3D Hybrid Point of Care Chip and Analysis Method Using the Same

Abstract

To provide a three-dimensional hybrid point of care chip that solves conventional problems, and can use advantages of superparamagnetic particle imaging technologies maximally.SOLUTION: The present invention relates to a three-dimensional hybrid point of care chip that includes: at least one or more sample introduction regions; at least one or more analytical regions; a fluid absorption region; and a reagent reservoir as necessary. A structure of the three-dimensional hybrid point of care chip is a lamination plate within a range of a single layer to ten layers, and the sample introduction region, reagent reservoir, analytical region and fluid absorption region are sequentially connected by a microchannel. The microchannel makes it possible to divide a sample including an analyte to guide the sample to a level of the lamination plate.SELECTED DRAWING: Figure 9A

Description

Technical Field

[0001] <Cross - Reference to Related Applications> This application claims the priority of U.S. Provisional Application No. 62 / 655,828 filed on April 11, 2018, U.S. Provisional Application No. 62 / 664,946 filed on May 1, 2018, and U.S. Utility Application No. 16 / 379,748 filed on April 9, 2019. The U.S. Utility Application claims the rights of U.S. Provisional Application No. 62 / 655,828 filed on April 11, 2018 and U.S. Provisional Application No. 62 / 664,946 filed on May 1, 2018. The subject matter and content of all the above - mentioned U.S. applications are incorporated herein by reference.

[0002] The present invention relates to biosensing technology, and in particular, to an analysis method for simultaneously measuring multiple analytes in a sample using superparamagnetic particle imaging or other technologies, and a chip and device used therein.

Background Art

[0003] Biosensing refers to any method for detecting biological elements and related software or computer technology for identifying the biological characteristics of a sample, and is an essential part of medical diagnosis, environmental monitoring, and food safety assurance. A biosensing system incorporates biological substances (such as tissues, enzymes, and nucleic acids) and chemical analysis into electrical, electronic, and photonic devices to generate detectable signals for monitoring or identifying biological phenomena. Biosensing is increasingly applied in biomedical, food production and processing, and the detection of bacteria, viruses, and biotoxins, and is also applied in the defense against biological warfare, representing a new scientific case where advanced biology, nanotechnology, and information technology are significantly integrated.

[0004] Biosensing technologies can be classified into optical, electrochemical, and magnetic biosensing. First, based on the light conversion mechanism, optical biosensing can be classified into luminescence methods including fluorescence, phosphorescence, fluorescence resonance energy transfer (FRET), chemiluminescence, bioluminescence, quantum dots, absorption, and scattering, and surface methods including surface plasmon resonance (SPR), surface-enhanced Raman scattering (SERS), and interference. Generally, optical biosensing methods are highly sensitive and easy to multiplex.

[0005] Second, electrochemical biosensing includes methods using enzyme-linked assays, field-effect sensors, electroactive tags, nanoparticle-based sensors, and sensors based on electrochemiluminescence. These methods and assays are essentially interfacial, where biological or physical changes caused by recognition events directly change the electrical properties of the contacting materials. These assays are simple and highly sensitive, and the localization of binding events at the interface enhances the discrimination between specific analytes and background analytes. Also, these assays are compatible with expansion into array formats and integration with microfluidic structures.

[0006] Third, magnetic biosensing generally includes methods based on alternating current susceptibility measurements, Hall effect measurements, giant magnetoresistance, superconducting quantum interference devices, and excitation inductance. Compared with optical and magnetic biosensing methods, magnetic particle-based sensing methods improve biocompatibility and environmental safety and reduce synthesis costs. Furthermore, since there is little or no magnetic signal from biological samples, the background noise in magnetic particle-based sensing methods is low. Therefore, it has received great attention in the development of biosensing and diagnostic tools. See Issadore, D et al., "Magnetic sensing technology for molecular analyses", Lab Chip. 14(14), 2385 - 2397 (2014).

[0007] The AC susceptibility measurement method is an accurate detection technique that utilizes the diffusion characteristics of magnetic nanoparticles (MNPs) in solution. See Park, K et al., "Multiplexed sensing based on Brownian relaxation of magnetic nanoparticles using a compact AC susceptometer," Nanotechnol. 22(8), 085501 (2011). This technique is based on the principle of a Brownian relaxation detection scheme that uses the random rotational motion of magnetically labeled sensors determined by measuring the collective susceptibility as a function of the frequency of the applied magnetic field. When the excitation frequency approaches the frequency of the rotational motion of the magnetically labeled sensor, the loss component of the complex susceptibility increases significantly. This phenomenon is observed as the peak frequency (out-of-phase 90o: χ”) of the imaginary component of the complex susceptibility. The application of the technique for biological diagnosis depends on the shift of the peak frequency of χ” due to the binding of the target to the labeled MNP. Subsequently, when the target molecule binds to a specific receptor on the sensor, the hydrodynamic size of the sensor effectively increases, and due to the cubic relationship with the hydrodynamic radius, there is an easily measurable shift in the maximum value of the frequency to a lower value. The AC susceptometer showed high sensitivity at a low magnetic field of 10 μT with a concentration of 1 mg / ml and a volume of 5 μl, but the application of this method is limited to solution media.

[0008] Hall sensors based on Hall effect measurements are defined as crosses with an arm width w of approximately 1 μm by photolithography and dry etching with an argon ion mill. See Mihajlovic, G. n., "Detection of single magnetic bead for biological applications using an InAs quantum-well micro-Hall sensor", Appl. Phys Lett. 87, 112502 (2005), and Landry, G. et al., "Characterization of single magnetic particles with InAs quantum-well Hall devices", Appl. Phis Lett. 85, 4693 (2004). Some crosses are further defined by focused ion beam milling with arm widths of 500, 600, and 700 nm. Each sensor is characterized by van der Pauw and Hall measurements. After processing, the values of the Hall coefficient and sheet resistance are 0.031 < R H < 0.046 Ω / Oe and 150 < R H<It is in the range of 600 Ω / Oe. When the sensor chip is placed in a vertical alternating excitation magnetic field ~B0 that varies at a frequency f0, the sensor is biased by a direct current I0, and the Hall voltage is measured at the frequency f0 using a lock-in amplifier. Since the beads are superparamagnetic, their magnetization follows Langevin behavior. The alternating current signal depends on the DC magnetic state of the beads because it substantially measures the slope of the Langevin curve. Thus, when the beads are exposed to a DC magnetic field B1, their magnetic state shifts in the direction of low magnetic susceptibility, reducing the alternating magnetization induced in the beads, thereby reducing the average alternating levitation magnetic field sensed by the cross, which is itself indicated as a decrease in the AC Hall voltage signal. The linearity of the Hall sensor ensures that no change in the AC Hall signal occurs in the empty Hall cross when B1 has no beads on top. Therefore, this decrease is a decisive signal indicating the presence of beads in the Hall cross. The drawback of this method is the large shift caused by the direct Hall response of the sensor to the alternating excitation magnetic field, which is usually several orders of magnitude larger than the small signal from the beads.

[0009] Giant magnetoresistance (GMR) is a quantum mechanical magnetoresistance effect observed in multilayers consisting of alternating ferromagnetic and nonmagnetic conductive layers. See Hall, D et al., "GMR biosensor arrays - a system perspective", Biosens Bioelectron. 25(9), 2051 - 2057 (2010), and Baselt, D, "A biosensor based on magnetoresistance technology", Biosens Bioelectron., 13, 731 - 739 (1998). The observed effect is that a significant change in resistance depends on whether the magnetizations of adjacent ferromagnetic layers are aligned parallel or antiparallel. When aligned parallel, the total resistance is relatively low, but when aligned antiparallel, the total resistance is high. The magnetization direction can be controlled, for example, by applying an external magnetic field. This effect is based on the dependence of electron scattering on spin orientation. The development of magnetoresistive materials enables the optical patterning of high - sensitivity micrometer - scale magnetic field sensors. Magnetoresistive materials generally have a film - metal multilayer structure whose resistance changes in response to a magnetic field. Several quite different types of magnetoresistive materials have been disclosed, including anisotropic magnetoresistive materials and giant magnetoresistive materials. Magnetoresistive sensors are commercially used for reading magnetic tapes or disks, portable magnetic field sensors, and position sensors. By using magnetoresistive materials, small detectors for magnetic bead assays can be fabricated. Such detectors can be embedded in an assay substrate to detect beads in their vicinity. The main advantage of this method over optical or micro - mechanical detection is that thousands of detectors can be fabricated on a single chip with a side length of about 1 cm. The disadvantages of GMR sensors are their non - linearity and monolayer characteristics. They are very sensitive to the distance between the surface of an object and the magnetic beads and the sensor.

[0010] A superconducting quantum interference device (SQUID) is a highly sensitive magnetometer used to measure very small magnetic fields based on a superconducting loop containing Josephson junctions. See Kotitz, R et al., "Determination of the binding reaction between avidin and biotin by relaxation measurements of magnetic nanoparticles", J. Magn. Magn. Mater., 194, 62-68 (1999); Hathaway HJ, "Detection of breast cancer cells using targeted magnetic nanoparticles and ultra-sensitive magnetic field sensors", Breast Cancer Research, 13, R108 (2011), and De Haroa. L et al., "Magnetic relaxometry as applied to sensitive cancer detection and localization", Biomed. Eng.-Biomed. Tech., 60(5), 445-455 (2015), and Perez. J et al., "Magnetic relaxation switches capable of sensing molecular interactions", Nat Biotechnol., 20, 816-820 (2002). The sensitivity of SQUIDs is sufficient to measure low magnetic fields as low as 5 aT (5×10-18 T) in average measurements over several days. Their noise levels are as low as 3 fT·Hz-1 / 2.For comparison, a typical refrigerator magnet generates 0.01 tesla (10-2T), and some processes in animals generate very small magnetic fields between 10-9T and 10-6T. There are two main types of SQUIDs: direct current (DC) and radio frequency (RF). An RF SQUID can operate with only one Josephson junction (superconducting tunnel junction). Although SQUIDs are very sensitive, they require cryogenic conditions and expensive equipment and are thus not suitable for routine analysis.

[0011] The excitation inductance refers to the phenomenon that when passing through a coil, the inductance of the coil changes due to the change in the relative permeability of magnetic particles. See Makiranta.J et al., "Magnetic relaxation switches capable of sensing molecular interactions," 28th Annual International Conference of the IEEE Engineering in Medicine and Biology Society, New York, USA, Conf Proc IEEE Eng Med Biol Soc. 4598-411 (2006). The change in the inductor can be used to quantify the amount of magnetic particles entering the coil. This method has been applied to many devices for in vitro diagnosis, but this method cannot be multiplexed and has low reproducibility.

[0012] Magnetic particle imaging (MPI) is a new non-invasive and highly sensitive tomography technique, as disclosed in U.S. Patent No. 7,778,681 B2. The first prototype of an MPI scanner was published in Gleich, B., "Tomographic imaging using the nonlinear response of magnetic particles," Nature, 435(7046), 1214-1217 (2005). MPI utilizes the non-linear response of magnetic particles to a changing external magnetic field, and its basic theory was first invented in 1908 by Paul Langevin, as described in Lemons, D., "Paul Langevin’s 1908 paper ‘On the Theory of Brownian Motion’," Am. J. Phis., 65, 1079 (1997). It is disclosed that signals generated by the magnetization of magnetic particles (tracers) in an alternating magnetic field are spatially encoded, and after reconstructing the spatially encoded signals by combining signals proportional to the concentration and position of the tracers, successful imaging of a phantom is achieved.The main applications of MPI are in vivo imaging (Weizencker, J, "Three dimensional real-time in vivo magnetic particle imaging", Phys. Med. Biol. 54(5), L1-L10 (2009), and Zhou, X, "First in vivo magnetic particle imaging of lung perfusion in rats", Phys. Med. Biol. 62(9), 3510-3522 (2017)", cancer diagnosis (Yu, Yu) E et al., "Magnetic Particle Imaging: A Novel in Vivo Imaging Platform for Cancer Detection", Nano Lett. 17(3)1648-654 (2017), and cell tracking (Zheng, B et al., "Quantitative Magnetic Particle Imaging Monitors the Transplantation, Biodistribution, and Clearance of Stem Cells In Vivo", Theranostics 6(3), 291-301 (2016)).The principles of MPI and the construction methods of general MPI devices are described in detail in Knopp et al., "Magnetic Particle Imaging - An Introduction to Imaging Principles and Scanner Instrumentation", Springer Science & Business Media (2012), and Bugz, T et al., "Magnetic Nanoparticles - Particle Science, Imaging Technology, and Clinical Applications", World Scientific Publishing (2010).

[0013] Magnetic biosensing methods and techniques have many advantages, for example, since human samples are naturally lacking in ferromagnetic materials, there are few interferents (different from electron and optical techniques where a large amount of interferents exist). MNPs are used in biomedical separation techniques and imaging. For a detailed discussion of the current advantages and disadvantages of magnetic biosensing, see Lee, H et al., "Recent Developments in Magnetic Diagnostic Systems", Chem. Rev. 115(19), 10690 - 10724 (2015). So far, different from optical sensing methods, the main disadvantage of existing magnetic sensing methods used in diagnosis is that multiple analytes cannot be measured simultaneously. Another problem that magnetic sensing methods face is usually dealing with homogeneous media or single layers.

[0014] In recent years, with the increasing clinical demand, various point-of-care (POCT) sensing methods have been explosively increasing. See Cheng, M et al., "Nanotechnologies for Biomolecular Detection and Medical Diagnostics", "Curr. Opin. Chem. Biol." 10(1), 11-19 (2006), and Giljohann, D et al., "Drivers of Biodiagnostic Development", "Nature" 462(7272) 461-464 (2009). These point-of-care methods are often based on electrical impedance, colorimetric, optical, and magnetic sensing strategies, and in particular, in cell, molecular, and genetic tests, they face many challenges including further improvement of sensitivity and specificity, increased test complexity, complex early-stage purification (where precious samples may be lost), specific problems related to small batch testing, higher training needs, higher quality control costs, regulatory burden and expenses.

[0015] Lateral flow immunoassay (LFIA) is one of the most widely used formats in point-of-care devices. LFIA uses a porous membrane, antibodies (monoclonal and / or polyclonal), and typically a visible signal generation system to perform highly sensitive, disposable, and easy-to-use tests. This technology is used in rapid diagnostic tests for pregnancy, fertility, drug abuse, infectious diseases, and DNA detection. Both general-use pharmaceuticals and point-of-care can perform similar tests. It has become one of the most widely used formats in point-of-care assays due to its ease of use and low cost. However, due to its design and structure, the sample binding of LFIA is inefficient, the connection between sections is insufficient, the films do not match, the sample leaks, and the capture area is variable (Wang, R et al., "Lateral Flow Immunoassay", Humana Press, 2009). These problems result in a large coefficient of variation (CV), mainly restricting LFIA to qualitative analysis. The large CV of LFIA is mainly due to the insufficient connection between each section, the mismatch of the films used for the immobilization of the capture material and sample transport, the leakage of the sample from the edge of the test strip, and the variable capture material being banded in the analysis area. In many cases, manual reading is unclear.

[0016] Microfluidics is another form widely used in point-of-care devices. A microfluidic chip is a pattern of molded or etched microchannels. By guiding, mixing, separating, or manipulating fluids within the microchannels, multiplexed, automated, high-throughput systems are realized. To achieve desired functions such as lab-on-a-chip, pathogen detection, electrophoresis, DNA analysis, etc., it is necessary to accurately and detailedly describe the microchannel network design. Microfluidic technologies used in chemical or biological analysis reduce reagent consumption, have short analysis times, are small in scale, and have versatility and high sensitivity. In the past 30 years, microfluidics-based miniaturized analysis systems and technologies used in chemical analysis, biological analysis, and clinical diagnosis have increased explosively. However, using microfluidics in chemical and biological analysis faces quite significant challenges such as complex and rigorous sample pretreatment and processing, difficult and complex design and manufacturing. Analytes measured with microfluidic chips are usually in solution, and methods for detecting analytes, as well as the cost and complexity of development and manufacturing, are limited (Noh, J et al., "Topics in Current Chemistry (Top. Curr. Chem.)" 304, 117 - 152 (2011)). Due to the complexity, an external driving force is always required to complete the process, which tends to be more expensive.

[0017] The EISA format is rarely used in point-of-care devices. Enzyme-linked immunosorbent assay (ELISA) is a plate-based assay technology designed to detect and quantify substances such as peptides, proteins, antibodies, and hormones. In ELISA, it is necessary to immobilize an antigen on a solid surface and complex it with an antibody conjugated to an enzyme. Detection is performed by evaluating the activity of the conjugated enzyme through incubation with a substrate to generate a measurable product. The most important element in the detection strategy is the highly specific antibody-antigen interaction. The limitations of the EISA format are that it operates in multiple steps, reagent handling is difficult, and the equipment is large. This is not applicable to point-of-care applications. Due to the limitations of this format, although ELISA has high sensitivity and specificity, it is usually used in large clinical analysis devices rather than point-of-care devices.

[0018] The construction and design of disposable and biodegradable assay formats are restricted by the types and formats of samples that are usually analyzed. Such restrictions are imposed not only by the technology of large-scale production facilities, but also by the analytical environment, the analyte, the materials, and the physical conditions of the analytical methods used for measurement, all of which are driven by market price, competition, and performance. Summary of the Invention Problems to be Solved by the Invention

[0019] Most point-of-care devices are linear. For example, common plastic cassettes are used to hold lateral flow tests (LFTs) such as early pregnancy tests (EPT). These are composed of different arrangements of a backing card, a lateral flow membrane (nitrocellulose), a sample introduction pad, a filter, and an absorption membrane. Some known designs of plastic cases are convenient for holding components, applying a liquid sample, developing, and then reading the results. One of the latest problems when using such tests is the disposal of the test equipment used. In the past two years, more than 650 million assays have been performed for malaria, HIV, and dengue fever on the African continent alone. This has become a problem in developing countries with limited capacity for biological waste treatment. Devices made of biodegradable materials are strongly demanded.

[0020] The present invention combines superparamagnetic particle imaging technology with a hybrid point-of-care (HY-POC) chip to provide solutions to all problems while maintaining and expanding the advantages of magnetic biosensing technology. Furthermore, the hybrid point-of-care chip of the present invention not only solves the problems of conventional formats, but also maximally utilizes the advantages of superparamagnetic particle imaging technology. The present invention also provides an analytical device used for the analytical method and the chip. Means for Solving the Problems

[0021] The analytical method based on superparamagnetic nanoparticles of the present invention includes the steps of providing a sample containing at least one analyte in a sample matrix, providing a point-of-care chip having at least one analysis region which is a stationary phase each having at least one section, labeling each of the analytes in the sample with superparamagnetic nanoparticles and immobilizing the labeled analytes on the stationary phase, providing an analytical apparatus comprising an apparatus for exciting the superparamagnetic nanoparticles in vitro and an apparatus for sensing, receiving and transmitting the response of the excited superparamagnetic nanoparticles, placing the point-of-care chip and the analysis region including the stationary phase in the analytical apparatus and exciting the superparamagnetic nanoparticles in vitro, sensing, receiving and transmitting the response of the superparamagnetic nanoparticles, and analyzing the response of the superparamagnetic nanoparticles to determine the characteristics of the analyte, wherein the response of the superparamagnetic nanoparticles includes harmonics.

[0022] In the present invention, the analytical method based on the superparamagnetic nanoparticles further includes, in the analytical apparatus, the steps of providing a changing external magnetic field and a field-free point, or a field-free line, or a field-free space which may be a field-free region within the changing external magnetic field, placing the point-of-care chip in the analytical apparatus, and the field-free region scanning the entire analysis region, and the excitation coil exciting the superparamagnetic nanoparticles on the stationary phase in the field-free region to generate the spatially encoded response, wherein the stationary phase of the analysis region includes two or more sections, the superparamagnetic nanoparticles in the sections generate a spatially encoded response, and the characteristics of the analyte are quantitatively determined from the spatially encoded response whether or not removal or reconstruction of the unbound analyte is performed.

[0023] In the present invention, the number of sections in the stationary phase ranges from 1 to 20, and preferably, the stationary phase consists of a single section. In the present invention, the stationary phase may be employed to immobilize at least one or more different superparamagnetic nanoparticles within the range of 1 to 20.

[0024] In the present invention, each of the superparamagnetic nanoparticles may correspond to each of the labeled analytes, and may be different from other superparamagnetic nanoparticles on the labeled analytes in the sample matrix. In the present invention, the particle size of the superparamagnetic nanoparticles may be in the range of 1 nm to 1000 nm. The superparamagnetic nanoparticles may be made of Fe, CoFe, Co, Co alloy, ferrite, cobalt nitride, cobalt oxide, Co-Pd, Co-Pt, iron, iron alloy, Fe-Au, Fe-Cr, Fe-N, FeO, Fe-Pd, Fe-Pt, Fe-Zr-Nb-B, Mn-N, Nd-Fe-B, Nd-Fe-B-Nb-Cu, Ni or Ni alloy material. Further, the superparamagnetic nanoparticles may be spherical, elliptical, flattened or tubular in shape, and a material that changes the response of the superparamagnetic nanoparticles to the external magnetic field may be coated.

[0025] In the present invention, the sample is in a sample matrix that may be a liquid, solid extract, liquid, air sample, or a mixture thereof. Further, the sample matrix may be whole blood, serum, plasma, urine, saliva, feces, tears or sweat.

[0026] In the present invention, the analyte may be an organic molecule, biomolecule, peptide, polymer, amino acid, protein, enzyme, antibody, DNA, RNA, virus, cell, bacterium, pathogen, inorganic molecule, drug, or a mixture thereof. In the present invention, the analysis region may be an assay format that is a hybrid point-of-care, lateral flow, microfluidic beads, or ELISA monolayer.

[0027] The present invention further provides a three-dimensional hybrid point-of-care chip including at least one or more sample introduction regions, at least one or more analysis regions, a fluid absorption region, and optionally a reagent reservoir. The structure of the three-dimensional hybrid point-of-care chip is a laminate within the range of 1 to 10 layers, and the sample introduction region, the reagent reservoir, the analysis region, and the fluid absorption region are sequentially connected by microchannels that enable splitting a sample containing an analyte and guiding it to the levels of the laminate.

[0028] In the present invention, when the three-dimensional hybrid point-of-care chip further has two or more levels in the laminate, it may include a switching column, and the switching column is positioned between the sample introduction region and the analysis region, connecting the levels of the laminate and enabling splitting the sample containing the analyte and guiding it to different levels of the laminate.

[0029] In the present invention, the three-dimensional hybrid point-of-care chip may further include a liquid driving mechanism such as a diaphragm pump connected to the sample introduction region. In the present invention, at least one or more levels of the laminate are laminated layers of films through which the sample containing the analyte flows by itself, and the surface of the film is optionally modified. The film is made of plastic, adhesive, paper, wood, fiber, silicon, polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), glass fiber, cellulose, polysaccharide, protein polymer, or rolled particulate material.

[0030] In the present invention, the number of the sample introduction regions may be in the range of 1 to 5. The sample introduction region may further include a red blood cell separation mechanism such as the apparatus described in Aunet (US4933092, 1990 of Aunet, D). Further, the sample introduction region may further contain a reagent that helps the labeled analyte identification material and / or the sample to flow into the microchannel, controls the pH of the sample, and enhances the reaction between the analyte and the identification material or the capture material in the analysis region. Generally, the sample introduction region can accommodate the sample with a volume in the range of 1 to 200 microliters, and the sample may be whole blood, plasma, serum, urine, saliva, tears, sweat, fecal extract, DNA / RNA extract, antigen-containing solution, antibody, enzyme, protein, peptide, amino acid, hormone, organic molecule, inorganic molecule, biomarker, industrial pollutant, pathogen, virus, cell, cell culture extract or environmental sample.

[0031] In the present invention, the number of the switching columns in the chip may be in the range of 1 to 5.

[0032] In the present invention, the number of the reagent reservoirs in the chip may be in the range of 0 to 10. The reagent reservoir can accommodate one or more reagents necessary for identifying and immobilizing the analyte in the sample.

[0033] In the present invention, the number of the analysis regions in the chip may be in the range of 1 to 20. The analysis region is a stationary phase including one or more sections assembled integrally, the number of the sections in the analysis region is in the range of 1 to 20, and the form of each section is a particle, a porous membrane, a water-insoluble gel, or a colloid. The particles of the stationary phase may be made of plastic, silica, glass, aluminum oxide, organic polymer, inorganic polymer, or biodegradable polymer.

[0034] In the present invention, the pore membrane may be composed of plastic, fiber, polymer, polysaccharide, cellulose, paper, wood, biological construct, biomatrix, glass fiber, biodegradable polymer, or protein polymer, and the pore membrane is woven, non-woven or rolled particles.

[0035] In the present invention, the stationary phase may be functionalized by physical adsorption or covalent bonding with an identification reagent specific to the analyte in the sample. In the present invention, the stationary phase may be pre-formed into an appropriate shape and size for direct placement or distribution in the analysis region. And the analysis region may be structured within one level or across multiple levels of the laminate.

[0036] In the present invention, the number of the fluid absorption regions in the chip ranges from 1 to 5. The fluid absorption region may include a chamber having a fluid absorption pad. The fluid absorption pad may be made of hydrogel, particles, rolled particles, or a pore membrane, and the pore membrane may be composed of plastic, fiber, polymer, polysaccharide, cellulose, paper, wood, biological construct, biomatrix, glass fiber, biodegradable polymer, or protein polymer.

[0037] The present invention also provides an analysis method using the hybrid point-of-care chip of the present invention, comprising the steps of identifying an analyte in a sample with a reagent in a reagent reservoir, immobilizing the identified analyte in an analysis region, and determining the characteristics of the analyte by a detection method which is a magnetic, acoustic, radioactive, fluorescent, chemiluminescent detection method or a combination thereof. The reagent may include magnetic particles, fluorescent particles, chemiluminescent particles, radioactive particles, or a mixture thereof functionalized with an antibody, protein, DNA / RNA probe or chelating agent, and the antibody, protein, DNA / RNA probe or chelating agent labeled with magnetic, fluorescent, chemiluminescent or radioactive labels binds to and identifies the analyte in the sample, and the reagent is placed directly in the reagent reservoir or absorbed on a solid support and placed in the reagent reservoir, and in the analysis region, there is a section of a stationary phase functionalized with an identification reagent for immobilizing the identified analyte.

[0038] In the present invention, the hybrid point-of-care chip may have a plurality of analysis regions provided along an arc. The plurality of hybrid point-of-care chips are arranged to form an array and share the same sample introduction region.

[0039] In the present invention, the hybrid point-of-care chip is designed to automatically run a sample without external assistance functioning by a capillary.

[0040] In the present invention, the magnetic detection method may include superparamagnetic imaging, total accumulation of magnetic particles, excitation inductance, alternating current magnetic susceptibility measurement method, complementary metal oxide semiconductor (CMOS) alternating current magnetic susceptibility measurement method, Hall effect, magnetoresistance, giant magnetoresistance (GMR), colossal magnetoresistance (CMR), superconducting quantum interference device (SQUID), magnetic relaxation method, or magnetic resonance imaging (MRI) spin relaxation time.

[0041] The present invention further provides a superparamagnetic particle imaging analyzer, which includes a housing arranged along a horizontal axis and having an internal volume, a pair of permanent magnets arranged along the horizontal axis and adapted to the internal volume of the housing, and maintained such that the matching magnetic poles face each other to form a field-free region therebetween, a pair of excitation coils arranged between the pair of permanent magnets along the horizontal axis, each approaching the field-free region to generate an alternating current in the field-free region, and a pair of receiving coils arranged between the pair of excitation coils along the horizontal axis, each approaching the field-free region. A sample having an analyte labeled with superparamagnetic nanoparticles fixed in an analysis region is placed in the internal volume of the housing, and the superparamagnetic nanoparticles are excited to pass through the field-free region that transmits a paramagnetic response detected and transmitted by the pair of receiving coils for analysis.

[0042] In the present invention, the permanent magnet may be made of NdFeB. In one embodiment of the analyzer of the present invention, the housing is cylindrical and the internal volume is a cylindrical internal volume. The pair of permanent magnets are cylindrical and adapted to the cylindrical internal volume of the housing. The pair of excitation coils are alternating modulation magnetic field coils that form an alternating current in the field-free region to excite the superparamagnetic nanoparticles. The sample is at a hybrid point-of-care tip that moves in the same straight line in the cylindrical internal volume of the cylindrical housing.

[0043] In another embodiment of the analyzer of the present invention, the housing is a C-shaped frame with open sides. The pair of permanent magnets are linear and forced such that the anodes of each permanent magnet face each other to form a linear field-free region. The pair of excitation coils are a pair of Helmholtz coils for exciting the superparamagnetic nanoparticles. The sample containing an analyte labeled with fixed superparamagnetic nanoparticles moves from multiple directions and enters the linear field-free region in the housing.

[0044] In yet another further embodiment of the analyzer of the present invention, the pair of excitation coils are a pair of E-shaped sintered ferrite magnetic cores facing each other and separated by an insulator, and each leg of the E-shaped magnetic core has an electromagnetic coil winding, whereby a magnetic field is generated at two opposite poles of the E-shaped magnetic core short-circuited to form a gap. The pair of permanent magnets are forced to face each other anodically within the gap to generate a field-free region therein, and the sample moves through the gap and an accessible region for excitation and analysis.

[0045] In yet another further embodiment of the analyzer of the present invention, the analyzer is a single-sided analyzer without a permanent magnet and includes two concentrically arranged transmitting coils and receiving coils. The current directions of the two transmitting coils are opposite, forming magnetic field lines, and the field-free region is symmetric with the magnetic field lines. The sample having an analyte labeled with superparamagnetic nanoparticles is placed in the field-free region for excitation, and the paramagnetic response of the superparamagnetic nanoparticles is sensed and transmitted by the receiving coil.

[0046] In yet another further embodiment of the analyzer of the present invention, the analyzer is a Hall sensor analyzer, including a magnetically non-permeable hollow shaft, a cylindrical permanent magnet having a cylindrical interior on the inside and attached and supported by the magnetically non-permeable hollow shaft passing through it, a bias wire and a signal, and a Hall sensor disposed in the cylindrical interior of the permanent magnet and on the magnetically non-permeable hollow shaft. In this embodiment, the permanent magnet has theoretical magnetic field lines forming a magnetic force field, and the magnetic field lines leaving the cylindrical permanent magnet generate a zero region at the center of the cylinder and the center of the magnetic force field. The permanent magnet provides induction to the sample having an analyte labeled with superparamagnetic nanoparticles in the zero region. The Hall sensor is disposed in the zero region at the center of the cylindrical interior, senses and receives the paramagnetic response of the superparamagnetic nanoparticles, and the signal of the bias wire and the signal of the paramagnetic response are transmitted for signal processing.

[0047] Furthermore, the sample may be in a hybrid point-of-care chip including a plurality of analysis regions for the sample, and the analysis regions are arranged on the chip along an arc. In the present invention, a hybrid point-of-care chip having a configuration of a plurality of analysis regions along an arc operates particularly well in the single-sided analyzer and the hall sensor analyzer of the present invention. The present invention further provides a method of using a superparamagnetic particle imaging analyzer, including providing a sample having an analyte labeled with superparamagnetic nanoparticles, placing the sample in a field-free region of the analyzer, exciting the superparamagnetic nanoparticles in the field-free region to obtain a signal from the paramagnetic response of the superparamagnetic nanoparticles, sensing and transmitting the signal, and analyzing the signal to obtain characteristics of the analyte.

Brief Description of the Drawings

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DETAILED DESCRIPTION OF THE INVENTION

[0049] In the present invention, the term "format" or "assay format" refers to the collection of members, devices, and reagents that are necessary when performing an analysis method and that are used in the analysis method.

[0050] The analysis method of the present invention uses superparamagnetic particle imaging technology based on Langevin theory to detect and analyze the non-linear response of superparamagnetic nanoparticles (SPNP), mainly its harmonics, to a changing external magnetic field. In contrast to the measurement of free magnetic particles in a solution by magnetic particle imaging technology, the concentration of an analyte labeled with superparamagnetic nanoparticles fixed to a stationary phase is measured. As a result, the method of the present invention only needs to measure the total concentration of the analyte in the analysis region without the need to measure the distribution of the tracer. Since the analysis region is known, there is no need to reconstruct it to measure the concentration.

[0051] The method of the present invention uses superparamagnetic particle imaging technology that simultaneously and quantitatively assays multiple types of analytes in the analysis region of a chip by using the spatially encoded non-linear response of superparamagnetic nanoparticles to a changing external magnetic field.

[0052] In the present invention, superparamagnetic particle imaging technology is applied to in vitro diagnosis. The range of materials used for the production of superparamagnetic nanoparticles is much wider than those used for in vivo diagnosis, and may be Co, Fe, CoFe, Co alloy, ferrite, cobalt nitride, cobalt oxide, Co-Pd, Co-Pt, iron, iron alloy, Fe-Au, Fe-Cr, Fe-N, FeO, Fe-Pd, Fe-Pt, Fe-Zr-Nb-B, Mn-N, Nd-Fe-B, Nd-Fe-B-Nb-Cu, Ni, or Ni alloy.

[0053] In the present invention, particles having different shapes are used to provide different harmonics. Superparamagnetic nanoparticles can be manufactured in various geometric shapes including, but not limited to, spherical, elliptical, and flattened shapes. Some particles are coated with different paints to generate different harmonics for analysis.

[0054] In the present invention, since particles of different particle sizes provide different harmonics, the size of the superparamagnetic nanoparticles is between 1 and 1000 nm. In the present invention, fixed particles and free particles provide different harmonics. For this reason, a method for separating signals from the fixed superparamagnetic nanoparticles is provided. As a result, the analyte can be directly measured after the start of the assay without the need to wash the analysis region and remove unbound superparamagnetic nanoparticles. In the present invention, the analyte may be a substance that can be labeled with superparamagnetic nanoparticles and fixed to a stationary phase, including, but not limited to, organic molecules, biomolecules, peptides, polymers, amino acids, proteins, enzymes, antibodies, DNA, RNA, viruses, bacteria, cells, inorganic molecules, and drugs. In the present invention, measurable samples include, but are not limited to, body fluids such as whole blood, serum, plasma, urine, saliva, feces, tears, and sweat. They may also be liquid, solid, and gas extracts.

[0055] The analysis method of the present invention will be further described with reference to the drawings in the following embodiments. In the first embodiment of the analysis method of the present invention shown in FIG. 1, the assay format for performing the analysis includes one analysis region 10 consisting of one incubation chamber 11 for fixing the analyte 12 labeled with superparamagnetic nanoparticles 13.

[0056] When the assay format is arranged in the superparamagnetic particle imaging analyzer of the present invention, a changing excitation field is applied to the analysis region 10, and the superparamagnetic nanoparticles 13 in the analysis region 10 and the incubation chamber 11 respond to generate a harmonic signal in the receiving coil of the analyzer. The generated harmonic signal is directly proportional to the concentration of the superparamagnetic nanoparticles in the incubation chamber 11.

Number

[0057] Here, TIFF0007709777000002.tif1210 is the signal received from the partitioned studio 11, TIFF0007709777000003.tif1112 is the system function of the partitioned studio 11, and TIFF0007709777000004.tif118 is the concentration of superparamagnetic nanoparticles, or the concentration of the analyte in the partitioned studio 11. The system function is the response of a sample with a known concentration TIFF0007709777000005.tif1113 ( TIFF0007709777000006.tif1113) can be obtained by measuring:

Number

[0058] Once the system function is obtained, the concentration of the analyte in the format can be determined by measuring the signal in the receiving coil. In the second embodiment of the analysis method of the present invention shown in FIG. 2, the non-linear response of superparamagnetic nanoparticles to a changing external magnetic field is used to quantify multiple types of analytes in an assay format. The assay format includes one analysis region 10 consisting of one partitioned studio 11. In the partitioned studio 11, a total of Z analytes (the first analyte is shown as 12, and the Z-th analyte is shown as 12') are immobilized. The number Z is an integer from 1 to 20. Each analyte can be labeled with superparamagnetic nanoparticles (superparamagnetic nanoparticles 13 and 13' for labeling analytes 12 and 12' respectively are shown).

[0059] When the assay format is arranged in the superparamagnetic particle imaging analysis device of the present invention, a changing excitation field is applied to the analysis region 10 and the partitioned studio 11, and all the superparamagnetic nanoparticles in the partitioned studio 11 respond and generate a harmonic signal in the receiving coil of the analysis device. The concentration of the magnetic particles is proportional to the harmonic. The n-th harmonic measured in the partitioned studio 11 The amplitude of TIFF0007709777000008.tif1217 is the amplitude of the nth harmonic of all the particles in the compartment 11 The sum of TIFF0007709777000009.tif1315 (see Rauwerdink, A, "Simultaneous quantification of multiple magnetic nanoparticles", Nanotechnology, 21(45), 455101 (2010)), [Number] The amplitude of the nth harmonic of particle i TIFF0007709777000011.tif2124 is the concentration of particle i TIFF0007709777000012.tif1419 is proportional to [Number] TIFF0007709777000014.tif1214 is a constant that can be obtained by measuring the nth harmonic of a solution of particle i with a known concentration ( TIFF0007709777000015.tif1214). [Number]

[0060] Since the ratio of all the harmonics of the particles has no relation with the concentration (see Rauwerdink, A, 2010), the concentration of each particle in the compartment 11 can be solved by the following formula [Number] Here [Number] In the formula, harmonics are used from the lower order to the higher order. This is because any harmonic can be used for calculation, but the amplitude of the harmonic rapidly decreases as the order increases. By measuring the third to (2Z + 1)th harmonics in the partition chamber 11, the concentrations of all Z types of analytes are determined by the formula. At most, 1 (analysis region) · 1 (partition chamber) · Z (SPNP) analytes can be analyzed simultaneously. In this case, 1 (analysis region) · 1 (partition chamber) × 20 (SPNP) = 20 analytes.

[0061] In the third embodiment of the analysis method of the present invention shown in FIG. 3, the non-linear response of superparamagnetic nanoparticles in a spatially encoded partition chamber to a changing external magnetic field is used to quantify multiple types of analytes in an assay format. The assay format includes one analysis region 10 consisting of Y partition chambers (the first partition chamber 11 and the Yth partition chamber 11' are shown). Y is an integer in the range of 1 to 20. Each partition chamber immobilizes a different analyte (the first analyte 12 in the partition chamber 11 and the Yth analyte 12' in the partition chamber 11' are shown). Each type of analyte is labeled with superparamagnetic nanoparticles (the particle label 13 of the analyte 12 in the partition chamber 11 and the particle label 13' of the analyte 12' in the partition chamber 11' are shown).

[0062] When the assay format is arranged in the superparamagnetic particle imaging analyzer of the present invention, the convergence field coil generates a field-free space (FFS) of the size of the cell compartment 11. Since the structure of the analysis region 10 (including the number, shape, and position of the cell compartments) is known, the FFS can be moved based on the position of the cell compartment and applied to one cell compartment. When the FFS is applied to the cell compartment 11, the changing excitation field generated by the drive field coil is applied to the cell compartment. The superparamagnetic nanoparticles 13 in the field-free cell compartment 11 respond and generate a harmonic signal in the receiving coil of the analyzer. Analyze the analyte 12 fixed in the cell compartment 11 in the manner described in the first embodiment. After analyzing the cell compartment 11, move the FFS to the next cell compartment and analyze the analyte fixed in the next cell compartment. Repeat the same process until all Y cell compartments in the analysis region 10 are analyzed. In this way, all Y types of analytes (only the first analyte 12 and the Yth analyte 12' are shown) are all quantified. At most, 1 (analysis region) · Y (cell compartments) · 1 (SPNP) analytes can be analyzed simultaneously. In this case, 1 (analysis region) · 20 (cell compartments) · 1 (SPNP) = 20 analytes.

[0063] In the fourth embodiment of the analysis method of the present invention, the spatially encoded non-linear response of the SPNP to the changing external magnetic field is used to simultaneously quantify multiple types of analytes in the assay format. The assay format is the same as the third embodiment shown in FIG. 3, but a different analysis method is used in the configuration. When the assay format is arranged in the superparamagnetic particle imaging analyzer of the present invention, the convergence field coil generates a field-free point (FFP). (Since the structure of the analysis region 10 including the number of cell compartments (11, 11'...), their shapes, and their positions is known, the FFP moves within the analysis region 10 along a predetermined route. When a changing excitation field is applied to the FFP, it causes the magnetization of the SPNPs 13, 13'... inside the FFP. Since the FFP is spatially and temporally encoded, the signal generated from the FFP is also spatially and temporally encoded. The length of the induced magnetization is directly proportional to the concentration of the magnetic particles 13, 13'... The concentration of the magnetic particles ( TIFF0007709777000019.tif1827) and the position ( TIFF0007709777000020.tif1511) and the signal at that time TIFF0007709777000021.tif1511 and the relationship between them is

Number

Number

Number

[0064] In the fifth embodiment of the analysis method of the present invention shown in FIG. 4, the non-linear response of superparamagnetic nanoparticles in spatially encoded studios to a changing external magnetic field is used to quantify multiple types of analytes in an assay format. The assay format includes one analysis area 10 consisting of Y studios (Studio 11 in Zone 1 and Studio Y 11' are shown). Y is an integer in the range of 1 to 20. Each studio immobilizes Z types of analytes (the first analyte 12 and the Z-th analyte 12' are shown). Z is an integer in the range of 2 to 20. The Z types of analytes are each labeled with Z types of superparamagnetic nanoparticles (the first SPNP label 13 and the Z-th SPNP label 13' are shown).

[0065] When the assay format is arranged in the superparamagnetic particle imaging analysis device of the present invention, the converging field coil generates a field-free space (FFS) of the size of the studio. Since the structure of the analysis area 10 (including the number Y of studios 11, 11'..., their shapes, and their positions) is known, the FFS can be moved according to the position of the studio and applied to one studio at a time. When the FFS is applied to Studio 11 in Zone 1, the changing excitation field generated by the driving field coil is applied to the studio. The SPNP in the field-free studio 11 responds to generate a harmonic signal in the receiving coil.

[0066] The analytes immobilized in the first cell 11 are analyzed in the manner described in the second embodiment. Quantify the analytes 12, 12’... (the total number of analytes is Z) in the first cell 11. After analyzing the first cell 11, move the FFS to the second cell and analyze the cell, continuing until the Yth cell 11’ in the analysis region 10 is analyzed. The analysis method for each cell is the same as that for cell 11. At most, 1 (analysis region)·Y (cells)·Z (SPNPs) analytes can be analyzed simultaneously. In this case, 1 (analysis region)·20 (cells)·20 (SPNPs) = 400 analytes.

[0067] In the sixth embodiment of the analysis method of the present invention, the spatially encoded non-linear response of SPNPs to a changing external magnetic field is used to simultaneously quantify multiple analytes in an assay format. This embodiment is another method for analyzing the assay format shown in FIG. 4.

[0068] When the assay format is arranged in the superparamagnetic particle imaging analyzer of the present invention, the convergent field coil generates a field-free point (FFP). Since the structure of the analysis region 10 (including the number of cells, their shapes, and their positions) is known, the FFP moves within the analysis region along a predetermined route. When a changing excitation field is applied to the FFP, it causes the magnetization of the SPNPs inside the FFP. Since the FFP is spatially and temporally encoded, the signal generated from the FFP is also spatially and temporally encoded. Signals from the same cell are processed together.

[0069] The amplitude of the nth harmonic measured in the cell TIFF0007709777000031.tif1216 is the amplitude of the nth harmonic of all the particles in the cell as follows The sum of TIFF0007709777000032.tif1616 (Rauwerdink, A, "Simultaneous Quantification of Multiple Magnetic Nanoparticles", "Nanotechnology", 21(45), 455101 (2010)):

Number

[0070] The n-th amplitude measured in the experiment ( TIFF0007709777000034.tif1613) and by using the method described in the second embodiment, the amplitude of each particle i TIFF0007709777000035.tif1525 is determined. The amplitude of the n-th harmonic of particle i in the cell is the total concentration of particle i generated by FFP TIFF0007709777000036.tif1529 is proportional to

Number

Number

Number

[0071] In the seventh embodiment of the analysis method of the present invention shown in FIG. 5, the non-linear response of SPNP to a changing external magnetic field is used to simultaneously quantify multiple types of analytes in an assay format. The assay format includes X analysis regions (the first analysis region 10 and the Xth analysis region 10' are shown), and X is an integer in the range of 1 to 20. Each analysis region consists of one cell (the first cell 11 in the first analysis region 10 and the Xth cell 11' in the Xth analysis region 10' are shown). Each cell immobilizes different analytes (the first analyte 12 in cell 11 and the Xth analyte 12' in the Xth cell 11' are shown) labeled with SPNP (the first SPNP 13 in the first cell 11 and the Xth SPNP 13' in the Xth cell 11' are shown). The seventh embodiment of the analysis method is the same as the first embodiment, and the difference is that it is repeatedly executed X times. At most, X (analysis regions)·1 (cell)·1 (SPNP) analytes can be analyzed simultaneously, in which case 20 (analysis regions)·1 (cell)×1 (SPNP)=20 analytes.

[0072] In the eighth embodiment of the analysis method of the present invention shown in FIG. 6, the non-linear response of SPNP to a changing external magnetic field is used to simultaneously quantify multiple types of analytes in an assay format. The assay format consists of X analysis regions (the first analysis region 10 and the Xth analysis region 10' are shown), and X is an integer within the range of 1 to 20. Each analysis region consists of one confinement chamber (the confinement chamber 11 in the first analysis region 10 and the confinement chamber 11' in the Xth analysis region 10' are shown). Each confinement chamber immobilizes Z types of analytes (both the first analyte 12 and the Zth analyte 12' in the confinement chambers 11 and 11' are shown), and Z is an integer within the range of 1 to 20. Each type of analyte in one confinement chamber is labeled with a different SPNP (the first labeled SPNP 13 used for the first analyte and the Zth labeled SPNP 13' used for the Zth analyte are shown).

[0073] The analysis method used in the eighth embodiment is the same as that in the second embodiment, and the difference is that it is repeatedly executed X times. At most, X (analysis regions) · 1 (confinement chamber) · 1 (SPNP) analytes can be analyzed simultaneously. In this case, 20 (analysis regions) · 1 (confinement chamber) × 1 (SPNP) = 20 analytes.

[0074] In the ninth embodiment of the analysis method of the present invention shown in FIG. 7, the non-linear response of SPNPs in a spatially encoded compartment to a changing external magnetic field is used to quantify multiple analytes in an assay format. The assay format includes X analysis regions (shown as the first analysis region 10 and the Xth analysis region 10'), and X is an integer in the range of 1 to 20. Each analysis region consists of Y compartments (shown as the first compartment 11 and the Yth compartment 11' in the first analysis region 10, the first compartment 11a and the Yth compartment 11a' in the Xth analysis region 10'), and Y is an integer in the range of 1 to 20. Each compartment immobilizes different analytes (shown as the first analyte 12 in the first compartment 11 of the analysis region 10 and the Yth analyte 12' in the Yth compartment 11' of the analysis region 10, and the first analyte 12a in the first compartment 11a of the first analysis region 10' and the Yth analyte 12a' in the Yth compartment 11a' of the Xth analysis region 10') labeled with SPNPs (shown as the SPNP label 13 used for the analyte 12 in the first compartment 11 of the analysis region 10, the SPNP label 13' used for the analyte 12' in the Yth compartment 11' of the analysis region 10, the SPNP label 13a used for the analyte 12a in the first compartment 11a of the first analysis region 10', and the SPNP label 13a' used for the analyte 12a' in the Yth compartment 11a' of the Xth analysis region 10').

[0075] The analysis method used in the ninth embodiment is the same as that in the third embodiment, and the difference is that it is repeatedly executed X times. At most, X (analysis regions) · Y (compartments) · 1 (SPNP) analytes can be analyzed simultaneously, in which case 20 (analysis regions) · 20 (compartments) · 1 (SPNP) = 400 analytes.

[0076] In the tenth embodiment of the present invention, the spatially encoded non-linear response of SPNPs to a changing external magnetic field is used to simultaneously quantify multiple analytes in an assay format. The assay format is the same as that shown in FIG. 7 in the ninth embodiment, but this embodiment is a different method of analyzing the composition. The analysis method used in the 10th embodiment is the same as that in the 4th embodiment, and the difference is that it is repeatedly executed X times. At most, X (analysis regions) · Y (compartments) · 1 (SPNP) analytes can be analyzed simultaneously. In this case, 20 (analysis regions) · 20 (compartments) × 1 (SPNP) = 400 analytes.

[0077] In the 11th embodiment of the analysis method of the present invention shown in FIG. 8, the non-linear response of SPNPs in spatially encoded compartments to a changing external magnetic field is used to quantify multiple types of analytes in an assay format. The assay format includes X analysis regions (the first analysis region 10 and the Xth analysis region 10' are shown), and X is an integer in the range of 1 to 20. Each analysis region consists of Y compartments (the first compartment 11 and the Yth compartment 11' in the first analysis region 10, the first compartment 11a and the Yth compartment 11a' in the Yth analysis region 10' are shown), and Y is an integer in the range of 1 to 20. Each compartment immobilizes Z types of analytes (the first analyte 12 and the Zth analyte 12 in the compartment 11 of the first analysis region 10, the first analyte 12' and the Zth analyte 12z' in the compartment 11', the first analyte 12a and the Zth analyte 12b in the compartment 11a of the Xth analysis region 10', the first analyte 12a' and the Zth analyte 12b' in the compartment 11a' are shown), and Z may be an integer in the range of 1 to 20. Each analyte is labeled with a different SPNP (SPNP13 and 13z used for the analytes 12 and 12z in the compartment 11 of the analysis region 10 respectively, SPNP13' and 13z' used for the analytes 12' and 12z' in the compartment 11', and SPNP13a and 13b used for the analytes 12a and 12b in the compartment 11a of the analysis region 10' respectively, SPNP S 13a' and 13b' are shown).

[0078] The analysis method used in the 11th embodiment is the same as that in the 3rd embodiment, and the difference is that it is repeatedly executed X times. At most, X (analysis regions) · Y (compartments) · Z (SPNP) analytes can be analyzed simultaneously. In this case, it is 20 (analysis regions) · 20 (compartments) · 20 (SPNP) = 8,000 analytes.

[0079] In the 12th embodiment of the analysis method of the present invention, the spatially encoded non-linear response of SPNP to a changing external magnetic field is used to simultaneously quantify multiple types of analytes in an assay format. The assay format is the same as that shown in the 11th embodiment in FIG. 8, but this embodiment is a different method of analyzing the composition. The analysis method used in this embodiment is the same as that in the 6th embodiment, and the difference is that it is repeatedly executed X times. At most, X (analysis regions) · Y (compartments) · X (SPNP) analytes can be analyzed simultaneously. In this case, it is 20 (analysis regions) · 20 (compartments) · 20 (SPNP) = 8,000 analytes.

[0080] As shown in these embodiments, the analysis method of the present invention uses superparamagnetic particle imaging technology to quantitatively measure analytes immobilized on a stationary phase. The analysis method measures the change in the amount of magnetic flux caused by the magnetization of magnetic labels to simultaneously measure the concentrations of multiple types of magnetic label analytes in an analysis sample immobilized on a three-dimensional stationary phase in a multiplex assay.

[0081] In the present invention, the superparamagnetic particle imaging technology can cooperate well with the three-dimensional hybrid point-of-care chip of the present invention and can also be applied to other assay formats such as lateral flow tests, microfluidic assays, "lab-on-a-chip" devices, and independent biosensors. The demand for high-throughput and cost-effective detection platforms for chemical and biological reagents has not yet been met. These platforms can be applied to many analytical and diagnostic applications, including screening of chemical libraries used in drug development, toxicity studies, point-of-care diagnostics, and environmental monitoring. Ordinary sensors generally interpret biochemical phenomena using chemical, optical, spectroscopic, electrical impedance, or mass-based detection. Superparamagnetic particle imaging uses magnetic labels or magnetic nanoparticles that differ in size, shape, and composition.

[0082] The analytical method of the present invention using superparamagnetic particle imaging technology represents a new paradigm for performing direct and accurate detection according to many medical diagnostic assays and drug monitoring tests. The superparamagnetic particle imaging technology not only extends the effectiveness and sensitivity of magnetic-based diagnostic assays but also overcomes the main drawbacks of other magnetic-based assays, that is, simultaneously detecting multiple analytes in a single measurement and having the advantages of optical-based assays. The present invention enables the development of analytical methods that require rapid and sensitive devices without the associated costs of expensive modern analytical laboratories. Currently, tests such as DNA / RNA analysis, environmental testing, detection of chemical and biological warfare, screening for drug abuse, and quality detection of food supplies can be improved by utilizing superparamagnetic particle imaging technology and producing small portable handheld devices with biodegradable and disposable assay chips.

[0083] The present invention also provides a hybrid point-of-care chip that can be used in combination with the analytical method based on the superparamagnetic nanoparticles of the present invention.

[0084] The hybrid point-of-care chip of the present invention comprises a sample introduction region into which a sample is introduced, a switching column that guides the flow of the sample to different levels of the chip, an analysis region where an analyte is captured, concentrated, and cleaned, a waste chamber that collects excess sample and cleaning liquid, and microchannels through which different elements on the chip communicate with each other. In this design, a liquid sample can automatically flow from the sample introduction region to the absorption chamber by capillary force. It may also comprise a sample dispersion channel, a reagent storage tank, a mixing port, and a pump.

[0085] The analysis region of the hybrid point-of-care chip of the present invention houses a three-dimensional multi-compartment stationary phase loaded with an analyte capture material. The sample fluid flows in a three-dimensional route to and through the stationary phase of the analysis region, which is itself a three-dimensional region. Since their sizes and positions are known, the concentration of the analyte can be measured without the need for reconstruction.

[0086] The format of the hybrid point-of-care of the present invention is composed of a stationary phase in the flow path of a liquid sample. The stationary phase consists of materials directly dispensed into pores, grown by biological methods, or preforms of materials made of self-assembled plastics and polymers, silica, or equivalents (such as organic / inorganic structures). Examples include stromatolites biologically produced in nature, colloidal minerals such as agate and opal gel formed inorganically in hot springs, and honeycomb-like walls and compartments in bamboo shoots or banana tree stems. All natural structures are controlled by the design of DNA / RNA biological activity and are all applicable to the present invention.

[0087] Block or cast structures can be used in the manufacture of the structures of the present invention, and these structures are placed in the gaps formed within the preferred laminated structure. In the present invention, columns, capture regions, or analysis regions, which are actually porous materials with a very large surface area, allow the flow of sample liquid through the functional "stationary phase". The analysis regions are functionalized by sandwich or competitive immunoassays or similar chemical methods of DNA / RNA biochips known in the art. Analytes of interest are captured in these regions by using antibodies or antigens of immunoassay methods, or small DNA probes labeled with magnetic nanoparticles. Alternatively, in competitive assays, the analyte competes for binding sites on the surface structure of the functionalized stationary phase. The entire introduced sample flows through the respective vertical routes of the linear region or cylinder from the three-dimensional channels formed by the laminated combination of hydrophilic / hydrophobic films.

[0088] In a preferred form, the column gaps are formed by multiple laminated films which may be paper, polymer, plastic, or metallized plastic or metal materials. These films are available from Adhesive Research, 3M, DuPont Polymers, Pall, Coveme and Tesa. The films have different coatings and properties, for example, some are hydrophilic or hydrophobic. Their thickness may be between.001” and.020”, preferably between.002” and.015”, and most preferably between.002” and.009”. Depending on the requirements of the magnetic reading system, all of these laminations can be converted into the required format. The versions are the multiple linear format with a dispersion sample introduction device, capture or analysis regions, and a final storage core, the faster cylindrical format and the partial disk.

[0089] Each column is in liquid communication with each other, and the liquid flows from the bottom to the top of the column, then from the top to the bottom of the adjacent column, and then from the bottom to the top of the next adjacent column, and finally (depending on the number of analysis regions) to the core member. Each or all of the channels and columns may block the transported air to form air pockets, which may block the flow of the liquid. If the blocked air becomes a problem, it can be solved by providing small vents at the top of each column. Through these vents, the blocked air can leak from the fluid channels and columns, thereby preventing blocking of the liquid flow, but the vents are small enough to prevent liquid leakage. These vents may be provided with a porous plastic that closes when in contact with the liquid or is manufactured small enough so that the surface tension of the liquid sample prevents liquid leakage, which is known in the art. This size may be in the range of 0.1 to 10 microns, and preferably about 5 microns. As another method, the occurrence of this situation can be prevented by ensuring that the capillary force is sufficient to move the transported air before the fluid flows, which is achieved by selecting the material or coating film in the channel.

[0090] Between the sample introduction region and the communication channels that control the flow, velocity and three-dimensional level of the required analysis region, there is a small column that switches the flow of the liquid sample flowing into the fluid channel to the required first analysis column. The "switching column" in the present invention may or may not have a filter, a blocking agent or a complex (the complex is a magnetically labeled identification element, for example, an antibody or protein that can be captured and measured in the analysis column in a sandwich assay). With these loaded reagents, it is not necessary to pre-mix the sample with the complex before applying it to the sample introduction region. Therefore, the storage period is extended, the sensitivity is improved, and the coefficient of variation of the assay is reduced.

[0091] The sample introduction device in the present invention may be provided with a device for separating red blood cells. Since the device has the ability to change the shape of red blood cells and / or the ability to release internal fluid, which may cause interference with the capture chemicals in the analysis region during the hemolysis process, column blocking is prevented.

[0092] Each column is in liquid communication with each other. Whether in the same plane or intentionally separating the analysis regions by separating the sample flow into different regions, chemical interference can be avoided or the analysis of the measurements obtained can be made more accurate. As an example, when the analyte in the sample to be measured has a higher concentration compared to the low-concentration analyte from the same sample, the measured result will always obscure the reading value of the low-concentration analyte. By changing the sample flow to different three-dimensional levels of sample flow, any interference from these two types of analytes can be minimized.

[0093] The hybrid point-of-care chip of the present invention has a three-dimensional multi-level structure with a stationary-phase analysis region. It is manufactured by integrally laminating layers of film. Before lamination, certain characteristics of the hybrid point-of-care chip are combined with the film. Other characteristics are created after lamination. The layer through which the sample flows is called a level. Each chip may have 1 to 20 levels, preferably 1 to 10 levels.

[0094] The hybrid point-of-care chip consists of 1 to 5 sample introduction regions, 1 to 5 switching columns, 0 to 10 reagent reservoirs, 1 to 20 analysis regions, and 1 to 5 liquid absorption sites. The sample introduction regions, reagent reservoirs, analysis regions, and fluid absorption regions are connected by microfluidic channels and through-holes (channels that are perpendicular and connected to each other), thereby enabling the sample to be split and guided to different levels. The hybrid point-of-care chip may or may not have a liquid driving mechanism such as a diaphragm pump connected to the sample introduction region.

[0095] The film used for the hybrid point-of-care chip is manufactured from plastics, adhesives, paper, wood, fibers, silicon, polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), glass fibers, cellulose, polysaccharides, protein polymers, and rolled particles. The surface of the film can be modified to have the required properties including hydrophobicity and hydrophilicity. Each sample introduction region may or may not have a red blood cell separation device. It can further contain reagents necessary for identifying and immobilizing the target analyte in the sample. The sample introduction region can accommodate a sample of 1 to 200 microliters.

[0096] The sample may be whole blood, plasma, serum, urine, saliva, tears, sweat, fecal extract, DNA / RNA extract, antigen-containing solution, antibody, enzyme, protein, peptide, amino acid, hormone, organic and inorganic molecules, biomarker, industrial contaminants, pathogen, virus, cell, cell culture extract, and environmental sample. The reagent reservoir contains reagents necessary for identifying and immobilizing the target analyte, including magnetic particles, fluorescent particles, chemiluminescent particles, and radioactive particles, antibodies, proteins, DNA / RNA probes, or magnetic particles, fluorescent particles, chemiluminescent particles, and radioactive particles functionalized with chelating agents, fluorescent, chemiluminescent, or radioactively labeled antibodies, proteins, DNA / RNA probes, and chelating agents. The reagent reservoir may be placed in the same or different sample routes. The reagent can be placed directly in the reagent reservoir or adsorbed onto a solid support. Each analysis area is formed by combining 1 to 20 sections of the stationary phase. Different sections of the analysis area may consist of the same or different stationary phases. The stationary phase is made of particles manufactured from plastic, silica, glass, aluminum oxide, organic polymers, inorganic polymers, and biodegradable polymers, plastic, fibers, polymers, polysaccharides, cellulose, paper, wood, biological constructs, biomatrices, glass fibers, biodegradable polymers, and pore membranes manufactured from protein polymers, water-insoluble gels, and colloids. The membrane may be woven, non-woven, or rolled particles. The stationary phase is functionalized by physical adsorption or covalent bonding with a discrimination reagent specific to the analyte of interest. The stationary phase may be preformed in the shape and size of the analysis area and then placed in the area, or directly dispensed into the area. The analysis area may be structured within one level or across multiple levels.

[0097] Each absorption site consists of a chamber having a fluid absorption pad. The pad is made of particles structured from plastic, fibers, polymers, polysaccharides, cellulose, paper, wood, biological constructs, biomatrices, glass fibers, biodegradable polymers, and pore membranes manufactured from protein polymers, hydrogels, and rolled particles.

[0098] The laminated material is biodegradable and can be manufactured from multiple types of polymers, fibers, celluloid, and paper that can be rapidly decomposed without causing adverse effects on the environment. Examples include those without a conventional plastic cassette to support the test structure, where the support is integrated into the test design of the laminate and is self-supporting. As shown in FIGS. 9A to 9D, the structure of the hybrid point-of-care chip of the present invention will be further described in the following embodiments. In one embodiment of the hybrid point-of-care chip 20 of the present invention shown in FIG. 9A, the three-dimensional hybrid point-of-care chip 20 of the present invention includes a sample introduction region in the form of one sample introduction region 21, three analysis regions 210a, 210b, and 210c, a switching column 24, and a fluid absorption region in the form of an absorption chamber 26 having a vent 28. Subsequently, the sample introduction region 21 and the switching column 24 are connected by a microchannel 23a, the switching column 24 and the first analysis region 210a are connected by a microchannel 23b, the first analysis region 210a and the second analysis region 210b are connected by a microchannel 23c, and the second analysis region 210b and the third analysis region 210c are connected by a microchannel 23d. The first analysis region 210a is a multi-chamber analysis region having a first chamber 211a, a second chamber 211b, and a third chamber 211c. The arrows in FIG. 9A indicate the flow of the sample in the chip.

[0099] Reagents 22 required for the chemical and biological reactions of the assays occurring in the chip are pre-filled in the sample introduction region 21. The amount of the analysis sample is 1 to 500 microliters, usually 1 to 100 microliters, and preferably 5 to 10 microliters. The sample includes, but is not limited to, serum, plasma, saliva, sweat, tears, sputum, urine, and surface wipes extracted with a cotton swab, and further semi-solids such as cell cultures, feces, whole blood (when a red blood cell separation mechanism is incorporated into the structure of the sample introduction region 21 for whole blood analysis), and may or may not be pretreated.

[0100] A sample is added to the sample introduction region 21 and rapidly dissolved in the reagent 22. The sample is mixed with the reagent 22, passes through the microchannel 23a, and enters the switching column 24. The function of the switching column 24 is to guide the sample to different levels. In this embodiment, the switching column 24 guides the sample to a higher level in the analysis region 210a of the chip 20 via the microchannel 23b (it is also possible to switch the sample flow to a lower level if necessary). In the multi-level design of the present invention, the switching column 24 can switch the sample to any level as needed. The packing material 25 in the switching column 24 further functions as a filter that removes solid impurities in the sample and transports the cleaned sample to the analysis region. After passing through the switching column 24, the sample passes through the microchannel 23b and enters the first analysis region 210a.

[0101] Generally, the analysis region in the hybrid point-of-care chip of the present invention may be manufactured to have 1 to 20 compartments, usually 1 to 10 compartments, and preferably 1 to 5 compartments. In this embodiment, there are three compartments 211a, 211b, and 211c. Each compartment may be manufactured to have the same or different stationary phases depending on the assay. The stationary phase is made of particles, plastics, fibers, polymers, polysaccharides, cellulose, paper, wood, biological constructs, biomatrices, glass fibers, biodegradable polymers, and protein polymers made of plastics, silica, glass, aluminum oxide, organic polymers, inorganic polymers, and biodegradable polymers. The membrane may be woven, non-woven, or rolled particles. The stationary phase is functionalized by physical adsorption or covalent bonding with a discrimination reagent specific to the analyte of interest. The stationary phase may be placed in the subsequent region preformed by the shape and size of the analysis region, or may be directly dispensed into the region. The analysis region may be structured within one level or across multiple levels. Each compartment can immobilize different analytes.

[0102] When the sample passes through the first analysis region 210a, the analyte of interest is captured. Other samples leave the first analysis region 210a. In this process, the analytes are separated by a sectional chamber having a selective stationary phase. The analytes are also concentrated in the sectional chamber and cleaned by the passing sample liquid. The multi-sectional chamber design of the present invention enables multiplexing.

[0103] After passing through the microchannel 23c, the sample enters the second analysis region 210b. The analysis region 210b may have the same structure as the first analysis region 210a, including the number of sectional chambers, the material of the stationary phase, and the number of different capture materials loaded in the sectional chamber. After passing through the second analysis region 210b, a second set of analytes are separated and captured. The sample continues to move through the microchannel 23d and enters the third analysis region 210c. A third set of analytes are separated in the third analysis region 210c. The excess sample fluid and excess reagents are absorbed by the fluid absorption pad 27 in the absorption chamber 26. The absorption pad 27 is made of a porous membrane, hydrogel, particles, or rolled particles structured with plastic, fiber, polymer, polysaccharide, cellulose, paper, wood, biological constructs, bio scaffolds, glass fiber, biodegradable polymer, and protein polymer.

[0104] In the present invention, the hybrid point-of-care chip may be manufactured to have 1 to 20 analysis regions, usually 1 to 10 analysis regions, and preferably 1 to 6 analysis regions. FIG. 9B shows the structure of the hybrid point-of-care chip of the present invention. The hybrid point-of-care chip 20 of the present invention is a three-dimensional multi-level structure having stationary phase analysis regions 210a, 210b, and 210c. In this embodiment, the chip 20 is formed by integrally laminating layers of film (including the bottom layer 201).

[0105] In the present invention, before lamination, certain features of the hybrid point-of-care chip are combined with the film. Other features are created after lamination. The layer through which the sample flows is called a level. Each chip may have 1 to 20 levels, preferably 1 to 10 levels.

[0106] In the present invention, the hybrid point-of-care chip comprises 1 to 5 sample introduction regions, 0 to 10 reagent reservoirs, 1 to 20 analysis regions, and 1 to 5 fluid absorption regions. The sample introduction regions, reagent reservoirs, analysis regions, and fluid absorption regions are connected by microfluidic channels and through-holes (channels that are perpendicular and connected to each other), whereby the sample can be split and guided to different levels. The hybrid point-of-care chip may or may not have a liquid driving mechanism. The film used for the hybrid point-of-care chip is made of plastic, adhesive, paper, wood, fiber, silicon, polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), glass fiber, cellulose, polysaccharide, protein polymer, rolled particles. The surface of the film can be modified to have the required properties such as hydrophobicity and hydrophilicity.

[0107] In the structure of the chip 20 shown in FIG. 9B, the chip 20 consists of five layers of film, namely, a bottom layer 201, a bottom channel layer 202, an intermediate layer 203, a top channel layer 204, and a top layer 205. The thickness of the film is adjusted based on the design requirements. The microchannels are formed by the channel layers 202 and 204. When assembling the layers 201, 202, and 203, a microchannel 23a that connects the sample introduction region 21 to the switching column 24, a microchannel 23c that connects the first analysis region 210a to the second analysis region 210b, and a microchannel 23e that connects the third analysis region 210c to the absorption chamber 26 are formed. After adding the layers 204 and 205, a microchannel 23b that connects the switching column 24 to the first analysis region 210a and a microchannel 23d that connects the second analysis region 210b to the third analysis region 210c are formed. At the same time, the switching column 24 and the analysis region are structured. With this structure, it becomes possible for the sample to move horizontally between positions through the microchannels and vertically between levels through the switching column and the analysis region. Before adding the layers 204 and 205 to complete the structure of the chip, it is necessary to place the stationary phase material in the switching column and the analysis region by distributing particles or placing a preformed membrane. The chip 20 shown in FIG. 9B is for illustrative purposes only. Additional functions, such as adding more analysis regions or reagent reservoirs, can be added to the chip. The number of levels can be increased by adding more film layers.

[0108] Figure 9C shows another embodiment of the hybrid point-of-care chip of the present invention, which utilizes the flexibility that the hybrid point-of-care chip of the present invention communicates between positions using microchannels. Therefore, the features on the chip do not need to be arranged linearly, which is different from the lateral flow design. The structure of the chip 20 in this embodiment is the same as that described in FIGS. 9A and 9B, and the difference is that the analysis regions 210a, 210b, 210c, 210d and 210e are provided along an arc. This arrangement enables the chip 20 or the detector to move in a circular motion, thereby significantly reducing the design and manufacturing costs.

[0109] Another embodiment of the hybrid point-of-care chip of the present invention is in the form of a chip array shown in FIG. 9D, where the chip 20 is in the form of a chip array having eight individual chips 20a, 20b, 20c, 20d, 20e, 20f, 20g and 20h. These individual structures are the same as those described in FIGS. 9A and 9B, and the difference is that each layer of the film has multiple sets of chip features, and all the chips on the same disk mix one sample introduction region 21 at the centralized positions shown in FIG. 9D. In this embodiment, each individual chip has three sorted analysis regions 210a, 210b and 210c in sequence. This arrangement enables testing of multiple sets of analytes from a single sample. Each individual chip is completely independent of the other chips in the chip array 20. Therefore, multiple chemical and / or biocompatibility analyses can be performed in one run.

[0110] Depending on the structure, the hybrid point-of-care chip of the present invention can be used together with different types of detection systems. Preferably, the detection method used for the hybrid point-of-care chip is a detection method based on magnetism, acoustics or radioactivity because it does not depend on the transparency of the analysis region. They can measure signals from not only the surface but also the entire three-dimensional stationary phase object.

[0111] A more preferred method is magnetic-based detection, including superparamagnetic particle imaging (SPI), magnetic particle imaging (MPI), magnetic particle spectroscopy (MPS), total accumulation of magnetic particles, excitation inductance, alternating current magnetic susceptibility measurement, CMOS alternating current magnetic susceptibility measurement, Hall effect, magnetoresistance, giant magnetoresistance (GMR), colossal magnetoresistance (CMR), superconducting quantum interference device (SQUID), magnetic relaxation method, magnetic resonance imaging (MRI) spin relaxation time. More preferred magnetic detection methods are superparamagnetic particle imaging (SPI), magnetic particle imaging (MPI), magnetic particle spectroscopy (MPS), total accumulation of magnetic particles, excitation inductance, Hall effect.

[0112] The most preferred magnetic detection methods are superparamagnetic particle imaging, magnetic particle spectroscopy (MPS), total accumulation of magnetic particles, excitation inductance. Furthermore, when the stationary phase in the analysis region is made of a light-transmissive material (glass, agar, transparent plastic), optical-based detection methods including fluorescence, UV-Vis, laser, chemiluminescence can be used.

[0113] The hybrid point-of-care chip of the present invention has advantages such as the versatility and flexibility of a microfluidic device, the low cost and simplicity of a lateral flow test, the low background and high sensitivity of an Elisa assay, and can be multiplexed. The difference between the hybrid point-of-care chip and other designs is that the multi-level and multi-compartment analysis region is filled with a stationary phase. The multi-compartment structure enables stacking together multiple compartments filled with different capture materials, realizing multiplexing in an additional dimension. The stationary phase in the analysis region immobilizes the analyte labeled with an appropriate reporter, for example, magnetic particles, radioactive materials, acoustically active materials. Due to its very large surface area, the stationary phase concentrates the analyte in the sample, significantly improving the sensitivity. Due to its heterogeneous nature, interfering substances are washed away, and it has a much lower background than other assay designs.

[0114] The hybrid point-of-care chip of the present invention solves all problems related to the lateral flow format. By directly connecting the sample introduction region to the analysis region in the microfluidic channel, all intersections existing in the lateral flow format are eliminated. Since the sample is sent directly to the analysis region, the changes caused by the inconsistent film between the sample introduction region and the test line in the lateral flow format are significantly reduced. Since the microfluidic channel is sealed, there is no leakage problem caused by the open edge of the lateral flow strip. In the lateral flow format, the capture material is banded on the film. Due to changes in the film, banding conditions, and banding devices, the analysis region is highly inconsistent. In the hybrid point-of-care format, the analysis region is filled with a three-dimensional stationary phase made of a highly organized material. The variation between stationary phases is less than 0.1%. The hybrid point-of-care format overcomes all problems of the lateral flow format and retains the advantages of ease of use and low cost offered by the lateral flow format.

[0115] The hybrid point-of-care format of the present invention further provides answers to these problems in the format of microfluidics. By disposing a penetrable three-dimensional stationary phase in the route of the microchannel, only the analyte is captured and accumulated, and all other substances are allowed to pass through. In this process, the analyte is concentrated, thereby significantly improving the sensitivity. At the same time, all substances unrelated to the analyte are removed, significantly reducing the noise. This design eliminates the need for sample pretreatment and the need to add a sample purification process to the device. A multi-chamber stationary phase was designed using microfluidics. Each chamber can immobilize different analytes. By combining the hybrid point-of-care format with superparamagnetic particle imaging technology, simultaneous detection of multiple analytes can be completed. The multi-level design of the hybrid point-of-care format enables different analytes that are chemically and / or biologically incompatible to be guided to different routes and / or different levels for simultaneous analysis. Since the hybrid point-of-care format is manufactured with a laminate, it is suitable for large-scale automated production and cost reduction compared to the lateral flow format.

[0116] Furthermore, depending on the materials used, the surface area of the chemical and biological processes occurring in the stationary phase of the hybrid point-of-care format is 300 to 10,000 times that of the general ELISA assay format, which extremely improves the sensitivity, eliminates the need for reagent treatment and operation, and does not require a long waiting time.

[0117] The hybrid point-of-care of the present invention is a novel and innovative format that uses small amounts of biological, environmental, or chemical fluids to complete highly sensitive, quantitative, and rapid diagnostic assays. It can compete with binding sites that label the analyte of interest or measure the reduction of labels using various solutions, colloids, or suspended particles (including magnetic particles). It uses a stationary phase and a laminate (including laminates made of biodegradable materials) and has a three-dimensional heterogeneous design and function without using conventional medical plastic cassettes. The typical sample volume of serum, plasma, saliva, sweat, tears, sputum, urine, surface wipes extracted with cotton swabs, and even semi-solids (such as cell cultures and feces) is 10 microliters or less. Furthermore, integrated red blood cell separation technology and devices can be used to separate and prevent minimal hemolysis, and in the hybrid point-of-care format, not even a single drop of whole blood is required for analysis, and the analysis time is less than 5 minutes.

[0118] The present invention realizes a rapid development of quantitative analysis with a single-digit execution time, whereas the usual lateral flow tests, in contrast, have an execution time of 20 minutes or more and require any necessary normalization. The significant reduction in execution time is due to the reduction in sample volume and the minimization of the interaction between surface kinetics and fluid dynamics.

[0119] The format and chip of the hybrid point-of-care of the present invention enable the coefficient of variation of a normal assay to be 10% or less at the limit of detection (LOD) (generally at the picogram level).

[0120] The present invention further provides a superparamagnetic particle imaging analyzer designed based on superparamagnetic particle imaging technology, which creates a field-free point (FFP) and / or a field-free line (FFL), spatially and temporally encodes the signals generated by superparamagnetic nanoparticles, records the signals, and is used to convert the signals into the concentration of the analyte over the entire chip in a single measurement. According to one aspect of the present invention, another superparamagnetic particle imaging analyzer is used to create a field-free space (FFS) covering each cell chamber, record the total signal generated by each cell chamber, and convert the signal into the concentration of the analyte over the entire chip in a single measurement.

[0121] According to one aspect of the present invention, another superparamagnetic particle imaging analyzer is used to simultaneously analyze multiple types of analytes labeled with different SPNPs.

[0122] In the first embodiment of the superparamagnetic particle imaging analyzer of the present invention shown in FIG. 10A, the apparatus is a collinear superparamagnetic particle imaging analyzer. The analyzer 30 includes a housing 31, a pair of permanent magnets 32 and 32', a pair of excitation coils 35 and 35', and a pair of receiving coils 36 and 36'.

[0123] In this embodiment, the housing 31 is in the shape of a cylinder 310, and inside the cylinder 310, each permanent magnet is cylindrical with an inner cylindrical volume 33. The permanent magnets 32 and 32' are made of a rare earth alloy (e.g., NdFeB) and are mechanically forced by a fixing device and clamp 34 to face each other with their corresponding magnetic poles (i.e., the north pole of the first magnet faces the north pole of the second magnet, or the south pole of the first magnet faces the south pole of the second magnet). In this process, a field-free point (FFP) or field-free region (FFR) 37 is formed at the location where a neutral magnetic field exists. Theoretically, it is a single point, but in practice, it is a region formed by the competing magnetic fields colliding with the magnetic field lines. (See Gleich, B, 2005, Knopp, T, 2012). The structure and arrangement form a stable and uniform direct current (DC) field-free region (FFR), which can also be accomplished by a pair of Helmholtz coils driven by a DC amplifier. The pair of permanent magnets 32 and 32' enables the assembly of a simple and inexpensive platform, but there is no adjustability.

[0124] As shown in FIG. 10B, the hybrid point-of-care chip of the present invention includes functional mechanisms of sample processing, collection (functioning as a sample introduction region 21), dispersion, capture (functioning as an analysis region (also referred to as a region of interest (ROI)) 210), and waste collection (functioning as a fluid absorption region 26) in a single multiplexing device 20, and is a disposable member of the superparamagnetic particle imaging analysis device 30 of the present invention.

[0125] By providing an alternating drive field that forms a pair of Helmholtz excitation coils 35 and b35', the formation of alternating current (AC) within the field-free region 37 is completed. Usually, it is driven at 20KZ to 30KZ, and in this embodiment, it is 25KZ.

[0126] The term "excitation" is used to describe a magnetic field and a "drive field" and a "modulation field". Any magnetic material within this magnetic field vibrates at the frequency of the Helmholtz drive frequency and is thus modulated or driven.

[0127] To measure any response within the ambient magnetic field, a pair of receiving coils 36 and 36' (which are planar or circular) are simply attached, thereby physically approaching the material to be quantified as closely as possible. In this embodiment, in the case of a paramagnetic labeled biomolecule, the region of interest 210 or a plurality of regions are moved into a cylinder 310 provided with a pair of permanent magnets 32 and 32', a pair of excitation coils 35 and 35', and a pair of receiving coils 36 and 36' to measure the paramagnetic response and its harmonics within the cylinder 310. The field-free region 37 is such a region where the superparamagnetic particles are not magnetically saturated (i.e., not magnetized), but vibrate to overcome Brownian and Neel relaxation, and not only emit a fundamental excitation frequency of 25KZ, but also further emit harmonics of this frequency (identifiers of particle size and material). The signal is a linear quantification of the number of particles and their spatial positions.

[0128] In the second embodiment of the superparamagnetic particle imaging analysis device of the present invention shown in FIGS. 11A and 11B, the analysis device is an open-type analysis device. Similar to magnetic resonance imaging, superparamagnetic particle imaging requires a uniform magnetic field. In imaging during magnetic resonance imaging, the field of view (FOV) information from a detector or detector array is combined integrally (referred to as tomography) and displayed as a three-dimensional image generated from a contrast agent or the tissue itself. Magnetic resonance imaging requires a large magnetic field and a radio frequency (RF) coil to match approaching the center of gravity of these magnetic fields. In comparison, in the superparamagnetic particle imaging analyzer of the present invention, the behavior of the superparamagnetic label has a very large difference: it does not measure the proton array interfered by the RF magnetic field, quantifies the non-linear magnetization of the paramagnetic particles in the field-free region, and in the field-free region, only Brown and Neel relaxation control the direction of the field of each particle. The alternating field of the excitation coil at a specific frequency (for example, about 25 KZ) rapidly switches the magnetic poles of the particles from + to - at the basic rate M(t). The basic frequency of excitation generates not only the induction field but also harmonics that are a function of the properties and size of the particle material. The harmonic spectrum is the fingerprint and position of each magnetic label. (See Gleich, B., 2005, Goodwill, P. et al., "Multidimensional X-Space Magnetic Particle Imaging", IEEE Trans Med Imaging, 30(9):1581-1590 (2011), Knopp, T. et al., 2012).

[0129] In the second embodiment of the superparamagnetic particle imaging analyzer of the present invention shown in FIGS. 11A and 11B, the general geometric shape of the three-dimensional imaging device used for small animals and humans disclosed below (Gleich, B., 2005, Goodwill, P., 2011, Konkle J., "Magnetic Particle Imaging with Advanced Tomographic Reconstruction Methods", Ph.D. thesis, University of California, Berkeley (2014)) is improved and combined with a new method (the hybrid point of care tip of the present invention). Instead of the point or region without a field (FFP or FFR) 37 in the embodiment shown in FIGS. 10A and 10B, a linear portion of the neutral field-free region, called FFL37', is created. The second embodiment improves the access to FFL37' compared to the extended design of the cylindrical midpoint used for mechanical placement, quantification, and imaging. FFL37' can access anywhere other than the region of interest in the co-linear cylinder, which limits the mechanical movement of all axes.

[0130] As shown in FIG. 11A, the housing 31 is the mechanical support of the superparamagnetic particle imaging analyzer 30 of the present invention and consists of a C-shaped frame 311, which limits the region without a linear field - forming a pair of NdFeB permanent magnets 32 and 32', and mechanically forcing the anode or cathode of each magnet to face each other to form a region without a linear field.

[0131] In the second embodiment, the magnet is linear, whereby the FFL37’ is also linear. Next, a pair of Helmholtz coils 35 and 35’ composed of Helmholtz, and a pair of parallel receiving coils 36 and 36’ sense the induced magnetic field and the harmonics generated by the b from the paramagnetic label in the analysis region 210 within the basic excitation region. As shown in FIG. 14, the analysis region 210 of the hybrid point-of-care chip 20 of the present invention is in close contact with and transmitted to the induction coils (also called excitation coils) 35 and 35’, thereby generating a measurable signal. The hybrid point-of-care chip 20 has sample and reagent processing capabilities (sample introduction region 21) and is distributed to the capture analysis column 210 and the fluid absorption region by the switching column 24 (see FIG. 9A).

[0132] The analyzer and analysis method of the second embodiment exceed the limits of lateral flow and point-of-care design of microfluidic devices. Since the linearity required for most common assays is not required for the analyzer of the present invention, the analyzer of the present invention has no such limitations, which is accomplished by the embodiments shown in FIGS. 11A and 11B, where by approaching the mechanical scanning of the region of interest in the rapidly moving FFL or FFL space, a metric can be generated for the number, state (bound or unbound), and position of the paramagnetic label within that region of interest.

[0133] In the third embodiment of the superparamagnetic particle imaging analyzer of the present invention shown in FIGS. 12A and 12B, the analyzer 30 has an "E"-shaped magnetic core excitation field used in the superparamagnetic particle imaging analyzer. Simplifying the excitation coils 35 and 35' in the second embodiment shown in FIGS. 11A and 11B, in order to generate a very uniform alternating current transmission or modulation field, the analyzer 30 uses a pair of "E"-shaped sintered ferrite magnetic cores 35a and 35a' facing each other and separated by a ceramic insulator (not shown). The electromagnetic coil windings 35b and 35b' on each column of the "E"-shaped magnetic core generate a magnetic field at two opposite poles of the "E"-shaped magnetic cores 35a and 35a' short-circuited to form a gap. The electromagnetic windings 35b and 35b' operate at an alternating current electrical frequency and there is a feedback circuit to the drive electronics (similarly, see FIG. 14). As shown in FIG. 9A, this is a modulation field for exciting and vibrating the magnetic labels of the hybrid point-of-care chip. A pair of superimposed permanent magnets 32 and 32' are forced to face each other anodically, thereby generating an FFR or FFP 37 within the gap of the "E"-shaped magnetic core. Outside the neutral region of the FFR 37, all materials within the direct current field are magnetized. The hybrid point-of-care chip 20 moves through the gap and the accessible region. By the alternating current induction of the paramagnetic label, the fundamental frequency of the alternating current field and the resulting harmonics are generated, and these are measured by the receiving coils 36 and 36' and transmitted to the electronics of the signal chain. The third embodiment using the combination of the "E"-shaped magnetic core design and the hybrid point-of-care chip of the present invention is an improved design because it gives an excellent SNR (signal-to-noise ratio) in approaching the alternating current excitation and receiving coils.

[0134] In the fourth embodiment of the superparamagnetic particle imaging analyzer of the present invention shown in FIGS. 13A and 13B, the analyzer is a single-sided superparamagnetic particle imaging analyzer having a transmitting coil arranged in two concentric circles and a separated receiving coil. Different from other embodiments, no permanent magnet is used in this embodiment. In this embodiment, the analyzer 30 has a pair of single-sided Helmholtz coils, realizes the effectiveness of the hybrid point-of-care chip 20, and interacts with the formed 1D field of view (FOV) 38. The circular external DC drive field is surrounded by a Helmholtz pair of AC fields. The arrow 39 in FIG. 13A indicates the selected field generated by the DC current in the opposite direction. It generates opposite fields and forms a symmetric FFR 37. When the hybrid point-of-care chip 20 exceeds the field of view of the receiving coil 36, the SPNPs in the analysis region of the FFR 37 are excited to generate signals. The advantage of using Helmholtz coils is that they function as both excitation coils and receiving coils at the same time. The analysis region 210 is brought beyond the sensor or to a single position in a gradient meter fashion for measurement.

[0135] FIG. 14 shows the signal chain and communication with the end user of the superparamagnetic particle imaging analysis apparatus and the hybrid point of care technology based on the alternating current coil of the present invention such as the embodiments shown in FIGS. 10, 11, and 12. As shown in FIG. 14, the shield 41 is used to provide magnetic shielding field driving and includes an alternating current driving field 42 and a direct current driving field 43. Both the alternating current driving field 42 and the direct current driving field 43. The magnetic resonance signal is sensed and transmitted in the receiving coil 36, and one or more fundamental frequencies of the signal are processed. In the signal preamplification unit 44, the harmonics are separated from the transmitted signal, and in the signal preamplification unit, the harmonics with a very small voltage are converted by using a preamplifier equipped with a notch filter and a lock-in amplifier. Currently, many of these devices can be purchased as individual chip packages, thereby reducing the number of individual circuit boards. Usually, the signal noise is reduced through the low-pass filter 45 and the 1 / f thermal noise is reduced in the crosstalk step, and the preamplified voltage is converted by the analog-to-digital conversion circuit 46, linearly amplified by the signal amplifier 47, and transmitted to the external central processor (CPU) 49.

[0136] The two-dimensional barcode read by the barcode reader 48 identifies the type, manufacturing date, user, and importantly, the lock-in amplifier based on each manufacturing lot of the hybrid point of care, and determines the quality and sensitivity of the hybrid point of care assay. These quantitative results can be information of the Bluetooth (registered trademark) 50 or the wireless signal output 51, and may be used as a wired signal (used for the operator and the hospital information system (HIS)) to the graphical user interface (GUI) 54. The graphical user interface 54 processes inputs to a portable storage device or a database, such as in-vehicle GPS, assay execution time, recording, and data management.

[0137] The graphical user interface 54 controls the conventional display 52 on the touch screen panel, and the printing machine 53 wirelessly prints the results and hard copies of the database, and further communicates with a sophisticated mobile application 55 that provides results, positions, recommended interpretations, and data history. With this feature, it becomes possible to perform graphical management of multiple data points in the real-time mobile environment of the mobile application 55.

[0138] The fifth embodiment of the superparamagnetic particle imaging analysis apparatus of the present invention is shown in FIGS. 15A to 15D, where the analysis apparatus is an NdFeB permanent magnet having a Hall sensor. In the analysis apparatus 30, an NdFeB cylindrical permanent magnet 32 is attached (for illustration purposes, having virtual magnetic field lines 63). The lines of force 63 are located in a uniform field of quantum energy formed and surrounded by an arbitrary magnetic source, and like the gravitational field, cannot be observed. These fields and their distributions are explained only in FIGS. 15A and 15B.

[0139] The NdFeB magnet 32 has magnetic field lines 63 that leave the south pole of the magnet and fold around at the north pole of the permanent magnet. These poles are named only for normal purposes. The Hall probe sensor 60 is disposed in the zero-free region 37b, and the magnetic field lines 63 that leave the circular magnet 32 reach the theoretical zero region at the exact center of the cylinder 33a and the force field. By attaching the Hall probe inside the cylinder 33a and to the non-magnetizable hollow shaft 71, it becomes possible to transmit the bias wire and signal 62 to the signal processing electronics according to the route as in FIG. 14, a drive field is not required, and the permanent magnet provides a DC magnetic field corresponding to the coil set, thereby greatly simplifying the design and manufacture of the apparatus. Since this field is similar to the drive DC field that forms the field-free region 37, no harmonics are generated. In the zero region 37b, the theoretical magnetic region is very small or there is no theoretical magnetic region, and there is only a narrow scattered field.

[0140] In the present invention, the chip 20 and the analysis region 210 are transported onto the sensor 60 either in a gradiometer fashion or as a single measurement, and the superparamagnetic particle imaging analyzer operates together with the hybrid point-of-care chip. By attenuating the zero magnetic field 37b to generate an induction signal to the Hall magnetic sensor 60, the magnetic labels in the analysis region are magnetized. The signal can be quantified or processed to generate an image.

[0141] Figures 15C and 15D show structural images of the superparamagnetic particle imaging analyzer having the hybrid point-of-care chip of the present invention. The permanent magnet 32 is an NdFeB magnet supported by a hollow shaft 61 which is a hollow non-magnetic Hall sensor axis, and a force field as illustrated by line 63 in Figures 15A and 15B is formed in the cylinder 33a of the magnet 32. The external support and shield 41 of the apparatus 30 of the superparamagnetic particle imaging analyzer houses the hollow shaft 61. The hollow shaft 61 routes the voltage and output signal of the Hall magnetic sensor 60 and at the same time is the mount for the probe within the zero magnetic field 37b of the permanent magnet 32.

[0142] The hybrid point-of-care chip 20 of the present invention, described in detail in Figures 9A - 9D, is shown in Figures 15C and 15D in side and plan views, is transmitted over the Hall magnetic sensor 60 and generates a gradiometer or spatially encoded signal which, as shown in Figure 14, is then processed by the CPU 49 and displayed by the GUI 54. The plan view of Figure 15D illustrates the relationship when the analysis regions 210a, 210b, and 210c are statically measured beyond or above the Hall magnetic sensor 60.

[0143] The sixth embodiment of the superparamagnetic particle imaging analyzer 30 operating with the non-linear hybrid point-of-care chip 20 of the present invention is shown in FIG. 16. In the sixth embodiment, the analyzer 30 is supported by a support base 64. The analysis regions 210a to 210e of the chip 20 move in an arc sequence with respect to the accessible superparamagnetic particle imaging permanent magnet 32 and the Hall magnetic sensor 60 described in FIGS. 15A and 15B and enter the zero region 37b. The generated signal passes through the Hall probe bias wire and the output signal 62. The form of the permanent magnet 32 and the non-linear analysis chip 20 reduces the total cost, and the analysis region 210 is multiplexed and imaged using prior knowledge of the spatial construction. The reconstruction technique used in computed tomography shows possible 3D imaging using the Hall magnetic sensor 60 or a plurality of sensors in the zero region 37b, and each sensor has a field of view (FOV) contributing to the mathematical model. The support base 64 of the analyzer 30 is a primary non-magnetic material that induces and distributes the magnetic field lines of the NdFeB magnet 32, and the Hall probe bias wire and the output signal 62 pass through the support base 64.

[0144] As shown in FIG. 17, a signal chain 40 is outlined that illustrates an example for measuring a sensor signal from a magnetic field. In the signal chain 40, a plug-in transformer 56 from a 120 - 240 AC wall power supply or an internal battery 57 provides a DC bias voltage to a Hall sensor 60'. A thermistor 65 calibrates the ambient temperature of the device, and an additional Hall sensor 60 calibrates all magnetic fields around the device, including the Earth's magnetic field. When the Hall sensor generates a signal or voltage, an offset correction integrated circuit feedback circuit of a filter 66 filters it and transmits it to a preamplifier 67 that is tightly coupled to a linear amplifier 47. The amplified signal is low power and matches a gate driver 68 and overcurrent protection (OCP), and is input to a central processing unit (CPU) 49 that manages a graphical user interface (GUI) 54 or user input. The GUI 54 is a human interface that instructs to transmit the magnetic signal measured from the Hall element 60' to an application program or program 55, a printer 53, a display 52, Bluetooth (R) 50 or wireless communication 51, for example, the Internet or the cloud.

[0145] Example 1. Dosage response of rabbit IgG-binding SPNP on the HY-POC chip of the present invention Materials: In this example, the materials used include rabbit IgG 150K (Arista Bio, AGRIG - 0100, lot 091325551, 2.88 mg / ml), purified goat anti - rabbit IgG (H&L) antibody (BioSpacific: G - 301 - C - ABS, lot WEB08, 6.39 mg / ml), magnetic beads (MicroMod, 09 - 02 - 132, 130 nm, 10 mg / ml), silica beads (CORPUSCULAR C - SiO - 10COOH, 10 - micron spheres, 10 mg / ml), nitrocellulose membrane (Millipore HF180UBXSS, lot R6EA62198C), N - hydroxysulfosuccinimide sodium (Sulfo - NHS) (Combi - Blocks category: OR - 6941, CAS number 106627 - 54 - 7), 1 - ethyl - (3 - dimethylaminopropyl) carbodiimide hydrochloride (EDC) (AK Scientific catalog number 965299), bovine serum albumin (BSA), polysorbate 20, coupling buffer: 10 mM PBS pH 7.4, storage buffer (10 mM PBS, 0.6 mg / ml BSA, 0.05% NaN3), and sample running buffer (10 mM PBS, 1 mg / ml BSA, 0.1% polysorbate 20).

[0146] Method: 1. Preparation of rabbit IgG labeled with magnetic beads (R - IgG - SPNP): 0.1 ml of 10 mg / ml 130 - nm magnetic beads was added to a 1.5 - ml tube, followed by 0.2 ml of PBS coupling buffer. Then, 0.01 ml of EDC (10 mg / ml) and 0.01 ml of NHS - Sulfo (10 mg / ml) were added to the above solution. The mixture was reacted at room temperature for 20 minutes while continuously mixing. Next, 0.57 ml of rabbit IgG (2.88 mg / ml) was added to the mixture, and the mixture was rotated at room temperature for 2 hours. Thereafter, the beads were pulled down with a magnetic separator, washed with 2×0.5 ml of PBS, and suspended in 1 ml of storage buffer to obtain R - IgG - SPNP.

[0147] 2. Preparation of stationary phase: Silica beads covalently bound to anti-rabbit IgG (GAR) antibody: 0.1 ml of 10 mg / ml 10-micron carboxylic acid silica beads were added to a 1.5 ml tube, followed by 0.2 ml of PBS coupling buffer, 0.01 ml of EDC (10 mg / ml), and 0.01 ml of NHS-Sulfo (10 mg / ml) to the above solution to form a mixture. The mixture was reacted at room temperature for 20 minutes with continuous mixing. Next, 0.156 ml of goat anti-rabbit-IgG (6.39 mg / ml) was added to the mixture. The mixture was rotated at room temperature for 2 hours and then centrifuged at 13,000 RPM for 3 minutes to remove the supernatant. Thereafter, the silica beads were washed with 2 x 0.5 ml of PBS and dried at 35°C for 12 hours.

[0148] 3. Preparation of anti-rabbit IgG (GAR) antibody-adsorbed silica beads: 0.1 ml of 10 mg / ml 10-micron carboxylic acid silica beads were added to a 1.5 ml tube, followed by 0.2 ml of PBS coupling buffer and 0.005 ml of goat anti-rabbit IgG (6.39 mg / ml). The mixture was rotated at room temperature for 12 hours and centrifuged at 13,000 RPM for 3 minutes. Next, the supernatant was removed, the silica beads were washed with 2 x 0.5 ml of PBS, and dried at 35°C for 12 hours.

[0149] 4. Preparation of anti-rabbit IgG (GAR) antibody-adsorbed nitrocellulose disk: 0.2 ml of PBS was added to a 1.5 ml tube, followed by 0.003 ml of goat anti-rabbit IgG (6.39 mg / ml) and a preformed nitrocellulose disk. The mixture was rotated at room temperature for 2 hours and the solution was removed. The nitrocellulose disk was dried at 35°C for 12 hours.

[0150] 5. Preparation of Rabbit IgG Hybrid Point-of-Care Chip: As shown in FIG. 18, the hybrid point-of-care chip 20 of the present invention was assembled. In chip 20, the switching column 24 was filled with ordinary 10-micron carboxylic acid silica beads. The first analysis region 210a was filled with silica beads covalently bonded to the GAR antibody as the stationary phase. The second analysis region 210b was filled with GAR antibody-adsorbing silica beads as the stationary phase. The third analysis region 210c was filled with a GAR antibody-adsorbing nitrocellulose disk as the stationary phase.

[0151] The structure of this chip shows two different methods for manufacturing the stationary phase: distributing the particles in the first analysis region 210a and the second analysis region 210b, or placing the pre-formed material in the third analysis region 210c. It further shows two different methods for loading the immobilization matrix onto the stationary phase: covalent bonding in the case of the first analysis region 210a, or physical adsorption in the case of the second analysis region 210b and the third analysis region 210c.

[0152] 6. Preparation of Sample Running Solution: Rabbit IgG (1 mg / ml) labeled with 130-nm magnetic beads was diluted to an appropriate concentration using a sample running buffer. The sample concentrations were 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 ng / ml.

[0153] 7. Execution of Assay: 0.01 ml of the sample running solution was pipetted and the solution was added to the sample introduction region. Waited until the sample introduction region was empty (3 - 5 minutes). Next, 0.01 ml of the sample running buffer was added. Waited until the sample introduction region was empty (3 - 5 minutes), and then the chip was read with an SPI device.

[0154] 8. Reading of Hybrid Point-of-Care Chip: The hybrid point-of-care chip was analyzed with an SPI analyzer. All three analysis regions 210a, 210b, and 210c were analyzed in a single scan within 17 seconds.

[0155] <Experimental Results and Discussion> The experimental results are shown in Figure 19, indicating that different concentrations of SPNP-labeled rabbit IgG samples (R-IgG-SPNP) were analyzed using the hybrid point-of-care chip of the present invention. As can be seen from the results in Figure 19, rabbit IgG was captured in all three analysis regions, and the magnetic signal was proportional to the SPNP concentration. The sample automatically flowed through the hybrid point-of-care chip 20, without the need for any pump. The assay time was less than 10 minutes. The sample volume could be less than 10 microliters. Different materials (silica beads and nitrocellulose membrane) can be used as the stationary phase for immobilizing the analyte (rabbit IgG). Different methods of functionalizing the stationary phase (covalent bonding and physical adsorption) can be used for loading the capture material. Moreover, the hybrid point-of-care chip can be manufactured using different stationary phase filling methods (in-situ filling and pre-forming).

Description of Reference Numerals

[0156] 10 - Analysis region of the 10-assay format (10’ or 10a’ represents different analysis regions arranged continuously), 11 - Compartment (11’, 11a, 11a’, 11z represent different analysis regions arranged continuously), 12 - Analyte, 13 - Superparamagnetic nanoparticles for labeling the analyte 12, which in the present invention is also referred to as superparamagnetic particles, superparamagnetic nanoparticle label, or superparamagnetic particle label. 20 - Hybrid point-of-care chip or chip array (20a, 20b, 20c, 20d, 20e, 20f, 20g, and 20h represent each chip in the chip array), 210 - Analysis region in the chip 20 (210a, 210b, 210c, 210d, and 210e represent the first, second, third, fourth, and fifth analysis regions arranged successively), 21 - Sample port, 22 - Reagent, 23 - Microchannel (23a, 23b, 23c, 23d, 23e are different microchannels for connecting different sections of the chip), 24 - Switching column, 25 - Filling material in the switching column 24, 26 - Absorption chamber, 27 - Absorption pad, 28 - Ventilation port. 30 - SPI analysis device, 31 - housing, 310 - cylinder, 311 - frame, 32 - permanent magnet, 33 - internal cylinder volume, 33a - internal cylinder of the permanent magnet, 34 - clamp, 35 - excitation coil, 36 - receiving coil, 37 - point without a field (FFP) or field - free region (FFR), 37’ - point without a linear field in the region (FFL), 37b - zero - free non - magnetized region, 38 - field of view (FOV), 39 - arrow indicating the selected field. 40 - signal chain, 41 - shield, 42 - AC drive field, 43 - DC drive field, 44 - signal pre - amplification unit, 45 - low - pass filter, 46 - analog - to - digital conversion, 47 - signal amplifier, 48 - barcode reader, 49 - central processor (CPU), 50 - Bluetooth (registered trademark), 51 - wireless signal output (WIFI), 52 - display, 53 - (wireless) printer, 54 - graphical user interface (GUI), 55 - mobile application, 56 - external 12 - 240V wall transformer. 60 - Hall magnetic sensor, 60’ - Hall element, 61 - hollow shaft, 62 - bias wire and signal output, 63 - generated magnetic field line representation (phantom of uniform magnetic quanta), 64 - support base, 65 - thermistor, 66 - filter (for offset / removal), 67 - Hall signal pre - amplification stage.

Claims

1. A three-dimensional hybrid point-of-care chip, comprising: at least one sample introduction region into which one or more samples containing one or more analytes are introduced; a plurality of compartments, each compartment having a stationary phase that is the same as or different from the stationary phase in other compartments, the stationary phase being a three-dimensional structure made of a porous material, and each stationary phase being functionalized with the same or different analyte capture materials for capturing one or more analytes in the sample; one or more switching columns; one or more fluid absorption regions; optionally, a reagent reservoir; microchannels for connecting the sample introduction region, the analysis region, the fluid absorption region, and the reagent reservoir; the three-dimensional hybrid point-of-care chip is composed of a laminate of film layers, each layer of the film layer through which one or more samples flow is called a level, and the levels in the laminate are in the range of 1 to 10; the sample introduction region is connected to the switching column via the microchannel, and the switching column is configured to direct the flow of one or more samples to the analysis region via other microchannels; the one or more samples sequentially pass through each compartment in the analysis region, and one or more analytes in the sample are immobilized on the stationary phase in the compartment; reach the fluid absorption region via the microchannel; A three-dimensional hybrid point-of-care chip that is guided by capillary force from the sample introduction region through the analysis region to different levels of the laminate and automatically reaches the fluid absorption region.

2. The laminate has two or more levels, and at least one of the switching columns is connected to the microchannel and guides the flow of one or more samples vertically to different levels. The three-dimensional hybrid point-of-care chip according to Claim 1.

3. The surface of the film layer is modified. The three-dimensional hybrid point-of-care chip according to Claim 1.

4. The film layer is made of plastic, adhesive, paper, wood, fiber, silicon, polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), glass fiber, cellulose, polysaccharide, protein polymer, or rolled particle material. The three-dimensional hybrid point-of-care chip according to claim 3.

5. The sample introduction region can maintain the volume of the sample within a range of 1 to 200 microliters, and the sample is whole blood, plasma, serum, urine, saliva, tears, sweat, fecal extract, DNA / RNA extract, antigen-containing solution, antibody, enzyme, protein, peptide, amino acid, hormone, organic molecule, inorganic molecule, biomarker, industrial pollutant, pathogen, virus, cell, cell culture extract or environmental sample. The three-dimensional hybrid point-of-care chip according to claim 1.

6. The number of the compartments in the analysis region is in the range of 1 to 20, and the form of the stationary phase in each compartment is a particle, a porous membrane, a water-insoluble gel, or a colloid. The three-dimensional hybrid point-of-care chip according to claim 1.

7. An analysis method using the hybrid point-of-care chip according to claim 1, comprising: identifying an analyte in a sample by a reagent in the sample introduction region, or a reagent in the reagent reservoir, or reagents in both; fixing the identified analyte to the stationary phase of the compartment in the analysis region; determining the characteristics of the analyte by a detection method which is a magnetic, acoustic, radioactive, fluorescence, chemiluminescence detection method or a combination thereof; the reagent includes magnetic particles, fluorescent particles, chemiluminescent particles, radioactive particles, or a mixture thereof functionalized with an antibody, a protein, a DNA / RNA probe or a chelating agent, and the antibody, protein, DNA / RNA probe or chelating agent labeled with magnetic, fluorescence, chemiluminescence or radioactivity binds to and identifies the analyte in the sample; the reagent is directly placed in the reagent reservoir or absorbed on a solid support and then placed in the reagent reservoir; An analysis method using a hybrid point-of-care chip.

8. The magnetic detection method is superparamagnetic imaging, total accumulation of magnetic particles, excitation inductance, alternating current magnetic susceptibility measurement method, CMOS alternating current magnetic susceptibility measurement method, Hall effect, magnetoresistance, giant magnetoresistance (GMR), colossal magnetoresistance (CMR), superconducting quantum interference device (SQUID), magnetic relaxation method or magnetic resonance imaging (MRI) spin relaxation time. An analysis method using the hybrid point-of-care chip according to claim 7.

9. Further comprising a liquid driving mechanism. The liquid driving mechanism is connected to one or more of the sample introduction regions. The three-dimensional hybrid point-of-care chip according to claim 1.

10. The switching column further includes a packing material, The packing material functions as a filter for removing impurities in one or more of the samples before the sample reaches the analysis region. The three-dimensional hybrid point-of-care chip according to claim 1.

11. Further, a red blood cell separation device is provided in one or more of the sample introduction regions. The three-dimensional hybrid point-of-care chip according to claim 1.

12. The stationary phase is in the form of particles, and the particles are made of plastic, silica, glass, aluminum oxide, organic polymer, inorganic polymer, and biodegradable polymer. The three-dimensional hybrid point-of-care chip according to claim 6.

13. The stationary phase is in the form of a porous membrane, and the porous membrane is made of plastic, fiber, polymer, polysaccharide, cellulose, paper, wood, biological construct, biomatrix, glass fiber, biodegradable polymer, and protein polymer, and is made of woven, non-woven or rolled particles. The three-dimensional hybrid point-of-care chip according to claim 6.

14. The stationary phase is functionalized by physical adsorption or covalent bonding with one or more identification reagents specific to one or more of the analytes in one or more of the samples. The three-dimensional hybrid point-of-care chip according to claim 1.

15. The stationary phase is arranged in a rear region pre-formed to the shape and size of the analysis region. The three-dimensional hybrid point-of-care chip according to claim 1.

16. The fluid absorption region consists of a chamber with a fluid absorption pad, The fluid absorption pad is made of hydrogel, particles, rolled particles, or a porous membrane. The three-dimensional hybrid point-of-care chip according to claim 1.

17. The analysis region is provided along an arc. The three-dimensional hybrid point-of-care chip according to claim 1.

18. A plurality of the three-dimensional hybrid point-of-care chips according to claim 1 are provided as a chip array, Each three-dimensional hybrid point-of-care chip shares the same sample introduction region at the central position of the chip array. The three-dimensional hybrid point-of-care chip according to claim 1.

19. Further comprising one or more ventilation openings for leaking air, at least one of the ventilation openings being within the fluid absorption region, The three-dimensional hybrid point-of-care chip according to claim 1.

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