KR101890593B1 - Biosensor for simultaneous detection and death of pathogen and method for preparing the same - Google Patents

Abstract

The present invention relates to a biosensor using a discoloring substance to which a pathogen-specific enzyme is bound, and a method for producing the biosensor. The biosensor according to the present invention can simultaneously detect and kill pathogens by using a discoloring substance to which a pathogen-specific enzyme is bound. In the case of using the biosensor, And demonstrate excellent pathogen detection effects in an economical manner.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a biosensor capable of simultaneous detection and death of a pathogen,

The present invention relates to a micro-fluidic system and a biosensor for simultaneous detection and death of pathogens using a discoloring substance sensitive to an external stimulus and a method for producing the same.

A biosensor is a sensor that converts a reaction due to binding to a biochemical substance into a signal and enables quick inspection of a specific substance. This principle is based on the same principle as the enzyme-substrate complex of biological concept that one ligand reacts only to one substance having a specific component for the ligand and measures its degree of reactivity. The miniaturized biosensor minimizes human injury and enables the diagnosis of painless human body, and it has the advantage that it can directly analyze single cells. In addition, biosensors with improved operating characteristics such as high stability, fast response time, high sensitivity, and high selectivity enable continuous measurement of human diagnosis and can perform single molecule analysis.

Polydiacetylene (PDA) vesicles are materials exhibiting a color-evoking ability to change from blue to red by external disturbance, such as heat, pH, physical or chemical stimulation, or a solvent. Furthermore, the red polydiacetylene vesicle has the property of exhibiting fluorescence and has been developed as various sensors using such properties. For example, polydiacetylene sensors have been used for the detection of many other biologically relevant molecules of interest such as influenza viruses, cholera toxins, E. coli, oligonucleotides, lipopolysaccharides, antibodies and antigens. Microfluidic systems have provided improved methods of performing chemical, biochemical and biological analysis and synthesis. Such a microfluidic system enables the execution of multi-stage, multi-species chemical operations in a chip-based microchemical analysis system, and in particular includes a conventional microfluidic control element capable of processing and analyzing chemical and biological samples It is common. Typically, the term microfluidic in the art refers to a system or apparatus having a channel-connected processing node, wherein the channel has a cross-sectional dimension in the range of about 1.0 [mu] m to about 500 [mu] m. In the art, a channel having such a cross sectional dimension is referred to as a microchannel. Recently, such microfluidic systems have been applied in many fields such as cytodiagnostic tests, medical drug development, basic biology or cosmetology.

Therefore, it is urgently required to study a biosensor based on a discoloring material for simultaneous detection and death of pathogens using such a microfluidic system.

KR 10-2008-0047683

The inventors of the present invention have confirmed that when a chromogenic material-based biosensor is used, the detection and the death of a pathogen can be simultaneously performed by using a chromogenic material combined with a microfluidic system and a pathogen-specific enzyme, .

Accordingly, the present invention provides a micro-fluidic system and a biosensor using the discoloring substance sensitive to an external stimulus and a method of manufacturing the same.

In order to achieve the above object,

The present invention

A substrate on which a microchannel is formed; A coloring material responsive to an external stimulus coupled to the microchannel; And a pathogen-specific enzyme bound to the discoloration substance. The present invention also provides a biosensor for detecting and killing pathogens.

In addition,

(1) fabricating a substrate on which a microchannel is formed;

(2) combining the microchannel with a discoloring substance sensitive to an external stimulus; And

(3) a step of binding a pathogen-specific enzyme to the discoloring substance, and a method of manufacturing a biosensor for detecting and killing pathogens.

In addition,

And detecting a color change after applying a fluid including pathogens to the microchannel of the biosensor. The present invention also provides a method for detecting and killing pathogens using the biosensor.

Hereinafter, the present invention will be described in detail.

The present invention relates to a substrate having a microchannel formed therein; A coloring material responsive to an external stimulus coupled to the microchannel; And a pathogen-specific enzyme bound to the discoloration substance. The present invention also provides a biosensor for detecting and killing pathogens.

Further, the present invention provides a method of manufacturing a microchannel, comprising the steps of: (1) fabricating a substrate on which a microchannel is formed; (2) combining the microchannel with a discoloring substance sensitive to an external stimulus; And (3) binding a pathogen-specific enzyme to the discoloring substance. The present invention also provides a method for producing a biosensor for detecting and killing pathogens.

The microchannel is patterned on the substrate so that a fluid including pathogens can flow. The microchannel preferably has an average width of 100 to 1000 mu m and an average depth of 100 to 500 mu m, an average width of 300 to 600 mu m, It is more preferable that the average depth is 200 to 300 占 퐉, but it is not limited thereto. The microchannel can be applied to a medical tubing or a filter having a width of 0.1 mm to 10 mm, and is not limited to the inside diameter.

The microchannel may have various structures on the surface. Rectangular, square, or polygonal (triangular, rectangular, pentagonal, hexagonal, etc.) structures, and is not limited in its shape. According to the present invention, these structures can increase the killing rate of pathogens by increasing the reaction between pathogen-specific enzymes and pathogens.

The substrate may be a polymer or a silicon material, but is not limited thereto, and a microfluidic system can be manufactured from any material. For example, polydimethylsiloxane (PDMS), poly methyl methacrylate (PMMA), poly vinyl chloride (PVC), polycarbonate (PC), polyvinyl acrylate (PVA), polytetrafluoroethylene (PTFE), silicone, PE (polyethylene) or the like may be used, and PDMS is more preferable, but not limited thereto.

The external stimulus may be a color, such as temperature, pressure, light, pH, humidity, solvent, electric field, magnetic field, stress, etc., It is not.

The coloring material may be polydiacetylene (PDA) vesicles or gold nanoparticles (GNP), preferably polydiacetylene (PDA) vesicles.

The polydiacetylene (PDA) vesicle is a vesicle-type structure composed of a diacetylene-based lipid and has two triple bonds in the tail portion of the lipid, and has a color-shifting characteristic through crosslinking. Blue polydiacetylene vesicles are red polydiacetylenes phosphors as they are transformed to a red color having a maximum absorption wavelength at about 540 nm due to changes in the external environment such as temperature, pH, solvent, physical stimulus, and chemical stimulation .

The pathogen-specific enzyme is an enzyme capable of specifically binding to a pathogen to be detected and killed. Examples of the pathogen-specific enzyme include, but are not limited to, resourcetapine, beta glucuronidase, glucanase, glutathione, lysozyme, It is preferable that the enzyme is any one enzyme selected from the group consisting of mannanase, mutanolysin, zymolase, cellulase, phenothiolase, streptolysin, and combinations thereof, more preferably resourcetopine, but is not limited thereto .

Pathogens can be detected without limitation as long as they are capable of binding to the pathogen-specific enzyme by physical, chemical, or biological interactions with microorganisms, bacteria, or viruses that selectively bind to the pathogen-specific enzyme. That is, the pathogenic bacteria may be selected from the group consisting of staphylococcus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Serratia marcescens, Streptococcus pneumoniae, Haemophilus influenzae ), Enterobacter cloacae, Enterococcus faecalis, Salmonella, Campylobacter, and combinations thereof. However, the present invention is not limited thereto, But is not limited thereto.

In addition,

And detecting a color change after applying a fluid including pathogens to the microchannel of the biosensor. The present invention also provides a method for detecting and killing pathogens using the biosensor.

The method can simultaneously detect and kill pathogens.

When a fluid containing a pathogen is added to the microchannel, the pathogen binds to a pathogen-specific enzyme bound to the PDA vesicles present in the microchannel and is killed. Simultaneously, the PDA vesicles are discolored from blue to red So that pathogens can be detected visually.

The biosensor according to the present invention can simultaneously detect and kill pathogens by using a microfluidic system and a discoloring substance to which a pathogen-specific enzyme is conjugated. In the case of using the biosensor, It exhibits excellent pathogen detection effect by a simple and economical method through mutation.

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a diagram showing a substrate of a substrate on which a microchannel is formed according to Embodiment 1-1. FIG.
FIG. 2 is a view showing the bonding process of polydiacetylene (PDA) vesicles to the microchannel according to Example 1-2.
FIG. 3 is a conceptual diagram of a biosensor using a microfluidic system and PDA vesicles.
FIG. 4 is a diagram showing optical microscopic results of microchannels in which (a) circular structures and (b) rectangular structures according to Embodiment 1-1 are formed. FIG.
FIG. 5 is a diagram showing fluorescence microscopic results of PDA vesicles bound to microchannels according to Example 1-2. FIG.
6 is a graph showing the mortality rate of pathogens according to the flow rate of a pathogenic solution applied to the microchannel.
FIG. 7 is a graph comparing mortality rates of pathogens when a pathogen solution is added to a pathogen-specific enzyme bound to a PDA vesicle of a biosensor and when a pathogen solution is added to a pathogen-specific enzyme solution.
FIG. 8 is a graph showing (a) the killing rate of pathogens and (b) the color variation of the PDA vesicles according to the number of structures formed in the microchannels of the biosensor.
FIG. 9 is a graph showing the degree of killing of a pathogen according to the angle of a structure formed on a microchannel of the biosensor and the flow rate of a pathogen solution, and (b) color variation of a PDA vesicle.
10 is a diagram showing the pathogen-specificity of the biosensor.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples.

Example 1. Preparation of a biosensor

1-1. Fabrication of microchannel-formed substrate

A microfluidic system was fabricated using a soft lithography process. First, PDMS (polydimethylsiloxane) was used as a substrate, and a microchannel having a width of 500 μm and a depth of 300 μm was formed on the substrate by a soft lithography process. In addition, circular, elliptical and rectangular pillar structures having various angles (30 °, 45 ° and 60 °) were formed with respect to the flow direction of the fluid on the microchannels.

FIG. 1 is a schematic view of a substrate on which a microchannel is formed according to the embodiment 1-1.

1-2. Coupling of PDA vesicles to microchannels

Polya diacetylene (PDA) vesicles were bonded to the substrate on which the microchannel fabricated in Example 1-1 was formed. First, the substrate on which the microchannel was formed was surface-modified by treating with oxygen plasma, and APTES (3-Aminopropyltriethoxysilane) was added to form an amine functional group.

TCDA (10,12-Tricosadiynoic acid) was purchased from GFS Chemical (Columbus, OH, USA) and DMPC (1,2-dimyristoyl phosphatidylcholine) was dissolved in AvantiPolar Liquid (Alabaster, USA), and chloroform was purchased from Fluca (Buchs, Switzerland). HEPES was purchased from Sigma-Aldrich (St. Louis, MO, USA), dissolved in 5 mM of tertiary distilled water and adjusted to pH 7.5. Polydiacetylene (PDA) vesicles were prepared according to known methods (Su Y-l et al. (2004) Biointerfaces 38 (1-2): 29-33). First, two lipid monomers, TCDA and DMPC, were dissolved in chloroform at an 8: 2 molar ratio to a final concentration of 2 mM in the vial. The chloroform was then blown off with an argon gas stream and allowed to stand overnight in a vacuum to completely remove the remaining solvent. HEPES solution (5 mM, pH 7.5) was added to the final 2 mM lipid concentration. The solution was heated in a water bath at 80 ° C for 1 hour and sonicated for 15 minutes (probesonicator, Sonics & Materials, INC, VCX 500) Respectively. After the ultrasonic treatment, the solution was filtered using a 0.8 μm syringe filter to remove lipid aggregates and stored at 4 ° C. in a refrigerator for overnight crystallization. By irradiating the UV (Vilber Lourmat, VL-4. LC) at a wavelength of 365 nm, the diacetylene monomer was polymerized by 1,4-addition and the blue PDA vesicle solution was obtained.

Then, the polydiacetylene (PDA) vesicles were bound to the microchannel-formed substrate using an EDC / NHS coupling reaction according to a known method (Lim MC et al. (2011) Anal Bioanal Chem. 400: 777-785).

The binding process of polydiacetylene (PDA) vesicles to the microchannel according to Example 1-2 is shown in FIG.

1-3. Binding of pathogen-specific enzymes to PDA vesicles

Lysostaphin was purchased from Sigma-Aldrich (St. Louis, Mo., USA) to bind the microchannel-attached PDA vesicles and the pathogen-specific enzyme prepared in Example 1-2 above . In addition, the binding of the resource taspin enzyme to the surface of the polydiacetylene (PDA) vesicle was carried out using an EDC / NHS coupled reaction according to a known method (Lim MC et al. (2011) Anal Bioanal Chem 400 : 777-785). Subsequently, by irradiating UV at a wavelength of 365 nm, the PDA vesicles formed a polymer and turned blue.

FIG. 3 is a conceptual diagram of a biosensor using the microfluidic system and the PDA vesicle of the present invention.

Experimental Example 1. Observation of morphology of a microchannel in which a structure is formed

The microchannels in which the structures of (a) circular and (b) rectangles according to Example 1-1 were formed were observed using an optical microscope, and the results are shown in FIG.

As shown in FIG. 4, it can be seen that (a) a circular or (b) rectangular pillar is formed in the microchannel according to the shape of the structure. The structures are believed to increase the killing of pathogens when the pathogen solution passes through the microchannel.

Experimental Example 2. Observation of the shape of PDA vesicles bound to microchannels

PDA vesicles bound to the microchannel according to Example 1-2 were irradiated with UV at a wavelength of 365 nm and then converted into red PDA vesicles from blue PDA vesicles by adding NaOH. The fluorescence microscopic results thereof were shown in Fig. 5 Respectively.

As shown in FIG. 5, it can be confirmed that red PDA vesicles are bonded to the microchannels.

Experimental Example 3. Detection and Death of Pathogenic Bacteria According to Flow Rate of Pathogenic Bacterial Solution

After staphylococcus solution was added to the microchannels of the biosensor according to Example 1, the mortality of the pathogen was measured using absorbance at a wavelength of about 600 nm.

First, staphylococcus solution was added to the microchannels having no structure at different flow rates, and mortality was measured. The results are shown in FIG.

As shown in FIG. 6, it can be seen that as the flow rate of the staphylococcal solution decreases, the mortality rate of Staphylococcus aureus increases. This is because the longer the reaction time of Staphylococcus aureus binds to the PDA vesicles as the flow rate of the staphylococcal solution decreases, the longer the time required for the resource tapin to decompose staphylococci.

The mortality rate of pathogenic bacteria was compared between the case where the staphylococcal solution was added to the resource taffine enzyme bound to the PDA vesicles of the biosensor of Example 1 and the case where the staphylococcus solution was added to the resource taffine enzyme solution. 7.

As shown in FIG. 7, the addition of Staphylococcus aureus to the resource taffine enzyme bound to the PDA vesicles in the microchannel showed a higher rate of staphylococcal death than the addition of the staphylococcus solution to the resource taffine enzyme solution .

EXPERIMENTAL EXAMPLE 4. Detection and Destruction of Pathogenic Bacteria According to Structures in Microchannels

The mortality of Staphylococcus aureus according to the number of structures formed in the microchannels of the biosensor according to Example 1 was measured using absorbance at a wavelength of about 600 nm. The results are shown in FIG.

As shown in FIG. 8 (a), when the structure is present in the microchannel, the higher death rate of Staphylococcus aureus can be seen, and as the number of the structures increases, The mortality rate is further increased. Also, as shown in FIG. 8 (b), it can be seen that as the number of structures formed on the microchannels increases, the PDA vesicles are further red. This result is due to the fact that the structure formed in the microchannel increases the reaction time of the resource tapin and the staphylococci, thereby further increasing the detection and killing time of the staphylococci.

The mortality rate of the staphylococci according to the angle of the structure formed in the microchannel of the biosensor according to Example 1 and the flow rate of the staphylococcus solution was measured. The results are shown in FIG.

As shown in FIG. 9, as the angle of the structure tilts at an angle of 30 °, 45 °, and 60 ° with respect to the flow direction of the fluid, the greater the rate of death of the staphylococci and the lower the flow rate of the staphylococcal solution The mortality rate is further increased. This result is due to the fact that the structure formed in the microchannel further interferes with the flow of the fluid, further increasing the reaction time of the resource tapin and the staphylococcus to further increase the detection and death of Staphylococcus aureus.

Experimental Example 5. Detection and Death of Pathogenic Bacteria According to Pathogen-Specific Enzyme

In order to confirm the pathogen-specificity of the biosensor according to Example 1, the mortality was measured after adding E. coli solution instead of staphylococcus, and the results are shown in FIG.

As shown in Fig. 10, it can be seen that E. coli is not killed by the biosensor according to the first embodiment. This result is due to the fact that the resource tapin bound to the PDA vesicle does not react with E. coli. From these results, it can be seen that the biosensor of the present invention is pathogen-specific.

Claims (13)

A substrate on which a microchannel is formed; A discoloring material sensitive to an external stimulus directly coupled to the microchannel; And a pathogen-specific lytic enzyme directly coupled to said chromogenic material. The method according to claim 1,
Wherein the microchannel has an average width of 100 to 1000 占 퐉 and an average depth of 100 to 500 占 퐉. The method according to claim 1,
Wherein the microchannel comprises a circular, elliptical, rectangular, square or polygonal structure. The method according to claim 1,
The substrate may be formed of a material such as polydimethylsiloxane (PDMS), poly methyl methacrylate (PMMA), polyvinyl chloride (PVC), poly carbonate, polyvinyl acrylate (PVA), polytetrafluoroethylene (PTFE) Wherein the biosensor is one selected from the group consisting of polyethylene (PE) and a combination thereof. The method according to claim 1,
Wherein the external stimulus is any one selected from the group consisting of temperature, pressure, light, pH, humidity, solvent, electric field, magnetic field, stress and combinations thereof. For biosensors. The method according to claim 1,
Wherein the color change material is polydiacetylene (PDA) vesicles or gold nanoparticles (GNP). The method according to claim 1,
Wherein said pathogen-specific lytic enzyme is selected from the group consisting of resourcetapine, beta glucuronidase, glucanase, glutathione, lysozyme, lyticase, mannanase, mutanolysin, zymolase, cellulase, peptolytic, streptolysin And combinations thereof. ≪ Desc / Clms Page number 15 > The method according to claim 1,
A biosensor for detecting and killing pathogens, characterized in that the pathogens bind to the pathogen-specific lytic enzyme by physical, chemical or biological interactions. 9. The method of claim 8,
Such pathogens include, but are not limited to, staphylococcus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Serratia marcescens, Streptococcus pneuminiae, Haemophilus influenzae ), Enterobacter cloacae, Enterococcus faecalis, Salmonella, Campylobacter, and combinations thereof. The present invention also provides a method for producing A biosensor for detecting and killing pathogens. (1) fabricating a substrate on which a microchannel is formed;
(2) directly coupling a discoloring substance sensitive to an external stimulus to the microchannel; And
(3) directly binding a pathogen-specific lytic enzyme to the discoloring substance. 11. The method of claim 10,
Wherein the microchannels have an average width of 100 to 1000 占 퐉 and an average depth of 100 to 500 占 퐉. A method for detecting and killing pathogens, comprising applying a fluid comprising a pathogen to a microchannel of the biosensor of any one of claims 1 to 9, and then detecting a color variation. 13. The method of claim 12,
Wherein said method is capable of simultaneously detecting and killing pathogens.

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