Non-invasive device and method for detecting RNA-related diseases

Abstract

The present invention relates to a non-invasive device (100) and method (400) for detecting a disease associated with RNA from the activity of a biological sample. The device (100) and method (400) include passing a biological sample through one or more stacked layers (102) in a first portion (101), which in turn induces visually monitored enzymatic activity. In this way, the ability of the sample to digest and decompose the layer (102) can be measured, thereby determining a risk assessment. In addition, the same biological sample may be dropped through the permeable connector (103) into the integrated chamber (104) for analysis. The integrated chamber (104) is further connected to the light-tight compartment (105) to quantify miRNA levels in the biological sample to determine the RNA-based disease type. Thus, a single device (100) herein provides dual functions of risk assessment and disease detection, with minimal sample size.

Description

[0001] Cross - reference to related applications and priority

[0002] This application claims priority to Indian Provisional Patent Application No. 202221012042, filed on September 7, 2022, and PCT Application No. PCT / IB2023 / 058840, filed on September 7, 2023, which are incorporated herein by reference. Technical Field

[0003] The subject matter described herein generally relates to the identification and assessment of proteolytic entities and RNA species in biological fluids. More specifically, the subject matter relates to an apparatus and method for identifying and assessing enzyme activity and RNA species in biological fluids for screening and identifying disease - related pathogenic RNAs and severity. Background Art

[0004] Genetic diseases refer to any diseases caused by abnormalities in an individual's genetic makeup. Genetic diseases are diseases caused in whole or in part by a deviation of the DNA sequence from the normal sequence. Those skilled in the art recognize that genetic disorders can be caused by mutations in one gene, mutations in multiple genes, a combination of gene mutations and environmental factors, or chromosomal damage.

[0005] Genetic diseases can also be complex, multifactorial, or polygenic, meaning that they are likely related to polygenic effects as well as lifestyle and environmental factors. Multifactorial disorders include cancer, heart disease, and diabetes.

[0006] Some diseases are caused by gene mutations inherited from parents, which are present at birth, such as sickle - cell anemia. Other diseases are due to acquired mutations in one or a group of genes that occur during a person's lifetime. Such gene mutations are not inherited from parents but are random or due to certain environmental exposures. Examples of diseases caused by acquired gene mutations include, but are not limited to, cancers caused by smoking and neurofibromatosis.

[0007] Worldwide, the incidence of acquired genetic disorders is the highest. There are more than 6000 known genetic disorders, and approximately 65% of people have some health problem due to gene mutations. Examples of the most common genetic diseases include, but are not limited to, Huntington's disease, sickle - cell anemia, cystic fibrosis, phenylketonuria, glycogen storage diseases, galactosemia, hemophilia, and hereditary spherocytosis.

[0008] In developed countries, it has been recognized that genetic diseases are a major cause of defects and can have fatal consequences, such as long-term hospitalization or even the death of an individual. Due to the large number of known genetic disorders, the diagnosis varies greatly depending on the type of genetic disorder. Most genetic disorders are diagnosed before birth, at birth, in early childhood, and in adulthood. By early assessment and detection of genetic disorders, the dominance of genes can be avoided.

[0009] In daily life, a person is constantly exposed to many factors, such as environmental and human factors, which can cause genetic damage or lead to changes in an individual's genetic makeup, often affecting the expression of certain genes without any traceable changes in the DNA. This additional genetic regulation is called epigenetic control. Environmental factors that cause genetic and epigenetic changes may include compounds produced by viruses, plants, fungi, and bacteria, industrial chemicals, combustion products, alcohol, ultraviolet light, and ionizing radiation.

[0010] The management of genetic diseases can be divided into counseling, testing, diagnosis, and treatment. Briefly, the basic purpose of genetic counseling is to help an individual or family understand their risks and options so that they are able to make informed decisions. The diagnosis of genetic diseases is sometimes based on clinical diagnosis according to specific symptoms, and sometimes on molecular diagnosis according to recognized gene mutations, whether or not there are clinical symptoms. Although there are effective treatments for some genetic diseases, there are some that do not.

[0011] In some cases, either there are no obvious gene mutations but there are pathologies related to the disease, or although there are gene markers related to the disease, there are no obvious abnormalities in an individual's lifetime. Therefore, it is obvious that predicting disease manifestations based solely on traceable gene mutations is not an accurate solution in the field of pre-symptomatic detection.

[0012] In addition, detecting RNA-related diseases (such as cancer) at an early stage can significantly reduce the discomfort of patients and improve prognosis, treatment intervention, survival rate, and recurrence rate. However, detecting and monitoring diseases often requires painful invasive procedures, such as biopsies and repeated blood draws.

[0013] Therefore, some widely used techniques for detecting specific RNA samples, such as saliva-based microbial, immunological, and molecular biomarkers, RNA blotting, nuclease protection assay (NPA), in situ hybridization, and reverse transcription polymerase chain reaction (RT-PCR), etc., are used as alternative means to bypass invasive measures to evaluate the stage, type, and intensity of the disease.

[0014] Therefore, the expression of any gene mutation in DNA can be traced through RNA profiles and RNA activity, which is an accurate tool for early prediction of many diseases long before their clinical symptoms appear.

[0015] In the prior art, U.S. Application "US9353409B2" discloses compositions and methods containing trehalose and DNA polymerase for facilitating rapid and efficient amplification of nucleic acid molecules and detection and quantification of RNA molecules, and improving detection sensitivity and reliability by generating safe cDNA molecules prior to gene-specific primer-dependent amplification. The reagent mixture includes a ready-to-use reagent solution, where the solution includes: (a) trehalose at a concentration between about 5% and about 35%; (b) viral reverse transcriptase; and (c) at least one DNA polymerase, in a buffer suitable for reverse transcription reaction, where the buffer includes cofactor metal ions and nucleoside triphosphates.

[0016] In the prior art, another U.S. Application "US5945515A" discloses solutions and methods for effectively and simply separating / extracting DNA, RNA, and proteins from a single biological material sample (such as cells, tissues, and biological fluids). Preferred solutions include an effective amount of a chaotropic agent, buffer, reducing agent, and may or may not include an organic solvent. Using the solutions and methods of this invention, genomic DNA and total RNA can be separated within 20 minutes, and proteins can be separated within 30 minutes.

[0017] In the art, U.S. Application "US20090215102A1" discloses a method for assessing breast cancer. This method only determines the risk of having the disease, but does not disclose a method for identifying a specific disease.

[0018] Techniques for detecting RNA-related genetic diseases (such as cancer) include extracting RNA from a sample, enzymatically amplifying the genome, then preparing cDNA and performing real-time PCR. However, such techniques are time-consuming, require expensive real-time detection thermal cyclers and extensive pre-treatment of biological samples to enrich the target RNA preparation. In addition, these techniques also require large-volume samples and use large instrument setups and multiple devices, thus increasing the risk of errors.

[0019] Furthermore, advanced laboratory equipment is thus a prerequisite for this RT-PCR-based detection. These currently known traditional methods are expensive, time-consuming, and cannot be widely used due to the economic burden.

[0020] Therefore, there has long been a need to develop a key device that has a dual function of both determining disease risk assessment and identifying disease-related pathogenic RNAs from biological fluid samples, thereby quickly and conveniently screening and predicting the onset of RNA-related diseases. The present invention also solves the following problems: providing a tool for detecting target RNAs with extremely high specificity without the need for purification and amplification in a short period of time. Summary of the Invention

[0021] The main object of the present disclosure is to provide a detection device and method for determining risk assessment and detecting the types of RNA-related diseases by analyzing biological fluids.

[0022] Another object of the present invention is to provide a highly reliable detection tool for predicting the health status of users.

[0023] Still another object of the present disclosure is to develop a device for detecting health status and disease types by implementing an RNA-based biomarker profile.

[0024] In addition, another object of the present invention is to develop a device that uses a method that is amplification-free and extraction-free to detect RNA-related disorders.

[0025] However, another object of the present invention is to develop a device that is cost-effective, requires a minimal amount of sample, and can speed up the diagnostic process compared to traditional methods.

[0026] The present invention is directed to introducing concepts related to devices and methods for detecting RNA species in biological fluids. The present invention is not intended to identify the essential features of the claimed subject matter, nor is it intended to determine or limit the scope of the claimed subject matter.

[0027] In one embodiment, a non-invasive device for detecting RNA-related diseases is disclosed. The non-invasive device includes a first part having a funnel-shaped central member for determining the proteolytic activity of a biological sample, wherein the first part is cast from at least two stacked layers. The device may also include a second part for identifying proteolytic entities and target RNA species in the biological sample. In addition, the second part may include at least one integrated chamber containing an enzyme mixture for identifying and cleaving target RNA species in the biological sample.

[0028] In addition, the device may further include an opaque compartment that contains a fluorescence excitation light source to generate fluorescence in response to the presence of a target RNA species. The device may also include at least one permeable enclosure configured to drip a biological sample from a first part to a second part. If the biological sample digests at least two stacked layers of the first part, the permeable enclosure enables the biological sample to pass through the first part to the second part.

[0029] In another embodiment, a method of detecting an RNA-related disease by enabling the device is disclosed. The method includes a plurality of steps. The method may include the step of injecting a biological sample into an opening of a central funnel-shaped member of the first part. The method may include another step of passing the biological sample through the first part to interact with each stacked layer, wherein enzymes from the biological sample digest and break down each stacked layer. The method may include a further step of dripping the biological sample through at least one permeable enclosure into at least one integrated chamber of the second part. The method may include the step of reacting an enzyme mixture present in the integrated chamber with a target RNA species in the biological sample. The method may include the step of irradiating the integrated chamber with a fluorescence excitation light source of the opaque compartment to generate a chemiluminescence signal in response to a control and a fluorescence signal in response to the presence of the target RNA species.

[0030] Other features and advantages of the present invention will be apparent from the following detailed description of the invention, which illustrates the principles of the invention by way of examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The detailed description is described with reference to the accompanying drawings. In the figures, the leftmost digit of the reference numeral indicates the figure in which the reference numeral first appears. The same numerals are used throughout the figures to denote the same features and components.

[0032] Figure 1 An isometric view of a non-invasive device (100) for detecting RNA-related diseases according to an embodiment of the present disclosure is described.

[0033] Figure 2a A cross-sectional view of a first part (101) of a non-invasive device for detecting RNA-related diseases according to an embodiment of the present disclosure is described.

[0034] Figure 2b An isometric view of a first part (101) of a non-invasive device (100) for detecting RNA-related diseases according to an embodiment of the present disclosure is described.

[0035] Figure 3 An alternative arrangement of a non-invasive device (100) for detecting RNA-related diseases according to an embodiment of the present disclosure is described.

[0036] Figure 4 Describes a flowchart of a method (400) for detecting RNA-based diseases according to an embodiment of the present disclosure.

[0037] Figure 5 Describes an illustration of total RNA isolated from human saliva on a 1% agarose gel according to an embodiment of the present disclosure.

[0038] Figure 6 Describes in vitro transcription of miRNA, gRNA, and control RNA using template DNA from a kit according to an embodiment of the present disclosure.

[0039] Figure 7 a Describes RT-PCR amplification plots of GAPDH (purple curve) and miR145 (green curve) of cDNA synthesized using RNA isolated from saliva according to an embodiment of the present disclosure.

[0040] Figure 7 b Describes melting curves of RT-PCR of GAPDH (purple curve) and miR145 (green curve) of cDNA synthesized using RNA isolated from saliva according to an embodiment of the present disclosure.

[0041] Figure 8 a Describes RT-PCR amplification plots of GAPDH (purple curve) and miR145 (green curve) of cDNA synthesized using direct saliva according to an embodiment of the present disclosure.

[0042] Figure 8 b Describes melting curves of RT-PCR of GAPDH (purple curve) and miR145 (green curve) of cDNA synthesized using direct saliva according to an embodiment of the present disclosure.

[0043] Figure 9a Describes a Cas13 assay pilot with test and control assays according to an embodiment of the present disclosure.

[0044] Figure 9b Describes a Cas13 assay pilot with gRNA, miRNA, and total RNA controls according to an embodiment of the present disclosure.

[0045] Figure 10 a Describes a Cas13 assay performed with two different concentrations of Cas13 (Test 1: 1 μM; Test 2: 0.8 μM) and a standard miRNA target according to an embodiment of the present disclosure.

[0046] Figure 10b depicts a Cas13 assay using 1 μM Cas13 and a direct saliva target according to an embodiment of the present disclosure. Detailed Description

[0047] References to "various embodiments", "one embodiment", "some embodiments", "an embodiment", or "an embodiment" throughout this specification mean that a particular feature, structure, or characteristic associated with the embodiment is included in at least one embodiment. Thus, the phrases "in various embodiments", "in some embodiments", "in one embodiment", or "in an embodiment" appearing in this specification do not necessarily all refer to the same embodiment. Additionally, in one or more embodiments, the features, structures, or characteristics may be combined in any suitable manner.

[0048] For those skilled in the art, various modifications to the embodiments may be apparent, and the general principles herein may also be applied to other embodiments. However, it is readily apparent to those of ordinary skill in the art that the present disclosure is not limited to the illustrated embodiments, but rather should be accorded the broadest scope consistent with the principles and features described herein.

[0049] In the prior art, biological samples (interchangeably referred to as biological fluids, body fluids, bodily fluids, biological cancers) are analyzed to detect RNA-based diseases such as diabetes, cancer, Huntington's disease, sickle cell anemia, cystic fibrosis, phenylketonuria, glycogen storage disease, galactosemia, hemophilia, and hereditary spherocytosis, among others.

[0050] According to an embodiment of the present disclosure, a non-invasive device (100) for detecting RNA-related diseases is illustrated herein.

[0051] In one embodiment, according to an embodiment of the present disclosure, an enzyme composition is disclosed herein for identifying any specific target RNA species and thereby identifying the type of disease in the body fluid or biological fluid.

[0052] The enzyme composition may include a predetermined amount of enzymes such as glucose oxidase and / or Cas13a. The enzyme composition may also include a predetermined amount of peroxidase. The enzyme composition may also include a predetermined amount of luminol (for detecting the activity of peroxidase in response to the presence of glucose in a sample used as a control).

[0053] As used herein, biological samples include, but are not limited to, saliva secretions, semen, vaginal secretions, mucus, nasal secretions, sweat, pancreatic juice, gastric secretions, and urine. However, saliva may be more preferred because it contains a variety of molecular and microbial analytes and is an effective indicator of local and systemic conditions. In addition, proteomic and transcriptomic biomarkers related to cancer have recently been discovered in saliva.

[0054] As used herein, the term "body fluid" as described herein can be any standard test sample, including matrix metalloproteinases, biopsy samples, biological fluids, body fluids, saliva samples, urine samples, uterine samples, body tissues, swabs, blood samples, or any pathological fluid sample, physiological fluid sample, including interstitial fluid, sweat, milk, ascites, mucus, tissue extracts, cell samples, etc.

[0055] As used herein, proteolytic enzymes, also known as proteases, peptidases, or proteinases, such as collagenase, gelatinase, and proteinase, have the unique property of catalyzing protein hydrolysis, that is, these enzymes have the property of digesting, degrading, and decomposing proteins into smaller polypeptides or single amino acids. A specific group of 24 proteases, collectively called matrix metalloproteinases (MMPs), consists of zinc-dependent endopeptidases that can degrade proteins.

[0056] As used herein, CRISPR arrays are families of DNA sequences found in the genomes of prokaryotes (such as bacteria and archaea). These sequences are DNA fragments from phages that have previously infected the prokaryote. They are used to detect and destroy DNA from similar phages during subsequent infections as an adaptive immune response to the virus.

[0057] As used herein, Cas13a is an RNA-dependent Rnase that can be programmed to specifically bind to a target RNA sequence approximately 20 nucleotides long by designing a crRNA (CRISPR-RNA) or gRNA (guide-RNA) complementary to its target RNA sequence.

[0058] Cas13 enzymes target single-stranded RNA (ssRNA), and its characteristic effectors are Cas13a, Cas13b, Cas13d, Cas13X, and Cas13Y. The type VI CRISPR-Cas13 system has an RNA-guided Rnase domain that, when activated, induces collateral degradation of nearby ssRNA molecules in a promiscuous manner. After binding and cleaving the target, Cas13a produces non-specific collateral Rnase activity. Cas13 enzymes have been used in various next-generation CRISPR-based diagnostic applications by taking advantage of the highly specific target recognition and cleavage of Cas enzymes, followed by trans-cleavage of reporter molecules through collateral activity.

[0059] In one embodiment, the RNA can include, but is not limited to, transfer RNA (tRNA), ribosomal RNA (rRNA), microRNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA (snoRNA), Piwi-interacting RNA (piRNA), tRNA-derived small RNA (tsRNA), small tRNA-derived RNA (srRNA), long non-coding RNA (lncRNA), and messenger RNA (mRNA).

[0060] As used herein, the term "miRNA" as described herein may be a key molecular component in the process of tumorigenesis, affecting several cell signaling pathways necessary for carcinogenesis. miRNA can be detected in saliva and has shown potential as a non-invasive biomarker for various cancers, including breast cancer, oral cancer, and lung cancer.

[0061] As used herein, the term "integrated chamber" refers to a single integrated space or compartment that combines different functions or elements into a tight whole.

[0062] More specifically, the subject matter of the present invention exemplifies a non-invasive device (100) capable of detecting the risk of RNA-related diseases, including assessing and identifying the type of disease by analyzing biological samples. In one embodiment, the device (100) can include two main parts (101, 106), such as a first part (101) for disease risk assessment and a second part (106) for detecting the type of RNA-based disorder. The second part (106) contains a CRISPR / Cas13-based enzyme composition for detecting specific miRNA biomarkers in oral cancer.

[0063] In one embodiment, specific miRNA biomarkers for oral cancer are used, more preferably miR145 present in saliva samples. In addition, the proposed device (100) was compared with the RT-PCR gold standard method, and a comparison between the isolated RNA and direct saliva was shown.

[0064] In one embodiment, with reference to Figure 1 and Figure 2, the device (100) is configured for risk assessment and detecting / identifying disease-related RNA by analyzing biological samples. In addition, the device (100) includes a first part (101) to determine the risk level of the disease or the severity of the disease. In addition, the first part (101) has a funnel-shaped central member (202) that can be cast above or together with one or more stacked layers (102), preferably at least two layers (102), more preferably three layers (102), one stacked on top of the other.

[0065] In one embodiment, the central funnel-shaped member (202) of the first part (101) may include an inlet end configured to receive a biological sample and an outlet end configured to direct the biological sample towards the first layer (102).

[0066] In a related embodiment, each stacked layer (102) (interchangeably referred to as a gelatin layer) cast in the text is composed of at least one of albumin, gelatin, fibrin, and globulin, more preferably gelatin. The analysis in the first part (101) (interchangeably referred to as a disease risk assessment component) is based on the gelatinase activity of proteases in a biological sample (preferably a saliva sample containing MMP) to digest and decompose the gelatin layer in the first part (101). The gelatinase activity in the subject's saliva can also be used to detect and quantify RNA species at the onset of an individual's disease state. The individual disease state refers to a diabetic state, a cancer state, or any other individual glucose peroxidase disorder affected by genes.

[0067] According to proteolytic efficiency and compositional domains, this family can be divided into four major classes: gelatinases (MMP-2, MMP-9), collagenases (MMP-1, MMP-8, MMP-13), stromelysins (MMP-3, MMP-10, MMP-12), and membrane-type matrix metalloproteinases (MT1-MMP, MT2-MMP, MT3-MMP, MT4-MMP). A typical MMP has a multi-domain structure, including a signal peptide, a pro-domain, a catalytic domain, a hinge region, and a hemopexin-like domain. MMP-2 and MMP-9 are secreted gelatinases, also known as type IV collagenases. These enzymes mainly degrade type IV collagen, which is an important scaffold for basement membrane proteins.

[0068] In the prior art, matrix metalloproteinases (MMPs) and disintegrants are a family of proteolytic enzymes mainly produced in the saliva secretions of subjects. The present disclosure provides a predictive tool for evaluating the proteolytic activity of the enzyme matrix metalloproteinase (MMP), such as MMP-9 and MMP-2.

[0069] As used herein, matrix metalloproteinases (MMPs) may include, but are not limited to, collagenases (such as MMP-1, MMP-8, and MMP-13), gelatinases (such as MMP-2 and MMP-9), stromelysins (such as MMP-3, MMP-10, and MMP-11), enamelysin (such as MMP-20), matrilysins (such as MMP-7 and MMP-26), metalloelastase (such as MMP-12), membrane-type MMPs, and other MMPs, such as MMP-19, MMP-21, MMP-23A, MMP-23B, MMP-27, and MMP-28.

[0070] In another embodiment, with reference toFigure 1 The apparatus (100) may include a second part (106) for identifying proteolytic entities and target RNA species (interchangeably referred to as targeted RNA, target RNA) in a biological sample. Herein, if the enzyme in the sample exhibits sufficient proteolytic activity to digest at least three stacked layers (102), the biological sample is transferred to the second part (106). The second part (106) includes at least one integrated chamber (104).

[0071] Herein, the integrated chamber (104) containing the enzyme mixture is configured to collect the biological sample dripping from the first part (101). The enzyme mixture helps obtain information on the disease type based on the sequencing of RNA. In addition, different from other conventional methods, the apparatus (100) of the present disclosure can be directly performed on the biological sample without separating RNA from the biological sample.

[0072] The enzyme mixture is selected from a CRISPR / Cas13a reaction mixture, glucose oxidase-peroxidase, and combinations thereof. Herein, the CRISPR / Cas13a reaction mixture consists of a chloride salt, a sulfonic acid buffer, a reporter RNA (rRNA), a guide RNA (gRNA), and Cas13a.

[0073] More preferably, the CRISPR / Cas13a reaction mixture in the second part (106) may include a gRNA in the range of 20 nM - 5 μM; a Cas13 type enzyme in the range of 80 - 120 nM; a chloride salt in the range of 3 - 6 mM; a fluorescent reporter in the range of 1 μM - 250 nM; and a sulfonic acid buffer in the range of 0.01 - 0.2 M.

[0074] Herein, the sulfonic acid buffer is more preferably HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid). Herein, the assay buffer optimizes and adjusts the components to minimize the required amount of Cas13a, thereby achieving higher efficiency than conventional methods.

[0075] In one embodiment, the untreated biological sample in the integrated chamber (104) contains microRNA as a disease biomarker. When the presence of miRNA is detected, the gRNA in the integrated chamber (104) activates and guides Cas13a to bind to the target miRNA, resulting in the cleavage of the target miRNA. In addition, after being activated by the target miRNA, Cas13a starts to cleave the reporter RNA through its collateral activity.

[0076] In addition, the device (100) may further include at least one permeable connector (103) connecting the first part (101) and the second part (106). The permeable connector (103) is used to transfer body fluid from the first part (101) to the integrated chamber (104) of the second part (106). In this article, if the body fluid digests at least three stacked layers (102) of the first part (101), the permeable connector (103) can transfer the body fluid from the first part (101) to the second part (106) only.

[0077] In one embodiment, the permeable connector (103) is selected from at least one of capillary tubes, tubular channels, sieves, filter membranes, tubular openings with bottom threads, perforated structures, and perforated plates, but is not limited thereto, and more preferably capillary tubes or tubular openings with bottom threads.

[0078] In one embodiment, the permeable connector (103) is located between the first and second parts (101, 106) to enable risk assessment and disease type detection of a single biological sample simultaneously in the same device (100). If the gelatin layer (102) is not effectively digested by the enzyme in the biological sample, this arrangement can facilitate the removal and discard of the first part (101). In this article, the first part (101) is detachably connected to the second part (106).

[0079] In another embodiment, as Figure 3 shown, the device (100) may further include a plurality of permeable connectors (103) in the form of capillary tubes, arranged on the outer side of the first part (101).

[0080] In one embodiment, the permeable connector (103) connected to the first part (101) is configured to drip a part of the biological sample into the integrated chamber (104). The working principle of the integrated chamber (104) of the second part (106) is based on the CRISPR / Cas13a enzyme to detect a specific type of RNA, thereby detecting biomarkers related to a specific type of disease.

[0081] In one embodiment, the integrated chamber (104) may include a CRISPR / Cas13a reaction mixture. Cas13a is an RNA-dependent Rnase, which can be programmed by designing crRNA (CRISPR-RNA) or gRNA (guide-RNA) complementary to the sequence of its target RNA species to specifically bind to a target RNA sequence about 20 nucleotides long. After binding and cleaving the target, Cas13a generates non-specific collateral Rnase activity.

[0082] In one embodiment, the integrated chamber (104) may include luminol for control, peroxidase for verification and measurement of enzyme detection, or detect the glucose level in a biological sample by detecting hydrogen peroxide generated by a glucose oxidation reaction. Herein, luminol emits light when reacting with hydrogen peroxide in the presence of peroxidase.

[0083] In another embodiment, the device (100) may further include a light-tight compartment (105) containing a fluorescence excitation light source, including but not limited to a UV LED. The accompanying Rnase activity of the reporter RNA can generate fluorescence in response to the presence of cleaved biomarker RNA. The fluorescence can be quantified and evaluated to detect the type of biomarker present in the biological sample, thereby determining the disease type. The device (100) can also be customized to generate a chemiluminescence control signal in response to the biological sample, such as but not limited to the salivary glucose level in one of the integrated chambers (104). Herein, the glucose concentration is used as a control to verify whether the device (100) and the method are working properly.

[0084] In one embodiment, the integrated chamber (104) that generates chemiluminescence can further send a control signal to the display unit (301).

[0085] In another embodiment, the device (100) may include one or more optical fibers that pass through each integrated chamber (104) (alternatively referred to as an integrated cell) for transmitting the fluorescence signal to a photoresistor (not shown in the figure) or a simple photodiode detection unit (not shown in the figure).

[0086] In another embodiment, the device (100) may optionally include an external or internal power source, which can be used to combine the simple photodiode detection unit with a radio transmitter (not shown in the figure) to establish a wireless connection and facilitate reading on a display unit (301) including but not limited to a mobile phone, a computer, etc.

[0087] In another embodiment, the device (100) may optionally include a lid (201) for covering the opening of the first part (101) to prevent the entry of any impurities that may cause result errors during testing.

[0088] In one embodiment, the device (100) can be used as a "disease risk assessment kit" to detect proteolytic activity, such as the gelatin hydrolysis activity of gelatinase, and can also be used as a "disease type detection kit" to evaluate RNA biomarker species through a CRISPR / Cas13a-based composition mixed with a biological sample.

[0089] As used herein, the device (100) may be interchangeably referred to as a "kit", "testing device", "detection device", "activity detection device", "enzyme detection device", "MMP protease detection device", "proteolytic activity detection kit", "gelatin activity detection kit", "RNA detection device", "RNA detection equipment", "RNA detection unit", etc.

[0090] In one embodiment, the device (100) may include a central portion for visually monitoring enzyme activity (interchangeably referred to as gelatinase activity) and an opaque compartment (105). The integrated chamber (104) is adjacent to a UV LED serving as a fluorescence excitation source, and optical fibers pass through each CRISPR reaction unit to transmit the fluorescence signal to a photoresistor or a simple photodiode detection unit.

[0091] In one embodiment, the device (100) including a plurality of integrated chambers (104) may further include different types of gRNA in the reaction mixture, which can be used to identify specific RNA species in a given biological sample. This is unique to the proposed solution and is not obvious to those skilled in RNA detection techniques. By binding different gRNAs together, different mRNAs can be detected simultaneously at the same time, thereby utilizing the limited available sample to identify different diseases in a single device.

[0092] In one embodiment, the monitoring unit is used to determine the activity of the biological sample in the first part and the second part for risk assessment and RNA-related disease identification. The activity herein preferably includes, but is not limited to:

[0093] ● The proteolytic activity of enzymes in the sample;

[0094] ● Identifying RNA-related diseases based on the irradiated light through the integrated chamber;

[0095] ● Indicating the normal function of the device (100) based on the detection of peroxidase activity in the integrated chamber.

[0096] In another embodiment, according to an embodiment of the present disclosure, a method (400) for detecting RNA-based diseases based on CRISPR / Cas13a RNA in a biological sample is illustrated.

[0097] In a related embodiment, the method (400) includes Figure 4 the multiple steps provided in. Refer to Figure 4, the method (400) may include the step (401) of injecting a biological sample into the opening of the central funnel-shaped member (202) of the first part (101). Herein, the biological sample is injected into the first part (106) in the range of 50 μl to 500 μl, preferably 100 - 200 μl.

[0098] The method (400) may further include a step (402) of passing the biological sample through the first part (101) to interact with each stacked layer (102). Herein, the enzyme in the biological sample digests and decomposes each stacked layer (102) through the activity of the enzyme to move downward along the central funnel-shaped member (202) of the first part (101).

[0099] In a related embodiment, the method (400) may further include the step of visually monitoring the enzyme activity in the disease risk assessment component. The method (400) may further include the step of passing the biological sample through one or more permeable connectors (103) and simultaneously dripping it into respective integrated chambers (104), and the integrated chambers (104) contain a CRISPR / Cas13a-based enzyme composition for specific RNA detection.

[0100] In a related embodiment, the method (400) may further include the step (403) of dripping the biological sample into at least one integrated chamber (104) of the second part (106) through at least one permeable connector (103). In addition, the method (400) may further include the step (404) of reacting the enzyme mixture in the integrated chamber (104) with the target RNA species (which can be interchangeably referred to as targeted RNA, target RNA) in the biological sample. Herein, only 5 - 15 μl of the biological sample is required in the integrated chamber (104) for the enzyme reaction.

[0101] The method (400) may further include the step (405) of irradiating the integrated chamber (104) with a fluorescence excitation light source of the light-tight compartment (105) to generate a chemiluminescence signal in response to the control and a fluorescence signal in response to the presence of the target RNA species.

[0102] In a related embodiment, after the CRISPR / Cas13a-based enzyme composition binds to and cleaves the target, it generates non-specific collateral Rnase activity.

[0103] The fluorescence generated by the reaction of the RNA in the biological sample with the enzyme can be detected and quantified to determine the type of disease / condition. In one embodiment, a self-quenching fluorescent RNA beacon can be used to monitor the reaction.

[0104] In one embodiment, the method (400) may further include the step (205) of presenting a disease risk assessment level and a disease type based on collateral Rnase activity associated with CRISPR / Cas13a monitored using a self-quenching fluorescent RNA beacon or a reporter RNA.

[0105] In one example, in accordance with the disclosure of the present invention, a device (100) as a disease type detection kit may provide a preliminary estimate of the glucose level in a body fluid, thereby preliminarily analyzing the diabetes status of an individual.

[0106] In addition, the method only requires basic sample pretreatment, has low cost, simple operation, and extremely small sample volume required. Therefore, the disease risk assessment and detection kit (100) has lower cost and higher sensitivity, and is a better alternative to traditional RNA detection techniques. In addition, in order to better understand the present disclosure and related methods, the following examples will be discussed.

[0107] Example 1: Activity of biological samples in the first part

[0108] A: MMP normalization

[0109] To identify the digestion characteristics of MMP, three different culture media were prepared. These three different culture media included a 2% bovine serum albumin (BSA) control, MMP9 with 2% BSA, and MMP9 without BSA. In addition, different culture medium samples were used in the first part of the device, and the time required for the digestion of the layer was recorded.

[0110] Table 1: Digestion of 2% BSA control

[0111] Group number Pass through the first layer 1 11.55 minutes 2 12.43 minutes

[0112] Table 2: Digestion of MMP9 with 2% BSA

[0113] Group number Pass through the second layer 1 14.3 minutes 2 Did not pass through 3 Did not pass through

[0114] Table 3: Digestion of MMP9 without BSA

[0115] Group number Pass through the second layer 1 8.4 minutes

[0116] As can be seen from Table 1, Table 2, and Table 3, the BSA control group only digested the first layer of the first part, while MMP9 with 2% BSA and MMP9 without BSA digested two layers, indicating that MMP9 is active.

[0117] In addition, different amounts of MMP9 and MMP2 were also selected to test the time required for digesting the layer.

[0118] Table 4: Digestion of MMP9 and MMP2 over time

[0119]

[0120]

[0121] It can be concluded from Table 4 that when MMP9 and MMP2 are present in the sample simultaneously, three stacked layers can be digested, indicating the presence of proteolytic activity and oncogenic cancer.

[0122] B: Gelatin kit as the internal casting layer structure of the first part

[0123] The gelatin-agarose kit was prepared by the following method: First, 18% gelatin powder (type B bovine) was mixed with MMP buffer (50 mM Tris-HCl pH 7.6, 300 mM NaCl, 5 mM CaCl 2 , 1 mM ZnCl 2 , 20 mg% SDS), and then 0.25% agarose was added. The mixture was slowly heated to uniformity but not boiled. The resulting liquid gelatin was made into films on kit molds of different sizes and dried overnight in a controlled environment. Then the dried kits were carefully inspected and packaged to ensure their quality and usability. In addition, the stability and shelf life of the device (100) over time were also tested.

[0124] Table 5 (a - e): Activity of the first part (101) of the device (100) with BSA as the control and trypsin as the experimental group within one month

[0125] Table 5 (a):

[0126]

[0127]

[0128] Table 5 (b):

[0129]

[0130] Table 5 I:

[0131]

[0132]

[0133] Table 5 (d):

[0134]

[0135] Table 5 (e):

[0136]

[0137] Example 2: Activity of biological samples in the second part (106) A: Sample collection and processing and total RNA isolation

[0138] The procedure for collecting saliva samples follows established guidelines to ensure the accuracy and consistency of sample collection. Patients are required not to eat, drink, or smoke for at least 30 minutes before collection, as the fasting period helps minimize potential contaminants in the saliva.

[0139] Unstimulated morning saliva is collected from patients at different stages of oral squamous cell carcinoma (OSCC). Patients are instructed to rinse their mouths with water for 30 seconds. Subsequently, 10 ml of normal saline is poured into the patient's mouth and stirred for about 1 - 2 minutes, and then the unstimulated saliva is carefully collected into a 5 - ml sterile collection tube pre - filled with a buffer solution. Part of the sample is centrifuged at 10,000 rpm for 10 minutes, and the supernatant is discarded. The remaining pellet is resuspended in an appropriate buffer for RNA isolation. Saliva samples are isolated from four subjects to ensure suitable extraction.

[0140] In addition, total RNA is isolated from 5 ml of normal human saliva using the Qiagen miRNEasy Mini Kit (Cat. No.: 1038703) according to the manufacturer's instructions. The isolated RNA is subjected to qualitative and quantitative analysis on a 1% agarose gel at 80 V for 2 hours.

[0141] B: In vitro transcription of standard miRNA, gRNA, and GAPDH mRNA

[0142] Standard miRNAs and corresponding gRNAs are synthesized by in vitro transcription using the T7 High - Yield RNA Transcription Kit (Cat. No.: THY - 50rxn). Briefly, the laboratory ordered transcription templates attached with the T7 promoter region for RNA synthesis. 0.5 μg of dsDNA templates of miRNA, gRNA, and GAPDH mRNA are transcribed using high - yield T7 polymerase and 3 mM NTPs in the standard buffer provided by the kit in a 20 - μl reaction. The reaction is carried out at 37 °C for 16 hours. After the reaction, 10 μl of the prepared standard miRNAs and gRNAs are treated with 1 μl of DNase I to remove residual DNA and incubated at 37 °C for 15 minutes.

[0143] C: Reverse transcription to prepare cDNA

[0144] cDNA was synthesized using the Qiagen miRNEasy Mini Kit (reference number: 1038703) and miRCURY RT SYBR. The cDNA was synthesized from direct saliva samples, standard miRNAs, and total RNA for RT-PCR detection. When processing direct saliva samples, 10 μl of the sample was heated at 98 °C for 10 minutes, and 2 μl of it was reverse transcribed into a 10 μl reaction volume. 2 μl each of the transcribed miRNA and total RNA were used for cDNA preparation, with a reaction volume of 10 μl. All subsequent steps were carried out according to the protocol provided on the kit.

[0145] Example 3: Real-time PCR assay conditions

[0146] Using the miRCURY LNA RT (reference number: 339340) kit, locked nucleic acid primers targeting miR145 were used to perform RT-PCR to detect standard miRNAs and miRNAs in cDNA from direct saliva. GAPDH primers were used to amplify the endogenous control. The assay tubes for detecting standard miRNAs used GAPDH cDNA as a template to amplify the endogenous control. In the case of directly measuring saliva, no additional cDNA was added. The conditions for RT-PCR were as follows: -95 °C - 5 minutes, 40 cycles of -95 °C - 30 seconds / -58 °C - 1 minute / -72 °C - 01:30 minutes, and a final extension at -72 °C - 10 minutes. The amplification plots and melting curves of the two assays were analyzed.

[0147] Example 4: CRISPR / Cas13a detection conditions

[0148] The Cas13 assay was carried out in a nuclease assay buffer (100 mM HEPES buffer, pH 7.4) with 1 μM LwaCas13a, 10 μM gRNA, 2 μM target miRNA-145, and 5 mM MgCl 2 LwaCas13a and gRNA were pre-incubated at 37 °C for 15 minutes, then 2 μg of total RNA was added for a small-scale test, and 1250 nM fluorescent reporter RNA (based on FAM) was added. The fluorescence kinetics was measured every 5 minutes for 3 hours, and the excitation and emission ranges were set to 493 nm and 517 nm. The detection of miRNA-145 was confirmed by fluorescence detection of RNA biomarkers, thus identifying the cancer type as oral cancer. In addition, these findings were further applied to the Cas13 detection (enzyme mixture) in the second part of the cast device disclosed for detecting RNA-related diseases.

[0149] Example 5: Characterization test

[0150] A: Isolation of total RNA from saliva samples

[0151] Total RNA isolated from four saliva samples was processed by agarose gel electrophoresis. As Figure 5 shown, two distinct RNA bands were observed in samples at different concentrations.

[0152] B: In vitro transcription of standard miRNA and gRNA using T7 polymerase

[0153] Using the T7 polymerase kit, in vitro transcription was performed on standard miRNAs and gRNAs, as well as control RNAs in the kit. Any contaminating DNA was further removed by DNase I treatment. The in vitro transcribed RNAs before and after DNase I treatment were subjected to agarose gel electrophoresis ( Figure 6 ). The decrease in band intensity after treatment may be due to the removal of contaminating DNase or RNase contamination in the DNase I provided in the kit.

[0154] C: Detection of standard miRNA from in vitro transcribed template RNA and direct saliva samples based on RT-PCR technology

[0155] Figure 7 a, b, and Figure 8 a, b depict the amplification plots and melting curves for the miR145 and GAPDH amplicons using the standard miRNA template. The Ct values for the endogenous control and miRNA obtained using the standard miRNA were 16.3 and 18.8, respectively. For the assay using direct saliva samples for cDNA preparation and RT-PCR, the Ct values for the endogenous control and miRNA were 18.7 and 21.3, respectively. Although the melting curves showed a single peak for the amplicons, they were not very distinct.

[0156] When comparing the isolated total RNA as a template with the direct saliva sample as a template, a three-cycle difference in the Ct values under the miRNA standard was observed. The single peak in the melting curve describes the formation of a single product during RT-PCR, thus confirming the specificity of the primers. 1. The Ct values using direct saliva were 2 to 3 cycles lower than those using the standard miRNA template, and the curve showed a single peak.

[0157] D4: Detection of collateral activity of in vitro synthesized miRNA targets and total RNA using CRISPR / Cas13 assay

[0158] The in vitro test of the Cas13 assay initially used total RNA degradation as a test for collateral activity. The test assay was performed simultaneously with a control assay in which no specific gRNA was added. There was no degradation of total RNA in the control assay, indicating that Cas13 was not activated by cleavage with gRNA and target miRNA, although no collateral activity was detected (a, b in Figure 9).

[0159] D5: Detection of in vitro synthesized miRNA targets using standard miRNA and direct saliva samples, and detection of collateral activity of fluorescent reporters using CRISPR / Cas13 assay

[0160] Perform the Cas13 assay using a standard miRNA as the target and a direct saliva sample, and use a fluorescent probe for quantification. The fluorescence values are quantified in real time within 3 hours and plotted ([ Figure 10 a, b). It was observed that when using the standard miRNA target for the assay, the fluorescence values ranged from 2000 - 4000 units, while when using the direct saliva sample for the assay, the fluorescence values within the 3 hours of analysis ranged from 2000 - 3000 units. It was observed that the detection of miRNA in saliva was comparable to in vitro detection, only with slightly lower sensitivity. Compared to in vitro assays, it was found that in the assays performed with direct saliva tested, the fluorescence readings indirectly tested through collateral activity were relatively low.

[0161] Therefore, it can be concluded that the CRISPR / Cas13 assay is capable of detecting approximately 2 μM miRNA in the assay and in saliva. It was found that the miRNA detection between the control group and the test was detectable, i.e., the difference was 2000 u. It has been reported that the average concentration of miRNA found in saliva is approximately 22 μM. The fluorescence unit range for the standard miRNA assay was 2000 - 4000 U within 3 hours, while in direct saliva, the fluorescence unit range was 2000 - 3000 U from 0 - 3 hours.

[0162] Example 5: Method (400) for identifying RNA related to oral cancer by enabling device (100)

[0163] Inject 150 μl of the saliva sample into the opening of the central funnel-shaped part of the first section. Further, pass the biological sample through the first section to interact with three layers of gelatin layers. The enzymes in the biological sample digest and decompose each of the at least three gelatin layers, and in turn, the gelatin layers cause the biological sample to drip from one or more different integrated chambers of the second section through a permeable connector. Additionally, the gRNA, reporter RNA, and Cas13a in the enzyme mixture (see Table 6) react with the target RNA (a specific miRNA biomarker for oral cancer, more preferably miR145) in the biological sample that drips from the first section into the integrated chambers of the second section.

[0164] Table 6: Components of the enzyme mixture

[0165] Component Stock solution Volume Working concentration gRNA 50 μM 2 μl 5 μM, 10 μM, 50 nM, 20 nM Cas13 1 μM 1 μl 100 nM, 200 nM, 1 μM Magnesium chloride 50 mM 1 μl 5 mM Fluorescent reporter 12.5 μM 1 μl 1.25 μM, 125 nM, 250 nM HEPES buffer 1M 3 μl 0.1M

[0166] The second part is used to detect target miRNAs in biological samples in the range of as low as 1 μM - 20 nM. In addition, at least one integrated chamber is irradiated by a fluorescence excitation source from an opaque compartment (105), thereby generating a chemiluminescence control signal generated by components such as luminol and a fluorescence signal in response to the presence of target RNA, so as to identify the target RNA associated with oral cancer. Then, the chemiluminescence control signal and the fluorescence signal in response to the presence of target RNA are sent to a display unit through a detector unit (i.e., a fluorescence detection system). The total time required for detection in device 1 is less than 24 hours, preferably between 2 - 18 hours.

[0167] According to an embodiment of the present disclosure, the above-mentioned device (100) may have the following advantages, including but not limited to:

[0168] · The device (100) is a portable and inexpensive system.

[0169] · The device (100) is a reliable and precise tool that can simultaneously perform disease risk assessment and disease type detection according to the types of RNA present in body fluids without any pretreatment thereof.

[0170] · Different from traditional RT-PCR-based detections (which usually take 24 - 48 hours to obtain results), the assay of the device (100) can provide results within a time range of 30 minutes to 10 hours.

[0171] · The device (100) can evaluate RNA-based types with a minimum sample volume and the shortest analysis time.

[0172] · The device (100) operates by passing a biological sample through the first part, thereby activating the second part (106).

[0173] This design can effectively prevent sample waste and resource waste in the second part (106).

[0174] · The device (100) provides a portable and convenient tool that can be used to detect specific diseases and simultaneously evaluate the risk level in a single device.

[0175] The foregoing applications of the device / kits (100) developed and disclosed herein may include but not be limited to RNA-based disease types such as oral cancer identification, diabetes condition detection, virus type detection, gene expression research, epigenetic analysis, therapeutic intervention, etc.

[0176] Those of ordinary skill in the art will understand that certain modifications can be made within the scope of the present disclosure. To limit the scope of the present disclosure and the subject matter, a subsequent complete specification will be submitted. The true scope and content of the present disclosure will be determined in the subsequent complete specification.

Claims

1. A non-invasive device (100) for detecting RNA-related diseases, comprising: A first part (101) having a funnel-shaped central member (202) for determining the proteolytic activity of a biological sample, wherein the first part (101) is cast from at least two stacked layers (102); A second part (106) for identifying proteolytic entities and target RNA species in the biological sample, wherein the second part (106) comprises: At least one integrated chamber (104) containing an enzyme mixture for identifying and cleaving target RNA species in the biological sample, and An opaque compartment (105) comprising a fluorescence excitation light source to produce fluorescence in response to the presence of the target RNA species; And At least one permeable connector (103) configured to drip the biological sample from the first part (101) onto the second part (106), wherein if the biological sample digests the at least two stacked layers (102) of the first part (101), the permeable connector (103) enables the biological sample to pass through the first part (101) to the second part (106).

2. The device (100) according to claim 1, wherein, The stacked layer (102) of the first part (101) is a protein casting layer of at least one of albumin, gelatin, fibrin, and globulin.

3. The device (100) according to claim 1, wherein, The permeable connector (103) is selected from at least one of a capillary, a tubular channel, a sieve, a filter membrane, a tubular opening with a bottom thread, a perforated structure, and a porous plate, more preferably a capillary or a tubular opening with a bottom thread.

4. The device (100) according to claim 1, wherein, The enzyme mixture composition is selected from a CRISPR / Cas13a reaction mixture, glucose oxidase-peroxidase, and combinations thereof.

5. The device (100) according to claim 4, wherein, The CRISPR / Cas13a reaction mixture of the second part (106) comprises: gRNA in the range of 20 nM - 5 μM; Cas13 enzyme in the range of 80 - 120 nM; Chloride salt in the range of 3 - 6 mM; Fluorescent reporter in the range of 1 μM - 250 nM; and 0.01 - 0.2 M sulfonic acid buffer.

6. The device (100) according to claim 1, wherein, The first part (101) and the second part (106) are detachably arranged together.

7. The device (100) according to claim 1, comprising a monitoring unit for determining the activity of the biological sample in the first part (101) and the second part (106) for risk assessment and identification of RNA-related diseases.

8. A method (400) for detecting RNA-related diseases by enabling a device (100), comprising the following steps: Injecting a biological sample into the opening of the central funnel-shaped member (202) of the first part (101) (401); Cause the biological sample to pass through the first part (101) to interact (402) with each stacked layer (102), wherein one or more enzymes in the biological sample digest and decompose each stacked layer (102); Drop the biological sample into at least one integrated chamber (104) of the second part (106) through at least one permeable connector (103) (403); React the enzyme mixture present in the integrated chamber (104) with the target RNA species in the biological sample (404); Irradiate the integrated chamber (104) with a fluorescence excitation light source of an opaque compartment (105) to generate a chemiluminescence signal in response to a control and a fluorescence signal in response to the presence of the target RNA species (405).

9. The method (400) according to claim 8, wherein, The biological sample in the range of 100 - 200 μl is injected into the first part (101), and the reaction in the integrated chamber (104) requires 5 - 15 μl of the sample.

10. The method (400) according to claim 8, wherein, If at least two stacked layers (102) of the first part (101) are digested, the biological sample is dropped into the at least one integrated chamber (104).

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