TW201840854A - Method and device for accurate diagnosis of dental diseases - Google Patents

Abstract

The present invention provides a method and device for concentration and detection of a target analyte in a solution using a pre-coated porous material embedded with ATPS components. In one embodiment, the water soluble nanoparticles include, but are not limited to, silicon dioxide, iron oxide, titanium dioxide, silver oxide and any combinations thereof. In one embodiment, the present invention provides a device comprising ATPS as a concentration module linked with a lateral flow immunoassay (LFA) component as a detection module. In one embodiment, as the sample wicks up the device and ATPS components undergo phase separation, the analytes are concentrated substantially in the leading front, the concentrated analytes are then detected by the LFA module generating visual test results. In one embodiment, the present method and device are used to detect Streptococcus mutans and thereby dental caries.

Description

Method and apparatus for accurately diagnosing dental diseases

RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application Serial No. No. No. No. No. No. No. No The present application also incorporates a number of publications, the entire disclosure of which is hereby incorporated by reference in its entirety in its entirety in its entirety in the extent of the disclosure.

The present invention relates to a method and apparatus for improving detection accuracy and diagnostic performance. This method completely embeds the components of the two-liquid phase system (ATPS) in the porous material, allowing the phases to separate spontaneously, so that the user can purify and concentrate the analyte in one step. The present invention also provides a method of performing the present invention using a diagnostic test device.

Many new biomarkers and drugs related to diseases have been discovered or invented in recent years. However, many patients still die from these diseases every year. One of the causes of death is the inability to detect the disease at an early stage or not at all. Many diseases have no obvious symptoms in their early stages. When the symptoms develop to a significant extent, the patient is already in advanced or terminal stage. Healthcare professionals can do little to treat patients or save their lives. If these diseases are diagnosed early, the patient may be completely cured by applying the correct procedure or using the appropriate medication. An experienced health care provider may suspect that the patient has certain illnesses when examining the patient. However, the biomarker concentration of early patients is usually so small that it cannot be detected. If the diagnostic technique does not confirm the presence of the disease, the health care professional cannot produce the appropriate drug. The current dilemma is that disease-related biomarkers are present in patients but cannot be detected. Although the drug for treating the disease exists, it has not been prescribed by a doctor. All of these problems are caused by the inability to detect the disease early.

It is a huge challenge to detect the presence of very small analytes. The analyte can be a biomarker for a disease, such as free circulating DNA (ccfDNA), free circulating tumor DNA (ctDNA) or protein, which may be present in the patient's blood, saliva, urine or other body fluids. If the concentration is too low, many of the diagnostic or detection methods of the present invention may erroneously report the absence of the analyte. If the analyte concentration is extremely low, the gold standard for diagnostics such as PCR and ELISA may also produce false negative results.

Free DNA (cfDNA) extracted from liquid biopsy has become a popular biomarker for many pathologies. For example, cfDNA may be released from apoptotic or necrotic tumor cells, which can be used to monitor cancer progression and recurrence, and its potential sensitivity is better than current gold standard imaging methods, such as computed tomography (CT). Positron emission tomography (PET) and magnetic resonance imaging (MRI). Compared to tumor biopsies from solid tumor areas, liquid biopsy is non-invasive, can be collected at multiple time points, and can obtain cfDNA that is more representative of tumor diversity, which helps identify drug-resistant mutations.

In addition, the fetal cfDNA can show a variety of abnormalities during pregnancy, such as aneuploidy, which is earlier than current prenatal testing methods such as chorionic villus sampling and amniocentesis, and does not pose a risk of miscarriage. A liquid biopsy from a heart transplant patient can show the donor's cfDNA, a warning sign that the patient may have rejection within weeks or months after transplantation.

Despite the discovery and invention of many new biomarkers associated with disease in recent years, diagnostic tests for these new biomarkers, such as the cfDNA test, lack sensitivity and specificity and have not been used in routine clinical practice. When the biomarker is present in low amounts, accurate detection depends on the ability to separate and concentrate the biomarker from the background. Depending on the separation method, the problem may become more complicated due to differences in fragment size and the effects of the test inhibitor.

The literature reports a number of purified products that can concentrate free DNA (cfDNA) and other cancer biomarkers. However, these expensive kits are often subject to the maximum amount of sample that can be processed, as well as the amount of DNA that can be purified prior to clogging of the blood sample. A method of concentrating an analyte using a porous material embedded in ATPS is disclosed in the patent WO2017041030. Unfortunately, the multiples used to detect the concentration of the major bacteria (Streptococcus mutans) that cause dental caries are only about 10 to 60 times. This is difficult to meet the accuracy and sensitivity requirements of dental caries diagnosis. Optimization methods and equipment capable of achieving a multiple of 60 times the concentration are highly desirable.

To overcome these limitations, the present invention provides an improved method and apparatus for purifying a target analyte from a sample and concentrating the purified target analyte for further analysis, which can be accomplished simply and quickly in one step . No complicated equipment is required. Using a porous material embedded in ATPS, the methods and apparatus provided by the present invention can perform a number of processes simultaneously, including cell lysis, separation of target biomolecules, removal of non-target biomolecules and impurities, and concentration of target biomolecules. Concentrate biomolecules or remove inhibitors to improve detection accuracy and diagnostic performance.

Using a porous material embedded in ATPS, the method and apparatus of the present invention can purify the target analyte from the sample and concentrate it to 100 times or more.

In general, the methods and apparatus of the present invention can increase the accuracy, sensitivity, and efficiency of detection and quantification of biomolecules, thereby enabling improved performance of various technical assays or diagnostics that rely on biomolecules for detection and/or quantification. If a disease is discovered early, many life-threatening diseases will heal.

The present invention relates to an improved method and apparatus for improving detection accuracy and diagnostic performance. The method and apparatus of the present invention involves fully embedding the components of the two-liquid phase system (ATPS) in the porous material, allowing the phases to separate spontaneously, thus allowing the user to purify and concentrate the analyte in a single step.

In one embodiment, the present invention provides a method and apparatus for concentrating and detecting target analytes in solution using an embedded ATPS component and a precoated porous material. The water soluble nanoparticles are pre-coated on the porous material. Water soluble nanoparticles include, but are not limited to, ceria, iron oxide, titanium dioxide, silver oxide, and any combination thereof.

In one embodiment, the invention provides an apparatus comprising a concentration module ATPS coupled to a detection module lateral flow immunoassay (LFA). In one embodiment, the detection module is mounted in a plastic housing with a window and a gold nanoprobe. When the sample is drawn into the device with capillary action, it is phase separated with the ATPS component and the analyte is concentrated at the leading edge of the fluid flow. The concentrated analyte is then tested by the LFA module for visual test results.

The foregoing summary, as well as the following detailed description The accompanying drawings are merely illustrative of the invention and are not intended to be limiting. The invention is not limited to the precise arrangements and instrumentalities shown.

In the following description, several embodiments of the invention are described. For the sake of explanation, specific configurations and details are set forth to provide a thorough understanding of the embodiments. In addition, the plural or singular forms of a word, as well as the degree of orientation of the illustrated embodiment, are described as "top", "bottom", "front", "back", "left", "right", and the like. It is intended to assist the reader in understanding the embodiments and is not intended to limit the invention. The present invention may be practiced without specific details by those skilled in the art. The invention is further understood by the following examples, which are to be understood by those skilled in the art, It should be noted that the term "inclusive" or "including" is "inclusive" or "characteristic" and is inclusive or open-ended and does not exclude additional, unlisted elements or Method steps.

The present invention relates to an improved method and apparatus for improving detection accuracy and diagnostic performance. The method and apparatus of the present invention involves fully embedding the components of the two-liquid phase system (ATPS) in the porous material, allowing for spontaneous phase separation, thus allowing the user to purify and concentrate the analyte in a single step.

In one embodiment, the present invention provides a method and apparatus for concentrating and detecting target analytes in solution using precoated porous materials embedded in an ATPS component. The water soluble nanoparticles are pre-coated on the porous material. Water soluble nanoparticles include, but are not limited to, ceria, iron oxide, titanium dioxide, silver oxide, and any combination thereof.

In one embodiment, the invention provides an apparatus comprising a concentration module ATPS coupled to a detection module lateral flow immunoassay (LFA). In one embodiment, the detection module is mounted in a plastic housing with a window and a gold nanoprobe. When the sample is drawn into the device by capillary action, it is phase separated with the ATPS component and the analyte is concentrated at the leading edge of the fluid flow. The concentrated analyte is then tested by the LFA module to obtain visible test results.

In one embodiment, the present invention provides a method that can improve the performance of various detection and diagnostic techniques, for example, in terms of accuracy, sensitivity, and efficiency.

In one embodiment, the invention also provides a method of performing the invention using a diagnostic test device. Sample type and analyte

In one embodiment, the methods and devices of the present invention can separate target analytes from non-target molecules (eg, small molecules and macromolecules of natural origin may interfere with detection or quantification of target analytes), thereby Accurate detection and diagnosis.

In one embodiment, the method and apparatus are applicable to any sample type. In one embodiment, the sample includes, but is not limited to, blood, plasma, serum, tissue, bacteria, virus, RNA virus, smear, bacterial culture, cell culture (eg, cell suspension and adherent cells), urine, saliva, feces And body secretions (such as tears, sputum, nasopharyngeal mucus, vaginal secretions and semen), polymerase chain reaction (PCR) mixtures and in vitro nucleic acid modification reaction mixtures.

In one embodiment, the methods and apparatus of the invention are used to purify and concentrate target analytes from blood. The method is capable of dissolving blood cells to release intracellular material, separating the target from non-target molecules (including cell debris) while concentrating the target analytes together.

In one embodiment, the target biomarker is purified and concentrated from saliva or urine using the methods and devices of the invention. As shown in Figure 4, the invention is applicable to urine and saliva. Although additional organic and inorganic components are present in other samples, the concentration is similar to PBS. In addition, porous materials can filter out any large molecules that interfere with LFA performance.

In one embodiment, the target analytes include, but are not limited to, proteins, nucleic acids, carbohydrates, lipids, bacteria, viruses, nanoparticles, and the like.

In one embodiment, the target analyte is a free molecule, including but not limited to free DNA (ccfDNA), free protein, exosomes, and free molecules circulating in the body fluid of the subject. In one embodiment, the biomarker is a molecule that is attached to or contained in the cell surface.

In one embodiment, the target analyte is various types of nucleic acids (eg, DNA comprises cDNA; RNA comprises mRNA and rRNA), forms (eg, single-stranded, double-stranded, coiled, as a plasmid, non-coding or encoding) And length (such as oligonucleotides, genes, chromosomes, and genomic DNA), originating from the subject or exogenous or both.

In one embodiment, the target analyte is a protein that is a peptide or polypeptide comprising intact protein molecules, degraded protein molecules, and digested fragments of protein molecules. In one embodiment, the biomarkers include, but are not limited to, antigens, receptors, and antibodies, originating from a subject or a foreign body, or both.

In one embodiment, the target analyte is a small molecule, such as a metabolite. In one embodiment, the metabolite is a disease-associated metabolite that indicates the presence or extent or health of the disease. In one embodiment, the metabolite is a drug-related metabolite, such as a pharmaceutical by-product, the level of which varies with body depletion of the drug.

In one embodiment, the target analyte is derived from the subject itself (e.g., a molecule derived or released from any organ, tissue, or cell), a foreign body (a pathogen such as a virus associated with a disease, or a bacterium). Or from food or medicine taken by the subject.

In one embodiment, the target analyte is a molecule produced by a tumor or cancer, or a response of a subject's body to a tumor or cancer.

In one embodiment, target analytes are generally not found in healthy subjects. In one embodiment, the target biomarker is a molecule that is normally found in a healthy subject, but its concentration level reflects a disease or condition. Two-liquid phase system ( ATPS)

In one embodiment, a two-liquid phase system (ATPS) fully embedded in a porous material is provided for purifying and concentrating target analytes in a sample.

In one embodiment, the ATPS component is fully embedded in the porous material, allowing for spontaneous phase separation, thus allowing the user to purify and concentrate the analyte in a single step.

In one embodiment, the two components of the ATPS are fully embedded in the porous material precoated with the water soluble nanoparticles.

In one embodiment, the ATPS is used on a porous material such as paper, and when the two phase solution of the ATPS flows through the porous material, the two phases of the ATPS separate. Because the two phases have different physicochemical properties, they flow at different rates on the porous material. Different molecules in the mixture are distributed between the two phases due to their different properties, so ATPS can be used to separate and concentrate the target biomarkers with the simplest equipment and manpower. The phase can be separated because it relies on isothermal kinetics and capillary action to cause liquid to flow. The process does not require a power supply or equipment at all.

An advantage of the present invention is that high purity and concentration of target analytes can be readily obtained and can be directly applied to downstream analysis without further purification or concentration.

The method and equipment provided by the invention are strong in stability, low in price, simple, easy to handle, safe, convenient to use and fast. The method is capable of purifying and concentrating the target analyte, and ensures that the downstream analysis results are not affected by impurities in the original sample. Since the present invention does not require any additional power, complex instrumentation or electronic hardware to operate, it can provide a quick and easy method for rapid nucleic acid isolation and purification.

Due to the unique function of the present invention, the present invention facilitates the rapid purification and concentration of target analytes without the use of additional power sources or complex instruments, and is suitable for samples containing very low or small amounts of target analytes. In addition, the method is easy to use for automation and includes a high throughput screening system. Designing porous materials embedded in ATPS

In one embodiment, the invention provides a porous material embedded in an ATPS component. Various ATPS can be used in the present invention, including but not limited to polymer-polymers (e.g., PEG-dextran), polymer-salts (e.g., PEG-salt), and micelles (Triton X-114). The porous material can use any suitable porous material that can absorb and transfer liquids. Porous materials suitable for use in the present invention include, but are not limited to, fiberglass paper, cotton-based paper, other types of paper, polymer foam plastic, cellulose foam plastic, other types of foam plastic, rayon fabric, cotton fabric, other types of fabrics. , wood, stone and any other material that absorbs and transfers liquids.

In one embodiment, the porous material is commercially available or manufactured in-house.

Figure 1 illustrates the separation capabilities of several types of porous materials from three suppliers after embedding ATPS (glass fiber paper, cotton-based paper, single-layer matrix paper, and polyolefin foam mat). Target analytes are indicated in purple. As shown in Figure 1, fiberglass paper FG1 from supplier 1, fiberglass paper FG1 from supplier 2, and fiberglass paper FG2, and polyolefin foam pad from supplier 3, are deep at the leading edge of fluid flow. Purple, they are the best in concentration. Preparation of porous materials precoated with water-soluble nanoparticles

In one embodiment, the present invention provides a method and apparatus for concentrating and detecting target analytes in solution using precoated porous materials embedded in an ATPS component.

In one embodiment, the present invention provides a porous material that is embedded in an ATPS component and pre-coated with water soluble nanoparticles. The pre-coated porous material can significantly increase the concentration of the target analyte by increasing the adhesion of the ATPS to the porous material and promoting the degree of embedding of the ATPS in the porous material.

In one embodiment, the porous material of the present invention is pre-coated with water soluble nanoparticles. Water soluble nanoparticles include, but are not limited to, ceria, iron oxide, titanium dioxide, silver oxide, and any combination thereof.

In one embodiment, the porous material comprises a surface and a plurality of pore/capillary systems. In one embodiment, the surface of the porous material is pre-coated with water soluble nanoparticles. In another embodiment, the pore/capillary system of the porous material is pre-coated with water soluble nanoparticles. In another embodiment, both the surface of the porous material and the pore/capillary system are pre-coated with water soluble nanoparticles. In one embodiment, both the surface of the porous material and the pore/capillary system are pre-coated with cerium oxide.

Water-soluble nanoparticles (such as cerium oxide) have positive and negative charges and play an important role in the intermolecular interaction between ATPS and porous materials. The present inventors have found that porous materials precoated with water soluble nanoparticles can significantly promote the efficiency of ATPS embedding on porous materials. ATPS also has good stability on porous materials, ultimately increasing the concentration. In one embodiment, the concentration doubling achieved by the present invention can be up to 100 times or more.

In one embodiment, the porous material, such as porous glass paper, is coated with water soluble nanoparticles such as cerium oxide. Prepared by the following procedure: cerium oxide is dissolved in an aqueous solution at a concentration of 5M. The porous glass paper was immersed in a 5 M aqueous solution of cerium oxide and stirred vigorously for one hour. Hold at 80 ^o C for 24 hours. It is then taken out and rinsed twice with water and finally dried in compressed air. Adjusting the number of times concentrated

In one embodiment, the relative amount of ATPS components in the porous material can be adjusted.

By varying the amount of ATPS component embedded on the porous material, i.e., the volume ratio of the two phases, the target biomarker can be preferentially concentrated in one phase. In one embodiment, the target analyte is retained at the flow front of the ATPS and these analytes are then collected and optionally analyzed using appropriate techniques. In one embodiment provided herein, the target analyte was purified on top of the porous material in which the ATPS was embedded, and the concentration factor was increased by as much as 50-100 fold compared to the control paper stack without the embedded ATPS.

In one embodiment, the order of the ATPS components on the porous material can be adjusted as desired. In one embodiment, one or more components can be embedded in the porous material.

In order to better quantify the invention relating to the present invention, the present invention has developed an analytical method for evaluating the relationship between the relative amount of ATPS components embedded in a porous material and the concentration factor that can be achieved. The concentration factor can be selected and fine tuned by adjusting the relative amounts of the two components of the ATPS.

In one embodiment, the ATPS component is integrated into a porous material, and the ATPS component is soluble in water (or a suitable buffer) and applied to the porous material at a certain ratio. The porous material is then placed in a lyophilizer to remove water so that the ATPS component is directly embedded in the porous material. When the sample is added to the porous material, the ATPS component is immediately replenished to separate the molecules in the sample and concentrate the target analyte at the leading edge of the fluid flow without any external power or equipment to provide the driving force.

As shown in Figure 3, a combination of two ATPS (micelle ATPS and polymer/salt ATPS) was tested and the target analyte concentration was 100 times or more. The same amount of purple analyte was added to the porous material with different amounts of ATPS components embedded, and the porous material without ATPS was used as a control. In the control, the light purple analyte was evenly distributed on the porous material. On the other hand, in a porous material in which ATPS is embedded, the analyte can be concentrated at the leading edge of the fluid flow, as indicated by dark purple. The data in this paper shows that the concentration multiple can reach between 30 and 100 times. More importantly, the data herein demonstrates that by adjusting the ratio of ATPS components embedded in the porous material, the present invention can adjust the concentration multiples as needed. In addition, data indicate that a wide range of ATPS components (polymer/salt systems and micellar systems) can be embedded in porous materials to enable purification and concentration of analytes. Other polymers/salts, micelles, polymer/polymer systems can also be implemented and further optimized.

In one embodiment, the invention provides an apparatus comprising a porous glass paper embedded in an ATPS comprised of a polymer-polymer, such as PEG-dextran. When a sample containing multiple analytes is added to the ATPS on porous glass paper, the porous glass paper preferentially flows the analyte-containing components and runs in front of the other ATPS components. Thus, the ATPS component containing the target analyte is concentrated at the leading edge of the fluid flow.

In one embodiment, the invention provides an apparatus comprising pretreated porous glass paper embedded in an ATPS comprised of a polymer-salt base, such as a PEG salt. When a sample containing multiple analytes is added to the ATPS on pretreated porous glass paper, the porous glass paper preferentially flows the analyte-containing components and runs in front of the other ATPS components. Thus, the ATPS component containing the target analyte is concentrated at the leading edge of the fluid flow.

In one embodiment, the invention provides an apparatus comprising a porous glass paper embedded in an ATPS comprised of a micellar based surfactant solution. When a sample containing multiple analytes is added to the ATPS on porous glass paper, the porous glass paper preferentially flows the analyte-containing components and runs in front of the other ATPS components. Thus, the ATPS component containing the target analyte is concentrated at the leading edge of the fluid flow.

In one embodiment, the buffer used herein includes, but is not limited to, phosphate buffered saline (PBS), TE buffer. In the present invention, the buffer is preferably 9.57 mM PBS buffer (8 mM sodium chloride, 0.2 mM potassium chloride, 1.15 mM sodium phosphate phosphate, 0.2 mM potassium dihydrogen phosphate, solution pH 7.35-7.65). In the present invention, the preferred buffer is TE buffer containing 2% bovine serum albumin (BSA), 0.1% Tween-20, 0.1% PEG and 20 mM Tris, pH 7.5.

In one embodiment, multiple ATPS components are incorporated into the porous material to control the concentration multiple of the target analyte.

In one embodiment, the ratio of polymer/salt ATPS components is optimized to control concentration multiples.

In one embodiment, the ratio of polymer/polymer ATPS components is optimized to control concentration multiples.

In one embodiment, the ratio of the micelle ATPS components is optimized to control the concentration multiple.

In one embodiment, the ATPS embedded in the porous material allows the biomolecule to concentrate at the leading edge of the fluid flow. Adjust the ratio of the ATPS components to achieve a concentration multiple between 10 and 100 times.

In one embodiment, there are a variety of different ATPS systems including, but not limited to, polymers (e.g., PEG-dextran), polymer-salts (e.g., PEG-salt), and micelles (e.g., Triton X-114). The first and / or second component comprises a polymer. The polymer includes, but is not limited to, polyethylene glycol, such as hydrophobically modified polyalkylene glycol, poly(oxyalkylene) polymer, poly(oxyalkylene) copolymer, such as hydrophobically modified poly(oxyalkylene) copolymer. , polyvinyl alcohol pyrrolidone, polyvinyl alcohol, polyvinyl alcohol caprolactam, polyvinyl methyl ether, alkoxylated surfactant, alkoxylated starch, alkoxylated cellulose, alkyl hydroxyalkyl Cellulose, organogermanium modified polyether, poly N-isopropyl acrylamide. In another embodiment, the first polymer comprises polyethylene glycol, polypropylene glycol or dextran.

In one embodiment, the polymer concentration of the first component or the second component is in the range of from about 0.01% to about 90% (w/w) based on the total weight of the aqueous solution. In various embodiments, the concentration of the polymer solution is about 0.01% w/w, about 0.05% w/w, about 0.1% w/w, about 0.15% w/w, about 0.2% w/w, about 0.25%. w/w, about 0.3% w/w, about 0.35% w/w, about 0.4% w/w, about 0.45% w/w, about 0.5% w/w, about 0.55% w/w, about 0.6% w /w, about 0.65% w/w, about 0.7% w/w, about 0.75% w/w, about 0.8% w/w, about 0.85% w/w, about 0.9%) w/w, about 0.95% w /w, or about 1% w/w. In certain embodiments, the concentration of the polymer solution is about 1% w/w, about 2% w/w, about 3% w/w, about 4% w/w, about 5% w/w, about 6% w/w, about 7% w/w, about 8% w/w, about 9% w/w, about 10% w/w, about 11% w/w, about 12% w/w, about 13% w/w, about 14% w/w, about 15% w/w, about 16% w/w, about 17% w/w, about 18% w/w, about 19% w/w, about 20 % w/w, about 21% w/w, about 22% w/w, about 23% w/w, about 24% w/w, about 25% w/w, about 26% w/w, about 27% w/w, about 28% w/w, about 29% w/w, about 30% w/w, about 31% w/w, about 32% w/w, about 33% w/w, about 34% w /w, about 35% w/w, about 36% w/w, about 37% w/w, about 38% w/w, about 39% w/w, about 40% w/w, about 41% w/ w, about 42% w/w, about 43% w/w, about 44% w/w, about 45% w/w, about 46% w/w, about 47% w/w, about 48% w/w , about 49% w/w, about 50% w/w.

In one embodiment, the first and/or second component comprises a salt, including but not limited to a lyophilic salt, a chaotropic salt, a cationic containing inorganic salt, such as a linear or branched trimethylammonium, Triethyl ammonium, tripropyl ammonium, tributyl ammonium, tetramethyl ammonium, tetraethyl ammonium, tetrapropyl ammonium and tetrabutyl ammonium, and anions such as phosphates, sulfates, nitrates, chlorides And hydrogen carbonate. In another embodiment, the salt is selected from the group consisting of sodium chloride, sodium phosphate, potassium phosphate, sodium sulfate, potassium citrate, ammonium sulfate, sodium citrate, sodium acetate, and combinations thereof. Other salts, such as ammonium acetate, can also be used.

In one embodiment, the total salt concentration is in the range of 0.001 to 100 mM. Those skilled in the art will appreciate that the amount of salt required in the ATPS will be affected by the molecular weight, concentration, and physical state of the polymer.

In various embodiments, the salt phase is selected from a salt solution at a concentration of from about 0.001% to about 90% w/w. In various embodiments, the salt solution concentration is about 0.01% w/w, about 0.05% w/w, about 0.1. % w/w, about 0.15% w/w, about 0.2% w/w, about 0.25% w/w, about 0.3% w/w, about 0.35% w/w, about 0.4% w/w, about 0.45% w/w, about 0.5% w/w, about 0.55% w/w, about 0.6% w/w, about 0.65% w/w, about 0.7% w/w, about 0.75% w/w, about 0.8% w /w, about 0.85% w/w, about 0.9%) w/w, about 0.95% w/w, or about 1% w/w. In certain embodiments, the salt solution has a concentration of about 1% w/w, about 2% w/w, about 3% w/w, about 4% w/w, about 5% w/w, about 6% w. /w, about 7% w/w, about 8% w/w, about 9% w/w, about 10% w/w, about 11% w/w, about 12% w/w, about 13% w/ w, about 14% w/w, about 15% w/w, about 16% w/w, about 17% w/w, about 18% w/w, about 19% w/w, about 20% w/w , about 21% w/w, about 22% w/w, about 23% w/w, about 24% w/w, about 25% w/w, about 26% w/w, about 27% w/w, About 28% w/w, about 29% w/w, about 30% w/w, about 31% w/w, about 32% w/w, about 33% w/w, about 34% w/w, about 35% w/w, about 36% w/w, about 37% w/w, about 38% w/w, about 39% w/w, about 40% w/w, about 41% w/w, about 42 % w/w, about 43% w/w, about 44% w/w, about 45% w/w, about 46% w/w, about 47% w/w, about 48% w/w, about 49% w/w, about 50% w/w.

In one embodiment, the first component and/or the second component of the ATPS comprise a solvent that is incompatible with water. In certain embodiments, the solvent comprises a non-polar organic solvent. In certain embodiments, the solvent comprises an oil. In certain embodiments, the solvent is selected from the group consisting of pentane, cyclopentane, benzene, 1,4-dioxane, diethyl ether, dichloromethane, chloroform, toluene, and hexane.

In one embodiment, the first component and/or the second component of the ATPS comprises a micellar solution. In certain embodiments, the micellar solution comprises a nonionic surfactant. In certain embodiments, the micellar solution comprises a detergent. In certain embodiments, the micellar solution comprises Triton-X. In certain embodiments, the micellar solution comprises a polymer similar to Triton-X, such as Igepal CA-630 and Nonidet P-40. In certain embodiments, the micellar solution consists essentially of Triton-X.

In one embodiment, the first component of the ATPS comprises a micellar solution and the second component of the liquid phase comprises a polymer. In one embodiment, the second component in the liquid phase comprises a micellar solution and the first component in the liquid phase comprises a polymer. In one embodiment, the first component in the liquid phase comprises a micellar solution and the second component in the liquid phase comprises a salt. In one embodiment, the second component in the liquid phase comprises a micellar solution and the first component comprises a salt. In one embodiment, the micellar solution is a Triton-X solution. In one embodiment, the first component comprises a first polymer and the second component comprises a second polymer. In one embodiment, the first/second polymer is selected from the group consisting of polyethylene glycol and dextran. In one embodiment, the first component comprises a polymer and the second component comprises a salt. In one embodiment, the second component comprises a polymer and the first component comprises a salt. In certain embodiments, the first component comprises polyethylene glycol and the second component comprises potassium phosphate. In certain embodiments, the second component comprises polyethylene glycol and the first component is potassium phosphate. In one embodiment, the first component comprises a salt and the second component comprises a salt. In one embodiment, the first component comprises a lyophilic salt and the second component comprises a chaotropic salt. In certain embodiments, the second component comprises a lyophilic salt and the first component comprises a chaotropic salt.

In one embodiment, the ratio of the first component to the second component is between 1:1 and 1:1000. In certain embodiments, the ratio of the first component to the second component is selected from the group consisting of about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1: 6, about 1:7, about 1:8, about 1:9, about 1:10. In certain embodiments, the ratio of the first component to the second component is selected from the group consisting of about 1:20, about 1:30, about 1:40, about 1:50, about 1:60, about 1: 70, about 1:80, about 1:90, about 1:100. In certain embodiments, the ratio of the first component to the second component is selected from the group consisting of about 1:200, about 1:300, about 1:400, about 1:500, about 1:600, about 1: 700, about 1:800, about 1:900, about 1:1000.

In one embodiment, the ratio of the second component to the first component is selected from the group consisting of about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6. , about 1:7, about 1:8, about 1:9, about 1:10. In certain embodiments, the ratio of the second component to the first component is selected from the group consisting of about 1:20, about 1:30, about 1:40, about 1:50, about 1:60, about 1: 70, about 1:80, about 1:90, about 1:100. In certain embodiments, the ratio of the second component to the first component is selected from the group consisting of about 1:200, about 1:300, about 1:400, about 1:500, about 1:600, about 1: 700, about 1:800, about 1:900, about 1:1000. Downstream processing of isolated analytes

In various embodiments, the invention can be used in conjunction with one or more procedures or reagents for washing and eluting analytes retained in a porous matrix, or for storing analytes for post-treatment.

In one embodiment, after contacting the analyte with the ATPS, the ATPS is washed one or more times with a wash buffer to remove non-target analytes or impurities remaining in the porous matrix. The wash buffer can comprise different ionic strengths, pH values or wash solutions containing additives. The wash buffer comprises, but is not limited to, 20%-50% ethanol and 20%-50% isopropanol; a solution of about 0.1-4.0 M guanidine hydrochloride, a detergent and up to about 80% ethanol; or about 80% ethanol.

In one embodiment, the target analyte is applied to the porous substrate of the ATPS and to one end of the porous substrate. In one embodiment, the isolated target analyte of the invention is eluted with a suitable elution buffer or deionized water. The isolated target analyte in one embodiment does not elute but is stored in a porous matrix for future use. For example, after separation of an analyte using the present invention, a porous material (paper) containing a target analyte (e.g., DNA) is dried and stored. In one embodiment, the analyte stored on the porous substrate can be eluted for further analysis or processing. The choice of elution buffer may depend on the intended use of the purified biomarker. Examples of suitable elution buffers for nucleic acid analytes include, but are not limited to, TE buffer, heavy/double distilled water, and PCR buffer. In one embodiment, the purified analyte on the porous paper is eluted in a test tube containing TE buffer (10 mM Tris-Cl, 1 mM EDTA solution, pH 7.5), and the purified analyte is relatively small. Recycling within the volume, for example, less than 10 ul.

The analytes obtained by the present invention can be widely applied downstream, such as detection or analysis of biomarkers in forensic science, diagnostics, and therapeutics, as well as laboratory processing such as sequencing, amplification, reverse transcription, labeling, digestion. , immunoblotting and the like. It is contemplated that the present invention can increase the downstream characteristics or processing steps of the analyte.

In one embodiment, the analyte is applied downstream, including but not limited to any detection, analysis or diagnostic process, comprising detecting or quantifying the purified analyte. In one embodiment, the detection, analysis or diagnostic process in combination with the present invention includes, but is not limited to, performing in a commercial clinical laboratory, and performing sequencing, amplification (eg, PCR, reverse transcription PCR (RT-PCR)) in a laboratory. , real-time PCR, real-time RT-PCR, reverse transcription, labeling, digestion, immunoblotting, ELISA, radioimmunoassay (RIA), immunoassay, enzymatic analysis, GC /MS, protein biology and other methods, and so on.

In one embodiment, the analytes obtained in the present invention can be analyzed by LFA. Diagnostic testing equipment

In one embodiment, the invention provides an apparatus comprising a concentration module ATPS coupled to a detection module lateral flow immunoassay (LFA). In one embodiment, the detection module is mounted in a plastic housing with a window and a gold nanoprobe. When the sample is drawn into the device by capillary action, it is phase separated with the ATPS component and the analyte is concentrated at the leading edge of the fluid flow. The concentrated analyte is then tested by the LFA module to obtain visible test results.

In one embodiment, the device of the present invention is combined with other diagnostic tests such as LFA. The seamless interface between concentration and detection of target analytes allows the patient or user to obtain test results in a single, single step.

In one embodiment, the apparatus of the present invention is a diagnostic apparatus comprising an LFA detection module and a concentration module comprising an ATPS component.

Lateral flow immunoassay (LFA) methods and apparatus have been extensively described, for example, in US Pat. No. 4,956,302, Gordon and Pugh; WO 90/06511, H. Buck, et al.; US-6,764,825,T Wang; Patent US-5,008,080, W. Brown, et al.; US Pat. No. 6,183,972 and patent EP00987551A3, Kuo and Meritt. These assays involve the detection and determination of analytes, which are one of the specific combinations of ligands and receptors. The ligand and receptor are related, the receptor specifically binds to the ligand, is capable of distinguishing a particular ligand or distinguishing a ligand from other components of the sample that have similar characteristics. Immunoassays involve an example of a specific combination of reactions between antibodies and antigens. Other examples include DNA and RNA hybridization reactions as well as binding reactions involving hormones and other biological receptors.

In one embodiment, the embedded ATPS component is combined with a lateral flow immunoassay (LFA) on a precoated porous material as a concentration module.

Figure 7 shows an embodiment of the manufacturing process of the concentration module of the present invention. Porous materials (paper or foam pads of different material types) are precoated and treated with different solutions containing ATPS and dried with a lyophilizer. The precoated paper is cut into sheets (or other shapes) and has a pointed tip for easy collection of the concentrated phase. The foam pad material is also cut into pieces before treatment.

As shown in Figure 7, the ATPS component is embedded in the porous material. According to one embodiment shown in Figure 7, the polymer solution (PEG) in DI water is added to the narrow pores of various porous materials including glass fiber paper, cotton base paper, special single layer matrix paper and polyolefin. Foam pad. The paper has the same width but adjustable length to accommodate the difference in absorption. The protein-containing TE buffer (such as bovine serum albumin), surfactant (Triton X-114), Tween-20, and PEG polymer are then added to the first solution at a certain ratio. The porous material is then placed in a lyophilizer to remove the solvent to directly embed the ATPS component in the porous material.

In one embodiment, the paper is cut into sheets of 0.5 cm width. The sheets are folded up (for example, every 4 sheets of paper) and cut from the corners to form a pointed tip for easy collection of concentrated ingredients. The paper stack is then placed in a buffer containing the indicator (eg, colloidal gold) at a different salt concentration (5%, 4.5%, and 4% w/w potassium phosphate in phosphate buffer to achieve 30 times, Concentration of 50 times and 100 times. As the salt concentration decreases, the concentration factor increases. The minimum amount of salt required depends on each individual polymer formulation.

Figure 8 demonstrates the feasibility of changing the position and sequence of the ATPS components in the concentrating module, indicating that the precoated porous material can be immersed in the polymer or salt in different regions (left) or the polymer and salt cover each other into a continuous region ( right). The invention is processed in two steps. The first step is to dry the salt and polymer in different areas in different areas. This is similar to drying different solutions in the immediate vicinity. The second step is to first dry the polymer zone with a lyophilizer and then dry the salt solution directly on the dry polymer and dry it once every hour with a lyophilizer. This method concentrates the analyte. The flow buffer containing the analyte causes the two components of the ATPS to absorb water during fluid flow, resulting in concentration of the purple indicator at the leading edge of the device. In this case, the flow buffer does not require prior metering of the ATPS component, and any liquid sample, including urine, saliva, etc., can also be used to provide greater flexibility to the user (e.g., patient).

In one embodiment, two identical porous materials are prepared, embedded in the polymer/salt and micelle, respectively, except for the solution components and flowing solution components embedded in the porous material. In order to achieve concentration ratios of 40 times, 70 times and 100 times, the amount and concentration of the surfactant-containing solution embedded on the paper were adjusted. An increase in surfactant concentration results in a higher concentration factor. In one embodiment, the phosphate buffer containing the indicator acts as a flow buffer in three concentration multiples.

A variety of different porous materials can be used as a concentration module, and various designs can facilitate analyte concentration and transfer to LFA sample pads and test strips. In one embodiment, all of these designs can take a large volume by capillary action and optimize for a particular application.

The invention can be implemented in a variety of versions, including the one shown in FIG. The ATPS Concentration Module is designed to facilitate concentration and transfer of analytes to LFA sample pads and test strips. This design draws a large volume of sample. In addition, different porous materials have different designs depending on the specific application. In one embodiment, the present invention provides a paper stack design in which a plurality of porous materials are stacked side by side and the solution flows vertically from the bottom of the stack to the top. The paper stack design of the present invention is not limited to the detection of S. mutans or the assays described herein. As described herein, one of the main features of the present invention is that the ATPS component can be embedded in a porous material in any form and immersed in any sample (such as urine, saliva, blood, buffer, etc.), and the phases are automatically separated and concentrated. Remove the analyte of interest. Detection of Streptococcus mutans

Further provided herein diagnostic assays for detecting Streptococcus mutans (S. mutans) to demonstrate the present invention may be used to improve the detection limit of the LFA. Streptococcus mutans ( S. mutans ) is the main bacterium that causes dental caries (fangs) [1, 2]. Therefore, concentration is critical for diagnostic testing. In this test, the detection limit of this bacterium was increased by a factor of 100, from 10 ⁶ CFU/ml to 10 ⁴ CFU/ml, as shown in Figure 5. The ATPS micelles were directly embedded in the precoated porous material and the concentrated components were combined with the LFA test strip. This one-step test can also be used for polymer/salt ATPS.

Figure 5 demonstrates that the detection limit for S. mutans is 100 times higher than for LFA alone. The left panel shows one embodiment of a 3PS device for testing (including the ATPS two components for testing, one LFA detection module, one concentration module). The LFA detection module is combined upstream with the concentration module. The right panel display compares the test results for the specified concentration of S. mutans using the LFA detection module (top) and the 3PS device (bottom) without the concentration module . The presence of the control line indicates the validity of the test, and the presence of the test line indicates a positive result. The test line is indicated by a black arrow. "C" is the control line; "T" is the test line. In the LFA detection module alone, there is no visible test line for the negative sample to confirm the validity of the control. At a concentration of 10 ⁶ CFU/ml of S. mutans , the test line was visible and a positive result was confirmed. However, there are no visible test lines in 10 ⁴ CFU/ml. In the 3PS device, the test line for the negative sample is not displayed, confirming the validity of the control. At 10 ⁶ CFU / ml, the test lines are visible, and the test line intensities in 10 ⁶ CFU / ml is stronger than the LFA alone, which confirmed the successful concentration module was concentrated and displayed the S. mutans S. mutans density detection is concentrated About 100 times. Furthermore, the 3PS device of the present invention is capable of detecting bacteria at 10 ⁴ CFU/ml, which cannot be detected when LFA is used alone. Therefore, the 3PS device of the present invention can achieve a 100-fold improvement in the detection limit of S. mutans .

In one embodiment, the diagnostic test device of the present invention allows the user to perform both the concentration and detection of the analyte in only one step.

In one embodiment, the present invention provides a one-step diagnostic device comprising a concentration module of an ATPS component and a detection module of an LFA test. As shown in Figure 2, a diagnostic test device is provided to allow the user to concentrate and detect the target analyte in only one step.

The apparatus of the present invention allows the user to take only one step from placing the sample to the analyte collection point. The ATPS component is attached to the collection point of the analyte to form a concentration module. In one embodiment, the detection module further includes a plastic housing. The plastic case contains a window that shows the test results. In one embodiment, the LFA detection module and the gold nanoprobe are contained within a plastic housing. The concentration module is connected to the detection module to form a simple connection point. The two modules are tightly entangled with tape and promote flow. In one embodiment, the device can be slightly longer to accommodate more sample volume. After the user adds the sample to the analyte collection point, the sample enters the device via capillary action, and the ATPS component is rehydrated to cause phase separation. When flowing into the plastic housing, these analytes are concentrated primarily at the leading edge of the fluid flow. The concentrated analyte is combined with a gold nanoprobe and detected by an embedded conventional LFA detection module. The same visible test results as the LFA test will be displayed in the window of the plastic case. By using the apparatus of the present invention, the user can obtain intuitive and accurate test results in one step.

In one embodiment, the 3PS device of the present invention can be packaged in a cassette housing.

As shown in Figure 10, the 3PS device contains a box-type enclosure with many important features: 1) It protects the fragile 3PS device components from damage and avoids wasted or ineffective testing. 2) It provides an intuitive user interface to support the proper use of 3PS devices; 3) Pressure points provided at dedicated locations improve overall flow and fit release during testing; 4) 3PS device orientation ensures users It can be tested in the right way; 5) It prevents the user from coming into contact with any chemicals that are dehydrated on different pads; 6) The sample cell holding the sample prevents the user from coming into contact with the released sample due to continuous use.

In one embodiment, the cartridge housing design of the 3PS device may be similar to the design of a conventional LFA test cartridge housing. However, because the 3PS unit is capable of handling larger volumes and improving the concentration factor, the housing size and sample well are larger than conventional LFA test boxes to accommodate additional capacity and fluids. In one embodiment, the concentration module is integrated with the LFA detection module into a new device (3PS) that handles the seamless integration between concentration and detection, enabling the user to achieve results in one step. In one embodiment, the concentration module can be integrated into the LFA detection module in a variety of ways, including with tape, as shown in Figure 9, packaged within the housing. In one embodiment, the concentration modules can be of different sizes to accommodate different sample volumes. Due to the nature of the 3PS device, it can accommodate large volumes of sample (for example, 5 ml or more). In one embodiment, the format of the concentration module can be more varied, including the format shown by the present invention. In one embodiment, the device of the present invention is a platform device, and in addition to detecting S. mutans , there are many different applications including, but not limited to, detection of Chlamydia, malaria, Zika virus, and the like.

In one embodiment, the present invention provides various designs including two vertical directions and two horizontal orientation designs. In one design, the design is vertical, with a housing to protect the test strip and a separate sample slot that can be interlocked with the start flow (Figure 9A). In another design, it is vertical and consists of a single housing with the sample slot and housing inseparable (Figure 9B). In another design, the device is horizontal and has a large sample slot on the test strip (Figure 9C). In another design, the device is horizontal and has a large sample cell under the test strip, where the test strip is cantilevered (Figure 9D).

The main differences in the design in Figure 9 are: 1) In Figure 9A, the actual test strip may be exposed to the environment; 2) In Figure 9B, additional sealing tape, such as a rubber O-ring, is required; 3) In Figure 9C Add a sample to the top and gravity to allow liquid to flow from the sample cell down into the test strip. 4) In Figure 9D, add the sample to the sample well under the test strip and insert the cantilever test strip into the slot to allow Capillary guides the flow.

In one embodiment, the apparatus of the present invention for concentrating and detecting a target analyte in a sample solution comprises: (a) a sample tank for holding a sample solution; (b) a phase of the sample tank a connected concentration module comprising a component coated with water soluble nanoparticles and incorporated into a two-liquid phase system ATPS, wherein one of the phase solutions flows faster than the other phase solution as the aqueous solution flows through the porous material Forming two phase solutions, wherein the sample solution is introduced into the apparatus through the sample tank, the porous material is sucked up the sample solution, wherein the target analyte in the sample solution is distributed into the faster flowing phase solution and a leading edge position of the faster flowing phase solution is concentrated; and a detection module coupled to the concentration module for detecting the target analyte, the detection module comprising a lateral flow analysis test and a bonding pad, wherein the porous material of the concentration module and the bonding pad In combination, the concentrated target analyte flows from the porous material to the mating pad and is then analyzed by lateral flow analysis test, and a color reaction occurs. The presence of the target analyte.

In one embodiment described herein, the nanoparticles are selected from the group consisting of cerium oxide, iron oxide, titanium dioxide, silver oxide, and any combination thereof.

In one embodiment described herein, the porous material is selected from the group consisting of fiberglass paper, cotton-based paper, single layer matrix paper, and polyolefin foam pads.

In one embodiment described herein, the ATPS component is selected from the group consisting of polymers, salts, and surfactants.

In one embodiment described herein, the polymer is selected from the group consisting of polyethylene glycol, poly(oxyalkylene) polymers, poly(oxyalkylene) copolymers, polyvinyl alcohol pyrrolidone, polyvinyl alcohol, polyvinyl alcohol caprolactone. Amine, polyvinyl methyl ether, alkoxylated surfactant, alkoxylated starch, alkoxylated cellulose, alkyl hydroxyalkyl cellulose, organic hydrazine modified polyether, poly N-isopropyl Acrylamide. Polyethylene glycol, polypropylene glycol and dextran.

In one embodiment described herein, the salt is selected from the group consisting of a lyophilic salt, a chaotropic salt, containing, for example, a linear or branched trimethylammonium, triethylammonium, tripropylammonium, tributylammonium, tetramethyl Alkyl ammonium, tetraethylammonium, tetrapropylammonium and tetrabutylphosphonium cations, and inorganic salts such as phosphates, sulfates, nitrates, chlorides and hydrogencarbonate anions. In another embodiment, the salt is selected from the group consisting of sodium chloride, sodium phosphate, potassium phosphate, sodium sulfate, potassium citrate, ammonium sulfate, sodium citrate, sodium acetate, and combinations thereof.

In one embodiment described herein, the surfactant is selected from the group consisting of nonionic surfactants, detergents, Triton-X, Igepal CA-630 and Nonidet P-40.

In one embodiment described herein, the sample solution contains one or more of blood, plasma, serum, urine, saliva, feces, and body exudates.

In one embodiment described herein, the analyte is DNA, free DNA, circulating tumor DNA, proteins, nucleic acids, carbohydrates, lipids, bacteria, viruses or nanoparticles.

In one embodiment described herein, the ATPS components are completely or partially overlapping on the porous material.

In one embodiment described herein, the analyte can be concentrated to more than 100 times.

In one embodiment described herein, the porous material format is a multi-layered sheet of paper with one end of the strip being a pointed end.

In one embodiment described herein, the sample solution is a phosphate buffer or a Tris-EDTA buffer with an indicator.

In one embodiment described herein, the concentration module and the detection module are separable and are both within the housing, wherein the device can optionally include a rubber ring for sealing the joint between the housings.

In one embodiment described herein, the concentration module and the detection module are inseparable.

Throughout the application process, the transitional terms "comprising" and "comprising", "including" or "characterized" are synonymous, inclusive or open-ended, and do not exclude additional, non-exemplified elements or method steps.

The invention will be better understood by reference to the following examples. Those skilled in the art will readily appreciate that the examples are provided for illustrative purposes only and are not intended to limit the scope of the invention. The invention is defined by the scope of the subsequent patent application. DETAILED DESCRIPTION OF THE INVENTION Example 1 - Precoated porous material embedded in ATPS

Different porous materials, including fiberglass paper, cotton-based paper, special single-layer matrix paper, and polyolefin foam pads (width 2.2 cm and adjustable length) were each immersed in a 5 M aqueous solution of cerium oxide and stirred vigorously for one hour. Solution was maintained at 80 ^o C 24 h. The porous material is then removed and washed twice with water and finally dried in compressed air.

The prepared ATPS component was dehydrated on precoated porous paper with an ATPS component of 20% (w/w) PEG 4600, 2% (w/w) SDS, 16% (w/w) Triton-X114 and 18.5% ( w/w) sodium sulfate Na ₂ SO ₄ . A PEG solution (in DI water) is added to each narrow channel of the porous material. 50 uL TE buffer (containing 2% bovine serum albumin (BSA), 0.1% Tween-20 and 0.1% PEG, 20 mM TE, pH 7.5) was immediately added with the first solution. Figure 7 illustrates a typical process in accordance with an embodiment of the present invention.

The pre-coated porous material embedded in the ATPS was dried in a lyophilizer for 1 hour and cut into 4 cm x 0.5 cm sheets. Then stack the sheets (four sheets into a stack) and cut them from the corner to a point 0.5 cm from the end (form a triangle, see Figure 7). The porous material embedded in the ATPS was then immersed in a PBS buffer (pH 7.4) containing an indicator (colloidal gold) containing a salt (here, potassium phosphate). Flow occurs under capillary action. Example 2 - Concentrated DNA in Saliva

The precoated porous glass paper embedded in the ATPS component prepared in Example 1, blank paper (without ATPS or precoating) and control paper (ATPS embedded on paper without precoating) were smashed in the saliva mixture, including pH 7.4 in PBS buffer and 200 ng/ml DNA molecular weight standard. The sample was taken by capillary action for approximately 2 minutes until the solution reached the top position.

The solution was allowed to flow up on the paper, sampled at the leading edge of the fluid flow and analyzed by UV spectrophotometry. The measurement results were compared with similar experiments with blank paper (concentration multiple of 1). Please note that the indicator used is colloidal gold. Concentration times are summarized in Table 1. Table 1. DNA concentration multiples of the method of the invention Example 3 - testing equipment used 3PS

For the LFA detection module: Strip-shaped nitrocellulose membranes are: 1) anti- S. mutans antibody at a concentration of 1 mg/ml, 2) concentration of 0.2 mg/ml protein (bovine serum albumin, BSA). Colloidal gold nanoparticles were combined with anti- S. mutans antibodies and dried using a lyophilizer according to the supplier's instructions. The sample pad contained sample pad material immersed in Tris buffer containing 2% bovine serum albumin BSA, 16% surfactant (Triton X-114), 0.1% PEG polymer and 20 Mm Tris salt. The absorbent pad contains untreated paper. The LFA test was used alone without a concentration module, the various materials were assembled on a viscous plate, and the device was immersed in a solution containing different concentrations of S. mutans . The results were analyzed by direct observation.

For 3PS equipment: Prepare a concentration module and apply 300 pL of surfactant (Triton X-114) solution to pre-coated glass paper FG1 (size 4 cm X 2.2 cm). The sheet was then dried in a lyophilizer and cut into 0.5 cm long pieces of paper. Four sheets of paper are stacked into a stack to form a concentrating module.

The concentrating module is connected to the LFA detection module, and the sample pad and the concentrating module of the detection module are directly connected. In order to keep this connection and promote flow, the tape is tightly wrapped around the two modules. Although this new device is slightly longer and requires more volume, the running test is the same. As shown in Figure 5, S. mutans was successfully concentrated using a 3PS device and achieved a 100-fold concentration factor. The presence of the control line indicates the validity of the test and the presence of the test line indicates a positive result. The test line is indicated by a black arrow. "C" is the control line; "T" is the test line. When testing using only the LFA detection module, there is no visible test line for the negative sample, indicating the effectiveness of the control. At a concentration of 10 ⁶ CFU/ml for S. mutans , the test line was visible, indicating a positive result. However, there was no visible test line at 10 ⁴ CFU/ml. In the 3PS device of the present invention, the negative sample has no visible test line indicating the effectiveness of the control. When the concentration of S. mutans is 10 ⁶ CFU/ml, the test line is visible and the test line is more chroma than the LFA test alone. When the concentration of S. mutans was 10 ⁴ CFU/ml, the test line was still visible, confirming that the concentration module successfully concentrated S. mutans and when the S. mutans was detected, the concentration factor was 100. Overall, the experimental results demonstrate that the 3PS device of the present invention is capable of detecting bacteria at concentrations as low as 10 ⁴ CFU/ml, which is not detectable by LFA alone. The 3PS device of the present invention reduces the detection limit of S. mutans by a factor of 100. References: 1. Aas, JA, Paster, BJ, Stokes, LN, Olsen, I. and Dewhirst, FE, 2005. Defining the normal bacterial flora of the oral cavity. Journal of clinical microbiology, 43(11), pp.5721 -5732. 2. Corby, PM, Lyons-Weiler, J., Bretz, WA, Hart, TC, Aas, JA, Boumenna, T., Goss, J., Corby, AL, Junior, HM, Weyant, RJ and Paster, BJ, 2005. Microbial risk indicators of early childhood caries. Journal of clinical microbiology, 43(11), pp.5753-5759.

Figure 1 shows the phase separation of different porous materials embedded in two components of ATPS. The precoated porous materials used included glass fiber paper (FG), cotton base paper (C), single layer matrix paper (SM) and polyolefin foam mat (PO) from 3 different suppliers.

Figure 2 shows a schematic of the apparatus of the present invention in which the analyte is concentrated at the leading edge of the fluid flow and visible test results are detected.

Figure 3 shows an embodiment of the invention in which biomolecules are concentrated to different folds at the leading edge of the embedded ATPS porous material.

Figure 4 shows the use of the precoated porous material of the embedded ATPS component of the present invention to concentrate biomolecules in different types of samples (urine and saliva).

Figure 5 shows the apparatus of the present invention comprising a porous material embedded with two components of ATPS to enhance the sensitivity of detecting S. mutans . The results show that the detection limit of S. mutans is 100 times lower than that of the LFA module alone.

Figure 6 shows the apparatus of the present invention using a porous material embedded in two components of ATPS as various designs for the concentration module.

Figure 7 presents an embodiment illustrating the manufacturing process of the concentration module.

Figure 8 demonstrates how to change the feasibility and location of the ATPS components on the concentrating module.

Figure 9 shows various designs of the device of the present invention. Figure 9A shows a closed (assembled) and open vertical box housing design. The device contains separate sample compartments. Figure 9B shows a closed (assembled) and open vertical box housing design that includes a joined sample slot. Figure 9C shows a closed (assembled) and open horizontal box housing design that allows samples to be placed from above the test strip. Figure 9D shows a closed (assembled) and open horizontal box housing design that allows samples to be placed from under the test strip.

Figure 10 shows a CAD model of several cassette housing designs of the present invention.

Claims (15)

An apparatus for concentrating and detecting a target analyte in a sample solution, comprising: (a) a sample tank for holding a sample solution; (b) a concentration module coupled to the sample tank, the module comprising a water soluble solution a nanoparticle and a component that breaks into the two-liquid phase system ATPS, wherein when the aqueous solution flows through the porous material, one of the phase solutions flows faster than the other phase solution, thereby forming two phase solutions, wherein the sample solution Introducing the device through the sample tank, the porous material is sucked up with a sample solution, wherein the target analyte in the sample solution is distributed into the faster flowing phase solution and concentrated at the leading edge of the faster flowing phase solution And (c) a detection module coupled to the concentration module for detecting a target analyte, the detection module comprising a lateral flow analysis test and a bonding pad, wherein the porous material of the concentration module is combined with the bonding pad, wherein the concentrated The target analyte flows from the porous material to the mating pad and is subsequently analyzed by lateral flow analysis testing, with the appearance of a color reaction with the presence of the target analyte. The device of claim 1, wherein the nanoparticles are selected from the group consisting of cerium oxide, iron oxide, titanium dioxide, silver oxide, and any combination thereof. The apparatus of claim 1 wherein the porous material is selected from the group consisting of fiberglass paper, cotton-based paper, single-layered matrix paper, and polyolefin foam pads. The device of claim 1 wherein the ATPS component is selected from the group consisting of a polymer, a salt, and a surfactant. The device according to claim 4, wherein the polymer is selected from the group consisting of polyethylene glycol, poly(oxyalkylene) polymer, poly(oxyalkylene) copolymer, polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl alcohol Indoleamine, polyvinyl methyl ether, alkoxylated surfactant, alkoxylated starch, alkoxylated cellulose, alkyl hydroxyalkyl cellulose, organogermanium modified polyether, poly N- Isopropyl acrylamide. Polyethylene glycol, polypropylene glycol and dextran. The device of claim 4, wherein the salt is selected from the group consisting of a lyophilic salt, a chaotropic salt, containing trimethylammonium, triethylammonium, tripropylammonium, tributylammonium, tetramethylammonium, and tetra Ethyl ammonium, tetrapropylammonium or tetrabutylammonium cations and inorganic salts containing phosphates, sulfates, nitrates, chlorides or hydrogencarbonate anions, sodium chloride, sodium phosphate, potassium phosphate, sodium sulfate, citric acid Potassium, ammonium sulfate, sodium citrate, sodium acetate, and combinations thereof. The device of claim 4, wherein the surfactant is selected from the group consisting of a nonionic surfactant, a detergent, Triton-X, Igepal CA-630, and Nonidet P-40. The device of claim 1, wherein the sample solution comprises one or more of blood, plasma, serum, urine, saliva, feces, and body exudates. The device of claim 1, wherein the analyte is DNA, free DNA, circulating tumor DNA, protein, nucleic acid, carbohydrate, lipid, bacteria, virus or nanoparticle. The device of claim 1 wherein the ATPS components are completely or partially overlapping on the porous material. The device of claim 1, wherein the analyte can be concentrated to more than 100 times. The apparatus of claim 1, wherein the porous material format is a multi-layered paper strip, one end of which is a tip end. The device of claim 1, wherein the sample solution is a phosphate buffer or a Tris-EDTA buffer with an indicator. The device of claim 1, wherein the concentration module and the detection module are both within the housing and separable, wherein the device can optionally include a rubber ring for sealing the joint between the housings. The device of claim 13 or 14, wherein the concentration module and the detection module are inseparable.