WO2025042037A1 - Linker for detecting biomarker embedded with phase-shift probe - Google Patents

Abstract

The present invention provides a linker having a net or cage structure, the structure of which is modified by a biomarker operating under a biphasic system consisting of a water layer and an oil layer. The linker is structurally modified in the presence of a biomarker to release a phase-shift probe contained therein, and the released phase-shift probe can be detected in the oil layer. In detection of the biomarker under the biphasic system using the linker of the present invention, the recognition of a target and the detection of a signal by the recognition are separated into the water layer and the oil layer, and thus the target can be detected with high accuracy. In addition, the system of the present invention does not require pretreatment of a sample, and thus can rapidly and accurately detect a quantitative biomarker with high sensitivity without expensive equipment and expertise required for pretreatment of a clinical specimen.

Description

Linker for biomarker detection embedding a phase-mobile probe

The present invention relates to a linker having a mesh or cage structure for detecting a biomarker existing in a liquid biopsy under a biphasic system composed of a water layer and an oil layer. Specifically, the present invention can be used in a method for diagnosing a disease by detecting a biomarker identified or identified at a high concentration in a liquid biopsy of a urogenital disease, and more specifically, the present invention can be used as a hyaluronic acid hydrogel for diagnosing bladder cancer by detecting hyaluronidase detected in the urine of a patient.

A biomarker is a type of biomolecule that exists in a biological or medical sample, and serves as a marker that allows for the diagnosis of disease status and a comprehensive judgment of the therapeutic effect of a drug and its correlation with other diseases by qualitatively and/or quantitatively detecting changes in its structure or concentration.

Biological or medical samples are divided into liquid biopsy and tissue biopsy. Tissue biopsy is obtained by an invasive method and has the advantage of being clinically verified and providing histological evaluation, but has the disadvantages of high separation costs, patient pain during the sample acquisition process, risk of infection, and long analysis times. On the other hand, liquid biopsy is obtained by a non-invasive method such as blood, urine, or saliva, and although it does not provide histological evaluation, it has the advantages of being easy to separate, having no risk to the patient, and being able to be analyzed relatively quickly.

Meanwhile, bladder cancer (BC) is the most common urogenital cancer worldwide, with ~500,000 new cases and ~200,000 deaths reported annually. Depending on the stage of cancer, bladder cancer is divided into non-muscle-invasive BC (NMIBC) at stages Ta, T1, and Tis and muscle-invasive BC (MIBC) at stages T2 to T4, and approximately 70–80% of bladder cancer patients have non-muscle-invasive bladder cancer, which has a high recurrence rate and a high possibility of progressing to muscle-invasive bladder cancer. The 5-year survival rate of bladder cancer patients is 95% in the early stage (NMIBC) and decreases to 10% in patients with confirmed metastasis. In addition, the majority of patients with muscle-invasive bladder cancer require cystectomy, and cystectomy significantly reduces the quality of life of patients. Therefore, accurate early diagnosis of bladder cancer through periodic and repeated examinations is necessary.

Currently, the clinical diagnosis of bladder cancer relies on cystoscopy and urine cytology, which involves inserting an optical instrument into the bladder to visualize the lesion. Cystoscopy and cytology require skilled technicians, but the morphologic heterogeneity of non-muscle-invasive bladder cancer results in low accuracy in diagnosing bladder cancer, depending on the cell sample and the skill of the technician. In addition, cystoscopy is highly invasive and expensive, and has side effects such as significant pain and a high risk of infection. In addition, urine cytology is diagnosed based on the subjective judgment of the pathologist after urine cell culture and staining, resulting in a low sensitivity of 15.8%. Although both protocols are standard clinical procedures, they are invasive tests that require highly trained technicians, cause side effects such as pain, and have low diagnostic accuracy for early-stage bladder cancer, which makes them insufficient for follow-up tests for bladder cancer patients and for the diagnosis of patients suspected of having bladder cancer.

Recently, as the paradigm shifts from invasive to noninvasive methods and from qualitative to quantitative analysis in the diagnosis of diseases, various urine-based diagnostic methods, including PCR tests, are being developed and are showing potential for the diagnosis of early bladder cancer. Despite the ease of collecting urine samples, preprocessing of urine samples for testing is still required, which requires laboratory-level equipment and skilled technicians. Some point-of-care (POC) tests have been developed so that unskilled people can test using urine samples, but the accuracy and sensitivity are still low, and especially in the case of early bladder cancer, diagnosis is difficult due to low biomarker concentrations and many nontarget substances in urine samples. In particular, 85% of bladder cancer patients have hematuria, which contains approximately 20,000 proteins at concentrations greater than 1 mM, interfering with the generation and transmission of desired target signals. Therefore, development of a POC-based test with high sensitivity and accuracy that can diagnose bladder cancer early without skilled technicians despite the heterogeneity of urine samples is required.

The technical problem to be achieved by the present invention is to provide a biphasic system capable of diagnosing urogenital diseases using a liquid biopsy obtained by a non-invasive method.

In addition, the present invention provides a hydrogel used to detect a biomarker found to be related to a disease under the biphasic system, and aims to provide a method for quantitatively detecting a target with high sensitivity without pretreatment of a liquid biopsy using a hydrogel and a kit for performing the same.

In addition, the purpose is to provide a hyaluronic acid hydrogel capable of detecting a biomarker for diagnosing urogenital diseases.

However, the technical problems to be achieved by the present invention are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

To solve the above problem, the present invention provides a biphasic system for detecting biomarkers in a liquid biopsy without preprocessing the liquid biopsy.

The biphasic system of the present invention includes a water layer containing a liquid biopsy and an oil layer that does not mix with the water layer.

In addition, the present invention provides a phase-transfer probe (PTP) operating under the biphasic system, wherein the phase-transfer probe may be fixed to a water layer by a linker.

In addition, the present invention provides a linker capable of detecting a biomarker by operating under the biphasic system, wherein the linker has a mesh or cage structure and includes the phase-shift probe within the mesh or cage structure.

In one embodiment of the present invention, the linker changes its structure when in contact with a liquid biopsy containing a biomarker to change the phase-mobile probe into a non-immobilized state, and in the present specification, the change of the phase-mobile probe fixed to the water layer into a non-immobilized state in the presence of the biomarker is expressed as dissociation, release, or the like of the phase-mobile probe from the linker.

In another embodiment of the present invention, the phase-transfer probe may include a signal generating substance or may be the signal generating substance itself. The phase-transfer probe may float in water and move to the upper oil layer through low density after dissociation and release from the linker to move from the water layer to the oil layer (lower layer: water layer, upper layer: oil layer), or conversely, move to the lower oil layer through high density (lower layer: oil layer, upper layer: water layer).

The above-mentioned phase-transfer probe functions as a signal messenger that is fixed to the water layer by a linker and is released in the presence of a biomarker to transfer the signal generating substance to the oil layer. Therefore, the term "phase-transfer probe" in the present specification may be used interchangeably with the term "signal messenger".

In another embodiment of the present invention, the signal generating material is not limited as long as it generates an electrical or optical signal detectable in the oil layer, and non-limiting examples thereof include a fluorescent substance, a chromophore, a catalyst, a dye, and an electrolyte. The generation of the signal is used to mean inducing a chemical or physical change that is converted into an electrical or optical signal detectable in the oil layer.

In another embodiment of the present invention, the fixation by the linker means preventing the phase-transfer probe from moving to the interface between the oil layer or the water layer and the oil layer, and the linker may react with the biomarker to free the phase-transfer probe fixed to the water layer.

As another embodiment of the present invention, the linker may have a structure of a net or cage to fix the phase-mobile probe therein.

As another embodiment of the present invention, the linker may be in a gel phase, more specifically, may be a hydrogel.

In another embodiment of the present invention, the linker can react with the biomarker, and if the linker is in a gel form, the reaction of the linker with the biomarker can decompose the gel, and if the linker has a mesh or cage structure, it can loosen or decompose the structure.

In another embodiment of the present invention, when the phase-transfer probe immobilized in the water layer is dissociated (unimmobilized) by the reaction of the linker and the biomarker, all or part of the phase-transfer probe moves from the water layer to the oil layer or to the interface of the oil layers.

In another embodiment of the present invention, the movement of the phase-transfer probe from the water layer to the oil layer may be due to diffusion, buoyancy, surface tension, or magnetic force.

As another embodiment of the present invention, the phase-transfer probe may be composed of an amphiphilic molecule, and may be a micelle structure or an organogel formed by the amphiphilic molecule.

In addition, the present invention provides a kit for detecting a biomarker, which comprises a container having a linker embedded with the phase-mobile probe fixed to the bottom surface and a nonpolar solvent capable of forming an oil layer.

In one embodiment of the present invention, the non-polar solvent may have a lower density than water, and the non-polar solvent may be stored in a separate container from the container in which the hydrogel is fixed to the bottom surface. Alternatively, a non-polar solvent having a higher density than water is also possible, in which case the hydrogel may be fixed to the wall surface of the upper layer.

In addition, the present invention provides a hyaluronic acid hydrogel for diagnosing urogenital diseases that operates under the biphasic system.

In the present invention, the urogenital disease may be bladder cancer.

The hyaluronic acid hydrogel of the present invention comprises a phase-transfer probe having the ability to move from a water layer to an oil layer. Specifically, the phase-transfer probe is embedded within the hyaluronic acid network structure of the hydrogel.

The phase-transfer probe of the present invention may be a micelle structure or an organic gel made of amphiphilic molecules, and may include a signal-generating substance therein.

As one embodiment of the present invention, the phase-transfer probe may be a monodisperse organogel particle prepared by adding 5 wt% of 12-hydroxystearic acid to a mixed solvent of toluene and oleic acid in a volume ratio of 2:1 and then sonicating the mixture.

As another embodiment of the present invention, the phase-transfer probe may have a density lower than that of water, specifically, the density of the phase-transfer probe may be 870 to 990 kg/m ³ , and preferably 880 to 890 kg/m ³ .

As another embodiment of the present invention, the signal generating substance contained within the phase-shift probe may be at least one selected from the group consisting of a fluorescent substance, a chromophore, a catalyst, a dye, and an electrolyte, and preferably may be a solvable chromophoric dye, and the solvable chromophoric dye may be Nile Red.

As another embodiment of the present invention, the hyaluronic acid hydrogel may be crosslinked by mixing hyaluronic acid and glutaraldehyde in a weight ratio of 1:2, and more specifically, may be produced by crosslinking the mixture on glass modified with O2 plasma.

In addition, the present invention provides a kit for diagnosing urogenital diseases, including a container having a hyaluronic acid hydrogel for diagnosing urogenital diseases fixed to the bottom surface and a nonpolar solvent capable of forming an oil layer.

As one embodiment of the present invention, the nonpolar solvent may have a lower density than water, and the nonpolar solvent may be stored in a separate container from the container in which the hyaluronic acid hydrogel is fixed to the bottom surface.

As another embodiment of the present invention, the kit may additionally include a blue LED.

As another embodiment of the present invention, the kit may additionally include a battery capable of operating the LED.

As another embodiment of the present invention, the kit may additionally comprise an orange acrylic filter.

In addition, the present invention provides a method for providing information for diagnosing a urogenital disease, comprising the following steps:

(1) A step of preparing a urine sample separated from an object;

(2) A step of adding the urine sample to a container having the hyaluronic acid hydrogel for diagnosing urogenital diseases fixed to the bottom surface to form a water layer;

(3) a step of forming an oil layer by adding a non-polar solvent having a lower density than water to the container to which the urine sample is added; and

(4) A step of detecting an electrical or optical signal in the above oil layer.

As an embodiment of the present invention, the method may further include a step of determining that the subject has developed a urogenital disease or is likely to develop a urogenital disease if an electrical or optical signal by a signal material is detected in step (4).

As another embodiment of the present invention, the urine sample of step (1) may be separated from the subject and may not be subjected to any separate pretreatment.

The present invention provides a linker having a mesh or cage structure whose structure is transformed by a biomarker operating under a biphasic system composed of a water layer and an oil layer. The linker transforms its structure in the presence of a biomarker to release a phase-shift probe contained therein, and the released phase-shift probe can be detected in the oil layer. Biomarker detection under a biphasic system using the linker of the present invention enables target detection with high accuracy because target recognition and detection of a signal by the recognition are separated into a water layer and an oil layer. In addition, since the system of the present invention does not require sample preprocessing, it is possible to rapidly and accurately detect quantitative biomarkers with high sensitivity without expensive equipment and specialized personnel required for clinical specimen preprocessing.

Figure 1 is a schematic diagram of the BLOOM assay for diagnosing bladder cancer.

In Figure 1a, a is the workflow of the BLOOM assay, b is the organogel messenger released by the non-gel film that is degraded by the biomarker, and c is the solvatochromic dye that migrates into the organic layer via density-based flotation.

Figure 1b is a photograph showing the steps of performing a BLOOM assay on a biphasic system under UV light irradiation to detect biomarkers in urine samples.

Figure 1c is a photograph showing the steps of performing a BLOOM assay on a biphasic system under UV light irradiation to detect a biomarker targeting hematuria.

Figure 2a is a schematic diagram of a manufacturing process of a Boyant organogel messenger containing Nile Red, a solvation chromogenic dye, and a photograph of the messenger dispersed in water.

Figure 2b is a confocal microscope image of the above-mentioned Boyant Organogel Messenger (scale bar: 40 μm).

Figure 2c is a size distribution graph of Boyant organogel particles (n=100).

Figure 2d schematically illustrates the mechanism of autonomous signal transduction based on the Voyant organogel. Specifically, the organogel messenger containing the solvatochromic Nile Red rises due to buoyancy from a low-density material (ρ=885.7 kg/m ³ ). At the interface of dodecane and water, the organogel is decomposed by dodecane, and the Nile Red released from the organogel emits green fluorescence that is different from the organogel fluorescence.

Figure 2e is a photograph of a biphasic system with organogel particles dispersed. The left is taken without UV irradiation, and the right is taken under UV irradiation.

Figure 2f is a UV-Vis absorption wavelength graph.

Figure 2g is the wavelength of fluorescence of organogel dissolved in water or dodecane.

Figure 2h is a photograph of the BLOOM assay performed under a biphasic system on artificial hematuria samples (containing NPU and blood) containing different concentrations of blood.

Figure 2i is a schematic diagram showing the transition from a polar solvent to a nonpolar solvent according to the mixing volume ratio of oleic acid and toluene in the production of Boyant Organogel Messenger.

Figure 2j is a drawing confirming the signal transmission efficiency of the Boyant organogel messenger synthesized by changing the volume ratio of oleic acid and toluene.

Figure 3a is a schematic diagram of the synthesis of a non-gel film degraded by hyaluronidase.

Figure 3b is an optical image of the Bigel film (d=15 mm).

Figure 3c is a cross-sectional image of the bigel film using a scanning electron microscope. The encapsulated organogel particles are indicated in purple.

Figure 3d is a fluorescence image of the Boyant organogel within the Bigel film using a confocal microscope.

Figure 3e is a graph of the size distribution of the Boyant organogel within the bigel film confirmed using a confocal microscope (n=40).

Figure 3f is the UV-Vis absorption and fluorescence spectra of bigel films ((excitation: 510 nm; inset: photograph of bigel films under UV irradiation).

Figure 3g is an infrared spectrum of a non-gel film with or without glutaraldehyde cross-linking.

Figure 4a is a photograph of Hdase detection on a biphasic system irradiated with UV. The Hdase-reactive bigel film is located on the bottom of a glass vial, and the biphasic system has a dodecane layer on the top and NPU on the bottom (Left: control, Right: 1 U/mL of Hdase). After 20 minutes of incubation, the Boyant organogel captured on the bigel film floats due to buoyancy and is located at the interface of NPU-dodecane.

Figure 4b shows the results of fluorescence intensity measurement by a reader for 60 minutes in a BLOOM analysis using an Hdase-free sample under a biphasic system (excitation: 460 nm).

Figure 4c shows the results of fluorescence intensity measured by a reader in a BLOOM analysis using NPU containing Hdase at a concentration of 1 U/mL under a biphasic system (excitation: 460 nm).

Figure 4d shows the results of measuring the fluorescence intensity at 540 nm over time under a biphasic system for samples with different Hdase presence or absence.

Figure 4e is a standard curve of normalized fluorescence intensity in NPU at different concentrations of HDase from 0.1 mU/mL to 1 U/mL.

Figure 4f compares the normalized fluorescence intensities of HDase and non-target analytes (10 mM for ions, urea, glucose, and amino acids and 1.4 mg/mL for proteins) in urine.

Figure 4g is a schematic diagram of the system and the normalized fluorescence spectrum of Nile Red in the Voyant signal messenger in the monophasic BLOOM system for Hdase detection (urine only or hematuria sample).

Figures 4h and 4i are a schematic diagram of the system and the detection results of the normalized fluorescence spectrum of Nile Red in the dodecane layer in a biphasic BLOOM system for Hdase detection (upper: dodecane, lower: urine or hematuria sample). Fluorescence was measured during incubation of an artificial hematuria sample of 250 RBCs/HPF (red blood cells per high-power field) containing 1 U/mL of Hdase for 60 minutes (excitation: 460 nm wavelength).

Figure 4j is a comparative graph of normalized fluorescence intensities of urine and hematuria (250 RBCs/HPF) according to HDase concentration.

Figure 4k shows the comparison of normalized fluorescence intensities in the dodecane layer incubated with artificial hematuria samples containing increasing RBCs/HPF and 1 U/mL of Hdase (inset: photographs of artificial hematuria samples with different RBCs/HPF values). Comparisons were performed using two-tailed Student's t- tests, and are expressed as means±SD values of three replicate experiments (NS: not significant).

Figure 5a is a schematic diagram of the clinical diagnosis in a hospital and the confirmation test for diagnosis of clinical samples using BLOOM and NMP22BC® rapid kits.

Figure 5b shows cancer tissue biopsy, cystoscopy, and magnetic resonance images (MRI) of a patient with muscle-invasive bladder cancers (MIBCs) (T2) and a patient with non-muscle-invasive bladder cancers (NMIBCs) (Ta and T1).

Figure 5c shows the normalized fluorescence intensities obtained from BLOOM analysis performed using urine samples from bladder cancer (BC) patients compared with non-tumor subjects (patients with urogenital diseases and healthy subjects) using Two-tailed Student's t-tests (****p<0.0001).

Figure 5d shows the normalized fluorescence intensities obtained from BLOOM analysis performed using urine samples from NMIBC patients compared with non-tumor subjects (urogenital disease patients and healthy subjects) using Two-tailed Student's t-tests (****p<0.0001).

Figure 5e is an operating characteristic curve of the receiver for predicting the sensitivity and specificity of BC and NMIBC diagnosis.

Figure 5f shows the degree of agreement between the diagnostic results of BLOOM or NMP22BC® and clinical diagnostic results for 105 urine samples.

Figure 6 shows the BLOOM POC device and the BLOOM analysis results using it.

In conventional sample analysis methods, target recognition, signal generation, and detection are performed in a single step in the analyte. At this time, target recognition and detection of the generated signal are interfered with by substances other than the target contained in the analyte, making it difficult to secure clean results.

The inventors of the present invention, taking into account the fact that most liquid biopsies are composed of water and that most substances in the biopsies are water-soluble substances, developed a biphasic system in which target recognition and signal generation and detection occur in different phases to detect biomarkers with high sensitivity without interference from non-target substances and without sample preprocessing.

In the present invention, a biphasic system includes a water layer containing a liquid biopsy, an oil layer that is not mixed therewith, and a phase-transfer probe (PTP) that can move from the water layer to the oil layer. The phase-transfer probe includes a signal-generating substance, and moves from the water layer to the oil layer in the presence of a target substance to transfer the signal-generating substance to the oil layer.

The present invention develops and provides a linker of a net or cage structure whose structure is transformed in the presence of a biomarker, for biomarker detection performed under the biphasic system.

In the present invention, the phase-mobile probe is embedded in a linker having a mesh or cage structure, and is fixed by the linker to a water layer containing or made of a liquid biopsy. The water layer may be formed by administration of a liquid biopsy or may be a liquid biopsy administered, and when a biomarker is present in the liquid biopsy, the biomarker decomposes or loosens the structure of the linker, thereby dissociating the phase-mobile probe from the linker.

In the present invention, the biomarker that is the detection target is capable of changing the structure of the linker, and if the biomarker is a biomarker that has been found to be related to a disease, the linker of the present invention can be used for the purpose of diagnosing the disease.

For example, hyaluronidase (Hdase) is an enzyme that breaks down hyaluronic acid and is known to be increased in the urine of bladder cancer patients. Accordingly, when the detection target of the present invention is set to Hdase, the linker of the present invention can be designed as a gel containing or made of hyaluronic acid and used for the diagnosis of bladder cancer.

The present inventors have verified the action of the linker of the mesh or cage structure of the present invention under the biphasic system by demonstrating that the biphasic system can be applied to the diagnosis of bladder cancer by detecting hyaluronidase in urine samples.

Hyaluronidase can decompose hyaluronic acid, and the signal messenger was designed to be fixed in the water layer by hyaluronic acid hydrogel.

The urine of bladder cancer patients is frequently hematuria mixed with blood, and hematuria contains a large amount of non-target substances such as proteins in addition to red blood cells, so preprocessing is essential before testing. However, the system provided by the present invention enables target detection with high sensitivity by utilizing the substrate specificity of the target substance in the urine sample, i.e., hyaluronidase, without preprocessing the urine sample, and target recognition through enzymatic action and detection of a signal by the recognition are separated into a water layer and an oil layer, enabling target detection with high accuracy.

The biphasic system for diagnosing bladder cancer of the present invention comprises a water layer containing a liquid biopsy, an oil layer not mixed therewith, and a signal messenger embedded in a hyaluronic acid hydrogel and fixed in the water layer. When Hdase is contained in the liquid biopsy, the network structure of the hyaluronic acid hydrogel collapses and the signal messenger embedded in the hydrogel is naturally liberated and moves to the interface between the water layer and the oil layer, and transmits the signal substance to the oil layer. At this time, the presence or absence of Hdase in the liquid biopsy can be determined by detecting an electric or optical signal by the signal generating substance in the oil layer, and the intensity of the signal is proportional to the amount of Hdase present in the liquid biopsy, so that quantitative analysis of Hdase in the liquid biopsy is possible.

In the present invention, the “water layer” is not limited to any type of polar solvent that can be mixed with water, and may also be simply composed of a liquid biopsy.

In the present invention, the "oil layer" may include, without limitation, any non-polar solvent that is not mixed with the water layer and can be separated. In the specific experiment of the present invention, dodecane was used as the non-polar solvent, but is not limited thereto.

In the biphasic system of the present invention, a phase-transfer probe fixed to an aqueous layer dissociates in the presence of a biomarker, and the dissociated phase-transfer probe moves from the aqueous layer to the oil layer or the interface layer between the aqueous layer and the oil layer.

The "movement" of the above-described phase-transfer probe into the oil layer or interfacial layer may be due to an intrinsic property of the phase-transfer probe, may be due to simple diffusion, or may be due to an external force such as buoyancy, gravity, electric force, or magnetic force. For example, the ability of the phase-transfer probe to move from the water layer to the oil layer may be due to the density difference with respect to the liquid forming the water layer. Specifically, when the specific gravity of the water layer is higher than that of the oil layer, the specific gravity of the phase-transfer probe is lower than that of the water layer.

In a specific experiment of the present invention, the density of the phase-moving probe was made lower than that of water so that it could move into the oil layer by buoyancy.

The phase-shift probe of the present invention may be a nanoparticle or microparticle to which a signal generating substance is attached or contained, a polymer particle, or a metal particle.

In the present invention, the metal forming the metal particles is not limited to zinc (Zn), selenium (Se), tellurium (Te), cesium (Cs), copper (Cu), lead (Pb), nickel (Ni), manganese (Mn), mercury (Hg), cobalt (Co), chromium (Cr), cadmium (Cd), strontium (Sr), iron (Fe), gold (Au), silver (Ag), platinum (Pt), praseodymium (Pr), gadolinium (Gd), barium (Ba), zirconium (Zr), molybdenum (Mo), indium (In), tin (Sn), lanthanum (La), cerium (Ce), neodymium (Nd), samarium (Sm), yttrium (Y), aluminum (Al), and europium (Eu), but preferably, it may be formed of at least one metal selected from the group consisting of gold, silver, copper, platinum, silicon, and iron with low reactivity. there is.

In addition, the phase-transfer probe of the present invention may have a property of being able to remain at the interface between water and oil layers for a long time, including amphiphilic characteristics, by having a hydrophilic ligand and a hydrophobic ligand attached to its surface, and is also a material having a function of being able to transfer a signal-generating substance to the oil layer.

In the present invention, the "signal generating material" is not limited as long as it generates a detectable electrical or optical signal, and the generation of the signal includes a chemical or physical change convertible into an electrical or optical signal in an oil layer. Therefore, the signal generating material in the present invention is not limited as long as it causes an electrical or optical signal itself or a physical or chemical change convertible into the signal, and non-limiting examples thereof include a fluorescent substance, a chromophore, a catalyst, a dye, an electrolyte, an acidic or basic substance, and in a specific experiment of the present invention, a solvatochromic dye was used as a signal generating material.

In the present invention, a "solvatochromic dye" is a dye or fluorescent molecule that absorbs or emits a specific wavelength depending on the properties of a solvent, and in the specific experiments of the present invention, Nile red was used, but is not limited thereto.

In the present invention, liquid biopsy means a biological sample isolated from a human, and in bladder cancer diagnosis, liquid biopsy means urine.

According to one embodiment of the present invention, the phase-transfer probe comprises a solvatochromic dye (specifically, Nile Red) as a signal-generating substance in a buoyant organogel. The buoyant organogel is manufactured to have a lower density than water.

According to one embodiment of the present invention, the fixation of the phase-transfer probe to the water layer is performed by mixing hyaluronic acid and Boyant organogel, adding glutaraldehyde (GA) to crosslink, drop-casting the mixture onto a cover glass treated with O2 plasma to produce a bigel film decomposed by hyaluronidase, and placing the cover glass modified with the bigel film on the bottom surface of a container to form a biphasic system. In a specific experiment, the average thickness of the bigel film attached to the surface of the cover glass is 20 μm. The bigel film decomposes when hyaluronidase is contained in a urine sample, thereby releasing Boyant organogel into a free state. The released Boyant organogel floats at a lower density than water and moves to the interface between the water layer and the oil layer, and in the oil layer, Boyant organogel releases a signal substance into the oil layer, thereby enabling the detection of specific fluorescence in the oil layer (Fig. 1a).

According to one embodiment of the present invention, the crosslinking density of the bigel film affects the release of the organogel. Therefore, in order to form a bigel network structure that can be decomposed by a trace amount of Hdase but does not release the organogel by simple swelling in the absence of Hdase, bigel films were produced by mixing hyaluronic acid and glutaraldehyde at various weight ratios, and the swelling stability and the degree of signal generation according to the decomposition by Hdase were confirmed. As a result, it was found that a bigel film produced by mixing hyaluronic acid and glutaraldehyde at a weight ratio of 1:2 had high swelling stability and could be sensitively decomposed by Hdase.

According to one embodiment of the present invention, the voyant organogel is a micelle structure, and when hyaluronidase is present in a urine sample, it is liberated from the hyaluronic acid network structure, moves to the oil layer, and comes into contact with a non-polar solvent in the oil layer to release a signal substance contained therein into the oil layer by reverse micelle.

According to one embodiment of the present invention, the micelle-structured voyant organogel particles affect the signal transmission efficiency occurring at the interface of the water layer and the oil layer according to their wettability. In order to optimize the wettability of the organogel particles, 5 wt% of 12-hydroxystearic acid was added to a mixed solvent of toluene and oleic acid in a volume ratio of 2:1, and the mixture was sonicated to produce monodisperse organogel particles.

Hereinafter, in a specific embodiment of the present invention, a buoyant organogel capable of moving from a water layer to an oil layer by buoyancy is called BLOOM (buoyancy-lifted bio-interference-orthogonal organogel messenger), and a biomarker detection analysis through phase movement according to the presence or absence of a biomarker under a biphasic system using the BLOOM is called BLOOM analysis.

The POC test method for diagnosing bladder cancer proposed by the present inventors may utilize BLOOM analysis performed under a biphasic system.

The present invention can be modified in various ways and has various embodiments, and thus specific embodiments are illustrated in the drawings and described in detail in the detailed description below. However, this is not intended to limit the present invention to specific embodiments, and it should be understood that all modifications, equivalents, and substitutes included in the spirit and technical scope of the present invention are included. In describing the present invention, if it is determined that a specific description of a related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted.

[Experimental methods and materials]

1. Materials

Nile red (NR), 12-hydroxystearic acid (HAS), and glutaraldehyde (GA) were purchased from TCI Chemicals. Hyaluronidase (Hdase) from bovine tests, hyaluronic acid sodium salt (HA) from Streptococcus equi , cetyltrimethylammonium bromide (CTAB), dodecane, toluene (Tol), and oleic acid (OA) were purchased from Sigma-Aldrich. Sealing tape (clear polyolefin) and laked horse blood were purchased from Thermo Fisher Scientific. Microscope cover glass (15 mm, round) and normal pooled urine (NPU) from human donors were obtained from Marienfeld superior and LEE biosolutions. NPU was used after repeated freeze-thaw cycles. All chemicals were used directly without further purification.

2. Tools

The morphology and size of the emulsion and bigel were measured by a confocal microscope (LSM 800, Zeiss) and a cryogenic scanning electron microscope (Quanta 3D, FEI). The formation of the bigel film was confirmed by Fourier transform infrared spectroscope (FT-IR) (Nicolet® iS20 FTIR, Thermo Fisher Scientific). An ultraviolet-visible spectroscope (S-3100 spectrophotometer, Scinco) and a fluorescence spectroscope (SpectraMax® M2 Multi-Mode Microplate reader, Molecular Devices) were used for characterization and detection experiments.

3. Preparation of buoyant organogel particles containing solvatochromic dye

10 mM NR was dissolved in Tol and OA (Tol/OA [v/v] = 2:1; total 1 mL). The solution was incubated in a heating bath at 65°C for 5 min to completely dissolve the NR dye in the mixed solvent. Then, 5 wt% HSA (43.7 mg) was added to the Tol/OA mixture to prepare stock solution 1. Then, a mixture of stock solution 1 and deionized water in a volume ratio of 1:25 was prepared (200 μL of stock solution and 5 mL of deionized water; total 5.2 mL). Then, the oil-water mixture was sonicated using a tip sonicator (Branson 450 digital Sonifier®) to prepare an emulsion. The sonication conditions for forming a monodisperse emulsion were optimized to be 160 W amplitude and 5 min or more of generation time. The size of 100 randomly selected acquired emulsions from the confocal images is ~4.1±0.7 μm. Organogel particles were formed by cooling at 25°C.

4. Signal transduction under biphasic solvent system

The wettability of buoyant organogel particles affects the efficient signal transduction at the fluid-fluid interface in a binary system. To optimize the wettability of buoyant organogel particles, a mixture of OA and Tol in a volume ratio of 1:2 was used. The OA and Tol mixture was used without any further treatment. To evaluate the signal transduction/organogel dissolution in a binary system, 200 μL of organogel dispersion was poured into the water layer located at the bottom of the binary system (1 mL of deionized water and 1 mL dodecane). The NR fluorescence intensity of the dodecane layer was measured for 60 min (excitation: 460 nm, emission: 540 nm). Additionally, to measure signal transduction and organogel dissolution under the down-regulated binary system, 5 mM CTAB was added to the water layer, and the fluorescence intensity of NR in the dodecane layer was measured for 60 min (excitation: 460 nm, emission: 540 nm wavelength). Artificial hematuria samples consisting of NPU and 5% laked horse blood were prepared, and the signal transduction efficiency between NPU and artificial hematuria samples was evaluated.

5. Manufacturing of HDase degradable bigel film

For hydrogel modification, HA (1 wt%) was mixed with 1.6 mL of water, and the mixture was heated in a bath heated to 65 °C for 6 h to dissolve the HA. Then, the 1 wt% HA solution was mixed with 3.2 mL of organogel dispersion. GA was added to the mixture of the HA solution and the organogel dispersion, and the pH was adjusted to 3 using HCl (0.02 N) to induce HA cross-linking for hydrogel preparation (mass ratio of HA to GA = 1:2). After gentle mixing, 200 μL of the mixture was drop-casted onto a cover glass treated with O2 plasma (power supply, 70 W; generation time, 1.5 min; FEMTO Science). A Hdase-degradable bigel film of uniform thickness was formed by incubating the O2 plasma-treated cover glass in a drying oven (BF-80N, Biofree) at 30°C for 24 h to induce crosslinking of GA and HA.

The crosslinking of bigel films was optimized using mixed solutions of HA and GA at various weight ratios (mass ratio of HA and GA=1:2, 1:4, 1:6, and 1:8). To optimize the production of bigel films, bigel films were produced by incubating in a drying oven for 6, 18, or 24 h.

6. Detection of Hdase using BLOOM under biphasic system

To verify the quantitative detection ability of Hdase of BLOOM under the biphasic system, NPU containing Hdase at concentrations of 0.1 mU/mL to 1 U/mL were prepared. BLOOM bigel film was placed on a 24-well plate, and 1 mL of NPU solution containing Hdase at different concentrations was added to each well. Then, 1 mL of dodecane was gently added on top of the solution containing Hdase, and the fluorescence spectrometer was set to 37°C and the excitation (460 nm) and emission (540 nm) wavelengths of NR in the organic layer were measured.

7. Detection of Hdase in artificial hematuria

Artificial hematuria samples were prepared by adding laked horse blood of intended concentration to NPU. The number of red blood cells (RBCs) in the artificial hematuria samples was measured. The number of RBCs corresponding to the concentration of laked horse blood was calculated using a confocal microscope of ×400 HPF. The standard curve was calculated based on the RBCs/HPF value. The number of RBCs in 1% laked horse blood in NPU is 253±0.7. Then, Hdase was detected in the same manner as in the BLOOM analysis described above using an artificial hematuria sample containing Hdase instead of NPU under a biphasic system.

To verify whether the signal acquisition of the BLOOM assay in artificial hematuria is effective in a biphasic system, a monophasic system without an organic layer on the top was constructed. The monophasic system was fabricated by adding 2 mL of urine/artificial hematuria sample without dodecane to a quartz cuvette instead of the bilayer of 1 mL of urine/artificial hematuria sample and 1 mL of dodecane. The monophasic system experiment used an artificial hematuria sample of 250 RBCs/HPF at 1 U/mL. The fluorescence intensity under the biphasic and monophasic systems was measured using a fluorescence spectrometer in the upper 1 mL.

8. Collection of clinical samples

Urine specimens from BC-suspected patients (BC and GU patients) and healthy individuals were provided by Korea University Anam Hospital in a blinded manner. All clinical samples were collected according to the protocol approved by the Institutional Review Board (2018AN0332). All patients and healthy individuals signed a consent form before participation, and the age, sex, and pathologic diagnosis of all participants were recorded anonymously. None of the urine samples came from the same patient, and the clinical samples were used for the experiment as soon as possible after collection.

9. BLOOM analysis of clinical samples

To perform BLOOM analysis using clinical samples, 500 μL of each clinical sample was mixed with reaction buffer (sodium acetate buffer, pH 4, 0.1 M) in a 1:1 ratio to adjust the pH. Prior to analysis, the bigel film on a 24-well plate was washed three times with phosphate-buffered saline (PBS) to remove excessive GA that can decompose HDase. Subsequently, 1 mL of clinical sample and 1 mL of dedocan were added to the bigel film to form a biphasic system. As a control, NPU that had undergone several freeze-thaw cycles was used. After incubation at 37°C, the fluorescence intensity was measured at 460 nm (excitation) and 540 nm (emission) using a fluorescence spectrometer.

10. NMP22 BladderChek® test on clinical samples

The NMP22 BladderCheck® (Abbott) system was used according to the manufacturer’s protocol. Briefly, four drops of clinical sample were added to each sample well on the test cartridge. The cartridge was incubated at ambient temperature for at least 30 minutes and photographed using a mobile phone camera. A visual color line in the test area was considered positive.

11. Structure of BLOOM POC reader device

The BLOOM POC reader device was designed using Autodesk Fusion 360 3D CAD software. The housing and other components were printed using a JGMaker A5S printer (JGMaker). The specific structure of the device is shown in Fig. 6a. Black polylactic acid was used as the 3D printing material to minimize reflectance in the fluorescence intensity measurement. An orange acrylic sheet (Inventables) was used as a filter to remove light with a shorter wavelength than the emission wavelength (540 nm) of the NR in the dodecane layer when illuminated with a light-emitting diode (LED). A polyimide heater (Alibaba), LED lights (Adafruit), and a temperature controller (Digikey) were used. The internal temperature of the POC device is maintained at 37°C. All components and electronic circuits are mounted in a single housing device. The layout diagram of the components is shown in Fig. 6a, and an actual photograph is shown in Fig. 6c.

12. BLOOM analysis of clinical samples using a POC reader

To perform BLOOM analysis on urine/hematuria samples of NMIBC patients using a POC device, 1 mL of clinical sample was loaded into a sample vial containing 1 mL of reaction buffer (acetate buffer, pH 4, 0.1 M) and an Hdase-degradable bigel film on the bottom. A vial containing 2 mL of reaction buffer and an Hdase-reactive bigel film was used as a control. Then, 3 mL of dodecane was gently added to each of the clinical sample and the control. Then, the heater was turned on in the BLOOM POC device and incubated for 3 hours.

Step 1: Mount the smartphone in the detachable smartphone holder, turn on the LED light with the switch, and acquire the NR fluorescence intensity of the dodecane layer of the experimental and control groups using the smartphone.

Step 2: Quantitative analysis of the fluorescence intensity obtained from the vials of the experimental and control groups was performed using ImageJ 1.53e software, and the NFI (I _sample /I _control ) of the sample vial was used.

13. Statistical Analysis

To analyze the statistical differences in each sample, Student's t-test was used for comparison. Statistical analysis was performed using GraphPad Prism 9.1.0. Quantitative data were expressed as mean ± standard deviation. Differences of *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 were considered significant.

[Experimental Results]

1. Messenger production and solvatochromic signal transduction

To prepare a uniformly sized Boyant organogel messenger, distilled water, toluene (Tol) containing 12-hydroxystearic acid (HSA) as an organogelator, oleic acid (OA) as a surfactant, and Nile red (NR) as a fluorescent signal substance were emulsified by heating above the gelation temperature using tip sonication (Fig. 2a). The emulsion was cooled to 25°C, and the resulting organogel particles had an average diameter of 4.1±0.7 μm and could float in water at a density lower than that of water (895 kg/m ³ for OA and 867 kg/m ³ for Tol; calculated density of organogel=885.7 kg/m ³ ; Figs. 2b to 2e). When in contact with the dodecane layer, the organogel particles dissolve and release NR molecules, which then emit green fluorescence that is different from the red fluorescence observed in the organogel (Figs. 2d and 2). The reading wavelength was selected to be 540 nm to exclude overlap with the fluorescence of the organogel in the water layer (Figs. 2f and 2g).

Since the self-suspension process of the signal messenger is biochemically orthogonal, we hypothesized that the signal generation in the dodecane layer would be independent of the clinical sample environment according to the intended design. To verify this hypothesis, biphasic systems were formed using normal pooled urine (NPU) or artificial hematuria samples and dodecane (5% and 50% of blood concentration in NPU, respectively). When the voyant signal messenger was injected into the bottom of the water layer, suspended particles and bright green fluorescence were observed in the dodecane layer (Fig. 2h). The fluorescence intensities of all systems were consistent and did not show significant differences compared to clear urine. This demonstrates the stability of the BLOOM assay to generate signals directly from clinical hematuria samples without any pretreatment to remove interferences, etc.

2. Optimization of phase transport of Boyant organogel particles

)는 아래 식 1로 표현할 수 있다The hydrophilic-lipophilic balance of organogel particles was optimized based on the stabilized interfacial energy of the particles under a biphasic system to enhance the phase-transport capability of signal messengers. The interfacial stabilization by colloidal particles is closely related to the transport efficiency from the bulk phase to the oil-water interface. The free energy associated with the transport of spherical particles from the bulk to the interface (

) can be expressed by Equation 1 below.

[Formula 1]

)는 콜로이드 입자의 습윤성을 이용하여 조절할 수 있다. 오가노겔 입자의 습윤성을 최적화하기 위하여, OA:Tol 부피비를 조절하여 다양한 계명활성제를 입자 표면에 개질시킨 일련의 오가노겔을 제조하였다(도 2j). OA가 없는 경우, 오가노겔 입자는, 계면활성제 존재 하에서 합성된 오가노겔 입자와 달리, 불안정하고 비정질(amorphous) 응집체를 형성하였다. 오가노겔의 Tol 함량을 OA 부피의 2배로 증가시키면, 도데칸 층에서 형광강도는 Tol이 없는 것과 비교하여 1.7배 증가하였다. 상기 결과를 기초로, 추가실험에서 오가노겔의 비율을 결정하였다. 또한, 이온성 계면활성제인 CTAB(cetyltrimethylammonium bromide)을 biphasic system에 첨가함으로써 계면 장력을 낮추면 신호 전달이 개선되고 형광 강도가 3배 증가하였다.In the above equation 1, R is the radius of the particle, θ is the interfacial tension, and θ is the three-phase contact angle between the particle and the interface. According to the above equation 1, the interfacial free energy (

) can be controlled by utilizing the wettability of the colloidal particles. In order to optimize the wettability of the organogel particles, a series of organogels were prepared by modifying the particle surface with various surfactants by controlling the OA:Tol volume ratio (Fig. 2j). In the absence of OA, the organogel particles formed unstable and amorphous aggregates, unlike the organogel particles synthesized in the presence of surfactants. When the Tol content of the organogel was increased to twice the OA volume, the fluorescence intensity in the dodecane layer increased by 1.7 times compared to that without Tol. Based on the above results, the ratio of the organogel was determined in additional experiments. In addition, the addition of CTAB (cetyltrimethylammonium bromide), an ionic surfactant, to the biphasic system lowered the interfacial tension, which improved signal transmission and increased the fluorescence intensity by 3 times.

3. Manufacturing of HDase degradable bigel film

After optimization of organogel preparation, Hdase-degradable film was fabricated on copper glass to capture and encapsulate Boyant organogel particles (d = 15 mm; Fig. 3a). A mixture of aqueous solution containing 1 wt% HA and Boyant organogel dispersion was drop-casted onto oxygen plasma-treated glass. For gelation of HA, glutaraldehyde (GA) was used as a cross-linker at acidic pH. After drying the film on the glass, Hdase-degradable non-gel film was formed (Fig. 3b).

Using cryogenic scanning electron microscopy, the formation of the hydrogel and the uniform thickness (20 μm) of the bigel film were confirmed (Fig. 3c), and spherical organogel particles were confirmed inside it. Fluorescence images acquired using a confocal microscope showed the distribution of the organogel on the film (Fig. 3d). The diameter of the organogel particles on the bigel film was 4.8±1.4 μm, which was slightly larger than that of the freely dispersed organogel, indicating that not much aggregation occurred during the drying process (Fig. 3e). The absorption wavelength of the bigel film was 550 nm, and the emission fluorescence wavelength was 610 nm, which are consistent with those of the organogel in the aqueous dispersion (Fig. 3f). The infrared spectroscopy of the bigel film showed a peak at 2,940 cm ^-1 , which corresponds to the crosslinking of HA and GA, confirming the crosslinking network structure of the bigel film (Fig. 3g).

4. Detection of Hdase using BLOOM analysis

The Hdase detectability of the BLOOM assay was measured. When NPU containing Hdase (1 U/mL) was poured onto the bigel film, the film was degraded after 20 min of incubation. This suggests that the network structure including HA was degraded by Hdase (Fig. 4a). While the film was degrading, the boyant organogel trapped in the bigel film floated to the dodecane-NPU interface and conducted the fluorescence signal into the dodecane layer. The bigel film incubated with NPU without Hdase (control) swelled, but no organogel or fluorescence signal could be observed magnetically floating in the dodecane layer. Subsequently, the fluorescence intensity over time was measured in each experimental and control group (Figs. 4b and 4d). In contrast to the 540 nm emission wavelength, which was not observed during 60 min of incubation in the control experiment, the BLOOM analysis of NPU containing Hdase showed a gradual increase in fluorescence intensity, which did not overlap with the NR emission at 610 nm of the non-gel film (Fig. 4c and Fig. 4d). The results suggest the detection of Hdase by observation of the flotation process and solvation fluorescence in the BLOOM analysis using clinical samples.

Next, the bigel film was optimized for sensitive detection of Hdase. The dense network structure of the bigel requires a large amount of Hdase to sufficiently decompose the hydrogel and release the voyant organogel particles. In contrast, the loose network of the bigel can release the organogel even when it is simply swollen in the absence of Hdase. Therefore, the crosslinking density of the bigel was adjusted (HA:GA mass ratio = 1:2, 1:4, 1:6, and 1:8) to fabricate a bigel network that is decomposable by a small amount of Hdase and stable when swollen in water. The 1:2 ratio was confirmed to be the optimal condition because it showed swelling stability and strong fluorescence intensity toward Hdase. Considering the optimal activity of the enzyme, BLOOM analysis was performed at 37°C.

For quantitative analysis, BLOOM analysis was performed using a series of NPU containing various concentrations of Hdase (0.1 mU/mL to 1 U/mL). In the experiment, the fluorescence intensity increased with increasing concentration of Hdase, and the detection limit was 0.28 mU/mL (~6.44 pM, three standard deviations above the background; Fig. 4e).

In addition, to confirm the specificity for Hdase in the BLOOM assay, analyses were performed using various competitor substances present in human urine samples (Fig. 4f). Typically, excessive amounts of various non-target substances {e.g., ions (NaCl, KCl, MgCl2, CaCl2, NaHPO4, and KHPO4; 10 mM), small molecules (urea and glucose; 10 mM), amino acids (histidine and tyrosine; 10 mM), and proteins (bovine serum albumin, lysozyme, fibrinogen, cathepsin, caspase, trypsin, and thrombin; 1.4 mg/mL)} are contained in NPU. The BLOOM assay did not react with any substances except for Hdase, suggesting that the BLOOM assay is a sensitive and specific detection system for Hdase.

5. Detection of Hdase in artificial hematuria

Hematuria occurs in 85% of BC patients. However, only 5% of symptomatic patients are diagnosed with BC. This symptom is because an increase in error signals, such as background signals, overlaps with the real signal and interferes with accurate quantitative analysis. Even differences in blood concentration between samples interfere with accurate signal acquisition through compensation. Based on the system designed in the present invention, we investigated whether BLOOM analysis can alleviate the interference of blood and urine, and found that the biphasic system of the present invention could acquire consistent signals regardless of the hematuria status. As a control, a monophasic system without a dodecane layer was prepared, and the oligomer release of voyant oligomers dispersed on the upper part of the artificial hematuria sample was observed after the addition of Hdase to an artificial hematuria sample (NPU with 250 red blood cells per high-power field (RBCs/HPF)) (Fig. 4g). Despite the release of Boyant Organogel messenger from the bigel film on top of the artificial hematuria sample, a difference in fluorescence intensity at 610 nm was observed compared to the NPU sample without any treatment, indicating quenching of the fluorescence signal by the substance in the hematuria sample. In the biphasic system, green fluorescence of NR monomer emission at 540 nm was observed in the dodecane layer of the artificial hematuria sample, which did not differ significantly from that of NPU (Fig. 4h). Although the artificial hematuria sample with high blood content (1250 RBCs/HPF) exhibited red fluorescence of Boyant Organogel in the bigel film, the fluorescence of the dodecane layer of the biphasic system showed a discernible signal in the presence of Hdase (Fig. 4i). Next, we verified whether consistent signal acquisition of BLOOM analysis was possible by comparing the normalized fluorescence intensities (NFI) in NPU with those in artificial hematuria samples (250 RBCs/HPF) using various concentrations of Hdase. There was no significant difference between the NFI of NPU and artificial hematuria samples at 1 mU/mL to 1 U/mL of Hdase (Fig. 4j). Regardless of the RBC count in the artificial hematuria samples, the fluorescence signals in the artificial hematuria samples were similar under the BLOOM system (1 U/mL Hdase; Fig. 4k). These results demonstrate that the biphasic system can overcome the obstacles caused by heterogeneous hematuria conditions and efficiently generate sufficiently reliable signals.

6. BLOOM evaluation for BC diagnosis in clinical samples

To evaluate the clinical performance of the BLOOM assay using the test results, to evaluate the clinical diagnosis of urologists, and to evaluate the interpretation of the NMP22BC® rapid kit using 105 clinical urine samples (60 BC patients, 20 patients with genitourinary diseases other than BC (GU), and 25 healthy subjects). Urine samples from BC and GU patients were collected for double-blind testing with consent for clinical validation. The urologists performed hematoxylin and eosin (H&E) histological analysis, cystoscopy, and magnetic resonance imaging on an individual patient basis in the hospital for BC diagnosis and cancer staging (Fig. 5b). Of the obtained samples, 66.7% were measured as hematuria ≥5 RBCs/HPF, which interferes with the sensitive and accurate detection of the biomarker. In the translational study, the NFI value (NFIV) obtained from the BLOOM analysis of clinical urine samples was used directly and was not converted to the corresponding HDase value. The mean NFIV (I/I0=1.78±0.52) of urine samples from BC patients was higher than that of non-smokers (I/I0=1.21±0.19) including healthy controls and GU patients (p<0.0001). Compared with the NFIV of urine samples from healthy controls (I/I0=1.18±0.15, p<0.0001), it could be distinguished from the NFIV of samples from symptomatic GU patients requiring accurate diagnosis and active surveillance for BC (I/I0=1.23±0.22, p<0.0001). The mean NFIV of healthy controls was lower than that of GU patients, and there was a slight difference in the distribution of NFIV, which is consistent with a previous study showing that Hdase concentrations are increased in urine samples from BC patients. In particular, BLOOM analysis was able to identify NMIBC patients (I/I0=1.73±0.48), including early stages (Tis, Ta, and T1), from non-tumor individuals (p<0.0001 for healthy controls, p<0.0001 for GU patients, Fig. 5d).

Next, the receiver operating characteristic (ROC) curve of the BLOOM analysis was constructed and the area under the curve (AUC) was calculated to investigate the diagnostic accuracy of BLOOM (Fig. 5e). In the BLOOM analysis, the estimated AUC values of BC and NMIBC patients were 0.93 and 0.92, respectively, compared with no-tumor individuals. Similar values were obtained between BC and NMIBC patients. The above results from the study design set including hematuria and GU specimens suggest that the BLOOM analysis has the practical applicability as an at-home POC test for the early diagnosis of BC. The sensitivity and specificity of the BLOOM test for BC patients without tumor were 88.3% and 88.9%, respectively (Fig. 5f). In contrast, the FDA-approved NMP22BC® adopted NMP22 (nuclear matrix protein) as the biomarker and showed lower sensitivity than the BLOOM assay for urine samples (sensitivity: 20.0% and specificity: 97.8% against no-tumor individuals). The sensitivity of NMP22® was lower than previously reported (56%, from the 19 studies). This low sensitivity is likely due to the fact that the majority of the patient population in the trial had NMIBCs (78.3%), the inclusion of GU patients in the non-tumor control group (44.4%), and the high incidence of hematuria.

In addition, the present inventors hypothesized that the target biomarker Hdase in the BLOOM analysis is correlated with the stage of BC because more Hdase is secreted by BC epithelial cells during the progression of BC, and that Hdase is associated with the invasiveness and metastasis of cancer cells. Under the above assumption, the ability of BLOOM to distinguish BC patients at various T stages was investigated. The AUC of the BLOOM test increased with the T stage of BC (0.904 for Ta, 0.935 for T1, and 0.938 or less for T2), confirming that it is helpful in identifying the stage of BC.

7. BLOOM POC device for early NMIBC identification

Next, we built a user-friendly, smartphone-based, portable fluorescence reader for the BLOOM system. The reader includes a polyimide heating pad for temperature control, a blue light-emitting diode for fluorescence, an orange filter, and a smartphone holder to maintain a constant distance between the smartphone camera and the sample for accurate signal intensity readings. The components include the BLOOM POC device (fluorescence reader), two sample vials for reference solution (buffer) and urine sample, two BLOOM Bigel films, liquid dispensers for urine and dodecane, and two containers for dodecane. The low cost of the reusable components, including the fluorescence reader, and the disposable Bigel films (approximately $0.11), allows for aggressive surveillance and repeated, widespread routine testing of large populations at a low cost per test.

The BLOOM assay with a POC device is suitable for at-home testing because the diagnostic workflow of the device consists of two simple steps: Step 1: Load the urine sample into the sample vial containing the reaction buffer and dodecane, and load both the control (buffer solution) and the sample vial using the liquid dispenser; Step 2: Turn on the heater switch and incubate the vials at 37°C, and then acquire the fluorescence image using a smartphone (Fig. 6b, c). Next, to validate the analytical performance of the POC device, a series of diluted clinical samples from patients with NMIBC and patients without BC were used to extract the fluorescence signal (I/I _control ) from the smartphone photos of the BLOM POC device through image processing (Fig. 6d). The fluorescence signal intensity decreased with the degree of dilution of the clinical samples, indicating the quantitative detection of Hdase using our device. Subsequently, the I/I _control for clinical samples from patients with NMIBC (n=10) and patients without BC (n=5; Fig. 6e and Fig. 6f) were analyzed. The signal intensity of NMIBC samples (I/I0=3.11±0.83) was significantly higher than that of individual samples without BC (I/I0=1.55±0.24, p<0.0001), demonstrating the diagnostic capability of the BLOM POC device.

Although the embodiments have been described with limited drawings as described above, those skilled in the art can apply various technical modifications and variations based on the above. For example, appropriate results can be achieved even if the described techniques are performed in a different order than the described method, and/or components of the described system, structure, device, circuit, etc. are combined or combined in a different form than the described method, or are replaced or substituted by other components or equivalents.

Therefore, other implementations, other embodiments, and equivalents to the claims are also included in the scope of the claims described below.

Claims (21)

As a linker for detecting biomarkers in a net or cage structure, The above linker contains within its structure a phase-transfer probe having the ability to move from a water layer to an oil layer, The above linker is in contact with the biomarker and its structure is modified, A linker for detecting a biomarker, wherein the above-mentioned phase-shift probe comprises a signal generating substance. In the first paragraph, A linker for detecting a biomarker, wherein the signal generating substance generates an electrical or optical signal detectable in an oil layer. In the second paragraph, A linker for detecting a biomarker, wherein the signal generating substance is at least one selected from the group consisting of a fluorescent substance, a chromophore, a catalyst, a dye, and an electrolyte. In the first paragraph, The above-mentioned phase-mobile probe is a linker for biomarker detection, which has a lower specific gravity than the water layer, is amphiphilic, or is an organic gel. In the first paragraph, A linker for detecting a biomarker, wherein the linker is in a gel form. A hyaluronic acid hydrogel for diagnosing urogenital diseases including a phase-shifting probe, The above-mentioned phase-mobile probe has the ability to move from the water layer to the oil layer, The above-mentioned phase-shifting probe is a hyaluronic acid hydrogel for diagnosing urogenital diseases, which contains a signal material therein that generates an electrical or optical signal detectable in an oil layer. In Article 6, The above-mentioned phase-mobile probe is embedded in a hyaluronic acid hydrogel for diagnosing urogenital diseases, which is a micelle structure or an organic gel by an amphiphilic molecule. In Article 7, A hyaluronic acid hydrogel for diagnosing urogenital diseases, wherein the organic gel comprises at least one selected from the group consisting of 12-hydroxystearic acid, oleic acid, and mixtures thereof. In Article 8, The above-mentioned phase-transfer probe is a hyaluronic acid hydrogel for diagnosing urogenital diseases, which is in the form of a monodispersed emulsion prepared by adding 5 wt% of 12-hydroxystearic acid to a mixed solvent containing toluene and oleic acid in a volume ratio of 2:1 and then ultrasonicating the mixture. In Article 6, The above-mentioned phase-mobile probe is a hyaluronic acid hydrogel for diagnosing urogenital diseases, which has a density lower than that of water and thus has the ability to move from a water layer to an oil layer due to buoyancy. In Article 6, A hyaluronic acid hydrogel for diagnosing urogenital diseases, wherein the signal material is at least one selected from the group consisting of a fluorescent substance, a chromophore, a catalyst, a dye, and an electrolyte. In Article 10, A hyaluronic acid hydrogel for diagnosing urogenital diseases, wherein the signal substance is a solvation chromogenic dye. In Article 11, A hyaluronic acid hydrogel for diagnosing urogenital diseases, wherein the solvent-based chromogenic dye is Nile red. In Article 6, The above hyaluronic acid hydrogel is a hyaluronic acid hydrogel for diagnosing urogenital diseases, which is cross-linked by mixing hyaluronic acid and glutaraldehyde in a weight ratio of 1:2. In Article 6, Hyaluronic acid hydrogel for diagnosing urogenital diseases, wherein the above urogenital disease is bladder cancer. A kit for diagnosing urogenital diseases, comprising a container having a hyaluronic acid hydrogel for diagnosing urogenital diseases of any one of claims 6 to 15 fixed to the bottom surface, and a nonpolar solvent that does not mix with water. In Article 16, A kit for diagnosing urogenital diseases, wherein the nonpolar solvent is stored in a separate container from the container in which the hyaluronic acid hydrogel is fixed to the bottom surface. In Article 16, A kit for diagnosing urogenital diseases, wherein the nonpolar solvent is dodecane. In Article 16, A kit for diagnosing urogenital diseases, wherein the above kit additionally includes a blue LED. Method for providing information for diagnosing genitourinary diseases, comprising the following steps: (1) A step of preparing a urine sample separated from an object; (2) A step of adding the urine sample to a container having a hyaluronic acid hydrogel for diagnosing a urogenital disease among the first to ninth clauses fixed to the bottom surface to form a water layer; (3) a step of forming an oil layer by adding a non-polar solvent having a lower density than water to the container to which the urine sample is added; and (4) A step of detecting an electrical or optical signal in the above oil layer. In Article 19, The above information providing method is an information providing method that does not include preprocessing of a urine sample separated from an individual.

Images