knife, but the present invention is not limited thereto. Next, the upper substrate 200 may be bonded to the surface of the lower substrate 300 where the channel 10 is formed. In one embodiment, the upper substrate 200 and the lower substrate 300 are bonded by an oxygen plasma bonding method, but are not limited thereto.

FIGS. 6A and 6B are conceptual diagrams showing a process in which an antibacterial peptide-immobilized glass beads is bound to E. coli, and FIG. 6C is an electron micrograph showing a form in which glass beads and E. coli are actually bonded.

Referring to FIG. 6A, since the antimicrobial peptide has a positive charge, the glass beads 100 to which the antimicrobial peptide binds are also positively charged. However, when the pathogenic bacteria 400 having a negative charge such as E. coli are bound to the antimicrobial peptide of the glass beads 100, the positive charge of the glass beads 100 is reduced due to the binding of the pathogens 400 as shown in FIG. 6B . As shown in FIG. 6C, a plurality of pathogens 400 may be combined with one glass bead 100. When the number of the coliform bacteria 400 to be combined over time increases, Many parts are changed to non-chargeable, and when pathogens bind to the number of positively charged glass beads, they become completely uncharged.

FIGS. 7A and 7B are conceptual diagrams illustrating charge variation in the chamber formed in the channel due to the coupling process of FIGS. 6A and 6B and the variation of the flow potential.

7A and 7B, in the present embodiment, the channel walls are negatively charged while the glass beads 100 to which the antibacterial peptide is immobilized are filled in the chamber 140. The chamber 140 is initially positively charged due to the glass beads 100 as shown in FIG. 7A, but as pathogens having a negative charge are gradually coupled to the glass beads 100 as shown in FIG. 7B, (140) portion gradually becomes uncharged. This corresponds to a change from a state in which a part of the wall surface 120 described above with reference to FIG. 3A is positively charged to a state in which the partial region described above with reference to FIG. 2 is changed into a non-charge state.

Therefore, when the flow potential of the channel 140 is measured, the glass beads 100 of the chamber 140 can be observed with a lapse of time, since the flow rate of the channel increases as the portion of the chamber 140 changes from positive to non- You can sense the degree of association with this pathogen.

8 is a schematic diagram of an unlabeled sensing device in accordance with one embodiment.

Referring to Figure 8, the unlabeled sensing device comprises a channel 10 configured in accordance with the embodiment described above with reference to Figure 5, and a glass bead for filling the chamber within the channel 10. The inlet 20 is connected to the inlet tubing 25 and a syringe pump 27 injects the sample fluid containing pathogens into the inlet tubing 25. Pathogens of the sample liquid are bound to the glass beads of the chamber, and the remaining sample liquid excluding the pathogens can be discharged through the outflow portion 30 and the outflow tubing 25 connected thereto.

On the other hand, an electrode for measuring the flow potential of the channel 10 is provided in the non-labeling sensing device. The electrodes may be composed of a pair of electrodes disposed in front of and behind the chamber formed in the channel 10, respectively. For example, a hollow space having a diameter of the electrode lines 40 and 50 is formed at a position in the channel 10 separated by about 5 millimeters (mm) before and after the chamber of the channel 10, and the upper substrate and the lower substrate are bonded The electrode lines 40 and 50 may be inserted into the corresponding space and then sealed with an epoxy resin or the like. The electrode lines 40 and 50 may be made of silver (Ag) / silver chloride (AgCl) or other suitable conductive material.

In one embodiment, the unlabeled sensing device further comprises a detector for detecting and analyzing the flow potential across the electrode lines 40,50. For example, the detection portion may include a digital multimeter 60 for measuring the flow potential value in electrical connection with the electrode lines 40 and 50, and a digital multimeter 60 for analyzing, processing, and / or storing measured values of the digital multimeter 60 And a personal computer (PC) 70, but the present invention is not limited thereto. For accurate measurement, the digital multimeter 60 can be used with an accuracy of 10 -7 to 10 -8 V level.

In addition, in one embodiment, the unlabeled sensing device further comprises a fluorescence microscope (80). The fluorescence microscope 80 is capable of performing fluorescence image observation in a conventional manner in parallel with the pathogen detection by the flow electric potential measurement. The fluorescence microscope 80 is configured such that the objective lens is arranged so as to face the channel 10 from above the channel .

The inventors of the present invention conducted a detection experiment on E. coli using a microfluid chip-based non-labeling sensing method and apparatus constructed as described above to measure the detection rate and the detection efficiency, and compared the fluorescence images obtained by fluorescence microscopy according to the conventional method Was compared with the detection result by the test.

As the glass beads to which the pathogens are to be bound, those having a primary amine group and a diameter of about 30 to 38 micrometers (μm) were purchased and used. The antimicrobial peptide was fixed on glass beads by a known chemical method, And fixed with a scanning electron microscope (SEM). 5, inflow tubing and outflow tubing were installed in the prepared microfluidic chip, and hundreds of glass beads having the antibacterial peptide immobilized by the syringe pump were charged into the chamber of the channel.

Escherichia coli (DH5α) was obtained from an American Type Culture Collection (ATCC) and cultured and diluted. The microorganism was cultured by UV-Vis spectrophotometer (UV-VIS) And the number of E. coli was calculated by measuring the optical density (OD). As a sample solution containing E. coli, phosphate buffered saline (PBS) solution was used. The concentration of electrolyte ions, which are dissociated from commercial conditions, was lowered and the total ion concentration was adjusted to several mM.

The non-labeling sensing device is constructed as in the embodiment of FIG. 8, and the sample liquid prepared by the above-described process is injected into the channel at a flow rate of about 1.2 × 10 -3 mL / min by a syringe pump, The potential value was measured. From the channel cross-sectional area in the flow rate and the electrode line of the sample solution in the point, line speed, and it is possible to calculate the pressure difference, 10 3, 10 4, 10 5 / mL units of pressure over time for E. coli having a concentration per car flow The change of the electric potential (? E /? P) is shown in Fig.

As shown in Fig. 9, when the E. coli concentration is 10 3 cells / mL, there is a slight variation in the flow potential, but there is clearly a difference in detection for the three kinds of concentrations. Also, the flow potential gradually increased with time, and the rate of increase of the flow potential was saturated when about 30 minutes elapsed.

On the other hand, as a comparative example to the example according to the present invention, the present inventors observed that the binding of E. coli to glass beads was also observed through fluorescence images by fluorescence microscopy as in the prior art.

FIGS. 10A to 10D show images obtained by observing with time a fluorescence microscope for a concentration of 10 5 / mL of E. coli stained by a conventionally known method using a fluorescent dye, propidium iodide , Fluorescence images are shown immediately after pathogen injection, 5 minutes after injection, 20 minutes after injection, and 50 minutes after injection, respectively.

11 is a graph showing the change in cumulative fluorescence intensity per one bead with time, which is calculated from the image shown in Fig. Comparing the results shown in FIG. 11 with the results shown in FIG. 9 according to an embodiment of the present invention, it can be confirmed that the change characteristics of the detection speed with time and the detection characteristics according to the concentration of E. coli are similar. This means that the result of detection by the flow potential according to the embodiment of the present invention corresponds to the detection result through the conventional fluorescence image, and it can be understood that the sensing method based on the flow potential is an effective non-label sensing method.

While the invention has been shown and described with reference to certain embodiments thereof, it will be understood by those skilled in the art that various changes and modifications may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. However, it should be understood that such modifications are within the technical scope of the present invention. Accordingly, the true scope of the present invention should be determined by the technical idea of the appended claims.

Claims (16)

Injecting at least one glass bead with an antimicrobial peptide immobilized in a chamber in a channel positioned between the opposing lower substrate and the upper substrate;
Introducing a sample liquid containing pathogens into the channel by means of a pump to couple the glass beads in the chamber with pathogens; And
Measuring a change in flow potential in the channel to sense a pathogen bound to the glass bead while introducing the sample solution by the pump,
Wherein the lower substrate is made of polydimethylsiloxane,
Wherein the channel comprises an empty space of an electrode line diameter size at both ends of the chamber spaced a predetermined distance from the chamber within the channel and the inner wall of the channel is negatively charged,
Wherein the step of injecting the glass beads comprises preventing the glass beads from separating from the chamber using two banks formed in the lower substrate and located at opposite ends of the chamber in the channel,
Wherein measuring the change in the flow potential includes measuring a flow potential value by a digital multimeter electrically connected to the pair of electrode lines and the electrode lines inserted in the void space.
delete delete The method according to claim 1,
Wherein measuring the change in flow potential in the channel comprises measuring a change in flow potential per unit pressure difference over time.
5. The method of claim 4,
Wherein the pressure difference is determined based on a cross-sectional area of the channel and a flow rate of the sample liquid.
5. The method of claim 4,
Wherein the flow potential is generated by an inner wall of the channel and an electrical double layer formed on a surface of the glass bead filled in the chamber in contact with the sample solution.
The method according to claim 1,
Wherein the change in flow potential corresponds to a change in charge of the chamber filled with the glass beads due to bonding of the glass beads with pathogens over time.
A lower substrate made of polydimethylsiloxane;
A channel formed on the lower substrate and including a chamber for flowing a sample solution containing pathogens;
An upper substrate facing the lower substrate to cover the channel and the chamber;
A pump for injecting the sample solution into the chamber;
One or more glass beads filled into the chamber and having an antimicrobial peptide immobilized thereon for binding to pathogens;
Two banks formed in the lower substrate to prevent the glass beads from escaping from the chamber, the chambers being located at opposite ends of the chamber within the channel; And
And a detector configured to measure a change in flow potential in the channel to sense a pathogen bound to the glass bead in a state in which the sample liquid is injected into the channel by the pump,
Wherein the channel comprises an empty space of an electrode line diameter size at both ends of the chamber spaced a predetermined distance from the chamber within the channel and the inner wall of the channel is negatively charged,
Further comprising a pair of the electrode lines inserted in the empty space,
Wherein the detecting section comprises a digital multimeter electrically connected to the electrode line and configured to measure a flow potential value.
delete 9. The method of claim 8,
An inlet for introducing the sample solution using the pressure difference by the pump; And
Further comprising an outlet for discharging the sample liquid after pathogens are bound to the glass beads.
delete 9. The method of claim 8,
Wherein the detecting unit is configured to measure a change in the flow potential per unit pressure difference with the lapse of time.
13. The method according to claim 10 or 12,
Wherein the pressure difference is determined based on a cross-sectional area of the channel and a flow rate of the sample liquid.
9. The method of claim 8,
Wherein the flow potential is generated by an inner wall of the channel and an electric double layer formed on a surface of the glass bead filled in the chamber in contact with the sample liquid.
9. The method of claim 8,
Wherein the width of the channel is greater than the height of the channel.
9. The method of claim 8,
Wherein the change in the flow potential corresponds to a change in charge of the chamber filled with the glass beads due to bonding of the glass beads with pathogens over time.
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