Metal oxide thin film transistor-based biosensor for detecting MMP-9 in tears in order to diagnose dry eye syndrome

Abstract

The present invention relates to a metal oxide thin film transistor-based biosensor for detecting matrix metalloproteinase-9 (MMP-9) in tears in order to diagnose dry eye syndrome and, more particularly, to a surface modification method using 3-aminopropyltriethoxysilane (APTES) and an aptamer, and a biosensor for detecting matrix metalloproteinase-9 (MMP-9) in tears in order to diagnose dry eye syndrome, the method and the biosensor being based on a metal oxide electrolyte-gated thin-film transistor.

Description

Metal Oxide Thin Film Transistor-Based Biosensor for Detecting MMP-9 in Tears for Dry Eye Syndrome Diagnosis

[0001] The present invention relates to a biosensor for detecting MMP-9 in tears for the diagnosis of dry eye disease based on a metal oxide thin film transistor, and more specifically, to a surface modification method using 3-aminopropyltriethoxysilane (APTES) and an aptamer based on a metal oxide electrolyte gate thin film transistor and a biosensor for detecting Matrix metalloproteinase-9 (MMP-9) in tears for the diagnosis of dry eye disease.

[0002] Dry eye disease (DED) is the most common inflammatory disease of the ocular surface, characterized by an unstable tear film associated with symptoms of visual impairment. Globally, the prevalence of dry eye disease ranges from 5% to 50%, with an estimated 16 million patients in the United States and 128 million worldwide. Currently, the clinical diagnosis of dry eye disease is based on a combination of signs and symptoms, including tear secretion, tear separation time, corneal fluorescence staining, and the height of the tear lunula and inferior lunula. However, because the interpretation of results from these existing tests depends on the examiner's subjective judgment, irregular correlations between reported symptoms and observed signs have potentially limited the diagnosis and assessment of the severity of dry eye disease. Therefore, objective and accurate biomarkers for timely diagnosis and evaluation are essential for the prevention and treatment of dry eye disease.

[0003] Recent studies for the diagnosis of dry eye disease have focused on identifying tear fluid biomarkers, including proteins, lipids, and metabolites, through various analyses. Among these, matrix metalloproteinase-9 (MMP-9) is emerging as a promising biomarker due to its strong correlation with the clinical signs and symptoms of dry eye disease. MMP-9 is a proteolytic enzyme secreted by the corneal epithelium that can degrade the extracellular matrix and cell surface adhesion molecules; therefore, increased MMP-9 concentrations can impair the barrier function of the corneal epithelium, potentially leading to inflammation of the ocular surface. Consequently, the concentration of MMP-9 in tears can serve as an important diagnostic biomarker, acting as a reliable response factor for predicting dry eye disease and a prognostic tool for topical anti-inflammatory treatment.

[0004] Recently, amorphous metal oxide (MOx) semiconductors fabricated via the sol-gel process have been attracting significant attention in the field of flexible transparent electronic devices. In particular, active research is being conducted in various fields such as displays, sensors, memory, and photovoltaics. MOx semiconductors are gaining prominence due to the following advantages compared to silicon, a conventional semiconductor material. They can be fabricated using solution-based processes at low temperatures, which is advantageous for large-area, low-cost production, and enable the excellent realization of transparency essential for transparent electronic devices. Furthermore, they allow for the formation of high-quality thin films over large areas and exhibit superior electrical properties based on high mobility and carrier concentration. Thanks to these advantages, MOx semiconductors are evaluated as materials with the potential to replace or complement existing silicon-based electronic devices. Moreover, recent studies suggest that the potential of MOx semiconductors extends beyond traditional electronic device fields to the field of biosensors utilizing various biological fluids such as tears, urine, sweat, and saliva. However, biosensors utilizing MOx semiconductors remain in the early research stage, and several challenges must be resolved before they can be applied clinically.

[0005] [Prior Art Literature]

[0006] [Patent Literature]

[0007] Korean Intellectual Property Office Published Patent Application (A) No. 10-2024-0140478 (Published September 24, 2024)

[0008] One objective of the present invention is to provide a biosensor capable of operating at low voltage to minimize the electrolysis of tears and leakage current.

[0009] Another objective of the present invention is to design and manufacture a biosensor that can operate stably in tears without degradation.

[0010] Another objective of the present invention is to selectively detect only specific biomarkers using only a small amount of sample within tears containing a mixture of various types of substances.

[0011] According to one aspect of the present invention, an MMP-9 detection biosensor is provided based on an electrolyte-gate thin-film (EGTFT) comprising: a substrate; a source electrode and a drain electrode spaced apart and disposed on the substrate; an IGZO channel disposed between the source electrode and the drain electrode and on the source electrode and the drain electrode; and a gate electrode disposed on the IGZO channel, wherein the IGZO channel comprises a sensing layer having an aptamer immobilized on the surface of the IGZO (indium-gallium-zinc oxide) channel to detect MMP-9.

[0012] According to another aspect of the present invention, a method for manufacturing an MMP-9 detection biosensor is provided, based on a method for manufacturing an electrolyte thin film transistor (EGTFT) comprising the steps of: preparing a substrate; forming a source electrode and a drain electrode spaced apart on the substrate; forming an IGZO channel between the source electrode and the drain electrode and on the source electrode and the drain electrode; and forming a gate electrode on the IGZO channel, wherein the step of forming the IGZO channel comprises: hydroxylating the surface of the IGZO channel; treating the hydroxylated surface of the IGZO channel with APTES; and immobilizing an aptamer on the surface of the APTES-treated IGZO channel.

[0013] According to one embodiment of the present invention, a biosensor capable of operating at low voltage can be provided based on an electrolyte gate thin film transistor having an IGZO channel.

[0014] According to one embodiment of the present invention, a biosensor capable of operating stably without degradation can be provided based on an electrolyte gate thin film transistor having an IGZO channel.

[0015] According to one embodiment of the present invention, a biosensor based on an electrolyte gate thin-film transistor having an IGZO channel can be provided to selectively detect only specific biomarkers using only a small amount of sample in tears containing a mixture of various types of substances.

[0016] FIGS. 1a and FIGS. 1b are design conceptual diagrams of an MMP-9 detection biosensor according to one embodiment of the present invention.

[0017] FIGS. 2a, FIGS. 2b, FIGS. 2c, FIGS. 2d, FIGS. 2e, FIGS. 2f, FIGS. 2g, FIGS. 2h, and FIGS. 2i show the electrical characteristics of an electrolyte-gate type IGZO thin-film transistor (IGZO-EGTFT) using a PBS solution according to one embodiment of the present invention.

[0018] FIGS. 3a, 3b, and 3c show the design concept and operation mechanism of an MMP-9 detection biosensor based on IGZO-EGTFT according to one embodiment of the present invention.

[0019] FIGS. 4a, FIGS. 4b, FIGS. 4c, FIGS. 4d, FIGS. 4e, and FIGS. 4f show surface analysis of a functionalized IGZO surface of an MMP-9 detection biosensor according to one embodiment of the present invention.

[0020] FIGS. 5A, FIGS. 5B, FIGS. 5C, FIGS. 5D, FIGS. 5E, and FIGS. 5F show the electrical characteristics of an IGZO-EGTFT for MMP-9 detection in an MMP-9 detection biosensor according to one embodiment of the present invention.

[0021] FIG. 6 is a flowchart illustrating a method for manufacturing an MMP-9 detection biosensor according to one embodiment of the present invention.

[0022] The above objectives, other objectives, features, and advantages will be easily understood through the following preferred embodiments associated with the accompanying drawings. However, the embodiments described herein are not limited to those described herein and may be embodied in other forms. Rather, the embodiments introduced herein are provided to ensure that the disclosed content is thorough and complete and that the technical concept is sufficiently conveyed to a person skilled in the art.

[0023] In describing each drawing, similar reference numerals have been used for similar components. In the attached drawings, the dimensions of the structures are depicted enlarged from their actual size for clarity of the invention. Terms such as "first," "second," etc., may be used to describe various components, but said components should not be limited by said terms. These terms are used solely for the purpose of distinguishing one component from another. For example, without departing from the scope of the invention, the first component may be named the second component, and similarly, the second component may be named the first component. A singular expression includes a plural expression unless the context clearly indicates otherwise.

[0024] In this specification, terms such as "comprising" or "having" are intended to specify the existence of the features, numbers, steps, actions, components, parts, or combinations thereof described in the specification, and should be understood as not precluding the existence or addition of one or more other features, numbers, steps, actions, components, parts, or combinations thereof. Furthermore, when a part such as a layer, film, region, or plate is described as being "on" another part, this includes not only the case where it is "immediately above" the other part, but also the case where there is another part in between. Conversely, when a part such as a layer, film, region, or plate is described as being "below" another part, this includes not only the case where it is "immediately below" the other part, but also the case where there is another part in between.

[0025] Unless otherwise specified, all numbers, values, and / or expressions used herein to represent amounts of ingredients, reaction conditions, polymer compositions, and formulations should be understood to be modified by the term “approximately” in all cases, as these numbers are essentially approximations reflecting the various uncertainties of measurement that occur in obtaining these values ​​among other things. Furthermore, where numerical ranges are disclosed herein, such ranges are continuous and, unless otherwise indicated, include all values ​​from the minimum value of such range to the maximum value including said maximum value. Moreover, where such ranges refer to integers, they include all integers from the minimum value to said maximum value including said maximum value, unless otherwise indicated.

[0026] In this specification, where a range is described for a variable, it will be understood that the variable includes all values ​​within the described range, including the described endpoints of the range. For example, the range “5 to 10” will be understood to include not only the values ​​5, 6, 7, 8, 9, and 10, but also any sub-ranges such as 6 to 10, 7 to 10, 6 to 9, 7 to 9, etc., and any values ​​between integers valid for the category of the described range, such as 5.5, 6.5, 7.5, 5.5 to 8.5, and 6.5 to 9. Also, for example, the range “10% to 30%” will be understood to include all integers including values ​​such as 10%, 11%, 12%, 13%, etc. and up to 30%, as well as any sub-range such as 10% to 15%, 12% to 18%, 20% to 30%, etc., and any value between valid integers within the stated range category such as 10.5%, 15.5%, 25.5%, etc.

[0027] A biosensor for detecting the concentration of MMP-9 in tears according to one aspect of the present invention comprises a channel, an electrolyte disposed on the channel, and an aptamer targeting MMP-9 bound to the surface of the channel in contact with the electrolyte, wherein the aptamer reacts with MMP-9 in tears applied to the electrolyte to generate current modulation in the channel, thereby enabling the concentration of MMP-9 in tears to be detected from the degree of current modulation.

[0028] According to one embodiment of the present invention, an MMP-9 detection biosensor can be implemented based on an electrolyte thin-film transistor structure.

[0029] According to one embodiment of the present invention, an MMP-9 detection biosensor further comprises a substrate, a source electrode and a drain electrode spaced apart and disposed on the substrate, and a gate electrode disposed in contact with an electrolyte, wherein the source electrode, the drain electrode, and the gate electrode may be separated from each other by a channel.

[0030] According to one embodiment of the present invention, the channel may be an IGZO channel.

[0031] According to one embodiment of the present invention, the MMP-9 detection biosensor may further include a well formed around the electrolyte to accommodate the electrolyte within the channel, thereby allowing tears to be captured by the electrolyte. According to one embodiment of the present invention, the aptamer may be fixed to the surface of the channel by being bonded to a crosslinking agent that is covalently bonded to the surface of the channel.

[0032] According to one embodiment of the present invention, the crosslinking agent may include APTES.

[0033] According to one embodiment of the present invention, the gate electrode may be an Ag / AgCl reference electrode (RE).

[0034] According to one embodiment of the present invention, the source electrode and the drain electrode may include gold (Au).

[0035] According to one embodiment of the present invention, the source electrode and the drain electrode may be made of gold (Au) laminated on top of a layer comprising at least one of titanium, chromium, and platinum.

[0036] According to one embodiment of the present invention, the degree of current modulation can be detected through the drain electrode.

[0037] According to one embodiment of the present invention, a power supply unit having a gate electrode as a reference electrode is further included, and the power supply unit can supply a voltage in the range of -0.5V to 1.0V to the reference electrode and a voltage of 1V or less to the drain electrode. By adjusting the voltage range of the reference electrode to have a low operating range, the biosensor including it can operate with low power consumption, thereby achieving high energy efficiency, which is essential for extending the battery life of wearable devices or portable diagnostic devices, and enabling easy miniaturization. In addition, by adjusting the voltage value of the drain electrode to have a low operating range, the same effect as described above regarding the voltage range of the reference electrode can be obtained.

[0038] According to one embodiment of the present invention, the substrate may be a glass substrate or a silicon substrate.

[0039] A method for manufacturing an MMP-9 detection biosensor according to another aspect of the present invention comprises the steps of: binding an aptamer targeting MMP-9 to the surface of a channel; and placing an electrolyte on the channel with the aptamer in between. The aptamer reacts with MMP-9 in tears applied to the electrolyte to generate current modulation in the channel, thereby allowing the concentration of MMP-9 in tears to be detected from the degree of current modulation.

[0040] According to one embodiment of the present invention, an MMP-9 detection biosensor can be implemented based on an electrolyte thin-film transistor structure.

[0041] According to one embodiment of the present invention, a method for manufacturing an MMP-9 detection biosensor may further include the steps of preparing a substrate, arranging a source electrode and a drain electrode spaced apart on the substrate, forming a channel between the source electrode and the drain electrode, and forming a gate electrode to contact an electrolyte.

[0042] According to one embodiment of the present invention, the channel may be an IGZO channel.

[0043] According to one embodiment of the present invention, a method for manufacturing an MMP-9 detection biosensor may further include the step of forming a well formed around an electrolyte to accommodate an electrolyte on a channel, thereby allowing tears to be captured by the electrolyte.

[0044] According to one embodiment of the present invention, the step of bonding an aptamer to the surface of a channel may include the step of covalently bonding a crosslinking agent to the surface of the channel, and the step of bonding the aptamer to the crosslinking agent so that the aptamer is fixed to the surface of the channel.

[0045] According to one embodiment of the present invention, the crosslinking agent may include APTES.

[0046] According to one embodiment of the present invention, the step of covalently bonding a crosslinking agent to the surface of a channel includes the step of treating the surface of the channel with oxygen plasma to hydroxylate the surface of the channel, wherein treating with oxygen plasma is an essential process for fixing APTES to the surface of the channel, and hydroxyl groups can be generated on the surface of the channel through oxygen plasma treatment.

[0047] According to one embodiment of the present invention, the step of fixing an aptamer to the surface of a channel may include the step of covalently bonding the aptamer to the surface of the channel.

[0048] The present invention will be explained in more detail through the following examples. The following examples are merely illustrative to aid in understanding the present invention and do not limit the scope of the invention.

[0049] < Examples >

[0050] 1. Design Concept of MMP-9 Detection Biosensor

[0051] FIGS. 1a and 1b are conceptual design diagrams of an MMP-9 detection biosensor according to an embodiment of the present invention. Specifically, FIG. 1a illustrates the ocular surface composition and MMP-9 in the tear film, and FIG. 1b is a schematic diagram of an IGZO-EGTFT-based sensor developed to detect MMP-9 in a human tear sample.

[0052] Referring to Figures 1a and 1b, the present invention proposes a new method for diagnosing dry eye disease (DED) using IGZO-EGTFT, which exhibits highly sensitive and real-time detection capabilities in human tear samples.

[0053] The detection capability for MMP-9 was enhanced by carefully and sequentially performing a surface functionalization process on the IGZO surface using APTES and aptamers. This improvement was systematically evaluated through optical, physical, and electrical measurements. Furthermore, the results of chemical binding between the aptamer and MMP-9 obtained via electrical methods were found to be consistent with those derived from ELISA techniques. The systematic molecular modification of the IGZO surface enables stable detection of MMP-9, achieving a very low detection limit of 1 fg / ml and a wide detection range of 1 fg / ml to 100 ng / ml, along with excellent reproducibility. The performance and reliability of this biosensor were further verified using raw tear samples collected from DED patients. Compared to existing methods, the biosensor of this invention offers several key advantages in MMP-9 detection, including a much smaller sample volume (1 μl compared to the typical 100 μl used in ELISA), quantitative detection without interference from other biomolecules, ultra-sensitivity, and real-time detection. Furthermore, since the detection of biomarkers relies on aptamer sequences immobilized on the IGZO surface, this biosensor design can be easily adapted to identify other biomarkers by simply replacing them with DNA sequences that specifically target the desired biomarker. Considering ultra-sensitivity, high selectivity, and real-time detection, the proposed biosensor has the potential to be used as a point-of-care device to assess the severity of inflammation and ultimately facilitate the early detection and timely treatment of DED patients, going beyond simple diagnosis.

[0054] 2. Materials and Methods

[0055] (1) Preparation of IGZO precursor solution

[0056] In(NO3)3xH2O, Ga(NO3)3xH2O, and Zn(CH3COO)22H2O were used as IGZO precursor solutions, and the precursor solutions were prepared by uniformly dissolving In-Ga-Zn in 2-methoxyethanol with a total concentration of 0.1 M in a molar ratio of 0.1:0.15:0.0275. The mixture was stirred at 60°C for 4 hours, and the resulting precursor solution was filtered through a 0.2 μm hydrophobic polytetrafluoroethylene membrane-based syringe filter to remove undispersed precipitates before use.

[0057] (2) Fabrication of IGZO-EGTFT biosensor

[0058] To fabricate the MMP-9 sensor, the substrate was prepared by thermally oxidizing a 300 nm thick layer of silicon dioxide on a boron-heavy doped Si wafer. Then, source and drain electrodes were fabricated using a combination of 5 nm titanium and 50 nm gold metal via electron beam evaporation and photolithography. Before spin-coating the IGZO precursor solution onto the Si wafer, oxygen plasma treatment was performed at 100 W for 10 minutes to convert the device's hydrophobic surface into a hydrophilic surface. Subsequently, the IGZO precursor solution was spin-coated at 4000 rpm for 30 seconds, and the coated layer was annealed in air at 400°C for 1.5 hours to form a densified metal-oxide-semiconductor layer. Consequently, the IGZO thin film was patterned using conventional photolithography and wet etching processes. The final device was completed by passivation of epoxy-based SU-8 3008, excluding patterned IGZO channels. To evaluate the film thickness, cross-sectional micrographs of the IGZO thin film were obtained by a focused ion beam (Helios 5 UX, ThermoFisher).

[0059] 3. Design and Electrical Characterization Analysis of IGZO-EGTFT

[0060] FIGS. 2a, 2b, 2c, 2d, 2e, 2f, 2g, 2h, and 2i show the electrical characteristics of an electrolyte-gate type IGZO thin-film transistor (IGZO-EGTFT) using a PBS solution according to an embodiment of the present invention. Specifically, FIG. 2a is a photograph showing the measurement setup of an IGZO-EGTFT fabricated on a silicon substrate (a total of 20 devices are 15 × 15 mm). 2 It was fabricated as), and Fig. 2b is an optical microscope image of an IGZO-EGTFT, where an IGZO channel layer and SU-8 passivation are deposited on gold electrodes (source and drain) for electrical insulation, and the dimensions of the IGZO channel layer are 200 μm x 10 μm, Fig. 2c is a schematic diagram of the device structure and electrical connections of the IGZO-EGTFT, and Fig. 2d is V D = 0.5V(V G Transfer characteristics (I) measured at = -0.5-1V) D vs V G ) and FIG. 2e shows the output characteristics of the IGZO-EGTFT (I D vs V D )(V G = at 0, 0.2, 0.4, 0.6, 0.8, and 1 V), and as a statistical distribution in PBS solution, Fig. 2f shows the maximum transconductance (gm = Δ). D / Δ G Defined as), FIG. 2g is the sub-threshold swing (SS), FIG. 2h is the threshold voltage, and FIG. 2i is various gate biases (V) with the drain voltage at 0.5V. G Source-drain current (I) obtained every 1 second at = 0.2, 0.4, 0.6, 0.8, 1V) D It is a plot of time versus time.

[0061] Referring to Fig. 2a, a photograph of the completed IGZO-EGTFT is shown. Processed sol-gel IGZO was used as the channel material due to its high current driving capability, excellent manufacturability, excellent size scalability, and cost-effective process. Scanning electron microscope (SEM) images indicated that a thin (20–25 nm) IGZO film was uniformly formed on the substrate over a large area. Source and drain (S / D) electrodes are patterned beneath the IGZO channel, defining a large width-to-length ratio (20, width / length = 200 μm / 10 μm) to enhance channel current modulation. The fabricated device is 15 × 15 mm 2 Twenty TFTs were included in the area. PDMS wells were fixed on top of the TFTs to detect the target electrolyte in an aqueous environment without leakage during electrical measurement and to stabilize the potential while maintaining a constant volume of the electrolyte solution. Subsequently, an Ag / AgCl reference electrode (RE) was placed as the gate electrode to prevent the formation of an electric double layer (EDL) on the gate electrode and to apply a constant potential.

[0062] Referring to Fig. 2b, an enlarged optical microscope image of the IGZO channel layer and the S / D electrode is shown. The IGZO channel layer, selectively exposed within an active area of ​​2000 μm² via a conventional photolithography process, was simultaneously passivated by SU-8. This passivation prevents short circuits caused by direct interaction between the electrolyte and the S / D electrode. The structure and electrical connections of the IGZO-EGTFT are shown in Fig. 2c. For the device structure, a top gate bottom contact configuration was adopted to prevent direct contact between the S / D electrode and the electrolyte. The operating voltage applied to the Ag / AgCl RE (ranging from -0.5 V to 1 V) moved ions from the electrolyte to the IGZO channel layer, causing a change in electronic charges between the interfacial electrolyte and the IGZO channel layer. The accumulated electron charges form an electric double layer (EDL) composed of nanometer-thick, compact inner and outer Helmholtz layers, generating very high capacitance.

[0063] To verify the electrical performance, the key parameters of the IGZO-EGTFT were evaluated in PBS solution. Figures 2d and 2e illustrate representative transfer and output curves that provide the basis for the subsequent experimental design. From the transfer curves, the gate bias was set to Ag / AgCl RE and the constant drain voltage (V D Various gate voltage ranges (V) in (, 0.5V) GS Drain current (I) over -0.5 ~ 1V D Monitored ). The measured result was 10 8It exhibits impressive n-type electrical characteristics at low operating voltages, including a high on / off ratio, high transconductance of 3.97 mS (Fig. 2f), a sub-threshold swing of 65.77 mV / Dec with small average clockwise hysteresis cycles across more than 50 devices in a narrow distribution (Fig. 2g), and a threshold voltage of 0.17 V (Fig. 2h). Another important parameter of the IGZO-EGTFT is operational stability in biological solutions. Fig. 2i shows V D The dynamic plot of drain current at various gate voltages ranging from 0.2V to 1V, while remaining constant at 0.5V, shows that changes in electrical characteristics were minimal after 103 seconds of recording. This demonstrates the biological robustness of the IGZO channel layer, guaranteeing the extended reliability of the IGZO-EGTFT for at least 103 seconds. This period coincides with the critical settling period required for the accurate detection of biological analytes in potential applications. Finally, the uniformity of electrical performance on a large-area scale at the wafer level was also confirmed.

[0064] 4. Operating Mechanism of MMP-9 Detection Biosensor

[0065] FIGS. 3a, 3b, and 3c show the design concept and operation mechanism of an MMP-9 detection biosensor based on IGZO-EGTFT according to one embodiment of the present invention. Specifically, FIG. 3a is a structural illustration of the domain structure and motifs of MMP-9, FIG. 3b is a predicted secondary structure of an MMP-9 aptamer and a scrambled aptamer equipped with 6-fluorescein phosphoramidite (FAM) predicted by the RNAfold web server, FIG. 3c shows that surface functionalization for detecting MMP-9 includes a series of steps, namely, hydroxylation of the IGZO surface via oxygen plasma, treatment with APTES as a crosslinking agent, immobilization of a specific aptamer as a bioreceptor, and direct recognition of MMP-9 in the electrolyte, wherein when MMP-9 is present in the electrolyte, a conformational change occurs in the aptamer-MMP-9 complex and the aptamer-MMP-9 complex moves closer to the IGZO sensing layer, causing channel current modulation, and the transfer curve shifts dramatically in the positive direction in the presence of MMP-9.

[0066] Referring to Fig. 3a, the domain structure of MMP-9 including a signal peptide, a propeptide domain, a catalytic domain, and a flexible hinge region is illustrated. The major DNA sequence of an aptamer specifically targeting MMP-9 (5'-phosphate-TTT TTT TTT TCG TAT GGC ACG GGG TTG GTG TTG GGT TGG-3') was selected as a bioreceptor. Referring to Fig. 3b, to control non-specific interactions, a scrambled aptamer with a randomized DNA sequence of the same length (5'-phosphate-TGG TTG TTT GTT TGC CGT TGG AGG TCT TTG TGT TGG ATG-FAM-3') was used simultaneously. Here, A, T, C, and G represent the four chemical components that make up DNA, namely nucleic acid bases; more specifically, A represents adenine, T represents thymine, C represents cytosine, and G represents guanine.

[0067] Referring to Fig. 3c, the operating mechanism related to MMP-9 detection is illustrated through the step-by-step process flow of surface functionalization and bioreceptor immobilization, which are key processes for the development of a high-sensitivity selective MMP-9 sensor. First, chemical modification using APTES is based on the use of an organic functional alkoxysilane that forms a covalent bond with the IGZO surface. This bond is formed through a condensation reaction between the siloxane group of the silane molecule and the hydroxyl group generated on the IGZO surface during the oxygen plasma treatment process. To develop an aptamer-based sensing interface, an aptamer modified with a phosphate group at the 5' end was covalently bonded to the APTES-functionalized IGZO surface. When MMP-9 is directly recognized on the aptamer-immobilized IGZO surface, the aptamer undergoes a conformational change on the IGZO surface, forming a stable and compact G-quadruplex structure. This causes the negatively charged phosphate backbone of the aptamer to migrate closer to the IGZO surface. Consequently, the potential between the IGZO surface and the electrolyte is expected to decrease due to the increase in negative charge on the IGZO. The effective V resulting from this potential change of IGZO TH Changes in channel current and channel current are electronically quantified in a label-free manner during electrical measurement. Therefore, the degree of channel current modulation is used as an indicator representing the MMP-9 concentration of the sensor of the present invention.

[0068] 5. Binding analysis of designed aptamers and scrambled aptamers by ELASA

[0069] To evaluate the specificity of aptamers recognizing MMP-9, an aptamer-based sandwich assay (ELASA) was performed using aptamers specifically designed to target MMP-9 and scrambled aptamers. To account for non-specific interactions, scrambled aptamers with random DNA sequences of equal length were used concurrently as a control. Initially, the MMP-9 aptamers and scrambled aptamers were labeled with 6-fluorescein phosphoramidite (FAM: green) dye at their 3' ends. 96-well microplates were pre-coated with anti-human MMP-9 antibodies. MMP-9 present in the samples was captured in the microplate wells, and any excess was removed through thorough washing. After introducing the MMP-9 aptamers and scrambled aptamers into the prepared microplates, strong fluorescence intensity was detected, indicating the successful formation of complete aptamer-MMP-9 complexes. These results support the fact that the MMP-9 aptamer used in this study specifically recognizes MMP-9.

[0070] 6. Surface analysis of MMP-9 detection biosensor

[0071] FIGS. 4a, FIGS. 4b, FIGS. 4c, FIGS. 4d, FIGS. 4e, and FIGS. 4f show surface analysis of a functionalized IGZO surface of an MMP-9 detection biosensor according to one embodiment of the present invention. Specifically, FIG. 4a shows AFM surface morphologies for surface functionalization of an IGZO surface, where (i) is pristine IGZO, (ii) is APTES treatment, (iii) is aptamer immobilization, and (iv) is an AFM morphology scan (5 μm x 5 μm) acquired in a non-contact mode of MMP-9 recognition; FIG. 4b shows changes in surface roughness according to the surface functionalization process obtained by analyzing the AFM results; FIG. 4c shows droplet contact angles according to surface functionalization including pristine IGZO, APTES treatment, aptamer immobilization, and MMP-9 recognition, where, based on specific surface conditions, the insets correspond to the droplet shape of each stage; FIG. 4d shows Fourier Trans Infrared (FT-IR) spectra according to surface functionalization including pristine IGZO, APTES treatment, MMP-9 aptamer immobilization, and MMP-9 recognition, where the FT-IR spectrum is 1cm -1 It was obtained without correction from 32 transmission scans of the resolution. Fig. 4e schematically shows the experimental strategy for determining the aptamer-MMP-9 complex using ELASA, and Fig. 4f shows the fluorescence intensity according to the formation of the aptamer-MMP-9 complex.

[0072] To comprehensively evaluate the effects of surface functionalization and bioreceptor immobilization, a systematic series of experimental investigations was conducted at each process step using atomic force microscopy (AFM), water contact angle (WCA), and Fourier transform infrared (FT-IR) spectroscopy. Figures 4(i), (ii), (iii), and (iv) confirm the functionalization of the IGZO surface by showing AFM images and height histograms after physical characterization of the device microstructure at each step ((i) pristine IGZO surface, (ii) after APTES treatment, (iii) aptamer immobilization, (iv) MMP-9 recognition). As shown in Figure 4b, AFM topography reveals an initial pristine IGZO rms surface roughness (Rq) of ~0.11 nm. After modification with APTES and aptamers, it was observed that roughness gradually increased as silane and aptamer molecules were added on top, reaching 0.28 nm and 0.48 nm, respectively. In particular, the increase in the Rq value of IGZO compared to the aptamer-modified surface was similar to the roughness changes detected in similar studies. Finally, by drop-casting the MMP-9 solution and the aptamer-MMP-9 complex to recognize MMP-9, relatively clear molecular clusters were observed on the surface. Additionally, the Rq of the IGZO surface increased significantly to 4.06 nm, reflecting an increase of 3.58 nm, indicating that the aptamer-MMP-9 complex was completed.

[0073] Referring to Fig. 4c, the WCA results for the IGZO surface before and after APTES treatment, after immobilizing the aptamer on the APTES-modified IGZO surface, and after direct recognition of MMP-9 are illustrated. As shown, after APTES treatment and aptamer immobilization, the WCA increases significantly due to the formation of a monolayer of APTES and aptamer, a result consistent with previous studies using aptamers for surface functionalization. Before APTES treatment, the raw IGZO surface exhibited an average WCA of 52.45°. After APTES treatment, this value increased to 64.99°, which is attributed to amine-terminal surface modification that enhanced surface hydrophobicity. After immobilizing the aptamer, the WCA further increased to approximately 72.85°, which appears to be due to the hydrophobic carbon chains of the aptamer, indicating successful covalent bonding between APTES and the aptamer. Conversely, it was observed that the WCA decreased significantly to 44.91° immediately after the introduction of MMP-9. This significant decrease may be due to the increased hydrophilicity of the sensing surface caused by the presence of hydrophilic functional groups, such as carboxyl and amino groups, within the polypeptide chain of MMP-9.

[0074] To further confirm the surface functionalization process, FT-IR spectroscopy was performed as shown in Fig. 4d. After APTES treatment, the presence of silanes equipped with free amine groups on the IGZO surface was confirmed through a strong absorption peak at 1568 cm⁻¹, corresponding to the modification mode of the amine group. Additionally, at 2953 cm⁻¹ -1 and 2847 cm -1The peaks represent stretching vibrations of -CH3 and -CH2 from the APTES backbone, respectively, further confirming that APTES was successfully deposited on the IGZO surface. This pattern was consistently observed at all APTES concentrations ranging from 1% to 10%. Given that aptamers are synthetic nucleic acids, the appearance of spectroscopic peaks between 1450 and 1750 cm⁻¹ on the aptamer-functionalized surface, which represent heterocyclic compounds, provided additional evidence that the aptamers successfully formed covalent bonds on the IGZO surface. These peaks match well with previously reported DNA signatures, particularly those of adenine, thymine, guanine, and cytosine. After MMP-9 recognition, a decrease in transmittance was observed in two specific spectral regions of the aptamer-immobilized IGZO surface: 1659 cm⁻¹ and 3550 cm⁻¹. The 1659 cm-1 region is attributed to the stretching vibration of the C=O bond, while the 3550 cm-1 region is associated with the bonding vibration of the NH2 bond.

[0075] To further verify MMP-9 recognition, ELASA was performed (Fig. 4e). Scrambled aptamers with random DNA sequences of equal length were used to control non-specific interactions. First, both aptamers were labeled with 6-FAM fluorescent dye at their 3′ ends. When MMP-9 and the aptamers were added to a 96-well microplate pre-coated with a human anti-MMP-9 antibody, the specific aptamer exhibited strong fluorescence intensity, whereas the scrambled aptamer did not (Fig. 4f). This clearly demonstrates the formation of an aptamer-MMP-9 complex. Therefore, all experimental results support the successful achievement of surface functionalization of the IGZO surface at each stage of the process.

[0076] 7. Sensitivity, Selectivity, Stability, and Reproducibility of MMP-9 Sensor

[0077] FIGS. 5A, FIGS. 5B, FIGS. 5C, FIGS. 5D, FIGS. 5E, and FIGS. 5F show the electrical characteristics of an IGZO-EGTFT for MMP-9 detection in an MMP-9 detection biosensor according to one embodiment of the present invention. Specifically, Fig. 5a shows the delivery characteristics of IGZO-EGTFT after APTES treatment, MMP-9 aptamer immobilization, and MMP-9 recognition on a linear scale in PBS solution, where the inset shows the delivery characteristics on a logarithmic scale; Fig. 5b shows the delivery characteristics according to various concentrations of MMP-9 on a linear scale in PBS solution, where the inset shows a wide detection range and a linear response to MMP-9 concentrations from 1 fg / ml to 100 ng / ml (error bars indicate standard deviation, total N = 144); Fig. 5c is an overview of detection performance, including the limit of detection (LOD) and detection range, for various types of MMP-9 detection technologies; Fig. 5d is a real-time measurement of serially diluted MMP-9 concentrations in the range of 1 fg / ml to 100 ng / ml using aptamer-immobilized IGZO-EGTFT; and Fig. 5e is phosphate-buffered saline (PBS), human serum albumin (HAS), and bovine serum This is a selectivity test of the MMP-9 sensor against normal interference including albumin (BSA), lethal bovine serum albumin (FSA), lysozyme, glucose oxidase (oxidase) of Aspergillus niger, β-casein, lactoferrin, and glucose, where *** indicates significant (p < 0.001) and NS indicates non-significant, and Figure 5f is the long-term stability of the sensor for 100 ng / ml of MMP-9, where after immobilizing the aptamer, the sensor was placed in a sealed vacuum tube and stored at room temperature for 4 weeks.

[0078] Referring to Fig. 5a, to verify the validity of the MMP-9 sensor, the transfer curve (I) after each step including raw IGZO, APTES treatment, aptamer immobilization, and detection of MMP-9. D -VG ) was evaluated. First, the amino-terminal groups (-NH2) on the surface of IGZO deposited with APTES [affect] hydrogen ions (H₂) in the electrolyte + It changed easily due to the adsorption of ), inducing a change in drain current. After the aptamer covalently bonded to APTES, the transfer curve and V TH All of them showed a positive shift. This shift indicates that the charge density of the surface functionalized by the negatively charged phosphate groups of nucleotides was modulated, which is consistent with previous studies. This trend corresponds to V corresponding to the surface functionalization steps. TH This was further confirmed by a positive shift. Although aptamers possess high target recognition capacity, optimizing experimental conditions is essential to improve sensor performance, including sensitivity, detection limit, and response time, by reducing the influence of unreacted active sites. To this end, NR values ​​were measured at various aptamer concentrations ranging from 1 to 15 μM and response times ranging from 1 to 3 hours. It was found that as the aptamer concentration increased, the NR value decreased significantly, stabilized at 5 μM, and remained unchanged above this concentration. Furthermore, a response time of 3 hours was confirmed to be optimal for surface functionalization using aptamers. These results suggest that under the specific conditions of a 5 μM aptamer concentration and a 3-hour response time, all APTES active sites on the IGZO surface were successfully covalently bonded with the aptamer.

[0079] When considering the practical application of tear analysis, it is essential to determine the minimum sample volume required to minimize the risk of false-negative results. As the sample volume decreased from 3 μl to 1 μl, the NR value showed minimal change until it reached 1 μl. Conversely, when the sample volume was reduced from 1 to 0, the NR value changed significantly from -0.426 to -0.335. Therefore, it was concluded that the minimum value capable of ensuring accuracy without producing false-negative results is a sample volume of 1 μl.

[0080] To determine the sensitivity of the device proposed herein for MMP-9 detection, the response of the sensor to various MMP-9 concentrations was experimentally evaluated. As shown in Fig. 5b, the transfer curve was systematically recorded over a concentration range of 1 fg / ml to 100 ng / ml at a constant drain voltage of 0.5 V. The inset is a calibration plot of the NR values ​​derived from the transfer curve, showing a high linear correlation coefficient (R² = 0.996) between the NR values ​​and the logarithm of the MMP-9 concentration. As a result of deriving the correlation equation, the slope and intercept errors were calculated to be -0.047 and -0.745, respectively. Consequently, the device according to the present invention had a very low detection limit of MMP-9 at 1 fg / ml and a wide detection range extending from 1 fg / ml to 100 ng / ml. This performance is superior to existing methods reported in recent literature, as can be seen in Fig. 5c.

[0081] For further review regarding actual diagnostic applications, real-time responses must also be secured. Figure 5d shows the real-time response of the aptamer-functionalized IGZO-EGTFT V G and V D While keeping constant at 1V and 0.5V, I over time DThe results of tracking and measuring changes in NR are shown. As the MMP-9 concentration increased from 1 fg / ml to 100 ng / ml, instantaneous channel current modulation was observed, suggesting a very excellent dynamic range and fast response for MMP-9 detection.

[0082] Another key feature of this sensor is its ability to specifically interact with the target analyte to generate a significantly enhanced signal, even in the presence of structurally similar interfering biomolecules. To evaluate selectivity, NR values ​​and transfer curves (I D -V G ) was measured. In addition, electrical characteristics were measured in the presence of other biomarkers associated with DED diagnosis, such as PBS, glucose oxidase (oxidase) of Aspergillus niger, β-casein and glucose, lactoferrin and lysozyme (Fig. 5e).

[0083] In particular, the sensor of this institute showed a statistically significant decrease in NR values ​​(ΔI / I0 = -0.426) in response to 100 ng / ml of MMP-9, whereas no significant change in NR values ​​was observed for other tested biomolecules. Furthermore, V which is negligible in the transport curve in the presence of these interfering biomolecules TH A shift was observed. These results demonstrate that the selectivity of the sensor is achieved through the specific recognition of MMP-9 by the aptamer without interference from other components in complex biological solutions.

[0084] Finally, to evaluate the shelf life of the device, the performance of the sensor was evaluated after storage for a period ranging from 1 to 4 weeks following aptamer immobilization. After aptamer immobilization, the device was stored in a sealed vacuum tube at room temperature. As shown in Fig. 5f, the device maintained 96.7% of its initial performance for an MMP-9 concentration of 100 ng / ml even after 4 weeks of storage. This stability is attributed to the excellent biocompatibility of the IGZO channel and the stability of the biological coating, both of which contribute to the maintenance of MMP-9 detection performance. Additionally, the reproducibility of the sensor was investigated by using seven different aptamer-modified devices for more than 4 weeks. The relative standard deviation (RSD) obtained at an MMP-9 concentration of 100 ng / ml was 3.95%, indicating excellent reproducibility of the sensor. These results highlight the excellent stability and reproducibility of the device and are expected to expand into clinical applications.

[0085] FIG. 6 is a flowchart illustrating a method for manufacturing an MMP-9 detection biosensor according to one embodiment of the present invention.

[0086] Referring to FIG. 6, first, in step 110, a substrate can be prepared. For example, the substrate can be prepared by thermally oxidizing a 300 nm thick layer of silicon dioxide on a boron-heavy doped Si wafer. Then, in step 120, a source electrode and a drain electrode can be placed on the substrate. For example, the source electrode and the drain electrode can be formed using a combination of 5 nm titanium and 50 nm gold metal via electron beam evaporation and photolithography.

[0087] Next, in step 130, a channel may be formed between the source electrode and the drain electrode. Here, the channel may be an IGZO channel. For example, the channel may be formed by performing oxygen plasma treatment on a substrate, particularly a Si wafer, to convert a hydrophobic surface into a hydrophilic surface, spin-coating a precursor solution, annealing it, and then patterning it. Then, in step 140, an aptamer may be bonded to the surface of the channel. The aptamer may be immobilized on the surface of the channel by bonding to a crosslinking agent covalently bonded to the surface of the channel. Here, the crosslinking agent may include APTES. More specifically, the crosslinking agent may be covalently bonded to the surface of the channel, and then the aptamer may be bonded to the crosslinking agent. For example, the surface of the channel may be treated with oxygen plasma to hydroxylate the surface of the channel, and then the crosslinking agent may be covalently bonded to the surface of the channel.

[0088] Next, in step 150, an electrolyte may be placed on the surface of the channel. More specifically, the electrolyte may be placed on the surface of the channel with an aptamer in between. Additionally, a well may be formed around the electrolyte to contain the electrolyte within the channel, thereby allowing the tear to be captured by the electrolyte. For example, the well may be formed of PDMS. Then, in step 160, a gate electrode may be formed to contact the electrolyte. Here, the gate electrode may not be in direct contact with the channel. The gate electrode may be an Ag / AgCl reference electrode.

[0089] According to one embodiment of the present invention, a biosensor capable of operating at low voltage can be provided based on an electrolyte gate thin film transistor having an IGZO channel.

[0090] According to one embodiment of the present invention, a biosensor capable of operating stably without degradation can be provided based on an electrolyte gate thin film transistor having an IGZO channel.

[0091] According to one embodiment of the present invention, a biosensor based on an electrolyte gate thin-film transistor having an IGZO channel can be provided to selectively detect only specific biomarkers using only a small amount of sample in tears containing a mixture of various types of substances.

Claims

1. In an MMP-9 detection biosensor for detecting the concentration of MMP (matrix metalloproteinase)-9 in tears, channel; An electrolyte disposed on the above channel; and Aptamer targeting MMP-9 bound to the surface of the channel in contact with the electrolyte Includes, The above aptamer reacts with MMP-9 in tears applied to the electrolyte to generate current modulation in the channel, thereby allowing the concentration of MMP-9 in the tears to be detected from the degree of current modulation. MMP-9 detection biosensor.

2. In Paragraph 1, Implemented based on an electrolyte-gate thin-film (EGTFT) structure, MMP-9 detection biosensor.

3. In Paragraph 1, Substrate; A source electrode and a drain electrode spaced apart and disposed on the substrate; and A gate electrode positioned in contact with the above electrolyte Includes more, The source electrode, the drain electrode, and the gate electrode are separated from each other by the channel. MMP-9 detection biosensor.

4. In Paragraph 1, The above channel is an IGZO (indium-gallium-zinc oxide) channel, MMP-9 detection biosensor.

5. In Paragraph 1, A well formed around the electrolyte on the above channel to accommodate the electrolyte internally, thereby allowing the tears to be captured by the electrolyte. including more, MMP-9 detection biosensor.

6. In Paragraph 1, The above aptamer is bonded to a crosslinking agent that is covalently bonded to the surface of the channel and is fixed to the surface of the channel. MMP-9 detection biosensor.

7. In Paragraph 6, The above crosslinking agent comprises APTES (3-aminopropyltriethoxysilane), MMP-9 detection biosensor.

8. In Paragraph 3, The above gate electrode is an Ag / AgCl reference electrode (RE), MMP-9 detection biosensor.

9. In Paragraph 3, The degree of the above current modulation is detected through the drain electrode, MMP-9 detection biosensor.

10. A method for manufacturing an MMP-9 detection biosensor for detecting the concentration of MMP-9 in tears, A step of binding an aptamer targeting MMP-9 to the surface of a channel; and Step of placing an electrolyte on the channel with the aptamer in between Includes, The above aptamer reacts with MMP-9 in tears applied to the electrolyte to generate current modulation in the channel, thereby allowing the concentration of MMP-9 in the tears to be detected from the degree of current modulation. Method for manufacturing an MMP-9 detection biosensor.

11. In Paragraph 10, The above MMP-9 detection biosensor is implemented based on an electrolyte thin-film transistor structure, Method for manufacturing an MMP-9 detection biosensor.

12. In Paragraph 10, Step of preparing the substrate; A step of spaced-apartly arranging a source electrode and a drain electrode on the substrate; A step of forming the channel between the source electrode and the drain electrode; and Step of forming a gate electrode to contact the above electrolyte including, Method for manufacturing an MMP-9 detection biosensor.

13. In Paragraph 10, The above channel is an IGZO channel, Method for manufacturing an MMP-9 detection biosensor.

14. In Paragraph 10, A step of forming a well on the channel above, formed around the electrolyte to accommodate the electrolyte internally, so as to capture the tears by the electrolyte. including, Method for manufacturing an MMP-9 detection biosensor.

15. In Paragraph 10, The step of binding the aptamer to the surface of the channel is, A step of covalently bonding a crosslinking agent to the surface of the above channel; and A step of binding the aptamer to the crosslinking agent so that the aptamer is fixed to the surface of the channel. including, Method for manufacturing an MMP-9 detection biosensor.

16. In Paragraph 15, The above crosslinking agent comprises APTES (3-aminopropyltriethoxysilane), Method for manufacturing an MMP-9 detection biosensor.

17. In Paragraph 15, The step of covalently bonding the crosslinking agent to the surface of the channel is, A step of hydroxylating the surface of the channel by treating the surface of the channel with oxygen plasma; and Step of covalently bonding the crosslinking agent to the surface of the channel including, Method for manufacturing an MMP-9 detection biosensor.

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