KR20160042745A - Biosensor and wearable device for detecting information of living bodies comprising hybrid electronic sheets - Google Patents

Abstract

The present invention relates to a biosensor comprising a hybrid electronic sheet, and to a wearable device to detect bio-information. The biosensor has high electrochemical activities, and is able to detect an analyte in a DET-based sample. In addition, the biosensor uses an electrode harmless to the human body, is able to detect the analyte with high sensitivity and high selectivity, and can efficiently be used for a wearable device to detect bio-information. The biosensor comprises: a substrate; an electronic sheet formed on the substrate; and an analyte coupling material fixated on the electronic sheet.

Description

TECHNICAL FIELD [0001] The present invention relates to a biosensor including a hybrid electronic sheet and a wearable device for detecting biological information,

A biosensor including a hybrid electronic sheet, and a wearable device for biometric information detection.

Life expectancy is steadily increasing due to improvement of quality of life and medical technology, and interest and investment in self - diagnosis of health condition is increasing actively due to increase of elderly population.

In the biosensor field for self-diagnosis, it is not a method of extracting blood in an invasive manner, but a sensor worn by a patient continuously measures the concentration of glucose using the patient's body fluids (saliva, sweat, tears, etc.) A wearable biosensor capable of being transmitted to an external device via wireless communication has been attracting attention. Since the concentration of glucose in body fluids such as tears is 10 to 20 times lower than that of a normal blood phase, in order to develop a wearable biosensor that effectively measures low glucose levels, a higher sensitivity than a sensor for measuring blood glucose concentration A sensor of the type is required. In addition, since it must be attached to the human body, the size of the sensor must be greatly reduced by using a patternable material, and the characteristics should also be improved in terms of flexibility, transparency, and surface adhesion .

As an effort to fabricate a biosensor having improved characteristics, a 3rd generation biosensor, which directly detects the change of the redox state of the reagent immobilized on the surface of the sensor without a mediator, is receiving attention. In the case of the glucose sensor using the third-generation biosensor concept, direct electron transfer (DET) occurs, thereby effectively excluding the disturbing substances AA and UA. In addition, the effect of glucose oxidase can be developed in terms of the effect and accuracy of the sensor, since the reaction with glucose can be directly transferred to the electrode directly by DET without any mediator.

However, in order to manufacture such a DET-based third-generation biosensor, it is necessary to build a sensor platform using nanomaterials that require complicated processes, which results in poor reproducibility and difficulty in controlling the sensitivity of the sensor. in need. Furthermore, in order to implement a DET-based third-generation biosensor in a form that can be attached to a human body, nanomaterials must be patterned. In addition, the measurement method should be able to operate not only on the reference electrode which can dissolve in the body fluid but on the reference electrode which is harmless to the human body. Accordingly, there is a need for a biosensor in which manufacturing cost is reduced, patterning is possible, and sensitivity of the sensor is improved.

One aspect includes a substrate; An electronic sheet formed on the substrate; And an analyte binding material immobilized on the electronic sheet, wherein the electronic sheet comprises a graphical material and a phage coupled to the graphical material, wherein the combination of the graphical material and the phage is selected from the group consisting of a sheath of the phage And a biosensor comprising a protein or a peptide displayed on the fragment and the graft material.

Another aspect provides a wearable device for biometric information detection including the biosensor.

One aspect includes a substrate; An electronic sheet formed on the substrate; And an analyte binding material immobilized on the electronic sheet, wherein the electronic sheet comprises a graphical material and a phage coupled to the graphical material, wherein the combination of the graphical material and the phage is selected from the group consisting of a sheath of the phage And a biosensor comprising a protein or a peptide displayed on the fragment and the graft material.

Referring to FIG. 1, a substrate and an electronic sheet formed on the substrate are included in the biosensor. 1A, the electronic sheet 20 can be transferred onto the substrate 10, and the electronic sheet 20 transferred onto the substrate 10, as shown in FIGS. 1B and 1C, May be formed. The electronic sheet 20 may be patterned without a chemical etching process. The substrate may be a conductive substrate or an insulating substrate, and may be an insulating substrate on which at least one electrode 200 is disposed, as shown in FIG. 1D. The substrate may be a conductive substrate or an insulating substrate, and may be an insulating substrate on which at least one electrode is disposed. The at least one electrode may be a first electrode, a second electrode, or a third electrode, and may be a working electrode, a counter electrode, or a reference electrode. In addition to the working electrode, the counter electrode, or the reference electrode, the electrode may further include an auxiliary electrode and a recognition electrode. When the electronic sheet is formed on an insulating substrate on which at least one electrode is disposed, the electronic sheet may be disposed on the first electrode, the working electrode, or a part thereof.

The biosensor may further include a second substrate facing the substrate. The second substrate may be the same as or different from the substrate. When the second substrate is further included, the first electrode and the second electrode may be opposed to each other.

Examples of the substrate may include silver, silver, silver, silver, silver, silver, silver, silver, silver, silver, silver, silver, silver, silver, silver, Examples of the electrode may be silver, silver, epoxy, palladium, copper, gold, platinum, silver / silver chloride, silver / silver ion, or mercury / silver oxide.

Further, the substrate may be a transparent flexible substrate. Examples of transparent flexible substrates include polydimethylsiloxane (PDMS), polyethersulfone (PES), poly (3,4-ethylenedioxythiophene), poly (styrene sulfoxide) Polyurethane, polyester, perfluoropolyether (PFPE), polycarbonate, or a combination of the above polymers, for example, from poly (styrenesulfonate), polyimide, polyurethane, And may be a manufactured substrate.

The electronic sheet may comprise a graphical material and a phage coupled to the graphical material, wherein the binding is between a peptide displayed on the envelope protein of the phage or a fragment thereof and the graphical material.

As used herein, the term "sheet" may refer to a material having a certain width and thickness, and may be understood to include, for example, a film, web, membrane, or composite structure thereof.

In addition, the electronic sheet may be patterned using a substrate or a mask, and a person skilled in the art can form a pattern on the electronic sheet according to the intended use.

The area of the electronic sheet may be, for example, 0.0001 to 1000 cm ² , 0.0001 to 100 cm ² , or 1 to 20 cm ² , and the thickness may be, for example, 20 to 400 nm, 40 to 200, 100 nm. In addition, the internal structure of the graphitic material may have a percolated network structure. As used herein, the term " percolated network "may refer to a lattice structure composed of randomly conducting or non-conducting connections.

As used herein, the term "graphitic materials " may refer to a material having a graphitic surface, in which the carbon atoms are arranged in a hexagonal shape, It can be included in a graphical material regardless of its physical, chemical and structural properties. The graphitic material may be, for example, a graphene sheet, a highly oriented pyrolytic graphite (HOPG) sheet, a single-walled carbon nanotube and double-walled carbon nanotubes, multi-walled carbon nanotubes), or fullerenes. The graphitic material may be a metallic, semiconductive or hybrid material. For example, a mixture of a graphene sheet and a single-walled carbon nanotube may be used.

The peptide having specific binding ability to the above-mentioned graphitic substance is X ₂ SX ₁ AAX ₂ X ₃ P (SEQ ID NO: 1), X ₂ X ₂ PX ₃ X ₂ AX ₃ P (SEQ ID NO: 2), SX ₁ AAX ₂ X ₃ P (SEQ ID NO: 3) and X ₂ PX ₃ X ₂ AX ₃ P (SEQ ID NO: 4). In addition, the peptide may be a peptide or a peptide set comprising at least one selected from the group consisting of the amino acid sequences of SEQ ID NOS: 5 to 8. The N-terminal or C-terminal end of the amino acid sequence of the peptide or peptide set may be linked to the consecutive amino acid sequence of the phage coat protein. Thus, for example, the set of peptides or peptides may comprise a sequence of 5 to 60 amino acids, 7 to 55 amino acids, 7 to 40 amino acids, 7 to 30 amino acids, 7 to 20 amino acids, 7 to 10 amino acid sequences.

The peptide may be one which comprises a conservative substitution of the disclosed peptide. As used herein, the term "conservative substitution" refers to substitution of a first amino acid residue with a second, different amino acid residue, wherein the first and second amino acid residues have a side chain with similar biophysical characteristics It can mean. Similar biophysical properties may include the ability to provide or accept hydrophobic, charge, polar, or hydrogen bonds. Examples of conservative substitutions include, but are not limited to, basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, valine and methionine), hydrophilic amino acids Alanine, serine and threonine), aromatic amino acids (phenylalanine, tryptophan, tyrosine and histidine), and small amino acids (glycine, alanine, serine and threonine). In general, amino acid substitutions that do not alter specific activity are known in the art. Thus, for example, in the peptide X ₁ can be W, Y, F or H, X ₂ can be D, E, N or Q, and X ₃ can be I, L or V.

Peptides that bind to the graphitic agent can be selected through a library of peptides and can be selected, for example, through phage display techniques. The phage display technique allows the peptide to be displayed on the outside of the phage genetically linked, inserted, or substituted into the phage's coat protein, and the peptide can be encoded by the genetic information in the virion. By screening the proteins of various variants by the displayed proteins and the DNA encoding them, they can be screened and called "biopanning". Briefly, the bio-panning technique involves reacting a displayed phage with an immobilized target (e.g., a graphical material), washing the unbound phage, and then destroying the binding interaction between the phage and the target, And a method of eluting the combined phage. A portion of the eluted phage can be left for DNA sequencing and peptide identification, and the remainder can be amplified in vivo and a sub-library for the next round can be generated and repeated.

The term " phage "or" bacteriophage "is used interchangeably and can refer to a virus that infects bacteria and replicates in bacteria. Phage or bacteriophage may be used to display peptides that selectively or specifically bind to a graphical substance or a volatile organic compound. The phage may be one that has been genetically engineered such that a peptide capable of binding to the graphical substance is displayed on the envelope protein or fragment thereof of the phage. The term "genetic engineering" or "genetically engineered" in the context of the present invention is intended to include peptides having a binding capacity to a glycotic material, May refer to the act of introducing a genetic modification or a phage created thereby. The genetic modification includes introducing a foreign gene encoding the peptide. The phage may be a filamentous phage, for example, M13 phage, F1 phage, Fd phage, If1 phage, Ike phage, Zj / Z phage, Ff phage, Xf phage, Pf1 phage, or Pf3 It can be a phage.

The term "phage display " in the present invention may refer to the display of functional foreign peptides or proteins on the surface of phage or phagemid particles. The surface of the phage may mean the envelope protein of the phage or a fragment thereof. Also, the phage may be linked to the N-terminus of the phage coat protein of the phage, or the C-terminus of the functional peptide may be inserted between consecutive amino acid sequences of the phage coat protein of the phage or the consecutive amino acid sequence of the coat protein Which is a part of the phage. The position of the consecutive amino acid sequence in which the peptide is inserted or substituted in the coat protein is selected from the N-terminal of the coat protein at positions 1 to 50, positions 1 to 40, positions 1 to 30, positions 1 to 20, Position 10, position 2 to 8, position 2 to 4, position 2 to 3, position 3 to 4, or position 2. In addition, the envelope protein may be p3, p6, p8 or p9. For example, the C-terminal of any one of SEQ ID NO: 1 to SEQ ID NO: 8 may be linked to the N-terminal of p8 (SEQ ID NO: 14) of 50 amino acid residues present in the trunk of the M13 phage. For example, when the peptide of any one of SEQ ID NOS: 1 to 8 is an amino acid sequence (i.e., EGD) at position 2 to 4 of the coat protein p8 of M13 phage, position 2 to 3, position 3 to 4 Position, or in place of the amino acid sequence of position 2.

In one embodiment, the hybrid electronic sheet has excellent non-destructive binding ability to display a phage with a non-destructive binding ability, so that its electrical characteristics are excellent, but its characteristics are suitably controllable as required, and has semiconducting properties.

In another embodiment, the hybrid electronic sheet is structurally stable, transparent, flexible and can be transferred to various substrates or non-conventional substrates, as well as various patterning using a substrate or mask. .

In another embodiment, the hybrid electronic sheet is hybridized with a phage and has good affinity with a biomaterial, and further, can be functionalized with another biomaterial (for example, an analyte binding material).

Referring to FIG. 2A, a biosensor according to an embodiment includes an analyte binding material 100 immobilized on the electronic sheet.

In the present invention, the term "analyte binding materials" or " analyte binding reagents "are used interchangeably and refer to substances or analytes capable of imparting functionalization to electronic sheets- The analyte binding material may include a redox enzyme. The redox enzyme may mean an enzyme that oxidizes or reduces a substrate, and may be, for example, an oxidase Examples of the oxidoreductase include glucose oxidase, lactate oxidase, cholesterol oxidase, glutamate oxidase, horseradish peroxidase (HRP), alcohol oxidase, glucose (glucose oxidase), glucose oxidase Glucose oxidase (GOx), glucose dehydrogenase (GDH), cholesterol (S) may include an esterase, an esterase, an esterase, a ribose esterase, an esterase, an esterase, an esterase, an esterase, a esterase, a esterase, a ascorbic acid oxidase, an alcohol dehydrogenase, a laccase, a tyrosinase, a galactose oxidase or a bilirubin oxidase The analyte binding material may be immobilized on an electronic sheet and the term "immobilized" may refer to a chemical or physical bond between the analyte binding material and the electronic sheet.

As used herein, the term "analyte" may refer to a material of interest that may be present in a sample. Detectable analytes may include those that may be involved in a specific-binding interaction with one or more analyte binding substances that are capable of participating in a sandwich, competition, or displacement assay configuration. Examples of the sample may include blood, body fluids, cerebrospinal fluid, urine, manure, saliva, tears, sweat, and the like. Examples of analytes include, but are not limited to, sequence-specific hybridization reactions with antigen or haptens such as peptides (e.g., hormones), proteins (e.g., enzymes), carbohydrates, proteins, drugs, pesticides, microbes, antibodies, And may include nucleic acids that are capable of participating. More specific examples of such analytes may include glucose, cholesterol, lactate, hydrogen peroxide, catechol, tyrosine or galactose.

Referring to FIG. 2B, the biosensor may further include a protective layer 50 formed on the immobilized analyte binding material layer 100. The protective layer 50 may be used for protection of the biosensor, and may be any known to those of ordinary skill in the art or may be used as is apparent from the general knowledge in the art. For example, the protective layer 50 can be a tetrafluoroethylene-based copolymer, Nafion ( ^R ) or a second electronic sheet.

In addition, the electronic sheet or the protective layer may be modified such that the surface of the electronic sheet or the protective layer, which is in contact with the analyte binding material, has a positive charge or a negative charge opposite to that of the analyte binding material.

Referring to FIG. 2C, FIG. 2C shows that the surface of the electronic sheet 20 or the protective layer 50 is modified with a positive charge polymer or a negative charge polymer 30 so as to have a positive charge or a negative charge. When the orientation of the analyte binding material 100 is negative, the positively charged polymer 30 can be used to modify the analyte binding material 100 contact surface of the electronic sheet 20 or the protective layer 50 to a positive charge And the surface of the electronic sheet 20 or the protective layer 50 can be negatively charged by using the negative charge polymer 30 when the analyte binding material 100 has a positive charge. The surface of the electronic sheet 20 or the protective layer 50 may be modified with the positive charge polymer 30 and then the negative charge polymer 30 may be modified to form the negative charge polymer 30, The analyte binding material 100 having a positive charge tendency can be immobilized or bonded on the electronic sheet 20. [

Examples of the positive charge polymer include PAH (poly (allyamine), PDDA (poly (dimethyliminammonium)), PEI (polyimide), or PAMPDDA (acrylamide-co-diallyldimethylammonium) Examples of which are PSS (poly (4-styrenesulfonate), PAA (poly (acrylic acid), PAM (poly (acryl amide), poly (vinylphosphonic acid) propanesulfonic acid), PATS (poly (anetholesulfonic acid)), or PVS (poly (vinyl sulfate)).

2D, the biosensor may have a structure in which a plurality of repeating units including the electronic sheet 20 and the analyte binding material 100 or the analyte binding layer 40 are stacked. The repeating unit may be two or more, three or more, four or more, five or more, six or more, seven or more, or eight or more. In one embodiment, a biosensor having a structure in which a plurality of repeating units are stacked can increase the sensitivity of the sensor as compared with a single-layer structure.

Referring to FIG. 2E, FIG. 2E is a view illustrating that an electronic sheet on which an analyte binding material is immobilized is formed on an insulating substrate on which at least one electrode is disposed as shown in FIG. 1D. The at least one electrode may be a first electrode, a second electrode, or a third electrode, and may be a working electrode, a counter electrode, or a reference electrode. In addition to the working electrode, the counter electrode, or the reference electrode, the electrode may further include an auxiliary electrode and a recognition electrode. When the electronic sheet is formed on an insulating substrate on which at least one electrode is disposed, the electronic sheet may be disposed on the first electrode, the working electrode, or a part thereof.

The biosensor may further comprise a test cell for receiving the sample, the electronic sheet and the analyte binding material, and the test cell may be provided with a channel including an inlet or an outlet for injecting or discharging the sample.

3 to 8, a biosensor 60 according to an embodiment includes a channel formed on a substrate 10 on which a working electrode WE, a counter electrode CE, and a reference electrode RE are disposed. And may include a test cell 610 provided therein. The test cell 610 may be covered with a cover 60. The test cell 610 has an inlet 611 through which a sample is injected or an outlet 612 through which a sample is discharged. A sample enters through the inlet 611 and the analyte present in the sample undergoes a redox reaction with the analyte binding material 100 to cause a chemical potential gradient in the test cell. The term " chemical potential gradient "can refer to the concentration gradient of the redox active species. When such a gradient exists between the two electrodes, the potential difference will be detected when the circuit is opened, and the current will flow until the circuit is closed, until the slope is eliminated. The chemical potential gradient may refer to any potential gradient arising from the application of a potential difference or current flow between such electrodes arising from the asymmetry of the distribution of the redox enzyme (e. G., Analyte binding material). In the biosensor according to one embodiment, a strong redox reaction peak appears at the working electrode to which the electronic sheet 20 is transferred, and almost no redox peak appears at the non-electrode. Therefore, as shown in Fig. 9, in the biosensor according to one embodiment, the movement of electrons by the oxidation-reduction reaction of the analyte and the analyte binding material 100 is carried out without a medium It can be directly occurring (DET).

The channel in the test cell may be modified to facilitate capillary action of the sample. The modification may be modified with a hydrophobic substance. Examples of hydrophobic materials include, but are not limited to, glyceride, polystyrene, polymethylmethacrylate (PMMA), polyethylene terephthalate (PET), polyvinyl chloride (PVC), polyethylene (PE), polypropylene Poly (L-lactic acid) (PLLA), poly (D, L-lactic acid) (PDLLA), poly (glycolic acid) (PGA), and the like are used as the fluorine-ethylene (PTFE), silicon compound, wax, wax emulsion, ), Poly (caprolactone) (PCL), poly (hydroxyalkanoate), polydioxanone (PDS), or polytrimethylene carbonate, or poly (lactic acid-co-glycolic acid) ), Poly (L-lactic acid-co-caprolactone) (PLCL), or poly (glycolic acid-co-caprolactone) (PGCL).

The biosensor according to one embodiment may further include a meter for determination of the analyte. In the present invention, the term " determination of an analyte "may refer to qualitative, semi-quantitative and quantitative processes for evaluating a sample. In a qualitative evaluation, the results indicate whether an analyte is detected in the sample. In an semi-quantitative evaluation, the result indicates whether the analyte is present above a predefined threshold value. In quantitative evaluation, the result is a numerical indication of the amount of analyte present.

The measuring device may include an electronic device for measuring a potential difference or current at a predetermined time after introduction of the sample, and for converting the measured value into the displayed value. The measurement of the potential difference or the current may be performed by using cyclic voltammetry (CV) to determine the oxidation current reaction voltage value. The cyclic voltammetric method may be a method of measuring current by circulating the potential of the first electrode (for example, working electrode) at a constant rate. The conversion of the measured values may also use a look-up table that converts the specific value of the current or potential to a value of the analyte depending on the specific device structure and the correction value for the analyte. In addition, the meter may further include a frame having a display for displaying results and one or more control interfaces (e.g., power button, scroll wheel, etc.). The frame may include a slot for receiving the biosensor. Inside the frame, there may be a circuit for applying potential or current to the electrodes of the biosensor when the sample is provided. A suitable circuit that may be used in the meter may be, for example, an ideal voltage meter capable of measuring the potential across the electrode. A switch that is open when the potential is measured or closed for measuring current is also provided. The switch may be a mechanical switch (e.g., a relay) or a FET (field-effect transistor) switch, or a solid-state switch. This circuit can be used to measure potential difference or current difference. As can be appreciated by those skilled in the art, other circuits, including simpler and more complex circuits, can be used to achieve potential difference or current or both.

Another aspect includes a substrate; An electronic sheet formed on the substrate; And an analyte binding material immobilized on the electronic sheet, wherein the electronic sheet comprises a graphical material and a phage coupled to the graphical material, wherein the combination of the graphical material and the phage is selected from the group consisting of a sheath of the phage And a biosensor comprising a protein or a peptide displayed on the fragment and the graphitic substance.

The biosensor is as described above.

The wearable device may be one for detecting biometric information. The wearable device may be a patch, a watch, a contact lens, or the like.

The term "contact lens" may refer to any ophthalmic or cosmetic device that may be present in or on the eye. For example, the contact lens may be an intraocular lens, an overlay lens, an ocular insert, a tear point stopper, or a device that can improve or prevent the condition of the eye, where vision can be corrected or corrected, and / And other similar devices whose physiology can be improved cosmetically (e.g., iris color). The contact lens may comprise a soft contact lens comprised of a silicone elastomer or hydrogel (e. G., A silicone hydrogel) and a fluorohydrogel.

The contact lens for biometric information detection according to an embodiment further includes a controller for receiving and processing signal data generated from the biosensor in order to logically communicate with the biosensor and output data related to the control of the biosensor .

The biosensor in the contact lens for detecting biometric information is controlled by the controller so that the biosensor responds to the biosensor at a predetermined time interval or in response to a specific event (for example, a significant decrease or increase in glucose in the leakage) It is possible to receive and process the detected biometric information.

 The contact lens for biometric information detection may further include a memory capable of storing a processor for operation of the controller and temporarily storing input / output data (for example, biometric information). The memory may store information on an analyte (e.g., glucose) in the leakage fluid detected from the biosensor.

Further, the contact lens for biometric information detection transmits information processed by the controller or information stored in the memory to a contact lens wearer or another user (e.g., a doctor, a hospital, a wearer's family, etc.) having a wireless communication system The wireless communication unit may further include a wireless communication unit. For example, the wireless communication unit may include a broadcast receiving module, a mobile communication module, a wireless Internet module, and a short-range communication module. The information on the analyte in the leakage fluid detected by the biosensor can be transmitted to the wearer or another user through the wireless communication unit.

The contact lens for biometric information detection may further include an energy source capable of supplying energy or capable of putting the device into an active state. The energy source may be, for example, a lithium ion battery.

It may also be embedded in the contact lens via the biosensor, the controller, the memory, the wireless communication portion, or the energy source media insert, or may be attached on the surface of the contact lens.

In a wearable device according to a specific embodiment, the biosensor has remarkable electrochemical characteristics on a transparent and flexible substrate and on an electrode harmless to the human body. In addition, the biosensor does not need a mediator that is harmful to the human body and is remarkably sensitive enough to detect a small amount of analyte in the sample, and thus can be usefully used in a wearable device (for example, a contact lens for detecting biological information) have.

The biosensor according to one aspect has a high electrochemical activity, and it is possible to detect an analyte in a DET-based sample.

A wearable device according to one aspect has high sensitivity and high selectivity to analytes while being harmless to the human body, so it is possible to detect a small amount of analytes in a sample by a non-invasive method.

FIG. 1 is a diagram showing an electrode including a hybrid electronic sheet according to one embodiment.
FIG. 2 is a schematic diagram of an electrode including a hybrid electronic sheet on which an analyte binding material according to one embodiment is immobilized. FIG.
3 is a diagram illustrating a biosensor according to an embodiment of the present invention.
4 is a diagram illustrating a Y-axis cross-sectional view of a biosensor according to one embodiment.
5 is a diagram illustrating a cross-sectional view of an X-axis of a biosensor according to one embodiment.
6 is a perspective view of a cover of a biosensor according to one embodiment.
7 is a diagram showing a cross-sectional view of a biosensor according to one embodiment.
FIG. 8 is a diagram illustrating a cross-sectional perspective view of a cover of a biosensor according to one embodiment.
9 is a conceptual diagram showing a DET reaction principle on a hybrid electronic sheet of an analyte binding material according to one embodiment.
10A is a diagram illustrating a manufacturing process of a hybrid electronic sheet according to one embodiment.
Fig. 10B is a diagram showing the principle of formation of the hybrid electronic sheet according to one embodiment.
FIG. 10C is a graph showing the concentration polarization phenomenon among the formation principle of the hybrid electronic sheet according to one embodiment.
11 is a view showing an image of a large area free standing hybrid electronic sheet according to one embodiment.
12 is a view showing an image of a sample having only single-walled carbon nanotubes to which no phage is bonded.
13 is a scanning electron microscopy (SEM) image showing a nanostructure of a hybrid electronic sheet to which a phage is coupled and an electronic sheet to which a phage is not combined according to an embodiment.
FIG. 14 is a graph showing electrochemical characteristics of a hybrid electrode according to one embodiment.
FIG. 15 is a diagram illustrating a process of fabricating a transparent and flexible biosensor based on a hybrid electronic sheet-GOx having a multilayer structure according to an embodiment.
FIG. 16 is a CV graph showing a direct electron transfer (DET) reaction of a hybrid electronic sheet-GOx-based biosensor according to an embodiment in comparison with a GOx electrode formed on a gold electrode without a hybrid electronic sheet.
17 is a graph comparing the current response of glucose to a mixture of glucose and ascorbic acid and uric acid in a hybrid electronic sheet-GOx-based biosensor having a single layer structure according to an embodiment .
FIG. 18 is a graph showing a pure DET current response change according to a voltage scanning speed of a hybrid electronic sheet-GOx-based biosensor having a single-layer structure according to an embodiment.
19A is a graph showing that the degree of DET redox reaction of GOx increases as the immobilized GOx concentration is increased when immobilizing different concentrations of GOx on the hybrid electronic sheet according to one embodiment.
FIG. 19B is a graph showing that as the concentration of GOx immobilized on the hybrid electronic sheet according to one embodiment increases, the response of the current to the glucose concentration change increases, thereby increasing the sensitivity of the sensor.
20A is a graph showing that a multilayered hybrid electronic sheet-GOx-based biosensor according to an embodiment has a higher DET reduction current than a single-layer structure.
FIG. 20B is a graph showing that the sensitivity of the sensor in the case of multiple stacking can be increased because the sensitivity of the hybrid electronic sheet-GOx-based biosensor according to an embodiment to the glucose concentration is increased as compared with the single layer structure.
FIG. 21 is a graph showing a result of measuring the sensitivity of a hybrid electronic sheet-GOx-based biosensor having a multi-layer structure according to an embodiment on a reference electrode which is harmless to the human body.
22 is a diagram showing the sensitivity and flexibility of a transparent and flexible biosensor based on a hybrid electronic sheet-GOx of a multilayer structure according to an embodiment.
23A is a graph showing sensitivity of a hybrid electronic sheet-cholesterol oxidase based biosensor according to an embodiment.
23B is a graph showing the sensitivity of a hybrid electronic sheet-lactate oxidase based biosensor according to an embodiment.
24A is a graph showing the sensitivity of a hybrid electronic sheet-HRP-based biosensor according to an embodiment.
24B is a graph showing sensitivity of a hybrid electronic sheet-catalase-based biosensor according to an embodiment.
25A is a graph showing the sensitivity of a hybrid electronic sheet-galactose oxidase-based biosensor according to an embodiment.
25B is a graph showing the sensitivity of a hybrid electronic sheet-tyrosinase-based biosensor according to an embodiment.
25C is a graph showing the sensitivity of the hybrid electronic sheet-lacquer-based biosensor according to one embodiment.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1. Preparation and Characterization of Biosensor

1. Fabrication of electrode with hybrid electronic sheet

1.1. Preparation of colloidal solution

First, an aqueous solution was prepared by adding sodium surfactant, sodium cholate, to the distilled water at a concentration of 2% w / v. Then, carbon nanotubes (Nanointegris, SuperPure SWNTs, ml) was dialyzed for 48 hours to prepare a colloidal solution in which single-walled carbon nanotubes were stabilized with sodium cholate.

Assuming that the average length of carbon nanotubes (CNTs) is 1 μm and the average diameter is 1.4 nm, the calculation formula of the number of single-walled carbon nanotubes contained in the colloid solution is as follows.

[Equation 1]

Number of single-walled carbon nanotubes (dog / ml) = concentration / / ml X 3 X 10 ¹¹ CNT

According to the above equation, the number of single-walled carbon nanotubes contained in the colloidal solution is (7.5 × 10 ¹³ ) number / ml.

1.2. Preparation of a phage displaying a peptide capable of binding to a graphitic substance

(P8GB # 1) displaying DNWIADP (SEQ ID NO: 5) and a phage displaying a DNPIQAVP (SEQ ID NO: 6) (p8GB # 5), SWAADIP (SEQ ID NO: 7), and NPIQAVP (SEQ ID NO: 8) were produced in the following manner.

For the site-directed mutation of C in G1381 base pair of the M13KE vector (NEB, product # N0316S) (SEQ ID NO: 9), oligonucleotides of SEQ ID NOs: 10 and 11 Were used to construct the M13HK vector. The prepared M13HK vector was double-digested using restriction enzymes BspHI (NEB, product # R0517S) and BamHI restriction enzyme (NEB, product # R3136T), and was then digested with antarctic phosphatase Lt; / RTI > The dephosphorylated vector was incubated with double-cut DNA duplex overnight at 16 ° C. The product was then purified and concentrated. Electrocompetent cells (XL-1 Blue, Stratagene) were transformed by electroporation into 2 μl of concentrated, coupled vector solution at 18 kV / cml and a total of 5 transformations were performed for library construction Respectively. Subsequently, the transformed cells were cultured for 60 minutes, and the fraction of the multiple transformants was transformed with X-gal / isopropyl-beta-D-1-thiogalactopyranoside (IPTG) / tetracycline (Tet) ≪ / RTI > to determine the library ' s diversity. Remaining cells were amplified in shaking incubator for 8 hours. Oligonucleotides of SEQ ID NOS: 12 and 13 were used to construct the phage display p8 peptide library.

The base sequence of the phage display p8 peptide library prepared according to one embodiment has 4.8 x 10 ⁷ pfu (plaque form unit) kinds of diversity and has a copy number of about 1.3 × 10 ⁵ per each sequence .

Then, a highly ordered pyrolytic graphite (HOPG) substrate (manufacturer: SPI product # 439HP-AB) having a diameter of 1 cm was prepared. At this time, a relatively large HOPG substrate having a grain size of 100 μm or less was used as the HOPG substrate. Conventionally, since the surface of the carbon nanotube film, which is mostly damaged by the surface during the production process, is used as a graphical surface, it is difficult to derive a peptide having high binding strength. In order to compensate for this, HOPG, a material with a graphical surface, was peeled off the substrate from the substrate before the experiment to obtain a fresh surface to minimize defects such as oxidation of the sample surface. Then, a phage display p8 peptide library of 4.8 × 10 ¹⁰ pfu (4.8 × 10 ⁷ kinds of replicates, 1000 replications per each sequence) prepared above was prepared in 100 μL of Tris-buffered saline (TBS) buffer, For 1 hour at 100 rpm in a shaking incubator. After 1 hour, the solution was removed and then washed 10 times in TBS. The washed HOPG surface was reacted with Tris-HCl (pH 2.2) as an acid buffer solution for 8 minutes to elute the non-selectively reacting peptide, and then the peptide was eluted with a mid-log XL-1 blue E.I. coli < / RTI > culture for 30 minutes. A portion of the eluted culture was left for DNA sequencing and peptide identification and the rest amplified to create a sub-library for the next round. The above procedure was repeated using the created sub-library. (P8GB # 1), DNPIQAVP (SEQ ID NO: 5) displaying a peptide sequence having strong binding ability to a graphical substance (SEQ ID NO: 5), and the like were analyzed by DNA analysis to obtain p8 peptide sequences. (P8GB # 5), SWAADIP (SEQ ID NO: 7), and NPIQAVP (SEQ ID NO: 8) displayed in SEQ ID NO: 6 were displayed.

1.3 Manufacture of electrode with hybrid electronic sheet

The colloidal solution prepared above and a phage solution containing M13 phage (p8GB # 1) having strong binding force with the graphite surface were mixed at a molar ratio of 4: 1. The mixture was then placed in a tube of semipermeable dialysis membrane (SpectrumLab, MWCO 12,000 ~ 14,000, product # 132 700) for dialysis, and each membrane tube was dialyzed against the third distilled water. After about 16 hours from the start of the dialysis, a thin electronic sheet was formed along the surface of the membrane tube. Then, each membrane tube was transferred into third distilled water, and the membrane of the membrane tube was twisted to remove the electronic sheet and dried. The thickness of the prepared electronic sheet was about 100 nm. An image of the electronic sheet mixed at a molar ratio of 4: 1 in the formed electronic sheets is shown in Fig. Then, the prepared free standing hybrid electronic sheet film was placed on a 4 mm diameter gold electrode (SPE 250BT, DropSens) commercialized using a stencil mask having a desired 4 mm diameter pattern, Lt; / RTI > The hybrid film transferred onto the gold electrode was washed with deionized water after removing the stencil mask, and then dried using nitrogen gas. The thickness of the hybrid film thus produced was about 200 nm.

Further, as a comparative example of the present invention, an electronic sheet containing no phage was prepared as follows. First, an aqueous solution prepared by adding sodium cholate as a surfactant to a distilled water at a concentration of 2% w / v was prepared. Then, a single-wall carbon nanotube (manufactured by Nanointegris, SuperPure SWNTs, ml) was dialyzed for 48 hours to stabilize the single-walled carbon nanotubes with sodium cholate to prepare a colloidal solution. Next, 0.4 ml of the colloidal solution was diluted in 10 ml of 1% w / v sodium cholate aqueous solution in a tube of semipermeable dialysis membrane (SpectrumLab, MWCO 12,000 ~ 14,000, product # 132 700) for dialysis, The membrane tube was placed in the third distilled water and dialyzed. After about 24 hours from the start of dialysis, an electronic sheet was formed along the surface of the membrane tube. Then, the membrane tube was transferred into the third distilled water, and the membrane of the membrane tube was twisted to peel off the electronic sheet. A photograph of the removed electronic sheet and a scanning electron microscope image are shown in Fig. 12 and compared with Fig. 11, which is a hybrid electronic sheet with a grip. In addition, the nanostructures of the hybrid electronic sheet to which the phage was bonded and the electronic sheet to which the phage was not bonded were photographed by a scanning electron microscope, and the results are shown in FIG.

10A is a diagram illustrating a manufacturing process of a hybrid electronic sheet according to one embodiment.

As shown in FIG. 10 (a), it can be seen that the carbon nanotube is dispersed or dissolved in the colloidal material stabilized by adding it to the solution containing the surfactant. It can be confirmed that the single-walled carbon nanotube is combined with about one M13 phage to finally form a sheet having a structure of carbon nano-view and M13 phage percolated network.

Fig. 10B is a diagram showing the principle of formation of the hybrid electronic sheet according to one embodiment.

10C is a graph showing a concentration polarization phenomenon in the principle of forming a hybrid electronic sheet according to an embodiment.

As shown in FIGS. 10B and 10C, the formation of M13 phage-bound carbon nanotubes displaying a peptide according to one embodiment is formed by dialysis against a dialysis solution by adding a mixture of a phage solution and a colloid solution in a membrane tube . During the dialysis process, the concentration of carbon nanotubes on the surface of the colloidal material stabilizes the concentration of the surfactant in the tube due to the diffusion effect due to the concentration difference between the inside and the outside of the membrane. The neighborhood is the largest. On the other hand, since the M13 phage displaying a peptide having a strong binding force with the carbon nanotubes can start to react with the carbon nanotubes when the concentration of the surfactant surrounding the carbon nanobeads is lowered, The phenomenon occurs near the most active membrane. With this principle, a sheet can be formed using a dialysis method.

11 is a view showing an image of a large area free standing hybrid electronic sheet according to one embodiment.

12 is a view showing an image of a sample having only single-walled carbon nanotubes to which no phage is bonded.

13 is a scanning electron microscopy (SEM) image showing a nanostructure of a hybrid electronic sheet to which a phage is coupled and an electronic sheet to which a phage is not combined according to an embodiment.

As shown in the drawings, the hybrid electronic sheet combined with the phage according to one embodiment has a structure in which the electronic sheet is stably formed in a wide area by the combination of the carbon nanotubes and the phages, and the nanostructures in which the carbon nanotubes are uniformly dispersed As shown in Fig. On the other hand, it can be seen that the electronic sheet to which the phage is not bonded is shattered in the manufacturing process, and also has a microstructure in the form of a bundle. As a result, the free standing electronic sheet, which is a hybrid electronic sheet to which the phage is bonded according to one embodiment, has a uniformly distributed nanostructure while maintaining its shape due to the strong binding force between the carbon nanotube and the phage, If the dialysis is proceeded, the electronic sheet is formed near the membrane, but it is broken and the application is limited.

1.4. Electrochemical Characterization of Electrode with Hybrid Electronic Sheet

In order to analyze electrochemical characteristics of the electrode prepared in Example 1 1.1.3, an electrode (SPE 250BT, DropSens) including a gold colacator was purchased and used without surface modification as a comparative example.

Specifically, the electrode prepared in Example 1, 1.1.3, and the comparative electrode were used as a working electrode (WE), and a titanium-coated titanium chamber was treated with a counter electrode (CE) ) And 10 mM K ₃ [Fe (CN) ₆ ] (244023, Sigma Aldrich) using Ag / AgCl (3M KCl saturated, PAR, K0260) as a reference electrode pH = 7.4, 79383, Sigma Aldrich). The redox reaction was carried out by cyclic voltammetry (CV). The measurement voltage was applied between -0.2 V and 0.6 V with respect to the reference electrode, and the scan rate was 200 mV / s. The measurement results are shown in FIG. 14A. Also, electrochemical impedance was measured on the open circuit potential (OCP) of the electrochemical system cell by applying 10 mV AC to the electrodes within the range of 100,000 Hz to 0.1 Hz, The results are shown in Fig. 14B.

FIG. 14 is a graph showing electrochemical characteristics of a hybrid electrode according to one embodiment.

As shown in FIG. 14A, it can be seen that the redox current of the electrode to which the hybrid electronic sheet was transferred was increased by about 50% as compared with the gold electrode of the comparative example.

Also, as shown in FIG. 14B, it can be seen that the charge transfer resistance (Rct) measured on the electrode to which the hybrid electronic sheet was transferred was reduced by about 80% as compared with the gold electrode of the comparative example.

As a result, it can be seen that the electrochemical activity of the hybrid electrode according to one embodiment is generally higher than that of the gold electrode, which is generally used in electrochemical experiments. The result shows that the thickness of the hybrid electronic sheet is about 200 nm Together, it means that the hybrid electrode according to one embodiment can be used as a high-performance biosensor.

2. Fabrication and characterization of hybrid electronic sheet-GOx-based biosensor

2.1. Fabrication of single-layered hybrid electronic sheet-GOx-based biosensor

2.1.1. Fabrication and characterization of single-layered hybrid electronic sheet-GOx-based biosensor 1

10 μl of a solution of 6 mg of poly-allyamine hydrochloride (PAH, 43092, MW: 120000 to 200000, Alfa Aesar) having a positive charge tendency on the negative electrode having the negative charge tendency prepared in Example 1, 1.3, After the dropwise addition, it was dried in the air for 1 hour, dried and then washed with deionized water and dried using nitrogen gas. To this solution, 30 mg of GOx (glucose oxidase) was dissolved in 1 mL of PBS buffer, and then 20 μL of the solution was added to the solution, followed by immobilization in a refrigerator at 4 ° C for 12 hours. After immobilization, the plate was carefully washed with 10 mM PBS buffer, and 10 μl of PAH was added dropwise. The plate was dried in air for one hour, dried again, washed again with deionized water, and dried using nitrogen gas. To protect the immobilized GOx layer, 1 占 퐇 of 5% Nafion (70160, Sigma Aldrich) or another hybrid electronic sheet was coated once again as a protective layer.

2.1.2. Fabrication of single-layered hybrid electronic sheet-GOx-based biosensors 2 to 5

In order to analyze the sensitivity characteristics according to the concentration of the immobilized GOx, except for the case where the concentration of GOx was changed to 10 mg / mL, 25 mg / mL, 50 mg / mL or 100 mg / mL, Four additional bio-sensors based on a hybrid electronic sheet-GOx based on a single-layer structure were further fabricated in the same manner as in Example 1.

2.1.3. Production of Comparative Example

A biosensor in which GOx was immobilized was prepared in the same manner as in 2.1.1 except that the hybrid electronic sheet prepared in the above 1.3 was not transferred, and the biosensor immobilized with GOx was prepared as a comparative example of the biosensor according to one specific example Respectively.

2.2. Fabrication of multi-layered hybrid electronic sheet-GOx-based biosensor

The procedure of Example 2.1.1 was repeated once more to form a gold- (hybrid film / PAH / GOx / PAH) ₂ structure. On the last PAH layer, 1 占 퐇 of 5% Nafion (70160, Sigma Aldrich) or another hybrid electronic sheet was coated once again as a protective layer to prepare a multilayered hybrid electronic sheet-GOx-based biosensor.

2.3. Multi-layered hybrid electronic sheet -GOx-based transparent and flexible biosensor fabrication

In order to fabricate a transparent and flexible biosensor based on a hybrid electronic sheet-GOx having a multilayer structure, a process shown in Fig. 15 was made.

Specifically, a 100 nm thick 2 mm x 2 mm platinum electrode was deposited on a 5 cm x 2.5 cm polydimethylsiloxane (PDMS) film over a stencil mask by sputtering. Among the three platinum electrodes, the middle electrode was used as the working electrode (WE), and the left and right electrodes were used as the auxiliary electrode (CE) and the pseudo-reference electrode (RE), respectively. Further, the electronic sheet of Example 1.1.3 was connected to the working electrode using a stencil mask. Then, PEI (polyethyleneimine) having a positive charge tendency was dropped on the electronic sheet, and then 2 μl of 100 mg / ml GOx was immobilized. The above procedure was repeated one more time so that a platinum working electrode on the PDMS had a structure of (GOx / PEI / SWNT film) ₂ / Pt. On the other hand, PDMS was dropped onto a fluidic channel master (1.5 mm (L) x 2.5 mm (W) x 200 um (T)) based on SU-8 manufactured by a photolithography process on a silicon wafer, To make a PDMS cover, and then an inlet and an outlet were formed at both ends of the channel with a diameter of about 0.5 mm. The PDMS fluid cover thus fabricated was superimposed on the PDMS film having the two-layer structure working electrode and the reference electrode and the auxiliary electrode prepared above, and a microfluidic-based transparent flexible glucose sensor was fabricated. The SU-8 substrate may also be treated with a hydrophobic material to facilitate capillary action of the analyte in the channel.

FIG. 15 is a diagram illustrating a process of fabricating a transparent and flexible biosensor based on a hybrid electronic sheet-GOx having a multilayer structure according to an embodiment.

2.4. Comparison of electrochemical properties of single-layered hybrid electronic sheet-GOx-based biosensor 1

The electrode prepared in Example 2.1.1 and the electrode prepared in Comparative Example 2.1.3 were used as a working electrode and a Pt-coated titanium chamber was used as a counter electrode and Ag / AgCl (3M KCl saturated, PAR, K0260 ) Was used as a reference electrode and the solution was used as a electrolyte in a 10 mM PBS buffer (pH = 7.4, 79383, Sigma Aldrich). The measured voltage was applied between -0.6 V and 0.6 V with respect to the Ag / AgCl reference electrode to observe the DET of GOx, and the scanning speed was 200 mV / s. The above results are shown in Fig.

FIG. 16 is a CV graph showing a direct electron transfer (DET) reaction of a hybrid electronic sheet-GOx-based biosensor according to an embodiment in comparison with a GOx electrode formed on a gold electrode without a hybrid electronic sheet.

As shown in FIG. 16, it can be seen that a peak of a strong oxidation-reduction reaction is observed in the -0.4 V region in the case of the GOx electrode transferred with the hybrid electronic sheet having a single-layer structure according to one embodiment. On the other hand, no redox peak was observed in the -0.4 V region in the electrode where the hybrid electronic sheet was not transferred. As a result, it was found that not only the hybrid electronic sheet had high electrochemical activity but also effectively caused the transfer of GOx and electrons (DET) at a position closer to the gold electrode in which the hybrid electronic sheet was not used . In particular, in the case of GOx, the FAD redox center is blocked by a thick protein wall. Therefore, in order to perform effective DET, it is necessary to be located within 1 to 2 nm of the electrode. In the case of a hybrid electronic sheet, by-layer technique shows that GOx is immobilized on a hybrid electronic sheet within a very close range where DET can occur. In addition, a higher capacitive current was observed in the voltage range of -0.6 V to 0.6 V than in the comparative electrode. As a result, the hybrid electronic sheet had a higher surface area as well as higher electrochemical reactivity than the comparative electrode .

2.5. Comparison of electrochemical properties of single-layered hybrid electronic sheet-GOx-based biosensor 2

To determine whether the electrode prepared in Example 2.1.1 can effectively screen for ascorbic acid and uric acid, a 3-electrode system was used in the manner mentioned in 2.4. Above, and 10 mM PBS buffer (pH = 7.4, 79383, (Sigma Aldrich) and 1 mM ascorbic acid (A5963, Sigma Aldrich) were added to the same concentration of the solution (10 mM glucose in 10 mM PBS buffer), and 10 mM glucose (G7528, Sigma Aldrich) Were mixed to prepare a comparative solution. The measurement voltage was applied between -0.6 V and 0.6 V with respect to the Ag / AgCl reference electrode to observe the DET of the GOx, and the scan rate was 200 mV / s. The above results are shown in Fig.

17 is a graph comparing the current response of glucose to a mixture of glucose and ascorbic acid and uric acid in a hybrid electronic sheet-GOx-based biosensor having a single layer structure according to an embodiment .

As shown in FIG. 17, when the 1 mM or 10 mM glucose solution was injected into the electrolyte, it was confirmed that the reduction current was decreased as compared with the result of using 10 mM PBS (0 mM Glucose). As a result, the equilibrium reaction of GOX with FAD-FADH ₂ on the electrode proceeded with 0 mM glucose, and the addition of glucose to the electrolyte progressed FAD to FADH ₂ by the enzyme reaction, It can be seen that the amount of FAD to be consumed is reduced, so that the reduction current at the time of adding glucose is reduced than that at 0 mM glucose. As a result of the addition of 10 mM glucose and the addition of 1 mM ascorbic acid or uric acid on 10 mM glucose, the oxidation reaction of ascorbic acid and uric acid proceeds in the range of 0.2 V and 0.4 V, The oxidation current was detected in the presence of 10 mM glucose in the DET range, compared with the case where pure 10 mM glucose was added. As a result, the DET reaction of GOx detected at negative voltage (-0.4 V) was not influenced by ascorbic acid or uric acid, which is an interfering substance causing electrochemical reaction at positive voltage (0.2 V or 0.4 V) As shown in Fig.

2.6. Comparison of electrochemical properties of single-layered hybrid electronic sheet-GOx-based biosensor 3

To investigate whether the DET reaction of GOx on the electrode prepared in Example 2.1.1 was effectively immobilized and adsorbed on the electrode, a 3-electrode system was used in the manner mentioned in 2.4 above, and a 10 mM PBS buffer (pH = 7.4, 79383, Sigma Aldrich). Electrode properties were tested by applying various scanning speeds (50, 100, 200, 400, 600, 800 and 1000 mV / s) The measured voltage was limited to between -0.1 V and 0.6 V with respect to the Ag / AgCl reference electrode in order to measure pure DET only of GOx. The above results are shown in Fig.

FIG. 18 is a graph showing a pure DET current response change according to a voltage scanning speed of a hybrid electronic sheet-GOx-based biosensor having a single-layer structure according to an embodiment.

As shown in FIG. 18A, it is known that the redox current of DET increases when the scanning speed is changed under the condition of 0 mM glucose, that is, the equilibrium reaction of FAD-FADH ₂ of GOx of the electrode is maintained purely have. Further, as shown in Fig. 18B, it can be seen that both the oxidation current and the reduction current are linearly proportional to the scanning speed (R ² to 0.99). As a result, it can be seen that GOx is adsorbed / immobilized on the hybrid electronic sheet according to one embodiment at a very close distance.

2.7. Analysis of Sensitivity Characteristics of Hybrid Electronic Sheet-GaOx-based Biosensor with Single Layer Structure

To verify the characteristics of the four electrodes prepared in Example 2.1.2 above, a three-electrode system as described in 2.4 above was used, and a 200 mV / s (10 mM) PBS buffer (pH = 7.4, 79383, Sigma Aldrich) The characteristics of the electrodes were tested at the scanning speed of. The measured voltage was limited to between -0.1 V and -0.6 V compared to the Ag / AgCl reference electrode to measure pure DET only of GOx. The above results are shown in Fig.

19A is a graph showing that the degree of DET redox reaction of GOx increases as the immobilized GOx concentration is increased when immobilizing different concentrations of GOx on the hybrid electronic sheet according to one embodiment.

FIG. 19B is a graph showing that as the concentration of GOx immobilized on the hybrid electronic sheet according to one embodiment increases, the response of the current to the glucose concentration change increases, thereby increasing the sensitivity of the sensor.

As shown in FIG. 19A, when four electrodes were tested under the same conditions under the condition of 0 mM glucose, that is, in the state where the equilibrium reaction of FAD-FADH ₂ of the GOx of the electrode was maintained purely, It can be seen that the DET redox current of the GOx immobilized on the hybrid electronic sheet is increased.

As shown in FIG. 19B, when glucose of various concentrations (0.1, 0.25, 0.5, 0.75, and 1 mM) was added to an electrode immobilized with a GOx concentration of 25 mg / mL and 100 mg / mL GOx, (25 mg / mL) of GOx exhibited a sensitivity of about 38 uA / mM cm ² on the immobilized electrode, while the sensitivity of the immobilized electrode of GOx was about 66 uA / mM cm ² , It can be seen that the sensitivity of the sensor can be increased by immobilizing a high concentration of GOx on a hybrid electronic sheet having a high surface area fabricated using SWNT based nanomaterials.

2.8. Analysis of Sensitivity Characteristics of Multi-layered Hybrid Electronic Sheet-GOx-based Biosensor

To measure the sensitivity of the biosensor prepared in Example 2.2, the 3-electrode system described in 2.4 above was used. To the electrolyte of 10 mM PBS buffer (pH = 7.4, 79383, Sigma Aldrich), 200 mV / s The characteristics of the electrodes were tested at the scanning speed. The measured voltage was limited to between -0.1 V and -0.6 V compared to the Ag / AgCl reference electrode to measure pure DET only of GOx. The above results are shown in Fig.

In order to confirm applicability to the human body, a 13 mm ² area of Pt on SPE 250BT instead of Ag / AgCl as a reference electrode was used as a biocompatible pseudo-Pt reference electrode, The results are shown in FIG. 21. FIG.

20A is a graph showing that a multilayered hybrid electronic sheet-GOx-based biosensor according to an embodiment has a higher DET reduction current than a single-layer structure.

FIG. 20B shows that the sensitivity of the sensor in the case of multiple stacking can be increased because the multilayered hybrid electronic sheet-GOx based biosensor according to one embodiment increases sensitivity to glucose concentration changes compared to the single layer structure.

As shown in FIG. 20A, the DET reaction of GOx was increased about 140% as compared with the single layer structure at 0 mM glucose condition.

20B, when the glucose in the range of 0.1, 0.25, 0.5, 0.75, or 1 mM is added, the sensitivity of the multi-layer biosensor is about 81 uA / mM cm ² , The sensitivity of the biosensor is about 38 uA / mM cm ² , which is about 100% higher. As a result, it can be seen that the multilayered hybrid electronic sheet-GOx-based biosensor according to one embodiment can measure the glucose concentration with high sensitivity even in a body fluid such as a tear having a low glucose level.

FIG. 21 is a graph showing a result of measuring the sensitivity of a hybrid electronic sheet-GOx-based biosensor having a multi-layer structure according to an embodiment on a reference electrode which is harmless to the human body.

As shown in Figure 21a, and the value of the reduction current, as the glucose concentration increased to determine that the decrease in sequence, In addition, as shown in Figure 21b, the sensitivity is a Ag / AgCl to about 85 uA / mM cm ² When used, it is almost similar to the measured value of 81 μA / mM cm ² . In addition, the measurement value when adding 0.1 mM ascorbic acid or uric acid on 1 mM glucose showed an error value of only 1% or less as compared with that measured purely on 1 mM glucose, It can be seen that the DET reaction of the hybrid electronic sheet-GOx-based biosensor according to the example works effectively without interfering with ascorbic acid and uric acid.

2.9. Analysis of sensitivity and flexibility of transparent and flexible biosensor based on multi-layered hybrid electronic sheet-GOx

In order to measure the sensitivity of a transparent and flexible biosensor based on the hybrid electronic sheet-GOx based on the multilayer structure fabricated in Example 2.3, the 3-electrode system mentioned in Example 2.4 was used, and a 10 mM PBS buffer (pH = 7.4, 79383, Sigma Aldrich). Electrode properties were tested at a scanning rate of 200 mV / s. The measured voltage was limited to between -0.9 V and -0.2 V relative to the virtual-Pt reference electrode for purely DET measurement of GOx.

In order to analyze the flexibility of the biosensor, the biosensor was placed on a polyimide film, bent at an angle of about 50 ^o using a syringe pump, and CV was measured to determine the pre- CV.

The above results are shown in Fig.

22 is a diagram showing the sensitivity and flexibility of a transparent and flexible biosensor based on a hybrid electronic sheet-GOx of a multilayer structure according to an embodiment.

As shown in FIG. 22A, it can be seen that the CV when bent at an angle of 50 is not changed with the measured CV when it is placed flat. As a result, it can be seen that a transparent and flexible biosensor based on a hybrid electronic sheet-GOx of a multilayer structure according to an embodiment can be usefully used as a wearable device.

Further, as shown in FIG. 22B, when glucose in the range of 0.1, 0.25, 0.5, 0.75, or 1 mM is added, the multilayered hybrid electronic sheet-GOx-based transparent and flexible biosensor according to one embodiment is fine The reduction current was linearly decreased in the microfluidic system environment, and the sensitivity of the measured electrode was about 113 uA / mM cm ² . As a result, when measuring the concentration of glucose in a body fluid such as tears having a low glucose level, a transparent and flexible biosensor based on a multilayered hybrid electronic sheet-GOx based on one embodiment is used as a flexible element It can be understood that measurement with high sensitivity is possible.

3. Hybrid electronic sheet - Fabrication and characterization of biosensor based on cholesterol oxidase or lactate oxidase

3.1. Hybrid electronic sheet - Fabrication of biosensor based on cholesterol oxidase or lactate oxidase

Except that 5 占 퐇 of 10 mg / ml of cholesterol oxidase (CholOx) or 50 mg / ml of lactate oxidase (LOx) was mixed with a 100 mM PBS buffer solution and immobilized on electrodes having positive charges, respectively. A biosensor was fabricated in the same manner as in 2.1.1.

3.2. Hybrid electronic sheet - Sensitivity analysis of biosensor based on cholesterol oxidase or lactate oxidase

To measure the sensitivity of the hybrid electronic sheet-cholesterol oxidase or lactate oxidase-based biosensor, a 3-electrode system as described in Example 2.4 above was used, and a 10 mM PBS buffer (pH = 7.4, 79383, Sigma Aldrich) The characteristics of the electrodes were tested at a scan rate of 200 mV / s. The measured voltage was limited to between -0.1 V and -0.6 V relative to the Ag / AgCl reference electrode for pure DET-based determinations of FAD-based enzymes. The above results are shown in Fig.

23A is a graph showing sensitivity of a hybrid electronic sheet-cholesterol oxidase based biosensor according to an embodiment.

23B is a graph showing the sensitivity of a hybrid electronic sheet-lactate oxidase based biosensor according to an embodiment.

As shown in FIG. 23A, as the concentration of cholesterol is increased when cholesterol is added in the range of 0.01, 0.025, 0.05, 0.075, 0.1, 0.25, 0.5, 0.75, 1, or 2.5 mM, And the sensitivity of the electrode is about 28 uA / mM cm ^{2 in} the range of 0 to 1 mM.

When L-lactate in the range of 0.01, 0.025, 0.05, 0.075, 0.1, 0.25, 0.5, 0.75, 1, or 2.5 mM was added as shown in Fig. 23B, the reduction current decreased and decreased to 0 to 0.5 mM you can see the visible sensitivity of about 143 uA / cm ² mM range.

4. Fabrication and Characterization of Hybrid Electronic Sheet-HRP or Catalase-based Biosensor

4.1. Fabrication of Hybrid Electronic Sheet-HRP or Catalase Based Biosensor

Except that 5 占 퐇 of 10 mg / ml of catalase was mixed with 100 mM PBS buffer solution and immobilized on a positive electrode. In this manner, the hybrid electronic sheet-catalase Based biosensor.

5 μl of 10 mg / ml of HRP was dissolved in 100 mM PBS buffer (pH 7.0), and the surface of the electrode was electrochemically modified using 5 μl of 6 mg / ml polystyrene sulfonate (PSS) Solution, and then immobilized on the electrode modified with negative charge, a hybrid electronic sheet-HRP-based biosensor was produced in the same manner as in the above-mentioned 2.1.1.

4.2. Sensitivity analysis of hybrid electronic sheet-HRP or catalase-based biosensor

In order to measure the sensitivity of the hybrid electronic sheet-HRP or catalase-based biosensor, a 3-electrode system as described in Example 2.4 above was used, and 200 mV (pH 7.4, 79383, Sigma Aldrich) / s. < / RTI > The measured voltage was limited to between -0.1 V and -0.6 V relative to the Ag / AgCl reference electrode for pure Heme-based DET only measurements. The above results are shown in Fig.

24A is a graph showing the sensitivity of a hybrid electronic sheet-HRP-based biosensor according to an embodiment.

24B is a graph showing sensitivity of a hybrid electronic sheet-catalase-based biosensor according to an embodiment.

As shown in Figure 24A, as the concentration of H ₂ O ₂ increases with the addition of H ₂ O ₂ in the range of 0.01, 0.025, 0.05, 0.075, 0.1, 0.25, 0.5, 0.75, 1, or 2.5 mM, The reduction current is increased in the range of -0.4 V vs. the electrode, and the sensitivity of the electrode is about 230 uA / mM cm ^{2 in} the range of 0 to 5 mM.

As shown in Figure 24b, as H ₂ O ₂ concentration increases when H ₂ O ₂ is added in the range of 0.01, 0.025, 0.05, 0.075, 0.1, 0.25, 0.5, 0.75, 1, or 2.5 mM, The reduction current is increased in the range of -0.4 V vs. the electrode, and the sensitivity of the electrode is about 334 uA / mM cm ^{2 in} the range of 0 to 5 mM.

As a result of the above, the increase of the reduction current is caused by the oxidation of the Heme redox center in the enzyme by H ₂ O ₂ and the increase of the amount of oxidized heme during the increase of the H ₂ O ₂ concentration, It can be seen that the reduction current is increased by the reduction by the chemical reaction.

5. Fabrication and characterization of hybrid electronic sheet-based biosensor based on lactase, tyrosinase or galactose oxidase

5.1. Fabrication of hybrid electronic sheet-biosensor based on lactase, tyrosinase or galactose oxidase

Except that 5 μl of 50 mg / ml of laccase or 20 mg / ml of tyrosinase was mixed with 100 mM PBS buffer solution and immobilized on a positive electrode. A biosensor based on hybrid electronic sheet-lacquer or tyrosinase was prepared in the same manner as in (1).

In addition, 5 μl of 6 mg / ml polystyrene sulfonate (PSS) on the PAH-coated electrode was used to negatively charge the electrode surface and 5 μl of 3 mg / ml galactose oxidase (GalOx) A hybrid electronic sheet-GalOx-based biosensor was fabricated in the same manner as in the above-described 2.1.1, except that it was mixed with 100 mM PBS buffer solution and immobilized on a negatively charged electrode.

5.2. Sensitivity analysis of hybrid electronic sheet-biosensor based on lactase, tyrosinase or galactose oxidase

In order to measure the sensitivity of a hybrid electronic sheet-lacquer, tyrosinase or galactose oxidase-based biosensor, the 3-electrode system mentioned in Example 2.4 above was used, and 10 mM PBS buffer (pH = 7.4, 79383, Sigma Aldrich) electrolyte at a scan rate of 200 mV / s. The above results are shown in Fig.

25A is a graph showing the sensitivity of a hybrid electronic sheet-galactose oxidase-based biosensor according to an embodiment.

25B is a graph showing the sensitivity of a hybrid electronic sheet-tyrosinase-based biosensor according to an embodiment.

25C is a graph showing the sensitivity of the hybrid electronic sheet-lacquer-based biosensor according to one embodiment.

As shown in FIG. 25A, when galactose in the range of 0.01, 0.025, 0.05, 0.075, 0.1, 0.25, 0.5, 0.75, 1, or 2.5 mM was added, the concentration of galactose increased to -0.4 V And the sensitivity of the electrode is about 40 uA / mM cm ^{2 in} the range of 0 to 1 mM.

As shown in Fig. 25B, as the concentration of catechol increases when catechol is added in the range of 0.01, 0.025, 0.05, 0.075, 0.1, 0.25, 0.5, 0.75, 1, or 2.5 mM, The reduction current is increased in the range of -0.35 V to -30 V, and the sensitivity of the electrode is about 326 uA / mM cm ^{2 in} the range of 0 to 1 mM.

As shown in Fig. 25C, as the concentration of catechol increases when catechol is added in the range of 0.01, 0.025, 0.05, 0.075, 0.1, 0.25, 0.5, 0.75, 1, or 2.5 mM, The reduction current is increased in the range of -0.35 V to the contrast, and the sensitivity of the electrode is about 541 uA / mM cm ^{2 in} the range of 0 to 1 mM.

1: Electrode 2: Biosensor
10: Substrate 20: Hybrid electronic sheet layer
30: polymer layer 40: analyte binding layer
50: protective layer 100: analyte binding material
200: electrode 610: test cell
611: inlet 612: outlet 612:

<110> Korea Institute of Science and Technology <120> Biosensor and wearable device for detecting information of living          bodies comprising hybrid electronic sheets <130> PN109041 <150> KR 2014-0136992 <151> 2014-10-10 <160> 14 <170> Kopatentin 2.0 <210> 1 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Peptide selective binding to graphitic materials and volatile          organic compounds <220> <221> VARIANT <222> (3) <223> X is W, Y, F or H <220> <221> VARIANT <222> (1) <223> X is D, E, N, or Q <220> <221> VARIANT <222> (6) <223> X is D, E, N or Q <220> <221> VARIANT <222> (7) <223> X is I, L, or V <400> 1 Xaa Ser Xaa Ala Ala Xaa Xaa Pro   1 5 <210> 2 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Peptide selective binding to graphitic materials and volatile          organic compounds <220> <221> VARIANT <222> (1) <223> X is D, E, N, or Q <220> <221> VARIANT <222> (2) <223> X is D, E, N, or Q <220> <221> VARIANT <222> (4) <223> X is I, L, or V <220> <221> VARIANT <222> (5) <223> X is D, E, N, or Q <220> <221> VARIANT <222> (7) <223> X is I, L, or V <400> 2 Xaa Xaa Pro Xaa Xaa Ala Xaa Pro   1 5 <210> 3 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Peptide selective binding to graphitic materials and volatile          organic compounds <220> <221> VARIANT <222> (2) <223> X is W, Y, F, or H <220> <221> VARIANT <222> (5) <223> X is D, E, N, or Q <220> <221> VARIANT <222> (6) <223> X is I, L, or V <400> 3 Ser Xaa Ala Ala Xaa Xaa Pro   1 5 <210> 4 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Peptide selective binding to graphitic materials and volatile          organic compounds <220> <221> VARIANT <222> (1) <223> X is D, E, N, or Q <220> <221> VARIANT <222> (3) <223> X is I, L, or V <220> <221> VARIANT <222> (4) <223> X is D, E, N, or Q <220> <221> VARIANT <222> (6) <223> X is I, L, or V <400> 4 Xaa Pro Xaa Xaa Ala Xaa Pro   1 5 <210> 5 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Peptide selective binding to graphitic materials and volatile          organic compounds <400> 5 Asp Ser Trp Ala Ala Asp Ile Pro   1 5 <210> 6 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Peptide selective binding to graphitic materials and volatile          organic compounds <400> 6 Asp Asn Pro Ile Gln Ala Val Pro   1 5 <210> 7 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Peptide selective binding to graphitic materials and volatile          organic compounds <400> 7 Ser Trp Ala Ala Asp Ile Pro   1 5 <210> 8 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Peptide selective binding to graphitic materials and volatile          organic compounds <400> 8 Asn Pro Ile Gln Ala Val Pro   1 5 <210> 9 <211> 7222 <212> DNA <213> Artificial Sequence <220> <223> cloning vector M13KE <400> 9 aatgctacta ctattagtag aattgatgcc accttttcag ctcgcgcccc aaatgaaaat 60 atagctaaac aggttattga ccatttgcga aatgtatcta atggtcaaac taaatctact 120 cgttcgcaga attgggaatc aactgttata tggaatgaaa cttccagaca ccgtacttta 180 gttgcatatt taaaacatgt tgagctacag cattatattc agcaattaag ctctaagcca 240 tccgcaaaaa tgacctctta tcaaaaggag caattaaagg tactctctaa tcctgacctg 300 ttggagtttg cttccggtct ggttcgcttt gaagctcgaa ttaaaacgcg atatttgaag 360 tctttcgggc ttcctcttaa tctttttgat gcaatccgct ttgcttctga ctataatagt 420 cagggtaaag acctgatttt tgatttatgg tcattctcgt tttctgaact gtttaaagca 480 tttgaggggg attcaatgaa tatttatgac gattccgcag tattggacgc tatccagtct 540 aaacatttta ctattacccc ctctggcaaa acttcttttg caaaagcctc tcgctatttt 600 gt; aattcctttt ggcgttatgt atctgcatta gttgaatgtg gtattcctaa atctcaactg 720 atgaatcttt ctacctgtaa taatgttgtt ccgttagttc gttttattaa cgtagatttt 780 tcttcccaac gtcctgactg gtataatgag ccagttctta aaatcgcata aggtaattca 840 caatgattaa agttgaaatt aaaccatctc aagcccaatt tactactcgt tctggtgttt 900 ctcgtcaggg caagccttat tcactgaatg agcagctttg ttacgttgat ttgggtaatg 960 aatatccggt tcttgtcaag attactcttg atgaaggtca gccagcctat gcgcctggtc 1020 tgtacaccgt tcatctgtcc tctttcaaag ttggtcagtt cggttccctt atgattgacc 1080 gtctgcgcct cgttccggct aagtaacatg gagcaggtcg cggatttcga cacaatttat 1140 caggcgatga tacaaatctc cgttgtactt tgtttcgcgc ttggtataat cgctgggggt 1200 caaagatgag tgttttagtg tattcttttg cctctttcgt tttaggttgg tgccttcgta 1260 gtggcattac gtattttacc cgtttaatgg aaacttcctc atgaaaaagt ctttagtcct 1320 caaagcctct gtagccgttg ctaccctcgt tccgatgctg tctttcgctg ctgagggtga 1380 cgatcccgca aaagcggcct ttaactccct gcaagcctca gcgaccgaat atatcggtta 1440 tgcgtgggcg atggttgttg tcattgtcgg cgcaactatc ggtatcaagc tgtttaagaa 1500 attcacctcg aaagcaagct gataaaccga tacaattaaa ggctcctttt ggagcctttt 1560 ttttggagat tttcaacgtg aaaaaattat tattcgcaat tcctttagtg gtacctttct 1620 attctcactc ggccgaaact gttgaaagtt gtttagcaaa atcccataca gaaaattcat 1680 ttactaacgt ctggaaagac gacaaaactt tagatcgtta cgctaactat gagggctgtc 1740 tgtggaatgc tacaggcgtt gtagtttgta ctggtgacga aactcagtgt tacggtacat 1800 gggttcctat tgggcttgct atccctgaaa atgagggtgg tggctctgag ggtggcggtt 1860 ctgagggtgg cggttctgag ggtggcggta ctaaacctcc tgagtacggt gatacaccta 1920 ttccgggcta tacttatatc aaccctctcg acggcactta tccgcctggt actgagcaaa 1980 accccgctaa tcctaatcct tctcttgagg agtctcagcc tcttaatact ttcatgtttc 2040 agaataatag gttccgaaat aggcaggggg cattaactgt ttatacgggc actgttactc 2100 aaggcactga ccccgttaaa acttattacc agtacactcc tgtatcatca aaagccatgt 2160 atgacgctta ctggaacggt aaattcagag actgcgcttt ccattctggc tttaatgagg 2220 atttatttgt ttgtgaatat caaggccaat cgtctgacct gcctcaacct cctgtcaatg 2280 ctggcggcgg ctctggtggt ggttctggtg gcggctctga gggtggtggc tctgagggtg 2340 gcggttctga gggtggcggc tctgagggag gcggttccgg tggtggctct ggttccggtg 2400 attttgatta tgaaaagatg gcaaacgcta ataagggggc tatgaccgaa aatgccgatg 2460 aaaacgcgct acagtctgac gctaaaggca aacttgattc tgtcgctact gattacggtg 2520 ctgctatcga tggtttcatt ggtgacgttt ccggccttgc taatggtaat ggtgctactg 2580 gtgattttgc tggctctaat tcccaaatgg ctcaagtcgg tgacggtgat aattcacctt 2640 taatgaataa tttccgtcaa tatttacctt ccctccctca atcggttgaa tgtcgccctt 2700 ttgtctttgg cgctggtaaa ccatatgaat tttctattga ttgtgacaaa ataaacttat 2760 tccgtggtgt ctttgcgttt cttttatatg ttgccacctt tatgtatgta ttttctacgt 2820 ttgctaacat actgcgtaat aaggagtctt aatcatgcca gttcttttgg gtattccgtt 2880 attattgcgt ttcctcggtt tccttctggt aactttgttc ggctatctgc ttacttttct 2940 taaaaagggc ttcggtaaga tagctattgc tatttcattg tttcttgctc ttattattgg 3000 gcttaactca attcttgtgg gttatctctc tgatattagc gctcaattac cctctgactt 3060 tgttcagggt gttcagttaa ttctcccgtc taatgcgctt ccctgttttt atgttattct 3120 ctctgtaaag gctgctattt tcatttttga cgttaaacaa aaaatcgttt cttatttgga 3180 ttgggataaa taatatggct gtttattttg taactggcaa attaggctct ggaaagacgc 3240 tcgttagcgt tggtaagatt caggataaaa ttgtagctgg gtgcaaaata gcaactaatc 3300 ttgatttaag gcttcaaaac ctcccgcaag tcgggaggtt cgctaaaacg cctcgcgttc 3360 ttagaatacc ggataagcct tctatatctg atttgcttgc tattgggcgc ggtaatgatt 3420 cctacgatga aaataaaaac ggcttgcttg ttctcgatga gtgcggtact tggtttaata 3480 cccgttcttg gaatgataag gaaagacagc cgattattga ttggtttcta catgctcgta 3540 aattaggatg ggatattatt tttcttgttc aggacttatc tattgttgat aaacaggcgc 3600 gttctgcatt agctgaacat gttgtttatt gtcgtcgtct ggacagaatt actttacctt 3660 ttgtcggtac tttatattct cttattactg gctcgaaaat gcctctgcct aaattacatg 3720 ttggcgttgt taaatatggc gattctcaat taagccctac tgttgagcgt tggctttata 3780 ctggtaagaa tttgtataac gcatatgata ctaaacaggc tttttctagt aattatgatt 3840 ccggtgttta ttcttattta acgccttatt tatcacacgg tcggtatttc aaaccattaa 3900 atttaggtca gaagatgaaa ttaactaaaa tatatttgaa aaagttttct cgcgttcttt 3960 gtcttgcgat tggatttgca tcagcattta catatagtta tataacccaa cctaagccgg 4020 aggttaaaaa ggtagtctct cagacctatg attttgataa attcactatt gactcttctc 4080 agcgtcttaa tctaagctat cgctatgttt tcaaggattc taagggaaaa ttaattaata 4140 gcgacgattt acagaagcaa ggttattcac tcacatatat tgatttatgt actgtttcca 4200 ttaaaaaagg taattcaaat gaaattgtta aatgtaatta attttgtttt cttgatgttt 4260 gtttcatcat cttcttttgc tcaggtaatt gaaatgaata attcgcctct gcgcgatttt 4320 gtaacttggt attcaaagca atcaggcgaa tccgttattg tttctcccga tgtaaaaggt 4380 actgttactg tatattcatc tgacgttaaa cctgaaaatc tacgcaattt ctttatttct 4440 gttttacgtg caaataattt tgatatggta ggttctaacc cttccattat tcagaagtat 4500 aatccaaaca atcaggatta tattgatgaa ttgccatcat ctgataatca ggaatatgat 4560 gataattccg ctccttctgg tggtttcttt gttccgcaaa atgataatgt tactcaaact 4620 tttaaaatta ataacgttcg ggcaaaggat ttaatacgag ttgtcgaatt gtttgtaaag 4680 tctaatactt ctaaatcctc aaatgtatta tctattgacg gctctaatct attagttgtt 4740 agtgctccta aagatatttt agataacctt cctcaattcc tttcaactgt tgatttgcca 4800 actgaccaga tattgattga gggtttgata tttgaggttc agcaaggtga tgctttagat 4860 ttttcatttg ctgctggctc tcagcgtggc actgttgcag gcggtgttaa tactgaccgc 4920 ctcacctctg ttttatcttc tgctggtggt tcgttcggta tttttaatgg cgatgtttta 4980 gggctatcag ttcgcgcatt aaagactaat agccattcaa aaatattgtc tgtgccacgt 5040 attcttacgc tttcaggtca gaagggttct atctctgttg gccagaatgt tccttttatt 5100 actggtcgtg tgactggtga atctgccaat gtaaataatc catttcagac gattgagcgt 5160 caaaatgtag gtatttccat gagcgttttt cctgttgcaa tggctggcgg taatattgtt 5220 ctggatatta ccagcaaggc cgatagtttg agttcttcta ctcaggcaag tgatgttatt 5280 actaatcaaa gaagtattgc tacaacggtt aatttgcgtg atggacagac tcttttactc 5340 ggtggcctca ctgattataa aaacacttct caggattctg gcgtaccgtt cctgtctaaa 5400 atccctttaa tcggcctcct gtttagctcc cgctctgatt ctaacgagga aagcacgtta 5460 tacgtgctcg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg 5520 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt 5580 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg 5640 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga 5700 tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac 5760 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc 5820 tatctcgggc tattcttttg atttataagg gattttgccg atttcggaac caccatcaaa 5880 caggattttc gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc 5940 caggcggtga agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg 6000 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 6060 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct 6120 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat 6180 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagcttgc 6240 atgcctgcag gtcctcgaat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc 6300 ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata 6360 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc 6420 gctttgcctg gtttccggca ccagaagcgg tgccggaaag ctggctggag tgcgatcttc 6480 ctgaggccga tactgtcgtc gtcccctcaa actggcagat gcacggttac gatgcgccca 6540 tctacaccaa cgtgacctat cccattacgg tcaatccgcc gtttgttccc acggagaatc 6600 cgacgggttg ttactcgctc acatttaatg ttgatgaaag ctggctacag gaaggccaga 6660 cgcgaattat ttttgatggc gttcctattg gttaaaaaat gagctgattt aacaaaaatt 6720 taatgcgaat tttaacaaaa tattaacgtt tacaatttaa atatttgctt atacaatctt 6780 cctgtttttg gggcttttct gattatcaac cggggtacat atgattgaca tgctagtttt 6840 acgattaccg ttcatcgatt ctcttgtttg ctccagactc tcaggcaatg acctgatagc 6900 ctttgtagat ctctcaaaaa tagctaccct ctccggcatt aatttatcag ctagaacggt 6960 tgaatatcat attgatggtg atttgactgt ctccggcctt tctcaccctt ttgaatcttt 7020 acctacacat tactcaggca ttgcatttaa aatatatgag ggttctaaaa atttttatcc 7080 ttgcgttgaa ataaaggctt ctcccgcaaa agtattacag ggtcataatg tttttggtac 7140 aaccgattta gctttatgct ctgaggcttt attgcttaat tttgctaatt ctttgccttg 7200 cctgtatgat ttattggatg tt 7222 <210> 10 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> BamH I_SM_upper which is a primer used for site-directed mutation <400> 10 aaggccgctt ttgcgggatc ctcaccctca gcagcgaaag a 41 <210> 11 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> BamH I_SM_lower which is a primer used for site-directed mutation <400> 11 tctttcgctg ctgagggtga ggatcccgca aaagcggcct t 41 <210> 12 <211> 90 <212> DNA <213> Artificial Sequence <220> <223> BamM13HK_P8_primer which is an extension primer used for          preparation <400> 12 ttaatggaaa cttcctcatg aaaaagtctt tagtcctcaa agcctctgta gccgttgcta 60 ccctcgttcc gatgctgtct ttcgctgctg 90 <210> 13 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> M13HK_P8 which is a library oligonucleotide used for preparation <220> <221> variation <222> (1) <223> n is a, g, c or t <220> <221> variation <222> (1) <223> m is a or c <400> 13 aaggccgctt ttgcgggatc cnmnnmnnmnnmnnmn nmncagcagc gaaagacagc 60 atcggaacga gggtagcaac ggctacagag gcttt 95 <210> 14 <211> 50 <212> PRT <213> P8 protein of M13 phage <400> 14 Ala Glu Gly Asp Asp Pro Ala Lys Ala Ala Phe Asn Ser Leu Gln Ala   1 5 10 15 Ser Ala Thr Glu Tyr Ile Gly Tyr Ala Trp Ala Met Val Val Val Ile              20 25 30 Val Gly Ala Thr Ile Gly Ile Lys Leu Phe Lys Lys Phe Thr Ser Lys          35 40 45 Ala Ser      50

Claims (17)

Board;
An electronic sheet formed on the substrate; And
And an analyte binding material immobilized on the electronic sheet,
Wherein the electronic sheet comprises a graphical material and a phage coupled to the graphical material and wherein the binding of the graphical material and the phage is performed between a peptide displayed on the phage & And the biosensor. The biosensor according to claim 1, wherein the substrate is an insulating substrate on which at least one electrode is disposed. The biosensor of claim 2, wherein the at least one electrode is a first electrode and a second electrode. The biosensor according to claim 3, wherein the electronic sheet is disposed on the first electrode or a part thereof on the substrate. The biosensor according to claim 1, wherein the substrate is a transparent flexible substrate. The biosensor according to claim 1, wherein the electronic sheet is patterned on a substrate. The method of claim 1, wherein the graphitic material is selected from the group consisting of a graphene sheet, a highly orientated pyrolytic graphite (HOPG) sheet, a single-walled carbon nanotube, double-walled carbon nanotubes, Wherein the biosensor is at least one selected from the group consisting of a multi-walled carbon nanotube and a fullerene. The biosensor according to claim 1, wherein the graphitic material is a mixture of a graphene sheet and a single-walled carbon nanotube. The biosensor according to claim 1, wherein the peptide comprises at least one selected from the group consisting of amino acid sequences of SEQ ID NOS: 1 to 8. The phage according to claim 1, wherein the phage is M13 phage, F1 phage, Fd phage, If1 phage, Ike phage, Zj / Z phage, Ff phage, Xf phage, Pf1 phage or Pf3 phage. The biosensor according to claim 1, wherein the analyte binding substance is oxidase, peroxidase, reductase, catalase or dehydrogenase. The biosensor of claim 1, further comprising a protective layer formed on the immobilized analyte binding material. The biosensor of claim 1, wherein the electronic sheet is modified such that a surface in contact with the analyte binding material has a positive charge or a negative charge opposite to that of the analyte binding material. The biosensor according to claim 1, wherein the biosensor is a laminate of a plurality of repeating units including an electronic sheet and an analyte binding material. The test cell of claim 1, further comprising a test cell for receiving the sample, the electronic sheet, and the analyte binding material, wherein the test cell is a bioreactor comprising a channel including an inlet and an outlet for sample injection and discharge, sensor. A wearable device for biometric information detection comprising the biosensor of claim 1. 16. The wearable device of claim 15, wherein the wearable device is a contact lens.

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